Advances in Carbohydrate Chemistry and Biochemistry, Vol. 55

PREFACE

This issue of Advances features an extensive article by García Fernándezand Ortiz Mellet (Seville, Spain) on N-thiocarbonyl derivatives of carbohy-drates. The classic role of these highly reactive derivatives in the synthesisof heterocyclic structures attached to sugar residues has in recent years ex-panded greatly and now touches on many areas of glycobiology. These de-rivatives provide simple and efficient methods for tailored construction ofcomplex structural targets, as already well exemplified in established pro-cedures for preparation of neoglycoproteins; newer and developing aspectsinclude potential applications in such areas as solid-phase synthesis andcombinatorial chemistry.

A chapter contributed by Varela and Orgueira (Buenos Aires, Ar-gentina) deals with synthetic polyamides formed from sugar derivativescontaining amino and carboxyl functionalities. Such chiral analogues of ny-lon present interesting structural aspects stemming from stereochemicaldifferences in the monomers, and at the practical level they provide a modefor conferring hydrophilicity, biocompatibility, and biodegradability on thepolymers.

The classic work of Emil Fischer more than a century ago introduced thechemistry of sugars reacting with phenylhydrazine as a key tool in eluci-dating stereochemical relationships, and subsequent years revealed a richvariety of products arising from the reactions of different hydrazines withsugars. The structural identity of many of these compounds remained con-troversial for many years, however, until the advent of modern spectro-scopic and X-ray techniques. In a comprehensive article that reflects a career-long preoccupation with this subject, El Khadem, in collaborationwith Fatiadi (Washington, DC), surveys the entire modern literature on themultifarious compounds resulting from reactions of sugars and their carbo-cyclic analogues with hydrazine derivatives, emphasizing important appli-cations in synthesis and pointing out areas ripe for further exploration.

One of the oldest areas of enzymology is the action of glycosylases oncarbohydrate substrates, but precise mechanistic and stereochemical inter-pretation of the behavior of these enzymes is still lacking. With the aid inparticular of extensive recent X-ray structural work on glycoside hydro-lases and studies with substrate analogues, Hehre (New York) here presentsdetailed insight into the stereochemical factors at work in the action of the“inverting” and “retaining” enzymes.

Volume 52 of this series featured the comprehensive IUPAC-IUBMBdocument “Nomenclature of Carbohydrates” that meets a long-standingneed for up-to-date standardized nomenclature for sugar derivatives andcomplex saccharides. Ongoing work of the international committee has

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now brought about the updated set of recommendations “Nomenclature ofGlycolipids,” which is published in this volume.

The life and work of two Nobel Prize winners who made major contri-butions to the carbohydrate field are recorded here by Buchanan (Bath,UK) writing on Alexander Lord Todd and by Moses (London) with an ar-ticle on Melvin Calvin. Godshall (New Orleans) provides an appreciationof Margaret Clarke, whose global influence in the field of sugar technologywas but one aspect of her remarkable personal talents in bringing togethercarbohydrate scientists around the world in constructive synergy.

Washington, DC DEREK HORTON

September 1999

PREFACEx

4888 Horton FM 11/17/99 3:05 PM Page x

LORD TODD

1907–1997

Alexander Robertus Todd was born in Cathcart, a southern suburbanarea of Glasgow, Scotland, on October 2, 1907, second in a family of three.His father, Alexander Todd, then a clerk in the head office of the GlasgowSubway Railway Company, became its secretary and was later managing di-rector of the Drapery and Furnishing Cooperative Society Limited, a sub-stantial department store at Glasgow Cross. His paternal grandfather was ajobbing tailor living close to the Gorbals area of Glasgow. His maternalgrandmother (née Ramsay) was the daughter of a farm worker on theDuke of Hamilton’s estate at Cadzow in Lanarkshire who married RobertLowrie, a foreman in an engineering works in Polmadie. His mother, JaneLowrie, was born near to his father’s birthplace. Both parents had only anelementary education and Alexander was essentially self-taught, with thehelp of night classes. His parents believed passionately in the value of edu-cation for their children.

In 1912, Alex Todd went to Holmlea Public School in Cathcart, near tohis home. When, in 1914, the family moved a few miles to Clarkston, the ex-pectation that the tramway would be extended was not fulfilled and theyoung Alex had a long walk to and from school each day. In 1918 he enteredAllan Glen’s School in the center of Glasgow, accessible by train fromClarkston, and passed the Scottish Education Department’s Qualifying Ex-amination to enter the senior school in 1919. His parents were encouragedin this course of action and in his future education by his uncle, WalterTodd, who was a Glasgow University graduate. Allan Glen’s was alsoknown as the Glasgow High School of Science, and there was a strong em-phasis on the teaching of mathematics, physics, and chemistry. Alex’s inter-est in chemistry, awakened by a home chemistry set and encouraged by theproximity of Baird and Tatlock, the laboratory suppliers to the school lab-oratory, was fostered by the good teaching of Robert Gillespie. The samecould not be said for his physics instruction and Todd, in his autobiogra-phy,* blames this in part for his later attitude toward physical chemistry.

1

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 55

Copyright © 2000 by Academic Press0065-2318/00 $30.00 All rights of reproduction in any form reserved.

* “A time to remember: the autobiography of a chemist,” Cambridge University Press (1983).

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Alex Todd passed the Scottish Higher Leaving Certificate in the spring of1924 and entered Glasgow University a few days before his 17th birthday.In his first year he won the Joseph Black medal and Roger Muirhead prize,which provided him with a scholarship for the remainder of his course. Hedid not enjoy the strong emphasis on quantitative inorganic analysis in thepractical classes, but in his final year he worked with T. S. Patterson, Profes-sor of Organic Chemistry, on the reaction of phosphorus pentachloride withdiethyl tartrate. The B.Sc. course also involved the study of subsidiary sub-jects which included physics, mathematics, geology, bacteriology, and metal-lurgy. He showed a marked aptitude for mathematics and became very in-terested in geology, including paleontology, which gave him an introductionto biology. Todd graduated in June, 1928 with first class honors, top of hisyear, and was awarded a Carnegie Research Scholarship to work with Pro-fessor Patterson on the optical rotatory dispersion of mannitol derivatives.He soon realized that the organic chemistry of naturally occurring com-pounds was the subject which most appealed to him. With Patterson’s en-couragement he applied to and was accepted by Professor Walther Borschein the University of Frankfurt-am-Main, to work on bile acids, beginning inOctober, 1929. The move to Germany widened his horizons in many ways.The combination of natural product and synthetic organic chemistry inFrankfurt was much to his liking and the level of equipment and avail-ability of experimental techniques, particularly catalytic hydrogenation and microanalysis, was much better than he had known before. He foundthat he had a flair for languages, and much later in his career took greatpride in introducing speakers in their native language, including Russianand, in one case at least, Mandarin Chinese. In addition, he formed lifelongfrienships with other British chemists who had made the pilgrimage toGermany, particularly B. K. Blount and A. L. Morrison. Two of his Glasgowcontemporaries, T. F. Macrae and A. Lawson, went to Munich at the sametime.

Todd submitted his Dr. phil.nat. thesis in the early summer of 1931 and,on Patterson’s advice, applied for an 1851 Exhibition Senior Studentship.When this was successful he joined Oriel College, Oxford in September,1931 to work with Robert Robinson, who had recently moved from Man-chester. Following earlier synthetic studies by Alexander Robertson, hesynthesised the diglucoside anthocyanins hirsutin, malvin, pelargonin, pe-onin, and cyanin as their chloride salts.1 In his autobiography Todd recallsthe first crystallization of the intermediate crucial to the project, 2-O-(2,3,4,6-tetra-O-acetyl-D-glucopyranosyl)phloroglucinaldehyde, which hadproved intractable up to this point. His laboratory was adjacent to that ofRobinson, and during a tea break devoted to solving the Times crosswordwith Robinson and Blount, a methanolic solution was dropped accidentally

J. GRANT BUCHANAN2


into a hot water bath. The following morning crystals had appeared in thedirty flask, which awaited washing up. The almost daily contact with Robin-son also led to a collaboraiton with Professor Harold Raistrick on some an-thraquinone pigments isolated from plant pathogens of the Helminthospo-rium group.

In 1934 Todd, as a Research Assistant and then a Beit Memorial Re-search Fellow, joined George Barger, Professor of Medicinal Chemistry atthe University of Edinburgh, to study the chemistry of the antiberiberi vit-amin B1 (thiamine). The laboratory was a cosmopolitan one and he formedhis first research group, which included Franz Bergel, recently arrived fromFreiburg. Through Barger, Todd established contacts with Hoffmann-LaRoche of Basle, where concentrates of the vitamin were prepared. This washis first experience of an industrial collaboration and one which was to lastfor the remainder of his career. In competition with well-financed groups inGermany and the United States (Merck), Todd and Bergel produced a syn-thesis of the vitamin2 which was not only the most elegant of the first syn-theses but, in its essentials, is used industrially to this day.

In 1936, when J. M. Gulland left the Lister Institute of Preventive Medi-cine to take up the Chair of Chemistry in Nottingham, Todd was appointedin his place and was made Reader a few months later. The head of bio-chemistry at that time was Robert Robison, well known for his pioneeringwork on sugar phosphates, and the Institute itself had a strong reputationin the field of vitamins. At the Lister, Todd completed the work on thiamineand its analogs and began studies on vitamin E. Making use of the animalfacilities in the Institute he isolated b-tocopherol from rice-germ oil, helpedto establish the main features of its structure, and began its syntheses andthat of a-tocopherol. He also studied the active principle of Cannabis indica(C. sativa) and the hyaluronidase in testicular extracts.

In 1937 Todd was approached by R. A. Millikan, President of the Cali-fornia Institute of Technology, with a view to a new Professorship of Bio-organic Chemistry in Pasadena. In March the following year he and his wifemade the trip to California, by ship and train, spending several weeks thereand making plans for future equipment, courses, and research. He estab-lished a lasting friendship with Linus Pauling, who would have been his col-league. He returned to Britain with a firm offer in his hand, havingpromised to give Millikan a definite reply within 10 days of his arrivalhome. Unexpectedly, there was a telegram awaiting him from the Vice-Chancellor of Manchester University.A few days later he found himself be-ing interviewed to replace Professor I. M. Heilbron, who was moving to Im-perial College, London. At the age of 30, he was appointed to the SirSamuel Hall Chair of Chemistry in Manchester University. The decision togo to Manchester was a turning point in his career.

LORD TODD 3


Established in Manchester, Todd continued research on vitamin E andthe constituents of Cannabis. He had many wartime commitments, includ-ing research on chemical defence and antimalarials, and was a member ofthe British team on penicillin. He was, however, able to begin the workwhose ultimate objective was the synthesis of the nucleotide coenzymes,several of which are closely related to the B vitamins, and potentially lead-ing to a study of the nucleic acids.Attention was directed initially to each ofthe components, the heterocyclic bases, the nucleosides, and the chemistryof phosphoric esters. This broad strategic plan,3 begun in Manchester, wascontinued and expanded when Todd was appointed to the Chair of OrganicChemistry in Cambridge in 1944.

J. GRANT BUCHANAN4

There already existed a large body of structural work on nucleosides andnucleotides, mainly by P.A. Levene and his collaborators at the RockefellerInstitute in New York. The Lister Institute had an excellent library on itstop floor and it is said that while he was there Todd became familiar withthe Levene and Bass monograph “Nucleic Acids.” The structures of thepyrimidine nucleosides uridine and cytidine had been established, apartfrom their anomeric configurations. Gulland and his colleagues had shown,by UV spectroscopy, that in the purine nucleosides adenosine and guano-sine the point of attachment of the ribofuranosyl unit to adenine and gua-nine was N9 rather than N7, but again the anomeric configuration was indoubt. Confirmation of the ribonucleoside structures by chemical synthesiswas highly desirable, and an unambiguous synthesis of adenosine was anearly objective. The first “nucleoside,”D-glucopyranosyladenine, had beensynthesized as long ago as 1914 by Emil Fischer and B. Helferich. The UVspectrum, studied later by Gulland, indicated that, like adenosine, it had N9substitution and its synthesis from “acetobromoglucose” implied the b-configuration.A synthesis from a suitable acylated ribofuranosyl halide, notthen available, would undoubtedly have led to adenosine but this would nothave established rigorously the N9 substitution. In his earlier synthetic


work with thiamine (vitamin B1) Todd had gained experience of pyrimidinechemistry and of 4,5-diaminopyrimidines in particular. He envisaged a syn-thesis of adenosine from a 4(6)-amino-6(4)-glycosylaminopyrimidine,4–6

followed by introduction of a 5-amino group. In the well-known Traubepurine synthesis, a 4,5-diaminopyrimidine is heated with formic acid toform the imidazole ring, conditions which would certainly cause cleavage ofa glycosylamino compound.

LORD TODD 5

During the thiamine work Todd had prepared 4-amino-5-thioformamido-6-methylpyrimidine under mild conditions from the diamine and found thatit underwent cyclization when heated above its melting point to give 6-methylpurine,7 a reaction which he was now able to exploit for purine nucleoside synthesis. A practical difficulty at the time was the scarcity of D-ribose as a starting material. Under wartime conditions and for sometime thereafter, ribose was treated with great respect and most of the earlyexploratory work was carried out with D-xylose. The first adenine nucleo-side to be synthesised by the unambiguous route was 9-D-xylopyranosyl-adenine,8,9 to be followed by 9-D-ribopyranosyladenine.10 The lactol ringsize was determined by periodate oxidation.11 An important synthesis, anearly success after the move to Cambridge, was that of 9-D-mannopyra-nosyladenine.12 When subjected to periodate oxidation it gave the same di-aldehyde as from adenosine and Fischer’s glucopyranosyladenine,13

thereby establishing 9-substitution and the b-configuration for all threecompounds.The synthesis of adenosine itself by this route proved more dif-ficult, but was achieved14 in 1949.

The glucopyranosides corresponding to the pyrimidine nucleosides uri-dine and cytidine had been synthesized by G. E. Hilbert and his colleaguesin 1930 and 1936, and a similar comparison of their periodate-oxidationproducts with those from uridine and cytidine established the b configura-tions here as well.13 The preparation of the 2,3,5-tri-O-acetylribofuranosylhalides enabled the synthesis of uridine and cytidine15 by the Hilbert routeand adenosine and guanosine16 by the Fischer and Helferich method from2,8-dichloroadenine. At this point the assignment of the b configuration of


the natural nucleosides depended on the mechanism of reaction of the gly-cosyl halides. Conclusive evidence for the b configuration in the ribonucle-osides was obtained when 59-O-p-tolylsulfonyl derivatives of adenosine andcytidine were found to undergo intramolecular cyclization to give cyclonu-cleosides, a reaction only possible in the b-anomeric configuration and con-firmed by X-ray crystallographic studies.17

Analogous structures were established for the 29-deoxynucleosides. Theywere not oxidized by periodate18 and could be converted into cyclonucleo-sides,19,20 thus showing the b configuration. In addition the ribonucleosideuridine was converted into 29-deoxyuridine,21 obtained by deamination ofthe naturally occurring 29-deoxycytidine, via O2,29-cyclouridine,22,23 a ver-satile intermediate exploited in Cambridge and elsewhere for the synthesisof 3-b-D-arabinofuranosyluracil (spongouridine) and other nucleosides.24

This and other cyclonucleosides are capable of a remarkable number ofuseful transformations.

In the early 1940s the methods of phosphorylation were fairly primitive.C. Neuberg had used phosphorus oxychloride at the beginning of the cen-tury, and a major advance was the introduction of diphenyl phospho-rochloridate by P. Brigl and H. Müller in 1939. Dibenzyl triesters, in whichremoval of benzyl protecting groups by hydrogenolysis occurred morereadily, had been prepared in 1939 by L. Zervas by the reaction of silverdibenzyl phosphate with an appropriate bromo compound. Dibenzyl phos-phorochloridate was a key compound, said by Zervas to be too unstable tobe useful. The preparation of this relatively labile acid chloride wasachieved by chlorination of the corresponding phosphite.25 In the longerterm the choice of benzyl protecting groups was a happy one. Selective removal of one benzyl group in a triester could be achieved not only by partial hydrogenolysis25–27 but by acid hydrolysis,28 aminolysis,29 anionicdisplacement,30–32 or reaction with phenol.33 Reaction of a derived phos-phate salt, originally a silver salt, with a phosphorochloridate afforded a pyrophosphate.24 The synthetic manipulation of phosphoric esters and pyrophosphates became for the first time a science and not a mystical art. Although many and improved protecting groups are now available,the benzyl ester still has its place in phosphorylation studies. The first application to a nucleotide was a new synthesis of adenosine 59-phosphateand subsequently adenosine 59-diphosphate (ADP).28 A major achieve-ment was the synthesis of adenosine triphosphate (ATP),34 whose lineartriphosphate structure had been confirmed by periodate oxidation.35 Uri-dine 59-diphosphate,36 a degradation product of uridine diphosphate glucose (UDP-Glc), and uridine 59-triphosphate37 were synthesized.Successful syntheses of the nucleotide coenzymes flavin adenine dinu-

J. GRANT BUCHANAN6


cleotide (FAD)38–40, UDP-Glc,41,42 and nicotinamide adenine dinucleotide(NAD)43–45 required the development of new methods for unsymmetricalP1P2 diesters of pyrophosphoric acid. A major contribution to pyrophos-phate, and later phosphodiester, synthesis was the discovery by H. G. Kho-rana that carbodiimides, which he had prepared in connection with his workwith G. W. Kenner on the peptide hormone ACTH, could be used as con-densing agents for phosphates.46 Other imidoyl phosphates were pre-pared47–49 as potential intermediates for unsymmetrical pyrophosphates,together with mixed anhydrides50,51 and phosphoramidates.52–56 Benzylphosphorochloridates51,57,58 derived from suitably protected nucleosideswere also prepared via phosphite intermediates. Some of these methodswere applicable to phosphodiester synthesis, as is described later. The syn-thesis of phosphates by reaction of a phosphate salt with a sugar-derivedepoxide25,59 was also studied. It was found that oxidation of a quinol phos-phate gave a reactive phosphorylating species which could be used to obtain pyrophosphates and in particular ADP.60,61 The speculation that this was related to biochemical oxidative phosphorylation was not borneout.

In parallel with the research directed toward nucleotide coenzymes,Todd became increasingly interested in the nucleic acids. In 1949 C. E.Carter and W. E. Cohn, using ion exchange and paper chromatography, re-ported that the alkaline hydrolysis of ribonucleic acid (RNA) yielded twoseparable isomers (a and b) of adenosine monophosphate, neither ofwhich was the 59-phosphate, and in 1950 that the other mononucleotidesalso existed as mixtures of two isomers. The earlier work by Levene hadapparently shown that alkaline hydrolysis of yeast RNA yielded only fourmononucleotides, the 39-phosphates of the corresponding nucleosides.Todd’s laboratory was in an ideal position to resolve the puzzle.62,63 It was eventually shown that adenylic acids a and b were the 29- and 39-phosphates,64 respectively, and the conflict with Levene’s work could beexplained by phosphate migration under the conditions of acid hydrolysisof the nucleotides. The structures of the other a and and b mixtures werealso assigned.65–67

Most important was the realization by D. M. Brown and Todd68,69 thatthe alkaline hydrolysis of RNA was analogous to the behavior of esters ofglycerol phosphates described by E. Baer and M. Kates and involved theformation of 29,39-cyclic phosphates which were further hydrolyzed irre-versibly to give a mixture of the 29- and 39-monophosphates. The monoben-zyl esters of the 29- and 39-phosphates were prepared and related to the par-ent nucleotides by hydrogenolysis, conditions which did not causephosphate migration. The action pattern of various diesterases, particularly

LORD TODD 7


pancreatic ribonuclease70,71 and a spleen nuclease,72 on these monobenzylesters showed that the phosphodiester linkage in RNA involved the 39- and59-positions of adjacent ribofuranose units.This was consistent with an anal-ogous (39→59)phosphodiester structure for DNA, which lacks a 29-hydroxylgroup and is therefore stable to dilute aqueous alkali. The primary struc-tures of RNA and DNA were thus determined. The work was sufficientlyadvanced to be reported to the 75th Anniversary Meeting of the AmericanChemical Society in New York in the autumn of 1951 and provided a firmbasis for the entirely independent X-ray structural work of Watson, Crick,Franklin and Wilkins on DNA in 1953.

Todd then turned his attention to the synthesis of the remaining mononu-cleotides66,73 and the dinucleoside phosphates and dinucleotides. (59→59)-Linked diesters of ribonucleosides were synthesised,74,75 but the crucial(39→59)-linkage was much more difficult and was first achieved in the de-oxyribose series.76 The “unnatural” adenosine 29-(uridine 59-)phosphatewas synthesised77 by the phosphorochloridate method and there were fur-ther studies on nucleoside phosphites.78,79 Although Todd himself did notfully exploit the methods for nucleotide phosphodiester synthesis, his worklaid the foundation for the spectacular advances in the synthesis of polynu-cleotides, particularly the phosphate triester and H-phosphonate routes. Heconsidered that that his natural successors in this field were A. M. Michel-son, F. Cramer, H. G. Khorana, and C. B. Reese. Methods for sequence de-termination in oligonucleotides were developed.80,81 The b-eliminationmethod,80 proposed independently by R. Markham and P. R. Whitfeld, hasbeen developed and applied successfully to individual ribonucleic acids bythese and other authors.

In Cambridge Todd pursued new research interests. When vitamin B12

was isolated from liver by Lester Smith in Glaxo Laboratories in 1948 hewas asked to undertake a chemical study. With A. W. Johnson the productsof hydrolysis were shown to contain a benzimidazole nucleotide82,83 andlater one of the crystalline degradation products84 gave an important leadin the X-ray studies of Dorothy Hodgkin, which finally solved the structureof the vitamin. Some synthetic work with V. M. Clark was directed towardthe corrin ring system, but this was not pursued.

A major project was the study of aphid pigments, a topic whose originscan be traced to Todd’s experience with anthraquinones in his Oxforddays. The compounds, though quinonoid in character, were much morecomplicated and only with the advent of NMR were the structures finallysolved. A. W. Johnson, S. F. MacDonald, and, later, D. W. Cameron, led anenthusiastic international group, whose work involved not only chemistrybut the detection and harvesting of aphid infestations from the broad bean

J. GRANT BUCHANAN8


fields of Cambridgeshire. In Manchester, and later in Cambridge with A. W. Johnson, he attempted to isolate and identify the “hatching factor”of the potato eelworm. There were other related problems of obligate par-asitism which were studied in Cambridge, but with little overall success,due mainly to problems of chromatographic fractionation and biologicaltesting. He was also involved with tropolone natural products, some alka-loid chemistry, and peptide antibiotics of the actinomycin and ostreogrycingroups.

Alex Todd was a master in the strategy of research. In his own words:“I always tell youngsters looking for a field of research that they should(1) choose an important one; (2) choose one large enough to give roomfor changes in direction; and (3) avoid fashionable fields and choose ifpossible one in which they themselves would be the authors of all or mostof the relevant literature.” His view of natural product chemistry em-braced biochemically important molecules, however intractable, and notjust those immediately amenable to the experimental techniques cur-rently available. He had a genius for organization and delegation, select-ing the right person for a particular job, and, in the case of young men, giv-ing them the confidence to succeed. When he moved to Cambridge fromManchester in 1944, at the age of 36, he found a Department requiringcomplete reorganization and renovation. He brought with him a band ofenthusiastic colleagues and research students who helped him to achievethis transformation. In spite of the upheaval and the wartime conditions,the momentum of research was not lost. The material changes werebrought about by the Laboratory Superintendent, A. R. Gilson, later re-sponsible for much of the planning for the new University Chemical Lab-oratory in Lensfield Road. When Todd retired from the Chair of OrganicChemistry in 1971, the new University Chemical Laboratory had been inoperation for 15 years and the Department had attained a high interna-tional reputation.

Todd applied his managerial skills to all of his committee work. He hadbeen elected a Fellow of Christ’s College, Cambridge in 1944 and becameMaster in 1963. During his Mastership (1963–1978) a major building pro-gram was undertaken and statutes changed to admit women students forthe first time. In Cambridge he played a major part in founding ChurchillCollege (including his proposal of the College motto “Forward”) and Dar-win College.As Chairman of the Syndics of the Cambridge University Presshe turned the Press into a profit-making organization.

Todd played a full part in national and international affairs. From1952–1964, under successive Conservative governments, he was Chairmanof the Advisory Council on Scientific Policy and Chairman of the Royal

LORD TODD 9


Commission on Medical Education (1965–1968). He was president of theChemical Society (1960–1962), of the Society of Chemical Industry(1981–1982), and of the British Association for the Advancement of Science(1969–1970). He was a managing trustee of the Nuffield Foundation(1950–1973) and its Chairman (1973–1979). He was a director of Fisons Ltd.(1963–1978) and a member of the National Research Development Cor-poration (1968–1976). He was much sought after as a consultant to major chemical and pharmaceutical firms. He was chairman (1980–1988)and later president of the Croucher Foundation in Hong Kong. He waspresident of the International Union of Pure and Applied Chemistry(1963–1965).

While working in Edinburgh Todd met Alison Dale, who was doing post-doctoral research in the Department of Pharmacology of the University.They were married in London in 1937. In his autobiography, with charac-teristic understatement, he speaks of his marriage to Alison as “perhaps thebest thing I ever did.” She gave up her own scientific career and was a greatsupport to him in all of his later enterprises. They had a son and two daugh-ters, and when Alison died in 1987, the year of their golden wedding, he feltthe loss very deeply. Alison’s father was Sir Henry Dale, the physiologistand Nobel prizewinner, and both he and Sir Robert Robinson were very in-fluential in advising Alex in his younger days.

Alex Todd was a gregarious man, greatly encouraged by his wife, Alison.When Master of Christ’s he and his wife made a point of meeting everymember of the College. The group that accompanied him to Cambridgefrom Manchester became the Toddlers, a dining club which met annually.He was a keen sportsman, a county-class tennis player in his youth, and anenthusiastic golfer and fisher. He eagerly participated in the annual labora-tory cricket match, and the writer remembers the determination with whichhe took a running catch on the boundary in 1949. He enjoyed traveling, of-ten with his wife, and had a wide circle of friends overseas.

Alex Todd was a dominating figure, both in stature and in personality.He was never afraid, after due consideration, to take unpopular or un-palatable decisions. In 1943 he was given the opportunity to succeed F.Gowland Hopkins as Professor of Biochemistry in Cambridge, with thestrong encouragement of Sir Henry Dale, Sir Robert Robinson, and SirCharles Harrington. He analyzed the situation with great care and turneddown the offer, to the great benefit of Organic Chemistry in Cambridgethe following year. Although Todd left Scotland in 1929, apart from his pe-riod in Edinburgh in 1934–36, he remained a Scotsman in temperamentand outlook. His appointment as the first Chancellor of Strathclyde Uni-versity in his home city of Glasgow (1965–91) gave him particular plea-sure. He received many honors, beginning with the Meldola Medal in

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1936. He was elected to the Fellowship of the Royal Society in 1942, be-came its president in 1975–80 and received three of its medals (Davy,Royal, and Copley). His work on nucleotides and nucleic acids led to theaward of the Nobel Prize for Chemistry in 1957. He was knighted in 1954and was made a Life Peer, Baron Todd of Trumpington, in 1962. In 1977he was appointed to the Order of Merit. He was a member of several Na-tional Academies worldwide and the holder of many honorary degrees athome and overseas.

Many of Alex Todd’s co-workers and students have taken up senior po-sitions in academic life and industry in Britain and overseas. In Cambridge,Dan Brown developed his interests in phospholipid and nucleoside chem-istry and later in molecular biology. Basil Lythgoe, who played an importantpart in the early stages of the nucleoside program, became Professor of Or-ganic Chemistry in Leeds. James (later Sir James) Baddiley held the Chairof Organic Chemistry (and later Chemical Microbiology) in Newcastleupon Tyne; Alan Johnson in Nottingham and Sussex; George Kenner andlater Ian Sutherland in Liverpool; Jan Michalski in L– odz, Poland; Ted Cor-bett in Dunedin, New Zealand; Charles Dekker in Berkeley, California;Malcolm Clark and David Hutchinson in Warwick; Stuart Trippett inLeicester; Grant Buchanan in Heriot-Watt University, Edinburgh; GordonKirby in Loughborough and Glasgow; Don Elmore in Queen’s University,Belfast; Colin Reese in King’s College, London; Cedric Hassall in the Uni-versity of the West Indies and Swansea; Len Haynes in the University of theWest Indies and the Open University; Hugh Forrest in Austin, Texas; RayBonnett in Queen Mary and Westfield College, London; Rod Quayle inSheffield and later Vice-Chancellor Bath University; Mike Blackburn inSheffield; Neil Hughes in Newcastle upon Tyne. Franz Bergel, and sometime later Cedric Hassall, became Director of Research at Roche Productsin Welwyn Garden City, where Frank Atherton continued his pioneeringwork in organophosphate chemistry. Herchel Smith became Director ofResearch at Wyeth in Philadelphia.

Following Alison’s death Alex Todd suffered several setbacks to hishealth. During that time Barbara Mann, his former secretary, who hadmoved to Cambridge from Manchester in 1944, looked after him with greatcare; he died on January 10, 1997. Although to some he appeared austere,the lasting impression of Alex Todd is one of directness, loyalty, and hu-manity. The world of science has lost one of its major figures.

I am greatly indebted to Professor Sir James Baddiley and Dr. Daniel M.Brown for their helpful comments and suggestions in the preparation ofthis memoir.

J. GRANT BUCHANAN

LORD TODD 11


REFERENCES

0(1) R. Robinson and A. R. Todd, J. Chem. Soc., (1932) 2293–2299; (1932) 2299–2303; (1932)2488–2496.

0(2) A. R. Todd and F. Bergel, J. Chem. Soc., (1937) 364–367.0(3) A. R. Todd, J. Chem. Soc., (1946) 647–653 (Pedler Lecture).0(4) J. Baddiley, B. Lythgoe, D. McNeil, and A. R. Todd, J. Chem. Soc., (1943) 383–386.0(5) J. Baddiley, B. Lythoge, and A. R. Todd, J. Chem. Soc., (1943) 386–387.0(6) J. Baddiley, B. Lythgoe, and A. R. Todd, J. Chem. Soc., (1943) 571–574.0(7) A. R. Todd, F. Bergel, and Karimullah, J. Chem. Soc., (1936) 1557–1559.0(8) G. W. Kenner, B. Lythgoe, and A. R. Todd, J. Chem. Soc., (1944) 652–656.0(9) G. A. Howard, B. Lythgoe, and A. R. Todd, J. Chem. Soc., (1945) 556–833.(10) J. Baddiley, G. W. Kenner, B. Lythgoe, and A. R. Todd, J. Chem. Soc., (1944) 657–659.(11) B. Lythgoe and A. R. Todd, J. Chem. Soc., (1944) 592–595.(12) B. Lythgoe, H. Smith, and A. R. Todd, J. Chem. Soc., (1947) 355–357.(13) J. Davoll, B. Lythgoe, and A. R. Todd, J. Chem. Soc., (1946) 833–838.(14) G. W. Kenner, C. W. Taylor, and A. R. Todd, J. Chem. Soc., (1949) 1620–1624.(15) G. A. Howard, B. Lythgoe, and A. R. Todd, J. Chem. Soc., (1947) 1052–1054.(16) J. Davoll, B. Lythgoe, and A. R. Todd, J. Chem. Soc., (1948) 967–969; (1948) 1685-1687.(17) V. M. Clark, A. R. Todd, and J. Zussman, J. Chem. Soc., (1951) 2952–2958.(18) D. M. Brown and B. Lythgoe, J. Chem. Soc., (1950) 1990–1991.(19) W. Andersen, D. H. Hayes, A. M. Michelson, and A. R. Todd, J. Chem. Soc., (1954)

1882–1887.(20) A. M. Michelson and Sir A. R. Todd, J. Chem. Soc., (1955) 816–823.(21) D. M. Brown, D. B. Parihar, C. B. Reese, and Sir A. Todd, J. Chem. Soc., (1958) 3035–3038.(22) D. M. Brown, Sir A. Todd, and S. Varadarajan, J. Chem. Soc., (1956) 2388–2393.(23) D. M. Brown, W. Cochran, E. H. Medlin, and S. Varadarajan, J. Chem. Soc., (1956)

4873–4876.(24) D. M. Brown, D. B. Parihar, and Sir A. Todd, J. Chem. Soc., (1958) 4242–4244.(25) F. R. Atherton, H. T. Openshaw, and A. R. Todd, J. Chem. Soc., (1945) 382–385.(26) V. M. Clark, G. W. Kirby, and Sir A. Todd, J. Chem. Soc., (1958) 3039–3043.(27) F. R. Atherton, H. T. Howard, and A. R. Todd, J. Chem. Soc., (1948) 1106–1111.(28) J. Baddiley and A. R. Todd, J. Chem. Soc., (1947) 648–651.(29) J. Baddiley, V. M. Clark, J. J. Michalski, and A. R. Todd, J. Chem. Soc., (1949) 815–821.(30) V. M. Clark and A. R. Todd, J. Chem. Soc., (1950) 2023–2030; (1950) 2030–2034.(31) J. Lecocq and A. R. Todd, J. Chem. Soc., (1954) 2381–2384.(32) R. J. W. Cremlyn, G. W. Kenner, J. Mather, and Sir A. Todd, J. Chem. Soc., (1958) 528–530.(33) G. W. Kenner and J. Mather, J. Chem. Soc., (1956) 3524–3531.(34) J. Baddiley, A. M. Michelson, and A. R. Todd, J. Chem. Soc., (1949) 582–586.(35) B. Lythgoe and A. R. Todd, Nature, 155 (1945) 695–696.(36) N. Anand, V. M. Clark, R. H. Hall, and A. R. Todd, J. Chem. Soc., (1952) 3665–3669.(37) G. W. Kenner, A. R. Todd, R. F. Webb, and F. J. Weymouth, J. Chem. Soc., (1954)

2288–2293.(38) H. S. Forrest and A. R. Todd, J. Chem. Soc., (1950) 3295–3299.(39) H. S. Mason, H. S. Forrest, and A. R. Todd, J. Chem. Soc., (1952) 2530–2535.(40) S. M. H. Christie, G. W. Kenner, and A. R. Todd, Nature, 170 (1952) 923; J. Chem. Soc.,

(1954) 46–52.(41) G. W. Kenner, A. R. Todd, and R. F. Webb, J. Chem. Soc., (1954) 2843–2847.(42) A. M. Michelson and Sir A. Todd, J. Chem. Soc., (1956) 3459–3463.(43) L. J. Haynes and A. R. Todd, J. Chem. Soc., (1950) 303–308.

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LORD TODD 13

(44) L. J. Haynes, N. A. Hughes, G. W. Kenner, and Sir A. Todd, J. Chem. Soc., (1957)3727–3732.

(45) N. A. Hughes, G. W. Kenner, and Sir A. Todd, J. Chem. Soc., (1957) 3733–3738.(46) H. G. Khorana and A. R. Todd, J. Chem. Soc., (1953) 2257–2260.(47) B. H. Chase, G. W. Kenner, Sir A. R. Todd, and R. F. Webb, J. Chem. Soc., (1956)

1371–1376.(48) G. W. Kenner, C. B. Reese, and Sir A. Todd, J. Chem. Soc., (1958) 546–551.(49) R. J. Cremlyn, G. W. Kenner, and Sir A. Todd, J. Chem. Soc., (1960) 4511–4514.(50) H. S. Mason and A. R. Todd, J. Chem. Soc., (1951) 2267–2290.(51) S. M. H. Christie, D. T. Elmore, G. W. Kenner, A. R. Todd, and F. J. Weymouth, J. Chem.

Soc., (1953) 2947–2953.(52) F. R. Atherton, H. T. Openshaw, and A. R. Todd, J. Chem. Soc., (1945) 660–663.(53) F. R. Atherton and A. R. Todd, J. Chem. Soc., (1947) 674–678.(54) V. M. Clark, G. W. Kirby, and Sir A. Todd, J. Chem. Soc., (1957) 1497–1501.(55) D. M. Brown, J. A. Flint, and N. K. Hamer, J. Chem. Soc., (1964) 326–335.(56) V. M. Clark, Lord Todd, and S. G. Warren, Biochem. Z., 338 (1963) 591–598.(57) N. S. Corby, G. W. Kenner, and A. R. Todd, J. Chem. Soc., (1952) 3669–3675.(58) G. W. Kenner, A. R. Todd, and F. J. Weymouth, J. Chem. Soc., (1952) 3675–3681.(59) W. E. Harvey, J. J. Michalski, and A. R. Todd, J. Chem. Soc., (1951) 2271–2278.(60) V. M. Clark, D.W. Hutchinson, G.W. Kirby, and Sir A.Todd, J. Chem. Soc., (1961) 715–721.(61) V. M. Clark, D. W. Hutchinson, and Sir A. Todd, J. Chem. Soc., (1961) 722–725.(62) D. M. Brown, L. J. Haynes, and A. R. Todd, J. Chem. Soc., (1950) 408; (1950) 3299–3304.(63) D. M. Brown and A. R. Todd, J. Chem. Soc., (1952) 44–51.(64) D. M. Brown, G. D. Fasman, D. I. Magrath, and A. R. Todd, J. Chem. Soc., (1954)

1448–1455.(65) D. M. Brown, D. I. Magrath, and A. R. Todd, J. Chem. Soc., (1952) 2708–2714; (1954)

1442–1447; (1954) 1442–1447; (1955) 4396–4401.(66) A. M. Michelson and A. R. Todd, J. Chem. Soc., (1954) 34–40.(67) F. Baron and D. M. Brown, J. Chem. Soc., (1955) 2855–2860.(68) D. M. Brown and A. R. Todd, J. Chem. Soc., (1952) 52–58.(69) D. M. Brown, D. I. Magrath, A. H. Neilson, and A. R. Todd, Nature, 177 (1956) 1124–1125.(70) D. M. Brown, C. A. Dekker, and A. R. Todd, J. Chem. Soc., (1952) 2715–2721.(71) D. M. Brown and A. R. Todd, J. Chem. Soc., (1953) 2040–2049.(72) D. M. Brown, L. A. Heppel, and R. J. Hilmoe, J. Chem. Soc., (1954) 40–46.(73) D. H. Hayes, A. M. Michelson, and A. R. Todd, J. Chem. Soc., (1955) 808–815.(74) D. T. Elmore and A. R. Todd, J. Chem. Soc., (1952) 3681–3686.(75) R. H. Hall, Sir A. Todd, and R. F. Webb, J. Chem. Soc., (1957) 3291–3296.(76) A. M. Michelson and Sir A. R. Todd, J. Chem. Soc., (1955) 2632–2638.(77) A. M. Michelson, L. Szabo, and Sir A. R. Todd, J. Chem. Soc., (1956) 1546–1549.(78) J. A. Schofield and Sir A. Todd, J. Chem. Soc., (1962) 2316–2320.(79) G. M. Blackburn, J. S. Cohen, and Lord Todd, J. Chem. Soc., (1966) 239–245.(80) D. M. Brown, M. Fried, and Sir A. Todd, J. Chem. Soc., (1955) 2206–2210.(81) G. P. Moss, C. B. Reese, K. Schofield, R. Shapiro, and Lord Todd, J. Chem. Soc., (1963)

1149–1154.(82) J. G. Buchanan, A. W. Johnson, J. A. Mills, and A. R. Todd, Chem. Ind. (London), (1950)

426; J. Chem. Soc., (1950) 2845–2855.(83) A. W. Johnson, G. W. Miller, J. A. Mills, and A. R. Todd, J. Chem. Soc., (1953) 3061–3066.(84) R. Bonnett, J. R. Cannon,A.W. Johnson, and Sir A.Todd, J. Chem. Soc., (1957) 1148–1158.


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MELVIN CALVIN

(1911–1997)

Inspiration, competence, confidence, being in the right place at the righttime and sheer luck can help toward success in any endeavor. In his unrav-eling of the path of carbon in photosynthesis, Melvin Calvin was blessedwith them all.

Born in 1911 to an immigrant family in St. Paul, Minnesota, Calvin be-came interested in natural phenomena early in life: he collected rocks,watched birds and, in the family grocery store, mused about the physicalcomposition of all those products he saw on the shelves. Inevitably he be-came drawn to chemistry, earning his B.S. at the Michigan College of Min-ing and Technology in 1931 and a Ph.D. in 1935 from the University of Min-nesota.

There he worked with George Glockler on the electron affinity of halo-gens (initially iodine and later bromine and chlorine as well) from space-charge effects—Calvin’s problem was to measure the amount of energy re-leased when a halogen atom captures an electron and for that he had firstto devise the methods for doing so.

With personal savings and a postdoctoral fellowship from the NationalResearch Council, he went to England to spend 2 years with MichaelPolanyi at Manchester University. There he immersed himself in the inter-actions between quantum mechanical theory and chemical experimenta-tion, starting with platinum–hydrogen activation systems. It was an impor-tant formative period for Calvin: his first experience of living in Europe,meeting new and different kinds of people and, via the activation of hydro-gen by phthalocyanine and copper phthalocyanine, being introduced to pigment chemistry and light absorption which, 10 years later, was to lead him directly into biology and photosynthesis. While he was withPolanyi, Joel Hildebrand of the Berkeley Chemistry Department visitedManchester and Calvin’s future was discussed. Polanyi recommended him highly, letters passed and Gilbert Lewis invited Calvin to join theBerkeley faculty. His own inheritance and experience were the sources of Calvin’s inspiration, competence and confidence; he was soon to benefitfrom being in the right place at the right time, and good luck followed indue course.

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Ingen-Housz in the late 18th century showed that, in the light, greenplants fixed carbon dioxide. Sugars, including starch and sucrose, were laterrecognized as being among the main products of photosynthesis in differ-ent plants but the biochemical details of carbon dioxide fixation were en-tirely mysterious. Because the empirical formula for carbohydrates is(HCHO)n, one theory as late as the 1930s supposed a primary reduction ofcarbon dioxide to formaldehyde with subsequent polymerization. It was allsupposition: there was no supporting evidence.

Hope came with the discovery of radioisotopes. Here was a method thatoffered the opportunity of distinguishing between the carbon atoms of thesubstrate and those preexisting in the plant. Feeding radiolabeled carbondioxide to green plants should allow the biochemical sequences leading tosugars to be monitored. Early attempts to do so were made in prewarBerkeley by Sam Ruben and Martin Kamen but the experimental difficul-ties were immense because the only radioisotope of carbon then availablewas 11C, with a half-life of little more than 20 min; experiments had to becompleted within 2 or 3 h of manufacturing the nuclide.

Their work attracted the attention of Ernest Lawrence, the founder anddirector of the Radiation Laboratory which was later to become pivotal inthe Manhattan project and famous as a center for nuclear research and de-velopment. From his understanding of nuclear physics, Lawrence reasonedthat there should exist a long-lived carbon isotope with a mass number of14 which, if used as a tracer, would greatly aid such biochemical work. Earlyin 1940, in cans of ammonium nitrate solution used as a shield aroundLawrence’s cyclotron, Ruben and Kamen discovered 14C with a half-life of5700 years. Minute though the quantities were, the two scientists, joinedlater by Andy Benson, began to use it in an exploration of the path of car-bon in photosynthesis until the events of December 7, 1941 put all suchnonessential investigations on hold.

By the end of the war, Ruben was dead and Kamen and Benson had bothleft Berkeley; Lawrence, however, had forgotten neither 14C nor the prob-lem of photosynthesis. With radiocarbon now much more plentiful from nuclear reactors, Lawrence suggested to Calvin that he might like to un-dertake a twofold program: to develop the synthetic chemistry of 14C-labeled compounds and to resume the work on photosynthesis. Adequatesupplies of 14C as well as a building (the Old Radiation Laboratory—“ORL” as it was eventually known round the world) were made availableand funding was offered by the U.S.Atomic Energy Commission to take theproject forward. Then an Associate Professor in the Chemistry Depart-ment, Calvin grasped the opportunity with both hands; acting as overall di-rector himself, he invited Benson back to Berkeley to manage the photo-synthesis laboratory.

VIVIAN MOSES16


Calvin and Benson were both chemists and saw experimentation inchemical terms; their sort of chemistry was usually done in solution, accu-rately weighing or pipetting samples. Rather than using plant material inthe form of excised portions of leaves, with all the implied variability be-tween samples, they opted for a system based on microscopic algae culturedunder constant conditions. Whenever an experiment was to be performed,algae would be taken from the culture vessel with the experimenters confi-dent that the biological material would always be the same. Though theymight not have been quite right about that in those early days, they were alot closer than if they had used bits of leaf.

In their standard experimental conditions, aqueous suspensions of algalcells in flat-sided vessels (the famous “lollipops”) were illuminated by spot-lights and allowed to metabilize 14CO2 (supplied in solution as NaH14CO3)for short periods before the reactions were rapidly brought to a halt bydropping the reaction mixture into boiling ethanol. The identification ofproduct molecules was difficult because their actual quantities in chemicalterms were minute; it was the radioactivity label they carried which madepossible their separation and characterization.

It became clear that an early product of carbon dioxide fixation, perhapsthe very first in the sequence, was acidic and, from its behavior on ion-exchange resins, more likely to be a phosphoric than a carboxylic acid. (Inthe end it turned out to be both.) Intelligent guesses were made as to whatit might be.Years later, Calvin told the story of how its identity came to himas he sat nervously in his car in a red zone at the corner of Cedar and GroveStreets in Berkeley, waiting for his wife who was buying supplies in the lo-cal frozen-food outlet. Using isotope dilution and reisolation, this first com-pound was unequivocally identified as 3-phosphoglyceric acid, the “PGA”of so many subsequent presentations.

Had Calvin and his colleagues had forever to rely on ion exchange, it isdoubtful whether they could have progressed very fast or very far. Luckilyhelp was at hand—around 1948, Bill Stepka, then a graduate student in theBotany Department in Berkeley, introduced them to paper chromatogra-phy and radioautography. There was no looking back; analysis improved byleaps and bounds. Using both classical and novel methods, a whole range ofcompounds incorporating 14C from labeled bicarbonate was identified, al-ways on minute chemical quantities which were revealed and made acces-sible by virtue of their 14C labeling. The kinetic relationships between thesecompounds was explored: how fast and in which order did these substancesacquire 14C? Degradation methods were developed that allowed the accu-mulation of radiocarbon to be measured within the individual atoms of theproduct molecules.

It soon became clear that carbon dioxide was converted into hexoses by

MELVIN CALVIN 17


a reversal of the glycolysis reactions, the reducing power necessary to drivethe process deriving from the capture of sunlight by the energy-conversionmechanism of chloroplasts. A new problem arose which turned out to berather more difficult to solve: if the first stable compound of carbon dioxidefixation was the 3-carbon molecule PGA, what was the 2-carbon acceptorwith which carbon dioxide combined? Since, in short-term labeling experi-ments, this acceptor would not itself be labeled, there was no obvious wayto find out. The clues came form the kinetics of the intramolecular distrib-ution of 14C within many of the compounds already shown to be productsof carbon dioxide fixation. As it gradually became clear that the biochem-istry of carbon fixation was a cyclic process, Calvin and his colleaguesmoved toward the idea of a 5-carbon acceptor which, in accepting one molecule of carbon dioxide, would be split into two molecules of PGA,each identical with the other except for its labeling pattern. In two classicexperiments in 1954 and 1955, Peter Massini from Switzerland and Alex Wilson from New Zealand showed that when the light was switchedoff and then on again, or the carbon dioxide concentration suddenly de-creased, the kinetic behavior of compounds in the putative cycle was con-sistent with the operation of a cycle. By the late 1950s, the mystery of thepath of carbon in photosynthesis had essentially been solved, save for somemopping-up activities. A detailed understanding of the enzymology, partic-ularly of the primary fixation reaction, came later from work in a variety oflaboratories.

In 1961, Calvin was awarded the Nobel Prize in Chemistry for his workon photosynthesis. In many ways that changed his life. Until then he hadbeen so totally dedicated to his scientific research and teaching that he hadnot had much contact with the other minor affairs of mankind. Naive in pol-itics, with little time for culture, though having some industrial experiencevia consultancies, he had no need to concern himself with domestic mattersor even his own health as his wife, Genevieve, took care of those things forhim. Thus he had little to distract him from chemistry in general and pho-tosynthesis in particular, and this was what he lived for. It was not to last; in1962, invited by Kennedy to join the President’s Science Advisory Com-mittee, Calvin discovered Washington and the big world outside Berkeley.He became drawn into more and more activities away from his laboratoryand his scientific colleagues.

However, things in Berkeley were not standing still. In 1959, ORL, whichhad been within yards of Calvin’s office in the Chemistry Department, wasdemolished to make room for a major new chemistry building. The photo-synthesis group was displaced to a basement a quarter of a mile away downthe hill and inevitably its link with Calvin weakened; no longer was he inthe lab morning and evening, poring over chromatograms and graphs with

VIVIAN MOSES18


the experimentalists, working out what it might all mean. The thematicdrive that had been so important in keeping the group together had, in anycase, grown fainter as the primary problem had been solved. It was an un-satisfactory state of affairs and time to try to develop a new home. Using hiscontacts and extensive power of persuasion, Calvin succeeded in raising thefunding for a new building on the Berkeley campus reserved entirely forthe use of his group which, for the first time since its founding in 1945, wasto shelter under a common roof.

The new building, styled the Laboratory of Chemical Biodynamics(LCB), was remarkable for its circular form and internal architecture in-tended, in a modern format, to recapture the intimacy and open informal-ity of ORL. Occupied in November, 1963, the three floors were largeenough to house some 90 people and the group inexorably grew to fillthem. It was still a rosy time for science: for several years more, minimumeffort kept the funding coming from the Atomic Energy Commission andits successors, the Energy Research and Development Agency and the De-partment of Energy. Calvin’s own horizons seemed to expand in step withthe availability of space. While many of the earlier activities, including pho-tosynthesis, continued to develop, new ones came (and sometimes went):more work on the physics of light absorption and energy transduction inphotosynthesis; the biochemistry of learning in Planaria (including tests ofthose intriguing claims that cutting a trained worm in two would producetwo trained individuals, or that naive worms fed the ground-up corpses oftheir trained fellows would themselves acquire training—leading to storiesof a future in which students would be fed minced professors); genetic con-trol of protein synthesis; the biochemistry of aging; and, eventually, theproblems of cancer. But there was not the cohesion of the path of carbondays. Calvin himself could not keep up in detail with all this activity; never-theless, such was the nature of the group and of his colleagues’ relationshipsboth with him and among themselves, that there was virtually no tendencyto fragmentation. That came later, mainly after Calvin retired from the di-rectorship of the group in 1979.

Never after the close of the 1950s did the group experience the constantexcitement that participants in those early photosynthesis days still re-member with such pleasure. In part this resulted from the ending of a uni-fying theme. The senior staff had, of course, grown older and the gap in agegrew more pronounced between them and the graduate students and post-doctoral visitors who continued to flood into the group. (When the groupwas founded in 1945, Calvin, at 34, was by far the oldest member.) And, ofcourse, the big unified biological sciences research group, which had beenso extraordinary in the 1950s, was not longer quite such a novelty later on.Fragmentation of the Calvin group did begin to become more obvious

MELVIN CALVIN 19


when funding grew tighter in the 1970s and individual senior scientistsstarted to acquire their own grants. Unfortunately, it has now progressed sofar that, in that marvelous round building, the open spaces with commonequipment which once expressed a unitary organization are now divided byscreens and cabinets marking off individual territories.

Calvin early recognized the value of publicity. With a splendid programgoing very well indeed, he made sure the world knew what was happeningin his laboratory. Students and postdoctoral scientists from around theworld began to be attracted to that old wooden building in Berkeley (andto the people who worked in it) as well as to the other wing of the groupthat had started out developing the chemistry of 14C and progressed intoatomic recoil chemistry, brain biochemistry, the origin of life on earth and ahost of other activities. While it is difficult now to be accurate, the recordsshow that during the period of 1947–1971 at least 77 advanced degrees wereawarded to students in the group and between 230 and 250 postdoctoral vis-itors came from all corners of the globe. Nor did the pace slacken subse-quently. All this was achieved with the help of no more than 14 senior sci-entific staff, not all of them present during the whole of those 23 years.Benson, initially Calvin’s main collaborator in the photosynthesis studies,left Berkeley in 1954; he was replaced in that role by Al Bassham, who re-mained with the group for the rest of his working life.

With his wide and deep knowledge of chemistry, his ebullient personalityand his total dedication to his work, Calvin’s leadership qualities stood re-vealed. New ideas, not a few of them outrageous, flowed thick and fast. Heshowed a rare skill in choosing his collaborators and in inspiring all his col-leagues with his own enthusiasm. An excellent coordinator, it was often un-clear whether those new ideas were totally original or were derived by in-teraction of his chemical understanding with what he heard from the manypeople with whom he spoke or whose papers he read. Perhaps it did notmatter. What he did was to provide and question, in private and in public,much to the discomfort of his junior colleagues at the ritual Friday morningseminars: many a graduate student cringed as only halfway through his first,carefully prepared sentence, Calvin popped up with the first searchingquery.

He was never a man to stand on ceremony. A postdoc arriving with wifeand child from another continent would be invited to stay in the Calvinhousehold until they found their own place. And then Calvin might be seencarrying a sofa on his head into the new postdoc’s flat to help with its fur-nishing. There were always parties: parties for people leaving, parties forparticular events, parties at Christmas, parties for skiing, parties for hik-ing—and just parties. But in the lab the talk was overwhelmingly of science,results and interpretations, invariably with enthusiasm.

VIVIAN MOSES20


After stepping down from the directorship of LCB, Calvin maintained asmall research group in the Chemistry Department where he continued hisown explorations. He became interested in the supposed world shortage ofhydrocarbons, proposing the mass cultivation in desert regions of Euphor-bia from which, he claimed, an oil could be expressed usable in diesel en-gines without being refined. Whenever anyone visited, he wanted to knowand to understand what they were doing, why they were doing it and couldit not perhaps be done better some other way.

In 1942 he married Genevieve Jemtegaard. She supported him mar-velously as his career developed during the early years of their marriage,taking the domestic burdens off his shoulders so that he could get on withhis work largely unencumbered. In some of his projects she became his pro-fessional collaborator and together they published a number of papers.Sadly, she died in 1987 and Calvin never recovered from his loss. Not phys-ically robust and carrying the legacy of a severe heart attack that occurredwhen he was some 40 years old, he gradually became ever more frail andbegan to lose his intellectual vigor. Still, until the very last few days of hislife, he spent several hours each working day in his office, assisted by Mar-ilyn Taylor, that most remarkable of secretaries who had been with himsince 1948. She was still there in the office when he died at the age of nearly86 on January 8, 1997.

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MARGARET A. CLARKE

1942–1998

Margaret Alice Clarke, the Managing Director of Sugar Processing Re-search Institute, Inc., died of cancer on June 18, 1998. Margaret was inter-nationally known, not only within the specialized field of sugar processing,but in the much wider world of carbohydrate chemistry. Her outstand-ing ability to integrate and synthesize the industrial–technological aspectsof carbohydrate chemistry with theoretical and basic carbohydrate re-search, to bring people together from these disparate worlds for meaning-ful exchanges, and her intense support of research in new uses of agricul-tural byproducts and search for new products distinguished her among herpeers.

Margaret was born in Northern Ireland on May 8, 1942, the oldest childand only daughter among four siblings. At an early age, around 12, her fam-ily emigrated to Canada. Her mother, Peggy, describes a little girl who lovedto read and was able to do so as early as 3 years of age. She learned all ofthe domestic arts from her mother, including sewing, cooking, baking, gar-dening, and the fine craft of gift wrapping, skills she carried throughout herlife, to great advantage. She also learned ballet, drama, and piano, skills shedid not carry on with, but maintained a great love of each. From her father,she learned the love of socializing and the satisfactions of generosity towardothers.

Her visionary leadership qualities were demonstrated early on.When herfamily was leaving Ireland for Canada, Margaret wanted to leave a cup tobe won by a pupil in her school, Cambridge House, and so she created anaward of a silver cup, the Margaret Clarke Cup, to be won by a junior stu-dent who had striven to achieve a better grade. This was not for the studentwith the highest marks, but rather meant for a student who had shown themost improvement.

She received a B.Sc. (Honors) in Chemistry form the University of West-ern Ontario in 1963 and her Ph.D. in physical inorganic chemistry at TulaneUniversity in New Orleans in 1970. In 1980, she received an MBA fromLoyola University. She often told the story of how she came to choose NewOrleans for her graduate school. It happened toward the end of her term at

23




Western Ontario, on an icy winter day on campus, when she slipped for thethird time. Upon picking herself up, she decided then and there that shewould never again live in such a cold climate and set about finding a goodschool in a more hospitable climate. She chose New Orleans and Tulane. Itwas evidently a perfect fit, as she loved the city of New Orleans and thestate of Louisiana, immersing herself in its culture and cuisine and quicklymaking lifetime friends.

She was hired as a chemist with the Cane Sugar Refining Research Pro-ject in 1969 and in 1972 was named the administrator of the Project. In1981, she became the Managing Director of the organization, which at thetime expanded its scope and was renamed Sugar Processing Research In-stitute, Inc. She remained in this position until the time of her death.

In 1991, she married Per J. Garegg. With her marriage to Per, a distin-guished professor of chemistry at the Arrhenius Institute, University ofStockholm, came the full maturing of her unique talent for bringing to-gether carbohydrate chemistry groups in small meetings and to make connections among diverse carbohydrate interests. Per encouraged and supported her in this and was also a source of advice in scientific matters.

In 1984, during a Christmas visit to New Orleans, Margaret invited PerGaregg and Donald E. Kiely, University of Alabama at Birmingham, to giveseminars at the Southern Regional Research Center, where SPRI is lo-cated. The twin seminars were repeated the next year. Thus were born theNew Orleans Carbohydrate Symposia. In 1987, the program expanded to aday-and-a-half meeting called the Third New Orleans Carbohydrate Sym-posium. It was again held August 27–28 at the Southern Regional ResearchCenter, utilizing a Gordon Conference format. The speakers at this first formalized meeting, which quickly became nicknamed “Carbo Days”and later became affectionately known as “Carbodaze,” included JohnRobyt of Iowa State University, Yu-Teh Li of Tulane University School ofMedicine, Bertil Samuelsson of Astra AB, Geoffrey N. Richards of the University of Montana, Grant Taylor of the University of Louisville,Michael R. Ladisch of Purdue University, Roger Laine of Louisiana StateUniversity, Alfred D. French of the Southern Regional Research Cen-ter, and, of course, Per Garegg and Don Kiely, the “founding fathers.” In1989, the meeting was held prior to the spring meeting of the AmericanChemical Society to take advantage of European carbohydrate chemiststraveling to the United States, and this format continued until the Four-teenth New Orleans Carbohydrate Symposium, held April 2–4, 1998, the final meeting.

The list of participants at the New Orleans Carbohydrate Symposia reads

MARY AN GODSHALL24


like a “Who’s Who” of carbohydrate chemistry, and the group photographstaken at the end of each session are an excellent archive of the prominentcarbohydrate chemists of our time. Margaret expressed surprise and grati-fication when the meeting became so well considered that people traveledfrom Europe and other parts of the United States to New Orleans just toattend her meeting and not because they were then going on to attend theACS meeting. These meetings were always by invitation only.

A biography of Margaret Clarke is not complete without mention of thehistory of the company she helped to shape. The original incarnation of theSugar Processing Research Institute, Inc. (SPRI, Inc.) was founded in 1939under Dr. Victor R. Deitz in Washington, DC, as the Bone Char ResearchProject (BCRP) and under the auspices of the National Bureau of Stan-dards (NBS). At the time, Mr. Frederick J. Bates (“Sugar“ Bates) was headof the Sugar and Polarimetry Divisions, and he encouraged the formationof an industry group operating in close cooperation with the government towork on industrial problems. In 1937, there were a total of 26 projects at theNBS working through its Research Associated Plan of Cooperation ofGovernment with Industry. At the time, the charge of the BCRP was to re-search the chemistry and mechanisms of action of bone char, a newly de-veloped adsorbent for decolorizing sugar. In 1948, Dr. Frank G. Carpenterjoined the organization as a research chemical engineer. The reports andproceedings of the seven Technical Sessions on Bone Char are still impor-tant sources of information on decolorizing carbon and bone char in sugarrefining.

In 1963, NBS changed its policy and stopped housing industry-sponsoredresearch. However, the sugar industry wanted to continue the profitable in-teraction with government and instituted a cooperative research effort withthe U.S. Department of Agriculture in New Orleans at their Southern Re-gional Research Center. The BCRP was expanded in scope and renamedthe Cane Sugar Refining Research Project, Inc. (CSRRPI), and Dr. FrankCarpenter moved to New Orleans as its new director. He remained thereuntil his retirement in 1984. He hired Margaret Clarke as a researchchemist in 1969. While technically a refiners group, the work of CSRRPI in-evitably encompassed more and more work on raw cane sugar productionand quality from both the refiners’ and producers’ viewpoints. In the early1980s, beet sugar producers showed interest in membership, leading to a re-organization of the group into Sugar Processing Research, Inc. in 1981. (Itbecame the Institute in 1984.)

The organization is funded by annual subscriptions from sponsoringsugar companies, suppliers to the industry, and users of sugar, ranging from35–45 industrial sponsors at any one time. One of Margaret’s major duties

MARGARET A. CLARKE 25


as Managing Director of SPRI, Inc. was to seek out new sponsors and, ofcourse, to keep current sponsors on board. A major source of SPRI, Inc.support also comes from the USDA in the form of housing, supplies, equip-ment, and cooperation with resident scientists.

The goals of SPRI are (a) To examine the fundamentals of sugar produc-tion and refining processes in order to gain understanding of the chemicaland physical bases of these processes to improve operation and to developnew processes and products; (b) to study the chemical nature of sugars andnonsugars in sugar-producing plants to explain processing problems andproduct quality problems; (c) to develop new analytical methods for thesugar industry, to improve and expand methods in current use, and to applyrapid, practical methods to industrial needs; (d) to serve as an informationresource and database on sugar production, process problems, sugar manu-facturing byproducts, sweeteners, and associated areas for sponsoring com-panies of SPRI, Inc.; and (e) to assist in problem solving in research-relatedareas for sponsoring companies of SPRI, Inc.

During her tenure as the Managing Director, Margaret had almost com-plete fiscal and operational responsibility for the operation of the SugarProcessing Research Institute, Inc. She sometimes chafed at working withina government bureaucracy and hated having SPRI, Inc. confused with agovernment agency and often lamented the financial constraints that keptthe staff smaller than she would have liked, but she genuinely appreciatedthe cooperation with USDA scientists that allowed SPRI to be so produc-tive.

During her career with the sugar industry, Margaret authored or coau-thored over 200 technical papers, magazine articles, encyclopedia articles,books, and proceedings.A review of her titles will show that she did, indeed,meet the goals of research enumerated above for SPRI, Inc. Among themost important of her contributions was her leadership in applying new an-alytical technology to the sugar industry; namely, she pioneered the use ofhigh-performance liquid chromatography (HPLC) and ion chromatogra-phy (IC) systems in the 1980s and the use of Near Infrared (NIR) spec-troscopy in the 1990s. She was indefatigable in her pursuit of these applica-tions for the sugar industry, giving many talks all over the world on their useand carrying the instruments around with her to visit many cane and beetfactories and refineries. Her able coauthors in much of the HPLC workwere Mary Ann Brannan and, later, Dr. Charles Tsang. Her co-workers inthe area of NIR were Dr. Charles Tsang until his early death and then Dr.Leslie A. Edye.

Among other areas in which she made very important contributions tothe sugar industry are the development, along with Earl Roberts and Mary

MARY AN GODSHALL26


An Godshall, of the now Official AOAC method for dextran in raw sug-ars.1,2 She also developed a rapid assay for dextran3 in cane juice whichcould be used in laboratories with very limited equipment and which is nowin use throughout the Louisiana sugar industry as well as a quick test forstarch in cane juice, also applicable in poorly equipped laboratories. Shehelped to educate the sugar industry on the dangers of dextran formationfrom the ubiquitous soil microorganism Leuconostoc mesenteroides andsuggested ways to prevent or ameliorate it. She was very interested in elu-cidating the role of vegetative and microbial polysaccharides in causingprocessing problems for both cane and beet sugar producers. She devel-oped a series of simple tests that could distinguish the type of colorant thatwas in a raw sugar,4 which could then be used to determine how it wouldrefine and whether special attention would be needed in the refining of thesugar. Her most recent accomplishment was the elucidation of the role ofoleanolic acid in the formation of acid beverage floc from beet sugar. Shehad also become intensely interested in new products and value-addedproducts from sugar crops and had been involved in patenting a microbiallevan from sucrose5 and a diethylaminoethyl derivative of bagasse with de-colorizing properties.6

As already mentioned, the cooperation that SPRI, Inc., received fromUSDA scientists at the Southern Regional Research Center, where SPRI is housed, was very beneficial to SPRI. Margaret had successful and productive interactions with Frederick Parrish,7 Yuan W. Han,5,8 John R.Vercellotti,9 Wilton Goynes,7 Gillian Eggleston,10,11,15 and Armand Pepperman.15 A long-term working relationship was forged with BenjaminL. Legendre of the USDA Sugarcane Research Unit in Houma, Louisiana,in which work done at SPRI under Margaret’s direction was useful for thesugarcane breeding program.12–14 Additionally, she encouraged and men-tored a number of young scientists and exchange students, both in SPRIand at SRRC.

The tradition from the old Bone Char Research Project days was to or-ganize a conference on the research every 2 years, a custom that continuesto this day. Margaret edited the Proceedings of the Conference on SugarProcessing Research from about 1974 and was solely responsible forputting together the programs since about 1984. These proceedings containa treasure trove of information about all aspects of sugar processing and areunparalleled for the scope of information they contain relating to the areaof sugar processing.

In 1984, Margaret instituted a series of workshops to be held followingthe Conferences on Sugar Processing Research. These also have been pub-lished and represent an excellent resource for practical areas of concern



within the sugar industry. The workshops organized by Margaret Clarkewere 1984 Workshop on Dextran, 1986 Workshop on Raw Sugar Quality,1988 Workshop on White Sugar Quality, 1992 Workshop on Analysis of Sug-ars in Foods, 1994 Workshop on New Products of Sugarbeet and Sugarcane,1996 Workshop on Separation Processes in the Sugar Industry, and 1998Workshop on Sugar Around the World.

It is worthwhile to include here a quote from Dr. Steve Clarke, which ap-peared in the July, 1998 Sugar Journal, “It is not possible to write about thework of the Sugar Processing Research Institute without referring to the re-cent death of Dr. Margaret Clarke. Her leadership transformed the rathernarrowly focused Cane Sugar Refiners group into the more broadly-basedSPRI. Others will write more detailed and formal accounts of her work, butone essential and constant feature was the importance of good science, orgetting the chemistry right.”

She served on the editorial boards of Sugar Industry Abstracts, SugarTechnology Reviews, and the recently started Seminars in Food Analysis.The June, 1998 issue of Seminars in Food Analysis on applications of nearinfrared spectroscopy in the food industry was put together by her and con-tained a comprehensive review of her activity in the field of NIR analysis inthe sugar industry.14

She consulted for a number of organizations, including the United Na-tions Industrial Development Organization (UNIDO), the Food andChemicals Codex Committee, the National Academy of Sciences, the Hu-man Nutrition Service of the U.S. Department of Agriculture, Bioraf Den-mark, and the Fauji Foundation. She had an espeically close working rela-tionship with the Chinese sugar industry, making at least four trips to visitand consult, forging many personal and professional friendships and subse-quently supporting several Chinese scientists in fellowship-type programsat SPRI. It was in her work with Bioraf Denmark that she began to con-ceive of an integrated system for utilization of agricultural materials, specif-ically to treat and fractionate key crops to isolate new products, especiallyfor nonfood uses.

During her career, Margaret Clarke received a number of prestigiousawards and recognitions. In 1984 she received the Honorary Crystal Awardof the Sugar Industry Technologists (SIT), given for contributions to sugartechnology and the highest award given by this group of industrial peers.Perhaps her favorite honor was the Dyer Memorial Award, up until thenknown as the Sugar Man of the Year award, received in 1987, making herthe only woman and youngest person to have received either award and theonly recipient of both awards. She always insisted on calling it the SugarMan of the Year award, since it was not until after she received it that the

MARY AN GODSHALL28


name was changed. She was the 1996 Melville L. Wolfrom awardee, whichis given for service to the American Chemical Society Division of Carbo-hydrate Chemistry and outstanding contributions to the field of carbohy-drate chemistry. In 1998 she received a Meritorious Service Award fromICUMSA (International Commission for Uniform Methods of SugarAnalysis) for her years of dedication to the organization. She was made anHonorary Lifetime Member of the American Society of Sugar Beet Tech-nologists in 1997. Also in 1997, she was appointed an Honorary Member ofthe Italian Association of Sugar Technologists on the occasion of their 90thanniversary.

She maintained active professional memberships in numerous organiza-tions, among them, the American Chemical Society, the International Asso-ciation of Official Analytical Chemists (AOAC Internationsl), Sigma Xi, theHonorary Scientific Research Society, the Institute of Food Technologists,the United States National Committee on Sugar Analysis (USNC), the In-ternational Commission for Uniform Methods of Sugar Analysis(ICUMSA), Commission Internationale Technique de Sucrerie (CITS),Sugar Industry Technologists, American Society of Sugar Beet Technolo-gists, International Society of Sugar Cane Technologists, American Societyof Sugar Cane Technologists, International Carbohydrate Society, and theChemists’ Club of New York.

Herein are some of her activities with several of these organizations.AOAC International: In 1994 she was appointed as a member of the

newly formed committee, the AOAC Peer-Verified Methods AdvisoryCommittee. This committee was set up to respond to the expressed needfor many more analytical methods than could be routinely accommo-dated through the Official Methods Program and to avoid the perceivedbureaucratic burden of the Official Methods Program. This was a good fitfor Margaret, as she was especially interested in the peer-verified methodas a possible route for incorporation of sugar industry-approved methodsinto AOAC. She updated and rewrote much of the chapter on Sugars andSugar Products in the AOAC Official Methods Book. She had been theGeneral Referee for Sugar and Sugar Products since 1985, the duties ofwhich entailed coordinating numerous associate referees in areas as dis-parate as lactose purity testing, authenticity testing of honey, oligosac-charides in sugar products, sugars in cereals, sugar alcohols, stable iso-tope ratio analyses, screening of sulfites, and polarimetric methods, toname several. Margaret was able to use venues such as these to stayabreast of analytical technology as it may have related to the sugar in-dustry. She also served as the Chair of the Methods Committee on FoodNutrition.



American Chemical Society: She was a long-term member of the Amer-ican Chemical Society and was especially active as a member of the Divi-sion of Carbohydrate Chemistry, including terms as Chairman of the Divi-sion and, at the time of her death, a Councilor of the Division. She wasactive in raising funds for speakers to attend symposia and in organizing thevarious Divisional social functions, especially those that involved banquets,as she could always find an elegant venue. She presented at many of the na-tional meetings. In 1987, she organized two symposia for the Division ofCarbohydrate Chemistry, at the spring meeting in Denver on the Chemistryand Processing of Sugarbeet and at the fall meeting in New Orleans on theChemistry and Processing of Sugarcane. This resulted in a book, publishedin 1988, which was part of the Elsevier Sugar Series (Number 9), entitledChemistry and Processing of Sugarbeet and Sugarcane, edited by M. A.Clarke and M. A. Godshall.

Institute of Food Technologists: She became a member of IFT in 1988,the year she helped to organize a symposium, along with Mary An God-shall, on the role of carbohydrates in food. She was a member of four divi-sions, each reflecting an area of intense interest—the Biotechnology, Car-bohydrate, International, and Food Laws and Regulations Divisions.

USNC and ICUMSA: These sister organizations are involved with thedevelopment and collaborative testing of uniform methods of sugar analy-sis that can then be used in commerce, contracts, and governmental regu-lations. Margaret was involved in both, with the USNC as a long-time mem-ber of the Executive Committee and as a Referee or Associate Referee forvarious subjects, including Color and Turbidity and Oligosaccharides andPolysaccharides. She organized the workshop on The Role of the NationalCommittee in 1986 and was the chair of the ICUMSA Working Group onCollaborative Studies, whose report in 1990 led to the ICUMSA’s accep-tance of the IUPAC Harmonized Guidelines on Collaborative Studies,thereby making ICUMSA methods acceptable to other validating societies,such as AOAC and ISO.

Along with her activities in international and U.S. organizations, she wasalso involved in the specific carbohydrate communities in other countries.Her activities with Italy and Portugal were especially dear to her heart.

The First International Meeting of the Portuguese Carbohydrate Chem-istry Group, which was nicknamed “Glupor 1,” was held in Lisbon, PortugalSeptember 26–30, 1995. Margaret had been deeply involved in encouragingthe organization of this group, which was formed in 1994. She presented theopening plenary lecture of the industrial session on September 28, 1995,with a talk entitled, “Sugar Crops: Food, Feed and Industrial Resources.”The second meeting of this organization, “Glupor 2” was held in Porto, Por-tugal, September 21–24, 1997, and Margaret again participated by present-

MARY AN GODSHALL30


ing the opening plenary lecture of the industrial session on September 23,“New Compounds from Microbiological Products of Sucrose,’’ coauthoredby her, Earl J. Roberts, and Per J. Garegg. The third meeting, Glupor 3,scheduled for September, 1999, is dedicated to the memory of MargaretClarke.

Quoting from a letter from the coordinator of the Portuguese Carbohy-drate Chemistry Group, Amelia Rauter, upon hearing of her death, “Shewas a dedicated friend, an enthusiast for all types of carbohydrate chem-istry. Her scientific contribution was very important in stimulating a sym-biosis between sugar chemists and industrial scientists.”

She was also very involved with the Italian carbohydrate community. OnOctober 20, 1990, she was invited by the Italian Association of Sugar Tech-nologists (A.N.T.Z.A.) to give a lecture at Ferrara on research conducted atthe Sugar Processing Research Institute, with emphasis on polysaccharidesand coloring matter. On July 30th, 1996, she was invited by the Sugar Schoolat the University of Ferrara to lecture on the microbiological formation ofpolysaccharides from sucrose. Upon her death, six different Italian carbo-hydrate groups sent condolences.

Her prodigious activity was exhausting even to contemplate, yet seemedto invigorate her. In March and April of 1998, only months before herdeath, she organized three major international meetings simultaneously—the 1998 Sugar Processing Research Conference, the 1998 Workshop onSugar Around the World, and the Fourteenth New Orleans CarbohydrateSymposium. All three meetings were very successful.

To summarize her professional activities, it may be said that MargaretClarke had a special ability to recognize the potential of a laboratory dis-covery and to encourage and develop it to its maximum potential and tothen make it known to the scientific community. She moved easily betweenthe practical, technological world of sugar processing and the academicworld of scientific discovery and was able to bring items of that world intopractical use within the more technological universe. The benefit to thesugar industry of this type of synergy was immense.

A danger in writing about Margaret Clarke is that one can use up all theallotted space cataloguing her many activities and accomplishments with-out touching on the personal side. It should, however, be noted that forMargaret, her professional and personal lives, like those of many outstand-ing scientists, were indistinguishable one from each other—her work washer life and her professional colleagues were also her personal friends. Nev-ertheless, she also had many other friends who had nothing whatsoever todo with chemistry or carbohydrates, and they ranged from clerks in theFrench Quarter grocery store where she shopped, her hairdresser of manyyears, artists, jazz musicians and historians, and her beloved maid, Maggie,



whom Margaret kept employed even when Maggie reached a quite ad-vanced age and became somewhat infirm.

She was a host with no parallel, giving small parties and receptions at herFrench Quarter apartment for her Board of Directors, members of the ACSCarbohydrate Division, and other distinguished groups who were in townin addition to an annual Christmas party for friends and colleagues. Theseparties were often entertained by a jazz trio, made up of personal friends inthe New Orleans jazz scene, jazz being another of her interests. She alsoquite often threw a Christmas party at the Arrhenius Institute. An ex-ample was one held on December 8, 1995 in which a partial listing of themenu, all prepared by Margaret, consisted of eggnog, oyster patties in pas-try shells (prepared and brought over from New Orleans), artichokesquares, Mexican corn bread, chicken salad, beer cheese, blue cheesespread, deviled ham sandwiches, curried eggs, homemade bread, bourbonballs, brownies, almond toffee, chocolate peanut butter squares, and heav-enly hash.

Margaret was very hospitable and always wanted to do something foreveryone. She kept track of innumerable birthdays, and when she was intown, made home-baked cakes for many people, including those on theSPRI staff as well as for USDA friends. She had a tremendous repertoire ofbakery items, including traditional hot cross buns that she would bring onGood Friday.

The ICUMSA meeting, its 22nd Session and 100th Anniversary, held inMay, 1998 in Berlin, was the last meeting Margaret attended. She was veryill at that time, certainly too ill to be traveling so much, but she was cheer-ful and involved, never admitting to anyone her condition. It was my im-pression in retrospect that she wanted to see as many of her dear “sugarpeople” as she could in the time allotted her.

The outpouring of sympathy from all continents of the world upon Mar-garet’s death was stunning in its sheer volume and attested to the very per-sonal impact she had on many hundreds of people during her professionallife.

ACKNOWLEDGMENTS

The author is extremely grateful to Mrs. Margaret (Peggy) Clarke Sloane, Margaret’smother, for the information on her early childhood. Much helpful information, encourage-ment, and advice were also received from her husband, Per Garegg, and her colleague, John R.Vercellotti.

MARY AN GODSHALL

MARY AN GODSHALL32


REFERENCES

0(1) E. J. Roberts, M. A. Clarke, and M. A. Godhsall. Proc. Internat. Soc. Sugar Cane Technol,18 (1983) 104–126.

0(2) M. A. Clarke and M. A. Godshall. J. Assoc. Off. Anal. Chem. 71 (1988) 276–279.0(3) M. A. Clarke, J. Bergeron, and F. Cole. Sugar y Azucar, 82 (1987) 23–24.0(4) M. A. Clarke, R. S. Blanco, and M. A. Godshall. Proc. 1984 Sugar Process. Res. Conf.,

(1986) 284–303.0(5) Y. W. Han and M. A. Clarke. U.S. Patent 5,547,863, August 20, 1996.0(6) M. A. Clarke Garegg and E. J. Roberts. U.S. Patent 5,504,196, April 2, 1996.0(7) F.W. Parrish,W. R. Goynes, E. J. Roberts, and M.A. Clarke. Proc. 1986 Sugar Process. Res.

Conf., (1986) 53–59.0(8) Y. W. Han and M. A. Clarke. J. Agric. Food Chem., 38 (1990). 393–396.0(9) J. R.Vercellotti, M.A. Clarke, and L.A. Edye, Proc. 1996 Sugar Process. Res. Conf., (1996),

321–349.(10) G. Eggleston, J. R. Vercellotti, L. Edye, and M. A. Clarke. J. Carbohydr. Chem. 14 (1995)

1035–1042.(11) G. Eggleston, J. R. Vercellotti, L. A. Edye, and M. A. Clarke, J. Carbohydr. Chem. (1996)

81–94.(12) B. L. Legendre, W. S. C. Tsang, and M. A. Clarke. Proc. 1986 Sugar Process. Conf., (1986),

92–107.(13) B. L. Legendre, M. A. Clarke, M. A. Godshall, and M. P. Grisham. Proc. 1998 Sugar

Process. Res. Conf., (1998), 160–175.(14) M. A. Clarke, B. L. Legendre, and L. A. Edye. Semin. Food Anal. 3 (1998) 141–153.(15) G. Eggleston, M. A. Clarke, and A. B. Pepperman. Proc. 1998 Sugar Process. Res. Conf.,

(1998), 212–232.



CHEMISTRY AND DEVELOPMENTS OFN-THIOCARBONYL CARBOHYDRATE DERIVATIVES:

SUGAR ISOTHIOCYANATES, THIOAMIDES, THIOUREAS,THIOCARBAMATES, AND THEIR CONJUGATES

BY JOSE9 MANUEL GARCI9A FERNA9 NDEZ AND CARMEN ORTIZ MELLET*

Instituto de Investigaciones Químicas, CSIC, Américo Vespucio s/n, Isla de la Cartuja, E-41092Sevilla, Spain, and * Departamento de Química Orgánica, Facultad de Química, Universidad

de Sevilla, Apartado 553, E-41071 Sevilla, Spain

IIIII. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36IIIII. Energetics and Structure of N-Thiocarbonyl as Compared to N-Carbonyl

Compounds: Implications for Reactivity, Conformations, and Electronic Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

IIIII. Sugar Isothiocyanates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401. Methods of Synthesis of Sugar Isothiocyanates . . . . . . . . . . . . . . . . . . . . . . . . 402. Reactions of Sugar Isothiocyanates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643. Spectroscopic Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

IIIV. Sugar Thioamides and Thiolactams. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691. Addition of Carbon Bases to Sugar Isothiocyanates . . . . . . . . . . . . . . . . . . . . 692. Thionation of N-Carbonyl Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713. Thioacylation of Amino Sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 724. Miscellaneous Sugar Thioamide Syntheses . . . . . . . . . . . . . . . . . . . . . . . . . . . . 735. Conformational Properties of Sugar Thioamides . . . . . . . . . . . . . . . . . . . . . . . 73

IIIV. Sugar Thioureas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751. Coupling of Sugar Isothiocyanates with Amine Nucleophiles . . . . . . . . . . . . . 752. Coupling of Amino Sugars with Isothiocyanates . . . . . . . . . . . . . . . . . . . . . . . 793. Coupling of Sugar Isothiocyanates with Amino Sugars . . . . . . . . . . . . . . . . . . 824. Sugar Thioureas from Sugar Carbodiimides . . . . . . . . . . . . . . . . . . . . . . . . . . . 865. Functional Group Transformations in Sugar Thioureas . . . . . . . . . . . . . . . . . . 876. Spectroscopic and Conformational Properties . . . . . . . . . . . . . . . . . . . . . . . . . 87

IIVI. Sugar Thiocarbamates and Dithiocarbamates . . . . . . . . . . . . . . . . . . . . . . . . . . . . 881. Linear Sugar Thiocarbamates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 892. Cyclic Sugar Thiocarbamates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 913. Linear Sugar Dithiocarbamates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 954. Cyclic Sugar Dithiocarbamates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

IVII. Miscellaneous N-Thiocarbonyl Carbohydrate Derivatives . . . . . . . . . . . . . . . . . . 99VIII. Naturally Occurring N-Thiocarbonyl Carbohydrate Derivatives . . . . . . . . . . . . . 101IIIX. N-Thiocarbonyl Sugars in Molecular Recognition . . . . . . . . . . . . . . . . . . . . . . . . 102

1. Interactions with Membrane Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

35


Copyright © 2000 by Academic Press.All rights of reproduction in any form reserved.0096-5332/00 $30.00


2. Neoglycoconjugates, Glycodendrimers, and Glycoclusters. . . . . . . . . . . . . . . . 1043. Enzyme Inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1094. Artificial Receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

IIIX. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

I. INTRODUCTION

Investigations on N-thiocarbonyl carbohydrate derivatives go back al-most a century. As early as 1903, Schoorl1 reported on the reaction of D-glucose and thiourea as a model system to understand the condensation ofD-glucose and urea under physiological conditions. Although formation ofthe corresponding N-D-glucosylthiourea occurred, he did not succeed onisolating the pure product. Eleven years later, Emil Fischer2 described thesynthesis of the first sugar isothiocyanate, namely 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl isothiocyanate, and its transformation into sugar thio-carbamates and thioureas, including Schoorl’s thioureido sugar. Since then,a vast literature on N-thiocarbonyl sugar derivatives has accumulated andthis continues to be a very active field in carbohydrate chemistry.A key rea-son for this activity is obviously the diversity of reactions and the availabil-ity of reactants. Moreover, thiocarbonyl compounds are close isosters ofcarbonyl compounds and may thus be useful for structure–activity studiesin connection with naturally occurring, biologically active N-carbonylamino sugars.

The purpose of this chapter is to present the recent developments in thechemistry of N-thiocarbonyl-containing carbohydrate derivatives, includ-ing isothiocyanates, thioamides, thioureas, thiocarbamates, and dithiocarba-mates. Emphasis has been placed on the mutual interactions between thevarious functional groups that may coexist in a given molecule; their conse-quences in the structural and chemical properties; and the synthetic, bio-logical, and technical applications of these families of compounds. Glucosi-nolates,3 the precursors of the natural isothiocyanates and derivedN-thiocarbonyl compounds in plants, have been omitted from this chaptersince they constitute a rather homogeneous class of molecules in which theisothiocyanate functionality is masked by formation of a b-D-glucopyra-nosylthiohydroxamate O-sulfonate derivative. Nevertheless, we have in-cluded all types of glycoconjugates having these functional groups, eithersynthetic or isolated from natural sources, independent of the glyconic oraglyconic position of the N-thiocarbonyl segment.

The chemistry of sugar isothiocyanates and their reactions has been thesubject of a specialized article in this series by Witczak4 in 1986 with 143 ref-erences dating up to 1984. The reader is also referred to the earlier work ofGoodman5 on ureido sugars for a thorough historical perspective. It should

GARCÍA FERNÁNDEZ AND ORTIZ MELLET36


be noted that this chapter does not duplicate their material unless requiredfor the sake of congruity.The literature has been surveyed up to April, 1998.The preparation, properties, and reactions of N-thiocarbonyl compoundshave been the subject of valuable comprehensive6,7 as well as specializedaccounts,8–14 which should be consulted for details.

II. ENERGETICS AND STRUCTURE OF N-THIOCARBONYL AS COMPARED TO

N-CARBONYL COMPOUNDS: IMPLICATIONS FOR

REACTIVITY, CONFORMATIONS, AND ELECTRONIC PROPERTIES

The exchange of oxygen by sulfur in a molecule in general, and in car-bonyl compounds in particular, leads to close analogs which, however, mayshow notable differences in their physicochemical and biological propertiesbecause of the greater bulk and polarizability of the sulfur atom (covalentratio: O, 0.66; S, 1.04 Å) and its decreased electronegativity (Pauling scale:O, 3.5; S, 2.4).

Isothiocyanates, the esters of isothiocyanic acid, can be considered as theheteroallene representatives of N-thiocarbonyl compounds and their uni-versal progenitors. In spite of their strong electrophilic character, isothio-cyanates are less reactive and less hazardous to work with than the homol-ogous isocyanates. Dipole moments, Raman spectra, and structural dataindicate that the electron distribution in both families can be summarizedin structures 1a–1d (Fig. 1).9,15 While structure 1d contributes little if any-thing in the case of isocyanates, it is a significant contributor in the case ofthe sulfur analogs. The higher contribution of structures 1b and 1c to theelectron distribution of isocyanates probably accounts for the differences inthe electron-withdrawing capacity of the central carbon atom, responsiblefor the reactivity toward nucleophiles.

The coupling reactions of isothiocyanates with carbon, nitrogen, oxygen,and sulfur nucleophiles containing a labile hydrogen afford thioamides,thioureas, thiocarbamates, and dithiocarbamates, respectively. Since boththe N and S atoms of the resulting N-thiocarbonyl adducts may act as newnucleophilic centers, subsequent intramolecular reactions may take place,and these have been used for the preparation of a plethora of heterocyclicderivatives.12,13 In addition, the NCS group reacts with suitable agents toform 1,2-, 1,3-, and 1,4-cycloadducts. It may be assumed that one of the po-

N-THIOCARBONYL CARBOHYDRATE DERIVATIVES 37

FIG. 1. Resonance model for iso(thio)cyanates.


lar resonance structures 1b or 1c is involved at the stage of chemical reac-tion in these cases.9

In N-thiocarbonyl compounds, conjugation of the f-system of the CSgroup and the lone pair of nitrogen can take place. This electron-donationeffect of N-substituents at thiocarbonyl groups is quite important and sta-bilizing, more than in the related N-carbonyl derivatives.16 Consequently,and in contrast to many other thiocarbonyl compounds, thioamides,thioureas, and thiocarbamates are generally very stable and frequentlycharacterizable as crystalline solids. All available X-ray data show unam-biguously that the key atoms of N––C(S) functional groups are situated ina plane, as in the ligands at normal alkenic double bonds, suggesting largecontributions from sp2-hybrid atomic orbitals of the central carbon and ni-trogen atoms to the corresponding molecular orbital, that is, a preferencefor polar resonance structures such as 2b (Fig. 2).10

As a corollary, N-monosubstituted and unsymmetrically N-disubstitutedderivatives are expected to exhibit (E)-(Z) geometrical isomerism. In thecase of N,N9-unsymmetrically disubstituted thioureas, up to four differentconfigurational arrangements can be envisaged, the (E,E) isomer beinggenerally absent because of steric repulsion between the nitrogen sub-stituents (Fig. 3).

The typical activation energy values (Ea) for rotating the N-group out ofthe plane in N-thiocarbonyl derivatives fall in the range of 25–27 kcal mol21

for thioformamides, 20–25 kcal mol21 for other thioamides, 10–13 kcalmol21 for thioureas, 16–18 kcal mol21 for thiocarbamates, and 14–16 kcalmol21 for dithiocarbamates.17 A further consequence is that the corre-sponding NMR spectra exhibit temperature dependence, with tempera-tures of coalescence (Tc) for the signals of the N-substituents ranging from110–1708C for thioamides to 0–408C for thioureas.17–20

Although the most widely accepted explanation for the hindered rotationin N––C(S) bonds employs the foregoing 2a–2b resonance model, this can-not satisfactorily explain the fact that the rotational barrier is greater, typi-cally by 2–3 kcal mol21, for N-thiocarbonyls than for the carbonyl analogs.16

It has been suggested that this traditional picture is actually more appropri-


FIG. 2. Resonance model for N-(thio)carbonyl compounds.


ate for N––C(S) systems, charge transfer from N to S being favored by thesmall difference in electronegativity between carbon and sulfur and thelarge size of the sulfur atom.21 In carbonyls, charge transfer from C to O(structure 2c) would be more important. A model based on the greater do-nation of charge from the softer CS group as compared to CO to the ni-trogen atom has been proposed, based on theoretical calculations.22 The ori-gin of the rotational barrier would be then in the cost of pyrimidalization ofN, that is, rehybridation from sp2 (more electronegative) to sp3.

The greater inherent ability of sulfur over oxygen to stabilize an anion isevident in the species derived from the amides on account of the weakerCS over the CO bond (by about 30 kcal mol21). Consequently, replace-ment of oxygen by sulfur in carboxamides and ureas causes striking decreases23 in the N—H bond pKHA values. Conversely, the thiocarbonylbases are, in water solution, weaker bases than the corresponding carbonylbases on the pKBH

1 scale by 1.5–2.0 pK units, the basic center being the sulfur atom.24–27 Notwithstanding, thiocarbonyl compounds have beenshown to be consistently more basic than their carbonyl homologs in the gas phase.16,27,28 This variance has been ascribed to the strong attenu-ation of polarizability effects in water solutions and the poorer solva-tion of the protonated thiocarbonyl compounds. Regarding the nitro-gen center, the thiocarbonyl group acts as a powerful electron sink which decreases the basicity by 14.1 pK units as compared to the parentamine.29

The diffuseness of the electronic charge in the lone pairs of sulfur leadsto CS compounds being substantially weaker hydrogen-bonding accep-tors than the homologous CO derivatives.16,24,28,30 In contrast, the higheracidity of NH protons in N-thiocarbonyl derivatives correlates, in general,with an enhanced hydrogen-bonding donor capability. The potential for hy-drogen bonding is increased for thioureas because of the possibility to formtrans-bidentated bonds (3, Fig. 4). An NMR investigation of this type of as-sociation, using acetate anion as acceptor, has indicated, however, that a fastequilibrium between monodentated complexes (4a–4b, Fig. 4) is also com-patible with the present evidence.31

FIG. 3. Possible rotameric forms about the N––C(S) bonds for N,N9-unsym-metrically disubstituted thioureas.



III. SUGAR ISOTHIOCYANATES

Sugar isothiocyanates are among the most versatile synthetic intermedi-ates in carbohydrate chemistry. They play a pivotal role in the preparationof a broad series of functional groups such as amide, isonitrile, carbodi-imide, and N-thiocarbonyl derivatives allowing, simultaneously, the cova-lent coupling of a quite unrestricted variety of structures to the saccharidepart. Moreover, isothiocyanates are important reagents in heterocyclicchemistry, which may be exploited in the synthesis of nucleosides and otherN-glycosyl structures.The development of several efficient general methodsfor the introduction of the isothiocyanate functionality at different posi-tions of the carbohydrate molecule has translated these considerations intopractical approaches. Nowadays, sugar isothiocyanates can be preparedroutinely from inexpensive starting materials on the gram scale and someof them are commercially available. This has obviously contributed to thetremendous expansion of work on N-thiocarbonyl carbohydrate derivativesduring the past decade, including both synthetic aspects and biomedical as-says.

For the purpose of this chapter, three types of isothiocyanate derivativesof sugars are considered, depending on the location of the NCS group in themolecule: glycosyl isothiocyanates, deoxyisothiocyanato sugars, and isothio-cyanate conjugates. For a complete list of structures reported, see TablesI–III.

1. Methods of Synthesis of Sugar Isothiocyanates

a. Reaction of a Glycosyl Donor with an Inorganic Thiocyanate.—Thereaction of an O-protected glycosyl halide with an inorganic thiocyanatehas been the most widely used procedure for the synthesis of glycosylisothiocyanates.4,8 Problems with this synthesis arise from the ambidentcharacter of the thiocyanate anion. Whereas the use of silver thiocyanate


FIG. 4. Bidentated (3) versus monodentated hydrogen bonds (4a–4b)between disubstituted thioureas and acetate anion.


TABLE I

Glycosyl Isothiocyanates

Rotation

MP (8C) [a]D Solvent Reference

Monosaccharidesb-D-Arabinofuranosyl

2,3,4-Tri-O-benzoyl 149–150 — — 402-Deoxy-a-D-arabino-

hexopyranosyl4,6-Di-O-acetyl-3-bromo-

3-deoxy Amorphous — — 503,4,6-Tri-O-acetyl Syrup/70–72 1168.8/1142 CHCl3–CH2Cl2 49,503,4,6-Tri-O-benzoyl Syrup 184.4 CHCl3 493,4,6-Tri-O-(4-

nitrobenzoyl) Amorphous 173.0 CHCl3 492-Deoxy-b-D-arabino-

hexopyranosyl4,6-Di-O-acetyl-3-bromo-

3-deoxy Amorphous 146 CH2Cl2 503,4,6-Tri-O-acetyl 100–101/90–92 215.7/226 CHCl3–CH2Cl2 49,503,4,6-Tri-O-benzoyl Syrup 111.0 CHCl3 493,4,6-Tri-O-(4-

nitrobenzoyl) Amorphous 213.3 CHCl3 49a-D-Arabinopyranosyl

2,3,4-Tri-O-acetyl Syrup/175–178 — — 40,1612,3,4-Tri-O-benzoyl Foam 2114 CHCl3 34

b-L-Arabinopyranosyl2,3,4-Tri-O-acetyl Syrup 245.5 CHCl3 39

a-L-Fucopyranosyl2,3,4-Tri-O-acetyl 102 2173.6 CH2Cl2 47

b-D-Fucopyranosyl2,3,4-Tri-O-acetyl 94 120 CHCl3 152

b-L-Fucopyranosyl2,3,4-Tri-O-acetyl 102–104 — — 1522,3,4-Tri-O-benzoyl 58–60 2174 CH2Cl2 65

b-D-Galactofuranosyl2,3,5,6-Tetra-O-benzoyl Syrup 236 CHCl3 53,54

a-D-Galactopyranosyl2,3,4,6-Tetra-O-benzyl Syrup 1103 CHCl3 48

b-D-Galactopyranosyl2,3,4,6-Tetra-O-acetyl 92–94/97–99 110 CHCl3 37,38,47,1523,4,6-Tri-O-acetyl-2-deoxy-

2-iodo 145–150 1110 CHCl3 573,6-Di-O-benzoyl Amorphous — — 692,3,4,6-Tetra-O-benzoyl 68–71 1137 CHCl3 632,3,6-Tri-O-benzoyl 150–152 12 CH2Cl2 692,3,4,6-Tetra-O-benzyl 98–99 15.1 CHCl3 48

D-Glucosyl2,3,4,5,6-Penta-O-acetyl 132–135 — — 162




a-D-Glucopyranosyl2,3,4-Tri-O-acetyl-6-bromo-

6-deoxy 164.5 116.2 CCl4 1633,4,6-Tri-O-acetyl-2-deoxy-

2-thiocyanate 94.5–96 1249 CHCl3 562,3,4,6-Tetra-O-benzyl Syrup 173 CHCl3 342,3,4,6-Tetra-O-benzyl-5a-

carba 103–104 199 CHCl3 48,862,3,4-Tri-O-benzyl-6-deoxy-

6-fluoro Syrup 1125 CHCl3 4083,4,6-Tri-O-benzyl Oil 120 CHCl3 52

b-D-Glucopyranosyl Syrup 172 MeOH 58,59,1642-Acetamido-2-deoxy Syrup 125 MeOH 602-Acetamido-3,4,6-tri-O-

acetyl-2-deoxy 161 19.5 CHCl3 342,3,4,6-Tetra-O-acetyl 113–115 14.4 CHCl3 2,34,37,38,632,3,4-Tri-O-acetyl Amorphous 15.0 CH2Cl2 702,3,6-Tri-O-acetyl Syrup 246.0 CH2Cl2 703,4,6-Tri-O-acetyl-2-deoxy-

2-iodo 103–105 14 CHCl3 572,3,4,6-Tetra-O-benzoyl 147–148 117 CH2Cl2 642,3,4-Tri-O-benzoyl Foam 216.2 CH2Cl2 702,3,6-Tri-O-benzoyl 197–199 146 CH2Cl2 692,3,4,6-Tetra-O-benzyl Syrup 112 CHCl3 342,3,4-Tri-O-benzyl-6-deoxy-

6-fluoro 109–110 210.6 CHCl3 4082-Deoxy-a-D-lyxo-

hexopyranosyl3,4,6-Tri-O-acetyl Syrup 1129 CH2Cl2 50

2-Deoxy-b-D-lyxo-hexopyranosyl3,4,6-Tri-O-acetyl Syrup 125 CH2Cl2 50

a-D-Mannopyranosyl2,3,4,6-Tetra-O-acetyl 92–94 / Syrup 1132/1144 CHCl3 34,47,483,4,6-Tri-O-acetyl-2-deoxy-

2-iodo Syrup 159 CHCl3 57Methyl (a-D-glycero-D-

galacto-2-nonulopyranosyl)onate5-Acetamido-4,7,8,9-tetra-

O-acetyl-3,5-dideoxy 95–99 217.06 MeOH 51a-D-Quinovosyl

2,3,4-Tri-O-benzyl Syrup 1118 CHCl3 48b-D-Quinovosyl

2,3,4-Tri-O-benzyl 114–115 22.6 CHCl3 48b-L-Quinovosyl

3,4-Di-O-acetyl-2-deoxy-2-iodo 62–65 25 CHCl3 57

TABLE I—Continued

Rotation



a-L-Rhamnopyranosyl3,4-Di-O-acetyl-2-deoxy-2-

iodo Syrup 2115 CHCl3 572,3,4-Tri-O-acetyl 106.7–107.3 2185 CHCl3 42

b-L-Rhamnopyranosyl2,3,4-Tri-O-benzoyl 55–57 1182 CH2Cl2 65

b-D-Ribofuranosyl2,3,5-Tri-O-acetyl Syrup 23 CHCl3 34,412,3,5-Tri-O-benzoyl Syrup/96–97 291 CHCl3 53,165

3-Deoxy-a-D-ribo-hexopyranosyl2,3,4-Tri-O-benzyl Syrup 1110 CHCl3 48

3-Deoxy-b-D-ribo-hexopyranosyl2,3,4-Tri-O-benzyl 65–66 142.5 CHCl3 48

a-D-Ribopyranosyl2,3,4-Tri-O-benzoyl Syrup 257 CH2Cl2 72

b-D-Ribopyranosyl2,3,4-Tri-O-benzoyl Syrup 2187 CH2Cl2 72

a-D-Talopyranosyl3,4,6-Tri-O-acetyl-2-deoxy-

2-iodo 116–119 1110 CHCl3 57a-D-xylo-Hex-5(5a)-

enopyranosyl5a-Carba-2,3:4,6-di-O-

isopropylidene Syrup 1260 CHCl3 48b-D-Xylofuranosyl

2,3,5-Tri-O-benzoyl Syrup 236 CHCl3 53b-D-Xylopyranosyl

2,3,4-Tri-O-acetyl 72–73 231.2 CHCl3 39

Disaccharidesa-Cellobiosyl

2,3,6,29,39,49,69-Hepta-O-benzyl Syrup 166.7 CHCl3 48

b-Cellobiosyl2,3,6,29,39,49,69-Hepta-O-

acetyl 191–195 29.0 CH2Cl2 4,47,662,3,6,29,39,49,69-Hepta-O-

benzyl 114–115 128.5 CHCl3 48b-Chitobiosyl

N,N’-Diacetyl-3,4,39,49,69-penta-O-acetyl 157–160 19 CHCl3 42

b-D-Galp-(1→6)-b-D-Glcp-(1→NCS)2,3,4,39,49,69-Hexa-O-

acetyl-29-deoxy-29-isothiocyanato Amorphous 0.0 CH2Cl2 67

TABLE I—Continued

Rotation





TABLE I—Continued

Rotation


b-D-Galp-(1→4)-a-D-Manp-(1→NCS)a

3,6,29,39,49,69-Hexa-O-acetyl-2-deoxy-2-iodo 57–59 147 CHCl3 57

Gentiobiosyl2,3,6,29,39,49,69-Hepta-O-

acetyl 173–174 13 CHCl3 68a-D-Glcp-(1→4)-a-D-Manp-

(1→NCS)a

3,6,29,39,49,69-Hexa-O-acetyl-2-deoxy-2-iodo 60–62 1120 CHCl3 57

b-D-Glcp-(1→4)-a-D-Manp-(1→NCS)a

3,6,29,39,49,69-Hexa-O-acetyl-2-deoxy-2-iodo 127–129 133 MeOH 57

b-Lactosyl — — — 622,3,6,29,39,49,69-Hepta-O-

acetyl 157–159/167–169 -18.5 CHCl3 4,37,38,47,66a-Maltosyl

2,3,6,29,39,49,69-Hepta-O-benzyl Syrup 13.1 CHCl3 48

b-Maltosyl — — — 582,3,6,29,39,49,69-Hepta-O-

acetyl 120–123 157.7 CH2Cl2 4,47,662,3,6,29,39,49,69-Hepta-O-

benzyl Syrup 12.6 CHCl3 48b-Melibiosyl

2,3,4,29,39,49,69-Hepta-O-acetyl Syrup 192.8 CH2Cl2 66

2,3,4,39,49,69-Hexa-O-acetyl-29-deoxy-29-isothiocyanato Amorphous 160.0 CH2Cl2 67

Peracetyl trisaccharidesb-Chitotriosyl Amorphous 216.5 CHCl3 42b-D-GlcNAcp-(1→4)[a-L-

Fucp-(1→6)]-b-D-GlcNAcp-(1→NCS) 155–157 274.2 CHCl3 44

a-D-Glcp-(1→4)-b-D-Glcp-(1→6)-b-D-Glcp-(1→NCS) Amorphous 162 CHCl3 68

b-D-Manp-(1→4)- b-D-GlcNAcp-(1→4)-b-D-GlcNAcp-(1→NCS) — — — 46

a Erroneously named as glucopyranosyl isothiocyanate derivatives in the original reference.


TABLE II

Deoxyisothiocyanato Sugars

Rotation

MP(8C) [a]D Solvent Reference

Monosaccharidesa-D-Allofuranose

5,6-Di-O-acetyl-3-deoxy-1,2-O-isopropylidene-3-isothiocyanato Syrup 1183 CH2Cl2 84

3-Deoxy-1,2-O-isopropylidene-3-isothiocyanato 60–62 1151 CH2Cl2 84

3-Deoxy-1,2:5,6-di-O-isopropylidene-3-isothiocyanato 58–59 1151.1 CHCl3 84

b-D-Fructopyranose4,5-Di-O-acetyl-1-deoxy-2,3-O-

isopropylidene-1-isothiocyanato 107–109 219.0 CHCl3 831-Deoxy-2,3:4,5-di-O-

isopropylidene-1-isothiocyanato Oil 265.8 CHCl3 83a-D-Galactofuranose

5,6-Di-O-acetyl-3-deoxy-1,2-O-isopropylidene-3-isothiocyanato Syrup 110.0 CH2Cl2 84

3-Deoxy-1,2-O-isopropylidene-3-isothiocyanato Syrup 131.3 CH2Cl2 84

3-Deoxy-1,2:5,6-di-O-isopropylidene-3-isothiocyanato Oil 275.0 CHCl3 84

a-D-Galactopyranose6-Deoxy-1,2:3,4-di-O-

isopropylidene-6-isothiocyanato Oil 283 CHCl3 81,82a-D-Galactopyranoside, methyl

2,3,4-Tri-O-acetyl-6-deoxy-6-isothiocyanato Syrup 1105 CHCl3 75,82

2,3,6-Tri-O-benzyl-4-deoxy-4-isothiocyanato Syrup 171.0 CHCl3 89

6-Deoxy-6-isothiocyanato 120–122 1110 MeOH 826-Deoxy-6-isothiocyanato-2,3,4-tri-

O-trimethylsilyl Syrup 172.8 CH2Cl2 85D-Glucitol

2,3,4,5,6-Penta-O-acetyl-1-deoxy-1-isothiocyanato Syrup 158.8 CH2Cl2 75

a-D-Glucofuranose5,6-Di-O-acetyl-3-deoxy-1,2-O-

isopropylidene-3-isothiocyanato Amorphous 260.0 CHCl3 843-Deoxy-1,2:5,6-di-O-

isopropylidene-3-isothiocyanato 64–65 275.0 CHCl3 846-Deoxy-1,2:3,5-di-O-

isopropylidene-6-isothiocyanato 68 245 Me2CO 80D-Glucopyranose

1,2,4,6-Tetra-O-acetyl-3-deoxy-3-isothiocyanato Oil 155.0 CHCl3 84

1,3,6-Tri-O-acetyl-4-O-benzyl-2-deoxy-2-isothiocyanato — — — 97




a-D-Glucopyranose1,3,4,6-Tetra-O-acetyl-2-deoxy-2-

isothiocyanato 65–66 1142 CHCl3 73a-D-Glucopyranoside, methyl

2,3,4-Tri-O-acetyl-6-deoxy-6-isothiocyanato 98–99 1129 CHCl3 75,82



3,4,6-Tri-O-benzyl-2-deoxy-2-isothiocyanato 75–77 1164 CHCl3 876-Deoxy-6-isothiocyanato 52–53 1117 Me2CO 82

6-Deoxy-6-isothiocyanato-2,3,4-tri-O-trimethylsilyl Syrup 176.9 CH2Cl2 85

b-D-Glucopyranose3-O-Acetyl-1,6-anhydro-4-O-benzyl-

2-deoxy-2-isothiocyanato 73–75 1200 CHCl3 971,3,4,6-Tetra-O-acetyl-2-deoxy-2-

isothiocyanato 72–73 173 DMF 761,6-Anhydro-4-O-benzyl-2-deoxy-2-

isothiocyanato 89–90 1115 CHCl3 971,6-Anhydro-4-O-benzyl-2-deoxy-2-

isothiocyanato-3-O-p-tolylsulfonyl 82–84 1234 CHCl3 97b-D-Glucopyranoside, benzyl


b-D-Glucopyranoside, methyl 2,3,4-Tri-O-acetyl-6-deoxy-6-

isothiocyanato Syrup 228 CHCl3 826-Deoxy-6-isothiocyanato 108–110 2196 MeOH 82

a-D-glycero-L-gluco-Heptopyranose1,3,4,6,7-Penta-O-acetyl-2-deoxy-2-

isothiocyanato 75–76 2121 CHCl3 74b-D-glycero-L-gluco-Heptopyranose

1,3,4,6,7-Penta-O-acetyl-2-deoxy-2-isothiocyanato 136–138 223 CHCl3 77

D-arabino-Hex-1-enitol3,4,6-Tri-O-acetyl-1,5-anhydro-2-

deoxy-2-isothiocyanato Syrup 260 CHCl3 166D-erythro-Hex-3-enopyranoside, ethyl

2,3,4-Trideoxy-2-isothiocyanato-6-O-methylsulfonyl 68–69 2105 CHCl3 167,168

D-threo-Hex-3-enopyranoside, ethyl2,3,4-Trideoxy-2-isothiocyanato-6-

O-methylsulfonyl Syrup 1375 CHCl3 167,168

TABLE II—Continued

Rotation



a-D-Mannopyranoside, methyl2,3,4-Tri-O-acetyl-6-deoxy-6-

isothiocyanato 114–115 162 CHCl3 75,822,3,4-Tri-O-benzoyl-6-deoxy-6-

isothiocyanato 227 229.6 CHCl3 1446-Deoxy-6-isothiocyanato Syrup 171 Me2CO 826-Deoxy-6-isothiocyanato-2,3,4-tri-

O-trimethylsilyl Syrup 147.8 CH2Cl2 85a-D-Mannopyranoside, p-nitrophenyl

6-Deoxy-6-isothiocyanate Amorphous 185.2 MeOH 144D-Threose diethylacetal

2,4-O-Isopropylidene-3-deoxy-3-isothiocyanato oil — — 88

DisaccharidesD-Cellobitol

2,3,5,6,29,39,49,69-Octa-O-acetyl-1-deoxy-1-isothiocyanato Syrup 125 CH2Cl2 75

a-Gentiobioside, ethyl3,4,29,39,49,69-Hexa-O-acetyl-2-

deoxy-2-isothiocyanato 151 190.6 CH2Cl2 7829,39,49,69-Tetra-O-acetyl-3,4-di-O-

benzoyl-2-deoxy-2-isothiocyanato 125 148.3 CH2Cl2 78a-Kojibioside, methyl

2,4,6,39,49,69-Hexa-O-benzyl-29-deoxy-29-isothiocyanato Syrup 193 CHCl3 87

a-Melibioside, ethyl3,4,29,39,49,69-Hexa-O-acetyl-2-

deoxy-2-isothiocyanato — — — 6729,39,49,69-Tetra-O-acetyl-3,4-di-O-

benzoyl-2-deoxy-2-isothiocyanato — 1149 CH2Cl2 67b-D-Galp-(1→6)-b-D-Glcp-(1→OEt)

39,49,69-Tri-O-acetyl-3,4-di-O-benzoyl-2,2’-dideoxy-2,29-diisothiocyanato — 142.9 CH2Cl2 67

a-D-Fructofuranose b-D-fructopyranose1,29:2,19-dianhydride3,4-Di-O-acetyl-6-deoxy-6-

isothiocyanato 39,49,59-tri-O-acetyl Amorphous 234.0 CHCl3 986-Deoxy-6-isothiocyanato Amorphous 216.0 MeOH 98

SucroseN-Acetyl-2,3,19,39,49-penta-O-acetyl-

6-amino-6,69-dideoxy-69-isothiocyanato 6,4-(cyclicthiocarbamate) Syrup 226.3 CHCl3 98


Rotation





2,3,4,19,39,49-Hexa-O-acetyl-6,69-dideoxy-6,69-diisothiocyanato Syrup 172.7 CHCl3 94

6-Amino-6,69-dideoxy-69-isothiocyanato 6,4-(cyclicthiocarbamate) Syrup 1150.1 MeOH 98

6,69-Dideoxy-6,69-diisothiocyanato Syrup 174.3 MeOH 98a,a9-Trehalose

N-Acetyl-2,3,29,39,49-penta-O-acetyl-6-amino-6,69-dideoxy-69-isothiocyanato 6,4-(cyclicthiocarbamate) Amorphous 112.0 CH2Cl2 98

2,3,4,29,39,49-Hexa-O-acetyl-6,69-dideoxy-6,6’-diisothiocyanato 75–77 1113.8 CHCl3 98

6-Amino-6,69-dideoxy-69-isothiocyanato 6,4-(cyclicthiocarbamate) Amorphous 110 MeOH 98

6,69-Dideoxy-6,69-diisothiocyanato Amorphous 1100.7 MeOH 982,3,4,29,39,49-Hexa-O-

(trimethylsilyl)-6,69-dideoxy-6,69-diisothiocyanato 106–108 185.1 CH2Cl2 100

NucleosidesAdenosine

39-Azido-29-O-t-butyldimethylsilyl-39,59-dideoxy-59-isothiocyanato Amorphous — — 269

Thymidine59-Amino-39,59-dideoxy-39-

isothiocyanato-59-N-triphenylmethyl — — — 270

39-Azido-39,59-dideoxy-59-isothiocyanato Oil — — 268

29,39-Dideoxy-39-isothiocyanato — — — 93Thymidine

39-Deoxy-39-isothiocyanato 110–112 — — 94,96Thymidine

59-O-t-Butyldimethylsilyl-39-deoxy-39-isothiocyanato 147–149 — — 94

Uridine29,39-Dideoxy-29-isothiocyanato — — — 96

Uridine29-Deoxy-29-isothiocyanato-39,59-di-

O-(1,1,3,3-tetraisopropyldisiloxyl) — — — 96

OligosaccharidesCyclomaltoheptaose

6I-Deoxy-6I-isothiocyanato Amorphous 1112 Pyridine 99


Rotation




Rotation


Heptakis(6-deoxy-6-isothiocyanato) .255 (dec.) 146 Me2SO 98Cyclomaltohexaose

Hexakis(6-deoxy-6-isothiocyanato) .255 (dec.) 120 Me2SO 98Cyclomaltooctaose

Octakis(6-deoxy-6-isothiocyanato) .240 (dec.) 185 Me2SO 98

in apolar solvents following the classical Fischer method1 leads to the glycosylically linked isothiocyanate, the use of the (less expensive)alkali metal thiocyanate salts affords preferentially the kineticallyfavored thiocyanate isomer.32,33 This important drawback has beenovercome by the use of phase-transfer catalysts that promote in situthiocyanate→isothiocyanate conversion under mild conditions.34,35

Generally, per-O-protected glycosyl bromides in reaction with potas-sium or ammonium thiocyanate are used as precursors.36–43 The reactionis conducted in polar aprotic solvents (such as acetonitrile) in thepresence of cyclic polyethers or tetraalkylammonium salts as catalysts(Scheme 1).

In the case of glycopyranosyl donors bearing a participating group at C-2, glycopyranosyl isothiocyanates having a 1, 2-trans relative dis-position are obtained in pure anomeric form. By this method, a broad series of isothiocyanate derivatives of per-O-acetylated and per-O-ben-zoylated aldohexoses, 2-amino-2-deoxyaldoses, and aldopentoses havebeen prepared in 60–80% yields. This approach has also been success-fully applied to the synthesis of oligosaccharide glycosyl isothiocyanates


SCHEME 1



TABLE III

Sugar Isothiocyanate Conjugates

Sugar Bridging Arm Reference

MonosaccharidesN-Acetylneuraminic acid

2-Aminoalditol-N→ 6-Isothiocyanatohexyl 1373-Deoxy-D-manno-2-octulosonic acid

2-Aminoalditol-N→ 6-Isothiocyanatohexyl 137a-L-Fucopyranoside p-Isothiocyanatophenyl 126

2-O-tert-Butyldimethylsilyl-3,4-O-isopropylidene 4-Isothiocyanatobutyl 131

b-L-Fucopyranoside p-Isothiocyanatophenyl 117a-D-Galactopyranoside

2-Acetamido-2-deoxy p-Isothiocyanatophenyl 126b-D-Galactopyranoside p-Isothiocyanatophenyl 103,126

2-Acetamido-2-deoxy p-Isothiocyanatophenyl 1262-Acetamido-2-deoxy, p-Isothiocyanatophenyl 135

1-aminoalditol-N→a-D-Glucopyranoside p-Isothiocyanatophenyl 103,126a-D-Glucopyranoside, methyl

2-Amino-2-deoxy-N→ p-Isothiocyanatobenzoyl 103b-D-Glucopyranoside p-Isothiocyanatophenyl 103,126

2-Acetamido-2-deoxy p-Isothiocyanatophenyl 1263-O-Methyl p-Isothiocyanatophenyl 124

a-D-Mannopyranoside 2-Isothiocyanatoethyl 130a-D-Mannopyranoside p-Isothiocyanatophenyl 126

6-Phosphate p-Isothiocyanatophenyl 126b-D-Mannopyranoside p-Isothiocyanatophenyl 126Methyl (a-D-glycero-D-galacto-2-

nonulopyranosid)onate5-Acetamido-4,7,8,9-tetra-O-

acetyl-3,5-dideoxy-2-thio-(2S→ p-Isothiocyanatophenyl 116

Nojirimycin2,3,4,6-Tetra-O-benzyl-5-(N→ 2-Isothiocyanatoethyl 140

a-L-Rhamnopyranoside p-Isothiocyanatophenyl 126

Disaccharidesa-Abep- (1→3)-a-D-Manp (1→a p-Isothiocyanatophenyl 120Chitobioside

N,N9-Diacetyl Isothiocyanatoglycyl 138a-D-Glcp-(1→6)-a-D-Manp-(1→ 2-(p-Isothiocyanatophenyl) ethyl 127b-D-Glcp3OMe-(1→3)-a-L-Rhap-(1→ p-Isothiocyanatophenyl 125b-Lactoside p-Isothiocyanatophenyl 126b-Lactoside 2-(p-Isothiocyanatophenylthio)

ethyl 129b-Maltoside p-Isothiocyanatophenyl 126a-D-Manp-(1→6)-a-D-Glcp-(1→ 2-(p-Isothiocyanatophenyl)ethyl 127a-D-Manp-(1→2)-a-D-Manp-(1→ p-Isothiocyanatophenyl 114a-D-Manp-(1→2)-a-D-Manp(1→ 2-(p-Isothiocyanatophenyl)ethyl 127


a-D-Manp-(1→6)-a-D-Manp-(1→ 2-(p-Isothiocyanatophenyl)ethyl 127a-Parp-(1→3)-D-Manp-a-(1→b p-Isothiocyanatophenyl 121a-L-Rhap-(1→2)-6-deoxy-a-L-Talp-(1→ p-Isothiocyanatophenyl 125a-Tyvp-(1→3)-a-D-Manp (1→c p-Isothiocyanatophenyl 118

TrisaccharidesChitotriose

1-Aminoalditol-N→ p-Isothiocyanatophenyl 1352-O-(a-D-Glcp)isomaltoside 2-(p-Isothiocyanatophenyl)ethyl 1283-O-(a-D-Glcp)isomaltoside 2-(p-Isothiocyanatophenyl)ethyl 1284-O-(a-D-Glcp)isomaltoside 2-(p-Isothiocyanatophenyl)ethyl 128a-D-Neu5Ac-(2→6)-b-D-Galp-(1→4)-b-D-

GlcpNAc-(1→ Isothiocyanatoglycyl 13839-O-a-Sialyllactose

1-Aminoalditol-N→ p-Isothiocyanatophenyl 1351-Aminoalditol-N→ 2-(p-Isothiocyanatophenyl)ethyl 134

69-O-a-Sialyllactose1-Aminoalditol-N→ 2-(p-Isothiocyanatophenyl)ethyl 134

b-D-Galp-(1→6)-b-D-Galp-(1→6)- p-Isothiocyanatophenyl 111b-D-Galp-(1→

b-D-Glcp3OMe-(1→3)-a-L-Rhap-(1→2)-6-deoxy-a-L-Talp-(1→ p-Isothiocyanatophenyl 125

a-D-Manp-(1→2)-[a-D-Manp-(1→6)]-a-D-Manp-(1→ 2-(p-Isothiocyanatophenyl)ethyl 127

Higher oligosaccharidesb-D-GlcpNAc-(1→2)-a-D-Manp-(1→3)-[b-D-

GlcpNAc-(1→2)-a-D-Manp-(1→6)]-b-D-Manp-(1→4)-b-D-GlcpNAc-(1→4)-b-D-GlcpNAc-(1N→ Isothiocyanatoglycyl 138

b-D-Galp(1→4)-b-D-GlcpNAc-(1→2)-a-D-Manp-(1→3)-[b-D-Galp-(1→4)-b-D-GlcpNAc-(1→2)-a-D-Manp-(1→6)]-b-D-Manp-(1→4)-b-D-GlcpNAc-(1→4)-b-D-GlcpNAc-(1N→ 6-Isothiocyanatohexanoyl 139

a-D-Neu5Ac-(2→6)-b-D-Galp-(1→4)-b-D-GlcpNAc-(1→2)-a-D-Manp-(1→3)-[a-D-Neu5Ac-(2→6)-b-D-Galp-(1→4)-b-D-GlcpNAc-(1→2)-a-D-Manp-(1→6)]-b-D-Manp-(1→4)-b-D-GlcpNAc-(1→4)-b-D-GlcpNAc-(1N→ 6-Isothiocyanatohexanoyl 139

a-D-Neu5Ac-(2→3)-b-D-Galp-(1→4)-b-D-GlcpNAc-(1→2)-a-D-Manp-(1→3)-[a-D-Neu5Ac-(2→3)-b-D-Galp-(1→4)-b-D-GlcpNAc-(1→2)-a-D-Manp-(1→6)]-b-D-Manp-(1→4)-b-D-GlcpNAc-(1→4)-b-D-GlcpNAc-(1N→ 6-Isothiocyanatohexanoyl 139

A-Tetrasaccharide1-Aminoalditol-N→ p-Isothiocyanatophenyl 135

TABLE III—Continued




Lacto-N-difucohexaose I1-Aminoalditol-N→ p-Isothiocyanatophenyl 1351-Aminoalditol-N→ 2-(p-Isothiocyanatophenyl)ethyl 134

Lacto-N-fucopentaose I1-Aminoalditol-N→ p-Isothiocyanatophenyl 135

Lacto-N-fucopentaose II1-Aminoalditol-N→ p-Isothiocyanatophenyl 1351-Aminoalditol-N→ 2-(p-Isothiocyanatophenyl)ethyl 134

Lacto-N-fucopentaose III1-Aminoalditol-N→ p-Isothiocyanatophenyl 135

Lacto-N-hexaose1-Aminoalditol-N→ p-Isothiocyanatophenyl 135

Lacto-N-tetraose1-Aminoalditol-N→ p-Isothiocyanatophenyl 135

Salmonella-specific oligosaccharides1-Aminoalditol-N→ 2-(p-Isothiocyanatophenyl)ethyl 106

PolysaccharidesAeromonas-specific polysaccharides

KDO-2-Aminoalditol-N→ 6-Isothiocyanatohexyl 137Vibrio anguilarum polysaccharide

Oxidized heptose-6-NH→ 6-Isothiocyanatohexyl 137Cellulose-O→ (CH2)2-NH-CS-NH-R-NCSd 141Cellulose-O→ (CH2)2-NCS 141Cellulose-O→ CH2-CO-NH-R-NCSe 141Cellulose-O→ CO-NH-R-NCSf 141,143

a Abe, abequose (3,6-dideoxy-D-xylo-hexose).b Tyv, tyvelose (3,6-dideoxy-D-arabino-hexose).c Par, paratose (3,6-dideoxy-D-ribo-hexose).

d

e

f

TABLE III—Continued



of biological importance, such as lactose, chitobiose, and chitotriose de-rivatives.36–38,42 Krepinski and coworkers44 have synthesized in a similarway the branched trisaccharide glycosyl isothiocyanate b-D-GlcpNAc-(1→4)-[a-L-Fucp-(1→6)]-b-D-GlcpNAc-(1→NCS), structurally relatedto the oligosaccharide core portion of N-glycoproteins. The syntheticscheme involved activation of the peracetylated trisaccharide with tita-nium tetrabromide and further treatment with potassium thiocyanate.

The use of oxazolinium cations as glycosyl donors (such as 5) is an inter-esting alternative for the preparation of glycosyl isothiocyanate derivativesof 2-acetamido-2-deoxy-D-glucose and oligosaccharides thereof.45 Followingthis approach, Günther and Künz46 prepared the linear trisaccharide isothio-cyanate 6, which was further used in the first synthesis of the core region unitof N-glycoproteins by coupling to the amino acid asparagine (Scheme 2).

Recently, Lindhorst and Kieburg47 have reported a novel, solvent-freepreparation of glycopyranosyl isothiocyanates by reaction of peracetylatedglycosyl bromides with potassium thiocyanate in the molten state. In con-trast to previous attempts of thermal isomerization of glycosyl thiocyanates,this procedure affords exclusively the thermodynamic isothiocyanate com-pounds. The method was effective with hexoses, 6-deoxyhexoses, pentoses,and disaccharides, with isolated yields ranging between 41 and 73%(Scheme 3). The 1,2-trans-configured glycosyl isothiocyanates were exclu-sively obtained in all cases, except for the D-galactopyranosyl derivative,where a 1 : 9 a : b anomeric mixture was formed. The readily available inex-pensive reagents and the lack of a need for additives make this procedurevery convenient for bulk preparation of glycosyl isothiocyanates from com-mercially available sugars.

The preparation of 1,2-cis-configured glycopyranosyl isothiocyanates

SCHEME 2




SCHEME 4

requires the use of nonparticipating groups. Several perbenzylated mono-and disaccharide glycosyl isothiocyanates have been thus obtained as a,b-anomeric mixtures by treatment of the corresponding glycosyl chlorideswith potassium isothiocyanate in the presence of a tetrabutylammoniumsalt.34,35,48 Analogously, peracetylated 2-deoxy-a-D-glycosyl bromidesyielded mixtures of 2-deoxy-a- and b-D-glycosyl isothiocyanates upon re-action with either potassium thiocyanate–[18]-crown-6 or silver thio-cyanate.49,50 In all cases, the a : b ratio was close to 1 : 1, and both anomerswere separated after column chromatography. A notable exception is thereaction of methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-a-D-glycero-D-galacto-2-nonulopyranosyluronate) chloride (7) with potassiumthiocyanate under conditions of phase-transfer catalysis, which affords ex-clusively the a-configured glycosyl isothiocyanate derivative of N-acetyl-neuraminic acid 8 (Scheme 4).51

To overcome the problems derived from formation of anomeric mixtureswhen O-benzylated glycosyl halides are used as precursors, Ledford andCarreira52 have disclosed an elegant synthesis of 2,3,4-tri-O-benzyl-a-D-glucopyranosyl isothiocyanate (10) based on the use of 1,6-anhydro-2,3,4-tri-O-benzyl-b-D-glucopyranose (9) as the glycosyl donor. Treatment of 9with tetra-n-butylammonium thiocyanate and boron trifluoride–etheratecomplex provided 10 in 50% yield with total control of the anomeric con-figuration (Scheme 5).

The reaction of fully acetylated glycofuranosyl halides with KSCN fol-lows an analogous pattern to that just described for their pyranosyl coun-terparts, namely, glycofuranosyl thiocyanates are formed as the major

SCHEME 3


reaction products,53 which can be isomerized to the corresponding isothio-cyanates by using a phase-transfer catalyst.41 Remarkably, replacement ofthe acetyl protecting groups by benzoyl groups results in the direct forma-tion of glycofuranosyl isothiocyanates with no need for any additive.53,54

The reaction is totally diastereoselective, affording the 1,2-trans-configuredisothiocyanates as the sole reaction products under mild conditions (roomtemperature, 2 h). Fully benzoylated b-D-galacto- (14), b-D-ribo-, and b-D-xylo-furanosyl isothiocyanates were thus prepared in 80–90% yield. Crudeglycofuranosyl chlorides (such as 12) or bromides (for example, 13), ob-tained from the perbenzoylated sugars (for instance, 11) by treatment withacetyl chloride–HCl or bromotrimethylsilane, respectively, were used asprecursors (Scheme 6).

The trimethylsilyl isothiocyanate–tin tetrachloride system has been pro-posed55 as an alternative to the use of inorganic thiocyanate salts for thesynthesis of glycosyl isothiocyanates. By applying this reagent, 1,2,3,4,6-penta-O-acetyl-a-D-glucopyranose was directly transformed into the corre-sponding b-isothiocyanate in 80% yield.

SCHEME 6

SCHEME 5


4888 Horton Chapter 4-2 11/17/99 6:54 AM Page 55

b. Electrophilic Addition to Glycals.—The possibility of using glycals,namely 1,2-unsaturated sugars, as glycosyl isothiocyanate precursors was explored by Igarashi and Honma56 in the 1960s. However, formation ofa complex mixture of diastereomers as well as of isothiocyanate–thiocyanate isomers was observed using thiocyanogen as reagent, and theapproach was no longer investigated.

In 1994, Santoyo-González et al.57 reported a very convenient route forthe simultaneous introduction of the iodo and isothiocyanate functionali-ties in a sugar molecule starting from glycals. Electrophilic addition of iodine (I) thiocyanate, generated in situ from silica-supported KSCN and iodine, to the double bond leads exclusively to trans-2-deoxy-2-iodoglycopyranosyl isothiocyanates. In the case of monosaccharide glycals,a mixture of the trans-diaxial (major) and trans-diequatorial product (mi-nor) was obtained, whereas the trans-diaxial vic-iodoisothiocyanate was thesole product in the case of disaccharide glycals (Scheme 7). The high yield,good stereoselectivity, and simplicity of the method make it very attractivefor the preparation of highly functionalized sugar derivatives.

c. Isothiocyanation of Sugar Amines.—From the variety of preparativemethods available for the synthesis of isothiocyanates, the reaction of aprimary amine with an isothiocyanation reagent is probably the mostgenerally useful. Formation of the C––N bond occurs here prior togeneration of the isothiocyanate group and, therefore, formation of theisomeric thiocyanate is prevented. Depending on the location of the aminefunctionality in the sugar precursor, this approach allows access to glyco-syl isothiocyanates, deoxyisothiocyanato, sugars, and isothiocyanateconjugates.

Although the transformation of glycosylamines (such as 15) into fully un-protected glycosyl isothiocyanates by reaction with thiophosgene was al-ready reported in the 1970s,58–61 these compounds were later shown to beunstable,62 and no further chemistry has been reported. The synthesis ofstable, fully protected hexopyranosyl isothiocyanates by the thiophosgenereaction was first reported by Fuentes Mota and coworkers.63–65 The reac-tion sequence involves a glycosyl enamine (for example, 16) as key inter-mediate which, after O-protection (→17), is hydrolyzed under mild condi-


SCHEME 7


tions using chlorine or bromine in moist chloroform (→18). By this proce-dure, peracetylated and perbenzoylated glycosyl isothiocyanates (such as19) of several mono-63–65, di-66–68, and trisaccharides68 were obtained fromthe commercial sugars in five steps and 50–60% overall yields. Some par-tially acylated derivatives have been also prepared following an analogousstrategy (Scheme 8).69,70

Since the preparation of glycosylamines from reducing sugars is undercontrol of the reverse anomeric effect, the foregoing methodology affordsb-configured glycopyranosyl isothiocyanates independent of the orienta-tion of the acyloxy group at C-2, thus complementing the glycosyl halide–inorganic thiocyanate approach already mentioned. Following either one orthe other method, both 2,3,4,6-tetra-O-acetyl-a- and b-D-mannopyranosylisothiocyanates (22 and 23) may be prepared with total stereocontrol fromthe corresponding a-bromide 20 or b-mannosylamine 21, respectively(Scheme 9).71

SCHEME 8

SCHEME 9



Some deviations from this general pattern have been reported72 in thealdopentose series. Thus, 2,3,4-tri-O-benzoyl-b-D-ribopyranosylamine hy-drobromide partially anomerized on treatment with thiophosgene, leadingto a 4 : 1 b : a anomeric mixture of the corresponding tri-O-benzoyl-D-ribopyranosyl isothiocyanates 24 and 25. It is noteworthy that, in bothanomers, the NCS group adopts an axial disposition, in accord with theanomeric effect.

The synthetic value of the enamine strategy for temporary amine protec-tion in the preparation of sugar isothiocyanates is further underlined by thepossibility of access to peracylated derivatives bearing the NCS group at a nonanomeric secondary73,74 or primary position.75 No O→N acyl migra-tion occurred, either in the halogenolysis or in the isothiocyanation steps,even in the case of the acyclic glucitol (26) and cellobiitol (27) derivatives.Other N-protecting methodologies applied to the synthesis of 2-deoxy-2-isothiocyanatohexoses from D-glucosamine and oligosaccharides thereofinclude the formation of a Schiff base76,77 or a benzyloxycarbonyl deriva-tive.67,78,79


Aldose and ketose derivatives bearing an isothiocyanate group at a pri-mary carbon atom have also been obtained from cyclic acetal deriva-tives.80–83 In this case, introduction of the amino group follows hydroxylprotection and no N-protecting group is needed. 6-Deoxy-1,2 : 3,5-di-O-isopropylidene-6-isothiocyanato-a-D-glucofuranose,80–82 6-deoxy-1,2 : 3,4-di-O-isopropylidene-6-isothiocyanato-a-D-galactopyranose,81,82 and 1-de-oxy-2,3 : 4,5-di-O-isopropylidene-1-isothiocyanato-b-D-fructopyranose83

have been thus prepared in three steps from the corresponding readily ac-cessible selectively O-acetalated monosaccharides. An analogous syntheticpathway has been employed84 in the preparation of 3-deoxy-1,2 : 5,6-di-O-isopropylidene-3-isothiocyanato derivatives of kanosamine (3-amino-3-deoxy-D-glucose) and of its C-3 and C-4 epimers (with the D-allo and D-galacto configurations, respectively).

Although thiophosgene is generally preferred, since it provides clean andvery fast reactions, other isothiocyanation reagents may also be employedfor the transformation of amino sugars into sugar isothiocyanates. Carbondisulfide–N,N9-dicyclohexylcarbodiimide85 and N,N9-thiocarbonyldiimida-zole48,85–88 have proved particularly useful in the case of derivatives bear-


ing acid-sensitive silyl ether protecting groups.85 Other reported examplesinclude Mukaiyama89,90 (CS2, Et3N, then 2-chloro-1-methylpyridinium io-dide, Et3N) and Wadsworth–Emmonds89,91,92 [(EtO)2P(5O)Cl, Et3N, thenNaH, n-Bu4NBr, carbon disulfide] isothiocyanation protocols.

The synthesis, chemistry, and pharmacological properties of deoxy-isothiocyanato ribonucleosides93–96 have been the subject of recent atten-tion. Cech and coworkers96 examined different systems for the isothio-cyanation of the aminodeoxy precursors. Thiophosgene in pyridine,N,N9-thiocarbonyldiimidazole, and CS2–HgO were found effective for thetransformation of 39-amino-29,39-dideoxythymidine 28 into the corre-sponding isothiocyanate 29, the later reagent having some practical ad-vantages (Scheme 10). This was also the method of choice for the prepa-ration of 29-deoxy-29-isothiocyanato derivatives in the uridine series.Interestingly, compound 29 was found to be cytotoxic to different mouseleukemic cell lines.94

The stability of monosaccharide derivatives bearing both isothiocyanateand free hydroxyl groups is dramatically dependent on the configurationand conformation of the sugar template. As a general rule, b- and c-hydroxyisothiocyanates undergo spontaneous or base-induced annelationreaction to the corresponding five- and six-membered cyclic thiocarb-amates, respectively.81–84, 96 Nevertheless, the partially protected derivatives30–32 were stable compounds which could undergo further transformations(such as acylation) without affecting the NCS functionality.84, 97 In all these


SCHEME 10


cases, the intramolecular nucleophilic cycloaddition would lead to a five-membered–six-membered trans-fused bicyclic system, an unfavorablearrangement.

Fully unprotected 6-deoxy-6-isothiocyanato aldopyranosides (33) havebeen found to be stable in the absence of base.82 Per-O-acyl and per-O-trimethylsilyl derivatives were also prepared after conventional O-protecting reactions,82,85 although some thiocarbamate formation was observed in the latter derivatization. The foregoing results have been ex-panded to the preparation of stable unprotected and O-protected sugarisothiocyanates of nonreducing oligosaccharides of economic and biologi-cal significance, such as sucrose (34), a,a9-,trehalose (35), and cyclomal-tooligosaccharides (a-, b-, and c-cyclodextrins; 36, 37) by reaction of thecorresponding (poly)-amines with thiophosgene.98–100


Isothiocyanate and hydroxyl groups separated by three carbon atoms ormore do not interfere between them, even in the presence of base catalystsor after prolonged heating. Consequently, homologation of primary amino-deoxy sugar methyl glycosides via the corresponding glycurononitrile (38),following the method reported by Defaye and Gadelle (→39),101 and fur-


ther isothiocyanation resulted in very stable fully unprotected, 6,7-dideoxy-7-isothiocyanatoheptose derivatives 40 (Scheme 11).102

The thiophosgene reaction has been broadly employed for the prepara-tion of isothiocyanate conjugates, an important family of compounds inwhich the NCS group is located in a noncarbohydrate moiety covalentlylinked to the saccharide core. The isothiocyanate segment of the moleculeis usually called the “handle’’ or “bridging arm’’ when such conjugates aregenerated as intermediates for the attachment of an oligosaccharide to amacromolecular carrier or solid matrix. In 1968, Buss and Goldstein103 re-ported a synthesis of monosaccharide–phenyl isothiocyanate conjugates(43) as convenient intermediates for coupling reactions with proteins, byanalogy with the well-known use of phenyl isothiocyanate (Edman’sreagent) in protein analysis. The reaction scheme involved catalytic hydro-genation of the nitro group in commercially available p-nitrophenyl glyco-sides (41) and reaction of the resulting amine 42 with thiophosgene in aque-ous ethanol (Scheme 12). Since this pioneering work, phenyl isothiocyanateconjugates have become very popular tools in the hands of glycobiologistsfor the preparation of neoglycoconjugate probes.104–117 The amine precur-sor can alternatively be liberated from suitable N-protected aminophenyl

SCHEME 12


SCHEME 11


glycosides, such as 4-(p-toluenesulfonamido)118 or p-trifluoroacetamido de-rivatives (44).119–123 The isothiocyanation reaction may also be performedunder mild alkaline conditions (pH 8) that would not affect the labile gly-coside linkages of oligosaccharides containing deoxy sugar or sialic acidresidues. By using this technique, several oligosaccharide–phenyl isothio-cyanate conjugates related to the O-antigenic polysaccharide chains of theSalmonella119–123 and Mycobacterium124,125 bacteria have been prepared.The final sugar–isothiocyanate conjugates are usually employed for proteinconjugation without further characterization, the presence of the NCSgroup being confirmed by chromatographic or spectroscopic (IR) tech-niques.126

In addition to the phenyl derivatives, a variety of aglycons have been pro-posed from which in a final synthetic step the reactive isothiocyanate func-tionality is generated. The choice of the arm and the mode of attachment tothe sugar part of the conjugate depends mainly on the immunogenic re-quirements of the oligosaccharide to be coupled as a hapten to the protein.The reported examples include p-isothiocyanatophenethyl (45),127,128 2-(p-isothiocyanatophenylthio)ethyl (46),129 2-isothiocyanatoethyl,130 4-isothio-cyanatobutyl,131 and 6-isothiocyanatohexyl132,133 (47) glycosides.


If the terminal reducing sugar residue can be sacrificed, which is usuallythe case for large haptenic oligosaccharides, reductive amination becomesan attractive method for the coupling of the handle to the carbohydrate part.Several bifunctional diamine spacers, such as 4-aminophenetylamine,134 p-trifluoroacetamidoaniline (TFAN),135,136 and 1,6-hexanediamine137 havebeen employed for this purpose.The isothiocyanate group is generated aftertreatment of the adduct with thiophosgene (Scheme 13). This general strat-egy has proved useful for the preparation of isothiocyanate conjugates ofcomplex oligosaccharides of biological importance, including those incorpo-rating reducing ketose residues (for example, N-acetylneuraminic acid and3-deoxy-D-manno-2-octulosonic acid).137 The (6-isothiocyanatohexyl)aminobridging arm has also been inserted into the core oligosaccharide of bacte-


rial glycolipids allowing further conjugation to bovine serum albumin (BSA)protein. In this case, the isothiocyanate portion of the conjugate is linked toC-6 of a partially oxidized terminal heptosyl residue.137

The major drawbacks associated with the reductive amination couplingprocedure are the loss of structural and biological information at the re-ducing end and the possibility of undesirable immunogenicity of theadducts. The reaction of b-glycosylamines with heterobifunctional reagentsconstitutes an interesting alternative. Importantly, oligosaccharide b-glycosylamine derivatives have structural integrity and can be obtainedvery efficiently, thus making the method suitable for derivatizing complexoligosaccharides, either synthetic or from biological sources and availableonly in limited quantities. Two notable examples of this approach are thepreparation of N-glycyl138 and N-(6-aminohexanoyl)139 glycosylamines,which were modified with thiophosgene to form the corresponding isothio-cyanate conjugates 48. A further original version of the N-linked spacerstrategy has been used in the preparation of deoxynojirimycin–trehalamineconjugates.140 The spacer arm was introduced by a reaction sequence in-volving the formation of an N-glycyl derivative, which was further reducedto the N-(2-aminoethyl)imino sugar. Isothiocyanation following theMukaiyama methodology afforded the corresponding N-(2-isothiocyana-toethyl)-1-deoxynojirimycin derivative 49.


SCHEME 13


Several macromolecular cellulose–isothiocyanate polyconjugates havebeen reported141–143 and characterized by their binding capacity with re-spect to amine and sulfur nucleophiles. Functionalization occurred at thecellulose fiber surfaces, and a variety of aromatic, aliphatic, and mixed-typespacers were used.

d. Reaction of Sugar Iminophosphoranes with Carbon Disulfide.—Staudinger reaction of azido groups with a phosphine and subsequent aza-Wittig-type condensation of the resulting iminophosphorane with carbondisulfide is an attractive synthetic strategy for the direct transformation of sugar azides into sugar isothiocyanates (Scheme 14).144 Two maindrawbacks for these routes are the high tendency of the formingisothiocyanate to react with the remaining iminophosphorane to give acarbodiimide145 and the separation of the final product from excessphosphine and phosphine thioxide. The use of polymer-bound triphenyl-phosphine, recently reported in the nucleoside series,96 constitutes apromising alternative to overcome such problems. By using this reagent, 39-azido-3-deoxythymidine (AZT) was transformed into the correspondingisothiocyanate 29 in 93% yield after a purification step that involved asimple filtration process.

2. Reactions of Sugar Isothiocyanates

a. Reactions with C-, N-, O-, and S-Nucleophiles.—The reaction of sugarisothiocyanates with C-, N-, O-, and S-nucleophiles bearing a labilehydrogen atom leads, at least in a first stage, to N-monosubstituted adductsin which the electronegative residue is linked to the carbon atom of theheteroallene group. The resulting sugar thioamides, thioureas, thiocar-bamates, and dithiocarbamates are of interest in view of their syntheticvalue, especially in heterocyclic chemistry, and their wide spectrum oftechnical and biological applications. Nucleophilic addition of amino,hydroxy, and mercapto groups to the NCS functionality also plays animportant role in the conjugation of sugar isothiocyanates withbiomolecules. The literature concerning this important aspect of sugarisothiocyanate reactivity has been discussed in part in several reviews.4,8,146

The growing interest in these reactions is further corroborated by thenumerous new results reported in the past few years. A detailed survey ofthese results has been included in sections of this chapter.


SCHEME 14


b. Condensation with Carboxylic Acids.—The triethylamine-catalyzedreaction of glycosyl isothiocyanates with carboxylic acids to giveglycosylamides was first investigated by Khorlin and coworkers147,148 andfurther implemented149 for the construction of the N-glycosylic linkagebetween 2-acetamido-2-deoxy-b-D-glucopyranosylamine and suitablyprotected L-aspartic acid derivatives, analogous to that existing in naturalN-glycoproteins. Under optimal reaction conditions, which involve strictexclusion of water and the use of 0.1 molar equiv. of Et3N, yields higherthan 70% of the desired glycosylamide are obtained,44,46 thus favorablycomparable to other N-glycopeptide synthetic methodologies.150,151 Thereaction has been applied to monoprotected dicarboxylic acids152–154 and tothe selectively functionalized glycosyl isothiocyanate 51 (Scheme 15).155

The glycosylamides thus obtained incorporated a reactive group either inthe aglycon (50) or in the sugar moiety (52), which was subsequently usedin their coupling to b-cyclodextrin derivatives.

c. Self-Condensation Reactions.—Bis(glycosyl)thioureas are oftenformed as secondary products during the synthesis and reactions of glycosylisothiocyanates. Different mechanisms have been proposed to explain theirformation, such as the coupling of unreacted amine with isothiocyanateduring the isothiocyanation step or the hydrolysis of a mixed anhydride inthe aforementioned condensation with carboxylic acids.149 The problem hasbeen examined in detail71 and shown to involve two isothiocyanatemolecules. In a first step, base-catalyzed addition of a water molecule to theNCS group takes place to give a thiocarbamic acid derivative. Subsequentaddition to a second NCS group and elimination of COS affords thecorresponding symmetric thiourea (Scheme 16). The reaction proceeds atroom temperature in 10 : 1 pyridine–water in the case of glycosyl

SCHEME 15



isothiocyanates or at 608C in the case of deoxyisothiocyanato sugars and isof synthetic value for the preparation of a variety of symmetric sugarthioureas.

d. Desulfurization Reactions.—Desulfurization of glycosyl isothio-cyanates with tributyltin hydride at room temperature in the absence of afree-radical initiator affords glycosyl isocyanides (53) in 58–76% yields.39

The outcome of the reaction may strongly depend on the reagent ratio andthe presence of moisture, since formation of the corresponding glycosylthioformamides has been reported by other authors156 under apparentlyidentical reaction conditions. Under more strenuous conditions and in thepresence of azobis(isobutyronitrile) (AIBN) as a free-radical initiator,reduction of isothiocyanates leads to the formation of 1,5-anhydroalditols(54) via glycosyl isocyanide intermediates in virtually quantitative yield.Hassel and Müller157,158 have reported the preparation of glycosylisocyanide dichlorides (55) by chlorination of mono- and disaccharideperacetyl glycosyl isothiocyanates. Interestingly, via these highly reactiveintermediates, the isothiocyanate group can be transformed into a varietyof other functionalities and heterocyclic derivatives, thus widening thealready broad spectrum of synthetic applications of sugar isothiocyanates(Scheme 17).

e. Aza-Wittig-Type Reactions.—The intermolecular aza-Wittig-typereaction of sugar isothiocyanates and sugar iminophosphoranes has beenthe subject of a detailed study159,160 aimed at the preparation ofpseudooligosaccharide structures incorporating (1→6)-carbodiimideintersaccharide bridges. Coupling of glycosyl iminophosphoranes with 6-deoxy-6-isothiocyanato sugars afforded the desired compounds in


SCHEME 16


25–35% yields. A converse strategy implying a one-pot Staudinger reactionof 6-azido-6-deoxy sugars (56) and triphenylphosphine and subsequent insitu aza-Wittig condensation with per-O-acetylated glucosyl (58) andcellobiosyl (59) isothiocyanates proved much more convenient, leading tothe (1→6)-linked glycosyl carbodiimido sugars 60 in yields higher than 80%after 10 min of reaction time at room temperature. On the basis of kineticconsiderations, the authors suggested a transient phosphazide (57), formedat the early stages of the Staudinger reaction, as the reactive intermediate(Scheme 18).160

SCHEME 18


SCHEME 17


f. Cycloaddition Reactions.—The ability of the NCS group to reactthrough the CS or the CN bond in cycloaddition reactions makes sugarisothiocyanates important precursors in the synthesis of heterocyclicderivatives of carbohydrates. From the body of results collected in theliterature,4 it appears that cycloaddition reactions of glycosyl isothio-cyanates show, preferentially, the more electron-rich thiocarbonyl doublebond to afford glycosylamino heterocycles instead of the isomeric N-nucleosides. Accordingly, the cycloaddition of 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl isothiocyanate 58 and the 1-aza-2-azoniaallene ions 61 and62 furnished glycosylimino-1,3,4-thiadiazoles 63 and 64, respectively(Scheme 19).55 The reactive heterocumulenic cations were generated in situfrom the correspondig a-chloro-azo compounds by treatment withantimony pentachloride.

3. Spectroscopic Properties

The main spectroscopic features of sugar isothiocyanates are the charac-teristic strong IR absorption (VNCS 2100–1990 cm-1) and the 13C NMRchemical shift of the isothiocyanate functionality (dNCS 145–135 ppm). Thefirst one appears as a strong wide band and is extensively used both forstructural confirmation and monitoring reactions involving sugar isothio-cyanate derivatives. Its position is very characteristic and allows discrimina-tion from the isomeric thiocyanates. Other NMR spectroscopic data showthe upfield shift of the 13C resonance of the carbon atom directly attachedto the heteroallene group (by 15 ppm) and the 1H resonance of the corre-sponding a-located protons (by 1 ppm) as compared to the parent sugar.4,8


SCHEME 19


The UV spectra of acetylated sugar isothiocyanates show a low-intensityabsorption at 250–254 nm, which allows UV detection in the course of chro-matographic separations.50,70,78 The band is frequently overlapped whenstronger chromophores (such as benzoyl groups) are present in the mole-cule.65,69,70,72

The main primary fragmentation of glycosyl isothiocyanates in electron-impact (EI) or chemical-ionization (CI) mass spectrometry consists in theloss of the NCS radical to give an oxocarbenium cation (m/z M 2 58). Theloss of thiocyanic acid (m/z M 2 59) is also frequently observed, whereasthe molecular peak is either absent or of very low intensity.50,57,65,70,78 Sig-nificantly, EI mass spectra of peracetyl 6-deoxy-6-isothiocyanato glycopyra-nosides show intense molecular peaks and losses of CH2NCS and MeNCSas the main primary fragmentation.82,83 An analogous fragmentation path-way was observed in the fast-atom bombardment (FAB1) mass spectra ofthe unprotected derivatives.82 These data probably reflect the higher stabil-ity of deoxyisothiocyanato sugars as compared to glycosyl isothiocyanates.

IV. SUGAR THIOAMIDES AND THIOLACTAMS

Despite the important role that N-acylated amino sugars play in many bi-ological processes and the long-known influence that replacement of oxy-gen by sulfur may exert in the biochemical properties of a sugar molecule,only a few reports on the synthesis and reactivity of N-thioacylated aminosugars appeared4 before 1985. Since that time the chemistry of sugarthioamides has expanded significantly, fueled by both their synthetic po-tential, especially in nucleoside chemistry, and their biological applications.

1. Addition of Carbon Bases to Sugar Isothiocyanates

Formally, the reaction of carbon nucleophiles and sugar isothiocyanatesprovides a general route to sugar thioamides. However, the instability of theNCS functionality under the strongly basic conditions needed for the gen-eration of carbanions results, generally, in rather low to moderate couplingyields. The reported examples are limited to the use of enamines or activemethylene compounds as carbanion precursors and glycosyl isothio-cyanates as electrophiles.

The reaction of monosaccharide isothiocyanates with enamines leads tothe formation of a,b-unsaturated thioamides4,169 (67), which can be cy-clized to isothiazole derivatives.4 Formation of additional products arisingfrom attack of the amino group to the isothiocyanate functionality has alsobeen observed, the outcome of the reaction being dependent on the natureof the enamine reagent and on reaction conditions. Thus, nucleophilic addi-tion of a series of 3-amino-3-penten-2-ones (65) and ethyl 3-aminocroto-



nates (66) to 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl isothiocyanate (58)proceeded through carbon (the hard site) in the case of N-aryl substituents,whereas it involved preferentially the amino group (soft site) for N-alkylderivatives (Scheme 20).169

Nucleophilic addition of diethyl malonate derivatives 68 to the glucosylisothiocyanate 58 in the presence of sodium hydride afforded the corre-sponding N-thioacylglucosylamines 69 in 40–60% yields.170 Adducts incor-porting an a-phenacyl substituent (68, R 5 Ar) were further cyclodehy-drated using phosphoric acid and acetic anhydride to give N-nucleosidederivatives of 2-pyrroline 70. Under identical reaction conditions, the a-acetonyl derivative (68, R 5 Me) led to the tetrahydropyridine heterocycle71 (Scheme 21).

Carbanions derived from ethyl cyanoacetate, phenylthioacetonitrile, andcyanoacetamide likewise reacted with 58 to give a thioenolic sodium salt in-termediate (72).170 Attempts to isolate the related thioamide were, how-


SCHEME 20

SCHEME 21


ever, unsuccessful. After in situ reaction with phenacyl bromide the corre-sponding thioethers (such as 73, 74) were formed which, in some cases, un-derwent spontaneous cyclization to give glucosylamino-2-thioxopyrrolinederivatives (such as 75–77; Scheme 22).

2. Thionation of N-Carbonyl Derivatives

Thionation of N-acylated amino sugars is the method most frequentlyused for the preparation of sugar thioamides. The O→S exchange has beentraditionally accomplished by using phosphorus pentasulfide as the thiona-tion reagent.171–175 The procedure is of general applicability for O-pro-tected carbohydrates, regardless of the anomeric or nonanomeric characterof the amide substituent, and has been successfully applied to the prepara-tion of N-thioformyl, N-thioalkanoyl, and N-thiobenzoyl derivatives.176–178

In recent years Lawesson’s reagent, namely, 2,4-bis(4-methoxyphenyl)-1,3-dithiadiphosphetane-2,4-disulfide, has become the reagent of choice forthionation of carbonyl compounds. By this methodology, a series of gly-conothiolactams (79), used as key synthetic intermediates in the prepara-tion of a variety of glycosidase inhibitors, were obtained in high yield fromthe corresponding lactams 78 (Scheme 23).179–185

Thionation of N-acetylated amino sugars provides a convenient route forN-deacetylation in compounds sensitive to strongly basic conditions such asnucleosides, since the thioacetyl group can be removed under mild basicconditions using methanolic ammonia.171,172 Alternatively, S-methylationand subsequent mild acid hydrolysis of the resulting thioiminoether allowsremoval of the N-thioacetyl group in the presence of O-acetyl groups.173

SCHEME 22



The reverse CS→CO transformation of N-alkyl thioacetamido sugars hasbeen achieved by treatment with silver acetate.186

3. Thioacylation of Amino Sugars

In contrast to the acylation of amines, for which acyl halides and acid an-hydrides are the predominant reagents, direct introduction of a thioacylsubstituent on an amino group is generally achieved by using thionocar-boxylates or dithiocarboxylates as thioacylating reagents.187 Brossmer andIsecke188 have reported the direct thioformylation, thioacetylation, andthiopropanoylation of fully unprotected 2-amino-2-deoxyaldoses in the D-gluco, D-galacto, and D-manno series by reaction with O-ethyl thiofor-mate, methyl dithioacetate, and methyl dithiopropionate, respectively(→80). The method was further extended to the preparation of sialic acidderivatives bearing thioamido groups at C-5 (81) or C-9 (82) as inhibitorsof influenza virus hemagglutinin.189 Thioacetylation of O-protected amino

SCHEME 23



sugars has alternatively been effected by treatment with dithioacetic acid inthe presence of N,N9-dicyclohexylcarbodiimide.190

4. Miscellaneous Sugar Thioamide Syntheses

O-Protected sugar thioformamides have also been prepared in 44–87%yields by tri-n-butyltin hydride reduction of isothiocyanate precursors inether.156 For the preparation of 5-N-thioacylneuraminic acids (84), achemoenzymatic route based on the N-acetylneuraminate pyruvate lyase-mediated condensation of the corresponding N-thioacyl-D-mannosaminederivatives (83) and sodium pyruvate has been reported.189 The enzyme-catalyzed aldol reaction was performed at pH 6.8 and afforded the desiredcompound in 55% yields (Scheme 24).

Masson and co-workers191 have reported a different type of sugarthioamide in which the carbohydrate moiety forms part of the thioacyl rad-ical, namely D-mannofuranosyl-ethanethioamides (87, 89). Their synthesisinvolved a Horner–Wadsworth–Emmons reaction of thiocarbamoyl-methylphosphonates (86) with 2,3 : 5,6-di-O-isopropylidene-D-mannofura-nose (85). The coupling yields were higher than 90% for N-alkyl and N,N-dialkylthioamides, while decreasing to 15% or even zero for amino acidderivatives. An alternative route for such compounds was devised consist-ing of the methylation–sulfhydrolysis of a preformed thioamide. The result-ing methyl dithioate (88) was further used as thioacylating reagent forglycine and its methyl ester (Scheme 25).Though the prior formation of theC-glycosylic bond led to a mixture of the a- and b-anomers, they could bereadily separated by silica gel chromatography in all cases.

5. Conformational Properties of Sugar Thioamides

The well-known rotational isomerism of thioamides has been studied indetail for monosaccharide derivatives bearing a thioacetamido substituentat a secondary or primary position.176,177,188,189 As common features,

SCHEME 24



formamido and N,N-disubstituted thioamides exist at room temperature asa mixture of unequally populated Z and E rotamers at the N––C(S) bond,and two sets of signals can be seen in the corresponding 1H and 13C NMRspectra. In contrast, the Z rotamer exclusively occurs in the case ofNHC(S)R substituents for R other than H. The relative dispositionaround the contiguous N––CH bond has been determined to be antiperi-planar for thioamide groups at secondary positions in a pyranose ring.Several useful rules for configurational assignment in these compoundshave been given176 on the basis of 1H and 13C NMR spectral parameters(Fig. 5).


SCHEME 25

FIG. 5. Spectral parameter relationships for (Z) and (E) rotamers of sugar thioamides.


V. SUGAR THIOUREAS

A large body of work in the area of N-thiocarbonyl carbohydrate deriv-atives has been directed toward the synthesis and applications of sugarthioureas. The early contributions focused on their use as synthetic inter-mediates in heterocyclic chemistry,4,8 an approach that continues to be ex-plored intensively for the preparation of nucleoside and other N-glycosylcompounds. More recently, the extension toward glycobiology in the carbo-hydrate field and the promise of seminatural or unnatural carbohydrate-containing substances for basic biomedical research and practical medicalapplications has spurred an aggressive effort to design neoglycoconjugates,among which those incorporating thiourea linkers are of prime importance.Further uses of the thiourea technology include the attachment of saccha-ride portions to pharmaceuticals and the preparation of diastereomericconjugates from enantiomeric mixtures for analytical purposes.

1. Coupling of Sugar Isothiocyanates with Amine Nucleophiles

Sugar isothiocyanates are, probably, the most powerful starting materialsfor preparing sugar thioureas and derivatives. In a general manner, glycosylisothiocyanates37,54,57,66,73,74 and deoxyisothiocyanato sugars97,192–194 reactwith ammonia, primary amines, and secondary amines to give 1-substituted,1,3-disubstituted, and trisubstituted thioureas, respectively (Scheme 26).This reaction generally takes place with good yields and leads to well-defined, frequently crystalline compounds of high stability. One of the mainadvantages of the method is its versatility, being compatible with a widerange of protecting and functional groups both in the amine and in thesugar isothiocyanate reagents.

The new examples reported include the preparation of antiviral, antibac-terial, and antitumor agents by coupling sugar isothiocyanates and such bi-ologically active amines as triazole derivatives,195 mitomycin,196 isothia-zolopyrimidines,40 and also platinum compounds.197 Other N-nucleophilessuch as hydrazine,37 isothiourea,198 and guanidine derivatives199 have beensimilarly coupled to sugar isothiocyanates.

Isothiocyanate groups located at anomeric positions are distinctly more reactive than nonanomeric isothiocyanates toward addition of N-nucleophiles. Thus, whereas the reaction of glycosyl isothiocyanates with


SCHEME 26


ammonia proceeds in ether or ethanol to give the corresponding N-glyco-sylthiourea in quantitative yield, no reaction was observed in the case of 6-deoxy-6-isothiocyanatoaldose derivatives under identical reaction condi-tions. Nevertheless, high coupling yields were obtained when the reactionwas performed in pyridine as solvent and catalyst.85

Sugar thioureas containing an N-azolyl substituent, such as the thiazole,thiazoline, or benzoxazole rings, have been the subject of attention in con-nection with the interest in azole nucleoside analogs as antineoplastic and antiviral compounds.200–202 Their preparation involves the reaction of O-protected glycosyl isothiocyanates or 2-deoxy-2-isothiocyanatoaldoseswith the corresponding 2-aminoheterocycles. A complete spectroscopic (UV,IR, NMR, and MS) study has shown the existence of six-membered in-tramolecular hydrogen bonding in chloroform solution, with the anomericNH group acting, generally, as the donor (90). Notable exceptions are 4,4-diphenyloxazoline derivatives, which exist as tautomeric mixtures of thiourea(91a), and isothiourea (91b) derivatives, with the thiocarbonyl and the thiolgroups acting as hydrogen bond acceptor and donor, respectively.201


The reaction of 39-deoxy-39-isothiocyanatothymidine (29) with selec-tively protected diamines (→92) has been used96 to introduce thioureaspacers that allow further attachment with fluorescent dyes. Alternatively,7-amino-4-methylcoumarin was added to 29 to give the fluorescentthiourea conjugate 93 (Scheme 27). The reaction is chemoselective and noprotection of the primary OH group was needed.

Amino acids and peptides have been coupled to glycosyl isothiocyanatesto give N-(glycosylthiocarbamoyl)peptides which, in some cases, haveshown immunoadjuvant and antitumor activities.42 The interest of this re-

4888 Horton Chapter 4-3 11/18/99 3:05 PM Page 76

action for the chiral derivatization of a-amino acid enantiomers was al-ready realized by Nimura and co-workers in the early 1980s.203–205 Cou-pling of a glycosyl isothiocyanate with an enantiomeric mixture of D- and L-amino acids or the corresponding ethyl esters affords diastereomericglycosylthiourea derivatives (Scheme 28), which can be separated using re-verse-phase high-performance liquid chromatography (HPLC). The reac-tion is complete in 20–30 min in acetonitrile–water as the solvent at roomtemperature and the crude product can be injected directly into the chro-matograph. 2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl isothiocyanate and2,3,4-tri-O-acetyl-a-D-arabinopyranosyl isothiocyanate have been mostwidely used for this purpose, although other sugar isothiocyanates, such as2,3,4,6-tetra-O-benzoyl-b-D-glucopyranosyl,206 2,3,4,6-tetra-O-pivaloyl-b-D-galactopyranosyl,206 and 2,3,5,6-tetra-O-benzoyl-b-D-galactofuranosyl54

isothiocyanates have been also proposed as chiral derivatization reagents.A porous graphitic carbon (PGC) column has proved to be a convenient al-ternative to the classic reverse-phase columns for the separation of the di-astereomeric thioureas.207

SCHEME 28


SCHEME 27


The same derivatization protocol has been applied to the optical resolu-tion of other chiral physiologically active agents and pharmaceuticals con-taining amino groups, such as catecholamines,208 b-adrenergic antago-nists,206,209 adrenergic agents,210–212 amphetamines,213 amino alcohols,214

mexiletine,215 epinephrine,216 oxiranes,206,217 and unusual aromatic aminoacids,218,219 thus illustrating the scope of glycosyl isothiocyanate-derivedthioureas in HPLC analysis.220 Some problems related to the presence of anunidentified reactive impurity in commercial as well as locally prepared 58have been pointed out.221 Nevertheless, the side reaction could be com-pletely eliminated by pretreatment of the isothiocyanate reagent with an-other amine prior to the derivatization reaction.

The thiourea adduct resulting from the addition of one equivalent ofamine to a sugar isothiocyanate might still undergo a subsequent intramo-lecular cyclization if adequate functionalization is present. Amino aldehydeand amino ketone derivatives are among the many reagents employed forthis purpose. The corresponding oxothioureas (94) may undergo sponta-neous or acid-promoted cyclization to give imidazoline (95),50,70,72,222–226

thiazole (96),50,64,65,70,72,223–226 or tetrahydropyrimidine227–229 (97) hetero-cycles (Scheme 29). Closely related is the addition of semioxamazide to gly-cosyl isothiocyanates.230 Treatment of the adducts 98 with mercury(II) ox-ide affords glycosyloxadiazole derivatives 99 (Scheme 30).

Although a rationalization of the ambident sulfur-versus-nitrogen nucle-ophilicity in thioureas is problematic, from the ensemble of results availablein the literature it appears that nitrogen is generally involved in nucle-ophilic addition to carbonyl groups, whereas heterocyclic ring closures in-volving nucleophilic displacement generally proceed through sulfur. Inagreement with this generalization, the reaction of glycosyl isothiocyanateswith 2-chloroethylamine yielded 2-glycosylamino-2-thiazolines 101 viatransient 2-chloroethylthioureas 10070,231–234 and not imidazolidine-2-thiones as erroneously reported in a previous paper.235 Eventually, the


SCHEME 29


adduct may be added to a second isothiocyanate molecule to give trisubsti-tuted thioureas. Bicyclic thiazolines (103 and 105) have also been obtainedby the spontaneous cyclization of 1,2-trans-2-deoxy-2-iodoglycopyra-nosylthioureas (102) and from 2-deoxy-2-thioureido sugars (104) after gen-erating the corresponding glycosyl bromide (Scheme 31).73,194

The electron withdrawing -I effect of the pyranose ring decreases the nu-cleophilicity of the anomeric N-atom in glycosylthioureas, which is, conse-quently, less prone to participate in counterattack-type reactions. Thus,the attempted cyclization of N-(2-cyanoethyl)-N-ethyl-N9-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)thiourea (106) to the corresponding 5,6-dihydro-2-thiouracil has been reported236 to be unsuccessful, in contrast to what has been found for noncarbohydrate thioureas. Instead, hydrol-ysis of the cyano group to the corresponding amide 107 was observed(Scheme 32).

2. Coupling of Amino Sugars with Isothiocyanates

Sugar thioureas can be prepared, conversely, by nucleophilic addition ofthe amino group of amino sugars to alkyl and aryl isothiocyanates. Since the

SCHEME 31


SCHEME 30


reaction of amino alcohols with heterocumulenes is chemoselective, involv-ing exclusively the amine functionality, fully unprotected amino sugars may be used as precursors. Following this approach, Plusquellec and co-workers237,238 have reported the preparation of amphiphilic di- and trisub-stituted thioureas (109) by direct coupling of b-lactosylamine and its N-octyl derivative (108, R 5 H, n-octyl) with phenyl and long-chain alkylisothiocyanates in N-methylpyrrolidine (NMP) (Scheme 33).

Nonreducing amino sugars likewise afford sugar thioureas upon reactionwith isothiocyanates. Reducing sugar thioureas, however, generally undergofurther cyclization reactions entailing the acyclic carbonyl form to give het-erocyclic compounds. Although the study of the reaction of unprotected 2-amino-2-deoxyaldoses (such as 110) and 1-amino-1-deoxy-2-ketoses withisothiocyanates began almost 100 years ago, the structure of the adducts hasbeen the subject of frequent controversy,239 being unequivocally estab-lished in later years by chemical and spectroscopic methods240–247 as well asby X-ray diffraction.248–250 The reaction mechanism has been also posi-tively identified by isolation of the early intermediates and their transfor-mation into the final reaction products.251,252 The thiourea formed in thefirst step spontaneously undergoes intramolecular nucleophilic attack of anitrogen atom to the sugar carbonyl group to give an imidazoline-2-thionederivative (such as 111). When the reaction is carried out at acidic pH, fur-ther b-elimination of water and cyclodehydration reactions take place lead-


SCHEME 33

SCHEME 32


ing, respectively, to imidazoline-2-thiones (for example, 112) and bicyclic(aldoses; such as 113) or spiro (2-ketoses) furanoid compounds (Scheme34). The relative proportion of both types of compounds depends on reaction conditions. Bicyclic pyranoid derivatives were also obtained251 byblocking the hydroxyl group at C-4.

An important exception to the foregoing reaction scheme is the reactionof 2-amino-2-deoxy-D-glucose (110) with benzoyl and ethoxycarbonyl iso-thiocyanates. Stable acylthioureas were isolated in these cases, probably be-cause of the decreased nucleophilicity of the imide-type nitrogen atom.251

Despite their aforementioned instability, the adducts resulting from conden-sation of 2-amino-2-deoxy-D-glucose and 2-amino-2-deoxy-D-galactose withphenyl isothiocyanate have been proposed for the quantitative determina-tion of these amino sugars in glycoproteins.253 The authors claimed to havedeveloped conditions to avoid further transformation, preserving the stabil-ity of the phenylthiocarbamoyl derivatives by quenching the reaction mix-ture with acetic acid–triethylamine buffer. Nevertheless, it must be stressedthat the structural characterization of the adducts was rather poor and thatthe thiourea structure is improbable in view of the whole literature back-ground on this and related condensations.251,252 Alternatively, the aminosugars were reduced to the corresponding amino alcohols, which, after de-rivatization with phenyl isothiocyanate, yielded stable thioureas.253

The reaction of carbohydrate derivatives bearing amino groups with flu-orescein isothiocyanate has been widely used to introduce a fluorescent dye into a sugar molecule. Several strategies have been proposed for the

SCHEME 34




preparation of the amine precursors: reductive amination of reducingoligosaccharides,254 copolymerization of allyl glycosides with allylamine oracrylamine,255 incorporation of amine-containing spacers,256,257 replace-ment of hydroxyl by amino,258,259 or direct derivatization of natural aminosugars.258 Particularly noteworthy examples are the synthesis of thiourea-tethered fluorescein-labeled nucleotides256 (for instance 114) and sialic acidderivatives258–263 (for example, 115), which were enzymatically incorpo-rated into DNA and oligosaccharide chains of glycoproteins, respectively.The use of 7-isothiocyanato-4-methylcoumarin has also been proposed forthe identical purpose in the nucleoside series.96

3. Coupling of Sugar Isothiocyanates with Amino Sugars

The reaction of O-acylated sugar isothiocyanates with selectively O-acylated amino sugars leads to pseudooligosaccharide derivatives in whichboth saccharide subunits are joined through a thiourea spacer. A variety ofpseudodi-, pseudotri-, and pseudotetrasaccharide structures containing asingle (1→1), (1→2), or (2→2) thiourea tether have been prepared in thisway,4,63,77,78 but in no case were the final adducts deacylated. It is possiblethat solubility problems under standard Zemplén deacetylation conditions,rather than instability of the thiourea functionality in the presence of base,prevented the deprotection step, according to the experience of the authors.Unsymmetrical N,N9-bis(glycosyl)thioureas with benzylated and acetylatedmoieties have also been prepared in a similar way.264

2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl isothiocyanate (58) has beencoupled with unprotected amino sugar derivatives of neuraminic acid265


and daunorubicin266 to give the corresponding glucosylthiocarbamoyladducts 116 and 117. Compound 116 exhibited immunomodulating activity,whereas compound 117 was successfully deacetylated using sodium car-bonate in water–acetone to give 118, which showed antileukemic and anti-tumor activities with IC50 values in the submicromolar range.


To avoid the use of the somewhat problematic acyl O-protecting groupsduring the preparation of (1→6)-thiourea-linked pseudodisaccharides, thereactions of O-acetalated and O-trimethylsilylated 6-deoxy-6-isothio-cyanato sugars (for example, 119 and 120, respectively) with b-D-glucopy-ranosylamine (15) were studied.85 The coupling yields ranged from 30 to65%; in hydrolysis of 15 the splitting of ammonia which eventually reactedwith NCS groups was identified as the main competing side-reaction.The fi-nal deprotection steps (on, for example, 121 and 122) were effected follow-ing standard protocols and afforded the reducing (for instance 123) ornonreducing derivatives (such as 124) in ,100% purity (Scheme 35).

Upon further investigation, it was realized that a converse strategy, usinga per-O-acetylated glycopyranosyl isothiocyanate (58, 126, 127) and fullyunprotected methyl 6-amino-6-deoxy-a-D-glucopyranoside (125), affordedmuch higher coupling yields. Deacetylation of the thiourea adducts (128 →129) was effected in quantitative yield by Zemplén (sodium methoxide inmethanol) or mixed Zemplén–saponification (sodium methoxide inmethanol and then water) methodologies. The latter was effective for com-pounds that precipitate in methanol on treatment with base (Scheme 36).267

A main interest of thiourea-linked pseudooligoaccharides stems from thestructural analogy of the thiourea group and related pseudoamide func-tionalities with other groups of atoms, such as phosphate and urea, that oc-cur in nature linking monosaccharide frameworks in biologically importantcompounds.This structural feature, together with the synthetic versatility ofthioureas, has been notably exploited by Bruice and co-workers95,268–270 inthe preparation of a series of antisense oligonucleotides with various back-



bone modifications.Two basic strategies were developed for the synthesis ofthe (deoxy)ribonucleic thioureas. The first one involves 39-azido-39,59-dideoxy-59-isothiocyanato nucleosides (130) as key building blocks.95,268,269

In every cycle, the amine nucleophile (as in 132) is generated by reductionof the 39-azido group with hydrogen sulfide, chain extension occurring bysubsequent coupling with 130, as depicted in Scheme 37, to give the DNA(133) or RNA analog (134).

In a different approach, the 39-deoxy-39-isothiocyanatothymidyl deriva-tive 135 was used as chain-extending intermediate, which in reaction with

SCHEME 35

SCHEME 36


the 59-amine 136 afforded270 the (39→59)-thiourea-linked dimer 137. Chainextension followed a cyclic two-step process involving deprotection of theamino group with acetic acid (→138) and coupling, in quantitative yield,with another equivalent of 135 to give, after three cycles, the thymidyl pen-tamer 139 (Scheme 38).

The heterobifunctional oligosaccharide hapten 140, consisting of a group

SCHEME 37


SCHEME 38



A-active tetrasaccharide (A-tetra) and a blood Lea-active pentasaccharide(lacto-N-fucopentaose II, LNF II) linked to each other with a phenyl-aminothiourea spacer, was prepared by reaction of the N-(p-isothio-cyanatophenyl) derivative of the 1-amino-1-deoxyalditol of A-tetra withthe 1-amino-1-deoxyalditol of LNF II.271 The hapten retained both bloodgroup activities and was successfully applied to affinity purification of monoclonal anti-Lea and anti-A antibodies.

Glycosyl isothiocyanates have also been allowed to react with unpro-tected 2-amino-2-deoxyaldoses and 1-amino-1-deoxy-2-ketoses.68 This re-action leads to the formation of heterocyclic derivatives resulting from cy-clization involving the carbonyl group of the amino sugar moiety followingthe mechanistic pathway already discussed for similar condensation reac-tions with alkyl and aryl isothiocaynates.

4. Sugar Thioureas from Sugar Carbodiimides

Carbodiimides are the main alternative to isothiocyanates for preparingthioureas, nucleophilic addition of hydrogen sulfide to the heteroallene func-tionality generating the thiocarbonyl group, usually in high yield. This ap-proach has been, however, little exploited in the carbohydrate field, probablydue to the lack of synthetic methodologies leading to the sugar carbodiimideprecursors.272 Very recently, this transformation has been studied in 6-deoxy-


6-carbodiimido sugars159 and (1→6)-carbodiimide-linked pseudodisaccha-rides.273 The process was accomplished by bubbling dry H2S through a solu-tion of the carbodiimide in toluene, using silica gel as an acidic catalyst, and af-forded the target thiourea in yields over 80% (Scheme 39).

5. Functional Group Transformations in Sugar Thioureas

The thiourea group of linear and cyclic sugar thioureas can be trans-formed into other functional groups including urea,97,274,275 carbodi-imide,276,277 guanidine,95,268,269 and isothiourea270,278–280 by classic stan-dard procedures, thus opening a versatile route to a variety of other sugarderivatives (Scheme 40). The last two transformations are particularly effi-cient and have been successfully applied to the preparation of polycationanalogs of DNA and RNA.268–270,276–278,281

6. Spectroscopic and Conformational Properties

The more characteristic spectroscopic feature of sugar thioureas is the13C NMR chemical shift of the thiocarbonyl carbon atom at 180–185 ppm.

SCHEME 39

SCHEME 40



The UV f–f* absorption of the CS group appears at 252–256 nm formonosubstituted thioureas and at 242–247 nm for N,N9-disubstituted deriv-atives. In the electron-impact mass spectra of glycosylthioureas, three basicgroup of ions are analytically significant: (a) rupture of the glycosylic C-1––NH bond, (b) cleavage of the sugar NH––C(S) bond, and (c) cleavageof the (CS)––NH aglycon bond.

The E-Z rotameric interconversion rates at the pseudoamide N––C(S)bonds in sugar thioureas fall in the range of the chemical-shift time scale,which generally results in large broadening of the NMR signals. Variable-temperature NMR studies have shown that the thiourea group in per-O-acylated glycosylthioureas adopts the Z configuration at the sugarNH––C(S) bond, the only rotameric form in chloroform-d solution, withthe NH proton and H-1 in anti relative disposition.77,78,176,273 A similar sit-uation is found for thiourea groups located at secondary nonanomeric po-sitions.77,78 In stark contrast, both Z and E rotamers are detected when thethiourea functionality is placed at a primary carbon atom, resulting in muchmore complex 1H and 13C NMR spectra.85,192 Examination of the temper-ature-dependence of the NMR chemical shifts of the thiourea proton androtational barrier calculations supported a stabilization of the E rotamer byseven-membered intramolecular NH…O hydrogen bonding. This foldingpattern seems to be a main structural feature of thioureido sugars, as seenfrom specifically designed models,193 probably favored by the inherent tor-sional preferences of the covalent bonds that connect the hydrogen-bonddonor (N9H proton) and acceptor (endocyclic oxygen atom) centers. Theexistence of six-membered intramolecular hydrogen bonding in galactofu-ranosyl thioureas has also been suggested to explain the conformationalpreferences of the furanose ring (Fig. 6).54


FIG. 6 Postulated seven- (A) and six-membered in-tramolecular hydrogen bonds (B) in sugar thioureas.

VI. SUGAR THIOCARBAMATES AND DITHIOCARBAMATES

The chemistry of sugar-derived thiocarbamic and dithiocarbamic esterswas initially much less developed than that of the thiocarbamides alreadydiscussed, being limited to the condensation of reducing monosaccharides


or amino sugars with thiocyanic acid282 or carbon disulfide,283 respectively.The reasons are mainly related to the lower nucleophilicity of alcohols andthiols as compared to amines and the reversible character of the nucle-ophilic addition of the derived anions to isothiocyanates. This situation sig-nificantly changed when Barton and McCombie conceived their radical de-oxygenation of hydroxyl groups via thiocarbonyl esters.284,285 Manyexamples of the application of this procedure in the carbohydrate field,involving thiocarbamate derivatives, may be found in more general re-views.7,286–288 The ability of dithiocarbamates to form stable complexeswith divalent metals and free-radical species has been a further stimulus forinvestigating the preparation, reactivity, and biological properties of sugarderivatives incorporating thiocarbamoyl and dithiocarbamoyl functionalgroups.

1. Linear Sugar Thiocarbamates

The addition of alcohols to carbohydrate isothiocyanates is a generalmethod for the preparation of linear N-sugar, O-alkyl thiocarbamates. Thisreaction is frequently used as a tool for structure confirmation.34,53,54 It re-quires the use of a large excess of the alcohol and reflux conditions to shiftthe reaction to the desired thiourethane.

Elbert and Cerny97 have reported the condensation of 1,6-anhydro-4-O-benzyl-2-deoxy-2-isothiocyanato-3-O-p-tolylsulfonyl-b-D-glucopyranoside(141) with methanol. The presence of a good leaving group vicinal to thethiocarbamate functionality resulted in spontaneous cyclization, the out-come of the reaction being dependent on the reaction conditions.Thus, withsodium methoxide in 1,4-dioxane the reaction involved the nitrogen atomto give an epimine (142) as the sole product. In contrast, with methanolictriethylamine a bicyclic thiazoline (143) was obtained (Scheme 41).

The kinetics of the reaction of hydroxyl groups of monosaccharides andpolyols with benzyl isothiocyanate has been checked by Augustín and Balázusing UV spectroscopy.289 However, because of the reversibility of the re-action, this procedure is not of synthetic utility. O-Sugar thiocarbamateshave been more conveniently prepared by aminolysis of thiocarbonate de-rivatives290,291 or by thiocarbamoylation of free hydroxyl groups with 1,19-thiocarbonyldiimidazole.7,286–288

The main synthetic utility of O-sugar thiocarbamates is the aforemen-tioned radical deoxygenation of secondary hydroxyl groups. The method isbased on the radical-trapping ability of the thiocarbonyl group, leading toan intermediate carbon radical which fragments into a sugar radical and anS-thiocarbamate, as depicted in Scheme 42. In the presence of an H-donor,the corresponding deoxy sugar is formed, the energy gained on the changefrom thiocarbonyl to carbonyl driving the transformation.




Several reports on the use of this methodology for the synthesis of de-oxynucleosides292–295 as well as their carbocyclic analogs296,297 are onrecord. It is noteworthy that, unlike other procedures which involve anionic mechanism, this radical process is not subjected to steric hindrance orelectrostatic repulsion. Although N,N-diethylaminothiocarbonyl deriva-tives were originally employed, the O-(imidazolylthiocarbonyl) analogshave proved more efficient.7,286–288,298–301 The methodology is compatiblewith the presence of nitrogen and phosphorus functionalities in the mole-cule, as shown for the deoxygenation of the HO––C––P unit in the phos-phinyl derivatives 144 and 145 (Scheme 43), a key step in the synthesis ofxylopyranose mimics having phosphorus instead of oxygen in the ring (146and 147).302,303

SCHEME 41

SCHEME 42


In the case of vicinally disubstituted sugars with a pair of radical leavinggroups, such as thiocarbamate and halogen (148), treatment with tri-n-butyltinhydride and a,a9-azobis(isobutyronitrile) (AIBN) led to the correspondingunsaturated derivatives (for example, 149) in high yield without observed sideproducts (Scheme 44).304 Thioacylimidazole esters have been found, however,less effective than thiocarbonates and xanthates in promoting radical C-allylation, even in the presence of AIBN as radical initiator.305

Ley and co-workers306,307 have investigated the use of glycosyl thiocar-bonylimidazolides as glycosyl donors using silver perchlorate as promotor.The method was successfully applied to the total synthesis of the antipara-sitic agent Avermectin B1a by coupling of the oleandrose disaccharide thio-carbamate 150 with the monoacetylated aglycon 151 (Scheme 45). The a-glycoside 152 was isolated in 64% yield together with 11% of thecorresponding b-anomer.

2. Cyclic Sugar Thiocarbamates

The simultaneous presence of isothiocyanate and free hydroxyl groups ina sugar molecule may lead to formation of intramolecular cyclic thiocarba-

SCHEME 43


SCHEME 44


mates, depending on their relative disposition and on conformational bias.The following rules apply when several isomeric structures can be foreseen:(a) formation of five-membered cyclic thiocarbamates is favored againstsix-membered analogs, (b) furanoid rings are more prone to afford bicyclicheterocycles than their pyranoid tautomers, and (c) hydroxyl groups intrans-diaxial orientation with respect to the NCS group or distant by fivecovalent bonds or more are, generally, unreactive.

According to this general scenario, the recently revised275 reaction of re-ducing aldoses with thiocyanic acid provides furanoid bicyclic oxazolidine-2-thiones (153) as the major reaction products, probably via an open-chainisothiocyanate (Scheme 46). Under the same reaction conditions, ketosesafforded mixtures of spiro- and bicyclic-thiocarbamate derivatives.308

When the isothiocyanate group is vicinal to the anomeric position in re-ducing monosaccharides, the more acidic hemiacetalic hydroxyl group is in-


SCHEME 46

SCHEME 45


volved in thiocarbamate ring closure. Different sugar tautomeric structurescan thus react with the corresponding loss of product purity. In the case of1-deoxy-1-isothiocyanato-D-fructose (156), generated either from the cor-responding amino sugar309 154 or from the diacetonated derivative83 155, a5 : 1 mixture of the b-pyranoid and b-furanoid spironucleosides 157 and 158was obtained (Scheme 47).

If the cyclization process does not implicate the anomeric position,the regioselectivity of the reaction, regarding both the tautomeric form of the sugar and the functional groups involved, is self-controlled by theconfiguration of the sugar template. A variety of enantiomerically pure ox-azolidine- and tetrahydrooxazine-2-thione heterocycles have been thusprepared by spontaneous or base-induced cyclization of carbohydrate-derived hydroxyisothiocyanates. The reported structures include 6,5-(159),6,4-(160), 3,4-(161), 3,2-(162), 3,5-(163), and 5,6-(164) cyclic thiocarba-mates.81,82,84,310 The sucrose derivative 165 illustrates the selectivity in the

SCHEME 47



formation of 1,3-O,N-heterocycles from unprotected sugar isothiocyanates.Whereas the trans-decalin-type system at the glucopyranosyl moiety read-ily originates from the corresponding c-hydroxyisothiocyanate segmentprecursor (see 34) upon treatment with base, the NCS group at C-6 of thefructofuranosyl moiety remains unreactive98 even in the presence of tri-ethylamine in N,N-dimethylformamide solution at 808C.

The formation of a seven-membered thiocarbamate ring system (168) inlow yield has been observed during the attempted reduction156 of the se-lectively protected b-D-galactopyranosyl isothiocyanate 166 to the corre-sponding glycosyl thioformamide 167 (Scheme 48).

Uzan and co-workers311 have reported an alternative route for thepreparation of pyranose and furanose cis-1,2-fused oxazolidine-2-thionesthat uses 1,2-O-sulfinyl sugar derivatives (169) as precursors. After treat-ment with sodium thiocyanate in DMF at 808C, the bicyclic thiocarbamates(172) were isolated in 60–90% yield. The proposed mechanism involves formation of a b-configured thiocyanate (170) that isomerizes to the a-isothiocyanate derivative (171) under the reaction conditions (Scheme 49).

Five-membered cyclic thiocarbamates have also been reported to origi-nate during the reduction of vic-azidothiocarbonates, nucleophilic attack ofthe intermediate amine to the thiocarbonyl group being faster than reduc-tion of the ester.96,312

Recently, Pintér and co-workers313 have reported the use of cyclic thio-carbamates as precursors of cyclic isoureas by sequential S-p-chlorobenzy-


SCHEME 48


lation and nucleophilic displacement of the p-chlorobenzylthio group withmorpholine. Preparation of oxazoline heterocycles by desulfurization withRaney nickel has also been effected.308

3. Linear Sugar Dithiocarbamates

The reaction of isothiocyanates with thiols is, in general, impractical forthe preparation of dithiocarbamates, since N-monosubstituted derivativesreadily decompose into the starting materials. Nevertheless, the method issynthetically useful in the case of aryl isothiocyanates. Thus, cellulose–arylisothiocyanate conjugates exhibited a substantial thiol binding capacitywhich was absent for nonaromatic analogs.142,143

A valuable alternative for the preparation of N-sugar dithiocarbamatesis the reaction of a sodium dithiocarbamate salt obtained from the conden-sation of an amino sugar with carbon disulfide in the presence of base, withan excess of alcohol97 or alkyl iodide190 (Scheme 50).

To overcome the problems associated with purification of dithiocarba-mate salts, Giboreau and Morin314 have disclosed a synthetic procedurethat employs stannyl dithiocarbamates as precursors. The method is com-patible with the presence of hydroxyl groups and has been applied to thehigh-yielding preparation of the sodium N-methyl-D-glucamine dithiocar-bamate salt 175. Thus, treatment of the secondary amine 173 with carbondisulfide and bis(tri-n-butyltin)oxide achieved the stannyl dithiocarbamate174 in 83% yield after purification on neutral alumina. Cleavage to thedithiocarbamate salt was effected in quantitative yield by using sodium hy-drogensulfide (Scheme 51).

N-Methyl-D-glucamine dithiocarbamate (175, MGD) was first conceivedas a water-soluble metal chelating agent and both the sodium and ammo-nium salts were found to be effective antagonists of cadmium toxicity.315


SCHEME 49

SCHEME 50


Closely related are the 1-benzylamino-1-deoxylactitol dithiocarbamatesalts developed by Eybl and co-workers316,317 for the same purpose. How-ever, the most important application of 175 is, probably, its use as a non-toxic, water-soluble nitric oxide probe in vivo. In view of the central impor-tance that this gaseous free-radical species plays in regulating a broad rangeof important biological functions,* its detection and quantification near itssite of production and action is of prime importance. For this purpose, theferrous salt of MGD, which forms a stable water-soluble mononitrosyliron–dithiocarbamate complex (176) with a characteristic electron spin res-onance (ESR) spectrum at room temperature, is currently used.318–323

Cao and co-workers324 have reported the preparation of a b-cyclodextrinderivative containing an N-linked dithiocarbamate group at the C-2 posi-tion (177). The corresponding Mn(II) and Cu(II) complexes showed super-


SCHEME 51

* The discoverers of nitric oxide as a signal transmitter in the mediation of a variety of im-portant cellular functions, in particular in the cardiovascular system, R. F. Furchgott, F. Mu-rad, and L. J. Ignarro, were awarded the 1998 Nobel Prize in Physiology and Medicine.


oxide dismutase mimetic activity, promoting the disproportionation of su-peroxide radical (O2˙2) into O2 and H2O2.

S-Glycosyl-N,N-dialkyldithiocarbamates have attracted attention be-cause of the known fungicidal, insecticidal, and anticarcinogenic propertiesof related dialkyldithiocarbamates. The classic synthetic methodology fortheir preparation relies on the reaction of a per-O-acyl protected glycosylbromide with the sodium salt of a dialkyl dithiocarbamate.325 By this pro-cedure, Bertram and co-workers326,327 prepared peracetylated N,N-diethyland N,N-diallyl S-sugar dithiocarbamate derivatives of D-glucose, lactose,and cellobiose, which were further deprotected with methanolic ammonia.The S-glycosidic bond was found to be stable under physiological condi-tions in vitro and some of the compounds exhibited in vivo inhibition of nitrosamine-induced DNA damage. An original approach for the prepara-tion of these types of compounds, developed by Szeja and Bogusiak,328 es-pecially suited for acetal and benzyl protecting groups, involves in situ-generated glycosyl tosylates under phase-transfer conditions (Scheme 52).

Mention should also be made of the application of Mukaiyama’s method-ology to the synthesis of S-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-N,N-dimethyldithiocarbamate using a pyridinium derivative as theanomeric leaving group.329

Fügedi and co-workers330 have examined the potential of glycosyl 1-piperidinecarbodithioates as glycosyl donors in oligosaccharide synthesis.Such thiophilic promotors as methyl or silver triflate were efficient activa-tors for the glycosylation reaction, although other less expensive salts suchas tin(IV) or iron(III) chloride also gave good yields. Of note is the fact thatunder conditions used by the authors, thioglycoside acceptors, which are

SCHEME 52



themselves potential glycosyl donors, remained stable (Scheme 53). S-Glycosyl-N,N-dimethyldithiocarbamate derivatives have been analogouslyused as glycosyl donors in the 2-amino-2-deoxyhexose series.331

Carbohydrate derivatives bearing S-linked dithiocarbamate functionali-ties at nonanomeric positions have been obtained by nucleophilic displace-ment of suitable leaving groups by N,N-dialkyldithiocarbamate anions.332

Interestingly, the glucose-derived bis(dithiocarbamoyl) esters 179 and 180,obtained from the 3-iodo-6-O-tosyl derivative 178 (Scheme 54), exhibitedsignificant antifungal activity.333

4. Cyclic Sugar Dithiocarbamates

The reaction of fully unprotected 2-amino-2-deoxyaldoses or 1-amino-1-deoxyketoses with carbon disulfide leads to five-membered cyclic dithio-carbamates (182) involving the open-chain tautomeric form of the sugar(181), as unequivocally proved by Avalos and co-workers.251 N-Alkylglyco-sylamines undergo a similar transformation via an Amadori rearrange-ment. The monocyclic derivatives can be dehydrated to thiazoline deriva-


SCHEME 53

SCHEME 54


tives (183), which were used as precursors of the 5-carbaldehyde structures184 (Scheme 55).334

VII. MISCELLANEOUS N-THIOCARBONYL CARBOHYDRATE DERIVATIVES

Bis(thiocarbonyl)hydrazide derivatives of galactaric acid (186, 187) havebeen prepared by reaction of 2,3,4,5-tetra-O-acetyl-D-galactaroyl dichlo-ride (185) with 4-arylthiosemicarbazides335 or S-methyl(benzyl) hy-drazinecarbodithioates336 (Scheme 56). The adducts were subsequentlyconverted into a variety of bis(heterocyclic) compounds including thiadia-zole, triazole, and oxadiazole derivatives. The tetraacetates 186 have been

SCHEME 55

SCHEME 56



also reacted with different bifunctional amines (piperazine, tetraethyl- andtetramethyl-ethylendiamine, and tetramethyl-1,3-propanediamine) to givemonomeric or polymeric bis(hydrazide) salts, depending on the ratio of thereagents.337

Opening of aldonolactons by thiosemicarbazide affords transient N-glyconoyl adducts that undergo spontaneous cyclodehydration to the cor-responding acyclic C-nucleosides of 1,2,4-triazole (188, Scheme 57). Dou-ble-headed analogs were also prepared using diethyl D-galactarate as precursor.338

Tweeddale and co-workers339,340 have investigated the binding of sugarsto a polymeric support through thiosemicarbazone spacers (190). Althoughthiosemicarbazones have been prepared in good yield by direct condensa-tion of reducing carbohydrates with arylthiosemicarbazides, the reactionwas found unsuitable in the solid phase because of the unfavorable ther-modynamics. Nevertheless, coupling of glycosylhydrazines (189) with iso-thiocyanate-substituted polystyrene achieved good immobilization within afew hours at room temperature (Scheme 58). A main advantage of themethod is that the bound sugar may be further released by reaction with ei-ther hydrazine hydrate or benzaldehyde.

Sugar thiosemicarbazones have been the subject of further interest be-cause of their capability to act as water-soluble tetradentated ligands for di-valent transition-metal cations. Horton and co-workers341 reported the syn-thesis of 3-deoxyaldos-2-ulose bis(thiosemicarbazones) by the reaction ofreducing aldoses with thiosemicarbazide in the presence of p-toluidine(Scheme 59). The authors observed in vivo antitumoral activity in the


SCHEME 57


murine L-1210 assay for the copper(II) complex of the D-glucose deriva-tive, namely 3-deoxy-D-erythro-hexos-2-ulose bis(thiosemicarbazone)(191). More recently, the bis(thiosemicarbazones) obtained from D-glucose,D-galactose, and D-ribose as well as their copper(II) and nickel(II) com-plexes were fully characterized by spectroscopic (1H and 13C NMR, UV, IR,ESR) methods.342

In order to preserve the cyclic form of the sugar, glycosyl derivatives of2-hydroxyacetaldehyde (192) have been proposed as precursors ofthiosemicarbazone ligands343 (193, Scheme 60). The corresponding cop-per(II) and manganese(II) complexes exhibited superoxide dismutasemimetic activity, with IC50 values ranging from 0.2 to 0.8 eM.

VIII. NATURALLY OCCURRING N-THIOCARBONYL

CARBOHYDRATE DERIVATIVES

The only carbohydrate derivatives incorporating N-thiocarbonyl func-tional groups of natural origin appear to be isothiocyanate and thiocarba-mate glycosides embodying a-L-rhamnopyranose and 4-hydroxybenzyl

SCHEME 59


SCHEME 58



isothiocyanate or thiocarbamate as the glyconic and aglyconic moieties, re-spectively.These compounds are mustard oil glycosides, which are very rarein nature and were isolated from plants of the Moringaceae family. Themonosaccharide is found fully unprotected and as its 4-O-acetyl and 2,3,4-tri-O-acetyl derivatives. The isothiocyanates 194 and 195 have been identi-fied as the antibiotic elements of the plant,344,345 whereas the thiocarba-mates 196–202, which exist in both E and Z configurations at thepseudoamide bond, showed hypotensive and spasmolytic properties.346–351

Recently, chemical synthesis of the niazinin (196), niazimicin (197), niazicin(198), and niaziminin (199) thiocarbamate glycosides has been achieved.352

IX. N-THIOCARBONYL SUGARS IN MOLECULAR RECOGNITION

The critical role of carbohydrate recognition in cellular functioning hasimpelled aggressive research directed toward the understanding and con-trol of the intermolecular processes involved in carbohydrate metabolismand signaling. Modified saccharide structures incorporating N-thiocarbonyl

SCHEME 60


functionalities have proved to be very useful tools for such goals. A mainfactor is that attaching sugars to the amino groups of proteins, their puta-tive receptors in biological systems, can be achieved very efficiently bymeans of N-thiocarbonyl linkers. This approach has been further extendedto the new concepts of glycodendrimers and glycoclusters, highly orderedpolyglycosyl structures whose preparation requires high-yielding couplingstrategies. Moreover, the synthetic versatility of N-thiocarbonyl functionalgroups, allowing access to many other functionalities, and their particularstructural and electronic properties, can be exploited in the preparation ofsugar mimetics and artificial receptors useful for enzymatic and supramol-ecular studies aimed at elucidating the structural requirements for affinityand specificity in carbohydrate interactions.

1. Interactions with Membrane Receptors

The ability of isothiocyanate groups to react with the amino group of ly-sine residues makes sugar isothiocyanates attractive candidates for labelingthe specific proteins involved in the transport of carbohydrate substratesacross cell membranes.59 Fully unprotected b-D-glucopyranosyl60 and b-maltosyl isothiocyanates61 were first used for this purpose. Both werefound to behave as potent irreversible inhibitors of glucose translocation inthe human erythrocyte. Using b-[14C]maltosyl isothiocyanate-labeled ery-throcyte membranes, the question of carrier identity could be addressed.353

A main drawback for these applications is the low stability of fully unprotected glycosyl isothiocyanates under physiological conditions,so that results may be perturbed by decomposition of the reagent.62

6-Deoxy-6-isothiocyanato-D-glucopyranose was proposed as an alternativeaffinity reagent.80 However, it has been shown subsequently that this com-pound is actually unstable and undergoes spontaneous cyclization to give afuranoid 6,5-cyclic thiocarbamate.82 Stable fully unprotected phenyl iso-thiocyanate glycosides have been successfully used instead. Thus, p-isothiocyanatophenyl b-D-glucopyranoside is a nontransported inhibitor ofthe Na1–glucose cotransporter and has found application in studies di-rected to elucidate the geometry and nature of the glucoside–transport pro-tein interaction.117,354 Under mild conditions, not favorable for covalentlinkage formation, p-isothiocyanatophenyl 2-acetamido-2-deoxy-b-D-glucopyranoside reversibly inhibited N-acetyl-D-glucosamine countertrans-port in lysozomal membrane vesicles by 70%.355

Replacement of N-acyl and O-acyl by N-thioacyl groups in sialo-sugarchains of cell surface glycoproteins can influence certain biological func-tions mediated by sialic acids, such as cellular adhesion phenomena or virusspecificity for host cells. Interestingly, sialyltransferases specific for different




acceptor sequences exhibited a low enzyme specificity with respect to vari-ous N-thioacyl-modified neuraminic acid derivatives.189,356,357 This allowed,after sequential enzymatic desialylation–resialylation, substitution of N-acetyl-9-O-acetylneuraminic acid subunits by 9-deoxy-9-thioacetamido N-thioacetylneuraminic acid residues in intact erythrocytes.358 The syntheticanalog was recognized by the receptor-destroying enzyme (acetylesterase)of this virus, so that the virus particles attached to the cells but were unableto infect them. N-Thioacetylneuraminic acid glycosides likewise are recog-nized by influenza virus hemagglutinin and have been used in the prepara-tion of polymeric multisialylated structures (203–205) showing increasedactivity against different virus strains.359–361 Moreover, the thioglycosidepolymer 204 was resistant to viral neuraminidase, which is an intrinsic ad-vantage for the inhibitor efficiency.360

2. Neoglycoconjugates, Glycodendrimers, and Glycoclusters

Neoglycoconjugates are synthetic compounds that emulate the behavior ofthe natural conjugates, being of direct application in practically every aspectof glycobiology.Those most often used are neoglycoproteins, obtained by co-valent attachment of conveniently functionalized oligosaccharides to a car-rier protein and are conceived as a way to remedy the lack of carbohydratehomogeneity in natural glycoproteins. Not surprisingly, generation of athiourea bridge between the saccharide hapten and the protein is probablythe most popular method to generate neoglycoproteins.104,114 Comparativedescriptions of this technique and other conjugation methodologies can befound in several reviews.362–366 Sugar–isothiocyanate conjugates have beengenerally used as precursors because of their higher stability and the possi-bility of manipulating the spacer arm for optimal geometric or immunogenicproperties. The major initial reaction that occurs under mild conditions atneutral or slightly alkaline pH is the formation of thiourea derivatives withterminal amino groups and with e-amino groups of lysine residues (Scheme


SCHEME 61


61). Bovine serum albumin (BSA),114,138,139 keyhole limpet hemocyanin(KLH),367 inmunoglobulin A (IgA),111 human serum albumin (HSA),368 andR-phycoerythrin,113 have been used as the conjugated proteins, frequently la-beled with fluorescein residues. The technique has permitted the raising ofantibodies against specific carbohydrate moieties; detection and quantifica-tion of oligosaccharide receptors in tissues, cells, or extracts; selection of a ligand structure for subsequent purification by affinity chromatography;and detailed analysis of carbohydrates and carbohydrate-binding proteins.

In most cases, interactions involving carbohydrates in biological systemshave a multivalent character, requiring a local density and a precise spatialorientation of the active saccharide epitopes that can be better achieved byusing well-defined artificial templates. Application of the thiourea method-ology for coating polyamine scaffolds and dendrimer carriers with carbo-hydrates has shown considerable promise in this respect.369–373 Lindhorst and co-workers374,375 have reported the synthesis of thiourea-bridged gly-coclusters and glycodendrimers by coupling peracetylated glycosyl isothio-cyanates with tris(2-aminoethyl)amine (→206) and dendritic multivalentcores of the polyamidoamine (PAMAM) family (→207–209). The adductswere further deacetylated in a final step. The strategy was also implementedfor glycocoating other polyamine templates of different geometries, such asazamacrocycles376 (→210) and an a-D-glucose-centered pentaamine337

(→211). In addition, protected and fully unprotected spacer-armed sugarisothiocyanate conjugates130,131 (→212, 213) as well as deoxyisothiocyanatosugars114 (→214) may be used as precursors. Fully unprotected thiourea-bridged glycodendrimers and glycoclusters with a-D-mannopyranosylresidues were tested for their binding capacity to mannose-specific lectin ontype 1 fimbriae of Escherichia coli,114,374,376 while the 2-acetamido-2-deoxy-b-D-glucopyranosyl derivatives were checked against the dimeric receptor of rat natural killer cells, sNKR-P1A protein.375






Roy and co-workers have also used tetra-, octa-, hexadeca-, and 32-valent PAMAM cores to build a-D-mannopyranosyl- and a-D-thiosialodendrimers116 (217) by coupling them with the correspondingphenyl isothiocyanate glycoside (215) and thioglycoside378 (216), respec-tively. After deacetylation, these multivalent neoglyconjugates wereshown to exhibit a high lectin-binding avidity and can be used as biochro-matography materials for isolating carbohydrate-binding proteins(Scheme 62).

Other reports from the same laboratory deal with the effect of shape,size, and valency of multivalent mannosides on their binding properties toconcanavalin A and pea lectins.379–381 To optimize lectin–carbohydrate in-teraction, a series of thiourea-bridged di-, tri-, tetra-, and hexaantennaedclusters derived from different aliphatic and aromatic scaffolds were pre-pared. Coupling isothiocyanate 215 with mono-, di-, and hexaamines379,380

(→218–220) or reacting fully unprotected amine-functionalized manno-sides with polyisothiocyanates381 (→221–223) afforded the target glyco-clusters. Both the intramannosyl distance and valency were proved to beimportant factors for optimum protein binding.

The use of cyclodextrins as biocompatible scaffolds for adjusting glyco-cluster molecular weight and topology using the thiourea technology hasbeen explored by the groups of García Fernández and Defaye.382,383 Nu-cleophilic addition of cyclomaltoheptaose (b-CD) derivatives bearing oneor seven amino groups at the primary face to peracetylated b-D-glucopyra-nosyl, b-cellobiosyl, and b-lactosyl isothiocyanates provides efficient accessto the corresponding mono- and heptaantennary conjugates. The reactionconditions (pyridine and water–acetone at pH 8, respectively) as well as thelocation of the reactive functional groups were found to be critical. Thus, areverse strategy involving b-CD-derived isothiocyanates and glycosyl-amines proved much less satisfactory. The subsequent deacetylation stepfollowing a mixed Zemplén–saponification procedure was quantitative. Anoteworthy exception to this general pattern was reported, however, for a-D-mannopyranosylthiourea substituents.382 The authors observed an unprecedented anomerization reaction upon base treatment, which is incontrast with previously reported results on the preparation of a-D-mannopyranosylthiourea glycoclusters and glycodendrimers. A thorough


investigation of the anomeric behavior of a-configured glycosylthioureas insolution should throw some light onto this question.

3. Enzyme Inhibitors

Even though the use of N-thiocarbonyl carbohydrate derivatives in en-zyme studies was already devised in the mid-1970s, their prominent positionboth as precursors and as targets in the design and synthesis of glycosidaseinhibitors was firmly established only in the beginning of the 1990s and runsparallel to the development of the concept of transition-state analogs. Theunique properties of N-thiocarbonyl functional groups as versatile sourcesof planar resonance structures that resemble the incipient oxocarbeniumcation postulated as the transition state in enzymatic glycoside cleavage isthe main reason for this situation.

Fully unprotected glycosyl isothiocyanates have been reported to act as spe-cific irreversible inhibitors of glycosidases. Thus, b-D-glucopyranosyl isothio-cyanate inactivated the action of sweet-almond b-glucosidase,384,385 while 2-acetamido-2-deoxy-b-D-glucopyranosyl isothiocyanate likewise inhibited the human and boar N-acetyl-b-D-hexosaminidase.60 Competitive-inhibitionassays indicated that covalent binding to the protein occurs at the active site. The structural analogy of glycosyl isothiocyanates and the b-glycoside substrates suggested that the NCS groups would interact with one of


SCHEME 62



the catalytic groupings of the enzyme. However, because of the instability andhigh reactivity of fully unprotected glycosyl isothiocyanates, decompositionand reaction at different positions in the protein may be a serious perturba-tion in these studies. Stable, unprotected deoxyisothiocyanato sugars mightconstitute an interesting alternative to overcome such drawbacks.82

Thioamides 226 and 227, resulting from replacement of the amide car-bonyl by thiocarbonyl in p-nitrophenyl 2-acetamido-2-deoxy-b-D-gluco-and galacto-pyranosides, were shown to inhibit 2-acetamido-deoxy-b-D-glucosidase from Turbatrix aceti.174 In contrast, the related p-nitrophenylthioglycosides exhibited almost no inhibitory effect. In the light of the re-cent results of Knapp and co-workers,386 using the 4-methylumbelliferyl de-


rivative 228, it is probable that the glycoside is converted in situ into a bi-cyclic thiazoline (229), which would be the real active species (Scheme 63).Structure 229 with D-gluco configuration was eventually prepared by refluxing 1,3,4,6-tetra-O-acetyl-2-deoxy-2-thioacetamido-b-D-glucopyra-nose in toluene and proved to be a very potent inhibitor of jack bean N-acetylhexosaminidase.386

Since the first communication by Ganem and co-workers179 in 1990 on thebroad-spectrum inhibition of glycosidases by amidine-type carbohydratemimics, several reports on the synthesis of these derivatives using thio-lactams as the key precursors have been published. The groups ofGanem,179,180,182,387,388 Vasella,181,184,185 and Tellier183,389,390 have been in-strumental in developing these transformations that include access to ami-dine, amidrazone, amidoxime, and amidine N-arylcarbamate structures,which rank among the most potent competitive inhibitors of glycosylhydro-lases reported. The approach has been extended to pentoses388,391 with thepreparation of the D-ribonamidoxime 235 and the D-ribonamidhyrazones236–238 from the corresponding thiolactam 234. Compound 237 is the mostpotent nucleoside hydrolase inhibitor known to date (Schemes 64 and 65).



Closely related to the just-discussed transformation of sugar thiolactamsinto amidines is the preparation392 of the cyclic guanidinium glycomimetics241 and 242 from the cyclic thiourea precursor 239. Replacement of thethiocarbonyl sulfur atom by nitrogen was effected after treatment withethyl iodide and aminolysis or hydrazinolysis, respectively, of the corre-sponding S-ethyl derivative 240 (Scheme 66). Alternatively, analogs of glycosides (244) and disaccharides (245) containing a cyclic guanidinium


SCHEME 63


SCHEME 64

SCHEME 65

SCHEME 66


structure have been obtained from acyclic c-aminothioureas (243) upontreatment with lead(II) oxide (Scheme 67).393–395

These two synthetic strategies are not compatible with the presence of ahydroxyl group contiguous to the guanidine functionality in the final gly-comimetic. To overcome this limitation, Wong and coworkers88 have pro-posed a different approach involving thiourea derivatives of D-threose inthe anchored acyclic form 246, which, after transformation into the guani-dine analogs (247) and deprotection, afforded the target cyclic guanidiniumhexose mimics 248 (Scheme 68).

An impressive number of papers has appeared in the past few yearsbased on the use of sugar thioureas as intermediates in the synthesis of thenaturally occurring potent trehalase inhibitor trehazolin (251) and of sev-eral isomers.52,396–404 The key reaction step involved the cyclization of anN,N’-disubstituted a-D-glucopyranosyl–aminocyclitol (a-D-glucopyranosyl-


SCHEME 67

SCHEME 68


trehalosamine in the natural compound) thiourea (249) with partic-ipation of a b-located hydroxyl group. Either mercury(II) oxide or 2-chloro-3-ethylbenzoxazolium tetrafluoroborate promoted the de-sired transformation (→250) with generation of the aminoxazole ring(Scheme 69).

The cyclocondensation reaction proceeds via a transient hydroxycar-bodiimide that leads, selectively, to the cis-fused five-membered cyclicsourea. In addition to the preparation of a range of diastereomers, a thor-ough systematic modification of the sugar and aminocyclitol moieties has-been effected48,86,405–409 that allowed confirmation of the trehazolin struc-ture, identification of new potent trehalase inhibitors, and determination ofstructure–inhibitory activity relationships. Some anomalous results havebeen reported, however, in the attempted preparation of tetrahydropy-rano[2,3-d]oxazole analogs of trehazolin (e.g., 254) from N,N9-bis(D-glucopyranosyl)thioureas.264 Whereas the di-b-configured derivative 252afforded the expected cyclocondensation compound 253 in low yield, thea,b-isomer 255 yielded a furanose-fused structure (256) and the di-a-epimer a complex mixture of unidentified products (Scheme 70).

The reaction is not restricted to glycosylthioureas. Starting from carbo-hydrate derivatives bearing the cyclitolthioureido substituent at anonanomeric position87,89,409 (for example, 258→259), new trehalozoid gly-cosidase inhibitors have been designed that, in some cases410 (260), exhib-ited aglycon selectivity (Scheme 71).

Mixed 1-deoxynojirimycin–trehalamine inhibitors have also been pre-pared from the corresponding iminosugar b-hydroxythioureas 261–263.140

The versatility of this approach has been further illustrated by the synthe-sis of imidazoline analogs of trehazoline starting from sugar-derived b-aminothioureas (263).408

The aminocyclitols allosamizoline (266) and demethylallosamizoline(267), which are found in the pseudotrisaccharide chitanase inhibitorsknown as allosamidin and demethylallosamidin, have been prepared fromcyclic thiocarbamate411 (264) and b-hydroxythiourea412 (265) precursors,


SCHEME 69


SCHEME 71

SCHEME 70


respectively. A glucosamine–aminothiazoline analog (269), obtained from1,3,4,6-tetra-O-acetyl-2-deoxy-2-(3,3-dimethylthioureido)-b-D-glucopyra-nose (268), has also been reported413 (Scheme 72).

The glycosidase-inhibitory properties of sugar-shaped cyclic thioureaswere first explored by Lehmann and co-workers,414 who obtained the hexa-hydropyrimidine-2-thione glucomimetic 271 by thiocarbonylation of the di-amine precursor 270. Compound 271 was a very weak inhibitor of sweet-almond b-glucosidase, and this was ascribed to the lack of the OH group atthe homologous position of C-3 in D-glucopyranosides. Moreover, an unex-pected preference for the trans-diaxial conformation in deuterium oxide so-lution was observed (Scheme 73).

García Fernández and co-workers415,416 have developed a different syn-thetic route to carbohydrate mimics having a cyclic thiourea structure basedon the intramolecular nucleophilic addition of thiourea groups to themasked carbonyl group of reducing monosaccharides. Thus, starting from 3-deoxy-3-thioureido sugars, the iminooctitol analogs 273 were obtained(Scheme 74). Likewise, a preference for structures with axial disposition of


SCHEME 72

SCHEME 73


the carbon substituent was observed, a formal “deoxoanomeric effect’’which seems to be a common feature to these azaheterocycles. On the otherhand, the aminoketalic pseudoanomeric hydroxyl group adopted exclusivelyaxial orientations in water solution, in accord with the anomeric effect.

The possibility of controlling the conformation and configuration ofanomeric hydroxyl groups by a vicinal N-thiocarbonyl functionality wasfurther exploited in the design of bicyclic “azasugar’’ glycomimetics struc-turally related to the iminosugar (“azasugar’’) glycosidase inhibitor fam-ily.310 Acid hydrolysis of the isopropylidene group in the 5,6-(cyclic thio-carbamate) 274 resulted in tautomeric rearrangement to the bicyclicderivative 275 (Scheme 75). In contrast to data for reducing iminosugars,compound 275 is stable in water solution and exists exclusively in the a-anomeric configuration, probably due to a very efficient delocalization in-teraction between the f-type lone-pair orbital of the sp2-hybridized N-atomin the ground state of N-thiocarbonyl functionalities and the s* antibond-ing orbital of the contiguous C––O bond. It is of note that this control of theanomeric configuration resulted in a dramatic increase (104-fold) in theyeast a-glucosidase (Ki 5 40 eM) versus sweet-almond b-glucosidase se-lectivity as compared with the parent 5-amino-5-deoxy-D-glucopyranose(nojirimycin).


SCHEME 75

SCHEME 74


Monocyclic N-thiocarbonyl iminosugars are highly prone to undergo in-tramolecular glycosylation reactions when the resulting bicyclic structurescan accommodate the anomeric effect at the aminoketalic center. Thus, thesynthesis of inhibitors of the calystegine family has been accomplished bytandem tautomerization–intramolecular glycosylation of 5-deoxy-5-thioureido-L-idose derivatives (276, Scheme 76).417 The resulting com-pounds (277) acted as specific inhibitors of b-glucosidases, analogous to theparent natural alkaloids.

4. Artificial Receptors

Modification of cyclodextrins (CDs) with thioureido substituents is avery convenient way for conjugating these natural cyclooligosaccharide re-ceptors with a variety of molecules. The resulting semisynthetic compounds(so-called second-generation cyclodextrins) may exhibit different inclusion,solubility, or toxicity properties as compared to their native counterparts. b-Cyclodextrin derivatives bearing thiourea-bridged oligosaccharide– andglycopeptide–thiourea antennae at the primary face have been proposed asdrug-carrier systems, endowed with the capability of molecular recognitionat the cell membrane level.99,155,382,383,418 A significant increase in water sol-ubility, initially ascribed to the hydrophilic saccharide branch, was mea-sured for such adducts. However, the same behavior was observed when hy-drophobic substituents such as peracetylated sugars or alkyl groups wereincorporated. It was concluded that the increase in water solubility is actu-ally imparted by the thiourea group, which probably forms hydrogen bondswith water molecules breaking the intercyclodextrin hydrogen-bond net-work, thus preventing aggregation. Moreover, thiourea-modified b-CDs ex-hibited an about fourfold decrease in their hemolytic properties withoutany significant modification of the inclusion capability as determined forthe anticancer drug Taxotere.

Thiourea segments have also been incorporated into macrocyclic a,a9-trehalose-based pseudocyclooligosaccharides (278–280) obtained100,419 by

SCHEME 76



the coupling reaction of bifunctional isothiocyanate and amine precursors.The geometrical and conformational properties of the new host moleculeswere governed by the strong hydrogen-bond donor character of thethiourea NH protons and the presence of four slow rotating N––C(S)pseudoamide bonds. Low-temperature NMR experiments revealed thepresence of two configurational patterns in solution, namely the Z,E : E,Zalternate conformation and the Z,E : Z,E parallel conformation, both hav-ing C2 symmetry and involving two intramolecular NH…O hydrogenbonds.

The hydrogen-bonding donor capability of thiourea groups has been fur-ther exploited in the design of multitopic podantlike sugar thiourea recep-tors suitable for recognition and complexation of complementary func-tional groups such as carboxylate or phosphate (for instance, 281, 282).Preliminary results420–422 have shown association constants up to 105 M21

for neuraminic acid derivatives as guests using the tritopic tris(glucopyra-



nosylthiourea) receptor 282 in chloroform-d solution and above 102 M21 ina polar competitive solvent such as dimethyl sulfoxide.

X. CONCLUSIONS

From the previous discussion, it is clear that the study of the chemistryof N-thiocarbonyl carbohydrate derivatives is at present experiencing atremendous expansion, both quantitatively and qualitatively. In additionto the classic uses as synthetic intermediates in heterocyclic chemistry orin neoglycoprotein preparation, the new applications on record are ofconcern to virtually every aspect of glycobiology. A major reason is un-doubtedly the possibility of access to highly functionalized complex de-rivatives with tailored structural properties employing relatively simpleand very efficient synthetic procedures. The wide range of structures re-ported demonstrates that the basis for construction and manipulation ofN-thiocarbonyl carbohydrate compounds is now well established, and fur-ther challenges are ready to be undertaken in the near future. Thus, thesynthetic methodologies seem to be mature for solid-phase and combina-torial chemistry, which should allow preparation and testing of numerousnovel linear and branched pseudooligosaccharides. The chelating and hydrogen-bonding capabilities of N-thiocarbonyl derivatives in combina-tion with the chirality of carbohydrates remain almost unexplored and de-serve thorough investigation in connection with molecular recognitionand catalysis.

ACKNOWLEDGMENTS

The authors thank Dr. José L. Jiménez Blanco, Juan M. Benito, and M. Isabel GarcíaMoreno for assistance in obtaining references and in proofreading the manuscript. This workwas supported by the Dirección General de Investigación Científica y Técnica of Spain undercontract PB 97/0747.

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and H. Haruyama, J. Org. Chem., 59 (1994) 4450–4460.(404) J. Li, F. Lang, and B. Ganem, J. Org. Chem., 63 (1998) 3403–3410.(405) C. Uchida, H. Kimura, and S. Ogawa, Bioorg. Med. Chem. Lett., 4 (1994) 2643–2648.(406) C. Uchida, H. Kitahashi, T. Yamagishi, Y. Iwaisaki, and S. Ogawa, J. Chem. Soc., Perkin

Trans. 1 (1994) 2775–2785.(407) H. Miyazaki, Y. Kobayashi, M. Shiozaki, O. Ando, M. Nakajima, H. Hanzawa, and

H. Haruyama, J. Org. Chem., 60 (1995) 6103–6109.(408) C. Uchida, T. Yamagishi, H. Kitahashi, Y. Iwaisaki, and S. Ogawa, Bioorg. Med. Chem., 3

(1995) 1605–1624.(409) C. Uchida, H. Kimura, and S. Ogawa, Bioorg. Med. Chem., 5 (1997) 921–939.(410) S. Knapp, A. Purandare, K. Rupitz, and S. G. Withers, J. Am. Chem. Soc., 116 (1994)

7461–7462.(411) B. K. Goering and B. Ganem, Tetrahedron Lett., 35 (1994) 6997–7000.(412) S. Takahashi, H. Terayama, and H. Kuzuhara, Tetrahedron Lett., 35 (1994) 4149–4152.(413) S. Knapp, B. A. Kirk, D. Vocadlo, and S. G. Withers, Synlett (1997) 435–436.(414) R. Jiricek, J. Lehmann, B. Rob, and M. Scheuring, Carbohydr. Res., 250 (1993) 31–43.(415) J. L. Jiménez Blanco, C. Ortiz Mellet, J. Fuentes, and J. M. García Fernández, Chem.

Commun. (1996) 2077–2078.



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(418) J. Defaye, C. Ortiz Mellet, J. M. García Fernández, and S. Maciejewski, Thioureido b-cyclodextrins as molecular carriers for the anticancer drug Taxotère, in MolecularRecognition and Inclusion, A. W. Coleman (Ed.), Kluwer, Dordrecht, 1998, 313–316.

(419) J. M. García Fernández, C. Ortiz Mellet, J. L. Jiménez Blanco, J. Fuentes, M. Martín-Pastor, and J. Jiménez-Barbero, Macrocyclic sugar thioureas: Cyclooligosaccharidesmimicking cyclopeptides, in Molecular Recognition and Inclusion, A. W. Coleman (Ed.),Kluwer, Dordrecht, 1998, 103–108.

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(422) J. L. Jiménez Blanco, J. M. Benito, C. Ortiz Mellet, and J. M. García Fernández, Org. Lett.,1 (1999) in press.



SYNTHESIS OF CHIRAL POLYAMIDES FROMCARBOHYDRATE-DERIVED MONOMERS

OSCAR VARELA* AND HERNAN A. ORGUEIRA

CIHIDECAR (CONICET), Departamento de Química Orgánica, Facultad de CienciasExactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria,

1428-Buenos Aires, Argentina

IIII. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137IIII. Chiral Polyamides: Properties and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . 138IIII. Chiral Polyamides Based on Diamino Saccharides. . . . . . . . . . . . . . . . . . . . . . . . . 143I IV. Chiral A,B-Type Polyamides (Nylons-n) Based on “Amino Acids”IIIII Derived from Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147II V. Polyaldaramides: Polyhydroxy Chiral Analogs of Nylon-n,6 and Nylon-n,5 . . . . . 155IVI. Polytartaramides: Polyhydroxy Chiral Analogs of Nylon-n,4 . . . . . . . . . . . . . . . . . 162VII. Chiral Analogs of Nylon-3 Prepared from Carbohydrate-Based Aspartic IIIII Acid-like Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170IIIII References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

I. INTRODUCTION

Polyamides are condensation products that contain recurring amidegroups as integral parts. Polyamides are usually referred to as “nylons,” ageneric term.1 The first polyamide was synthesized by Gabriel and Maas.2

Thirty years later, in 1929, this study was renewed by Carothers.3 Immedi-ately after the first patents were issued,4 nylon stockings were introduced tothe public in 1940 and were an immediate and outstanding commercial suc-cess. Since that time, the production of polyamides has greatly expandedthroughout the world.1 However, in the near future, access to fossil raw ma-terials is expected to become increasingly difficult and more expensive. Theneed for conservation of petroleum feedstocks and the increased awarenessin recent years of the low biodegradability of petroleum-based polymershas drawn attention to the utilization of natural regrowing resources for thechemical synthesis of polymers.5 They are also promising materials with

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* E-mail: [email protected].

0096-5332/00 $30.00

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novel technical possibilities and improved properties, such as biocompati-bility and biodegradability.

Among the different natural sources that are inexpensive and readilyavailable, carbohydrates, because of their great stereochemical diversity,stand out as highly convenient materials, especially for the synthesis ofpolymers containing several stereocenters in the main chain.6,7 In this chap-ter we discuss the synthesis of monomeric precursors derived from carbo-hydrates, their subsequent polymerization, and the physicochemical char-acterization of the resulting polyamides. A short introduction on thedifferent types of polyamides and their common nomenclature is pre-sented. Current and potential applications and properties of chiralpolyamides derived from sugars are detailed. We have also included a com-ment on some of the requirements for control of the regio- and stereoreg-ularity in the growing chains during the polymerization. The pioneeringwork on carbohydrate-based polymers is concisely described, although amore detailed historical overview can be found in the review article byThiem and Bachmann.6 As the interest for new polyamides based on car-bohydrates has steadily increased during the past 2 decades, we have fo-cused attention mainly on the work published during this period. The sub-sequent sections of this chapter have been organized taking into accountthe class of carbohydrate-derived monomer (for example, a diamine, anamino acid, a dibasic acid, and so on) that is employed as precursor in thesynthesis of a given type of polyhydroxy polyamide (analogs of nylon-3,nylon-5, nylon-6, nylon-n,5, nylon-n,6, and so on). Tartaric acids constitutethe aldaric acid derivatives of tetroses and therefore polytartaramides havebeen included in this chapter.

II. CHIRAL POLYAMIDES: PROPERTIES AND APPLICATIONS

Linear polyamides are formed as the products of condensation of bi-functional monomers. If the monomers are amino acids or their lactams, theresulting polyamides are called the AB type, A representing amine groupsand B, carboxyl groups. Polyamides formed by condensation of a diamineand a dicarboxylic acid are called AABB types. A common “shorthand”symbolism is the use of numbers that signify the number of carbon atomsin the respective monomers. For AABB polyamides, two numbers (m,n) areused. The first, m, gives the number of carbon atoms separating the aminogroups of the diamine, and the second (n) gives the number of carbons be-tween the acid groups in the dicarboxylic acid (including the carbonyl car-bon). The self-condensation polymer derived from an amino acid or lactam(AB polyamide) is known as an n-type polyamide (nylon-n), where n givesthe number of straight-chain carbon atoms separating the amine and theacid functions (Scheme 1).

OSCAR VARELA AND HERNAN A. ORGUEIRA138


The physical properties of conventional nylons are essentially deter-mined by the distance between the polar groups, namely, the length of thepolymethylene chain. As a consequence of this constitutional uniformity,the nylons show a rather monotonous behavior, whereas peptides and pro-teins display a great diversity of physical and biological properties and func-tions. Therefore, conventional polymers are being replaced with high per-formance materials, designed to manifest improved biocompatibility,biodegradability, and other desired properties.7 These materials, useful forapplications in medicine, include synthetic polymers that mimic naturalsubstances, naturally occurring macromolecules, and chemically modifiednatural polymers.8 One advantage of using biodegradable polymers as drugdelivery agents, suture filaments, ligature clamps, and the like is that the de-vice does not have to be removed after its purpose has been fulfilled.9,10

Ideally, polymer degradation in an aqueous environment, such as the hu-man body, should occur with the formation of natural metabolites or yieldharmless degradation products.8

Polymer biodegradation includes various mechanisms, such as pho-todegradation, hydrolysis, enzymatic degradation, and thermooxidativedegradation. Polyesters, polyamides, and polyester amides may undergo hy-drolytic degradation because of the presence of hydrolytically unstablebonds, hydrophilic enough to allow for water access.9 Polyhydroxy alka-noates derived from glycolic (PGA), lactic (PLA), and 3-hydroxybutanoicacids (PHB) and from copolymers PGA–PLA are the most successful classof biodegradable polymers.11–14 Attempts have been made to synthesizebiodegradable derivatives of nylon.15 Thus, some polyamides derived from

SYNTHESIS OF CHIRAL POLYAMIDES 139

SCHEME 1


natural amino acids have shown bioelastic properties and a certain degreeof biodegradability.16,17 Polyamides containing methyl and hydroxyl groupsare biodegradable,18 and hydroxypropyl methacrylamide polymers havingpeptide linkages are hydrolyzed by papain.19 Furthermore, the presence ofelectron-withdrawing groups vicinal to the carbonyl group of a polyamideenhances its rate of hydrolysis.20

In addition to chemical hydrolysis, hydrolysis by enzymes can operate asan alternative degradation process. It has become widely accepted thatbiodegradable synthetic polymers tend to be designed to mimic those struc-tures prevailing in nature, since enzymes produced by microbial popula-tions may not discriminate between polymers of similar structure.11 Syn-thetic nonpolypeptidic, chiral polyamides could mimic natural peptides orproteins, resulting in biodegradable products useful in biomedicine.

Besides the aforementioned uses and promising potential developmentsfor polyamides containing stereogenic centers, these materials are also en-visaged as a class of polymers in which chiroptical and particularly desirablephysical properties may be encompassed. In order to establish the relationexisting between constitution and conformation in optically activepolyamides, a fair number of them have been prepared and their opticalproperties studied in solution.21 It has been reported that the presence of astereocenter in the polyamide chain may promote important conforma-tional changes. For example, helical arrangements in the solid state, similarto those known as typical of polypeptides and proteins have beenfound.22,23 These helical nylons displayed particular properties such as liquid-crystalline behavior and piezoelectricity.24,25

From the synthetic point of view, the introduction of asymmetric carbonsin the repeating unit of a polyamide makes it possible to adjust the physi-cal properties by controlling the tacticity and also to study the effect of chi-rality on biological activity.9 In fact, stereo- and regioregularity areachieved in AB-type polyamides, regardless of the configuration of themonomer, as polycondensation is not restricted by the occurrence of direc-tional isomerism. However, the stereo- and regioselectivity of chains of theAABB types rely upon the existence of C2 symmetry in the monomersfrom which the polymer is generated; otherwise regioisomerism will prob-ably occur, giving rise to aregic polyamides. These topics are discussed inthe next sections (Scheme 2).

Carbohydrate-based synthetic polymers can be prepared by polymeriza-tion of small, activated carbohydrate-derived monomers. A pioneeringstudy in this field was the preparation and polymerization26 of methyl2,3,4,6-tetra-O-allyl-a-D-glucopyranoside (1). Under the influence of oxy-gen and heat, compound 1 gradually polymerizes, first to a viscous liquidand finally to a colorless, transparent resin. Similarly, acrylate and



methacrylate derivatives of 1,4 : 3,6-dianhydro-D-glucitol (2) and 1,4 : 3,6-dianhydro-D-mannitol (3) gave, on polymerization, transparent, colorlessresins of the thermosetting type.27 Condensation of 1,2 : 5,6-dianhydro-3,4-O-isopropylidene-D-mannitol (4) with phthalic acid gave, on heating, a so-lution that became thicker and finally set to a gel. If the heating is stoppedbefore the gel stage is reached, brittle fibers of polyester could be drawnfrom the cooling melt.28 The first polyamides based on a carbohydrate wereobtained from the crystalline salts of 1,6-diamino-2,3 : 4,5-dimethylene-D-mannitol (5) in reaction with such dibasic acids, as oxalic, hexanedioic(adipic), and decanedioic (sebacic) acids.29 These salts polymerized onheating above their melting points to afford polyamides, which did not giveoriented fibers when cold-drawn. The preparation of salts of 5 with aldaricacid derivatives was somewhat difficult.

Owing to the lack of thermal stability of the monomers derived from car-bohydrates, the melt-polycondensation process led only to brittle fibers oflow molecular weights and poor physical properties. As a consequencepolymerization in solution came into practice. In an attempt to benzoylate2,4 : 3,5-di-O-methylene-D-gluconic acid (6) with an equivalent amount ofbenzoyl chloride or benzoic anhydride in pyridine solution, rapid polymer-ization of the hydroxy acid was observed. The polyesterification of 6 underthose conditions afforded a white, amorphous polymer in 57% yield.30

Condensation of 2,3,4,5-tetra-O-acetyl-galactaroyl dichloride (7) with eth-ylenediamine or piperazine, with subsequent acetylation, gave the corre-sponding polyamides, which decomposed above 2508C with no evidence ofmelting.31 Deacetylation was effected (with probable hydrolytic degrada-tion) to yield polymers having 30–40 repeating units.A patent has described


SCHEME 2


the condensation at high temperatures of diamines with acetals and ketalsof galactonic acid, to give linear polyamides32 (Scheme 3).

During the 1960s new polyamides were obtained employing the tech-nique of interfacial polycondensation. This technique is well suited to car-bohydrate monomers because of the low temperatures required for poly-merization. Thus, Bird et al.33–36 synthesized carbohydrate polyamides withhigh inherent viscosities, comparable to those of commercial nylons. Twoseries of high-viscosity polyamides have been prepared35,36: (a) from 1,6-diamino-1,6-dideoxy-di-O-methylenehexitols (such as 8) and decanedioyl(sebacoyl) or hexanedioyl (adipoyl) dichlorides and (b) from hexamethyl-enediamine (1,6-diaminohexane) or decamethylenediamine and a di-O-methylenehexaroyl dichloride (such as 9). A patent on these products hasbeen issued.37 The following sections deal with the synthesis and propertiesof nylons derived from carbohydrates, work that has been published mainlyduring the past 2 decades (Scheme 4).


SCHEME 3


III. CHIRAL POLYAMIDES BASED ON DIAMINO SACCHARIDES

Diamino derivatives of carbohydrates have been employed for polycon-densation reactions with carboxyl-activated aliphatic and aromatic dicar-boxylic acids. The resulting polyamides are of the AABB type (nylons-m,nanalogs), and therefore, the regio- and stereoregularity in the polyamidechain is determined by the configuration of the carbohydrate precursor.When such a molecule lacks a C2 axis of symmetry, random polymerizationleads to nonstereoregular polyamides.

As the presence of two amino groups in a natural sugar is rare, diaminosaccharides are usually obtained by synthesis. However, the most commonamino sugar, D-glucosamine, has been used as precursor of polymers havingamide linkages. Thus, Kurita et al.38 prepared poly(ester amide)s by directpolycondensation of D-glucosamine and aliphatic and aromatic dicar-boxylic acid chlorides. The polymerization took place in polar solvents, inthe presence of pyridine, and involved the primary hydroxyl and the aminogroups of glucosamine. The resulting poly(ester amide)s of the type 10 hadinherent viscosities between 0.11 and 0.23 dL.g-1 and were soluble in polarsolvents. Thermogravimetric analysis revealed that the polymers decom-posed on heating, showing 10% weight loss at 180–1978C.

Chitobiose, a disaccharide of glucosamine, possesses two amino func-tions, at C-2 and C-29. These amino groups reacted with aliphatic and aro-matic diacid chlorides to give linear polyamides (11).39 These polymers af-forded transparent films, which showed high permeability, indicatingprobable utility as permeation-dialysis membranes (Scheme 5).

Starting from D-glucosamine or common saccharides, diamino sugar derivatives may be prepared by conversion of hydroxyl groups into amines.The procedure usually employed involves sulfonylation of an OH group,followed by nucleophilic substitution by azide and subsequent hydro-genation. An alternative “one-pot” procedure for the conversion of a primary hydroxyl group into azide is the reaction with N-bromosuccin-imide, triphenylphosphine, and sodium azide.40 From the nonreducing disaccharide sucrose, a diamino derivative (6,69-diamino-6,69-dideoxysu-crose) was synthesized by means of the first-mentioned general procedure.


SCHEME 4


This synthesis and the polycondensation of the resulting diamine withadipoyl dichloride has been patented.41 With oligosaccharides, the synthe-sis of bifunctional diamino monomers is more complicated. However,cyclodextrins have been successfully employed for the synthesis ofpolyamides. Cyclodextrins are cyclic oligosaccharides formed with glucoseresidues having a-(1 → 4) linkages. The diamino derivatives of cyclodex-trins were prepared from ditosylate precursors and polycondensed with di-carboxylic acid dichlorides in a mixture of N,N-dimethylformamide (DMF)and water. A patent concerning the synthesis and use of cyclomaltohep-taose (b-cyclodextrin) polyamides as membranes has been issued.42

2,6-Diamino saccharides, obtained from D-glucosamine, have been usedas monomers for polycondensation reactions with acid dichlorides. Methyl(12) and benzyl (13) glycosides of N-benzyloxycarbonyl-D-glucosaminewere prepared and the primary hydroxyl groups converted into azides bythe Appel procedure.40 Compounds 14 and 15 were the precursors of thediamino derivatives 16 and 17, respectively. Interfacial and solution poly-condensations of these diamino derivatives 16 and 17 with aliphatic andaromatic acyl dichlorides yielded the corresponding polyamides (18).Employing the 3-O-pivaloyl and 3,4-di-O-pivaloyl derivatives of 16 asmonomers, the corresponding pivaloylated polyamides were obtained.43

The partially substituted polyamides had similar properties as the unpro-tected polyamides (18). Number-average molecular weights for these mate-rials, synthesized by interfacial polycondensation, ranged between 10,300and 24,000, as determined by gel-permeation chromatography (GPC), withnumber-average degrees of polymerization up to Pn 5 64. Polycondensa-tion in solution afforded only higher oligomers. As the polycondensations


SCHEME 5


were random, these AABB-type polyamides were nonstereoregular. How-ever, their melting points were rather high (Tm . 2008C), and they meltedwith decomposition (Scheme 6).

In order to synthesize a polyamide containing the more stable C-glycosyllinkage, instead of O-glycoside units in the polymer backbone, a C-glyco-sylic diamino saccharide (22) was prepared from D-glucal triacetate (19).Addition of trimethylsilyl cyanide to 19, with boron trifluoride etherate ascatalyst, afforded the cyano compound 20 as the main product. Upon hy-drogenation of the double bond and deacetylation, the OH group at C-6was replaced by azide to give 21, which underwent hydrogenolysis, afford-ing 22. In contrast with the other diamino saccharides (16 and 17), com-pound 22 had two primary amino groups, which are more reactive. Poly-condensation of 22 with the same dicarboxylic acid dichlorides as before ledto polymeric materials. However, neither structural determinations (exceptfor the IR spectra) nor molecular-weight measurements for the polymerswere reported43 (Scheme 7).

Anhydroalditols, which are thermostable, proved to be suitable carbohy-drate precursors for polymer synthesis. Thus chiral, cis-fused bicyclic


SCHEME 6


1,4 : 3,6-dianhydrohexitols, having only two remaining hydroxyl groups ineither exo or endo orientations, have been employed for the synthesis ofpolyesters44 and polyurethanes.45 1,4-Anhydroalditols have also been usedfor the synthesis of polyethers.46 Thiem and Bachmann47 reported the syn-thesis of polyamides stating from D-mannitol (23) and D-glucitol (26) viathe corresponding 1 : 4,3 : 6-dianhydrohexitols of the D-manno (24), D-gluco(27), and L-ido (29) configurations. The three stereoisomeric dianhy-droalditols were functionalized to their respective diamino derivatives 25,28, and 30 by sulfonylation of the hydroxyl groups, azide substitution, andhydrogenolysis (Scheme 8).

The same synthetic route was also applied to the transformation of 1,4-anhydro-D,L-threitol (31) and 1,4-anhydroerythritol (33) into the corre-sponding diamines 32 and 34. The reaction of diamines 25, 28, 30, 32, and 34 with aromatic and aliphatic dicarboxylic acid dichlorides was performedby interfacial polycondensation in emulsions of organic solvents and water,with sodium carbonate as base and sodium dodecyl hydrogensulfate asemulsifier. The resulting polyamides (35–39), obtained in 60–80% yields,were characterized by IR and NMR spectroscopy. Number-average molec-ular weights were determined by gel-permeation chromatography andrange between 5,000 and 25,000, corresponding to a number-average de-gree of polymerization up to Pn ,100. Inherent viscosities were measuredand correlated with the observed average molecular weights. The thermalbehavior of the polyamides was determined by differential scanningcalorimetry (DSC) and indicated that many of the polyamides were crys-talline (Scheme 9).

The polyamides 35 and 37 are stereoregular, as the precursor diamines 25and 30 posses a C2 axis of symmetry (a symmetric distribution of the stereo-centers) and the two amino functions are topologically and stereochemi-


SCHEME 7


cally equivalent. The polyamide 36 is nonstereoregular, as polymerizationwas random and the precursor 28 has two nonequivalent amino functions(one endo and the other exo). Polyamides 38 and 39, having tetrahydro-furan residues in the polymer backbone, were optically inactive, as 31(precursor of 38) is racemic and 33 (precursor of 39) is a meso form(Scheme 10).

Diamine derivatives 25, 28, 30, 32, and 34 were also condensed with2,3,4,5-tetra-O-acetylgalactaroyl dichloride (7) employing the facial poly-condensation technique.48 The conclusions from these studies are reportedin Section V.

IV. CHIRAL A,B-TYPE POLYAMIDES (NYLONS-n) BASED ON “AMINO

ACIDS” DERIVED FROM CARBOHYDRATES

The polyamides obtained by polycondensation of bifunctional monomerswhich contain the amino and the acid functions in the same molecule(“amino acids”) are stereoregular, regardless of the configuration of the


SCHEME 8


SCHEME 9

SCHEME 10


monomer. These polymers are also regioregular, as regioisomerism cannotoccur in the formation of the amide linkage.

Tokura et al.49 synthesized a polyamide of the Perlon type (40) startingfrom a 6-O-carboxymethyl-D-glucosamine derivative as monomer. This com-pound was obtained by carboxylmethylation of chitin with monochloroaceticacid, followed by hydrolysis of the glycosidic linkage. The molecular weight(Mw 5 15,000) of the resulting water-soluble polyamide 40 was determinedby gel-permeation chromatography and electrophoresis (Scheme 11).

Galbis and co-workers50 reported the synthesis of stereoregular AB-typepolyamides based on a- and g-amino acids derived from carbohydrates.Thus, starting from N-acetyl-D-glucosamine (41), 2-amino-2-deoxy-3,4,5,6-tetra-O-methyl-D-gluconic acid (43) was prepared51 via the lactone 42.Compound 43 was activated for the polycondensation with trichloromethylchloroformate, to give the N-carboxy anhydride 44. The polymerization wasconducted in N,N-dimethylformamide or dichlorometane and in the pres-ence of N-ethyldiisopropylamine (EDPA). The resulting polyamide 45 is achiral analog of nylon-2, having a polymethoxylated lateral chain in the re-peating unit (Scheme 12).

Methyl a-D-glucopyranoside (46) was employed as a chiral template forthe synthesis of 6-azido-6-deoxy-2,3,4,5,-tetra-O-methyl-D-glucono-1,5-lactone (47), which was converted into the open-chain derivative 48. For thepolymerization of 48, the active ester method was employed.52 The aminofunction was protected as the N-Boc derivative and the carboxylic acid wasactivated as the pentachlorophenyl or p-nitrophenyl ester to give 50. How-ever, attempted polymerization of such a compound led to the lactam 51 in-stead of the polymer. In order to avoid this intramolecular reaction, thedimer 52 was prepared by condensation of 49 with the pentachlorophenylester 52 obtained from 48. Compound 53 was conveniently derivatized forthe polymerization as the active ester 54. Thus, polycondensation of 54 un-der alkaline conditions afforded the chiral nylon-6 analog 58, having atetramethoxypentamethylene chain spacing the amido groups along thepolymer backbone50 (Scheme 13A).


SCHEME 11


The molecular weights of the polyamides 45 and 55 were estimated as25,000 and 67,000, respectively, on the basis of viscosimetric measurements.Both polyamides displayed high optical activity; they were highly hy-drophilic and readily soluble in water and in organic solvents, includingchloroform. Polyamide 55 was crystalline and yielded resistant films with aspherulitic texture (Scheme 13B).

Derivatives of 5-amino-5-deoxy-L-arabinonic (58) and D-xylonic (59)acids were prepared from the respective per-O-methyl aldono-1,5-lactones(56 and 57). Opening of the lactone by alcoholysis followed by tosylation ofHO-5, azide substitution, and hydrogenolysis led to the precursors of 58 and59, respectively. Also, the (S)-5-amino-5-deoxy-4-methoxypentanoic acid(60) was prepared from D-ribono-1,4-lactone (via its 2-butenolide) andfrom L-glutamic acid, as depicted in Scheme 14.53

Compound 60 was N-protected and carboxylate activated to give 61. The


SCHEME 12


polymerization of 61 competed with the intramolecular lactamization andtherefore the dimer of 62 was prepared by condensation of 60 and 61 in or-der to increase the distance between the reacting groups. The dimers of theamino acid derivatives having the L-arabino and D-xylo configuration weresimilarly prepared. Removal of the N-protecting groups under acidic con-ditions gave the corresponding hydrochlorides (for example, 63), whichpolymerized when the amino group was released with a base.54 The poly-[(S)-5-amino-4-methoxypenathonic acid] (64), poly(5-amino-5-deoxy-2,3,4-tri- O-methyl-L-arabinonic acid) (65), and poly (5-amino-5-deoxy-2,3,4-tri-O-methyl-D-xylonic acid) (66) were obtained in very good yields (,90%)and were characterized by elemental analysis, IR and NMR spectroscopies,and powder X-ray diffraction. They had a pronounced affinity for water, al-though they were not soluble in this solvent. Their intrinsic viscosities weremeasured in dichloroacetic acid at 258C, and their molecular weights (Mw 5 7.800–11.700) were determined by gel-permeation chromatographyanalysis. Polyamide 64 was highly crystalline and afforded films with aspherulitic texture (Scheme 15).

Stereoregular, AB-type polyamides, containing a natural amino acid


SCHEME 13A


(glycine) and a synthetic component (60), have been prepared by the activeester polycondensation procedure.55 Reaction of 60 with the N-Boc deriv-ative of p-nitrophenyl glycinate (67) in N,N-dimethylformamide and N-ethyldiisopropylamine afforded 68, which was readily converted into 69.The polycondensation reaction of 69 was carried out in N,N-dimethylform-amide or dichloromethane and in the presence of N-ethyldiisopropylamine.The expected polyamide 70 was formed, accompanied by a considerableamount (,30%) of a cyclic product (71).The formation of the macrolactam71 was attributed to the glycine moiety, which, being achiral, permits abroader range of allowable NH-CaH dihedral angles. For this reason, a con-formation could be adopted that favors the fomation of cyclic 71 by bring-ing together the reactive groups located at the extremeties of theoligoamide chain (Scheme 16).

The formation of cyclic by-products was avoided by introducing an addi-tional molecule of 60 into the polymerizing unit. Therefore, the oligoamide

SCHEME 13B



72 was prepared by condensation of 60 with the pentachlorophenyl ester of68, followed by usual functional group interconversions. Polymerization of72 (N,N-dimethylformamide–N-ethyldiisopropylamine) gave the polyamide73 in 85% yield, free of macrocyclic by-products. This polymer was spectro-scopically characterized, and thermal studies and polarized optical mi-croscopy indicated that it was crystalline. Films obtained by slow evapora-tion of formic acid solutions of 73 displayed birefrigence associated with aspherulitic texture (Scheme 17).

Tri-O-methyl-L-arabinono-1,5-lactone (56), previosly used for the syn-thesis of 65, underwent ammonolysis to the corresponding amide, which onreduction gave the 1-aminoarabinitol derivative.The activated monomer 74was obtained via blocking group manipulations, selective succinylation at C-5, and ester activation of the carboxyl group. Polymerization of 74afforded the stereoregular poly(ester amide) 75, derived from a sugar


SCHEME 14


SCHEME 15

SCHEME 16



template.56 The polymerization was carried out in various polar solvents.The molecular weight of 75 was estimated by viscosimetry and gel-perme-ation chromatography. Poly(ester amide)s 77 and 79 were prepared bypolycondensation of 1-amino-1-deoxy-5-O-glutaryl-L-arabinitol (76) and 1-amino-1-deoxy-5-O-succinyl-D-xylitol (78) derivatives, respectively.57 Thepolymers were hydrophilic and exhibited moderate optical activity. Ther-mal and X-ray diffraction studies showed that they were slightly crystallineand stable up to 2508C under nitrogen. The hydrolytic degradation ofpoly(ester amide)s 75, 77, and 79 was studied and was shown to occur by hy-drolysis of the ester linkages58 (Scheme 18).

Replacement of the aliphatic acid residue in the 1-amino-1-deoxy-L-arabinitol derivatives by an aromatic dicarboxylic acid gave the precur-sor 80. Attempted polymerization of 80 afforded, instead of the expectedpoly(ester amide), a mixture of oligomers (81) and the monomeric 1-deoxy-1-phthalimido-L-arabinitol derivative 82. When the reaction wasconducted in hexamethylphosphoric triamide, these products were ob-tained in 30 and 51% yields, respectively, indicating that intramolecularcondensation competes with the intermolecular reaction.59 This case illustrates a limitation of the “active ester” method of polymerization(Scheme 19).

V. POLYALDARAMIDES: POLYHYDROXY CHIRAL ANALOGS OF

NYLON-n,6 AND NYLON-n,5

Polyaldaramides are hydroxylated, linear polyamides wherein the diacidmonomer unit of a typical nylon copolymer, such as nylon-6,6, is replaced


SCHEME 17

SCHEME 18


by an aldaric acid. As already described for other AABB-type polyamides,the regio- and stereoregularity in the construction of the polymer chain de-pends on the configuration of the comonomers. Thus, random polymeriza-tion of asymmetric aldaric acid derivatives with common aliphatic or aro-matic diamines leads to nonstereoregular polyaldaramides. Althoughaldaric acid derivatives have been selected as monomers suitable for the synthesis of polyamides since the 1940s,29,31,32,35,36 polymerization pro-cesses employing active diesters of aldaric acids and diamines were not developed until the decade of 1970. Pioneer work of Ogata and coworkersestablished the order of reactivity of active esters of adipic acid with hexa-methylenediamine60 or diols61 in a variety of solvents, and the conditionsfor the synthesis of the respective polyamides and polyesters were opti-mized. It was shown that heteroatom groups (such as ether or hydroxylgroups) greatly enhanced the reactivity of the diester in polycondensationreactions in polar solvents when they were located at the a- or b-positionsto the ester carbonyl group.62 The same authors also reported the polycon-densation of diethyl galactarate with several diamines.63 These reactionswere conducted in polar solvents such as methanol, dimethyl sulfoxide, andN-methylpyrrolidone under mild conditions. Polymerization of diethylgalactarate with hexamethylenediamine gave 83, a hydroxylated analog ofnylon-6,6, which did not melt and decomposed at 2008C. The copolycon-densation behavior of diethyl tartrate, diethyl galactarate, and diphenyl adi-pate with hexamethylenediamine was interpreted in terms of the solvent ef-fect on the forming copolymer chain.64 The copolymerization of diethylgalactarate and diphenyl adipate with hexamethylenediamine in methanolyielded an almost homopolyamide of the former. The enhancement effectof the hydroxyl group on the reactivity of the ester toward aminolysis was


SCHEME 19


attributed to hydrogen bonding of the OH group with the approachingamino group at an intermediate reaction stage. The polymerization of di-ethyl galactarate with 1,6-diaminohexane in dimethyl sulfoxide at 608C andin the presence of poly(4-vinylpyridine) gave a polyamide having highermolecular weight than that prepared in the absence of the matrix65

(Scheme 20).Hoagland brought further understanding to the condensation of aldaric

acid esters with diamines when he established the mechanism of aminolysisof six-carbon galactaric diesters66 and five-carbon xylaric acid diesters.67

Such a mechanism requires a two-step sequence at each diester function: afast, base-catalyzed five-membered lactonization step followed by a slowerstep, the aminolysis of the lactone. From these studies it was clear that acti-vation of five- or six-carbon aldaric acid diesters results from the facile for-mation and high reactivity of these five-membered aldarolactones. Thus, inthe case of diethyl di-O-isopropylidenegalactarate, a lower rate of polycon-densation with ethylenediamine was observed owing to the more difficultlactonization.

Acetylation of D-galactaric acid afforded the 2,3,4,5-tetra-O-acetyl deriv-ative (84), which was activated for condensation as the dichloride 7.Polyamides were obtained by solution polycondensation of 7 with variousaliphatic and aromatic amines.68 Similar yields of polyamides were ob-tained by the interfacial polycondensation of the dichloride with di-amines.48 When 1,6-diaminohexane was used, the resulting polyamide 85 resembles a nylon-6,6 in which half of the hydrogens have been sub-stituted by acetoxy groups. However, the polycondensation of 7 with the diamino derivatives of dianhydrohexitols having the L-ido (25) and D-manno (30) configurations afforded respectively the stereoregularpolyamides 86 and 87, completely constructed from carbohydrate precur-sors. The polyamides derived from diamines 28, 32, and 34 were also pre-pared. These polymers showed molecular weights (Mn), determined by gel-permeation chromatography, between 4,000 and 17,000 and their inherentviscosities ranged between 0.05 and 0.34 dL.g-1. Polyamides having free hydroxyl groups were obtained by deacylation with aqueous ammonia(Scheme 21).


SCHEME 20


The preparation of unprotected, activated D-glucaric acid estersmonomers and their polymerization to poly(alkylene D-glucaramide)s werereported69,70 and patented71 by Kiely et al. D-Glucaric acid (monopotas-sium salt, 88) was esterified with alcohols, such as methanol, to give mix-tures that contained varying amounts of dimethyl D-glucarate (89), methylD-glucarate 1,4-lactone (90), and methyl D-glucarate 6,3-lactone (91). Theseester forms of D-glucaric acid are also in equilibrium under the conditionsof the polymerization with diamines. However, in order to obtain good stoichiometric control, which influences the degree of polymerization, com-pound 90 was isolated from the mixture in crystalline form, in 58% overallyield and employed for the polymerization (Scheme 22).

The polycondensation of 90 with alkylenediamines was carried out inmethanol solution and in the presence of triethylamine, at room tempera-


SCHEME 21


ture, for several hours. Isolated yields of polyamides 92 ranged from 67%for poly(49-azaheptamethylene D-glucaramide) to 96% for poly(m-xylenylD-glucaramide). The linear aliphatic and arylalkylenediamines producedpolyamides of higher crystallinity and high melting points. Heteroatom- orbranch-containing poly(alkylene D-glucaramide)s were more soluble in wa-ter and alcohols, but they were not as crystalline as their straight-chaincounterparts and had correspondingly lower melting points and highermolecular weights. Furthermore, the polymers of glucaric or xylaric acidswith diprimary diamines containing at least one heteroatom in the mainchain formed films when cast from aqueous solution and showed adhesiveproperties. The analogous polymers prepared from galactaric acid deriva-tives did not form satisfactory films and exhibited poor or no adhesiveproperties72 (Scheme 23).

As D-glucaric acid is not a symmetrical diacid (2R, 3S, 4S, 5S), polycon-densation of its esterified forms with diamines generated nonstereoregularpolymers with randomly oriented glucaric acid units in the polymer chain.In order to prepare stereoregular polyglucaramides, the same authors de-scribed73 and patented74 a simple procedure starting from sodium D-glucarate 6,3-lactone (93). Compound 93 was synthesized in two steps from88. The two carboxylic acid ends of 93 have different reactivities and hencethey underwent regioselective aminolysis with diamines at the activatedcarbon (lactone), affording the N-(aminoalkyl)-D-glucaramide salts (94).The carboxylate group was converted into a mixture of ester 95 and lac-tone 96 on treatment with methanolic HCl. When the mixture was made basic the polymerization occurred spontaneously. The products, head-, tail-poly(alkylene D-glucaramide)s (97), precipitated from methanol, were


SCHEME 22


isolated in 82–91% yields. The stereoregular polymers 97 exhibited similarproperties to those of the random polymers 92, including high meltingpoints (185–2008C), comparable solubilities, and also relatively low molec-ular weights (Mn average 2400, determined by NMR) (Scheme 24).

Hashimoto and co-workers reported the use of hexaric-1,4 : 6,3-dilactones as monomers in related polycondensations.75,76 Ring-openingpolyaddition of D-glucaro-1,4 : 6,3-dilactone (98) with several alkylenedi-amines proceeded at room temperature in N,N-dimethylformamide or di-methyl sulfoxide, with no catalyst.The resulting polyamides (99) were moreamorphous and hydrophilic than the corresponding nylons, having no hy-droxyl groups, and were hydrolyzed more readily than the latter in acidicconditions. This fact was attributed to the neighboring-group effect of the hydroxyl groups of the chain upon protonation of the amide group(Scheme 25).

Another hexarodilactone, D-mannaro-1,4 : 6,3-dilactone, underwent ring-opening polyaddition when treated with diamines having two-, four-, andsix-carbon chains.76 The polymerizations were conducted in dimethyl sulfoxide solution at 258C. As the mannarodilactone has a symmetricalarrangement of hydroxyl groups (all S) along the backbone, the resultingpolyamides were stereoregular. Therefore, their melting points were higherthan those of nonstereoregular poly(alkylene D-glucaramide)s. However,both the polymer yield and the molecular weight of the polyamides frommannarodilactone were lower than those obtained from 98. In our labora-tory,77 the yield of poly(alkylene D-mannaramide)s was markedly enhanced


SCHEME 23


SCHEME 24

SCHEME 25


by employing diamines having larger polymethylene chains and by con-ducting the polycondensations in methanol.

We have reported78 a stereocontrolled synthesis of stereoregular, chiralanalogs of nylon-5,5 and nylon-6,5. The key chiral intermediate in the syn-thesis of both polyamides was the pentachlorophenyl ester of (S)-2-hydroxypentanedioic acid 5,2-lactone (100). This compound, prepared from D-ribonolactone or L-glutamic acid, possess a stereocenter at C-2 that impedes the construction of AABB-type polyamides, having orderedspatial configurations. However, stereocontrol in the synthesis of the poly-mers was achieved by chemoselective condensation of the ester group oflactone 100 with amino alcohols.The primary alcohol group of the resultingN-(hydroxyalkyl)amides (101) was converted into tosylate (102) and thesulfonate replaced by azide. Hydrogenolysis of 103 afforded the “amino lac-tone” 104, conveniently functionalized for the polycondensation. This reac-tion took place in N,N-dimethylformamide, after deprotonating the aminofunction with N-ethyldiisopropylamine, to give polyamides 105 and 106. Asobserved for other stereoregular polymers, these two polyamides displayedrelatively high optical rotations compared with the monomers. Powder X-ray diffraction showed that they are highly crystalline products, havingviscosimetric molecular weights of about 5000 (Scheme 26).

VI. POLYTARTARAMIDES: POLYHYDROXY CHIRAL ANALOGS OF NYLON-n, 4

Polytartaramides are polyaldaramides deriving from tartaric (tetraric)acids. Stereoregular polytartaramides may be obtained, depending on theconfiguration of the starting tartaric acid derivative employed in the poly-condensation. Since either of the chiral forms of tartaric acid (D- or L-threo)are disymmetric (containing a twofold axis normal to the backbone), theygive on polymerization a stereoregular polymer, as the substituents will beequally oriented in every repeating unit of the polytartaramide chain. Incontrast, if the meso (erythro) form is used for the polymerization, the sub-stituents become oriented at random, and an aregic structure is formed.

The synthesis of polytartaramides was first reported by Minoura and co-workers.79 During the 1970s, Ogata et al.80,81 described the polycondensa-tion of tartaric acid itself or its 2,3-O-methylene derivative with diamines,by a variety of procedures. These authors also studied the copolymeriza-tions of diethyl-L-tartrate with other diesters64 and reported that the rate ofpolymerization of dimethyl-L-tartrate with 1,6-diaminohexane in dimethylsulfoxide at 608C increased when the reactions were conducted in the pres-ence of such polymer matrices as poly(vinyl pyrrolidone), pullulan, andpoly(vinyl alcohol). The rate increased with increasing molecular weight ofthe matrix.82



A renewed interest in polytartaramides has emerged in the past fewyears, mainly due to the work of Muñoz-Guerra and coworkers, who pre-pared a series of stereoregular polyamides by the polycondensationmethod developed by Katsarava et al.83 This procedure is based on activa-tion of the diacid as the bis(pentachlorophenyl) ester and the diamine asthe bis(trimethylsilyl) derivative, and the polymerization reactions may beconducted under very mild conditions, affording linear polyamides havingacceptable molecular weights. Starting from L-tartaric acid, the bis(pen-tachlorophenyl) 2,3-O-methylene derivative (107) was prepared and polycondensed with the N,N9-bis(trimethylsilyl) derivatives of 1,9- and1,12-alkanediamines.84 The polymerization was carried out in several solvents; the highest degree of polymerization was achieved for 1,12-dode-canediamine in chloroform.The polyamides 108 were soluble in chloroformand in warm dimethyl sulfoxide or N-methylpyrrolidone. Both viscosimetryand gel-permeation chromatography were used to estimate the molecular


SCHEME 26


weights, which ranged between 6,000 and 44,000. Well-defined diagramswere obtained by powder X-ray diffraction for samples of the polyamidesprepared by slow precipitation (or evaporation of solvents), indicating highcrystallinity (Scheme 27).

A series of poly(alkylene-2,3-O-dimethyl-L-tartaramide)s (110) of thenylon-n,4-type have been prepared by the same polymerization procedureas just described.85 The activated 2,3-di-O-methyl-L-tartaric acid derivative109 was used to provide the two chiral backbone carbons. The number ofmethylene units in the polymethylene sequence of polyamides 110 rangedfrom 2 to 9 and 12. Polycondensations were conducted in chloroform attemperatures from ambient up to 608C. By these means, polyamides havinglimiting viscosity numbers between 0.6 and 2.3 were obtained in yields ex-ceeding 90% in most cases. Their optical activities were found to decreasewith the length of the polymethylene chain, as the density of chiral centersalso decreased. Number-average molecular weights, measured by gel-permeation chromatography, were between 8,000 and 50,000, with polydis-persities ranging from 1,4 to 2,2. Polytartaramides 110 are hydrophilic andare hydrolyzed faster than conventional polyamides. Melting points fellwithin the range 185–3128C, with glass transitions (Tg) between 84 and1238C (Scheme 28).

The degradation of polytartaramides derived from alkanediamines hav-ing 6, 8, and 12 methylene groups was investigated. The polymers, in theform of disks, were placed in buffered solutions of pH 2.3, 7.4, and 10.6 attemperatures of 37, 55, and 708C. The polytartaramides degraded slowly at378C, with the degradation rate depending on the number of methylenegroups in the diamine unit.86 Thus, the polytartaramide derived from 1,6-diaminohexane was degraded considerably faster than the others,87 show-ing a decrease of 30% in viscosity after 180 days of incubation in phosphatebuffer (pH 7.4) at 378C.


SCHEME 27

SCHEME 28


L-Tartaric acid was also used for the synthesis of (2S,3S)-2,3-dimethoxy-1,4-butanediamine (113), which was employed in the preparation ofpolyamides of the 4,n-type.88 Diethyl L-tartrate (111) was reduced withlithium aluminium hydride to the corresponding diol 112, which was converted into 113 by conventional tosylation, azide substituion, and hydrogenation. The diamine 113 was activated as the bis(trimethylsilyl) derivative 114 and polycondensed with the pentachlorophenyl esters ofeven-numbered aliphatic diacids (ranging from 4 to 12 carbons) to give thestereoregular poly[(2S,3S)-2,3-dimethoxybutylenealkanamide]s (116). Thesehighly crystalline polyamides melted over the range of 150–1908C, had apronounced affinity to water, and exhibited moderate optical activity.Theseproperties were investigated in relation to the molecular structure andcompared with those of tartaramides of the type of 110 (Scheme 29).

The polycondensation of the trimethylsilyl derivative of 1,6-hexanedi-amine with mixtures of bis(pentachlorophenyl)-2,3-di-O-methyl-D- and L-tartrates in chloroform at room temperature afforded a series of polytar-taramides, such as 117, with enantiomeric D:L ratios ranging from 1 : 9 to1 : 1.89 Polymerization of mixtures of enantiomers is a method frequently


SCHEME 29


applied to generate stereochemical microheterogeneities in a polymerchain. By this means, the degree of crystallinity is depressed and relatedproperties may be conveniently modified.9,90 In the absence of stereoselec-tive catalysts or chiral solvents, atactic polymers following Bernouillian statistics are usually obtained. Thus, the microstructure of copolyamides,determined by NMR, appeared to consist of a statistical distribution of D-and L-configurations. The copolyamides were highly crystalline materials,with melting points close to that of the optically pure polymer 110 (x 5 4),which is about 2308C. However, the Tg values steadily decreased from 106to 688C as the D:L ratio increased from 0 to 1. Powder X-ray diffraction in-dicated a crystal structure very similar to that described for the pure enan-thiomer and that the replacement of L- by D-units is feasible over the wholerange of enantiomeric compositions, without much distortion of the crystallattice and properties. Consistent with the moderate decay in crystallinity ofthe stereocopolyamides, the susceptibility toward the hydrolytic degrada-tion was not significantly enhanced with respect to that displayed by opti-cally pure 110 (Scheme 30).

As an efficient approach for increasing the degradability of a polyamidewithout losing good physical properties, poly(ester amide)s were preparedemploying 1,6-diaminohexane, 1,6-hexanediol, 2,3-di-O-methyl-L-tartaricacid, and succinic acid as building blocks.91 The ester linkages were intro-duced in pairs, using as diacid compound 118, the product of the esterifica-tion of 1,6-hexanediol with 2 molar equivalents of succinic anhydride. Thecarboxyl groups of compound 118 were activated as the pentachlorophenylesters, affording 119. The carboxyl-activated derivatives 109 and 119 werepolycondensed with N,N9-bis(trimethylsilyl)-1,6-diaminohexane in chloro-form at room temperature. The ester:amide content of the copolymer wasadjusted by fixing the composition of the feed monomers 109 and 119. Theresulting poly(ester amide)s 120 had number-average molecular weights inthe range 10,000–40,000 and were found to be highly crystalline, with melt-ing points above 2008C. They were degraded by aqueous buffer at pH 7.4 ata rate that increased with the content of succinic acid units in the copoly-mer (Scheme 31).

The crystal structures of a series of polytartaramides, such as 110, derivedfrom 2,3-di-O-methyl-L-tartaric acid and 1,n-alkylenediamines (n 5 2, 4, 6,


SCHEME 30


and 8), were studied by X-ray diffraction of powders and fibers as well asby electron diffraction of single crystals.92 The lattice parameters were es-tablished for each polymer. A triclinic unit-cell with space group P1 wasfound to be shared by the whole series. Semiempirical quantum-mechanicalcalculations revealed that the favored conformation for these polyamidesinvolves the tartaric acid moiety in a gauche arrangement with the amidegroups rotated out of the plane containing the all-trans polymethylene seg-ment. Crystal models compatible with the crystallographic data suggested afavored structure consisting of hydrogen-bonded pleated sheets packedwith a staggered arrangement similar to that found in nylon-6,6.

In addition, the crystal structures of both the racemic copolyamide 117and the equimolecular mixture of the two configurationally homogeneousD- and L-polyamides were studied and compared with that of optically pure110.93 This study combined X-ray, electron microscopy, and 13C CP-MASNMR measurements with computational methods. The two optically com-pensated and the optically pure polymers were shown to be highly crys-talline systems; the melting point of the racemic mixture was 2508C, consid-erably higher than those of the homopolymer (2328C) and the racemicpolymer (2268C). The crystal structure of the racemic mixture could be


SCHEME 31


represented by a monoclinic unit-cell containing two enantiomeric chainsrelated by a glide plane. A similar model appeared to be adequate for theracemic copolyamide, assuming that the crystal lattice was composed ofconfigurationally averaged identical chains. This arrangement of the chainsin the crystal seems to be substantially the same as that adopted in the tri-clinic structure of pure 110. Energy calculations corroborated the ability ofD- and L-tartaric units to cocrystallize without altering the side-by-sidepacking of the chains of the three systems, which are of comparable energy.

The potential development of polytartaramides such as 108 and 110 asdegradable biomaterials has some limitations, since aliphatic diamines re-main relatively toxic. In order to obtain fully biocompatible polytar-taramides, Bou and Muñoz-Guerra94 employed L-lysine as a diamine ofnatural origin. Polycondensation of the L-tartaric acid derivative 109with ethyl N,N9-bis(trimethylsilyl)-L-lysinate (121) in chloroform solutionfor 3 days afforded poly[(S)-1-(5)-ethoxycarbonyl-pentamethylene-di-O-methyl-L-tartaramide] (122) in almost quantitative yield (Scheme 32).

Both optically active monomers behave in a different manner regardingthe regioisomerism of the polymer chain being formed. As already ex-plained, a unique arrangement is possible for the L-tartaric acid residue inthe polymer chain. In contrast, two orientations are allowed for the L-lysineresidue, depending on which of the two NH (a or «) is implicated in eachamide group and, as shown in the following scheme, the peptide linkagecould be aa, ««, or a«. Pure syndioregic chains will consist of an alternatingsequence of aa and «« structures, but in an isoregic polymer only a«


SCHEME 32


sesquiads should be present. The regioregularity of 122 was examined by13C NMR with the support of model compounds, which facilitated the as-signment of the splitting resonances of the carbons of the diacid moiety.This study revealed that the chain is predominantly syndioregic. Polytar-taramide 122 had a molecular weight of 6000 with a polydispersity of 1.9 (asdetermined by gel-permeation chromatography), displayed high optical ac-tivity, and was soluble in water (Scheme 33).

Two stereoregular polyamides based entirely on tartaric acid have beensynthesized.95 Tartaric acid derivatives having the D- and L-configurationwere polymerized with (2S, 3S)-2,3-dimethoxy-1,4-butanediamine (113), af-fording respectively the polyamides 123 and 124. Each diastereoisomericpolymer posses two pairs of stereocenters in the repeating unit, one in thediamine and the other in the diacid counterpart (Scheme 34).

Number-average molecular weights around 30,000 were estimated forcompounds 123 and 124, by gel-permeation chromatography and vis-cosimetry. Circular dichroism and 1H NMR data in chloroform suggestedthe presence of definite secondary structures in this solvent. Crystals ofboth 123 and 124 were obtained upon annealing, and their structures were studied by X-ray diffraction of powders and oriented fibers.Polyamide 123 seemed to adopt a P1 triclinic structure as observed for


SCHEME 33


other polytartaramides, whereas 124 crystallized in an orthorombic latticein the space group P22121. In both cases, the polymer chain appeared to bein a folded conformation, but more contracted than in the c-form of con-ventional nylons.

VII. CHIRAL ANALOGS OF NYLON-3 PREPARED FROM

CARBOHYDRATE-BASED ASPARTIC ACID-LIKE DERIVATIVES

Synthetic polyamides containing b-aspartyl moieties in the main chainhave shown a character intermediate between those of polypeptides andnylons. In this way, such nylons as poly(a-isobutyl L-aspartate) (136), whichbear an alkoxylcarbonyl group stereoregularly attached to the polymerbackbone, are able to adopt helical conformations of the type commonlyfound in polypeptides and proteins.22,23 X-Ray diffraction of 136 indicatedtwo crystalline forms, tetragonal and hexagonal, depending on the condi-tions used for preparation of the sample. In both forms, the polymer isarranged in intramolecular hydrogen-bonded helices, very similar to the a-helix of polypeptides.

Polyamide 136 was first synthesized by polycondensation of active es-ters96 and then by polymerization of the b-lactam of a-isobutyl-L-asparate(isobutyl 4-oxo-2-azetidinecarboxylate) (135).97,98 In this case, the poly-merization was conducted either in solution or thermally. Anionic poly-merization in solution, using potassium tert-butoxide as catalyst, yielded apolymer, of better quality having a higher molecular weight (intrinsic vis-cosity 3.0) than that obtained by the active ester method, although itshowed lower optical activity, as it was partially racemized (Scheme 35).

Just one example of an asparate-type polyamide derived from a carbo-hydrate precursor poly[isobutyl(2S,3R)-3-benzyloxyaspartate] (133), has


SCHEME 34

SCHEME 35


been reported.99 Compound 133 was synthesized from the chiral b-lactam132, which bears a new stereocenter at C-3. For preparation of 132, 2,3-O-isopropylidene-D-glyceraldehyde (127), derived from D-mannitol,was converted into the Schiff base 128. This compound reacted with 2-benzyloxyacetyl chloride in the presence of triethylamine to give the lac-tam 130, as a unique diastereoisomer (45% yield), probably via the inter-mediate 129. The b-lactam 130 was readily converted, by successive re-moval of the ketal protecting group, oxidation, and esterification, intocompound 181. The p-anisyl group was removed by oxidation with Ce(IV)under mild conditions, affording lactam 182. Polymerization of 182 was per-formed in dichloromethane with potassium tert-butoxide as catalyst, atroom temperature for 3 days. The polyasparatate 133 was obtained as a col-orless, powdery solid of high molecular weight (Mw 5 232,000 as deter-mined by gel-permeation chromatography) and of an intrinsic viscosity of1.23 dL.g-1. No epimerization took place during the polymerization(Scheme 36).

ACKNOWLEDGMENTS

We thank the University of Buenos Aires (Project 01/TW70), the Agency for the Promo-tion of Science and Technology of República Argentina (ANPCYT-PICT 01698), and the Na-tional Research Council (CONICET) for financial support of the project on synthesis of chi-ral polyamides. O.V. is a Research Member of CONICET.


SCHEME 36


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219–227.



HYDRAZINE DERIVATIVES OF CARBOHYDRATESAND RELATED COMPOUNDS

BY HASSAN S. EL KHADEM AND ALEXANDER J. FATIADI*

Department of Chemistry, The American University, Washington, DC. 20016, USA; and*Biotechnology Division, National Institute of Science and Technology,

Gaithersburg, Maryland 20899, USA

III. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175III. Saccharide Azines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176III. Saccharide Hydrazones and Glycosylhydrazines . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

1. General Aspects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1772. Formation of Saccharide Hydrazones and Glycosylhydrazines . . . . . . . . . . . . . 1793. Structure of Saccharide Hydrazones and Glycosylhydrazines . . . . . . . . . . . . . . 1814. Reactions of Saccharide Hydrazones and Glycosylhydrazines . . . . . . . . . . . . . . 184

IV. Saccharide Osazones and Poly(hydrazones). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1961. Formation of Osazones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1962. Structure of Osazones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2033. Reactions of Osazones. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2104. Saccharide Poly(hydrazones) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

I V. Hydrazones of Carba-Sugars and Related Compounds. . . . . . . . . . . . . . . . . . . . . . 2281. Importance of Carba-Sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2282. Formation of Carba-Sugar Hydrazones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2293. Structure of Carba-Sugar Hydrazones and Polyhydrazones . . . . . . . . . . . . . . . . 2364. Reactions of Carba-Sugar Hydrazones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

VI. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248

I. INTRODUCTION1

Hydrazine (H2N-NH2), hydroxylamine (HO-NH2), and hydrogen perox-ide (HO-OH) are highly reactive nucleophiles that add to carbonyl com-pounds to give N- and O-adducts, which are not usually isolated. The N-adducts of hydrazines and hydroxylamines readily undergo elimination togive condensation products of the type CN-NH-R and CN-O-R,whereas the O-adducts of hydroxylamine and of hydrogen peroxide do notdo so because of valence considerations. Instead, the O-adducts of hydrox-ylamine remain in equilibium with the substrates and the O-adducts of per-oxide adducts split up their weak O–O bonds to give oxidation products.

175


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Hydrazines and hydrazones exhibit important biological activities; for ex-ample, arylhydrazines inhibit oxidases and redox systems such as hemoglo-bin,2 myoglobin,3 cytochrome P-450,4 and lactoperoxidase.5 The reactionseems to proceed via carbon-centered free radicals6,7 that lead to the for-mation of s-aryliron(III) complexes,8–10 generated from diazenes11 and di-azonium salts.12–14 Other hydrazines inhibit mitochondrial systems by act-ing as uncouplers of oxidative phosphorylation.15 Several hydrazones havebeen found to possess antiviral16 and antimicrobial activity.17–20

The hydrazine derivatives of saccharide are here discussed in sections onsaccharide azines, which are formed when one hydrazine molecule reactswith two saccharide molecules; saccharide hydrazones and glycosylhy-drazines, which are the tautomeric acyclic and cyclic products formed whenone hydrazine molecule reacts with one sugar residue; saccharide osazonesand poly(hydrazones), which are formed when two or more hydrazine mol-ecules are linked to a saccharide residue; and, finaly, hydrazones of carba-sugars and related compounds.

II. SACCHARIDE AZINES

Monosaccharides and reducing disaccharides react with unsubstitutedhydrazine to give azines, such as 2, which on prolonged treatment with anexcess of reagent yield hydrazones (3 see Scheme 1).21,22 Azines exist asequilibrated mixtures of tautomeric cyclic and acyclic forms, and on acetyl-ation they yield acyclic acetates that possess ester, but no amide groups.22

Hydrazone azines (6) may be obtained by treating glycos-2-ulose N-methyl-N-phenylhydrazone (4) with hydrazine and acetic acid or frommixed bis(arylhydrazone) (5) on treatment with acetic anhydride.23

KHADEM AND FATIADI176

SCHEME 1. Formation of azines, hydrazones, and azine hydrazones.


X-Ray crystallography has revealed that the N–N bonds of azines assumes-trans conformations and that phenyl rings, if present, are usually coplanarwith the adjacent CN groups.24–26 However, bulky groups in the ortho po-sition of phenyl rings may cause deviation from coplanarity. Thus the planeof the phenyl ring in (ZE)-o-nitroacetophenone azine was found by X-raycrystallography to deviate by some 388 from coplanarity with the CNgroup.27 Photolysis of the azine of D-galactose regenerates D-galactose andyields some D-lyxose by chain degradation.28

III. SACCHARIDE HYDRAZONES AND GLYCOSYLHYDRAZINES29

1. General Aspects

Originally, all condensation products formed by combination of one moleof hydrazine with one mole of a mono- or disaccharide were referred to ashydrazones, and when more than one form of the product was isolated, asin the case of D-glucose phenylhydrazone, the isomers were differentiatedby the Greek letters a, b, and so on. With the advent of modern methods ofstructure elucidation, and in particular of NMR spectroscopy and X-raycrystallography, more accurate structure determinations became possible.These revealed that hydrazones, like their parent saccharides, exist inacyclic and cyclic forms. The products that proved to be acyclic retained thename hydrazone (for example, D-galactose phenylhydrazone), whereas thecyclic isomers were designated as glycosylhydrazines [for example, 1-(b-D-glucopyranosyl)-2-acetylhydrazine, depicted in Scheme 2 in the 4C1 confor-mation].30 Glycosylhydrazines may exist in pyranose or furanose forms andmay possess a- or b-configurations, although the equatorially substitutedderivatives such as the b-D-hexopyranosyl-hydrazines depicted are usuallyfavored.

It must be emphasized that the term hydrazone is still used to desig-nate products whose structures have not been fully established. For exam-ple, many of the arylhydrazones that have been used in the isolation and

HYDRAZINE DERIVATIVES OF CARBOHYDRATE COMPOUNDS 177

SCHEME 2. Acyclic hydrazones and cyclic glycosylhydrazines.


characterization of mono- and disaccharides have never been subjected torigorous structure elucidation, and yet they are refered to as hydrazones. Itis to be expected that when the structure of all hydrazones have been de-termined, this ambiguity in terminology will vanish and only correct nameswill be used.

Saccharide hydrazones have often been used to separate and character-ize saccharides. Nowadays this is usually done by subjecting the free sac-charides or their hydrazones to such chromatographic techniques asgas–liquid chromatography (GC), GC mass spectrometry (GCMS), andhigh-performance liquid chromatography (HPLC), which permit their sep-aration identification, and estimation. In the last method, conversion of sac-charide mixtures to hydrazones before analysis enables the use of ultravio-let (UV) detectors (which are much more sensitive than refractive-indexones).31–33 Similar conversions into hydrazones enable the separation ofsaccharide mixtures by electrophoresis (because, in buffered solutions, theelectric charge on the nitrogen allows derivatives to migrate in electricfields).34,35 Nucleophilic addition of hydrazines to sugars is mostly used inthe synthesis of cyclic and acyclic sugar derivatives and of amino and amidocompounds,36–53 and ozonolysis has been used in the preparation of alde-hydo sugars from hydrazones.54

Although the hydrazones of organic aldehydes have been studied moreextensively than their saccharide counterparts, the use of Cu(II) salts to hy-drolyze hydrazones and hydrazides efficiently (by decomposing the gener-ated hydrazine to benzene, water, and nitrogen) was first introduced by car-bohydrate chemists55 and used much later in terpene chemistry.56 The following organic reactions have rarely been performed on saccharide substrates and ought to receive more attention by carbohydrate chem-ists: (a) carbonyl-group reactions,57–59 such as in the Fischer indole synthe-sis,60 and other means of converting carbonyl compounds into hydrocar-bons61 as well as the synthesis of 1,2,4-triazoles;62 (b) the conversion of 1,2-disubstituted hydrazines and hydrazones into the corresponding di-azenes and diazonium compounds by two-electron oxidations;63 (c) hy-drazines and hydrazones have been metallated and used in synthesis tomake use of their reactivity, resistance to proton transfers, and propensityto alkylate exclusively on carbon64 (the complex with gold has been studiedin detail65 and that with Ru3(CO)12 was found to cause fixation of the N–Nunit and to bridge the ligand to the metal framework);66,67 (d) tosylhydra-zones react with alkyllithiums to form anionic adducts,68 which decomposeby sigmatropic rearrangement of allylic diazene intermediates to affordalkenes69–70 (similar sigmatropic rearrangements have been invoked in thereduction and elimination71 of tosylhydrazones as well as in the oxidationof alkylhydrazines);72 (e) the stereoselective synthesis of alkenes from sily-



lated sulfonylhydrazones has been studied,73–75 and charge-transfer com-plexes of hydrazones have been examined by UV and infrared (IR) spec-troscopy and by X-ray crystallography;76 and (f ) the photochromic proper-ties of phenylhydrazones have been studied by laser flash spectroscopy,77

and 13C NMR spectroscopy has been used to study electron distribution inhydrazones, phenylhydrazones, oximes, and oxime ethers.78 Key papers andreview articles detailing these reactions are presented here in the hope thatsome of the reactions mentioned might be applied to saccharide hydra-zones and more spectroscopic studies would be directed toward carbohy-drate derivatives.

2. Formation of Saccharide Hydrazones and Glycosylhydrazines

Kinetic studies79,80 have clarified the mechanism of formation of phenyl-hydrazones of aromatic aldehydes. It was found that, during the formationof benzaldehyde phenylhydrazone,79 attack of the nucleophile is the rate-determining step under slightly acidic conditions, whereas dehydration ofthe carbinolamine intermediate is the rate-determining step under neutraland basic conditions.

Saccharide hydrazones are prepared by treating aldoses, ketoses, al-dosuloses, or reducing disaccharides with unsubstituted hydrazine;81,82

with monosubstituted hydrazines having alkyl,81 acyl,30,83,84 aroyl,85 sul-fonyl,36,39,40,86–88 aryl,89–93 or disubstituted N,N-dialkyl-,81 N,N-diaryl-,94 orN-alkyl-N-arylhydrazines39,40,43,95–99 or heterocyclic hydrazines;93,100–108 orwith semicarbazide83 and thiosemicarbazide.109 Arylhydrazines have beenextensively used for the characterization and identification of saccharides.In contrast, alkylhydrazines have been seldom used because the alkylhy-drazones formed are difficult to crystallize and possess unfavorable equi-librium constants for their formation.

Unsubstituted saccharide hydrazones are prepared by prolonged heatingof sugars with an excess of hydrazine; the excess is needed to obviate theformation of azines.81,82 Substituted hydrazones are prepared by heatingequimolar amounts of saccharide and substituted hydrazines in the free-base form36,39,40,42 or by treating cold, aqueous solutions of a sugar with aweakly acidic solution of hydrazine acetate. Aldosulose monohydrazonesare prepared either by treating aldosuloses with one mole of hydrazine110

or by removing one hydrazone residue from osazones with nitrous acid111

or copper(II) sulfate (see Scheme 3).112 Both reactions must be performedunder mild conditions to prevent formation of the bis(hydrazone) in thefirst case and the aldosulose in the second.

Hydrazones are formed most rapidly in media that are slightly acidic (pH4 to 5) in order to protonate the substrate. Kinetically, the reaction is



pseudounimolecular113,114 when acetate ions are present. Since saccharidesexist in solution as equilibrium mixtures of various cyclic and acyclic forms,nucleophilic addition to the carbonyl groups of the acyclic form and nucle-ophilic substitution of the cyclic forms (a- or b-furanoses or a- or b-pyranoses) or on the hydrate of the acyclic form can occur concurrently, butthe nucleophilic addition is usually faster. For example, it was found that therate of nucleophilic addition to aldehydo-D-galactose pentaacetate is muchhigher than that of nucleophilic substitution of the two isomeric cyclictetraacetates of D-galactose.113,114 This is why the preponderance of cyclicforms can only partly compensate for the rapidity of the nucleophilic addi-tion reaction. Both reactions seem to be initiated by protonation. Protona-tion of the carbonyl group of the acyclic form of a saccharide (15), followedby nucleophilic addition of phenylhydrazine, results in the formation of a 1-phenylhydrazine-1-ol (16), which, upon elimination, affords a protonatedhydrazone (not shown) and then an acyclic hydrazone (17). The nucleo-philic substitution reaction is also initiated by protonation of the anomerichydroxyl group of the saccharide 12, which facilitates elimination of waterand formation of a carbocation (13 in Scheme 4). The latter, when attackedby a substituted hydrazine, affords a cyclic hydrazone (14) having the samering size as the starting cyclic sugar.

It should be emphasized that, in solution, the acyclic hydrazones and allof the glycosylhydrazines, having various ring sizes, anomeric config-urations, and different conformations, exist in equilibrium. They are in-terconvertible with one another and with the acyclic form by reactionsquite similar to those of anomerization of free saccharides. It is not surpris-ing therefore to find that the nature of the preponderant form of a hydra-zone in solution and the structure of the solid isolated from this mixture isnot determined by the mechanism of formation, but by the relative stabil-ity of the isomers. For example it is now known that D-glucose hydrazone is


SCHEME 3. Formation of aldosulose monohydrazones from aldosuloses andfrom osazones.


formed initially when D-glucose is treated with aqueous hydrazine at roomtemperature and it then gradually cyclizes to afford predominantly b-D-glucopyranosylhydrazine30 (like the one depicted in Scheme 2).

3. Structure of Saccharide Hydrazones and Glycosylhydrazines

Although saccharide hydrazones exist in solution as equilibrium mix-tures of several forms, the crystalline hydrazones isolated from solution areusually composed of only one form, which is often the most stable one inthe medium. It is to be expected that substituted hydrazines would affordtwo types of products, one formed by nucleophilic attack of N-1 and one byN-2. This expectation was confirmed when phenylazostyrene (18) wastreated with phenylhydrazine (19) at low temperature and the rate-controlled N-1 adduct (21) was isolated from the reaction mixture and con-verted into benzil bis(phenylhydrazone) (20) by heating. It may be assumedthat the reaction proceeds by the conversion of the rate-controlled N-1adduct to the equilibrium-controlled N-2 adduct (not depicted), followedby an oxidation step (Scheme 5).115,116

The hydrazones of sugars are capable of existing in various cyclic forms,whose presence is apparent from their nuclear magnetic resonance (NMR)spectra and from the complex mutarotation curves they exhibit110,111,117,118

(which seldom follow first-order kinetics). The principal structures encoun-tered in saccharide hydrazones are the acyclic, Schiff base-type true hydra-zones and the cyclic hydrazino forms, namely glycopyranosyl- and gly-cofuranosylhydrazines. For example, three isomeric forms of D-glucosephenylhydrazone have been isolated.119 The Schiff base derivatives can be


SCHEME 4. Formation of phenylhydrazones from cyclic and acyclic saccharides.


recognized by polarographic120 and spectrophotometric (ultraviolet and in-frared) analysis121–123 as well as by X-ray crystallography.124–126 Protonmagnetic resonance can also distinguish between (cyclic) glycopyranosylhydrazines and acyclic saccharide hydrazones.127–130 However, in the caseof arylhydrazones, the IR spectra are difficult to interpret because the C5Nabsorption band at 1620 cm-1 may be masked by the phenyl ring absorp-tions.59,130,131 Similar difficulties may be encountered with NMR spectra,where some signals may be buried under aromatic multiplets.82 Despitethese difficulties it was possible to show by 1H and 13C NMR spectroscopythat the conformation of arylhydrazones is syn129,132 and by X-ray crystal-lography that the product obtained by reacting D-glucose or L-arabinosewith p-bromophenylhydrazine possesses a cyclic structure; both are glycopyranosyl-p-bromophenylhydrazines.124–126 A chemical method forestablishing the cyclic or acyclic structure of hydrazones depends on theirreaction with benzenediazonium chloride. Acyclic phenylhydrazones gen-erally form crystalline diphenylformazans (see Scheme 17), whereas nowell-defined product is obtained from the cyclic isomers.133 For example,the so called “b-form” of D-glucose phenylhydrazone yields a crystallinediphenylformazan, whereas its two cyclic isomers do not. This reaction canbe used to estimate the proportions of acyclic forms during mutarota-tion.133

Chemical evidence for the open-chain structure of D-galactose hydra-zones was first provided by the reaction of N-methyl-N-phenylhydrazinewith 2,3,4,6-tetra-O-acetyl-D-galactopyranose (22); 2,3,5,6-tetra-O-acetyl-D-galactofuranose (24); and penta-O-acetyl-aldehydo-D-galactose (27). Thehydrazones (23 and 25) formed from 22 and 24 could be converted byacetylation into the same penta-O-acetyl-D-galactose N-methyl-N-phenyl-


SCHEME 5. Formation of the rate-controlled adduct of a phenylhydazone.


hydrazone (26) obtained from 27, and so it was concluded that hydrazones23 and 25 have open-chain structures (see Scheme 6).114

Acetylation of acyclic hexose arylhydrazones yields tetra-O-acetyl deriv-atives, whereas cyclic structures may afford derivatives acetylated on bothnitrogen and oxygen.134 Benzoylation of acyclic hydrazones yields N- andO-benzoylated derivatives. The structures of several hydrazones and semi-carbazones have been determined by studying the IR spectra of their ac-etates.135–142 The Schiff base structure of D-glucose semicarbazone was fur-ther confirmed when the same penta-O-acetyl derivative (29) was obtainedby acetylating 28 and by treating penta-O-acetyl-aldehydo-D-glucose (30)with semicarbazide (Scheme 7).

Aldosuloses react with substituted hydrazines to give mono- and bis-hydrazones. It is possible to remove one hydrazone residue from the latterto obtain aldosulose monohydrazones having the hydrazone residues at-tached to C-1.The position of substitution was established when D-arabino-hexosulose mono-(N-methyl-N-phenyl)hydrazone (31) was converted intoD-mannose N-methyl-N-phenylhydrazone (32) on catalytic hydrogenation(Scheme 8).143

The sign of optical rotation or of the Cotton effect is affected moststrongly by the chiral center nearest to the chromophore. It is not surprising


SCHEME 6. Formation of the same hydrazone from tetra-O-acetyl-b-D-galactopyranose,tetra-O-acetyl-b-D-galactofuranose and penta-O-acetyl-aldehydo-D-galactose.


therefore that several rules have been formulated that correlate the config-uration of the C-2 hydroxyl group of acyclic aldose hydrazones with theirsign of rotation. Thus the “Benzyl Rule” states that if, in the Fischer projec-tion formula, the C-2 hydroxyl group is to the right, the corresponding N-benzyl-N-phenylhydrazone is levorotatory; if the group is to the left, the de-rivative is dextrorotatory.144–147 The circular dichroism (CD) and opticalrotatory dispersion (ORD) spectra of benzoylhydrazones were also relatedin a similar manner to their configurations.148

Saccharide hydrazones can exist in several tautomeric forms, which in-clude the phenylazo and enehydrazine forms.

The glycosylhydrazines can exist in the furanose and pyranose forms andmay possess a- or b-configurations, although usually the equatorially sub-stituted D-hexopyranosylhydrazines are favored.149 Most of these cyclicforms have not been isolated, but their amounts can be calculated fromtheir NMR spectra and their mutarotation curves.150,151

4. Reactions of Saccharide Hydrazones and Glycosylhydrazines

Because of the similarity between the C5O group of acyclic monosac-charides and the C5N group of their hydrazones, the latter exhibit many ofthe reactions of their parent saccharides; for example, they are susceptible


SCHEME 7. Formation of the same penta-O-acetyl semicarbazone from D-glucose, semi-carbazone, and penta-O-acetyl-aldehydo-D-glucose

SCHEME 8. Formation of D-mannose N-methyl-N-phenylhydrazone by hydrogenating D-arabino-hexos-ulose mono-(N-methyl-N-phenyl)hydrazone.


to nucleophilic attack and to the action of oxidizing and reducing agents.However, they behave differently in basic media; reducing saccharides un-dergo rapid epimerization, whereas saccharide hydrazones epimerize muchmore slowly. Furthermore, reducing saccharides undergo degradative oxi-dation in the presence of oxygen and base to give lower aldonic acids,whereas hydrazones do not undergo such oxidative degradation. These dif-ferences have been attributed to the ability of free saccharides to enolize inbasic media and to the resistance of hydrazones to do so, as detailed next.

a. Action of Alkalies.—It has already been noted that, in contrast toaldoses, ketoses, and reducing disaccharides, which undergo epimerizationin dilute alkaline solutions and are degradatively oxidized in the presenceof oxygen, saccharide phenylhydrazones do not epimerize and are notappreciably degraded during oxidation. The reason for the difference is thelower acidity of the a-hydrogen of saccharide hydrazones, relative to theimino proton. This is because the hydrazone anion, formed by ionization ofthe imino group, is more stable, that is, it is stabilized by more resonanceforms (33, 34, and 35) than is the ene-hydrazine anion, formed byabstraction of the a-hydrogen (forms 36 and 37). In addition, the par-tial negative charge on the hydrazone carbon facilitates elimination of aleaving group (not a proton) from the adjacent carbon atom. The pref-erential ionization of the NH protons of phenylhydrazones suppressesenolization and slows epimerization and degradative oxidation of aldosephenylhydrazones.152–154 The fact that phenylhydrazones do not enolize inbasic media does not preclude their enolization in acidic media. The acid-catalyzed enolization of hydrazones is initiated by protonation of the iminonitrogen of a hydrazone (38), followed by elimination of the a-proton withwater to give intermediate 39. Shifts of electrons then form a CC doublebond and neutralize the charge on the nitrogen to give 40 (see Scheme 9).The ability of hydrazones to enolize in acid media explains why osazoneformation (which requires enolization at one stage) occurs in acidic but notin basic media. The inability of aldose phenylhydrazones to enolize in basicmedia also explains why their acetates, which possess good leaving groupsattached to C-2, undergo elimination in basic media to give azoalkenes(instead of eliminating a proton), and why the oxidation of aldosephenylhydrazones does not result in degradation, but leads instead to N-phenyl-N-aldonohydrazono-1,4-lactones,152–154 as is shown later.

Literature surveys reveal several studies47,130,152–157 on the action ofstrong bases on sugar phenylhydrazones. These includes the isomerizationof phenylhydrazones in akali to give alkyl phenyldiimides,158 the effect ofalkali metals on nitrogen–nitrogen bond breaking in arylhydrazones,159 andthe thermolysis of arylhydrazones in the presence of alkaline agents.160,161



Generally, alkali causes homolytic fission of the saccharide chain adjacentto the hydrazone moiety to form two radicals (initiation). This is followedby combination of a radical (43) with another hydrazone molecule to formglyoxal bis(phenylhydrazone) (44) and a new radical (propagation).162–169

Another product of this free-radical reaction is 1-phenylpyrazole (41),formed by aromatization of a phenylhydrazone residue and a three-carbonsugar moiety (Scheme 10).165


SCHEME 9. The resonance hybridization of an ionized NH group (upper) and an Ionizeda-CH group (lower) in hydrazones.

SCHEME 10. Homolysis of phenylhydrazones in alkali.


Early studies in the sugar series reveal that glyoxal bis(benzoylhydra-zone) is a major product when, for example, D-glucose is treated with ben-zoylhydrazine in aqueous alkali.161 Hydrogen–tritium exchange experi-ments reveal that, in alkaline medium, hydrazones undergo a series of rapidtautomerizations which may lead to degradation products.162–164 Experi-ments with D-[2-14C]glucose benzoylhydrazone show that, in forming gly-oxal bis(benzoylhydrazone), a labeled entity derived from C-1 and C-2 com-petes with glycolaldehyde fragments (derived from the nonlabeled atomsC-3 and C-6) for reaction with the benzoylhydrazone group.170 This wouldsuggest that fragmentation is achieved by a reversed aldol mechanism.170–172

b. Free-Radical Oxidation Reactions.—The autoxidation of aromaticphenylhydrazones at room temperature to produce phenylazo-hydroperoxides is well documented.173–175 It is also known176,177 thatoxidative degradation of free sugars with oxygen in alkaline solution is afree-radical process and that anion radicals of the semiquinone type areformed during alkaline, oxidative degradation of saccharides, polysaccha-rides, and wood (ESR studies).178,179 Advances in free-radical chemistrymake it possible to generate and to trap short-lived radicals,180 includingfree radicals of the anomeric carbon atom (whose behavior is influenced bystereoelectronic effects).181,182 Electron-transfer pathways that involverearrangements by free radicals183,184 or radical ions185 can be inferredfrom the structure of rearranged products. Of the methods available for thedetection of free radicals, spin trapping offers the opportunity to measuresimultaneously and distinguish among a variety of important, biologicallygenerated free radicals.186,187 A review on spin-labeled carbohydrates,including mono-, di-, and polysaccharides, glycoproteins, and nucleosides,has appeared.188

(i) Formation of Hydrazono Lactones.—It is known that aldehydephenylhydrazones undergo oxidation by free-radical mechanisms whenbrought in contact with oxygen or air and are converted into phenylazo-hydroperoxides.173,175,189–191 The same reaction in basic media probablystarts with ionization followed by removal of an electron by a radical(initiation) to give a resonance-stabilized radical. Reaction with oxygenaffords a peroxide radical, which is needed for propagation. The latter isconverted into a peroxy anion, which on protonation gives a phenylazo-hydroperoxide.

Hydrazono-1,4-lactones have been isolated153,154 when solutions of aldose phenylhydrazones in aqueous ethanol containing potassium hy-droxide were kept at room temperature in contact with air (bubbling air or oxygen into such solutions may lead to spontaneous explosion of



peroxides). A plausible mechanism for this free-radical peroxidation startswith an attack by oxygen on C-1 of a phenylhydrazone (45) to give a hy-droperoxide (not shown), which rearranges to a phenylazo-hydroperoxideform (46), which is tautomeric with the phenylhydrazono-hydroperoxideform (47). The latter then undergoes nucleophilic attack by O-4 and elimi-nation of a hydroperoxide ion to give the isolated N-phenyl-aldonohydra-zono-1,4-lactone (48). The electron-spin resonance (ESR) evidence re-ported in the literature192 and the explosive nature of the intermediatesobserved in this work suggest that a significant part of the peroxidation pro-ceeds via a free-radical mechanism (Scheme 11).146,147

(ii) Formation of Oxadiazoles.—Saccharide benzoylhydrazones, semi-carbazones, and thiosemicarbazones undergo free-radical oxidations,similar to the one just described, with iodine193 or Fe3+ to give 3-acetoxyalkyl-5-aryl-oxadiazoles and thiadiazoles.194–199 The same com-pounds can be obtained from aldonic acids or aldonic acid chlorides. Forexample, D-galactose benzoylhydrazone pentaacetate (49) affords, ontreatment with iodine, an acyclic 1,3,4-oxadiazole (50).193 This product,upon deacetylation, yields a cyclic hydrazono-lactone (52), which is ananalog of compound 48. Analogous hydroximo- and hydrazino-1,5-lactoneswere obtained by Vasella and coworkers by oxidation of sugar oximes,sulfonylhydrazones and phenylsemicarbazones.200 These authors were ableto convert the first compounds into diazirines, and then, by heating orirradiation, to carbenes, which were successfully used as intermediate inglycosidation reactions. Reviews of this last work have appeared.201 Theelectro-chemical oxidation of N-acylhydrazones to give oxadiazolines andoxadiazoles has been described202 and reviewed.203,204 Oxadiazolines havealso been obtained by acetylation of aroylhydrazones (Scheme 12).205

c. Elimination Reactions (Formation of Azoalkenes).—One result of therelative acidity of the imino protons of hydrazones is the facile eliminationof leaving groups (such as acetate esters located on C-2, which are


SCHEME 11. Formation of N-phenyl-aldonohydrazono-1,4-lactones by the action of air onD-galactose phenylhydrazone in dilute alkali.


eliminated as acetic acid). Thus, the penta-O-acetyl derivatives of D-galactose and D-mannose arylhydrazones (53), when warmed with pyridine,undergo elimination to give azoalkenes, for example, 3,4,5,6-tetraacetoxy-1-phenylazo-trans-1-hexene (55).The structure of this and similar compoundshas been confirmed by NMR spectroscopy.164,206–208 The reaction wasexplained by Wolfrom and Blair in terms of an ionic mechanism whichstarts with the ionization of the imino proton of a hydrazone acetate (53) inbases to give a resonance-stabilized anion (54). This anion readily under-goes elimination of the leaving group (OAc) to afford the azoethylenederivative 55. A free-radical mechanism was later proposed157 which startswith the formation of anion 54 in base. Initiation of the free-radical reactionis achieved with oxygen to generate a resonance-stabilized radical anion(forms 56 and 57) (deprotonation of hydrazobenzene by molecular oxygenin alkaline solution proceeds via a dianion which also loses an electron tooxygen).209–211 Reaction of oxygen with the resonance form 57, having theradical on C-1,212,213 affords a peroxide radical (not shown)29,173–175 that isneeded for propagation and to produce a peroxide anion (58) by additionof an electron. The peroxy anion attacks the carbonyl group of the acetateon C-2 to form a six-membered ring (59). Finally, fission by electron shiftsaffords the observed product 60 (Scheme 13).

The reaction of phenylhydrazine with phenylazoalkenes at low tempera-ture yields labile 1-phenylhydrazino-phenylhydrazones, which are con-verted at higher temperature to the thermodynamically more stable (2-phenylhydrazino)-phenylhydrazones.115 In the saccharide series, treatmentof 3,4,5,6-tetraacetyl-1-phenylazo-trans-1-hexenes (61) with phenylhy-drazine was shown to yield the free osazones (63), denoting that oxidationof the addition product (62) must have occured during the reaction. Itshould be noted that the ester groups are eliminated by transamidation toform acetic phenylhydrazide.152,214 Several azoalkenes and azoalkene in-termediates have been subjected to conjugate nucleophilic additions(Scheme 14).215–217


SCHEME 12. Formation of oxadiazoles and hydrazono lactones by oxidation of benzoylhy-drazones.



SCHEME 13. Formation of azoalkenes by ionic and free-radical mechanisms.


d. Derivatives of Saccharide Hydrazones and Glycosylhydrazines.—Acetylation of saccharide hydrazones (65) with acetic anhydride inpyridine affords per-O-acetylated derivatives (such as 66) with acyclichydrazones and N-acetyl-O-acetyl derivatives with the cyclic ones.85,114,

134,165 In the last case, the NH group attached to the aryl moiety is notacetylated, but the more basic NH group attached to the sugar moiety isacetylated. The O-acetyl groups are split off by base more readily than theN-acetyl groups,218 and methods for selective saponification have beendevised. With acetyl chloride in N,N-dimethylaniline, the NH groupattached to the aryl residue becomes acylated and acyclic hexosehydrazones give N-acetyl-penta-O-acetyl derivatives (64);165 similarly,benzoylation with benzoyl chloride in pyridine affords N-benzoyl-O-benzoyl derivatives (Scheme 15).219

e. Electrophilic Substitution—(i) Formation of Bromino and DibromoDerivatives.—Arylhydrazones having electron-attracting groups such as p-nitro that deactivate the aromatic ring may undergo electrophilicsubstitution on the hydrazone residues by an SE29 mechanism.213,214 Theproducts are tautomeric (azo-hydrazono) pairs of monobromo derivatives,such as 68 and 69. With an excess of bromine a gem dibromo phenylazoderivative (72) having the E configuration is isolated (Scheme 16).220–222

(ii) Formation of Formazans.223—Aldose phenylhydrazones undergoelectrophilic substitution with aryldiazonium salts to give brilliant red,


SCHEME 15. Acetylation and benzoylation of hydrazones.

SCHEME 14. Nucleophilic addition of phenylhydrazine to azoalkenes.


crystalline formazan derivatives.98,134 Two structural features are requiredto form formazans: the presence of an aldehyde arylhydrazone (a Schiffbase type of structure) and the presence of a free methine hydrogen atom on the arylhydrazone group. In consequence, aldose phenylhydra-zones in the cyclic hemiacetal forms, ketose phenylhydrazones, and N,N-disubstituted hydrazones fail to yield formazans.

Formazans are known to have a chelated structure which permits theequilibration of monosubstituted isomers.224–227 For example, the tau-tomeric pair structure of N-phenyl-N9-p-bromophenylformazan (74 and 76)can be formed from either the p-bromophenylhydrazone 73 and a ben-zenediazonium salt or from a phenylhydrazone (75) and a p-bromoben-zenediazonium salt.228 The neutral D-mannose diphenylformazan229 mole-cule has a chelate structure in which the imino proton spans thephenylhydrazono and the phenylazo groups (Scheme 17).230

Treatment of a formazan such as 77 with a strong acid, such as perchloricacid, results in a color change from orange-red to purple-blue. Apparently,the strong acid ruptures the chelate ring and forms a resonance-stabilizedcation. Delocalization of the positive charges over the resonance system(78a and 78b) may account for the bathochromic shift observed in the spec-trum of on protonation.230 Saccharide formazan acetates, upon deacetyla-tion, undergo some epimerization (Scheme 18).231


SCHEME 16. Bromination of hydrazones.



SCHEME 17. Formation of formazans.

SCHEME 18. Protonation of formazans.


Circular dichroism studies of saccharide formazans and their acetateswere used to establish the configuration at the C-2 atom and the confor-mation of the sugar chain.232

Formazans react with salts of heavy metals to give stable complexes;233

and they are also oxidized by N-bromosuccinimide or (after acetylation) bylead tetraacetate to yield tetrazolium salts.234–236 Reduction of the lattergive back the formazans. Reductive decomposition of sugar formazans (79)with hydrogen sulfide leads to aldothionic acid phenylhydrazides(82).237–240 Treatment of these with benzaldehyde in the presence of hy-drochloric acid yields sugar thiadiazolines (83) (Scheme 19).237

Because of their sharp melting points, sugar formazans can be used toidentify aldoses233 (ketoses do not give formazans) and to purify them afterconversion to the thioaldonic phenylhydrazides.241,242 Fluoroboric acid inacetic anhydride transforms penta-O-acetyl-D-galactose diphenylformazaninto mono-N-acetyl-penta-O-acetyl-D-galactose diphenylformazan, which,on saponification, yields 2,6-anhydro-D-galactose diphenylformazan.231

f. Cycloaddition.—Aldose aryl and alkylhydrazones react with acetyl-enic compounds to give pyrazole derivatives.243–246 If the reaction is


SCHEME 19. Action of hydrogen sulfide on formazans.


carried out with 2,5-anhydropentose 1-bromo-arylhydrazones (84), itaffords C-nucleoside analogs, such as 86 (Scheme 20).247,248

g. Reduction.—In theory, reduction of hydrazones should yieldhydrazino derivatives and then, under more vigorous conditions, thecorresponding aminodeoxy derivatives. However, most aldose hydrazonesafford the aminodeoxy derivatives directly. For example, reduction ofhydrazone 87 yields the 1-amino-1-deoxyalditol (88).249 The hydrazinoderivatives of carbohydrates can be obtained by nucleophilic substitution,for example, by hydrazinolysis of sulfonic esters of monosaccharides,250

oligosaccharides,251 and polysaccharides.252,253

The following two examples illustrate the use of hydrazone reductions inthe preparation of amines. (a) Formation of 5-amino-5-deoxy-D-ribofura-nose, a so-called “aza-sugar,” so named because its furanose ring exists inequilibrium with the more stable six-membered azacyclohexane ring. Thisgroup of amino and imino sugars has attracted considerable attention be-cause many examples exhibit interesting biologically properties; some act asenzyme inhibitors,254–256 such as glycosidase inhibitors,257 others have po-tential antidiabetic properties,258 and a few show anticancer259 or anti-HIVactivity.260 As noted, 5-amino-5-deoxy-D-ribofuranose (93) is in equilibriumwith its six-membered isomer 94. Its synthesis starts with the periodate oxi-dation of 1,2-O-isopropylidene-D-allofuranose (89) to form aldehyde 90,which is converted into a phenylhydrazone 91 and reduced to 92 and thendeblocked to give a mixture of the desired 5-amino-5-deoxy-D-ribofuranose(93) and the a- and b-anomers of 5-amino-5-deoxy-D-ribopyranose (94).255

The other example (b) involves stereoselective amination of the monohy-drazone (96) of glyoxal. This synthon is converted into a chiral aminal (97)


SCHEME 20. Cycloaddition to hydrazones.


SCHEME 30. Conversion of squaric acid bis(phenylhydrazone) to a dianilide.

SCHEME 31. Fischer structure of D-arabino-hexulose phenylosazone.


tris(phenylhydrazones). For example, squaric acid (163), upon treatmentwith phenylhydrazine, yields a tris(phenylhydrazone), which exists prepon-derantly in the azoenehydrazine form (165) rather than in the tautomerictris(hydrazone) form (164) (Scheme 34).314

b. Chelated Structures of Osazones.—Fieser and Fieser315 predicted, ontheoretical grounds, that sugar arylosazones exist as equilibrium mixturesof four tautomeric forms (166–169). However, only one pair of tautomerscould be detected, namely the chelated pair (166 and 167), which is clearlyevident by X-ray crystallography (see Fig. 1).307,308,313 It is probable thatthe other pair (168 and 169) is not formed because its members would be


FIG. 1. Electron-density projection in the direction of the b axis of D-erythro-pentulose p-bromophenylosazone. Note that C-1 is linked two bulky atoms only (C and N).

SCHEME 32. Periodate oxidation and acetylation products of a hexuloseosazone.


destabilized by the close proximity of the bulky saccharide residue and theanilino group (Scheme 35).

It should be noted that the members of the chelated pair have never beenseparated in the case of saccharide osazones, but this has been achievedwith noncarbohydrate bis(phenylhydrazones). In at least one case theywere found to be interconvertible, either by irradiation with light (pho-tochromism) or by treatment with acids or bases (see Fig. 2).316

Further evidence for the chelated structure of osazones was obtained byphysical methods,317 such as polarography318 and ultraviolet317,319–327 and1H NMR307–312,326 spectroscopy, which clearly showed that the two NHgroups (attached to C-1 and C-2) were not in identical environments. Sim-ilarly 15N NMR spectroscopy revealed significant differences in the chemi-cal shifts of the two H-coupled nitrogen atoms, attributable to chela-tion.328,329

The presence of stable chelated rings in osazones is also evident from theslowing down, and sometime inhibition, of certain reactions. Thus, the for-mazan reaction223 requires potassium hydroxide to break the chelated ringof osazones and to effect coupling with the diazonium ion.319,320 Similarly,the formation of polyhydrazones is inhibited by chelation accross the C-1


SCHEME 33. Tautomeric forms of osazones.

SCHEME 34. Tautomeric structures of squaric acid tris(phenylhydrazone).


and C-2 hydrazone residues. This is the reason why N,N-disubstituted hy-drazines, which lack the imino protons necessary for cheation, afford tris-and tetrakis-arylhydrazones, whereas monosubstituted arylhydrazines whichcan form osazones with a chelated ring do not yield such polyhydrazones.

c. Mutarotation of Osazones.—It is agreed that during mutarotation, theinitial form of the saccharide osazone is the chelated pair of tautomericbis(phenylhydrazones) (170) and their tautomers. However, differentstructures have been suggested for the final form(s).324–336 These includeunchelated azoenehydrazine forms and C-3 chelated forms (171).325,337–342

The following observations regarding mutarotation of osazones have been made: (a) osazones mutarotate in basic solvents by a first-orderprocess, differing in this respect from phenylhydrazones (see Section III.3);(b) base-catalyzed mutarotation is shown by sugar osazones having achelated structure and not by bis N-alkyl-N-phenyl bis(hydrazones), whichlack chelated ring structures; (c) mutarotation is accompanied by a hyp-sochromic shift in the ultraviolet absorption maximum; (d) mutarotation isreversible, and the product recovered from solution at equilibrium isidentical to the starting osazone; and (f ) the NMR spectra of osazonesundergoing mutarotation exhibit325 changes expected by the conversion ofN-chelated structures such as 170 to O-chelated structures such as 171.

Rules have been fomulated to corrolate the configuration at C-3 with thesign of optical rotation311 and the sign of the Cotton effect at l 250–270 nm(Scheme 36).325,326


SCHEME 35. Two pairs of tautomeric chelated osazones arepossible, but only the top pair is formed.



3. Reactions of Osazones

a. Nucleophilic Substitution (Hydrolysis and Transhydrazonation).—Fischer110 was the first to report that sugar osazones (172) are hydrolyzedby the action of hydrochloric acid. This hydrolysis is an example of

FIG. 2. Ultraviolet absorption spectra of two tautomeric bis(phenyl-hydrazones), taken at different intervals to show their interconversion.

SCHEME 36. The starting and ending chelated structures of osazones in basic media.


nucleophilic substitution; it is initiated by protonation of the hydrazone,followed by attack of a molecule of water to give an adduct. The latter loses phenylhydrazine to give first the corresponding aldos-2-ulose 1-phenylhydrazone (173) and then the aldos-2-ulose (originally termed aglycosone) (174). The bis(hydrazone) residues may also be removed bytranshydrazonation with an aldehyde such as benzaldehyde343,344 or a keto acid such as pyruvic acid.345 In addition, the groups may also beremoved completely or partially by the oxidative action of nitrous acid346

or Cu2+ salts,112 which decompose the hydrazine generated and afford, first,aldos-2-ulose 1-phenylhydrazones (173) and then aldosuloses (174)(Scheme 37).

Aldos-2-ulose 1-phenylhydrazones react with differently substituted hy-drazines to yield mixed arylosazones.347–352 In addition, they react with o-phenylenediamine to give quinoxalines (as seen in Scheme 29).347,352 Itseems that the phenylhydrazone residue undergoes nucleophilic substitu-tion with o-phenylenediamine, either directly or after hydrolysis to the al-dosulose, to give a quinoxaline lacking a hydrazone residue. If the hydra-zone residue is stabilized by chelation, as in the case of compound 175, thereaction proceeds with retention of the hydrazone residue in the quinoxa-line formed (176) (Scheme 38).314

b. Electrophilic Substitution.—D-arabino-Hexulose phenylosazone (177)reacts with diazotized aniline to give a formazan (178).The same compoundis obtained by treating D-arabino-hexosulose 1-phenylhydrazone formazan(179) with phenylhydrazine (Scheme 39).319,347,350

Because of the chelated structure of sugar osazones, the osazone for-mazan 181, obtained by coupling diazotized [14C]aniline to a saccharidephenylosazone (180) loses 42% of the label (as aniline) upon conversion toan osotriazole (182), whereas the formazan of an unchelated osazone, suchas pyruvaldehyde osazone loses exactly half of the radioactivity as anilineduring osotriazole formation (Scheme 40).168

c. Action of Bases (Formation of Free Radicals and Degradation).—Treatment of a solution of glyoxal bis(phenylhydrazone) with base causes


SCHEME 37. Stepwise hydrolysis of bis(phenylhydrazones).



SCHEME 38. Formation of a quinoxaline with retention of the hydrazone residue.

SCHEME 39. Formation of formazans.

SCHEME 40. Reaction of formazans.



ionization of the imino proton and produces a deep-purple color.29 Thepresence of oxygen causes such solutions to become paramagnetic353

because of the formation of resonance-stabilized anionic free radicals. Thedianionic character of these radicals was shown by their conversion intoN,N-dimethyl and N,N-dibenzoyl derivatives with dimethyl sulfate and withbenzoyl chloride. The resolution of the ESR spectrum of glyoxalbis(phenylhydrazone) is greatly enhanced when certain positions of thebenzene rings are substituted. For example, the spectrum of glyoxalbis[(2,5-dichlorophenyl)hydrazone] in the presence of tert-butoxide gives a44-line ESR spectrum (see Fig. 3). The hyperfine structures are attributedto the interaction of unpaired electrons with equivalent nitrogen atoms,353

as in the case of a hydrazine cation radical.354 Saccharide phenylhydrazonesand phenylosazones do not show evidence of the paramagnetic species thatare characteristic of glyoxal bis(phenylhydrazone). Instead they show 3-lineESR spectra characteristic of nitroxide radicals having a-hydrogenatoms,157 unlike nitroxide radicals having no a-hydrogen atoms.355 Thenitroxide radicals are generated by oxidation of the phenylhydrazineliberated by hydrolysis.

Saccharide osazones (183) are relatively stable in cold concentrated al-kalies, but degrade progressively with time. The degradation starts at the

FIG. 3. Radical anion from glyoxal bis[(2,5-dichlorophenylhydrazone)] in base.


hydroxyalkyl chain of saccharide phenylosazones and gives rise to glyoxalbis(phenylhydrazone) (184) (Scheme 41).162,163,168

d. Oxidation of Bis(hydrazones)—(i) Formation of 1,2-Bis(phenylazo)-ethene.—Oxidation of glyoxal bis(phenylhydrazone) with Fe3+, Cu2+, orCr3+ yields an intense red-colored compound, 1,2-bis(phenylazo)ethene(185).This compound has an extended, conjugated f-electron system that isdisrupted by protonation in some of the resonance forms of the ion 186,causing a hypsochromic shift (Scheme 42).230

(ii) Formation of Dehydroosazones.—Treating saccharide phenylosa-zones in alkaline media with atmospheric oxygen gives rise to dehydro-osazones,356 which are very similar in appearance to the parent osazones.Structural investigations357,358 have shown that the dehydro derivativeobtained from D-arabino-hexulose phenylosazone has a pyranoid ring (itconsumes one mole of periodate to give a dialdehyde and does not afford aformazan). The NMR spectrum of its tri-O-acetyl derivative suggests thatthe hydroxyl group attached to C-3 is not equatorial, as would be expectedfrom the D-arabino configuration (187), but axial (D-ribo configuration)(188). D-Glucose gives the same dehydro-osazone as D-allose and D-galactose gives the same derivative as D-gulose, suggesting that an inversionat C-3 occurs in one of each pair (Scheme 43).

(iii) Formation of Phenylazo-Phenylhydrazones.—This oxidation wasfirst observed359 with dehydroascorbic acid bis(phenylhydrazones) (189). Itproceeds by cyclization of the side chain to give a bicyclic hydrazinohydra-zone (190). A facile hydrazino-azo oxidation then takes place with Cu2+ oriodine to give a phenylazo-phenylhydrazone (191). The reaction isreversible and the product may be reduced to the starting bis(phenylhydra-zone) (Scheme 44).359–364


SCHEME 41. Degradation of saccharide phenylosa-zones in alkali to glyoxal bis(phenylhydrazone).



SCHEME 42. Effect of protonation on the resonance forms of 1,2-bis(phenylazo)ethene.



SCHEME 42. Continued


e. Aromatization of the Bis(phenylhydrazone) Residues.—The hydra-zone residues of bis(hydrazones) readily undergo conversion into aromaticheterocycles. If one hydrazone residue is involved in aromatization, apyrazole results, but if two hydrazone residues are involved a phenyl-triazole is usually formed.

(i) Formation of Pyrazoles from Bis(hydrazones).—Mesoxaldehydebis(phenylhydrazone) (193), obtained by periodate oxidation of saccharideosazones (192) is readily cyclized in the presence of acids to give 1-phenyl-4-phenylazo-pyrazole (195).162,365 Hexulose phenylosazones (192) are alsodisproportionated in the presence of acidic salts of carbonyl reagents, such as hydroxylamine hydrochloride, to give 1-phenyl-4-phenylazo-pyrazolin-5-one (196). The reaction probably proceeds via mesoxalic acid 1,2-bis(phenylhydrazone) (194).365 The hydroxalkyl derivatives of 196 areproduced from dehydroascorbic acid bis(phenylhydrazone) by treatment withbase to open the lactone ring and permit the conversion of 197 to 199.351, 366

Another type of pyrazole that is formed by dehydrating osazones with aceticanhydride is discussed later under anhydroosazones (see Schemes 45, 53).

(ii) Formation of Saccharide Triazoles.367,368—Two types of 1,2,3-triazoles are obtained by treatment of saccharide bis(hydrazones) with mild


SCHEME 43. Dehydro-osazone derived from D-glucose.

SCHEME 44. Formation of a phenylazo-phenylhydrazone from a bis(phenylhydrazone).


oxidants. The first are the 2-aryl-4-(hydroxyalkyl)-1,2,3-triazoles, alsoknown as saccharide osotriazoles, and the other are the 1-arylamino-4-(hydroxyalkyl)-1,2,3-triazoles, obtained from saccharide bis(aroylhydra-zones). Other types of carbohydrate triazoles can be prepared by cycloaddi-tion of azides to acetylenic sugars.


SCHEME 45. Different types of pyrazoles formed by aromatization of hydrazone residues.


Saccharide Phenylosotriazoles. These 2-aryl-1,2,3-triazoles (201) areby far the most extensively studied triazoles. They were first prepared by Hann and Hudson369 by refluxing arylosazones (200) with aqueous copper (II) sulfate. Subsequently, numerous osotriazoles have been pre-pared to characterize the osazones of monosaccharides,369–379 disaccha-rides,380–386 and anhydroosazones.387–391 The conversion of arylosazonesinto the corresponding osotriazoles and aniline necessitates the presence ofan oxidizing agent, which suggests that the process is not as the structure ofthe reactants and products might suggest, a simple elimination of anilinefrom an osazone. Apart from copper(II) sulfate, which is the regent mostcommonly used, other oxidizing heavy-metal salts, such as mercuric ace-tate392 and ferric sulfate and choride,393 have been used. In addition, halo-gens394 and nitrososulfonates have also been used.395 The acetylated osa-zones are converted by nitrous acid into osotriazoles,396 and this reagentdecomposes unacetylated osazones to give aldosuloses.397 The structure ofsaccharide osotriazoles was confirmed by oxidation of the hydroxyalkylchain with periodate to yield 2-phenyl-1,2,3-triazole-4-carboxaldehyde(202) and with permangante, giving 2-phenyl-1,2,3-triazole-4-carboxylicacid (203) (Scheme 46).

Mechanism of Osotriazole Formation. A mechanism was proposed byEl Khadem398 to explain (a) why the triazole reaction requires oxidizingagents, (b) why the formation of an osazone–Cu(II) complex must precedetriazole formation, (c) how aniline is eliminated to form a triazole, and (d)how Cu(II)2+ is converted to Cu0 during the reaction. The first step is thereaction of the osazone (204) with Cu(II) ions to form an osazone–Cu(II)


SCHEME 46. Formation and structure of saccharide phenylosotriazoles.


complex, such as 205, which undergoes a one-electron shift from the nitro-gen atom of the ligand to the metal in the complex. It should be noted thata monomeric complex such as 206 in Scheme 47 would achieve the same re-


SCHEME 47. Mechanism of formation of osotriazoles.


sult. In either case the intramolecular oxidation–reduction results in reduc-tion of the Cu(II) complex to a Cu(I) complex (208) and formation of a ligand radical (207). This undergoes a set of one-electron shifts to form thetriazole (209) by elimination of a phenylimine radical (Ph-NH); which isquickly converted in water to aniline.The Cu(I) complex 208 obtained fromthe dimer 205 decomposes to regenerate 1 mole of osazone and Cu(I)+,whereas the Cu(I) complex from the monomer yields directly the triazole,aniline, and Cu(I)+. The Cu(I)+ produced in either way is converted toCu(II)2+ and Cu0 by disproportionation. This is a well-known reaction ofCu(I) ions,399 which undergo disproportionation upon boiling in acidifiedwater to give Cu(II)2+ and Cu0. It should be noted that the exact amount ofcopper precipitated depends of the ratio of at least two competing reac-tions, triazole formation and hydrolysis to the glycosulose.400 The role ofCu(II) complexes in triazole formation is to facilitate the oxidation bybringing the electron-rich part of the ligand (nitrogen) next to the oxidant.Stronger oxidizing agents, such as chlorine or bromine in water, can directlyattack the hydrogen-bonded chelated ring of osazones and do not requiremetal-complex intermediates.

The oxidation of labeled osotriazoles has been used to determine the po-sition of 14C-label in aldoses401 and to ascertain that the unchelated anilineof the C-1 phenylhydrazone residue (and not that of C-2) is eliminated dur-ing triazole formation. Similar results were obtained by using 82Br-labeledD-arabino-hexulose p-bromophenylosazone,291 mixed arylosazones,397 and1-phenylazo-osotriazoles.402 The nitrogen atom of the triazole ring activatesthe phenyl ring by resonance and electrophilic substitution occurs mainly inthe para position393,394 because the ortho positions are crowded.

Correlations have been made between the configuration of the hydroxylgroup attached to C-3 of sugar osotriazoles and the sign of their optical rota-tion403,404 and the sign of their Cotton effect.405,406 Comparative NMR stud-ies of osotriazoles as a function of the configuration of the side chain demon-strated that the chain adopts a “sickle” conformation such as 210 if the planarzig-zag arrangement of carbon atoms would have given rise to a paralled 1,3-interaction between hydroxyl37 or acetoxyl groups (Scheme 48).38


SCHEME 48. The sickle conformation of saccharide osotriazoles.


Saccharide 1-benzamidotriazole enolbenzoates (212) have been obtainedby the free-radical oxidation of bis(benzoylhydrazone) acetates (211), withiodine and hydrolysis of the ester groups of the resulting 1-a-benzoyloxy-benzylideneamino-1,2,3-triazoles (213).407,408 The structure of these tri-azoles was determined by periodate oxidation, which gave 1-benzamido-4-formyl-1,2,3-triazole (214), and by NMR spectroscopy (Scheme 49).

f. Reduction of Bis(phenylhydrazones).—Fischer409 obtained 1-amino-1-deoxy-D-fructose (215) by reduction of D-arabino-hexulose phenylosa-zone (216) with zinc and acetic acid and called it “iso-D-glucosamine.” Theyield of this product was increased when hydrogenation over a palladium-on-carbon catalyst was used.310,410

When D-arabino-hexulose phenylosazone (216) was reduced over Raneynickel in 2M alcoholic potassium hydroxide (added to open the chelatedring), the reduction proceeded to the alditol stage and yielded 1,2-diamino-1,2-dideoxy-D-mannitol and -D-glucitol (217 and 218).249,411,412 Similar re-ductions have been carried out on disaccharide phenylosazones244,393,394, 413

and dehydroascorbic acid osazone (Scheme 50).414,415

g. Formation of Anhydroosazones.—The formation of anhydroosazonesis initiated by an elimination reaction, followed by a cyclization. The latterinvolves nucleophilic attack by a chain oxygen or a suitably locatedhydrazone nitrogen atom. Three types of anhydroosazones have beenisolated, namely (i) monocyclic 3,6-anhydroosazones, (ii) monocyclicdianhydroosazones, and (iii) bicyclic dianhydroosazones. All three types ofanhydroosazone are formed from a common intermediate, a 2-phenylazo-1-phenylhydrazono-1-alkene (220).168 These intermediates are similar to


SCHEME 49. Formation and reactions of 1-a-benzoyloxybenzylideneamino-1,2,3-triazoles.


the azoalkenes formed from hydrazones. It is postulated that, in thepresence of acids or bases, such osazones as 219 undergo conjugateelimination to afford 2-phenylazo-1-phenylhydrazono-1-alkenes (220). Inthe presence of acids the reaction is initiated by protonation of the OHgroup attached to C-3 to facilitate the elimination of water and in basicmedia by ionization of the imino protons to shift electrons toward theleaving group (Scheme 51).

(i) Formation of 3,6-Anhydroosazones.—These compounds were firstdiscovered by Fischer286, 416 and were later obtained by boiling osazones inmethanol containing some sulfuric acid as catalyst.417 Their structure (forexample, compound 223) was studied by many investigators,162,418 and theywere finally shown to be 3,6-anhydrohexulose phenylosazones.419–421

Mechanistically, they are formed by nucleophilic attack of O-6 of the sugarchain on the double bond at C-2 of 2-phenylazo-1-phenylhydrazono-1-alkenes (221). During the formation of 3,6-anhydroosazones, two 3-epimers


SCHEME 50. Reduction of osazones.

SCHEME 51. Formation of azoalkene intermediates.



are produced. The major product usually has the hydroxyl groups at C-3and C-4 cis to each other, and the minor product has these hydroxyl groupstrans oriented.388 However in the case of heptulose osazones,422–424 thisrule is not so strictly observed. 3,6-Anhydroosazones are also formed whenosazones are boiled with acetic anhydride169,425–427; the products in thiscase are accompanied by dianhydroosazones having pyrazole rings such as,for example, compound 225, discussed in the next section (Scheme 52).

(ii) Dianhydroosazones of the Pyrazole Type.—These dianhydroosa-zones are obtained by boiling saccharide phenylosazones in aceticanhydride. They are formed from the same intermediate, 2-phenylazo-1-phenylhydrazono-1-alkenes (224), by migration of the double bond,followed by nucleophilic attack by nitrogen to give an acetoxy-pyrazoline(226), which is aromatized by elimination of the acetoxy group to yield thepyrazole (228). The structure of this type of dianhydroosazone (228) wasestablished by degradation, including oxidation to a known pyrazoledi-carboxylic acid (227), and by NMR spectroscopy.219,312, 425–427 An analog ofpyrazole was obtained by heating 3,4,5-tri-O-acetyl-pentulose phenylosa-zones with pyridine (Scheme 53).169,427

(iii) Bicyclic Dianhydro-Osazones.—This third type of anhydroosazonewas first prepared by Percival when deacetylating a hexulose phenyl-osazone tetraacetate with sodium hydroxide. Two enantiomeric compoundsare otainable from hexose precursors, one from D-hexoses and the other fromL-hexoses.166 Tricyclic structures were first assigned to these compounds, butwere later revised.428 Finally, NMR data provided evidence for a chelatedbicyclic structure 231 which possesses one imino proton.429 Thesedianhydroosazones are formed from the same intermediate, 2-phenylazo-1-phenylhydrazono-1-alkenes (229) by conjugate elimination to give 230,followed by nucleophilic attack of O-6 to afford the product 231 (Scheme 54).

SCHEME 52. Formation of 3,6-anhydroosazones.


h. Derivatives—(i) Esters.—The fully acetylated and benzoylated sugarphenylosazones have acyclic structures. Mild acetylation of D-arabino- andD-lyxo-hexulose phenylosazones leads to tetra-O-acetyl derivatives(232).218,294,313 Stronger acetylating agents, such as acetyl chloride, yield a 1-N-acetyl-tetra-O-acetyl derivative (234).313 To acetylate the chelated


SCHEME 53. Dianhydroosazone of the pyrazole type.

SCHEME 54. Formation of Percival’s dianhydroosazonee.


NH group on C-2, the acetylation requires the presence of Lewis acids(Scheme 55).215,216

Benzoylation of hexulose osazones with benzoyl chloride in pyridine af-fords crystalline pentabenzoates312,326 which possess one N-benzoyl andfour O-benzoyl groups.

(ii) Ethers.—Methylation of D-arabino-hexulose phenylosazone (174)under mild conditions yields preferentially the 6-methyl ether.430,431

Vigorous methylation leads to a mixture of methylated products. Mixed 1-N-methyl-N-phenyl-2-phenylosazones are obtained either by directmethylation of the unchelated nitrogen432,433 or by transhydrazonation ofbis(N-methyl-N-phenyl)osazones with phenylhydrazine.434 Two mono-N-methyl derivatives of D-arabino-hexulose phenylosazone, designated mixedosazones A and B, have been described in the literature. Mixed osazone Awas later identified as 1-(N-methyl-N-phenyl)-2-phenylosazone, whereasmixed osazone B proved to be a mixture of compound A and D-arabino-hexulose phenylosazone.157,317,320

When treated with acetone in the presence of an acidic catalyst, the os-azone 235 yields a 5,6-isopropylidene acetal (236), which is converted intothe O-isoprophylidene-N-methyl-di-O-methyl derivative (237) on methyla-tion with dimethyl sulfate.432 These reactions also provide evidence in favorof the acyclic structure of the sugar osazones. (Scheme 56).

4. Saccharide Poly(hydrazones)

If the assumption is correct that the chelated ring in osazones pre-vents the osazone-forming reaction from proceeding beyond C-2, it wouldbe expected that osazones incapable of forming such chelated rings would undergo an extended reaction which ultimately would involve the whole poly(hydroxyalkyl) chain. This expectation was realized in the


SCHEME 55. O-Acetylated and O,N-acetylated osazones.


case of the N-methyl-N-phenylosazones, which are incapable of formingchelated rings. Treating trioses, tetroses, and pentoses with N-methyl-N-phenylhydrazine afforded tris-, tetrakis-, and pentakis-hydrazones. The re-sulting polyhydrazones (238) were given the generic name of alkazones(Scheme 57).435,436

Triose tris(hydrazones) may also be obtained from periodate-oxidizedsaccharide bis(hydrazones)313,365 (239) by treating the resulting mesoxalde-hyde bis(benzoylhydrazone) (240) with benzoylhydrazine. Mesoxalalde-hyde tris(benzoylhydrazone) (241) was found to react with iodine in a man-ner similar to that of hexosulose bis(benzoylhydrazones) to give a triazolederivative408 242 (Scheme 58).

Alkazones are very reactive compounds; they readily cyclize and aroma-tize. This renders alkazones of hexoses and higher sugars difficult to isolatefrom the dark products they form. Other highly reactive species of bis(hy-drazones) are the derivatives of mesoxaldehyde and of ascorbic acid, which


SCHEME 56. Isopropylidene derivatives of osazones.

SCHEME 57. Structure of an alkazone.



contain additional carbonyl groups. Most monosaccharides contain morenucleophiles than nucleophile acceptors (they contain several OH groupsand only one CO group at the anomeric carbon). The reactivity of theirderivatives is greatly enhanced by the presence of additional CC, CN, orCO groups to which nucleophiles can add.An empirical rule has been de-veloped398 to quantitate the reactivity of polyfunctional molecules such assugars and measure their susceptibility to cyclize by nucleophilic addition.According to this rule, sugars and their oximes and hydrazones have a nu-cleophilicity quotient of about 1, the (more reactive) osazones and bis(hy-drazones) have a quotient of 2, and alkazones and ascorbic acid bis(hydra-zones), which are still more reactive, have a quotient of about 3. This rulewould predict that esters of phenylosazones and bis(benzoylhydrazones)should exhibit higher reactivities because of the additional CO groupsthat they contain. This is indeed the case; the osazone acetates form twotypes of aromatized dianhydroosazones (see Schemes 53 and 54) whichcannot be formed without acetylation, and benzoylhydrazones show manyreactions not given by osazones (see Schemes 49 and 58).

V. HYDRAZONES OF CARBA-SUGARS* AND RELATED COMPOUNDS

1. Importance of Carba-Sugars

Carbocyclic analogs of monosaccharides in which the ring-oxygen atomis replaced by a methylene group were first synthesized by McCasland andco-workers437 and named “pseudo-sugars”; today, they are termed “carba-sugars.”* Some members of this important group of compounds occur naturally,438–440 others inhibit enzymes441,442 or possess antibacterial properties (for example, carba-a-D-galactopyranose).443 Interest in the use of carba-sugars as artificial sweeteners444,445 led to the synthesis of

SCHEME 58. Formation and reactions of mesoxaldehyde tris(hydrazones).

* IUPAC-IUBMB, Nomenclature of Carbohydrates, Adv. Carbohydr. Chem. Biochem., 52(1997) 43–177; see also Nomenclature of Cyclitols, Eur. J. Biochem., 57 (1975) 1–7.


carba-b-D-fructopyranose. At first, only racemic mixtures were synthe-sized,440 but later the synthesis of enantiomerically pure carba-sugars wasachieved.442,445–448 Carba-b-D-fructopyranose was synthesized from achemically resolved Diels–Alder adduct of furan445 and carba-a-D-mannopyranose from L-(2)-quebrachitol (1-L-2-O-methyl-chiro-inosi-tol).449 (2)-Quinic acid was converted into optically active carba-b-D-fructopyranose and carba-b-D-mannopyranose,450 and the synthesis ofcarba-a-D-glucopyranose and carba-a-D-mannopyranose has been re-ported.451 Keto intermediates derived from L-(2) quebrachitol were uti-lized to synthesize an antifungal metabolite, (2)-isoavenaciolide,452 and re-lated products,453–456 and the first total synthesis of simondsin, acyanoglucoside, was achieved.454 A free-radical cyclization of sugar deriva-tives to chiral aminodeoxy carba-sugars has been described,457 and valio-lamine (an aminodeoxy carba-sugar that is a strong a-D-glucosidase in-hibitor) has also been synthesized.458

Synthesis of (1)-pinitol, a natural product that possesses hypoglycemicactivity,459 and of related compounds was achieved via microbial oxidationof benzene460 or halobenzenes461 with Pseudomonas putida and other mi-croorganisms.461–464 Azidoinososes, intermediates for the sythesis of amino-glycoside antibiotics, have also been synthesized.465,466 A method intro-duced by Ferrier,467 involving mercury salt-mediated ring transformationsof 6-deoxy-5-enopyranosides into deoxyinososes, has been used in the syn-thesis of cyclitols, aminocyclitols,468,469 and carba-sugars470,471 as well asenantiomerically pure inositols and inososes.472–482

2. Formation of Carba-Sugar Hydrazones

a. Preparation and Uses of Phenylhydrazones.—The chemistry ofcarba-sugars is quite similar to that of cyclitols. Both groups lack thelatent carbonyl groups of their saccharide counterparts and therefore donot exhibit many of the reactions characteristic of monosaccharides. Thus,carba-sugars and cyclitols do not form hydrazones or osazones whentreated with hydrazines,439 nor do they mutarotate or reduce heavy-metalsalts in base. Their hydrazones are prepared from hydroxycyclohexanones(inososes, ketocyclitols) by procedures analogous to those used for thepreparation of saccharide hydrazones. The hydrazones of inososes,483–488

like those of monosaccharides,47,130,155,156 may be used to isolate andpurify substrates. For example, 2,4,6/3,5-pentahydroxycyclohexanone(myo-inosose-2) is purified by treatment with phenylhydrazine,recrystallization of the resulting phenylhydrazone, and regeneration ofthe inosose with benzaldehyde489 or with a sulfonic acid-type cation-exchange resin.490 Table I lists some protected inososes and hydro-xycyclohexanones that have been converted into phenylhydrazones toproduce carba-sugars.



TA

BL

EI

Ket

o-C

yclo

hexa

ne D

eriv

ativ

es P

urifi

ed b

y C

onve

rsio

n to

Hyd

razo

nes

and

Use

d to

Pre

pare

Car

ba-S

ugar

s

Ket

o-C

yclo

hexa

neC

arba

-Sug

arR

efer

ence

Ket

o-C

yclo

hexa

neC

arba

-Sug

arR

efer

ence

439

449

449

447

439,

440

439,

440

438

carb

a-a

DL

-tal

opyr

anos

e

437

carb

a-a

-D-m

anno

pyra

nose

carb

a-b

-DL

-ga

lact

opyr

-an

ose

carb

a-a

- D-g

alac

topy

rano

se

carb

a-a

-D-a

llopy

rano

seca

rba-

b-D

-man

nopy

rano

se

carb

a-a

- DL

-al

trop

yran

ose

carb

a-b

-DL

-allo

pyra

nose

carb

a-b

-D-

gluc

opyr

anos

eca

rba-

a-L

-id

opyr

anos

e

230


447

450

439,

440

439,

448

439,

463

carb

a-a

-DL

-ga

lopy

rano

seca

rba-

a- D

-gl

ucop

yran

ose

carb

a-b

- L-i

dopy

ra-

nose

carb

a-b

-DL

-ta

lopy

rano

se

carb

a-b

-D-

man

nopy

rano

seca

rba-

b-D

-fr

ucto

pyra

nose

231



b. Formation of 1,2-Bis(phenylhydrazones).—Inosose phenylhydra-zones, such as myo-inosose-2 phenylhydrazone,491 are converted withdifficulty into bis(phenylhydrazones); for this reason, many cyclitolosazones are prepared directly from cyclic 1,2-diketones.485,488,492 Investi-gation of the rate of formation of cyclohexane-1,2-dione bis(phenylhydra-zone) from 2-hydroxycyclohexanone phenylhydrazone and of the corre-sponding 2-methoxy-, 2-acetoxy-, and 2-chloro-derivatives revealed thatazoalkenes, formed by 1,4-elimination, are key intermediates in thereaction.299 Other studies299,493 have confirmed the formation of azoalkeneintermediates during the conversion of a-acetoxycyclohexanone and of a-substituted oxosteroids into the corresponding bis(phenylhydrazones)(Scheme 59).494

c. Formation of 1,3-Bis- and 1,2,3-Tris(phenylhydrazones).—Reaction ofcyclohexane-1,3-dione (251–252) with phenylhydrazine yields cyclohexane-trione bis- and tris-(phenylhydrazones). The formation of bis(phenylhydra-zone) 256 and tris(phenylhydrazone) 257 proceeds by an ionic mechanism(see Scheme 60), whereas that of cyclohexanetrione bis(phenylhydrazone)(263–264) from the enol form of 1,3-cyclohexanedione (259) involves free-radical intermediates (see Scheme 61). The formation of free radicals in thefirst reaction became evident when a solution containing the cyclohexane-1,3-dione (251–252) and phenylhydrazine revealed a five-line ESRspectrum.495,496 The presence of a paramagnetic species, identified as a

SCHEME 59. Formation of inosose 1,2-bis(phenylhydrazones).



SCHEME 60. Formation of 1,2,3-tris(phenylhydrazones) by an ionic mechanism.

(phenylhydrazone) structure, for instance 265, and the red one has anenolic phenylhydrazono-phenylazo structure (266 and 267). The deep red color of 2-oxo-1,3-bis(phenylhydrazones) and the strong f → f*

absorptions they display suggest that they have conjugated phenyl-hydrazono-phenylazo groups, similar to the diphenylformazans of sugars.223,240 The latter were studied by 15N NMR on 15N-labeledformazans,527 which confirmed the formazan ring structure.528 Themarked similarity in the absorption spectra of the 2-oxo-1,3-bis(phenylhydrazones) and of formazans clearly supports thephenylhydrazono-enol-phenylazo structures (266–267) assigned to thesecompounds. This was confirmed by 1H NMR studies, which reveal thepresence of both chelated and nonchelated imino protons in thetautomeric forms 265–267 (Scheme 62).526

An analogous structure for 2-oxo-1,3-bis(phenylhydrazono)cyclopen-tane (268) was likewise based on spectroscopic evidence.526 Quantum-mechanical calculation (HMO) of the bonding energies of various tau-tomers (such as 268, 269, and 270) indicates505 that the most stable tautomeric structure is the chelated bis(phenylhydrazone) 269. It seemsthat interconversion of the tautomers 268, 269, and 270 occurs in polar sol-vents and that the dichelated structure 271 is preponderant in the solid stateor in nonpolar solvents (Scheme 63).526

b. Structure of Tris(phenylhydrazones).—Tris(phenylhydrazones) re-semble 2-oxo-1,3-bis(phenylhydrazones) in possessing chelated ring struc-tures. The NMR spectra of 1,2,3-tris(phenylhydrazono)cyclopentane (272),1,2,3-tris(phenylhydrazono)cyclohexane (273), and 2,3,4-tris(phenylhydra-zono) cyclohexanecarboxylic acid (274) all show the presence of twochelated rings. Similarly, the NMR spectra of the tris(phenylhydra-zono)propane (275–276) show no low-field NMR signals immediately after


SCHEME 62. Chelation and resonance forms of enolic, phenylhydrazono-phenylazo struc-tures of cyclohexanetrione bis(phenylhydrazones).


dissolution, but only after a few minutes of equilibration.526 These resultswere later substantiated (Scheme 64).365

4. Reactions of Carba-Sugar Hydrazones

a. Action of Acids and Bases—(i) Protonation and Formation ofAnions.—Protonation of 2-oxo-1,3-bis(phenylhydrazones), diphenylform-azans, and 1,2-bis(phenylazo)ethene produces purple, blue, or green


SCHEME 63. Chelation and resonance forms of cyclopentanetrione bis(phenylhydrazones).

SCHEME 64. Chelated structures of tris(phenylhydrazones).


cations.230 On the other hand, cyclitol phenylosazones and bis(phenyl-hydrazones) that cannot form resonance-stabilized cations on protonationdo not usually give a blue coloration. For example, when perchloric acid isadded to a solution of 2-oxo-1,3-bis(phenylhydrazono)cyclohexane inacetic acid, a protonated, blue, crystalline salt is obtained.230 The observedbathochromic shift and the intense color were found to arise from theformation of a conjugated, resonance-stabilized cation having structure277. Electron-spin resonance measurements of solutions of colored 2-oxo-1,3-bis(phenylhydrazones) and diphenylformazans do not show thepresence of radical species, thus indicating the ionic character of theproducts formed on protonation.

Treatment of 1-phenylhydrazino-3-phenylazo-2-cyclohexene with astrong base also produces a blue coloration. The amphoteric character ofthis compound and of other formazan vinylogs has been ascribed502 to theformation of such resonance-stabilized anions as 278a,b. The absorptionspectrum of this anion resembles the spectrum of the resonance-stabilizedcation of 2-oxo-1,3-bis(phenylhydrazones), indicating some amphotericcharacter in these compounds (Scheme 65).

(ii) Formation of Stable Free-Radicals.—The ESR probe can be used to distinguish between the hydrazine derivatives of saccharides andnonsaccharides. Saccharide phenylhydrazones, which are much less stablein basic media than inosose phenylhydrazones, exhibit 3-line ESR patterns


SCHEME 65. Resonance-stabilized structures of 2-oxo-1,3-bis(phenylhydrazono)cyclo-hexane in acid and in base.


characteristic of nitroxide radicals, produced by oxidation of thephenylhydrazine moiety generated by hydrolysis. On the other hand,inosose phenylhydrazones and, in particular, their esters, exhibit well-defined spectra. For example, 2,4,5,6/3-pentahydroxycyclohexanonephenylhydrazone pentapropanoate (DL-epi-inosose-2 phenylhydrazonepentapropanoate) (280c), shows a 30-line ESR spectrum.157,353 The hyper-fine structure observed in the spectrum of 280c has been attributed to thegreat stability of this inosose phenylhydrazone ester toward alkali.

Elimination (Formation of Phenylazo-Cycloalkenes). Eliminations, sim-ilar to the Wolfrom reaction of acylated saccharide phenylhydrazones, havebeen observed with some, but not all, inosose phenylhydrazone acetates.Thus,acetylation of 2,4,6/3,5-pentahydroxycyclohexanone phenylhydrazone (myo-inosose-2 phenylhydrazone) (279a) with pyridine–acetic anhydride at 10–158Cyields a pale yellow elimination product, namely 6-phenylazo-5-cyclohexene-DL-ido-1,2,3,4-tetrol tetraacetate (281a). In contrast, treatment of the samephenylhydrazone with propanoic anhydride and pyridine gives the pen-tapropanoate 279c without elimination. Similarly, treatment of 2,4,5,6/3-pentahydroxycyclohexanone phenylhydrazone (DL-epi-inosose-2 phenylhydra-zone, (280a) with acetic or propanoic anhydride gives pentaacetate 280b orpropanoate 280c without elimination. However, if the reaction temperaturefor 279a or 280a is raised, elimination occurs, with formation of aromatic azocompounds. The major factors influencing the course of the reaction seem tobe (i) thermodynamic stability, (ii) the nature of the acyl group (acetyl,propanonyl, benzoyl, and the like), and (iii) temperature (Scheme 66).529

b. Nucleophilic Substitution.—When a leaving group present in the a-position relative to a hydrazone residue undergoes elimination, theresulting azo-cycloalkene is able to add a nucleophile in the same position.Thus if a 2-benzoyloxyinosose p-nitrophenylhydrazone is treated with azideanions, the benzoyloxy group is replaced by an azide group.207,472–474 Forexample, when 2L-(2,4,5/3)-4-benzamido-2,3-dibenzoyloxy-5-hydroxycy-


SCHEME 66. Formation of phenylazo-cycloalkenes.


clohexanone (283a), prepared from 282 via a Ferrier transformation, istreated with p-nitrophenylhydrazine it gives a p-nitrophenylhydrazone(283b);475,476 this compound, upon treatment with sodium azide, affords theazide 286. An analogous product is obtained from the corresponding oxime(283c) by treatment with tetrabutylammonium azide. The product in thiscase is a mixture of the epimers of 2-azido-4-benzamido-3-benzoyloxy-5-hydroxycyclohexanone (E)-oxime (284 and 285), which are convenientlyseparable by column chromatography.475 In this situation, the hydroximinogroup shows477,478 an activating effect similar to that of the arylhydrazonegroup, and affords a nitroso-cycloakene intermediate. Preparation ofazidoinososes from derivatives 284–286 may be achieved by use of a cation-exchange resin490 or by acid hydrolysis (Scheme 67).


SCHEME 67. Nucleophilic substitution.


c. Aromatization—(i) Aromatization of the Cyclohexane Ring.—Thebase-catalyzed acetylation of 279a shown in Scheme 66 is accompanied byelimination to give an arylazocyclohexene derivative (281a).529 However,the thermodynamically less stable 4,6/5-trihydroxy-1,3-bis(phenylhydra-zono)cyclohexanone (287), shown in Scheme 68, upon similar treatmentundergoes complete aromatization at ambient temperature to give asubstituted benzene (291).530 Compound 287 is prepared from 4,6/5-trihydroxy-1,2,3-cyclohexanetrione by treatment with phenylhydrazine atroom temperature.531 The formation of 291 from 287 probably proceeds viaan ionic pathway that involves (i) acetylation of the hydroxyl groups to give288, (ii) ionization of the imino hydrogen atom with base and formation


SCHEME 68. Aromatization of the cyclohexane ring.


of an enol (289), (iii) sequential cleavage of the acetoxy group to give 290, and, finally, (iv) aromatization with acetic anhydride to give the pro-duct 291.

(ii) Aromatization of the Hydrazone Residues, Formation of Phenyl-osotriazoles.—A number of cyclitol phenylosotriazoles have been preparedfrom the corresponding bis(phenylhydrazones). For example, 1D (292a), 1-L-chiro (293), and DL-inositol phenylosotriazoles have been prepared532

from the corresponding inosose phenylosazones492 by using mercuric ace-tate as the oxidant.532

Unlike sugar phenylosotriazoles which possess a flexible side chain,130

some cyclitol phenylosotriazoles possess symmetrical structures, as indi-cated by the simplicity of their proton-decoupled 13C NMR spectra.392,532

Thus the 1H NMR spectra of inositol phenylosotriazoles, 292a, and their es-ters revealed the presence of a simple, twofold axis of symmetry and thering protons were symmetrical about a midpoint (see 294), making them ex-amples of four-nucleus AA9BB9 systems.

In solution, the favored conformation for the osotriazole tetra-isobu-tanoate 292c is 5H4, as depicted in structure 295. A small coupling-constantfor H-3–H-6 is attributed to the influence of the neighboring planar oso-triazole ring and is consistent with a half-chair conformation.392,532 TheNMR spectra of alkyl-substituted inositol phenylosotriazoles [from D-(1)-pinitol (1-D-3-O-methyl-chiro-inositol, 296a) or L-(2)-quebrachitol (1L-2-O-methyl-chiro-inositol, 297a)] or those from (1)-quercitol [(1)-proto-quercitol (1 D-1,3,4/2,5-cyclohexanepentol), 298a] or their acetates (296b,297b, and 298b), which are not symmetric, show the ring-proton signals aspart of an ABX system (Scheme 69).392

d. Oxidation and Reduction.—In an early investigation, Magasanik andChargaff533 showed that cyclitol osazones, for example, 1-D-chiro-inositolphenylosazone (299) consume the expected amount of periodate (threemoles), but that the product, namely, 2,3-bis(phenylhydrazono) succindial-dehyde (300), cyclized to give a pyrazole (301). Similarly, the oxidation ofsuch cyclitol phenylhydrazones533 as the 2-oxo-1,3-bis(phenylhydrazone)303 (prepared upon the reaction of 302 with phenylhydrazine),531 upontreatment with sodium periodate, yields 3-oxo-2,4-bis(phenylhydrazono)-glutaraldehyde (304), which was not isolated, but was directly convertedinto the methyl hemiacetal of 4-oxo-1-phenyl-5-(phenylazo)-3-pyrazinecar-boxaldehyde (307).225 This conversion presumably involves formation of305, followed by dehydration to aldehyde 306 and reaction with methanolto give 307 (Scheme 70).

The oxidation of 308 by periodic acid provides a synthetic route to




SCHEME 69. Aromatization of the hydrazone residues, formation of phenylosotriazoles.



SCHEME 70. Periodate oxidation.


interesting pyrazine derivatives. However, reduction with sodium amalgamin ethanol gives534 streptamine (309), a degradation product of the antibi-otic streptomycin. The synthesis of 309 offers an additional proof of thestructure of 308. When 308 is reduced catalytically,534 the product is the an-tibiotic actinamine 310. The exclusive formation of a 1,3-diamine (310),with two equatorial amino groups, by catalytic hydrogenation over plat-inum is noteworthy because phenylhydrazones of inososes535,536 or hex-uloses537 generally form products having axial amino groups.The differencemay be due to the fact that compound 308 exists514 mainly in the phenyl-hydrazono-phenylazo form and not the bis(phenylhydrazono) form526 orthat the phenyl group plays a directing role during catalytic hydrogena-tion.534 The rotation of the phenyl group in the chair conformers has beenthe subject of numerous studies (Scheme 71).538–540

VI. CONCLUSION

The study of the hydrazine derivatives of sugars has played an importantrole in the development of carbohydrate chemistry since its inception.Phenylhydrazine, first described in Emil Fischer’s doctoral thesis, was al-


SCHEME 71. Reduction of 2-oxo-1,3-bis(phenylhydrazones).


lowed to react with sugars to form hydrazones and osazones. The lattercompounds proved invaluable in establishing the configuration of D-glucose, which won Fischer the Nobel Prize in 1902, and in the synthesis ofL-ascorbic acid, which won Sir Norman Haworth the prize in 1937. Hydra-zones and osazones have been extensively used in analysis to separate andcharacterize mono- and disaccharides and in the preparation of new sugarssuch as glyculoses (ketoses), aldos-2-uloses (“osones”), and amino- andimino-deoxy sugars as well as countless aromatic nitrogen heterocycles.

As in other fields of chemistry, research in the area of carbohydrates hasbenefited from the advent of such instrumental methods of structure eluci-dation, as NMR, MS, and X-ray crystallography. The impact of these toolscan be realized by considering the time it took chemists to determine thestructure of two hydrazine derivatives of about equal complexity, beforeand after the advent of NMR spectroscopy: it took 65 years (from 1887 to1952) and the efforts of four of the best chemists to prove by classic meth-ods that the anhydro derivative obtained in acid media from what was thencalled “glucosazone” was 3,6-anhydro-D-ribo-hexulose phenylosazone and only a few months in 1990 to determine that the oxidation products obtained from saccharide phenylhydrazones in basic media were in fact N-phenylaldonohydrazono-1,4-lactones (see schemes 52 and 11, respec-tively).

In this chapter an attempt has been made to present an account of therich chemistry of the hydrazine derivatives of sugars, the versatility oftheir structures, and their availability, which makes them valuable enan-tiomerically pure synthons for chiral products. For example, reduction ofaldose hydrazones affords an important class of chiral amino- and imino-deoxy sugars which contain nitrogen in place of the oxygen present in therings of natural sugars. These imino sugars exhibit a wide spectrum of bi-ological activities, mainly attributable to their ability to act as enzyme in-hibitors. Also of considerable interest are the carba-sugars, the carbo-cyclic analogs of monosaccharides, which are readily available frominosose phenylhydrazones. Members of both these classes of compounds,the carba-sugars and the imino sugars (“aza sugars”) are capable of in-hibiting enzymes because they mimic the enzyme’s natural substrates(the sugars). For the same reason they, and other members of bothgroups, often exhibit antibacterial, antiviral, or antitumor properties. It isnot surprising, therefore, to find that the study of these compounds is at-tracting the interest of numerous researchers from the field of carbohy-drate chemistry as well as from other disciplines. This area of research isunder rapid development and is producing an ever-increasing number ofpapers that enrich the chemical literature. Another area currently at-tracting great interest is the enantioselective synthesis of a-aminoalde-hydes and b-lactam intermediates, obtained by reduction or conjugatereduction of hydrazine derivatives.




In this chapter on the hydrazine derivatives of sugars and related com-pounds, an attempt has been made to offer more than a review of what hasalready been achieved by focusing on reactions that have not yet been triedon carbohydrate substrates (examples of these are given in Sections III.3and IV.4). In summary, a serious effort has been made to point to novel ap-proaches for the synthesis of, and new applications for, the reactions of sac-charide hydrazones and their carbocyclic analogs.

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(1988) 227–239.(480) S. L. Bender and R. J. Budhu, J. Am. Chem. Soc., 113 (1991) 9883–9884.(481) V. A. Estevez and G. D. Prestwich, J. Am. Chem. Soc., 113 (1991) 9885–9887.(482) S. K. Chung and S.-H. Moon, J. Chem. Soc. Chem. Commun. (1992) 77–79.(483) H. G. Fletcher, Jr., Adv. Carbohydr. Chem., 3 (1948) 45–77.(484) S. J. Angyal and L. Anderson, Adv. Carbohydr. Chem., 14 (1959) 135–212.(485) T. Posternak, The Cyclitols (Fr. ed.), Herman, Paris, 1962; Engl. ed. (updated), Herman,

Paris, and Holden-Day, San Francisco, California, 1965.(486) L. Anderson, The Carbohydrates, Chemistry, and Biochemistry, Vol. 1A, W. Pigman and

D. Horton (Eds.), Academic Press, New York, 1972, Chapter 15, 519–579.(487) T. Hudlicky and M. Cebulak, Cyclitols and Derivatives, VCH, New York, 1993.(488) B. Magasanik and E. Chargaff, J. Biol. Chem., 174 (1948) 173–188.(489) T. Posternak, Biochem. Prep., 2 (1952) 57–64.(490) A. J. Fatiadi, Carbohydr. Res., 1 (1966) 489–491.(491) H. E. Carter, C. Belinskey, R. K. Clark, Jr., E. H. Flynn, B. Lyttle, G. E. McCasland, and

M. Robins, J. Biol. Chem., 174 (1948) 415–426.(492) A. J. Fatiadi, Carbohydr. Res., 8 (1968) 135–147.(493) L. Caglioti, G. Rosini, and F. Rossi, J. Am. Chem. Soc., 88 (1966) 3865–3866; L. Caglioti

and A. G. Giumanini, Bull. Chem. Soc. Jpn., 44 (1971) 1048–1050; A. G. Giumanini, L.Caglioti, and W. Nardini, Bull. Chem. Soc. Jpn., 46 (1973) 3319–3320.

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147–335.(502) B. Eistert, G. Kilpper, and J. Goring, Chem. Ber., 102 (1969) 1379–1396.(503) E. S. Gould, Mechanism, and Structure in Organic Chemistry, Henry Holt, New York,

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(510) A. A. Frime. (Ed.), Singlet Oxygen Reaction Modes and Products, Vol. II, Part 1, CRCPress, Boca Raton, FL, 1985.

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(1982) 1–9.(526) A. J. Fatiadi and H. S. Isbell, Carbohydr. Res., 5 (1967) 302–319.(527) R. P. Larsen and L. E. Ross, Anal. Chem., 31 (1959) 176–178; E. D. Marshall and R. R.

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CARBOHYD 2.(531) A. J. Fatiadi and H. S. Isbell, J. Res. Natl. Bur. Std., 68A (1964) 287–299.(532) A. J. Fatiadi, Carbohydr. Res., 20 (1971) 179–184.(533) B. Magasanik and E. Chargaff, J. Am. Chem. Soc., 70 (1948) 1928–1929; see also

B. Magasanik, in Essays in Biochemistry, S. Graff (Ed.), Wiley, New York, 1956, 180–190.(534) F. W. Lichtenthaler, H. Leiner, and T. Suami, Chem. Ber., 100 (1967) 2383–2388.(535) L. Anderson and H. A. Lardy, J. Am. Chem. Soc., 72 (1950) 3141–3147.(536) T. Posternak, Helv. Chim. Acta, 33 (1950) 1597–1604.(537) C. F. J. Chittenden and R. D. Guthrie, J. Chem. Soc. (1966) 1508–1510.(538) M. Oki, Top. Stereochem., 14 (1983) 1–81; M. Oki, Applications of Dynamic NMR Spec-

troscopy to Organic Chemistry, VCH, Deerfield Beach, FL., 1985.(539) C. Jaime, M. Rubiralta, M. Feliz, and E. Giralt, J. Org. Chem., 51 (1986) 3951–3955; D.

Kost, K. Aviram, and M. Raban, J. Org. Chem., 54 (1989) 4903–4908.(540) For more on conformational and steric factors, see J. March, Advanced Organic Chem-

istry, 4th Edition, Wiley, New York, 1992, pp. 143, 275; E. Juaristi, Introduction to Stereo-chemistry and Conformational Analysis, Wiley, New York, 1991, Chapter 3.



A FRESH UNDERSTANDING OF THE STEREOCHEMICALBEHAVIOR OF GLYCOSYLASES: STRUCTURAL DISTINCTION

OF “INVERTING” (2-MCO-TYPE) VERSUS “RETAINING” (1-MCO-TYPE) ENZYMES

BY EDWARD J. HEHRE

Department of Microbiology and Immmunology, Albert Einstein College of Medicine,New York, USA

III. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265III. The Extraordinary Catalytic Abilities of Glycosylases. . . . . . . . . . . . . . . . . . . . . . . 267

1. Use of Minisubstrates of Forbidden Configuration . . . . . . . . . . . . . . . . . . . . . . . 2682. Nonretaining Reactions Catalyzed by Retaining Enzymes, Noninverting

Reactions by Inverting Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2693. Glycosyl Transfer by “Inverting” Glycosidases . . . . . . . . . . . . . . . . . . . . . . . . . . 272

III. X-Ray Findings That Support Catalytic Group Versatility and Identify the Structures Controlling Stereochemical Outcome . . . . . . . . . . . . . . . . . . . . . . . . . . . 2741. Residues Potentially Able to Protonate Minisubstrates of “Improper”

Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2742. Structures That Limit the Stereochemical Outcome in Glycosylase-

Catalyzed Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2763. Structural Basis for Separating Glycosylases into 1-MCO (“Retaining”)

and 2-MCO (“Inverting”) Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286IV. Relation of Stereochemical Behavior to Catalytic Mechanism . . . . . . . . . . . . . . . . 296

1. Do 1-MCO (“Retaining”) Glycosylases Invariably Act via Double Displacements? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296

2. Relationship of Transition-State Structure to the Stereochemistry and Mechanism of Glycosylase Reactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298

3. Structures That May Help Keep Solvent from the Catalytic Center inGlycosylase–Substrate Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301

4. Unresolved Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304

I. INTRODUCTION

This is a time of an unprecedented merging of different broad streams ofnovel findings that are reshaping the traditional understanding of the re-lation of protein structure to the stereochemical behavior and catalytic

265


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workings of glycosylases. Reported crystallographic studies reveal the over-all active-site geometry of some 30 glycosyl mobilizing enzymes and iden-tify, with differing degrees of certainty, the nature and disposition of com-ponents deemed important to the catalytic process.1–3 Gene and amino acidsequencing with analysis for homology4–6 has allowed the assembly ofmany glycosidase families whose members share structural features and thestereochemical outcome of their catalyzed reactions.5 Again, computationaltreatment of multiple kinetic isotope-effect data has allowed the derivationof highly refined transition-state structures for a number of hydrolytic re-actions.7 Finally, studies of reactions promoted with small glycosyl (butnonglycosidic) substrates have yielded much evidence at odds with tradi-tional views about the catalytic scope and stereochemical behavior of glycosylases.8–10 The origins of this approach, and the ways in which re-sults obtained with it depart from long accepted assumptions, form a pref-ace to the main present theme, which is to examine the reported crystalstructures of various glycosylases in an effort to identify the structural fea-tures that underlie the stereochemical behavior of “inverting” and “retain-ing” enzymes. Such findings could be significant in terms of mechanisticfunctioning.

In the traditional view, the catalytic groups of an individual glycosylaseact always to invert (or always to retain) substrate configuration and possi-bly do so by effecting single (or double) nucleophilic displacements. How-ever, the key question of how a given enzyme’s catalytic groups “know”which way they are to function has only recently begun to be addressed ex-perimentally through (a) studies of reactions catalyzed with small nongly-cosidic substrates8–10, (b) by measurements of the average distance be-tween the catalytic carboxyl oxygens of glycosidases11, and (c) by presentobservations on the contribution of structures in addition to the catalyticgroups to the stereochemical behavior of individual enzymes.

Glycosidases and glycosyltransferases are here considered together as catalysts of glycosylation (glycosylases) despite their formal assign-ment to separate the hydrolase [EC 3.2] and transferase [EC 2.4] classes;all of their reactions are assumed to effect, by one means or another, a simple type of chemical change whereby the glycosyl moiety of a substratereplaces a proton of a cosubstrate and is itself replaced by a proton.12–14

The stoichiometry predicts something long overlooked—that a compoundmay need only to bind appropriately at an active site and to yield a glyco-syl group in exchange for a proton in order to serve as a substrate. This isabundantly confirmed; many well-known enzymes can use an appropri-ate glycosyl fluoride and/or enolic glycosyl donor as substrate (see re-views8–10,14–18).

EDWARD J. HEHRE266


II. THE EXTRAORDINARY CATALYTIC ABILITIES OF GLYCOSYLASES

Glycosyl fluorides of appropriate structure and anomeric configurationserve as substrates for diverse glycosylases, and the catalyzed reactions usu-ally are closely similar in kind and in rate to those promoted with the en-zyme’s best substrates. Yet, on occasion, large departures from this close-ness do occur. Thus alpha-amylases, which usually are thought of as lackingsignificant glycosyl transferring ability, convert a-maltosyl fluoride to maltooligsaccharides in great preference to hydrolyzing this substrate,demonstrating that alpha-amylases have a large latent potential for glyco-syl transfer.19,20 Again, glucoamylase hydrolyzes a-D-glucosyl fluoridemuch faster than it cleaves the nonreducing-end D-glucosyl group frommaltosaccharides, suggesting that release of the bulky residual saccharidemay perhaps limit the latter substrate’s rate of hydrolysis.21 Finally, cy-clodextrin glycosyltransferase, which produces cyclo-dextrins from mal-tosaccharides and starch, does so more effectively from a-maltosyl fluoridethan from maltoteraose; it also transfers all of the glucosyl units of a-maltoheptaosyl fluoride in forming the cyclic heptaose, a “total transfer” ca-pability not demonstrated with natural substrates.22 These and other com-parable observations offer clues as to possible types of structures that mayhave a role in the process of catalysis by glycosylases.

The present main focus, however, is not on reactions with anomericallycorrect glycosyl donors, but on those catalyzed with nonglycosidic sub-strates lacking proper configuration. These unusual reactions reveal aspectsof catalytic behavior long thought to be forbidden for particular types ofglycosylases.8–10

Traditional views of the catalytic scope of glycosidases and glycosyltrans-ferases arose from decades of studies of reactions with glycosidic type sub-strates (saccharides, glycosides, nucleosides, glycosyl phosphates, sugar nu-cleotides, and so on). The accumulated findings gave rise to threeconfidently accepted rules about the limits of catalytic activity, namely (a) agiven glycosidase uses substrates only of a- (or only of b-) anomeric con-figuration23, (b) a glycosidase or transferase always retains (or always in-verts) substrate configuration15,16, and (c) glycosidases which hydrolyzesubstrates with inversion never promote glycosyl transfer to compoundsother than water.10,15,24 These confidently assumed limits of catalytic scopeand steric course can be of practical usefulness when applied to reactionswith glycosidic substrates. However, the lack of generality of each of therules is evident in their failure to hold for many reactions promoted withsmall nonglycosidic substrates. Although the latter reactions are not foundin living matter and generally show a very low order of catalytic efficiency,

STEREOCHEMICAL BEHAVIOR OF GLYCOSYLASES 267


their occurrence in contradiction to long-standing belief recalls experiencesin other disciplines where unusual effects of very small magnitude have al-tered basic understanding and opened new opportunities for progress.

1. Use of Minisubstrates of Forbidden Configuration

Various glycosidases react slowly with small substrates lacking the par-ticular a- (or b-) anomeric configuration required of their glycosidic sub-strates. Some use a glycosyl fluoride whose configuration is opposite that ofthe enzyme’s natural substrates (reviewed in refs.8,9, see also25); amongthese, a- and b-glucosidase,26–28 for example, also use an enolic glycosyldonor of one or more structural type lacking the anomeric

I II III IV Vb-D-glycoside b-D-glycosyl D-glycal “D-glyco- “D-glyco-

fluoride heptenitol” octenitol”

configuration and ring conformation of glycopyranosides. Others such as a-and b-galactosidase29–35 and cellulases of several types36,37 act on appro-priate enolic glycosyl donors; still others, including a b-xylosidase38 and theamylo-(1 → 6)-glucosidase component of glycogen-debranching enzyme,39

use the wrong anomer of an appropriate glycosyl fluoride. In most cases, theforbidden substrate is protonated from a different direction and by a dif-ferent catalytic source than the enzyme’s glycosidic substrates.

How is it that such familiar enzymes as a- and b-glucosidases,26 b-galactosidase,29 beta-amylase,40 and glycogen phosphorylase41 use very smallglycosyl compounds lacking correct anomeric configuration and/or ringconformation as substrates (albeit poor ones), whereas they never act onglycosidic compounds of the wrong configuration, and only in rare cases usefuranosides?a Appropriate glycosyl fluorides (both anomers), glycals, andexocyclic enitols presumably have the potential to bind productively at an

EDWARD J. HEHRE268

a One laboratory group has reported that almond b-glucosidase hydrolyzes aryl b-D-glucofuranosides as well as the corresponding b-D-glucopyranosides42; that limpet b-glucosiduronase hydrolyzes 2-naphthyl b-D-glucofuranosiduronic acid as well as 2-naphthyl b-D-glucopyranosiduronic acid43; and that almond b-galactosidase (but not E. colior bovine liver) b-galactosidase hydrolyzes various b-D-galactofuranosides.44


enzyme’s donor site because they lack an unsuitably oriented bulky sub-stituent at C-1, in contrast to glycosides of the wrong configuration. For ex-ample, various a-glucosidases hydrolyze compounds of types II–V such asb-D-glucopyranosyl fluoride, D-glucal, 2,6-anhydro-1-deoxy-D-gluco-hept-1-enitol (“D-gluco-heptenitol”), and (Z)-3,7-anhydro-1,2-dideoxy-D-gluco-oct-2-enitol (“D-gluco-octenitol”), whereas no a-glucosidase is known toact on a compound of type I. Even the smallest of the latter, methyl b-D-glucopyranoside, carries a sizeable equatorial substituent at C-1 that pre-sumably precludes the productive binding of I to a-glucosidases by clashingwith protein residues lining the active-site cavity.

Finally, the means whereby an enzyme activates a suitably bound mini-substrate of forbidden configuration is suggested by the finding noted ear-lier that, in most cases, the protonation of such substrates involves a differ-ent catalytic source than that which protonates natural substrates. Presum-ably, an enzyme’s paired catalytic carboxyl groups, one of which acts onglycosidic substrates as general acid with the other as a base or nucleophile,would function in the reverse way—with the latter carboxyl group (un-charged) serving to protonate a bound minisubstrate of forbiddenanomeric configuration.

2. Nonretaining Reactions Catalyzed by Retaining Enzymes,Noninverting Reactions by Inverting Enzymes

Glycosylases are commonly referred to as inverting or retaining enzymesbased on whether the reaction–product configuration is opposite to or thesame as that of the enzyme’s glycosidic substrates.15 Yet, these neat charac-terizations of the two types of enzymes are not free from ambiguity. Theyimply that a given glycosylase catalyzes all reactions with inversion (or allwith retention) and that product configuration derived from that of sub-strates by the number of displacements effected by the catalytic groups isthe only way the steric course of reactions can be effected. However, glyco-sylases cannot be considered to catalyze retaining or inverting reactionswith glycals or exocyclic enolic glycosyl donors, as these substrates have noa- or b-anomeric configuration to retain or invert.

Beginning with the first NMR studies of the hydration of D-glucal pro-moted by an a- and a b-glucosidase, carried out with Fred Brewer,26 thestereochemistry of more than 25 such reactions by various “retaining” or“inverting” enzymes with different enolic substrates has been elucidated bywork in our own and other laboratories, particularly that of JochenLehmann.The latter’s syntheses of the heptenitol and octenitol analogs of D-galactal and D-glucal opened the way to stereochemical studies of a criticallywidened range of glycosylation reactions catalyzed with prochiral substrates.



Table I shows that, in every reaction examined, the hydration (or trans-fer) products formed from an enitol have the same configuration as the hy-drolytic (or transfer) products formed by the same enzyme from its morereactive chiral substrates. The conservation of stereochemical outcomeholds whether the enzyme protonates the enitol from the same or oppositedirection than its glycosidic substrates. The finding that a given glycosylasecan create, de novo, a particular product configuration from a prochiral sub-strate indicates that the stereochemical outcome of all its reactions could beunder topological control.

This possibility is further suggested by the finding that hydrolysis of a gly-cosyl fluoride of the wrong configuration is catalyzed by certain en-zymes. For example, a-glucosidases of several origins slowly hydrolyze b-D-glucosyl fluoride to form a-D-glucose,52,53 while sweet almond b-

EDWARD J. HEHRE270

TABLE I

Stereochemical Course of Reactions Catalyzed by Various “Retaining” and “Inverting”Glycosylases With Prochiral Glycosyl Donors

Prochiral ConfigurationEnzyme Donora of Product(s)b Reference

Candida tropicalis, buckwheat, D-Glucala,c aH 26,45rice, Aspergillus niger a-glucosidase

Candida tropicalis, rice a-glucosidase D-gluco-Heptenitol aH, aT 28,46Rice, Aspergillus niger a-glucosidase D-gluco-Octenitolc aH 47Sweet almond b-glucosidase D-Glucalc bH 26Sweet almond b-glucosidase D-gluco-Heptenitol bH 28Coffee bean a-galactosidase D-galacto-Octenitol aH 33Escherichia coli b-galactosidase D-Galactalc bT 31Escherichia coli b-galactosidase D-galacto-Heptenitol bH 30Escherichia coli b-galactosidase D-galacto-Octenitol bH 32Iapex lacteus Ex-1, Trichoderma reesei Cellobialc bH 36,37CBH I cellulaseTrichoderma reesei CBH I cellulase Lactalc bH 37Muscle, potato, Escherichia coli D-Glucal aT 41

phosphorylaseMuscle, potato phosphorylase D-gluco-Heptenitol aT 48,49Sweet potato, soybean, b-amylase Maltalc bH 50,51Trichoderma reesei trehalase D-gluco-Octenitolc bH 47Arthrobacter globiformis D-gluco-Heptenitol bH, aT 28,46

glucodextranase

a The “enitol” names refer to 2,6-anhydro-1-deoxyhept-1-enitol or (Z)-3,7-anhydro-1,2-dideoxyoct-2-enitol.

b H, anomeric configuration of the hydration product; T, configuration of the mobilized glycosyl unit inthe transfer product(s).

c Substrate found to be protonated from a direction opposite to that generally assumed for glycosidicsubstrates.


glucosidase very slowly hydrolyzes a-D-glucosyl fluoride to form b-D-glucose.53 These inverting reactions obey Michaelis–Menten kinetics andshow no evidence of transfer-product formation. In a related situation, theCBH II cellulase of Trichoderma reesei (which hydrolyzes substrates of b-configuration with inversion)54 is found to hydrolyze a-cellobiosyl fluorideto form a-cellobiose with no evidence of transfer-product formation or de-parture from Michaelian kinetics.25

A minimal mechanism whereby a-glucosidases hydrolyze b D-glucopyra-nosyl fluoride, a-D-glucopyranosides, and D-glucal (in each case yielding theproduct as the a-anomer) is illustrated53 in Fig. 1. This mechanism assumesthat in each case the lone pair on the substrate’s ring oxygen assists cleav-age of the C-1 glycosyl bond to form an oxocarbonium ionlike transitionstate and that this is stabilized by negatively charged carboxylates and fi-nally attacked by water from a structurally restricted (a) direction to pro-vide an SN1-type mechanism. For the CBH II-catalyzed hydrolysis of b-glycosidic substrates with inversion and of a-cellobiosyl fluoride with retention, Konstantinidis et al.25 suggested a different mechanism. Theyconsidered that the reported crystal structure of the catalytic domain of cellulase CBH II (of family 6)55 shows no plausible candidate for a catalyticbase and that hydrolysis of a-cellobiosyl fluoride by the enzyme involvesmerely a pattern of electrostatic fields that indiscriminantly stabilize a

FIG. 1. Proposed mechanisms of reactions catalyzed by rice a-glucosidase with substratesof different anomeric configuration. Reproduced from H. Matsui et al., Carbohydr. Res., 250(1989) 45–56, with permission from Elsevier Science, Ltd.



cationlike transition state for SNi displacement of the (small) departing flu-oride ion.b Regardless of the mechanism(s) involved, these examples of “re-taining” enzymes promoting hydrolysis with inversion and “inverting” en-zymes promoting hydrolysis with retention again point to the existence ofprotein structural features that control the stereochemical outcome of re-actions independent of substrate configuration.

3. Glycosyl Transfer by “Inverting” Glycosidases

The literature records a consistent failure of such hydrolytic enzymes asbeta-amylase,58,59 mold glucoamylase,60,61 glucodextranase,62 trehalase,63,64

and Bacillus pumilus b-xylosidase65 to promote glycosyl transfer.c Thesenegative results of tests using glycosidic substrates led to the widely heldview that “inverting” glycosidases are unable to form transfer products, asthe latter would lack the anomeric configuration required of the enzyme’ssubstrates. Were glucoamylase, for example, to transfer the a-D-glucosylresidue of maltose to the 4-OH group of D-glucose, one would expect for-mation of cellobiose, which is not a substrate for the enzyme.15,10 Yet, malt-ose phosphorylase—which, like glucoamylase, acts on maltose but not oncellobiose—catalyzes D-glucosyl transfer from maltose to inorganic phos-phate in a reaction that proceeds with inversion of configuration.68,69

Actually, each of the five just cited hydrolytic enzymes acts with inversionon both a- and b-anomers of a glycosyl fluoride—hydrolyzing the correctanomer and catalyzing glycosyl transfer with the wrong anomer21,38,40,70,71;the glycogen debranching enzyme of rabbit muscle [specifically, its amylo-(1 → 6)-glucosidase (EC 3.2.1.33) component] behaves similarly,39 cat-alyzing D-glucosyl transfer from b-D-glucosyl fluoride to cyclomalto-heptaose as acceptor. Rhizopus nireus glucoamylase acts on b-D-glucosylfluoride plus methyl a-D-glucopyranoside (a nonsubstrate) as acceptor

EDWARD J. HEHRE272

b Rouvinen et al.55 considered that Asp401 of cellulase CBH II might possibly serve as a gen-eral base in cellodextrin hydrolysis, but presented no data on the distances from its carboxylgroup to the reaction center or to the carboxyl group of the putative general acid residue.The possibility that Asp401 might serve as the catalytic base appeared to be supported bythe tentative assignment of this function to its counterpart Asp265 in family 6 Ther-momonospora fusca cellulase E2.56,24 New findings with a mutant of Asp265, however, arenot consistent with this residue’s role as a catalytic base in E2.57

c The first report proposing the glycosyl moiety to be a functional group of biochemical sig-nificance12 introduced the term “transglycosylation” for nonhydrolytic reactions catalyzedby enzymes subsequently classed as glycosyltransferases [EC 2.4]. The more general stoi-chiometric view of grouping together all enzymes that catalyze glycosyl–proton interchange(or glycosylation)13,66,14 further covers hydrolytic reactions and their reversals as well as re-actions promoted with small nonglycosidic glycosyl donors. At present, some authors use“glycosyl transfer” in this broad sense.15


to yield methyl a-maltoside and a little methyl a-isomaltoside.21 Gluco-dextranase acts similarly to form mainly methyl a-isomaltoside; in addi-tion, it catalyzes both the hydration of “D-gluco-heptenitol” to form b-D-gluco-heptulose and transfers a glycosyl group from the enitol to forma-D-gluco-heptulosyl-“2,7-heptenitol” and a-D-gluco-heptulosyl-“2,7-D-gluco-heptulose.”28,46 Thus, it is not that “inverting” glycosidases lack the ability to catalyze nonhydrolytic glycosylation reactions but that small substrates having an “anomeric” configuration other than that of hydrolyzable glycosidic substrates often allow the ability to be demon-strated.

How they do so is suggested by the behavior of Rimiveus glucoamylase.The catalytic groups of this enzyme promote transfers from b-D-glucosylfluoride in a way that parallels their actions in condensing two tandemlybound D-glucose molecules to form maltose. In condensation, b-D-glucose(the anomer-specific donor substrate)66 is protonated at the si-face by thesame catalytic group (now uncharged) that functions as a base in the hy-drolysis of maltose—as required by the principle of microscopic reversibil-ity. However, since the stereocomplementary hydrolysis and transfer reac-tions catalyzed by glucoamylase with a- and b-D-glucosyl fluoride are notreversals of each other, the reactions observed clearly indicate that the en-zyme’s catalytic groups are functionally flexible beyond needs of the prin-ciple of microscopic reversibility.21 The small size of b-D-glucopyranosylfluoride, which is comparable to that of the b-D-glucose that undergoes con-densation, would permit it to bind productively and to allow a second mol-ecule (or other acceptor) to bind simultaneously in tandem at the reactioncenter. In contrast, the equatorial aglycon group at C-1 of any b-glucosidewould clash with protein residues at the active site, preventing b-glucosidessuch as cellobiose from achieving the productive binding alignment re-quired of glucoamylase substrates.

That “inverting” glycosidases catalyze complementary hydrolysis andtransfer reactions with the opposed anomers of a glycosyl fluoride has beenconsidered15,72,73 to be strong evidence for Koshland’s74 single nucleophilicdisplacement mechanism. However, the large intrinsic secondary 3H kineticisotope effects obtained by Matsui et al.75 for a-D-glucopyranosyl fluoridehydrolysis with inversion, catalyzed by glucoamylases of different bio-logical origins, do not support the inversion mechanism proposed byKoshland74 in which: “the significant feature is that the covalent bond be-tween B and X [of substrate] is broken simultaneously with or after the nu-cleophilic attack on B by molecule A.” The large secondary kinetic isotopeeffects point instead to a mechanism in which substantial glucosyl bondbreaking and development of a carbonium ionlike enzyme transition stateoccur before appreciable product is formed.



III. X-RAY FINDINGS THAT SUPPORT CATALYTIC GROUP VERSATILITY AND

IDENTIFY THE STRUCTURES CONTROLLING STEREOCHEMICAL OUTCOME

Interest centers on the extent to which the reported crystal structures ofindividual glycosylases (a) are able to identify an active-site residue which,by nature and spatial disposition, might serve to protonate a bound enolicsubstrate or wrong glycosyl fluoride anomer and (b) are able to identifyprotein structural features that could dictate the direction of approach ofacceptor cosubstrates to the reaction center.

1. Residues Potentially Able to Protonate Minisubstrates of “Improper” Configuration

The catalysis of reactions by glycosidases acting on glycosidic substratesis commonly considered to depend on a pair of spatially fixed carboxylgroups disposed across the reaction center from each other—one acting asa general acid, the other as a general base or nucleophile. That many gly-cosidases use enolic substrates or the wrong glycosyl fluoride anomer, pro-tonating them from a direction opposite that for glycosidic substrates, sug-gested that their carboxyl groups are able to function flexibly and toactivate such disfavored substrates.9,40

Recent crystal structure studies confirm the presence of two fixed and op-posed active-site carboxyl groups in nearly all glycosidases, including b-galactosidase;77 cellulasesd of families 6,7,9,10, and 45;56,80–88 xylanases;89–93

alpha-amylases;94–100 beta-amylase;101,102 and glucoamylase.103,104e Hev-amine, a chitinase from Hevea braziliensis, is exceptional in lacking a cat-

EDWARD J. HEHRE274

d A cellulase is classed either as an endo b-glucanase [EC 3.2.1.4] or a b-glucan cellobiohy-drolase [EC 3.2.1.91] distinguished by cleaving mostly cellobiose from the chain ends. Eachis made up of a set of genetically distinct families characterized by the steric course of its re-actions. Thus an “endoglucanase” of family 10 hydrolyzes cellulosic linkages with retention;one of family 9 does so with in version.5,6 Likewise, a “cellobiohydrolase,” CBH I, of family7 hydrolyzes cellulosic chains from the reducing end with retention78–80; CBH II of family 6does so from the nonreducing end with inversion.55,80 A point to be noted is that theanomeric configuration of a cellobiose molecule released on hydrolysis by CBH I is not cre-ated by the reaction, whereas, the a-anomeric configuration of the cellobiose released on hy-drolysis by CBH II is so created; CBH II is found to use cellobiose (presumably the a-form)as a glycosyl donor to the terminal 4-OH group of cellulose.67

e Independent evidence for the highly frequent presence of a pair of oppositely disposed car-boxyl groups at the catalytic center of glycosidases has come from hydrophobic clusteranalysis.105 Motifs formed by contiguous Val, Ile, Leu, Met, Phe, Trp, and Val residues, corre-sponding to a-helical or strand structures, indicate that a pair of glutamic acid residues char-acterizes all (.150) members of families 1, 2, 5, 10, 23, 30, 35, 39, and 42, which hydrolyze b-D-glycosidic substrates with retention of configuration.105 These motifs are not observed forenzymes that hydrolyze a-D-glycosidic substrates, whether with retention (alpha-amylases)or inversion of configuration (beta-amylases).


alytic base.106,107 Possibly, as previously noted, this lack may also character-ize cellulase CBH II.55,25 Among glycosyltransferases, this arrangement ofopposed carboxyl groups across the reaction center appears not to be thenorm. It is found Bacillus circulans cyclodextrin glucanosyltransferase (analpha-amylase family member),108–110 but not in orotate 59-phosphoribosyltransferase,111,112 purine nucleoside phosphorylase,113 NAD1 nucleosidase(oxidized nicotinamide adenosine diphosphate ribosylase),114 or glycogenphosphorylase.115–119

In learning how glycosylases may protonate a substrate lacking correctanomeric configuration it is of note that opposing catalytic carboxyl groupsexist at the active site of two inverting glycosidases (glucoamylase and beta-amylase), which use a glycosyl fluoride of disfavored (b) configuration, pro-tonating it differently than their a-linked substrates21,40; likewise, in two re-taining glycosidases—cellulase CBH I, which hydrates cellobial andlactal,37 and b-galactosidase, which hydrates D-galactal31—each enzymeprotonating these glycals differently from its b-linked substrates.

a. Glucoamylase and Beta-Amylase.—That the probable source ofprotonation of b-D-glucosyl fluoride by glucoamylase is Glu400, whichnormally functions as the catalytic base in hydrolyzing a-linkedmaltosaccharides, is evident from the crystal structure of Aspergillusawamori glucoamylase complexed with 1-deoxynojirimycin.103 Thecarboxyl group of Glu400, presumably uncharged in some proteinmolecules, appears properly positioned to assist departure of fluoride fromb-D-glucosyl fluoride with formation of a transient oxocarbonium ion-type–enzyme complex.103 Likewise, the crystal structure of soybean beta-amylase complexed with b-maltose shows the catalytic site with Glu186 andGlu380 suitably disposed to serve as general acid and base catalyst,respectively, in maltosaccharide hydrolysis (Fig. 2, see color plate). Thecarboxyl group of Glu380, presumably present in the uncharged state insome molecules, is properly located to serve as the source of protonation101

in reactions catalyzed by beta-amylase with b-maltose, b-maltosyl fluoride,and maltal.

b. CHB I and b-Galactosidase.—Similarly, the structure of cellulaseCHB-I of T. reesei complexed with o-iodobenzyl 1-thio-b-cellobiosidecontains two opposed glutamic acid residues. Glu217, near the labile b-glycosidic oxygen bridge atom, is judged to be the general acid catalyst withGlu212, across the reaction center, the probable nucleophile in theretaining reactions catalyzed with b-glucosidic substrates.79 Glu212 appearsto be suitably spatially disposed to account for the observation that CBH Iprotonates cellobial and lactal from a direction opposite that for



protonating the glycosidic bridge oxygen of cellulose chains.37 It wouldappear likely that Glu212 exists in uncharged form in some proteinmolecules, possibly with its pKa elevated by proximity of the double bondof a bound glycal, providing it with the flexibility to function as a generalacid with prochiral substrates. Again, the crystal structure of the very largetetrameric Escherichia coli b-galactosidase molecule shows the active sitelocated in a deep pocket present at the end of the TIM-like barrel of eachmonomer.77 The conserved catalytic residues found in the walls of thispocket comprise Glu461, the presumed acid catalyst, and Glu537, thecatalytic nucleophile.120,121 The latter residue appears suitably positoned tofunction as the general acid catalyst in the observed protonation of thedouble bond of D-galactal from a direction opposite that for protonating b-D-galactosides.31

In sum, each of these four glycosidases possesses a suitably disposed glu-tamic acid carboxyl group which, in the uncharged state, could potentiallyactivate a small glycosyl donor of disfavored configuration. Figure 2 illus-trates the geometry of the catalytic site in one of these enzymes, soybeanbeta-amylase, complexed with b-maltose. The latter binds both as as donorsubstrate (violet and green glucose units) and cosubstrate (red and yellowunits).101

2. Structures That Limit the Stereochemical Outcome in Glycosylase-Catalyzed Reactions

The different anomeric configuration of products formed from starch byalpha- and beta-amylase led to the early proposal that these and other en-zymes catalyzing reactions with retention or inversion act by clearly differ-ent direct nucleophilic displacement mechanisms74 and later to the ideathat they might act by a common carbonium-ion mechanism if they vary thedirection whereby water approaches the reaction center.122–124 Experimen-tal indications that glycosidases may, indeed, control product configurationtopologically first emerged from studies of reactions of such enzymes withenolic glycosyl substrates (Table I). The enzymes a- and b-glucosidase, forexample, both catalyze hydration of D-glucal; each leads to a product of thesame anomeric configuration as formed from its usual substrates, eventhough each enzyme protonates D-glucal differently from maltosaccha-rides.26 Again, certain “retaining” a-glucosidases and the “inverting” glu-codextranase of Arthrobacter globiformis act on D-gluco-heptenitol, yield-ing both D-gluco-heptulose and D-gluco-heptulosyl transfer products. Ineach case the anomeric configuration of the hydration product and that ofthe transfer products matches the configuration of the corresponding hy-drolysis and transfer products formed from chiral substrates.28,46 The stereo-

EDWARD J. HEHRE276


chemical outcome of these reactions catalyzed with heptenitol, createdwithout guidance from substrate configuration, indicates that the a-glucosi-dases must somehow direct all incoming cosubstrates to the catalytic centerin the same (a) orientation; whereas the glucodextranase must ensure thatwater approaches the center from the b direction, carbohydrate cosub-strates from the opposite (a-) direction.28,46 Although the tertiary struc-tures of the a-glucosidases and the glucodextranase used have not yet beendescribed, those reported for various other “retaining” and “inverting” en-zymes add independent new information concerning the structural basis ofthe stereochemical behavior of these enzymes.

a. Reported Findings with “Retaining” Glycosylases.—The centralquestion now to be asked regarding reported crystallographic findings forindividual (ligand complexed) glycosylases of this type is whether the active-site cavity orients all acceptor cosubstrates (water, carbohydrates, andothers) to reach the catalytic center (namely, C-1 of the reactive glycosylmoiety) from the same direction. This is a narrower and more realistic goalthan seeking to derive a mechanism from extended time-averaged X-raycrystal structures that may or may not reveal the disposition of all orderedwater molecules and that do not resolve proton positions. For example, inthe case of hen’s egg-white lysozyme, where the proposed oxycarboniumion-stabilizing role of Asp52 has been questioned,73,125,126 subsequent X-rayfindings show not only that that Asp52 and its interactions with boundsubstrate help strain the pyranose ring into a more reactive conformationbut that this residue’s interactions with neighboring residues block allacceptors from approaching the center from the a-side.

(i) Hen’s Egg-White Lysozyme.—The active site is located in a cleft inthe protein surface. Glu35 functions as the general acid, with Asp52possibly implicated in stabilizing the transition state.1,76 The structure of theenzyme complexed with a reaction product, N-acetyl-b-muramic acid–N-acetyl-b-D-glucosamine—N-acetyl-b-muramic acid (MurNAc-GleNAc-MurNAc) shows the trisaccharide bound at subsites B, C, and D in theactive-site cleft.1 The reducing MurNAc unit in subsite D has a sofa-typeconformation and b-anomeric configuration; its equatorial O-1 atom is H-bonded to the OE1 atom of Glu35. There is no room for binding the a-anomer of this MurNAc, as its axially oriented 1-OH would clash withAsp52 and other closely interacting components in the region. Thesefeatures indicate that water and other cosubstrate molecules can only ap-proach the reaction center from the b (Glu35)-side. Studies with a .99%inactive Asp52Ser mutant, cocrystallized with b-(1 → 4)-N-acetyl-D-glucosamine hexasaccharide, confirm the importance of the position of the



side chain of Asp52 in relation to substrate in blocking an a-directedapproach of acceptors.127

(ii) CBH I Cellulase, b-Galactosidase, and Glycogen Phosphorylase.—X-Ray structures have been reported for these three “retaining”glycosylases, which convert enolic substrates into products of the sameconfiguration as formed from their glycosidic substrates. The reportedcrystallographic findings are consistent with the view that the conservedstereochemical outcome of reactions catalyzed by each enzyme is due tosubstrate and cosubstrate-binding structures in the active-site cavity thatorient all reactants, including water, to the catalytic center from the samedirection with no cosubstrate access to the center from the oppositedirection.

The structure of cellulase CBH I of Trichoderma reesei complexed witho-iodobenzyl 1-thio-b-cellobioside shows the active site to be located in along tunnel flattened to accomodate glucose units in subsites A to G.79 Thistunnel directs all reactants, including water, toward the presumed generalacid catalyst, Glu217, on the b-side of the reaction center. In theenzyme–ligand complex, Glu217 is H-bonded to the O-4 atom of cellobio-side’s O-glycosidic linkage between subsites B and C; Glu212, across thecenter and proximate to it, is assumed to be the nucleophile. The data arehighly suggestive of water entry from the b-side, even though bound wateris not found near the nucleophile and reaction center. Whether residues onthe a-side of the center (around Glu212) have zero access to solvent is notestablished. The authors concluded that the catalytic groups necessary for adouble displacement reaction are present,79 but the data do not establishthat a covalent a-glycosyl–Glu217 intermediate is necessarily formed. Theentry of water via the active-site cavity is consistent with the enzyme’s ob-served creation of a hydration product of b-configuration from cellobial byway of a carbonium ion type-mediated reaction.37

The crystal structure of the very large tetrameric E. coli b-galactosidasemolecule has been reported77 at relatively low (3.5 Å) resolution and onlyfor the unliganded enzyme; it does not inform as to whether the catalyticsite has access to solvent from the a- as well as from the b-side of the reac-tion center. However, the data are consistent with the idea that all accep-tors, including water, gain access to the center from the b-side, which en-sures that products of b-configuration are formed from various enitols30–32

as they are from b-D-galactosides.Crystallographic studies of the structures of rabbit muscle glycogen phos-

phorylase b, complexed with D-gluco-heptenitol or a maltosaccharide plusinorganic phosphate (Pi), confirm that the a-anomeric configuration of theproduct of these phosphorolytic reactions is dictated topologically. As illus-trated in Fig. 3, the enitol binds at the catalytic site of phosphorylase in a

EDWARD J. HEHRE278


position essentially occupied by the glucose of bound a-D-glucopyranosylphosphate.115 The attacking inorganic phosphate (Pi) binds between the enolic bond of heptenitol and the 59-phosphate of the pyridoxal 59-phosphate catalytic cofactor; that is, as is found for the axial 1-PO4 group ofa-D-glucosyl phosphate and related compounds.115–118 Figure 3 illustratesthat the active-site cavity of phosphorylase provides for the conserved (a-)


FIG. 3. Reported mechanism of catalysis by glycogen phosphorylase for thephosphorolysis of D-gluco-heptenitol to form a-D-heptulosyl phosphate (above)and the phosphorolysis of malto-oligosaccharides to form a-D-glucosyl phosphate(below). Adapted from McLaughlin et al., Biochemistry, 23(1984) 5862–5873, withpermission of the American Chemical Society.


binding orientation to the catalytic center of all cosubstrates, including theinorganic phosphate (or arsenate) in lytic reactions and the maltosaccha-ride acceptors in syntheses from a-D-glucosyl phosphate.115,117–119 Sinceglycogen phosphorylase has no ionizable group near the catalytic cen-ter,115,117 neither binding nor activation of Pi can occur on the b-side of thecenter.

(iii) Pancreatic Alpha-Amylase and Cyclodextrin Glycosyltransferase.—Active-site structures have been described for these related glycosylaseswhose reactions with maltosidic substrates and a-maltosyl fluoride proceedwith retention of configuration. Their stereochemical behavior withminisubstrates of improper configuration has not been described.

The active center of porcine pancreatic alpha-amylase (family 13) is foundin a long cleft in the protein surface that provides easy access to solvent anddissolved ligands. The crystal structure of the enzyme soaked with 1 mMacarbose (A,B,G1,G2), an a-linked inhibitor in which A,B 5 acarviosideand G1,G2 5 maltose, shows cyclic units bound in five subsites. These areidentified as G,G,A,B,G with G,G representing maltose a-(1 → 4)-linked toA, the nonreducing end unit of acarbose. The catalytic center, located be-tween units A and B, comprises a trio of conserved carboxylates. Those ofGlu233 and Asp300, each on the a-side of the active-site cleft and 3 Å fromthe glycosidic bridge N-atom of the acarvioside moiety, presumbly providefor the protonation of the anomeric carbon atom of an a-linked moiety ofa substrate. The presumed base or nucleophile, Asp197, lies opposite in thecleft, across the reaction center.98,99 An ordered water molecule is not ob-served in the region of Asp197, suggesting that solvent as well as carbohy-drate ligands can reach the reaction center only from the a-direction via theactive-site cavity. The authors found OD2 of Asp197 and OE2 of Glu233 tobe located 3.3 and 3.5 Å from the anomeric C-I atom of cyclitol A—dis-tances ,1.8 Å longer than expected for covalent bonds. They consideredthat the crystal structure does not provide evidence for a covalent b-linkedglycosyl–enzyme intermediate99 but supports, instead, a mechanism forporcine alpha-amylase involving a carbonium-ion transition state as pro-posed for lysozyme.1,76 For the alpha-amylase of Asperillus oryzae, a watermolecule is found hydrogen bonded to the presumed acid catalyst, each ori-ented alpha to the reaction center.95

Cyclodextrin glycosyltransferases [EC 2.4.1.19] share family 13 with alpha-amylases, although the latter are formally classed as hydrolases [EC3.2.1.1]. The CGTases utilize starch, maltodextrins, and a-maltosyl fluorideto synthesize cyclic compounds of six, seven, or eight a-(1 → 4)-linked glu-copyranose units as well as related noncyclic dextrins.The Bacillus circulansstrain 251 enzyme complexed with acarbose shows an active-site structure

EDWARD J. HEHRE280


with the inhibitory ligand bound in the same orientation as maltosaccha-rides.110 Glu257, found within H-bonding distance of the glycosidic oxygenbetween the B and C units of acarbose, is considered to be the protondonor; Asp229, on the opposide (b-) side of the reaction center, is the pre-sumed general base or nucleophile. It interacts closely with a water mole-cule in a pocket formed by the main-chain atoms of residues 139 and 229,but this water does not appear to be catalytically significant as it is not inclose contact with the reactive C-1 atom of the B unit of acarbose. Thoughproof is lacking that this C-1 atom lacks access to solvent from the b-side,the crystallographic data strongly suggest that water, as well as carbohy-drate cosubstrates, approach the catalytic center from only one (a-) direc-tion, by way of the active-site cavity.

(iv) Cex b-Cellulase/Xylanase.—Crystallographic studies have beenreported for the catalytic domain of the Cex b-glycanase of Cellulomonasfimi, which hydrolyzes cellulase and xylan with retention of configuration.The active-site structure of the complex of this enzyme with 2,4-dinitrophenyl 2-deoxy-2-fluoro-b-cellobioside shows the presence of bound2-deoxy-2-fluorocellobiose, with C-1 covalently a-linked to the nucleophilicresidue, Glu233.88 The intermediate is catalytically competent. Boundwater is not observed in proximity to Glu233, but measurement of theaccessibility of the nucleophile to solvent is not reported. An ordered watermolecule within H-bonding distance of the acid catalyst Glu127 and ofresidue Gln203 is considered to be the likely nucleophile in the terminalstep of catalysis. This water has the same (b-) orientation to the catalyticcenter as the enzyme’s substrates.87,88

b. Reported Findings with “Inverting” Glycosylases.—(i). CellulaseCBH II, Beta-Amylase, and Glucoamylase.—X-Ray diffraction findingshave been reported for these three glycosidases, which had been found,through studies of their reactions with minisubstrates, to control productconfiguration topologically. Their crystal structures provide significantinsights into how protein structural features control the stereochemicaloutcome of reactions by enzymes of this type.

Trichoderma reesei cellulase CBH II directly hydrolyzes cellodextrinsand both a-and b-cellobiosyl fluoride to form a-cellobiose in each case.25,73

The crystal structure of the catalytic domain shows the active site to be lo-cated in a tunnel-like cavity of restricted volume, affording four subsites intandem.55 After the nonreducing end-unit of a cellulose chain has enteredand traversed the tunnel, hydrolysis of the penultimate b-(1 → 4) linkageoccurs at the reaction center; the a-cellobiose product is then extruded intosolvent. Rouvinen et al.55 reported that a narrow tubular passage, roughly



orthogonal to the tunnel, channels water from the protein surface to the re-action center specifically from the a-direction. It is not evident whether awater molecule needs to be bound to be activated, but no ordered watermolecule is found near (and alpha) to the reaction center. A catalyticresidue serving to help water attack is not apparent, but the presumed acidcatalyst, Asp221, is reportedly buried and inaccessable to solvent. Hence,the structure of CBH II is consistent with the view that formation of a-cellobiose from b-cellobiosyl fluoride54 and from methyl b-cellotetraoside37

results from a structure-limited a-orientation of water to the reaction center.55

A different specific mode is envisioned for beta-amylase, based on thecrystal structures of soybean enzyme, unliganded or treated with b-maltoseor with maltal.101 Beta-amylase does not hydrolyze maltose but uses b-maltose as a glycosyl donor, condensing it to a small extent with a secondmaltose to form maltotetraose and water, as expected from the principle of microscopic reversibility.66 Beta-amylase also catalyzes the slow hydra-tion of maltal [a-D-glucopyranosyl-(1 → 4)-D-glucal] to form 2-deoxy-b-maltose.50,51 The crystal structure locates the catalytic site in a deep pocketin the protein surface. The b-maltose-treated enzyme shows two moleculesof the substrate/cosubstrate bound in tandem, with some maltotetraosecondensation product detectable in the same subsites. The maltal-treatedenzyme shows two molecules of the 2-deoxymaltose hydration productbound in tandem (the nonreducing end of each saccharide points to thebase of the active-site pocket).101 In unliganded beta-amylase the pocket isopen for substrate binding or product release, but one of its walls is formedby a mobile hinged loop that closes down on bound substrates (Fig. 4, seecolor plate). The closed loop’s Asp101 residue interacts with the two mostdeeply positioned glucose units in the active-site pocket and also (indi-rectly) with its Val99 residue; Asp101 is too far removed from the reactioncenter to be part of the catalytic chemistry.f

Through contacts between the methyl groups of Val99 and those ofLeu383 the closed loop also forms a hydrophobic surface across the reaction center, shielding it from solvent (Fig. 5, see color plate). In the b-maltose-treated enzyme this surface extends over the area between the two bound maltose molecules (or middle a-D-glucosidic linkage of

EDWARD J. HEHRE282

f Totsuka et al. initially proposed, based on finding site-directed mutants of Asp101 andGlu186 to be nearly inactive, that these two moieties are the essential catalytic residues forbeta-amylase.128 However, later reports confirm the crystallographic evidence that Glu186and Glu380 are suitably disposed to function as the catalytic residues,101 since activity is lostin mutants of these residues.129,130 The importance of Asp101 and of Leu383 (whoseLeu383Ser mutant shows remarkably decreased activity)130 lies in their demonstrated rolesin binding or recognizing substrate and in their contributions, via interactions with Val99,that allow the closed loop to form a hydrophobic surface over the reaction center.101


maltotetraose) and over both catalytically active carboxyl groups, namelyof Glu186 on the a-side of the reaction center and of Glu380 on the b-side.101 There is then no measurable solvent access to the side chains of thecatalytic residues or to the potentially reactive carbon and oxygen atoms ofthe sugars at the catalytic center. This means that the water molecule in-volved in starch hydrolysis or maltal hydration must exist beneath this hy-drophobic surface. In unliganded beta-amylase an ordered water molecule(H2O 866) is indeed found within H-bonding distance of OE2 of Glu380,the presumed nucleophile, and the main-chain O of Asn381. In the enzymetreated with maltal, this water molecule is displaced by the equatorial O-1atom of the 2-deoxy-b-maltose hydration product. Moreover, when mal-totetraose is formed by the enzymic condensation of maltose, the by-product appears as an ordered water in the same position as occupied byH2O 866 in the unliganded enzyme, H-bonded to the carboxyl group ofGlu380 and main-chain O of Asn 381. No ordered water is found nearAsp221, the general acid catalyst in maltosaccharide hydrolysis, located onthe a-side of the reaction center.

An apparently similar situation is found with fungal glucoamylase, whichcatalyzes the hydrolysis of a-D-glucosyl fluoride and maltosaccharides toyield b-D-glucose, but forms a-linked transfer products from b-D-glucose66

or b-D-glucosyl fluoride.21 The crystal structure of Aspergillus awamori glu-coamylase complexed with 1-deoxynojirimycin103 shows the active site tobe located in a pocket. A water molecule (water500) is found hydrogen-bonded to OE1 of Glu400, the putative general base located across the re-action center from the presumed general acid catalyst, Glu179, indicatingthat hydrolysis of an a-D-glucosylic substrate will yield b-D-glucose. Onbinding b-D-glucose or b-D-glucosyl fluoride as substrates, the equatorial 1-OH or 1-F would displace water500.103 Glucosyl transfer from these twodonors would then occur by reversal of the catalytic roles of the two car-boxylate side chains; a-linked products would be formed as the 4-OH groupof a D-glucosyl acceptor molecule would face the reaction center in thesame orientation as that of the second a-D-glucosyl unit of a maltosac-charide substrate. Although the mode whereby solvent is purged from the active-site pocket (and kept from Glu179 in particular) is not clear,Harris et al.103 suggested that a transient compression of the active-sitestructure may allow water to slide by the substrate (or inhibitor) as it entersthe close-fitting recess of the active-site pocket. Perhaps water in the deep-est part of the pocket may be displaced through lateral diffusion as sub-strate enters.104

(ii) Cellulases of Families 9 and 45.—Crystal structures are reportedfor two different endocellulases that hydrolyze b-(1 → 4)-linked substrateswith inversion but whose actions on minisubstrates are not known. The



structural findings strongly suggest that product configuration is controlledtopologically, although they do not fully establish that reactant waterreaches the reaction center only from the a-direction or that it binds closeto both the nucleophile and the center. The active-site structure of eachenzyme is an open cleft with two oppositely disposed catalytic carboxylgroups reported to resemble the arrangement in such groups at the reactioncenter of hen’s egg-white lysozyme.

In CelD (family 9) of Clostridium thermocellum, the presumed nucle-ophile Asp201 is located too far from the catalytic center to react directlywith a positively charged reaction intermediate.83 The authors suggestedthat a pocket occupied by the «-amino group of Lys38 from an abuttingmolecule in the crystal could accomodate a water molecule betweenAsp201 and the reaction center.83 With the enzyme in solution the openpocket might allow water to bind near Asp201 and to be activated by it soas to become a specifically a-directed nucleophile. However, the data donot indicate how the reaction center might be shielded from solvent presentin the active-site cavity.

The crystal structure of endocellulase EGV (family 45) from Humicolainsolens, with cellobiose in the leaving-group site, shows an ordered watermolecule near Asp10, the proposed general base.85 The authors indicatedthat this water, if moved ,1 Å, would be suitably positioned to make a nu-cleophilic attack leading to inversion of configuration. Were it bound toAsp10 and sufficiently near C-1 of the glucosyl unit at the reaction center(distances not noted) it could attack without need for prior product depar-ture. The structure of an inactive Asp10Asn mutant of EGV complexedwith cellohexaose is almost isomorphous to the native enzyme; but a localconformational change involving a loop segment (disordered in the apoen-zyme) moves residues Asp114 and Leu115 some 7Å to enclose the activesite at the point of cleavage. The authors considered that the data show awater molecule suitably disposed to participate in a single-displacement re-action,85 with loop closure possibly preventing access of other potential nu-cleophiles to the active site.86

(iii) Orotate Phosphoribosyltransferase.—A different example is pro-vided by the inverting orotate phosphoribosyl transferase of Salmonellatyphimurium, representative of the various phosphoribosyl transferasesinvolved in nucleotide synthesis. This enzyme catalyzes the reversibletransfer of the 59-phosphoribosyl moiety from a-phosphoribosyl pyrophos-phate (a-PRPP), the light-atom structure at the bottom of Fig. 6, to oroticacid (light-atom structure at the top of the figure), acting as specific base, toform b-phosphoribosyl orotate (dark atoms) plus pyrophosphate, PPi. Theposition of reactants, plotted directly from the crystal structures by Scapin

EDWARD J. HEHRE284


et al.111,112 shows both the free orotate and 59-b-phosphoryl orotate boundfar from pyrophosphate and a-PRPP. In the reverse (lytic) reaction, dashedlines mark the b-D-ribosyl C–N bond cleaved and the a-D-ribosyl C–Obond formed with the PPi consubstrate. In either direction, the boundorotate and PPi cosubstrates are thus oriented oppositely to C-1 of theribosyl moiety.The 59-phosphoribosyl unit, upon being transferred betweenO-19 of PPi and N-1 of orotate, rotates about 608 around the pivot of its 59-phosphate—with the C-1 atom moving about 7 Å between the twocosubstrates.

Figure 7 shows the electrostatic potential surface of the active-site regionfor the enzyme complexed with orotate b-phosphoriboside. The orotatering is deep in its binding site, with the b-ribosyl unit near the surface and


FIG. 6. Diagram from Scapin et al. (Biochemistry, 34 (1995)10744–10754) illustrating the location of bound orotic acid (light atoms), of orotic acid glycosidically linked in orotidyl 59-phospho-b-D-riboside (dark atoms), and of pyrophosphate(light atoms) oriented to form the a-59-phosphoriboside in oro-tate 59-phosphoribosyl transferase.


its 59-phosphate group visible on the surface.111,112 The binding site for thePPi cosubstrate molecule is evident as a separate depression in the surface.In short, although the organic orotate and inorganic pyrophosphate cosub-strates reach their binding sites via a common active-site cavity, they be-come oppositely oriented to the ribosyl C-1 atom by the different disposi-tions of their binding sites. Details of the catalytic mechanism, and of howwater is kept out, are not known.

3. Structural Basis for Separating Glycosylases into 1-MCO(“Retaining”) and 2-MCO (“Inverting”) Types

As noted earlier (compare Table I), studies made using small prochiralsubstrates gave the first concrete indications that product configuration insome glycosylase-catalyzed reactions does not depend on that of the sub-strate but is determined topologically by protein structures which deter-mine how incoming cosubstrates approach the catalytic center.26,32,36,45–48

EDWARD J. HEHRE286

FIG. 7. Electrostatic potential surface of the active-site region of orotate 59-phosphoribo-syl transferase complexed with orotidyl b-59-phosphoriboside. Reproduced from Scapin et al.,(1995) Biochemistry 34, 10744–10754, with permission of the American Chemical Society.


Reported crystal structures of the liganded enzymes already reviewed hereexpand this conclusion by identifying the probable controlling structures ina number of “lytic” glycosylases.

The observations summarized in Table II indicate that the cosubstrates ofa particular “retaining” enzyme all approach the catalytic center only fromits a- (or only from its b-) side. Examination of the available structures ofsuch enzymes gave no indication that water or phosphate is directed to thecenter differently from carbohydrate or nitrogenous cosubstrates. On theother hand, the findings with the “inverting” glycosylases in Table III pro-vide evidence for, or at least strongly suggestive of, the presence of a spe-cial structural feature which ensures that the inorganic or lytic cosubstrate(water, phosphate, or pyrophosphate) is positioned so as to reach the reac-tion center from a direction opposite that required for carbohydrate or ni-trogenous cosubstrates. Both soybean beta-amylase101 and A. awamori glu-coamylase103 show an ordered water molecule at a specific site closelyproximate to both a nucleophilic residue and the C-1 atom defining the re-action center. This water not only is positioned to attack from the b-side,opposite the direction of approach of carbohydrate acceptors but, most sig-nificantly, it is poised for activation and preemptory attack on substrate,whereas organic cosubstrates can bind and react only after prior release ofthe substrate’s aglycone.

A specific water-binding site would appear to exist in several other “in-verting” glycosidases (Table III). Structural evidence also is found for acomparable special pyrophosphate binding site in orotate phosphoribosyltransferase and, presumably, in various other inverting PRtransferases. Thissite, which is part of a common PRPP binding motif, places O-19 of the in-organic acceptor close to and on the a-side of the 59-phosphoribosyl C-1atom. It is far separated from the binding site for orotate (or other specificnitrogenous bases used by different PRtransferases) whose N-1 atom is ori-ented to the b-side of the 59-phosphoribosyl C-1 atom. Although the py-rophosphate and orotic acid cosubstrates gain access to their specific bind-ing sites from a common cavity, their selective binding affinities positionthem at sites which orient them oppositely with respect to the C-1 atom ofthe phosphoribosyl moiety.

Kinetic studies indicate that a special binding site, this time for inorganicphosphate, likely exists in maltose phosphorylase, which catalyzes the phos-phorolysis of maltose to form b-D-glucosyl phosphate plus glucose as wellas maltose synthesis from b-D-glucosyl phosphate plus glucose.68 A crystalstructure has not been reported for this enzyme but, as found in kineticstudies68 and illustrated in Fig. 8,69 Pi most likely binds on the b-side of C-1 of the maltose undergoing phosphorolysis. In contrast, in the reverse re-action with b-D-glucosyl phosphate as substrate, the glucose cosubstrate



288

TA

BL

EII

Obs

erve

d Fe

atur

es o

f th

e C

ryst

al S

truc

ture

s of

“R

etai

ning

” E

nzym

es T

hat

Pro

vide

but

One

Mod

e of

App

roac

h of

All

Cos

ubst

rate

s to

the

Enz

yme–

Subs

trat

e R

eact

ion

Cen

ter

Enz

yme

Porc

ine

panc

reat

ic

Alp

ha-a

myl

ase

Cyc

lode

xtri

n

glyc

osyl

tran

sfer

ase

Gly

coge

n

phos

phor

ylas

e

a-S

ide

a-S

ide

a-s

ide

Act

ive-

site

Res

idue

s D

irec

t All

Inco

min

g C

osub

stra

tes

to t

heC

atal

ytic

Cen

ter

From

One

Sid

ea

No

Indi

cati

on o

f a

Spec

ial S

truc

ture

Tha

t D

irec

ts S

mal

l Ino

rgan

ic C

osub

stra

tes

(Wat

er o

r P

hosp

hate

) to

the

Cat

alyt

ic C

ente

r W

ith

a D

iffe

rent

Ori

enta

tion

Tha

nC

arbo

hydr

ate

Acc

epto

rs

The

str

uctu

re o

f P

PA t

reat

ed w

ith

acar

bose

sho

ws

no o

rder

ed w

ater

bet

wee

n th

epr

esum

ed n

ucle

ophi

le A

sp19

7,on

the

b-s

ide

of t

he c

atal

ytic

cen

ter,

and

the

C-1

ato

m o

f th

e cy

clit

ol u

nit.

Acc

ess

of t

he c

ente

r to

sol

vent

fro

m t

he b

-sid

e,th

ough

not

mea

sure

d,ap

pear

s un

likel

y.99

Asp

229,

the

pres

umed

nuc

leop

hile

on

the

b-s

ide

of t

he r

eact

ion

cent

er,i

s st

abili

zed

by a

2.8

Å in

tera

ctio

n w

ith

a w

ater

mol

ecul

e.T

he la

tter

,loc

ated

in a

pock

et f

orm

ed b

y re

sidu

es 1

39 a

nd 2

29,i

s no

t ne

ar t

he r

eact

ion

cent

er.

Alt

houg

h th

e da

ta d

o no

t es

tabl

ish

that

the

cat

alyt

ic c

ente

r la

cks

acce

ss t

o so

lven

t fr

om t

he b

-sid

e,al

l inc

omin

g co

subs

trat

es a

ppea

r ab

le t

o re

ach

the

cent

er f

rom

the

a-s

ide

only

.110

Cry

stal

str

uctu

re o

f th

e en

zym

e co

mpl

exed

wit

h a

-D-g

luco

syl p

hosp

hate

indi

cate

sth

at P

ibi

nds

only

on

the

axia

l (a

-) s

ide

of t

he c

atal

ytic

cen

ter.

No

phos

phat

esi

teis

obs

erve

d on

the

b-s

ide

and

no io

niza

ble

resi

due

exis

ts in

that

reg

ion

tose

rve

as a

nuc

leop

hile

to h

elp

activ

ate

a ph

osph

ate

grou

p.11

5–11

7


289

Hen

’s e

gg-w

hite

lyso

zym

e (H

EW

L)

T.re

esei

cellu

lase

CB

H I

Cel

lulo

mon

as fi

mi

Cex

cel

lula

se–

xyla

nase

aT

his

wou

ld a

ccou

nt f

or t

he o

bser

vati

on t

hat

all

reac

tion

s by

a g

iven

“re

tain

ing”

enz

yme

have

the

sam

e st

eric

out

com

e. T

he s

truc

tura

l fin

ding

s in

sev

eral

in-

stan

ces

do n

ot s

uffic

e to

est

ablis

h th

at in

com

ing

cosu

bstr

ates

can

rea

ch t

he c

atal

ytic

cen

ter

only

fro

m t

he s

ide

spec

ified

, but

the

y ar

e co

nsis

tent

wit

h th

is v

iew

.

b-S

ide

b-S

ide

b-S

ide

HE

WL

com

plex

ed w

ith

a pr

oduc

t,M

urN

Ac-

Glc

NA

c-M

urN

Ac,

show

s th

e lig

and

boun

d at

sub

site

s B

–D.T

here

is n

o ro

om a

t su

bsit

e D

for

the

a-

anom

er o

f M

urN

Ac

as it

s ax

ial 1

-OH

wou

ld c

lash

wit

h A

sp52

and

oth

erre

sidu

es in

the

reg

ion.

The

se s

truc

tura

l fea

ture

s co

nfirm

tha

t w

ater

and

oth

erac

cept

ors

reac

h th

e re

acti

on c

ente

r on

ly f

rom

the

b-s

ide.

1

The

com

plex

wit

h o-

iodo

benz

yl 1

-thi

o-b

-cel

lobi

osid

e sh

ows

no o

rder

ed w

ater

betw

een

the

pres

umed

nuc

leop

hile

Glu

212

and

the

reac

tion

cen

ter.

The

dat

asu

gges

t bu

t do

not

est

ablis

h th

at t

he c

ente

r la

cks

acce

ss t

o so

lven

t fr

om t

hea

-sid

e.T

he c

atal

ytic

gro

up p

osit

ions

are

con

sist

ent

wit

h a

doub

le-d

ispl

acem

ent

mec

hani

sm,79

but

they

als

o ca

n ac

coun

t fo

r ce

llobi

al h

ydra

tion

by

a di

ffer

ent

mec

hani

sm.37

The

com

plex

wit

h 2,

4-di

nitr

ophe

nyl-

2-flu

oro-

b-c

ello

bios

ide

show

s88bo

und

2-flu

oroc

ello

bios

e w

ith

C-1

cov

alen

tly

a-l

inke

d to

the

OE

1 of

the

nuc

leop

hilic

resi

due

Glu

233.

No

mea

sure

men

ts o

f th

e ac

cess

ibili

ty o

f G

lu23

3 to

sol

vent

are

repo

rted

.


TA

BL

EII

I

Obs

erve

d Fe

atur

es o

f th

e C

ryst

al S

truc

ture

s of

“In

vert

ing”

Gly

cosy

lase

s T

hat

Pro

vide

Tw

o M

odes

of A

ppro

ach

of D

iffe

rent

Cos

ubst

rate

s to

the

Enz

yme–

Subs

trat

e R

eact

ion

Cen

ter

Enz

yme

Soyb

ean

Bet

a-am

ylas

e

A.a

wam

ori

gluc

oam

ylas

e

T.re

esei

cellu

lase

CB

H I

I

C.t

herm

ocel

lum

cellu

lase

Cel

D

H.i

nsol

ans

cellu

lase

EG

V

Oro

tate

59p

hosp

ho-

trib

osyl

rans

fera

se

aT

he s

truc

ture

of

each

enz

yme

indi

cate

s th

at o

rgan

ic (

carb

ohyd

rate

, alc

ohol

ic, n

itro

geno

us)

cosu

bstr

ates

rea

ch t

he r

eact

ion

cent

er f

rom

one

(a

- or

b-)

sid

e by

one

rout

e on

ly, b

y w

ay o

f a

com

mon

bin

ding

sit

e or

sim

ilarl

y or

ient

ed b

indi

ng-s

ites

.b

Any

hyd

roly

tic,

pho

spho

roly

tic,

or

pyro

phos

phor

olyt

ic p

rodu

ct fo

rmed

by

a lis

ted

enzy

me

wou

ld th

us h

ave

a co

nfigu

rati

on o

ppos

ite

to th

at o

f tra

nsfe

r pr

oduc

tsfo

rmed

wit

h or

gani

c co

subs

trat

es.

Wat

er86

6 is

fou

nd H

-bon

ded

to O

E2

of G

lu38

0 (t

he n

ucle

ophi

le),

to t

he O

ato

m o

fA

sn38

1,an

d to

C-1

of

the

reac

tion

inte

rmed

iate

.Loo

p cl

osur

e on

bou

nd s

ubst

rate

shie

lds

the

Glu

186

(aci

d ca

taly

st)

regi

on a

nd a

ll po

tent

ially

rea

ctiv

e at

oms

from

so

lven

t.101

An

inhi

bito

r co

mpl

ex s

how

s or

dere

d w

ater

500

in p

roxi

mit

y to

OE

1 of

Glu

400

(gen

eral

bas

e) a

nd t

o N

5 of

the

inhi

bito

r (e

quiv

alen

t to

C-1

of

a m

alto

sacc

hari

de).

Wat

er is

pre

sum

ably

pur

ged

from

the

Glu

179

(aci

d ca

taly

st)

regi

on b

y cl

osel

y fit

ting

into

the

act

ive

site

.103,

104

A n

arro

w t

ube

conv

eys

H2O

to

the

a-s

ide

of t

he c

atal

ytic

cen

ter;

but

resi

dues

on

the

b-s

ide

lack

acc

ess

to s

olve

nt.S

truc

tura

l con

trol

of

ster

ic o

utco

me

is c

lear

,yet

no

orde

red

wat

er is

fou

nd b

etw

een

the

reac

tive

C-1

ato

m a

nd a

pot

enti

al n

ucle

ophi

licre

sidu

e.55

Wat

er a

ppar

entl

y re

ache

s th

e ca

taly

tic

cent

er o

nly

from

the

a-s

ide.

The

Asp

201

base

is t

oo f

ar f

rom

the

cen

ter

to r

eact

dir

ectl

y w

ith

C-1

.Bet

wee

n th

em is

a s

ite

occu

pied

by

the

ε-gr

oup

of L

ys38

fro

m a

nea

rby

prot

ein

in t

he la

ttic

e,w

hich

pe

rhap

s co

uld

in s

olut

ion

beco

me

a H

2O a

ctiv

atin

g si

te.83

The

act

ive-

site

str

uctu

re o

f th

e co

mpl

ex w

ith

cello

bios

e sh

ows

an o

rder

ed H

2O n

ear

to b

ut n

ot H

-bon

ded

to A

sp10

(ge

nera

l bas

e).T

he a

utho

rs s

tate

d th

at a

n in

acti

ve

cello

hexa

ose

com

plex

has

wat

er s

uita

bly

boun

d to

par

tici

pate

in a

sin

gle

disp

lace

men

t re

acti

on.85

,86

Pyr

opho

spha

te b

inds

in a

PP

RP

sit

e on

the

a-s

ide

of t

he r

ibos

yl C

-1 a

tom

.Oro

tate

bids

in a

sep

arat

e si

te o

n th

e b

-sid

e of

the

cen

ter,

tailo

red

to t

he b

indi

ng o

f sp

ecifi

c ni

trog

enou

s ba

ses

in o

ther

suc

h tr

ansf

eras

es.11

1,11

2

a-S

ide

a-S

ide

b-S

ide

b-S

ide

b-S

ide

b-S

ide

Org

anic

Cos

ubst

rate

s A

ppro

ach

the

Rea

ctio

n C

en-

ter

From

One

Dir

ecti

on O

nlya

Thr

ough

a S

peci

al S

truc

tura

l Fea

ture

,Ino

rgan

ic C

osub

stra

tes

(H2O

,Pi,

PP

i) A

ppro

ach

the

Cat

alyt

ic C

ente

r Fr

om a

Dir

ecti

on O

oppo

site

to T

hat R

equi

red

for

Org

anic

Acc

epto

rsb


binds at a separate site with its O-4 atom a-oriented to the reaction center.This contrasts with glycogen phosphorylase, where both phosphate and thenonreducing-end glucose unit of dextrin cosubstrates bind in the same ori-entation to the reaction center and lead to reaction products of a-anomericconfiguration. X-Ray studies of human purine nucleoside phosphorylase, aninverting D-ribofuranosylase, show that the Pi cosubstrate is bound in an a-orientation, purines in the opposite (b-) orientation to the reaction cen-ter.113 Carboxyl groups apparently are not involved in the catalytic event.

As summarized in Table IV, the evidence thus far obtained with variousglycosidases, phosphorylases, and pyrophosphorylases indicates that “re-taining” glycosylases would probably be more precisely characterized asproteins with a 1-MCO type of structure, which restricts both substrates andcosubstrates to one mode of orientation to the reaction center. The struc-turally directed approach of all incoming cosubstrates, either always fromthe a- or always from the b-side of the center, can account for the constancy


FIG. 8. Stereochemistry of reactions catalyzed by maltose phosphorylase with inversion ofconfiguration. (A) a-Maltose synthesis from b-D-glucopyranosyl phosphate and the reversephosphorolysis of a-maltose; (B) a-maltose synthesis from b-D-glucopyranosyl fluoride plus a-D-glucose. X represents a protein component whose interaction with the axial 1-OH of a-D-glucopyranose is required to activate all reactions promoted by the enzyme. Reproduced fromTsumuraya et al., Arch. Biochem. Biophys., 281 (1990) 58–65, with permission of AcademicPress.


TA

BL

EIV

Pro

pose

d Su

bdiv

isio

n of

Gly

cosy

lase

s B

ased

on

Stru

ctur

al F

eatu

res

Tha

t D

icta

te t

he S

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of product configuration in all reactions promoted by the lytic 1-MCO en-zymes in Table IV, including the hydration of enitols and slow hydrolysis ofb-D-glucosyl fluoride by a-glucosidases to form a-products.

In contrast, the lytic 2-MCO glycosylases are distinguished by the pos-session of a second structural feature which ensures that the inorganic co-substrate approaches the center from a direction opposite that of incomingorganic cosubstrates. In CBH II cellulase, a narrow channel restricts the ap-proach of water so that the hydrolytic product(s) are of a-configuration55;in other enzymes, a binding site positions the appropriate inorganic cosub-strate (H2O, Pi, or PPi) proximate to both the catalytic nucleophile and re-action center. That the findings with representative phosphorylases and py-rophosphorylases are comparable to those obtained with glycosidases is asign of the potential generality of the subdivision presented in Table IV.

Whether the same structural distinction extends to all enzymes classed asglycosyltransferases [EC 2.4]c is not known. Evidence on this point mustawait description of the positioning of cosubstrates in crystal structures ofrelevant enzyme–ligand complexes. However, one can envision, for exam-ple, that the many enzymes which act on uridine 59-(a-D-glucopyranosyldiphosphate) (UDP-a-D-glucose) plus a cosubstrate to form transfer prod-ucts of a-configuration may have a 1-MCO structure—with the UDP leav-ing group and the cosubstrate binding separately but with each a-orientedto the reaction center. Lactose synthase, which acts on UDP-a-D-galactoseto catalyze an inverting transfer reaction to glucose to form lactose plusUDP, may well have a 2-MCO structure with the UDP leaving-group (in-organic at its reactive pyrophosphate end) and D-glucose cosubstratebound in opposite orientations to the reaction center.

The evidence summarized in Tables II and III and presently used to char-acterize certain individual glycosidases as having a 1-MCO or 2-MCOstructure is enhanced by, and further extends, Withers’ observation3,11 thata simple parameter derived from crystal structures distinguishes “invert-ing” from “retaining” glycosidases.The authors found a greater average dis-tance between the opposed catalytic carboxyl groups in enzymes of the firsttype and assumed that this provides room for a water molecule to inter-vene.3,11 A separation between the opposed carboxyl oxygens (average offour possible distances), amounting to 4.8 6 0.5 Å for four a-amylases and5.3 6 0.2 Å for several “retaining” b-glycosidases, is recorded; in contrast, acombined distance of 9.0 6 1 Å is reported for beta-amylase and glu-coamylase and 9.5 Å for a third “inverting” enzyme, the E2 cellulase of Tri-choderma fusca.

In glycosidases, the calculated distance between the catalytic carboxyloxygen atoms correlates well with their stereochemical behavior in react-ing with glycosidic substrates. The averaged distance between the opposing



carboxyl oxygen atoms does not, however, necessarily represent the dis-tance between the two oxygens actually involved in catalyzing a reaction.For example, in soybean beta-amylase, OD2 of the nucleophile Glu380 andOD2 of the acid catalyst Glu186 on the opposite (a-) side of the reactioncenter are well positioned for catalyzing hydrolysis. However, whereas thecalculated average of possible distances between the carboxyl oxygens ofGlu380 and of Glu186 is 7.5 6 1.0 Å, the measured distance between theOE2 atoms presumably involved in catalysis is only 5.7 Å.101 Again, in therecently reported crystal structure of cellulase CelA of Clostridium ther-mocellum,81 shorter distances of 5.8 Å (or 7.5 Å) separate the carboxylgroup of the acid catalytic residue, Glu95, from that of the likely nucle-ophile, Asp152 (or Asp278) respectively, than projected3 for “inverting”enzymes. Nevertheless, an averaged distance extending across the centerevidently does identify “inverting” glycosidases, even though the measure-ments include some less directly relevant parts. The present interpretation(Tables II and III) emphasizes the location, in several inverting glycosi-dases, of an ordered water molecule between and closely proximate to thenucleophile and the reaction center. This supports the assumption3 that thegreater distance between the carboxyl oxygen atoms in enzymes of this typeis associated with the positioning of reactant water.

In a further study of broad significance, Withers and his associates11 ex-amined the activity of two mutants of Agrobacterium fecalis b-glucosidasein which the nucleophile Glu358 was replaced by Asp or Ala. TheGlu358Asp mutant, which presumably increased the average separation ofthe enzyme’s active-site carboxylate atoms by 1 Å, lowered by 2500-fold theactivity for 2,4-dinitrophenyl b-D-glucopyranoside but did not change thestereochemistry. The activity of the Glu358Ala mutant was some 107-foldlower than that of the wild-type enzyme, but the addition of an azide or formate nucleophile increased kcat 105-fold, that is, most of the way back toward the wild-type value. The reaction product was identified as a-D-glucosyl azide. Further checks confirmed the change-of-reaction ste-reochemistry from one of retention to one of inversion: wild-type b-glucosidase failed to release fluoride from a-D-glucopyranosyl fluoride,whereas the Glu358Ala mutant rapidly catalyzed an inverting D-glucosyltransfer reaction from one a-fluoride molecule to a second serving as co-substrate, to yield a product identified as a cellobiose derivative, presum-ably a-cellobiosyl fluoride.11

The observed change in the stereochemical behavior of the A. fecalis b-glucosidase mutant acting on an aryl b-D-glucoside11 does not arise solelyfrom the elimination of assistance to a b-directed attack by water, but de-pends also on the creation of a new site of access located as to permit an a-directed attack by azide. One would wish to know the relative proportion

EDWARD J. HEHRE294


of transfer product formation to hydrolysis in the altered reaction andwhether wild-type A. fecalis b-glucosidase forms detectable glucopyranosylazide from the glycosidic substrate in presence of azide.

A similar modification of the catalytic nucleophile, Glu233, of the Cex xy-lanase/cellulase of Cellulomonas fimi in the mutant Glu233Ala eliminatesthis enzyme’s ability to hydrolyze (with retention) 2,4-dinitrophenyl b-cellobioside. Catalytic activity is restored in the presence of azide, with theformation of a transfer product of inverted configuration, a-cellobiosylazide.133 A probable (obverse) example involves a covalent glycosyl–en-zyme adduct isolated from a digest of E. coli cell wall by the Thr26Glu mu-tant of phage T4 lysozyme.134,135 The authors considered this adduct to bea stabilized intermediate (a-linked to Glu26) in a double displacement in-volving final water attack from the b-side of the reaction center. On directstructural comparison, native unliganded T4 lysozyme shows a water mole-cule close to both Thr26 and the center. The authors suggested that naturalT4 lysozyme is an “inverting” enzyme which is converted by the Thr26Glumutation into one acting with retention.134,135 Direct establishment of thesteric course of hydrolysis catalyzed by native T4 lysozyme would appeardesirable, though difficult to achieve. The enzyme does not use the simplechito-oligosaccharides that allow the observation of hydrolysis with inver-sion by papaya lysozyme.136

In short, there is now a merging of basic information on the relation ofstructural elements to stereochemical behavior.The degree of separation ofthe two catalytic carboxyl groups of a glycosidase correlates with such be-havior,3 as does replacing the carboxyl group of the catalytic base by a smallspace open to solvent.11,133 The stereochemical behavior of a glycosidase orglycosyltransferase further correlates with its 1-MCO or 2-MCO type struc-ture (Table IV), even when the substrate lacks the proper configuration.The notable point is that the structural findings for several enzymes of 2-MCO type (Tables III and IV) confirm the assumption3 that the wider cat-alytic carboxyl-group separation in “inverting” glycosidases provides forthe intervention of a water molecule; it also confirms the significance of thealtered stereochemical behavior of mutant b-glycosidases whose nucle-ophilic carboxyl group is replaced by a space for a small entering nucle-ophile.11,133 The correspondence of these different observations about thedisposition of these several significant structural features, relative to eachother, is strongly supportive when other evidence shows that a glycosidase-catalyzed reaction occurs through a single or double “displacement” mech-anism. However, the findings assembled in Table IV can also accomodateother mechanisms, and this would encourage further investigation of reac-tions whose features do not readily accord with the view that “retaining”enzymes act only by way of a double-displacement mechanism.



IV. RELATION OF STEREOCHEMICAL BEHAVIOR TO CATALYTIC MECHANISM

1. Do 1-MCO (“Retaining”) Glycosylases Invariably Act via Double Displacements?

Over the past few decades the reactions of a number of “retaining” gly-cosylases have been reported to involve an oxocarbonium ion rather thana covalent intermediate. During the same period, however, a revised ver-sion of the traditional double-displacement mechanism has become widelyaccepted as the way “retaining” glycosidases function. Here, the requiredcovalent intermediate is reached via an oxocarbonium ionlike transitionstate in which the anomeric C-1 atom remains partly bonded to its originalaxial or equatorial substituent, with a second transition state interveningbetween the intermediate and cosubstrate. According to this model, theidea of an ion-pair intermediate is untenable—with the questionable ex-ception of hen’s egg-white lysozyme.137,15

The existence of a covalent intermediate has been inferred for a numberof reactions.15 Clear direct evidence has been presented by Withers and hisassociates, who recovered a covalent a-D-glucopyranosyl–enzyme adduct inthe hydrolysis of 2,4-dinitrophenyl 2-deoxy-2-fluoro-b-D-glucopyranosyideby the b-glucosidase of Agrobacterium fecalis138,139 and in the hydrolysis ofsimilar b-D-glycosides by the Cex cellulase/xylanase of C. fimi.133,140 Crys-tallographic studies confirm the structure of the intermediate in the lattercase87,88 and of a comparable intermediate observed for the complex ofwhite mustard seed myrosinase with 2-deoxy-2-fluoro-b-D-glucotrope-olin.141 Further covalent intermediates are demonstrated in reactions ofSaccharomyces cerevisiae a-glucosidase with 5-fluoro-a-D-glucopyranosylfluoride142,143 and in a reaction catalyzed by the a-D-glucanotransferasepart [EC 2.4.1.25] of glycogen debranching enzyme using 4-deoxy-a-maltotriosyl fluoride as a probe.144

The recovery of covalent intermediates in reactions involving transition-state destabilizing substrates provides clear evidence of the nucleophile’sidentity and mode of functioning in the reactions studied. Probes that dras-tically perturb the catalytic process through carbonium ion destabilizationallow the possibility that the catalytic nucleophile might act differently withordinary substrates. Thus far, however, kinetic studies and 2H kinetic iso-tope effects of reactions catalyzed with a range of aryl b-D-glucosides by theA. fecalis and C. fimi b-glycosidases give findings consistent with the pres-ence of a covalent intermediate.145,146

Yet, a number of investigators report the catalysis of reactions by various“retaining” enzymes that they consider to be inconsistent with a mechanismrequiring a covalent glycosyl–enzyme intermediate. For lysozyme, the crys-tallographic data show that OE2 of Asp52 and C-1 of b-MurNAc-GlcNAc-

EDWARD J. HEHRE296


MurNAc cannot approach one another closer than 2 Å and that the synlone pair orbitals on the Asp52 carboxylate are not favorably oriented toform a covalent bond with of C-1 of MurNAc. These findings appear tomake it most unlikely that a covalent a-linked glycosyl–enzyme intermedi-ate is formed.1,127,147,148 The counterargument that Asp52 is catalyticallyunimportant since the equivalent residue in bacteriophage-T4 lysozyme isreplaceable with little loss of activity73 appears fragile, since the stereo-chemical behavior of T4 lysozyme evidently differs from that of the hen’segg-white enzyme.134, 135 For glycogen phosphorylase, kinetic findings andcrystallographic evidence of the lack of a suitably disposed carboxylategroup that might act as a nucleophile have led to the view that catalysis oc-curs via an ion pair that collapses without involving a covalent b-D-glucosylintermediate117 or by a concerted mechanism without requiring a sequen-tial double inversion of configuration.119

The Glu461 mutant of E. coli lacZ b-galactosidase lacking the ionizableside chain of the catalytic residue, Glu46, has been reported to vigorouslycatalyze galactosyl transfer from 2-nitrophenyl b-D-galactopyranoside to azide to form b-D-galactopyranosyl azide (second-order rate constant4900 M21 s21). Wild-type enzyme gives no detectable reaction. The equilib-rium constant for galactosyl transfer by the mutant is .8000-fold higherthan for the wild-type enzyme.149 These findings demonstrate the predomi-nant reactivity of azide as nucleophile over water. In the presence of formateion, the galactosylated Glu461Gly mutant gives only D-galactose, suggestingthat carboxylate anions can provide general base catalysis of reaction inter-mediates with water. These and other findings indicate that replacement ofthe anionic side chain of Glu461 by hydrogen exposes an enzyme-stabilizedoxocarbonium ion to approach and attack by an external nucleophile andthat b-galactosidase acts via an ionic rather than a covalent intermediate.149

Again, the structure of porcine pancreatic a-amylase, in complex with thepseudotetrasaccharide acarbose shows the OD2 of Asp197 and the OE2 ofGlu233 (the presumed catalytic components) to be located 3.3 and 3.5 Å,respectively, from the cleavable glycosyl bond. These are much greater dis-tances than the length of a glycosylic bond and are reported to be support-ive of an ionic mechanism.99 On the other hand, a nuclear magnetic reso-nance study of a pancreatic alpha-amylase–[1-3H] maltotetralose mixture,set up in cold (2208) buffer containing 40% dimethyl sulfoxide and exam-ined under cryoscopic conditions, gave findings pointing to the presence ofa b-linked covalent adduct.150

Several other “retaining” glycosidases promote reactions where the find-ings are reported to be unsupportive of a mechanism requiring a covalentintermediate. For example, the a-glucosidases of A. niger, ungerminatedrice, and sugar beet seed catalyze the slow hydrolysis of the wrong (b) D-



glucopyranosyl fluoride anomer with the formation of a-D-glucose.52,53 Onemay argue that, on protonation of the disfavored (b-) substrate, a carbo-nium ionlike structure is formed which interacts with the catalytic nucle-ophile to form the same (b-linked) covalent intermediate as in the case ofa-linked substrates. However, such an intermediate is not expected to arisevia a transition state where C-1 remains weakly bonded to the original b-oriented fluorine substituent. The suggested (and more likely) mechan-ism involves one of the lone pairs on the pyranose ring oxygen assisting in cleavage of the C-1–F bond to form a stabilized carbonium ion inter-mediate, with attack by the structurally positioned water cosubstrate (a-oriented to the reaction center in 1-MCO a-glucosidases) assisted by thegeneral base catalyst (Fig. 1).53 Again, it is possible that the hydration ofglycals and other prochiral glycosyl donors catalyzed by various “retaining”glycosidases (Table I) also may not require a covalent intermediate. A gly-cosyl carbonium ion transition-state structure arising on protonation of aglycal would have neither an a- nor a b-substituent at C-1, unlike the tran-sition state specified15 for bimolecular reactions with chiral glycosyl donors.In the hydration of cellobial by CBH I cellulase, for example, an a-linkedcovalent intermediate might be reached via a carbonium ion or ion-typestructure—although the enzyme’s catalytic nucleophile, presumably havingjust protonated the glycal, may be ionized. Alternatively, an oxycarboniumion intermediate could be stabilized and attacked by a structurally posi-tioned water, assisted by the acid–base catalyst, to give a product of b-configuration as found for the hydration of cellobial or lactal.37

Finally, intestinal sucrase/isomaltase shows large secondary 2H isotopeeffects for hydrolysis of p-chlorophenyl a-D-glucopyranoside and a nearlyzero b1g value for p-substituted phenyl a-D-glucopyranosides, 151,152 sug-gesting the essentially complete protonation of the leaving group at thetransition state.15 Other examples of hydrolytic reactions showing an ex-tremely high ratio of bond breaking to bond formation at the transitionstate are discussed next.

2. Relationship of Transition-State Structure to the Stereochemistry andMechanism of Glycosylase Reactions

Transition-state structures computed from multiple kinetic isotope-effectdata have provided important information on the catalytic mechanisms ofglycosylases. This is exemplified by the refined transition-state structuresderived by Schramm and his associates and used to probe the hydrolytic re-actions catalyzed by nucleosidases and the ADPR (adenosine diphosphori-bosyl) transferase toxins of Vibrio cholerae and Corynebacterium diphthe-

EDWARD J. HEHRE298


riae.153–156 The methods employed in these studies153,154 were used byTanaka et al.157 to derive refined transition-state structures for the hydroly-sis of a-D-glucopyranosyl fluoride by two enzymes that differ in stereo-chemical behavior. The aim was to determine the relationship of transition-state character to the steric course of each reaction as well as to gaininformation about the catalytic mechanism(s). The hydrolysis promoted bythe a-glucosidase of sugar beet seed yields a-D-glucose; that catalyzed byRhizopus niveus glucoamylase yields b-D-glucose and shows large andequivalent a-secondary 3H and 2H kinetic isotope effects.75 To determinethe transition state for each enzyme, [1-3H, 6-14C]-, [2-3H, 6-14C]-, [6-3H, 6-14C]-, and [1-14C, 6-3H]-a-D-glucopyranosyl fluoride were hydrolyzed byeach; near-intrinsic a-, b-, and remote-secondary 3H and primary 14C kineticisotope effects were concurrently determined and subjected together tobond energy–bond order analysis.157

The modeled transition-state structures for hydrolysis by the a-glucosi-dase and the glucoamylase show significant oxocarbonium ion characterwith the D-glucosyl unit of each having a flattened 4C1 conformation con-sistent with a C-1–O-5 bond order of 1.92, even though opposite D-glucoseanomers are formed from the substrate. The transition-state structures showmodest differences but they do not predict the stereochemical outcome of thecatalyzed reactions.157

Table V lists transition-state features of special relevance to the mech-anism of hydrolysis with fluoride release by the a-glucosidase and


TABLE V

Features of the Anomeric Carbon Atom at the Transition State for the Hydrolysis of b-D-Ribofuranosyl–N and a-D-Glucopyranosyl–F Bonds

Bond Order (Bond Length, A)

Substrate Stereochemical C-1–F orGlycosylase Hydrolyzed Course C-1‘–N C-1‘–O’ Reference

Glucoamylase, a-Glucosyl a → b 0.045 (2.3) 0.010 (2.8) (157)R. niveus fluoride

a-Glucosidase, a-Glucosyl a → a 0.27 (1.7) 0.001 (3.5) (157)sugar beet seeds fluoride

ADP- NAD1 b → ?a 0.107 (2.2) 0.002 (3.3) (155)Ribosyltransferase,V. cholerae

a ADP-D-Ribosyl transfer products formed from NAD1 are of b-configuration.158 Configuration of theADP-D-ribose hydrolysis product has not been established.


glucoamylase as well as to that of the hydrolysis of oxidized nicotinamideadenine dinucleotide (NAD1) with cation release catalyzed by the V.cholerae toxin.The findings for each of the three reactions appear most con-sistent with an SN1 rather than an SN2 mechanism. The independently de-rived transition states for the sugar beet seed a-glucosidase157 and ADPR-transferase-catalyzed reactions155 have a notable point in common, namely,a barely detectable (0.001 or 0.002) C-1–O bond order and a too-great C–O“bond length” (3.5 or 3.3 Å) to constitute a bond. In each case, the extentof bond formation relative to substrate bond breaking is miniscule,,0.25%. Considering that each transition-state structure represents the av-erage of a range of structures for individual reactant complexes, a fair pro-portion of these may represent sufficiently stabilized oxocarbonium ions tohave a real existence until they undergo attack by a structurally positionedwater molecule.

Kinetic isotope-effect findings157 for hydrolysis of a-D-glucopyranosylfluoride by the sugar beet seed a-glucosidase indicate that the reaction oc-curs by other than the concerted SN2 mechanism found by Banait andJencks159 for the nonenzymatic and nonhydrolytic reactions of the samesubstrate in water containing added anionic nucleophiles as well as releasedfluoride. For this model, these authors found159 that an intimate ion paircould not exist as an intermediate species as its lifetime would be too short.The refined transition-state structure derived for hydrolysis of this sub-strate in hot water also indicates that the D-glucopyranosyl cation is too un-stable to exist in solution contact with the fluoride leaving group.160

Yet, it is not entirely appropriate to dismiss the mechanism reported forthe hydrolysis catalyzed by the sugar beet seed a-glucosidase157 on the ba-sis of its departure from the findings of respected nonenzymic solvolysismodels. For one thing, this particular enzymic reaction is but one of a clus-ter of mechanistically extreme reactions catalyzed by a group of closely re-lated enzymes and not an isolated example. Later studies show that the a-glucosidase of sugar beet seed shares the conserved amino acid sequencesof the aspartic acid catalyst region with A. niger and rice a-glucosidase as well as with rabbit intestinal sucrase and isomaltase.161–163 As previ-ously noted, the three a-glucosidases slowly hydrolyze the wrong (b-) D-glucopyranosyl fluoride anomer via a mechanism unlikely to involve a covalent intermediate;52,53 each also catalyzes the slow hydration of D-glucal by a reaction where the possibility of an ion-pair intermediate is notexcluded.45 Further, the large a-secondary kinetic 2H isotope effects ob-served for hydrolysis of [1,1-2H]-isomaltose by A. niger and sugar beet seeda-glucosidase under optimal-rate conditions are reported to support thepresence of a carbonium-ion intermediate at the transition state.10 In addi-tion, reactions promoted by the closely related mammalian sucrase and iso-

EDWARD J. HEHRE300


maltase with p-substituted aryl a-D-glucopyranosides also appear unusualin showing essentially complete protonation of the leaving group at thetransition state.151,152

As to the V. cholerae toxin-catalyzed hydrolysis of NAD1, the kinetic isotope-effect findings point to a mechanism providing nearly unimolecularN-glycosylic bond cleavage with minimal nucleophilic participation at thetransition state.155 These findings, when compared with a nonenzymic solu-tion model,164 also suggest that a glycosyl carbonium ion would be on theborderline of having a short but significant lifetime in water.155 A quantumstudy of the hydrolysis of b-D-ribofuranosyl nicotinamide1 in the gasphase165 finds that the C-1–N glycosylic bond must be broken almost en-tirely before solvent can react with the oxocarbonium-ion intermediate.Further, the transition state for hydrolysis with inversion shows a lower C-1–O bond order than that for hydrolysis with retention, contrary to indica-tions that inverting and retaining reactions pass through a single activatedcomplex.166 This distinction, however, is not observed in the enzymicallycatalyzed hydrolysis of a-D-glucopyranosyl fluoride (Table V).157

Indications exist that the hydrolysis of a-D-glucosyl fluoride by sugarbeet a-glucosidase157 and of NAD1 by V. cholerae toxin155,167 may involvea relatively desolvated reaction center at the transition state. The view alsohas been expressed that solvation may be critical to the stability of the b-D-ribosyl–nicotinamide linkage in solution since rapid dissociation into oxo-carbonium ion and nicotinamide occurs in the gas phase.167 The questionarises whether the findings of studies of nonenzymic reactions in aqueoussolution are always directly applicable to glycosylase-catalyzed reactions.Careful studies are needed to determine whether the structures of someglycosylase–substrate complexes may possibly shield the reaction centerfrom solvent during the catalytic process.

3. Structures That May Help Keep Solvent from the Catalytic Center inGlycosylase–Substrate Complexes

Glycosidases are extensively hydrated during their formation. Finalstructures contain many individual water molecules hydrogen-bonded tovarious amino acid residues and also may show localized pools of disor-dered solvent. Further, the catalytic groups of unliganded glycosidases gen-erally are sufficiently exposed to solvent to allow slow replacement of theirreactive carboxyl protons by deuterons in enzyme dissolved in 2H2O. Yet,all hydrolysis reactions catalyzed by a given glycosidase with whetheversubstrate yield product(s) of the same anomeric configuration. This resultindicates that the reaction center of a glycosidase–substrate complex is ac-cessible to a water molecule positioned to attack C-1 from one structurally



specified direction. The key question is whether C-1 and the catalytic car-boxyl groups of an enzyme–substrate complex are as exposed to bulk sol-vent as the catalytic groups of the unliganded enzyme. One can envisionthat, in some complexes, the close apposition of substrate and the many in-teractive protein residues may transiently displace disordered water fromproximity to those atoms involved in the catalytic chemistry.

One apparent example has been observed crystallographically.The struc-ture of soybean beta-amylase complexed with b-maltose shows two tandemmaltose molecules and some maltotetraose condensation product bound atthe same subsites. A mobile hinged loop at the active site, present mainly inan open conformation in the unligated enzyme, is found in closed confor-mation in the complex. It participates in ligand binding and also in seques-tering the reaction center from bulk solvent (Figs. 4 and 5).101 Measure-ments on the structure of the complex made with a 1.4-Å-diameterspherical probe168 show values of O Å2 (zero access to solvent) for theanomeric carbon atom and equatorial O-1 atom of the b-maltose donor;likewise, zero access to solvent for the reactive O-4 atom of the adjacentmaltose cosubstrate; also for the side chain of each of the catalytic residues.In unliganded beta-amylase (loop open), the side chain of each catalystresidue has free access to solvent.

Thus far, few other glycosidase active sites have been found to include amobile loop that closes on the ligand and adds to its binding. Porcine pan-creatic alpha-amylase shows such a loop sequence. On binding to acarbose,it moves toward the ligand and narrows the breadth of the active site cleft.One loop residue binds to a unit of the ligand from the solvent side, partlyprotecting the bound fragment from solvent.99 In other glycosidases, sim-pler structural means, such as the narrow close-fitting substrate bindingpocket in glucoamylase,103,104 could have a role, not yet critically evaluated,in displacing solvent from the reaction center.

Phosphorylases offer other examples of how the catalytic process may beprotected from solvent. In glycogen phosphorylase, water cannot act as acosubstrate since the phosphate of a-D-glucopyranosyl phosphate or thatused as D-glucosyl acceptor, when bound to the enzyme, has an essentialcatalytic role,117,119 which water bound in its place cannot assume. In thecase of maltose phosphorylase, only the a- but not the b-anomer of maltose(or analogs lacking an axial 1-OH group) serves as a donor substrate. Also,in reversal, the enzyme uses b-D-glucopyranosyl phosphate or fluoride asthe donor only when the D-glucose cosubstrate is the a-anomer.69 The axial1-OH required of a disaccharide substrate, and of the cosubstrate in rever-sal, binds to an unidentified protein component, X, located remote from thecatalytic center (Fig. 8). This required binding interaction limits the enzymeto the synthesis and phosphorolysis (arsenolysis) of a-maltose-type disac-

EDWARD J. HEHRE302


charides. Hydrolysis is negligible, as water bound at X is too far removed toattack C-1 at the reaction center.

4. Unresolved Issues

Analysis of the structures of various liganded glycosylases indicates thatspecific structural features of an enzyme limit the stereochemical outcomeof its reactions and provide a rational basis for distinguishing glycosylasesdiffering in stereochemical behavior. Glycosylases of the 1-MCO typestructurally orient all cosubstrates to C-1 at the reaction center in one par-ticular way; those of 2-MCO type provide one mode of orientation to C-1for water (or other appropriate inorganic cosubstrate) and a second, oppo-site, orientation mode for organic cosubstrates. Although present evidencefalls short of establishing the complete generality of this structural subdivi-sion, the assumption is not unreasonable. Both 1-MCO and 2-MCO en-zymes correspond to “retaining” and “inverting” glycosidases, respectively,when the comparison involves reactions where other evidence demon-strates nucleophilic substitution(s) to be effected by the catalytic groups.According to the latter (mechanistic) subdivision,15 reaction-product con-figuration derives from that of the substrate via the action(s) of the en-zyme’s catalytic groups. That the stereochemical outcome of a retaining re-action catalyzed via a double-displacement mechanism corresponds to theoutcome required by an enzyme’s 1-MCO structure does not mean that thisparticular mechanism is needed to meet the enzyme’s structural constraintas to cosubstrate orientation.The latter allows for the possibility that the re-actions of some “retaining” glycosylases may not proceed via a covalent gly-cosyl–enzyme intermediate. As discussed earlier, such reactions are re-ported to be catalyzed by lysozyme, b-galactosidase, pancreatic alpha-amylase,glycogen phosphorylase, and several genetically related a-glucosidases.Proposals that these reactions may proceed by way of an oxycarbonium-ionintermediate have been opposed in general on the basis that the reactionsare bimolecular and that the very short lifetime of oxocarbonium ions inwater precludes their existance as transient intermediates in enzyme-catalyzed reactions.15,169

One observation raises the question of whether enough is known aboutthe carbonium-ion lifetime in enzymic sites to be certain that it is always asshort as estimated for reactions in water. The crystal structure of soybeanbeta-amylase complexed either with b-maltose or maltal shows featuresstrongly suggesting that the reaction center is shielded from solvent.101 Amechanism involving a carbonium-ion transition state with C-1 unsubsti-tuted and subject to attack by a structurally positioned water molecule,to form a product of b-configuration, is likely for the inverting reactions



catalyzed by this enzyme. Studies of the structures of “retaining” enzyme complexes would appear desirable in order to gain some insight into the extent to which ligand binding may displace solvent from the catalytic center in such complexes. Techniques are available for measuring access tosolvent of the reactive groups at the catalytic center169 as well as for inves-tigating the electrostatic fields generated at the protein surface that might,as reported for lysozyme170 and suggested for cellulase CBH II,25,73 helpstabilize a transient carbonium ion. Among the glycosidase-substrate com-plexes which might be studied are several showing noncovalently boundligand with the reactive glycosyl unit in a distorted (sofa-type) conforma-tion1,127,132,171 and others showing a covalently linked glycosyl–enzyme in-termediate.88,132,141

A final point concerns the progress made on aspects of stereochemistryuncovered in earlier studies. The findings showed that small nonglycosidic(especially enolic) glycosyl donors are protonated by certain glycosidasesfrom a different direction than the enzyme’s glycosidic substrates, yet pro-vide reactions having the same stereochemical outcome as similar reactionscatalyzed with “normal” substrates.26,31 The indication from these findingsthat an enzyme’s catalytic groups can function flexibly but that reactionproduct configuration is ultimately controlled topologically is presentlyconfirmed. The reported active-site structures of representative “inverting”and “retaining” glycosidases show that a residue identified as the catalyticnucleophile in the tested enzymes is suitably positioned to protonate anearby double bond. The overall crystal structures further show featuresthat specifically orient cosubstrates to the catalytic groups and reaction cen-ter in ways consonant with the stereochemical outcome of reactions cat-alyzed with substrates of either disfavored or proper configuration.Whether the presently established mechanisms for some glycosylase-catalyzed reactions are applicable to all reactions to the point of excludingother mechanisms is not yet settled, and further investigations are to be en-couraged in efforts to do so. At least the time has come for the terms “re-taining” and “inverting” enzymes to be put in quotation marks.

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INTERNATIONAL UNION OF PURE AND APPLIED CHEMISTRYAND

INTERNATIONAL UNION OF BIOCHEMISTRY AND MOLECULAR BIOLOGY JOINT COMMISSION ON

BIOCHEMICAL NOMENCLATURENOMENCLATURE OF GLYCOLIPIDS*

(Recommendations, 1997)

Prepared for publication by M. Alan Chester, Blodcentralen,Universitetssjuhuset i Lund, Sweden

GL-1. General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310GL-2. Generic Terms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

2.1. Glycolipid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3112.2. Glycoglycerolipid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3112.3. Glycosphingolipid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3112.4. Glycophosphatidylinositol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3112.5. Psychosine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3122.6. Other names . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

GL-3. Principles of Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3123.1. Number of Monosaccharide Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

311


* These are recommendations of the IUPAC-IUBMB Joint Commission on BiochemicalNomenclature (JCBN), whose members are A. Cornish-Bowden (Chairman, France), A. J.Barrett (UK), R. Cammack (UK), M. A. Chester (Sweden), D. Horton (USA), C. Liébecq(Belgium), K. F. Tipton (Ireland), and B. J. Whyte (Secretary, Switzerland).

JCBN thanks a panel convened by C. C. Sweeley (USA), whose members were S. Basu(USA), H. Egge (Germany), G. W. Hart (USA, co-opted), S. Hakomori (USA), T. Hori(Japan: deceased 1994), P. Karlson (Germany), R. Laine (USA), R. Ledeen (USA), B.Macher (USA), L. Svennerholm (Sweden), G. Tettamanti (Italy), and H. Wiegandt (Ger-many), for drafting the recommendations; and other present or former members of theNomenclature Committee of IUBMB (NC-IUBMB), former members of JCBN, and invitedobservers, namely A. Bairoch (Switzerland), H. Berman (USA), C. R. Cantor (USA), H. B.F. Dixon (UK), M. A. C. Kaplan (Brazil), K. L. Loening (USA), A. McNaught (UK), G. P.Moss (UK), J. C. Rigg (The Netherlands),W. Saenger (Germany), N. Sharon (Israel), P.Vene-tianer (Hungary), and J. F. G. Vliegenthart (The Netherlands).

Acknowledgement. This document was first published in Pure Appl. Chem. 69, 2475–2487(1997): © 1997 IUPAC.

A World Wide Web version, prepared by G. P. Moss, is available at http://www.chem.qmw.ac.uk/iupac/misc/glylp.html.

0096-5332/00 $30.00


3.2. Naming of Monosaccharide Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3123.3. Use of Symbols for Defining Oligosaccharide Structures . . . . . . . . . . . . . . 3123.4. Ring Size and Conformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313

GL-4. Classification of Glycolipids Based on Their Lipid Moieties. . . . . . . . . . . . . . . . 3144.1. Glycoglycerolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3144.2. Glycophosphatidylinositols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3154.3. Glycosphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316

GL-5. Neutral Glycosphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3175.1. Monoglycosylceramides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3175.2. Diosylceramides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3175.3. Neutral Glycosphingolipids with Oligosaccharide Chains . . . . . . . . . . . . . . 317

GL-6. Acidic Glycosphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3196.1. Gangliosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3196.2. Glycuronoglycosphingolipids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3206.3. Sulfatoglycosphingolipids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3206.4. Phosphoglycosphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3206.5. Phosphonoglycosphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321

GL-7. Short Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3217.1. Recommended Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3227.2. The Svennerholm Abbreviations for Brain Gangliosides . . . . . . . . . . . . . . 322

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323

GL-1. GENERAL CONSIDERATIONS

Glycolipids are glycosyl derivatives of lipids such as acylglycerols, cer-amides, and prenols. They are collectively part of a larger family of sub-stances known as glycoconjugates. The major types of glycoconjugates areglycoproteins, glycopeptides, peptidoglycans, proteoglycans, glycolipids, andlipopolysaccharides. The structures of glycolipids are often complex anddifficult to reproduce in the text of articles and certainly cannot be referredto in oral discussions without a nomenclature that implies specific chemicalstructural features.

The 1976 recommendations1 on lipid nomenclature contained a sec-tion (Lip-3) on glycolipids, with symbols and abbreviations as well as triv-ial names for some of the most commonly occurring glycolipids. Since then, more than 300 new glycolipids have been isolated and character-ized, some having carbohydrate chains with more than 20 monosaccha-ride residues and others with structural features such as inositol phos-phate. The nomenclature needs to be convenient and practical as well as extensible to accommodate newly discovered structures. It should also be consistent with the nomenclature of glycoproteins, glycopep-tides, and peptidoglycans,2 oligosaccharides,3 and carbohydrates in general.4

This chapter supersedes the glycolipid section in the 1976 Recommenda-tions on lipid nomenclature.1

NOMENCLATURE OF GLYCOLIPIDS312


GL-2. GENERIC TERMS

GL-2.1. Glycolipid

The term glycolipid designates any compound containing one or moremonosaccharide residues bound by a glycosidic linkage to a hydrophobicmoiety such as an acylglycerol, a sphingoid, a ceramide (N-acylsphingoid),or a prenyl phosphate.

GL-2.2. Glycoglycerolipid

The term glycoglycerolipid is used to designate glycolipids containingone or more glycerol residues.

GL-2.3. Glycosphingolipid

The term glycosphingolipid designates lipids containing at least onemonosaccharide residue and either a sphingoid or a ceramide. The gly-cosphingolipids can be subdivided as follows.

A. Neutral glycosphingolipids1. Mono-, oligo-, and polyglycosylsphingoids2. Mono-, oligo-, and polyglycosylceramides

B. Acidic glycosphingolipids1. Sialoglycosphingolipids (gangliosides, containing one or more

sialic acid residues)2. Uronoglycosphingolipids (containing one or more uronic acid

residues)3. Sulfoglycosphingolipids (containing one or more carbohydrate-

sulfate ester groups)4. Phosphoglycosphingolipids (containing one or more phosphate

mono- or diester groups)5. Phosphonoglycosphingolipids [containing one or more (2-

aminoethyl)hydroxyphosphoryl groups]

GL-2.4. Glycophosphatidylinositol

The term glycophosphatidylinositol is used to designate glycolipids which contain saccharides glycosidically linked to the inositol moiety ofphosphatidylinositols (e.g., diacyl-sn-glycero-3-phosphoinositol), inclusiveof lyso- (Lip-2.6 in ref. 1) species and those with various O-acyl-, O-alkyl-,O-alk-1-en-1-yl- (e.g., plasmanylinositols5), or other substitutions on theirglycerol or inositol residues.

NOMENCLATURE OF GLYCOLIPIDS 313


GL-2.5. Psychosine

Psychosine was coined historically to designate a monoglycosylsphingoid(i.e., not acylated). The use of this term is not encouraged (Lip-3.4 in ref. 1).

GL-2.6. Other Names

Other terms such as fucoglycosphingolipid, mannoglycosphingolipid, xy-loglycosphingolipid, and so on may be used when it is important to high-light a certain structural feature of the glycolipid.

GL-3. PRINCIPLES OF NOMENCLATURE

GL-3.1. Number of Monosaccharide Residues

The number of monosaccharide residues in an oligosaccharide is indi-cated by suffixes such as “diosyl,” “triaosyl,” “tetraosyl,” and so on.1,6

Thus, the general name for the oligosaccharide residue of all glycosphin-golipids containing 10 monosaccharide residues is “glycodecaosyl”; itmight be a glycodecaosylceramide or a 3-glycodecaosyl-1,2-diacyl-sn-glycerol.

Note 1: “diosyl” not “biosyl” is the correct suffix.Note 2: The “a” in “tetraosyl,” and so on is not elided in order to differ-

entiate a tetrasaccharide residue (tetraosyl) from a four-carbon sugar (tet-rose), and so on. The “a” in “triaosyl” is added for a similar reason.

Recommendations have been made for the nomenclature of oligosac-charides.3,4

GL-3.2. Naming of Monosaccharide Residues

Monosaccharide residues are named and abbreviated (Table I) accordingto the proposed nomenclature recommendations for carbohydrates4 (seealso the nomenclature of glycoproteins2).The D- and L-configurational sym-bols are generally omitted; all monosaccharides are D with the exception offucose and rhamnose, which are L unless otherwise specified.

GL-3.3. Use of Symbols for Defining Oligosaccharide Structures

Using the condensed system of carbohydrate nomenclature (ref. 2, sec-tion 3.7: ref. 4, 2-Carb-38.5), positions of glycosidic linkages and anomericconfigurations are expressed in parentheses between the monosaccharideresidues that are thus linked. This principle should be adhered to with fullnames as well as with the abbreviated structures. A “short form” for repre-



senting sequences more briefly can be used for specifying large structures.Positions of glycosidic linkages are still given, but the number of theanomeric carbon is omitted, since this is invariable for each monosaccha-ride, i.e., C-1 for Glc, and so on; C-2 for Neu5Ac, and so on.

Examplea-D-Galp-(1 → 3)-a-D-Galp- (extended form)

or Gal(a1-3)Gal(a- (condensed form)or Gala3Gala- or Gala-3Galaa- (short form)

GL-3.4. Ring Size and Conformation

Ring size and conformation should be designated only when firmly es-tablished from NMR or other experimental data. Previously published


TABLE I

Recommended Abbreviations for Some Monosaccharides,Derivatives, and Related Compounds

Name Symbol

N-Acetylgalactosamine GalNAcN-Acetylglucosamine GlcNAcN-Acetylneuraminic acida Neu5Ac or NeuAc5,9-N,O-Diacetylneuraminic acida Neu5,9Ac2

Fucose (6-deoxygalactose) FucGalactitol Gal-olGalactosamine GalNGalactopyranose 3-sulfate Galp3SGalactose GalGalacturonic acid GalAGlucitol Glc-olGlucosamine GlcNGlucose GlcGlucose 6-phosphate Glc6PGlucuronic acid GlcAN-Glycoloylneuraminic acida Neu5Gc or NeuGcmyo-Inositolb InsMannose Man4-O-Methylgalactose Gal4MeRhamnose RhaXylose Xyl

a Acylated neuraminic acids and other derivatives of neuraminicacid may also be called sialic acids (abbreviated Sia) when the na-ture of the N-acyl substituent(s) is not relevant or is unknown.7

b myo-Inositol with the numbering of the 1D-configuration.8


recommendations on the specification of conformation should be con-sulted.9,10

Examplea-D-galactopyranosyl-4C1-(1 → 3)-a-D-galactopyranosyl-4C1-

or Galp4C1a3Galp4C1a-

Subsequently, examples will usually be in the more traditional form withparentheses and both anomeric locants, as, for example, Gal(b1-4)Glc-, butit is understood that the short form (i.e., Galb4Glc-) is also acceptable.

GL-4. CLASSIFICATION OF GLYCOLIPIDS BASED ON THEIR LIPID MOIETIES

GL-4.1. Glycoglycerolipids

Esters, ethers, and glucose derivatives of glycerol are designated by a pre-fix, denoting the substituent, preceded by a locant. As previously discussedin detail1, the carbon atoms of glycerol are numbered stereospecifically,with carbon atom 1 at the top of the formula shown below. To differentiatethis numbering system from others that have been used, the glycerol is al-ways accompanied by the prefix sn (for stereospecifically numbered, Lip-1.13 in ref. 1) in systematic and abbreviated names.


Example

1,2-di-O-acyl-3-O-b-D-galactosyl-sn-glycerol


GL-4.2. Glycophosphatidylinositols

4.2.1.—Glycophosphatidylinositol (GPI) nomenclature should incorpo-rate the accepted IUB-IUPAC recommendations1,2 for the naming ofphospholipids and the glycan portions of glycolipids or glycoproteins.Whilethe diversity of glycophosphatidylinositol structures is only beginning to berealized (for reviews see refs. 11 and 12), many appear to have a common“core.”

“Core” structure of glycophosphatidyinositols


Xaa 5 C-terminal residueR 5 acyl, alkyl, etc., side chains

4.2.2.—Glycophosphatidylinositols covalently attached to polypeptidesare termed “GPI-anchors.” Generally, such anchors are covalently attachedto the C-terminus of a polypeptide via an amide linkage to 2-aminoethanol,which is linked to the terminal core mannose residue via a phosphodiesterbond on O-6 of the mannose. A core Mana2Mana6Mana4GlcNa6 glycanstructure is attached to the inositol (generally D-myo-inositol) of phos-phatidylinositol. The nonacetylated GlcN is a characteristic feature ofglycophosphatidylinositols. Anchor structures appear to vary considerablyboth in terms of modifications on the core glycan and with respect toadditional modifications of the inositol residue. Free glycophosphatidyl-inositols have generically been termed “glycoinositolphospholipids” todistinguish them from those covalently attached to proteins or largerglycan structures.


GL-4.3. Glycosphingolipids

4.3.1.—A glycosphingolipid is a carbohydrate-containing derivative of asphingoid or ceramide. It is understood that the carbohydrate residue isattached by a glycosidic linkage to O-1 of the sphingoid.

4.3.2.—Sphingoids are long-chain aliphatic amino alcohols. The basicchemical structure is represented by the compound originally called“dihydrosphingosine” [2S,3R)-2-aminooctadecane-1,3-diol]. This sphingoidshould now be referred to1 as sphinganine (I).

The terms sphinganine, sphing-4-enine, and so on imply a chain length of18 carbon atoms. Chain-length homologs are named by the root chemicalname of the parent hydrocarbon. For example, the sphingoid with 20 car-bon atoms is icosasphinganine and the sphingoid with 14 carbon atoms istetradecasphinganine.

Unsaturated derivatives of sphinganine and other sphingoids should bedefined in terms of the location and configuration of each olefinic center.The most commonly occurring unsaturated sphingoid was originally called“sphingosine” [(2S,3R,4E)-2-aminooctadec-4-ene-1,3-diol]. It should nowbe referred to as (E)-sphing-4-enine (II). The trivial name “sphingosine”can be retained. As a second example, a C18 sphingoid with two trans dou-ble bonds at 4,14 should be called (4E,14E)-sphinga-4,14-dienine.

Substituents such as hydroxy, oxo, methyl, and so on are referred to byappropriate suffixes that denote the position of each substituent.The sphin-goid containing a hydroxyl group at C-4 of sphinganine was originallycalled phytosphingosine. According to the nomenclature adopted in 19761,it should be called (2S,3S,4R)-2-aminooctadecane-1,3,4-triol. A trivial (butincorrect) name is (R)-4-hydroxysphinganine (III).



4.3.3.—Ceramides are N-acylated sphingoids. The fatty acids of naturallyoccurring ceramides range in chain length from about C16 to about C26

and may contain one or more double bonds and/or hydroxy substituents at C-2. The complete chemical name for a specific ceramide includes thesphingoid and fatty acyl substituents. For example, a ceramide containing 2-hydroxyoctadecanoic acid and sphing-4-enine should be called (E)-N-(2-hydroxyoctadecanoyl)sphing-4-enine.

GL-5. NEUTRAL GLYCOSPHINGOLIPIDS

GL-5.1. Monoglycosylceramides

The trivial name “cerebroside” was historically used for the substancefrom brain, b-galactosyl(1 ↔ 1)ceramide, and was later modified to includeb-glucosyl(1 ↔ 1)ceramide from the spleen of a patient with Gaucher’s disease. It has become a general term for these two kinds of monoglycosyl-ceramides. However, since other monosaccharides are found in this class,the more structurally explicit terms such as glucosylceramide (GlcCer or,better, Glcb1Cer), galactosylceramide (GalCer), xylosylceramide (XylCer),and so on should be used.

GL-5.2. Diosylceramides

Diosylceramides may be named systematically, e.g., b-D-galactosyl-(1 ↔ 4)-b-D-glucosyl-(1 ↔ 1)-ceramide. However, it is often more conve-nient to use the trivial name of the disaccharide and call the structure givenabove lactosylceramide (LacCer).

GL-5.3. Neutral Glycosphingolipids with Oligosaccharide Chains

5.3.1.—Systematic names for glycosphingolipids with larger oligosac-charide chains become rather cumbersome. It is therefore recommended touse semisystematic names in which trivial names for “root” structures areused as a prefix. The recommended root names and structures are given inTable II.

The name of a given glycosphingolipid is then composed of (rootname)(root size)osylceramide. Thus, lactotetraosylceramide designates thesecond structure listed in Table II linked to a ceramide. When referring toparticular glycose residues Roman numerals are used (Lip-3.9 in ref. 1),counting from the ceramide (see Table II).

The use of the prefix “nor-” for unbranched oligosaccharide chainsshould be abandoned since this prefix has a well-defined meaning (“onecarbon atom less”) in organic chemistry nomenclature.



5.3.2.—The root name applies also to structures that are shorter than theroot given in Table II. Thus, gangliotriaosylceramide is the name for thestructure GalNAcb4Galb4GlcCer, where the fourth, terminal residue ismissing. The trisaccharides obtained from the lacto and neolacto series areidentical and in this case the former (shorter) name should be used.

5.3.3.—In the lacto series, the residues III and IV can form a repeatingunit. Thus, names like neolactohexaosylceramide (not recommended) havebeen used, even though the chemical nature of the two glycose residues atthe nonreducing end are not explicit in the name.

Exampleb-D-Galp-(1 → 4)-b-D-GlcpNAc-(1 → 3)-b-D-Galp-(1 → 4)-b-D-GlcpNAc-(1 → 3)-b-D-Galp-(1 → 4)-b-D-Glcp-(1 ↔ 1)Cer

or Galb4GlcNAcb3Galb4GlcNAcb3Galb4GlcCeror Galb-4GlcNAcb-3Galb-4GlcNAcb-3Galb-4GlcCer

The correct name is b-(N-acetyllactosaminyl)-(1 → 3)-neolactotetraosyl-ceramide, where N-acetyllactosaminyl is b-D-Galp-(1 → 4)-D-GlcNAc-.

5.3.4.—Substances containing glycose residues that are not part of a rootstructure should be named by referral to the root oligosaccharide andlocating the additional substituents by a Roman numeral designating theposition of the substituent in the root oligosaccharide (counting from theceramide end) to which the substituent is attached, with an arabic numeral


TABLE II

Root Names and Structures

Root Symbol Root Structure

IV III II IGanglio Gg Galb3GalNAcb4Galb4Glc-Lactoa Lc Galb3GlcNAcb3Galb4Glc-Neolactob nLc Galb4GlcNAcb3Galb4Glc-Globo Gb GalNAcb3Gala4Galb4Glc-Isoglobob iGb GalNAcb3Gala3Galb4Glc-Mollu Mu GlcNAcb2Mana3Manb4Glc-Arthro At GalNAcb4GlcNAcb3Manb4Glc-

a Lacto as used here should not be confused with lactose.b The prefix “iso-” is used here to denote a (1 → 3) versus (1 → 4)

difference in the linkage position between the monosaccharide residuesIII and II, while the term “neo-” denotes such a difference [(1 → 4) ver-sus (1 → 3)] between residues IV and III. This scheme should be usedalso in other cases where such positional isomers occur and only in suchcases.


superscript indicating the position on that residue which is substituted. Theanomeric configuration should also be specified.

Examples(i) III2-a-fucosylglobotriaosylceramide

or Fuca2Gala4Galb4GlcCeror III2-a-Fuc-Gb3Cer

(ii) II2-b-xylosylmollutetraosylceramideor GlcNAcb2Mana3(Xylb2)Manb4GlcCeror II2-b-Xyl-Mu4Cer

5.3.5.—Branched structures should be designated in a systematic manner,locating substituents in correlation with the Haworth structure of the multiplysubstituted monosaccharide.This principle should be applied in full structuresas well as linear formulations, wherein substituents are in one or more sets ofsquare brackets. Such names and abbreviations should refer to the substituenton the highest carbon number of the branched monosaccharide first andproceed toward the substituent on the lowest carbon number.This recommen-dation is consistent with the nomenclature of glycoproteins, glycopeptides, andpeptidoglycans,2 although not explicitly stated therein.

Note: When root names (see GL-5.3.1) are used, the branches should betreated as side chains and named accordingly even when linked to a carbonatom with a higher number than the member of the root oligosaccharide. Inoligosaccharide nomenclature4 the longest chain is the parent structure. Iftwo chains are of equal length the one with lower locants at the branchpoints is preferred, although some oligosaccharides are traditionally de-picted otherwise—frequently NeuAc and Fuc derivatives.

ExampleGalNAcb4Galb4Glc-

|Neu5Aca3

or GalNAcb4(Neu5Aca3)Galb4Glc-.Otherwise in ref. 4: Neu5Aca3(GalNAcb4) Galb4Glc-

or II3-a-Neu5Ac-Gg3-

GL-6. ACIDIC GLYCOSPHINGOLIPIDS

GL-6.1. Gangliosides

Gangliosides are sialoglycosphingolipids.They are named as N-acetyl- or N-glycoloyl-neuraminosyl derivatives of the corresponding neutral glycosphin-golipid, using the nomenclature given in GL-5.3.The position of the sialic acidresidue(s) is indicated in the same way as is the case of a branched structure.



ExampleIV3-a-N-II3-a-N-acetylneuraminosylgangliotetraosylpceramide

or Neu5Gca3Galb3GalNAcb4Galb4GlcCer|

Neu5Aca3or IV3-a-Neu5Gc,II3-a-Neu5Ac-Gg4Cer

Gangliosides containing neuraminic acid residues (with O-acyl or othersubstituents) should be named accordingly, with the positions of the sub-stituents given.

ExampleIV3-a-N-N-acetylneuraminosylgangliotetraosylceramide

or Neu5,9Ac2a3Galb3GalNAcb4Galb4GlcCer|

Neu5Aca3or IV3-a-Neu5,9Ac2,II3-a-Neu5Ac-Gg4Cer.

GL-6.2. Glycuronoglycosphingolipids

These are best named according to the guidelines of GL-5.2 and GL-5.3.Special root names have not yet been assigned.

GL-6.3. Sulfoglycosphingolipids

These are glycosphingolipids carrying a sulfate ester group, formerlycalled “sulfatides.” They are sometimes termed sulfatoglycosphingolipids.

Sulfoglycosphingolipids may also be named as sulfate esters (sulfates) ofthe neutral glycosphingolipids (see GL-5).

ExampleII3-sulfo-LacCer

or lactosylceramide II3-sulfate

GL-6.4. Phosphoglycosphingolipids

Two types of glycosphingolipids containing phosphodiester bonds areknown: (i) those containing a 2-aminoethyl phosphate residue esterified toa monosaccharide residue, and (ii) those with a phosphodiester bridge be-tween an inositol residue and the ceramide moiety.

Those of the first type can be easily named by analogy to the sulfogly-cosphingolipids.

ExampleIII6-(2-aminoethanolphospho)arthrotriaosylceramide

or 6(EtnP)-GlcNAcb3Manb4GlcCeror III6-Etn-P-At3Cer



The second type can be named as inositolphosphoceramide derivatives

Examplea-(N-acetyllactosaminyl)-(1 → 4)-a-glucuronosyl-(1 → 2)-inositolphosphoceramide

or Galb4GlcNAca4GlcAa2Ins-1-P-Cer

GL-6.5. Phosphonoglycosphingolipids

These are glycolipids esterified with an alkylphosphono acid, i.e., a com-pound containing a C–P bond. Their nomenclature is best derived using theprefix phosphoryl that denotes the trivalent radical O5P←. The residue


may be termed (2-aminoethyl)hydroxyphosphoryl. The location of thisgroup is given in the same way as other ester groups.

Example(4-O-methyl-b-D-galactopyranosyl)-(1 → 3)-(2-acetamido-2-deoxy-b-D-galac-topyranosyl)-(1 → 3)-[a-L-fucopyranosyl-(1 → 4)]-(2-acetamido-2-deoxy-b-D-glucopyranosyl)-(1 → 2)-a-D-mannopyranosyl-(1 → 3)-[a-D-xylopyranosyl-(1 → 2)]-6-[(2-aminoethyl)hydroxyphosphoryl]-b-D-mannopyranosyl-(1 → 4)-b-D-glucopyranosyl-(1 ↔1 )-ceramide

or Gal4Meb3GalNAcb3(Fuca4)GlcNAcb2Mana3(Xyla2)-6-(NH2-CH2CH2-P(OH)5O)Manb4GlcCer

or OH|

NH2CH2CH2-P5O|6

Gal4Meb3GalNAcb3GlcNAcb2Mana3Manb4GlcCer| |

Fuca4 Xyla2

GL-7. SHORT ABBREVIATIONS

There are no easy solutions to the dilemma that has arisen from the dis-covery of so many (nearly 300) glycosphingolipids of diverse structures.Short abbreviations are so attractive that a logical system, with broad ap-plication to more complex compounds, is desirable.


GL-7.1. Recommended Abbreviations

A system already used (GL-5.3) is based on the abbreviated root namesof the oligosaccharide structures. The full root structures are tetrasaccha-rides, and sequential removal of terminal monosaccharide residues givessmaller, precisely defined structures. Elongation of root tetrasaccharides is,on the other hand, undefined and hence ambiguous. The root name may beused, followed by an arabic number indicating the total number of mono-saccharide residues. A lowercase letter can be added to differentiate be-tween particular compounds.

Example(i) Galb3GalNAcb3Gala4Galb4GlcCer

or IV3-b-Gal-Gb4Cer(ii) GalNAca3GalNAcb3Gala4Galb4GlcCer

or IV3-a-GalNAc-Gb4Cer.

Either of these compounds could, after definition, be referred to as Gb5Cer.In the presence of both structures the abbreviations Gb5a and Gb5b may bedefined and used. It is recommended that the use of “Ose,” as inGbOse4Cer, be discontinued.

Since this short form sometimes leads to ambiguities, the full structureshould be given once in a paper or in a footnote, using the abbreviated fromaccording to GL-5.3.

GL-7.2. The Svennerholm Abbreviations for Brain Gangliosides

In this system, the fact that we are dealing with gangliosides is indicatedby the letter G and the number of sialic acid residues is stated by M formono-, D for di-, T for tri-, and Q for tetra-sialoglycosphingolipids. A num-ber is then assigned to the individual compound which referred initially toits migration order in a certain chromatographic system.13

Though these designations are far from being systematic, and it is impos-sible to derive the structure from them, they have the advantage of beingshort and well understood since they have been in use for a long time.A listof these abbreviations is given in Table III.

Since there is no clear-cut system in these abbreviations, it is not recom-mended to extend the list by coining new symbols of this kind.As a result, thefollowing two cases are examples of abbreviations that should not be used.

1. A disialoganglioside, Neu5Aca3Galb3(Neu5Aca6)GalNAcb4Galb4GlcCer, has been abbreviated GD1a. This practice should be discontinued. Therecommended abbreviation for this compound is IV3-a-Neu5Ac,III6-a-Neu5Ac-Gg4Cer.




2. The system has been extended to gangliosides of other “root” types,such as those derived from lactotetraosylceramide. An example of this kindis the widely distributed ganglioside called sialoparagloboside, Neu5Aca-3Galb4GlcNAcb3Galb4GlcCer, which has at times been abbreviated LM1,but should be referred to as IV3-a-Neu5Ac-nLc4Cer.

Attempts to abbreviate more complex glycosphingolipids derived fromthese examples have resulted in other illogical abbreviations, such as Fuc-39-LM1 for Neu5Aca3Galb4(Fuca3)GlcNAcb3Galb4GlcCer (IV3-a-Neu5Ac,III3-a-Fuc-nLc4Cer).

More information on the structures of various glycolipids and the bio-logical material from which they were obtained may be found in several re-views.14–16

REFERENCES

(1) IUPAC-IUB Commission on Biochemical Nomenclature (CBN). The nomenclature oflipids (Recommendations 1976). Eur. J. Biochem., 79 (1977) 11–21; Hoppe-Seylers Z.Physiol. Chem., 358 (1977) 617–631; Lipids, 12 (1977) 455–468; Mol. Cell. Biochem., 17(1977) 157–171; Chem. Phys. Lipids, 21 (1978) 159–173; J. Lipid Res., 19 (1978) 114–128;Biochem. J., 171 (1978) 21–35.

(2) IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). Nomenclature ofglycoproteins, glycopeptides and peptidoglycans (Recommendations 1985). Eur. J.Biochem., 159 (1986) 1–6; Glycoconjugate J., 3 (1986) 123–134; J. Biol. Chem., 262 (1987)13–18; Pure Appl. Chem., 60 (1988) 1389–1394; Royal Society of Chemistry Specialist Pe-riodical Report, Amino Acids and Peptides, Vol. 21, 1990, 329.

(3) IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN).Abbreviated ter-minology of oligosaccharide chains (Recommendations 1980). Eur. J. Biochem., 126

TABLE III

Some Abbreviations Using the Svennerholm System

Structure Abbreviationa

Neu5Aca3Galb4GlcCer GM3GalNAcb4(Neu5Aca3)Galb4GlcCer GM2Galb3GalNAcb4(Neu5Aca3)Galb4GlcCer GM1aNeu5Aca3Galb3GalNAcb4Galb4GlcCer GM1bNeu5Aca8Neu5Aca3Galb4GlcCer GD3GalNAcb4(Neu5Aca8Neu5Aca3)Galb4GlcCer GD2Neu5Aca3Galb3GalNAcb4(Neu5Aca3)Galb4GlcCer GD1aGalb3GalNAcb4(Neu5Aca8Neu5Aca3)Galb4GlcCer GD1bNeu5Aca8Neu5Aca3Galb3GalNAcb4(Neu5Aca3)Galb4GlcCer GT1aNeu5Aca3Galb3GalNAcb4(Neu5Aca8Neu5Aca3)Galb4GlcCer GT1bGalb3GalNAcb4(Neu5Aca8Neu5Aca8Neu5Aca3)Galb4GlcCer GT1cNeu5Aca8Neu5Aca3Galb3GalNAcb4(Neu5Aca8Neu5Aca3)Galb4GlcCer GQ1b

a Previously written using subscripts, e.g., GM3, and so on.


(1982) 433–437; J. Biol. Chem., 257 (1982) 3347–3351; Pure Appl. Chem., 54 (1982)1517–1522; Arch. Biochem. Biophys., 220 (1983) 325–329.

(4) IUPAC-IUBMB Joint Commission on Biochemical Nomenclature (JCBN). Nomencla-ture of carbohydrates (Recommendations 1996). Pure Appl. Chem., 68 (1996) 1919–2008;Carbohydr. Res., 297 (1997) 1–90; Adv. Carbohydr. Chem. Biochem., 52 (1997) 43–177;J. Carbohydr. Chem., 16 (1997) 1191–1280.

(5) W. L. Roberts, S. Santikarn, V. N. Reinhold, and T. L. Rosenberry, J. Biol. Chem., 263(1988) 18776–18784.

(6) C. C. Sweeley and B. Siddiqui, in The Glycoconjugates, Vol. 1, M. I. Horowitz, and W. Pig-man (Eds.), Academic Press, New York, 1977, 459–540.

(7) G. Reuter and R. Schauer, Glycoconjugate J., 5 (1988) 133–135.(8) Nomenclature Committee of IUB (NC-IUB). Numbering of atoms in myo-inositol (Rec-

ommendations 1988). Biochem. J., 258 (1989) 1–2; Eur. J. Biochem., 180 (1989) 485–486.(9) IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). Conformational

nomenclature for five- and six-membered ring forms of monosaccharides and their de-rivatives (Recommendations 1980). Eur. J. Biochem., 111 (1980) 295–298; Arch. Biochem.Biophys., 207 (1981) 469–472; Pure Appl. Chem., 53 (1981) 1901–1905.

(10) IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). Symbols forspecifying the conformation of polysaccharide chains (Recommendations 1981). Eur.J. Biochem., 131 (1983) 5–7; Pure Appl. Chem., 55 (1983) 1269–1272.

(11) M. G. Low and A. R. Saltiel, Science, 239 (1988) 268–275.(12) M. A. J. Ferguson and A. F. Williams, Annu. Rev. Biochem., 57 (1988) 285–320.(13) L. Svennerholm, J. Neurochem., 10 (1963) 612–623.(14) H. Wiegandt, in New Comprehensive Biochemistry: Glycolipids, A. Neuberger, and L. M.

van Deenen (Eds.), Vol. 10, Elsevier, New York, 1985, 28.(15) S. Hakomori, in Handbook of Lipid Research, Vol. 3, J. N. Kanfer, and S. Hakomori (Eds.),

Plenum, New York/ London, 1983, 1–165.(16) C. L. M. Stults, C. C. Sweeley, and B. A. Macher, Methods Enzymol., 179 (1989) 167–214;

see also B. A. Macher and C. C. Sweeley, Methods Enzymol., 50 (1978) 236.



Abashev, Yu. P., 43, 44, 75, 122(42)Abass, T. M., 97, 98, 104, 131(336; 337)Abbas, I. M., 234, 261(525)Abboud, J.-L. M., 38–39, 121(16; 27)Abdallah, A. A., 186, 251(195; 198)Abdelfattah, N. F., 234, 261(525)Abdel Rahman, M. M., 184, 186, 196, 215,

225, 236, 250(167), 251(195; 196; 201),254(283), 256(365)

Abelson, J. N., 226, 227, 258(442)Acharya, K. R., 264, 273, 277, 278, 286, 295,

300, 302(2), 306(117)Adachi, H., 226, 227, 258(442)Adachi, M., 280, 306(129)Aebersold, R., 274, 294, 306(121), 307(140)Afanas’ev, V. A., 41, 74, 122(37; 38)Aftab, K., 101, 131(346–350)Ágoston, K., 50, 51, 61, 124(125)Ahnoff, M., 77, 127(221)Akiya, S., 224, 258(432)Albers-Schonberg, G., 226, 258(443)Al-Daher, S. S., 193, 253(257)Alemán, C., 165, 172(92; 93)Aleshin, A. E., 272, 273, 281, 285, 288, 300,

306(103; 104)Alesker, A., 227, 259(470)Alexis, A., 194, 253(261)Alfes, H., 217, 257(396)Alföldi, J., 180, 249(129)Algrim, D. J., 39, 121(23)Ali, M. A., 96, 131(332)Ali, R. S., 177, 249(101)Alizari, P. M., 272, 292, 305(81)Alla, A., 164, 172(91)Allan, Z., 182, 186, 250(146)Allen, A., 183, 185, 250(154)

327

Allgire, J. F., 77, 127(216)Allison, W. S., 174, 246(5)Almarsson, Ü., 86, 129(278)Al-Masoudi, N., 55, 66, 122(55)Al-Soud, Y. A., 55, 66, 122(55)Alzari, P. M., 272, 282, 288, 305(83)Amaral, L., 177, 248(79)Ambartsumova, R. F., 75, 127(200)Amer, A., 55, 66, 122(55)Ames, M. M., 77, 127(213)Amit, A. G., 272, 282, 288, 305(83)Amyes, T. L., 295, 307(149)Anand, N., 6, 13(36)Andersen, W., 6, 13(19)Anderson, L., 227, 230, 244, 260(485; 486),

261(535)Ando, O., 45, 46, 58, 113, 114, 123(89),

133(407)Ando, T., 270, 304(61)André, S., 51, 62, 103, 125(139)Andres, C. J., 193, 253(256)Andronova, T. M., 43, 44, 75, 122(42)Angulo, M., 68, 69, 126(169)Angyal, S. J., 227, 260(484)Antonopoulos, C. A., 176, 247(31)Anumula, K. R., 80, 128(253)Anzeveno, P. B., 193, 253(258)Apel, R., 177, 249(93)Aplin, J. D., 102, 132(363)Appel, R., 141, 142, 171(40)Arai, M., 113, 133(402; 403)Aravind, S., 106, 132(379)Archer, D. B., 276, 295, 302, 306(127)Ardagh, E. G. R., 176, 247(42)Areces, P., 86, 129(280)Areces Bravo, P., 79, 86, 128(247), 129(279)

Author Index

4888 Horton Auth Index 11/17/99 3:21 PM Page 327

Arison, B. H., 226, 258(443)Armand, S., 273, 306(107)Armstrong, A., 89, 130(307)Armstrong, E. F., 209, 256(343)Armstrong, R. K., 193, 220, 253(249)Arndt, F., 194, 254(263)Arnold, A., 174, 177, 246(22), 248(81)Arrese, F., 175, 246(25)Arya, D. P., 82, 83, 86, 129(270)Asano, N., 193, 253(258)Ashare, R., 37, 121(13)Ashiura, M., 46, 47, 58, 114, 123(87)Ashry, S. H., 212, 256(360)Atherton, F. R., 6–7, 13(25; 27), 14(52; 53)Atta, K., 212, 256(364)Aubert, J.-P., 272, 282, 288, 305(83)Augé, C., 102, 132(357)Augustín, J., 37, 38, 52, 62, 88, 94, 121(9),

125(141–143), 129(289)Augusto, O., 174, 246(7)Augy, S., 227, 259(471)Avalishvili, L. M., 161, 172(83)Avalos, M., 46, 58, 65, 70–73, 77, 79–81,

86–87, 90, 92, 97, 123(77), 125(156),126(176; 186), 128(233; 234; 251),129(275; 280)

Avalos González, M., 46, 58, 74, 77–79, 86,123(73; 74), 126(194), 128(231; 243; 246),129(274)

Aviram, K., 244, 261(539)Awad, L. F., 98, 131(338)Awata, M., 227, 259(466)Ayad, M., 206, 255(322)Aymamí, J., 138, 170(22; 23)Aymes, T. L., 299, 308(164)Azimov, V. A., 183, 250(159)

Babiano, R., 43, 46, 58, 65, 68, 70–73, 77,79–81, 86–87, 90, 92, 97, 123(72; 77),125(156), 126(176; 186), 128(233; 234;251), 129(275; 280)

Babiano Caballero, R., 41, 42, 56–57, 68, 77,81, 122(63), 123(69), 127(224), 128(231)

Bachmann, F., 142–145, 155, 171(43; 47; 48)Backinowski, L. V., 61, 125(132; 133)Baddiley, J., 5–6, 13(4–6; 10; 28; 29; 34)Badicke, G., 209, 222, 256(348), 258(428)Baehler, B., 189, 252(220–222)Bahl, O. P., 70, 109, 126(174)

AUTHOR INDEX328

Bairoch, A., 264, 272, 303(6)Baker, J. D., 176, 248(70)Bakina, E. V., 81, 129(266)Baláz, S., 88, 129(289)Balding, P., 60, 103, 124(113)Ballesteros, E., 39, 121(27)Balmér, K., 77, 127(221)Bamba, T., 137, 170(12)Bamford, W. R., 194, 254(281)Banait, N. S., 298, 307(159)Bandas, E. M., 198, 254(302), 255(303; 304)Banoub, J. H., 50, 52, 61, 125(137)Baptista, J. A. B., 44, 64, 122(44)Barawkar, D. A., 86, 129(277)Barbalat-Rey, F., 193, 253(247)Barker, H. A., 217, 256(381), 257(382)Barker, R., 194, 254(266)Baron, F., 7, 14(67)Barr, B. K., 272, 305(80)Barrett, D. A., 76, 127(207)Barrows, T. H., 137, 170(13)Barry, J. A., 232, 233, 260(499)Barry, V. C., 197, 254(290)Barton, D. H. R., 87–89, 129(284; 286),

226–227, 258(442), 259(471)Barzilay, M., 60, 124(110)Bastos, M. P., 177, 248(79)Batley, M., 98, 131(339)Batta, G., 50, 51, 61, 124(125)Battioni, P., 174, 246(11)Baudat, A., 193, 253(256)Bauer, C., 209, 256(352)Baum, H., 174, 246(15)Baxter, E. W., 193, 253(256)Bayne, S., 209, 217, 256(344), 257(389)Beaupère, D., 92, 130(311)Becker, R. S., 177, 248(77)Beckwith, A. L. J., 185, 251(184)Bedi, G. S., 70, 109, 126(174)Beguin, P., 264, 272, 282, 288, 302(5), 305(83)Behrend, R., 181, 189, 190, 250(134)Beintema, J. J., 273, 306(106)Belaich, A., 272, 305(82)Belaich, J.-P., 272, 305(82)Belal, S., 206, 255(322)Belinskey, C., 230, 260(491)Bellamy, A. J., 185, 187, 251(173; 190)Benárquez Fonseca, F., 79, 128(244)Bender, D. R., 176, 248(60)Bender, H., 273, 306(108)


Bender, S. L., 227, 260(480)Bendiak, B., 175, 177, 179, 247(30), 248(83)Bendnyagina, N. P., 189, 206, 235, 252(223)Benitez, L. V., 174, 246(5)Benito, J. M., 57, 60, 64, 82, 106, 107, 118, 120,

123(71), 124(102), 129(267), 132(382),133(417), 134(421)

Bennani, Y. L., 194, 253(261)Bensen, D., 183, 185, 250(153; 154)Berad, B. N., 74, 127(198)Beraldo, H., 100, 131(342)Beránek, J., 70, 71, 126(171)Bergel, F., 3, 5, 13(2; 7)Bergeron, J., 27, 33(3)Bergfors, T., 269, 270, 272, 273, 279, 280, 288,

291, 304(55)Berkebile, J. M., 194, 254(271)Berliner, L. J., 185, 251(188)Bernstein, J., 181, 250(141)Berthelot, M., 39, 121(24)Berthod, T., 80, 81, 128(256)Berti, P. J., 297, 307(156)Bertram, B., 95, 131(326; 327)Bezouska, K., 104, 132(375)Bhattacharjee, A. K., 51, 60, 103, 124(111)Biely, P., 272, 305(78)Billault, I., 226, 227, 258(442)Binkley, R. W., 175, 247(28)Binkley, W. W., 175, 247(28)Binte, H. J., 174, 206, 246(23), 255(321)Bird, T. P., 140, 154, 170(33–35), 171(36)Bjamer, K., 180, 202, 205, 206, 249(125),

255(307)Black, W. A. P., 140, 154, 170(33–35), 171(36;

37)Blackadder, D. A., 187, 252(209)Blackburn, G. M., 8, 14(79)Blair, H. S., 180, 249(121; 122)Blair, M. G., 187, 252(206)Blake, C. C. F., 295, 307(147)Blanchard, J. S., 268–269, 271, 296–299,

304(53), 305(75), 307(157)Blanco, R. S., 27, 33(4), 68Blanke, S. R., 297, 307(156)Blasco López, A., 91, 97, 130(309), 131(334)Blattner, R., 227, 258(446), 259(467)Blériot, Y., 70, 110, 126(183), 133(389; 390)Bloom, S. H., 177, 248(74)Blummel, F., 176, 177, 247(36)Bobrova, N. I., 183, 250(160)

AUTHOR INDEX 329

Bocelli, G., 176, 177, 248(69; 76)Bock, W., 181, 250(142)Bode, G., 220, 257(414)Bodenheimer, T. S., 217, 256(375)Bodley, J. W., 307(131)Boel, E., 272, 305(96)Bognár, R., 71, 94, 126(190), 227, 259(468)Bogusiak, J., 95, 131(328)Boink, G. J., 197, 198, 223, 254(294)Bols, M., 193, 253(256)Bommuswamy, J., 298, 307(160)Bonaly, R., 64, 125(154)Bonfils, E., 60, 124(115)Bonnett, R., 8, 14(84)Borders, L. L., Jr., 293, 307(136)Bordwell, F. G., 39, 121(23)Bou, J. J., 161–163, 165–167, 172(84–88;

92–95)Bouab, W., 39, 121(27)Boutellier, M., 110, 133(391)Bovin, N. V., 70, 102, 126(175; 178), 132(361)Bowden, C. R., 74, 127(199)Boyd, D. R., 227, 259(464)Boyer, J. H., 234, 260(511)Bracke, B. R. F., 39, 121(30)Brady, L., 272, 305(96; 97)Brandi, A., 193, 253(256)Brannon-Peppas, L., 137, 170(14)Braude, E. A., 197, 254(289)Braun, C., 266, 270, 294, 303(39), 307(144)Braun, W., 176, 177, 247(39)Brauns, O., 193, 253(250)Brenken, H., 176, 177, 247(39)Brewer, C. F., 265–276, 280, 281, 284, 285,

287, 296, 298, 300, 302, 303(21; 26; 28; 33;36–38; 40; 45–47; 50–53; 69–71; 75)

Brock, J., 177, 189, 248(85)Brockhaus, M., 266, 268, 276, 303(30)Broder, S., 89, 130(292; 293)Brossmer, R., 71–72, 81, 102, 126(188; 189),

129(258–262), 132(356; 358–360)Brown, D. M., 6–8, 13(18; 21–24), 14(55;

62–65; 67–72; 80)Brown, E., 79, 128(237; 238)Brown, R. L., 194, 254(272; 273; 275; 276)Browne, K. A., 58, 82–83, 86, 123(95),

129(268; 281)Brozozowski, A. M., 308(172)Bruice, T. C., 58, 82–83, 86, 123(95),

129(268–270; 276–278; 281)


Brull, L., 209, 256(345)Bruni, P., 176, 248(69)Bruse, G., 60, 61, 124(122)Brzozowski, A. M., 272, 302, 305(96),

307(132)Buchanan, J. G., 8, 14(82), 192, 220,

253(244–246)Buchmann, F., 136, 170(6)Buckingham, J., 175, 182, 187, 211, 230,

247(29), 250(150), 260(494)Buckley, N., 299, 308(165)Budhu, R. J., 227, 260(480)Buehner, M., 273, 278, 295, 300, 306(119)Bueno, M., 147–150, 171(50; 53–55)Bueno Martínez, M., 147, 153, 171(52; 56; 57;

59)Buisson, G., 272, 278, 286, 295, 300, 305(99)Bunin, B. A., 226, 227, 258(442)Burgess, K., 193, 253(254)Burke-Laing, M., 175, 246(24)Burneister, W. P., 294, 302, 307(141)Buss, D. H., 50, 60, 124(103)Butler, C. L., 179, 249(118)Butler, K., 140, 154, 170(32)Butters, T. D., 193, 253(259)Buzykin, B. I., 189, 206, 235, 252(223)Buzykin, B. J., 176, 180, 247(59)Byramova, N. E., 102, 132(361)Byranova, N. E., 70, 126(178)

Caglioti, L., 230, 260(493)Callebaut, I., 272, 306(105)Camarasa, M. J., 41–43, 54, 55, 88, 122(34; 41)Camarasa Rius, M. J., 54, 122(35)Cameron, L. M., 39, 121(22)Campadelli-Fiume, G., 174, 246(16)Campbell, R. L., 272, 305(91)Cañada, F. J., 92, 117, 130(310)Canard, B., 80, 81, 128(256)Cane, D. E., 176, 248(71)Cannon, J. R., 8, 14(84)Cantoni, A., 176, 248(69)Cao, R., 95, 100, 131(342; 343)Cardellini, L., 177, 248(76)Cardillo, B., 176, 248(69)Carless, H. A. J., 227, 259(451)Carnovskii, A. D., 189, 206, 235, 252(223)Caro, H. N., 106, 107, 118, 132(382)Carothers, W. H., 135, 170(3; 4)

AUTHOR INDEX330

Carpenter, N. C., 193, 253(257)Carreira, E. M., 42, 54, 113, 122(52)Carroll, P. J., 193, 253(256)Carson, J. F., 217, 256(380)Carter, H. E., 230, 260(491)Cascio, D., 272, 305(100)Casiraghi, G., 193, 253(256)Castillon, S., 227, 259(470)Castro, C., 234, 244, 261(514)Catar, G., 174, 246(18)Cavallaro, C. L., 176, 247(54)Cebulak, M., 227, 260(487)Cech, D., 48, 58, 63, 81, 92, 123(96)Ceni de Bello, J., 193, 253(257)Cerny, M., 45, 46, 59, 74, 86, 88, 94, 123(97)Cert Ventulá, A., 79, 128(240; 242; 245)Chaby, R., 80, 128(257)Chacon-Fuertes, M. E., 192, 220, 253(244;

245)Chamberlin, A. R., 177, 248(74)Chan, A. W.-Y., 111, 133(392)Chan, W. C., 76, 127(207)Chandler, J. H., 176, 248(70)Chaney, A., 139, 154, 170(31)Chang, Y.-T., 226, 227, 258(442)Chaplin, A. F., 185, 251(191)Chaplin, D. A., 193, 253(254)Chapman, O. L., 206–207, 225, 255(325; 332),

258(435)Chargaff, E., 202, 206, 227, 230, 241,

255(309), 260(488), 261(533)Charon, D., 80, 128(257)Chase, B. H., 7, 14(47)Chatterjee, M., 193, 253(259)Chen, D., 193, 253(256)Chen, L., 156–157, 172(69; 70; 72–74), 193,

253(257)Chernyak, A. Ya., 102, 132(364)Chiba, H., 96, 131(331), 177, 249(108)Chiba, S., 264, 265, 268, 270, 280, 284, 298,

303(10), 304(45; 47; 51), 307(161; 163)Chiba, T., 176, 186, 206, 248(63), 251(202),

255(316)Chida, N., 227, 259(452–454)Chilton, W. S., 182, 219, 250(148), 257(405)Chima, J., 227, 259(464)Chittenden, C. F. J., 244, 261(537)Chmurski, K., 82, 106, 107, 118, 129(267),

132(382)Chow, H.-F., 104, 132(373)


Christ, D. D., 77, 127(211; 212)Christie, S. M. H., 7, 13(40), 14(51)Chung, S. K., 226–227, 258(442), 260(482)Churn, S. C., 176, 247(32)Ciajola, M. R., 175, 246(26)Cintas, P., 46, 58, 77, 79–81, 86, 87, 90, 97,

123(77), 128(233; 234; 251), 129(275)Cintas Moreno, P., 74, 77, 78, 86, 126(194),

128(231), 129(274)Claeyssens, M., 264, 266, 268, 270, 272, 273,

276, 280, 282, 287, 288, 296, 302(5),303(37; 38), 305(83)

Clark, R. K., Jr., 230, 260(491)Clark, V. M., 6–7, 13(17; 26; 29; 30; 36), 14(54;

56; 60; 61)Clarke, A. E., 264, 303(18)Clarke, M. A., 27, 28, 33(1–5; 7–10),

34(11–15), 68Clarke Garegg, M. A., 27, 33(6)Clennan, E. L., 234, 261(513)Cléophax, J., 227, 259(469)Cluss, E., 212, 256(356)Cochran, W., 6, 13(23)Cogoli, A., 296, 299, 307(151)Cohen, J. S., 8, 14(79)Cole, F., 27, 33(3)Coleman, A. W., 41, 64, 125(152; 153)Collins, P., 176, 247(54)Collins, P. M., 227, 238, 259(472)Colman, P. M., 193, 253(258)Compton, J., 178, 181, 189, 249(114)Conde, A., 79, 128(248–250)Conde, C. F., 79, 128(248; 249)Conn, R. S. E., 176, 248(60)Connerton, I., 272, 305(90)Connolly, M. L., 300, 308(168)Cook, J. D., 234, 261(520)Cooper, P. W., 194, 197, 254(274)Corby, N. S., 7, 14(57)Cordona, F., 193, 253(256)Corey, E. J., 176, 248(71; 72)Corley, E. G., 176, 248(60)Cortesi, R., 36, 120(3)Coste-Sarguet, A., 47–49, 60, 92, 124(98)Cottaz, S., 294, 302, 307(141)Cotton, F. A., 38, 121(20)Courtois, J. E., 270, 304(63)Cox, R. A., 39, 121(25)Coxon, B., 199, 201, 205, 209, 234, 255(306;

314), 261(523)

AUTHOR INDEX 331

Cremlyn, R. J., 6, 7, 13(32), 14(49)Cretcher, L. H., 179, 249(118)Crich, D., 37, 88, 89, 121(7), 186, 187, 189,

251(203), 252(213)Crossman, A., 183, 185, 187, 250(152–154)Crum, J. D., 194, 254(266; 269)Cuevas Lorite, T., 75, 127(201)Cueves, T., 47, 58, 68, 81, 87, 123(78; 79)Cui, Y., 227, 258(451)Cumming, D. A., 177, 248(83)Cummings, N., 272, 305(90)Czarnik, A. W., 226, 227, 258(442)Czjzek, M., 272, 305(82)Czubarow, P., 219, 257(400)Czyzewski, Z., 209, 256(346)

Dahlquist, F. W., 293, 295, 307(136; 148)Dahm, S., 202, 205, 206, 255(307)Dahmén, J., 50, 61, 124(129)Dakour, J., 84, 129(271)Daley, L., 92, 130(312)Dalko, P., 226, 227, 258(442)Dalton, D. R., 193, 253(256)Damo, C. P., 77, 127(216)Daniel, J. K., 193, 253(258)Dao-Pin, S., 302, 308(170)Dauter, M., 302, 307(132), 308(172)Dauter, Z., 272, 282, 288, 302, 305(85),

308(171)Davidis, E., 174, 246(21)Davies, G., 272, 282, 288, 300, 302, 305(82; 85;

86), 306(105), 307(132), 308(169; 172)Davoll, J., 5, 13(13; 16)Davoodi, J., 272, 305(91)Day, J., 272, 305(100)Debacher, N. A., 39, 121(29)de Bernardo, S., 89, 130(298)De Bruyne, C. K., 270, 304(65)De Clercq, E., 74, 127(195)Defaye, J., 47–49, 60, 65, 82, 92, 106, 107, 118,

124(98; 99; 101), 125(155), 129(267),132(382; 383), 133(418), 270, 304(64)

Degano, M., 272–274, 280, 281, 285, 288, 292,300, 301, 306(101)

Dekker, C. A., 8, 14(70)de las Heras, F. G., 41–43, 54, 55, 88, 89,

122(34; 41), 130(299)de las Heras Martín Maestro, F. G., 54,

122(35)


Delaumney, J.-M., 227, 259(469)de Lederkremer, R. M., 41, 43, 55, 74, 75,

87–88, 122(53; 54)de Marco, A. M., 176, 248(60)Demchenko, A. V., 41, 54, 121(33), 122(49)Demes, P., 174, 246(18)Dempcy, R. O., 58, 82–83, 86, 123(95),

129(268; 269; 278; 281)de Paz, J. L. G., 39, 121(27)de Paz, M. V., 147, 169, 171(50), 172(99)de Paz-Bañez, M. V., 147, 171(51)Derdall, G. D., 39, 121(26)Derewenda, U., 272, 305(89)Derewenda, Z. S., 272, 305(89; 96; 97)de S. Sierra, M. M., 39, 121(29)Deshmukh, S. P., 74, 127(198)de Vries, G. E., 273, 306(109)Dewar, E. T., 140, 154, 170(33–35), 171(36;

37)Deyn, W., 227, 258(447; 449)Dhekne, V. V., 227, 259(459)Diánez, M. J., 74, 79, 80, 87, 126(193),

128(252)Días-Martín, D., 89, 130(307)Díaz, A., 100, 131(342; 343)Díaz Arribas, J. C., 92, 117, 130(310)Díaz Pérez, V. M., 45–47, 58, 59, 65, 66, 75,

81, 85, 87, 92, 117, 123(85), 125(159;160), 129(273), 130(310)

Diels, O., 184, 185, 212, 215, 221, 250(162),256(356), 257(417)

Dijkhuizen, L., 273, 279, 286, 306(109; 110)Dijkstra, B. W., 273, 279, 286, 306(106; 107;

109; 110)Dijong, I., 197, 198, 201, 219, 254(291)Dintinger, T., 110, 133(389; 390)Divne, C., 272, 273, 276, 287, 305(79)Dixon, M., 234, 244, 261(514)do Amaral, L., 177, 248(80)Doane, W. M., 89, 130(290; 291)Dobson, C. M., 276, 295, 302, 306(127)Dodson, E. J., 272, 305(97)Dodson, G. G., 272, 282, 288, 305(85; 96)Dominguez, R., 272, 292, 305(81)Dong, X., 51, 62, 103, 125(139)Dorman, L. C., 207, 255(331)Doudoroff, M., 217, 256(381), 257(382), 270,

285, 304(68)Douglas, A. W., 176, 248(60)Doyle, M. P., 174, 246(13)

AUTHOR INDEX332

Drewniak, J., 227, 239, 260(478)Driguez, H., 270, 294, 302, 304(64), 307(141)Driscoll, J. S., 89, 130(292; 293; 296)Drobnica, L., 37, 38, 52, 62, 94, 121(9),

125(141–143)Druet, L. M., 39, 121(25)Drumright, R. E., 185, 251(185)Dubber, M., 104, 132(377)Dubose, R. F., 272, 274, 276, 305(77)Ducros, V., 272, 305(82)Duddeck, H., 176, 247(54)Duee, E., 272, 278, 286, 295, 300, 305(99)Duke, E. M. H., 273, 277, 278, 306(118)Duke, J., 60, 124(105)Dukefos, T., 180, 249(124)Duplishcheva, A. P., 77, 128(229)Durán, C. J., 70–73, 87, 126(176; 186)Durham, L. J., 226, 258(437)Dursun, K., 177, 249(106)Duus, F., 37, 121(6)Dwek, R. A., 50, 51, 62, 103, 125(138), 193,

253(259)

Ealick, S. E., 273, 289, 306(113)Eberson, L., 233, 260(504)Ebisu, S., 60, 124(107)Eby, R., 50–51, 61, 124(127; 128)Eckhart, E., 190, 192, 252(228; 234)Edvabnaya, L. S., 61, 125(132)Edward, J. T., 39, 121(26)Edye, L. A., 27, 28, 34(9–11; 14)Eggleston, G., 27, 33(10), 34(11; 15)Ehlers, S., 47, 63, 125(144)Eichenauer, H., 233, 260(508)Eilert, U., 101, 131(345)Eistert, B., 194, 233, 237, 254(263), 260(502)Ekborg, G., 51, 60, 61, 103, 124(111; 119)Eklind, K., 50, 60, 61, 124(120)Ekwall, E., 60, 61, 124(122)El Adl, S., 206, 255(322)El Ashry, E. H., 186, 251(195–201)El Ashry, E. S. H., 98, 131(338), 196, 254(283)El Ashry, H., 212, 256(363)El Ashry, S., 184, 189, 212, 215, 250(165),

256(359; 361; 366)El Badry, S. M., 177, 249(101)Elbein, A. D., 110, 133(387), 193, 253(254; 258)Elbert, T., 45, 46, 59, 74, 86, 88, 94, 123(97)El Ghomari, M. J., 39, 121(24)


Elguero, J., 234, 261(517)Elion, M. D., 273, 289, 306(113)El Khadem, H. S., 176, 177, 180, 183, 184,

185, 186, 187, 189, 193, 196, 199, 201,202, 205–207, 209, 212, 215, 217,219– 220, 222–228, 233–234, 236, 241,247(37; 38; 47; 48), 249(112; 130),250(152–154; 165; 167), 251(193),252(219), 253(251), 255(306; 311–314;326; 341), 256(359–363; 365–368; 378),257(388; 393; 394; 396; 398; 400; 403;407; 408; 413; 422), 258(425; 426; 429)261(523)

El Kheir, A. A., 206, 255(322)El Kilany, Y., 186, 251(196; 198–200)El-Menshawi, B., 101, 131(344)El Meslouti, A., 92, 130(311)Elmore, D. T., 7, 8, 14(51; 74)El-Mouhtadi, M., 39, 121(27)El Sadek, M., 212, 256(363)El Sayed, 212, 256(363)El-Sekeli, M. A., 212, 256(361)El Sekily, M., 220, 257(415)El Shadem, H., 220, 257(415)El-Shafei, Z. M., 184, 189, 202, 206, 212,

217, 219, 222, 224, 234, 250(165),255(312), 256(363), 257(393; 394;422), 258(425)

El-Toukhy, A. A., 97, 98, 104, 131(336; 337)Emoto, S., 70, 71, 126(173)Enders, D., 233, 260(508)Engel, L. L., 207, 255(336)Enholm, E. J., 89, 130(305)Entwhistle, D. A., 193, 253(257)Erden, I., 234, 244, 261(514)Ergonenc, P., 234, 244, 261(514)Ermolaev, K. E., 198, 255(303)Ermolaev, K. M., 198, 254(302; 304)Esposito, E., 36, 120(3)Esseffar, M., 39, 121(27)Estabrook, R. W., 174, 246(4)Estevez, V. A., 227, 260(481)Estrada, M. D., 74, 79, 87, 126(193), 128(250)Estrwan, E. I., 222, 258(423)Estupinan, B., 297, 307(154)Ettel, V., 217, 256(377)Evans, E. F., 194, 254(267; 268; 272)Evans, W. L., 185, 250(171)Ewing, D., 91, 93, 130(308)Eybl, V., 94, 130(316; 317)

AUTHOR INDEX 333

Fabrega, S. E., 272, 306(105)Fadeeva, N., 137, 170(8)Faillard, H., 42, 54, 68, 122(51)Fairbanks, A. J., 193, 253(257)Faizi, S., 101, 131(346–351)Falk, I., 176, 177, 247(39)Fasman, G. D., 7, 14(64)Fatiadi, A. J., 177, 180, 183, 186, 187, 190, 199,

201, 205, 209, 211, 212, 217, 224, 227,230, 233–241, 248(75), 250(131; 157),251(192), 252(225; 230), 255(306; 314),256(353), 257(392), 260(490; 492; 495;496; 500; 501; 526), 261(526; 529–532)

Feather, M. S., 196, 254(284)Fechtner, E., 176, 177, 247(39)Feizi, T., 193, 253(257)Félix, C., 64, 125(154)Feliz, M., 244, 261(539)Ferguson, M. A. J., 315, 324(12)Fernandez, R., 176, 247(54)Fernández-Bolaños, J., 79, 80, 91, 97,

128(239–245; 252), 130(309), 131(334)Fernández-Bolaños Guzmán, H, 79, 80,

128(252)Fernández García-Hierro, J. I., 79, 86,

128(247), 129(279)Fernández-Resa, P., 41–43, 54, 88, 122(34; 35)Fernández-Santín, J. M., 138, 170(22; 23)Ferrier, R., 46, 126(167; 168), 176, 227,

247(54), 258(446), 259(467)Fessner, W. D., 227, 259(463)Fiandor, J., 89, 130(299)Fierobe, H.-P., 272, 305(82)Fieser, L. F., 205, 255(315)Fieser, M., 205, 255(315)Figueroa Pérez, S., 106, 132(381)Filippatos, E. C., 74, 127(195)Fink, C., 51, 62, 103, 125(139)Finkelstein, E., 185, 211, 251(187), 256(355)Finney, N. S., 176, 248(72)Firsov, L. M., 272, 273, 281, 285, 288, 300,

306(103; 104)Fischer, E., 36, 42, 120(2), 126(163), 176, 177,

179, 196, 197, 202, 208, 209, 220–221,247(52), 249(110), 254(285; 286),256(343), 257(409; 416)

Fischer, P. B., 190, 252(227)Fishman, M. L., 135, 170(5)Fitting, C., 270, 285, 304(68)Fitz, W., 227, 259(463)


Fleet, G. W. J., 193, 253(257)Fletcher, H. G., 227, 260(483)Flint, J. A., 7, 14(55)Flowers, H. M., 265, 303(23)Flynn, E. H., 230, 260(491)Forbes, E. C., 197, 254(289)Ford, M. J., 89, 130(306; 307)Forrest, H. S., 7, 13(38; 39)Forrester, A. B., 232, 233, 260(498)Fox, H. H., 181, 250(140)Fox, J. J., 70, 71, 126(171)Fraenkel, G., 187, 252(208)Fragoso, A., 95, 100, 131(324; 343)Franck, R. W., 193, 253(257)Franco, J. D., 39, 121(29)Frank, N., 95, 131(326)Frank, V., 174, 246(19)Frazier, J., 89, 130(295)Frejd, T., 50, 61, 124(129)Freudenberg, K., 176, 177, 193, 247(36),

253(250)Fricke, T., 104, 132(376)Fried, M., 8, 14(80)Friedberg, F., 198, 254(296)Friedman, H. A., 70, 71, 126(171)Friedrich, E., 233, 260(508)Friendman, R. B., 135, 170(5)Frime, A. A., 234, 260(510)Fritz, H., 268, 271, 274, 275, 284, 304(46)Fuentes, J., 41–48, 54, 57–60, 64, 65, 68–70,

72, 74, 75, 77, 81, 84, 85, 87, 92, 101, 102,108, 117–119, 122(50), 123(66–68; 70; 71;75; 77; 79; 81; 84; 85), 124(100), 125(159),126(169; 170; 177; 192), 128(233),130(310), 133(415–417), 134(419)

Fuentes Mota, J., 41–43, 45–49, 56–60, 68, 74,75, 77–79, 81, 86, 87, 91–92, 97,122(63–65), 123(69; 72–74; 78; 83),124(98), 126(194), 127(201; 222–226),128(231; 240; 242; 244; 245; 247),129(274; 279), 130(309), 131(334)

Fügedi, P., 95, 131(330)Fukase, H., 193, 227, 253(258), 259(458)Fukazawa, C., 280, 306(128)Fukui, K., 78, 128(236)Fukui, S., 176, 247(56)Fukuzawa, C., 280, 307(130)Funes, J. L., 150, 171(55)Furberg, S., 180, 202, 205–206, 249(125; 126),

255(307; 308)

AUTHOR INDEX334

Furuhata, K., 81, 129(265)Furukawa, Y., 113, 133(403)Furuta, S., 226, 258(437)Fuska, J., 174, 246(19)Fuskova, A., 174, 246(19)

Gaafar, A. E. M., 55, 66, 122(55)Gabius, H.-J., 51, 62, 103, 125(139)Gabriel, S., 135, 170(2)Gadelle, A., 47–49, 60, 92, 124(98; 101)Gafner, G., 235, 261(528)Gal, J., 77, 127(209; 213; 214; 217; 220)Galbis Pérez, J. A., 41, 43, 56–58, 68, 77, 79,

81, 86, 122(63), 123(72), 128(241; 243;246; 247), 129(279), 147–150, 153, 163,169, 171(50; 51; 52; 53–55; 56; 57–59; 89;99)

Galons, H., 64, 125(153)Gambaryan, A. S., 70, 102, 126(178),

132(361)Ganem, B., 70, 110, 111, 113, 115, 126(179;

180; 182), 133(387; 388; 391; 392; 404;411), 272, 305(80)

Gao, Z., 109, 110, 133(386)García, I., 100, 131(342)García Alvarez, M., 168, 172(98)Garcia-Blanco, S., 175, 246(25)García-Calvo-Flores, F., 41–44, 56, 68, 74,

122(57)García Fernández, J. M., 37, 40–49, 56–60,

63–66, 68, 70, 72, 74–75, 77, 81, 82, 87,91–92, 102, 106–108, 117–120, 121(8),122(64), 123(66; 69; 71; 72; 75; 81–85),124(98–100; 102), 125(155; 159; 160),126(177; 192; 193), 127(201; 223–226),129(267), 130(310), 132(382; 383),133(415–418), 134(419–421)

García Gómez, M., 42, 56, 57, 77, 122(64)García González, F., 79, 128(239)García-López, M. T., 41–43, 54, 55, 88,

122(34; 35; 41)García-Martín, M. G., 147, 169, 171(50; 51),

172(99)García-Mendoza, P., 41–44, 56, 68, 74,

122(57)Garcia-Mina, M., 175, 246(25)García Rodríguez, S., 79, 80, 128(252)García-Verdugo, C., 65, 71, 125(156)Gardiner, D., 227, 238, 259(472)


Garegg, P. J., 50–52, 60–61, 95, 103, 124(106;107; 118–121; 123), 131(330), 132(367)

Garg, H. G., 64, 125(150)Gätzi, K., 194, 254(264)Gaudin, C., 272, 305(82)Gaudino, J. J., 226, 227, 258(442)Gautheron-Le Narvor, C., 102, 132(356)Gebler, J., 264, 272, 274, 302(5), 306(121)Gelin, S., 234, 261(524)Gemeiner, P., 52, 62, 94, 125(141–143)Genghof, D. S., 264–268, 270–274, 280–281,

284, 302, 303(14; 19–21; 26), 304(40; 66)Genre-Grandpierre, A., 70, 110, 126(183)Genre-Granpierre, A., 110, 133(389)Georges, L. W., 181, 250(137; 138)Gero, S. D., 226–227, 238, 239, 258(442),

259(469; 471; 474), 260(475)Gibas, J. T., 181, 250(140)Giboreau, P., 94, 130(314)Gibson, S. T., 137, 170(13)Giese, B., 185, 251(180; 181)Gijsen, H. J. M., 227, 259(463)Gilani, A. H., 101, 131(346–350)Gilbert, H. J., 272, 305(90)Gilding, D. K., 137, 170(10)Gilkes, N. R., 264, 272, 279, 294, 302(5),

305(87), 307(140)Gillet, G., 174, 246(11)Gingsburg, V., 51, 52, 61, 125(134)Giorgioni, E., 176, 248(69)Giralt, E., 244, 261(539)Giumanini, A. G., 230, 260(493)Glaudemans, C. P. J., 51, 60, 103, 124(111)Gliemann, J., 44, 56, 101, 122(62)Gnewuch, T., 185, 251(188)Godshall, M. A., 27, 33(1; 2; 4), 34(13), 68Goering, B. K., 115, 133(411)Goldstein, I. J., 50, 60, 102–104, 124(104; 105;

107; 108; 114)Gomez Guillen, M., 192, 252(243)Gómez Monterrey, I. M., 46, 58, 74, 77–78,

86, 123(73; 74), 126(194), 127(225),129(274)

Gonsalves, K. E., 136, 137, 170(7)González, L., 86, 100, 129(280), 131(342)Goodman, I., 36, 121(5)Gordon, M. S., 177, 248(78)Goring, J., 233, 237, 260(502)Goti, A., 193, 253(256)Goto, R., 176, 247(31)

AUTHOR INDEX 335

Gottammar, B., 50, 51, 60–61, 124(119–121)Gould, E. S., 233, 260(503)Goynes, W. R., 27, 33(7)Graham, R. W., 264, 270, 291–293, 303(11)Graham Shipley, G., 273, 306(114)Grandberg, I. L., 183, 250(160)Grech-Bélanger, O., 77, 127(215)Greemer, L. J., 193, 253(258)Green, D. C., 74, 127(197)Green, J. W., 185, 251(177)Green, R., 272, 305(89)Greenwood, A., 272, 305(100)Greenwood, C. T., 270, 304(58)Gregory, H., 139, 170(27)Grice, P., 89, 130(307)Griffin, T. S., 37, 121(11)Grisebach, H., 198, 254(295)Grisham, M. P., 27, 34(13)Gross, B., 220, 257(415)Gross, H. J., 81, 102, 129(258–263), 132(357)Grossman, A., 187, 189, 252(214)Groullier, A., 91, 93, 130(308)Grubmeyer, C., 273, 283–284, 288, 306(111;

112)Grussing, D. M., 137, 170(13)Guerreiro, C., 80, 81, 128(256)Guida, W. C., 273, 289, 306(113)Guile, G. R., 50, 51, 62, 103, 125(138)Guillo, N., 110, 133(390)Guilloux, E., 270, 304(63)Günther, W., 44, 64, 122(46)Guthrie, R. D., 182, 185, 187, 230, 244,

250(150), 251(173; 190), 260(494),261(537)

Gyorgydeak, Z., 176, 247(54)

Haas, H. J., 198, 255(305)Haas, J. W., Jr., 180, 249(120)Hadfield, A. T., 273, 276–278, 295, 302,

306(118; 127)Hadzic, A., 177, 249(106)Hadzic, M., 177, 249(106)Haering, T., 144, 171(46)Hagneav, F., 177, 248(77)Hague, C., 176, 247(34)Hahner, E., 176, 177, 247(39)Haines, S. R., 227, 259(467)Hajivarnava, G. S., 227, 238, 259(473)Hakomori, S., 323, 324(15)


Hall, R. H., 6, 8, 13(36), 14(75)Hamer, N. K., 7, 14(55)Hammer, C. F., 74, 127(197)Han, O., 176, 248(61)Han, Y. W., 27, 33(5; 8)Hand, M. V., 227, 259(464)Hanessian, S., 194, 253(261)Hanisch, G., 217, 257(391)Hann, R. M., 176, 217, 247(55), 256(369; 370;

374)Hansen, A., 193, 253(256)Hantschel, H., 181, 250(143)Hanzawa, H., 81, 113, 114, 129(264),

133(407)Hara, H., 226, 258(441)Harada, N., 266, 304(42)Harada, W., 272, 305(94)Hardegger, E., 217, 221, 222, 256(378),

257(387; 388; 390; 419)Hardy, L. W., 275, 306(126)Hare, J. B., 140, 154, 170(34; 35), 171(36)Harrelson, J. A., Jr., 39, 121(23)Harris, E. M. S., 272, 273, 281, 285, 288, 300,

306(103; 104)Harris, G. W., 272, 305(90)Hartwig, W., 88, 89, 129(287)Haruyama, H., 50, 62, 81, 113–114, 125(140),

129(264), 133(402; 403; 407)Harvey, D. J., 276, 295, 302, 306(127)Harvey, W. E., 7, 14(59)Hase, S., 80, 128(254), 176, 247(33)Haselhorst, M., 198, 254(295)Haser, R., 272, 278, 286, 295, 300, 305(82; 98;

99)Hashimoto, K., 158, 172(75; 76)Haskins, W. T., 217, 256(370)Hassan, H. H. A. M., 155, 171(68)Hassan, N. A., 55, 66, 122(55)Hassel, T., 65, 122(43), 125(157; 158)Hassid, W. Z., 217, 256(381), 257(382)Hassner, A., 87, 129(285)Hatano, K., 206, 255(316)Haushalter, K. A., 39, 121(31)Havukainen, R., 272, 305(93)Hawkins, E. G. E., 185, 187, 251(175)Haworth, W. N., 139, 154, 170(27; 29), 207,

255(335)Hay, J. M., 232, 233, 260(498)Hayashi, M., 227, 259(466)Hayashi, T., 138, 170(16)

AUTHOR INDEX336

Hayauchi, Y., 193, 253(254)Hayes, C. E., 50, 60, 102–104, 124(114)Hayes, D. H., 6, 8, 13(19), 14(73)Haynes, L. J., 7, 13(43), 14(44; 62)Hazalwood, G. P., 272, 305(90)Hearn, M. J., 176, 247(58)Heath, R. L., 139, 154, 170(29)Heaton, B. T., 176, 248(67)Hefnawy, M. M., 77, 127(219)Hegendus-Vajda, J., 190, 252(231)Hehre, E. J., 264–276, 280–281, 284, 285, 287,

296–300, 302, 303(8; 9; 12–14; 19–22; 26;28; 33; 36–38), 304(40; 45–47; 50–53; 66;69–71), 305(75), 306(102), 307(157)

Heightman, T. D., 70, 110, 126(185)Heiker, F. R., 227, 259(455)Helferich, B., 42, 126(163), 177, 248(83)Helmreich, E. J., 266, 268, 273, 278, 284, 295,

300, 304(41; 48), 306(119)Henderson, G., 185, 187, 251(174)Henderson, I., 193, 253(254)Henre, E. J., 272–274, 280, 281, 285, 288, 292,

300, 301, 306(101)Henrissat, B., 264, 270, 272, 273, 282, 288,

294, 302, 303(6), 304(64), 305(86),306(105; 107), 307(141)

Henseke, G., 174, 177, 179, 181, 206–207, 209,217, 219, 222, 246(23), 249(111),250(143), 255(321; 339; 340),256(346–349; 352), 257(391; 397),258(428)

Heo, C., 295, 307(149)Heppel, L. A., 8, 14(72)Herczegh, P., 71, 94, 126(190)Hernández-Mateo, F., 41–44, 56, 68, 74,

122(57)Herreros, M., 39, 121(27)Herrier, G., 102, 132(358)Hester, M. R., 174, 246(13)Hetterich, P., 102, 132(359; 360)Hetzheim, A., 177, 249(104)Heubach, G., 198, 254(300)Hey, D. H., 185, 251(191)Heyns, K., 46, 126(166)Hickmott, P. W., 176, 248(64)Hida, N., 177, 249(105)Hilditch, C. M., 60, 103, 124(113)Hill, H. A. O., 174, 246(6)Hill, M. L., 89, 130(294)Hilmoe, R. J., 8, 14(72)


Hinselwood, D., 187, 252(209)Hintz, P. J., 211, 256(354)Hiramaya, B. A., 50, 60, 102, 124(117)Hirose, M., 272, 306(102)Hirst, E. L., 177, 189, 248(85)Hjort, C., 272, 282, 288, 305(85; 86)Hoagland, P. D., 155, 171(66; 67)Hodder, H. J., 50, 52, 61, 125(137)Hohlweg, R., 46, 126(166)Hojo, K., 95, 131(329)Hojo, M., 176, 247(57)Holker, J. R., 180, 249(128)Hollender, J., 273, 306(108)Holman, G. D., 44, 56, 101, 122(62)Holmberg, B., 177, 189, 248(85)Holton, H., 227, 259(464)Holzach, V., 224, 258(431)Homan, H., 39, 121(27)Homma, I., 176, 248(71)Honda, S., 177, 249(88; 108)Honeyman, J., 185, 251(191)Honjou, N., 158, 172(75)Honma, M., 298, 307(161)Honma, T., 42, 56, 122(56)Honzatko, R. B., 272, 273, 281, 285, 288, 300,

306(103; 104)Hoos, R., 70, 110, 126(181; 184)Hoque, A. K. M. M., 176, 248(62)Horenstein, B. A., 110, 133(391), 264, 297,

303(7), 307(154)Horii, F., 227, 259(458)Horii, J., 193, 253(258)Horton, D., 99, 131(341), 176, 185, 193–194,

196, 202, 206–207, 219, 224, 247(37; 38;48), 251(177), 253(252), 254(279; 280),255(311; 326)

Hoshino, M., 227, 258(448)Hosoda, Y., 154, 160, 171(63), 172(80; 81)Hough, L., 96, 131(332), 176, 177, 247(40)Houptmann, S., 212, 256(364)Howard, F. B., 89, 130(295)Howard, G. A., 5, 13(9; 15)Howard, H. T., 6, 13(27)Howard, S. T., 39, 121(30)Hsieh, Y.-L., 272, 305(80)Hsu, L.-Y., 175, 246(27)Huang, S. J., 135, 170(5)Huang, Y., 193, 253(256)Hubbard, F. E., 272, 282, 288, 305(85)Huber, R. E., 295, 307(149)

AUTHOR INDEX 337

Hudlicky, T., 193, 227, 253(257),259(460–463), 260(487)

Hudson, C. S., 176, 182, 217, 247(55),250(144), 256(369–374)

Hughes, D. L., 176, 248(60)Hughes, N. A., 7, 14(44; 45)Hull, S. R., 176, 247(31)Humeres, E., 39, 121(29)Husain, S., 101, 131(351)Hutchins, R. O., 176, 248(70)Hutchinson, D. W., 7, 14(60; 61)Hutton, A., 235, 261(528)

Igaki, S., 298, 307(163)Igarashi, K., 42, 56, 122(56)Ignatova, L. A., 77, 127(227–229)Ihlo, B., 177, 249(91)Iijima, K., 154, 171(60)Iimo, N., 266, 304(44)Iimura, Y., 227, 258(448)Ikeda, S., 74, 127(195)Ikemoto, N., 48, 58, 123(93)Ikeuchi, Y., 147, 171(49)Illig, H.-K., 266, 303(27)Im, M. J., 268, 304(49)Imoto, I., 272, 275, 278, 305(76)Ingold, K. U., 185, 251(184)Iori, R., 36, 120(3), 294, 302, 307(141)Iribarren, I., 163, 172(88; 89; 92; 93; 95)Irving, H. M. N. H., 235, 261(528)Irwin, D., 265, 270, 272, 274, 290, 303(24),

305(84), 306(120)Isac-García, J., 41–44, 56, 68, 74, 122(57)Isbell, H. S., 176, 185, 190, 212, 234–237,

240–241, 247(49; 51; 53), 251(176),252(225; 230), 261(526; 530; 531)

Isecke, R., 71–72, 102, 126(188; 189),132(358–360)

Ishiguro, S., 95, 131(325)Ishihara, T., 177, 249(107)Ishikawa, Y., 193, 253(256)Ismailov, N., 41, 74, 122(37)Isogai, A., 273, 306(107)Itano, H. A., 174, 246(2; 3)Ito, H., 96, 131(331), 298, 307(161)Ito, M., 81, 129(265)Ito, Y., 234, 260(509)Itoh, M., 102, 132(360)Itoh, Y., 280, 306(128)


IUPAC-IUB Commission on BiochemicalNomenclature, 226, 310, 312, 314–317,319, 323(1–3), 324(4; 9; 10)

Iversen, T., 60, 124(107)Iwaisaki, T., 113, 133(406)Iwaisaki, Y., 113, 114, 133(408)Iwakura, Y., 141, 171(38)Iwanami, S., 298, 307(161)Iwatsuki, M., 138, 170(16)Izumi, M., 193, 253(256)

Jackman, L. M., 38, 121(20)Jacob, C., 176, 248(67)Jacob, G. S., 193, 253(260)Jacobi, H., 179, 249(117)Jacobs, W. A., 207, 255(333)Jacobson, G., 293, 307(136)Jacobson, R. H., 272, 274, 276, 305(77)Jaime, C., 244, 261(539)Jambor, B., 206, 224, 255(317)James, K., 227, 239, 260(477)James, M. N. G., 264, 275, 278, 287, 295, 302,

302(1)Jamieson, J. C., 193, 253(259)Janzen, E. G., 185, 251(186)Jaquier, R., 234, 261(517)Jardine, F. H., 219, 257(399)Jayamma, Y., 193, 253(259)Jayaraman, N., 104, 132(370)JCBN, 310, 312, 319, 324(4)Jeanloz, R. W., 64, 125(150)Jeenes, D. J., 276, 295, 302, 306(127)Jellinek, G., 217, 257(395)Jencks, W. P., 298, 299, 307(159), 308(164; 166)Jenke, B. T., 176, 248(66)Jenkins, J. A., 272, 305(90)Jenkins, R., 60, 103, 124(113)Jensen, V. J., 272, 305(96)Jeong, J.-H., 47, 58, 112, 123(88)Jermyn, M. A., 270, 304(60)Jiménez, J. L., 46, 58, 65, 70–73, 77, 79–81,

86–87, 90, 97, 123(77), 125(156),126(176; 186), 128(233; 234; 251),129(275; 280)

Jímenez-Barbero, J., 119, 134(419)Jiménez Blanco, J. L., 43–49, 57–60, 64, 68,

74, 75, 81, 87, 91–92, 117, 119, 123(66; 71;75; 83–85), 124(98; 100), 126(192; 193),130(310), 133(415; 416), 134(419)

AUTHOR INDEX338

Jiménez Requejo, J. L., 46, 58, 74, 77–79, 86,123(73; 74), 126(194), 128(231; 243; 246),129(274)

Jimeno, M. I., 227, 259(457)Jiricek, R., 115, 133(414)Jobe, H., 60, 102, 124(112), 132(355)Jochims, J. C., 46, 55, 58, 66, 87, 122(55),

123(76), 129(282; 283)Johns, B. A., 193, 253(256)Johns, K., 272, 279, 287, 294, 302, 305(88)Johnson, A. W., 8, 14(82–84)Johnson, C. R., 193, 253(256)Johnson, J. D., 137, 170(13)Johnson, L. M., 273, 277–278, 286, 295, 300,

306(115–117)Johnson, L. N., 264, 272, 275, 276, 278, 295,

302, 302(2), 305(76), 306(127), 307(147)Jonas, A. J., 60, 102, 124(112), 132(355)Jonen, H. G., 174, 246(4)Jones, J. K. N., 176–177, 189, 247(40), 248(85)Jones, M. M., 94, 130(315–318)Jones, S. G., 94, 130(315)Jones, T. A., 269, 270, 272–273, 276, 279, 280,

287, 288, 291, 304(55), 305(79)Jordan, A. D., Jr., 74, 127(199)Jordan, M. D., 273, 277, 278, 286, 295, 300,

306(117)Joshi, D. D., 227, 259(459)Jotterand, A., 189, 252(222)Jourdan, F., 176, 247(52)Juaristi, E., 244, 261(540)Judkins, B. D., 89, 130(294)Juenge, E. C., 77, 127(216)Jung, S.-H., 193, 253(257)Junqua, S., 60, 124(110)Just, E. K., 194, 254(280)Juy, M., 272, 282, 288, 305(83)

Kabalka, G. W., 176, 248(70)Kacher, M., 176, 248(70)Kadowaka, H., 280, 306(128)Kahlenberg, A., 45, 58, 102, 123(80)Kakehi, K., 177, 249(88; 108)Kakudo, M., 272, 278, 305(94; 95)Kalk, K. H., 273, 279, 286, 306(106; 107; 109;

110)Kallin, E., 50–52, 61, 84, 103, 125(135; 136),

129(271), 132(368)Kalyanaraman, B., 95, 131(321)


Kamada, T., 266, 304(42; 44)Kameda, Y., 193, 253(258)Kamitori, Y., 176, 247(57)Kamiya, H., 176, 247(31)Kanaya, E. N., 89, 130(295)Kanda, T., 266, 268, 270, 284, 303(36), 304(70)Kandil, S. H., 155, 171(68)Kaplan, L., 198, 254(296)Karabinos, J. V., 217, 256(374)Karaday, S., 176, 248(60)Karamanos, N. K., 176, 247(31)Karimullah, 5, 13(7)Karlson, G. B., 193, 253(259)Karpellus, P., 179, 187, 249(115; 116)Karplus, A., 265, 270, 272, 303(24), 305(84)Karplus, P. A., 270, 272, 274, 290, 304(56),

306(120)Kasahara, Y., 75, 76, 127(204; 208)Kassab, R., 64, 125(154)Kassem, A. A., 97, 98, 104, 131(336; 337)Kasube, Y., 272, 306(102)Kasumi, T., 266, 270, 303(38), 304(71)Kasuya, A., 113, 133(403)Kato, K., 206, 255(316), 266, 304(42–44)Kato, N., 141, 171(39)Katsarava, R. M., 161, 172(83)Katsuya, K., 141, 171(39)Kaul, B. L., 190, 252(227)Kaushal, G. P., 110, 133(387)Kayama, Y., 154, 160, 171(64)Kayser, K., 51, 62, 103, 125(139)Keck, G. E., 89, 130(305)Keeffe, J. R., 234, 244, 261(514)Kegel, W., 177, 249(94)Keil, K. D., 198, 254(298)Kelley, J. A., 89, 130(292)Kempton, J. B., 294, 307(139; 145)Kenner, G. W., 5–7, 13(8; 10; 14; 32; 33; 37; 40;

41), 14(44; 45; 47–49; 51; 57; 58)Kenner, J., 197, 254(288)Kenny, D. H., 175, 246(27)Keopsel, H. J., 217, 257(385)Kerékgyártó, J., 50, 51, 61, 124(124; 125)Kerr, D. E., 174, 246(9)Kersters-Hilderson, H., 266, 270, 303(38),

304(65)Kettner, M., 174, 246(19)Khan, R. A., 142, 171(41)Khan, S. H., 226, 227, 258(442)Kharadze, D. P., 161, 172(83)

AUTHOR INDEX 339

Kharitonenkov, I., 102, 132(359)Kholodova, E. V., 61, 125(132)Khorana, H. G., 7, 14(46)Khorlin, A. Ya., 42–44, 56, 64, 70, 75, 108,

122(36; 42; 60), 125(147–149), 126(164;175), 132(384), 133(385)

Kieburg, C., 41–44, 50, 53, 61, 104, 122(47),124(130), 132(374; 375; 377)

Kiely, D. E., 156, 157, 172(69–74)Kihlberg, J., 102, 132(364)Kilburn, D. G., 264, 272, 294, 302(5), 307(140)Kilpper, G., 233, 237, 260(502)Kim, C.-H., 89, 130(293)Kim, C.-S., 280, 306(128)Kim, Y., 104, 132(372)Kimber, S. J., 103, 132(368)Kimura, A., 298, 307(161–163)Kimura, H., 89, 113, 114, 130(301), 133(405;

406; 409)King, C. H. R., 193, 253(258)King, R. W., 206, 207, 225, 255(325), 258(435)Kinoshita, T., 75, 76, 127(203–205; 208)Kirby, G. W., 6, 7, 13(26), 14(54; 60)Kirchhoefer, R. D., 77, 127(216)Kirchner, K. D., 198, 254(295)Kirk, B., 109, 110, 115, 133(386; 413)Kirsch, J. F., 275, 306(125)Kirschenlohr, W., 220, 257(412)Kishi, H., 155, 171(65)Kitaev, Yu. P., 176, 180, 247(59)Kitahashi, H., 41–44, 54, 58, 113, 114,

122(48), 133(408)Kitahata, S., 265, 268, 270, 271, 273, 280, 281,

303(21; 22), 304(50; 51)Kjær, A., 101, 131(344)Klapötke, T. M., 39, 121(28)Klausner, A. E., 39, 121(25)Klayman, D. L., 37, 121(11)Klebe, J., 198, 233, 254(297)Klein, C., 273, 306(108)Klein, H. W., 266, 268, 273, 277–278, 284, 286,

295, 300, 304(41; 48; 49), 306(115; 119)Klein, R. A., 102, 132(359)Klenk, H.-D., 102, 132(359; 360)Klimov, E. M., 41, 54, 122(49)Klimov, E. V., 61, 121(33)Kline, P. C., 264, 303(7)Klobusiky, M., 174, 246(18)Klock, J. C., 176, 247(34)Klockow, A., 176, 247(35)


Kluepfel, D., 272, 305(89)Knapp, S., 58, 109, 110, 114, 115, 123(91),

133(386; 410; 413)Knecht, E., 197, 254(287)Knight, E. C., 197, 254(288)Knight, J. G., 89, 130(306; 307)Knirel, Yu. A., 61, 125(132)Knowles, J. K. C., 269, 270, 272–273, 276,

279–280, 287, 288, 291, 304(54; 55),305(79)

Knutsen, L. J. S., 89, 130(294)Kobayashi, H., 137, 170(12)Kobayashi, J., 89, 130(303)Kobayashi, Y., 45, 46, 58, 113, 114, 123(89),

133(396; 399; 401–403; 407)Koch, K. R., 235, 261(528)Kocharova, N. A., 61, 125(132)Kochetkov, N. K., 61, 121(33), 125(132; 133)Kocourek, J., 80, 128(255)Koepsel, H. J., 217, 257(383)Kohler, H., 207, 255(339)Kojima, S., 51, 62, 103, 125(139)Kolb, H. C., 89, 130(307)Kolkaila, A. M., 217, 219, 257(394)Kollmam, P. A., 299, 308(165)Komarov, A. M., 95, 130(318; 319), 131(323)Komitsky, F., Jr., 187, 252(208)Konaka, R., 232, 233, 260(497)Kondo, S., 226, 227, 258(442)Kong, C.-T., 102, 132(354)König, B., 104, 132(376)Konig, R., 212, 256(356)Konigsberg, M., 189, 202, 223, 252(218)Kononov, L. D., 102, 132(364)Konstantinidis, A. K., 266, 269, 271, 273, 279,

302, 303(25), 304(72)Kopecek, J., 138, 170(19)Koshinen, A. M. P., 194, 254(262)Koshland, D. E., Jr., 271, 274, 304(74),

306(123)Kost, D., 244, 261(539)Kötter, S., 47, 63, 125(144)Kotyzová, D., 94, 130(316; 317)Koutensky, J., 94, 130(316; 317)Kovác, P., 51, 60, 103, 124(111)Kovacik, V., 176, 247(31)Kovács, J., 65, 66, 85, 93, 125(159; 160),

130(313), 227, 238, 259(474)Koyama, Y., 141, 171(39)Krahn, R. C., 219, 257(405)

AUTHOR INDEX340

Krallmann-Wenzel, U., 47, 63, 104, 125(144),132(376)

Kramer, J. H., 95, 131(323)Kraus, A., 183, 184, 207, 209, 212, 220, 225,

227, 233, 234, 250(156; 168), 256(342),258(436)

Krause, J. G., 177, 248(78)Krausz, P., 227, 259(469)Kren, V., 104, 132(375)Krepinsky, J. J., 44, 64, 122(44)Kristian, P., 37, 38, 121(9)Kubrina, L. N., 95, 131(322)Kucar, S., 174, 246(20)Kuhn, R., 220, 257(412)Kukkola, P., 177, 248(73)Kumar, G. S., 138, 170(20)Kumar, S., 227, 238, 259(472)Kunz, H., 44, 64, 101, 122(46), 125(151),

131(352)Kunze, K. L., 174, 246(8)Kuo, E. Y., 176, 248(72)Kuppusamy, P., 95, 131(320)Kurita, K., 141, 171(38; 39)Kuroki, R., 293, 295, 307(134; 135)Kusunoki, M., 272, 278, 305(94; 95)Kuwahara, M., 160, 172(82)Kuznetsov, M. A., 183, 250(158)Kuzuhara, H., 70, 71, 115, 126(173),

133(412)Kyono, K., 234, 260(509)

Lai, C.-S., 95, 130(318; 319)Laidig, K. E., 39, 121(22)Laing, M., 175, 246(24)Laitinen, T., 272, 305(93)Lakhtina, O. E., 42, 56, 108, 122(60),

126(164), 132(384)Lamprecht, H., 177, 249(90)Lang, F., 113, 133(404)Langdon, R. G., 42, 44, 56, 101, 102, 122(58;

59; 61), 132(353)Lantos, I., 39, 121(26)Larcheveque, M., 194, 254(262)Lardy, H. A., 244, 261(535)Larner, J., 274, 306(122)Larsen, R. P., 235, 261(527)Larson, A., 272, 305(100)Lassaletta, J.-M., 176, 247(54)Laszlo, P., 227, 260(479)


Lau, J., 39, 121(31)Laurence, C., 39, 121(24)Laurie, J. I., 272, 305(90)Laus, G., 77, 127(218)Law, H., 82, 106, 107, 118, 129(267), 132(382)Lawrence, D. R., 140, 154, 170(32)Lawson, C. L., 273, 306(109)Lawson, S. L., 272, 305(92)Lawson, T., 174, 246(12)Lay, L., 227, 258(451)LeBlay, K., 80, 128(257)Lebold, S. A., 176, 247(58)Lecocq, J., 6, 13(31)Ledford, B. E., 42, 54, 113, 122(52)Lee, B.-H., 95, 131(326; 327)Lee, H. H., 44, 64, 122(44)Lee, P. H., 193, 253(256)Lee, R. T., 102, 132(365)Lee, Y. C., 63, 102, 125(146), 132(362; 365)Lefeuvre, M., 79, 128(238)Legendre, B. L., 27, 28, 34(12–14)Legler, G., 193, 253(257), 264–266, 303(16;

27)Lehmann, G., 177, 249(94)Lehmann, J., 112, 115, 133(393–395; 414),

266, 268, 271, 273–276, 284, 302,303(28–33), 304(46; 47)

Lehn, P., 272, 306(105)Le-Hong, N., 189, 252(220)Leiner, H., 244, 261(534)Lelièvre, Ph., 92, 130(311)Lemieux, R. U., 185, 227, 239, 250(172),

260(477)Leminger, O., 182, 250(145)Len, C., 96, 131(333)Lenaz, G., 174, 246(15)Lensen, N., 194, 253(261)Lentovaara, P., 269, 280, 304(54)Lenz, R. W., 164, 172(90)Leong, K.-W., 137, 170(15)Leoni, O., 36, 120(3)Le Questel, J.-Y., 39, 121(24)Leray, E., 41, 64, 125(152)Le Roy-Gourvennec, S., 71, 126(191)Leuck, M., 101, 131(352)Leupold, F., 193, 253(255)Levene, P. A., 194, 207, 254(278), 255(333)Lever, J. E., 102, 132(354)Lewis, G. E., 233, 260(507)Ley, S. V., 89, 130(306; 307), 227, 259(460)

AUTHOR INDEX 341

Li, J., 113, 133(404), 193, 253(256)Liao, D.-I., 302, 308(170)Libit, L., 176, 248(71)Lichtenthaler, F. W., 244, 261(534)Liebenow, W., 177, 179, 249(111)Liebster, J., 217, 256(377)Lin, J. K., 176, 247(31)Lin, S., 295, 307(149)Lin, T. H., 156, 172(69; 71)Lin, T.-S., 89, 130(304)Lindberg, A. A., 51, 52, 60, 61, 103, 124(106;

109; 118; 122), 132(367)Lindenberg, S., 103, 132(368)Lindhorst, T. K., 41–44, 47, 50, 53, 61, 63, 104,

122(47), 124(130; 131), 125(144),132(374–377), 294, 307(144)

Lindman, Y., 77, 127(221)Lineback, D. R., 187, 238, 252(207; 208)Linek, K., 174, 176, 180, 246(17–20), 247(31),

249(129)Linkletter, B., 86, 129(276; 277)Lipunova, G. N., 189, 206, 235, 252(223)Little, N., 60, 124(105)Liu, H., 176, 248(61)Liu, M.-C., 89, 130(304)Liu, P. S., 193, 253(258)Liu, W., 266, 270, 303(39)Livio, P. F., 189, 252(220)Ljunggren, A., 52, 60, 124(106)Lloyd, D. M. G., 207, 255(330)Lobell, M., 75, 127(206)Lohray, B. B., 193, 253(259)Lo Leggio, L., 272, 305(90)Lolsing, M., 181, 250(141)Lönn, H., 50–52, 61, 125(135)Loo, D. D. F., 50, 60, 102, 124(117),

132(354)López Aparcio, F. J., 79, 128(239)López-Barba, E., 41, 43, 56, 57, 68, 77,

122(65)López-Castro, A., 74, 79, 80, 87, 126(193),

128(252)López-Mardomingo, C., 39, 121(27)Losee, K., 181, 250(141)Lostao, M. P., 50, 60, 102, 124(117)Lou, J., 109, 110, 133(386)Lovell, A. V., 176, 248(60)Low, M. G., 315, 324(11)Lowe, G., 276, 295, 302, 306(127)Lubineau, A., 226, 227, 258(442)


Luckas, G., 227, 259(470)Ludewig, M., 50, 61, 104, 124(131)Lueders, H., 144, 171(44; 45)Luna, H., 227, 259(463)Lund, H., 176, 248(63)Lundblad, A., 84, 129(271)Luo, J., 82, 83, 86, 129(269)Lutz, W., 233, 260(508)Lycka, A., 206, 207, 255(329)Lyle, G. G., 219, 257(406)Lyons, R., 185, 250(170)Lythgoe, B., 5, 6, 13(4–6; 8–13; 15; 16;

18; 35)Lyttle, B., 230, 260(491)

MacCleod, A. M., 293, 294, 307(133)MacDonald, D. L., 194, 254(266)Machado, A. S., 227, 259(470)Macharadze, R. G., 64, 122(36), 125(147–149)Macher, B. A., 323, 324(16)Machida, H., 48, 58, 123(94)Machón, Z., 41, 74, 122(40)Machytka, D., 102, 132(359)Maciejewski, S., 48, 60, 65, 118, 124(99),

125(155), 133(418)Mackawy, K., 186, 251(197–199; 201)MacKenzie, D. A., 276, 295, 302, 306(127)Mackenzie, G., 91, 93, 130(308)MacKenzie, L., 302, 307(132), 308(172)Madhavan, G. V. B., 89, 130(297)Madin, A., 89, 130(307)Madi-Puskas, M., 227, 259(468)Madsen, N. B., 266, 270, 294, 303(39),

307(144)Madson, M. A., 196, 254(284)Maeda, H., 227, 258(448)Maetinez-Ripoll, M., 175, 246(25)Magasanik, B., 202, 206, 227, 230, 241,

255(309), 260(488), 261(533)Magnusson, G., 50, 61, 102, 124(129),

132(364)Magrath, D. I., 7, 14(64; 65; 69)Mahapatro, S. N., 174, 246(13)Mahmoud, M. A., 186, 251(201)Mahuteau, J., 64, 125(153)Mahy, J. P., 174, 246(11)Maimind, V. I., 198, 254(301; 302), 255(303;

304)Mair, G. A., 295, 307(147)

AUTHOR INDEX342

Major, A., 180, 192, 206, 207, 209, 221, 224,235, 250(133), 252(240), 255(320; 337),256(350), 257(420)

Mak, I. T., 95, 131(323)Makarenko, T. A., 61, 125(132)Malik, Sh. S., 227, 259(451)Mal’kova, V. P., 43, 44, 75, 122(42)Malver, O., 101, 131(344)Malysheva, N. N., 41, 54, 121(33), 122(49)Manasek, Z., 185, 251(178; 179)Mancy, S., 220, 257(415)Mangeney, P., 194, 253(261)Manger, I. D., 50, 51, 62, 103, 125(138)Manners, D. J., 270, 304(59)Mansour, E. M. E., 97, 98, 104, 131(336; 337),

155, 171(68)Mansuy, D., 174, 246(11)Maples, K. R., 233, 235, 260(505)Marby, C. A., 89, 130(307)March, J., 244, 261(540)Marchione, C. S., 74, 127(199)Marco-Contella, J., 227, 259(457)Margolis, S. A., 174, 246(15)Marino, C., 41, 43, 55, 74, 75, 87–88, 122(53;

54)Marino, S., 55, 66, 122(55)Markley, J. L., 222, 258(423)Márquez, R., 79, 128(248–250)Marquez, V. E., 89, 130(292; 293; 296)Marsden, I., 266, 269, 273, 279, 302, 303(25)Marshalkin, M. F., 183, 250(159)Marshall, D. R., 207, 255(330)Marshall, E. D., 235, 261(527)Martin, G. J., 227, 239, 260(476)Martin, J. C., 89, 130(297)Martin, J. L., 273, 277, 286, 306(116)Martin, M. L., 227, 239, 260(476)Martínez de Ilarduya, A., 163–165, 167,

172(89; 91; 93; 95)Martinez-Grav, A., 227, 259(457)Martín-Pastor, M., 119, 134(419)Martins, J., 181, 250(141)Martnez, L., 227, 259(457)Marubayashi, Y., 168, 172(96)Maryanoff, B. E., 74, 127(199), 176, 227,

248(70), 259(451)Masada, R. I., 176, 247(34)Mason, H. S., 7, 13(39), 14(50)Mass, T. A., 135, 170(2)Masson, S., 71, 126(191)


Masuda, R., 176, 247(57)Mather, J., 6, 13(32; 33)Matrosovich, M. N., 70, 102, 126(178),

132(361)Matsuda, A., 48, 58, 123(94)Matsui, H., 266, 268–269, 271, 284, 296, 298,

303(33), 304(45; 52; 53), 305(75),307(161)

Matsui, K., 193, 253(258)Matsumura, I., 275, 306(125)Matsuo, T., 193, 253(258)Matsuura, M., 158, 172(76)Matsuura, T., 234, 260(509)Matsuura, Y., 272, 278, 305(94; 95)Matsuzaki, K., 78, 128(236)Matteson, J. L., 174, 246(2)Matthews, B. W., 272, 274, 276, 293, 295,

305(77), 307(134; 135)Mattson, D., 95, 130(318)Maurer, K., 202, 206, 220, 255(310)Mayer, F. C., 274, 306(122)Mayer, R., 37, 121(14)McBroom, C. R., 60, 102, 124(104)McCarter, J. D., 264, 291–294, 302(3),

307(142; 143)McCasland, G. E., 226, 230, 258(437),

260(491)McCombie, S. W., 87, 129(284)McComsey, D. F., 227, 259(451)McDonald, G. G., 155, 171(67)McGlynn, S. P., 37, 121(15)McLaughlin, M. A., 193, 253(256)McLaughlin, P. J., 273, 277–278, 286, 295, 300,

306(115; 117)McNeil, D., 5, 13(4)McPherson, A., 272, 305(100)McPherson, E., 74, 127(196)Medlin, E. H., 6, 13(23)Meeks, J. L., 37, 121(15)Meguso, T., 217, 256(379)Mehltretter, C. L., 139, 170(30)Mellies, R. L., 139, 170(30)Mendes, C., 60, 124(115)Méndez-Castrillón, P. P., 41–43, 54, 88,

122(34; 35)Menegatti, E., 36, 120(3)Mentch, F., 297, 307(153)Merle-Subrey, L., 138, 170(21)Meshreki, M. H., 176, 196, 212, 217, 219,

247(48), 256(361), 257(394)

AUTHOR INDEX 343

Messmer, A., 63, 85, 125(145), 129(272), 177,190, 192, 206, 224, 227, 238, 239, 249(98),252(228; 231; 235–239; 241), 255(318;320), 259(474), 260(475)

Mester, L., 177, 180, 182–183, 189–190, 192,194, 202, 206–207, 209, 212, 219, 221,224, 227, 233–235, 241, 249(98; 127; 130),250(133; 151; 155), 252(223; 226; 228;232; 242), 254(282), 255(311; 317–320;323; 324; 327; 328; 337; 338; 341),256(350; 357; 358), 257(402; 420)

Mester, M., 192, 235, 252(232; 233–240)Mészáros, P., 93, 130(313), 227, 239, 260(475)Meuwly, R., 89, 130(300)Meyer, R., 184, 185, 212, 215, 221, 250(162),

257(417)Meyers, A. I., 193, 253(256)Michalski, J. J., 6, 7, 13(29), 14(59)Micheel, F., 197, 198, 201, 219, 220, 254(291),

257(414)Michelson, A. M., 6–8, 13(19; 20; 34; 42),

14(66; 73; 76; 77)Micklewright, R., 76, 127(207)Mickova, V., 94, 130(317)Middleton, S., 227, 259(467)Midoux, P., 50, 61, 124(110; 115; 126)Mielczarek, I., 41, 74, 122(40)Mikami, B., 272–274, 280, 281, 285, 288, 292,

300, 301, 306(101; 102; 129)Mikkelsen, J. M., 272, 282, 288, 305(85)Mikoyan, V. D., 95, 131(322)Milde, K., 177, 249(92)Miles, H. T., 89, 130(295)Mileski, C. A., 176, 248(70)Mil’grom, Yu. M., 75, 127(200)Millan, M., 79, 128(248; 249)Miller, G. W., 8, 14(83)Miller, J. B., 194, 254(268; 269; 277)Miller, K. J., 77, 127(213)Miller, R. C., Jr., 264, 272, 302(5)Miller, T. W., 226, 258(443)Miller, V. P., 176, 248(61)Mills, J. A., 8, 14(82; 83), 219, 257(404)Mino, T., 176, 247(56)Minor, J. L., 220, 257(411)Minoura, Y., 160, 172(79)Miocque, M., 64, 125(153)Mitchell, A., 234, 261(519)Mitchell, P. W. D., 197, 254(290)Mitchell, W. L., 89, 130(294)


Mitsuya, H., 89, 130(292; 293)Miwa, I., 226, 258(441)Miyajima, K., 141, 171(38)Miyamato, Y., 227, 258(451)Miyamoto, S., 113, 133(403)Miyazaki, H., 113, 133(396; 401; 407)Miyumdar, A. M., 227, 259(459)Mizokami, K., 265, 303(22)Mó, O., 38–39, 121(16; 17)Mochalova, L. V., 70, 126(178)Mochizuki, T., 81, 114, 129(264)Moczar, E., 182, 206, 207, 212, 250(151),

255(323; 324), 256(357; 358)Modro, T. A., 39, 121(25)Mohammed, M. A. A., 186, 251(201)Mohammed, Y. S., 217, 219, 257(394)Mohammed-Ali, M. M., 222, 258(425; 426)Mokhlisse, R., 39, 121(27)Moldenhauer, H., 184–185, 187, 212, 250(163;

164)Moldenhauer, W., 198, 225, 230, 254(299),

258(436)Molina, I., 148, 149, 171(53; 54)Molina, J. L., 43–44, 47, 57, 58, 68–70, 84,

123(67; 68), 126(169; 170)Molina, M. T., 38, 39, 121(16)Molina Molina, J., 41, 43, 56, 57, 68, 77,

122(65)Molina Pinilla, I., 153, 171(56–59)Mondange, M., 80, 128(257)Mong, T. K.-K., 104, 132(373)Monneret, 92, 130(312)Monsigny, M., 50, 60, 61, 124(110; 115; 126)Montserrat, J. M., 138, 170(24)Moon, S.-H., 227, 260(482)Moorhouse, S. J., 192, 253(246)Mooser, G., 264, 303(17)Mordarski, M., 41, 74, 122(40)Moreda, W., 42, 57, 68, 77, 123(70)Moreno Marín, A., 70, 72, 74, 87, 126(177; 192)Mori, H., 298, 307(161)Mori, O., 70, 71, 126(173)Morin, C., 94, 130(314)Morita, E., 217, 256(379)Morita, S., 96, 131(331)Morita, Y., 272, 306(102)Mornon, J.-P., 272, 306(105)Morosoli, R., 272, 305(89)Morton, D. W., 157, 172(72)Moss, G. P., 8, 14(81)

AUTHOR INDEX344

Mostad, A., 180, 249(124)Motherwell, W. B., 88, 89, 129(286), 186,

187, 189, 251(203), 252(213)Mousaad, A., 186, 251(200)Mufti, K. S., 142, 171(41)Mukaiyama, T., 58, 95, 123(90),

131(329)Mukerjee, A. K., 37, 121(13)Mukherjee, S., 89, 130(307)Müller, H. P., 65, 122(43), 125(158)Muller, V., 222, 258(428)Mullins, R. E., 56, 101, 102, 122(61), 132(353)Mungara, P. M., 136, 137, 170(7)Muñoz-Guerra, S., 138, 147, 161–168,

170(22–24), 171(50), 172(84–89;91–95; 97; 98)

Murali Dhar, T. G., 58, 123(91)Muramoto, K., 176, 247(31)Murkami, K., 141, 171(39)Murphy, R. C., 77, 127(220)Murphy, T. J., 206, 207, 225, 255(325),

258(435)Murray, B. W., 47, 58, 112, 123(88)Murray, M., 269, 280, 304(54)Murray, R. W., 234, 260(512)Muto, N., 227, 259(466)Myers, A. G., 176, 177, 248(72; 73)

Nagabushan, T. L., 227, 239, 260(477)Nagano, H., 177, 249(107)Nagy, Z., 61, 124(124)Nahrstedt, A., 101, 131(345)Nair, K. M., 234, 261(518)Nair, P. M., 234, 261(522)Nair, V., 177, 249(87)Naito, T., 43, 126(165), 177, 249(107)Najib, B., 91, 93, 130(308)Nakahashi, G., 193, 253(257)Nakajima, M., 113, 133(407)Nakamura, H., 154, 155, 160, 171(62; 63; 65),

172(82)Nakamura, S., 176, 248(71)Nakano, M., 270, 304(70)Nakata, K., 177, 249(105)Nakhla, N. A., 50, 52, 61, 125(137)Nance, S., 273, 306(114)Narayanan, C. R., 227, 259(459)Nardini, W., 230, 260(493)Nashed, M. A., 176, 196, 247(48)


Nasr, A. Z., 97, 131(335)Nasr, M. A. M., 97, 98, 104, 131(336; 337),

177, 186, 220, 225, 249(102), 251(193;194; 200; 201), 257(408)

Nassimbeni, L. R., 235, 261(528)Nastruzzi, C., 36, 120(3)Naughton, A. B., 58, 70, 110, 123(91),

126(181)Neilson, A. H., 7, 14(69)Neilson, T., 220, 257(410)Neimann, W., 177, 249(109)Nelson, S. F., 211, 256(354)Nemogova, E., 174, 246(18)Nepogodiev, S. A., 104, 132(370)Nesmzmelyi, A., 190, 252(231)Neuberg, C., 177, 249(109)Newton, R. F., 89, 130(294)Nguyen-Xuan, T., 193, 253(247)Nichols, P. L., Jr., 138, 170(26)Nicholson, L. K., 270, 304(57)Nickol, R. G., 99, 131(341)Nicotra, F., 227, 258(451)Nieforth, K. A., 193, 253(257)Nikolajewski, H. E., 177, 249(96)Nimura, N., 41, 75, 76, 126(162),

127(203–205; 208)Nishi, N., 147, 171(49)Nishimura, S., 147, 171(49)Nishimura, Y., 177, 226, 227, 249(108),

258(442)Noda, Y. J., 160, 172(79)Noe, L. J., 234, 261(513)Nomenclature Committee of IUB, 313,

324(8)Nong, V. H., 280, 306(128)Nongrum, M. F., 104, 132(373)Nonhebel, D. C., 234, 261(519)Noor, F., 101, 131(351)Noori, G., 50, 61, 124(129)Norberg, T., 50–52, 61, 84, 125(135), 129(271)Nordman, C. E., 175, 246(27)North, A. C. T., 272, 275, 278, 295, 305(76),

307(147)Notario, R., 38–39, 121(16; 27)Novotna, Z., 174, 246(20)

Obereigner, B., 138, 170(19)Ochoa, E., 100, 131(342)O’Connor, R., 185, 187, 251(174)

AUTHOR INDEX 345

Ogata, N., 154, 155, 160, 171(60–65),172(80–82)

Ogawa, S., 41–44, 46, 47, 54, 58, 89, 113–114,122(48), 123(86; 87), 130(301), 133(397;398; 400; 405; 406; 408; 409), 226–227,258(439–441; 444; 451), 259(452–454)

Ogawa, Y., 96, 131(331)Ogueira, H. A., 158, 160, 172(77; 78)Ogura, H., 41, 75, 77, 81, 125(161), 126(162),

127(203), 128(232; 235), 129(265), 177,249(105)

Oguri, S., 177, 249(88)Ohara, S., 176, 247(57)Ohashi, T., 89, 130(301)Ohle, H., 209, 215, 256(346; 351)Ohta, K., 78, 128(236)Ohtake, T., 154, 171(62)Ohtsuka, M., 227, 259(452)Ohya, T., 270, 304(62)Oikonomakos, N. G., 264, 302(2)Okada, G., 264–266, 268, 270, 271, 280, 281,

284, 298, 303(14; 19; 20; 36), 304(45; 66)Okada, M., 158, 172(75; 76)Okamoto, S., 154, 171(61)Okamoto, Y., 168, 172(96)Oki, M., 38, 121(18), 244, 261(538)Okimoto, M., 176, 186, 248(63), 251(202)Okuda, J., 226, 258(441)Olano, D., 44, 57, 84, 123(68)Olejniczak, B., 58, 123(92)Olin, S. M., 194, 254(267)O’Neill, R. A., 226, 227, 258(442)Onnen, O., 184, 185, 212, 215, 221, 250(162)Openshaw, H. T., 6–7, 13(25), 14(52)Oppenheim, A., 302, 308(171)Oppenheimer, N. J., 299, 307(131; 158),

308(165; 167)Orgueira, H. A., 148–150, 171(53–55)Oritz de Montellano, P. R., 174, 246(7–10)Orning, A., 176, 247(44)Ortiz, C., 42, 57, 68, 77, 123(70)Ortiz de Montella, P. R., 187, 252(210)Ortiz Mellet, C., 37, 40–49, 56–60, 63–66, 68,

70, 72, 74–75, 77, 81, 82, 85, 87, 91–92,102, 106–108, 117–120, 121(8), 122(64),123(66; 69; 71; 72; 75; 81–85),124(98–100; 102), 125(155; 159; 160),126(177; 192; 193), 127(201; 222–226),129(267), 130(310), 132(382; 383),133(415–418), 134(419; 420)


Ortiz Mellet, M. C., 46, 58, 74, 78, 123(73)Oscarson, S., 95, 131(330)Oshikawa, T., 89, 130(303)Osipov, C. A., 189, 206, 235, 252(223)Ostmann, P., 42, 126(163)O’Sullivan, M. L., 106, 107, 118, 132(382)Ottembrite, R. M., 137, 170(8)Ouchi, T., 137, 170(12)Overend, W. G., 227, 238, 259(472; 473)Oztruk, D. H., 273, 283, 284, 288,

306(112)

Padrines, M., 110, 133(389)Pagé, D., 103, 104, 106, 132(378–381)Page, P., 176, 248(67)Page, T. F., Jr., 176, 219, 247(38)Pakulski, Z., 121(32)Palacios, J. C., 46, 58, 65, 70–73, 77, 79–81,

86–87, 90, 97, 123(77), 125(156),126(176; 186), 128(233; 234; 251),129(275; 280)

Palacios Albarrán, J. C., 46, 58, 74, 77–79, 86,123(73; 74), 126(194), 128(231; 243; 246),129(274), 192, 252(243)

Palm, D., 266, 268, 273, 278, 295, 300, 304(41;49), 306(119)

Palmieri, S., 36, 120(3), 294, 302, 307(141)Palovcik, R., 180, 249(129)Pan, Y.-T., 110, 133(387), 193, 253(254)Panayotova-Heiermann, M., 102, 132(354)Pangrazio, C., 227, 258(451)Panza, L., 227, 258(451)Papadaki-Valirki, A. E., 74, 127(195)Papandreou, G., 70, 110, 126(179; 180; 182),

133(387)Paranjpe, M. G., 74, 127(198)Parello, J., 190, 206, 207, 252(226), 255(323;

324; 328)Parihar, D. B., 6, 13(21; 24)Parkanyi, C., 234, 261(525)Parker, K. J., 142, 171(41)Parkin, D. W., 297, 307(153; 154)Parrish, F. W., 27, 33(7)Parrot-Lopez, H., 41, 64, 125(152–154)Patai, S., 176, 248(63)Paulsen, H., 22, 58(449), 72, 177, 193, 227,

249(100), 253(254; 255), 258(447),259(455; 456)

Paulus, A., 176, 247(35)

AUTHOR INDEX346

Pausacker, K. H., 185, 197, 198, 223,251(189), 254(294)

Pavlisko, A., 138, 170(18)Paxton, J., 211, 256(355)Payen, F., 272, 278, 286, 295, 300, 305(98; 99)Pazur, J. H., 270, 304(61)Peciar, C., 180, 249(129)Pegorier, L., 194, 254(262)Pelyvas, I., 176, 227, 247(54), 259(468),

260(479)Penadés, S., 120, 134(420; 421)Penninga, D., 273, 279, 286, 306(109; 110)Pepperman, A. B., 27, 34(15)Percheron, F., 270, 304(63)Percival, E. E., 224, 258(430; 433)Percival, E. G. V., 173, 184, 221, 222, 224,

246(1), 250(166), 257(418), 258(430; 433)Pérez, R., 138, 170(25)Pérez-Garrido, S., 74, 87, 126(193)Perrakis, A., 302, 308(171)Perret, F., 189, 193, 252(221; 222), 253(247; 248)Perry, F. M., 181, 250(141)Pessen, H., 155, 171(67)Péter, A., 77, 127(218)Peter, B., 177, 189, 248(85)Petersen, C. S., 180, 202, 205, 206, 249(125;

126), 255(307)Petersen, S. B., 272, 305(96)Petit, Y., 194, 254(262)Petsko, G. A., 174, 246(9)Pettersson, G., 272, 273, 276, 287, 305(79)Philips, K. D., 194, 254(279)Phillips, D. C., 272, 275, 278, 295, 305(76),

307(147)Piazza, M. J., 219, 257(406)Picasso, S., 193, 253(256)Pickersgill, R. W., 272, 305(90)Pierozynski, D., 121(32)Pigman, W., 185, 251(177)Pihko, P. M., 194, 254(262)Pilo, M. D., 86, 129(280)Pingli, L., 227, 259(451)Pinkus, G., 183, 185, 250(161)Pinna, L., 193, 253(256)Pintér, I., 63, 65, 66, 85, 93, 125(145; 159; 160),

129(272), 130(313), 190, 192, 227, 238,239, 252(231; 241), 259(474), 260(475)

Pitzer, K. K., 193, 253(257)Placek, J., 185, 251(178; 179)Platt, R. M., 193, 253(259)


Platts, J. A., 39, 121(30)Pleshkova, A. P., 75, 127(202)Ploven, S., 190, 252(224)Plusquellec, D., 79, 128(237; 238)Poljak, R. J., 272, 282, 288, 305(83)Pollet, P., 234, 261(524)Poncet, J., 189, 252(221; 22)Pons, J.-F., 80, 128(257)Porco, J. A., Jr., 193, 253(257)Portal Olea, D., 41, 43, 56, 57, 68, 77, 122(65)Postel, D., 96, 131(333)Posternak, T., 227, 230, 244, 260(485; 489),

261(536)Postovskii, I. Y., 189, 206, 235, 252(223)Potapova, N. P., 81, 129(266)Poteete, A. R. E., 275, 306(126)Potter, A. L., 217, 257(382)Povarov, L. S., 81, 129(266)Pozuelo, C., 227, 259(457)Pradera, M. A., 41–44, 47, 54, 57–58, 68–70,

77, 81, 84, 87, 101, 122(50), 123(67; 68;78; 79), 126(169; 170)

Pradera Adrián, M. A., 41–43, 56–58, 68, 75,77, 122(64), 123(69; 72), 127(201;222–226)

Pratt, J. S., 217, 256(373)Prean, M., 179, 187, 249(115; 116)Preobrazhenskaya, M. N., 81, 129(266)Prestwich, G. D., 227, 260(481)Price, J. D., 227, 259(461–463)Prieto, A., 138, 170(25)Prough, R. A., 174, 246(4)Provencher, D. H., 193, 253(257)Purandare, A., 114, 133(410)

Qian, M., 272, 278, 286, 295, 300, 305(98; 99)Qiao, L., 227, 259(463)Quiclet-Sire, B., 227, 259(469; 471)Quintanilha, A., 174, 246(7)Quintero, L., 37, 88, 89, 121(7)

Raban, M., 244, 261(539)Rablen, P. R., 39, 121(21)Rademacher, T. W., 50, 51, 62, 103, 125(138)Radford, S. E., 276, 295, 302, 306(127)Raftery, M. A., 295, 307(148)Rahman, M. A. A., 202, 205, 206, 222–225,

234, 255(312; 313), 258(429)

AUTHOR INDEX 347

Rakhmatullaev, I., 41, 74, 122(37; 38)Ramaiah, M., 88, 89, 129(288)Ramamuroothy, V., 176, 248(65)Ramirez, J., 193, 253(256)Ramjeesingh, M., 45, 58, 102, 123(80)Ramos Montero, M. D., 79, 128(246)Ramsden, N. G., 193, 253(257)Rand-Meir, T., 295, 307(148)Rao, J. M., 234, 261(522)Rashed, N., 186, 196, 251(195; 196; 201),

254(283)Rashkes, Ya. V., 75, 127(200)Rasmussen, G., 272, 282, 288, 305(85)Rassau, G., 193, 253(256)Rauckman, E. J., 211, 256(355)Ravenscroft, P., 89, 130(294)Rebolledo Vicente, F., 79, 86, 128(247),

129(274; 279)Reckhaus, M., 197, 198, 254(293)Redmond, J. W., 98, 131(339; 340)Rees, W. D., 44, 56, 101, 122(62)Reese, C. B., 6–8, 13(21), 14(48; 81)Reese, E. T., 270, 304(71)Regaño, C., 163, 172(89)Regna, P. P., 217, 256(376)Rehpenning, W., 176, 247(46)Reichert, C. M., 50, 60, 102–104, 124(108;

114)Reichstein, T., 194, 254(264)Reidez, P., 176, 248(60)Reilly, P. J., 295, 307(150)Reinhold, V. N., 311, 324(5)Reinikainen, T., 272, 273, 276, 287, 305(79)Reinsberg, W., 181, 189, 190, 250(134)Reisch, J., 101, 131(344)Reitz, A. B., 74, 127(199), 193, 253(256)Remington, S. J., 302, 308(170)Repte, E., 177, 249(89)Resek, J. E., 193, 253(256)Resnick, P., 233, 260(506)Reuben, J., 185, 251(188)Reuter, G., 313, 324(7)Richard, J. P., 295, 307(149)Richardson, A. C., 96, 131(332)Richtmyer, N. K., 217, 256(371–373; 375),

257(386)Rickard, R. R., 235, 261(527)Rienäcker, C. M., 39, 121(28)Ringe, D., 174, 246(9)Rising, K. A., 297–299, 307(155)


Rist, C. E., 89, 130(290)Rob, B., 112, 115, 133(393–395; 414)Roben, W., 227, 258(449)Roberts, E. J., 2, 27, 33(1; 6; 7)Roberts, G. A. F., 180, 234, 249(122; 123),

261(521)Roberts, G. P., 180, 249(121)Roberts, J. D., 39, 121(31)Roberts, S. M., 227, 259(463)Roberts, W. L., 311, 324(5)Robina, I., 41–43, 54, 56–57, 68, 77, 101,

122(50; 65), 123(70)Robina Ramírez, I., 79, 128(244)Robins, M., 230, 260(491)Robinson, B., 176, 248(60)Robinson, E. A., 174, 246(3)Robinson, R., 2, 13(1), 176, 248(60)Robles-Díaz, R., 41–44, 56, 68, 74, 122(57)Robyt, J. F., 295, 307(150)Roche, A.-C., 50, 60, 61, 124(110; 115; 126)Rodríguez-Galán, A., 138, 161–164, 168,

170(22; 23), 172(84–87; 89; 91; 97; 98)Roeser, K. R., 266, 303(27)Roger, P., 92, 130(312)Rogers, L. J., 60, 103, 124(113)Rollin, P., 71, 126(191)Ronco, G., 96, 131(333)Rose, D. R., 272, 279, 287, 294, 302, 305(87; 88)Rosén, G., 95, 131(330)Rosen, G. M., 185, 211, 251(187), 256(355)Rosenberg, L. T., 52, 60, 124(106)Rosenberry, T. L., 311, 324(5)Rosenfeld, D. A., 217, 256(371)Rosini, G., 230, 260(493)Ross, L. E., 235, 261(527)Rossi, F., 230, 260(493)Rossi, M. H., 177, 248(80)Roth, J. S., 89, 130(292)Rothermel, J., 42, 54, 68, 122(51)Roulleau, F., 79, 128(237; 238)Rouvinen, J., 269, 270, 272, 273, 279, 280, 288,

291, 304(55), 305(93)Roy, R., 50, 60, 102, 104, 106, 124(116),

132(366; 369; 371; 378–381)Rozeboom, H. J., 273, 279, 286, 306(109; 110)Rozynov, B. V., 81, 129(266)Rua, L., 176, 248(70)Rubin, M. B., 234, 261(515)Rubiralta, M., 244, 261(539)Ruiz-Donaire, P., 162, 172(86)

AUTHOR INDEX348

Rulin, F., 227, 259(461; 463)Ruohonen, L., 272, 273, 276, 287, 305(79)Rupitz, K., 70, 110, 114, 126(181; 184),

133(410), 293, 294, 307(133)Rupley, J. A., 272, 275, 278, 305(76)Rupley, M. A., 293, 307(136)Rusinova, L. I., 189, 206, 235, 252(223)Russell, C. R., 89, 130(290)Russell, C. S., 185, 250(170)Russo, G., 227, 258(451)Rutherford, D., 140, 170(33), 171(37), 217,

257(386)Rutherford, F. C., 176, 247(42)

Sacchettini, J. C., 272–274, 280, 281, 283–285,288, 292, 300, 301, 306(101; 102; 111; 112)

Saeed, M., 177, 248(84)Saitz Barría, C., 57, 64, 123(71)Sakaguchi, M., 177, 249(105)Sakon, J., 274, 290, 306(120)Salberg, M. M., 100, 131(342)Saleem, R., 101, 131(346–351)Sallaiova, Z., 174, 246(20)Sallam, A. E. M., 222, 258(423; 424)Sallam, M., 222, 257(422)Salminen, O. M., 270, 304(57)Saltiel, A. R., 315, 324(11)Samanen, C. H., 60, 102, 124(104)Sandrinelli, F., 71, 126(191)Sandsted, C., 219, 257(400)Sandström, J., 38, 121(19)Sandterova, R., 176, 247(31)San Félix, A., 41–43, 54, 88, 122(34; 35)Sannan, T., 141, 171(38)Sano, M., 43, 126(165)Santikarn, S., 311, 324(5)Santoyo-González, F., 41–44, 56, 57, 64, 68,

74, 122(57), 123(71)Sanui, K., 154, 155, 160, 171(60; 62–65),

172(82)Sarfati, R. S., 80, 81, 128(256)Sarma, V. R., 295, 307(147)Sasaki, N., 226, 227, 258(445)Sasaki, T., 48, 58, 123(94)Sasso, G. J., 89, 130(298)Sato, K., 227, 258(451)Sato, M., 272, 306(102)Sato, O., 77, 128(235)Sato, S., 96, 131(331)


Sato, T., 176, 248(71)Satoh, M., 48, 58, 123(94)Sawai, T., 265, 270, 271, 273, 281, 303(21),

304(62; 70)Sawyer, D. T., 187, 252(211)Scapin, G., 273, 283–284, 288, 306(111; 112)Schaffer, R., 176, 247(50)Schantl, J. G., 179, 187, 249(115; 116)Scharnow, H.-G., 174, 177, 246(22), 248(81)Schauer, R., 313, 324(7)Schaukellis, H., 177, 189, 248(85)Schaumann, E., 71, 126(187)Schein, P. S., 74, 127(196; 197)Scheithauer, S., 37, 121(14)Scheuring, M., 115, 133(414)Schiedt, B., 202, 206, 220, 255(310)Schinzel, R., 273, 278, 295, 300, 306(119)Schirp, H., 177, 248(83)Schlesselmann, P., 266, 268, 271, 274–276,

284, 303(28; 32), 304(46)Schmezer, P., 95, 131(326; 327)Schmidt, E. W., 174, 246(14)Schmidt, O. T., 224, 258(431)Schmidt, P., 55, 66, 122(55)Schneider, M. P., 75, 127(206)Schoch, A., 55, 66, 122(55)Schofield, J. A., 8, 14(78)Schofield, K., 8, 14(81)Schoorl, M. N., 36, 120(1)Schramm, V. L., 110, 133(391), 264, 297–299,

303(7), 307(153–156)Schreiber, S. L., 48, 58, 123(93)Schreier, E., 217, 221, 222, 256(378),

257(387–390; 419)Schrörder, S., 299, 308(165)Schuerch, C., 50–51, 61, 124(127; 128)Schülein, M., 272, 282, 288, 302, 305(85; 86),

307(132), 308(172)Schultz, G. E., 273, 306(108)Schutz, A., 39, 121(29)Schwartz, J., 176, 247(54)Scopes, D. I. C., 89, 130(294)Scott, D. L., 273, 306(114)Scott, F. L., 232, 233, 260(499)Scott, M., 272, 305(90)Searles, S., 190, 252(224)Sedman, A. J., 77, 127(209)Seeliger, A., 46, 58, 87, 123(76), 129(282),

198, 255(305)Sekiguchi, H., 168, 172(97)

AUTHOR INDEX 349

Selegny, E., 138, 170(21)Semenyaka, A., 110, 133(391)Semenza, G., 296, 299, 307(151; 152)Seo, K., 89, 130(302; 303)Serafini-Cessi, F., 174, 246(16)Serezhenkov, V. A., 95, 131(322)Serrano, J. L., 175, 246(25)Shaban, M. A. E., 97, 131(335), 155, 171(68),

177, 186, 220, 225, 249(101–103),251(193; 194; 204), 257(407; 408)

Shafizadeh, F., 193, 220, 253(249)Shah, R. H., 70, 109, 126(174)Shaik, S. S., 233, 260(504)Shalaby, M. A., 199, 201, 205, 209, 255(306;

314)Shapiro, R., 8, 14(81)Sharma, N. D., 227, 259(464)Sharma, S., 37, 121(12)Sharon, N., 60, 124(110), 265, 303(23)Sharp, P. R., 176, 248(65)Sharpe, E. S., 217, 257(383; 385)Sharshira, E. M., 186, 251(204)Shasha, B. S., 88–89, 130(290; 291), 193,

253(253)Shateil, F., 272, 305(89)Shaw, A. N., 89, 130(307)Shaw, D. H., 50, 52, 61, 125(137)Shaw, G., 91, 93, 130(308)Shawali, A. S., 234, 261(525)Sheldrake, C. N., 227, 259(464)Shemyakin, M. M., 198, 254(301; 302),

255(303; 304)Shibanuma, T., 58, 123(90)Shidori, Y., 81, 129(265)Shine, H. J., 176, 248(62)Shing, T. K. M., 227, 258(150; 451), 259(458)Shinkai, I., 176, 248(60)Shinobu, L. A., 94, 130(315)Shinozaki, M., 113, 133(396)Shiono, M., 58, 123(90)Shiozaki, M., 45, 46, 50, 58, 62, 81, 113–114,

123(89), 125(140), 129(264), 133(399;401–403; 407)

Shiyan, S. D., 42, 108, 126(164), 132(384),133(385)

Shröter, E., 266, 303(29)Shulman, M. L., 42, 56, 108, 122(60),

126(164), 132(384), 133(385)Shutalev, A. D., 77, 127(227), 128(228; 229)Siddall, T. H. III, 38, 121(17)Siddiqui, B. S., 101, 131(346–351), 312, 324(6)


Siddiqui, S., 101, 131(346–350)Siebert, R., 220, 257(414)Silwanis, B. A., 95, 131(330)Simkovic, I., 185, 251(178; 179)Simon, H., 183–185, 187, 198, 207, 209, 212,

219, 220, 225, 227, 230, 233, 234, 250(156;163; 164; 168), 254(297–300), 256(342),257(401), 258(436)

Singh, A., 176, 248(72)Singh, P. K., 94, 95, 130(316–318)Sinha, A., 176, 247(58)Sinilova, N. G., 77, 128(229)Sinnott, L. M., 193, 253(257)Sinnott, M. L., 264–267, 269–271, 273, 275,

279, 280, 294–296, 298, 299, 301–302,303(15; 25), 304(54; 67; 72; 73), 307(137;160), 308(166; 169)

Sinnwell, V., 193, 253(254)Sirigu, A., 175, 246(26)Skop, E., 176, 247(34)Skraup, Z. H., 179, 249(119)Slawing, A. M. Z., 89, 130(307)Smiatacz, Z., 227, 239, 260(478)Smith, A. J., 60, 103, 124(113)Smith, D. F., 51, 52, 61, 125(134)Smith, H., 5, 13(12)Smith, P., 233, 235, 260(505)Smith, P. A. S., 176, 247(60)Snobl, D., 206, 207, 255(329)Sohn, K.-H., 226, 227, 258(442)Sojka, S. A., 177, 248(78)Soliman, F. M., 192, 252(241)Soliman, R., 186, 251(197)Soltzberg, S., 181, 189, 202, 223, 250(137;

139), 252(218)Somogyi, A., 227, 260(479)Somogyi, L., 184, 186–187, 221–222, 224,

250(169), 252(205; 215–217), 257(421),258(427)

Soro, P., 193, 253(256)Sorokina, I. B., 43, 44, 75, 122(42)Sosnovsky, G., 185, 251(188)Souchon, H., 272, 292, 305(81)Spangler, B. D., 273, 306(114)Spanu, P., 193, 253(256)Spearman, M. A., 193, 253(259)Spencer, G. I., 233, 260(507)Spencer, R. P., 176, 247(54)Spezio, M., 265, 270, 272, 303(24), 304(56),

305(84)

AUTHOR INDEX350

Stachissini, A. S., 177, 248(80)Stahlberg, J., 272, 273, 276, 287, 305(79)Stanislavski, E. S., 61, 125(132)Starr, C. M., 176, 247(34)Stbie, A., 192, 253(245)Steensma, D. H., 193, 253(257)Steffen, J., 219, 257(401)Stempel, G. H., Jr., 176, 177, 247(43; 44)Stephan, H. J., 212, 256(356)Stephen, A., 190, 206, 207, 252(226), 255(328)Sternfeld, F., 227, 259(460)Sternlicht, H., 266–268, 274, 284, 302, 303(26)Stevens, T. S., 194, 254(281)Stewart, J. T., 77, 127(219)Stewart, L. C., 217, 256(372)Stewart, W. E., 38, 121(17)Sticher, U., 81, 129(261)Stock, H. W., 177, 249(104)Stöckl, W. P., 49, 122(45)Stoddart, J. F., 104, 132(370)Stodola, A. H., 217, 257(385)Stodola, F. H., 217, 257(383)Stoeckler, J. D., 273, 289, 306(113)Stoeckli-Evans, H., 176, 248(66)Stohr, G., 217, 222, 257(388; 390)Stolle, W. T., 176, 248(68)Stone, B. A., 264, 303(18)Stout, E. I., 89, 130(290)Stowell, C. P., 63, 102, 125(146), 132(362)Stoye, D., 177, 249(100)Street, I. P., 294, 307(138; 139)Strietholt, W. A., 144, 171(46)Stroh, H.-H., 174, 176–178, 189, 246(22),

247(41; 45), 248(81; 85), 249(89–97; 113)Strokopytov, B., 273, 279, 286, 306(109; 110)Strynadka, N. C. J., 264, 275, 278, 287, 295,

302, 302(1)Stuart, D. J., 273, 277, 278, 286, 306(115)Stukan, R. A., 95, 131(322)Stults, C. L. M., 323, 324(16)Stumer, C., 87, 129(285)Suami, T., 226–227, 244, 258(438; 439; 441;

444; 445; 448), 261(534)Subirana, J. A., 138, 170(22–25)Sugar, A., 226, 258(444)Suhaza, Y., 193, 253(256)Sukhovitsky, A., 234, 244, 261(514)Sullivan, G. M., 77, 127(216)Sung, W. L., 272, 305(91)Sunia, A., 101, 131(349)


Suss-Fink, G., 176, 248(66)Suvorov, A. A., 183, 250(158)Suwama, M., 227, 259(452; 453)Suzuki, M., 227, 259(453)Svennerholm, L., 322, 324(13)Svenson, S. B., 60, 61, 124(109; 122)Svensson, B., 264, 302(4)Svensson, S., 52, 60, 124(106)Swain, L. C., 174, 246(5)Swanson, B. A., 174, 187, 246(10),

252(210)Sweeley, C. C., 312, 323, 324(6; 16)Sweeney, W., 135, 154, 170(1)Sweilem, N. S., 270, 271, 304(67)Swenson, L., 272, 305(89)Swift, G., 137, 138, 170(11)Swift, H. J., 272, 305(96; 97)Sy, K., 176, 247(58)Sysoeva, L. P., 189, 206, 235, 252(223)Szabo, L., 8, 14(77)Szegö, F., 63, 85, 125(145), 129(272)Szeja, W., 95, 131(328)Szilagyi, L., 176, 227, 247(54), 260(479)Szneler, E., 234, 261(513)Sztaricskai, F., 227, 260(479)Szurmai, Z., 50, 51, 61, 124(124; 125)Szweda, R., 227, 239, 260(478)

Tagmose, T. M., 193, 253(256)Taha, A. M., 186, 251(204)Taha, M. A. M., 97, 131(335), 177, 186,

249(102; 103), 251(194)Taha, M. I., 193, 253(252)Taigel, G., 87, 129(282)Takahashi, H., 41, 77, 125(161), 126(162),

128(235)Takahashi, S., 115, 133(412)Takata, M., 226, 258(444)Takayama, S., 47, 58, 112, 123(88)Takayama, T., 113, 133(403)Takayanagi, H., 77, 128(232)Takeda, K., 41, 77, 126(162), 128(232)Takeda, T., 206, 255(316)Takeda, Y., 182, 250(149)Takei, K., 226, 258(444)Taketani, Y., 168, 172(96)Takeuchi, H., 177, 249(105)Takeuchi, T., 226, 227, 258(442)Talebian, A., 74, 127(197)

AUTHOR INDEX 351

Tanaka, Y., 268–269, 296–299, 304(53),307(157)

Tandano, K., 227, 258(448)Tang, P. W., 177, 248(83)Tang, Y., 227, 258(450; 451)Tanikawa, T., 227, 259(454)Tanko, J. M., 185, 251(185)Tanner, E. M., 234, 261(516)Tao, B. Y., 295, 307(150)Tao, W., 297–299, 307(157)Tarrago, G., 234, 261(517)Tashpulatov, A. A., 41, 74, 122(37)Tashpulatov, O. A., 41, 122(38)Tauber, H. J., 217, 257(395)Taverna, R. D., 42, 44, 56, 122(58)Taylor, C. W., 5, 13(14)Taylor, J., 265, 270, 272, 303(24), 305(84)Taylor, P. B., 80, 128(253)Teeri, T., 269, 270, 272–273, 276, 279, 280,

287, 288, 291, 304(55), 305(79)Teijima, S., 224, 258(432)Tejima, S., 95, 131(325), 206, 255(316)Tellier, C., 70, 110, 126(183), 133(389; 390)Tengi, J. P., 89, 130(298)Tengler, H., 177, 189, 248(85)Terabe, S., 232, 233, 260(497)Terayama, H., 115, 133(412)Terwisscha van Scheltinga, A. C., 273,

306(106; 107)Tesler, I. D., 122(36)Tews, I., 302, 308(171)Thankarajan, N., 234, 261(518)Thiel, W., 70, 110, 126(181)Thiem, J., 50, 61, 104, 124(131), 136, 142–145,

155, 170(6), 171(43–48)Thim, L., 272, 305(96)Thoma, J. A., 274, 306(122–124)Thompson, A., 187, 194, 217, 238, 252(207),

254(271), 257(384)Thompson, F. P., 197, 254(287)Thompson, R. H., 232, 233, 260(498)Thornally, P. J., 174, 246(6)Thorpe, A. J., 193, 227, 253(257), 259(460)Tichá, M., 80, 128(255)Tiers, G. V. D., 190, 252(224)Tino, J., 185, 251(178; 179)Tipson, R. S., 177, 180, 248(82)Tobe, T., 227, 259(452; 454)Todd, A. R., 2–8, 13(1–17; 19–43), 14(44–66;

68–71; 73–84)


Todoulou, O. G., 74, 127(195)Tokura, S., 147, 171(49)Tolley, S. P., 272, 282, 288, 305(85; 86)Tomme, P., 266, 268, 273, 276, 280, 287, 296,

303(37)Tong, M. K., 70, 110, 126(179; 182)Török, G., 77, 127(218)Törrönen, A., 272, 305(93)Tosi, G., 176, 177, 248(69; 76)Toth, B., 174, 246(12)Toth, G., 77, 93, 127(218), 130(313), 227,

238, 239, 259(474), 260(475)Totsuka, A., 280, 306(128), 307(130)Tourwé, D., 77, 127(218)Toy, M. S., 139, 154, 170(31)Toyama, A., 75, 127(205)Tracy, S. M., 174, 246(5)Tran, S. V., 174, 246(13)Trancier, J.-P., 194, 253(261)Treanor, S. P., 89, 130(296)Trimbue, D., 264, 270, 291–293, 303(11)Tronchet, J. M. J., 189, 193, 252(220–222),

253(247; 248)Trujillo Pérez-Lanzac, M., 79, 128(240;

242; 245)Tsang, W. S. C., 27, 34(12)Tsegenidis, T., 176, 247(31)Tseng, C. K. H., 89, 130(292; 296)Tsubota, T., 168, 172(96)Tsukamoto, H., 266, 304(42)Tsumuraya, Y., 266, 270, 285, 300, 303(38),

304(69)Tsunoda, T., 227, 259(461)Tsvetkov, Yu. E., 61, 125(132; 133)Tull, D., 272, 279, 287, 293–294, 302, 305(88),

307(133; 140; 146)Tuman, R. W., 74, 127(199)Turco, S. J., 176, 247(31)Turgeon, J., 77, 127(215)Turkenburg, J. P., 272, 305(97)Turner, M. K., 227, 259(463)Turner, N. J., 227, 259(463)Tuzi, A., 175, 246(26)Tuzikov, A. B., 70, 102, 126(178), 132(361)Tweeddale, H. J., 98, 131(339; 340)

Ubukata, O., 50, 62, 114, 125(140)Uchida, C., 41–44, 46, 47, 54, 58, 89, 113–114,

122(48), 123(86; 87), 130(301), 133(397;398; 400; 405; 406; 408; 409)

AUTHOR INDEX352

Uchiyama, T., 266, 268, 271, 274–275, 284,303(28), 304(46)

Uedo, T., 48, 58, 123(94)Uematsu, Y., 226, 227, 258(444; 445)Uemura, M., 217, 256(379)Ugalde Donoso, M. T., 147, 171(52)Ulbrich, K., 138, 170(19)Umezawa, H., 227, 259(465)Umezawa, Y., 226, 227, 258(442)Unkovskii, B. V., 77, 127(227), 128(228; 229)Uno, T., 206, 255(316)Unverzagt, C., 51, 62, 103, 125(139)Uppugunduri, S., 84, 129(271)Urayama, S., 160, 172(79)Urry, D. W., 138, 170(17)Uspenskaya, M. N., 75, 127(202)Utsumi, S., 280, 306(129)Uzan, R., 92, 130(311)

Valencia, C., 79, 80, 86, 90, 97, 128(251),129(275)

Valent, M., 174, 246(18)Valentin, F., 182, 250(145)Valenza, S., 193, 253(256)Van Den Nest, W., 77, 127(218)van der Haar, A. W., 177, 249(99)Vandewalle, M., 227, 259(451)van Doorslaer, E., 270, 304(65)Vanin, A. F., 95, 131(322)van Montfort, R., 273, 306(109)Van Zyl, C. M., 174, 246(13)Varadarajan, S., 6, 13(22; 23)Varela, O., 41, 43, 55, 74, 75, 87–88, 99,

122(53; 54), 131(341), 148–150, 158,160, 171(53–55), 172(77; 78)

Varrot, A., 302, 307(132), 308(172)Vasella, A., 70, 89, 110, 126(181; 184; 185),

130(300)Vass, G., 180, 182, 192, 206–207, 227,

249(127), 250(151), 252(232), 255(328;341), 259(469)

Vasu, S., 177, 249(87)Vazquez de Miguel, L. M., 192, 252(243)Vedejs, E., 176, 248(68)Vercellotti, J. R., 27, 33(9; 10), 34(11), 193,

220, 253(251), 257(413)Vérez, V., 100, 131(343)Vérez Bencomo, V., 106, 132(381)Vert, M., 137, 170(9)


Vethaviyasar, N., 46, 126(167; 168)Viguera Robio, F. J., 79, 128(242)Vile, S., 89, 130(306; 307)Villa, P., 96, 131(333)Villalonga, R., 95, 131(324)Viratelle, O. M., 266, 303(35)Vives, J., 168, 172(97)Vocadlo, D., 109, 110, 115, 133(386; 413)Vogel, P., 193, 253(256)Volkovich, S. V., 75, 127(202)von Deyn, W., 227, 259(456)Vondracek, R., 224, 258(434)Vonhoff, S., 70, 110, 126(185)von Pechmann, H., 190, 252(229)Vorgias, C. E., 302, 308(171)Voss, J., 37, 38, 121(10)Votocek, E., 182, 186, 224, 250(145–147),

258(434)Vranesic, B., 51, 60, 103, 124(111)Vrsanskà, M., 272, 305(78)Vu, C. B., 234, 261(520)

Wacker, H., 198, 254(300)Wagenknecht, H.-A., 112, 133(395)Waisbrot, S. W., 194, 254(265; 273; 275)Wakarachuk, W. W., 272, 305(91; 92)Wakarchuk, W. W., 264, 272, 302(5)Wakatsuki, S., 273, 277, 278, 306(118)Walle, T., 77, 127(211; 212)Walle, U. K., 77, 127(211)Wallin, N.-H., 52, 60, 124(106)Walling, C., 187, 252(212)Walter, R. L., 273, 289, 306(113)Walter, W., 37, 38, 121(10)Walters, C., 227, 259(463)Waltuch, R., 207, 255(334)Wan, C.-W., 104, 132(373)Wan, L. H., 227, 259(458)Wan, P., 39, 121(25)Wander, J. D., 176, 219, 247(37)Wang, P., 95, 131(320)Wang, P. G., 193, 253(256)Wang, Q., 264, 270, 291–293, 303(11)Wang, Y.-F., 193, 253(257)Warren, A. J., 293, 294, 307(133)Warren, R. A. G., 264, 270, 291–294, 303(11),

307(140)Warren, R. A. J., 264, 272, 302(5)Warren, S. G., 7, 14(56)

AUTHOR INDEX 353

Wasserman, H. H., 234, 260(512), 261(520)Wassmann, A., 104, 132(376)Watanabe, K. A., 70, 71, 126(171)Watanabe, S., 41–44, 54, 58, 113, 122(48)Weaver, L. H., 293, 295, 307(134; 135)Webb, R. F., 6–8, 13(37; 41), 14(47; 75)Weber, B., 42, 54, 68, 122(51)Weber, W., 70, 110, 126(181)Weglicki, W. B., 95, 131(323)Wehrmuller, J. O., 193, 220, 253(249)Wei, Y., 272, 305(89)Weidmann, H., 49, 122(45)Weigel, T. M., 176, 248(61)Weigele, M., 89, 130(298)Weinber, H. R., 176, 248(63)Weinberg, N. L., 176, 248(63)Weinreb, S. M., 227, 259(467)Weisblat, D. I., 194, 254(265; 268; 269)Weiser, W., 266, 268, 284, 303(33), 304(47)Welsh, C., 42, 57, 64, 123(70), 7768Welstead, W. J., Jr., 206, 207, 225, 255(325),

258(435)Wen, T., 234, 261(513)Weng, M., 55, 66, 122(55)Wentworth, D. F., 266, 303(34)Werringloer, J., 174, 246(4)Wess, G., 176, 248(72)West, D. X., 100, 131(342)Westbrook, E. W., 273, 306(114)Westbrook, M. L., 273, 306(114)Westphal, G., 176, 247(41)Weygand, F., 197–198, 219, 233, 254(292; 293;

295; 297; 298), 257(402)Weymouth, F. J., 6, 7, 13(37), 14(51; 58)Whistler, R. L., 193, 222, 253(253), 258(423)White, A., 272, 279, 287, 294, 302, 305(87; 88)White, A. D., 89, 130(307)Wiberg, K. B., 39, 121(21)Wibullucksanakul, S., 158, 172(76)Wichterle, O., 182, 186, 250(147)Wieczorek, J., 41, 74, 122(40)Wiegandt, H., 323, 324(14)Wiessler, M., 95, 131(326; 327)Wiggins, L. F., 139, 154, 170(27–29)Wightman, R. H., 192, 220, 253(244–246)Wilcox, C. S., 226, 227, 258(442)Wilde, H., 212, 256(364)Wiley, M. R., 89, 130(305)Wilkinson, A. J., 272, 305(97)Willetts, A. J., 227, 259(463)


Williams, A. F., 315, 324(12)Williams, D. J., 89, 130(307)Williams, J. M., 177, 180, 248(83; 84),

250(132)Williams, N. R., 185, 227, 238, 251(182),

259(473)Wilson, D. B., 264, 265, 270, 272, 274, 290,

302(5), 303(24), 304(56; 57), 305(80; 84),306(120)

Wilson, K. S., 272, 282, 288, 302, 305(85),308(171)

Wilson, M. J., 77, 127(211)Wilson, S. R., 226, 227, 258(442)Wilt, J. W., 185, 251(183)Winchester, B. G., 193, 253(257)Winkley, M. W., 70, 71, 126(172)Winter, M., 209, 217, 219, 256(347; 349),

257(397)Witczak, Z. J., 36, 40, 41, 43, 44, 63, 65, 66, 74,

81, 120(4), 122(39), 193, 253(257)Withers, S. G., 70, 109–110, 114, 115,

126(181; 184), 133(386; 410; 413), 264,266, 270, 272–274, 277, 279, 286, 287,291–294, 301–302, 302(3; 5), 303(11; 39), 305(87; 88; 92), 306(116; 121),307(132; 133; 138–140; 142–146),308(169; 172)

Wittel, K., 37, 121(15)Wojtowicz, M., 77, 128(230)Woldike, H. F., 272, 305(96)Wolfenden, R., 266, 303(34)Wolff, H., 177, 189, 248(85)Wolfgang, D. E., 270, 304(57)Wolfrom, M. L., 70, 71, 126(172), 139, 154,

170(31), 178, 181, 184, 187, 189,193–194, 197, 202, 206, 207, 217, 220,223, 224, 238, 249(114), 250(135–139;165), 252(206–208; 218), 253(249; 251;252), 254(265; 267–277), 255(326),257(384; 396; 411; 413)

Wollin, R., 60, 61, 124(122)Wolters, B., 101, 131(345)Wong, C.-H., 47, 58, 112, 123(88), 193, 227,

253(257), 259(463)Wong, R., 193, 253(257)Wong, S. C., 39, 121(26)Wong, S. Y. C., 50, 51, 62, 103, 125(138)Wood, H. B., 194, 254(270)Wood, H. C. S., 220, 257(410)Woodall, C. C., 193, 253(256)

AUTHOR INDEX354

Woods, E. A., 177, 189, 248(85)Woods, M., 89, 130(307)Woods, T. S., 37, 121(11)Wright, E. M., 50, 60, 102, 124(117), 132(354)Wright, J. A., 193, 253(259)Wriston, J. C., Jr., 102, 132(363)Wu, S. S., 176, 247(31)Wu, Z., 176, 248(65)

Xiang, Y. B., 176, 248(72)

Yaginuma, S., 227, 259(466)Yaguchi, M., 272, 305(91)Yakhontov, L. N., 183, 250(159)Yale, H. L., 181, 250(141)Yamada, K., 176, 227, 247(57), 259(454)Yamagishi, T., 113, 114, 133(398; 400; 406;

408)Yamaki, T., 270, 304(62)Yamamoto, I., 78, 128(236)Yamamoto, S., 78, 128(236)Yamashita, H., 177, 249(86)Yamashita, M., 89, 130(303), 176, 177,

247(56), 249(86)Yáñez, M., 38–39, 121(16; 27)Yang, D., 176, 248(61)Yang, D. T. C., 176, 248(70)Yang, J.-H., 89, 130(304)Yankeelov, J. A., Jr., 274, 306(123)Yanovsky, E., 138, 170(26)Yao, H. C., 233, 260(506)Yasuda, K., 226, 258(444)Yasui, T., 177, 249(107)Yates, J. B., 89, 130(305)Yates, K., 39, 121(25)Yi, Y., 176, 248(65)Yon, J. M., 266, 303(35)Yoshida, K., 266, 304(42–44)Yoshida, S., 226, 227, 258(445)Yoshida, T., 176, 247(57)Yoshiike, R., 50, 62, 114, 125(140)Yoshikawa, N., 176, 247(57)Yoshinaga, M., 142, 171(42)Yoshino, H., 95, 131(329)Yu, H. K. B., 176, 247(54)Yu, L., 193, 253(256)Yu Cui, U., 227, 258(451)Yuki, H., 168, 172(96)


Zaalishvili, M. M., 161, 172(83)Zach, K., 221, 257(416)Zacharieva, E. I., 138, 170(19)Zamojski, A., 121(32)Zamora, F., 147–149, 171(50; 53; 54)Zamora Mata, F., 79, 128(241; 244), 147, 153,

171(52; 57; 58)Zanini, D., 50, 60, 124(116)Zehl, A., 48, 58, 63, 81, 92, 123(96)Zemek, J., 174, 246(17; 20)Zemplén, G., 190, 192, 206, 224, 252(228;

234; 237), 255(320)Zerner, E., 207, 255(334)Zhang, R.-G., 273, 306(114)Zhang, X.-J., 272, 274, 276, 305(77)Zhang, Y., 298, 307(160)Zhao, D., 176, 248(60)Zhu, J.-L., 89, 130(304)

AUTHOR INDEX 355

Zhu, Y.-H., 193, 253(256)Ziegler, B., 266, 268, 273, 274, 276, 302,

303(31)Zimmermam, S. C., 104, 132(372)Zimmerman, J., 135, 154, 170(1)Zinardi, F., 193, 253(256)Zinke-Allmang, G., 224, 258(431)Zinner, H., 176–177, 181, 189, 247(39; 46),

248(85), 250(142)Zollinger, H., 190, 252(227)Zopf, D. A., 51, 52, 61, 84, 125(134), 129(271)Zophy, W. H., 194, 254(265)Zsolldos-Mady, V., 190, 252(231)Zurabyan, S. E., 43, 44, 64, 70, 75, 122(36;

42), 125(147–149), 126(175)Zussman, J., 6, 13(17)Zweier, J. L., 95, 131(320)Zwierzak, A., 58, 123(92)


Acetylation, D-galactaric acid, 155Acetylenic compounds, reactions, 192–193Acid dichlorides, polycondensation reactions

with, 142–143Acids, action in carba-sugar hydrazones,

236–238Aldose arylhydrazones, reaction with

acetylenic compounds, 192–193Aldoses, for saccharide hydrazone

preparation, 177Aldosuloses, for saccharide hydrazone

preparation, 177Alexander Robertus Todd

aphid pigment research, 8–929-deoxynucleosides, 6dibenzyl phosphorochloridate, 6–7education, 1–3glucopyranoside research, 5–6managerial skills, 9marriage, 10nucleic acids, 7–8nucleotide coenzymes, 4–7personality and achievements, 10–11professorships, 3research strategy, 9role in national and international affairs,

9–10student careers, 11thiamine work, 5vitamin B12, 8

Alkaliesglycosylhydrazine reactions, 183–185saccharide hydrazone reactions, 183–185

Alkylenediamines, polycondensation,156–157

Alkylhydrazones, reaction with acetyleniccompounds, 192–193

357

Alpha-Amylase, pancreatic, structure,278–279

Amine nucleophiles, coupling with sugarisothiocyanates, 74–78

Amino acidscarbohydrate-derived, for chiral A, B-type

polyamides, 145–153coupling to glycosyl isothiocyanates,

75–762-Amino-2-deoxyaldose reactions

with carbon disulfide, 97with glycosyl isothiocyanates, 84

5-Amino-5-deoxy-L-arabinonic acid,derivative preparation, 148

2-Amino-2-deoxy-D-glucose, reaction withisothiocyanates, 80

1-Amino-1-deoxyketose, reaction withcarbon disulfide, 97

1-Amino-1-deoxy-2-ketose, reaction withglycosyl isothiocyanates, 84

4-Aminophenethylamine, reductiveamination, 61

Amino sugarsderivatives of neuraminic acid, 81–isothiocyanate coupling, 78–81–sugar isothiocyanate coupling, 81–84thioacylation, 71

Ammonolysis, tri-O-methyl-L-arabinono-1,5-lactone, 151–153

Anhydroalditols, for polymer synthesis,143–144

1, 6-Anhydro-4-O-benzyl-2-deoxy-2-isothiocyanato-3-O-p-toliylsulfonyl-b-D-glucopyranoside, condensation withmethanol, 88

1,4-Anhydroerythritol, transformation, 144Anhydroosazones, formation, 220–222

Subject Index

4888 Horton Sub Index 11/17/99 3:07 PM Page 357

3,6-Anhydroosazones, formation, 221–2221, 4-Anhydro-D,L-threitol, transformation,

1441, 6-Anhydro-2,3,4-tri-O-benzyl-b-D-

glucopyranose, as glycosyl donor, 54Anions, formation in carba-sugar hydrazone

reactions, 236–237Antineoplastic compounds, azole nucleoside

analogs as, 75Antiviral compounds, azole nucleoside

analogs as, 75Aphid pigments, research by Lord Todd,

8–9Aromatization

bis(phenylhydrazone) residues, 215–220cyclohexane ring, 240–241hydrazone residues, 241

Artificial receptors, N-thiocarbonyl sugars,118–120

Aspartic acid, carbohydrate-basedderivatives, 168–169

Aza-Wittig-related reactions, sugarisothiocyanates, 65–66

6-Azido-6-deoxy-2,3,4,5-tetra-O-methyl-D-glucono-1,5-lactone, synthesis, 147

39-Azido-39,59-dideoxy-59-isothiocyanatonucleosides, in (deoxy)ribonucleicthiourea synthesis, 82–83

Azoalkenes, formation, 186–188Azole nucleoside, analogs, 75

Bases, action in carba-sugar hydrazones,236–238

Benzoylisothiocyanates, reaction with 2-amino-2-deoxy-D-glucose, 80

Benzylisothiocyanates, reaction withhydroxyl groups, 88–89

Beta-Amylaseresidues for protonation, 273X-ray studies, 279–281

Bicyclic dianhydro-osazones, preparation, 222Biocompatible scaffolds, cyclodextrins as,

106–107Biodegradation, polymer, 137–138Bis(glycosyl)thioureas, as secondary

products, 64–65Bis(hydrazones)

oxidation, 212pyrazole formation, 215

SUBJECT INDEX358

2,4-Bis(4-methoxyphenyl)-1,3-dithiadiphosphetane-2,4-disulfide, seeLawesson’s reagent

1, 2-Bis(phenylazo)ethene, formation, 212Bis(phenylhydrazone)

reduction, 220residue aromatization, 215–220

1,2-Bis(phenylhydrazones), formation, 2301,3-Bis(phenylhydrazones), formation,

230–234Bis(thiocarbonyl)hydrazide, derivatives of

galactaric acid, 97–100

Carba-sugar hydrazonesacid and base action, 236–238aromatization, 240–2411,2-bis(phenylhydrazones), 2301,3-bis(phenylhydrazones), 230–234importance, 226–227nucleophilic substitution, 238–239oxidation, 241, 244phenylhydrazone preparation, 227phenylhydrazones, 227reduction, 241, 244structure, 234–2361,2,3-tris(phenylhydrazones), 230–234

Carbohydrate derivativesamino acids, for chiral A,B-type

polyamides, 145–153aspartic acid-like derivatives, 168–169reaction with fluorescein isothiocyanate,

80–81Carbon-14, Melvin Calvin research, 16–18Carbon bases, addition to sugar

isothiocyanates, 68–70Carbon disulfide reactions

with deoxyaldoses, 97with deoxyketoses, 97with sugar iminophosphoranes, 63

N-Carbonyl compoundscomparison to N-thiocarbonyl

compounds, 37–39thionation, 70–71

Carboxylic acids, condensation with sugarisothiocyanates, 64

Cellulase, X-ray studiesCBH-I, 273–274, 276–278CBH-II, 279–281family 9, 281–282family 45, 281–282


Cex b-cellulase, X-ray studies, 279Chiral polyamides

A,B-type, from carbohydrate-derived amino acids, 145–153

based on diamino saccharides, 141–145properties and applications, 136–140

Chitobiose, amino functions, 141Circular dichroism

glycosylhydrazines, 182saccharide hydrazones, 182

Condensationpolycondensation

with alkylenediamines, 156–157with 2,6-diamino saccharides, 142–143with hexaric-1,4:6,3-dilactones, 158interfacial, polyamides, 140

self-condensation, 64–65sugar isothiocyanates with carboxylic

acids, 64Conformations

N-carbonyl compounds, 37–39glycolipids, 313–314sugar thioamides, 72–73sugar thiourea, 86–87N-thiocarbonyl compounds, 37–39

Cyclic guanidinium glycomimetics,preparation, 111–112

Cyclic sugar dithiocarbamates, synthesis, 97Cyclic sugar thiocarbamates, synthesis, 90–933,2-Cyclic thiocarbamates, preparation,

92–933,4-Cyclic thiocarbamates, preparation,





92–93Cycloaddition

as glycosylhydrazine reaction, 192–193as saccharide hydrazone reaction, 192–193sugar isothiocyanates, 66

Cyclodextrin glycosyltransferase, pancreatic,278–279

Cyclodextrinsas biocompatible scaffolds, 106–107modification, 118–120

SUBJECT INDEX 359

Cyclohexane, ring aromatization, 240–241Cyclohexane bis(phenylhydrazone), free

radical mechanism, 233–234Cyclohexane 1,2,3-trione

tris(phenylhydrazone), ionicmechanism, 233

Degradationosazones, 209–212polymer, 137–138

Dehydroosazones, formation, 2126-Deoxy-6-isothiocyanato aldopyranosides,

stability, 59–6039-Deoxy-39-isothiocyanatothymidyl

derivative, in (deoxy)ribonucleicthiourea synthesis, 83–84

Deoxynucleosides, synthesis, 8929-Deoxynucleosides, research by Lord

Todd, 6(Deoxy)ribonucleic thiourea synthesis

with 39-azido-39, 59-dideoxy-59-isothiocyanato nucleosides, 82–83

with 39-deoxy-39-isothiocyanatothymidyl derivative, 83–84

Desulfurization reactions, glycosylisothiocyanates, 65

Diamino saccharides, chiral polyamidesbased on, 141–145

2,6-Diamino saccharides, forpolycondensation reactions, 142–143

Dianhydroosazones, pyrazole type, 222Diazo derivatives, reactions, 194Dibenzyl phosphorochloridate, research by

Lord Todd, 6–7Dimethyl D-glucarate, preparation, 156Diosylceramides, nomenclature, 317Disaccharides, for saccharide hydrazone

preparation, 177Dithiocarbamates

cyclic sugar dithiocarbamates, 97linear sugar dithiocarbamates, 94–96

Electron affinity, halogens, 15Electronic properties

N-carbonyl compounds, 37–39N-thiocarbonyl compounds, 37–39

Electrophilic addition, to glycals, 56


Electrophilic substitutionas glycosylhydrazine reactions, 189–192osazones, 209as saccharide hydrazone reactions,

189–192Elimination

in azoalkene formation, 186–188in carba-sugar hydrazone reactions, 238

Enzymatic degradation, polymer, 137–138Enzyme inhibitors, studies with N-

thiocarbonyl sugars, 107–118Enzymes

inverting, catalysis of noninvertingreactions, 267–270

retaining, catalysis of nonretaining reactions, 267–270

Esters, derivatives of saccharide osazones,223–224

Ethers, derivatives of saccharide osazones,224

Ethoxyisothiocyanates, reaction with 2-amino-2-deoxy-D-glucose, 80

Flurescein isothiocyanate, reaction withcarbohydrate derivatives, 80–81

Formazans, formation, 189–192Free radicals

formation, 209–212formation in carba-sugar hydrazone

reactions, 237–238in mechanism of cyclohexane

bis(phenylhydrazone), 233–234oxidation

glycosylhydrazines, 185–186saccharide hydrazones, 185–186

Furanose cis-1,2-fused oxazolidine-2-thione,preparation, 92

Galactaric acid, bis(thiocarbonyl)hydrazidederivatives, 97–100

D-Galactaric acid, acetylation, 155b-Galactosidase

residues for protonation, 273–274X-ray structure, 276–278

Gangliosidesbrain, Svennerholm abbreviations,

322–323nomenclature, 319–320

D-Glucaric acid esters, preparation, 156

SUBJECT INDEX360

Glucoamylaseresidues for protonation, 273X-ray studies, 279–281

Glucopyranosides, research by Lord Todd,5–6

Glycals, electrophilic addition to, 56Glycofuranosyl halides, acetylated, reaction

with KSCN, 54–55Glycogen phosphorylase, X-ray structure,

276–278Glycoglycerolipids

classification, 314definition, 311

Glycolipidsacidic glycosphingolipids, 319–320classification by lipid moiety, 314–317definition, 311monosaccharide residue naming, 312monosaccharide residue number, 312neutral glycosphingolipids, 317–319oligosaccharide structure definition,

312–313other names, 312recommended abbreviations, 322ring size and conformation, 313–314Svennerholm abbreviations, 322–323

Glycophosphatidylinositolsclassification, 315definition, 311

Glycopyranosyl donors, in sugarisothiocyanate synthesis, 49

Glycopyranosyl isothiocyanates1, 2-cis-configured, preparation, 53–54solvent-free preparation, 53

Glycosidasesglycosyl isothiocyanates as inhibitors,

108inverting, in glycosyl transfer, 270–271X-ray studies, residues for protonation,

272–274Glycosphingolipids

classification, 316–317definition, 311nomenclature

acidic glycosphingolipids, 319–320neutral, with oligosaccharide chains,

317–319neutral glycosphingolipids, 317–319

Glycosylaminespreparation, 57transformation, 56–57


Glycosylasescatalyzed reactions

inverting glycosylases, 279–284retaining glycosylases, 275–279

glycosyl transfer by inverting glycosidases,270–271

1-MCO and 2-MCO types, separation,284–293

1-MCO type, stereochemical behavior,294–296

minisubstrates of forbidden configuration,266–267

noninverting reactions by inverting enzymes, 267–270

nonretaining reactions by retaining enzymes, 267–270

role of transition-state structure,296–299

–substrate complexes, catalytic center,solvent proximity, 299–301

S-Glycosyl-N,N-dialkyldithiocarbamates,synthesis, 95

Glycosyl donor, in sugar isothiocyanatesynthesis, 41, 49, 53–55

Glycosylhydrazinesalkali action, 183–185bromo and dibromo derivative

formation, 189characteristics, 175–177cycloaddition reactions, 192–193derivatives, 189diazo derivative reactions, 194elimination reactions, 186–188formation, 177–179formazan formation, 189–192hydrazono lactone formation, 185–186oxadiazole formation, 186reduction reactions, 193–194structure, 179–182

Glycosyl isothiocyanatescoupling reactions, 75–76desulfurization reactions, 65as glycosidase inhibitors, 108reaction with 2-amino-2-deoxyaldoses, 84reaction with 1-amino-1-deoxy-2-

ketoses, 84N-(Glycosylthiocarbamoyl)peptides, by

glycosyl isothiocyanates coupling, 75–76Glycosyl transfer, by inverting glycosidases,

270–271

SUBJECT INDEX 361

Glycuronoglycosphingolipids, nomenclature,320

Halogens, electron affinity, 15Hapten, preparation, 84Hen’s egg-white lysozyme, structure,

275–2761, 6-Hexanediamine, reductive amination, 61Hexaric-1,4:6,3-dilactones, in

polycondensations, 158Hexarodilactones, polyaddition, 158–160Hydrazones

carba-sugar, see Carba-sugar hydrazonesresidue aromatization, 241saccharide, see Saccharide hydrazones

Hydrazono lactone, formation, 185–186Hydrolysis

osazones, 208–209polymer, 137–138

Hydroxyl groups, reaction with benzylisothiocyanate, 88–89

Infrared spectroscopyglycosylhydrazines, 181saccharide hydrazones, 181sugar isothiocyanates, 66

Inorganic thiocyanate, in sugarisothiocyanate synthesis, 41, 49, 53–55

Isothiocyanate conjugates, preparation,60–61

Isothiocyanatescoupling with amino sugars, 78–81energetics and structure, 37–39

Isothiocyanationreagents, 58sugar amines, 56–62

Isotopes, radioisotopes, Melvin Calvinresearch, 16

Ketoses, for saccharide hydrazonepreparation, 177

Laboratory of Chemical Biodynamics,Melvin Calvin leadership, 19

Lawesson’s reagent, for thionation, 70–71LCB, see Laboratory of Chemical

Biodynamics


Linear sugar dithiocarbamates, synthesis,94–96

Linear sugar thiocarbamates, synthesis,88–90

Lipid, in glycolipid classification, 314–317Lord Todd, see Alexander Robertus ToddLysozyme, hen’s egg-white, structure,

275–276

D-Mannaro-1,4:6,3-dilactone, polyaddition,158–160

Margaret Alice Clarkeawards and recognitions, 28–29consulting work, 28early years, 23education, 23–24friendships, 31–32marriage, 24New Orleans Carbohydrate Symposia,

24–25professional memberships, 29–30role in international carbohydrate

communities, 30–31Sugar Processing Research Institute, Inc.,

25–26Mass spectrometry, sugar isothiocyanates, 68Melvin Calvin

carbon-14 research, 16–18early years, 15generosity, 20–21LCB leadership, 19Nobel Prize in Chemistry, 18radioisotope research, 16use of publicity, 20

Membrane receptors, interaction with N-thiocarbonyl sugars, 101–102

Methanol, condensation with 1,6-anhydro-4-O-benzyl-2-deoxy-2-isothiocyanato-3-O-p-toliylsulfonyl-b-D-glucopyranoside, 88

Methyl 6-amino-6-deoxy-a-D-glucopyranoside, coupling reactions,81–82

Methyl D-glucarate 1, 4-lactone, preparation,156

Methyl D-glucarate 6,3-lactone, preparation,156

Methyl a-D-glucopyranoside, for lactonesynthesis, 147

SUBJECT INDEX362

Methyl 2,3,4,6-tetra-O-allyl-a-D-glucopyranoside, preparation andpolymerization, 138–139

Molecular recognition, N-thiocarbonylsugars

artificial receptors, 118–120enzyme inhibitors, 107–118glycoclusters, 102–107glycodendrimers, 102–107interactions with membrane receptors,

101–102neoglycoconjugates, 102–107

Monoglycosylceramides, nomenclature, 317Monosaccharides

hydroxyl groups, reaction with benzyl isothiocyanate, 88–89

residue naming, 312residue number, 312

Mutarotation, osazones, 207

Neuraminic acid, amino sugar derivatives, 81New Orleans Carbohydrate Symposia, and

Margaret Alice Clarke, 24–25Nobel Prize, Melvin Calvin, 18Nuclear magnetic resonance

glycosylhydrazines, 179–180saccharide hydrazones, 179–180sugar isothiocyanates, 66–68sugar thiourea, 86–87

Nucleic acids, research by Lord Todd, 7–8Nucleophiles, amine, coupling with sugar

isothiocyanates, 74–78C-Nucleophiles, reactions with sugar

isothiocyanates, 63N-Nucleophiles, reactions with sugar

isothiocyanates, 63O-Nucleophiles, reactions with sugar

isothiocyanates, 63S-Nucleophiles, reactions with sugar

isothiocyanates, 63Nucleophilic addition, in sugar thiourea

synthesis, 78–81Nucleophilic substitution

carba-sugar hydrazones, 238–239osazones, 208–209

Nucleotide coenzymes, research by LordTodd, 4–7

Nylonsnylon-3, chiral analogs, preparation,

168–169


nylon-n, based on carbohydrate-derived amino acids, 145–153

nylon-n, 4, polyhydroxyl chiral analogs,160–168



physical properties, 137

Oligosaccharidesneutral glycosphingolipids with chains,

317–319structure definition, 312–313

Optical rotatory dispersionglycosylhydrazines, 182saccharide hydrazones, 182

Orotate phosphoribosyltransferase, X-raystudies, 282–284

Osazonesaction of bases, 209–212chelated structures, 205–207electrophilic substitution, 209formation, 194–196formation mechanism, 196–197mutarotation, 207nucleophilic substitution, 208–209tautomeric structures, 202–205

Osotriazole, mechanism of formation,217–220

Oxadiazole, formation, 186Oxazolidine-2-thione heterocycles,

preparation, 92Oxazolinium cations, as glycosyl donors,

49, 53Oxidation, free radical


Oxidation–reductioncarba-sugar hydrazones, 241, 244intermolecular, saccharide osazones,

197–198intramolecular, saccharide osazones,

198–2022-Oxo-1,3-bis(phenylhydrazones), structure,

234–235

Peptides, coupling to glycosylisothiocyanates, 75–76

SUBJECT INDEX 363

Phenylazo-cycloalkenes, formation in carba-sugar hydrazone reactions, 238

Phenylazo-phenylhydrazones, formation,212

1-Phenylhydrazino-phenylhydrazones,preparation, 187

Phenylhydrazones, preparation, 227Phenylosotriazoles, formation, 241Phosphoglycosphingolipids, nomenclature,

320–321Phosphonoglycosphingolipids,

nomenclature, 321Photodegradation, polymer, 137–138Photosynthesis, Melvin Calvin research,

16–19Pigments, aphid, research by Lord Todd,

8–9Polyaddition, hexarodilactones, 158–160Polyaldaramides, polyhydroxy chiral

analogs, 153–160Polyamides

chiralA,B-type, from carbohydrate-derived

amino acids, 145–153based on diamino saccharides, 141–145properties and applications, 136–140

from interfacial polycondensation, 140Polycondensation

with alkylenediamines, 156–157with 2,6-diamino saccharides, 142–143with hexaric-1, 4:6, m3-dilactones, 158interfacial, polyamides, 140

Polyhydroxy chiral analogsnylon-n, 4, 160–168nylon-n, 5 and nylon-n, 6, 153–160

Polymersbiodegradation, 137–138synthesis, with anhydroalditols, 143–144

Polyols, hydroxyl groups, reaction withbenzyl isothiocyanate, 88–89

Polytartaramides, polyhydroxy chiralanalogs of nylon-n, 4, 160–168

Potassium thiocyanate, reaction withacetylated glycofuranosyl halides, 54–55

Protonationin carba-sugar hydrazones, 236–237glycosidase residues for, 272–274

Psychosine, definition, 312Pyranose cis-1, 2-fused oxazolidine-2-thione,

preparation, 92


Pyrazolesformation from bis(hydrazones), 215type of dianhydroosazones, 222

Radioisotopes, Melvin Calvin research, 16Reduction


Reduction–oxidationcarba-sugar hydrazones, 241, 244intermolecular, saccharide osazones,

197–198intramolecular, saccharide osazones,

198–202Reductive amination, for isothiocyanation,

61–62

Saccharide azines, synthesis, 174–175Saccharide hydrazones

alkali action, 183–185bromo and dibromo derivative

formation, 189characteristics, 175–177cycloaddition reactions, 192–193derivatives, 189diazo derivative reactions, 194elimination reactions, 186–188formation, 177–179formazan formation, 189–192hydrazono lactone formation, 185–186oxadiazole formation, 186reduction reactions, 193–194structure, 179–182

Saccharide osazonesaction of bases, 209–212anhydroosazone formation, 220–222bis(hydrazone) oxidation, 212bis(phenylhydrazone) reduction, 220bis(phenylhydrazone) residue

aromatization, 215–220chelated structures, 205–207electrophilic substitution, 209ester derivatives, 223–224ether derivatives, 224formation, 194–196intermolecular oxidation–reduction,

197–198intramolecular oxidation–reduction,

198–202

SUBJECT INDEX364

mutarotation, 207nucleophilic substitution, 208–209saccharide poly(hydrazones), 224–226tautomeric structures, 202–205

Saccharide phenylosotriazoles, formation,217

Saccharide poly(hydrazones), preparation,224–226

Saccharide triazoles, formation, 215–220Self-condensation reactions, sugar

isothiocyanates, 64–65Solvent, proximity to catalytic center,

299–301SPRI, Inc., see Sugar Processing Research

Institute, Inc.Sugar amines, isothiocyanation, 56–62Sugar carbodiimides, in sugar thiourea

synthesis, 84–85Sugar iminophosphoranes, reaction with

carbon disulfide, 63Sugar isothiocyanates

addition of carbon bases, 68–70aza-Wittig-type reactions, 65–66condensation with carboxylic acids, 64coupling with amine nucleophiles,

74–78coupling with amino sugars, 81–84cycloaddition reactions, 66desulfurization reactions, 65reactions

with C-nucleophiles, 63with N-nucleophiles, 63with O-nucleophiles, 63with S-nucleophiles, 63

self-condensation reactions, 64–65spectroscopic properties, 66–68synthesis

by electrophilic addition, 56by isothiocyanation, 56–62by reaction of glycosyl donor, 41, 49,

53–55by reaction of sugar imino-

phosphoranes, 63Sugar Processing Research Institute, Inc.,

and Margaret Alice Clarke, 25–26Sugar thioamides

conformational properties, 72–73synthesis

by carbon base addition, 68–70miscellaneous methods, 71–72


by thioacylation, 71by thionation, 70–71

Sugar thiocarbamatescyclic sugar thiocarbamates, 90–93linear sugar thiocarbamates, 87–90

Sugar thiosemicarbazones, synthesis, 99–100Sugar thiourea

conformational properties, 86–87functional group transformation, 86spectroscopic properties, 86–87synthesis

by amino sugar coupling, 78–81from sugar carbodiimides, 84–85by sugar isothiocyanate coupling, 74–78,

81–84Sulfoglycosphingolipids, nomenclature, 320Svennerholm abbreviations, brain

gangliosides, 322–323

Tautomers, osazones, 202–2052,3,4,5-Tetra-O-acetyl derivative, from D-

galactaric acid acetylation, 155S-(2,3,4,6-Tetra-O-acetyl-b-D-

glucopyranosyl) N,N-dimethyldithiocarbamate, synthesis, 95

2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosylisothiocyanate, coupling reactions, 81

Tetrahydrooxazine-2-thione heterocycles,preparation, 92

Thermooxidative degradation, polymer,137–138

Thiamines, research by Lord Todd, 5Thioacylation, amino sugars, 71N-Thiocarbonyl carbohydrate derivatives,

synthesis, 97–101N-Thiocarbonyl compounds, comparison to

N-carbonyl compounds, 37–39N-Thiocarbonyl sugars

artificial receptors, 118–120enzyme inhibitors, 107–118glycoclusters, 102–107glycodendrimers, 102–107

SUBJECT INDEX 365

interaction with membrane receptors,101–102

neoglycoconjugates, 102–107Thionation, N-carbonyl derivative, 70–71Thioureido substituents, in cyclodextrin

modification, 118–120Tin tetrachloride–trimethylsilyl

isothiocyanate system, 55Transformation, sugar thiourea functional

groups, 86Transhydrazonation, osazones, 208–209Transition state, glycosylase reaction,

296–299Tri-O-benzoyl-D-ribopyranosyl

isothiocyanates, preparation, 582,3,4-Tri-O-benzyl-a-D-glucopyranosyl

isothiocyanate, synthesis, 54p-Trifluoroacetamidoaniline, reductive

amination, 61Tri-O-methyl-L-arabinono-1, 5-lactone,

ammonolysis, 151–153Trimethylsilyl isothiocyanate–tin

tetrachloride system, 55Tris(phenylhydrazones), structure,

235–2361, 2, 3-Tris(phenylhydrazones), formation,

230–234

Ultraviolet spectroscopy, sugarisothiocyanates, 68

Vitamin B12, research by Lord Todd, 8

X-Ray studiesglycosidases, residues for protonation,

272–274inverting glycosylases, 279–284retaining glycosylases, 275–279

Xylanase, X-ray studies, 279D-Xylonic acid, derivative preparation, 148


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Advances in Carbohydrate Chemistry and Biochemistry, Vol. 55

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