229
The Synthesis of Several Azasugars, Glycosylated Azasugars and Disaccharides of Biological Interest Peter J. Meloncelli B.Sc. (Hons) (2007)
The Synthesis of Several Azasugars, Glycosylated Azasugars and
Disaccharides of Biological Interest
Peter J. Meloncelli B.Sc. (Hons) (2007)
This thesis is presented for the degree of Doctor of Philosophy to the University of Western Australia. The work described in this thesis was carried out by the author in the School of Biomedical, Biomolecular and Chemical Sciences at the University of Western Australia under the supervision of Professor Robert V. Stick. Unless otherwise referenced, the work described in this thesis is original. Peter Meloncelli January 2007
Contents
Summary iii
Acknowledgements vi
Glossary vii
Part 1 Introduction 1
References 15
Chapter 1 An Improved Synthesis of Isofagomine,
and Other Related Moleceules 19
Introduction 21
Discussion 35
Experimental 55
References 73
Appendix 76
Chapter 2 Synthesis of 3- and 4-O-β-D-Glucopyranosyl
Derivatives of Isofagomine and Noeuromycin 83
Introduction 85
Discussion 88
Experimental 109
References 134
Appendix 135
ii
Part 2
Chapter 3 Synthesis of Some
α-D-Glucopyranosyl-α-D-Galactopyranoses 139
Introduction 141
Discussion 151
Experimental 162
References 182
Chapter 4 Development of an Alternative Carbohydrate
Source for Pre-term Infants 185
Introduction 187
Discussion 195
Experimental 203
References 211
Appendix 214
iii
Summary
The development of several carbohydrate-based pharmaceuticals has stimulated an
increased interest in the field of carbohydrate chemistry. The discovery of Acarbose and
invention of Miglitol, treatments for type II diabetes, as well as the influenza treatments,
Relenza and Tamiflu, have been largely responsible for this increased interest. These
treatments operate by the inhibition of glycoside hydrolases, a group of enzymes
important in a variety of biological processes. This thesis involves the study of a group of
glycoside hydrolase inhibitors known as azasugars, which are nitrogen-containing sugar
mimics.
The thesis consists of two parts: Part 1 (Chapters 1 and 2) and Part 2 (Chapters 3 and 4).
Chapter 1 outlines the synthesis of several known azasugars, from the central key
imidazylate (22), namely isofagomine (13), noeuromycin (14), isofagomine lactam (24),
azafagomine (23) and the hydrazone (25). The synthesis of two new azasugars,
azanoeuromycin (27) and ‘guanadine’ isofagomine (26) is also reported.
OImSO2O
OO
OBn
NHHOHO
OH NHOH
HOHO
OH
NHOHO
OH
NH2
NH
NH
NHHOHO
OH
NH
NHOHO
OH
NH
NHOH
HOHO
OH
NHHOHO
OH
O
(13)
(14)
(26)
(24)
(23)
(27)
(25)(22)
iv
Based on the excellent glycoside hydrolase inhibition by the previously reported
glucosylated derivatives (28) and (15) of isofagomine, it was thought that the
glucosylated derivatives (20) and (21) of noeuromycin may prove to be even more potent
inhibitors.
O
HOHO
OH
OHO NH
HO
OH
OH
(20)
O
HOHO
OH
OHO NH
HO
OH
(28)
HO NH
O
OH
OH
(21)
O
HOHO
OH
OH
HO NH
O
OH
O
HOHO
OH
OH
(15)
Chapter 2 describes the synthesis of 3-O-β-D-glucopyranosylisofagomine (28) and 3-O-β-
D-glucopyranosylnoeuromycin (20). This was achieved by glycosylation of the diol (101)
using the trichloroacetimidate donor (172), followed by a sequence similar to that used
for the preparation of isofagomine and noeuromycin. For the two regioisomers (15) and
(21), it was decided to use a non-selective glycosylation of the diol (191), with a late
introduction of the required nitrile group. This more efficient route also gave access to
the previously prepared (28) and (20).
O
BnOBnO
OBn
OAcOTCA
(172) (191)(101)
OAllO
HOOH
OBnO
NCOH
OHOBn
The second part of the thesis focuses on the preparation and biological testing of the
disaccharides (203), (204), (205) and (206). Chapter 3 describes a synthesis of the four
disaccharides and offers a direct comparison of several methods used to prepare α-D-
v
glucosides, namely the use of glycosyl iodides, glycosyl iodides/triphenylphosphine
oxide, trichloroacetimidates and thioglycosides.
O
HOHO
OH
OHO
OH OH
OHO
OH
(204)
(205)
O
HO
OH
OH(206)
O
HOHO
OH
OHO
OH
O
HO
O
OHOH
O
HOHO
OH
OH
OH
O
HOHO
OH
OH
O
O
(203)
OH
HO
OH OH
The final chapter, Chapter 4, focuses on the testing of these disaccharides as a possible
alternative carbohydrate source for pre-term infants. Initially, commercially available
glycoside hydrolases were used to detect any hydrolysis of the four disaccharides, with
(206) exhibiting the most promising results (to provide D-glucose and D-galactose).
Detailed kinetic studies were then conducted using homogenates obtained from pig
intestinal mucosa. Unfortunately, the results indicated that (206) was unsuitable as an
alternative carbohydrate source for pre-term infants.
vi
Acknowledgments
I would like to thank Professor Bob Stick for his excellent supervision and patience,
without his help this work would not have been possible.
Professor Peter Hartmann for suggestion of the idea for Part 2 and for supervision of the
biochemical component.
Gideon Davies and Tracey Gloster at the University of York for providing X-ray
crystallographic data and enzyme inhibition studies.
Dr Lindsay Byrne for comprehensive n.m.r. spectroscopy instruction and to Dr Anthony
Reeder for all mass spectra.
The technical staff for providing me with the equipment and services to run things
smoothly, including Sarah Davis, Nigel Hamilton and Greg Cole.
The Hartmann lab, including Ching Tat Lai, Tracey Williams, Danielle Prime, Wei Wei
Pang, Holly McClellen, Nadia Khaldoune and Charles Czank for welcoming me into
their lab.
The people who have been great friends throughout the years including Nigel Lengkeek,
Sally Hyslop, Wendy Gilchrist, Maddie Jodrell, Emma Thomas, Sarah Ovenden, Sylvia
Sze, Emma Pearson and Phil Schauer.
The financial assistance of an Australian Postgraduate Award and a Completion
Scholarship was greatly appreciated.
vii
Glossary
(Boc)2O di-tert-butyl dicarbonate 9-BBN 9-borabicyclo[3.3.1]nonane ABTS 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) Ac2O acetic anhydride AcOH acetic acid BMSCl tert-butyldimethylsilyl chloride BSP 1-benzenesulfinylpiperidine BzOBT 1-(benzoyloxy)benzotriazole CSA 10-camphorsulfonic acid d day(s) DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DDI double deionised water DMAP 4-(dimethylamino)pyridine DMF N,N-dimethylformamide DPS diphenyl sulfoxide DSS 2,2-dimethylsilapentane-5-sulfonic acid Et2O diethyl ether Et2OBF3 boron trifluoride diethyl etherate EtOH ethanol gal D-galactose glc D-glucose h hour(s) Hünigs base N-ethyldiisopropylamine IC50 the half maximal inhibitory concentration ImH imidazole LDA lithium diisopropylamide LiHMDS lithium bis(trimethylsilyl)amide MeOH methanol min minute(s) n.m.r. nuclear magnetic resonance NMO N-methylmorpholine N-oxide o.n. overnight pyr pyridine PyrOTs pyridinium tosylate rt room temperature TBAF tetrabutylammonium fluoride
viii
TBAI tetrabutylammonium iodide TBDPSCl tert-butyldiphenylsilyl chloride Tf2O trifluoromethanesulfonic anhydride THF tetrahydrofuran TMSCN trimethylsilyl cyanide TMSI iodotrimethylsilane TMSOTf trimethylsilyl trifluoromethanesulfonate TPAP tetrapropylammonium perruthenate TTBP 2,4,6-tri-tert-butylpyrimidine Wilkinson's catalyst tris(triphenylphosphine)rhodium(I) chloride
Functional Group Abbreviations
Ac CH3CO All CH2CHCH2 BMS (CH3)3C(CH3)2Si Bn PhCH2 Boc (CH3)3COCO Bz PhCO Et CH3CH2 Me CH3 Ms CH3SO2 TBDPS (CH3)3CPh2Si TCA Cl3CC(NH) Tf CF3SO2 Troc Cl3CCH2OCO Ts 4-CH3C6H4SO2
2
3
DECLARATION FOR THESES CONTAINING PUBLISHED WORK AND/OR WORK PREPARED FOR PUBLICATION
This thesis does not contain work that I have published, nor work under consideration for publication. The thesis is completely the result of my own work, and was substantially conducted during the period of candidature, unless otherwise stated in the thesis. Signature……………………………….
This thesis contains sole-authored published work and/or work prepared for publication. The bibliographic details of the work and where it appears in the thesis is outlined below. Signature………………………………
This thesis contains published work and/or work prepared for publication, some of which has been co-authored. The bibliographic details of the works and where they appear in the thesis are set out below. (The candidate must attach to this declaration a statement detailing the percentage contribution of each author to the work. This must been signed by all authors. Where this is not possible, the statement detailing the percentage contribution of authors should be signed by the candidate’s Coordinating Supervisor).
1.) Meloncelli, P. J.; Stick, R. V. Aust. J. Chem. 2006, 59, 827-833 (Major Contributor) 2.) Gloster, T. M.; Meloncelli, P. J.; Stick, R. V.; Zechel, D.; Vasella, A.; Davies, G. J.; J. Am. Chem. Soc.
2007, 129, 2345-2354 (Minor Contributor) 1.) The majority of work conducted in Chapter 1 is discussed in this paper. 2.) Two X-ray crystal structures from Chapter 1 are discussed in this paper and are clearly
acknowledged. All work not conducted by the author is clearly acknowledged within the text of this thesis. Signature……………………………… (Candidate) Signature……………………………… (Supervisor)
1
Part 1
Introduction
2
3
Carbohydrates, Beyond the Function of Energy Source
Carbohydrates have been traditionally recognized as a source of biological energy and as
the structural polymer in plants, with cellulose being the most abundant bio-polymer in
existence. In reality carbohydrates perform a wide variety of other functions in cells
including roles in signaling, cell-cell communication, infection by pathogens and the
binding of viruses and toxins.1-3 The development of the field of glycobiology has greatly
expanded the understanding of the role of carbohydrates and has thus resulted in the
formation of a symbiotic relationship with synthetic carbohydrate chemistry.4-6
Glycoside Hydrolases
Of particular interest in glycobiology is a group of enzymes called glycoside hydrolases,
otherwise known as glycosidases, responsible for the hydrolysis of the glycosidic linkage.
The glycosidic linkage is quite stable, in particular that of cellulose, which is the most
stable naturally occurring biopolymer with a half life of around five million years.7
Hydrolysis of the glycosidic linkage via enzymatic means can increase the rate by a
factor of 1017, ranking glycosidases as one of the most efficient catalysts.7,8 Glycosidases
are essential not only for the hydrolysis of stored glycosides but also for the development
of eukaryote and prokaryote cell walls,9 defence against bacterial infection, and viral
replication.10,11 The absence of certain glycosidases is responsible for several serious and
debilitating disorders such as the lysomal storage disorders.12 On a commercial level
glycosidases are used in food processing, bio-stoning of textiles and in the pulp and paper
industry as bio-bleaching agents.10
4
Classification of Glycosidases
The traditional classification of glycosidases, developed in 1984 by the International
Union of Biochemistry, is based primarily on function, with classification according to
the following criteria:13
i) The nature of the substrate, that being the carbohydrate for which the
hydrolase is most active.
ii) The stereochemistry of the glycosidic linkage processed (α or β).
iii) The anomeric stereochemistry of the product relative to the substrate
(inverting or retaining).
iv) The region of the oligosaccharide chain where glycosidic cleavage occurs,
whether it is at the reducing or non-reducing terminus (exo-) or at internal
points within the chain (endo-).
Unfortunately this basic classification does not take into account the structural features of
the enzyme or events such as divergent or convergent evolution.14 In 1991 Henrissat and
Davies proposed a classification based on amino acid sequence similarities that takes into
account these factors and, through CAZY (http://www.cazy.org), has enabled the rapid
publication of, to date, 105 families of glycosidases.12,14,15
Mechanism of Action of Glycoside Hydrolases
The mechanism of action of glycosidases is generalized according to the two major
classes, inverting and retaining. The mechanism of action of retaining glycosidases,
proposed by Koshland in 1953, involves a covalent glycosyl-enzyme intermediate and a
5
double displacement.16 This proposed mechanism was confirmed by X-ray diffraction
studies by Davies et al., producing crystal structures of the five states along the
enzymatic reaction coordinate.17
OO
H
O O
O
OO
OO
H
O O
R O R
δ
δ
δ
δ
OO
O O
O
HO
H
O
OO
H
O O
O H
δ
δ
δ
δ
OHO
O O
O
OH
‡
‡
Proposed mechanism of action of a β-retaining glycosidase
Protonation of the aglycon by the acidic carboxylic acid results in the formation of the
oxacarbenium-ion-like transition state, leading to a covalent glycosyl-enzyme
intermediate.18 The carboxylate ion then deprotonates an incoming water molecule that
attacks the anomeric carbon of the glycosyl-enzyme intermediate to give the product,
with retention of configuration.18
6
Inverting glycosidases possess the same pair of carboxylate residues as those present in
retaining glycosidases, however, they are separated by 9.5 Å, which allows the
intervention of a water molecule.18 Cleavage occurs via a concerted process with the
protonation of the glycosidic oxygen by the catalytic acid accompanied by a catalytic-
base-assisted attack of water at the anomeric carbon.18
OO
H
O O
O
OO
OO
H
O OH
OH
R O R
H
O H
δ
δ
δ
δ
OO
O OH
O
OH
HOR
‡
δ
Proposed mechanism of action of an inverting glycosidase
Therapeutic Applications Based on Glycosidase Inhibition
The biological importance of glycosidases has opened the pathway for the development
of therapeutic treatments that target this class of enzyme. Progress has been rather slow,
with significant difficulties in the translation of in vivo results into clinical treatments.1
Poor bioavailability and high clearance rates, whether actual or perceived, plague the
development of carbohydrate-based pharmaceuticals.1,19
NH
HOHO
OH
(1)
N
HOHO
OH
(2)OH OH
The treatment of HIV-1 infected lymphocyte cultures with 1-deoxynojirimycin (1) and N-
butyl-1-deoxynojirimycin (2) has resulted in an inhibition of the ability of the virus to
spread.20 The most likely mechanism for this inhibition is the interference with the
7
glycosidation state of various glycoproteins necessary for entry into the host cell, in
particular the ability of HIV-1 to bind to the CD4 receptor, an essential process for
infection.20
CO2H
NH2
OAcHN
O CO2H
NHAcHN
HNNH2
HO
HO OH
(3) (4)
COOH
HO
HNNH
NH2
NHO
(5)
Influenza, an acute and highly infectious respiratory illness caused by the influenza virus,
causes patients to exhibit fever, headache, muscular pain and sore throat. Whilst the
disease is not generally lethal, a higher mortality rate is observed in the elderly and those
with compromised immune systems.21 With the advent of avian flu, a sub-type of
influenza A, significant interest has evolved towards the development of new anti-
influenza drugs, particularly those that are active against this new strain of influenza.
Two drugs are currently available for the treatment of influenza, Relenza (3) and Tamiflu
(4), and a third, known as Peramivir (5), is in clinical trials; these drugs are carbohydrate-
based and operate by the inhibition of viral sialidase.21,22 Sialidases are responsible for
the cleavage of sialic acid from the host cell-surface glycoproteins, enabling the release
of viral progeny from the infected cell.23,24
8
OH
HOHO
OH
HN O
HO
CH3
OHO O
HO
OH
OH O
HO
OH
OH
O
OH
N
HOHO
OH
OH
OH
(6) (7)
Diabetes, a disease in which the body does not produce or properly use insulin, affects
approximately seven percent of the population.25 Insulin is a hormone that is needed to
regulate the metabolism of glucose, starches and other carbohydrates required for healthy
function. It has been observed that inhibition of some intestinal glycosidases and
pancreatic α-amylase can regulate the absorption of carbohydrates and thus be used as a
therapeutic treatment.26,27 Two currently used medications for diabetes are acarbose (6)
and miglitol (7). Acarbose is a potent inhibitor of sucrase with an IC50 of 50 μM, enabling
its use as a medication to lower the post-prandial glucose concentration.27,28 In 1999 a
more effective inhibitor of α-glucosidase, miglitol, was introduced that not only relies on
α-glucosidase inhibition but also lowers blood glucose concentration, presumably by its
effect on insulin regulating factors.29
Mechanism of Action of Glycosidase Inhibitors
The inhibition of glycosidases has been broadly classed into three catagories.30 ‘Affinity
label’ is a classification given to inhibitors containing a chemically reactive group that
binds irreversibly to the target enzyme. One such example is an epoxyalkyl glycoside,
shown to bind irreversibly to the active site of a retaining β-glycosidases.10,31,32 A
9
disadvantage of affinity labels is that they are prone to indiscriminate labeling of non-
catalytic residues.30
O
HO
OH
OH
HOO
O
OO
O O
H
O
HO
OH
OH
HOO
OH
OO
O O
‘Mechanism-based inhibition’ involves the processing of the inhibitor into an
intermediate by the target enzyme and the formation of a stable covalent bond between
enzyme and inhibitor.30 Excellent examples of this type of inhibitor are the 2-fluoro and
5-fluoro sugars developed by Withers and co-workers: the 2-deoxy-2-fluoro-glycosides
(8)33 and (9)34, 2-deoxy-2-fluoro-β-D-glucopyranosyl fluoride (10)35 and 5-fluoro-β-D-
glucopyranosyl fluoride (11)36. These substrates are processed readily in the active site of
the enzyme to form a stable covalent-enzyme intermediate.30
O
HO
OH
F
HOO
NO2
NO2
O
HO
OH
F
OO
NO2
NO2
O
HO
OH
OH
HO
O
HO
OH
F
HOF
O
HO
OH
OH
HOF
F
(10)
(8)
(11)
(9)
The third category, known as a ‘tight-binding complement’, occurs when the inhibitor
mimics the transition state or a high energy intermediate along the reaction pathway.30
10
These inhibitors are both competitive and reversible and, in the case of glycosidases, they
are usually in the form of small molecules that exhibit some similarity to the purported
oxacarbenium ion ‘intermediate’ or transition state.37 The most important factors
attributed to the transition state character of a potential inhibitor are charge, trigonal
anomeric centre, half-chair conformation and relative configuration.37
One effective way of mimicking the transition state has been through the introduction of
a nitrogen into the (pyranose) ring, to act as a source of positive charge (when
protonated) and thus mimicking the oxacarbenium ion.37 The first inhibitor of this class,
often referred to as an azasugar, was nojirimycin (12), an effective inhibitor of α-
glycosidases.38 Other examples of this class include 1-deoxynojirimycin (1),39
isofagomine (13),40,41 noeuromycin (14)42,43 and 4-O-β-D-glucopyranosylisofagomine
(15).44
HO
NHHOOH
(12)OHOH
NH
HOHO
OH
(1)
HO NH
HOOH
(13)
HO NH
HOOH
(14)OH
HO NH
OOH
O
HOHO
OH
OH
(15)
OH
11
Inhibition of the action of glycosidases by these molecules has been proposed to occur
via protonation of the nitrogen by the carboxylate residue, resulting in a strong
electrostatic interaction.37,45,46 X-ray crystallographic data of (15) in complexation with
Cel5A (a family 5 glycosidase) showed in detail the protonation states of both the
enzyme active-site and the inhibitor, supporting the proposed mechanism. The data also
showed the presence of a water molecule in roughly the correct position for the
deglycosylation step of the subsequent reaction.46
HO N
OOH
OO
O
O
H
H
HO
H
O
HOHO
OH
OH
Glu139
Glu228
OH Tyr202
O
Ala234
Schematic representation of the binding of (15) with Cel5A.46
Weaker non-covalent interactions also play a significant role in binding (substrate-
enzyme or inhibitor-enzyme), in particular hydrogen bonding between the hydroxyl
groups and the enzyme.8,47-49 Interactions of the various hydroxyl groups have been
measured through the use of modified substrates in which the individual hydroxyl groups
were replaced by either hydrogen or a fluorine.49 This enabled the contribution to binding
energy of the hydroxyl groups in both the ground state and the transition state to be
calculated.49 The most important interaction for Cel5A was shown to be the hydroxyl
group at C2, contributing 18 and 22 kJ mol-1 to the ground and transition state binding
12
energy, respectively.49 Noeuromycin (14), in contrast to isofagomine (13), has the
hydroxyl group still present at C2, resulting in a 2- and 4000-fold increase in inhibition
of β- and α-glucosidase, respectively, highlighting how important this interaction is to
inhibitor design.43
HO NH
HOOH
(13)
HO NH
HOOH
(14)OH
The orientation of the hydroxyl groups is also crucial with isofagomine (13),
isogalactofagomine (16) and isofucofagomine (17) demonstrating the strongest inhibition
in glucosidases, galactosidases and fucosidases, respectively.43
HO NH
HO
OH
(13)
HO NH
OH OHNH
HOHO(16) (17)
In the case of the enzymes with multiple binding sites, such as the endo-glycanase Cex
from Cellulomonas fimi, non-covalent interactions play an even greater role in binding. In
the case of isofagomine (13), the addition of a β-D-glucosyl residue at C4, compound
(15), increased the inhibition by 1000-fold.44 Further addition of β-D-glucosyl residues,
generating compounds (18) and (19), resulted in minor increases in inhibitor efficacy.44
HO NH
OOH
O
HOHO
OH
OHO
HOO
OH
OH
n
(15) n=0(18) n=1(19) n=2
13
The combination of both the important interaction at C2 and the non-covalent interactions
provided by the introduction of β-D-glucosyl residues in glucosylated isofagomine (15)
suggests that the glucosylated derivatives (20) and (21) of noeuromycin could exhibit
better glycosidase inhibition than the parent molecule. Of particular interest would be the
comparison of inhibition between (15) and (21), using the endo-glycanase Cex from
Cellulomonas fimi.
O
HOHO
OH
OHO NH
HO
OH
OH
(20)
HO NH
O
OH
OH
(21)
O
HOHO
OH
OH
Overview of Part 1
Chapter 1 discusses the utilization of the imidazylate (22) to prepare easily a wide range
of azasugars, including the previously reported isofagomine (13),41,50 noeuromycin
(14),42,43 azafagomine (23),51-53 isofagomine lactam (24)54 and the hydrazone (25).55 Two
new azasugars were also prepared, namely the “guanidine” derivative (26) of
isofagomine, and azanoeuromycin (27). Whilst many syntheses exist for the preparation
of some of these compounds, there has never been an efficient synthesis from just the one
common precursor.
14
OImSO2O
OO
OBn
NHHOHO
OH NHOH
HOHO
OH
NHOHO
OH
NH2
NH
NH
NHHOHO
OH
NH
NHOHO
OH
NH
NHOH
HOHO
OH
NHHOHO
OH
O
(13)
(14)
(26)
(24)
(23)
(27)
(25)(22)
Chapter 2 details the synthesis of the known glucosylated derivatives (28) and (15) of
isofagomine;44,56 this Chapter also presents the synthesis of the previously unknown
glucosylated derivatives (20) and (21) of noeuromycin.
O
HOHO
OH
OHO NH
HO
OH
OH
(20)
O
HOHO
OH
OHO NH
HO
OH
(28)
HO NH
O
OH
OH
(21)
O
HOHO
OH
OH
HO NH
O
OH
O
HOHO
OH
OH
(15)
15
References
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(27) Asano, N. Glycobiology 2003, 13, 93R.
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E. Naturwiss. 1977, 64, 535.
(29) Joubert, P. H.; Foukaridis, G. N.; Bopape, M. L. Eur. J. Clin. Pharmacol. 1987,
31, 723.
(30) Krantz, A. Bioorg. Med. Chem. Lett. 1992, 2, 1327.
(31) Sulzenbacher, G.; Schülein, M.; Davies, G. J. Biochemistry 1997, 36, 5902.
17
(32) Keitel, T.; Simon, O.; Borriss, R.; Heinemann, U. Proc. Natl. Acad. Sci. USA
1993, 90, 5287.
(33) Withers, S. G.; Street, I. P.; Bird, P.; Dolphin, D. H. J. Am. Chem. Soc. 1987, 109,
7530.
(34) Mackenzie, L. F.; Wang, Q.; Warren, R. A. J.; Withers, S. G. J. Am. Chem. Soc.
1998, 120, 5583.
(35) Withers, S. G.; Rupitz, K.; Street, I. P. J. Biol. Chem. 1988, 263, 7929.
(36) McCarter, J. D.; Withers, S. G. J. Am. Chem. Soc. 1996, 118, 241.
(37) Legler, G. Adv. Carbohydr. Chem. Biochem. 1990, 48, 319.
(38) Niwa, T.; Inouye, S.; Tsuruoka, T.; Koaze, Y.; Niida, T. Agr. Biol. Chem. 1970,
34, 966.
(39) Schmidt, D. D.; Frommer, W.; Müller, L.; Truschiet, E. Naturwiss. 1979, 66, 584.
(40) Dong, W.; Jesperson, T. M.; Bols, M.; Skrydstrup, T.; Sierks, M. R. Biochemistry
1996, 35, 2788.
(41) Jespersen, T. M.; Dong, W.; Sierks, M. R.; Skyrdstrup, T.; Lundt, I.; Bols, M.
Angew. Chem. Int. Ed. Engl. 1994, 33, 1778.
(42) Andersch, J.; Bols, M. Chem. Eur. J. 2001, 7, 3744.
(43) Liu, H.; Liang, X.; Søhoel, H.; Bülow, A.; Bols, M. J. Am. Chem. Soc. 2001, 123,
5116.
(44) Macdonald, J. M.; Stick, R. V.; Tilbrook, D. M. G.; Withers, S. G. Aust. J. Chem.
2002, 55, 747.
18
(45) Zechel, D. L.; Boraston, A. B.; Gloster, T.; Boraston, C. M.; Macdonald, J. M.;
Tilbrook, D. M. G.; Stick, R. V.; Davies, G. J. J. Am. Chem. Soc. 2003, 125,
14313.
(46) Varrot, A.; Tarling, C. A.; Macdonald, J. M.; Stick, R. V.; Zechel, D. L.; Withers,
S. G.; Davies, G. J. J. Am. Chem. Soc. 2003, 125, 7496.
(47) Notenboom, V.; Birsan, C.; Nitz, M.; Rose, D. R.; Warren, R. A. J.; Withers, S.
G. Nature Struct. Biol. 1998, 5, 812.
(48) White, A.; Tull, D.; Johns, K.; Withers, S. G.; Rose, D. R. Nature Struct. Biol.
1996, 3, 149.
(49) Namchuk, M. N.; Withers, S. G. Biochemistry 1995, 34, 16194.
(50) Jesperson, T. M.; Bols, M.; Sierks, M. R.; Skrydstrup, T. Tetrahedron 1994, 50,
13449.
(51) Bols, M.; Hazell, R. G.; Thomsen, I. B. Chem. Eur. J. 1997, 3, 940.
(52) Liang, X.; Bols, M. J. Org. Chem. 1999, 64, 8485.
(53) Ernholt, B. V.; Thomsen, I. B.; Lohse, A.; Plesner, I. W.; Jensen, K. B.; Hazell, R.
G.; Liang, X.; Jakobsen, A.; Bols, M. Chem. Eur. J. 2000, 6, 278.
(54) Lillelund, V. H.; Liu, H.; Liang, X.; Søhoel, H.; Bols, M. Org. Biomol. Chem.
2003, 1, 282.
(55) Hansen, S. U.; Bols, M. J. Chem. Soc., Perkin Trans. 2 2000, 665.
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57, 187.
Chapter 1
An Improved Synthesis of Isofagomine, and
Other Related Molecules
20
21
Introduction
Isofagomine and Noeuromycin
Isofagomine (13) was first prepared by Lundt and Bols in 19941,2 and has subsequently
become an avidly sought after target, primarily owing to its excellent glycosidase
inhibition.1 Bols’ first synthesis began from the readily available levoglucosan (29) and,
via a four step sequence, provides the known epoxide (30). A regioselective Grignard
addition with vinylmagnesium bromide gave the 2-exo-alkene; ozonolysis followed by a
reductive workup afforded the hydroxymethyl compound (31). Acid-catalysed hydrolysis
of the anhydro linkage of (31) yielded the hemiacetal (32). Periodate cleavage between
C5 and C6 of (32) gave the dialdehyde (33), which was then reductively aminated to give
the piperidine with the requisite stereochemistry. Hydrogenolysis under acidic conditions
furnished isofagomine (13).
O
O
OBn
O
(30)
O
O
OH
OH
(29)
OH
O
O
OBn
OH
(31)OH
OBnO
HOOH
OH
HO
(32)
O OH O
OBnOH
(33)
NHHO
HO
OH
(13)
22
Ichikawa’s synthesis3 of isofagomine (13) started from the commercially available, albeit
expensive, D-lyxose (34), which was converted into the acetonide (35) via an
isopropylidenation, tosylation, benzoylation and displacement of the tosyl group with
sodium azide. Removal of the benzoyl group using sodium methoxide followed by a Ho
aldol reaction with formaldehyde gave the hydroxymethyl hemiacetal (36).
Hydrogenolysis of (36) using palladium hydroxide resulted in reduction of the azido
group, followed by an intramolecular cyclisation to afford (37) and the requisite
piperidine ring system. Protection of the amine followed by selective benzoylation gave
(38). Treatment of (38) with methyl oxalyl chloride resulted in conversion into the
unstable oxalate (39). Deoxygenation followed by removal of the benzoyl and benzyl
carbamate protecting groups gave a separable mixture of isofagomine (13) and the L-ido
derivative (40) in a 2:1 ratio.
OHOHO
OH
OH
NH
HOHO
OH
OHNBoc
BzOBzO
OBz
OH
NH
HOHO
OH
(13)
(34) (35)(36)
(37) (38)
NBoc
BzOBzO
OBz
OCO2Me
NH
(40)
OH
OH
HO
(39)
OOON3
OH
OH
O OON3
OBz
23
Ichikawa’s second synthesis4 started from the protected glyceraldehyde (41); a Horner-
Emmons condensation with trimethyl phosphonoacetate gave mainly the E-methyl ester,
with subsequent reduction giving the allylic alcohol (42). Introduction of the necessary
stereocenters was achieved using a Sharpless asymmetric epoxidation, affording the
epoxy alcohol (43) in excellent yield. Ring opening of the epoxide (43) successfully
introduced the cyano group with good regioselectivity and excellent yield (>90%). The
mixture was then silylated, enabling the separation of the dominant isomer (44). Removal
of the cyclohexylidene group under mildly acidic conditions, followed by tosylation,
produced the acyclic nitrile (45). Reduction of the nitrile using Raney-Nickel, followed
by an intramolecular displacement of the tosylate, yielded the piperidine (46), with acid-
catalysed hydrolysis of the silyl ether yielding isofagomine (13).
O
O CHO
O
O OH
O
O OH
O
O
O OH
OH
CNO
O OH
CN
OH
NH
HOHO
OBPS
NHHO
HO
OH
(41) (42) (43)
(47) (48)
(46) (13)TsO
HO OBPS
OH
CN
(45)
O
O OBPS
OH
CN
(44)12:1
24
Pandey’s synthesis5 of isofagomine commenced from (−)-tartaric acid (49), with
conversion into the aldehyde (50) via the procedure of Kibayashi and co-workers.6 The
aldehyde (50) was then converted into the alkyne (51) via Corey’s aldehyde-to-alkyne
chain extension procedure.7 Removal of the silyl ether, followed by treatment with CBr4
and Ph3P, gave the bromide; subsequent displacement of the bromide afforded the tertiary
amine (52). The most captivating aspect of this synthesis was the subsequent use of a
photo-induced electron transfer to afford the piperidine derivative (53). Hydroboration of
the alkene (53) using 9-BBN gave the primary alcohol (54) as the only isomer,
unfortunately, in average yield (45%); subsequent removal of the protecting groups
yielded isofagomine (13).
CO2H
CO2HHO
HOOBMSO
O
OOBMS
O
O
N
O
O SiMe3
Bn
N
O
O
CH2
Bn
NHHO
HO
OH
(13)
(49) (50) (51)
(52) (53)
N
O
O Bn
(54)
OH
25
Bols’ second synthesis started from the achiral arecoline (55) and commenced with a
known LDA isomerisation to afford (56), mainly.8 Reduction of the methyl ester
followed by N-demethylation and treatment with 2,2,2-trichloroethyl chloroformate
yielded the N,O-bis(Troc) protected derivative (57). The carbonate was selectively
saponified using potassium carbonate and then the alcohol silylated to give (58).
Epoxidation of the alkene (58), with subsequent hydrolysis, the Achilles’ heel of the
synthesis, yielded a mixture of diastereoisomers, (±)-(59) and (±)-(60). Finally, removal
of the Troc group gave a separable mixture of (±)-isofagomine (13) and (±)-(61).
N
CH3
CO2CH3
N
CH3
CO2CH3
N
O O
O
CCl3
O CCl3O
N
OBPS
O CCl3O
N
OBPS
O CCl3O
O
N
OH
O CCl3O
HO
OH
N
OH
O CCl3O
HO
OH
NH
OHHO
OH
NH
OHHO
OH
(55) (56) (57)
(58) (62) (±)-(59) (±)-(60)
(±)-(61)(±)-(13)
26
Ganem devised a graceful approach to isofagomine from the achiral methyl nicotinate
(63).9 Reduction of the pyridine (63) in the presence of sodium borohydride and phenyl
chloroformate gave the diester (64). Treatment of (64) with m-chloroperoxybenzoic acid
resulted in the regioselective formation of (65). Reduction of (65) followed by oxidation
with chromium trioxide gave the achiral enone (66). An asymmetric reduction of the
enone (66) with LiAlH4 in the presence of (−)-N-methylephedrine afforded the optically
active alcohol (67) (83% e.e.); hydrolysis of the methyl ester gave the hydroxy acid (68).
Hydroboration of the hydroxy acid (68) followed by an oxidative workup gave the triol
(69); subsequent hydrolysis of the carbamate yielded isofagomine (13).
N
CO2CH3
N
CO2CH3
CO2Ph
N
CO2CH3
CO2Ph
ArCO2
HO
N
CO2CH3
CO2Ph
O
N
CO2CH3
CO2Ph
HO
H
N
CO2H
CO2Ph
HO
H
N
CH2OH
CO2Ph
HO
OH
NH
HOHO
OH
(13)
(63) (64) (65)
(66) (67) (68)
(69)
27
Bols’ final synthesis of isofagomine10 was definitely his most efficient, starting from
benzyl β-D-arabinoside (70). A regioselective mono-oxidation of the benzyl glycoside at
C4 gave the ketone (71) that was treated with nitromethane via a Henry reaction to afford
a mixture of epimers (72) and (73) in respectable yield (55-65%). These two epimers
were acetylated to give the triacetates (74) and (75), with a reductive elimination of the
acetate and deacetylation to afford the diol (76). Catalytic hydrogenation of the diol (76)
gave a separable mixture of isofagomine (13) and isoidofagomine (77), in a 2:1 ratio.
O OBn
OH
OH
HO
O OBn
OH
OH
O
O OBn
OH
OHHO
O2N
O OBn
OH
OHHO
O2N
O OBn
OAc
OAc
AcOO2N
O OBn
OAc
OAcAcO
O2N
O OBn
OH
OH
O2N
(70) (71) (72) (73)
(74) (75) (76)
(77)
NHHO
HO
OH
(13)
NHHO
HOHO
Catalytic hydrogenation of (76) under slightly basic conditions resulted in reduction of
the nitro group without debenzylation; protection of the amine with di-tert-butyl
dicarbonate enabled the isolation of the diol (78). Subsequent hydrogenolysis followed by
acid treatment gave the hemiaminal, noeuromycin (14).10
28
O OBn
OH
OH
BocHN
(78)
O OBn
OH
OH
O2N
(76) (14)
NHHO
HO
OH
OH
Macdonald and co-workers’ synthesis of isofagomine (13) in 2002 was both efficient and
elegant, starting from the acetonide (79), prepared from L-xylose.11 Treatment of the
acetonide (79) with N,N΄-sulfuryldiimidazole and sodium hydride resulted in formation
of the imidazylate (22) in moderate yield (72%). Displacement of the imidazylate (22)
with trimethylsilyl cyanide and TBAF gave the nitrile (80). Reduction of the nitrile (80),
followed by protection with di-tert-butyl dicarbonate, gave the carbamate (81),
unfortunately in only 56% yield.
OHO
OO
OBn
(79) (22) (80)
OImSO2O
OO
OBn O
OO
OBn
CN
(82)(81)
NHHO
HO
OH
(13)
O
OO
OBn
BocHN
O
HOOH
OBn
BocHN
Removal of the isopropylidene group afforded the diol (82), similar to Bols’ diol (78),
with the exception of the anomeric configuration. Removal of the carbamate followed by
acidic hydrogenolysis yielded isofagomine (13).
29
Guanti and Riva’s synthesis12,13 of isofagomine (13) started from the readily available
chiral acetate (83). Conversion of the alcohol of (83) into a mesylate, followed by
displacement with sodium azide, successfully introduced an azido group (84). Removal
of the acetyl group, followed by reduction and protection of the resulting amine using di-
tert-butyl dicarbonate, gave (85). Protection of the primary alcohol using a silyl ether,
followed by N-allylation under basic conditions, gave the O-protected allyl derivative
(86). The key and unique step in this sequence was the use of ring-closing metathesis to
generate the piperidine ring (87).
OH N3
OAc OAc
NHBoc
OH
NBoc
OTIPS
NBoc
OTIPS
NBoc
OH
O NBoc
OH
O
(90)
(83) (84) (85)
(86) (87) (88) (89)
NHHO
HO
OH
(13)
NH
OH
OH
OH
Removal of the silyl group, followed by epoxidation, gave an inseparable mixture of the
diastereoisomers (88) and (89) in disappointing yield (40%) and poor selectivity (58:42).
Hydrolysis of the mixture of epoxides and removal of the tert-butyloxycarbonyl
protecting group afforded a separable mixture of isofagomine (13) and isogulofagomine
(90).
30
Ouchi and co-workers developed an elegant synthesis14,15 directly from the readily
available chiral piperidine (91). Silylation gave the protected piperidine (92) with
subsequent epoxidation yielding the desired diastereoisomer (93) in favourable yield. The
nucleophilic ring-opening of the epoxide (93) with an organocuprate gave (95) in 71%
yield. Oxidative cleavage of the allyl group using osmium tetraoxide followed by
reduction and protecting group removal, yielded isofagomine (13) in excellent yield
(85%).
N
HO
Boc
N
BPSO
Boc
N
BPSO
Boc
O
N
PBSO
Boc
O
N
BPSO
Boc
O
N
BPSO
Boc
OH
(91) (92) (93) (94)
(93) (95)
NHHO
HO
OH
(13)
A more recent synthesis reported by Zhu and co-workers16 provided the most efficient
route to isofagomine (13) to date, starting from the easily accessed L-xyloside (96).
Treatment of (96) with 2,2-dimethoxypropane afforded the protected derivative (97) in
moderate yield. Treatment of (97) with triflic anhydride yielded the triflate, followed by
displacement with KCN to afford the nitrile (98). Hydrogenoloysis followed by acid
treatment resulted in debenzylation, reductive amination and deacetonation to yield
isofagomine (13). This synthesis follows very closely the sequence to isofagomine
reported by Macdonald and co-workers.11
31
(97)(96) (98)
OHO
HOOH
OBn
OHO
OO
OBn
O
OO
OBn
CN
NHHO
HO
OH
(13)
Isofagomine Lactam
Bols first prepared isofagomine lactam (24) in 2003, taking advantage of the diol (78)
previously used for the synthesis of noeuromycin (14).17 Removal of the benzyl group
under standard hydrogenolysis conditions gave the hemiacetal (99); TEMPO oxidation
afforded the lactone (100) in a poor (30%) yield. Removal of the protecting group saw
spontaneous rearrangement to the lactam (24).
O OBn
OH
OH
BocHN
(78)
O OH
OH
OH
BocHN
(99)
O O
OH
OH
BocHN
(100)
NHHOHO
OH
O(24)
Macdonald and co-workers, shortly after, in 2004 offered an alternative synthesis of the
lactam.18 Starting from the nitrile (80), removal of the isopropylidene protecting group
followed by silylation gave the disilyl ether (102).
32
(80)
O
OO
OBn
CN
(101)
O
OBn
BMSO
OBMS
NC
O
OH
BMSO
OBMS
NCO
O
BMSO
OBMS
NC
CO2Me
BMSO
OBMS
CN
CH2OH
CO2Me
BMSO
OBMS
CH2NH3Cl
CH2OH
NHHOHO
OH
O
(24)
NHBMSOBMSO
OH
O
(102)
(103) (104) (105)
(106) (107)
O
HOOH
OBn
NC
Hydrogenolysis of the disilyl ether followed by oxidation gave the lactone (104);
treatment with sodium methoxide then yielded the acyclic methyl ester (105). Reduction
of the nitrile gave the amine (106) that was then treated with a base resin to effect an
intramolecular attack and afford the lactam (107), however, in disappointing yield (36%).
Removal of the silyl ethers afforded isofagomine lactam (24).
Azafagomine
The preparation of azafagomine (23) has been a fruitful area for Bols, with no competing
synthesis having been reported.19 Racemic azafagomine, (±)-(23) was prepared via a
short sequence starting with a Diels-Alder reaction between the dienol (108) and the
triazoline (109) to give the adduct (110).
33
N
NN
CH2OH
O
O
PhN
NN
O
O
PhN
NN
O
O
PhN
NN
O
O
PhO O
N
NN
O
O
PhON
NN
O
O
Ph
HO
HO
(±)-(23)
(108) (109) (110) (±)-(111) (±)-(112)
OH OH OH
OH OH
NH
NHHO
HO
OH
(±)-(111) (±)-(113)
Epoxidation of the alkene (110) gave the epoxides (±)-(111) and (±)-(112) with moderate
stereoselectivity (3:1). Ring-opening of the epoxide (±)-(111) resulted in the formation
of the triol (±)-(113) in modest yield (73%). Finally, hydrazinolysis gave azafagomine
(±)-(23).
Bols’ second synthesis produced azafagomine (23) in enantiomerically pure form with
the key step being the use of a lipase to acetylate selectively the (S)-isomer of (114),
unfortunately in poor yield.20 Deacetylation of (115) followed by treatment with oxone
gave a separable mixture of epoxides (116) and (117) in a moderate (68%) yield. Ring-
opening of the epoxide (116) followed by hydrazinolysis gave enantiomerically pure
azafagomine (23).
34
N
NN
O
O
Me
(114)
N
NN
O
O
MeN
NN
O
O
MeO N
NN
O
O
MeO
N
NN
O
O
MeO N
NN
O
O
Me
HO
HO
NH
NHHOHO
OH
(23)
(115) (116) (117)
(118)(116)
OH OAc OAc OAc
OAc OAc
The third synthesis of azafagomine was definitely the most elegant, with the use of L-
xylose removing the difficulties associated with the generation of stereogenic centres.21
Preparation of the hemiacetal (120) was achieved through a simple two-step process from
L-xylose. The hemiacetal (120) was subjected to a reductive amination with tert-butyl
carbazate and sodium cyanoborohydride to give the acyclic hydrazide (121). Acetylation
of the hydrazide followed by introduction of a mesylate afforded (122). Removal of the
carbamate using acid treatment, followed by an intramolecular cyclisation and removal of
the remaining protecting groups, gave azafagomine (23) in moderate yield.
O OH
OH
OH
HO
(119)
O OH
OBnBnO
OBn
OBn OBn
NHBoc
HN
OBnOH
OBn OBn
NHBocN
OBnOMs AcNH
NHHOHO
OH
(23)
(120) (121)
(122)
35
An interesting paper published by Bols highlighted the fate of azafagomine with
prolonged exposure to air, which resulted in the formation of the diazene (123), followed
by isomerisation to give a mixture of the hydrazones (124) and (25).22
NH
NHHOHO
OH
(23)
N
N
HO
HO
OH
N
NH
HO
HO
OH
NH
N
HO
HO
OH
(123) (124) (25)
Discussion
Isofagomine
One of the disappointing aspects of our group’s previous synthesis of isofagomine was
the moderate yield achieved in the preparation of the imidazylate (22), namely 72%
yield.11 Substitution of the insoluble sodium hydride for the soluble hindered base,
lithium bis(trimethylsilyl)amide resulted in an increase in yield to an excellent 90%.
Again, displacement of the imidazylate (22) with trimethylsilyl cyanide gave the nitrile
(80) in good yield.11
OHO
OO
OBn(a) (b)
(79) (22) (80)
OImSO2O
OO
OBn O
OO
OBn
NC
a) (Me3Si)2NLi, (Im)2SO2, THF, 90%; b) Me3SiCN, Bu4NF, MeCN, 80%.
36
Another downfall in the previous synthesis was the reduction of the nitrile (80) to the
amine using lithium aluminium hydride, isolated as the carbamate (81), in disappointing
yield (56%).11 Lithium aluminium hydride has been shown to present significant issues in
the reduction of nitriles, primarily owing to the removal of the hydrogen at the α-position,
manifested in hydrogen evolution.23-26 Alane, on the other hand, does not suffer this
drawback, making it far more appropriate for reducing nitriles to the corresponding
amine.23,25 Treatment of the nitrile (80) with alane offered clean reduction to the amine,
again converted into a carbamate; unfortunately, a reductive cleavage of the acetonide
was also observed, giving the isopropyl ether (125) with the regiochemistry confirmed by
acetylation [(126)].
(125) (126)
(a)
(b)
(80)
O
OO
OBn
NC
(80)
O
OO
OBn
NC
O
HOO
OBn
(81)
(c)
BocHN
O
OO
OBn
BocHN
O
OAcO
OBn
BocHN
a) i) LiAlH4, Et2O; ii) (Boc)2O, CHCl3, 56%; b) i) AlH3, THF;
ii) (Boc)2O, 58%; c) Ac2O, pyridine, 91%.
Reductive cleavage of acetonides using a variety of aluminium and boron agents has been
well reported.27,28 Takana took advantage of this reductive cleavage using trimethylalane
to produce the 3-tert-alkoxy-1,2-glycol (128) and provided a rational justification for this
conversion.27
37
R
O
O
OHR2
R1
R
O
O
OR2
R1
AlMe2
R
O
O
O
R2
R1
AlMe2
R
O
OH
OH
R2
R1MeMe3Al
− CH4
i) Me3Al
ii) H2O
(127) (128)
Extension of this rationale to the reduction of the acetonide (80) yields the intermediate
(131), with subsequent reduction and hydrolysis affording the isopropyl ether (125).
(80)
O
OO
OBn
C
N
(125)
O OBn
O
O
NH2Al
O OBn
O
ONH2Al
H2Al
O OBn
O
ONH2Al
AlH2
(129) (130)
(131)
AlH3
O
HOO
OBn
BocHN
The retention of the acetonide during the reduction of the nitrile was solely to facilitate
the isolation of the amine prior to protection with di-tert-butyl dicarbonate. It was
proposed that circumventing the isolation process prior to protection of the amine would
render the acetonide superfluous. Thus, removal of the isopropylidene group was
achieved under standard conditions to yield the diol (101). Subsequent reduction of the
diol (101) followed by in-situ treatment with di-tert-butyl dicarbonate afforded the
carbamate (82) in excellent yield (85%).
38
(82)(101)(80)
O
OO
OBn
NC
(a) (b)O
HOOH
OBn
NC
O
HOOH
OBn
BocHN
a) CSA, MeOH, 90%; b) i) AlH3, THF; ii) (Boc)2O, THF, H2O, 85%.
The conversion of the diol (82) into isofagomine (13) proceeded as previously reported.11
NHHOHO
OH
(13)(82)
(a)O
HOOH
OBn
BocHN
a.) i) CF3COOH, ii) MeOH, Amberlite IRA 400 (OH−),
ii) Pd/C, H2, MeOH, 89%.
The two major improvements in this sequence (imidazylate formation and nitrile
reduction) now provide an excellent preparation of isofagomine; we routinely make 0.4 g
of isofagomine from 2.3 g of L-xylose in the space of ten days.
Noeuromycin
Hydrogenolysis of the diol (82) in ethanol as reported by Bols never provided a
satisfactory result, with small amounts of the ethyl glycoside always present.10,29
Substitution to a more appropriate tetrahydrofuran and water mixture allowed the
isolation of the hemiacetal (99) in a very pure form, allowing thorough characterization.
(99)
(a)
(82)
O
HOOH
OBn
BocHN
O
HOOH
OH
BocHN
a) Pd/C, H2, THF, H2O, 83%.
39
With the hemiacetal (99) in hand, treatment with 1M hydrochloric acid afforded
noeuromycin (14) in quantitative yield and in high purity, as shown by high resolution
n.m.r. spectroscopy.
NHOH
HOHO
OH
(14)(99)
(a)O
HOOH
OH
BocHN
a) 1 M HCl.
ppm2.002.503.003.504.004.505.00
NHOH
HOHO
OH
(14)
H2β-(14)
H2α-(14)
H5αH5β
H6α-(14)
-(14)H1-(132)H1-(133)
1H n.m.r. (600 MHz) spectrum of noeuromycin (14) hydrochloride in D2O
Although (14) bears little resemblance to a sugar, the ‘α/β’ nomenclature is retained and
refers to the hydroxyl at C2 being ‘down’ (α) or ‘up’ (β).
40
Bols suggested that the piperidine form of noeuromycin (14) existed in equilibrium with
the pyranose forms (132) and (133).29 The significantly higher field n.m.r. spectra of
noeuromycin obtained here confirmed the presence of these tautomeric forms indicated
by H1 (δ 5.06 and δ 4.47).
(14)
(132) (133)
NHOH
HOHO
OH
O
HOOH
OH
H2N
O
HOOH
OH
H2N
A collaboration with Gideon Davies and Tracey Gloster of the University of York led to
inhibition data and X-ray crystallographic determination of noeuromycin in complex with
a family 1, retaining β-glucosidase from Thermotoga maritima (EC 3.2.1.21).30 At pH
5.8, the pH for optimum catalysis, noeuromycin was shown to have Ki 88 nM; the
optimum inhibition was measured at pH 6.8 (Ki 37 nM), consistent with that reported by
Bols.29,30 X-Ray crystallographic determination was achieved only to a resolution of 1.8
Å so that information regarding the protonation states could not be determined; however,
it is anticipated that the nitrogen atom would be doubly protonated.
41
Glu166acid/base
Glu351nucleophile
Three-dimensional structure of the β-glucosidase from Thermotoga maritima (TmGH1)
and its complex with noeuromycin (14).
Isofagomine Lactam
The synthesis here takes advantage of the spontaneous, acid-induced conversion of the
purported lactone (100), derived from the carbamate (82), into the lactam (24), as
observed by Bols.31
NHHOHO
OH
O(24)(100)(82)
O
HOOH
OBn
BocHN
O
OHOH
O
BocHN
42
Access to the lactone (134) should be possible from the protected hemiacetal (135);
access to this hemiacetal might be possible through the hydrogenolysis of the glycoside
(136), derived from the diol (82), already at hand.
NHHOHO
OH
O(24)
(82)
(134) (135)
(136)
O
HOOH
OBn
BocHN
O
OPOP
OBn
BocHN
O
OPOP
OH
BocHN
O
OPOP
O
BocHN
Retrosynthetic analysis of isofagomine lactam.
Initial attempts to prepare isofagomine lactam started with the diol (82) and used tert-
butyldimethylsilyl protecting groups. Thus, treatment of the diol (82) with BMSCl and
imidazole gave the disilyl ether (137) in good yield. Unfortunately, owing to line
broadening effects in the n.m.r. spectra, it was not possible to determine unequivocally
the conformation of most of the compounds in this section.18
(82) (137)
(a)O
HOOH
OBn
BocHN
O
OBMSOBMS
OBn
BocHN
a) BMSCl, ImH, DMF, 85%.
Removal of the benzyl glycoside gave the hemiacetal (138) in good yield. Attention now
turned to the oxidation of the hemiacetal to obtain the lactone (139). Benhaddou and co-
43
workers reported a mild oxidising agent in the combination of TPAP and N-
methylmorpholine N-oxide (NMO) that has been shown to be effective in the conversion
of hemiacetals into lactones.32 Oxidation of the hemiacetal (138) provided the lactone
(139) in excellent yield. However, it was subsequently not possible to remove the
hindered silyl ethers from (139).
(138)
NHHOHO
OH
O
(139)
(24)
(a) (b)
(137)
O
OBMSOBMS
OBn
BocHN
O
OBMSOBMS
OH
BocHN
O
OBMSOBMS
O
BocHN
a) H2, Pd/C, THF, H2O; b) Pr4NRuO4,
N-methylmorpholine N-oxide, CH2Cl2, 86% (steps a and b).
Whilst silyl ethers seemed to be most appropriate for the current synthesis, a far more
labile ether was required to ensure that it could be effectively removed. Trimethylsilyl
ethers have proved to be highly labile to acid, to the extent that they have not seen much
synthetic use, mainly relegated as a tool in derivatisation, increasing the volatility for gas
chromatography and mass spectrometry. Conversion of the diol (82) into the disilyl ether
(140) proceeded in quantitative yield and without the need for chromatography.
44
(140)
(a)
(82)
O
HOOH
OBn
BocHN
O
OSiMe3
OSiMe3
OBn
BocHN
a) Me3SiCN, DMF.
Hydrogenolysis of the benzyl glycoside under anhydrous conditions prevented hydrolysis
of the silyl ethers and provided the hemiacetal (141). Oxidation using TPAP and NMO
gave the lactone (142) in a moderate yield, mainly owing to some silyl ether cleavage.
Acid treatment of the lactone (142) resulted in both protecting group removal and
formation of the lactam (24).
(a)
(142)
NHHOHO
OH
O
(24)
(c)
(141)
(b)
(140)
O
OSiMe3
OSiMe3
OBn
BocHN
O
OSiMe3
OSiMe3
OH
BocHN
O
OSiMe3
OSiMe3
O
BocHN
a) H2, Pd/C, THF; b) Pr4NRuO4,
N-methylmorpholine N-oxide, CH2Cl2, 65% (steps a and b);
c) i) CF3COOH, H2O; ii) MeOH, Amberlite IRA 400 (OH−), 80%.
Azafagomine
Retrosynthetic analysis suggested that access to azafagomine (23) should be possible
through the formation of the protected pyridazine (143). Preparation of the pyridazine
may occur through a reductive amination of a suitably protected hydrazide (144).
45
Protection of the hydrazide prior to the reductive amination was a necessity to prevent the
more favoured five-membered ring formation.21 Access to the hydrazide (144) should be
possible through a displacement of the imidazylate (22).
(22)
OImSO2O
OO
OBn
NH
NHHOHO
OH
(23) (144)
NBoc
NHPOPO
OH
(143)
O
Boc(H2N)NOP
OPOBn
Retrosynthetic analysis of azafagomine.
The Boc-protected hydrazine (145) was chosen owing to the ease of removal of the Boc
group and resistance to cleavage in the subsequent synthetic sequence. Treatment of tert-
butyl carbazate (145) with LiHMDS in THF and then subsequent addition of the
imidazylate (22) resulted in the formation of the hydrazide (146). Confirmation of the
structure of (146) was aided by proton-coupled 15N n.m.r. spectroscopy, showing a
singlet (N) and a triplet (NH2).
(22) (146)
(a)OImSO2O
OO
OBn O
OO
OBn
Boc(NH2)N
H2N NH Boc
(145)
a) (Me3Si)2NLi, THF, 69%.
46
The selectivity of the above transformation presumably results from deprotonation of the
more acidic proton, with the resulting nitrogen anion directly displacing the imidazylate
(22) to give the hydrazide (146).
H2N NH Boc H2N N Boc
Li
LiHMDS
(22)
OImSO2O
OO
OBn
(146)
O
OO
OBn
Boc(NH2)N
Reductive amination of (146) proceeded as expected, forming the hydrazide (147) in
excellent yield; subsequent deprotection yielded azafagomine (23).
NBoc
NHOO
OH
(147)
NH
NHHOHO
OH
(23)
(b)
(146)
O
OO
OBn
Boc(NH2)N
(a)
a) Pd/C, H2, THF, 88%; b) CF3COOH, 99%.
Inhibition and crystallographic data were again provided by Gideon Davies and Tracey
Gloster of the University of York. Inhibition by azafagomine was measured with the
same family 1, retaining β-glucosidase from Thermotoga maritima (TmGH1) (EC
3.2.1.21).30 At pH 5.8, the pH for optimum catalysis, azafagomine was an excellent
inhibitor (Ki 66 nM). X-Ray crystallographic determination was achieved only to a
resolution of 1.95 Å so that information regarding the protonation states again could not
be determined.
47
Glu166acid/base
Glu351nucleophile
Three-dimensional structure of the β-glucosidase from Thermotoga maritima (TmGH1)
in complex with azafagomine (23).
Azanoeuromycin
With several known derivatives prepared from the key imidazylate (22) it was decided to
prepare the novel azanoeuromycin (27) as a possible glycosidase inhibitor. A suitable
pathway would share strong similarity to the sequence used to prepare azafagomine.
Preparation of the hemiacetal (148) should be possible through a fully protected
hydrazide (149), thus preventing reductive amination from occurring. Preparation of the
hydrazide (149) should be possible from the central imidazylate.
48
(22)
OImSO2O
OO
OBn
NH
NHHOHO
OH
(27) (148)
OH
(149)
O
P(PN)NOP
OPOBnO
P(PN)NOH
OH
OH
Retrosynthetic analysis of azanoeuromycin.
Initial thoughts suggested that the easily prepared trisubstituted hydrazine (150) would
provide the best approach; subsequently, treatment of the imidazylate with the lithium
salt of the trisubstituted hydrazide (150) gave (151) in good yield.
(22)
OImSO2O
OO
OBn
(151)
O
OO
OBn
Boc[N(Boc)2]N
(a)Boc2N NH Boc
(150)
a) LiHMDS, THF, 89%.
Removal of the acetonide from (151) was achieved using the relatively mild acid,
pyridinium tosylate, to yield the diol (152) in quite good yield; great care was required in
order to prevent the undesired removal of one of the Boc groups. The 1H and 13C n.m.r.
spectra of the diol (152) in CDCl3 were complicated by the presence of rotamers. To
prove conclusively the presence of rotamers, a variable temperature experiment was
performed in D6-DMSO, with a coalescence of some signals at 348K, confirming the
presence of a single compound.
49
(152)
(a)
(151)
O
OO
OBn
Boc[N(Boc)2]N
O
OHOH
OBn
Boc[N(Boc)2]N
a) PPTS, MeOH, 89%.
Direct hydrogenolysis of (152) proved troublesome, giving a complex mixture of
products, presumably owing to the lability of (at least) one of the Boc groups.
Magnesium perchlorate has been shown to be a very mild Lewis acid capable of
removing one Boc group from a disubstituted amine.33 Treatment of the diol (152) with
magnesium perchlorate resulted in the smooth removal of one of the Boc groups, giving,
presumably, the diol (153), in excellent yield. Unfortunately, the structure of this
compound was impossible to confirm using 1H n.m.r. spectroscopy owing to significant
line broadening.
(152) (153)
(a)O
OHOH
OBn
Boc[N(Boc)2]N
O
OHOH
OBn
Boc[NHBoc]N
a) Mg(ClO4)2, CH3CN, 85%.
Hydrogenolysis of (153) proved to be temperamental, requiring a trace of water for
success, to give presumably the hemiacetal (154) in moderate yield.
(154)
(a)
(153)
O
OHOH
OBn
Boc[NHBoc]N
O
OHOH
OH
Boc[NHBoc]N
a) H2, Pd/C, THF/H2O, 75%.
50
Treatment of the hemiacetal (154) with 1 M hydrochloric acid provided a mixture of
three compounds, the two ‘anomers’ of azanoeuromycin (27) and a compound consistent
with the hydrazone (25), as reported by Bols.22 Azanoeuromycin is characterized by
having two doublets, δH 4.50 (J 8.5 Hz) and δH 4.88 (J 3.3 Hz), attributable to H3; the
hydrazone is characterised by a downfield doublet, δH 6.85 (J 1.2 Hz). Interestingly, there
was no hint of pyranose forms observed as in noeuromycin.
NH
NHOHO
OHNH
NHOH
HOHO
OH
(27) (25)
(a) 3
(154)
O
OHOH
OH
Boc[NHBoc]N
a) 1 M HCl.
In our recent paper it was suggested that the name azadeoxynojirimycin would be more
appropriate for (27) given that (23) is referred to as azafagomine and not azaisofagomine;
however such a name is associated with a hydroxyl group of fixed configuration at ‘C2’,
which is not the case with (27).34 Thus we decided to name (27) azanoeuromycin.
51
NH
NHOHO
OHNH
NHOH
HOHO
OH
(27) (25)
ppm4.05.06.07.0
H3
H3β
H3α
H4
H6α
H5H6β
1H n.m.r. spectrum of azanoeuromycin (27) and the hydrazone (25) (as the
hydrochlorides).
One would assume that azanoeuromycin exists in aqueous acid solution in equilibrium
with the hydrazone. In order to confirm this proposition, the hemiacetal (154) was treated
with neat CF3COOH in an attempt to force the equilibrium in favour of the hydrazone
(25). This proved to be successful, with the 1H n.m.r. spectrum in dry D6-DMSO
indicating the hydrazone as the predominant product.
NH
NHOHO
OH
(25)
(a)
(154)
O
OHOH
OH
Boc[NHBoc]N
a) CF3COOH.
52
NH
NHOHO
OH
(25)
ppm2.503.003.504.004.505.005.506.006.50
H3
H4 CH2OCH2O
H5
H6
1H n.m.r. spectrum of the hydrazone (25) (as the hydrochloride) in dry D6-DMSO.
Treatment of the hydrazone (25) with dilute mineral acid saw the equilibrium re-
established.
NH
NHOHO
OH
(25)
(a) NH
NHOHO
OHNH
NHOH
HOHO
OH
(27) (25)
a) 1 M HCl.
A ‘Guanidine’ Derivative of Isofagomine
A colleague, Professor Ian Jenkins (Griffith University), suggested that the guanidine
derivative (26) of isofagomine could provide interesting biological activity, based on the
success of Relenza® (3).35
53
NHOHO
OH
NH2
NH
(26)
O CO2H
NHAcHN
HNNH2
HO
HO OH
(3)
Retrosynthetic analysis suggested that the ‘guanidine’ derivative could be prepared
simply via the protected derivative (155), with the guanidine moiety introduced onto the
protected piperidine (156) obtained from isofagomine (13).
NPOPO
OP
NHP
NP
NHOHO
OH
NH2
NH(26)
NHHOHO
OH
(13)
NHPOPO
OP
(155)
(156)
The preparation of (26) commenced from isofagomine, with protection of the amine
using a Boc group, followed by acetylation to give (157).
NHHOHO
OH
NBocAcOAcO
OAc
(13) (157)
(a)
a) i) NaHCO3, (Boc)2O, Me2CO, H2O; ii) Ac2O, pyridine, CHCl3, 83%.
54
Treatment of the Boc protected piperidine (157) with anhydrous trifluoroacetic acid
smoothly yielded the amine (158); a subsequent treatment with the guanidinylating
reagent, N,N′-di-Boc-N′′-triflylguanidine36 yielded, presumably, the protected guanidine
derivative (159) in excellent yield. Confirmation of the structure of (159) at this stage
proved to be quite difficult owing to line broadening in both the 1H and 13C n.m.r.
spectra.
NBocAcOAcO
OAc
NH.CF3COOHAcOAcO
OAc
(157) (158)
(b)NAcO
AcO
OAc
NHBoc
NBoc(159)
(a)
a) CF3COOH; b) (BocNH)2C=NTf, EtPri2N, CH2Cl2, 95%.
Subsequent removal of the acetyl groups from (159) under acidic conditions, followed by
removal of the Boc groups, gave the ‘guanidine’ derivative (26) of isofagomine.
Preliminary results suggest that this ‘guanidine’ isofagomine is not an effective
glycosidase inhibitor.30
NAcOAcO
OAc
NHBoc
NBoc
NHOHO
OH
NH2
NH(159) (26)
(a)
a) i) HCl, MeOH; ii) CF3COOH, 95%.
55
Experimental
General
Melting points were determined on a Reichert hot stage apparatus. Optical rotations were
performed with a Perkin-Elmer 141 polarimeter in a microcell (1 mL, 10 cm path length)
in CHCl3 at room temperature, unless stated otherwise.
1H- and 13C-nuclear magnetic resonance (n.m.r.) spectra were obtained on a Bruker
AM300, ARX500 or AV600 spectrometer. Unless stated otherwise, deuteriochloroform
(CDCl3) was used as the solvent with CHCl3 (δH 7.26) or CDCl3 (δC 77.16) as the internal
reference. N.m.r. spectra run in D2O were calibrated with 2,2-dimethylsilapentane-5-
sulfonic acid (δH 0.00; δC 0.00).
Mass spectra were recorded with a VG-Autospec spectrometer using the fast-ion
bombardment technique (f.a.b.) and 3-nitrobenzyl alcohol as a matrix, unless stated
otherwise. Microanalyses were performed by M-H-W laboratories, Phoenix, Arizona or
the Microanalytical Unit, Australian National University, Canberra, ACT. Flash
chromatography was performed on BDH silica gel or Geduran silica gel 60 with the
specified solvents. Thin layer chromatography (t.l.c.) was performed on Merck silica gel
60 F254 aluminium-backed plates that were stained by heating (>200º) with either 5%
sulfuric acid in EtOH or 10% ammonium molybdate in 10% sulfuric acid.
All solvents were distilled prior to use with the exception of DMF, MeCN and PriOH.
56
Dry THF was prepared by distillation over potassium; dry Et2O was prepared by
distillation over Na wire; dry CH2Cl2 was prepared by distillation over calcium hydride.
HCl in MeOH was generated by the addition of acetyl chloride to anhydrous MeOH.
57
(22)
OImSO2O
OO
OBn
Benzyl 4-O-(Imidazolyl-1-sulfonyl)-2,3-O-isopropylidene-β-L-xyloside (22)
Lithium bis(trimethylsilyl)amide in THF (1 M, 30 mL, 30 mmol) was added dropwise to
the alcohol (79)37 (7.22 g, 26.1 mmol) in dry THF (150 mL, 0ºC) and the solution stirred
(30 min, 0ºC). The solution was cooled (–30°C) and freshly prepared N,N΄-
sulfuryldiimidazole38 (5.94 g, 30.0 mmol) was added portionwise (30 min); the solution
was then stirred (30 min, rt). Methanol (3 mL) was added and, after 30 min, the solution
was poured into EtOAc, washed with saturated NaHCO3 and dried. Flash
chromatography (EtOAc/petrol, 1:4 containing 0.5% Et3N) gave the imidazylate (22)
(9.53 g, 90%) as a colourless solid, m.p. 89-90°C (lit.11 90-92°C), [α]D +85.5° (lit.11
+86.6°). The 1H (300 MHz) and 13C (75.5 MHz) n.m.r. spectral data were in good
agreement with those reported.11
(80)
O
OO
OBn
CN
Benzyl 4-C-Cyano-4-deoxy-2,3-O-isopropylidene-α-D-arabinoside (80)
The imidazylate (22) (9.53 g) was treated according to the procedure of Best et al.37 to
give the nitrile (80) (5.14g, 80%) as pale yellow needles, m.p. 85-88°C (lit.11 89-90ºC),
[α]D –23.5° (lit.11–21.2°). The 1H (300 MHz) and 13C (75.5 MHz) n.m.r. spectral data
were in good agreement with those reported.11
58
(125)
O
HOO
OBn
BocHN(126)
O
OAcO
OBn
BocHN
Benzyl 4-C-[(tert-Butoxycarbonyl)amino]methyl-4-deoxy-2-O-isopropyl-α-D-arabinoside
(125) and Benzyl 3-O-Acetyl-4-C-[(tert-butoxycarbonyl)amino]methyl-4-deoxy-2-O-
isopropyl-α-D-arabinoside (126)
Freshly prepared AlH3 in THF25 (1.1 M, 3.6 mL, 4.0 mmol) was added to the nitrile (80)
(610 mg, 2.2 mmol) in dry THF (30 mL, 0°C) and the solution stirred (2 h). The mixture
was quenched by the addition of H2O (2.0 mL) and NaOH (1.0 M, 2.5 mL), followed by
treatment with (Boc)2O (640 mg, 2.8 mmol) (5 h, rt). The mixture was filtered, the filtrate
concentrated and the residue subjected to flash chromatography (EtOAc/petrol, 3:7) to
give the alcohol (125) (500 mg, 58%) as a colourless oil, [α]D +68.7º. δH (300 MHz) 1.13,
1.15 (2×d, 6H, J 5.9, J 5.9, CH3CH), 1.44 (s, 9H, CH3C), 2.21-2.31 (br m, H4), 3.18-3.25
(br s, CH2NH), 3.32 (br d, J2,3 8.3, H2), 3.44 (br s, OH), 3.57 (dd, J5,5 11.5, J4,5 3.9, H5),
3.70-3.80 (m, 3H, H3, H5, CH3CH), 4.52, 4.80 (AB, J 11.8, PhCH2), 4.71 (br s, H1), 4.90
(br m, NH), 7.30-7.40 (m, Ph). δC (75.5 MHz) 22.46, 22.57 (2C, CH3CH), 28.33 (CH3C),
36.20 (C4), 39.54 (CH2NH), 58.39 (PhCH2), 69.55 (C5), 68.62, 73.32, 71.83 (C2, C3,
CH3CH), 79.20 (CH3C), 98.98 (C1), 136.79-127.89 (Ph), 156.12 (C=O). m/z (FAB)
396.2379 (C21H34NO6 [M]+• requires 396.2386).
A small sample of (125) (10 mg) was dissolved in pyridine (1 mL) and Ac2O (0.5 mL)
and stirred (2 h). The reaction was quenched with MeOH (2 mL), the solution
concentrated and subjected to flash chromatography (EtOAc/petrol, 3:7) to give (126) (10
mg, 90%) as a colourless oil. δH (300 MHz) 1.13, 1.15 (2×d, 6H, J 5.9 Hz, CH3CH), 1.44
(s, 9H, CH3C), 2.02 (s, CH3CO), 2.35 (m, H4), 3.25 (m, CH2NH), 3.45-3.55 (m, 2H, H2,
59
H5), 3.70-3.90 (m, 2H, H5, CH3CH), 4.62 (m, H1), 4.48, 4.81 (AB, J 12.1, PhCH2), 4.85
(m, H3), 7.25-7.35 (m, Ph).
(101)
O
HOOH
OBn
NC
Benzyl 4-C-Cyano-4-deoxy-α-D-arabinoside (101)
The nitrile (80) (1.46 g) in MeOH (30 mL) was treated with CSA (10 mg) and stirred (2
h), followed by the addition of Et3N (1 mL). Evaporation and flash chromatography
(EtOAc/petrol, 1:1) of the residue gave (101) as a colourless solid (1.18 g, 90%), m.p.
134-136°C (lit.18 138-139ºC). The 1H (300 MHz) and 13C (75.5 MHz) n.m.r. spectral data
were in good agreement with those reported.18
(82)
O
HOOH
OBn
BocHN
Benzyl 4-C-[(tert-Butoxycarbonyl)amino]methyl-4-deoxy-α-D-arabinoside (82)
Freshly prepared AlH3 in THF25 (0.66 M, 5.6 mL, 3.6 mmol) was added to the nitrile
(101) (297 mg, 1.19 mmol) in dry THF (15 mL, 0ºC) and the solution stirred (2 h).
Further portions of AlH3 (0.66 M, 5.6 mL, 3.6 mmol) were added (2 h and 4 h) and the
solution stirred overnight. The mixture was quenched by the addition of H2O (5.0 mL)
and NaOH (5.0 mL, 1.0 M), followed by treatment with (Boc)2O (523 mg, 2.4 mmol) (5
h, rt). The mixture was then filtered, diluted with EtOAc (200 mL), dried and the filtrate
concentrated to give a colourless oil, which was subjected to flash chromatography
(EtOAc/petrol, 1:1) to give the carbamate (82) (350 mg, 85%) as a colourless solid, m.p.
157-159ºC (lit.11 158-160ºC), [α]D +75.9° (lit.11 +77.8°). The 1H (300 MHz) and 13C (75.5
MHz) n.m.r. spectral data were in good agreement with those reported. 11
60
NH2ClHOHO
OH
(3S,4R,5R)-5-(Hydroxymethyl)piperidine-3,4-diol (isofagomine) (13) hydrochloride
The diol (82) (30 mg) was treated according to the procedure of Best et al.37 to give
isofagomine (13) hydrochloride (14 mg, 89%), [α]D +15.5° (lit.11 +16.5°). The 1H (600
MHz) and 13C (150.9 MHz) n.m.r. spectral data were in good agreement with those
reported.2
(99)
O
HOOH
OH
BocHN
4-C-[(tert-Butoxycarbonyl)amino]methyl-4-deoxy-D-arabinose (99)
A stirred solution of the carbamate (82) (120 mg, 0.340 mmol) in THF/H2O (1:1, 15 mL)
was treated with Pd/C (10%, 10 mg) and H2 (1 atm, 12 h). The mixture was filtered,
concentrated and subjected to flash chromatography (THF/petrol, 7:3) to give the
hemiacetal (99) (74 mg, 83%) as a colourless oil. δH (500 MHz) 1.45 (s, 9H, CH3C),
1.94-2.04 (m, H4α, H4β), 2.95-3.10 (m, CH2N), 3.21-3.28, 3.42-3.48, 3.52-3.56, 3.60-
3.64, 3.69-3.76, 3.86-3.91, (m, H2, H3, H5) 4.45 (d, J1,2 6.9, H1β), 4.96 (d, J1,2 1.9, H1α).
δC (125.8 MHz) 27.55 (CH3C), 36.76, 36.03 (CH2NH), 40.30, 37.40 (C4), 61.99, 61.25
(C5), 71.60, 71.34, 69.16, 61.25 (C2, C3), 80.99 (CH3C), 96.73, 92.17 (C1), 158.20
(C=O). m/z (FAB) 264.1445 (C11H22NO6 [M+H]+• requires 264.1447).
61
NH2ClOH
HOHO
OH
(14)
(3S,4R,5R)-5-(Hydroxymethyl)piperidine-2,3,4-triol (noeuromycin) (14) hydrochloride
The triol (99) (14 mg) was treated with hydrochloric acid (1 M, 1 mL) and allowed to
stand (10 min). The solvent was removed to give noeuromycin (14) (10.5 mg) as a
colourless oil. δH (600 MHz, D2O) 1.90-2.02 (br m, H5α, H5β), 2.98 (dd, 1H, J6,6 13.0,
J5,6 13.1, H6α), 3.25-3.33 (m, H6β), 3.46 (dd, 1H, J5,6 4.6, H6α), 3.53-3.57 (m, H3α,
H4α), 3.63-3.66 (m, H3β), 3.70-3.85 (m, 5H, CH2O, H4β), 4.61 (d, J2,3 7.5, H2α), 5.26
(d, J2,3 2.7 Hz, H2β). δC (150.9 MHz) 41.26 (C6β), 43.47 (C5α), 43.74 (C5β), 44.15
(C6α), 61.66, 61.79 (CH2O), 69.54 (C4β), 72.53 (C4α), 74.60 (C3β), 76.99 (C3α), 80.79
(C2β), 83.85 (C2α). m/z (FAB) 164.0908 (C6H14NO4 [M+H]+ requires 164.0922).
(137)
O
OBMSOBMS
OBn
BocHN
Benzyl 4-C-[(tert-Butoxycarbonyl)amino]methyl-2,3-di-O-(tert-butyldimethylsilyl)-4-
deoxy-α-D-arabinoside (137)
The carbamate (82) (145 mg, 0.411 mmol) in dry DMF (10 mL) was treated with
imidazole (280 mg, 4.11 mmol) and BMSCl (308 mg, 2.05 mmol) and stirred (4 d, 75ºC).
The solution was concentrated and subjected to flash chromatography (EtOAc/petrol,
3:7) to give the disilyl ether (137) as a colourless oil (204mg, 85%), [α]D +69.2º
(CH2Cl2). δH (600 MHz) -0.01, 0.029, 0.05, 0.06 (4×s, 12H, CH3Si), 0.84, 0.85 (2×s,
18H, CH3CSi), 1.44 (s, 9H, CH3C), 2.21-2.25 (m, H4), 3.04-3.08 (m, 2H), 3.38-3.42 (m,
1H), 3.58-3.60 (m, 1H), 3.69-3.72 (br s, 1H), 3.92-3.97 (m, 1H), 4.43, 4.71 (AB, J 11.9
Hz, PhCH2), 4.54 (br s, 1H), 4.58-4.64 (m, 1H), 7.28-7.35 (m, Ph). δC (150.9 MHz) –4.83
62
, –4.66, –4.48, –3.82 (4C, CH3Si), 18.12, 18.20 (2C, CH3CSi), 25.81, 25.92 (CH3CSi),
28.55 (CH3CO), 36.73 (C4), 40.24 (CH2NH), 58.11, 71.28, 70.43 (C2, C3, C5), 69.35
(PhCH2), 79.24 (CH3CO), 99.95 (C1), 127.57-128.34 (Ph), 156.07 (C=O). m/z (FAB)
582.3620 (C30H56NO6Si2 [M+H]+ requires 582.3646).
(139)
O
OBMSOBMS
O
BocHN
4-C-[(tert-Butoxycarbonyl)amino]methyl-2,3-di-O-(tert-butyldimethylsilyl)-4-deoxy-D-
arabinono-1,5-lactone (139)
The disilyl ether (137) (80 mg, 0.138 mmol) in THF/H2O (1:1, 15 mL) was treated with
Pd/C (10%, 15 mg) and H2 and stirred (1 d). The suspension was filtered through Celite
and freeze-dried to give a colourless oil. This oil in dry CH2Cl2 was treated with 4Å
sieves (100 mg) and N-methylmorpholine N-oxide (22 mg, 0.19 mmol) and stirred (1 h).
The mixture was then treated with Pr4NRuO4 (4.7 mg, 0.013 mmol), stirred (4 h, 25ºC),
filtered through Celite and concentrated. Flash chromatography (EtOAc/petrol, 3:7) gave
the lactone (139) as a colourless solid (55 mg, 86%), m.p. 96-97ºC, [α]D –22.9º. υmax
(film) 1747, 1716 (cm-1). δH (600 MHz) 0.11, 0.11, 0.13, 0.15 (4×s, 12H, CH3Si), 0.89 (s,
18H, CH3CSi), 1.44 (s, 9H, CH3C), 2.57-2.66 (m, 1H), 3.09-3.15 (m, 2H), 3.85-3.88 (br
s, 1H), 3.98-4.01 (m, 1H), 4.25-4.33 (m, 2H), 4.57-4.66 (m, 1H). δC (150.9 MHz) –5.16,
–4.78, –4.75, –4.28 (4C, CH3Si), 18.06, 18.17 (2C, CH3CSi), 25.77, 25.79 (CH3CSi),
28.49 (CH3CO), 34.08 (CH2NH), 39.27 (C4), 68.13 (C5), 71.09, 71.63 (C2, C3), 79.86
(CH3CO), 156.07 (C=O), 169.55 (C1). m/z (FAB) 490.2989 (C23H48NO6Si2 [M+H]+
requires 490.3020).
63
(140)
O
OSiMe3
OSiMe3
OBn
BocHN
Benzyl 4-C-[(tert-Butoxycarbonyl)amino]methyl-4-deoxy-2,3-bis-O-trimethylsilyl-α-D-
arabinoside (140)
The carbamate (82) (197 mg, 0.558 mmol) in dry DMF (2 mL) was treated with
Me3SiCN (138 mg, 1.40 mmol) and stirred (40 min). The solution was concentrated to
give the disilyl ether (140) as a colourless oil (277 mg, 100%), [α]D –3.85º. δH (300 MHz)
0.08, 0.14 (2×s, 18H, CH3Si), 1.43 (s, 9H, CH3C), 2.01-2.10 (m, H4), 3.20-3.4 (m,
CH2NH), 3.43 (dd, J5,5 11.9, J4,5 2.6, H5), 3.51 (dd, J2,3 7.0, J1,2 5.7, H2), 3.70 (dd, J3,4
4.7, H3), 3.89 (dd, J4,5 4.7, H5), 4.30 (d, H1), 4.51, 4.83 (AB, J 11.9 Hz, PhCH2), 4.94 (br
s, NH), 7.25-7.37 (m, Ph). δC (75.5 MHz) 0.40, 0.63 (CH3Si), 28.56 (CH3C), 39.62
(CH2NH), 40.22 (C4), 62.41 (PhCH2), 70.31 (C5), 72.28, 73.81 (C2, C3), 79.11
(CH3CO), 102.58 (C1), 128.31-137.89 (Ph), 156.00 (C=O). m/z (FAB) 498.2744
(C24H44NO6Si2 [M+H]+ requires 498.2707).
(142)
O
OSiMe3
OSiMe3
O
BocHN
4-C-[(tert-Butoxycarbonyl)amino]methyl-4-deoxy-2,3-bis-O-trimethylsilyl-D-arabinono-
1,5-lactone (142)
The disilyl ether (140) (50 mg) in dry THF (20 mL) was treated with Pd/C (10%, 10 mg)
and H2 (1 atm, 4 d, 40ºC). The solution was then filtered and concentrated to give a
colourless oil (40 mg). The oil was dissolved in dry CH2Cl2 (10 mL) and stirred with 4Å
sieves (100 mg). N-Methylmorpholine N-oxide (22 mg, 0.186 mmol) in CH2Cl2 (3 mL)
was also stirred with 4Å sieves (100 mg). The two solutions were combined and then
64
treated with Pr4NRuO4 (4 mg, 0.012 mmol), stirred (1 h) and then filtered, concentrated
and subjected to flash chromatography (EtOAc/petrol, 1:4 containing 2% Et3N) to give
(142) essentially pure as a colourless oil (26 mg, 65%). A small sample was further
purified using flash chromatography to give (142), [α]D –14.5º (CH2Cl2), υmax (film)
1740, 1717 (cm-1). δH (500 MHz) 0.17, 0.19 (2×s, 18H, CH3Si), 1.46 (s, 9H, CH3C),
2.51-2.57 (m, H4), 3.12-3.27 (m, CH2NH), 3.91-3.94, 4.03-4.05, 4.25-4.34 (3×m, 4H,
H2, H3, H5), 4.71 (br s, NH). δC (125.8 MHz) 0.01 (CH3Si), 28.67 (CH3C), 35.62 (C4),
39.04 (CH2NH), 67.69 (C5), 72.14, 72.26 (C2, C3), 79.83 (CH3C), 156.01 (C=O), 170.36
(C1). m/z (FAB) 406.2100 (C30H56NO6Si2 [M+H]+ requires 406.2081).
NHHOHO
OH
O(24)
(3S,4R,5R)-3,4-Dihydroxy-5-hydroxymethylpiperidin-2-one (isofagomine lactam) (24)
The disilyl ether (142) (18 mg) was treated with CF3COOH/H2O (1:1, 3 mL) and stirred
(1 h). The solution was then concentrated and the residue taken up in MeOH and treated
with resin (Amberlite IRA 400, OH–) until neutral. The solution was filtered,
concentrated and subjected to flash chromatography (EtOAc/MeOH/H2O, 17:2:1) to give
isofagomine lactam (24) as an amorophous solid (5.5 mg, 80%), [α]D +9.5° (MeOH; lit.11
+11.0°). The 1H (300 MHz) and 13C (75.5 MHz) n.m.r. spectral data were in good
agreement with those reported.11
65
(146)
O
OO
OBn
Boc(NH2)N
Benzyl 4-[N-Amino-N-(tert-butoxycarbonyl)amino]-4-deoxy-2,3-O-isopropylidene-α-D-
arabinoside (146)
Lithium bis(trimethylsilyl)amide in THF (1 M, 6.10 mL, 6.10 mmol) was added dropwise
to tert-butyl carbazate (805 mg, 6.10 mmol) in dry THF (10 mL, –78ºC) and the solution
stirred (30 min, –30ºC). The imidazylate (22) (490 mg, 1.20 mmol) was added and the
solution heated to reflux (5 h). The solution was concentrated and the residue dissolved in
ethyl acetate, washed with water and dried. Flash chromatography (EtOAc/petrol, 3:7)
gave the carbazate (146) (290 mg, 69%) as a colourless oil, [α]D +59.1°. δH (600 MHz)
1.44 [s, (CH3)3C], 1.47 [s, (CH3)2C], 3.71 (m, H3), 3.76 (dd, 1H, J5,5 11.9, J4,5 6.7, H5),
3.83 (s, NH2), 4.18 (dd, 1H, J4,5 6.9, H5), 4.65, 4.86 (AB, J 12.0, PhCH2), 4.67 (dd, J2,3
10.5, J1,2 6.8, H2), 4.72-4.77 (br m, H4), 4.80 (d, H1), 7.25-7.40 (m, Ph). δC (150.9 MHz)
27.08, 27.23 [(CH3)2C], 28.44 [(CH3)3C], 51.83 (C4), 62.01 (C5), 69.84 (PhCH2), 74.70
(C3), 76.16 (C2), 81.33 [(CH3)3C], 101.42 (C1), 111.92 [(CH3)2C], 127.86-137.53 (Ph),
157.31 (C=O). δN (60.82 MHz) –316.64 (t, J 65.3 Hz, NH2), –274.73 (s, N). m/z (FAB)
394.2131 (C20H30N2O6 [M]+• requires 394.2131).
66
NBoc
NHOO
OH
(147)
(3aR,4R,7aR)-5-N-(tert-Butoxycarbonyl)-2,2-dimethyl-1,3-dioxolo[4,5-d]
hexahydropyridazine-4-methanol (147)
The hydrazide (146) (13.3 mg) in THF (15 mL) was treated with Pd/C (10%, 5 mg) and
H2 (1 atm , 12 h). The mixture was filtered, concentrated and subjected to flash
chromatography (EtOAc/petrol, 7:3) to give (147) (8.4 mg, 88%) as a colourless oil, [α]D
–82.4°. δH (600 MHz) 1.46, 1.49 (s, CH3C), 2.92 (dd, 1H, J7,7 9.8, J7,7a 9.1, H7), 3.64 (dd,
1H, J7,7a 7.4, H7), 3.71 (ddd, J3a,7a 8.2, H7a), 3.83-3.87 (m, 2H, H4, CH2O), 3.91-3.96 (m,
2H, H2, CH2O). δC (150.9 MHz) 27.06, 27.16, 28.51 (CH3C), 48.97 (C7), 62.61 (CH2O),
62.67 (C4), 75.52, 75.62 (C3a, C7a), 81.97 [(CH3)3C], 112.77 [(CH3)2C], 155.81 (C=O).
m/z (FAB) 288.16852 (C12H24N2O5 [M]+• requires 288.16852).
NH
NHHOHO
OH
(23)
(3S,4S,5S)-4,5-Dihydroxy-3-(hydroxymethyl)hexahydropyridazine (azafagomine) (23)
The pyridazine (147) (8 mg) was treated with CF3COOH (20 min, rt). The solution was
concentrated, the residue dissolved in MeOH and neutralised with resin (Amberlite IRA
400, OH–), filtered and concentrated. The residual gum was dissolved in HCl (1 M, 1
mL) and applied to a cation-exchange column (Dowex 50W-X2, H+). The column was
washed with water and eluted with aqueous NH3 (1.5 M). The eluate was concentrated to
give azafagomine (23) (5.1 mg, 99%) as a colourless oil, [α]D +38° (H2O, pH 2.5). δH
(600 MHz, D2O, pH 2.5) 2.89 (dd, 1H, J6,6 12.5, H6), 3.02 (ddd, J 9.6, 6.2, 2.7, H3),
67
3.45-3.49 (m, 2H, H6, H4), 3.70-3.75 (m, 2H, H5, CH2O), 3.84 (dd, 1H, J 12.4, 2.7,
CH2O). δC (150.9 MHz, D2O, pH 2.5) 48.52 (C6), 57.97 (CH2O), 60.78 (C3), 67.91 (C5),
69.17 (C4).
(151)
O
OO
OBn
Boc[N(Boc)2]N
Benzyl 4-[N-tert-Butoxycarbonyl-N-bis(tert-butoxycarbonyl)amino]amino-4-deoxy-2,3-
O-isopropylidene-α-D-arabinoside (151)
Lithium bis(trimethylsilyl)amide in THF (1 M, 3.50 mL, 3.50 mmol) was added dropwise
to the carbazate (150)39(1.36 g, 3.95 mmol) in dry THF (10 mL, –30ºC) and the solution
stirred (30 min). The imidazylate (22) (200 mg, 0.488 mmol) was added and the solution
heated to reflux (14 h). The solution was concentrated and the residue dissolved in
EtOAc, washed with H2O and dried. Flash chromatography (EtOAc/petrol, 3:7) gave the
hydrazide (151) (240 mg, 89%) as a colourless oil, [α]D −4.3°. δH (300 MHz) 1.39, 1.44,
1.50 (3×s, 33H, CH3) 3.49 (dd, J5,5 13.2, J4,5 2.1, H5), 3.65 (dd, J3,4 4.1, J2,3 10.1, H3),
3.85 (dd, J1,2 7.5, H2), 4.36-4.43 (m, H4, H5), 4.52 (d, H1), 4.64, 4.88 (AB, J 11.7,
PhCH2), 7.25-7.37 (m, Ph). δC (75.5 MHz) 26.51, 26.65 [(CH3)2C], 27.74, 27.85
[(CH3)3C], 56.24 (C4), 65.36 (C5), 69.79 (PhCH2), 73.53, 77.12 (C2, C3), 81.47
[(CH3)3C], 102.09 (H1), 110.21 [(CH3)2C], 127.54-137.06 (Ph), 154.03 (C=O). m/z
(FAB) 595.3217 (C36H46N2O10 [M+H]+ requires 595.3231).
68
(152)
O
OHOH
OBn
Boc[N(Boc)2]N
Benzyl 4-[N-tert-Butoxycarbonyl-N-bis(tert-butoxycarbonyl)amino]amino-4-deoxy-α-D-
arabinoside (152)
The hydrazide (151) (240 mg, 0.404 mmol) in MeOH (20 mL) was treated with
pyridinium tosylate (30 mg) and the mixture refluxed (2 h). The solution was
concentrated and the residue dissolved in EtOAc, washed with H2O and dried. Flash
chromatography (EtOAc/petrol, 1:1) gave the hydrazide (152) (201 mg, 89%) as a
colourless oil, [α]D +64.3°. δH (600 MHz) 1.48, 1.50, 1.53 (3×s, 27H, CH3), 3.57 (dd, J5,5
12.3, J4,5 3.5, H5), 3.72 (dd, J2,3 7.9, J1,2 5.4, H2), 3.84 (dd, J3,4 4.7, H3), 4.21 (dd, J4,5
4.8, H5), 4.39 (d, H1), 4.57, 4.82 (AB, J 11.9, PhCH2), 4.71 (ddd, H4), 7.28-7.37 (Ph).
δC (150.9 MHz) 27.97, 28.14, 28.17 (CH3), 54.94 (C4), 61.63 (C5), 70.05 (PhCH2),
71.84, 71.56 (C2, C3), 82.27, 84.03, 85.03 (CH3C), 101.43 (C1), 128.00-137.22 (Ph),
151.37, 153.44, 154.61 (C=O). m/z (FAB) 555.2906 (C27H43N2O6 [M+H]+ requires
555.2918).
(153)
O
OHOH
OBn
Boc[NHBoc]N
Benzyl 4-[N-tert-Butoxycarbonyl-N-(tert-butoxycarbonyl)amino]amino-4-deoxy-α-D-
arabinoside (153)
The hydrazide (152) (185 mg, 0.335 mmol) in CH3CN was treated with Mg(ClO4)2 (74.0
mg, 0.335 mmol) and the solution heated (50°C, 3 h). The solution was concentrated and
the residue dissolved in EtOAc, washed with H2O and dried. Flash chromatography
(EtOAc/petrol, 1:1) gave the hydrazide (153) (129 mg, 85%) as a colourless solid, a small
69
portion of which was recrystallised, m.p. 166-167°C (CH2Cl2/petrol), (Found C, 58.3; H,
7.6; N, 6.1. C22H34N2O8 requires C, 58.1; H, 7.5; N, 6.2).
(154)
O
OHOH
OH
Boc[NHBoc]N
4-[N-tert-Butoxycarbonyl-N-(tert-butoxycarbonyl)amino]amino-4-deoxy-α-D-arabinose
(154)
A stirred solution of the hydrazide (153) (16 mg, 0.340 mmol) in THF/H2O (100:0.1, 15
mL) was treated with Pd/C (10%, 4 mg) and H2 (12 h, 1 atm). The mixture was filtered,
concentrated and subjected to flash chromatography (THF/petrol, 4:1) to give,
presumably, the hemiacetal (10 mg, 75%) as a colourless oil.
NH
NHOHO
OHNH
NHOH
HOHO
OH
(27) (25)
(4S,5S,6S)-3,4,5-Trihydroxy-6-(hydroxymethyl)hexahydropyridazine (azanoeuromycin)
(27) hydrochloride and
(4S,5S,6S)-4,5-Dihydroxy-6-hydroxymethyl-1,4,5,6-tetrahydropyridazine (25)
hydrochloride
a) The hemiacetal (154) (10.0 mg, 0.026 mmol) was treated with hydrochloric acid (1 M,
2 mL) and kept (15 min). The solvent was removed to give a mixture of azanoueromycin
(27) and the hydrazone (25) (4.3 mg) as a colourless oil. δH (600 MHz, D2O) 3.05-3.08
(m, H6α), 3.17-3.23 (m, H6β, H5), 3.48 (dd, J4,5 9.0, J3,4 8.5, H4α), 3.53 (dd, J5,6 9.2,
H5α), 3.62-3.73, 3.76-3.81 (m, 8H, CH2α, CH2β, CH2, H4β, H5β, H5), 3.95 (dd, 1H, J
12.8, 3.15, CH2β), 4.19 (dd, J4,5 8.0, J3,4 1.2, H4), 4.50 (dd, H3α), 4.88 (d, J3,4 3.3, H3β),
70
6.85 (d, H3). δC (150.9 MHz, D2O) 57.21 (CH2β), 57.74 (CH2α), 58.37, 61.99 (C6β, C6),
58.79 (CH2), 60.59 (C6α), 64.94, 67.14, 71.06 (C4β, C5β, C5), 68.15, 73.85 (C4α, C5α),
69.46 (C4), 78.29 (C3β), 81.75 (C3α), 146.00 (C3). (27) m/z (FAB) 165.0865
(C5H13N2O4 [M+H]+ requires 165.0875); (25) m/z (FAB) 146.0690 (C5H10N2O3 [M]+•
requires 146.0691).
b) The hemiacetal (154) (5.5 mg) was treated with CF3COOH (1 mL, 15 min). The
solvent was removed, the residue dissolved in a little hydrochloric acid (1 M) and the
solvent again removed. The 1H (600 MHz) and 13C (150.9 MHz) n.m.r. spectra were
consistent with those reported for (27) and (25) in (a).
NH
NHOHO
OH
(25)
(4S,5S,6S)-4,5-Dihydroxy-6-hydroxymethyl-1,4,5,6-tetrahydropyridazine (25) (salt with
CF3COOH)
The hemiacetal (154)(5.0 mg) was treated with CF3COOH (2 mL); (20 min). The solvent
was removed to give predominantly the hydrazone (25) (2.1 mg) as a colourless oil. δH
(600 MHz, d6-DMSO) 2.85 (ddd, J5,6 10.2, J 7.7, 3.0, H6), 3.27 (dd, J4,5 7.6, H5), 3.36
(dd, 1H, J 11.1, CH2), 3.74 (dd, 1H, CH2), 3.82 (dd, J3,4 1.4, H4), 6.31 (d, H3). δC (150.9
MHz, d6-DMSO) 59.91 (C6), 60.52 (CH2O), 69.11 (C5), 70.55 (C4), 140.41 (C3).
71
NBocAcOAcO
OAc
(157)
(3R,4R,5R)-3,4-Diacetoxy-5-acetoxymethyl-N-(tert-butoxycarbonyl)piperidine (157)
Isofagomine (13) (110 mg, 0.601 mmol) in Me2CO/H2O (7:3, 10 mL) was treated with
NaHCO3 (500 mg, 6.0 mmol) and (Boc)2O (310 mg, 1.2 mmol) and stirred (2 h, rt). The
mixture was then somewhat concentrated, extracted with EtOAc, the extract dried and
concentrated and the residue dissolved in CHCl3 (10 mL). This solution was treated with
pyridine (4 mL), Ac2O (3 mL) and DMAP (10 mg) and stirred (2 h). Treatment with
MeOH (5 mL, 1 h), followed by concentration of this mixture and flash chromatography
(EtOAc/petrol, 1:4), gave the carbamate (157) (220 mg, 83%) as a colourless oil, [α]D
+31.2º. δH (600 MHz) 1.45 (s, 9H, (CH3C)), 2.02, 2.04, 2.05 (3×s, 9H, CH3C=O), 2.02-
2.05 (m, H5), 2.72-3.14 (m, 2H, H2, H6), 3.97-4.15 (m, 4H), 4.78 (br m, 1H), 4.97 (t,
1H). δC (150.9 MHz) 20.61, 20.64, 20.70 (3C, CH3CO), 28.14 (CH3C), 39.32 (C5),
43.90, 45.50 (C2, C6), 61.64 (CH2O), 70.04, 71.27 (C3, C4), 80.55 (CH3C), 154.23
(NC=O), 170.55, 169.98 (CH3C=O). m/z (FAB) 374.1804 (C17H28NO8 [M+H]+ requires
374.1815).
NAcOAcO
OAc
NHBoc
NBoc(159)
(3R,4R,5R)-3,4-Diacetoxy-5-acetoxymethyl-N,N′-bis-(tert-butyloxycarbonyl)piperidine-1-
carboxamidine (159)
The carbamate (157) (33 mg, 0.088 mmol) was treated with CF3COOH (1 mL); (5 min).
The solution was then concentrated and azeotropically dried with CH2Cl2/PhMe and
72
redissolved in CH2Cl2 (1 mL). This solution was then treated with ethyldiisopropylamine
(45 mg, 0.35 mmol), (BocN)2C=NTf36 (34 mg, 0.087 mmol) and stirred (5 d, rt).
Concentration of the mixture followed by flash chromatography (EtOAc/petrol, 1:4) gave
the triacetate (159) (38 mg, 95%) as a colourless oil, [α]D +1.1º (CH2Cl2). δH (600 MHz)
1.49 (s, 18H, CH3C), 2.06, 2.05, 2.03 (3×s, 9H, CH3C=O), 2.34-3.41 (br s, H5), 2.97-3.04
(m, 2H), 4.01 (dd, 1H, J 11.7, 3.3, CH2O), 4.05-4.12 (br s, 1H), 4.11 (dd, 1H, J 11.7, 5.7,
CH2O), 4.23-4.35 (br s, 1H), 4.99-5.03 (m, 1H), 5.08 (t, 1H, J 8.8). δC (150.9 MHz)
20.86, 20.89, 20.94 (3C, CH3CO), 28.22 (CH3C), 38.93 (C5), 61.71 (CH2O), 70.23, 71.54
(C3, C4), 155.64 (NC=O), 170.86, 170.40, 169.97 (3C, CH3CO). m/z (FAB) 516.2545
(C23H38N3O10 [M+H]+ requires 516.2557).
NHOHO
OH
NH2
NH2Cl(26)
(3R,4R, 5R)-3,4-Dihydroxy-5-(hydroxymethyl)piperidine-1-carboxamidine (26)
hydrochloride
The triacetate (159) (62 mg, 0.120 mmol) was treated with hydrogen chloride in MeOH
(1 M, 4 mL) and stirred (12 h). The solution was concentrated and treated with
CF3COOH (1 mL), then allowed to stand (20 min) and concentrated. The residue was
dissolved in hydrochloric acid (1 M, 2 mL) and the mixture evaporated (this procedure
was repeated another two times) to give the hydrochloride (26) as a pale, colourless oil
(26 mg), [α]D +101º (H2O). δH (600 MHz) 1.72-1.76 (m, H5), 2.89 (dd, 1H, J 13.4, 11.4,
H2), 2.95 (dd, 1H, J 14.0, 11.8, H6), 3.38 (dd, J 10.1, 8.8, H4), 3.51-3.55 (m, H3), 3.76
(dd, 1H, J 11.6, 3.4, CH2O) 3.59 (dd, 1H, J 6.9, CH2O), 3.79-3.84 (m, 2H, H2, H6). δC
73
(150.9 MHz) 42.79 (C5), 46.55 (C6), 49.02 (C2), 59.53 (CH2O), 69.93 (C3), 72.65 (C4),
156.15 (C=N). m/z (FAB) 190.1201 (C7H16N3O3 [M–Cl]+ requires 190.1201).
References
(1) Jespersen, T. M.; Dong, W.; Sierks, M. R.; Skyrdstrup, T.; Lundt, I.; Bols, M.
Angew. Chem. Int. Ed. Engl. 1994, 33, 1778.
(2) Jesperson, T. M.; Bols, M.; Sierks, M. R.; Skrydstrup, T. Tetrahedron 1994, 50,
13449.
(3) Ichikawa, Y.; Igarashi, Y.; Ichikawa, M.; Suhara, Y. J. Am. Chem. Soc. 1998,
120, 3007.
(4) Kim, Y. J.; Ichikawa, M.; Ichikawa, Y. J. Org. Chem. 2000, 65, 2599.
(5) Pandey, G.; Kapur, M. Tetrahedron Lett. 2000, 41, 8821.
(6) Iida, H.; Yamazaki, N.; Kibayashi, C. J. Org. Chem. 1987, 52, 3337.
(7) Corey, E. J.; Fuchs, P. L. Tetrahedron Lett. 1972, 36, 3769.
(8) Hansen, S. U.; Bols, M. J. Chem. Soc., Perkin Trans. 1 2000, 911.
(9) Zhao, G.; Deo, U. C.; Ganem, B. Org. Lett. 2001, 3, 201.
(10) Andersch, J.; Bols, M. Chem. Eur. J. 2001, 7, 3744.
(11) Best, W. M.; Macdonald, J. M.; Skelton, B. W.; Stick, R. V.; Tilbrook, D. M. G.;
White, A. H. Can. J. Chem. 2002, 80, 857.
(12) Guanti, G.; Riva, R. Tetrahedron Lett. 2003, 44, 357.
(13) Banfi, L.; Guanti, G.; Paravidino, M.; Riva, R. Org. Biomol. Chem. 2005, 3, 1729.
(14) Ouchi, H.; Mihara, Y.; Takahata, H. J. Org. Chem. 2005, 70, 5207.
74
(15) Ouchi, H.; Mihara, Y.; Wantanabe, H.; Takahata, H. Tetrahedron Lett. 2004, 45,
7053.
(16) Zhu, X.; Sheth, K. A.; Li, S.; Chang, H.-H.; Fan, J.-Q. Angew. Chem. Int. Ed.
2005, 2005, 7450.
(17) Lillelund, V. H.; Liu, H.; Liang, X.; Søhoel, H.; Bols, M. Org. Biomol. Chem.
2003, 1, 282.
(18) Macdonald, J. M.; Stick, R. V. Aust. J. Chem. 2004, 57, 449.
(19) Bols, M.; Hazell, R. G.; Thomsen, I. B. Chem. Eur. J. 1997, 3, 940.
(20) Liang, X.; Bols, M. J. Org. Chem. 1999, 64, 8485.
(21) Ernholt, B. V.; Thomsen, I. B.; Lohse, A.; Plesner, I. W.; Jensen, K. B.; Hazell, R.
G.; Liang, X.; Jakobsen, A.; Bols, M. Chem. Eur. J. 2000, 6, 278.
(22) Hansen, S. U.; Bols, M. J. Chem. Soc., Perkin Trans. 2 2000, 665.
(23) Yoon, N. M.; Brown, H. C. J. Am. Chem. Soc. 1968, 90, 2927.
(24) Nystrom, R. F. J. Am. Chem. Soc. 1954, 77, 2544.
(25) Brown, H. C.; Yoon, N. M. J. Am. Chem. Soc. 1966, 88, 1464.
(26) Soffer, L. M.; Katz, M. J. Am. Chem. Soc. 1956, 78, 1705.
(27) Takano, S.; Ohkawa, T.; Ogasawara, K. Tetrahedron Lett. 1988, 29, 1823.
(28) Coutts, L. D.; Cywin, C. L.; Kallmerten, J. Synlett 1993, 696.
(29) Liu, H.; Liang, X.; Søhoel, H.; Bülow, A.; Bols, M. J. Am. Chem. Soc. 2001, 123,
5116.
(30) Davies, G. J.; Gloster, T. unpublished results.
(31) Søhoel, H.; Xifu, L.; Bols, M. J. Chem. Soc., Perkin Trans. 1 2001, 1584.
75
(32) Benhaddou, R.; Czernecki, S.; Farid, W.; Ville, G.; Xie, J.; Zegar, A.
Carbohydrate Res. 1994, 260, 243.
(33) Burkhart, F.; Hoffmann, M.; Kessler, H. Angew. Chem. Int. Ed. 1997, 36, 1191.
(34) Meloncelli, P. J.; Stick, R. V. Aust. J. Chem. 2006, 59, 827.
(35) Kiefel, M. J.; von Itzstein, M. Prog. Med. Chem. 1999, 36, 1.
(36) Feichtinger, K.; Sings, H. L.; Baker, T. J.; Matthews, K.; Goodman, M. J. Org.
Chem. 1998, 63, 8432.
(37) Macdonald, J. M.; Stick, R. V.; Tilbrook, D. M. G.; Withers, S. G. Aust. J. Chem.
2002, 55, 747.
(38) Staab, H. A. Angew. Chem. Int. Ed. Engl. 1962, 1, 351.
(39) Mäeorg, U.; Pehk, T.; Ragnarsson, U. Acta Chem. Scand. 1999, 53, 1127.
76
Appendix
ppm2.002.503.003.504.004.505.00
ppm4050607080
NHOH
HOHO
OH
(14)
NHOH
HOHO
OH
(14)
H2β
H2α
H5αH5β
H6α
C2α
C2β
C3α
C3β
C4α
C4β
CH2O
C5αC5βC6α
C6β
1H and 13C n.m.r. spectra of noeuromycin (14) hydrochloride.
77
NHOHO
OH
NH2
NH(26)
ppm2.002.503.003.504.00
NHOHO
OH
NH2
NH(26)
H5
H2, H6
H4
H3
H2H6
CH2O
ppm50100150
N=C
C4C3
CH2OC2
C6
C5
1H and 13C n.m.r. spectra of guanidine derivative (26) of isofagomine (as the
hydrochloride).
78
NHHOHO
OH
O
(24)
ppm2.503.003.504.00
H4
DSS
H5H5
H3CH2O
CH2O
H2
ppm50100150
NHHOHO
OH
O(24)
C1
C2
C3 CH2O
DSS
C4C5
1H and 13C n.m.r. spectra of isofagomine lactam (24).
79
NH
NHHOHO
OH
(23)
ppm45.050.055.060.065.070.0
ppm3.003.504.00
NH
NHHOHO
OH
(23)
H6
H3
H4, H6
H5CH2O
CH2O
C6
CH2O
C3
C5
C4
1H and 13C n.m.r. spectra of azafagomine (23).
80
NH
NHOHO
OHNH
NHOH
HOHO
OH
(27) (25)
ppm50100150
NH
NHOHO
OHNH
NHOH
HOHO
OH
(27) (25)
ppm4.05.06.07.0
H3
H3β
H3α
H4
H6α
H5H6β
C3
C3α
C3β
1H and 13C n.m.r. spectra of azanoeuromycin (27) and the hydrazone (25) (as the
hydrochlorides).
81
NH
NHOHO
OH
(25)
ppm2.503.003.504.004.505.005.506.006.50
NH
NHOHO
OH
(25)
ppm60708090100110120130140
H3
H4 CH2OCH2O
H5
H6
C3
C4
C5
CH2O
C6
1H and 13C n.m.r. of the hydrazone (25) (salt with CF3COOH).
82
Chapter 2
Synthesis of 3- and 4-O-β-D-
Glucopyranosyl Derivatives of Isofagomine
and Noeuromycin
84
85
Introduction
3-O-β-D-Glucosylated Derivatives of Isofagomine
The first and only synthesis of 3-O-β-D-glucosylated derivatives (28) and (160) of
isofagomine (13) came from Macdonald and co-workers.1
O
OHO
OH
OHO
NH
HOOH
O
HOHO
OH
OH
O
HOHO
OH
OHO
NH
HOOH
(28) (160)
HO NH
HO
OH
(13)
Preparation of these derivatives (28) and (160) proceeded via the direct glycosylation of a
protected isofagomine acceptor (162). Commencing from isofagomine (13), protection of
the amine by treatment with benzyl chloroformate gave the carbamate (161), whilst the
selective protection of the 4-OH and the 6-OH groups was effected by subsequent
conversion into the 4,6-O-benzylidene derivative (162).1
HO NH
HOOH
HO NCO2Bn
(13) (162)
HO NCO2Bn
HOOH
(161)
OO
Ph
86
Treatment of the protected isofagomine derivative (162) with the trichloroacetimidate
(163) resulted in the formation of the pseudo-disaccharide (164). Removal of the
protecting groups yielded 3-O-β-D-glucopyranosylisofagomine (28).1
O
AcOAcO
OAc
OAcOTCA
O
O
AcOAcO
AcO
OAc
(163)
(162)
(164)
O
HOHO
OH
OHO
NH
HOOH
(28)
HO NCO2Bn
OO
PhNCO2Bn
OO
Ph
Preparation of the pseudo-trisaccharide (160) proceeded in an analogous manner, with the
α-laminaribiosyl trichloroacetimidate (165) as the donor, followed by deprotection to
give the pseudo-trisaccharide (160).1
O
OAcO
OAc
AcO
O
AcOAcO
OAc
OAc OTCA(165)
O
OHO
OH
OHO
NH
HOOH
O
HOHO
OH
OH
(160)
4-O-β-D-Glucosylated Derivatives of Isofagomine
The preparation of the 4-O-β-D-glucosylated derivatives (15), (18) and (19) of
isofagomine was again achieved by Macdonald and co-workers but this time employing
enzymatic methodology.2
87
HO NH
OOH
O
HOHO
OH
OHO
HOO
OH
OH
n
(15) n=0(18) n=1(19) n=2
The enzyme used to prepare the above glucosylated derivatives was the mutant enzyme
(AbgGlu358Ser), otherwise know as a ‘glycosynthase’.3 The acceptor (161) was treated
with α-D-glucopyranosyl fluoride (166) in the presence of the glycosynthase, followed by
acetylation, to produce a separable mixture of the pseudo- di- (167), tri- (168) and tetra-
saccharide (169). Deprotection gave the desired 4-O-glucosylated derivatives (15), (18)
and (19).
HO NCO2Bn
HOOH
(161)
O
HOHO
OH
OHF
(166)
AcO NCO2Bn
OOAc
O
AcOAcO
OAc
OAc O
AcOO
OAc
AcO
n
(167) n=0(168) n=1(169) n=2
(15) n=0(18) n=1(19) n=2
HO NH
OOH
O
HOHO
OH
OHO
HOO
OH
OH
n
88
Discussion
Synthesis of 3-O-β-D-Glucopyranosylisofagomine and
3-O-β-D-Glucopyranosylnoeuromycin
Initial exploration focused on the direct glycosylation of the nitrile (101), derived from
the imidazylate (22) already pivotal to the synthesis of several azasugars.
(101)
OImSO2O
OO
OBn
(22)
O
NCOH
OHOBn
Treatment of the nitrile (101) with the readily available trichloroacetimidate (163)
resulted in selective formation of the desired disaccharide (170). Regioselectivity was
established by an analysis of the 13C n.m.r. spectrum, with a downfield shift of C2 (δ
80.20) relative to C3 (δ 68.49) in the product. This selectivity can be rationalized in terms
of steric hindrance within the acceptor in the 1C4 conformation, with unfavorable
diequatorial and axial/equatorial interactions reducing the reactivity of the 3-OH. To a
lesser extent an inductive effect from the electron-withdrawing nitrile reduces the
nucleophilicity of the 3-OH, further reducing its reactivity.
89
(170)
O
AcOAcO
OAc
OAc
(a)(163)
OTCA
(101)
O
NCOH
OHOBn
O
O
NCOH
OOBn
OAc
OAcOAc
AcO
a) Et2OBF3, CH2Cl2, 76%.
Further confirmation of the regiochemistry came from the acetylation of (170) to yield
the pentaacetate (171), with 1H n.m.r. spectroscopy showing a downfield shift of H3 from
δ 3.85 in (170) to δ 5.09 in (171).
(171)
(a)
(170)
O
O
NCOH
OOBn
OAc
OAcOAc
AcO
O
O
NCOAc
OOBn
OAc
OAcOAc
AcO
a) Pyr, DMAP, Ac2O, CH2Cl2, 55%.
The next step in the synthesis required the removal of the acetyl groups from (170) but,
under either basic or acidic conditions, this proved surprisingly difficult.
In order to reduce the number of ester groups, the tri-O-benzyl trichloroacetimidate (172),
with the requisite 2-O-acetyl group to ensure formation of the β-glycoside, was used to
glycosylate the nitrile (101). This reaction proved highly selective, yielding the
disaccharide (173) with the regiochemistry again indicated by a downfield shift of C2 (δ
90
79.98). Removal of the acetyl protecting group was possible under acidic conditions,
yielding the diol (174) in moderate yield.4
(174)
O
BnOBnO
OBn
OAcOTCA
(172) (a)
(b)
(101)
O
NCOH
OHOBn
O
O
NCOH
OOBn
OAc
OBnOBn
BnO
(173)
O
O
NCOH
OOBn
OH
OBnOBn
BnO
a) Et2OBF3, CH2Cl2, 74%; b) HCl, MeOH, 77%.
Reduction of the nitrile (174) using alane, followed by in situ protection with di-tert-butyl
dicarbonate afforded the carbamate (175) in good yield. Acetylation of the diol (175)
yielded the diacetate (176), with the regiochemistry of the earlier glycosylation confirmed
by 1H n.m.r. spectroscopy, showing a downfield shift of H3 from δ 3.89 in (175) to δ 5.07
in (176). A change in conformation from 1C4 to 4C1 was also observed, indicated by a
large coupling for J4,5 (9.3Hz) in (176).
91
(175)
(a)
(174)
O
OBn
O
OAc
O
BnOBnO
OBn
OAc
(b)
(176)
BocHN
O
O
NCOH
OOBn
OH
OBnOBn
BnO
O
O
OHO
OBn
OH
OBnOBn
BnOBocHN
a) i) AlH3, THF; ii) (Boc)2O, 76%; b) Pyr, DMAP, Ac2O, CH2Cl2, 85%.
Removal of the Boc protecting group followed by treatment with hydrogen resulted in
debenzylation and reductive amination to afford 3-O-β-D-glucopyranosylisofagomine
(28), consistent in all respects with the material prepared by Macdonald and co-workers.1
O
HOHO
OH
OHO NH
HO
OH
(28)
(a)
(175)
O
O
OHO
OBn
OH
OBnOBn
BnOBocHN
a) i) Et2OBF3, CH2Cl2; ii) Amberlite IRA 400 (OH−);
iii) Pd/C, H2, MeOH, AcOH, 66%.
92
Attention then turned to the preparation of 3-O-β-D-glucopyranosylnoeuromycin (20).
Hydrogenolysis of (175) presumably yielded the hemiacetal (177); subsequent treatment
with 1 M hydrochloric acid then yielded 3-O-β-D-glucopyranosylnoeuromycin (20).
O
HOHO
OH
OHO NH
HO
OH
OH
(20)
(a)
(177)
(b)
(175)
O
O
OHO
OBn
OH
OBnOBn
BnO
O
O
OHO
OH
OH
OBnOBn
BnOBocHNBocHN
a) Pd/C, H2, THF, H2O; b) 1 M HCl, 79%.
O
HOHO
OH
OHO NH
HO
OH
OH
(20)
ppm2.002.503.003.504.004.505.005.50
H2(man)H1′(glc)
H6(glc)
H1(pyr)H3(pyr) CH2N(pyr)
1′
5
H1′(man), H2(glc)
H5
1H n.m.r. (600 MHz) spectrum of 3-O-β-D-glucopyranosylnoeuromycin (20)
hydrochloride.
93
As with the preparation of noeuromycin (14) itself, trace amounts of the pyranose form
(178) were found to be present, as indicated in the 1H n.m.r. spectrum. Confirmation of
the structure was helped by comparison with the 1H n.m.r. spectrum of noeuromycin (14),
with the shift of H2 (δ 5.34 and δ 4.69) and H5 (δ 1.85-1.95) characteristic of this ring
system.
(178)
O
O
OHO
OH
OH
OHOH
HOH2N
ppm405060708090100
O
HOHO
OH
OHO NH
HO
OH
OH
(20)
C1′(glc)
C1′(man)
C2(glc), C3(glc)
C3(man)
C5, C6(man)
C6(glc)
C6′, CH2OC2′
13C n.m.r. (600 MHz) spectrum of 3-O-β-D-glucopyranosylnoeuromycin (20)
hydrochloride.
94
The 13C n.m.r. spectrum showed the characteristic resonances of the noeuromycin ring
system with C2 (δ 76.00 and δ 80.75), C5 (δ 40.42 and δ 40.71) and C6 (δ 38.23 and δ
41.08) all closely coinciding to the values in noeuromycin (14). Confirmation of the
position of the glycosidic linkage was made by the observance of the downfield shift of
C3 (δ 77.97 and δ 80.75). Contributions from the pyranose form (178) were evident in the
13C n.m.r. spectrum.
X-Ray crystallographic data were again provided through a collaboration with Gideon
Davies and Victoria Money of the University of York, with determination of 3-O-β-D-
glucopyranosylnoeuromycin (20) in complex with a family 26 lichenase from
Clostridium thermocellum (EC 3.2.1.4) at pH 6.5 and 1.20 Å resolution.5 Although not
directly observed, it would be anticipated that the nitrogen would be ‘doubly’ protonated.
Inhibition data was also measured at pH 6.5 and shown to exhibit excellent inhibition (Ki
168 nM)
95
acid/base
nucleophile
Three-dimensional structure of the lichenase (CtLic26A) from Clostridium thermocellum
in complex with 3-O-β-D-glucopyranosylnoeuromycin (20).
96
Synthesis of 4-O-β-D-glucopyranosylisofagomine and
4-O-β-D-glucopyranosylnoeuromycin
It was anticipated that glycosylation of a protected nitrile (179), prepared from (101),
would provide access to 4-O-β-D-glucopyranosylisofagomine (15) and 4-O-β-D-
glucopyranosylnoeuromycin (21).
(179)
HO NH
O
OH
OH
(21)
O
HOHO
OH
OH
HO NH
O
OH
O
HOHO
OH
OH
(15)
(101)
O
NCOH
OHOBnO
NCOH
OPOBn
Benzoyl protecting groups are commonly used to effect the selective protection of a diol.
Treatment of (101) with benzoyl chloride provided (180) exclusively, in good yield, a
very surprising result in light of the previous selective glycosylation to form the alcohol
(170).
(180)
(181)
(182)
O OBn
OBnNC
(a) (b)
54%
15%
(101)
O
NCOH
OHOBn O
NCOBz
OHOBn
O
NCOBz
OBnOBn
a) BzCl, Et3N, CH2Cl2, 78%; b) BnBr, Ag2CO3, CH2Cl2.
97
This contrasting selectivity can be rationalized by the more acidic 3-OH of (101)
deprotonating in the presence of triethylamine, with the subsequent anion reacting with
the benzoyl chloride.
In an attempt to make use of the easy preparation of (180), and so obtain a compound
with just the 3-OH free, a subsequent benzylation was attempted. The benzylation of
(180) proved to be temperamental, with the standard conditions of sodium hydride and
benzyl bromide providing poor results. Treatment of (180) with silver carbonate and
benzyl bromide provided predominantly (182), with elimination arising owing to the
acidity of H4.
An alternative method of benzylation has been reported under acidic conditions using
benzyl trichloroacetimidate, specifically for cases where basic conditions are
incompatible.6 Treatment of (180) under these conditions yielded (181), unfortunately in
only modest yield. Removal of the benzoyl group from (181) under standard Zemplin
conditions resulted in negligible formation of the desired product (183), whilst the neutral
potassium cyanide induced debenzoylation gave (183) in moderate yield.7
O
OBn
BnO
OH
NC
(183)(181)
(a) (b)
(180)
O
NCOBz
OHOBn O
NCOBz
OBnOBn
a) CCl3C(NH)OBn, CF3SO3H, CH2Cl2 50%; b) KCN, MeOH, 67%.
98
Circumvention of the low yields observed in the above sequence led to the preparation of
the triethylsilyl ether (184) and the tetrahydropyran acetal (185). Unfortunately,
debenzoylation of (184) and (185) proved to be futile under a range of conditions.
O
OBn
Et3SiO
OBz
NC
(184)
(a)
(185)
(b)
(180)
O
NCOBz
OHOBn
O
NCOBz
OOBn
OO
NCOH
OOBn
O
O
NCOH
OSiEt3OBn
a) Et3SiCl, DMAP, pyr, 60%; b) 3,4-dihydro-2H-pyran, CSA, CH2Cl2, 94%.
Another approach hoped for the direct preparation of a 3-O-silyl derivative and an
investigation into its subsequent glycosylation. Treatment of (101) with BMSCl and
imidazole yielded a mixture of the 2-O-silyl ether (187) and 3-O-silyl ether (186) in a
favourable 7:3 ratio, with the regiochemistry confirmed by acetylation. Interestingly, the
2-O-silyl ether (187) was found to exist in a 1C4 conformation whilst the 3-O-silyl ether
(186) was found to exist in a 4C1 conformation.
99
(101) (186)
O
OBn
HO
OBMS
NC
(187)
(a)
(186)
O
OBn
HO
OBMS
NC (b)
(188)
O
OBn
AcO
OBMS
NC
(189)
(c)
70%30%
O
NCOH
OHOBn O
NCOH
OBMSOBn
(187)
O
NCOH
OBMSOBn O
NCOAc
OBMSOBn
a) BMSCl, ImH, DMF; b) Ac2O, DMAP, pyr, 95%;
c) Ac2O, pyr, DMAP, 65%.
Unfortunately, glycosylation of (187) using the trichloroacetimidate (172) was
unsuccessful. Thioglycosides have emerged as powerful donors for glycosylation,
operating via a highly reactive glycosyl triflate.8 Unfortunately, treatment of (187) with
the thioglycoside (190) under the conditions developed by van Boom and co-workers
again failed to provide the desired disaccharide, presumably due to steric hindrance of the
3-OH.9
(187)
O
BnOBnO
OBn
OAcOTCA
(172)
O
BnOBnO
OBn
OAcSPh
(190)
O
NCOH
OBMSOBn
O
NC
BMSOBnO
O
BnOBnO
OBn
OAc
O
100
Glycosylation prior to the introduction of the nitrile would be expected to circumvent
some of the difficulties observed with the above approaches. Allylation of (79) followed
by removal of the isopropylidene group provided the diol (191) in good yield.
OHO
OO
OBn
(79)
(a)
(191)
OAllO
HOOH
OBn
a) i) CH2=CHCH2Br, NaH, DMF; ii) CSA, MeOH, 89%.
Direct glycosylation of the diol (191), followed by deacetylation and benzylation to
facilitate separation, gave the 3-O-β-D-glucosyl (192) and 2-O-β-D-glucosyl (193)
derivatives in good yield and in a 1:1 ratio. The regiochemistry of glycosylation was
unable to be indubitably confirmed until the final stage of the synthesis. The lack of
regioselectivity here, in contrast to the glycosylation of (101), can be rationalized by
equal steric hindrance at both the 2- or 3-OH position, and the absence of the deactivating
electronic effect from the nitrile.
O
OAll
BnOBnO
O
BnOBnO
OBn
OAcOTCA
(172)
(191)
(a)O
BnOBnO
OBn
OBn
O
(192) (193)
O
BnOBnO
OBn
OBnO O
BnO
OAllBnO
40% 40%
OAllO
HOOH
OBn
a) i) Et2OBF3, CH2Cl2; ii) KCN, MeOH; iii) BnBr, NaH, DMF.
101
Although glycosylation at O-2 had been successfully concluded in the previous section,
culminating in a synthesis of 3-O-β-D-glucopyranosylisofagomine (28) and 3-O-β-D-
glucopyranosylnoeuromycin (20), it seemed worthwhile transforming (193) into 3-O-β-
D-glucopyranosylnoeuromycin (20) (for a second synthesis).
Removal of the allyl group from (193) under standard conditions provided the alcohol
(194) in excellent yield. Treatment of the alcohol (194) with LiHMDS and sulfuryl
diimidazole gave the imidazylate (195), which was found to be quite unstable and used
immediately.
O
BnOBnO
OBn
OBnO
(196)
(d)
OBn
OOBn
CN
O
BnOBnO
OBn
OBnO
(197)
OBn
OOBn
(a)
(194)
(b)
(195)
(c)
(193)
O
BnOBnO
OBn
OBnO O
BnO
OAllBnO O
BnOBnO
OBn
OBnO O
BnO
OHBnO
O
BnOBnO
OBn
OBnO O
BnO
OSO2ImBnO
BocHN
a) i) Wilkinson’s catalyst, EtOH; ii) 1 M HCl, 91%;
b) (Me3Si)2NLi , (Im)2SO2, THF, 88%; c) Me3SiCN, Bu4NF, MeCN, 75%;
d) i) AlH3, THF; ii) (Boc)2O, 65%.
102
Treatment of the imidazylate (195) with TMSCN and TBAF gave the nitrile (196) in
moderate yield. The only modification to the previously used procedure (for the
introduction of the nitrile) was the introduction of a trace of TBAF prior to reflux to
prevent decomposition of the imidazylate. A conformational change from 1C4 to 4C1 was
observed, indicated by a large value for J4,5 (10 Hz), and small values for J2,3 (3.5 Hz)
and J1,2 (2.1 Hz). Reduction of the nitrile (196) using alane, followed by in situ treatment
with di-tert-butyl dicarbonate, gave the carbamate (197) in moderate yield.
Debenzylation of (197), followed by treatment with 1 M hydrochloric acid yielded 3-O-
β-D-glucopyranosylnoeuromycin (20) consistent with the material just previously
prepared.
O
BnOBnO
OBn
OBnO
(197)
OBn
OOBn
O
HOHO
OH
OHO NH
HO
OH
OH
(20)
(a)
BocHN
a) i) Pd/C, H2, THF, H2O; ii) 1 M HCl, 97%.
The other allyl ether (192) was subjected to an identical sequence: removal of the allyl
group, formation of the imidazylate (199), displacement to give the nitrile (200) and
subsequent reduction and protection to afford (201).
103
O
OAll
BnOBnO
O
BnOBnO
OBn
OBn
O
(192)
(a)
O
OH
BnOBnO
O
BnOBnO
OBn
OBn
O
(198)
O
OSO2Im
BnOBnO
O
BnOBnO
OBn
OBn
O
(199)
(201)
(200)
O
BnOBnO
O
BnOBnO
OBn
OBn
O
NC
O
BnOBnO
O
BnOBnO
BnO
OBn
O
(b)
(c)
(d)
BocHN
a) i) Wilkinson’s catalyst, EtOH; ii) 1 M HCl, 98%;
b) (Me3Si)2NLi, (Im)2SO2, THF, 72%; c) Me3CN, Bu4NF, MeCN, 60%;
d) i) AlH3, THF; ii) (Boc)2O, 81%.
Removal of the Boc protecting group from (201), followed by treatment with hydrogen,
resulted in debenzylation and reductive amination to afford the known 4-O-β-D-
glucopyranosylisofagomine (15).2
(a)
HO NH
O
OH
O
HOHO
OH
OH
(15)(201)
O
BnOBnO
O
BnOBnO
BnO
OBn
O
BocHN
a) i) CF3COOH; ii) Amberlite IRA 400 (OH−), MeOH;
iii) Pd/C, H2, MeOH, AcOH, 75%.
104
Attention then turned to the preparation of 4-O-β-D-glucopyranosylnoeuromycin (21).
Hydrogenolysis of (201) presumably yielded the hemiacetal; subsequent treatment with 1
M hydrochloric acid then gave 4-O-β-D-glucopyranosylnoeuromycin (21), together with
substantial amounts of the pyranose tautomer (202).
HO NH
O
OH
OH
(21)
O
HOHO
OH
OH
(202)
(a)
(201)
O
BnOBnO
O
BnOBnO
BnO
OBn
O
BocHN
O
OHOH
O
OHHO
HO
OH
O
H2N
a) i) Pd/C, H2, THF, H2O; ii) 1 M HCl, 72%.
High resolution 1H and 13C n.m.r. spectroscopy enabled the full identification and
characterization of (21) and partial characterization of (202), denoted using the following
nomenclature:
HO NH
O
OH
OHO
HOHO
OH
OH
(glc)
HO NH
OHO OH
O
HOHO
OH
OH
(man) (α or β)
1′5
1′
4
O
OHOH
O
OHHO
HO
OH
O
H2N
Again, the conformation of only one of the pyranose rings of (202) was known with
certainty.
105
2.002.503.003.504.004.505.005.50
(21) (202)
H2(man)
H1β
H2(glc)
H1′(glc), H1′(man)
H5(glc),H5(man)
H4α, H4βH3β
H3α
H6(glc)H4(man)
HO NH
O
OH
OHO
HOHO
OH
OH
H1′α, H1′βCH2Nα, CH2Nβ
O
OHOH
O
OHHO
HO
OH
O
H2N
1H n.m.r. (600 MHz) spectrum of 4-O-β-D-glucopyranosylnoeuromycin (21) and the
tautomeric form (202) (as the hydrochlorides).
The characteristic resonances of (21) compared closely to those of noeuromycin,
especially H2 (δ 4.57 and δ 5.18) and H5 (δ 2.00-2.11). Confirmation of the structure of
(202) was a little more difficult, however, H1′ (δ 4.52), H3 (δ 4.04 and δ 4.14) and H4 (δ
2.41-2.48) were characteristic of this ring system.
106
HO NH
O
OH
OH
(21)
O
HOHO
OH
OH
(202)
C1′(glc)C1′(man)
C1′α C1α
C1β
405060708090100
C4α
C6(man)
CH2Nα
C5(glc), C5(man)C6(glc)
C6′(glc), C6′(man)CH2O(glc), CH2O(man)
C2(glc)
C4(glc)
C3α
C2(man)
C4(man)C5αC6′α
O
OHOH
O
OHHO
HO
OH
O
H2N
13C n.m.r. (600 MHz) spectrum of 4-O-β-D-glucopyranosylnoeuromycin (21) and the
tautomeric form (202) (as the hydrochlorides).
The 13C n.m.r. spectrum provided more conclusive evidence for the structure of (21),
showing the characteristic peaks for C2 (δ 77.73 and δ 80.88), C5 (δ 39.76 and δ 40.08)
and C6 (δ 37.11 and δ 40.96), with a characteristic downfield shift of C4 (δ 76.63 and δ
79.22). The structure of (202), α-anomer was supported by the shift for C1 (δ 96.91), C4
(δ 36.19) and a downfield shift of C3 (δ 78.59). The β-anomer was supported by the shift
for C1 (δ 92.15); unfortunately its low abundance made conclusive characterization
difficult.
X-Ray crystallographic data were again provided through a collaboration with Gideon
Davies and Tracey Gloster of the University of York, with determination of 4-O-β-D-
107
glucopyranosylnoeuromycin (20) in complex with a family 5 endocellulase Cel5A from
Bacillus agaradhaerens at pH 6.0 and 1.50 Å resolution.5 Although not directly observed,
it would be anticipated that the nitrogen would be ‘doubly’ protonated. At pH 6.0, the pH
for optimum catalysis, 4-O-β-D-glucopyranosylnoeuromycin (21) was shown to have Ki
24 nM. Inhibition was also measured at pH 7.0 with the endo-glycanase Cex from
Cellulomonas fimi and shown to exhibit excellent inhibition (Ki 28 nM). In both case the
inhibition was slow-onset and required pre-incubation for one hour to effect inhibition.
acid/base
nucleophile
Three-dimensional structure of the endocellulase (Cel5A) from Bacillus agaradhaerens
in complex with 4-O-β-D-glucopyranosylnoeuromycin (21).
108
The inhibition of the endocellulase Cel5A from Bacillus agaradhaerens with 4-O-β-D-
glucopyranosylisofagomine (15) has been previously reported (Ki 700 nM).10 The
inhibition of the endo-glycanase Cex from Cellulomonas fimi with (15) has also been
reported (Ki 2000 nM).2 Comparison to the above inhibition values for (21) conclusively
shows that the additional interaction provided by the hydroxyl at C2, in 4-O-β-D-
glucopyranosylnoeuromycin (21) plays a significant role in binding, with a 30- and 70-
fold increase in inhibition respectively.
HO NH
O
OH
OH
(21)
O
HOHO
OH
OH
HO NH
O
OH
O
HOHO
OH
OH
(15)
109
Experimental
(170)
O
O
NCOH
OOBn
OAc
OAcOAc
AcO
Benzyl 4-C-Cyano-4-deoxy-2-O-(tetra-O-acetyl-β-D-glucosyl)-α-D-arabinoside (170)
A mixture of tetra-O-acetyl-β-D-glucopyranosyl trichloroacetimidate (163) (254 mg,
0.531 mmol),11 the nitrile (101) (110 mg, 0.443 mmol) and 4Å molecular sieves (100 mg)
in dry CH2Cl2 was stirred (rt, 3 h). The mixture was cooled (−60°C), treated with
Et2OBF3 (20 μL) and allowed to warm (rt); treatment with Et3N (100 μL), followed by
filtration, concentration of the filtrate and flash chromatography (EtOAc/petrol, 1:1) gave
the disaccharide (170) (195 mg, 76%) as a colourless oil, [α]D +25.1°. δH (600 MHz)
1.83, 1.98, 2.02, 2.10 (4×s, 12H, CH3), 3.20 (m, H4), 3.58 (dd, J5,5 12.3, J4,5 2.9, H5),
3.66 (dd, J2,3 7.7, J1,2 6.2, H2), 3.76-3.79 (m, H5′), 3.85 (dd, J3,4 5.0, H3), 4.13 (dd, J4,5
4.2, H5), 4.14 (dd, J6′,6′ 12.3, J5′,6′ 4.2, H6′), 4.20 (dd, J5′,6′ 2.4, H6′), 4.45 (d, H1), 4.54,
4.87 (AB, J 11.6, PhCH2), 4.68 (d, J1′,2′ 8.1, H1′), 4.99 (m, H2′, H4′), 5.20 (dd, J 9.5, 9.5,
H3′), 7.31-7.37 (m, Ph). δC (150.9 MHz) 20.50, 20.63, 20.75 (CH3), 34.21 (C4), 60.44
(C5), 61.85 (C6′), 68.38, 68.49 (C3, C4′), 70.91 (PhCH2), 71.08 (C2′), 72.18 (C5′), 72.54
(C3′), 80.20 (C2), 99.94 (C1), 101.05 (C1′), 117.69 (CN), 127.77-136.50 (Ph), 169.44,
169.52, 170.15, 170.89 (4C, C=O). m/z (FAB) 580.2045 (C27H34NO13 [M+H]+ requires
580.2030).
110
(171)
O
O
NCOAc
OOBn
OAc
OAcOAc
AcO
Benzyl 3-O-Acetyl-4-C-cyano-4-deoxy-2-O-(tetra-O-acetyl-β-D-glucosyl)-α-D-
arabinoside (171)
The nitrile (170) (40 mg) in CH2Cl2 (1.5 mL) was treated with pyridine (200 μL), acetic
anhydride (100 μL) and DMAP (2 mg) and allowed to stand (rt, 2h). The solution was
treated with MeOH, concentrated and subjected to flash chromatography (EtOAc/petrol,
1:1) to give the pentacetate (171) (32 mg, 55%) as a colourless oil, [α]D +9.8°. δH (600
MHz) 1.95, 1.99, 2.02, 2.08, 2.09 (5×s, 15H, CH3), 3.36-3.39 (m, H4), 3.68 (dd, J5,5 11.8,
J4,5 3.5, H5), 3.68-3.73 (m, H5′), 3.90 (dd, J2,3 6.4, J1,2 4.5, H2), 4.08 (dd, J6′,6′ 12.3, J5′,6′
2.3, H6′), 4.19 (dd, J4,5 6.7, H5), 4.30 (dd, J5′,6′ 5.7, H6′), 4.51, 4.83 (AB, J 11.4, PhCH2),
4.54 (d, H1), 4.73 (d, J1′,2′ 8.0, H1′), 4.96 (dd, J2′,3′ 9.6, H2′), 5.03 (dd, J4′,5′ ≈ J3′,4′ 9.8,
H4′), 5.09 (dd, J3,4 4.3, H3), 5.17 (dd, H3′), 7.25-7.35 (m, Ph). δC (150.9 MHz) 20.70,
20.72, 20.74, 20.80 (CH3), 30.88 (C4), 58.85 (C5), 62.01 (C6′), 67.75 (C4′), 68.45 (C3),
70.77 (PhCH2), 71.41 (C2′), 72.12 (C5′), 72.85 (C3′), 74.00 (C2), 99.53 (C1), 100.33
(C1′), 116.78 (CN), 127.96-136.73 (Ph), 169.30, 169.55, 170.13, 170.33, 170.81 (5C,
C=O). m/z (FAB) 622.2147 (C29H36NO14 [M+H]+ requires 622.2135).
111
O
O
NCOH
OOBn
OAc
OBnOBn
BnO
(173)
Benzyl 2-O-(2-O-Acetyl-3,4,6-tri-O-benzyl-β-D-glucosyl)-4-C-cyano-4-deoxy-α-D-
arabinoside (173)
A mixture of 2-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucosyl trichloroacetimidate (172)
(1.165 g, 1.835 mmol),12 the nitrile (101) (457 mg, 1.84 mmol) and 4Å molecular sieves
(1.0 g) in dry CH2Cl2 (15 mL) was stirred (rt, 3 h). The mixture was cooled (−60°C),
treated with Et2OBF3 (100 μL) and allowed to warm (rt); treatment with Et3N (100 μL),
followed by filtration, concentration of the filtrate and flash chromatography
(EtOAc/petrol, 1:1) gave the disaccharide (173) (910 mg, 74%) as a colourless oil, [α]D
+8.5°. δH (600 MHz) 1.77 (s, CH3), 3.20-3.22 (m, H4), 3.53-3.57 (m, H5′), 3.58 (dd, J5,5
12.2, J4,5 2.9, H5), 3.64-3.75 (m, 5H, H2, H3′, H4′, H6′), 3.88 (dd, J3,4 5.0, J2,3 7.7, H3),
4.13 (dd, J4,5 4.1, H5), 4.46 (d, J1,2 6.0, H1), 4.52, 4.88 (AB, J 11.8, PhCH2), 4.54, 4.78
(AB, J 10.9, PhCH2), 4.56 (d, J1′,2′ 7.8, H1′), 4.57, 4.60 (AB, J 12.2, PhCH2), 4.67, 4.79
(AB, J 11.4, PhCH2), 5.01 (dd, J2′,3′ 9.1, H2′), 7.25-7.37, 7.16-7.19 (2×m, Ph). δC (150.9
MHz) 20.78 (CH3), 34.22 (C4), 60.45 (C5), 68.37 (C6′), 68.66 (C3), 70.83, 73.65, 75.17,
75.19 (4C, PhCH2), 72.96 (C2′), 74.83 (C5′), 77.64, 82.69 (C3′, C4′), 79.98 (C2), 100.03
(C1), 101.27 (C1′), 117.73 (CN), 127.82-138.10 (Ph), 169.64 (C=O). m/z (FAB)
722.2936 (C42H44NO10 [M−H]+ requires 722.2965).
112
(174)
O
O
NCOH
OOBn
OH
OBnOBn
BnO
Benzyl 4-C-Cyano-4-deoxy-2-O-(3,4,6-tri-O-benzyl-β-D-glucosyl)-α-D-arabinoside (174)
The nitrile (173) (270 mg) in MeOH (40 mL) was treated with HCl in MeOH (1 M, 2
mL) and stirred (40°C, 14 h). The solution was neutralized with Et3N, concentrated and
subjected to flash chromatography (EtOAc/petrol, 1:1) to give the nitrile (174) as a
colourless oil (195 mg, 77%), [α]D −4.0°. δH (600 MHz) 3.22-3.26 (m, H4), 3.50-3.69 (m,
8H, H2, H2′, H3, H3′, H4′, H5, H5′, H6′), 3.87 (dd, J6′,6′ 7.8, J5′,6′ 5.0, H6′), 4.18 (dd, J5,5
12.2, J4,5 4.0, H5), 4.40 (d, J1′,2′ 7.6, H1′), 4.50, 4.57 (AB, J 12.2, PhCH2), 4.51 (d, J1,2
6.9, H1), 4.53, 4.92 (AB, J 11.0, PhCH2), 4.57, 4.81 (AB, J 12.0, PhCH2), 4.82, 4.88
(AB, J 11.8, PhCH2), 7.15-7.18, 7.25-7.38 (2×m, Ph). δC (150.9 MHz) 34.27 (C4), 60.71
(C5), 68.54 (C6′), 68.80 (C3), 70.92, 73.65, 75.18, 75.29 (4C, PhCH2), 74.75, 75.02 (C2′,
C5′), 77.13, 80.99, 84.22 (C2, C3′, C4′), 99.83 (C1), 103.90 (C1′), 117.91 (CN), 127.91-
138.64 (Ph). m/z (FAB) 681.2979 (C40H43NO9 [M]+• requires 681.2938).
(175)
O
O
OHO
OBn
OH
OBnOBn
BnOBocHN
Benzyl 4-C-[(tert-Butoxycarbonyl)amino]methyl-4-deoxy-2-O-(3,4,6-tri-O-benzyl-β-D-
glucosyl)-α-D-arabinoside (175)
Freshly prepared AlH3 in THF13 (1.23 M, 2 mL, 2.46 mmol) was added to the nitrile
(174) (160 mg, 0.236 mmol) in dry THF (4 mL, 0°C) and the solution stirred (2 h). The
mixture was quenched by the addition of NaOH (1 mL, 1.0 M), followed by treatment
with (Boc)2O (180 mg, 0.826 mmol) (2 h, rt). The mixture was then filtered, diluted with
113
EtOAc (100 mL), dried, concentrated and subjected to flash chromatography
(EtOAc/petrol, 1:1) to give the carbamate (175) (140 mg, 76 %) as a colourless solid. A
small amount was further purified by recrystallisation, m.p. 128-129ºC (MeOH) (Found
C, 68.7; H, 7.1; N, 1.9. C45H55NO11 requires C, 68.8; H, 7.0; N, 1.9%), [α]D +20.8°. δH
(600 MHz) 1.44 (s, 9H, CH3), 2.19-2.23 (m, H4), 3.30-3.37 (br m, CH2N), 3.48-3.60 (m,
6H, H2, H2′, H3′, H4′, H5, H5′), 3.62 (dd, J6′,6′ 10.7, J5′,6′ 5.0, H6′), 3.68 (dd, J5′,6′ 2.0,
H6′), 3.89 (dd, J3,4 5.0, J2,3 7.7, H3), 3.91 (dd, J5,5 12.6, J4,5 5.1, H5), 4.35 (d, J1′,2′ 7.6,
H1′), 4.51, 4.54 (AB, J 12.2, PhCH2), 4.52 (d, J1,2 6.5, H1), 4.54, 4.83 (AB, J 12.2,
PhCH2), 4.58, 4.83 (AB, J 11.5, PhCH2), 4.80, 4.94 (AB, J 11.2, PhCH2), 7.15-7.17,
7.25-7.37 (2×m, Ph). δC (150.9 MHz) 28.33 (CH3), 39.05 (C4), 39.18 (CH2N), 62.33
(C5), 68.53 (C6′), 70.35, 73.37, 74.95, 74.96 (4C, PhCH2), 70.72 (C3), 74.71, 74.86 (C2′,
C5′), 76.99, 79.06, 84.07 (C2, C3′, C4′), 80.22 (CH3C), 99.67 (C1′), 103.56 (C1), 127.59-
138.49 (Ph), 156.00 (C=O). m/z (FAB) 786.3897 (C45H56NO11 [M+H]+ requires
786.3853).
O
OBn
O
OAc
O
BnOBnO
OBn
OAc
(176)
BocHN
Benzyl 3-O-Acetyl-2-O-(2-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucosyl)-4-C-[(tert-
butoxycarbonyl)amino]methyl-4-deoxy-α-D-arabinoside (176)
The diol (175) (6.0 mg) in CH2Cl2 (1.5 mL) was treated with pyridine (100 μL), acetic
anhydride (50 μL) and DMAP (0.5 mg) and stirred (rt, 2 h). The solution was treated with
MeOH, concentrated and subjected to flash chromatography (EtOAc/petrol, 1:1) to give
the diacetate (176) (5.9 mg, 85%) as a colourless oil, [α]D +60.0°. δH (600 MHz) 1.41 (s,
114
9H, CH3C), 1.88, 1.98 (2×s, 6H, CH3CO), 2.27-2.33 (m, H4), 3.05-3.10 (m, CH2N), 3.45-
3.48 (m, 2H, H5, H5′), 3.62 (dd, J3′,4′ 9.3, J2′,3′ 8.7, H3′), 3.69-3.81 (m, 4H, H2, H4′, H6′),
3.87 (dd, J5,5 11.4, J4,5 9.3, H5), 4.42, 4.80 (AB, J 11.7, PhCH2), 4.50 (d, J1′,2′ 8.1, H1′),
4.55, 4.64 (AB, J 12.2, PhCH2), 4.56, 4.64 (AB, J 10.7, PhCH2), 4.59-4.62 (m, H1, NH),
4.65, 4.77 (AB, J 11.4, PhCH2), 4.96 (dd, H2′), 5.06-5.08 (br m, H3), 7.16-7.18, 7.25-
7.34 (2×m, Ph). δC (150.9 MHz) 20.69, 20.78 (2C, CH3CO), 28.66 (CH3C), 35.29 (C4),
37.90 (CH2N), 58.90 (C5), 68.44 (C6′), 68.64 (C3), 69.33, 73.44, 74.85, 74.90 (4C,
PhCH2), 72.91 (C2′), 73.31 (C2), 75.00 (C5′), 77.64 (C4′), 79.20 (CH3C), 82.64 (C3′),
98.24 (C1), 100.72 (C1′), 127.32-138.03 (Ph), 155.72 (NC=O), 169.26, 170.38 (2C,
CH3C=O). m/z (FAB) 870.4078 (C49H60NO13 [M+H]+ requires 870.4065).
O
HOHO
OH
OHO NH2Cl
HO
OH
(28)
(3R,4R,5R)-3-(β-D-Glucopyranosyloxy)-5-(hydroxymethyl)piperidin-4-ol (3-O-β-D-
Glucopyranosylisofagomine) (28) hydrochloride
The disaccharide (175) (0.032 mmol) in CH2Cl2 (1 mL) was treated with Et2OBF3 (100
μL) and stirred (rt, 2 h). The solution was then treated with resin (Amberlite IRA 400,
OH–) until neutral, filtered and then concentrated. The residue was taken up in
MeOH/AcOH (100:1, 15 mL), then treated with Pd/C (10%, 5 mg) and H2 (1 atm , 12 h).
Filtration followed by concentration of the filtrate gave a colourless foam that was
dissolved in hydrochloric acid (1 M, 1 mL) and applied to a cation-exchange column
(Dowex 50W-X2, H+ form). The column was washed with water and eluted with aqueous
NH3 (1.5 M), the eluate was concentrated and the residual foam taken up in a little
hydrochloric acid (1 M, 1 mL) and again concentrated to give 3-O-β-D-
115
glucopyranosylisofagomine (28) hydrochloride (6.5 mg, 66%) as a colourless glass. The
1H (600 MHz) and 13C (150.9 MHz) n.m.r. spectral data were in good agreement with
those reported.1
O
HOHO
OH
OHO NH2Cl
HO
OH
OH
(20)
(2R/2S,3R,4R,5R)-3-(β-D-Glucopyranosyloxy)-5-(hydroxymethyl)piperidine-2,4-diol (3-
O-β-D-Glucopyranosylnoeuromycin) (20) hydrochloride
The disaccharide (175) (25 mg) in THF/H2O (1:1, 15 mL) was treated with Pd/C (10%,
10 mg) and H2 (1 atm, 48 h). The mixture was filtered, concentrated and subjected to
flash chromatography (THF/H2O, 20:1) to give presumably the hemiacetal as a colourless
oil. This was then dissolved in hydrochloric acid (1 M, 1.5 mL) and allowed to stand (15
min); concentration of the mixture then gave 3-O-β-D-glucopyranosylnoeuromycin (20)
hydrochloride (9.1 mg, 79%) as a colourless glass. δH (600 MHz, D2O) 1.85-1.95 (m,
H5), 2.90 (dd, J 13.1, 13.1, H6glc), 3.22-3.85 (H2′, H3, H3′, H4, H4′, H5′, H6, H6man,
H6′, CH2O), 4.51 (d, J1′,2′ 7.9, H1′glc), 4.67-4.73 (m, H1′man, H2glc), 5.34 (d, J2,3 2.4,
H2man). δC (150.9 MHz, D2O) 38.23 (C6glc), 40.42, 40.71 (C5), 41.08 (C6man), 58.75,
58.85, 60.52 (C6′, CH2O), 65.18, 68.25, 69.32, 69.42, 75.36, 75.45, 75.80, 75.96 (C3′,
C4, C4′, C5′), 72.69, 73.11 (C2′), 76.00 (C2man), 77.97 (C3man), 80.64 (C3glc), 80.75
(C2glc), 101.00 (C1′glc), 102.19 (C1′man). m/z (FAB) 326.1454 (C12H24NO9 [M−Cl]+
requires 326.1451).
116
(180)
O
NCOBz
OHOBn
Benzyl 3-O-Benzoyl-4-C-cyano-4-deoxy-α-D-arabinoside (180)
The nitrile (101) (39 mg, 0.16 mmol) in dry CH2Cl2 (2 mL) was treated with benzoyl
chloride (26 mg, 0.19 mmol) and triethylamine (19 mg, 0.19 mmol) and stirred (rt, 12 h).
The solution was then concentrated and subjected to flash chromatography
(EtOAc/petrol, 1:1) to give the benzoate (180) (42 mg, 78%) as a colourless oil, [α]D
−24.8°. δH (600 MHz) 3.48-3.51 (m, H4), 3.75 (dd, J5,5 12.1, J4,5 2.9, H5), 4.02-4.04 (m,
H2), 4.29 (dd, J4,5 4.3, H5), 4.50 (d, J1,2 5.9, H1), 4.60, 4.92 (AB, J 11.5, PhCH2), 5.21
(dd, J3,4 4.9, J2,3 8.0, H3) 7.20-7.30 (m, Ph). δC (150.9 MHz) 32.11 (C4), 60.46 (C5),
68.81, 70.02 (C2, C3), 70.76 (PhCH2), 101.28 (C1), 116.81 (CN), 128.00-136.48 (Ph),
165.73 (C=O). m/z (FAB) 354.1347 (C20H20NO5 [M+H]+ requires 354.1341).
(182)
O OBn
OBnNC
(181)
O
NCOBz
OBnOBn
Benzyl 3-O-Benzoyl-2-O-benzyl-4-C-cyano-4-deoxy-α-D-arabinoside (181) and
(5S,6S)-5,6-Di(benzyloxy)-5,6-dihydro-2H-pyran-3-carbonitrile (182)
a) The nitrile (180) (43 mg, 0.12 mmol) and benzyl bromide (31 mg, 0.18 mmol) in
CH2Cl2 (2 mL) were treated with Ag2CO3 (50 mg, 0.18 mmol) and stirred (reflux, 12 h).
The suspension was then filtered, the filtrate was concentrated and subjected to flash
chromatography (EtOAc/petrol, 1:3) to give firstly the alkene (182) (21 mg, 54%) as a
colourless oil, [α]D +80.5°. δH (600 MHz) 3.86 (m, H5), 4.23 (ddd, J2,2 16.3, J 1.5, 1.5,
H2), 4.27 (ddd, J 2.1, 2.1, H2), 4.61, 4.68 (AB, J 11.8, PhCH2), 4.61, 4.81 (AB, J 11.9,
PhCH2), 4.93 (d, J5,6 2.4, H6), 6.56-6.58 (m, H4), 7.28-7.39 (m, Ph). δC (150.9 MHz)
117
60.10 (C2), 70.09 (C5), 70.44, 72.44 (2C, PhCH2), 97.74 (C6), 115.24, 115.61 (C3, CN),
128.04-127.37 (Ph), 139.05 (C4). m/z (FAB) 321.1348 (C20H19NO3 [M]+• requires
321.1365).
Further elution (EtOAc/petrol, 1:2) gave the benzyl ether (181) (8 mg, 15%) as a
colourless oil, [α]D −19.8°. δH (600 MHz) 3.50-3.57 (m, H4), 3.78 (dd, J5,5 11.8, J4,5 3.5,
H5), 3.83 (dd, J2,3 6.4, J1,2 4.5, H2), 4.31 (dd, J4,5 6.6, H5), 4.57, 4.88 (AB, J 11.6,
PhCH2), 4.71 (d, H1), 4.74, 4.78 (AB, J 11.6, PhCH2), 5.33 (dd, J3,4 4.4, H3), 7.25-7.30,
7.37-7.41, 7.55-7.60, 7.96-8.00 (4×m, Ph). δC (150.9 MHz) 31.40 (C4), 58.99 (C5), 68.48
(C3), 70.71, 74.19 (2C, PhCH2), 74.32 (C2), 100.26 (C1), 116.96 (CN), 128.02-137.40
(Ph), 165.71 (C=O). m/z (FAB) 444.1806 (C27H26NO5 [M+H]+ requires 444.1811).
Further elution (EtOAc/petrol, 1:2) gave unreacted starting material (180) (8 mg).
b) The nitrile (180) (83.6 mg, 0.237 mmol) and benzyl trichloroacetimidate6 (238 mg,
0.947 mmol) in dry CH2Cl2 (3 mL) were stirred with 4Å molecular sieves (200 mg) (rt, 1
h). The mixture was treated with CF3SO3H (20 µL) (rt, 4 h), diluted with CH2Cl2,
filtered, and the filtrate washed with saturated NaHCO3 and brine. Concentration of the
organic layer followed by flash chromatography (EtOAc/petrol, 1:3) gave (181) (49.5
mg, 50%), with properties identical to those reported in (a).
O
OBn
BnO
OH
NC
(183)
Benzyl 2-O-Benzyl-4-C-cyano-4-deoxy-α-D-arabinoside (183)
The benzyl ether (181) (20 mg, 0.045 mmol) in MeOH (2 mL) was treated with KCN (3
mg, 0.046 mmol) and the mixture stirred (rt, 4 h). The solution was then concentrated,
adsorbed onto silica and subjected to flash chromatography (EtOAc/petrol, 1:2) to afford
118
the alcohol (183) (10 mg, 67%) as a colourless oil, [α]D +134.3°. δH (600 MHz) 3.30-3.40
(m, H4), 3.55 (dd, J2,3 4.6, J1,2 3.0, H2), 3.78 (dd, J5,5 11.8, J4,5 4.4, H5), 4.05 (m, H3),
4.11 (dd, J4,5 9.7, H5), 4.55, 4.81 (AB, J 11.7, PhCH2), 4.58, 4.68 (AB, J 11.8, PhCH2),
4.78 (d, H1), 7.20-7.30 (m, Ph). δC (150.9 MHz) 31.16 (C4), 56.23 (C5), 66.56 (C3),
70.24, 73.00 (2C, PhCH2), 73.82 (C2), 97.70 (C1), 117.51 (CN), 127.76-130.77 (Ph). m/z
(FAB) 340.1545 (C20H32NO4 [M+H]+ requires 340.1549).
O
OBn
Et3SiO
OBz
NC
(184)
Benzyl 3-O-Benzoyl-4-C-cyano-4-deoxy-2-O-triethylsilyl-α-D-arabinoside (184)
The alcohol (180) (18.6 mg, 0.053 mmol) in pyridine (1 mL) was treated with DMAP
(0.6 mg), Et3SiCl (15.9 mg, 0.105 mmol) and the mixture stirred (rt, 4 h). Concentration
of the mixture, followed by flash chromatography (EtOAc/petrol, 1:3) gave the silyl ether
(184) (15 mg, 60%) as a colourless oil, [α]D +3.1°. δH (600 MHz) 0.60 (t, 9H, J 7.9, CH3),
0.91 (q, 6H, CH2Si), 3.54 (m, H4), 3.80 (dd, J5,5 11.5, J4,5 3.7, H5), 3.99 (dd, J2,3 5.0, J1,2
3.3, H2), 4.33 (dd, J4,5 8.2, H5), 4.51, 4.83 (AB, J 11.5, PhCH2), 4.59 (d, H1), 5.20 (dd,
J3,4 4.8, H3), 7.26-7.28, 7.35-7.40, 7.55-7.59, 7.99-8.03 (4×m, Ph). δC (150.9 MHz) 4.82
(CH3), 6.78 (CH2Si), 30.05 (C4), 57.94 (C5), 67.36 (C3), 69.83 (C2), 70.44 (PhCH2),
100.37 (C1), 117.15 (CN), 127.99-137.00 (Ph), 165.72 (C=O). m/z (FAB) 468.2176
(C26H34NO5Si [M+H]+ requires 468.2206).
119
(185)
O
NCOBz
OOBn
O
Benzyl 3-O-Benzoyl-4-C-cyano-4-deoxy-2-O-(tetrahydro-2H-pyran-2-yl)-α-D-
arabinoside (185)
The alcohol (180) (50.6 mg, 0.143 mmol) in dry CH2Cl2 (2 mL) was treated with 3,4-
dihydro-2H-pyran (19.3 mg, 0.229 mmol) and CSA (2 mg) and the solution stirred (1 h).
The solution was neutralized with Et3N (200 μL), diluted with CH2Cl2 (50 mL) and
washed with saturated NaHCO3; the organic layer was dried, concentrated and subjected
to flash chromatography (EtOAc/petrol, 1:3) to give the tetrahydropyranyl ether (180) (58
mg, 94%) as a colourless oil and a mixture of diastereoisomers. Further purification
enabled partial separation of the diastereoisomers. Diastereoisomer A: δH (600 MHz)
1.45-1.60, 1.70-1.75, 1.78-1.85 (3×m, 6H, CH2), 3.46-3.50 (m, 1H, CH2O), 3.54 (m, H4),
3.82-3.87 (m, 2H, H5, CH2O), 4.04 (dd, J2,3 3.8, J1,2 2.1, H2), 4.37 (dd, J4,5 ≈ J5,5 11.1,
H5), 4.51, 4.81 (AB, J 11.5, PhCH2), 4.84 (dd, J 4.6, J 3.0, OCHO), 4.94 (d, H1), 5.34
(dd, J3,4 3.7, H3), 7.24-7.35, 7.53-7.57, 7.95-7.97 (3×m, Ph). δC (150.9 MHz) 19.60,
25.27, 30.87 (3C, CH2), 29.32 (C4), 56.53 (C5), 63.20 (CH2O), 67.38 (C2), 70.20 (C3),
70.26 (PhCH2), 99.70 (C1), 99.87 (OCHO), 116.83 (CN), 127.90-137.19 (Ph), 165.66
(C=O). Diastereoisomer B: δH (600 MHz) 1.38-1.45, 1.46-1.59, 1.65-1.75 (3×m, 6H,
CH2), 3.44-3.49 (m, H5), 3.51-3.54 (m, H4), 3.79-3.82 (m, 2H, CH2O, H5), 4.08 (dd, J2,3
6.1, J1,2 4.1, H2), 4.33 (dd, 1H, J 7.3, 11.7, CH2O), 4.55, 4.85 (AB, J 11.6, PhCH2), 4.69
(d, H1), 4.89 (dd, J 3.7, 3.7, OCHO), 5.42 (dd, J3,4 4.2, H3), 7.27-7.30, 7.35-7.38, 7.54-
7.57, 8.03-8.04 (4×m, Ph). δC (150.9 MHz) 19.30, 25.25, 30.59 (3C, CH2), 31.04 (C4),
58.58 (CH2O), 62.79 (C5), 68.52 (C3), 70.62 (PhCH2), 71.20 (C2), 99.03 (OCHO), 99.64
120
(C1), 117.02 (CN), 127.96-133.58 (Ph), 165.69 (C=O). m/z (FAB) 438.1887 (C25H28NO6
[M+H]+ requires 438.1917).
(186)
O
OBn
HO
OBMS
NC
(187)
O
NCOH
OBMSOBn
Benzyl 3-O-(tert-Butyldimethylsilyl)-4-C-cyano-4-deoxy-α-D-arabinoside (186) and
Benzyl 2-O-(tert-Butyldimethylsilyl)-4-C-cyano-4-deoxy-α-D-arabinoside (187)
The nitrile (101) (20.2 mg, 0.081 mmol) in DMF was treated with imidazole (27.6 mg,
0.406 mmol) and BMSCl (37 mg, 0.24 mmol) and the solution stirred (rt, 14 h). The
solution was then concentrated and subjected to flash chromatography (EtOAc/petrol,
1:3) to give the 3-O-silyl ether (186) (8.1 mg, 29%) as a colourless oil, [α]D +4.2°. δH
(600 MHz) 0.01, 0.05 (2×s, 6H, CH3Si), 0.09 (s, 9H, CH3C), 3.34 (ddd, J4,5 10.9, 4.7, J3,4
2.7, H4), 3.75 (dd, J2,3 3.8, J1,2 2.0, H2), 3.79 (dd, J5,5 11.6, J4,5 4.7, H5), 3.90-3.92 (m,
H3), 4.09 (dd, J4,5 10.9, H5), 4.53, 4.79 (AB, J 11.7, PhCH2), 4.63 (br m, H1), 7.31-7.38
(m, Ph). δC (150.9 MHz) −5.15 (CH3Si), 17.83 (CH3C), 25.50 (CH3C), 30.00 (C4), 55.22
(C5), 67.30 (C2), 68.56 (C3), 70.08 (PhCH2), 98.63 (C1), 117.79 (CN), 128.05-135.81
(Ph). m/z (FAB) 364.1937 (C19H24NO4Si [M+H]+ requires 364.1944).
Further elution gave the 2-O-silyl ether (187) (19.6 mg, 70%), [α]D +98.1°. δH (600 MHz)
0.01 (s, 6H, CH3Si), 0.91 (s, 9H, CH3C), 3.03 (ddd, J4,5 3.2, 2.7, J3,4 5.3, H4), 3.57 (dd,
J5,5 12.1, J4,5 2.7, H5), 3.66 (dd, J2,3 7.8, J1,2 6.4, H2), 3.78 (dd, H3), 4.20 (dd, J4,5 3.2,
H5), 4.33 (d, H1), 4.58, 4.89 (AB, J 11.7, PhCH2), 7.32-7.36 (m, Ph). δC (150.9 MHz)
−5.17, −4.64 (2C, CH3Si), 17.92 (CH3C), 25.50 (CH3C), 35.91 (C4), 60.90 (C5), 70.40
(C3), 70.60 (PhCH2), 72.26 (C2), 101.62 (C1), 117.69 (CN), 127.91-136.79 (Ph). m/z
(FAB) 364.1939 (C19H24NO4Si [M+H]+ requires 364.1944).
121
(189)
O
NCOAc
OBMSOBn
Benzyl 3-O-Acetyl-2-O-(tert-butyldimethylsilyl)-4-C-cyano-4-deoxy-α-D-arabinoside
(189)
The alcohol (187) (4.5 mg) was treated with pyridine (100 μL), acetic anhydride (50 μL)
and DMAP (0.5 mg) and left to stand (rt, 2 h). The solution was then treated with MeOH,
concentrated and subjected to flash chromatography (EtOAc/petrol, 1:1) to give the
acetate (189) (3.3 mg, 65%) as a colourless oil, [α]D +22.0°. δH (600 MHz) 0.04, 0.10
(2×s, 6H, CH3Si), 0.85 (s, 9H, CH3C), 2.11 (s, CH3CO), 3.37-3.42 (m, H4), 3.67 (dd, J5,5
11.8, J4,5 3.4, H5), 3.85 (dd, J2,3 6.6, J1,2 4.7, H2), 4.20 (dd, J4,5 6.2, H5), 4.44 (d, H1),
4.52, 4.84 (AB, J 11.7, PhCH2), 4.81 (dd, J3,4 4.4, H3), 7.32-7.35 (m, Ph). δC (150.9
MHz) −5.07, −4.84 (2C, CH3Si), 17.85 (CH3C), 20.73 (CH3CO), 25.50 (CH3C), 30.76
(C4), 58.92 (C5), 67.96, 70.22 (C2, C3), 70.35 (PhCH2), 100.82 (C1), 116.97 (CN),
127.80-136.80 (Ph), 170.11 (C=O). m/z (FAB) 406.2052 (C21H32NO5Si [M+H]+ requires
406.2050).
(188)
O
OBn
AcO
OBMS
NC
Benzyl 2-O-Acetyl-3-O-(tert-butyldimethylsilyl)-4-C-cyano-4-deoxy-α-D-arabinoside
(188)
The alcohol (186) (5 mg) was treated with pyridine (100 μL), acetic anhydride (50 μL)
and DMAP (0.5 mg) and left to stand (rt, 2 h). The solution was then treated with MeOH,
concentrated and subjected to flash chromatography (EtOAc/petrol, 1:1) to give the
acetate (188) (5.3 mg, 95%) as a colourless oil, [α]D +55.2°. δH (600 MHz) 0.12, 0.15
122
(2×s, 6H, CH3Si), 0.87 (s, 9H, CH3C), 2.09 (s, CH3CO), 3.13 (ddd, J4,5 9.5, 3.7, J3,4 3.5,
H4), 3.66 (dd, J5,5 10.8, J4,5 3.7, H5), 4.04 (dd, J2,3 3.4, H3), 4.31 (dd, J4,5 9.5, H5), 4.50,
4.74 (AB, J 11.8, PhCH2), 4.64 (d, J1,2 2.1, H1), 4.81 (dd, H2), 7.30-7.36 (m, Ph). δC
(150.9 MHz) −5.29, −4.83 (2C, CH3Si), 17.82 (CH3C), 20.80 (CH3CO), 25.50 (CH3C),
32.39 (C4), 56.11 (C5), 66.20 (PhCH2), 69.14, 70.22 (C2, C3), 96.56 (C1), 117.41 (CN),
127.80-136.84 (Ph), 169.27 (C=O). m/z (FAB) 406.2057 (C21H32NO5Si [M+H]+ requires
406.2050).
(191)
OAllO
HOOH
OBn
Benzyl 4-O-Allyl-β-L-xyloside (191)
The alcohol (79) (1.97 g, 7.09 mmol) in dry DMF (25 mL, 0°C) was treated with NaH
(60% in mineral oil, 480 mg, 12 mmol), allyl bromide (1.70 g, 14.2 mmol) and the
suspension stirred (0°C, 30 min). Methanol (5 mL) was added and the solution stirred
(0°C, 30 min), concentrated and taken up in MeOH (20 mL). The solution was treated
with CSA (200 mg) and stirred (rt, 30 min), followed by treatment with Et3N (1 mL).
Concentration of the solution followed by an aqueous workup and flash chromatography
(EtOAc/petrol, 3:2) gave the diol (191) as a colourless solid (1.76 g, 89%), m.p. 55-57ºC,
[α]D +116.5°. δH (600 MHz) 3.48-3.50 (m, 2H, H4, H5), 3.55 (dd, J2,3 5.8, J1,2 4.9, H2),
3.76 (dd, J3,4 6.1, H3), 4.08 (dd, J5,5 14.4, J4,5 5.3, H5), 4.13-4.15 (m, CH2O), 4.62 (d,
H1), 4.58, 4.84 (AB, J 11.7, PhCH2), 5.21-5.23, 5.28-5.32 (2×m, CH2CH), 5.87-5.94 (m,
CH2CH), 7.30-7.37 (m, Ph). δC (150.9 MHz) 60.44 (C5), 70.62 (PhCH2), 70.88 (C2),
71.01 (C3), 71.39 (CH2O), 76.44 (C4), 101.09 (C1), 117.99 (CH2CH), 128.26-136.79
(Ph), 134.25 (CH2CH). m/z (FAB) 281.1367 (C15H21O5 [M+H]+ requires 281.1389).
123
O
OAll
BnOBnO
O
BnOBnO
OBn
OBn
O
(192) (193)
O
BnOBnO
OBn
OBnO O
BnO
OAllBnO
Benzyl 4-O-Allyl-2-O-benzyl-3-O-(tetra-O-benzyl-β-D-glucosyl)-β-L-xyloside (192) and
Benzyl 4-O-Allyl-3-O-benzyl-2-O-(tetra-O-benzyl-β-D-glucosyl)-β-L-xyloside (193)
A solution of 2-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucosyl trichloroacetimidate (172) (3.95
g, 6.23 mmol)12 and the diol (191) (1.55 g, 5.56 mmol) in dry CH2Cl2 (15 mL) was
treated with 4Å molecular sieves (1.0 g) and stirred (rt, 3 h). The mixture was cooled
(−60°C), treated with Et2OBF3 (200 μL) and allowed to warm (rt, 1h); treatment with
Et3N (400 μL), followed by filtration and flash chromatography (EtOAc/petrol, 1:1) gave
a colourless oil. The oil in MeOH (15 mL) was treated with KCN (50 mg) and stirred
(50°C, 12 h). Concentration of the solution followed by flash chromatography
(EtOAc/petrol, 2:1) gave a colourless oil. The oil in dry DMF (25 mL, 0°C) was treated
with NaH (293 mg, 12.2 mmol) and BnBr (3.48 g, 20.3 mmol) and stirred (0°C, 30 min).
Treatment with MeOH (2 mL), followed by concentration of the mixture, an aqueous
workup and flash chromatography (EtOAc/toluene, 1:20) yielded firstly the 2-O-β-D-
glucosyl derivative (193) (1.92 g, 39%) as a colourless oil, [α]D +23.0°. δH (600 MHz)
3.24 (dd, J5,5 11.4, J4,5 9.8, H5), 3.38-3.40 (m, H5′), 3.46 (dd, J2′,3′ 8.5, J1′,2′ 8.0, H2′),
3.52-3.56 (m, H3, H4), 3.64 (dd, J3′,4′ 9.3, H3′), 3.70 (dd, J6′,6′ 11.5, J5′,6′ 5.3, H6′), 3.71
(dd, J4′,5′ 9.6, H4′), 3.76 (dd, J5′,6′ 1.7, H6′), 3.90 (dd, J2,3 8.1, J1,2 8.0, H2), 4.03 (dd, J4,5
4.3, H5), 4.14 (dd, 1H, J 12.7, 5.9, CH2O), 4.25 (dd, 1H, J 12.7, 5.4, CH2O), 4.40 (d, H1),
4.44, 4.77 (AB, J 11.0, PhCH2), 4.52, 4.55 (AB, J 12.2, PhCH2), 4.62, 4.85 (AB, J 10.8,
PhCH2), 4.76, 5.03 (AB, J 11.3, PhCH2), 4.81, 4.92 (AB, J 11.5, PhCH2), 4.85, 4.99 (AB,
124
J 10.8, PhCH2), 4.98 (d, H1′), 5.19 (m, 1H, CH2CH), 5.29 (m, CH2CH), 5.87-5.95 (m,
CH2CH) 7.20-7.45 (m, Ph). δC (150.9 MHz) 64.07 (C5), 69.04 (C6′), 71.67, 74.90, 74.96,
75.12, 75.85 (PhCH2), 72.50 (CH2O), 75.09 (C5′), 77.01 (C4′), 78.16, 78.27 (C2, C4),
82.40 (C3), 83.09 (C2′), 85.01 (C3′), 101.96 (C1′), 103.45 (C1), 117.23 (CH2CH),
127.46-139.11 (Ph), 134.99 (CH2CH). m/z (FAB) 893.4271 (C56H61O10 [M+H]+ requires
893.4205).
Further elution (EtOAc/toluene, 1:20) yielded the 3-O-β-D-glucosyl derivative (192) as a
colourless solid (1.93 g, 40%). A small amount was further purified by suspension in
petrol and filtration, m.p. 81-82ºC, [α]D +35.4°. δH (600 MHz) 3.17 (dd, J 11.1, 11.1, H5),
3.41 (dd, J2,3 8.3, J1,2 7.8, H2), 3.43 (dd, J2′,3′ 8.0, J1′,2′ 7.8, H2′), 3.45-3.54 (m, H4, H5′),
3.65-3.69 (m, 3H, H3′, H4′, H6′), 3.78 (dd, J6′,6′ 10.3, J5′,6′ 1.2, H6′), 3.95-4.03 (m, 4H,
H3, H5, CH2O), 4.47 (d, H1), 4.51 (s, 2H, PhCH2), 4.62, 5.08 (AB, J 10.7, PhCH2), 4.65,
4.93 (AB, J 11.9, PhCH2), 4.77, 4.81 (AB, J 11.4, PhCH2), 4.84, 4.85 (AB, J 10.0,
PhCH2), 4.89, 4.97 (AB, J 11.1, PhCH2), 4.98 (d, H1′), 5.06-5.07, 5.12-5.15 (2×m,
CH2CH), 5.80-5.86 (m, CH2CH), 7.20-7.40 (m, Ph). δC (150.9 MHz) 63.32 (C5), 69.13
(C6′), 71.22 (CH2CHCH2O), 71.99, 73.55, 74.69, 74.87, 75.12, 75.83 (6C, PhCH2), 75.19
(C5′), 78.37 (C4′), 78.64 (C4), 79.37 (C3), 80.12 (C2), 83.07 (C2′), 85.07 (C3′), 102.56
(C1′), 102.88 (C1), 117.61 (CH2CH), 127.42-138.94 (Ph), 134.78 (CH2CH). m/z (FAB)
893.4244 (C56H61O10 [M+H]+ requires 893.4205).
125
(194)
O
BnOBnO
OBn
OBnO O
BnO
OHBnO
Benzyl 3-O-Benzyl-2-O-(tetra-O-benzyl-β-D-glucopyranosyl)-β-L-xylopyranoside (194)
The allyl ether (193) (180 mg) in EtOH (5 mL) was treated with Wilkinson’s catalyst (20
mg) and the solution refluxed (3 h). The solution was then treated with hydrochloric acid
(1 M, 1 mL) and refluxed further (1 h). Treatment with Et3N (0.5 mL) followed by
concentration of the mixture and flash chromatography (EtOAc/toluene, 1:4) yielded the
alcohol (194) as a colourless oil (155 mg, 91%), [α]D +40.8°. δH (600 MHz) 3.36 (dd, J5,5
11.5, J4,5 8.2, H5), 3.38-3.41 (m, H5′), 3.48 (dd, J2′,3′ 8.5, J1′,2′ 8.0, H2′), 3.55 (dd, J3,4 7.4,
J2,3 7.3, H3), 3.65 (dd, J3′,4′ 8.9, H3′), 3.69 (dd, J4′,5′ 9.3, H4′), 3.70-3.76 (m, 3H, H4, H6′),
3.94 (dd, J1,2 6.1, H2), 4.10 (dd, J4,5 4.4, H5), 4.47 (A OF AB, 1H, J 11.2, PhCH2), 4.53,
4.58 (AB, J 12.2, PhCH2), 4.56 (d, H1), 4.60, 4.98 (AB, J 10.8, PhCH2), 4.63, 4.98 (AB,
J 10.9, PhCH2), 4.80-4.86 (m, 5H, H1′, PhCH2), 4.93 (A OF AB, 1H, J 11.3, PhCH2),
7.25-7.37 (m, Ph). δC (150.9 MHz) 64.08 (C5), 68.61 (C4), 68.99 (C6′), 71.19, 73.55,
73.82, 75.08, 75.16, 75.84 (6C, PhCH2), 75.04 (C5′), 77.20 (C2), 78.08 (C4′), 80.91
(C2′), 82.85 (C3), 84.94 (C3′), 102.18, 102.52 (C1, C1′), 127.60-138.73 (Ph). m/z (FAB)
853.3954 (C53H57O10 [M+H]+ requires 853.3952).
126
(195)
O
BnOBnO
OBn
OBnO O
BnO
OSO2ImBnO
Benzyl 3-O-Benzyl-4-O-(imidazolyl-1-sulfonyl)-2-O-(tetra-O-benzyl-β-D-
glucopyranosyl)-β-L-xyloside (195)
Lithium bis(trimethylsilyl)amide in THF (1 M, 0.15 mL, 0.15 mmol) was added dropwise
to the alcohol (194) (105 mg, 0.123 mmol) in dry THF (5 mL, 0ºC) and the solution
stirred (30 min, 0ºC). The solution was cooled (–30°C) and freshly prepared N,N΄-
sulfuryldiimidazole14 (30 mg, 0.15 mmol) was added, then the solution was allowed to
stir (3 h, 35°C). Methanol (0.3 mL) was added and, after 30 min, the solution was
concentrated, diluted with EtOAc, washed with saturated NaHCO3 and dried. Flash
chromatography (EtOAc/toluene, 1:9 containing 0.5% Et3N) gave the imidazylate (195)
(107 mg, 88%) as a colourless oil, [α]D +29.2° (CH2Cl2). δH (600 MHz) 3.35 (m, 3H, H2′,
H5, H5′), 3.57-3.71 (m, 5H, H3, H3′, H4′, H6′), 3.90 (dd, J2,3 6.5, J1,2 6.1, H2), 4.07 (dd,
J5,5 12.2, J4,5 4.5, H5), 4.43, 4.72 (AB, J 11.2, PhCH2), 4.47 (d, H1), 4.49-4.58 (m, 5H,
H4, PhCH2), 4.76 (A OF AB, 1H, J 11.1, PhCH2), 4.76 (d, J1′,2′ 8.0, H1′), 4.81-4.85 (m,
4H, PhCH2), 4.95 (A OF AB, 1H, J 10.9, PhCH2), 6.84-6.85 (m, 1H, Im), 7.16-7.18,
7.24-7.32 (2×m, Ph, Im), 7.89-7.91 (m, 1H, Im). δC (150.9 MHz) 61.01 (C5), 68.97 (C6′),
71.24, 73.51, 74.14, 75.14, 75.17, 75.85 (6C, PhCH2), 75.10 (C5′), 76.59 (C2), 77.08,
78.02 (C3′, C4′), 80.81 (C4), 82.82 (C3), 84.84 (C2′), 101.49 (C1), 102.24 (C1′), 118.11,
137.64 (Im), 127.65-138.78 (Ph). m/z (FAB) 983.3725 (C56H59N2O12S [M+H]+ requires
983.3789).
127
O
BnOBnO
OBn
OBnO
(196)
OBn
OOBn
CN
Benzyl 3-O-Benzyl-4-cyano-4-deoxy-2-O-(tetra-O-benzyl-β-D-glucopyranosyl)-α-D-
arabinoside (196)
The imidazylate (195) (459 mg, 0.467 mmol) in CH3CN (10 mL) was treated with
TMSCN (92.0 mg, 0.934 mmol) and TBAF (1 M, 70 μL, 0.07 mmol) in CH3CN (1 mL)
and the mixture heated to reflux. A further solution of TBAF (1 M, 700 μL, 0.70 mmol)
in CH3CN (1 mL) was added dropwise (30 min) and the solution refluxed (30 min). The
solution was then concentrated somewhat, diluted with EtOAc (100 mL), washed with
H2O and dried. Concentration of the solution followed by flash chromatography
(EtOAc/petrol, 1:3) gave the nitrile (196) as a colourless oil (292 mg, 75%), [α]D +35.6°.
δH (600 MHz) 3.32 (ddd, J4,5 10.0, 4.0, J3,4 3.5, H4), 3.36-3.38 (m, H5′), 3.42 (dd, J2′,3′
9.5, J1′,2′ 7.5, H2′), 3.58-3.65 (m, 4H, H3′, H4′, H6′), 3.70 (dd, J5,5 11.1, J4,5 4.0, H5), 3.91
(dd, J2,3 3.5, J1,2 2.1, H2), 4.11 (dd, H3), 4.33 (dd, J4,5 10.0, H5), 4.42 (d, H1′), 4.54 (s,
PhCH2), 4.57, 4.80 (AB, J 10.8, PhCH2), 4.67, 4.73 (AB, J 11.7, PhCH2) 4.70, 4.86 (AB,
J 10.8, PhCH2), 4.78-4.82 (m, 3H, H1, PhCH2), 4.85 (A OF AB, 1H, J 11.5, PhCH2),
4.93 (A OF AB, 1H, J 11.0, PhCH2), 7.18-7.20, 7.26-7.33 (2×m, Ph). δC (150.9 MHz)
30.41 (C4), 56.49 (C5), 68.75 (C6′), 69.87, 72.62, 73.68, 75.19, 75.23, 75.88 (6C,
PhCH2), 72.72 (C2), 73.17 (C3), 75.00 (C5′), 77.71 (C4′), 82.13 (C2′), 84.68 (C3′), 98.23
(C1), 103.92 (C1′), 117.96 (CN), 127.68-138.60 (Ph). m/z (FAB) 862.3952 (C54H56NO9
[M+H]+ requires 862.3955).
128
O
BnOBnO
OBn
OBnO
(197)
OBn
OOBn
BocHN
Benzyl 3-O-Benzyl-4-C-[(tert-butoxycarbonyl)amino]methyl-4-deoxy-2-O-(tetra-O-
benzyl-β-D-glucopyranosyl)-α-D-arabinoside (197)
The nitrile (196) (210 mg) was treated identically as for (174) to yield the carbamate
(197) (151 mg, 65%) as a colourless oil, [α]D +44.6°. δH (600 MHz) 1.43 (s, 9H, CH3),
2.30-2.35 (m, H4), 3.19-3.26 (m, CH2N), 3.41-3.49 (m, 3H, H2′, H5, H5′), 3.58-3.64 (m,
H3′, H4′), 3.66 (dd, J6′,6′ 10.9, J5′,6′ 4.7, H6′), 3.70 (dd, J5′,6′ 2.1, H6′), 3.83 (dd, J3,4 ≈ J2,3
3.8, H3), 3.98-4.04 (m, H2, H5), 4.43, 4.50 (AB, J 11.6, PhCH2), 4.54-4.57 (m, 4H, H1′,
PhCH2), 4.70 (A OF AB, 1H, J 10.8, PhCH2), 4.74-4.84 (m, 5H, H1, PhCH2), 4.95, 4.97
(AB, J 10.5, PhCH2), 7.17-7.19, 7.25-7.37 (2×m, Ph). δC (150.9 MHz) 28.56 (CH3),
36.61 (CH2N), 39.60 (C4), 59.50 (C5), 69.02 (C6′), 69.74, 71.72, 73.60, 75.11, 75.18,
75.87 (6C, PhCH2), 73.13 (C2), 74.98 (C5′), 75.80 (C3), 77.91 (C4′), 79.15 (CH3C),
82.28 (C2′), 84.79 (C3′), 99.15 (C1), 103.42 (C1′), 127.62-138.73 (Ph), 156.13 (C=O).
m/z (FAB) 966.4788 (C59H68NO11 [M+H]+ requires 966.4792).
129
O
HOHO
OH
OHO NH2Cl
HO
OH
OH
(20)
(2R/2S,3R,4R,5R)-3-(β-D-Glucopyranosyloxy)-5-(hydroxymethyl)piperidine-2,4-diol (3-
O-β-D-glucopyranosylnoeuromycin) (20) hydrochloride
The carbamate (197) (40 mg) was treated identically as for (175) to again yield 3-O-β-D-
glucopyranosylnoeuromycin hydrochloride (20) (14 mg, 97%) as a colourless glass, with
properties identical to those of the sample prepared earlier.
O
OH
BnOBnO
O
BnOBnO
OBn
OBn
O
(198)
Benzyl 2-O-Benzyl-3-O-(tetra-O-benzyl-β-D-glucopyranosyl)-β-L-xylopyranoside (198)
The allyl ether (192) (505 mg) was treated according to the preparation of (194) to yield
the alcohol (198) as a colourless solid (472 mg, 98%). A small amount was further
purified by recrystallisation, m.p. 145-147ºC (CH2Cl2/petrol), [α]D +15.6°. δH (600 MHz)
3.24 (dd, J5,5 11.6, J4,5 8.8, H5), 3.39-3.44 (m, H5′), 3.45 (dd, J2,3 7.7, J1,2 6.3, H2), 3.54
(dd, J2′,3′ 8.1, J1′,2′ 8.0, H2′), 3.57-3.61 (m, H4), 3.64-3.75 (m, 5H, H3, H3′, H4′, H6′), 3.93
(dd, J4,5 4.6, H5), 4.40, 4.56 (AB, J 12.0, PhCH2), 4.53 (d, H1), 4.56, 4.80 (AB, J 10.8,
PhCH2), 4.61 (A OF AB, 1H, J 12.0, PhCH2), 4.65 (d, H1′), 4.76 (A OF AB, 1H, J 11.2,
PhCH2), 4.84-4.89 (m, 6H, PhCH2), 7.13-7.16, 7.24-7.36 (2×m, Ph). δC (150.9 MHz)
64.07 (C5), 68.84 (C6′), 69.67 (C4), 70.88, 73.67, 74.30, 75.10, 75.69, 75.70 (6C,
PhCH2), 75.20 (C5′), 78.08 (C4′), 78.75 (C2), 82.41 (C2′), 84.76, 85.35 (C3, C3′), 102.02
130
(C1), 103.98 (C1′), 127.56-138.68 (Ph). m/z (FAB) 851.3812 (C53H55O10 [M−H]+
requires 851.3795).
O
OSO2Im
BnOBnO
O
BnOBnO
OBn
OBn
O
(199)
Benzyl 2-O-Benzyl-4-O-(imidazolyl-1-sulfonyl)-3-O-(tetra-O-benzyl-β-D-
glucopyranosyl)-β-L-xyloside (199)
The alcohol (198) (132 mg) was treated according to the preparation of (195) to yield the
imidazylate (199) as a colourless oil (110 mg, 72%), [α]D +18.8° (CH2Cl2). δH (600 MHz)
3.32-3.39 (m, 3H, H2′, H5, H5′), 3.54-3.62 (m, H2, H3′, H4′), 3.63 (dd, J6′,6′ 11.3, J5′,6′
4.8, H6′), 3.69 (dd, J5′,6′ 2.0, H6′), 3.99 (dd, J5,5 12.8, J4,5 3.8, H5), 4.02 (dd, J2,3 ≈ J3,4 6.0,
H3), 4.41 (d, J1′,2′ 7.7, H1′), 4.49, 4.53 (AB, J 12.1, PhCH2), 4.55-4.58 (m, PhCH2), 4.61-
4.70 (m, 5H, H1, H4, PhCH2), 4.76, 4.78 (AB, J 12.3, PhCH2), 4.80, 4.83 (AB, J 10.9,
PhCH2), 4.90 (A OF AB, 1H, J 11.0, PhCH2), 7.05-7.07, 7.93-7.95 (2×m, 2H, Im), 7.18-
7.32 (m, Ph, Im). δC (150.9 MHz) 59.90 (C5), 68.95 (C6′), 70.52, 73.56, 73.57, 74.88,
75.10, 75.78 (6C, PhCH2), 74.18 (C3), 75.15 (C5′), 76.75, 77.77 (C2, C4′), 80.92 (C4),
82.16 (C2′), 84.62 (C3′), 100.31 (C1), 101.98 (C1′), 118.23, 137.27 (Im), 127.82-138.60
(Ph). m/z (FAB) 983.3765 (C56H59N2O10 [M+H]+ requires 983.3789).
131
(200)
O
BnOBnO
O
BnOBnO
OBn
OBn
O
NC
Benzyl 2-O-Benzyl-4-cyano-4-deoxy-3-O-(tetra-O-benzyl-β-D-glucopyranosyl)-α-D-
arabinoside (200)
The imidazylate (199) (255 mg) was treated according to the preparation of (196) to yield
the nitrile (200) as a colourless oil (141 mg, 60%), [α]D +14.9°. δH (600 MHz) 3.27-3.30
(m, H4), 3.49-3.70 (m, 4H, H3′, H5′, H6′), 3.52 (dd, J2′,3′ 9.0, J1′,2′ 7.6, H2′), 3.57 (dd, J5,5
11.6, J4,5 3.4, H5), 3.60 (dd, J4′,5′ 9.6, J3′,4′ 9.6, H4′), 3.81 (dd, J2,3 5.9, J1,2 4.2, H2), 4.17-
4.20 (m, 2H, H3, H5), 4.50 (s, PhCH2), 4.57-4.71 (m, 8H, H1, H1′, PhCH2), 4.77, 4.91
(AB, J 11.0, PhCH2), 4.84 (A OF AB, 1H, J 11.4, PhCH2), 5.06 (A OF AB, 1H, J 11.1,
PhCH2), 7.19-7.21, 7.26-7.39 (2×m, Ph). δC (150.9 MHz) 31.17 (C4), 58.52 (C5), 69.34
(C6′), 70.28, 73.57, 74.04, 75.10, 75.23, 75.73 (6C, PhCH2), 73.25 (C3), 75.31 (C5′),
76.49 (C2), 77.99 (C4′), 82.45 (C2′), 84.71 (C3′), 100.10, 102.68 (C1, C1′), 117.74 (CN),
127.71-138.74 (Ph). m/z (FAB) 862.3951 (C54H56NO9 [M+H]+ requires 862.3955).
(201)
O
BnOBnO
O
BnOBnO
BnO
OBn
O
BocHN
Benzyl 2-O-Benzyl-4-C-[(tert-butoxycarbonyl)amino]methyl-4-deoxy-3-O-(tetra-O-
benzyl-β-D-glucopyranosyl)-α-D-arabinoside (201)
The nitrile (200) (110 mg) was treated according to the preparation of (175) to yield the
carbamate (201) as a colourless oil (100 mg, 81%), [α]D +19.1°. δH (600 MHz) 1.43 (s,
9H, CH3), 2.28-2.33 (m, H4), 3.19-3.25 (m, CH2N), 3.37-3.44 (m, 2H, H5, H5′), 3.46 (dd,
132
J 8.0, 7.9, H2′), 3.62-3.70 (m, 5H, H2, H3′, H4′, H6′), 3.88-3.95 (m, 1H, H5), 4.06 (dd,
J3,4 ≈ J2,3 5.8, H3), 4.48-4.61 (m, 6H, H1, H1′, PhCH2) 4.62, 4.69 (AB, J 10.3, PhCH2),
4.77 (A OF AB, 1H, J 11.3, PhCH2), 4.81-4.91 (m, 5H, PhCH2), 4.93-4.97 (br s, NH),
7.18-7.40 (m, Ph). δC (150.9 MHz) 28.34 (s, CH3), 37.18 (C4), 38.71 (CH2N), 61.35
(C5), 68.68 (C6′), 69.85, 73.30, 73.59, 74.93, 74.97, 75.55 (6C, PhCH2), 75.08 (C5′),
76.07 (C3), 77.10, 77.95 (C2, C4′), 78.85 (CH3C), 82.12 (C2′), 84.93 (C3′), 100.87 (C1′),
101.31 (C1), 127.40-138.58 (Ph), 155.88 (C=O). m/z (FAB) 966.4780 (C59H68NO11
[M+H]+ requires 966.4792).
HO NH2Cl
O
OH
O
HOHO
OH
OH
(15)
(3R, 4R, 5R)-4-(β-D-Glucopyranosyloxy)-5-(hydroxymethyl)piperidin-3-ol (4-O-β-D-
Glucopyranosylisofagomine) (15) hydrochloride
The carbamate (201) (26 mg) was treated with CF3COOH (2 mL) and allowed to stand
(10 min). The solution was then concentrated, taken up in MeOH (5 mL) and treated with
resin (Amberlite IRA 400, OH–) until neutral, filtered and then concentrated. The residue
was taken up in MeOH/AcOH (99:1, 15 mL), then treated with Pd/C (10%, 5 mg) and H2
(1 atm , 12 h). Filtration followed by concentration of the solution gave a colourless
foam. The foam was dissolved in hydrochloric acid (1 M, 1 mL) and applied to a cation-
exchange column (Dowex 50W-X2, H+). The column was washed with water and eluted
with aqueous NH3 (1.5 M), the eluate was concentrated, then taken up in hydrochloric
acid (1 M, 1 mL) and concentrated to give 4-O-β-D-glucopyranosylisofagomine (15)
hydrochloride (7 mg, 72%) as a colourless glass. The 1H (600 MHz) and 13C (150.9
MHz) n.m.r. spectral data were in good agreement with those reported.2
133
HO NH2Cl
O
OH
OH
(21)
O
HOHO
OH
OH
(202)
O
OHOH
O
OHHO
HO
OH
O
ClH3N
(2R/2S,3R,4R,5R)-4-(β-D-Glucopyranosyloxy)-5-(hydroxymethyl)piperidine-2,3-diol
(21) hydrochloride and 4-C-Aminomethyl-4-deoxy-3-O-(β-D-glucopyranosyl)-D-
arabinose (202) hydrochloride
The carbamate (201) (26 mg) was treated identically as for the preparation of (20) to
yield a mixture of (21) and (202) as a colourless glass (7 mg, 75%). δH (600 MHz) 2.00-
2.11 [m, H5(glc), H5(man)], 2.41-2.48 (m, H4α, H4β), 2.93 [dd, J 13.2, 13.2, H6(glc)],
3.04-3.13 (m, CH2Nα, CH2Nβ), 3.22-3.45 [m, H2′(glc), H2′(man), H2′α, H2′β, H3′(glc),
H3′(man), H3′α, H3′β, H4′(glc), H4′(man), H4′α, H4′β, H5′(glc), H5′(man), H5′α, H5′β,
H6(glc), H6(man), CH2Nα, CH2Nβ], 3.57-3.66, 3.73-3.89 [2×m, H2α, H2β, H3(glc),
H3(man), H4(glc), H5α, H5β, H6′(glc), H6′(man), H6′α, H6′β, CH2O(glc), CH2O(man)],
3.95 [dd, J 8.9, 8.8, H4(man)], 4.04 (dd, J3,4 5.3, J2,3 9.7, H3α), 4.14 (dd, J3,4 4.4, J2,3 7.9,
H3β), 4.45-4.47 [m, H1′(glc), H1′(man), H1α], 4.52 (d, J1′,2′ 8.0, H1′β), 4.52 (d, J1′,2′ 7.9,
H1′α), 4.57 [d, J2,3 9.0, H2(glc)], 5.11 (d, J1,2 2.9, H1β), 5.18 [d, J2,3 2.8, H2(man)]. δC
(150.9 MHz) 36.19 (C4α), 37.11 [C6(man)], 38.42 (CH2Nα), 39.76, 40.08 [C5(glc),
C5(man)], 40.96 [C6(glc)], 58.04, 58.37, 60.43, 60.49 [C6′(glc), C6′(man), CH2O(glc),
CH2O(man)], 60.55, 62.64 (C5α, C6′α), 69.29-75.95 [C2α, C2′(glc), C2′(man), C2′α,
C3(glc), C3(man), C3′(glc), C3′(man), C3′α, C4′(glc), C4′(man), C4′α, C5′(glc),
C5′(man), C5′α], 76.63 [C4(man)], 77.73 [C2(man)], 78.59 (C3α), 79.22 [C4(glc)], 80.88
[C2(glc)], 92.15 (C1β), 96.91 (C1α), 99.82 (C1′α), 102.53, 102.65 [C1′(glc), C1′(man)].
m/z (FAB) 326.1431 (C12H24NO9 [M−Cl]+ requires 326.1451).
134
References
(1) Macdonald, J. M.; Hrmova, M.; Fincher, G. B.; Stick, R. V. Aust. J. Chem. 2004,
57, 187.
(2) Macdonald, J. M.; Stick, R. V.; Tilbrook, D. M. G.; Withers, S. G. Aust. J. Chem.
2002, 55, 747.
(3) Mackenzie, L. F.; Wang, Q.; Warren, R. A. J.; Withers, S. G. J. Am. Chem. Soc.
1998, 120, 5583.
(4) Stanĕk, J.; Černá, J. Tetrahedron Lett. 1963, 4, 35.
(5) Davies, G. J.; Gloster, T. unpublished results.
(6) Wessel, H. P.; Iversen, T.; Bundle, D. R. J. Chem. Soc., Perkin Trans. 1 1985,
2247.
(7) Hezig, J.; Nudelman, A.; Gottlieb, H. E.; Fischer, B. J. Org. Chem. 1986, 51, 727.
(8) Crich, D.; Smith, M. J. Am. Chem. Soc. 2001, 123, 9015.
(9) Codée, J. D. C.; Litjens, R. E. J. N.; den Heeten, R.; Overkleeft, H. S.; van Boom,
J. H.; van der Marel, G. A. Org. Lett. 2003, 5, 1519.
(10) Varrot, A.; Tarling, C. A.; Macdonald, J. M.; Stick, R. V.; Zechel, D. L.; Withers,
S. G.; Davies, G. J. J. Am. Chem. Soc. 2003, 125, 7496.
(11) Schmidt, R. R.; Michel, J.; Roos, M. Liebigs Ann. Chem. 1984, 1343.
(12) Schmidt, R. R.; Effenberger, G. Liebigs Ann. Chem. 1987, 825.
(13) Brown, H. C.; Yoon, N. M. J. Am. Chem. Soc. 1966, 88, 1464.
(14) Staab, H. A. Angew. Chem. Int. Ed. Engl. 1962, 1, 351.
135
Appendix
ppm2.002.503.003.504.00
H5
H1′
H2, H6
H6
ppm405060708090100
C1′
C3
C3′, C5′ C2′
C4′,C4
C6′, CH2O
C2, C6
C5
O
HOHO
OH
OHO NH
HO
OH
(28)
O
HOHO
OH
OHO NH
HO
OH
(28)
1H and 13C n.m.r. spectra of 3-O-β-D-glucopyranosylisofagomine (28) hydrochloride.
136
O
HOHO
OH
OHO NH
HO
OH
OH
(20)
ppm2.002.503.003.504.004.505.005.50
ppm405060708090100
O
HOHO
OH
OHO NH
HO
OH
OH
(20)
H2(man)H1′(glc)
H6(glc)
C1′(glc)
C1′(man)
C2(glc), C3(glc)
C3(man)
C5, C6(man)
C6(glc)
C6′, CH2O
H1(pyr)H3(pyr) CH2N(pyr)
1′
5
H1′(man), H2(glc)
C2′
H5
1H and 13C n.m.r. spectra of 3-O-β-D-glucopyranosylnoeuromycin (20) hydrochloride.
137
(15)
2.503.003.504.004.50
405060708090100
HO NH
O
OH
O
HOHO
OH
OH
(15)
H1′
H5
H2, H6H6H2′
H3′
C1′
C4
C4′, C5′
C2′
C3′
C3C6′, CH2O
C2
C6
C5
HO NH
O
OH
O
HOHO
OH
OH
1H and 13C n.m.r. spectra of 4-O-β-D-glucopyranosylisofagomine (15) hydrochloride.
138
HO NH
O
OH
OH
(21)
O
HOHO
OH
OH
(202)
2.002.503.003.504.004.505.005.50
(21) (202)
H2(man)
H1β
H2(glc)
H1′(glc), H1′(man)
H5(glc),H5(man)
H4α, H4β
C1′(glc)C1′(man)
C1′α C1α
C1β
H3βH3α
H6(glc)H4(man)
HO NH
O
OH
OHO
HOHO
OH
OH
405060708090100
H1′α, H1′βCH2Nα, CH2Nβ
C4α
C6(man)
CH2Nα
C5(glc), C5(man)C6(glc)
C6′(glc), C6′(man)CH2O(glc), CH2O(man)
C2(glc)
C4(glc)
C3α
C2(man)
C4(man)C5αC6′α
O
OHOH
O
OHHO
HO
OH
O
H2N
O
OHOH
O
OHHO
HO
OH
O
H2N
1H and 13C n.m.r. spectra of (21) and (202) (as the hydrochloride).
Part 2
Chapter 3
Synthesis of Some
α-D-Glucopyranosyl-α-D-Galactopyranoses
140
141
Chapter 3 details the synthesis of four α-linked disaccharides (203), (204), (205) and
(206), used for subsequent investigations as described in the following Chapter. This
present Chapter also directly compares several techniques used to prepare the α-D-
glucosides.
O
HOHO
OH
OHO
OH OH
OHO
OH
(204)
O
HO
OH
OH(206)
O
HOHO
OH
OHO
OH(205)
O
HO
O
OHOH
O
HOHO
OH
OH
OH
O
HOHO
OH
OH
O
O
(203)
OH
HO
OH OH
Introduction
Formation of the glycosidic linkage has played a pivotal role in the development of
modern synthetic carbohydrate chemistry. From the initial work of Emil Fischer in 1893,
significant progress has been made on the efficient preparation of the glycosidic linkage.1
142
One method of classifying the glycosidic linkage is based on the orientation of the
hydroxyl group at C2 relative to the orientation of the glycosidic linkage, denoted by the
terminology 1,2-cis or 1,2-trans.2 This chapter will focus on the preparation of the more
difficult 1,2-cis linkages, in particular the α-glucosides.
OOH
OHOR
OOH
OHOR
OOHHO
ORO
OHHO
OR
α-D-gluco, galacto α-D-mannoβ-D-manno β-D-gluco, galacto
Difficult (1,2-cis) Easy (1,2-trans)
Glycosyl Iodides
Glycosylations using glycosyl halides were first reported in disaccharide synthesis in
1975 by Lemieux where glycosyl bromides and chlorides were found to be effective
glycosyl donors capable of preparing α-D-glucosides with high selectivity.3
The use of glycosyl iodides for the preparation of glycosides was sparked by the efficient
preparation of the α-D-glucosyl iodide (208) from the corresponding acetate (207).4
O
BnOBnO
OBn
OBn OAc
O
BnOBnO
OBn
BnOI
(a)
(207) (208)
a) TMSI, CH2Cl2.
143
Mechanistically, the D-glycosyl iodide forms via O-silylation of the acetyl group to give
an acetoxonium ion intermediate, followed by displacement to give the D-glycosyl
iodide; the anomeric effect favors formation of the α-D-glycosyl iodide.5,6
O
OAc
OOAc
O
O Me
OSiMe3
OO Me
OSiMe3
O
I
OI
Me3SiI
Me3SiI
I
I
I
Treatment of the α-D-glucosyl iodide (208) with tetrabutylammonium iodide and an
alcohol, in the absence of a participating group at C2, results in the highly selective
formation of the α-D-glucoside (214).7 Based on the halide-catalysis work of Lemieux,
the tetrabutylammonium iodide catalyzes the rapid interconversion of the α- and β-D-
glucosyl iodides.3 The α-D-glucoside is then formed by attack of the neutral alcohol on
the more reactive β-D-glucosyl iodide (212), via an SN2 process or through preferential α-
attack on the oxonium ion (213):5
O
BnOBnO
OBn
BnOI
O
BnOBnO
OBn
BnOI
I
O
BnOBnO
OBn
BnO
O
BnOBnO
OBn
BnOOR
ROH
(208)(213)
(214)
(212)
144
This simple method for the preparation of glycosyl iodides was extended by Gervay-
Hague into a formidable glycosylation technique, where it has proved very effective in
preparing α-linked oligosaccharides.8 Treatment of the iodide (209)∗ with
tetrabutylammonium iodide and the alcohol (210) yielded the α-linked disaccharide (211)
exclusively. Subsequent removal of the acetyl group from (211) enabled further
glycosylation.
O
BnOBnO
OAc
BnOI
O
BnOBnO
OH
BnOSCH2CO2Me
O
BnOBnO
OAc
BnOO
O
BnOBnO
BnOSCH2CO2Me
(209)
(210)
(211)
(a)
a) TBAI, EtPri2N, 4Å ms, PhMe, 93%.8
Glycosyl Iodide/Triphenylphosphine Oxide Methodology
An extension of the glycosyl iodide procedure by Mukaiyama was achieved through the
use of a trialkylphosphine oxide as a promoter, purportedly forming a
glycosyloxyphosphonium iodide intermediate. The phosphine oxide also serves to
neutralize hydrogen iodide, the by-product of the glycosylation, removing the need for
the addition of a base and maintaining near-neutral conditions.9
∗ The iodide (209) is simpler to prepare and far less susceptible to hydrolysis than (208).
145
O
BnOBnO
OBn
BnOX
X= Br, I
O
RHO
Ph3POCH2Cl2MS 5A
O
BnOBnO
OBn
BnO O
RO
In the D-glucopyranose series, the α-selectivity most likely arises owing to an SN2 attack
on the more reactive β-D-glucosyloxyphosponium iodide. The glucosyloxyphosphonium
iodide has not been observed in NMR studies, which perhaps indicates that very small
amounts of this reactive intermediate exist in equilibrium with the α-D-glucosyl iodide
and phosphine oxide:9
O
BnOBnO
OBn
BnOOR
O
BnOBnO
OBn
BnOOPPh3
O
BnOBnO
OBn
BnOI
I
O
BnOBnO
OBn
BnOI
O
BnOBnO
OBn
BnOOPPh3
I
ROH
Trichloroacetimidates
The dominant method of preparing the glycosidic linkage currently employs
trichloroacetimidates, initiated by Sinaÿ who reported the first use of an acetimidate in a
glycosylation.10,11 It was Schmidt, however, who was responsible for the development of
the trichloroacetimidate methodology into a formative technique for the efficient
preparation of the glycosidic linkage.12
146
Preparation of the trichloroacetimidate donor is typically achieved by the treatment of a
free sugar (215) with trichloroacetonitrile in a base-mediated reaction. The strength of the
base used dictates the anomeric configuration of the product.13
O
BnOBnO
OBn
BnO OH
O
BnOBnO
OBn
BnOO
NH
CCl3
CCl3CNK2CO3
CH2Cl2
CH2Cl2
CCl3CNDBU
O
BnOBnO
OBn
BnOO
NH
CCl3
(215)
(216)
(217)
Promotion of the trichloroacetimidate as a glycosyl donor is achieved under mild
conditions, typically with boron trifluoride diethyl etherate, trimethylsilyl triflate or triflic
anhydride.14 In the absence of a participating group at C2, several factors influence the
stereochemistry of the resulting glycosidic linkage. Generally the trichloroacetimidate
method results in the formation of a glycoside with inversion of configuration at the
anomeric carbon, presumably through an SN2 process.15 This is demonstrated by the
treatment of (218) with the acceptor (219) resulting in the formation of the α-D-glucoside
(220). This inversion is favored in conditions by low temperatures, a mild promoter such
as boron trifluoride diethyl etherate, and non-polar solvents such as diethyl ether.15
Preparation of both 1,2-trans and 1,2-cis glycosides is possible using this methodology,
ratifying its status as one of the most flexible and versatile techniques for the preparation
of glycosides.
147
O
BnO
OBn OBn
BnOO
NH
CCl3
O
BnOHO
OBn
BnO OPh
O
BnO
OBn OBn
BnO O
BnO
OBn
BnO OPh
O
(218)
(219)
(220)
(a)
a) TMSOTf, Et2O, 65%.16
Thioglycosides
Thioglycosides are another well-established method, capable of not only preparing 1,2-
cis and 1,2-trans glycosides, but also offering temporary protection to the anomeric
centre. This has resulted in thioglycosides becoming a dominant method in the
preparation of oligosaccharides. Preparation of the thioglycoside donor can be achieved
via a number of methods, however synthesis through a glycosyl halide (221)17 or a
glycosyl acetate (223)7 is the most common:
O
AcO
OAc OAc
AcOO
AcOO
OAc
AcOBr
(a)
(b)O
AcO
OAc OAc
AcO OAc
O
AcO
OAc OAc
AcOSMe
(221) (222)
(223) (224)
O
AcO
OAc OAc
AcOO
AcOO
OAc
AcOSPh
a) Bu4NHSO4, Na2CO3, PhSH, EtOAc, H2O, 92%;
b) Me3SiSiMe3, Me2S2, I2, MeCN, 90%.
148
In recent years two reagent combinations have emerged to promote thioglycosides,
developed by Crich and van Boom. Crich’s method uses 1-benzenesulfinylpiperidine
(BSP) and triflic anhydride (Tf2O) as the promoter with 2,4,6-tri-tert-butylpyrimidine
(TTBP) as the base, whilst van Boom’s method replaces BSP with diphenyl sulfoxide
(DPS). 1-Benzenesulfinylpiperidine (225) is a shelf-stable reagent that, in the presence of
triflic anhydride, results in the formation of the salt (226).18 The treatment of diphenyl
sulfoxide (227) with triflic anhydride produces a similar sulfonium species (228):19
NS
O
NS
OTf
Tf2O
TfO
S
O
S
OTf
Tf2O
TfO
(225) (226)
(227) (228)
Both sulfonium species, (226) and (228) are powerful thiophiles capable of converting
thioglycosides into glycosyl triflates.18,19 In the D-glucopyranose series, the promotion of
thioglycosides with either reagent combination occurs via the generation of D-glucosyl
triflates, where the α-D-glucosyl triflate (230) exists in equilibrium with the β-D-glucosyl
triflate (229).20 The selectivity observed is a result of the higher reactivity of the less
stable β-D-glucosyl triflate (229), resulting in the preferential formation of the α-D-
glucoside (231).20 As with the glycosyl iodide methodology, the α-D-glycoside can also
be formed through preferential α-attack on the oxonium ion (213):
149
O
BnOBnO
BnOOTf
O
BnOBnO
BnOOTf
O
BnOBnO
BnOOR
ROH
(230)
(229) (231)
OBn
OBn OBn
O
BnOBnO
OBn
BnO(213)
ROH
The key difference between the two promoters is that (228) lacks the nitrogen lone-pair
stabilization of (226), rendering the former a far more powerful electrophile.19 This
reactivity difference has been exploited in the synthesis of the tri-saccharide (236) where
the BSP/Tf2O promoter system is capable of activating only the armed thiogalactoside
donor (232), resulting in the formation of the disaccharide (234).19 Subsequent activation
of the disarmed thiomannoside (234) with DPS/Tf2O resulted in formation of the
trisaccharide (236).18,19
150
O
BnO
OBn OBn
BnOSPh
O
HO
N3
SPh
(a) ON3
SPh
O
BnO
OBn OBn
BnOO
O
AcOAcO
AcOOMe
(b)
ON3
O
O
BnO
OBn OBn
BnOO
O
AcOAcO
OH
AcOOMe
(232)(234)
(236)
(233)
(235)
OO
OO
Ph
Ph
OOPh
a) BSP, Tf2O, TTBP, CH2Cl2, (EtO)3P quench, 73%;
b) Ph2SO, Tf2O, TTBP, CH2Cl2, 64%.
151
Discussion
Key to the comparison of the various glycosylation strategies for the synthesis of the
disaccharides (203), (204), (205) and (206) was the selection and preparation of the
appropriate acceptors (237), (238), (239) and (240). All were readily prepared and, with
the exception of (237), all previously reported in the literature.
O
AllO
OMeOH
(237)
O
HO
OBn OBn
OMeBnO
(238)
O
BnO
OH OBn
OMeBnO
(239)
O
O
O
OO
OH
(240)
OO
Ph
Glycosyl Iodide Methodology
The well-established glycosyl iodide methodology was logically the initial choice for the
preparation of the required disaccharides, with the donor (209), previously shown on
several occasions capable of preparing α-D-glucosides with excellent selectivity.8 The
glycosyl iodide methodology was adapted from that reported by Lam and Gervay-Hague,
used in the preparation of several oligosaccharides.8 Treatment of the easily prepared
donor (209) with the acceptor (240) yielded the α-D-glucoside (241), together with
significant quantities of the alkene (242).
152
O
BnOBnO
OAc
(209)
BnOI
O
O
O
OO
OH
(240)
(a)
O
BnOBnO
OAc
(242)BnO
O
O
O
OO
(241)
O
BnOBnO
OAc
BnOO
a) TBAI, EtPri2N, 4Å ms, benzene.
The donor (209) was then treated with the acceptors (237), (238) and (239), with the
results summarized in Table 3.1. It is evident that only (237) and (240) were reactive
enough to undergo glycosylation; forming the α-D-glucosides (243) and (241)
respectively (the actual α/β ratio was 97:3 in both cases). The major disadvantage of this
technique was that excessive quantities of the donor (209) were required to compensate
for the production of the alkene (242).
Acceptor Yield % Product (α:β) (237) 70 (243) 97:3 (238) 0 (239) 0 (240) 90 (241) 97:3
Table 3.1 Glycosylations using the donor (209) and various acceptors.
(243 )
O
O
O
OO
(241)
O
BnOBnO
OAc
BnOO
O
BnOBnO
OAc
BnO
O
OOMe
AllO
OO
Ph
153
Glycosyl Iodide/Triphenylphosphine Oxide Methodology
The relatively new glycosyl iodide/triphenylphosphine oxide methodology was adapted
from that reported by Kobashi and Mukaiyama.9,21 Treatment of the acetate (207) with
iodotrimethylsilane yielded the α-D-glucosyl iodide (208); subsequent treatment with the
acceptor (240) and Ph3PO yielded the disaccharide (244).
O
O
O
OO
OH
(240)(a)O
BnOBnO
OBn
(207)
OBn OAc(b)
O
BnOBnO
OBn
(208)
BnOI
O
O
O
OO
O
BnOBnO
OBn
BnOO
(244)
a) TMSI, CH2Cl2; b) Ph3PO, 4Å ms, CHCl3.
The donor (207) was then treated with the acceptors (237), (238) and (239); forming the
disaccharides (245), (246) and (247) in good yield (Table 3.2). The diastereoselectivity
of (245) was determined by an analysis of the 1H n.m.r. spectrum of the mixture, and
confirmed by the isolation of the α- and β-anomers. The diastereoselectivity of (246) and
(247), where the anomers were inseparable by chromatography, was initially determined
by 1H n.m.r. spectroscopy, and subsequently confirmed by debenzylation and acetylation
of the reaction mixture, which facilitated the separation of the α- and β-anomers. For
(244), where the anomers were again inseparable by chromatography, the
diastereoselectivity was determined by direct analysis of the 1H n.m.r. spectrum.
Reactivity problems were less pronounced here than with the glycosyl iodide method,
with only (239) possessing a reduced reactivity, reflected in the lower yield of (247).
154
Also the neutral conditions resulted in no observable formation of the alkene, largely
responsible for the inefficiency of the previous glycosyl iodide method.
Acceptor Yield % Product (α:β) (237) 70 (245) 90:10 (238) 86 (246) 95:5 (239) 57 (247) 93:7 (240) 90 (244) 94:6
Table 3.2 Glycosylations using the iodide generated from (207), and various
acceptors.
(246)
(247 )
(245)
(244)
O
O
O
OO
O
BnOBnO
OBn
BnOO
O
BnOBnO
OBn
BnO
O
OOMe
AllO
OO
Ph
O
BnOBnO
OBn
BnOO
OBn OBn
OMeBnO
O
O
BnO
O
BnOOBn
O
BnOBnO
OBn
BnO
OMe
Trichloroacetimidate Methodology
The trichloroacetimidate methodology was adapted from that reported by Wegmann and
Schmidt.16 Treatment of the acceptor (240) with the β-D-glucosyl trichloroacetimidate
(248) yielded the disaccharide (244).
155
O
O
O
OO
OH
(240)
O
BnOBnO
OBn
(248)
OBnO
NH
CCl3
(a)
(244)
O
O
O
OO
O
BnOBnO
OBn
BnOO
a) TMSOTf, 4Å ms, Et2O.
The donor (248) was then treated with the acceptors (237), (238) and (239) affording the
disaccharides (245), (246) and (247) (Table 3.3). The diastereoselectivity was
determined in a manner identical to that for the glycosyl iodide/triphenyl phosphine oxide
methodology.
Acceptor Yield % Product (α:β) (237) 82 (245) 85:15 (238) 72 (246) 75:25 (239) 72 (247) 65:35 (240) 71 (244) 80:20
Table 3.3 Glycosylations using the donor (248) and various acceptors.
Unlike the two previous techniques, no problem with the reactivity of the donor was
observed. Unfortunately the selectivity was significantly poorer, particularly in the case
of (247). Another disadvantage of this method was the poor stability of β-D-glucosyl
trichloroacetimidate (248) upon prolonged storage, with hydrolysis giving the hemiacetal.
156
Thioglycoside Methodology
Two methods of promotion were examined here, firstly that reported by van Boom
(DPS/Tf2O) and, secondly, that of Crich and Smith (BSP/Tf2O).18,19
O
O
O
OO
OH
(240)
(a) or (b)
O
BnOBnO
OBn
(249)
OBnSPh
(244)
O
O
O
OO
O
BnOBnO
OBn
BnOO
a) DPS, TTBP, Tf2O, CH2Cl2;
b) BSP, TTBP, Tf2O, CH2Cl2.
Both sets of glycosylations using the thioglycoside donor (249) produced similar results,
with the DPS/Tf2O promoter producing slightly higher yields (Tables 3.4 and 3.5).
Unfortunately, poor diastereoselectivity was observed in two of the preparations [(245)
and (244)], with the β-D-glucoside in fact slightly favored.
Acceptor Yield % Product (α:β) (237) 76 (245) 45:55 (238) 75 (246) 75:25 (239) 85 (247) 75:25 (240) 85 (244) 40:60
Table 3.4 Glycosylations using donor (249) and various acceptors with DPS and Tf2O as
promoter.
157
Acceptor Yield % Product (α:β) (237) 71 (245) 45:55 (238) 65 (246) 70:30 (239) 74 (247) 70:30 (240) 65 (244) 35:65
Table 3.5 Glycosylations using donor (249) and various acceptors with BSP and Tf2O as
promoter.
Conclusions
The glycosyl iodide method offered the highest diastereoselectivity for the two successful
preparations [(243) and (241)]. This was particularly important in the case of (241) where
the separation of (241) from its β-anomer was unsuccessful. The inefficiency of using a
large excess of the donor (209) was outweighed by the high diastereoselectivity obtained.
Fortunately Lam and Gervay-Hague describe a preparation of the iodide (209) that is
amenable to large scale synthesis.8
The glycosyl iodide/triphenylphosphine oxide methodology is relatively new and hence
somewhat underrated; it is clearly very effective in the preparation of α-D-glucosides.
Excellent diastereoselectivity, moderate reactivity and efficiency provide a balanced and
attractive technique for the preparation of α-D-glucosides. Operationally, this method
proved to be more difficult when compared to a thioglycoside or trichloroacetimidate
donor.
158
The trichloroacetimidate method has proved to be effective and versatile, often
considered the benchmark glycosylation technique. Unfortunately, the
diastereoselectivity was not as good as that obtained from either the glycosyl iodide or
glycosyl iodide/triphenylphosphine oxide methodology. With the exception of the poor
stability of the trichloroacetimidate donor, this proved to be the simplest from an
operational perspective.
A thioglycoside proved to be less effective in the preparation of the four glycosides, with
poor diastereoselectivity generally observed. However, in the preparation of (246) and
(247), this method offered comparable results to a trichloroacetimidate. The similarity
between the two promoters used was expected, with the armed thioglycoside (249)
presumably forming the same glycosyl triflate in both systems. Operationally, this
method was slightly more difficult than with the trichloroacetimidate; methodology;
however, all of the reagents, with the exception of Tf2O, exhibited excellent long-term
stability.
In the end for the preparation of the glycosides (203) and (206), the glycosyl iodide
methodology was found to be the most appropriate, offering excellent selectivity (97:3)
in both cases. For the preparation of (204) and (205), the glycosyl
iodide/triphenylphosphine oxide methodology was found to be superior, offering
excellent selectivity (95:5 and 93:7, respectively).
159
Preparation of Disaccharides for Biological Testing
With effective techniques at hand for the preparation of the four α-D-glucosides, all that
remained was to remove the protecting groups to yield the free sugars. Removal of the
4,6-O-benzylidene group from (243), by treatment with aqueous acetic acid, gave the diol
(250). Subsequent removal of the allyl group using Wilkinson’s catalyst followed by acid
treatment and acetylation, gave the tetraacetate (251). Debenzylation followed by
acetylation gave (252), with subsequent acetolysis yielding the per-acetylated derivative
(253); deacetylation afforded the known but uncharacterized disaccharide (203).
O
BnOBnO
OAc
BnO
(250) (251)
(252) (253) (203)
(a) (b)
(c) (d) (e)
O
OOMe
AllO
OH OH
O
BnOBnO
OAc
BnO
O
OOMe
AcO
OAc OAc
O
AcOAcO
OAc
AcO
O
OOMe
AcO
OAc OAc
O
AcOAcO
OAc
AcO
O
OOAc
AcO
OAc OAc
O
HOHO
OH
OH
O
OOH
HO
OH OH
(243)
O
BnOBnO
OAc
BnO
O
OOMe
AllO
OO
Ph
a) AcOH/H2O, 78%; b) i) Wilkinson’s catalyst, EtOH; ii) 3 M HCl;
iii) Ac2O, pyridine, DMAP, 84%; c) i) H2, Pd/C, MeOH; ii) Ac2O, pyridine, DMAP, 84%;
d) Ac2O, H2SO4, 95%; e) NaOMe, MeOH 89%.
160
Debenzylation and subsequent acetylation of (246) enabled the chromatographic
separation of (254) and (255). Acetolysis of (254) gave the per-acetylated derivative
(256), with subsequent deacetylation affording the known, but poorly characterized
disaccharide (204).
O
AcOAcO
OAc
AcOO
OAc OAc
OMeAcO
O
O
AcOAcO
OAc
OAc
O
OAc OAc
OMeAcO
O
(255)(254)
O
AcOAcO
OAc
AcOO
OAc OAc
OAcO
(256)OAc
O
HOHO
OH
OHO
OH OH
OHO
OH
(204)
O
BnOBnO
OBn
OBn
O
OBn OBn
OMeBnO
O
(246)
(c)O
AcOAcO
OAc
AcOO
OAc OAc
OMeAcO
O
(254)
(a)
(b)
a) i) Pd/C, H2, MeOH; ii) Ac2O, pyridine, DMAP, 88%;
b) Ac2O, H2SO4, 94%; c) NaOMe, MeOH, 96%.
Debenzylation and subsequent acetylation of (247) facilitated the separation of the
anomers (257) and (258). Acetolysis of (257) then yielded the per-acetylated derivative
(259), with subsequent deacetylation yielding the known, but poorly characterized
disaccharide (205).
161
O
BnO
O OBn
OMeBnO
O
BnOBnO
OBn
OBn
(247)
O
AcO
O OAc
OMeAcO
O
AcOAcO
OAc
OAc
(257)
(259) (205)
(258)
(a)
(b) (c)O
HO
O
OHOH
O
HOHO
OH
OH
OH
O
AcO
O
AcOOAc
O
AcOAcO
OAc
AcO
OMe
(257)
O
AcO
O
AcOOAc
O
AcOAcO
OAc
AcO
OMeO
AcO
O
AcOOAc
O
AcOAcO
OAc
AcO
OAc
a) i) Pd/C, H2, MeOH; ii) Ac2O, pyridine, DMAP, 75%;
b) Ac2O, H2SO4, 99%; c) NaOMe, MeOH, 96%.
The preparation of (206) was achieved by hydrogenolysis of (241) in methanol, effective
at both debenzylation and removing the labile 6-O-acetyl group to give (260). Removal
of the isopropylidene groups under acidic conditions provided the known disaccharide
(206) in good yield.
O
O
O
OO
(241)
O
BnOBnO
OAc
BnOO
O
O
O
OO
(260)
O
HOHO
OH
OHO
O
HO
OH
OH
(206)
O
HOHO
OH
OHO
OH
(a) (b)
a) Pd/C, H2, MeOH, 89%; b) CF3COOH, H2O, 89%.
162
Experimental
O
AllO
OMeOH
(237)
O
AllO
OHOH
OMeOH
(261)
OO
Ph
Methyl 3-O-Allyl-4,6-O-benzylidene-α-D-galactoside (237)
The triol22 (261) (3.08 g, 13.2 mmol) in DMF (30 mL) was treated with benzaldehyde
diethyl acetal (3.55 g, 19.7 mmol) and CSA (100 mg) and the solution stirred (35°C, 36
h). The solution was neutralized with Et3N and concentrated; flash chromatography
(EtOAc/petrol, 1:1) gave the alcohol (237) (2.88 g, 68%) as a colourless solid, m.p. 159-
161°C, [α]D +216.0° (Found C, 63.4; H, 6.7. C17H22O6 requires C, 63.3; H, 6.9%). δH
(500 MHz) 3.47 (s, CH3), 3.66 (d, J 0.6, H4), 3.75 (dd, J2,3 10.0, J1,2 3.7, H2), 4.09 (dd,
J6,6 12.4, J5,6 1.7, H6), 4.16-4.31 (m, 5H, CH2O, H3, H5, H6), 4.96 (d, H1), 5.21-5.23,
5.32-5.36 (2×m, CH2CH), 5.56 (s, PhCH), 5.93-6.03 (m, CH2CH), 7.32-7.39, 7.52-7.54
(2×m, Ph). δC (125.8 MHz) 55.62 (CH3), 62.82 (C5), 67.88, 73.64, 76.01 (C2, C3, C4),
69.50, 70.51 (CH2O, C6), 100.18, 101.00 (PhCH, C1), 117.54 (CH2CH), 126.26-137.76
(Ph), 134.97 (CH2CH).
O
HO
OBn OBn
OMeBnO
(238)
Methyl 2,4,6-Tri-O-benzyl-α-D-galactoside (238)
The alcohol (238) was prepared by the method of Kong and Lu22 as a colourless solid,
m.p. 123-124°C (lit.22 125ºC), [α]D +38.5° (lit.22 +40.3°). The 1H (300 MHz) and 13C
(75.5 MHz) n.m.r. spectral data were in good agreement with those reported.22
163
O
BnO
OH OBn
OMeBnO
(239)
Methyl 2,3,6-Tri-O-benzyl-α-D-galactopyranoside (239)
The alcohol (239) was prepared by the method of Garegg and coworkers23 as a colourless
oil, [α]D +35.0° (lit.23 +40.0°) The 1H (300 MHz) and 13C (75.5 MHz) n.m.r. spectral data
were in good agreement with those reported.23
O
BnOBnO
OAc
(209)
BnOI
6-O-Acetyl-2,3,4-tri-O-benzyl-α-D-glucosyl iodide (209)
The iodide (209) was prepared by the method of Lam and Gervay-Hague and used
without purification.8
O
BnOBnO
OBn
(207)
OBn OAc
1-O-Acetyl-2,3,4,6-tetra-O-benzyl-D-glucose (207)
The acetate (207) was prepared by the method of Schmidt and Michel, with 1H (300
MHz) and 13C (75.5 MHz) n.m.r. spectral data in good agreement with those reported.24
164
O
BnOBnO
OBn
(248)
OBnO
NH
CCl3
Tetra-O-benzyl-β-D-glucopyranosyl Trichloroacetimidate (248)
The trichloroacetimidate (248) was prepared by the method of Rathore and coworkers,
with 1H (300 MHz) and 13C (75.5 MHz) n.m.r. spectral data in good agreement with
those reported.25
O
BnOBnO
OBn
(249)
OBnSPh
Phenyl Tetra-O-benzyl-1-thio-β-D-glucopyranoside (249)
The thioglycoside was prepared by the method of Fairbanks and coworkers, with 1H (300
MHz) and 13C (75.5 MHz) n.m.r. spectral data in good agreement with those reported.26
Glycosyl Iodide Method of Disaccharide Synthesis
The acceptor (0.32 mmol) in dry benzene (2 mL) was treated with EtNPri2 (130 mg, 0.96
mmol), TBAI (350 mg, 0.96 mmol) and 4Å molecular sieves (300 mg) and the mixture
stirred (rt, 2 h). The iodide (209)8 (800 mg, 1.28 mmol) in benzene (3 mL) was added and
the mixture refluxed (6 h). The mixture was filtered, the filtrate concentrated and
subjected to flash chromatography (EtOAc/petrol, 1:2) to give the disaccharide.
165
(243)
O
BnOBnO
OAc
BnO
O
OOMe
AllO
OO
Ph
Methyl 2-O-(6-O-Acetyl-2,3,4-tri-O-benzyl-α-D-glucosyl)-3-O-allyl-4,6-O-benzylidene-α-
D-galactoside (243)
The acceptor (237) (100 mg) gave the disaccharide (243) (172 mg, 70%) as a colourless
solid and as predominantly the α-anomer (97:3), m.p. 134-136°C, [α]D +111.5°. δH (600
MHz) 2.00 (s, CH3CO), 3.43-3.45 (m, H5), 3.45 (s, CH3O), 3.54 (dd, J2,3 9.6, J1,2 3.6,
H2), 3.65 (s, H4), 3.92 (dd, J5′,6′ 10.2, 3.5, H5′), 4.06-4.09 (m, 2H, H3, H6), 4.16-4.19 (m,
3H, CH2O, H6), 4.21-4.31 (m, 5H, H2′, H3′, H4′, H6′), 4.56, 4.85 (AB, J 11.2, PhCH2),
4.69, 4.78 (AB, J 12.0, PhCH2), 4.80, 5.00 (AB, J 10.8, PhCH2), 4.90 (d, H1), 4.96 (d,
J1′,2′ 3.4, H1′), 5.11-5.15, 5.27-5.35 (2×m, CH2CH), 5.54 (s, PhCH), 5.91-5.94 (m,
CH2CH), 7.23-7.38, 7.49-7.51 (2×m, Ph). δC (150.9 MHz) 20.55 (CH3CO), 55.17
(CH3O), 62.43 (C4), 62.94 (CH2O), 68.53 (C2′), 69.26 (C6), 71.02 (C6′), 71.33, 74.02,
74.26 (C3′, C4′, C5′), 72.87, 74.38, 75.52 (3C, PhCH2), 77.40 (C5), 79.40 (C2), 81.81
(C3), 94.50 (C1), 97.60 (C1′), 101.02 (PhCH), 117.14 (CH2CH), 126.22-138.62 (Ph),
134.98 (CH2CH), 170.82 (C=O). m/z (FAB) 795.3374 (C46H51O12 [M−H]+ requires
795.3380).
A significant quantity (300 mg) of the alkene (242) was also isolated, with 1H (300 MHz)
and 13C (75.5 MHz) n.m.r. spectral data consistent with those reported.8
166
O
O
O
OO
(241)
O
BnOBnO
OAc
BnOO
6-O-(6-O-Acetyl-2,3,4-tri-O-benzyl-α-D-glucosyl)-1,2:3,4-di-O-isopropylidene-α-D-
galactose (241)
The acceptor (240) (0.43 mmol) gave the disaccharide (241) (280 mg, 90%) as a
colourless oil and as predominantly the α-anomer (97:3), [α]D +16.1º. δH (600 MHz) 1.32,
1.33, 1.45, 1.55 (4×s, 12H, CH3C), 2.03 (s, CH3CO), 3.51 (dd, J4′,5′ ≈ J3′,4′ 9.8, H4′), 3.56
(dd, J2′,3′ 9.6, J1′,2′ 3.6, H2′), 3.74 (dd, 1H, J6,6 10.3, J5,6 7.3, H6), 3.79 (dd, 1H, J5,6 6.3,
H6), 3.93-3.96 (m, H5′), 4.01-4.06 (m, 2H, H3′, H5), 4.24 (dd, 1H, J6′,6′ 11.9, J5′,6′ 1.9,
H6′), 4.31-4.37 (m, H2, H3, H4), 4.57, 4.88 (AB, J 10.8, PhCH2), 4.62 (dd, 1H, J5′,6′ 1.3,
H6′), 4.71, 4.76 (AB, J 11.9, PhCH2), 4.82, 5.02 (AB, J 10.8, PhCH2), 4.96 (d, H1′), 5.53
(d, J1,2 5.0, H1), 7.25-7.40 (m, Ph). δC (150.9 MHz) 21.00 (CH3CO), 24.75, 25.04, 26.18,
26.25 (4C, CH3C), 63.20, 66.76 (C6, C6′), 66.00, 68.76 (C5, C5′), 70.70, 70.77, 70.99
(C2, C3, C4), 72.52, 74.99, 75.80 (PhCH2), 77.30 (C4′), 79.92 (C2′), 81.97 (C3′), 96.42,
97.12 (C1, C1′), 108.75, 109.37 (2C, CH3C), 127.76-138.82 (Ph), 170.91 (C=O). m/z
(FAB) 734.3325 (C41H50O12 [M]+• requires 734.3302).
A significant quantity (500 mg) of the alkene (242) was also isolated, with 1H (300 MHz)
and 13C (75.5 MHz) n.m.r. spectral data consistent with those reported.8
167
Glycosyl Iodide/Triphenylphosphine Oxide Method of Disaccharide Synthesis
The acetate (207) (290 mg, 0.51 mmol) in dry CH2Cl2 (5 mL) was treated with 4Å
molecular sieves (300 mg) and the mixture stirred (0°C, 1 h). The mixture was treated
with TMSI (122 mg, 0.608 mmol) and stirred (30 min); the mixture was then
concentrated and the excess TMSI removed by azeotropic distillation with PhMe (4×10
mL). The residue in dry CHCl3 (2 mL) was treated with Ph3PO (280 mg, 1.0 mmol) and
the acceptor (0.22 mmol) and the mixture stirred (rt, 14 h). The mixture was filtered, the
filtrate concentrated and subjected to flash chromatography (EtOAc/petrol, 1:2) to give
the disaccharide.
(245)
O
BnOBnO
OBn
BnO
O
OOMe
AllO
OO
Ph
Methyl 3-O-Allyl-4,6-O-benzylidene-2-O-(tetra-O-benzyl-α-D-glucosyl)-α-D-galactoside
(245)
The acceptor (237) (65 mg) gave the α-linked disaccharide (105 mg, 63%) as a colourless
oil, [α]D +91.7°. δH (500 MHz) 3.46 (s, CH3O), 3.56-3.60 (m, 2H, H2′, H6′), 3.65-3.72
(m, 3H, H4′, H5, H6′), 3.94 (dd, J2,3 10.3, J3,4 3.4, H3), 4.04 (dd, J3′,4′ ≈ J2′,3′ 9.4, H3′),
4.08 (dd, J6,6 12.6, J5,6 1.3, H6), 4.15-4.24 (m, 3H, CH2O, H5′), 4.25-4.31 (m, 2H, H4,
H6), 4.32 (dd, J1,2 3.4, H2), 4.39, 4.57 (AB, J 12.1, PhCH2), 4.49, 4.79 (AB, J 11.3,
PhCH2), 4.69, 4.81 (AB, J 12.0, PhCH2), 4.82, 4.96 (AB, J 10.8, PhCH2), 4.96 (d, J1′,2′
2.9, H1′), 5.01 (d, H1), 5.07-5.13 (m, CH2CH), 5.55 (s, PhCH), 5.88-5.97 (m, CH2CH),
7.10-7.50 (m, Ph). δC (125.8 MHz) 55.41 (CH3O), 62.73 (C5), 68.32, 69.47 (C6, C6′),
168
70.01, 71.18, 74.37, 74.83, 77.82, 79.57 (C2, C2′, C3, C4, C4′, C5′), 71.79 (CH2O),
73.18, 73.35, 74.64, 75.72 (4C, PhCH2), 82.17 (C3′), 94.77 (C1′), 97.84 (C1), 101.19
(PhCH), 117.43 (CH2CH), 126.52-138.55 (Ph), 135.17 (CH2CH). m/z (FAB) 844.3731
(C51H56O11 [M]+• requires 844.3823).
Also obtained was the β-linked disaccharide (15 mg, 7%) as a colourless oil, [α]D +98.2°.
δH (500 MHz) 3.44 (s, CH3O), 3.55-3.75 (m, 7H, H2′, H3′, H4′, H5, H5′, H6′), 3.98 (dd,
J2,3 10.3, J3,4 3.5, H3), 4.08-4.15 (m, 3H, CH2O, H6), 4.25 (dd, J1,2 3.5, H2), 4.31 (dd, J6,6
12.4, J5,6 1.4, H6), 4.34 (dd, J4,5 0.3, H4), 4.52, 4.60 (AB, J 12.0, PhCH2), 4.56, 4.82 (AB,
J 10.8, PhCH2), 4.66 (d, J1′,2′ 7.7, H1′), 4.74, 5.15 (AB, J 11.3, PhCH2), 4.77, 4.91 (AB, J
11.0, PhCH2), 5.04-5.09, 5.19-5.23 (2×m, CH2CH), 5.17 (d, H1), 5.58 (s, PhCH), 5.81-
5.89 (m, CH2CH), 7.15-7.58 (m, Ph). δC (125.8 MHz) 55.60 (CH3O), 62.26 (C5), 69.01,
69.55 (C6, C6′), 70.59 (CH2O), 73.41, 74.29, 74.92, 75.40 (4C, PhCH2), 74.44 (C5′),
75.60, 76.58 (C2, C3, C4), 77.62, 82.02, 84.65 (C2′, C3′, C4′), 100.42 (C1′), 101.08
(PhCH), 104.95 (C1), 117.09 (CH2CH), 126.32-138.90 (Ph), 135.23 (CH2CH). m/z
(FAB) 844.3812 (C51H56O11 [M]+• requires 844.3823).
(246)
O
BnOBnO
OBn
BnOO
OBn OBn
OMeBnO
O
Methyl 3-O-(Tetra-O-benzyl-D-glucosyl)-2,4,6-tri-O-benzyl-α-D-galactoside (246)
The acceptor (238) (110 mg) gave an inseperable mixture of the α-linked and β-linked
disaccharide (246) (200 mg, 86%) as a colourless oil.
169
(247)
O
BnO
O
BnOOBn
O
BnOBnO
OBn
BnO
OMe
Methyl 4-O-(Tetra-O-benzyl-D-glucosyl)-2,3,6-tri-O-benzyl-α-D-galactoside (247)
The acceptor (238) (105 mg) gave an inseperable mixture of the α-linked and β-linked
disaccharide (247) (127 mg, 57%) as a colourless oil.
(244)
O
O
O
OO
O
BnOBnO
OBn
BnOO
1,2:3,4-Di-O-isopropylidene-6-O-(tetra-O-benzyl-D-glucosyl)-α-D-galactose (244)
The acceptor (240) (70 mg) gave an inseperable mixture of the α-linked and β-linked
disaccharide (247) (201 mg, 90%) as a colourless oil, δH (600 MHz) 1.32, 1.33, 1.34,
1.46, 1.54 (5×s, CH3), 3.45-3.49 (m, H2′β, H5′β), 3.60 (dd, J2′,3′ 9.6, J1′,2′ 3.6, H2′α), 3.62-
4.75 (10×m, H3, H3′β, H4α, H5′α, H6, H6′, PhCH2), 4.00 (dd, J3′,4′ 9.6, H3′α), 4.04-4.06
(m, H5α), 4.10-4.11 (m, H5β), 4.32-4.34 (m, H2), 4.44 (d, J1′,2′ 7.5, H1′β), 4.63, 4.72
(AB, J 12.0, PhCH2α), 4.71 (A of AB, J 11.1, PhCH2β), 4.71, 4.76 (AB, J 11.9, PhCH2α),
4.83, 4.99 (AB, J 10.9, PhCH2α), 4.96 (A of AB, 1H, J 11.0, PhCH2β), 5.01 (d, H1′α),
5.06 (A of AB, J 11.1 , PhCH2β), 5.54 (d, J1,2 5.02, H1α), 5.58 (d, J1,2 5.0, H1β), 7.25-
7.40, 7.12-7.17 (2×m, Ph). δC (150.9 MHz) 24.35, 24.54, 24.82, 24.93, 25.90, 25.94,
25.96, 26.06 (CH3), 65.63 (C5α), 66.11, 68.28, 68.64, 69.60 (C6α, C6′α, C6β, C6′β),
70.13-77.61 (C2, C3, C4, C4′, C5′α, C5β, C5′β), 72.30-75.55 (PhCH2), 79.70 (C2′α),
170
81.53 (C2′β), 81.84 (C3′α), 84.43 (C3′β), 96.20 (C1α), 96.28 (C1β), 96.88 (C1′α), 104.26
(C1′β), 108.52, 109.15, 109.30 (CH3C), 127.41-138.81 (Ph).
Trichloroacetimidate Method of Disaccharide Synthesis
The trichloroacetimidate (248) (37 mg, 0.055 mmol) and the acceptor (0.046 mmol) in
dry ether (2 mL) were treated with 4Å molecular sieves (50 mg) and the mixture stirred
(rt, 3 h). The mixture was cooled (−40°C), treated with TMSOTf (20 μL) and allowed to
warm slowly (rt). The mixture was then treated with Et3N (100 μL) and filtered, the
filtrate was concentrated and subjected to flash chromatography (EtOAc/petrol, 1:2) to
give the disaccharide as a colourless oil. The results of the four separate glycosidations
are presented in Table 3.3.
Thioglycoside Method of Disaccharide Synthesis
(a) DPS/Tf2O promotion
The thioglycoside (249) (55 mg, 0.095 mmol), TTBP27 (65 mg, 0.265 mmol) and Ph2SO
(54 mg, 0.265 mmol) in dry CH2Cl2 (2 mL) were treated with Tf2O (45μL, 0.265 mmol)
and the solution stirred (−60°C, 10 min). The acceptor (0.142 mmol) in dry CH2Cl2 (1
mL) was added and solution allowed to warm (0°C). The solution was then treated with
Et3N (100 μL) and filtered, the filtrate concentrated and subjected to flash
chromatography (EtOAc/petrol, 1:2) to give the disaccharide as a colourless oil. The
results of the four separate glycosidations are presented in Table 3.4.
171
(b) BSP/Tf2O promotion
The thioglycoside (249) (84.7 mg, 0.146 mmol), BSP (30.5 mg, 0.146 mmol), and TTBP
(65 mg, 0.265 mmol) in dry CH2Cl2 (2 mL) were treated with Tf2O (45μL, 0.265 mmol)
and the solution stirred (−60°C, 10 min). The acceptor (0.219 mmol) in dry CH2Cl2 (1
mL) was added and solution allowed to warm (0°C). The solution was then treated with
Et3N (100 μL) and filtered, the filtrate concentrated and subjected to flash
chromatography (EtOAc/petrol, 1:2) to give the disaccharide as a colourless oil. The
results of the four separate glycosidations are presented in Table 3.5.
2-O-(α-D-Glucopyranosyl)-D-galactopyranose (203)
O
BnOBnO
OAc
BnO
(250)
O
OOMe
AllO
OH OH
(i) The disaccharide (243) (246 mg) was stirred in AcOH/H2O (4:1, 3 mL) (1 h, 50°C).
The solution was concentrated and subjected to flash chromatography (EtOAc/petrol,
1:1) to give the diol (250) (168 mg, 78%) as a colourless oil, [α]D +80.4°. δH (600 MHz)
2.00 (s, CH3CO), 3.43 (s, CH3O), 3.49 (dd, J 9.5, 9.3, H3′), 3.55 (dd, J2,3 9.6, J1,2 3.5,
H2), 3.80-3.85 (m, 3H, H4, H5, H6), 3.95 (dd, J6,6 10.9, J5,6 5.2, H6), 4.05-4.08 (m, H2′,
H3), 4.12-4.25 (m, 6H, CH2O, H4′, H5′, H6′), 4.58, 4.80 (AB, J 11.1, PhCH2), 4.68, 4.78
(AB, J 12.6, PhCH2), 4.86, 5.01 (AB, J 10.6, PhCH2), 4.87 (d, H1), 4.89 (d, J1′,2′ 3.4,
H1′), 5.13-5.17, 5.57-5.31 (2×m, CH2CH), 5.89-5.96 (m, CH2CH), 7.26-7.37 (m, Ph). δC
(150.9 MHz) 20.95 (CH3CO), 55.20 (CH3O), 63.07, 63.17 (C6, C6′), 68.61, 68.63 (C4′,
172
C5′), 69.10, 75.76 (C4, C5), 71.15 (C2′), 71.76 (CH2O), 73.13, 74.73, 75.79 (3C, PhCH2),
77.38 (C3′), 79.46 (C2), 82.09 (C3), 94.54 (C1), 96.95 (C1′), 118.34 (CH2CH), 127.77-
138.55 (Ph), 134.22 (CH2CH), 170.88 (C=O). m/z (FAB) 708.3177 (C39H48O12 [M]+•
requires 708.3146).
(251)
O
BnOBnO
OAc
BnO
O
OOMe
AcO
OAc OAc
(ii) The diol (250) (170 mg) in EtOH (20 mL) was treated with Wilkinson’s catalyst (27
mg) and the mixture refluxed (12 h); the mixture was then treated with hydrochloric acid
(3 M, 1 mL) and refluxed (1 h). The mixture was neutralized with Et3N (1 mL) and
concentrated to give a pale coloured oil that was dissolved in CH2Cl2 (3 mL) and treated
with pyridine (8 mL), Ac2O (3 mL) and DMAP (10 mg) and the solution stirred (10 h, rt).
The solution was then treated with MeOH (4 mL) (1 h, rt); concentration of the mixture
followed by flash chromatography (EtOAc/petrol, 1:1) gave the tetraacetate (251) (160
mg, 84%) as a colourless oil, [α]D +81.35° (Found C, 63.3; H, 6.6. C42H50O15 requires C,
63.5; H, 6.3%). δH (600 MHz) 2.01, 2.05, 2.06, 2.11 (4×s, 12H, CH3CO), 3.42 (s, CH3O),
3.48 (dd, J 9.8, 9.3, H4′), 3.52 (dd, J2′,3′ 9.6, J1′,2′ 3.5, H2′), 3.95-4.00 (m, H3′, H5), 4.07
(dd, J2,3 10.7, J1,2 3.5, H2), 4.09-4.10 (m, 2H, H6), 4.17-4.20 (m, 2H, H5′, H6′), 4.24 (dd,
J6′,6′ 12.0, J5′,6′ 3.8, H6′), 4.56, 4.86 (AB, J 11.1, PhCH2), 4.66, 4.78 (AB, J 11.9, PhCH2),
4.80, 4.98 (AB, J 10.7, PhCH2), 4.84 (d, H1′), 4.91 (d, H1), 5.36 (dd, J3,4 2.7, H3), 5.46
(d, H4), 7.22-7.36 (m, Ph). δC (150.9 MHz) 20.78, 20.86, 20.96 (CH3CO), 55.56 (CH3O),
62.03, 62.89 (C6, C6′), 66.37 (C5′), 68.50 (C4), 68.80 (C3), 69.19 (C5), 70.69 (C2),
173
73.32, 74.94, 75.80 (3C, PhCH2), 77.07 (C4′), 79.60 (C2′), 81.78 (C3′), 95.50 (C1′),
97.32 (C1), 127.85-138.67 (Ph), 169.87, 170.21, 170.61, 170.79 (4C, C=O).
(252)
O
AcOAcO
OAc
AcO
O
OOMe
AcO
OAc OAc
(iii) The tetraacetate (251) (140 mg) in MeOH was treated with Pd/C (10%, 10 mg) and
H2 and the mixture stirred (1 atm, 12 h, 35°C). The mixture was filtered, the filtrate
concentrated and treated with pyridine (3 mL), Ac2O (1 mL) and DMAP (10 mg) (5 h,
rt). Treatment with MeOH (5 mL) (1 h, rt), followed by concentration of the mixture and
flash chromatography (EtOAc/petrol, 1:1), gave the heptaacetate (252) (103 mg, 90%) as
a colourless oil, [α]D +108°. δH (600 MHz) 1.98, 1.99, 1.99, 2.02, 2.06, 2.11 (6×s, 21H,
CH3CO), 3.38 (s, CH3O), 4.01 (dd, J2,3 10.6, J1,2 3.6, H2), 4.04-4.11 (m, 4H, H5′, H6,
H6′), 4.13 (dd, J5,6 6.8, 6.4, H5), 4.22 (dd, J6′,6′ 12.3, J5′,6′ 3.9, H6′), 4.71 (dd, J2′,3′ 10.1,
J1′,2′ 3.7, H2′), 4.83 (d, H1), 5.02 (dd, J 9.9, 9.8, H4′), 5.26 (dd, J3,4 3.5, H3), 5.27 (d,
H1′), 5.40 (dd, H3′), 5.42 (d, H4). δC (150.9 MHz) 20.68, 20.72, 20.77 (CH3CO), 55.49
(CH3O), 61.67, 61.84 (C6, C6′), 66.26 (C5), 67.65 (C5′), 68.28, 68.37 (C4, C4′), 68.86
(C3), 70.02 (C3′), 71.34, 71.60 (C2, C2′), 94.02 (C1), 97.15 (C1′), 169.67-170.65 (7C,
C=O). m/z (FAB) 651.2145 (C27H39O18 [M+H]+ requires 651.2136).
174
(253)
O
AcOAcO
OAc
AcO
O
OOAc
AcO
OAc OAc
(iv) The heptaacetate (252) (103 mg) was stirred with Ac2O (3 mL) and H2SO4 (50 μL) (6
h, 0°C); the solution was then poured onto ice and allowed to stand (rt, 12 h). The
mixture was extracted with EtOAc, the extract washed with water, saturated NaHCO3 and
dried; concentration of the organic extract gave the octaacetate (253) (102 mg, 95%) as a
colourless oil and a mixture of anomers (α:β, 4:1). δH (600 MHz) 1.97-2.19 (CH3CO),
4.02-4.20 (m, H2β, H5′, H5β, H6, H6′), 4.19 (dd, J2,3 10.6, J1,2 3.6, H2α), 4.24 (dd, J6,6
12.2, J5,6 3.5, H6α), 4.25-4.28 (m, H6β), 4.32 (dd, J5,6 7.3, H5α), 4.75 (dd, J2,3 10.3, J1,2
3.9, H2′β), 4.92 (dd, J2′,3′ 10.4, J1′,2′ 3.5, H2′α), 5.03-5.05 (m, H3β, H4′β), 5.07-5.09 (m,
H4′α), 5.10 (d, H1′α), 5.31 (dd, J2,3 10.6, J3,4 2.5, H3α), 5.33-5.35 (m, H3′β), 5.35 (dd,
J3′,4′ 10.3, H3′α), 5.45-5.40 (m, H1′β, H4β), 5.52 (d, J3,4 2.5, H4α), 5.65 (d, J1,2 8.1, H1β),
6.29 (d, H1α). δC (150.9 MHz) 20.65-21.10 (CH3) 61.11, 61.27, 61.61 (C6′, C6) 66.99-
72.12 (C2, C2′, C3, C3′, C4, C4′, C5, C5′), 89.21 (C1α), 93.75 (C1β), 95.17 (C1′β), 95.72
(C1′α), 169.08-170.73 (C=O). m/z (FAB) 619.1857 (C26H35O17 [M–OAc]+ requires
619.1874).
175
(203)
O
HOHO
OH
OH
O
OOH
HO
OH OH
(v) The octaacetate (253) (90 mg) in MeOH (3 mL) was treated with NaOMe in MeOH
(1mL) and the solution stirred (4 h, rt). The solution was neutralized with resin
(Amberlite IR-120, H+), filtered and the filtrate concentrated to give the disaccharide
(203) (41 mg, 89%) as a colourless oil and a mixture of anomers (α:β, 1:1.4), [α]D
+129.0° (H2O). δH (600 MHz, D2O) 3.41-3.45 (m, H5), 3.52 (m, H2, H2′β), 3.67-3.92,
4.01-4.04, 4.07-4.11 (3×m, H2′α, H3, H3′, H4, H4′, H5′β, H6, H6′), 3.96 (dd, J 10.2, 3.3,
H5′α), 4.69-4.71 (m, H1β), 5.09 (d, J1,2 3.7, H1α), 5.37 (d, J1′,2′ 3.8, H1′β), 5.46 (d, J1′,2′
3.5, H1′α). δC (150.9 MHz, D2O) 63.12, 63.66 (C6β, C6′β), 63.15, 63.82 (C6α, C6′α),
70.34-79.71 (C2, C2′, C3, C3′, C4, C4′, C5, C5′), 92.35 (C1′α), 98.91 (C1α), 99.33 (C1β),
100.64 (C1′β). m/z (FAB) 343.1253 (C12H23O11 [M+H]+ requires 343.1240).
3-O-(α-D-Glucopyranosyl)-D-galactopyranose (204)
O
AcOAcO
OAc
AcOO
OAc OAc
OMeAcO
O
O
AcOAcO
OAc
OAc
O
OAc OAc
OMeAcO
O
(255)(254)
(i) The disaccharide (246) (156 mg) in MeOH (20 mL) was treated with Pd/C (10 %, 15
mg) and H2 and the mixture stirred (1 atm, 12 h). The mixture was filtered, the filtrate
concentrated and treated with pyridine (2 mL), Ac2O (1 mL) and DMAP (5 mg) and
stirred (rt, 8 h). Treatment with MeOH (2 mL), followed by concentration of the mixture
and flash chromatography (EtOAc/petrol, 1:2), gave first methyl 3-O-(tetra-O-acetyl-α-D-
176
glucopyranosyl)-2,4,6-tri-O-acetyl-α-D-galactoside (254) (65 mg, 64%) as a colourless
oil, [α]D +154.2°. δH (600 MHz) 1.98, 2.01, 2.05, 2.05, 2.12, 2.14, 2.14 (7×s, 21H,
CH3CO), 3.36 (s, CH3O), 4.06-4.10 (m, 4H, H5, H6, H6′), 4.27-4.29 (m, H5′), 4.30 (dd,
J2,3 10.5, J3,4 3.0, H3), 4.34 (dd, J6′,6′ 12.2, J5′,6′ 1.5, H6′), 5.00-5.04 (m, H2′, H4′), 5.05 (d,
J1,2 3.5, H1), 5.08 (dd, J1,2 3.4, H2), 5.19 (d, H1′), 5.36 (dd, J3′,4′ ≈ J2′,3′ 9.8, H3′), 5.40 (d,
H4). δC (150.9 MHz) 20.44, 20.49, 20.53, 20.58, 20.61 (CH3CO), 55.25 (CH3O), 61.68,
61.81 (C6, C6′), 65.68 (C4), 66.19 (C5), 67.74, 68.07 (C3, C5′), 68.66, 69.18 (C2′, C4′),
69.31 (C2), 69.69 (C3′), 91.88 (C1′), 96.98 (C1), 169.39-170.32 (C=O). m/z (FAB)
651.2179 (C27H39O18 [M+H]+ requires 651.2136).
Further elution gave methyl 3-O-(tetra-O-acetyl-β-D-glucopyranosyl)-2,4,6-tri-O-acetyl-
α-D-galactoside (255) (25 mg, 24%) as a colourless oil, [α]D +69.0° (lit28 +75.0°). The 1H
(600 MHz) and 13C (150.9 MHz) n.m.r. spectral data were in good agreement with those
reported.29
O
AcOAcO
OAc
AcOO
OAc OAc
OAcO
(256)OAc
(ii) The heptaacetate (254) (46 mg) in Ac2O (2 mL) was treated with H2SO4 (100 μL) and
the solution stirred (0°C, 6 h). The solution was then poured onto ice and stirred (12 h),
the mixture was then extracted with EtOAc, the extract washed with saturated NaHCO3,
brine and then dried. Concentration of the organic extract followed by flash
chromatography (EtOAc/petrol, 1:3) gave (256) (45 mg, 94%) as a colourless oil and
predominantly as the α-anomer (α:β, 1:0.05). δH (600 MHz) 1.98, 2.00, 2.04, 2.06, 2.07,
2.11, 2.14, 2.27 (8×s, 24H, CH3CO), 4.05-4.25 (m, 6H, H5, H5′, H6, H6′), 4.27 (dd, J2,3
10.8, J3,4 3.0, H3), 5.02 (dd, J2′,3′ 10.0, J1′,2′ 3.3, H2′), 5.09 (dd, J4′,5′ ≈ J3′,4′ 9.4, H4′), 5.22
177
(d, H1′), 5.30 (dd, J1,2 3.6, H2), 5.35 (dd, H3′), 5.45 (d, H4), 6.42 (d, H1). δC (150.9
MHz) 20.56-21.05 (8C, CH3), 61.19, 61.58 (C6, C6′), 65.28 (C4), 67.51, 68.52, 68.75,
68.83, 69.17, 69.52 (C2, C2′, C3, C4′, C5, C5′) 67.69 (C3′), 89.64 (C1), 92.57 (C1′),
168.94-170.71 (8C, C=O). m/z (FAB) 619.1835 (C26H35O17 [M–OAc]+ requires
619.1874).
O
HOHO
OH
OHO
OH OH
OHO
OH
(204)
(iii) The octaacetate (256) (39.0 mg, 0.058 mmol) in MeOH (3 mL) was treated with a
solution of NaOMe in MeOH (1mL) and the solution stirred (4 h, rt). The solution was
neutralized with resin (Amberlite IR-120, H+), filtered and the filtrate concentrated to
give the disaccharide (204) (20 mg, 96%) as a colourless oil and as a mixture of anomers
(α:β, 1:1.4), [α]D +146.2° (H2O). δH (600 MHz, D2O) 3.42-3.46, 3.56-3.62, 3.67-3.86,
3.92-3.96 (4×m, H2α, H2′, H3, H3′, H4′, H5, H5′, H6, H6′), 4.07 (dd, J2,3 6.1, J1,2 7.0,
H2β), 4.15 (d, J 2.5 Hz, H4β), 4.21 (s, H4α), 4.62 (d, H1β), 5.10 (d, J1′,2′ 3.5, H1′β), 5.12
(d, J1′,2′ 3.6, H1′α), 5.28 (d, J1,2 1.4, H1α). δC (150.9 MHz, D2O) 63.03, 63.08 (C6β,
C6′β), 63.14, 63.87 (C6α, C6′α), 67.74 (C4β), 68.31 (C4α), 69.42-76.82 (C2, C2′, C3α,
C3′, C4′, C5α, C5′), 77.57 (C5β), 80.20 (C3β), 95.04 (C1α), 97.69 (C1′α), 97.98 (C1′β),
99.10 (C1β). m/z (FAB) 343.1230 (C12H23O11 [M+H]+ requires 343.1240).
178
4-O-(α-D-Glucopyranosyl)-D-galactose (205)
O
AcO
O OAc
OMeAcO
O
AcOAcO
OAc
OAc
(257) (258)
O
AcO
O
AcOOAc
O
AcOAcO
OAc
AcO
OMe
(i) The disaccharide (247) (150 mg) in MeOH (20 mL) was treated with Pd/C (10 %, 15
mg) and H2 and the mixture stirred (12 h, 1 atm). The mixture was filtered, the filtrate
concentrated and treated with pyridine (2 mL), Ac2O (1 mL) and DMAP (5 mg) and the
solution stirred (rt, 8 h). Treatement with MeOH (2 mL) followed by concentration and
flash chromatography (EtOAc/petrol, 1:2) gave first methyl 4-O-(tetra-O-acetyl-α-D-
glucopyranosyl)-2,3,6-tri-O-acetyl-α-D-galactoside (257) (57 mg, 55%) as a colourless
oil, [α]D +32.2° (lit29 +36.0°). The 1H (600 MHz) and 13C (150.9 MHz) n.m.r. spectral
data were in good agreement with those reported.29
Further elution gave methyl 4-O-(tetra-O-acetyl-β-D-glucopyranosyl)-2,3,6-tri-O-acetyl-
α-D-galactoside (258) (20 mg, 20%) as a colourless oil, [α]D +8.5° (lit29 +7.0°). The 1H
(600 MHz) and 13C (150.9 MHz) n.m.r. spectral data were in good agreement with those
reported.29
(259)
O
AcO
O
AcOOAc
O
AcOAcO
OAc
AcO
OAc
(ii) The heptaacetate (257) (40 mg) was dissolved in Ac2O (2 mL) was treated with
H2SO4 (100 μL) and the solution stirred (rt, 6 h). The solution was poured onto ice and
stirred (6 h); the mixture was then extracted with EtOAc, the extract washed with
179
saturated NaHCO3, brine and then dried. Concentration of the organic extract gave the
octaacetate (259) (40 mg) as a colourless oil and as a mixture of anomers (α:β, 5:1). δH
(600 MHz) 1.98-2.15 (CH3CO), 3.93 (dd, J5,6 6.6, 6.4, H5β), 4.09-4.29 (m, H4, H5α, H5′,
H6, H6′), 4.36 (dd, J6,6 11.1, J5,6 6.7, H6α), 4.41 (dd, J6,6 11.4, H6β), 4.84 (dd, J3,4 2.6,
J2,3 10.7, H3β), 4.93-4.96 (m, H1′, H3α), 5.14-5.17 (m, H2′, H4′), 5.30 (dd, J1,2 8.1, H2β),
5.44 (dd, J2,3 11.2, J1,2 3.7, H2α), 5.47 (dd, J3′,4′ ≈ J2′,3′ 9.6, H3′α), 5.48 (dd, J3′,4′ ≈ J2′,3′
9.7, H3′β), 5.71 (d, H1β), 6.36 (d, H1α). δC (150.9 MHz) 20.65-21.19 (CH3), 61.17, 62.04
(C6β, C6′β), 61.30, 61.96 (C6α, C6′α), 66.07 (C2α), 67.74 (C2β), 68.12, 68.74, 69.64,
70.09, 70.48, 71.12 (C2′α, C3α, C3′α, C4′α, C5α, C5′α), 68.18, 68.47, 70.26, 71.18,
72.83, 73.07 (C2′β, C3β, C3′β, C4′β, C5β, C5′β), 77.94 (C4β), 78.43 (C4α), 89.99 (C1′α),
92.05 (C1′β), 99.43 (C1α), 99.54 (C1β), 168.98-170.81 (C=O). m/z (FAB) 619.1847
(C26H35O17 [M–OAc]+ requires 619.1874).
(205)
O
HO
O
OHOH
O
HOHO
OH
OH
OH
(iii) The octaacetate (259) (37 mg) in MeOH (4 mL) was treated with NaOMe in MeOH
(1 mL) and the solution stirred (rt, 1 h). The solution was neutralized with resin
(Amberlite IR-120, H+), filtered and the filtrate concentrated to give the disaccharide
(205) (18 mg, 96%) as a colourless oil and as a mixture of anomers (α:β, 0.4:1), [α]D
+116.5° (H2O). δH (600 MHz, D2O) 3.45 (dd, J 9.7, 9.7, H3′β), 3.50-3.55, 3.70-3.93,
4.09-4.15 (3×m, H2, H2′, H3, H3′α, H4′, H5, H5′, H6, H6′), 4.00 (d, J 2.9, H4β), 4.07 (d,
J 2.7, H4α), 4.64 (d, J1,2 7.8, H1β), 4.92-4.93 (m, H1′), 5.29 (d, J1,2 3.8, H1α). δC (150.9
MHz, D2O) 62.79, 62.82, 62.87, 63.08 (C6, C6′), 71.17-77.81 (C2, C2′, C3, C3′, C4′α,
180
C5, C5′), 80.09 (C4β), 81.39 (C4α), 95.08 (C1α), 99.35 (C1β), 102.70 (C1′β), 102.90
(C1′α). m/z (FAB) 342.1235 (C12H23O11 [M+H]+ requires 343.1240).
6-O-(α-D-Glucopyranosyl)-D-galactopyranose (206)
O
O
O
OO
(260)
O
HOHO
OH
OHO
(i) The acetate (241) (139 mg) in MeOH (25 mL) was treated with Pd/C (10%, 20 mg)
and H2 and stirred (2 d, 1 atm). The mixture was filtered, the filtrate was concentrated
and subjected to flash chromatography (EtOAc/petrol, 2:1) to give the tetrol (260) (60
mg, 89%) as colourless oil. δH (600 MHz) 1.30, 1.32, 1.41, 1.52 (4×s, 12H, CH3), 3.53-
3.58, 3.62-3.63, 3.73-3.76 (3×m, 5H, H2′, H3′, H4′, H5, H6), 3.69 (dd, J6′,6′ 10.8, J5′,6′ 5.2,
H6′), 3.80 (dd, J5′,6′ 7.2, H6′), 3.84-3.86 (m, 1H, H6), 3.96 (dd, H5′), 4.25 (dd, J3,4 8.0, J4,5
1.5, H4), 4.29 (dd, J1,2 4.9, J2,3 2.4, H2) 4.59 (dd, H3), 4.86 (d, J1′,2′ 3.55, H1′), 5.51 (d,
H1). δC (150.9 MHz) 24.64, 25.02, 26.09, 26.13 (4C, CH3C), 61.35, 67.25 (C6, C6′),
66.65, 69.62 (C5, C5′), 70.56 (C2), 70.71 (C3), 71.24 (C4), 71.99, 72.11, 74.24 (C2′, C3′,
C4′), 96.34 (C1′), 99.38 (C1), 108.96, 109.58 (2C, CH3C). m/z (FAB) 423.1873
(C18H31O11 [M+H]+ requires 423.1866).
181
O
HO
OH
OH
(206)
O
HOHO
OH
OHO
OH
(ii) The tetrol (260) (26 mg) in CF3COOH/H2O (4:1, 2 mL) was stirred (0ºC, 1 h). The
solution was then concentrated, applied to a Sephadex (IR 120) column and eluted with
H2O. Concentration of the eluant gave the free sugar (206) (18.5 mg, 89%) as a
colourless oil and as a mixture of anomers (α:β, 0.6:1), [α]D +119.0° (H2O). δH (600
MHz, D2O) 3.39 (m, H3′), 3.48 (dd, J2,3 9.8, J1,2 7.9, H2β), 3.54-3.56 (m, H2′), 3.65 (dd,
J3,4 3.4, H3β), 3.67-3.77, 3.82-3.88 (2×m, H3α, H4′, H5β, H5′, H6, H6′), 3.79 (dd, J2,3
10.3, J1,2 3.9, H2α), 3.97 (d, H4β), 4.02 (d, J3,4 3.0, H4α), 4.26 (dd, J5,6 6.4, 5.9, H5α),
4.58 (d, H1β), 4.93-4.95 (m, H1′), 5.25 (d, H1α). δC (150.9 MHz, D2O) 63.22 (C6′),
69.25, 69.55 (C6), 71.04 (C2α), 71.25 (C5α), 71.50, 71.79, 72.05, 72.26 (C4, C4′, C5β,
C5′β), 74.00, 74.03 (C2′), 74.52, 74.55, 75.44, 75.70, 75.75 (C2β, C3, C3′α,), 95.07
(C1β), 99.17 (C1α), 100.95, 100.98 (C1′). m/z (FAB) 343.1239 (C12H23O11 [M+H]+
requires 343.1240).
182
References.
(1) Fischer, E. Chem. Ber. 1893, 26, 2412.
(2) Stick, R. V. Carbohydrates: The Sweet Molecules of Life (Academic Press:
London, 2001).
(3) Lemieux, R. U.; Hendriks, K. B.; Stick, R. V.; James, K. J. Am. Chem. Soc. 1975,
97, 4056.
(4) Thiem, J.; Meyer, B. Chem. Ber. 1980, 113, 3075.
(5) Gervay, J. Org. Synth. 1998, 4, 121.
(6) Gervay, J.; Nguyen, T. N.; Hadd, M. J. Carbohydr. Res. 1997, 300, 119.
(7) Kartha, K. P. R.; Field, R. A. J. Carbohydr. Chem. 1998, 17, 693.
(8) Lam, S. N.; Gervay-Hague, J. Carbohydr. Res. 2002, 337, 1953.
(9) Kobashi, Y.; Mukaiyama, T. Chem. Lett. 2004, 33, 874.
(10) Pougny, J.-R.; Sinaÿ, P. Tetrahedron Lett. 1976, 17, 4076.
(11) Pougny, J.-R.; Jacquinet, J.-C.; Nassr, M.; Duchet, D.; Milat, M.-L.; Sinaÿ, P. J.
Am. Chem. Soc. 1977, 99, 6762.
(12) Schmidt, R. R.; Michel, J. Angew. Chem. Int. Ed. 1980, 19, 731.
(13) Schmidt, R. R.; Michel, J. Tetrahedron Lett. 1984, 25, 821.
(14) Osborn, H. M. I. Carbohydrates (Elsevier Science: Oxford, 2003).
(15) Schmidt, R. R.; Michel, J. J. Carbohydr. Chem. 1985, 4, 141.
(16) Wegmann, B.; Schmidt, R. R. J. Carbohydr. Chem. 1987, 6, 357.
(17) Tropper, F. D.; Andersson, F. O.; Grand-Maître, C.; Roy, R. Synthesis 1991, 734.
(18) Crich, D.; Smith, M. J. Am. Chem. Soc. 2001, 123, 9015.
183
(19) Codée, J. D. C.; Litjens, R. E. J. N.; den Heeten, R.; Overkleeft, H. S.; van Boom,
J. H.; van der Marel, G. A. Org. Lett. 2003, 5, 1519.
(20) Crich, D.; Cai, W. J. Org. Chem. 1999, 64, 4926.
(21) Mukaiyama, T.; Kobashi, Y. Chem. Lett. 2004, 33, 10.
(22) Kong, F.; Lu, D. Carbohydr. Res. 1990, 198, 141.
(23) Garegg, P. J.; Hultberg, H.; Wallin, S. Carbohydr. Res. 1982, 108, 97.
(24) Schmidt, R. R.; Michel, J. J. Org. Chem. 1981, 46, 4787.
(25) Rathore, H.; Hashimoto, T.; Igarashi, K.; Nukaya, H.; Fullerton, D. S.
Tetrahedron 1985, 41, 5427.
(26) France, R. R.; Rees, N. V.; Wadhawan, J. D.; Fairbanks, A. J.; Compton, R. G.
Org. Biomol. Chem. 2004, 2, 2188.
(27) Crich, D.; Smith, M.; Yao, Q.; Picione, J. Synthesis 2001, 323.
(28) Wozney, Y. V.; Backinowsky, L. V.; Kochetkov, N. K. Carbohydr. Res. 1979,
73, 282.
(29) Müller, M.; Huchel, U.; Geyer, A.; Schmidt, R. R. J. Org. Chem. 1999, 64, 6190.
184
Chapter 4
Development of an Alternative
Carbohydrate Source for Pre-term Infants
186
187
Introduction
Breast Milk as a Nutrient Source
Breast milk is universally considered to be the best source of nutrition for all newborn
infants.1 Along with providing all the nutritional requirements, breast milk contains a
large number of bioactive compounds that protect the infant against infection and provide
a range of other benefits, including protection against diarrhea,2-4 lower respiratory tract
infection,3,5 diabetes6-8 and necrotizing enterocolitis.9 Infants raised on breast milk also
show an eventual increase in intelligence quotient.10
Metabolism of Lactose
Lactose is the primary carbohydrate present in breast milk; it is hydrolysed by lactase,
present in the brush border region in the small intestine, to produce D-glucose and D-
galactose:11
O
HO
OH OH
OH OH
O
HOHO
OH
OH OHD-glucose
D-galactose
O
HO
OH OH
OHO
HOO
OH
OH OH
lactose
lactase
188
D-Glucose and D-galactose are then transported across the brush border membrane of the
small intestine by two Na+ D-glucose co-transporters and are then delivered to the liver
via the portal vein.12,13 Transportation across the liver cell membrane is achieved through
a transporter, followed by the initial process of metabolism, phosphorylation. Hexokinase
is responsible for the conversion of D-glucose into D-glucose-6-phosphate whilst
glucokinase converts D-glucose into α-D-glucose-1-phosphate; galactokinase converts D-
galactose into α-D-galactose-1-phosphate:14
O
HO
OH OH
OH OH
O
HOHO
OH
OH OHD-glucose D-galactose
galactokinase
O
HO
OH OH
OHOPO3
2-
α-D-galactose-1-phosphate
O
HOHO
OPO32-
OH OH
O
HOHO
OH
OHOPO3
2-
α-D-glucose-1-phosphateD-glucose-6-phosphate
glucokinasehexokinase
Hepatic processing of D-glucose and D-galactose.
Hexokinase has a relatively low Michaelis constant (KM) and therefore cannot augment
the hepatic phosphorylation of D-glucose during alimentation. In addition, hexokinase is
also inhibited by the formation of D-glucose-6-phosphate.15 Glucokinase, in contrast, has
a significantly higher KM enabling it to function at higher D-glucose concentrations,
particularly during alimentation; unfortunately, glucokinase activity is low in most
newborn infants.14,15 It has been shown that low birth weight infants often develop a
189
diabetic-like D-glucose tolerance curve and that D-glucose intolerance is also a
contributing factor to the higher mortality rates of pre-term infants.16 Galactokinase, on
the other hand, shows relatively high activity and is responsible for the rapid clearance of
D-galactose by the newborn.13,15,17
Lactase Deficiency
Lactase deficiency, a particular problem in pre-term and low birth weight infants, hinders
the metabolism of lactose and hence presents significant difficulties in regards to
achieving adequate nutrition.18,19 Lactase is the last enzyme to develop, present at 30% of
its full term level at 34 weeks;20,21 infants born pre-term have significantly lower lactase
activity compared to those born at term.19-21 As a consequence of the reduced lactase
activity, lactose is often only partially digested.22 Poor absorption of carbohydrates has
been strongly linked to the malabsorption of calcium and other minerals important to pre-
term infants.23
Necrotising enterocolitis is a disease of the gastrointestinal tract of unknown origin with
clinical symptoms including the development of necrotic lesions in the gastrointestinal
tract and disposition for intestinal perforation.24 Poor absorption of carbohydrates leads to
colonic fermentation with subsequent production of gas and bowel distention producing
ischemia, increasing the risk of necrotizing enterocolitis.24,25
190
Calorie Requirement of Pre-term Infants
In addition to the problem of lactase deficiency, pre-term infants require a significantly
higher calorie intake to offset the absence of the rapid growth period observed in the third
trimester of pregnancy.26 Treatment for both the intolerance to lactose and the inadequate
calorie content involves the replacement or supplement of lactose with an alternative
carbohydrate source. The most common lactose replacement is based on a glucose
polymer known as maltodextrin, with two examples being “Polyjoule” and “S26/SMA”.27
Hydrolysis of glucose polymers by the pre-term infant occurs mainly via the
glucoamylase-maltase complex in the small intestine, with a preference for glucose
polymers of less than ten glucose units. 28,29 A significant number of commercial glucose
polymer preparations are derived from the hydrolysis of cornstarch, which typically
contains around 35% glucose polymer of a chain length greater than ten units.29 Often
these longer chain glucose polymers are not fully hydrolyzed and remain partially
undigested.29
O
HOO
OH
OH
O
HO
OH
OH
O
Maltodextrin
O
HOO
OH
OH
O
HOHO
OH
OH
OH
Maltose
O
HOHO
OH
OH OH
D-glucose
glucoamylase-maltase
glucoamylase-maltase
n
Hydrolysis of maltodextrin by the glucoamylase-maltase complex
191
The major downfall of maltodextrin-based supplements is that they provide a source only
of D-glucose; pre-term infants lack the enzymatic activity to process D-glucose as their
sole carbohydrate source and often develop a diabetic like response.14,16
Issues with Osmolality and Feeding of Pre-term Infants
Treatment of milk containing lactose with a β-galactosidase is another possible treatment
for lactose intolerant infants, however significant issues exist with the subsequent
increase in osmolality.30 Osmolality greatly influences the feeding tolerance of pre-term
infants, with the recommended maximum of 425 mOsm/kg a significant restriction to the
supplementation and treatment of milk.26,31 Hyperosmolality of infant formulas has been
strongly associated with an increased risk of necrotizing enterocolitis as well as pulling
fluid into the bowel, resulting in diarrhea.26,30-33
Osmolality issues also come into play when using maltodextrin-based supplements since
routine handling of the supplemented breast milk can result in a significant increase in
osmolality owing to the hydrolysis of maltodextrin to maltose and D-glucose by the α-
amylase present in breast milk.31
Alternative Forms of Nutrition
An ideal nutrient source for pre-term infants would involve the removal of lactose from
breast milk through ultrafiltration, a technique commonly used in the dairy industry.34 A
superlative carbohydrate supplement would essentially mimic lactose in every respect
with the exception of the enzyme responsible for hydrolysis. Lactose is the only β-linked
192
disaccharide that humans can hydrolyse; α-linked disaccharides such as maltose,
isomaltose and sucrose make up the vast majority of hydrolysable carbohydrates. This
leaves α-linked disaccharides of D-glucose and D-galactose as the most logical target for
breast milk supplementation. It is also important that the sugar be resistant to hydrolysis
by the enzymes present in breast milk, most importantly α-amylase, to prevent any
possibility of hydrolysis during routine handling.
Enzymes in the Digestive Tract
The gamut of suitable enzymes is fairly limited with the sucrase-isomaltase and
glucoamylase-maltase complexes both present in the brush border region of the small
intestine.21,22 To a smaller extent α-galactosidase and α-mannosidase are also present in
the small intestine; however, detailed study on the levels present particularly in pre-term
infants is lacking.35
Sucrase-isomaltase is an exo-hydrolase that acts at the non-reducing end of
oligosaccharides and specifically cleaves α,β-(1→2), α-(1→4) and α-(1→6) bonds.36 The
sucrase-isomaltase complex contains two active sites: the sucrase active site contains two
subsites, only being able to bind two monomeric parts of the substrate, whilst the
isomaltase active site contains four subsites, able to bind di-, tri-, and tetra-saccharides.
28,37,38 Activity of the sucrase-isomaltase complex in newborn infants varies from three
times greater than lactase in the duodenum to eight times greater in the proximal end of
the small intestine.21 Sucrase-isomaltase isolated from the pig intestine has been shown to
have a large degree of flexibility in the linkages that it is able to process.36
193
Glucoamylase-maltase in an exoamylase that catalyses the hydrolysis of maltose, and
maltodextrins from the non-reducing end.39 The glucoamylase-maltase complex
specifically cleaves α-(1→4) bonds with a strong specificity for maltooligosaccharides up
to maltoheptaose.36 In contrast to the sucrase-isomaltase complex, the two active sites of
glucoamylase-maltase cannot be distinguished with respect to substrate specificity.38
Activity of glucoamylase-maltase in the pre-term infant is at least three times greater than
that of lactase.21,36 Glucoamylase-maltase is significantly more selective than sucrase-
isomaltase, hydrolysing only a small range of substrates with nearly all examples
containing α-(1→4) links.36
O
HOOH
O
HOHO
OH
OHO
HO
CH2OH
OH
OH
OHOO
O
HOOH
OH
HO
OH
OH
OHOO
OHO
OH
OH
OH
O
sucrase-isomaltase: 97% sucrase-isomaltase: 99%glucoamylase-maltase: 63%
α(1→6)
α(1→1)
O
HOOH
OH
HO
O
OHOH
OH OH
OH
sucrase-isomaltase: 60%glucoamylase-maltase: 34%
α(1→1)
O
HOOH
O
HOHO
OH
OHO
HO
α(1→6)
OHOH
OH OH
OHO
sucrase-isomaltase: 95%
Position of hydrolysis indicated by arrows, and as a % after 24 h.36
194
New Synthetic Sugars for Infants
Both sucrase-isomaltase and the glucoamylase-maltase complex exhibit α-glucosidase
activity, indicating that the most suitable disaccharide structure would be a D-galactosyl
α-D-glucoside. Four possible candidate disaccharides, synthesized in Chapter 3, are
presented below:
O
HOHO
OH
OHO
OH OH
OHO
OH
(204)
O
HO
OH
OH(206)
O
HOHO
OH
OHO
OH(205)
O
HO
O
OHOH
O
HOHO
OH
OH
OH
O
HOHO
OH
OH
O
O
(203)
OH
HO
OH OH
.
The hydrolysis of such a synthetic disaccharide in the brush border region of the small
intestine would produce D-glucose and D-galactose, identical in every respect to the
product from the hydrolysis of lactose:
OHO
OH
OH
HO
O
OH
OH
HO
OH
OH OH
synthetic sugar
α-glucosidaselactase
D-glucose D-galactose
O
HO
OH OH
OHO
HOO
OH
OH OH
lactose
O
HO
O
OHOH
O
HOHO
OH
OH
OH
195
Discussion
Initial Study
The preliminary work consisted of incubation of each of the four synthetic sugars with a
commercially available enzyme. Commercial sources of α-amylase, isomaltase, α-
glucosidase and α-galactosidase were proposed for the initial testing. Measurement of
hydrolysis was achieved via a standard colorimetric glucose assay. This enabled the
feasibility of the proposal to be determined in the most rapid and cost effective fashion.
In the example shown maltose produces two D-glucose units. The D-glucose is then
oxidised to D-gluconic acid, producing H2O2 in the process that then oxidises
ABTS(reduced) in the presence of a peroxidase. The coloured, oxidised ABTS(oxidised) is then
detected at 405 nm using a UV/Vis spectrophotometer:
maltose H2O D-glucoseα-glucosidase
glucose oxidase
peroxidase
D-glucose H2OO2 H2O2D-gluconic acid
H2O2 ABTS(reduced) ABTS(oxidised) 2 H2O
D-glucose
Mechanism of colorimetric glucose assay.
Allowance has to be made in situations where the disaccharide produces two units of
glucose and hence two units of oxidised ABTS:
196
melibiose H2O
isomaltose H2O
maltose H2O
α-D-glcp-(1→2)-D-gal H2O
H2O
H2O
H2O
D-glucose D-glucose
D-glucose D-glucose
D-glucose D-galactose
D-glucose D-galactose
D-glucose D-galactose
D-glucose D-galactose
D-glucose D-galactose
α-D-glcp-(1→3)-D-gal
α-D-glcp-(1→4)-D-gal
α-D-glcp-(1→6)-D-gal
Preliminary Tests Using Commercially Available Enzymes
Results from the initial study are shown below:
Enzyme Disaccharide α-amylase α-galactosidase α-glucosidase isomaltase
α-D-glcp-(1→2)-D-gal (203) N N N P α-D-glcp-(1→3)-D-gal (204) N N N N α-D-glcp-(1→4)-D-gal (205) N N N N α-D-glcp-(1→6)-D-gal (205) N N N H
Table 4.1 Hydrolysis of the synthetic disaccharides using commercially available
enzymes (N = no hydrolysis, P = partial hydrolysis, H = hydrolysis).
The results indicate that (206) appears to be the most suitable for further exploration in a
mammalian model. The disaccharide (206) clearly showed no hydrolysis with α-amylase,
indicating it would be stable in breast milk, whilst it showed good hydrolysis with
isomaltase. The activity of (203) with isomaltase was not completely unexpected
considering that the sucrase-isomaltase complex is known to cleave α-(1→2) bonds.36
197
Unfortunately the activity was insufficient to warrant further testing. Both (204) and
(205) showed no signs of hydrolysis and thus were rejected for further testing.
Mammalian Enzyme Study
Whilst the preliminary results suggested that (206) may be suitable as a milk supplement,
a detailed kinetic study on a mammalian model was needed. Despite the obvious external
differences, pigs are physiologically similar to humans with a near identical gasto-
intestinal tract, cardiovascular system, muscular structure and biliary system.40 This,
combined with the relative ease in obtaining pig intestinal samples resulted in the pig
being selected as the mammalian model for kinetic studies.
Despite the measurement of disaccharidase activity being used in the clinical
environment to diagnose associated disorders, only two techniques have been developed
to measure this activity. The first technique available was developed by Dahlqvist,
relying on the measurement of glucose produced in a one hour period of incubation of a
disaccharide with an intestinal mucosa sample.41 This technique relies on the colour
change produced by the oxidation of o-dianisidine, analogous to the ABTS assay
previously reported. The second technique reported was a continuous photometric
method, reported by Hansen and coworkers.42 This assay operates by converting β-D-
glucose into D-glucono-δ-lactone using glucose dehydrogenase, producing NADH in the
process. The concentration of glucose is then measured on a continuous basis via the UV
absorbance of NADH at 334 nm:
198
sucrose H2O D-glucose D-fructosesucrase
D-glucose β-D-glucosemutarotase
β-D-glucose NAD+ NADHD-glucono-δ-lactone H+
glucosedehydrogenase
Continuous measurement of glucose production via NADH.
Unfortunately, the availability of the mutarotase (aldose-1-epimerase) was quite limited,
forcing a resort to the method of Dahlqvist, albeit with some improvements. The method
of Dalhqvist makes use of the colorimetric change of o-dianisidine, a suspected
carcinogen, which has been replaced by ABTS as the colorimetric reagent in glucose
assays. Therefore it was decided to develop a new assay, based on ABTS as the
colorimetric reagent. Also of concern was the methodology itself: a mixture of the
enzyme and substrate was incubated for one hour, upon which the reaction was halted by
immersion in boiling water. At the same time an identical mixture that had not been
subjected to incubation was immersed in boiling water to act as the blank. This procedure
does not take into account the lag time of the enzyme, as well as the time required for the
equilibration to the desired temperature (37°C). Incubation of the sample for a period of
two hours and the blank for a period of one hour provided more consistent results.
The small intestinal mucosa of a 12 week old pig was collected at 50 cm intervals, to
provide data of disaccharidase activity throughout the entire length of the small intestine.
The mucosae were prepared as a homogenate, in a manner identical to the method of
Dahlqvist.41
199
Intestinal Section
Maltase (U/g)
Sucrase (U/g)
Lactase (U/g)
Disaccharidase acting on α-D-glcp-(1→6)-D-gal(U/g)
Isomaltase (U/g)
1 48 3.4 16.6 0.70 2 136 20.4 48.9 1.20 3 142 13.6 39.7 1.06 4 143 32.7 48.9 1.51 19.8 5 146 36.4 41.9 1.86 17.6 6 145 39.9 22.0 1.34 27.3 7 152 27.2 18.1 1.50 39.9 8 155 31.0 10.1 1.36 10.1 9 151 24.4 9.3 1.08 3.7 10 91 19.9 1.5 0.42 11 142 19.8 1.0 1.18 12 141 22.1 5.4 1.12
Table 4.2 Summary of pig intestinal disaccharidase activity.
The results for the hydrolysis of (206) were disappointing, with 1,6-α-glucosidase activity
approximately 40 times lower than that of sucrase, indicating poor suitability as a
nutritional supplement for breast milk. Comparison of sucrase and isomaltase activity
against the 1,6-α-glucosidase activity suggested that sucrase was responsible for the
hydrolysis. The stronger correlation of 1,6-α-glucosidase activity against sucrase activity
(R2 = 0.523) and, with the removal of the obvious outlier (R2 = 0.703), when compared to
isomaltase (R2 = 0.138), provides good evidence to support this hypothesis.
Unfortunately the lack of data points for isomaltase, primarily due to the cost of
isomaltose, prevented indubitable confirmation.
200
0 5 10 15 20 25 30 35 40 45
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
1,6-α
-Glu
cosi
dase
(U/g
)
Sucrase (U/g)
R2 = 0.523
Fig 4.1 Sucrase activity against 1,6-α-glucosidase
0 5 10 15 20 25 30 35 40 450.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
1,6-α
-Glu
cosi
dase
(U/g
)
Sucrase (U/g)
R2 = 0.703
Fig 4.2 Sucrase activity against 1,6-α-glucosidase with outlier removed
201
0 5 10 15 20 25 30 35 40 451.0
1.2
1.4
1.6
1.8
2.0
1,6-α
-Glu
cosi
dase
(U/g
)
Isomaltase (U/g)
R2 = 0.138
Fig 4.3 Isomaltase activity against 1,6-α-glucosidase
General Disaccharidase Activity
Whilst many studies have measured the disaccharidase activity in pigs and humans, they
almost always have consisted of a small isolated biopsy of the duodenum or jejunum.43-45
The study of Raul and co-workers is a notable exception, providing data for maltase,
sucrase and lactase for three sections of the small intestine in pre-term infants.21 The lack
of data on intestinal disaccharidase activity was surprising when one considers that the
two major pharmaceutical treatments for type II diabetes, Acarbose and Miglitol, act
primarily by inhibiting intestinal disaccharidase activity.46-49
The results here provide an overall picture of disaccharidase activity throughout the
entirety of the small intestine. Maltase activity appears to be quite high and constant
throughout the length of the small intestine. Sucrase activity on the other hand peaks
around the area of the jejunum and then declines to approximately 50% of its peak level.
202
Lactase activity appears to peak quite early in the small intestine and then quickly
declines to low levels. These observations were shown to have relevance in the human
model with similar patterns observed by Raul and co-workers.21
Conclusions
Despite the poor results for the hydrolysis of (206) in a mammalian model, insight was
gained into the hydrolysis of maltose, sucrose, lactose and isomaltose within the small
intestine. Further work is needed on the determination of disaccharidase levels
throughout the small intestine of pre-term infants, which was unfortunately outside the
scope of this project. The modifications to the original technique for disaccharidase
measurement by Dahlqvist offer several improvements and increase the reproducibility,
whilst maintaining the simplicity, of the method.
203
Experimental
Chemicals
Chemical Abbreviation Supplier isomaltose Sigma Chemicals melibiose Sigma Chemicals maltose Sigma Chemicals potassium dihydrogen orthophosphate KH2PO4 BDH sodium chloride NaCl Sigma Chemicals sodium dihydrogen orthophosphate NaH2PO4 BDH
Enzymes
Enzyme Source E.C. Number Units Supplier α-amylase Human saliva 3.2.1.1 99.8 U/mg Sigma Chemicals α-galactosidase Green coffee beans 3.2.1.22 12.5 U/mg Sigma Chemicals glucose oxidase Aspergillus niger Type II 1.1.3.4 15 500 U/mg Sigma Chemicals α-glucosidase Bakers yeast 3.2.1.20 7.6 U/mg Sigma Chemicals isomaltase Bakers yeast 3.2.1.10 7 U/mg Sigma Chemicals peroxidase Horseradish Type II 1.11.1.7 240 U/mg Sigma Chemicals
204
Initial Study
Preparation of Standards
A set of standards was prepared by dissolving the appropriate amount of sugar in 10 mL
of DDI. From this stock solution standards with the following concentration ranges were
prepared:
maltose 0-1.2 mM isomaltose 0-2.9 mM melibiose 0-2.3 mM α-D-glcp-(1→2)-D-gal 0-2.3 mM α-D-glcp-(1→3)-D-gal 0-1.8 mM α-D-glcp-(1→4)-D-gal 0-2.25 α-D-glcp-(1→6)-D-gal 0-1.9 mM
α-Amylase
α-Amylase reagent[Reagent] [Well]
Potssium phosphate buffer pH 6.9 20 mM 4.88 mMNaCl 67 mM 16.3 mMα-amylase 8 U/mL 1.95 U/mL
Glucose oxidase reagent[Reagent] [Well]
Potssium phosphate buffer pH 6.9 20 mM 14.6 mMNaCl 67 mM 49 mMGlucose oxidase 10.4 U/mL 7.6 U/mLPeroxidase 3.1 U/mL 2.3 U/mLABTS 300 μg/mL 218 μg/mL
Concentrations of reagents for α-amylase assay.
205
Method for Glucose Measurement
The α-amylase reagent (50 μL) was added to samples and standards (5 μL) in the wells of
a microtiter plate, and the plate mixed on a plate shaker. The plate was incubated at 37°C
for 60 minutes. After this time the glucose oxidase reagent (150 μL) was added to each
well and the plate mixed. The absorbance was monitored at 405 nm every 5 min until
peak absorbance reached a maximum. Raw data are provided in the Appendix.
α-Galactosidase
α-Galactosidase reagent [Reagent] [Well] potssium phosphate buffer pH 5 100 mM 24.4 mM α-galactosidase 4 U/mL 0.975 U/mL Glucose oxidase reagent [Reagent] [Well] potssium phosphate buffer pH 5 100 mM 73.2 mM glucose oxidase 10.4 U/mL 7.6 U/mL Peroxidise 3.1 U/mL 2.3 U/mL ABTS 300 μg/mL 218 μg/mL
Concentrations of reagents for α-galactosidase assay.
Method for Glucose Measurement
Activity was measured in a method identical to that for α-amylase.
206
α-Glucosidase
α-Glucosidase reagent [Reagent] [Well] potssium phosphate buffer pH 5 100 mM 24.4 mM α-glucosidase 8 U/mL 1.95 U/mL Glucose oxidase reagent [Reagent] [Well] potassium phosphate buffer pH 5 100 mM 73.2 mM glucose oxidase 10.4 U/mL 7.6 U/mL Peroxidise 3.1 U/mL 2.3 U/mL ABTS 300 μg/mL 218 μg/mL
Concentration of reagents for α-glucosidase assay.
Method for Glucose Measurement
Activity was measured in a method identical to that for α-amylase.
Isomaltase
Isomaltase reagent [Reagent] [Well] sodium phosphate buffer pH 6.7 50 mM 12.2 mM isomaltase 8 U/mL 1.95 U/mL Glucose oxidase reagent [Reagent] [Well] sodium phosphate buffer pH 6.7 50 mM 36.6 mM glucose oxidase 10.4 U/mL 7.6 U/mL Peroxidise 3.1 U/mL 2.3 U/mL ABTS 300 μg/mL 218 μg/mL
Concentration of reagents for isomaltase assay.
207
Method for Glucose Measurement
Activity was measured in a method identical to that for α-amylase.
Mammalian Enzyme Study
Processing of Pig Intestinal Samples
Intestinal mucosa samples were taken from a freshly euthanized 19.5 kg pig. The pig was
restricted from feed for 12 hours prior to death to minimise the quantity of undigested
material. Sampling commenced 20 cm into the small intestine with an interval of 50 cm
between samples. At each interval a 20 cm portion of small intestine was removed, the
sample was washed with isosmotic saline, cut open, patted dry with filter paper and the
mucosa scraped using a glass microscope slide. Approximately 1 g of the mucosa was
suspended in DDI (20 mL) and the sample homogenised by sonication. The volume was
made up to 50 mL with DDI and the sample centrifuged to remove cellular debris (2000
rpm, 10 min). The supernatant was collected and stored (−20°C) until required for
testing.
Protein Assay
Protein concentration was determined using the Bio-Rad assay dye reagent concentrate.
The assay was carried out according to the “Bio-Rad Protein Assay” technical
instructions provided. The dye concentrate was diluted in DDI (1:4) and was filtered prior
to use.
208
A portion of the intestinal homogenate was taken and diluted in DDI to a ratio of 1:4.
Standards were prepared using bovine serum albumin, with concentrations between 0-
1.03 mg/mL. In each well 5 µL of sample was treated with 250 µL of the diluted Bio-Rad
reagent and incubated for 30 min at 37°C. The absorbance was measured at 620 nm and
the absorbance of the homogenate compared to a protein standard curve. Protein
concentration values are provided in the Appendix.
Disaccharidase Assay Procedure
The following reagents were prepared according to the table below:
Maltose Reagent Glucose Reagent [Reagent] [Reagent]
maleate buffer pH 6.9 0.1 M tris buffer pH 7.02 0.5 M maltose 0.1 M ABTS 600 μg/mL
glucose oxidase 20.8 U/mL peroxidase 12.2 U/mL
Sucrose Reagent α-D-Glcp-(1→6)-D-Gal Reagent
maleate buffer pH 6.9 0.1 M maleate buffer pH 6.9 0.1 M sucrose 0.1 M 1,6 glu-gal 0.1 M
Lactose Reagent Isomaltose Reagent
maleate buffer pH 6.9 0.1 M maleate buffer pH 6.9 0.1 M lactose 0.1 M isomaltose 0.1 M
209
Maltase Assay
The maltose reagent (90 µL) was treated with the intestinal homogenate (10 µL) and
incubated at 37°C for 2 h. A blank was prepared by the treatement of the maltose reagent
(90 µL) with the intestinal homogenate (10 µL) and incubated for 1 h. At the end of the
incubation DDI (900 µL) was added to both samples and the solutions transferred to a
pyrex test tube. The solutions were then heated in a water bath at 90°C for 4 min and
subsequently cooled in an ice bath. An aliquot (100 µL) of each solution was treated with
the glucose reagent (900 µL) and the absorbance measured (405 nm). Glucose
concentration was measured by comparison of absorbance against a set of glucose
standards.
Sucrase Assay
The sucrose reagent (50 µL) was treated with the intestinal homogenate (50 µL) and
incubated at 37°C for 2 h. A blank was prepared by the treatement of the sucrose reagent
(50 µL) with the intestinal homogenate (50 µL) and incubated for 1 h. At the end of the
incubation DDI (900 µL) was added to both samples and the solutions transferred to a
pyrex test tube. The solutions were then heated in a water bath at 90°C for 4 min and
subsequently cooled in an ice bath. An aliquot (100 µL) of each solution was treated with
the glucose reagent (900 µL) and the absorbance measured (405 nm). Glucose
concentration was measured by comparison of absorbance against a set of glucose
standards.
210
Lactase Assay
The methodology was identical to the sucrase assay with the sucrose reagent substituted
by the lactose reagent.
Disaccharidase Acting on α-D-Glcp-(1→6)-D-Gal Assay
The α-D-glcp-(1→6)-D-gal reagent (50 µL) was treated with the intestinal homogenate
(50 µL) and incubated at 37°C for 6 h. A blank was prepared by the treatement of a 1,6
glc-gal reagent (50 µL) with the intestinal homogenate (50 µL) and incubated for 3 h. At
the end of the incubation DDI (900 µL) was added to both samples and the solutions
transferred to a pyrex test tube. The solutions were then heated in a water bath at 90°C for
4 min and subsequently cooled in an ice bath. An aliquot (100 µL) of each solution was
treated with the glucose reagent (900 µL) and the absorbance measured (405 nm).
Glucose concentration was measured by comparison of absorbance against a set of
glucose standards.
Isomaltase Assay
The methodology was identical to the sucrase assay with the sucrose reagent substituted
by the isomaltose reagent.
211
References
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214
Appendix Initial Study α-Amylase
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: 1,2 glc-galEnzyme: α-amylase
y= 0.0614 + 0.009xR2= 0.9355
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: 1,3 glc-galEnzyme: α-amylase
y= 0.0652 + 0.0049xR2= 0.501
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: 1,4 glc-galEnzyme: α-amylase
y= 0.0696 + 0.0036xR2= 0.2482
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: 1,6 glc-galEnzyme: α-amylase
y= 0.0652 - 0.001xR2= 0.0821
α-Galactosidase
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: mellibioseEnzyme: α-galactosidase
y= 0.1028 + 0.5485xR2= 0.9998
215
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: 1,2 glc-galEnzyme: α-galactosidase
y= 0.0917 + 0.0129xR2= 0.9683
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: 1,3 glc-galEnzyme: α-galactosidase
y= 0.0946 + 0.0123xR2= 0.914
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: 1,4 glc-galEnzyme: α-galactosidase
y= 0.0945 + 0.009xR2= 0.915
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: 1,6-glc-galEnzyme: α-galactosidase
y= 0.0888 + 0.004xR2= 0.1863
α-Glucosidase
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: maltoseEnzyme: α-glucosidase
y= 0.0884 + 0.7225xR2= 0.9974
216
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: 1,2 glc-galEnzyme: α-glucosidase
y= 0.0644 + 0.0354xR2= 0.9761
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: 1,3 glc-galEnzyme: α-glucosidase
y= 0.0708 + 0.0529xR2= 0.9794
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: 1,4 glc-galEnzyme: α-glucosidase
y= 0.0736 + 0.012xR2= 0.9072
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: 1,6 glc-galEnzyme: α-glucosidase
y= 0.0731 + 0.0636xR2= 0.9913
217
Isomaltase
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: isomaltoseEnzyme: isomaltase
y= 0.1288 + 0.8201xR2= 0.9987
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: 1,2 glc-galEnzyme: isomaltase
y= 0.0921 + 0.1032xR2= 0.9985
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: 1,2 glc-galEnzyme: no enzyme
y= 0.1102 + 0.008xR2= 0.8477
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: 1,3 glc-galEnzyme: isomaltase
y= 0.0891 + 0.0235xR2= 0.9754
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: 1,4 glc-galEnzyme: isomaltase
y= 0.0891 + 0.0195xR2= 0.9721
218
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: 1,6 glc-galEnzyme: isomaltase
y= 0.1039 + 0.3274xR2= 0.997
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.5
1.0
1.5
2.0
2.5
Abso
rban
ce
Concentration (mM)
Carbohydrate: 1,6 glc-galEnzyme: no enzyme
y= 0.1141 - 0.0019xR2= 0.1344
Mammalian Study Protein Assay
Intestinal Section
Protein Concentration (g/L)
1 0.567 2 0.985 3 1.155 4 1.377 5 1.366 6 0.837 7 0.698 8 1.059 9 0.996 10 0.935 11 1.062 12 0.735
Protein concentrations of intestinal homogenates.
