Journal of Scientific & Industrial Research Vol . 60, June 200 1 , pp 463-492
Structure Elucidation of Bacterial Polysaccharides by NMR
and Mass Spectrometry - A Review
Rashmi Sangh i l and Durga Nath Dhar'
·302 Southern Laboratories, Facil ity for Ecological and Analytical Testing,
Indian Institute of Technology, Kanpur 208 0 1 6, India 'Department of Chemistry, Indian Institute of Technology, Kanpur 208 0 1 6, India
The article highlights the use of NMR and Mass Spectrometry in the structural elucidation of bacterial polysaccharides.
Introduction
It was observed in twenties that the filtrate of culture of pathogenic pneumococci contains a substance which belongs to the i mportant class of compounds called polysaccharides. Subsequently, it was discovered that the type of specificity and the virulence of pneumococci was associated with the presence of polysaccharides which were the main components of the capsule surrounding the organism2.4 • S imilar bio-active polysaccharides have been obtained from other pathogenic bacteria, such as, Vibrio vulnificus. It produces a capsular polysaccharide which is reported5 to be essential for virulence in septicemia.The observations gave a great impetus to researchers to explore the structure of polysaccharides derived from bacterial sources (see, Table I ) . The article reviews structural studies carried out on the bacterial polysaccharides and covers the l i terature up to January 2000. Included in the article are the polysaccharides derived from n itrogen fixing bacteria (for example, Rhizobium fredii HH 1 036) which invade the roots of leguminous plants and trigger the formation of nodules that contain the n itrogen fixing microsymbiont.
The main theme of the article is to h ighlight the use of physical methods, with particular reference to the nondestructive techniques - nuclear magnetic resonance spectroscopy CH, DC, 2D) and mass spectrometry (E I , C I , MALDI, FAB) in the structural elucidation of bacterial polysaccharides . These methods in conjunction with the chemical methods (such as compositional analyses, partial degradation, methylation analysis, etc .) have
* Authors for correspondence
made it possible to assign unambigious three dimensional structures to bacterial polysaccharides (see, Table 2) . Introductory remarks have been incorporated about each method of spectroscopic analysis followed by examples of its appl ication. The polysaccharides are often subjected to chemical or enzymatic depolymerization to yield oligosaccharides which are readily amenable to structural analysis. A few i l lustrative examples have been included in the article to show the way bacteria can be cultivated in the laboratory, including the methods employed for the isolation and purification ( involving centrifugation, Iyophil isation, gel-permeation chromatography, h igh performance l iquid chromatography, etc.) .The homogeneity of the i solated polysaccharides can be gauged by taking recourse to thin-layer chromatography, ultra-centrifugation and gel-electrophoresis (in borate buffer).
Cultivation of Microorganisms
Streak Plate Method
The streak plate method is usually employed for the isolation and cultivation of the microorganisms; for example, the plant pathogen - Erwinia stewartii\ is reported to have been grown on p lates w i th CPGagar.Similarly, the pathogens - Vibrio cholerae sero type 0 1 39 (NRCC # 4740) are reportedX to have been grown on sheep's blood agar plates.
Gel Permeation Chromatography
It is a purification technique employed for an effective separation of molecules with low molecular weights ( 1 00-2,000) from molecules of larger molecular weights.
464 J SCI IND RES VOL 60 JUNE 200 1
In GPC, the largest molecules emerge out of the column first, fol lowed by the molecules of lower molecular weights. The column material employed for GPC of bacterial o l igo/, polysaccharides are: Sephadex G- J Ox,
G- I S� (buffer: Pyr-HOAc ; pH 5 .6) , G-2S ">, G-50" · 1 2, Superdex-30'3 , B i o-Gel p_2�· 14. 1 6, P-4 1 7 , P_6 I X, P- I O�
(Buffer: sodium acetate, pH S . 2).
High Performance Liquid Chromatography (HPLC)
HPLC has taken an important place in the armamentarium of organic chemists. It is the technique wherein the size and thermal labil ity of an organic compound is not a l iabi l i ty . It is an extremely rapid, sensitive and convenient method of analysis and offers a high resolution and excel lent reproducibi l i ty. HPLC is an invaluable tool and best suited to the separation of complex 0I igosaccharides ' �·2() and yields constituents in a high degree of purity.
Reverse Phase-HPLC
It is employed to separate low molecular weight compounds according to their hydrophobicity. In this type of chromatography, s i l ica gel particles, covered with chemical ly bonded hydrocarbon chains (C -C ) represent the l ipohi l ic phase. In reverse phase chroiilatography the hydrophi l ic compounds wi l l move faster than hydrophobic, s ince the mobi le phase is always more hydrophi l ic than the stationary phase.
The reverse phase HPLC has been exploited for purifying the Nod factor of Rhizobium frecfiiC', Rhizobium species2 1 NGR234. Likewise the monophosphoryl l ipid A was secured from the l i po -po ly sacc har ide of Rhodoseudomonas sphaeroides22 ATCC 1 7023 and Chlanydia trachomatis2J, respectively by the afore-mentioned technique.
Ultracentrifugation The range of u ltracentrifugation could vary from
1 0,000 (rpm) to 1 54,000 rpm and duration of spinning
from 4 h to 1 6 h, at 20 "c. 7.X. IS . I X,24.25
Dialysis The bio logical material i s extensively dialysed
against disti l l ed water (48 h). This i s the technique used for removing low molecular weight materials . 7. 1 5.25.26
Freez Drying
The d ia ly sed materi a l i s then freez-dried ( l yophi l ized)7 .. �. I I . I 5.24.2M�.5()
. The homogeneity is tested by any
of the fol lowing methods : TLC, gel electrophoresis, exc lus ion chromatography, and ul tra-centrifugation.
Gel Electrophoresi!-;24
The l ipopolysaccharide of Coxiella burnetii strain Nine Mi le is avirulent Phase II and is reported to g ive a single band on sodium dodecyl suphate polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 4 M urea. On the basis of this , the high degree of homogeneity of the material is estab l ished.
There is another report27 about the i solation of po l y sacchar ides from the mus h room, A Sfraeus hygrometricus and whose homogeneity was confirmed by high vol tage electrophoresis in borate buffer. The same objective has been achieved by taking recourse to exclusion chromatography and/or ultracentrifugal analysis2X .
Different S p ectrosc opic Studies on B acter ia l
Polysaccharides
Proton Nuclear Magnetic Resonance Spectroscopy ('H-NMR)
The 'H-NMR spectmm has been a source of valuable stmctural information of polysaccharides2� , for example, about the number and the configuration of anomeric protons. The s ignals arising due to anomeric protons usual ly appear in the range: 4.3-S .9 ppm, and also the protons of a-glycosides usual ly resonate 0 .3-0 .5 ppm downfield from those of the corresponding �-glycosides. The proton NMR spectmm also gives an idea about the constituent monosaccharides, based on chemical shifts and vicinal coupl ing constants (J ) . It may, however, be noted that the l imitation of 'H':�MR spectroscopy is that it i s difficu l t to determine the configuration of furanosides3(),3 1 .
Example: 'H-NMR analysis of the major fraction of l ipo-ch itin ol igosaccharide (obtained from Rhizohiwll fredii HH I 03) has been shown6 to have structure 1 . The NMR signals (ppm) corresponding to anomeric protons in the ol igosaccharide are given in Table 2 .
The proton NMR spectroscopic data of the extracel lu lar polysaccharide (deri ved from Sphingol11(}Jws paucimobilis l 7 strain 1-886) indicate that it consist of repeating units of six sugars (vide infra). This is arrived at by counting the number of proton s ignals in the anomeric region (5 .7-4.S ppm)32 33 ( vide infra) .
SANGHI & DHAR: STRUCTURE ELUCIDTION OF B ACTERIAL POLYSACCHAR IDES 465
Nuclear Overhauser Effect (NOE)
The NOE experiments are helpful in determining the linkages and the sequence of monosaccharides 14. The complete conformation of the individual monosaccharides can also be deduced from these experiments15.
Example: The ol igosaccharide (derived from Shigella dysenteriae Type 7 , strain 408, O-specific polysaccharide) on NaBH reduction is reported36 to yield another oligosaccharid� with the structure 2:
The sequencing of [A] , [B] , [C] (structure 2) has been arrived at by NOE experiments36. Thus, the irradiation of anomeric proton resonance of [A] at 5 . 1 3 ppm caused a NOE of the signal for H-4 of [B] at 4.49 ppm; whereas i rradiation of the anomeric proton resonance of [B] caused a NOE of the signal for one of the protons of the [C] at 4. 1 9 ppm. Conclusion was, therefore, drawn that [A] consequently is terminal sugar and 4- linked to [B] .
Another example which i l lustrates the lise of NOE experiments in elucidating (in part) the structure of bacterial polysaccharide is that of 0- polysaccharide derived from Hafnia alvei Strain 1 2 1 6. The constituents of the polysaccharide back-bone are sequenced as shown in structure 3.
2
n RI=unsatd. fatty acid
R2::methyl n=2
RCH CH(OH)CH CO OAt: ) Z
---14)-a-D-Quip)N( 1 ---14)j3-D-Galp( 1 -74)-j3-D-GIc-NAc-( 1 -74)-[AJ [BJ [CJ
j3-D-GlcpA-( 1 -73)- j3-D-GlcpNAc-( 1 -7
[DJ [EJ 3
The NOEIO experimental results pointed to the fact that the 0- poly-saccharide is l inear and the units comprising it are sequenced as A-B-C-D-E; with [A] , [B] and [D] substituted at position 4, [E] at position 3 and [C] at 3 or 4.
Nuclear Overhauser Effect Spectroscopy (NOESY) The NOESY experiments, the two dimensional
analogues of NOE experiments, provide information about the spatial structure of the molecule. NOESY cross peaks have been observed2·}.37.3x between proton pairs, for example, between H- I and H-3/H-5 for /3-glucopyranosyl residue and between H- I and H-2 for uglucosylpyranosyl configuration. It is also used in the assignment of sequence and for determining the site of glycosidic I inkages3�.4o in the oligo-I poly-saccharides. It may be noted, however, that NOESY experiments are superior to normal ID difference NOE experiments4 1 .
466 J SCI IND RES VOL 60 JUNE 200 1
Table 2 - N M R signals of anomeric proto n s in the olgosaccharide
N M R signals (ppm)
5.50 5 .42 5 .27 5 .04 4 .83 5 . 1 9 8 .67 8 . 3 8 7 .65
Coupling Constants (J, H z)
O Il . I .H-2) or (J 1 .2) 7 .8
3 .5 1 .7 8 .9
R e marks
P-D-glucopyranose res idue
P-D-glucopyranose res idue
L-Rhamnopyranose res i d u e
M ethyl ( R hamnose)
M ethylene
CONH2
-�
� to ,�(,� - - - - - - _ _ _ _ � (5. 1 3Ppm)
[AJ - - - - - -Ac N o � - - - - - - � (5.27 ppm)
(4.49,. - - - - - - �, leo ,� \ CH20H
\'H �_tf<H , � ' , � NHAc
o
NHAc OH [BJ - - - - - - - �
- OH
[C] - - - - - - - �
Example: The structure 4 has been suggested') for the repeating unit of Hafnia alvei 32 0 specifici polysaccharide.
" V I V
�4)-a-D-Galp-( I �2)-a-L-Rhap-( I �4)- p-D-GaIJl-( I �3)-P-DGalpNAc-
2,3
OAc
III ( 1 -)4 )-G1cpNAc-( 1 -)
4
,� - - - - - - � (4. 1 9ppm) , - - - - - - -.@
I3C-Nuclear Magnetic Resonance(CMR) Spectroscopy
The CMR spectroscopy has several advantages over the 'H-NMR spectroscopy of polysaccharides, such as
( i)
( i i )
( i i i )
Greater chemical shift dispersion and lack of complexities aris ing due to spin-spin coupl ing.
The proton noise decoupled is wel l resolved and easy to interpret, and
CMR spectral data provide almost al l the structural information l ike, for example, the number of sugar residues, constituent monosaccharides,
SANGHI & DHAR: STRUCTURE ELUCIDTION OF B ACTERIAL POLYSACCHARIDES 467
anomeric configuration, including the position of the appended groups.
Example: The oligosaccharide part of Vibrio salmonicida (Strain NCMB 2262) l ipo-polysaccharide has been reported 1 3 to have the structure 5 .
(PEA)
D G F C 2,7
a-D-Fuel' 4NBA-( I �4)-a-NonI'A-(2�6)-P-D-GIc-( I �4)-D-a-D-Hepfl-
( I �5)- i E B A 1
a-L-Rhap-( 1 -7 )-a-D-Glcp-( 1 -72)-L-a-D-Hepp
5
where:
L-a-D-Hep p L-glycero-a-D-manno-heptopyranose
D-a-D-Hep p = D-glycero-a-D-manno-heptopyranose
-a-D-Fucp4N = 4-amino-4,6-dideoxy-a-D-galactopyranose
a-NonA = 5-Acetamidino-7-acetam ido-3,5,7, 9-tetradeox y -L-g I ycereo-aD-galactononulosonic acid
Kdo = 3-deoxy-D-manno-oct-2-ulosonic acid
BA (R)-3-hydroxybutanoyl
PEA = phosphoethanolamine
P Phosphorylated
The above structure has been, in part, inferred l � from 1 3C-NMR spectroscopic data which is recorded in the Table 3 .
Example: The I 3C-NMR spectrum42 o f the polysaccharide isolated from Escherichia coli 0 1 26 l ipopolysaccharide exhibited four anomeric carbons located at 99.9, 1 02.3, 1 02.8 and 1 05 .4 ppm having IJ values of 1 73, 1 62, 1 60, 1 63 Hz. These data point t8'1he presence of one glycosidic l inkage, in the repeating unit of the 0-polysaccharide, with an a-configuration, while the remaining three have �-configuration. The signals at 1 7 .9 ppm are indicative of methyl group of fucose and the signals appearing at 63. 1 -63 .6 ppm arise due to three hydroxymethyl groups (C-6 of galactose, mannose and glucosamine). The C-2 atom of the amino-sugar shows a signal at 55.8 ppm. The other sugar carbons also show up in the region 69.3-8 1 .3 ppm as wel l as acetamido group (CH at 24 .6 ppm and CO at 1 79.2 ppm) . The above data �in conjunction with the other physico-chemi-
cal results) point to the fact that the polysaccharide is built up of a tetrasaccharide repeating un it of the structure 6.
�2)-�-D-Manp-( I �3)-a-D-Galp-( I �3)-�-D-G1c-NAcp( 1 � 2
i �-L-Fucp
6
Coorelation Spectroscopy (COSY), Total Correlation
Spectroscopy (TOCS Y), Rotating Frame Nuclear
Overhauser Effect Spectroscopy (R OES Y) and
Heteronuclear Multiple Quantum Coherence (HMQ)
In NMR spectroscopy, the hybrid methods involving the experimental techn iques l i ke COSY, TOCSY, ROESY and HMQC are useful in the assignment of various resonances leading to the structural elucidation of ol igo-/polysaccharides.
Example: The chemical depolymerization by HF of stewartan - the acidic exopolysaccharide of Eriwinia stewartii, is reported7 to yield a pentasaccharide. The structure 7 for the same has been arrived at by chemical and mass spectrometric analyses.
C
p-D-GlefJA
E F 4
p-D-Glcp-( 1 -73 )-p-D-Galp-a-D-Gal p-F
6 D
a-D-Glcp
G
7
The structure 7 has been confirmed by NMR spectroscopy (viz., 2D COSY, TOCSY and 2D ROESY).
In the structure 7, the four pyranose (three gluco and one galacto) monosaccharide spin systems, all with �-anomeric configuration were completely or partially assigned, using 2D COSY and TOCSY NMR spectra (vide data given above). For the anomeric proton of the fifth residue D (a galactopyranose with a-anomeric con-
468 J SCI IND RES VOL 60 JUNE 2001
Table 3 -- DC-NM R spectral data
DC Resonance ( ppm)
1 6.4, 1 7 .4 , 1 9. 1 , 1 9 .7, 22.7, 22.9
54.3, 54.5, 55.0
37.5, 45 .5
4 1 . 1 , 4 1 .2 (doublets)
['Jc.p - 7 Hz each]
60.9-66.2
62.6, 62.7
eJc.p - 5 Hz each]
95.6 ( I JC.H 1 73 Hz)
1 0 1 .3 ( I JC.H 1 74 Hz)
1 0 1 .6 eJC,H 1 72 Hz)
1 03.5 ( I JC.H 1 62 Hz)
- 98 and 1 0 1 (sma)) signals)
1 67.6, 1 73.3, 1 74.5, 1 76. 1
figuration), a characteristic signal with a large geminal coupling constant to fluorine (54.3 Hz) has been reported? The inter-residual cross peaks in the 2D-ROESY spectrum have been exploited to indicate the l inkages between the monosaccharide residues , as shown in structure 7.
Heteronuclear Multiple Quantum Coherence (HMQC)
B
-D-GlcpA
4
�-D-Galp-( I �3)-�-D-Galp-( I �3 )-a/�-D-Galp
D E C
8
Example: HF depolymerized amylovoran - the ac idic exopolysaccharide of Erwinia amylovora is reported:!) to yield a tetrasaccharide, which has the structure 8. This structure has been confirmed by NMR spectroscopy, for example, by using 2D COSY, TOCSY and 2D ROESY experiments. In addition, the DC chemical shifts of a l l carbon atoms in the tetrasaccharide have been determined
Interpretation
Six methyl carbons
Three carbons l inked to n i trogen
CH! carbons
Nitrogen bearing carbons of two PEA groups
Methylene groups
Hydroxymethyl of two PEA groups
Anomeric carbons
Carbonyl carbons
from a HMQC spectrum. The HMQC spectrum is reported25 to show the expected long range L 1C- 1H correlations for the proposed structure. Thus, H- I (d, 4.90 ppm) of residue B showed correlation to C-4 of residue C (a) at 75 .8 and C (�) at 75 .0 ppm; H- I (d, 4.66 ppm) of residue E showed correlation to C-3 of residue C(a) at 79.7 and C(�) at 82.2 ppm. H- I (a, 4.62 ppm) of res idue 0 showed a correlation to C-3 of residue E at 82.8 ppm.
Homonuclear Hartman-Hahn Spectroscopy (HOHAHA)
The experiment, known as HOHAHA; augumented by other NMR spectroscopic methods - gives a complete assignment of the resonances of each spin system
. to individual pyranoside rings in a o l igo-/poly-saccharide.
Example: The analysis I? of the partial ly hydrolysed ( i .e . , O-deacety l ated) exoce l \ u l ar po lysaccharide from Sphinogomonas paucimobilis strain 1-886, with one dimensional HOHAHA and various 2D NMR experiments yielded most of the IH NMR chemical shifts. Res idue A, B, C and 0 were assigned to the D-glucose, E to the
S ANGHI & DHAR: STRUCTURE ELUCIDTION OF BACTERIAL POLYSACCHARIDES 469
rhamnose residue, and F to deoxyglucuronic acid. Further, the spectral data show that one of the P-D-G1c residues is terminal . It also shows that the a-glucose residue is l inked to the 6-position of another glucose residue. The repeating units of the polysaccharide have the structure 9.
C E B
�4)-�-D-Glcp-( I �4)-a-L-Rhap-( I � 3 )-�- D-Glcp-( I �4 )-2-F 6
deoxy-�-D-arabino-HexpA-
�-D-Glcp-( I �6)-a-D-Glcp
9
Mass Spectrometry (MS)
In characterization of molecular structure of organic compounds (for example, polysaccharides23.43-46), mass spectrometry has become an ubiquitous research tool. In a mass spectrometer, the compound under investigation, is bombarded with a beam of electrons. The positive ion (molecular ion) and the fragments derived therefrom are quantitatively recorded as what is known as a mass spectrum.
Electron Impact Ionization Mass Spectrometry (EI-MS)
The technique of EI-MS is an old one and is applicable to a wide range of organic compounds (gas, liquid or solid) volatil ized at 400 lie . Electron beam (70 ev) is made to impinge on the organic molecule in the gas phase. In this process molecular ion and a large number of small fragments ions are formed, which provide useful structural information. The l imitation of EI-MS, however, is that the molecular ion may be weak or absent for some compounds. Moreover, thermally labile compounds are not suitable candidates for electron impact ionization mass spectrometry.
The oligosaccharides, obtained from Legionella pneumophila l l were subjected to GLC-MS (vide infra) using both EI and CI (with ammonia) modes .
When EI-MS on a particular organic compound fai ls to produce a molecular ion, CI-MS is then the method of choice. In CI, the so called "soft" ionization technique, an electron beam is impinged on to a reagent gas (methane, isobutane or ammonia) which ionizes and gently
transfers protons to the sample, usual l y producing (M+H)+ quasimolecular ion . Fewer fragmentation ions result in the process, and hence a simple mass spectrum is obtained. The fragmentation pattern, however, is not as much informative as that obtained by EI-MS .
Mass Spectrometry/Mass spectrometry (MS-MS) Tandem Mass Spectrometry
In tandem mass-spectrometry, two mass spectrometers are used in series. The first spectrometer is employed to produce the molecular ion and its fragment ions. These ions are selectively subjected to collision induced dissociation (CID) promoted by helium, under high pressure, fol lowed by analysis of the resulting product ion. MSI MS is particularly useful for obtaining structural information about the bacterial polysaccharide .. . There are reports about the structural investigations involving the use of MS-MS, on the oligosaccharides derived from Salmonella enteritidis47 and Rhizobium etli4K strain CE 358 and CE 359.
Gas Chromatography-Mass Spectrometry (GC-MS)
A powerful technique GC-MS (gas chromatograph directly coupled to a mass spectrometer) finds wide use, amongst other applications, in the structural investigations related to bacterial polysaccharides6.7.9. 10. 1 2. 1 (1.2 1 .24· 26,49-5 1 . The oligosaccharide (obtained by depolymerization of polysaccharide) is required to be derivatized prior to GC-MS analysis.
Example: The fol lowing example would i l lustrate the application of GC-MS employing both the ionization techniques, viz. CI and EI for investigating the structure of major polysaccharide (PSI) derived from the acidic exopol ysaccharide produced by mucoid strain of Burkholderia capacia49•
The GC-MS analysis of the trimethyl si lylated methyl g l ycos ide deri vati ve (o btai ned by the methanolysis of PSI) showed the abundant ions in the CI mass spectrum, at m/z 440 (M+NH )+, 408 (MMeOH+NH )+ which points to the molecblar mass of 422 (structute 10) corresponding to the trimethyl silyl derivative of a methyl-( I -carboxyethylidene methyl ester)-hexo-pyranoside.
Further structural information was provided by ElMS . Thus, rnIz 363 (M-COOMer and mlz 243 clearly
470 J SCI IND RES VOL 60 JUNE 200 1
o
N H A c N H A c :
point to 4,6-0( l -carboxyethylidene)-D-galactose. structure. The 4,6-linkage of the carboxyethylidene group is also deduced by the occurrence (in the EI-MS) of an ion at rnIz 204, requiring an adjacent trimethyl-silyl groups at positions 2 and 3, and establ ishing a pyranose form for the galactose unit.
Fast-Atom-Bombardment Mass Spectrometry (FAB-MS)
A simple and rapid ionization technique employed in organic mass spectrometry, for a large variety of compounds (including polar and/or thermally labile) is the FAB-MS, by high energy beam of neutral xenon atoms. FAB-MS has been employed for the structural investigat ions of several l ipo-o l igo/po ly- saccharideso,9, 1 3 , 1 5,24.36,47,5 I ,52 obtained from various bacteria.
Example: Smith degradation of O-specific polysaccharide, derived from Hafnia alvei strain 32 is reported!) to yield two core ol igosaccharides. One of the oligosacchari des on posit ive FAB-MS analysis yielded an (M+H)+ ion at rnIz 676. The high energy collision induced decomposition tandem-MS of (M+H)+ ion showed that the trisaccharide was l inked to a glycerol group (structure 11 ) .
0 ......... / CHI H ,C � ' : " 0 • C-O :/ : J /: O S iM e, M eO OC : .
\" O S iM e)
11
0
10
C O O H C H , O H
°1 O H C H 20 H
- -
Another example is the determination of structure of the liberated oligosaccharide, OS (derived from lipool igosacharide of Campylobaeter tdri strain PC 637) is, in part, based on the data obtained from FAB-MS . Thus, permethylated OS is reported 15 to yield a pseudo-molecular ion (M+Nht at 2555 amu corresponding to a composition shown in structure 12.
2225 . . .
464. . . 709 . . , 1 1 1 7 . . . 1 569. . . Hex :
He�HexNAc�HexNAc-----*Jal---�He�He�Kdo
I I Hex Hex Hex
[M+NaJ+=2555amu
1 2
Matrix-Assisted Laser Desorption/Ionization (MALDI)
The MALDI is an ionization technique used in the mass analysis of large and/or labile molecules.
Example: The MALDI-MS of Smith oligosaccharide of Vibrio vulnifieus ATCC 27562 is reported l � to exhibit two types of ions corresponding to m/z, 870.5 (M+Na)+ and mlz, 892.6 (M+2Na-H)+. The former ion was chosen to probe the structure of this molecule by high energy collision induced dissociation (CID) in the first field region of a double focussing mass spectrometer. The CID spectrum shows ions at m/z 666 (Nacetyl hexosamine) and mlz' 594 (Muramic acid) and a fragment ion at mlz, 392. These obse�vations point to the fact that the N-acetylhexosamine and muramic acid residues are linked independently to the molecule and are locked at the termini of the molecule. The fragment ions at rnIz, 782 and 766 are due to the loss of serine and tetritol, respectively from the precursor ion. The remain-
SANGHI & DHAR: STRUCTURE ELUCIDTION OF B ACTERIAL POLYSACCHARIDES 47 1
ing part of the molecule was i ntutively guessed as hexuronic acid. B ased on the above mass data, the structure of the (M+Na)+ is depicted as 13.
Serine
NH
666 782
�-GlpNAc-( 1 --4)-a-GaIA-( 1 -2)-tetritol
594 764
aMurNAc( 1 -3)
13
Example: The characterization of the 0 1 igosaccharide (derived from l ipopolysaccharide of Salmonella enteritidis47 has been achieved, in part, by taking recourse to MALDITOF-MS.
S i m i lar ly, the s truc tu re determina t ion of monophosphoryl l ipid A (prepared from l ipopolysaccharide of Chlamydia trachoma tis) involving the use of MALDI-MS and l iquid secondary ion mass spectrometry, has also been reported}:l .
Electrospray Ionization Mass Spectrometry (ESI-MS)
In this technique, the highly charged droplets, dispersed from a capil lary, and in an electric field, are evaporated, in a region maintained at high vacuum. This increases the charge on the droplets and the mult iple charged ions are drawn into a mass spectrometer. The outstanding feature ofESI spectrum is that the ions carry multiple charges which reduce their mlz ratio compared to a singly charged species. This allows mass spectra to be obtained for large molecules l ike, for example, the o l igosaccharide fraction N4 derived from Coxiella burnetii strain Nine Mile . ESI-MS is also reported} I to have been used in the structural investigation on the l ipooligosaccharides produced by symbiotic nitrogen fixing Rhizobium bacteria.
Example: The pyruvated repeating pentasaccharide of amylovoran (the acidic exopolysaccharide of Erwinia amylovora) on the positi ve ion mode electrospray ionisation mass spectrometry is reported25 to g ive an intense molecular ion at mlz, 1 1 76 [Pyr Hex Hex HexA Hex-ol + Na]+. MS-MS of the parent ion, mit, 1 1 76 shows a l inear arrangement of the molecule and is sequenced as fol lows25•
PyrHex-HexA-Hex-Hex-Hexol
Pyr = Pyruvate residue
Example: ESI-MS has also been employed in arriving at the structure of repeating unit of the nodular polysaccharide, NPS . The major fraction produced by Smith degradation, fol lowed by B ioGel P-2 chromatography of Bradyrhizobium japonicum50, NPS, on ESI-MS exhibited two major mass peaks, mlz, 562 and 584. These mass fragment ions have been assigned to [M+ 1 ] + and [M+Na]+ ions of the ol igomer containing one gal actosyl, two rhamnosyl residues, plus an additional component (90 amu)-deoxytetritol .
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1 0 Katzenellenbogen E, Romanowska E , Shahkov A S , Kocharova N A, Knirel Y A & Kochetkov N K, Carhoh.l'd Res, 259 ( 1 094) 67.
I I Knirel Y A, Moll H & Zahringer U, Carbohyd Res, 293 ( 1 096) 223.
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1 3 Edebrink P, Jansson P-E, B0gwald J & Hoffman J , Car!Jol/l'{1 Res, 287 ( 1 996) 225.
f .
1 4 Cox A D & Perry M B , Carhoh.l'd Res, 290 ( 1 996) 50. 1 5 Aspinall G 0, Monteiro M A, Pang H, Kurjanczyk L A & Penner
J L, Carbohyd Res, 279 ( 1 995) 227. 1 6 Dudman W F & Lacey M J, Carhoh.l'd Res, 145 ( 1 986) 1 75.
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20 Ng Ying Kin N M K & Wol fe L S , Anal Biochem, 102 ( 1 080) 2 1 3 .
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22 Qureshi N, Honovich H P, Hara H, Cotter R 1 & Takayama K, J Bioi Chem, 263 ( 1 2) ( 1 988) 5502.
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23 Qureshi N, Kaltashov I, Wal ker K, Doroshenko V, Cotter R J, 47 Rahman M M, Guard-Pettern 1 & Carlson R W, J Bacleriol, Takayama K, Sievert T R, Rice P A, Lin 1-S L & Golenbock D 179 (7) ( 1 997) 2 1 26. T, J Bioi Chern, 272 ( 1 6) ( 1 997) 1 0594.
48 Forsberg L S & Carlson R W, J BioI Chem, 273 (5) ( 1 998) 24 Toman R & Skultety L, Carbohyd Res, 283 ( 1 996) 1 75. 2747.
25 Nimtz M, Mort A, Domke T, Wray V, Zhang Y, Qui F, Coplin D 49 Cerantola ' S , Marty N & Montrozier H, Carhohyd Res, 285 & Geider K, Carbohyd Res, 287 ( 1 996) 59. ( 1 996) 59.
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52 Petersson C, 1achymek W, Klonowska A, Lugowski C, Niedziela P.L., Publisher: Suriya International Publications,( Dehra Doon, U,P, India) ( 1 997).
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28 Sharma S & Soni P L, I X Carbohydrate Conference held in the 53 Cherniak R, Morris L C & Meyer S A, Carbohyd Res, 225 ( 1 9(2)
Lucknow, Nov. 24-26, 1 993; in Trends in Carbohydrate Chem-33 1 .
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29 Agarwal P K, Phytochemistr)" 31 ( 1 0) ( 1 992) 3307, 55 Gamwn A, Katzenellenbogen E, Romanowska E, GrosskUl1h H
30 10sfeleau 1-P, Chambat G, Vignon M & Barnoud F, Carbohyd & Dabrowski 1, Carbohydr Res, 307 ( 1 -2) ( 1 998) 1 73,
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3 1 Usui T, Tsushima S , Yamaoka N , Matsuda K , Tazimufa K, ( I ) ( 1 995) 377.
Sugiyama H, Seto S, Fujieda K & Muyajima G, Agric BioI Chelll, 57 Senchenkova S N, Shashkov, A S , Toukach F V, Ziolkowski A,
38 ( 1 974) 1 409, Swierzko, A S, Amano K-I, Kaca W & Yu A, Bioc/'l'lIlislr\'
32 Shibuya H , Zhang R, Park 1 D, Baek N I , Takeda Y, Yoshikawa (Moscow), 62 (5) ( 1 997) 46 1 .
M & Kitagawa I , Chem Pharlll Bull, 40 ( 1 992) 2647, 5 8 Saksena S , Srivastav S S, Khare N K, Deepak D & Khare A,
33 Sri vastav S, Deepak D & Khare A, J Carbohyd Chelll, 13 ( 1 994) Proceedings XII Carbohydrate Conference, held at the Chem-istry Dept, Lucknow University, Lucknow, India, N () v ,
75, 20-2 1 , ( 1 997) pp, 32; 70, 34 Haslinger E, Korhammer S & Schubert-Zsilavecz M, Liebigs
59 Datta A K & Basu S, Plvceeding,I' XII CarhohydraTe CO/(/er-An/! Chern, ( 1 990) 7 1 3 .
ence, held at the Chemistry Dept, Lucknow University, Lucknow, 3 5 Kashman Y, Green D & Garcia, C, J Nat PlVd, 5 4 ( 1 99 1 ) 1 65 1 , India; Nov. 20-2 1 , ( 1 997), OP- I , p 33,
36 Knirel Y A, Dashunin V V, Shashkov A S & Kochetkov N K, 60 Dudman W F, Frazen L-E, Darvill 1 E, McNeil M, Darvi ll A G, Carbohyd Res, 179 ( 1 988) 5 1 . Albersheim P, Carbohyd Res, 117 ( 1 983) 1 4 1 .
37 Macura S & Ernst R R, Molec Phys, 41 ( 1 980) 95, 6 1 Aman P Franzen L-E, Darvill 1 E , McNeil A G , Darvill , A G &
3 8 Kumar A, Ernst R R & Wuthrich K, Biochelll Biophys Res Albersheim P, Carbohyd Res, 103 ( 1 982) 77,
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309, 63 Garegg P 1, Lindberg B, Onn T & Sutherland I W, Acta Chl'1Il Scand, 25 ( 1 97 1 ) 2 1 03 ,
42 Basu S, Bhattacharya T & Das S , I X Carbohydrate Conference
held in Lucknow, Nov, 24-26, 1 993; Trends ill Carbohydrate 64 Lindberg B, Lindh F, Lonngren 1 & Sutherland I W, Carhohyd
Chemistry, ( 1 995) 23, Edited by Soni , P.L., Surya International Res, 76 ( 1 979) 28 1 ,
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44 Venisse A, Riviere M, Vercauteren 1 & Puzo G, J BioI Chem, 67 Erbing G, Kenne L, Lindberg B, Lonngren 1 & Sutherland I W,
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45 Olsthoorn M M A, Peterson B 0, Schlecht S, Haverkamp 1, 68 (a) Dutton G G S , Personal Communication ( 1 982),
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69 (a) Lew 1 Y & Heidelberger M, Carhohyd Re.I·, 52 ( 1 976) 255, 46 Biely p, Vrsanska M, Tenkanen M & Kluepfel D, J BioTechnol,
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SANGHI & DHAR: STRUCTURE ELUCIDTION OF BACTERIAL POLYSACCHARIDES 473
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(b) Dutton G G S, Mackie K L, Savage A Y, Rieger-Hug D & Stinn S, Carbohyd Res, 84 ( 1 980) 1 6 1 .
7 1 Dutton G G S & Mackie K L , Carbohyd Res, 62 ( 1 978) 32 1 .
7 2 Linnerberg M , Weintraub A & Widmalm G , Carhohyd Re.�, 320 (3-4) ( 1 999) 200.
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74 Yang B Y, Gray J S & Montgomery R, Carbohyd Res, 316 ( 1 -4) ( 1 999) 1 38.
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76 Risberg A, Alvelium G & Schweda E K, Eur J Biochelll. 265 (3) ( 1 999) 1 067.
77 Risberg A, Masoud H , Hartin A, Richards J C, Moxon E R & Schweda E K, Eur J Biochem, 261 ( I ) ( 1 999) 1 7 1 .
7 8 Jaychymek W, Niedziela T, Petersson C , Lugowski C, Czaja J & Kenne L, Biochemistl)', 38 (36) ( 1 999) 1 1 788.
79 Gil-Serrano A M, Rodriguez-Carvajal M A, Tejero-Maleo P, Espartero J L, Menendez M, Corzo J , Ruiz-Sainz, J E & Buendi A-Claveria A M, Biochem J, 342 (Pt 3) ( 1 999) 527.
80 Rund S, Lindner B, Brade H & Holst 0, J Bioi Chell!, 274 (24) ( 1 999) 1 68 1 9.
8 1 Beukes M , Bierbaum G, Sahl H G & Hastings J W, App/ Environ Microbiol, 66 ( I ) (2000) 23.
474
Name of the organism
Bradyrhizobium aspalati
Bradyrhizobium japonicll111 and
B. elkanii
Burkhoderia capacia
Mucoid strain
Campylobacter lari strain PC 637
Campylobacter !ari type strain
ATCC 3522 1
Chlamydia trachomatis
Chlamydia trachoma tis serotype L2
Coxiella burnetii Strain Nine Mile
Clyptococcus neoformans
Sero type-C
Envinia amylovora
Envinia chrysanthellli CU643
Envinia stewartii
Escherichia coli
Escherichia coli 01 73
Eubacterium seburreulIl
Haemophilus influenza strain Rd
J SCI I ND RES VOL 60 JUNE 200 I
Table I - Some typical pathogenic and non-pathogenic bacteria
Remarks
Nitrogen-tixing symbionts (South African legumes)
Gram negati ve bacteria, n i trogen ti xation in soya bean
Ref No.
72
50
Pathogen in nosocomial infection (cystic tibrosis) 49
Human pathogen responsible for enteritis
Human pathogen
(enteri tis)
Human pathogen
Human pathogen
Etiological agent for Q-fever (Pneumonitis, hepati tis and neurologic complications)
Infection associated with AIDS, pulmonary pathogen
Plant pathogenic bacteria, causes fire bl ight on apple and pear trees
Gram negative phytopathogen responsible for soft rot in a number of plants
Corn pathogen
It is a complex group of bacteria and many of its sero-types are important human pathogens causing extra- intenstinal infections
Entetro-invastive organism
( Gram-negative bacteria)
1 5
5 1
23
73
24
5 3
25
74
7
54
75
1 2
7 6
Haemophilus influenza mutant strain RM 1 1 8-26 A genus of bacteria (Gram-negative) 77
Hafnia alvei, strain-32
Hafnia alvei, Strain 744
and PCM strain 1 1 94 and 1 2 1 0
Hafnia alvei Strain 1 2 1 6
Hafnia alvei S train 1 3337 & I 1 87
Klebsiella & Rhizobium species
Legionelia pneumophila
Sero group I
Mycobacterium bovis, BCG
Enterobacteriaceae - pathogens found in some nosocomial infections and septicc i l l i a
-do-
Bacteria
Pathogen causing severe respiratory infection in humans
Tubercle bac i l lus
9
52
1 0
55
16
I I
43
SANGHI & DHAR: STRUCTURE ELUCIDTION OF B ACTERIAL POLYSACCHARIDES
Table I - Some typical pathogenic and non-pathogenic bacteria - (Contd)
Name of the organism
Mycobacterium bovis, BCG
Mycobacterium xenopi
Proteus vulgaris OX 1 9 (Sero group 0 I )
Pseudomonas sp strain 1 . 1 5
Rhizobium etli Strain CE 358 and CE 359
RhizobiumJredtii HH 103
Rhizobium species NGR 234
Rhodopseudomonas sphaeroides ATCC 1 7023
Salmonella enterica SV Arizonae 062
Salmonella enteritidis
Shigella dysenteriae Type 7
SinorhizobiumJredii HH 103
Sphinogomonas paucimobilis Strain /-886 Streptococcus pyogenes
Vibrio cholerae 0 1 39
Vibrio cholerae 0 1 39
Vibrio salmonicida
Strain NCMB 2262
Vibrio vulnificus ATCC 27562
Yokenel la regenburgei strain PCM 2476, 2477, 2478 and 2494
Remarks
Tubercle baci l lus
Human pathogen
Fresh water biofi lm
Nitrogen fixing bacteria
Nitrogen fixing bacteria
Nitrogen tix ing bacteria
Human pathogen
Human pathogen
Human pathogen
Human pathogen
Pathogen responsible for cholera
Pathogen responsible for cholera
Gram negative bacteria, highly pathogenic to Atlantic salmon
Gram-negative bacterial pathogen (Septicemia)
Bacteria
475
S.No. 1 .
2
3
Oligo/ polysaccharide Lipo-chitin
oligosaccharide from Bradyrhizobium
aspalati nodule polysaccharide
derived from Bradyrhizobium species
repeating unit of the EPS of Burkholderia cepacia
Table 2 -Structures of oligo/polysaccharides (bacterial)
Structure/ Infonnation The l ipo-chitin oligosaccharides are highly substituted on the non-reducing-tenninal residue but unsubstituted
on the reducing tenninal residue. The molecular backbone consists of 3�5 �-(1�)- linked N-acetylD-glucosarnine residues substituted on the non-reducing terminus with a C 16:0, C 16: 1 , C1 8:0, C I 8 : 1 , C 19: 1 cy, or C20: 1 fatty acyl chain and are both N-methylated and 4 , 6- dicarbamoylated.
HO
�4 )-a-L-Rhap-(l �3)-�-L-Rhap-( 1 �4)- )-�-L-Rhap-( 1 �4)-D"Me-I3-D-Glcp( 1 �3)
OH
OCH) 2-0MeG1cA
Rha
OH n
�3 )-�-D-GJcp-( 1 �3)-a-D-Galp-( 1 � 4 6
X H3C COOH
Ref .. 73
50
49
- Contd
.j>. -...J 0-
'-Vl 0 Z 0 ;:I:l tTl Vl < 0 r 0-0 '-c:: Z tTl 8
4 extracellular polysaccharide obtained from Campytobacter tar; strain PC 637
�o I �"'/
.�,'O OH
/".o I O=P-O' I
H
OH
LOS: a-D-Glcp-(1�3)-�-D-GalpNAc-(1�3)-)-�-D-GalpNAc-( 1�3)-�-D-GaJp(l�3)-
2
�-D-Glcp J, 4
L-a-D-Man-Hepp-( 1 �3)-L-a-D-man-Hepp-( 1 �5)-Kdo 2 2 I I 1
a-D-Galp a-D-Galp
a-D-Galp
1 5
- Contd
til >
� == P:<> o ::J: > ?:l til :d B c:: � m m r c:: (") a -l o Z o "'Tl co >
� ;; r
a � til > (") (") ::J: > :=: o m til
""" -..I -..I
5
6
highly branched region of lipooligosaccharide of the Campylobacter lari type strain; ATCC
EPS: [-(P03-)�4 )-�-D-Glcp-( 1 �5)-6-d-a-L-Gal-Hepf-(1-)�
2
-6-d --a-L-gal-Hepp
I
2 I 1
6-d--a-L-Gal-Hepp
�-D-GlcpNAc-( 1 �3)-a-D-GalpNAc( 1 � 3 )-a-D-Galp-(l � 3 )-L-a-D-man-Hepp( 1 � 3)-. 2 I
3522 1 . �-D-Glcp a-D-Glcp
deactylated lipopolysaccharide from Chlamydia trachonwtic serotype L2.
1 AEP I I
4 4 L-a-D-man-Hepp(1�5)-a-Kdo( 1 �5)-Kdo
2
1 a-D-Galp
Abbreviation: AEP = 2-aminoethyl phosphate and for the tetraglycosyl phosphate repeating unit of the extracellular polysaccharide'� HP03)� 3)-�-D-GlcpNAc-( 1 �2)-6-d-a-L-gul-Hepp(1 �2)-3-d-P-Dthreo-Penp (1 �3) -6-d-L-gul-HepP-)n
Kdo a2�8 Kdo a2� Kdo a2�6D-GlcN� 1 �6D-Glcp N a 1 ,4'-bisphosphate
5 1
80
- Contd
.I>-..J 00
'-C/O 0 Z t:l � tTl C/O < 0 r 0-0 '-c:: Z tTl N :5
7
8
9
10
major lipid A ,component of Chlamydial lipopoly-saccharide '
Lipopolysaccharide from Coxiella burnetti strain Nine Mile in avirulent phase II
core repeating unit of the poLysaccharide of Cryptococcus neoformans serotype C
Erwinia amylovora
This is reported to be due to a glucosamine disaccharide that contains five fatty acids and a phosphate in the distal segment. Three fatty acyl groups are in the distal segment, and two are in the reducing end
'
segment. The acyloxylacyl group is located in the distal segment in amide linkage.
a-D-Manp-( 1 �2)-D-glycero-a-D-manno-Hepp-( 1 � 3)-D-glycero-a-D-manno-
a-D-Manp 1 I
4
-Hepp-( l � 5)- a-Kdo-(2� 4
�-D-GlcpA
i 2
a-Kdo-(4�2 )-a-Kdo
1 .l. 2
-3)-a-D-Manp( 1� 3)-a-D-Manp-(1� 3) )-a-D-Manp-( l � EPS a-D-Pyr4,6Ac I .JGalp
1 J, 4
1 J, 4
j3-D-GlcpA
6 t 1
[-+3)-a-D-Galp-(1 -+6)-j3-D-Galp-( 1 -+3)-I3-D-Galp-(1 -+ 1.
, (j3-D-Glcp}ci,�
23
24
' 53
75
- Contd
Vl ;l>
B ::: Ro o :I: ;l> ? Vl
;l c: Cl c: ;:tI ['T1 ['T1 r c
Q � a z o "Tl t:Xl ;l>
� ); r
� £< Vl ;l>
R :I: ;l> ;:tI 8 ['T1 Vl
� -:J '"
1 1
1 2
branched repeating unit with seven monosaccharide residues, all in their pyranose forms of the oligosaccharide derived from the Erwinia stewartii
Extracellular polysaccharide of Erwil1Uz chrysanthemi CU 643.
�-D-Glcp 1 J, 6
D-Galp 1 J,
�-D-Glcp 1 J, 4
[�3)-a-D-Ga)p-( 1 �6)-�-D-Glcp-(1 �3)-�-D-Ga)p-( 1 �]n 6 i 1
�-D-Glcp
---�3)-�-D-Ga)p-( 1 �2)-a-L-Rhap-(1 �4)-�-D�Glcp-(1 �2)-a-L-Rhap-(1 �2)-a-L-Rhap-(1 �2) )-a-LRhap-( l ---�( 1 )
7
74
- Contd
� 00 o
'-Vl 0 Z 0 A' m Vl
< 0 t"" 0\ 0 '-c:: z m N 8
1 3 The novel sugar constituent of the O-specific
. polysaccharides of Escherchia coli 0 1 14 has been reported54 to have the following structure
14 The repeating units of the polysaccharide obtained from Escherichia coli 0:25
1 5 O-Antigen polysaccharide from Escherichia coli 0173
1 6 The O-antigen (Escherichia coli 0125 : K72 : H19) is branched and is built up of repeating hexasaccharide unit
J4 1- OH AcNH H
OH
CH'zOH 3-[ (N -acetyl-L-seryl)amino ]-3 ,6-dideoxy-D-glucose
cx-L-Rhap 1 J, 3
�4)cx-L-FucNAc( 1 �3)-�-D-GlcNAc(1 �4)-cx-D-Glc-(1 ---+ 6 i 1
I3-D-Glcp
The repeating unit of the polysaccharide is the pentasaccharide. Its cleavage leads to the formation of a tetrasaccharide with the structure as shown:
cx-D-Glcp-(1 �2)-�-D-Glcp-(1 �3)-I3-D-Glcp NAc-(l �3)-D-Glc
� 2)-cx-0-Manp-( 1 � 3 )-cx-L-Fucp-( 1 � 3)-cx�D-GalpNAc( l ---+4 )-cx-D-GalpNAc-( 1 � 3 3 i i 1 I
cx-D-GIcp j3-D-Galp
54
58
72
59
- Contd
en > z C') :5 PI> 1:1 ::c > � en ;d c::
� c:: ::0 ttl ttl r-c:: (") a -l 0 z 0 "'rl a:J > � ::0 ): r-2l � en > (") (") ::c > ::0 a ttl en
.j>. 00
1 7
1 8
1 9
20
2 1
O-antigen froin enteropathogenic E.coli 0 1 26 .
Escherichin coli K 1 2 (S53)
O-specific polysaccharide of Hafizia alvei strain 1 2 1 6 is reported to be built up of l inear pentasaccharide units
Lipopolysaccharide of Haemophilus injluenza mutant strain RM 1 1 8-26
Lipopolysaccharide from Haemophilus injluenzae, strain Rd.
. The polysaccharide obtained from the above source is branched and built of tetrasaccharide repeating units �2)-�-D-Manp-( l �3)-a-D-Galp-( 1 �3)-�-D-GleNAcp( l �
---. 4-L-Fuc
2 i 1
�-L-Fudp
�3Glc 6 � 3-L-Fuc 4
Me 4 I � "C
/ 'Gal 6 � 4G1c A B � 3Gal HOOC
/ '6/
fL.
�4)-a-D-L-Rhap-« 1 �4)-�-D-Galp-(1 �3)-�-D-GalpNAc-( 1 �4)-a-D-GlepNAc-( I �
(R) CH3CH(OH)CH2CO OAC 1 3 :6
�4)-a-D-Quip 3N-(1 �4)-�-D-Galp-( 1 �4)-�-D-GlepNAc-( 1 �4)-�-D-Glep A-( 1 �3)-�-D-Glep NAc-( I �
One o f the major glycoforms (obtained by mild acid hydrolysis o f lipopolysaccharide) i s reported to contain three hexose moieties (Hex 3), in which a lactose unit, �-D-Ga\p-( l---�4)-�-D-Glep, is attached at the 0-2 position of the terminal heptose of the inner core element, L-a-D-Hepp-( l ---�2)-L-a-Hepp( 1 --�3)-[�-D-Glep-( l---� 4)-]-L-a-D-Hepp-(l �5)-a-Kdo. The glycoform is 40% substituted by an O-acetyl group attached to the 6-position of the glucose moiety in the lactose unit.
42
63
9, 1 0
76
The major lipopolysaccharide glycoforms are reported to contain three (two Glc and one Gal), four (two Gle 77 and two Gal) and five (two Glc, two Gal and one Gal NAc) hexoses-substituted by phosphocholine and phosphoethanol amine.
- Contd
.j>. 00 tv
til Q Z 0 � m til < 0 r 0-0 '-c z m tv 8
22 Klebsiella Kl
23 Klebsiella K3
24 Klebsiella K30
25 Klebsiella K32
26 Klebsiella K58
---' 4-L-Fuc . a. �3Gle � � 4GlcA B � 2A3 'C
/
H3C/ 'COOH
Contains Gal. Man. GaiA and pyruvic acid (10%) but the structure has not been determined. 4.6�O-( 1-. carboxyethylidene) D-mannose is known to be present
Gle
Ia. .... 4Gle B � 4Man � � 4Man B �
6
Me, ), t� /C
/Gal
HOOC '4
---.3Gal a � 2-L-Rha a. � 3-L-Rha � � 4-L-Rha a � 3A4 , / /C,
H3C COOH
--. 4-L-Fuc a �3GIc
fa Gal
�4GleA B � A 2, /3
/C H3C · 'COOH
67
68
64
70a. b
66
- Contd
en > z Cl :t
?<> I:) :t > �
� c: q c: � trl trl r c: (j � o z
� c:7 > � � > r
� � en > (j (j :t > � a f;l
� oc .....
27 Klebsiella K70
28 O-acetylated core of Legione,la pneumophila serogroup I lipopolysaccharide
29 The 6-0- methylglucose lipopolysaccharide secured from Mycobacte riltm bovis BCG
30 Two polysaccharides characterised from Mycobacte riulII xenopi
3 1 O-specific polysaccharide chain of Proteus vulgaris OX 19
-2Gl�3Gal:...1!. 2-L-Rha� 4GlcA--J}. 4-L-Rha�2-L�Rha 3� . " / /C
H3C 'COOH
Note: only 50%of the repeating units of this polysaccharide are pyruvated
ex-D-G1cpNAcll 1 ! 6
(X .. .
a-L-Rhapll-( 1 �3)-ex-L-RhapI -( 1 �3)-�-D-QuipNAc-( 1 �4)-�-D-G1cpNAcl-( 1 �4 )-ex-D-Manp-( 1 �5)-Kdo 2 2 4 3
I I I I Ac Ac Ac Ac
contain an unusual monosaccharide 2N-acetyl-2,6-di.deoxy-�-glucopyranose in its molecular backbone
The first one is composed of 1 6D glucopyranose residues, 1 1 of which are methylated. While the other contains 15D-glucopyranose residues 10 of which are methylated.
lipopolysaccharide branched pentasaccharide repeating units containing D-galactose, 2-acetami90-2-deoxy-D-glucose, 2-acctamido-2-deoxy-D-galactose and 2-acetamido-2,6-dideoxy-D-glucose (QuiNAc, two residues) which are connected to each other via a phosphate group.
7 1
1 1
43
56
57
- Contd
� 00 �
'-Vl 0 z 0
;:0 m Vl
< 0 r C'I 0 '-c: z m
8
32
33
34
35
Acidic exopolysaccharide from Pseudomonas sp. Strain 1 . 1 5.
intact lipopolysaccharide core region (obtained from Rhizobium etli strains CE 358 and CE 359)
Rhizobium phaseoli 127 K38
The repeating units of pyruvated polysaccharides Rhizobium phaseoli 1 27 K36
" The parent repeating unit of the 1 . 1 5 exopolysaccharide is a branched hexasaccharide. The main chain is 75 made up of a trisaccharide ---�4)-a-L-Fucp-( l ---�4)-a-L-Fucp)-(I ---�3)-P-D-Glcp�( I---� and the side chain consists of a-D-Galp-( l �4 )-P-D-GlcAp-( l ---�3)-a-D-Galp-( 1 � is attached to 0-3 of the first Fuc moiety. The terminal non-reducing Gal carries "a l -carboxy-ethylidene acetal in the R configuration at the position 4 and 6.
It consists of trisaccharide (2) attached to 0-4 of the Kdo residue in tetrasaccharide 1, and that an additional 48 Kdo residue is attached to 0-8 of the polymeric chain. It may be noted, however, that one mutant CE 358, completely lacks the O-chain polysaccharide, while the second mutant CE359 contains a truncated portion of this polysaccharide.
Kdo-p-(2�6)-a-D-Galp-( 1 �6)-[ a-D-Galp A-GaJp A( 1 �4 )-a-D-Manp-( 1 �6)-
-Kdop-(2 4 I
a-D-Galp A ( 1 �)-[a-D-GalpA-( l �6)-Kdop-[2
where Kdo =3-deoxy-D-manno-2-oct-2, ulo-pyranosonic acid.
Me 4 "C/ "
HOOC/ "6/Gk;
-. 4Glc A
Me 4 'C/ "
HOOC/ '-6/Ga1
-.4Glc A 13 . 4Glc A B • 4Glc B • 4Glc a .. 6 Ip 0-...6Gal a • 4Glc A B • 4GIc � • 4GIc � .. 4Glc
JL.4Glc A B .. 4Glc B .. 4GIc 6
I � �3Glc B • 4G1c � .. 4Glc 4A6 "C/
Mtf "COOH
�
61
1 6, 60
- Contd
en » z Cl :; It:> o :c » � en ;d c:
Q c: � m m r c: (') � (5 Z o "'1'1 co » (') trl � » r "" o � en » (') (') :c » � 6 m en
� 00 V>
36
37
38
Rhizobium phaseoli 127 K44
Rhizobium phaseoli 127 K87
RhizobiumJredii HH I 03
--. 4Glc A B • 4Glc A B • 4Glc B . 4Glc
Me" /4,, · fB /C" /Gal ex • 4Glc A B .3Glc B • 4Glc . 13 • 4Glc
HOOC 6
--. 4Glc A B • 4Glc A B • 4Glc B • 4Glc a • 6
t� i B • 6Glc 6 .6Gal a • 4Glo\ B • 4Glc 6 . 4Glc i !� /3" ... Me 6Gal B • Gal" /C
4 'COOH
�
� �OH �OH
�OCH3
H3C�0 OH
xo 0 0 0 x H 00 . 00
Y-Me �H �H
C=O COCH) COCH) I
H, /(CH2h c I I
/� H (CH2h Nod NG RB (R=H; RI=Acetate)
�H COCH3
I CH) Nod NGRA (R=S03H, R'=H)
X=H or Carbamoyl (O, lor 2)
62
65
2 1
OH
- Contd
� 00 0\
Vl Q z 0 :;:I:l tTl Vl < 0 r 0\ 0 .... c: Z tTl tv 0 0
------------ -- ------------... _-------------------------N Rl (fatty acid) R2
------------------------- ----------------------------
2 C16: 1�9 H 2 C16: 1�9 CH3 3 C16: 1�9 H 3 C16: 1 �9 CH3 1 C16: 1�9 CH3 3 C16:0 H 3 C16: 1�9 CH3 2 C16:O CH3 3 C18: 1�l l H 3 C18: 1�1 1 CH3 2 C18: 1�1 1 H 2 C18: 1�1 l CH3 3 C18: 1�1 1 CH3 1 C18: 1�t t CH3 1 C18:O CH3 2 C18:O CH3 3 C18:0 CH3
- Contd
.j::o 00 00
IZl Q Z t:J ;a::l trl IZl < 0 r 0-0 '-c::: Z trl IV 0 0
40
4 1
42
polysaccharide from the Shigella dysellteriae Type 7
The high molecular weight lipopolysaccharide obtained from Sall1wllella enteritidis (virulent isolate SE6-E2 l as well as avirulent isolate SE6-E5).
Polysaccharide from Siflorhizobium
fred;; HH 103
� ACNH W
AcGlyNH OH It shows differences in respect of repeating unit of the polymer Structure I: - [----�2)-[a-nV p-( l--- .. 3)}-p-D-Manp-( 1�4)-a-L-Rhap-( 1---�3)-<x-D-Galp-( l---�]Structure II: [----�2)-[a-1YVp-( 1---.. 3)] �-D-Manp-(l� 4)-a-L-Rhap-( 1 - - -�3)-[a-D-Glcp-( 1---. 4)]-a
-D-Galp-( 1 ---.]-
36
47
It consists of a homopolymer of 3 : 1 mixture of 5-acetamido-3.5.7.9-tetradeoxy-7-[(R)- and (S)-3-hydroxy 1 7 butyramido ]- I -glycero- l -manno-nonulosonic acid. The monomers are linked via both glycosidic and amide linkages
- Contd
til » z Cl :c
� R<>
o :c » ::0
� c:
� c: ::0 tTl tTl r c: n
a -l (5 Z
o "Tl til »
� » r
<3 £< til » n n :c » ::0
a tTl til
.j::o. 00 '-C>
43
44
45
Sphingomollas . pallcimobilis Strain 1-886; EPS
Streptococcus pneumoniae type IV
lipopolysaccharide of Vibrio choLerae 0 1 39
-4 )-�-D-Glcp-(1 �4 )-a-L-Rhap-( 1 �3)-�-D-Glcp( l �4)-2-deoxy-�-D�arabino-HexpA� 6 i 1
�-D-Glcp-(1 �6)-a-D-Glcp
----'4ManNAc � � 3-L-FucNAc a �3GaINAc a .. 4Gal a �
?A3 -, /
o O' P
4 6 a-Col-( 1 -2)-�-Gal
1 I
/C H3C 'COOH
a-Gle N 1 I
a-Hep I I
3 7 6 �-GIeN Ac-( 1 -4 )-a-GaIA -( 1 -3)-�-QuiNAc-( 1 -2 )-a-Hep-( 1 -2 )-a-Hep-( 1 -3)-
O-antigen Core a-GIe-( 1 -6)-a-Glc .
I 6
a-Hep-( l-5)-a-Kd04 P-(2-6)- �-GIeN4p-( l -6)-a-GlcNI P 4 I Lipid A 1
Fru f (2-6)-�-Glc Abbreviations: Fru= D-fructose; Kdo= 3-deoxy-D-manno-2-octulo-sonic acid, Gal= Galactose, GlcN= 2-
amino-2-deoxy-D-glucose, Qui N= 2-amino-2,6-dideoxy-D-glucose. GIe= D-glucose; Hep= Lglycero-D-manno-heptose. Gal N= D-galacturonic acid; col= 3,6-dideoxy-L-xylo-hexose.
1 7
69a, b
14
- Contd
"'" 'D o
V:l 0 Z 0 ;.:l m V:l < 0 c-O> 0 '-c z m tv 0 0
46 tetr�accharide triphosphate isolated from the lipopolysacc-haride of Vibrio cholerae 0139
�\ t4 NH2
H
H� Fru
H
H OI,OH
HO�
OH . OH 0
o ( OH
OH OH
�o OH � OH H203PO NH2 0 O�H2
NH2
8
- Contd
en >Z Cl ::t:
R<>
tl ::t: >;:tI
� c: (") --l c: ;:tI tTl tTl r c: (") � (5 Z
o "'I"l 0::1 >-
� ;:tI
:; r ""C o � en >(") (") ::t: >;:tI
6 tTl en
.j:>. �
47
48
49
oligosaccharide of Vibrio salmonicida (strain NCMB 2262)
Vibrio vulnificlls A TCC 27562
Carbohydrate O-specific side-chain moiety of the lipopolysaccharide of Yokenella regensburgli strains PCM 2476. 2477. 2478 and 2494
(PEA)2 J,
Z.7 o:-D-Fucp 4 NBA-(l �)- 0:-NonpA-(2�6)-�-D-Glcp-( 1 �)-D-o:-D-Hepp-( l �5)-Kdo
0:-L-Rhap-( 1 � )-0:-D-Glcp-( 1 � 2)-L-0:-D-Hepp CPS Serine
NH-amide I
{ �-D-GlcNAc( 1 -4)- 0:-D-GalA( I -4)-�-L-Rha( 1 -4) I
o:-Mur NAc( I -3)
3 4 i i
p
The aforesaid strains are reported to be composed of the same basic trisaccharide repeating unit of the following structure:
---�3 )-o:-D-Fucp NAc-( 1 ---�2)-L-0:-D-Hepp-( 1 ---�3)-6-deoxy-0:-L-Talp-( l---�. in which L-o:-D-Hepp is L-gi ycero-o:-D-manno-heptopyranose
13
1 8
78
- Contd
.j>. \0 tv
Vl Q Z 0 ;:0 m Vl < 0 r 0-0 '-C z m tv 8