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 .

References

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SANGHI & DHAR: STRUCTURE ELUCIDTION OF BACTERIAL POLYSACCHARIDES 473

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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

SANGHI & DHAR: STRUCTURE ELUCIDTION OF B ACTERIAL POLYSACCHARI DES 487

"0 = o u I

o

------------ -- ------------... _-------------------------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