Structural studies of S-7, another exocellular polysaccharide containing 2-deoxy-arabino-hexuronic acid.

The exocellular polysaccharide S-7, a heteropolysaccharide from Azotobacter indicus var. myxogenes has been studied using methylation analysis, Smith degradation, partial acid hydrolysis, NMR spectroscopy and mass spectrometry as the principal methods. It is concluded that the repeating unit has the following structure: [structure: see text] The absolute configuration of the deoxyhexuronic acid was deduced from 1H NMR chemical shifts and is most likely D. Approximately two O-acetyl groups per repeating unit are present, one of which is presumably on the Rha residue. The structure bears great resemblance to another polysaccharide, recently studied, produced by Sphingomonas paucimobilis I-886.

Structure of a D-glycero -D-manno-heptan from the lipopolysaccharide of Helicobacter pylori.

A lipopolysaccharide (LPS) was isolated by hot phenol-water extraction from Helicobacter pylori strain D4 and found to contain no fucosylated poly-N-acetyllactosamine chain typical of most H. pylori strains studied but a homopolymer of D-glycero-D-manno-heptose (DD-Hep). The heptan attached to a core oligosaccharide was released by mild acid degradation of the LPS, and the following structure of the trisaccharide-repeating unit was established by chemical methods and 1H and 13C NMR spectroscopy: --> 2)-D-alpha-D-Hepp-(1 --> 3)-D-alpha-D-Hepp-(1 --> 3)-D-alpha-D-Hepp-(1 -->. 1H NMR spectroscopy performed on small amounts of the intact LPS revealed the presence of the same polysaccharide in LPS of H. pylori strains D2 and D5, but not strain D10.

Structural studies of the O-specific polysaccharide of Vibrio cholerae O8 using solvolysis with triflic acid.

The O-specific polysaccharide (OPS) of Vibrio cholerae 08 was isolated by mild acid degradation of the lipopolysaccharide and studied by two-dimensional NMR spectroscopy, including NOESY and heteronuclear multiple-bond correlation (HMBC) experiments. The OPS was found to have a tetrasaccharide repeating unit with the following structure: --> 4)-beta-D-Glcp NAc3NAcylAN-(1 --> 4)-beta-D-Manp NAc3NAcAN-(1 --> 4)-alpha-L-Gulp NAc3NAcA-(1 --> 3) -beta-D-QuipNAc4NAc-(1 --> where QuiNAc4NAc is 2,4-diacetamido-2,4,6-trideoxyglucose, GlcNAc3NAcylAN is 2-acetamido-3-(N-formyl-L-alanyl)amino-2,3-dideoxyglucuronamide, ManNAc3NAcAN is 2,3-diacetamido-2,3-dideoxymannuronamide, and GulNAc3NAcA is 2,3-diacetamido-2,3-dideoxyguluronic acid. The OPS was stable towards acid hydrolysis and solvolysis with anhydrous hydrogen fluoride, but could be cleaved selectively with trifluoromethanesulfonic (triflic) acid by the glycosidic linkages of beta-QuiNAc4NAc and alpha-GulNAc3NAcA. The structures of the oligosaccharides obtained that were elucidated by electrospray ionization (ESI) MS and NMR spectroscopy, confirmed the OPS structure.

Structures of two cell wall-associated polysaccharides of a Streptococcus mitis biovar 1 strain. A unique teichoic acid-like polysaccharide and the group O antigen which is a C-polysaccharide in common with pneumococci.

The cell wall of Streptococcus mitis biovar 1 strain SK137 contains the C-polysaccharide known as the common antigen of a closely related species Streptococcus pneumoniae, and a teichoic acid-like polysaccharide with a unique structure. The two polysaccharides are different entities and could be partially separated by gel chromatography. The structures of the two polysaccharides were determined by chemical methods and by NMR spectroscopy. The teichoic acid-like polymer has a heptasaccharide phosphate repeating unit with the following structure: The structure neither contains ribitol nor glycerol phosphate as classical teichoic acids do, thus we have used the expression teichoic acid-like for this polysaccharide. The following structure of the C-polysaccharide repeating unit was established: where AAT is 2-acetamido-4-amino-2,4, 6-trideoxy-D-galactose. It has a carbohydrate backbone identical to that of one of the two structures of C-polysaccharide previously identified in S. pneumoniae. C-polysaccharide of S. mitis is characterized by the presence, in each repeating unit, of two residues of phosphocholine and both galactosamine residues in the N-acetylated form. Immunochemical analysis showed that C-polysaccharide constitutes the Lancefield group O antigen. Studies using mAbs directed against the backbone and against the phosphocholine moiety of the C-polysaccharide revealed several different patterns of these epitopes among 95 S. mitis and Streptococcus oralis strains tested and the exclusive presence of the group O antigen in the majority of S. mitis biovar 1 strains.

Structure of the acidic microcapsular glycan from the reference strain (C.D.C. 6320-58) for Serratia marcescens serotype O14:K12.

The acidic polysaccharide from Serratia marcescens serogroup O14:K12 was analyzed by means of chemical studies and NMR spectroscopy and its repeating unit structure found to be carbohydrate sequence [see text] O-Acetyl groups are proposed to be present in non-stoichiometric amounts on O-6 on one of the hexose residues in the main chain.

Structure of an atypical O-antigen polysaccharide of Helicobacter pylori containing a novel monosaccharide 3-C-methyl-D-mannose.

Lipopolysaccharides (LPS) were isolated by hot phenol-water extraction from Danish Helicobacter pylori strains D1, D3, and D6, which were nontypeable using a variety of anti-Lewis and anti-blood-group monoclonal antibodies. An atypical O-chain polysaccharide (PS) was liberated from the LPS of the three strains by acid under mild conditions and found to contain D-rhamnose (D-Rha), L-rhamnose (L-Rha), and a branched sugar, 3-C-methyl-D-mannose (D-Man3CMe). The last sugar, which has not hitherto been found in Nature, was identified using GLC-MS of the derived alditol acetate and the partially methylated alditol acetate, and (1)H and (13)C NMR spectroscopy, including NOESY and (1)H,(13)C HMBC experiments. The following structure of the trisaccharide repeating unit of the PS was established: -->2)-alpha-D-Manp3CMe-(1-->3)-alpha-L-Rhap-(1-->3)-alpha-D- Rhap-(1-- >. In contrast to the pathogenic importance of the Lewis antigen mimicry exhibited by the PS of H. pylori strains previously investigated, the biological relevance of the atypical PS for H. pylori pathogenesis is unclear. The production of a differing surface PS may represent a form of antigenic variation by these particular H. pylori strains and/or may reflect the adaptation of these strains to a particular human population.

Structural studies on lipopolysaccharides of serologically non-typable strains of Helicobacter pylori, AF1 and 007, expressing Lewis antigenic determinants.

In contrast to other Helicobacter pylori strains, which have serologically detectable Lewis(x)+ (Le(x)) and Lewis(y)++ (++Le(y)) antigenic determinants in the O-specific polysaccharide chains of the lipopolysaccharides, H. pylori AF1 and 007 were non-typable with anti-Le(x) and anti-Le(y) antibodies. The carbohydrate portions of the lipopolysaccharides were liberated by mild acid hydrolysis and subsequently studied by sugar and methylation analyses, 1H-NMR spectroscopy and electrospray ionization-mass spectrometry. Compared with each other, and with lipopolysaccharides of strains studied previously, the lipopolysaccharides of both AF1 and 007 showed similarities, but also differences, in the structures of the core region and O-specific polysaccharide chains. The O-specific polysaccharide chains of both strains consisted of a short or long polyfucosylated poly-N-acetyl-beta-lactosamine chains, which were distinguished from those of other strains by a high degree of fucosylation producing a polymeric Le(x)chain terminating with Le(x) or Le(y) units:[sequence: see text] where n = 0 or 1 in strain AF1 and 0 in strain 007, m = 0-2, 6-7 in strain AF1 and m = 0-2, 6-7 or approximately 40 in strain 007, the medium-size species being predominant. Therefore, compared with other strains, the lack of reactivity of lipopolysaccharide of H. pylori AF1 and 007 with anti-Le(x) and anti-Le(y) may reflect the presence of a polymeric Le(x) chain and has important implications for serological and pathogenesis studies. As the substitution pattern of a D-glycero-D-manno-heptose residue in the outer core varied in the two strains, and an extended DD-heptan chain was present in some lipopolysaccharide species but not in others, this region was less conservative than the inner core region. The inner core L-glycero-D-manno-heptose region of both strains carried a 2-aminoethyl phosphate group, rather than a phosphate group, as reported previously for other H. pylori strains.

The pneumococcal common antigen C-polysaccharide occurs in different forms. Mono-substituted or di-substituted with phosphocholine.

The structure of the pneumococcal common antigen, C-polysaccharide, from a noncapsulated pneumococcal strain, CSR SCS2, was studied using 1H-NMR, 13C-NMR and 31P-NMR spectroscopy. The dependence of NMR chemical shifts on the variation in pD was also studied. It was established that the C-polysaccharide is composed of a backbone of tetrasaccharide-ribitol repeating units that are linked to each other by a phosphodiester linkage between position 5 of a D-ribitol residue and position 6 of a beta-D-glucopyranosyl residue. The polysaccharide is substituted with one residue of phosphocholine at position 6 of the 4-substituted 2-acetamido-2-deoxy-alpha-D-galactopyranosyl residue. Both galactosamine residues in the polysaccharide are N-acetylated. O)-P-Cho | 6 6)-beta-D-Glcp-(1-->3)-alpha-AATp-(1-->4)-alpha-D-GalpNAc-(1-->3)- bet a-D-GalpNAc-(1-->1)-D-ribitol-5-P-(O--> where AAT is 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose and Cho is choline. This structure differs, concerning phosphocholine substituents and N-acetylation, from those reported previously for pneumococcal C-polysaccharide [Jennings, H.J., Lugowski, C. & Young, N.M. (1980) Biochemistry 19, 4712-4719; Fischer, W., Behr, T., Hartmann, R., Peter-Katalinic, J. & Egge, H. (1993) Eur. J. Biochem. 215, 851-857; Kulakowska, M., Brisson, J.-R., Griffith, D.W., Young, N.M. & Jennings, H.J. (1993) Can. J. Chem. 71, 644-648]. The structures of the C-polysaccharides present in three pneumococcal types were also examined. They contain one (in 18B) or two (in 32F and 32A) phosphocholine residues in the repeating unit. The degree of substitution was not determined. The backbone of all examined C-polysaccharides was identical and in all cases both galactosamine residues appeared to be N-acetylated.

The lipopolysaccharide of moraxella catarrhalis structural relationships and antigenic properties.

Moraxella catarrhalis has recently been shown to be both widespread and pathogenic, in contrast to previous reports. Several factors have been suggested as virulence factors, lipopolysaccharide (LPS) being one. Recent studies have shown the LPS to be without the O-chain, i.e. the polysaccharide part, and to have specific structural features corresponding to each of the three serogroups, A, B and C. The structures resemble in many respects those present in other Gram-negative nonenteric bacteria, with a galabiosyl element as a prominent common denominator. The presence of such common structures suggests that the LPS of these bacteria might be a part of a mechanism of survival for bacteria colonizing the human host.

More on the structure of Vibrio cholerae O22 lipopolysaccharide.

The structure of a short-chain lipopolysaccharide (LPS) of Vibrio cholerae O22 strain 169-68, that cross-reacts with V. cholerae O139 Bengal, was elucidated. The structure differs in detail from that reported on another strain of O22 [A.D. Cox, J-R. Brisson, P. Thibault and M.B. Perry, Carbohydr. Res., 304 (1997) 191-208]. The similarity and difference between the LPS structures of the two strains as well as between O22 and O139 are discussed.

The chemical structures of the capsular polysaccharides from Streptococcus pneumoniae types 32F and 32A.

The structures of the capsular polysaccharides from Streptococcus pneumoniae types 32F and 32A have been determined by means of NMR spectroscopy as the principal method. It is concluded that both polysaccharides are composed of tetrasaccharide repeating units with a phosphorylcholine (PCho) group linked to the 3-position of the 4-substituted beta-L-rhamnose (Rha) residue. Both polysaccharides are substituted with one O-acetyl group at the 2-position of the same beta-L-rhamnose residue. In addition, the type-32A polysaccharide is substituted with another O-acetyl group at the 4-position of the 2,3-disubstituted alpha-D-glucose residue, i.e. the branch-point residue. An unusual detail in the structure is that the side chain is composed of a rhamnosyl phosphate. [chemical structure: see text] In the type-32F polysaccharide R=H, and in the type-32A polysaccharide R=Ac. The structure of C-polysaccharide found in our preparations of type-32F and type-32A capsular polysaccharides is in agreement with that published previously for the pneumococcal common antigen C-polysaccharide [Fischer, W., Behr, T., Hartmann, R., Peter-Katalinic, J. & Egge, H. (1993) Eur. J. Biochem. 215, 851-857; Kulakowska, M., Brisson, J.-R., Griffith, D. W., Young, N. M. & Jennings, H. J. (1993) Can. J. Chem. 71, 644-648].

Computer-assisted structural analysis of oligo- and polysaccharides: an extension of CASPER to multibranched structures.

The CASPER program which is used for determination of the structure of oligo- and polysaccharides has been extended. It can now handle a reduced number of experimental signals from an NMR spectrum in the comparison to the simulated spectra of structures that it generates, an improvement which is of practical importance since all signals in NMR spectra cannot always be identified. Furthermore, the program has been enhanced to simulate NMR spectra of multibranched oligo- and polysaccharides. The new developments were tested on four saccharides of known structure but of different complexity and were shown to predict the correct structures.

Structure of the O-antigen of Vibrio cholerae O155 that shares a putative D-galactose 4,6-cyclophosphate-associated epitope with V. cholerae O139 Bengal.

The O-specific polysaccharide of Vibrio cholerae 0155 was studied by sugar and methylation analyses, dephosphorylation with 48% hydrofluoric acid, 1H- and 13C-NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, and heteronuclear single-quantum coherence (HSQC) experiments. The following structure of the pentasaccharide repeating unit of the polysaccharide was established: carbohydrate sequence [see text]. An unusual component, D-galactose 4,6-cyclophosphate, has been reported previously as a component of the capsular polysaccharide and O-antigen of V. cholerae O139 Bengal and appears to be responsible for the known serological cross-reactivity between V. cholerae O139 and O155.

The structure of the capsular polysaccharide from Klebsiella type 52, using the computerised approach CASPER and NMR spectroscopy.

The structure of the capsular polysaccharide from Klebsiella type 52 has been elucidated using an improved and extended version of the computerised approach CASPER and NMR spectroscopy as principal methods. A previous suggestion to the structure but without the anomeric prefixes, could be shown correct [H. Björndal et al., Carbohydr. Res., 31 (1973) 93-100]. The polysaccharide has a hexasaccharide repeat with the following structure: [formula: see text]

Structural studies on the short-chain lipopolysaccharide of Vibrio cholerae O139 Bengal.

A Vibrio cholerae O139 strain, MO10-T4, lacking capsular polysaccharide, produces a short-chain lipopolysaccharide (LPS), similar to enterobacterial SR strains. It was studied by acidic and alkaline degradation, dephosphorylation, sugar and methylation analysis, high-performance anion-exchange chromatography, one- and two-dimensional 1H-, 13C-, and 31P-NMR spectroscopy, and electrospray ionization mass spectrometry. The following structure was proposed for the core region of the LPS: [structure: see text] where PEtn stands for 2-aminoethyl phosphate, Fru for fructose, Hep for L-glycero-D-manno-heptose, and Kdo for 3-deoxy-D-manno-octulosonic acid; unless otherwise stated, the monosaccharide residues are D and present in the pyranose form. An O-acetyl group is present on a secondary position, tentatively O4 of the alpha-linked glucosyl group. Some LPS species contain an additional putative fructose residue whose location remains unknown. An O139-negative mutant strain, Bengal-2R, derived from V. cholerae O139, has also been investigated and shown to produce an O-antigen-lacking LPS similar to those from enterobacterial R strains, some of the LPS species containing the same core region as the strain MO10-T4 LPS and the other lacking the lateral heptose residue. The carbohydrate backbone core structure is the same for the V. cholerae O139 and V. cholerae O1 LPS, thus confirming the close relation between these bacteria; however, the 2-aminoethyl phosphate, the O-acetyl group, and the second fructose residue have not been reported for the O1 LPS. In the V. cholerae O139 strain MO10-T4 LPS, a short O-side chain is attached at position 3 of the 7-substituted heptose residue and has the same structure as one repeating unit of the V. cholerae O139 capsular polysaccharide. Some details of the structure proposed are at variance with those recently published for another V. cholerae O139 strain [Cox, A. D., Brisson, J.-R., Varma, V. & Perry, M. B. (1996) Carbohydr. Res. 290, 43-58; Cox, A. D. & Perry, M. B. (1996) Carbohydr. Res. 290, 59-65.]

Determination of the absolute configuration of sugar residues using gas chromatography. Method with potential for elimination of references.

The absolute configuration of a sugar can be determined by gas-liquid chromatography of the acetylated or trimethylsilylated dithioacetals from 1-phenylethanethiol. The isolation of both enantiomers of 1-phenylethanethiol is also described. Using the acetates and both thiol reagents the absolute configuration of C-2 can be determined, provided it is a hydroxyl group, with great certainty. A new way of determining the absolute configuration of sugars, without references, is thereby provided. The sugars analysed include aldoses, deoxyaldoses, 2-acetamido-2-deoxyaldoses and uronic acids. The analysis is made using columns with non-chiral stationary phase and the electron impact mass spectra of the acetylated and trimethylsilylated bis(1-phenylethyl)dithioacetals are described.

Structure of the O-polysaccharide from the LPS of a Hafnia alvei strain isolated from a patient with suspect yersinosis.

The carbohydrate backbone of the Hafnia alvei strain Y166/91 lipopolysaccharide (LPS) was isolated by mild acid hydrolysis followed by gel permeation chromatography and studied by NMR spectroscopy and methylation analysis. Treatment with periodate and hypoiodite gave a modified polysaccharide which was also characterised. It was concluded that the polysaccharide has the following structure with two distinct regions, which are connected. The chain length parameters m and n were not determined but the ratio m/n is approximately 2. [-->3)-beta-D-Galp-1(1-->3)-alpha-D-Galp-(1-->]m[-->3)-alpha-D-Galp-(1-- >3)- beta-D-Galf-(1-->]n From the present data it is not possible to determine whether it is the Galp-Galp chain or the Galp-Galf chain that is connected to the core. The structure found here is identical to that suggested for the O-specific polysaccharide chain of Klebsiella pneumoniae O1K2 (NCTC 5055) LPS [O. Kol, J.-M. Wieruszeski, G. Strecker, J. Montreuil, and B. Fournet, Carbohydr. Res., 217 (1991) 117-125; O. Kol, J.-M. Wieruszeski, G. Strecker, B. Fournet, R. Zalisz, and P. Smets, Carbohydr. Res., 236 (1992) 339-344].

Structural studies of the cell-envelope oligosaccharide from the lipopolysaccharide of Haemophilus influenzae strain RM.118-28.

The structure of the oligosaccharide part of the Haemophilus influenzae RM.118-28 lipopolysaccharide (LPS) has been investigated. The oligosaccharide was obtained from the LPS by mild acid hydrolysis followed by gel-permeation chromatography, and was studied by methylation analysis, NMR spectroscopy and mass spectrometry. The structure of the major compound, which is a hexasaccharide, is proposed as follows. [formula: see text] In the structure, Kdo is 3-deoxy-D-manno-octulosonic acid, PEtn is phosphoethanolamine, PCho is phosphocholine and L,D-Hep is L-glycero-D-manno-heptose. Electrospray-ionization mass spectrometry on O-deacylated LPS obtained after treatment with anhydrous hydrazine gave evidence for the presence of two minor compounds, which show additional substitution of the main structure with phosphate and PEtn, respectively. These substitutions have not been localized.

Structural elucidation of the capsular polysaccharide from Streptococcus pneumoniae type 18B.

The structure of the capsular polysaccharide from Streptococcus pneumoniae type 18B has been determined using NMR spectroscopy and methylation analysis as the principal methods. It is concluded that the polysaccharide is composed of pentasaccharide repeating units with a glycerol phosphate substituting the 3-position of the branch point residue. The carbohydrate backbone in type 18B is identical to that in S. pneumoniae type 18F but without the O-acetyl groups present in that type. [formula: see text] In this structure, the absolute configuration of the glycerol phosphate moiety has not been determined but should be D, in analogy with that determined for the capsular polysaccharide from S. pneumoniae type 18A [T. Rundlöf, G. Widmalm, Anal. Biochem., 243 (1996) 228-233].

The structures of oligosaccharides isolated from the lipopolysaccharide of Moraxella catarrhalis serotype B, strain CCUG 3292.

The oligosaccharides from the lipopolysaccharides of Moraxella catarrhalis serotype B, strain CCUG 3292, were isolated after mild acid hydrolysis and separated by high-performance anion-exchange chromatography. The structures of the oligosaccharides were established by fast atom bombardment mass spectrometry and nuclear magnetic resonance spectroscopy. It is concluded that the oligosaccharides comprise a mixture of mainly a nona- and a deca-saccharide. [formula: see text] Smaller amounts of undeca-saccharides and of truncated forms, namely, hexa-, hepta-, and octa-saccharides, were also detected.