Synthesis of an L-rhamnose tetrasaccharide, the common and major structure of the repeating unit of the O-antigenic polysaccharide of a strain of Klebsiella pneumoniae and Pseudomonas holci.

A tetrasaccharide, alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-L-Rhap, the common and major structure of the repeating unit of the O-antigenic polysaccharide of a strain of Klebsiella pneumoniae and Pseudomonas holci was synthesized as its methyl and octyl glycosides. Selective 3-O-glycosylation of allyl alpha-L-rhamnopyranoside with 2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl trichloroacetimidate gave allyl 2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl-(1-->3)-alpha-L-rhamnopyranoside (3). Benzoylation, deallylation, and trichloroacetimidation afforded 2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl-(1-->3)-2,4-di-O-benzoyl-alpha-L-rhamnopyranosyl trichloroacetimidate (6). Self condensation of 3,4-di-O-benzoyl-beta-L-rhamnopyranose 1,2-methyl orthoester or 1,2-octyl orthoester gave methyl or octyl 2-O-acetyl-3,4-di-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->2)-3,4-di-O-benzoyl-alpha-L-rhamnopyranoside (16 or 17), and subsequent selective deacetylation gave the disaccharide acceptor (18 or 19). Coupling of 6 with 18 (or 19), followed by deacylation in ammonia-saturated methanol, produced the target tetrasacharide.     

Structure of the group G streptococcal polysaccharide

The structure of the group-specific polysaccharide of group G Streptococcus was determined by means of methylation analysis and selective chemical degradations. The anomeric configurations and conformations of the sugar residues were studied by 1H- and 13C-n.m.r. spectroscopy. The tetrasaccharide repeating unit, ----3)-alpha-D-Galp-(1----2)-[alpha-L-Rhap-(1----3)-beta-D-GalpNAc - (1----4)]-alpha-L-Rhap-(1----, was determined.     

Structure of the O-specific, sialic acid containing polysaccharide chain and its linkage to the core region in lipopolysaccharide from Hafnia alvei strain 2 as elucidated by chemical methods, gas-liquid chromatography/mass spectrometry, and 1H NMR spectroscopy

Mild acid hydrolysis of Hafnia alvei strain 2 lipopolysaccharide released no O-specific polysaccharide but instead gave a monomeric octasaccharide repeating unit with N-acetylneuraminic acid as the reducing terminus. In addition, a dimer of the octasaccharide repeating unit, and also a decasaccharide composed of a fragment of the O-specific polysaccharide chain and the core region, were obtained in minute amounts. On the basis of the sugar and methylation analyses, periodate oxidation, and 1H NMR spectroscopy of the lipopolysaccharide hydrolytic products, the biological repeating unit of the O-specific polysaccharide was shown to be a branched octasaccharide: (Formula; see text) The linkage between the O-specific polysaccharide chain and core region has also been determined and has yield strong evidence that N-acetylneuraminic acid is an inherent lipopolysaccharide component. The lipopolysaccharide of H. alvei strain 2 is the first lipopolysaccharide reported to contain 4-substituted neuraminic acid in its O-specific polysaccharide region.     

Synthesis of a repeating unit and the dimer of the repeating unit of the major chain of Shigella flexneri O-antigen polysaccharide

Methyl glycoside of the tetrasaccharide GlcNAc(beta 1-2)Rha(alpha 1-2)Rha(alpha 1-3)Rha, which represents a repeating unit of the basic chain of Shigella flexneri O-antigenic polysaccharides, was synthesized using acylated monosaccharide synthons. A dimer of the repeating unit, octasaccharide [GlcNAc(beta 1-2)Rha(alpha 1-2) Rha(alpha 1-3)Rha(alpha 1-3)]2-OMe was obtained by TrClO4-catalyzed condensation of two tetrasaccharide blocks.     

Synthesis of an xylosylated rhamnose pentasaccharide, the repeating unit of the O-chain polysaccharide of the lipopolysaccharide of Xanthomonas campestris pv. begoniae GSPB 525

A xylosylated rhamnose pentasaccharide, alpha-L-Rhap-(1-->3)-[beta-L-Xylp-(1-->2)-]-alpha-L-Rhap-(1-->3)-[beta-L-Xylp-(1-->4)]-L-Rhap, the repeating unit of the O-chain polysaccharide (OPS) of the lipopolysaccharides of Xanthomonas campestris pv. begoniae GSPB 525 was synthesized by a highly regio- and stereoselective way. Thus coupling of 1,2-O-ethylidene-beta-L-rhamnopyranose (1) with 2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl trichloroacetimidate (2) to give (1-->3)-linked disaccharide (3), subsequent benzoylation, deethylidenation, acetylation, 1-O-deacetylation, and trichloroacetimidation afforded the disaccharide donor 11. Condensation of 11 with 1 yielded 2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->3)-2-O-acetyl-4-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->3)-1,2-O-ethylidene-beta-L-rhamnopyranose (12), and selective deacetylation of 12 yielded the trisaccharide diol acceptor 15. Coupling of 15 with 2,3,4-tri-O-benzoyl-alpha-L-xylopyranosyl trichloroacetimidate (16), followed by deprotection, gave the target pentasaccharide 19.     

Chemo-enzymatic synthesis of a tetra- and octasaccharide fragment of the capsular polysaccharide of Streptococcus pneumoniae type 14

The chemo-enzymatic synthesis is described of tetrasaccharide beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (1) and octasaccharide beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (2), representing one and two tetrasaccharide repeating units of Streptococcus pneumoniae serotype 14 capsular polysaccharide. In a chemical approach, the intermediate linear trisaccharide 3 and hexasaccharide 4 were synthesized. Galactose residues were beta-(1-->4)-connected to the internal N-acetyl-beta-D-glucosamine residues by using bovine milk beta-1,4-galactosyltransferase. Both title oligosaccharides will be conjugated to carrier proteins to be tested as potential vaccines in animal models.     

Conformation of the O-specific polysaccharide of Shigella dysenteriae type 1: molecular modeling shows a helical structure with efficient exposure of the antigenic determinant alpha-L-Rhap-(1-->2)-alpha-D-Galp.

The O-specific polysaccharide of Shigella dysenteriae type 1, which has the repeating tetrasaccharide unit -->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1-->3)-alpha-D-GlcNAcp-(1--> (A-B-C-D), is a major virulence factor, and it is believed that antibodies against this polysaccharide confer protection to the host. The conformational properties of fragments of this O-antigen were explored using systematic search with a modified HSEA method (GLYCAN) and with molecular mechanics MM3(96). The results show that the alpha-D-Gal-(1-->3)-alpha-D-GlcNAc linkage adopts two favored conformations, phi/psi approximately equal to -40 degrees /-30 degrees (I) and approximately 15 degrees /30 degrees (II), whereas the other glycosidic linkages only have a single favored phi/psi conformational range. MM3 indicates that the trisaccharide B-C-D and tetrasaccharides containing the B-C-D moiety exist as two different conformers, distinguished by the conformations I and II of the C-D linkage. For the pentasaccharide A-B-C-D-A' and longer fragments, the calculations show preference for the C-D conformation II. These results can explain previously reported nuclear magnetic resonance data. The pentasaccharide in its favored conformation II is sharply bent, with the galactose residue exposed at the vertex. This hairpin conformation of the pentasaccharide was successfully docked with the binding site of a monoclonal IgM antibody (E3707 E9) that had been homology modeled from known crystal structures. For fragments made of repetitive tetrasaccharide units, the hairpin conformation leads to a left-handed helical structure with the galactose residues protruding radially at the helix surface. This arrangement results in a pronounced exposure of the galactose and also the adjacent rhamnose in each repeating unit, which is consistent with the known role of the as alpha-L-Rhap-(1-->2)-alpha-D-Galp moiety as a major antigenic epitope of this O-specific polysaccharide.     

Characterization of five Shigella flexneri variant Y-specific monoclonal antibodies using defined saccharides and glycoconjugate antigens

The structural domains of the Shigella flexneri variant Y O-antigen epitopes 3,4 have defied definition, despite knowledge of the structure of the linear polysaccharide chain of the LPS molecule. The dual epitope designation of group antigen 3,4 is based on absorption data using polyvalent rabbit antisera. Five monoclonal antibodies specific for the Y antigen, generated after immunization of BALB/c mice or LOU/C rats, were selected on the basis of ELISA by using well-characterized S. flexneri Y LPS and chemically defined glycoconjugates. Chemically defined LPS from all S. flexneri serogroups, synthetic oligosaccharides, and saccharides obtained by phage Sf6-mediated hydrolysis of the O-polysaccharide were used either as free haptens or glycoconjugates in Farr assays and ELISA titrations. Two different patterns of antibody specificities were seen: two monoclonal antibodies had combining sites recognizing the terminal nonreducing end of the O-polysaccharide complementary to the tetrasaccharide repeating unit; and three antibodies bound to intrachain determinants and had larger combining sites, possibly accommodating at least an octasaccharide. The precise specificity of these two general types of antibodies indicate that variant Y polysaccharide generates more than two O-factors.     

Efficient synthesis of a heptasaccharide,the repeating unit of the O-chain lipopolysaccharide produced by Xanthomonas campestris strain 642

alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-[beta-D-Xylp-(1-->2)-][beta-D-Xylp-(1-->4)-]alpha-L-Rhap-(1-->3)-alpha-L-Rhap, the repeating unit of the O-chain lipopolysaccharide produced by Xanthomonas campestris strain 642 was synthesized as its methyl glycoside via 3-O-selective glycosylation of methyl alpha-L-rhamnopyranosyl-(1-->3)-2,4-di-O-benzoyl-alpha-L-rhamnopyranoside (9) with 2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->3)-2,4-di-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->2)-3,4-di-O-benzoyl-alpha-L-rhamnopyranosyl trichloroacetimidate (8), followed by dixylosylation with 2,3,4-tri-O-benzoyl-alpha,beta-D-xylopyranosyl trichloroacetimidate (12) and subsequent deacylation.     

The exopolysaccharide of Klebsiella aerogenes A 3 (Sl) (type 54). The isolation of O-acetylated octasaccharide, tetrasaccharide and trisaccharide

The exopolysaccharide slime produced by Klebsiella aerogenes A 3 (Sl) (type 54) is an O-acetylated polysaccharide, the components of which are glucose, glucuronic acid, fucose and acetyl in the molar proportions 4:2:2:1. A phage-induced fucosidase was obtained that hydrolyses the polysaccharide to give an octasaccharide having the same constituents in the same molar proportions. This octasaccharide (O3) is considered to be the repeating unit of the polysaccharide. It is hydrolysed by other phage-induced fucosidases described earlier to release two tetrasaccharides (O1 and O2). These differ only in that tetrasaccharide O2 is acetylated. An acetylated trisaccharide of structure beta-glucosylglucuronosylfucose was prepared from tetrasaccharide O2. A further unidentified group is present. Cell-free preparations were used to acetylate the disaccharide alpha-glucuronosylfucose. From these results the structure of the octasaccharide (O3) is postulated and its significance in the biosynthesis of the polysaccharide discussed.     

Elucidation of two O-chain structures from the lipopolysaccharide fraction of Agrobacterium tumefaciens F/1

Agrobacterium tumefaciens F/1 produces two different O-chains, both are constituted of rhamnose and glucosamine: the less abundant has a linear disaccharidic repeating unit 3)-alpha-L-Rhap-(1-->3)-beta-D-GlcpNAc-(1--> and the second one 4)-alpha-L-Rhap-(1-->3)-beta-D-GlcpNAc-(1-->. The two intact antigenic moieties were studied in mixture by 2D NMR. Additional supporting data were obtained by periodate degradation, the major component was cleaved selectively, leading to a glucosamine glycoside, whereas the minor one was recovered unaffected.     

Structure of a neutral polymer isolated from the lipopolysaccharide of the reference strain for Serratia marcescens serogroup O18

A neutral polymer (the putative O antigen) has been isolated from the lipopolysaccharide of the reference strain for Serratia marcescens serogroup 018. From the results of spectroscopic and degradative studies, the repeating unit of the polymer was identified as a linear tetrasaccharide having the structure shown. ----2)-alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----6)-alpha-D- GlcpNAc-(1----     

Structure of a neutral glycan isolated from the lipopolysaccharide of the reference strain for Serratia marcescens serogroup O22

Both a neutral and an acidic polymer have been isolated from a lipopolysaccharide extract of the reference strain for Serratia marcescens serogroup O22. The neutral polymer has a linear structure with the repeating unit shown. The same tetrasaccharide unit also forms the backbone of the branched neutral polymer isolated from the reference strain for serogroup O10, which cross-reacts strongly with O22. ----2)-alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----3)-alpha-L-+ ++Rhap-(1----3)-alpha- D-GlcpNAc-(1----     

A concise synthesis of two isomeric pentasaccharides, the O repeat units from the lipopolysaccharides of P. syringae pv. porri NCPPB 3364T and NCPPB 3365

A concise synthesis of two isomeric pentasaccharides, alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-[beta-D-GlcpNAc-(1-->2)]-alpha-L-Rhap (A) and alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-[beta-D-GlcpNAc-(1-->2)]-alpha-L-Rhap-(1-->3)-alpha-L-Rhap (B), the O repeats from the lipopolysaccharides of Pseudonomonas syringae pv. porri NCPPB 3364T and 3365 was achieved via assembly of the building blocks, allyl 3,4-di-O-benzoyl-alpha-L-rhamnopyranoside (1), 2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl trichloroacetimidate (2), allyl 4-O-benzoyl-3-O-chloroacetyl-alpha-L-rhamnopyranoside (6), 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate (7), and allyl 2,4-di-O-benzoyl-alpha-L-rhamnopyranoside (10). Coupling of 1 with 2 followed by deallylation and trichloroacetimidate formation gave the disaccharide donor 5, while condensation of 6 with 7, followed by dechloroacetylation, offered the disaccharide acceptor 9. Then, 5 was coupled with 10 to obtain the trisaccharide 11, and subsequent deallylation and trichloroacetimidate formation furnished the trisaccharide donor 13. Coupling of 9 with 13, followed by deprotection, afforded pentasaccharide 19, while condensation of 9 with 5, followed by deallylation and trichloroacetimidate formation, gave the tetrasaccharide donor 16, whose coupling with 10 and subsequent deprotection yielded another pentasaccharide 22.     

Structural studies of the O-antigen of the lipopolysaccharide from an avirulent strain (M4S) of Pseudomonas solanacearum

The structure of the O-antigen of the lipopolysaccharide from an avirulent strain (M4S) of Pseudomonas solanacearum has been investigated by methylation analysis, n.m.r. spectroscopy, and N-deacetylation-deamination, followed by analysis and controlled Smith-degradation of the product. These studies demonstrate that the O-antigen is composed of a tetrasaccharide repeating-unit having the following structure: ----3)-alpha-D-GlcpNAc-(1----2)-alpha-L-Rhap-(1----2)-alpha- L-Rhap-(1----3)- alpha-L-Rhap-(1----.     

Structural studies on O-specific polysaccharides of lipopolysaccharides from Yersinia enterocolitica serovars O:5 and O:5,27

Lipopolysaccharides of Yersinia enterocolitica serovars O:5 and O:5,27 were shown to have a similar sugar composition, consisting of L-rhamnose, D-glucose, D-galactose, D- and L-glycero-D-manno-heptose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, 3-deoxy-D-manno-octulosonate and D-threo-pent-2-ulose (D-xylulose). Partial hydrolysis of lipopolysaccharides with acetic acid produced rhamnans with the following repeating unit: ----3)-L-Rha rho(alpha 1----3)-L-Rha rho(alpha 1----3)-L-Rha rho(beta 1----. 13C-NMR and methylation studies of the lipopolysaccharides gave the following structure for the repeating unit of the two O-specific polysaccharides: ----3)-L-Rha rho(alpha 1----3)-L-Rha rho(alpha 1----3)-L-Rha rho(beta 1----. (formula; see text)     

Molecular recognition of oligosaccharide epitopes by a monoclonal Fab specific for Shigella flexneri Y lipopolysaccharide: X-ray structures and thermodynamics

The antigenic recognition of Shigella flexneri O-polysaccharide, which consists of a repeating unit ABCD [-->2)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-beta-D-GlcpNAc-(1-->], by the monoclonal antibody SYA/J6 (IgG3, kappa) has been investigated by crystallographic analysis of the Fab domain and its two complexes with two antigen segments (a pentasaccharide Rha A-Rha B-Rha C-GlcNAc D-Rha A' and a modified trisaccharide Rha B-Rha C-GlcNAc D in which Rha C* is missing a C2-OH group). These complex structures, the first for a Fab specific for a periodic linear heteropolysaccharide, reveal a binding site groove (between the V(H) and V(L) domains) that makes polar and nonpolar contacts with all the sugar residues of the pentasaccharide. Both main-chain and side-chain atoms of the Fab are used in ligand binding. The charged side chain of Glu H50 of CDR H2 forms crucial hydrogen bonds to GlcNAc of the oligosaccharides. The modified trisaccharide is more buried and fits more snugly than the pentasaccharide. It also makes as many contacts (approximately 75) with the Fab as the pentasaccharide, including the same number of hydrogen bonds (eight, with four being identical). It is further engaged in more hydrophobic interactions than the pentasaccharide. These three features favorable to trisaccharide binding are consistent with the observation of a tighter complex with the trisaccharide than the pentasaccharide. Thermodynamic data demonstrate that the native tri- to pentasaccharides have free energies of binding in the range of 6.8-7.4 kcal mol(-1), and all but one of the hydrogen bonds to individual hydroxyl groups provide no more than approximately 0.7 kcal mol(-1). They further indicate that hydrophobic interactions make significant contributions to binding and, as the native epitope becomes larger across the tri-, tetra-, pentasaccharide series, entropy contributions to the free energy become dominant.     

Molecular basis of S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus

The Gram-positive bacterium Geobacillus stearothermophilus NRS 2004/3a possesses a cell wall containing an oblique surface layer (S-layer) composed of glycoprotein subunits. O-Glycans with the structure [-->2)-alpha-L-Rhap-(1-->3)-beta-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->](n) (= 13-18), a2-O-methyl group capping the terminal repeating unit at the nonreducing end and a -->2)-alpha-L-Rhap-[(1-->3)-alpha-L-Rhap](n) (= 1-2)(1-->3)- adaptor are linked via a beta-D-Galp residue to distinct sites of the S-layer protein SgsE. S-layer glycan biosynthesis is encoded by a polycistronic slg (surface layer glycosylation) gene cluster. Four assigned glycosyltransferases named WsaC-WsaF, were investigated by a combined biochemical and NMR approach, starting from synthetic octyl-linked saccharide precursors. We demonstrate that three of the enzymes are rhamnosyltransferases that are responsible for the transfer of L-rhamnose from a dTDP-beta-L-Rha precursor to the nascent S-layer glycan, catalyzing the formation of the alpha1,3- (WsaC and WsaD) and beta1,2-linkages (WsaF) present in the adaptor saccharide and in the repeating units of the mature S-layer glycan, respectively. These enzymes work in concert with a multifunctional methylrhamnosyltransferase (WsaE). The N-terminal portion of WsaE is responsible for the S-adenosylmethionine-dependent methylation reaction of the terminal alpha1,3-linked L-rhamnose residue, and the central and C-terminal portions are involved in the transfer of L-rhamnose from dTDP-beta-L-rhamnose to the adaptor saccharide to form the alpha1,2- and alpha1,3-linkages during S-layer glycan chain elongation, with the methylation and the glycosylation reactions occurring independently. Characterization of these enzymes thus reveals the complete molecular basis for S-layer glycan biosynthesis.     

Structure of the O-specific polysaccharide of Proteus vulgaris O4 containing a new component of bacterial polysaccharides, 4,6-dideoxy-4

A high-molecular-mass O-specific polysaccharide was obtained by mild acid degradation of Proteus vulgaris O4 lipopolysaccharide followed by GPC. The polysaccharide was studied by chemical methods along with 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, H-detected 1H,13C HMQC, and 1H,13C HMBC experiments. Solvolysis of the polysaccharide with trifluoromethanesulfonic (triflic) acid resulted in a GlcpA-(1 --> 3)-GlcNAc disaccharide and a novel amino sugar derivative, 4,6-dideoxy-4-[N-[(R)-3-hydroxybutyryl]-L-alanyl]amino-D-glucose [Qui4N(HbAla)]. On the basis of the data obtained, the following structure of the tetrasaccharide repeating unit of the O-specific polysaccharide was established: --> 4)-beta-D-GlcpA-(1 --> 3)-beta-D-GlcpNAc-(1 --> 2)-beta-D-Quip4N(HbAla)-(1 --> 3)-alpha-D-Galp-(1 -->. This structure is unique among the O-specific polysaccharides, which is in accordance with classification of the strain studied in a separate Proteus serogroup.     

The structural basis for the serospecificity of Actinobacillus suis serogroup O:2.

Actinobacillus suis is an important bacterial pathogen of healthly pigs. An O-antigen (lipopolysaccharide; LPS) serotyping system is being developed to study the prevalence and distribution of representative isolates from both healthy and diseased pigs. In a previous study, we reported that A. suis serogroup O:1 strains express LPS with a (1-->6)-beta-D-glucan O-antigen chain polysaccharide that is similar in structure to a key cell-wall component in yeasts, such as Saccharomyces cerevisiae and Candida albicans. This study describes the O-antigen polysaccharide chemical structure of an O:2 serogroup strain, A. suis H91-0380, which possesses a tetrasaccharide repeating block with the structure: -->3)-beta-D-Galp-(1-->4)-[alpha-D-Galp-(1-->6)]-beta-D-Glcp-(1-->6)-beta-D-GlcpNAc-(1-->. Studies have shown that A. suis serogroup O:2 strains are associated with severely diseased animals; therefore, work on the synthesis of a glycoconjugate vaccine employing O:2 O-antigen polysaccharide to vaccinate pigs against A. suis serogroup O:2 strains is currently underway.