Structure of the O-specific polysaccharide of Mesorhizobium huakuii IFO15243T

The structure of the O-specific polysaccharide isolated by mild acid hydrolysis of the lipopolysaccharide of Mesorhizobium huakuii IFO15243T was studied using methylation analysis and various one- and two-dimensional 1H and 13C NMR experiments. The O-antigen polysaccharide was found to be linear polymer constituted by a trisaccharide repeating unit of the following structure: --> 2)-alpha-L-6dTalp-(1 --> 3)-alpha-L-6dTalp-(1 --> 2)-alpha-L-Rhap-(1 -->.     

The Shigella flexneri O-antigenic polysaccharide chain. Nature of the biological repeating unit

The sequence of monosaccharides in the biological repeating tetrasaccharide unit of Shigella flexneri variant Y O-antigenic polysaccharide chain was determined by subjecting three oligosaccharides of the polysaccharide, obtained by phage-Sf6-mediated enzymatic hydrolysis, to methylation analysis and proton nuclear magnetic resonance spectroscopy. The smallest saccharide was shown to be a tetrasaccharide with the structure alpha-L-Rhap-(1-2)-L-Rha. The next saccharide, an octasaccharide, was shown to be a dimer of the tetrasaccharide with the L-Rha residues linked alpha 1.3. The longest saccharide was shown to be a decasaccharide with the following structure: alpha-L-Rhap-(1-2)-alpha-L-Rhap-(1-3)-alpha-L-Rhap-(1- 3)-beta-D-GlcpNAc-(1-2)-alpha-L-Rhap-(1-2)-alpha-L-Rhap++ + +-(1-3)-alpha-L-Rhap-(1-3)-beta-D-GlcpNAc-(1-2)-alpha-L-R hap-(1-2)-L-Rha. Thus the decasaccharide differed from the octasaccharide and tetrasaccharide by having the alpha-L-Rhap-(1-2)-L-Rhap disaccharide added in the terminal non-reducing end of the saccharide chain. This shows that the alpha-L-Rhap-(1-2)-alpha-L-Rhap-(1-3)-alpha-L-Rhap-(1- 3)-D-GlcpNAc tetrasaccharide is the biological repeating unit of the O chain and that the repeating units are joined through a beta-D-GlcpNAc-(1-2)-L-Rhap linkage. Inhibition experiments utilizing the enzyme-linked immunosorbent assay (ELISA) with S. flexneri Y lipopolysaccharide/S. flexneri Y rabbit antiserum showed that the decasaccharide was the best inhibitor (threefold as active as the octasaccharide and sixtyfold as active as the tetrasaccharide); this supports the postulated structure of the biological repeating unit.     

Structural determination of a neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B332

The neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B332 in skimmed milk was found to be composed of d-glucose, d-galactose, and l-rhamnose in a molar ratio of 1:2:2. Linkage analysis and 1D/2D NMR (1H and 13C) studies carried out on the native polysaccharide as well as on an oligosaccharide generated by a periodate oxidation protocol, showed the polysaccharide to consist of linear pentasaccharide repeating units with the following structure: -->3-alpha-D-Glcp-(1-->3)-alpha-D-Galp-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1-->.     

O-specific polysaccharide structure of the aqueous lipopolysaccharide fraction from Xanthomonas campestris pv. vitians strain 1839

The structure of Xanthomonas campestris pv. vitians O-specific polysaccharide of the lipopolysaccharide fraction, extracted from the aqueous phase, was defined, on the basis of chemical and spectroscopical methods, as constituted by the following repeating unit: [--> 3)-alpha-L-Rhap-(1 -->]n 3)-beta-L-Rhap-(1 --> where n is more frequently equal to 2, but it also assumes values equal to 1 and to 3.     

Structure of the O-specific polysaccharide of the lipopolysaccharide of Azospirillum brasilense Sp245

An O-specific polysaccharide was isolated from the lipopolysaccharide of a plant-growth-promoting bacterium Azospirillum brasilense Sp245 and studied by sugar analyses along with one- and two-dimensional 1H and 13C NMR spectroscopy, including NOESY. The polysaccharide was found to be a new rhamnan with a pentasaccharide repeating unit having the following structure:-->2)-beta-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->2)-alpha-D-Rhap-(1-->2)-alpha-D-Rhap-(1-->     

X-ray structure determination and modeling of the cyclic tetrasaccharide cyclo

The cyclic tetrasaccharide cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] is the major compound obtained by the action of endo-alternases on the alternan polysaccharide. Crystals of this cyclo-tetra-glucose belong to the orthorhombic space group P2(1)2(1)2(1) with a = 7.620(5), b = 12.450(5) and c = 34.800(5) A. The asymmetric unit contains one tetrasaccharide together with five water molecules. The tetrasaccharide adopts a plate-like overall shape with a very shallow depression on one side. The shape is not fully symmetrical and this is clearly apparent on comparing the (phi, psi) torsion angles of the two alpha-(1-->6) linkages. There is almost 10 degrees differences in phi and more than 20 degrees differences in psi. The hydrogen bond network is asymmetric, with a single intramolecular hydrogen bond: O-2 of glucose ring 1 being the donor to O-2 of glucose ring 3. These two hydroxyl groups are located below the ring and their orientation, dictated by this hydrogen bond, makes the floor of the plate. Among the five water molecules, one located above the center of the plate occupies perfectly the shallow depression in the plate shape formed by the tetrasaccharide. Molecular dynamics simulation of the tetrasaccharide in explicit water allows rationalization of the discrepancies observed between the X-ray structures and data obtained previously by NMR.     

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.     

Structure of the O-specific polysaccharide of Hafnia alvei PCM 1196

An acidic O-specific polysaccharide was isolated from Hafnia alvei PCM 1196 lipopolysaccharide and studied by sugar and methylation analyses along with one- and two-dimensional 1H and 13C NMR spectroscopy, including NOESY and HMBC experiments. The following structure of the pentasaccharide repeating unit was established: -->4)-alpha-D-GalpA-(1-->3)-beta-D-GlcpNAc-(1-->2)-beta-D-Galp-(1-->6)-alpha-D-Glcp-(1-->6)-alpha-D-GlcpNAc-(1-->.     

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.     

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.     

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.     

Somatic antigens of shigella. Structural investigation on the O-specific polysaccharide chain of Shigella dysenteriae type 1 lipopolysaccharide.

The O-specific polysaccharide obtained from the lipopolysaccharide of Shigella dysenteriae type 1 (Shigella shiga) by mild acid hydrolysis followed by fractionation on Sephadex G-50 was found to be identical to that desribed by Morgan's group and was composed of L-rhamnose, D-galactose and N-acetyl-D-glycosamine in a ratio 2:1:1. On the basis of methylation analysis data the polysaccharide was proved to be a linear chain of monosaccharide residues in pyranose forms substituted at position 3, except for that of galactose substituted at position 2. Selective cleavage, based on the N-deacetylation reaction of the polymer, together with determination of linkage configurations by chromic anhydride oxidation showed that the O-specific polysaccharide is built up of repeating tetrasaccharide units whose proposed structure is given below -3)-alpha-L-Rhap (1-3)-alpha-L-Rhap(1-2)-alpha-D-Galp(1-3)-alphapD-GlcNAcp(1- where RHAP = rhamnopyranose, Galp = galactopyranose, and GlcNAcp = N-acetyl-glucosamine. The present findings confirmed the considerations of Heidelberger on the substitution patterns of L-rhamnose and D-galactose residues from the results of serological studies.     

Structural and serological studies on Hafnia alvei O-specific polysaccharide of alpha-D-mannan type isolated from the lipopolysaccharide of strain PCM 1223

On the basis of chemical and methylation analyses, one- and two-dimensional (1)H- and (13)C-NMR spectroscopy, including COSY, TOCSY, NOESY and (1)H, (13)C HSQC experiments, a neutral O-specific polysaccharide isolated from Hafnia alvei strain PCM 1223 lipopolysaccharide (LPS) was found to be an alpha-mannan composed of pentasaccharide repeating units having the following structure:-->3)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->. Immunoblotting showed a strong cross-reactivity between anti-H. alvei PCM 1223 serum and LPSs of Escherichia coli O9 and Klebsiella pneumoniae O3. The serological relationship of the LPSs of these bacteria is due to the structural identity of their O-specific polysaccharides, though the LPSs differ in their core regions.     

Synthesis of spacer-equipped phosphorylated di-, tri- and tetrasaccharide fragments of the O-specific polysaccharide of Vibrio cholerae O139

The synthesis of oligosaccharide fragments of the O-specific polysaccharide of Vibrio cholerae O139 containing a 4,6-cyclic phosphate galactose residue linked to GlcNAc is described. 8-Azido-3,6-dioxaoctyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-(1-->3)-2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D-glucopyranoside, obtained by condensation of 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide and 8-azido-3,6-dioxaoctyl 2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D-glucopyranoside, was converted to 8-azido-3,6-dioxaoctyl 3-O-benzyl-beta-D-galactopyranosyl-(1-->3)-2-acetamido-6-O-benzyl-2-deoxy-beta-D-glucopyranoside (6) by reductive opening of the acetal, followed by deacetylation and selective benzylation. Phosphorylation of 6 furnished two isomeric 4,6-cyclic 2,2,2-trichloroethyl phosphates. Glycosylation of the (S)-phosphate with 2,4-di-O-benzyl-3,6-dideoxy-alpha-L-xylo-hexopyranosyl bromide under halide-assisted conditions gave the desired tetrasaccharide, together with a trisaccharide. Global deprotection and reduction of the azide to an amine was effected by catalytic hydrogenation/hydrogenolysis to give the deprotected tetrasaccharide, which is functionalized for conjugation.     

Structure of the O-specific polysaccharide of Proteus vulgaris O45 containing 3-acetamido-3,6-dideoxy-D-galactose

An O-specific polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Proteus vulgaris O45 and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H-detected 1H,13C HSQC and HMBC experiments. The following structure of the pentasaccharide repeating unit of the polysaccharide was established:-->6)-alpha-D-GlcpNAc-(1-->4)-alpha-D-GalpNAc-(1-->4)-alpha-D-GalpA-(1-->3)-beta-D-GlcpNAc-(1-->2)-beta-D-Fucp3NAc4Ac-(1-->where Fuc3NAc4Ac is 3-acetamido-4-O-acetyl-3,6-dideoxygalactose. A cross-reactivity of anti-P. vulgaris O45 serum was observed with several other Proteus lipopolysaccharides, which contains Fuc3N derivatives.     

Structure of the sialic acid-containing O-specific polysaccharide from Salmonella enterica serovar Toucra O48 lipopolysaccharide.

Lipopolysaccharide was extracted from cells of Salmonella enterica serovar Toucra O48 and, after mild acid hydrolysis (1% AcOH, 1 h, 100 degrees C or 0.1 M NaOH-AcOH, pH 4.5, 5 h, 100 degrees C), the O-specific polysaccharide was isolated and characterized. The core and an oligosaccharide containing a fragment of the repeating unit linked to the core region were also obtained, depending on hydrolysis conditions. On the basis of sugar and methylation analyses and NMR spectroscopy of the hydrolysis products, the biological repeating unit of the O-specific polysaccharide was shown to be the following trisaccharide: -->4)-alpha-Neup5Ac(2-->3)-L-alpha-FucpNAc(1-->3)-D-beta-Glc pNAc(1--> The polysaccharide O-chain was substituted with a single molar equivalent of O-acetyl group, distributed between the Neu5Ac O-9 and O-7 positions, in an approximate ratio of 7 : 3.     

Synthesis of oligosaccharides related to the repeating unit of the capsular polysaccharide from Streptococcus pneumoniae type 37

A tetra- and a pentasaccharide were synthesized as analogues to the structure of the Streptococcus pneumoniae type 37 capsular polysaccharide, a homopolymer with a disaccharide-repeating unit of -->3)[beta-D-Glcp-(1-->2)]-beta-D-Glcp-(1-->. Synthesis of the tetrasaccharide employed a beta-(1-->2)-diglycosylation of a beta-(1-->3)-linked disaccharide. Subsequently, the pentasaccharide was synthesized from a suitably protected tetrasaccharide derivative by a beta-(1-->3)-extension at O-3'. Steric crowding was found to be an important factor in the formation of the pentasaccharide.     

Synthesis of alpha-Manp-(1-->2)-alpha-Manp-(1-->3)-alpha-Manp-(1-->3)-Manp, the tetrasaccharide repeating unit of Escherichia coli O9a, and alpha-Manp-(1-->2)-alpha-Manp-(1-->2)-alpha-Manp-(1-->3)-alpha-Manp-(1-->3)-Manp, the pentasaccharide repeating unit of E. coli O9 and Klebsiella O3

The tetrasaccharide repeating unit of Escherichia coli O9a, alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-D-Manp, and the pentasaccharide repeating unit of E. coli O9 and Klebsiella O3, alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-D-Manp, were synthesized as their methyl glycosides. Thus, selective 3-O-allylation of p-methoxyphenyl alpha-D-mannopyranoside via a dibutyltin intermediate gave p-methoxyphenyl 3-O-allyl-alpha-D-mannopyranoside (2) in good yield. Benzoylation (-->3), then removal of 1-O-methoxyphenyl (right arrow4), and subsequent trichloroacetimidation afforded the 3-O-allyl-2,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate (5). Condensation of 5 with methyl 4,6-O-benzylidene-alpha-D-mannopyranoside (6) selectively afforded the (1-->3)-linked disaccharide 7. Benzoylation of 7, debenzylidenation, benzoylation, and deallylation gave methyl 2,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-2,4,6-tri-O-benzoyl-alpha-D-mannopyranoside (11) as the disaccharide acceptor. Coupling of 11 with (1-->2)-linked mannose disaccharide donor 17 or trisaccharide donor 21, followed by deacylation, furnished the target tetrasaccharide and pentasaccharide, respectively.     

Structural characterization of the O-specific polysaccharide from the lipopolysaccharide of the fish pathogen Aeromonas bestiarum strain P1S

The O-specific polysaccharide obtained by mild-acid degradation of lipopolysaccharide of Aeromonas bestiarum P1S was studied by sugar and methylation analyses along with ¹H and ¹³C NMR spectroscopy. The sequence of the sugar residues was determined using ¹H,¹H NOESY and ¹H,¹³C HMBC experiments. The O-specific polysaccharide was found to be a high-molecular-mass polysaccharide composed of tetrasaccharide repeating units of the structureSince small amounts of a terminal Quip3N residue were identified in methylation analysis, it was assumed that the elucidated structure also represented the biological repeating unit of the O-specific polysaccharide.     

The structure of the O-specific polysaccharide of Escherichia coli O117:K98:H4

The primary structure of the O-antigen of Escherichia coli O117 was shown by monosaccharide analysis, methylation analysis, and by 1D and 2D 1H and 13C NMR spectroscopy to be composed of linear pentasaccharide repeating units with the structure: -->3)-alpha-D-GalpNAc-(1-->4)-beta-D-GalpNAc-(1-->3)-alpha-L-Rhap- (1-->4)- alpha-D-Glcp-(1-->4)-beta-D-Galp-(1-->