V. I. Torgov

Synthesis of O-β-d-mannopyranosyl-(1→4)-O-α-l-rhamnopyranosyl-(1→3)-d-galactopyranose, the trisaccharide repeating-unit of the O-specific polysaccharide from Salmonella anatum

Abstract Glycosylation of 1,2:5,6-di- O -isopropylidene-α- d -galactofuranose with 2,3-di- O -acetyl-4- O -(2,3,4,6-tetra- O -acetyl-β- d -mannopyranosyl)-α- l -rhamnopyranosyl bromide, followed by removal of the protecting groups, gave O -β- d -mannopyranosyl-(1→4)- O -α- l -rhamnopyranosyl-(1→3)- d -galactose, which is the trisaccharide repeating-unit of the O-specific polysaccharide chain of the lipopolysaccharide from Salmonella anatum . The formation of the β- d -mannopyranosyl linkage was achieved by a glucose-mannose conversion via stereoselective reduction of the corresponding oxo-disaccharide.

Biochemical characterization of WbdN, a β1,3-glucosyltransferase involved in O-antigen synthesis in enterohemorrhagic Escherichia coli O157

The enterohemorrhagic O157 strain of Escherichia coli, which is one of the most well-known bacterial pathogens, has an O-antigen repeating unit structure with the sequence [-2-d-Rha4NAcα1-3-l-Fucα1-4-d-Glcβ1-3-d-GalNAcα1-]. The O-antigen gene cluster of E. coli O157 contains the genes responsible for the assembly of this repeating unit and includes wbdN. In spite of cloning many O-antigen genes, biochemical characterization has been done on very few enzymes involved in O-antigen synthesis. In this work, we expressed the wbdN gene in E. coli BL21, and the His-tagged protein was purified. WbdN activity was characterized using the donor substrate UDP-[(14)C]Glc and the synthetic acceptor substrate GalNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. The enzyme product was isolated by high pressure liquid chromatography, and mass spectrometry showed that one Glc residue was transferred to the acceptor by WbdN. Nuclear magnetic resonance analysis of the product structure indicated that Glc was β1-3 linked to GalNAc. WbdN contains a conserved DxD motif and requires divalent metal ions for full activity. WbdN activity has an optimal pH between 7 and 8 and is highly specific for UDP-Glc as the donor substrate. GalNAcα derivatives lacking the diphosphate group were inactive as substrates, and the enzyme did not transfer Glc to GlcNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. Our results illustrate that WbdN is a specific UDP-Glc:GalNAcα-diphosphate-lipid β1,3-Glc-transferase. The enzyme is a target for the development of inhibitors to block O157-antigen synthesis.

Synthesis of branched arabinofuranose pentasaccharide fragment of mycobacterial arabinans as 2-azidoethyl glycoside.

Branched arabinofuranose pentasaccharide with 2-azidoethyl aglycon was prepared for the first time by [3+1+1] bis-(1,2-cis)-glycosylation of trisaccharide diol with silyl-protected thioglycoside glycosyl donor. The presence of 2-azidoethyl aglycon would enable the preparation of neoglycoconjugates using the click chemistry approaches.

Synthesis of the tetrasaccharide repeating-unit of the O-specific polysaccharide from Salmonella senftenberg

Abstract The oligosaccharide β- d -Man-(1 → 4)-α- l -Rha (1 → 3)- d -Gal-(6 ← 1)-α- d -Glc, which is the repeating unit of the O-specific polysaccharide chain of the lipopolysaccharide from Salmonella senftenberg , was obtained by glycosylation of benzyl 2,4-di- O -benzyl-6- O -(2,3,4-tri- O -benzyl-6- O - p -nitrobenzoyl-α- d -glucopyranosyl)-β- d -galactopyranoside or benzyl 2- O -acetyl-6- O -(2,3,4-tri- O -benzyl-6- O - p -nitrobenzoyl-α- d -glucopyranosyl)-β- d -galactopyranoside with 3- O -acetyl-4- O -(2,3,4,6-tetra- O -acetyl-β- d -mannopyranosyl)-β- l -rhamnopyranose 1,2-(methyl orthoacetate) followed by removal of protecting groups.

Biochemical characterization of the novel α-1, 3-galactosyltransferase WclR from Escherichia coli O3

Glycosyltransferases (GTs) catalyze the formation of regio- and stereo-specific glycosidic linkages between specific sugar donors and recipients. In this study, the function of the gene wclR from the Escherichia coli O3 O-antigen gene cluster that encodes an α 1, 3-galactosyltransferase (GalT) that acts on the linkage Gal α 1, 3-GlcNAc was biochemically characterized. WclR was expressed in E. coli BL21 (DE3), and the enzymatic product was identified by liquid chromatography-mass spectrometry (LC-MS), collision-induced dissociation electrospray ionization ion trap multiple tandem MS (CID-ESI-IT-MS(n)) and galactosidase digestion, using UDP-Gal as the donor substrate and the synthetic acceptor substrate GlcNAc-PP-De (decyl diphosphate N-acetylglucosamine). The physiochemical properties and the substrate specificity of WclR were investigated. WclR is the first bacterial GalT characterized that acts on the linkage Gal α 1, 3-GlcNAc. This study enhanced our knowledge of the diversified functions of GTs and provided a novel enzyme source for possible pharmaceutical application.

11-Phenoxyundecyl phosphate as a 2-acetamido-2-deoxy-α-d-glucopyranosyl phosphate acceptor in O-antigen repeating unit assembly of Salmonella arizonae O:59 ☆

A synthesis of 11-phenoxyundecyl phosphate and its biochemical transformation (using GlcNAc-P transferase from Salmonella arizonae O:59 membranes catalysing transfer of GlcNc-phosphate from UDP-GlcNAc on lipid-phosphate) into P(1)-11-phenoxyundecyl, P(2)-2-acetamido-2-deoxy-α-D-glucopyranosyl diphosphate are described.

Nuclear overhauser effects and conformations of branched trisaccharide methyl β-glycosides that contain a 2,3-disubstituted galactose residue

On the basis of the n.O.e. data and theoretical calculations, it was found that less than 90% of one conformer was present in aqueous solution for each of a series of trisaccharide methyl beta-glycosides with a 2,3-disubstituted galactose residue.

Synthesis of 3,6-di-O-methyl-β-d-glucopyranose conjugates

Abstract3,6-Di-O-methyl-β-d-glucopyranose neoglycoconjugates with bovine serum albumine and poly(acrylamide) carrier were obtained, which differ in the aglycon nature, linking pattern, and substitution degree.

Synthesis of glycosyl phosphates from acetylated glycosyl nitrates

Abstract Acetylated α-glycosyl nitrates were efficiently converted under mild conditions into protected β-glycosyl phosphates by treatment with cesium dibenzyl phosphate or into thermodynamically more stable α-glycosyl phosphate derivatives upon interaction with cesium diphenyl phosphate. These reactions were found to be applicable both to 2-azido-2,6-dideoxy- and 2-azido-2-deoxygalactopyranosyl nitrates as well as to 6-deoxygalactopyranosyl and galactopyranosyl derivatives.

Synthesis of a disaccharide of phenolic glycolipid from Mycobacterium leprae (PGL-I) and its conjugates with bovine serum albumin

Terminal disaccharide fragment of phenolic glycolipid from Mycobacterium leprae (PGL-I) was synthesized as a glycoside with 4-(2-aminoethoxy)phenyl aglycon. The obtained 4-(2-aminoethoxy)phenyl 4-O-(3,6-di-O-methyl-β-d-glucopyranosyl)-2,3-di-O-methyl-α-l-rhamnopyranoside was used for the synthesis of a series of neoglycoconjugates with bovine serum albumin with different degrees of substitution.