Total Synthesis of the Complete Protective Antigen of Vibrio cholerae O139.

The first chemical synthesis of the complete protective O-antigen of a human-disease-causing pathogenic bacterium is described. The synthesis involved a protecting-group strategy that facilitated the regioselectivity of the key transformations, stereoselective glycosylation reactions, and enabled the one-step global deprotection of the completely assembled, fully protected, phosphorylated hexasaccharide by hydrogenation/hydrogenolysis. The final amino-group-functionalized, linker-equipped antigen was obtained in a form ready for conjugation to suitable carriers, for example, proteins, to yield immunogens.

Chemical Synthesis of the Galacturonic Acid Containing Pentasaccharide Antigen of the O-Specific Polysaccharide of Vibrio cholerae O139 and Its Five Fragments.

Three pentasaccharides, two tetrasaccharides, and a trisaccharide fragment of the O-specific antigen of Vibrio cholerae O139 were synthesized by applying 1 + 1, 2 + 1, 3 + 1, and 4 + 1 coupling strategies. The most challenging tasks involved were the synthesis of the 1,2-cis-glycosidic linkage between galactose and the linker (spacer) molecule and final purification of the target multicharged substances. Difficulties with final deprotection by hydrogenation/hydrogenolysis caused by the presence of galacturonic acid were overcome by protecting the acid with a group inert to the treatment with hydrogen. Some intermediates described previously as incompletely characterized amorphous materials were obtained in the crystalline condition and were fully characterized for the first time.

Synthesis of a Conjugation-Ready, Phosphorylated, Tetrasaccharide Fragment of the O-PS of Vibrio cholerae O139.

A new pathway to the tetrasaccharide α-Colp-(1→2)-4,6-P-ß-d-Galp-(1→3)-[α-Colp-(1→4)]-ß-d-GlcpNAc-1-(OCH2CH2)3NH2 has been developed. Glycosylation of 8-azido-3,6-dioxaoctyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-ß-d-glucopyranoside with 3,4,6-tri-O-acetyl-2-O-bromoacetyl-α-d-galactopyranosyl bromide afforded the ß-linked disaccharide. Debromoacetylation followed by reductive opening of the benzylidene acetal afforded the disaccharide diol acceptor. Halide-assisted glycosylation with 2,4-di-O-benzyl-α-colitosyl bromide gave the 1,2-cis-coupling product. Deacetylation followed by regioselective phosphorylation gave isomeric (R,S)-(P)-4(II),6(II)-cyclic phosphates, which were globally deprotected by one-step catalytic (Pd/C) hydrogenation/hydrogenolysis. The target tetrasaccharide, obtained in high overall yield, is amenable for conjugation to proteins.

Preparation and immunogenicity of conjugate based on hydrazine-treated lipopolysaccharide antigen of Vibrio cholerae O139.

A glycoconjugate construct was based on attachment of V. cholerae O139 hydrazine-treated lipopolysaccharide (LPS) to carboxylated bovine serum albumin (CBSA) via its amino group. The immunological properties of the glycoconjugate were tested using BALB/c mice, injected subcutaneously without any adjuvant three times at 2 weeks interval. The immunogenicity of the conjugate was estimated by enzyme-linked immunosorbent assay, testing of anti-LPS IgG, IgM, and IgA antibodies. The conjugate elicited a statistically significant increase of LPS-specific IgG levels in mice (p < 0.001). The specific anti-LPS IgG and IgA response after the second booster dose was significantly higher compared with reference and unconjugated detoxified LPS response. Antibodies elicited by the dLPS-CBSA conjugate were vibriocidal.

Synthesis of the conjugation ready, downstream disaccharide fragment of the O-PS of Vibrio cholerae O:139.

The linker-equipped disaccharide, 8-amino-3,6-dioxaoctyl 2,6-dideoxy-2-acetamido-3-O-ß-D-galactopyranosyluronate-ß-D-glucopyranoside (10), was synthesized in eight steps from acetobromogalactose and ethyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-1-thio-ß-D-glucopyranoside. The hydroxyl group present at C-4(II) in the last intermediate, 8-azido-3,6-dioxaoctyl 4-O-benzyl-6-bromo-2,6-dideoxy-2-trichloroacetamido-3-O-(benzyl 2,3-di-O-benzyl-ß-D-galactopyranosyluronate)-ß-D-glucopyranoside (9), is positioned to allow further build-up of the molecule and, eventually, construction of the complete hexasaccharide. Global deprotection (9→10) was done in one step by catalytic hydrogenolysis over palladium-on-charcoal.

Conformation of the hexasaccharide repeating subunit from the Vibrio cholerae O139 capsular polysaccharide.

In the past decade, several outbreaks of cholera have been reported to be caused by Vibrio cholerae O139, a strain which differs from the more common O1 strain in that the former is encapsulated. The hexasaccharide repeating subunit has been isolated from the V. cholerae O139 capsular polysaccharide by digestion with a recently discovered polysaccharide lyase derived from a bacteriophage specific for this serogroup. It specifically cleaves at a single position of the 4-linked galacturonic acid producing an unsaturated sugar product in quantities for conformational studies by (1)H and (13)C NMR spectroscopy. We report conformational studies on this oligosaccharide by molecular modeling and NMR spectroscopy including nuclear Overhauser effects and residual dipolar coupling of a sample weakly oriented in liquid crystalline solution. The structure contains a tetrasaccharide epitope homologous to the human Lewis(b) blood group antigen, which adopts a relatively well-defined single conformation. Comparison of these results with those of a previously published study of the intact capsular polysaccharide indicates that the conformations of the epitope in the two cases are identical or at least closely similar. Thus, this epitope, which may be essential for the pathogenicity of this V. cholerae strain, is not a "conformational epitope" requiring a certain critical size for antigenicity as has been reported for several other bacterial capsular antigens.