Laurence A. Mulard

Synthesis of methyl O-α-L-rhamnopyranosyl-(1 → 2)-α-d-galactopyranosides specifically deoxygenated at position 3, 4, or 6 of the galactose residue☆

Abstract The title disaccharides were synthesized by condensation of 2,3,4-tri- O -benzoyl-α- l -rhamnopyranosyl bromide with suitably protected, deoxygenated derivatives of methyl α- d -galactopyranoside. Deoxygenation was achieved via activation of a protected methyl α- d -gluco- or galacto-pyranoside with N, N′-thiocarbonyldiimidazole followed by treatment with tributyltin hydride and azobisisobutyronitrile. At position 3, the deoxygenation was more successful when performed with the tri- O -benzoylated precursor, rather than the tri- O -benzylated one. The corresponding nucleophile was obtained by benzylidenation of the methyl 3-deoxy-α- d -xylo-hexopyranoside. The preparation of the glycosyl acceptor deoxygenated at position 4 could be pursued starting from derivatives having either the d - galacto or the d - gluco configuration. The pathway involving the former was found superior.

Synthesis of specifically monofluorinated ligands related to the O-polysaccharide of Shigella dysenteriae type 1☆

The synthesis is reported of galactopyranose nucleophiles monofluorinated at positions 3, 4, or 6 and protected by 4,6-O-benzylidene, 3,6-di-O-benzyl, or 3,4-O-isopropylidene groups, respectively. The condensation of these nucleophiles with 2,3,4-tri-O-benzoyl-alpha-L-rhamnosyl bromide gave, after deprotection, the disaccharide analogues of methyl O-alpha-L-rhamnopyranosyl-(1-->2)-alpha-D-galactopyranoside, monofluorinated at position 3, 4, or 6 of the galactoside residue.

Binding of modified fragments of the Shigella dysenteriae type 1 O-specific polysaccharide to monoclonal IgM 3707 E9 and docking of the immunodeterminant to its modeled Fv.

The O-specific polysaccharide (O-SP) of Shigella dysenteriae type 1 has been shown by others to have the structure-->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alp ha-D- Galp-(1-->3)-alpha-D-GlcpNAc-(1-->. We have shown in the past that IgM 3707 E9, an anti S. dysenteriae type 1 O-SP monoclonal antibody, binds specifically to the -alpha-L-Rhap-(1-->2)-alpha-D-Galp-determinant of the polysaccharide. In this report we show that determinant to have hydrogen bonds, necessary for binding to the antibody, involving positions 3, 4 and 6 of the galactopyranosyl residue. The hydroxyl groups of the rhamnopyranosyl moiety of the immunodeterminant appear not to partake in hydrogen-bond interactions with the antibody. A model is presented of the Fv of IgM 3707 E9 based on our previously established cDNA-sequence and two known, highly homologous immunoglobulin crystal structures. The methyl glycoside of the immunodeterminant alpha-L-rhamnopyranosyl-(1-->2)-alpha-D-galactopyranose is docked to the combining area of the Fv.

Synthesis of tri- and tetrasaccharide fragments of the Shigella dysenteriae type 1 O-antigen deoxygenated and fluorinated at position 3 of the methyl alpha-D-galactopyranoside terminus.

The blockwise synthesis of methyl alpha tri- and tetrasaccharide analogs of the biochemical repeating unit of the Shigella dysenteriae type 1 O-polysaccharide is described. Modifications include deoxygenation and deoxyfluorination at position 3 of the galactopyranoside residue. Methyl 4,6-O-benzylidene-3-deoxy-alpha-D-xylo-hexopyranoside (8) and methyl 4,6-O-benzylidene-3-deoxy-3-fluoro-alpha-D-galactopyranoside (9) were condensed with (2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl)-(1-->3) -2,4-di-O-benzoyl-alpha-L-rhamnopyranosyl chloride to give, after deprotection, the target trisaccharide methyl alpha-L-rhamnopyranosyl-(1-->3)-alpha-L- rhamnopyranosyl-(1-->2)-3-deoxy-alpha-D-xylo-hexopyranoside and the corresponding fluorinated oligosaccharide. For the tetrasaccharide synthesis, the glycosyl acceptors 8 and 9 were condensed with the temporarily protected (2,4-di-O-benzoyl-3-O-chloroacetyl-alpha-L- rhamnopyranosyl)-(1-->3)-2,4-di-O-benzoyl-alpha-L-rhamnopyranosyl chloride. Removal of the chloroacetyl group was followed by condensation of the resulting selectively deblocked trisaccharides with 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha-D-glucopyranosyl chloride. Reduction and deprotection then gave the free methyl 2-acetamido-2-deoxy- alpha-D-glucopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl- (1-->3)-alpha-L-rhamnopyranosyl-(1-->2)-3-deoxy-alpha-D-xylo-hexopyra noside and the fluorinated analog.

Investigation by NMR Spectroscopy and Molecular Modeling of the Conformations of Some Modified Disaccharide Antigens For Shigella Dysenteriae Type 1

Abstract The O-polysaccharide of Shigella dysenteriae type 1 is made up of multiple repeats of the linear tetrasaccharide 3)-α-L-Rhap-(1→2)-α-D-Galp-(1→3)-α-D-GlcpNAc-(1→3)-α-L-Rhap-(1→, for which the antigenic determinant for a murine monoclonal IgM antibody is the disaccharide α-L-Rhap-(1→2)-α-D-Galp. This disaccharide and various analogs have been studied by 2D NOESY, ROESY, and TOCSY NMR spectroscopy, in conjunction with proton spin-lattice relaxation rate measurements, restrained molecular mechanics, and restrained molecular dynamics with simulated annealing. It has been found that replacement of any single hydroxyl group in the determinant by a hydrogen atom, or replacement of any single hydroxyl group in the Gal residue by a fluorine atom has little if any influence on the conformation of the resulting derivatives. cPresent address: Institut Pasteur, Paris, France

SYNTHESIS OF SPECIFICALLY DEOXYGENATED DISACCHARIDE DERIVATIVES OF THE SHIGELLA DYSENTERIAE TYPE 1 O-ANTIGEN

The synthesis of methyl O-alpha-L-rhamnopyranosyl-(1-->2)-alpha-D-galactopyranosides specifically deoxygenated at position 2 (31), or 4 (21) of the rhamnopyranosyl residue was accomplished using methyl 3,4,6-tri-O-benzoyl-alpha-D-galactopyranoside (18) as the glycosyl acceptor. Phenyl thionocarbonate activation of the penta-O-benzoylated disaccharide precursor followed by Barton reduction and Zemplén transesterification gave 31, while 21 was obtained via condensation of the deoxygenated monosaccharide donor with 18, and subsequent debenzoylation of the product.