The structure of the carbohydrate backbone of the core-lipid-A region of the lipopolysaccharide from Vibrio cholerae strain H11 (non-O1).

Abstract

Lipopolysaccharide from Vibrio cholerae strain H11 (non-O1) was de-O-acylated, dephosphorylated, reduced, de-N-acylated, N-acetylated, and the products were separated by high-performance anion-exchange chromatography (HPAE). A decasaccharide, 1, was isolated as the major product, representing the core oligosaccharide attached to the reduced GlcN-disaccharide lipid A backbone. Its structure was established by compositional and methylation analyses, and extensive NMR investigations including 1H,1H correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), and nuclear Overhauser enhancement spectroscopy (NOESY), as well as heteronuclear 13C,1H COSY. In another reaction sequence the lipopolysaccharide was hydrolysed with dilute acetic acid and reduced with NaBH4. The resulting core fractions were separated by HPAE giving seven individual octasaccharides differing at the reducing 3-deoxy-D-manno-octulosonic acid (Kdo) residue. A major product, 2, was isolated and investigated by the same methods as described for the decasaccharide 1. The following structures are proposed for compounds 1 and 2: alpha-D-GlcNAcp-(1-7)-[beta-D-Galp-(1-3)-]-alpha-Hepp-(1-2)- alpha-Hepp- (1-3)-[beta-D-Glcp-(1-4)-]- [alpha-D-Glcp-(1-6)-]-alpha-Hepp-(1-5)-R, where R is alpha-Kdop-(2-6)-beta-D-GlcNAcp-(1-6)-D-GlcNAcol in 1 and 4,8-anhydro-Kdool in 2, and Hep is L-glycero-D-manno-heptose. In lipopolysaccharide, the terminal residue of alpha-D-glucosamine possessed a free amino group, as proved by deamination with nitrous acid and the 1H-NMR spectrum of de-O-acylated lipopolysaccharide. The conformational preferences of the terminal core heptasaccharide was assessed by Monte Carlo simulations combined with restrained calculations of side chains based on experimentally determined proton-coupling constants. These calculations, confirmed by NOE data, displayed several long-range interactions, which resulted in a well-defined three-dimensional structure of the core oligosaccharide.