THE STRUCTURE OF THE CARBOHYDRATE BACKBONE OF THE CORE-LIPID-A REGIONOF THE LIPOPOLYSACCHARIDE FROM VIBRIO-CHOLERAE STRAIN H11 (NON-O1)

Lipopolysaccharide from Vibrio cholerae strain H11 (non-O1) was de-O-a cylated, dephosphorylated, reduced, de-N-acylated, N-acetylated, and t he products were separated by high-performance anion-exchange chromato graphy (HPAE). A decasaccharide, 1, was isolated as the major product, representing the core oligosaccharide attached to the reduced GlcN-di saccharide lipid A backbone. Its structure was established by composit ional and methylation analyses, and extensive NMR investigations inclu ding H-1,H-1 correlation spectroscopy (COSY), total correlation spectr oscopy (TOCSY), and nuclear Overhauser enhancement spectroscopy (NOESY ), as well as heteronuclear C-13, H-1 COSY. In another reaction sequen ce the lipopolysaccharide was hydrolysed with dilute acetic acid and r educed with NaBH4. The resulting core fractions were separated by HPAE giving seven individual octasaccharides differing at the reducing 3-d eoxy-D-manno-octulosonic acid (Kdo) residue. A major product, 2, was i solated and investigated by the same methods as described for the deca saccharide 1. The following structures are proposed for compounds 1 an d 2: -(1-4)-]-[alpha-D-Glcp-(1-6)-]-alpha-Hepp-(1-5)-R, where R is alp ha-Kdop-(2-6)-beta-D-GlcNAcp-(1-6)-D-GlcNAcol in 1 and 4,8-anhydro-Kdo ol in 2, and Hep is L-glycero-D-manno-heptose. In lipopolysaccharide, the terminal residue of alpha-D-glucosamine possessed a free amino gro up, as proved by deamination with nitrous acid and the H-1-NMR spectru m of de-O-acylated lipopolysaccharide. The conformational preferences of the terminal core heptasaccharide was assessed by Monte Carlo simul ations combined with restrained calculations of side chains based on e xperimentally determined proton-coupling constants. These calculations , confirmed by NOE data, displayed several long-range interactions, wh ich resulted in a well-defined three-dimensional structure of the core oligosaccharide.