Determination of the structure and absolute configuration of the glycerolphosphate-containing capsular polysaccharide of Pasteurellahaemolytica serotype T3 by high-resolution nuclear magnetic resonance spectroscopy

The specific capsular polysaccharide antigen produced by Pasteurellahaemolytica serotype T3 was found to be a high molecular weight teichoic acid composed of equal molar amounts of D-galactose, glycerol, and phosphate and a nonstoichiometric amount of O-acetate. Structural analysis of the polymer employed a combination of one- and two-dimensional 1H and 13C nuclear magnetic resonance methods at high field. Homo- and heteronuclear chemical shift correlation techniques, together with J-resolved and nuclear Overhauser effect (nOe) difference experiments led to the unambiguous assignments of the 1H and 13C resonances corresponding to the D-galactose and glycerol structural units and established that the repeating unit of the polysaccharide antigen was composed of → 4)-α-D-Galp-(1 → 1)-L-glycerol-(3 → units joined by phosphate diester groups. The chirality of the glycerol moiety was established by conformational analysis of a 1-O-α-D-galactopyranosyl-S-glycerol fragment (2) obtained from the teichoic acid by depolymerization with HF, from which the stereochemistry of the glycerol methine carbon centre was related to that of the D-glycose from 1H–1H nOe and 3J values. The chirality was confirmed by gas–liquid chromatographic and mass spectral analysis of the (−)-camphanate ester of 2,3-di-O-methylglycerol derived from permethylated 2. The D-galactosyl residues are partially mono-O-acetylated at C-2 and C-3. Keywords: Pasteurellahaemolytica, capsular polysaccharide, NMR analysis.