GENERAL STRATEGY FOR STRUCTURAL-ANALYSIS OF THE OLIGOSACCHARIDE REGION OF LIPOOLIGOSACCHARIDES - STRUCTURE OF THE OLIGOSACCHARIDE COMPONENT OF PSEUDOMONAS-AERUGINOSA IATS SEROTYPE-06 MUTANT R5 ROUGH-TYPE LIPOPOLYSACCHARIDE

A general NMR-based strategy for the structural analysis of rough-type lipopolysaccharides, i.e., lipooligosaccharides, is introduced that involves initial deacylation of the glycolipids. The approach is illustrated here with the lipooligosaccharide (LOS) of the Pseudomonas aeruginosa serotype 06 rough-type mutant R5, which consists of a single major low molecular weight component. The LOS was isolated by using a modified phenol/chloroform/petroleum ether extraction method. Chemical analysis of the core oligosaccharide obtained from this LOS indicated that it was composed of D-glucose (D-Glc), 2-amino-2-deoxy-D-galactose (D-GalN), L-glycero-D-manno-heptose (L,D-Hep), 3-deoxy-D-manno-octulosonic acid (KDO), L-alanine (Ala), and phosphate. The glycan structure of the LOS was elucidated by employing a novel strategy that involved the use of one- and two-dimensional nuclear magnetic resonance techniques and mass spectrometric based methods on the backbone oligosaccharide obtained from the LOS by deacylation, dephosphorylation, and reduction of the terminal glucosamine. The location of phosphomonoester groups was unambiguously established by a 2D H-1-P-31 chemical shift correlation experiments on an O-deacylated sample of the LOS (LOS-OH). The LOS-OH carries amide-linked 3-hydroxydodecanoic acid groups and Ala on the two D-glucosamine residues and the D-galactosamine residue, respectively.