GENERAL STRATEGY FOR STRUCTURAL-ANALYSIS OF THE OLIGOSACCHARIDE REGION OF LIPOOLIGOSACCHARIDES - STRUCTURE OF THE OLIGOSACCHARIDE COMPONENTOF 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 i nvolves initial deacylation of the glycolipids. The approach is illust rated here with the lipooligosaccharide (LOS) of the Pseudomonas aerug inosa serotype 06 rough-type mutant R5, which consists of a single maj or low molecular weight component. The LOS was isolated by using a mod ified phenol/chloroform/petroleum ether extraction method. Chemical an alysis of the core oligosaccharide obtained from this LOS indicated th at 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-octuloso nic acid (KDO), L-alanine (Ala), and phosphate. The glycan structure o f the LOS was elucidated by employing a novel strategy that involved t he use of one- and two-dimensional nuclear magnetic resonance techniqu es and mass spectrometric based methods on the backbone oligosaccharid e obtained from the LOS by deacylation, dephosphorylation, and reducti on of the terminal glucosamine. The location of phosphomonoester group s was unambiguously established by a 2D H-1-P-31 chemical shift correl ation experiments on an O-deacylated sample of the LOS (LOS-OH). The L OS-OH carries amide-linked 3-hydroxydodecanoic acid groups and Ala on the two D-glucosamine residues and the D-galactosamine residue, respec tively.