Identification of 2-keto-3-deoxy-1,7-dicarboxyheptonic acid as a constituent of the lipopolysaccharide of Acinetobacter calcoaceticus NCTC 10305.

Abstract

A 3-deoxyaldulosonic acid, not previously described, was identified as a component of the lipopolysaccharide of Acinetobacter calcoaceticus NCTC 10305: 2-keto-3-deoxy-1,7-dicarboxyheptonic acid. This compound, whose steric configuration has not been determined, was released from lipopolysaccharide after acid hydrolysis as monosaccharide and in the form of a glucose-substituted disaccharide. It was identified by combined gas-liquid chromatography/mass spectrometry using various derivatization procedures. The presence of a 2-keto-3-deoxyaldulosonic acid was established from the typical fragmentation pattern of the C1-C6 moiety which was identical to that of similarly derivatized authentic 2-keto-3-deoxy-D-mannooctonic acid (dOclA). Location of the keto and deoxy function at C2 and C3, respectively, was deduced from the fragmentation pattern of the deuterolabelled derivatives after reduction of the keto and carboxy groups. The presence of two carboxy groups was ascertained after carboxy reduction with sodium boro-(2H)hydride of the carbonyl-reduced and permethylated derivative whereby the molecular masses were found to differ by 4 Da. Further proof for the presence of two carboxy groups was obtained by alkaline transesterification of the carbonyl-reduced and permethylated derivative with ethanolic sodium ethylate: it was shown that two methyl ester groups had been transesterified since the molecular mass was shifted higher by 28 Da. Substitution by a D-glucopyranosyl residue in position 4 was established by methylation analysis performed on the carbonyl- and carboxyl-reduced and permethylated disaccharide. The D-configuration of the glucosyl residue was determined by its reactivity with D-glucose oxidase. Thus, the structure of the disaccharide is 4-O-glucopyranosyl-2-keto-3-deoxy-1,7-dicarboxyheptonic acid.