Methylation analysis of the heptose/3-deoxy- d- manno-2-octulosonic acid region (inner core) of the lipopolysaccharide from Salmonella minnesota rough mutants

Abstract

A modified methylation analysis is described which allows the elucidation of the structure of the inner core region [heptose/3-deoxy- d- manno-2-octulosonic acid (KDO)] of enterobacterial lipopolysaccharides (LPS) of Salmonella minnesota rough mutants (Re, strain R595; and Rd 2P −, strain R4). Methylation, carboxyl-reduction, remethylation, hydrolysis, carbonyl-reduction, and acetylation of the Re-mutant LPS yielded the 2,6-di- O-acetyl and 2,4,6-tri- O-acetyl derivatives of partially methylated 3-deoxyoctitol in equimolar amounts, indicating the presence of a terminal and a 4-linked pyranosidic KDO residue. For Rd 2P − LPS, the hydrolysis step involved 0.1 m trifluoroacetic acid at 100° for 1 h which cleaved ketosidic linkages, and the final products included the foregoing acetyl derivatives in the molar ratio of 1:0.2 and a partially methylated and acetylated 3-deoxyoctitol derivative which was substituted at O-5 by a methylated heptopyranosyl residue. Trideuteriomethylation of the latter product followed by methanolysis and acetylation gave the 5- O-acetyl-3-deoxyl-1,7,8-tri- O-methyl-2,4,6-tri- O-trideuteriomethyl- d- glycero- d- talo/galacto-octitol and 1,5-di- O-acetyl-2,3,4,6,7-penta- O-methyl- l- glycero- d- manno-heptitol. These results prove the presence of a (1→4)-linked KDO disaccharide in Re LPS and show that the core region of Rd 2P − LPS contains a terminal α- l- glycero- d- manno-heptopyranosyl group and a non-substituted, a 4- O-, and a 4,5-di- O-substituted pyranosidic KDO residue in the molar ratios 1:1:0:2:1.