Isolation and structural analysis of phosphorylated oligosaccharides obtained from Escherichia coli J-5 lipopolysaccharide

The chemical structure of the phosphorylated lipopolysaccharide (LPS) of Es cherichia coli J-5 was investigated because it is of biomedical interest in the context of septic shock, a syndrome often encountered in nosocomial in fections with gram-negative pathogens. The successive de-O-acylation and de -N-acylation of J-5 LPS yielded phosphorylated oligosaccharides which repre sent the complete carbohydrate backbone. Five compounds were separated by h igh-performance anion-exchange chromatography and analysed by one-dimension al and two-dimensional homonuclear and heteronuclear H-1-NMR,C-13-NMR and P -31-NMR spectroscopy. The main product was a nonasaccharide of the structur e alpha-D-Glcp-(1-->3)-[alpha-D-GlcpN-(1-->7)-alpha-L,D-Hepp-( 1-->7)]-alph a-L,D-Hepp-(1--> 3)alpha-L,D-Hepp-4P-(1-->5)-[alpha-Kdop-(2-->4)]-alpha-Kdo p-(2--> 6)-alpha-D-GlcpN-1P wherein all sugars are present as D-pyranoses. Hep and Kdo represent L-glycero-D-manno-heptose and 3-deoxy-D-manno-oct-2-u losonic acid, respectively. In addition, two octasaccharides and two heptas accharides were isolated that were partial structures of the nonasaccharide . In one octasaccharide the terminal alpha-D-GlcpN was missing and an addit ional phosphate group linked to O4 of the branched heptose was present, whe reas in the other octasaccharide the sidechain Kdo was mis sing. In both he ptasaccharide s the side -chain alpha-D-GlcpN-(1-->7)-L-alpha-D-Hepp-disacc haride was absent; they differed in their phosphate substitution. Whereas b oth heptasaccharides contained two phosphates in the lipid-A backbone (beta -1,6-linked GlcpN-disaccharide at the reducing end) and one phosphate group at O4 of the first heptose, only one of them was additionally substituted with phosphate at O4 of the second heptose.