Isolation and structural analysis of phosphorylated oligosaccharides obtained from Escherichia coli J-5 lipopolysaccharide.
The chemical structure of the phosphorylated lipopolysaccharide (LPS) of Escherichia coli J-5 was investigated because it is of biomedical interest in the context of septic shock, a syndrome often encountered in nosocomial infections with gram-negative pathogens. The successive de-O-acylation and de-N-acylation of J-5 LPS yielded phosphorylated oligosaccharides which represent the complete carbohydrate backbone. Five compounds were separated by high-performance anion-exchange chromatography and analysed by one-dimensional and two-dimensional homonuclear and heteronuclear 1H-NMR, 13C-NMR and 31P-NMR spectroscopy. The main product was a nonasaccharide of the structure alpha-D-Glcp-(1-->3)-[alpha-D-GlcpN- (1-->7)-alpha-L,D-Hepp-(1-->7)]-alpha-L,D-Hepp-(1-->3)-alpha -L, D-Hepp-4P-(1-->5)-[alpha-Kdop-(2-->4)]-alpha-Kdop-(2-- >6)-beta-D-GlcpN-4p- (1-->6)-alpha-D-GlcN-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-ulosonic acid, respectively. In addition, two octasaccharides and two heptasaccharides were isolated that were partial structures of the nonasaccharide. In one octasaccharide the terminal alpha-D-GlcpN was missing and an additional phosphate group linked to O4 of the branched heptose was present, whereas in the other octasaccharide the side-chain Kdo was missing. In both heptasaccharides the side-chain alpha-D-GlcpN-(1-->7)-L-alpha-D-Hepp-disaccharide was absent; they differed in their phosphate substitution. Whereas both 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.
Müller-Loennies
S
S
Department of Chemistry, Carlsberg Laboratory, Valby, Denmark. sml@fz-borstel.de
Holst
O
O
Lindner
B
B
Brade
H
H
D002240:Carbohydrate Sequence; D004926:Escherichia coli / Q000737:chemistry*; D008070:Lipopolysaccharides / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification*; D010766:Phosphorylation
C089614:J-5 lipopolysaccharide; D008070:Lipopolysaccharides; D009844:Oligosaccharides