Chemical structure of lipid A isolated from Flavobacterium meningosepticum lipopolysaccharide

Abstract

The chemical structure of the lipid A of the lipopolysaccharide component isolated from Flavobacterium meningosepticum IFO 12535 was elucidated. Methylation and nuclear magnetic resonance analyses showed that two kinds of hydrophilic backbone exist in the free lipid A: a beta (1-->6)-linked 2-amino-2-deoxy-D-glucose, which is usually present in enterobacterial lipid A's, and a 2-amino-6-O-(2, 3-diamino-2,3-dideoxy-beta-D-glucopyranosyl)-2-deoxy-D-glucose, in a molar ratio of 1.00:0.35. Both backbones were alpha-glycosidically phosphorylated in position 1, and the hydroxyl groups at positions 4, 4', and 6' were unsubstituted. Liquid secondary ion-mass spectrometry revealed a pseudomolecular ion at m/z 1673 [M-H]- as a major monophosphoryl lipid A component carrying five acyl groups. Fatty acid analysis showed that the lipid A contained 1 mol each of amide-linked (R)-3-OH iC17:0, ester-linked (R)-3-OH iC15:0, amide-linked (R)-3-O-(iC15:0)-iC17:0, and both amide- and ester-linked (R)-3-OH C16:0. Fatty acid distribution analyses using several mass spectrometry determinations demonstrated that the former two constituents were distributed on positions 2 and 3 of the reducing terminal unit of the backbones and that the latter two were attached to the 2' and 3' positions in the nonreducing terminal residue.

FIG. 1

EI-mass spectrum and fragmentation pattern of the peracetylated derivative of GlcN3N found in F. meningosepticum lipid A.

FIG. 2

EI-mass spectrum and fragmentation pattern of methyl ester derivatives of (R)-3-O-(13-methyltetradecanoyl)-15-methylhexadecanoic acid found in F. meningosepticum lipid A.

FIG. 3

EI-mass spectrum and fragmentation pattern of the permethylated 2-acetamido-6-O-(2,3-diacetamido-2,3-dideoxy-d-glucopyranosyl)-2-deoxy-d-glucitol derivative originating from the hybrid backbone present in F. meningosepticum lipid A.

FIG. 4

LSI-mass spectrum of F. meningosepticum lipid A in the negative-ion mode. A mixture of triethanolamine and 3-nitrobenzyl alcohol (1:1 [vol/vol]) containing a small amount of Kryptofix 222 was used as the matrix.

FIG. 5

LSI-mass spectrum of F. meningosepticum de-O-acylated lipid A in the negative-ion mode. A mixture of triethanolamine and 3-nitrobenzyl alcohol (1:1 [vol/vol]) containing a small amount of Kryptofix 222 was used as the matrix.

FIG. 6

Negative-ion mode FAB-MS/MS of the molecular ion at m/z 1209 [M-H]⁻ detected in the LSI-mass spectrum of F. meningosepticum de-O-acylated lipid A.

FIG. 7

Proposed structure of F. meningosepticum lipid A.