Position of ester groups in the lipid A backbone of lipopolysaccharides obtained from Salmonella typhimurium

Abstract

Lipopolysaccharides extracted from the heptoseless mutant of Salmonella typhimurium G30/C21 were hydrolyzed with either 0.1 N HCl at 100 degrees C or treated twice with 20 mM sodium acetate, pH 4.5, at 100 degrees C for 45 min and finally purified by preparative thin layer chromatography to yield a structural series of mono- and diphosphoryl lipid A, respectively. Positive ion fast atom bombardment mass spectrometry of the diphosphoryl lipid A TLC-3 (a highly acylated major band) showed a major component with (M + H)+ ion of mass 1798, which fragmented to yield a (M - H2PO4)+ ion of mass 1700. Cleavage at the glycosidic bond gave rise to an oxonium ion fragment of mass 1087. In conjunction with other studies, this establishes the molecular formula and Mr of the major component to be C94H178N2O25P2 and 1797.2 (as the free acid), respectively. Similar analysis of monophosphoryl lipid A TLC-3 produced an (M + H)+ peak at m/z 1718, (M + Na)+ adduct peak at m/z 1740, and a fragment of mass 1087. The spectrum of monophosphoryl lipid A TLC-5 was devoid of the m/z 1087 peak and instead contained the phosphorylated oxonium ion of mass 876. This fragment ion is assigned as the distal subunit, and these results show that the distal subunit of the major lipid A TLC-3 contains two hydroxymyristoyl, one myristoyl, and one lauroyl residues, whereas the reducing end subunit contains two hydroxymyristoyl groups. A revised structure of the lipid A backbone in lipopolysaccharides of S. typhimurium is proposed.