Structural investigation of the lipopolysaccharide from Proteus mirabilis R45 (Re-chemotype)

Abstract

Five phosphorylated saccharides were isolated from the deacylated then N-acetylated lipopolysaccharide (LPS) of Proteus mirabilis R45 (Re-chemotype) by anion-exchange chromatography. Their structures were determined by 1- and 2-dimensional ¹H- and ³C-nuclear magnetic resonance spectroscopy as α-Kdo-(2-4)-[β-L-ArapNAc-(1-8)-]-α-Kdo-(2-6)-β-D-GIc pNAc-(1-6)-α-D-GIcpNAc 1-phosphate (1), α-Kdo-(2-4)-[β-L-Ara pNAc-(1-8)-]-α-Kdo-(2-6)-β-D-GlcpNAc-(1-6)-α-D-Glc pNAc 1,4'-bisphosphate (2), α-Kdo-(2-4)-α-Kdo-(2-6)-β-D-Glc pNAc-(1-6)-α-D-GlcpNAc 1-phosphate (3), α-Kdo-(2-4)-α-Kdo-(2-6)-β-D-Glc pNAc-(1-6)-α-D-GlcpNAc 1,4'-bisphosphate (4), and β-L-Ara pNAc 1-phosphate (5) (ArapNAc, 4-acetamido-4-deoxy-L-arabinopyranose; Kdo, 3-deoxy-D-manno-octulopyranosonic acid). Compounds 1-5 were obtained from deacylated LPS in a molar ratio of ~1.1:0.8:1.1:1.0:0.6 as determined by high-performance anion-exchange chromatography. Although we cannot exclude that in the deacylated LPS (without N-acetylation) the molar ratio may be different, the result can be taken as indication that in LPS about 50% of the 4'-phosphates of lipid A are substituted by (β-L-AraρN. Furthermore, our data prove that the first Kdo in the carbohydrate chain is partially substituted by a second residue of ArapN.

The de-O-acylated LPS was additionally investigated using fast atom bombardment and B/E linked scan mass spectrometry which confirmed the above findings and also identified minor amounts of another LPS species possessing a third pentosamine residue.