Preparation and structural analysis of oligosaccharide monophosphates obtained from the lipopolysaccharide of recombinant strains of Salmonella minnesota and Escherichia coli expressing the genus‐specific epitope of Chlamydia lipopolysaccharide

Abstract

The lipopolysaccharide of the recombinant strain Salmonella minnesota r595–207 expressing the genus‐specific epitope of Chlamydia lipopolysaccharide [Holst, O., Brade, L., Kosma, P. and Brade, H. (1991) J. Bacteriol. 173, 1862–1866] was sequentially de‐O‐ and de‐N‐acylated by mild hydrazinolysis and treatment with 4 M KOH, respectively. The resulting mixture of compounds was separated by high‐performance anion‐exchange chromatography and gel‐permeation chromatography, yielding four oligosaccharide phosphates two of which were readily identified by their 1H‐NMR‐ and 13C‐NMR spectra as α‐Kdo‐(2–4)‐α‐Kdo‐(2–6)‐β‐D‐GlcpN‐(1–6)‐α‐D‐GlcpN 1,4′‐bisphosphate (tetrasaccharide bisphosphate; Kdo = 3‐deoxy‐D‐manno‐octulopyranosonic acid) and α‐Kdo‐(2–8)‐α‐Kdo‐(2–4)‐α‐Kdo‐(2–6)‐β‐D‐GlcpN‐(1–6)‐α‐D‐GlcpN 1,4′‐bisphosphate (pentasaccharide bisphosphate) [Holst, O., Broer, W., Thomas‐Oates, J. E., Mamat, U. and Brade, H. (1993) Eur. J. Biochem. 214, 703–710]. The structures of the other two compounds were established by chemical analysis, NMR spectroscopy, and fast‐atom‐bombardment mass spectrometry as α‐Kdo‐(2–4)‐α‐Kdo‐(2–6)‐β‐D‐GlcpN‐(1–6)‐α‐D‐GlcpN 1‐phosphate (tetrasaccharide 1‐phosphate) and α‐Kdo‐(2–8)‐α‐Kdo‐(2–4)‐α‐Kdo‐(2–6)‐β‐D‐GlcpN‐(1–6)‐α‐D‐GlcpN 1‐phosphate (pentasaccharide 1‐phosphate). α‐Kdo‐(2–4)‐α‐Kdo‐(2–6)‐β‐D‐GlcpN‐(1–6)‐α/β‐D‐GlcpN 4′‐phosphate (pentasaccharide 4′‐phosphate) and α‐Kdo‐(2–8)‐α‐Kdo‐(2–4)‐α‐Kdo‐(2–6)‐β‐D‐GlcpN‐(1–6)‐α/β‐D‐GlcpN 4′‐phosphate (pentasaccharide 4′‐phosphate) were prepared from the 1,4′‐bisphosphates isolated from the recombinant strain Escherichia coli F515–207 by treatment with alkaline phosphatase and purification by high‐performance anion‐exchange chromatography and gel‐permeation chromatography. Their structures were characterised by chemical analysis, NMR spectroscopy, and fast‐atom‐bombardment mass spectrometry. Copyright © 1994, Wiley Blackwell. All rights reserved