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

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 rated by high-performance anion-exchange chromatography and gel-permeation chromatography, yielding four oligosaccharide phosphates two of which were readily identified by their H-1-NMR- and C-13-NMR spectra as alpha-Kdo-(2-4)-alpha-Kdo-(2-6)-beta-D-GlcpN-(1-6)-alpha-D-GlcpN 1,4'-bisphosphate (tetrasaccharide bisphosphate; Kdo = 3-deoxy-D-manno-octulopyranosonic acid) and alpha-Kdo-(2-8)-alpha-Kdo-(2-4)-alpha-Kdo-(2-6)-beta-D-GlcpN-(1-6)-alpha-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 alpha-Kdo-(2-4)-alpha-Kdo-(2-6)-beta-D-GlcpN-(1-6)-alpha-D-GlcpN 1-phosphate (tetrasaccharide 1-phosphate) and alpha-Kdo-(2-8)-alpha-Kdo-(2-4)-alpha-Kdo-(2-6)-beta-D-GlcpN-(1-6)-alpha-D-GlcpN 1-phosphate (pentasaccharide 1-phosphate). alpha-Kdo-(2-4)-alpha-Kdo-(2-6)-beta-D-GlcpN-(1-6)-alpha/beta-D-GlcpN 4'-phosphate (tetrasaccharide 4'-phosphate) and alpha-Kdo-(2-8)-alpha-Kdo-(2-4)-alpha-Kdo-(2-6)-beta-D-GlcpN-(1-6)-alpha/beta-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.