PREPARATION AND STRUCTURAL-ANALYSIS OF OLIGOSACCHARIDE MONOPHOSPHATESOBTAINED 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 lipopolysa ccharide [Holst, O., Brade, L., Kosma, P. and Brade, H. (1991) J. Bact eriol. 173, 1862-1866] was sequentially de-O- and de-N-acylated by mil d hydrazinolysis and treatment with 4 M KOH, respectively. The resulti ng mixture of compounds was separated rated by high-performance anion- exchange chromatography and gel-permeation chromatography, yielding fo ur oligosaccharide phosphates two of which were readily identified by their H-1-NMR- and C-13-NMR spectra as )-alpha-Kdo-(2-6)-beta-D-GlcpN- (1-6)-alpha-D-GlcpN 1,4'-bisphosphate (tetrasaccharide bisphosphate; K do = 3-deoxy-D-manno-octulopyranosonic acid) and )-alpha-Kdo-(2-6)-bet a-D-GlcpN-(1-6)-alpha-D-GlcpN 1,4'-bisphosphate (pentasaccharide bisph osphate) [Holst, O., Broer, W., Thomas-Oates, J. E., Mamat, U. and Bra de, H. (1993) Eur J. Biochem. 214, 703-710]. The structures of the oth er two compounds were established by chemical analysis, NMR spectrosco py, and fast-atom-bombardment mass spectrometry as )-alpha-Kdo-(2-6)-b eta-D-GlcpN-(1-6)-alpha-D-GlcpN 1-phosphate (tetrasaccharide 1-phospha te) and )-alpha-Kdo-(2-6)-beta-D-GlcpN-(1-6)-alpha-D-GlcpN 1-phosphate (pentasaccharide 1-phosphate). ha-Kdo-(2-6)-beta-D-GlcpN-(1-6)-alpha/ beta-D-GlcpN 4'-phosphate (tetrasaccharide 4'-phosphate) and ha-Kdo-(2 -6)-beta-D-GlcpN-(1-6)-alpha/beta-D-GlcpN 4'-phosphate (pentasaccharid e 4'-phosphate) were prepared from the 1,4'-bisphosphates isolated fro m the recombinant strain Escherichia coil F515-207 by treatment with a lkaline phosphatase and purification by high-performance anion-exchang e chromatography and gel-permeation chromatography. Their structures w ere characterised by chemical analysis, NMR spectroscopy, and fast-ato m-bombardment mass spectrometry.