DETERMINATION BY HETERONUCLEAR NMR-SPECTROSCOPY OF THE COMPLETE STRUCTURE OF THE CELL-WALL POLYSACCHARIDE OF STREPTOCOCCUS-SANGUIS STRAIN-K103

Although complete structures of complex polysaccharides have traditionally been determined by chemical degradative methods, a number of recent developments in instrumentation have greatly facilitated this task. We illustrate the application of several of these methods in a determination of the complete covalent structure of the polysaccharide from Streptococcus sanguis K103, which is composed of an octasaccharide repeating subunit linked by phosphodiester bonds. Carbohydrate analysis by HPAE-PAD and by reverse-phase chromatography of benzoylated derivatives of the hydrolysis products of the polysaccharide gave glucose (3 mol), galactose (1 mol), rhamnose (2 mol), N-acetylglucosamine (1 mol), and galactose 6-phosphate (1 mol). Circular dichroism of the O-benzoylated monosaccharides showed the absolute configurations to be D for all residues except for rhamnose, which is L. The H-1 NMR spectrum was completely assigned by two-dimensional homonuclear methods (DQF-COSY, NOESY, HOHAHA). The stereochemistry of pyranosides was assigned from 3J(HH) coupling constant values determined from these experiments. The C-13 spectrum was assigned by H-1-detected heteronuclear multiple-quantum correlation (H-1[C-13] HMQC) and by the hybrid method of HMQC-COSY. The glycosidic linkage positions of the polymer were determined by H-1-detected multiple-bond correlation (H-1[C-13] HMBC) and by 2D-NOESY spectra. The position of the phosphodiester linkage was determined by splitting observed In the C-13 resonances due to P-31 couplings leading to the overall structure given in Chart I.