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 1H NMR spectrum was completely assigned by two-dimensional homonuclear methods (DQF-COSY, NOESY, HOHAHA). The stereochemistry of pyranosides was assigned from 3JHH coupling constant values determined from these experiments. The 13C spectrum was assigned by 1H-detected heteronuclear multiple-quantum correlation (1H[13C] HMQC) and by the hybrid method of HMQC-COSY. The glycosidic linkage positions of the polymer were determined by 1H-detected multiple-bond correlation (1H[13C] HMBC) and by 2D-NOESY spectra. The position of the phosphodiester linkage was determined by splitting observed in the 13C resonances due to 31P couplings leading to the overall structure given in Chart I.