1H-NMR studies at 500 MHz of a neutral disaccharide and sulphated di-, tetra-, hexa- and larger oligosaccharides obtained by endo-beta-galactosidase treatment of keratan sulphate

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Abstract
In the preceding paper in this journal, the major oligosaccharides obtained by endo-β-galactosidase digestion of bovine corneal keratan sulphate were identified as a neutral disaccharide, GlcNAcβ1-3Gal, and sulphated di-, tetra-, hexa-, octa- and decasaccharides based on the sequence (-3/4GlcNAcβ1-3Galβ1-)n having 1, 3, 5, 7 and 9 sulphate groups, respectively. In the present study, these oligosaccharides have been analysed by 500-MHz 1H-NMR spectroscopy using spin-decoupling and two-dimensional correlated spectroscopy experiments. The NMR data confirm the β-configuration of all the interglycosidic linkages and are consistent with an alternating sequence of →4GlcNAc and →3Gal, a non-reducing-end N-acetylglucosamine residue and a reducing-end galactose residue. The NMR data have also established that a sulphate group is linked to the C6 position of all sugar residues except the reducing-end galactose as follows: The signals of the protons attached to the sulphated carbon atoms show marked downfield shifts (apprximately 0.4 ppm from equivalent protons of non-sulphated carbon atoms), while the protons at C5 vicinal to sulphated atoms show a change of 0.1–0.2 ppm and other protons of the sulphated monosaccharides show smaller changes in chemical shift (0.01–0.1 ppm). The proton at C4 of the non-sulphated reducing-end galactose linked at C3 also shows a significant change in chemical shift (0.03 ppm).

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