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.
In the preceding paper in this journal, the major oligosaccharides obtained by endo-beta-galactosidase digestion of bovine corneal keratan sulphate were identified as a neutral disaccharide, GlcNAc beta 1-3Gal, and sulphated di-, tetra-, hexa-, octa- and decasaccharides based on the sequence (-3/4GlcNAc beta 1-3Gal beta 1-)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 beta-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: (Formula: see text). The signals of the protons attached to the sulphated carbon atoms show marked downfield shifts (approximately 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).
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002417:Cattle; D003315:Cornea / Q000032:analysis; D004187:Disaccharides; D005696:Galactosidases / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000032:analysis*; D006026:Glycoside Hydrolases; D007632:Keratan Sulfate / Q000032:analysis*; D009682:Magnetic Resonance Spectroscopy; D009844:Oligosaccharides; D013431:Sulfates; D001616:beta-Galactosidase / Q000378:metabolism*
D004187:Disaccharides; D006025:Glycosaminoglycans; D009844:Oligosaccharides; D013431:Sulfates; D007632:Keratan Sulfate; D005696:Galactosidases; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D001616:beta-Galactosidase