Structural determination of sulfated tetrasaccharides and hexasaccharides containing a rare disaccharide sequence, -3GalNAc(4,6- disulfate)β1-4IdoAα1-, isolated from porcine intestinal dermatan sulfate

Abstract

In the course of structural studies on sulfated oligosaccharides isolated from porcine intestinal heparin after extensive digestion with Flavobacterium heparinase, we isolated several heparitinase-resistant unsaturated oligosaccharides. Amino sugar analysis of these oligosaccharides indicated that they contained galactosamine residues but no glucosamine residues. They were sensitive to chondroitinase ABC but resistant to chondroitinase AC-II, and therefore derived from dermatan sulfate, which was presumably contained as a minor component in the starting heparin preparation. The structures of these oligosaccharides were characterized by enzymatic digestions in conjunction with HPLC analysis of the digests and by one-dimensional and two-dimensional 500-MHz 1H-NMR spectroscopy. Structures of two tetrasaccharides and two hexasaccharides were determined as ΔHexAα1- 3GalNAc(4S)β1-4IdoAα1-3GalNAc(4S), ΔHexAα1-3GalNAc(4S,6S)β1-4IdoAα1- 3GalNAc(4S), ΔHexAα1-3GalNAc(4S)β1-4IdoAα1-3GalNAc(4S)β1-4IdoAα1- 3GalNAc(4S), and ΔHexAα1-3GalNAc(4S)β1-4IdoAα1-3GalNAc(4S,6S)β1- 4IdoAα1-3GalNAc(4S), where ΔHexA, IdoA, GalNAc, 4S and 6S represent 4- deoxy-α-L-threo-hex-4-enepyranosyluronic acid, L-iduronic acid, N-acetyl-D- galactosamine, 4-O-sulfate and 6-O-sulfate, respectively. The latter three compounds have never been reported as discrete structures. Since the four isolated oligosaccharides contained an unsaturated uronic acid residue at the nonreducing terminus, they appear to have been generated by eliminative cleavage by the action of Flavobacterium chondroitinase that was probably present as a minor contaminant in the Flavobacterium heparinase preparation used. Two out of the four oligosaccharides shared the rare disulfated disaccharide sequence, -3GalNAc(4S,6S)β1-4IdoAα1-. These oligosaccharides will be useful as authentic reference compounds for microanalyzing biologically active domains of dermatan sulfate.