Very-high-field n.m.r. studies of bovine lung heparan sulphate tetrasaccharides produced by nitrous acid deaminative cleavage. Determination of saccharide sequence, uronate composition and degrees of sulphation

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Abstract
Tetrasaccharides with the general structure UA-GlcNAc-GlcUA-aManOH (where UA represents uronate, GlcNAc N-acetylglucosamine, GlcUA glucuronate and aManOH anhydromannitol) were prepared from low-sulphated heparan sulphates of bovine lung origin by complete nitrous acid deaminative cleavage followed by reduction and fractionated by gel filtration. Ion-exchange chromatography of the tetrasaccharides yielded three major fractions in approximate yields of 37%, 45% and 14%. These were shown to be non-, mono- and di-sulphated respectively. Complete structural characterization of the tetrasaccharide fractions by quantitative high-field n.m.r. spectroscopy showed that each fraction contained only two discrete species and led to the following observations. (1) All of the uronate residues in the tetrasaccharides (and in larger oligosaccharides) are unsulphated, and hence sulphated iduronate [IdUA(2SO3)] must occur exclusively within -GlcNSO3-IdUA(2SO3)-GlcNSO3- sequences (where GlcNSO3 represents N-sulpho-glucosamine) in the parent polymers. (2) The GlcNAc residues in the tetrasaccharides are more highly C-6-O-sulphated than are the aManOH residues, and furthermore sulphation on the aManOH appears to occur only where the GlcNAc is also sulphated. (3) Where the GlcNAc is C-6-O-sulphated, iduronate is the major non-reducing terminal residue, whereas glucuronate predominates in this position if the GlcNAc is unsulphated. The quantitative data obtained are used to determine the degree of C-6-O-sulphation of glucosamine residues in specific sequences within the parent heparan sulphates.