Characterisation by LSI-MS and 1H NMR spectroscopy of tetra-, hexa-, and octa-saccharides of porcine intestinal heparin

The characterisation of oligosaccharide fragments isolated from enzymatically depolymerised porcine intestinal heparin is required in order to probe structure/function relationships of heparin in anticoagulation, antiangiogenesis and antiviral activity. We have used both LSI-MS and 600-MHz 1H NMR with chemical shift assignment by comprehensive 1H-1H TOCSY experiments to fully characterise the major oligosaccharide components including 4 tetrasaccharides, 3 hexasaccharides, and 2 octasaccharides. One of the octasaccharides has not been identified previously and has the structure: ΔUA(2S)-GlcNS(6S)-IdoA(2S)-GlcNS(6S)-IdoA(2S)-GlcNS(6S)-GlcA-GlcNS(6S), where ΔUA is 4,5-unsaturated uronic acid (4-deoxy-α-l-threo-hex-4-enopyranosyluronic acid), GlcN is → 4)-α-d-glucosamine, IdoA is → 4)-α-l-iduronic acid, GlcA is → 4)-β-d-glucuronic acid, and 2-O-, 6-O-, and 2-N-sulfate are abbreviated to 2S, 6S, and NS, respectively. Nearly complete NMR proton chemical shifts are reported for this data set. In addition a novel approach involving oxymercuration–lipid conjugation was used to independently assign sulfate substitution on the ΔUA residues.