CHARACTERIZATION BY LSI-MS AND H-1-NMR SPECTROSCOPY OF TETRA-SACCHARIDES, HEXA-SACCHARIDES, 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 H-1 NMR with chemical shift assignment by comprehensive H-1-H-1 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: Delta UA(2S)-GlcNS(6S)-IdoA(2S)-GlcNS(6S)-IdoA GlcNS(6S)-GlcA-GlcNS(6S), where Delta UA is 4,5-unsaturated uronic acid (4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid), GlcN is --> 4)-alpha-D-glucosamine, IdoA is --> 4)-alpha-L-iduronic acid, GlcA is --> 4)-beta-D-glucuronic acid, and 2-0-, 6-0-, 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 Delta UA residues.