NMR Analysis of Heparin Hydroxyl and Sulfamate Protons

Heparin is a long, unbranched, anionic polysaccharide whose basic structure consists of a repeating disaccharide of uronic acid and glucosamine.  Microheterogeneity is introduced during biosynthesis as various patterns of sulfonation and uronic acid epimerization.  Heparin is used pharmaceutically as an anticoagulant through binding of a specific pentasaccharide sequence to the protease inhibitor, antithrombin III.  Heparin has also been shown to bind to more than 300 proteins mediating a wide variety of biological and disease processes.  In order to study the role of heparin's microstructure in protein binding, it is first depolymerized, producing oligosaccharides of various lengths, which can then be purified chromatographically and characterized by NMR and mass spectrometry.

This work will demonstrate the use of the heparin hydroxyl and sulfamate (NHSO₃^-) protons to provide new information about the solution structure of heparin oligosaccharides.  Because these protons exchange with water, their solvent exchange rates can provide information about hydrogen bonding in heparin.  Measuring ¹H NMR chemical shifts as a function of temperature gives temperature coefficients; smaller temperature coefficients indicate protection from exchange with the solvent, usually in the form of a hydrogen bond.  We have demonstrated that the sulfamate NH in the internal N-sulfoglucosamine residue of Arixtra, a synthetic pentasaccharide that mimics the antithrombin III binding sequence, is involved in a hydrogen bond to the adjacent 3-O-sulfo group which is crucial for its antithrombin activity. Molecular dynamics simulations also suggest the presence of a hydrogen bond involving the glucuronic acid C3-OH.  NMR experiments are underway to explore this possibility experimentally.