fondaparinux and sulfamic-acid

Heparin is best known for its anticoagulant activity, which is mediated by the binding of a specific pentasaccharide sequence to the protease inhibitor antithrombin-III (AT-III). Although heparin oligosaccharides are thought to be flexible in aqueous solution, the recent discovery of a hydrogen bond between the sulfamate (NHSO3(-)) proton and the adjacent 3-O-sulfo group of the 3,6-O-sulfated N-sulfoglucosamine residue of the Arixtra (fondaparinux sodium) pentasaccharide demonstrates that definable elements of local structure are accessed. Molecular dynamics simulations of Arixtra suggest the presence of additional hydrogen bonds involving the C3-OH groups of the glucuronic acid and 2-O-sulfo-iduronic acid residues. NMR measurements of temperature coefficients, chemical shift differences, and solvent exchange rate constants provide experimental confirmation of these hydrogen bonds. We note that the extraction of rate constants from cross-peak buildup curves in 2D exchange spectroscopy is complicated by the presence of radiation damping in aqueous solution. A straightforward model is presented that explicitly takes into account the effects of radiation damping on the water proton relaxation and is sufficiently robust to provide an accurate measure of the proton exchange rate between the analyte hydroxyl protons and water.

Topics: Fondaparinux; Heparin; Hydrogen Bonding; Hydroxides; Kinetics; Magnetic Resonance Spectroscopy; Molecular Dynamics Simulation; Oligosaccharides; Polysaccharides; Protons; Solutions; Sulfonic Acids; Temperature; Water

Sulfamate groups (NHSO(3)(-)) are important structural elements in the glycosaminoglycans (GAGs) heparin and heparan sulfate (HS). In this work, proton nuclear magnetic resonance (NMR) line-shape analysis is used to explore the solvent exchange properties of the sulfamate NH groups within heparin-related mono-, di-, tetra- and pentasaccharides as a function of pH and temperature. The results of these experiments identified a persistent hydrogen bond within the Arixtra (fondaparinux sodium) pentasaccharide between the internal glucosamine sulfamate NH and the adjacent 3-O-sulfo group. This discovery provides new insights into the solution structure of the Arixtra pentasaccharide and suggests that 3-O-sulfation of the heparin N-sulfoglucosamine (GlcNS) residues pre-organize the secondary structure in a way that facilitates binding to antithrombin-III. NMR studies of the GlcNS NH groups can provide important information about heparin structure complementary to that available from NMR spectral analysis of the carbon-bound protons.

Topics: Anticoagulants; Antithrombin III; Binding Sites; Carbohydrate Conformation; Carbohydrate Sequence; Fondaparinux; Heparin; Heparitin Sulfate; Humans; Hydrogen Bonding; Hydrogen-Ion Concentration; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Oligosaccharides; Polysaccharides; Protein Binding; Protons; Solutions; Sulfonic Acids; Temperature

Sulfamate (NHSO(3)(-)) groups are critically important structural elements of the glycosaminoglycans heparin and heparan sulfate (HS). Experimental conditions are presented for detection of the sulfamate (1)H NMR resonances in aqueous solution. NMR spectra reported for N-sulfoglucosamine (GlcNS) and the synthetic pentasaccharide drug fondaparinux demonstrate the broad utility of the sulfamate group (1)H chemical shifts to reflect differences in molecular structure. The sulfamate protons also provide an efficient route for detection of (15)N chemical shifts through proton-nitrogen correlations measured with the heteronuclear single quantum coherence (HSQC) experiment. The HSQC spectra of GlcNS, fondaparinux, and the low-molecular weight heparin enoxaparin illustrate the power of the (1)H and (15)N chemical shifts of the sulfamate NH groups for the structural characterization of heparin and HS.

Topics: Enoxaparin; Fondaparinux; Heparin; Heparitin Sulfate; Hydrogen; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Nitrogen Isotopes; Polysaccharides; Protons; Sulfonic Acids; Water