A closer look at the nitrogen next door: 1H-15N NMR methods for glycosaminoglycan structural characterization

Recently, experimental conditions were presented for the detection of the N-sulfoglucosamine (GlcNS) NHSO3- or sulfamate H-1 and N-15 NMR resonances of the pharmaceutically and biologically important glycosaminoglycan (GAG) heparin in aqueous solution. In the present work, we explore further the applicability of nitrogen-bound proton detection to provide structural information for GAGs. Compared to the detection of N-15 chemical shifts of aminosugars through long-range couplings using the IMPACT-HNMBC pulse sequence, the more sensitive two-dimensional H-1-N-15 HSQC-TOCSY experiments provided additional structural data. The IMPACT-HNMBC experiment remains a powerful tool as demonstrated by the spectrum measured for the unsubstituted amine of 3-O-sulfoglucosamine (GlcN(3S)), which cannot be observed with the H-1-15N HSQC-TOCSY experiment due to the fast exchange of the amino group protons with solvent. The H-1-N-15 HSQC-TOCSY NMR spectrum reported for the mixture of model compounds GlcNS and N-acetylglucosamine (GlcNAc) demonstrate the broad utility of this approach. Measurements for the synthetic pentasaccharide drug Arixtra (R) (Fondaparinux sodium) in aqueous solution illustrate the power of this NMR pulse sequence for structural characterization of highly similar N-sulfoglucosamine residues in GAG-derived oligosaccharides. (C) 2012 Elsevier Inc. All rights reserved.