Determination of the primary structure and carboxyl pK (A)s of heparin-derived oligosaccharides by band-selective homonuclear-decoupled two-dimensional H-1 NMR

作者：Nguyen Khanh; Rabenstein Dallas L^*

来源：Analytical and Bioanalytical Chemistry, 2011, 399(2): 663-671.

DOI：10.1007/s00216-010-4224-4

• Heparin
• Heparin octasaccharide
• Oligosaccharides
• Acid dissociation constants
• Carboxylate pK(A)
• BASHD-NMR
• NMR

Determination of the structure of heparin-derived oligosaccharides by H-1 NMR is challenging because resonances for all but the anomeric protons cover less than 2 ppm. By taking advantage of increased dispersion of resonances for the anomeric H-1 protons at low pD and the superior resolution of band-selective, homonuclear-decoupled (BASHD) two-dimensional H-1 NMR, the primary structure of the heparin-derived octasaccharide a dagger UA(2S)-[(1 -> aEuro parts per thousand 4)-GlcNS(6S)-(1 -> aEuro parts per thousand 4)-IdoA(2S)-](3)-(1 -> aEuro parts per thousand 4)-GlcNS(6S) has been determined, where a dagger UA(2S) is 2-O-sulfated a dagger(4,5)-unsaturated uronic acid, GlcNS(6S) is 6-O-sulfated, N-sulfated beta-d-glucosamine and IdoA(2S) is 2-O-sulfated alpha-l-iduronic acid. The spectrum was assigned, and the sites of N- and O-sulfation and the conformation of each uronic acid residue were established, with chemical shift data obtained from BASHD-TOCSY spectra, while the sequence of the monosaccharide residues in the octasaccharide was determined from inter-residue NOEs in BASHD-NOESY spectra. Acid dissociation constants were determined for each carboxylic acid group of the octasaccharide, as well as for related tetra- and hexasaccharides, from chemical shift-pD titration curves. Chemical shift-pD titration curves were obtained for each carboxylic acid group from sub-spectra taken from BASHD-TOCSY spectra that were measured as a function of pD. The pK (A)s of the carboxylic acid groups of the a dagger UA(2S) residues are less than those of the IdoA(2S) residues, and the pK (A)s of the carboxylic acid groups of the IdoA(2S) residues for a given oligosaccharide are similar in magnitude. Relative acidities of the carboxylic acid groups of each oligosaccharide were calculated from chemical shift data by a pH-independent method.

• 出版日期2011-1