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

Nguyen, Khanh, Rabenstein, Dallas L.
Analytical and bioanalytical chemistry 2011 v.399 no.2 pp. 663-671
dissociation, nuclear magnetic resonance spectroscopy, oligosaccharides, protons, titration, uronic acids
Determination of the structure of heparin-derived oligosaccharides by ¹H 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 H¹ protons at low pD and the superior resolution of band-selective, homonuclear-decoupled (BASHD) two-dimensional ¹H NMR, the primary structure of the heparin-derived octasaccharide ∆UA(2S)-[(1 → 4)-GlcNS(6S)-(1 → 4)-IdoA(2S)-]₃-(1 → 4)-GlcNS(6S) has been determined, where ∆UA(2S) is 2-O-sulfated ∆⁴,⁵-unsaturated uronic acid, GlcNS(6S) is 6-O-sulfated, N-sulfated β-d-glucosamine and IdoA(2S) is 2-O-sulfated α-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 As of the carboxylic acid groups of the ∆UA(2S) residues are less than those of the IdoA(2S) residues, and the pK As 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.