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

Determination of the structure of heparin-derived oligosaccharides by H 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 protons at low pD and the superior resolution of band-selective, homonuclear-decoupled (BASHD) two-dimensional H NMR, the primary structure of the heparin-derived octasaccharide ΔUA(2S)-[(1→4)GlcNS(6S)-(1→4)-IdoA(2S)-]3-(1→4)-GlcNS(6S) has been determined, where ΔUA(2S) is 2-O-sulfated Δ-unsaturated uronic acid, GlcNS(6S) is 6-O-sulfated, N-sulfated β-Dglucosamine and IdoA(2S) is 2-O-sulfated α-L-iduronic acid. The spectrum was assigned, and the sites of Nand Osulfation 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 tetraand 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 pKAs of the carboxylic acid groups of the ΔUA(2S) residues are less than those of the IdoA(2S) residues, and the pKAs 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.