Determination of the primary structure and carboxyl pK (A)s of heparin-derived oligosaccharides by band-selective homonuclear-decoupled two-dimensional (1)H NMR.
Determination of the structure of heparin-derived oligosaccharides by (1)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(1) protons at low pD and the superior resolution of band-selective, homonuclear-decoupled (BASHD) two-dimensional (1)H 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 (A)s of the carboxylic acid groups of the ∆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.
Nguyen
Khanh
K
Department of Chemistry, University of California, Riverside, CA 92521, USA.
Rabenstein
Dallas L
DL
D000818:Animals; D002240:Carbohydrate Sequence; D002264:Carboxylic Acids / Q000737:chemistry; D006493:Heparin / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry; D011522:Protons; D013552:Swine; D014574:Uronic Acids / Q000737:chemistry
D002264:Carboxylic Acids; D009844:Oligosaccharides; D011522:Protons; D014574:Uronic Acids; D006493:Heparin