Analysis of Iduronic and Glucuronic Acid Residues in Heparin Polysaccharide Chains

Heparin is an anticoagulant comprised of long, unbranched, anionic polysaccharide chains.  Its basic structure consists of repeating disaccharides of uronic acid and glucosamine residues.  Microheterogeneity is introduced by various patterns of sulfonation and the presence of uronic acid epimers.  Iduronic acid (IdoA) and glucuronic acid (GlcA), required for antithrombin III binding, differ only by C5 epimerization.  In order to characterize heparin, it must first be depolymerized, producing oligosaccharides of various chain lengths.  Differences in the chemical properties of nearly identical tetrasaccharides, differing only by their uronic acid residue, were investigated by measuring the carboxylic acid pK_a values with ¹H NMR pH titrations.  We first determined the pK_a values of the IdoA and GlcA individually, as free monosaccharides.  The pK_a of α-IdoA was found to be 3.309 ± 0.023, and that of β-IdoA was 3.069 ± 0.010. For glucuronic acid, the pKa of α-GlcA was 3.116 ± 0.002, and that of β-GlcA was 3.019 ± 0.007.  Because the tetrasaccharides under investigation are isolated in small quantities, their titrations are performed directly in NMR tubes. The solution pH is determined indirectly through the chemical shifts of the NMR indicators: formate, acetate, and chloroacetic acid.  To further analyze small quantities of heparin, capillary isotachophoresis coupled with NMR microcoil detection will also be used.  This method couples the low limits of detection of the microcoil NMR with the sensitivity that comes from concentrating the sample by isotachophoresis, making it more feasible to characterize these oligosaccharides by 2D NMR.