Structural Characterization of the Oligosaccharides Formed by Depolymerization of Heparin with Nitrous Acid

Heparin was cleaved with nitrous acid at pH 1.5 and the products were reduced with Na+ boro[3H]hydride to generate a mixture of di- and tetrasaccharides having anhydro-D-[3H]mannitol (AManR) residues on their reducing terminals. The products were purified to homogeneity by gel filtration and high-performance liquid chromatography. For each oligosaccharide, the proportions of D-glucuronic acid (GlcUA), L-iduronic acid (IdoUA), N-acetyl-D-glucosamine (GlcNAc), and AManR and the monosaccharide sequence were determined by quantification of the products of acid hydrolysis. The tetrasaccharide sequences were determined by comparison of the disaccharide units formed by hydrazinolysis and deamination with previously characterized disaccharides. The following new oligosaccharides were identified: GlcUA(2-SO4)-AManR, GlcUA(2-SO4)-AManR(6-SO4), GlcUA-AManR(3,6-diSO4), GlcUA-GlcNAc-GlcUA-AManR, IdoUA-GlcNAc-GlcUA-AManR, GlcUA-GlcNAc(6-SO4)-GlcUA-AManR, IdoUA(2-SO4)-GlcNAc-GlcUA-AManR, IdoUA-GlcNAc(6-SO4)-GlcUA-AManR, IdoUA(2-SO4)-GlcNAc-GlcUA-AManR(6-SO4), IdoUA-GlcNAc(6-SO4)-GlcUA-AManR(6-SO4), IdoUA-GlcNAc(6-SO4)-GlcUA-AManR(3-SO4), IdoUA-GlcNAc(6-SO4)-GlcUA-AManR(3,6-diSO4), and IdoUA(2-SO4)-GlcNAc(6-SO4)-GlcUA-AManR(6-SO4). Then the disaccharides and the tetrasaccharides were readily resolved by high-performance anion-exchange liquid chromatography and were quantified on the basis of the amount of 3H counts/min in each. The structures are discussed in terms of their implications regarding heparin biosynthesis and anticoagulant activity.