Nearest neighbor analysis of heparin: identification and quantitation of the products formed by selective...

Heparin was carboxyl-reduced with sodium boro[3H]hydride and converted to a mixture of oligosaccharides by treatment with nitrous acid at pH 2. The oligosaccharide mixture was aldehyde-reduced with sodium boro[3H]hydride and the mixture of products, labeled both in the hexoses formed in the carboxyl-reduction step and in the reducing sugars formed in the nitrous acid reaction, was separated and analyzed. The major product, L-idosyl 2-sulfate leads to anhydro-D-mannitol 6-sulfate (I), contained 60% of hexoses derived from the hexuronic acid residues in the original heparin. A second product, which contained 15% of the hexoses derived from the hexuronic acid residues in the original heparin, was identified as a tetrasaccharide composed of two L-idosyl 2-sulfate residues, one anhydro-D-mannitol 6-sulfate residue (the reducing end),and a hydroxymethylpentose sulfate residue formed by deamination of a disulfated D-glucosamine residue without bond cleavage. Several additional disaccharides derived from the regions of the polymer which contained D-glucuronic acid residues and lower degrees of O-sulfation were also identified among the deamination products. The oligosaccharides that were obtained accounted for 100% of the original carboxyl-reduced heparin, and paper chromatographic profiles of the oligosaccharide separations can be used as a fingerprint of the heparin preparation. The properties of I were examined in greater detail. The glycosidic bond of the L-idosyl 2-sulfate residue was found to be extremely labile to 0.1 N HCl at 100 degrees C, hydrolyzing with a t 1/2 of 18 min to give high yields of L-idose 2-sulfate and anhydro-D-mannitol 6-sulfate. L-Idofuranose was also identified as an intermediate in the conversion of L-idose 2-sulfate to L-idosan. The acid lability of the L-idosyl 2-sulfate bond in I offers a new route for the selective cleavage of carboxyl-reduced heparin.