ISOLATION AND CHARACTERIZATION OF HEXASACCHARIDES DERIVED FROM HEPARIN - ANALYSIS BY HPLC AND ELUCIDATION OF STRUCTURE BY H-1-NMR

Four hexasaccharides representing major structural sequences of hepari n were isolated and characterized after degradation of heparin by hepa rinase. The structures were determined from two-dimensional H-1 NMR sp ectroscopy including TOCSY (total correlated spectroscopy), COSY (corr elated spectroscopy), and ROESY (rotating frame nuclear Overhauser enh ancement spectroscopy) methods, providing new data on hexasaccharides. One of the hexasaccharides, the last eluting component from anion exc hange chromatography, was derived from the tri-sulfated repeating disa ccharide, alpha-L-idopyranosyluronic acid 2-sulfate-(1-->4)-2-amino-2- deoxy-D-glucopyranose 6,N-disulfate, and having the structure Delta (1 -->4)-alpha-L-IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S. The second hexasaccha ride contained a nonsulfated D-glucuronic acid unit instead of the L-i duronic acid adjacent to the reducing end, and having the structure De lta 6S-(1-->4)-beta-D-GlcAp-(1-->4)-alpha-D-GlcNp2S6S. The last two he xasaccharides were obtained in lower yield and they have not been isol ated and characterized before. The structure of the third saccharide c orresponded to a trimer of the repeating disaccharide except for the l ack of a 6-O-sulfate group at the reducing end glucosamine residue; De lta S-(1-->4)-alpha-L-IdoAp2S-(1-->4)-alpha-D-GlcNp2S. The fourth and last hexasaccharide was less sulfated and the following structure was established Delta S-(1-->4)-alpha-L-IdoAp-(1-->4)-alpha-D-GlcNpAc6S. A nalysis of the ROESY spectra revealed conformational difference of the glucosidic linkage alpha-L-IdoAp-(1-->4)-alpha-D-GlcNp between the he xasaccharides and longer heparin chains.