Four hexasaccharides representing major structural sequences of heparin were isolated and characterized after degradation of heparin by heparinase. The structures were determined from two-dimensional 1H NMR spectroscopy including TOCSY (total correlated spectroscopy), COSY (correlated spectroscopy), and ROESY (rotating frame nuclear Overhauser enhancement spectroscopy) methods, providing new data on hexasaccharides. One of the hexasaccharides, the last eluting component from anion exchange chromatography, was derived from the tri-sulfated repeating disaccharide, a-L-idopyranosyluronic acid 2-sulfate-(1-o 4)-2-amino-2-deoxy-oglucopyranose 6,N-disulfate, and having the structure AUAp2S-
The second hexasaccharide contained a nonsulfated D-glucuronic acid unit instead of the L-iduronic acid adjacent to the reducing end, and having the structure AUAp2S-
. The last two hexasaccharides were obtained in lower yield and they have not been isolated and characterized before. The structure of the third saccharide corresponded to a trimer of the repeating disaccharide except for the lack of a 6-O-sulfate group at the reducing end glucosamine residue; AUAp2S-(1 ~ 4)-Ot-D-GlcNp2S6S-(1 ~ 4)-a-L-IdoAp2S-(1 ~ 4)-a-D-GlcNp2S6S-(1 ¢~ Abbreviations used.