Four octasaccharide serines and three octasaccharides were isolated after heparinase treatment of porcine intestinal heparin. Their structures were characterized by enzymatic digestion in conjunction with HPLC and 500 MHz H-1 NMR spectroscopy. Three of the four octasaccharide serines were structurally identical with those isolated previously, whereas one has the unreported structure Delta HexA(2-sulfate)alpha 1-4GlcN(N-sulfate)alpha 1-4GlcA beta 1-4G1cNAc alpha 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser (Delta HexA, GlcN, IdceA, and GlcA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid, D-glucosamine, L-iduronic acid, and D-glucuronic acid, respectively). The other three octasaccharides were isolated for the first time as discrete structures and shared the common core hexasulfated sequence Delta HexA(2-sulfate)alpha 1-4GlcN(N-sulfate)alpha 1-4IdceA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-4GlcN(N-sulfate)alpha 1-4IdceA(2-sulfate)alpha 1-4GlcN(N,6-disulfate) with one or two additional sulfate groups. The octasaccharides which were derived from the low-sulfated repeating disaccharide region of heparin contained the common trisaccharide sequence -4IdceA alpha 1-4GlcNAc alpha 1-4GlcA beta 1- [Yamada, S., Yamane, Y., Tsuda, H., Yoshida, K., and Sugahara, K. (1998) J. Biol, Chem. 273, 1863-1871], suggesting the programmed biosynthesis of heparin. These octasaccharides are the largest oligosaccharides isolated so far from the low-sulfated irregular region of heparin. Since oligosaccharides larger than a pentasaccharide appear to potentially exhibit binding activities toward growth factors or other functional proteins, they will be useful for investigating the structural requirement for molecular interactions between heparin and/or heparan sulfate and biologically active proteins. During the course of the present structural studies, we evaluated the NMR data accumulated thus far on heparin oligosaccharides and found several interesting rules on chemical shifts of proton signals affected by the neighboring sugar residues and their sulfation, which will be in turn useful for determining structures of unknown heparin and/or heparan sulfate oligosaccharides based on the proton resonances.
