PREPARATION AND STRUCTURAL CHARACTERIZATION OF LARGE HEPARIN-DERIVED OLIGOSACCHARIDES

Porcine mucosal heparin was partially depolymerized with heparin lyase I and then fractionated into low-molecular-weight (<5000) and high-mo lecular-weight (>5000) oligosaccharides by pressure filtration, The hi gh-molecular-weight oligosaccharide mixture (similar to 50 wt% of the starting heparin) also contained intact heparin, This intact polymer c omplicates oligosaccharide purification, Thus, the low-molecular-weigh t fraction was used to prepare homogeneous oligosaccharides for struct ural characterization. The low-molecular-weight oligosaccharide mixtur e was first fractionated by low-pressure gel permeation chromatography into size-uniform mixtures of disaccharides, tetrasaccharides, hexasa ccharides, octasaccharides, decasaccharides, dodecasaccharides, tetrad ecasaccharides and higher oligosaccharides. Each size-fractionated mix ture was then purified on the basis of charge by repetitive semi-prepa rative strong-anion-exchange high-performance liquid chromatography, T his approach has led to the isolation of 14 homogeneous oligosaccharid es from disaccharide to tetradecasaccharide, The purity of these hepar in-derived oligosaccharides was determined by gradient polyacrylamide gel electrophoresis, analytical strong-anion-exchange high-performance liquid chromatography, capillary electrophoresis and one-dimensional nuclear resonance spectroscopy, The structure of these oligosaccharide s was established using 600 MHz two-dimensional nuclear resonance spec troscopy, The spectral methods used included homonuclear correlation s pectroscopy, nuclear Overhauser effect spectroscopy and heteronuclear multiple quantum coherence spectroscopy, The H-1/H-1 connectivities of the protons of each sugar residue in an oligosaccharide were establis hed by two-dimensional homonuclear correlation spectroscopy, while H-1 /C-13 assignments were made using H-1 inverse detection, One- and two- dimensional nuclear resonance spectroscopic analysis of these heparin oligosaccharides showed two closely related groups of heparin-oligosac charides are afforded by enzymatic deporymerization of heparin, One gr oup is fully sulphated, having the structures Delta -->4)-alpha-L-IdoA p2S(1](n)-->4)-alpha-D-GlcNpS6S, where Delta UAp is 4-deoxy-alpha-L-th reo-hex-4-eno-pyranosyluronic acid, GlcNp is 2-deoxy-2-aminoglucopyran ose, IdoAp is idopyranosyluronic acid, S is sulphate and n = 0-6, The other group of oligosaccharides differ in that they contain beta-D-glu curonic acid in place of the alpha-L-iduronic acid residue nearest to the reducing end, The present study describes the isolation and struct ural elucidation of seven new oligosaccharides: an octasaccharide, two decasaccharides, two dodecasaccharides and two tetradecasaccharides. The utility of two-dimensional nuclear resonance spectroscopy to deter mine the structure of complex heparin oligosaccharides is also illustr ated.