Preparation and structural characterization of large heparin-derived oligosaccharides

Porcine mucosal heparin was partially depolymerized with heparin lyase I and then fractionated into low-molecularweight (<5000) and high-molecular-weight (>5000) oligosaccharides by pressure filtration. The high-molecular-weight oligosaccharide mixture (̃50 wt% of the starting heparin) also contained intact heparin. This intact polymer complicates oligosacsharide purification. Thus, the low-molecular-weight fraction was used to prepare homogeneous oligosaccharides for structural characterization. The low-molecular-weight oligosaccharide mixture was first fractionated by low pressure gel permeation chromatography into size-uniform mixtures of disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, decasaccharides, dodecasaccharides, tetradecasaccharides and higher oligosaccharides. Each size-fractionated mixture was then purified on the basis of charge by repetitive semi-preparative strong-anion-exchange high-performance liquid chromatography. This approach has led to the isolation of 14 homogeneous oligosaccharides from disaccharide to tetradecasaccharide. The purity of these heparin-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 oligosaccharides was established using 600 MHz two-dimensional nuclear resonance spectroscopy . The spectral methods used included homonuclear correlation spectroscopy, nuclear Overhauser effect spectroscopy and heteronuclear multiple quantum coherence spech-clscopy. The ¹H/¹H connectivities of the protons of each sugar residue in an oligosaccharide were established by two-dimensional homonuclear correlation spectroscopy, while ¹H/¹³C assignments were made using ¹H inverse detection. One- and two-dimensional nuclear resonance spectroscopic analysis of these heparin oligosaccharides showed two closely related groups of heparin-oligosaccharides are afforded by enzymatic depolymerization of heparin. One group is fully sulphated, having the structures △UAp2S(1[→4)-α-D-GlcNpS6S(1→4)-α-L-IdoAp2S(1]_n→4)-α-D-GlcNp is 2-deoxy 2-aminoglucopyranose, IdoAp is idopyranosyluronic acid, S is sulphate and n = 0-6. The other group of oligosaccharides differ in that they contain β-D-glucuronic acid in place of the α-L-iduronic acid residue nearest to the reducing end. The present study describes the isolation and structural elucidation of seven new oligosaccharides: an octasaccharide, two decasaccharides, two dodecasaccharides and two tetradecasaccharides. The utility of two-dimensional nuclear resonance spectroscopy to determine the structure of complex heparin oligosaccharides is also illustrated.