Conversion of N-sulfated glucosamine to N-sulfated mannosamine in an unsaturated heparin disaccharide by non-enzymatic, base-catalyzed C-2 epimerization during enzymatic oligosaccharide preparation

A novel disaccharide was isolated beside the predominant trisulfated disaccharide, ΔHexA(2-O-sulfate)(α1-4)GlcN(2-N-,6-O-disulfate) (ΔHexA and GlcN represent 4-deoxy-α-l-threo-hex-4-enepyranosyluronic acid and d-glucosamine, respectively) after treatment of porcine intestinal heparin with Flavobacterium heparinase. It accounted for 18% of total disaccharides. The structure was characterized by secondary ion mass spectrometry, enzymatic digestions, amino sugar analysis, and 500 MHz one- and two-dimensional 1H NMR spectroscopy as ΔHexA(2-O-sulfate) (α1-4)ManN(2-N-,6-O-disulfate), where ManN represents d-mannosamine. The C-2 epimerization from ΔHexA(2-O-sulfate)(α1-4)GlcN(2-N-,6-O-disulfate) to ΔHexA(2-O-sulfate) (α1-4)ManN(2-N-,6-O-disulfate) was also demonstrated to take place in vitro under very mild alkaline conditions. Hence, the latter compound is not a biosynthetic product, but is most likely an artifact generated by non-enzymatic, base-catalyzed C-2 epimerization during enzymatic preparation of heparin oligosaccharides. The present results warn that the formation of the C-2 epimerized compound has to be circumvented in the structural analysis of heparin/heparan sulfate.