title: 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.
abstract: A novel disaccharide was isolated beside the predominant trisulfated disaccharide, delta HexA(2-O-sulfate)(alpha 1-4)GlcN(2-N-,6-O-disulfate) (delta HexA and GlcN represent 4-deoxy-alpha-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 delta HexA(2-O-sulfate) (alpha 1-4)ManN(2-N-,6-O-disulfate), where ManN represents D-mannosamine. The C-2 epimerization from delta HexA(2-O-sulfate) (alpha 1-4)GlcN(2-N-,6-O-disulfate) to delta HexA(2-O-sulfate) (alpha 1-4)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.
authors.affiliation: Department of Biochemistry, Kobe Pharmaceutical University, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Yamada
authors.forename: M
authors.initials: M
authors.lastname: Watanabe
authors.forename: K
authors.initials: K
authors.lastname: Sugahara
mesh_terms: D002384:Catalysis; D004187:Disaccharides / Q000737:chemistry* / Q000302:isolation & purification; D006493:Heparin / Q000737:chemistry*; D019763:Heparin Lyase; D006595:Hexosamines / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009844:Oligosaccharides / Q000737:chemistry*; D011522:Protons; D013237:Stereoisomerism; D013463:Sulfuric Acid Esters / Q000737:chemistry*
chemical_list: C114972:4-deoxy-2-sulfooxy-threo-hex-4-enepyranosyluronic acid-1-4-(N,6-disulfooxyglucosamine); D004187:Disaccharides; D006595:Hexosamines; D009844:Oligosaccharides; D011522:Protons; D013463:Sulfuric Acid Esters; C008322:mannosamine; D006493:Heparin; D019763:Heparin Lyase