Structural Modification Induced in Heparin by a Fenton-Type Depolymerization Process

ABSTRACT

A low molecular weight heparin (LMWH) obtained by a depolymerization process induced by a Fenton-type reagent was characterized in depth by nuclear magnetic resonance (NMR) spectroscopy. The depolymerization involves the cleavage of glycosidic bonds, leading to natural terminal reducing end residues, mainly represented by N-sulfated glucosamine (A_NS). Natural uronic acids, especially the 2-O-sulfate iduronic acid (I_2S), are also present as reducing residues. A peculiar reaction results, such as the disappearance of the nonsulfated iduronic acid residues when followed by 6-O-nonsulfated glucosamine, and the decrease of the glucuronic acid when followed by the N-acetylglucosamine, were observed. Iduronic acid residues, followed by 6-O-sulfate glucosamine (A_Nx,6S), and the glucuronic acid residues, followed by A_NS residues, were not modified. A few minor internal chain modifications occur, possibly arising from oxidative breaking of the bond between C2-C3 of glucosamine and uronic acids, suggested by evidence of formation of new -COR groups. Finally, no change was observed in the content of the N-sulfated, 6-O-sulfated glucosamine bearing an extra sulfate on 3-O, which is considered the marker of the active site for antithrombin. With respect to the original heparin, this LMWH is characterized by a lower number of nonsulfated uronic acid residues, and as a consequence, by a lower degree of structural heterogeneity than LMWHs prepared with other procedures.

Low molecular weight heparin (LMWH) - radical depolymerization - nuclear magnetic resonance; Fenton-type reagent

REFERENCES

• 1 Casu B. Structure in biological activities of heparin.  Adv Carbohydr Chem Biochem. 1985;  43 51-134
• 2 Venkataraman G, Shriver Z, Raman R, Sasisekharan R. Sequencing complex polysaccharides.  Science. 1999;  286 537-542
• 3 Guerrini M, Raman R, Venkataraman G, Torri G, Sasisekharan R, Casu B. A Novel computational approach to integrate nmr spectroscopy and capillary electrophoresis for structure assignment of heparin oligosaccharides.  Glycobiology. 2002;  12 713-719
• 4 Ferro D R, Ragazzi M, Provasoli A et al.. Conformers population of l-iduronic acid residues in glycosaminoglycans sequences.  Carbohydr Res. 1990;  195 157-167
• 5 Hricovini M, Guerrini M, Bisio A, Torri G, Petitou M, Casu B. Conformation of heparin pentasaccharide bound to antithrombin III.  Biochem J. 2001;  359 265-272
• 6 Jacobsson I, Lindahl U, Jensen J W, Rodén L, Prihar H, Feingold D S. Biosynthesis of heparin. Substrate specificity of heparosan N-sulfate D-glucuronosyl 5-epimerase.  J Biol Chem. 1984;  259 1056-1063
• 7 Capila I, Linhardt R J. Heparin-protein interaction.  Angew Chem Int Ed. 2002;  41 390-412
• 8 Maccarana M, Casu B, Lindahl U. Minimal sequence in heparin/heparan sulfate required for binding of basic fibroblast growth factor.  J Biol Chem. 1993;  268 23898-23905
• 9 Casu B, Lindahl U. Structure and biological interactions of heparin and heparan sulfate.  Adv Carbohydr Chem Biochem. 2001;  57 159-208
• 10 Turnbull J E, Fernig D G, Ke Y, Wilkinson M C, Gallagher J T. Identification of the basic fibroblast growth factor binding sequence in fibroblast heparan sulfate.  J Biol Chem. 1992;  267 10337-10341
• 11 Kreuger J, Salmivirta M, Sturiale L, Gimenez-Gallego G G, Lindahl U. Sequence analysis of heparan sulfate epitopes with graded affinities for fibroblast growth factors 1 and 2.  J Biol Chem. 2001;  276 30744-30752
• 12 Ornitz D M, Herr A B, Nilsson M, Westman J, Svahn C M, Wachsman G. FGF binding and FGF receptor activation by synthetic heparan-derived di- and trisaccharides.  Science. 1995;  268 432-436
• 13 Kowenski J, Duchaussoy F, Bono M et al.. A synthetic heparan sulfate pentasaccharide, exclusively containing -iduronic acid, displays higher affinity for FGF-2 than its -glucuronic acid-containing isomers.  Bioorg Med Chem. 1999;  7 1567-1580
• 14 Guerrini M, Agulles T, Bisio A et al.. Minimal heparin/heparan sulfate sequences for binding to fibroblast growth factor-1.  Biochem Biophys Res Commun. 2002;  292 222-230
• 15 Naggi A, Casu B, Perez M et al.. Modulation of the heparanase-inhibiting activity of heparin through selective desulfation, graded N-acetylation, and glycol splitting.  J Biol Chem. 2005;  280(13) 12103-12113
• 16 Casu B. Structure and active domains of heparin. In: Garg H, Linhardt R, Hales C Chemistry and Biology of Heparin and Heparan Sulfate. Boston, MA; Elsevier Science & Technology 2005: 1-19
• 17 Casu B, Lindahl U. Structure and biological interactions of heparin and heparan sulfate.  Adv Carbohydr Chem Biochem. 2001;  57 159-208
• 18 Petitou M, Casu B, Lindahl U. 1976-1983, a critical period in the history of heparin: the discovery of the antithrombin binding site.  Biochimie. 2003;  85 83-89
• 19 Iacomini M, Casu B, Guerrini M, Naggi A, Pirola A, Torri G. “Linkage region” sequences of heparins and heparan sulfates: detection and quantification by nuclear magnetic resonance spectroscopy.  Anal Biochem. 1999;  274 50-58
• 20 Bisio A, De Ambrosi L, Gonella S et al.. Preserving the original heparin structure of a novel low molecular weight heparin by γ-irradiation.  Arzneim Forschung. 2001;  51(II) 806-813
• 21 Pejler G, Danielsson A, Björk I, Lindahl U, Nader H B, Dietrich C P. Structure and antithrombin binding properties of heparin isolated from the clams Anomalocardia brasiliana and Tivela macroides .  J Biol Chem. 1987;  262 11413-11421
• 22 Casu B, Guerrini M, Naggi A, Paterno M, Attolini M, Valle M G et al.. Characterization of sulfation patterns of beef and pig mucosal heparins by nuclear magnetic resonance spectroscopy.  Arzneimittelforshung. 1996;  46 472-477
• 23 Bianchini P, Liverani L, Mascellani G, Parma B. Heterogeneity of unfractionated heparins studied in connection with species, source, and production processes.  Semin Thromb Hemost. 1997;  23 3-10
• 24 Maccarana M, Sakura Y, Tawada A, Yoshida K, Lindahl U. Domain structure of heparan sulfates from bovine organs.  J Biol Chem. 1996;  271(30) 17804-17810
• 25 Casu B, Torri G. Structural characterization of low molecular weight heparins.  Semin Thromb Hemost. 1999;  25 17-25
• 26 Linhardt J R, Gunay N S. Production and chemical processing of low molecular weight heparins.  Semin Thromb Hemost. 1999;  25(suppl 3) 5-16
• 27 Bisio A, Guglieri S, Frigerio M et al.. Controlled γ-ray irradiation of heparin generates oligosaccharides enriched in highly sulfated sequences.  Carbohydr Polymers. 2004;  55 101-112
• 28 Guerrini M, Guglieri S, Beccati D, Torri G, Viskov C, Mourier P. Conformational transitions induced in heparin octasaccharides by binding with antithrombin III.  Biochem J. 2006;  399 191-198
• 29 Edwards J O, Curci R. Fenton type activation and chemistry of hydroxyl radical. In: Strukul G Catalytic Oxidations with Hydrogen Peroxide as Oxidant. London; Kluwer Academic 1992: 97-153
• 30 Casu B, Guerrini M, Torri G, Gonella S, Boveri G. Differentiation of beef and pig mucosal heparins by NMR spectroscopy.  Thromb Haemost. 1995;  74 1205
• 31 Casu B, Torri G. Structural characterization of low-molecular weight heparins.  Semin Thromb Hemost. 1999;  25(suppl 3) 17-25
• 32 Torri G. New NMR methods for the structural characterization of glycosaminoglycans. In: Harenberg J, Casu B Nonanticoagulant Glycosaminoglycans. New York; Plenum Press 1996: 15-25
• 33 Guerrini M, Bisio A, Torri G. Combined quantitative ¹H and ¹³C nuclear magnetic resonance spectroscopy for characterization of heparin preparations.  Semin Thromb Hemost. 2001;  27 473-482
• 34 Guerrini M, Naggi A, Guglieri S, Santarsiero R, Torri G. Complex glycosaminoglycans: profiling substitution patterns by two-dimensional nuclear magnetic resonance spectroscopy.  Anal Biochem. 2005;  337 35-47
• 35 Bertini S, Bisio A, Torri G, Bensi D, Terbojevich M. Molecular weight determination of heparin and dermatan sulfate by size exclusion chromatography with a triple detector array.  Biomacromolecules. 2005;  6 168-173
• 36 Yates E A, Santini F, Guerrini M, Naggi A, Torri G, Casu B. ¹H and ¹³C NMR spectral assignments of the major sequences of twelve systematically modified heparin derivatives.  Carbohydr Res. 1996;  294 15-27
• 37 Nonhebel D C, Walton J C. Oxidations and reductions. In: Free-Radical Chemistry, Structure and Mechanism. Cambridge, UK; Cambridge University Press 1974: 404-405