Low-molecular-weight heparin from Cu²⁺ and Fe²⁺ Fenton type depolymerisation processes

Summary

Hydrogen peroxide (H2O2) and Cu(OAc)2 or FeSO4 (Fenton type reagents) perform heparin (Hep) depolymerisation to low-molecular-weight heparin (LMWH) following a radical chain mechanism. Hydroxyl (OH) radicals which are initially generated from H2O2 reduction by transition metal ions abstract hydrogen atoms on the heparin chain providing carbon centred radicals whose decay leads to the depolymerisation process. The main depolymerisation mechanism involves Hep radical intermediates that cleave the glycosidic linkage at unsulphated uronic acids followed by a 6-O-nonsulphated glucosamine, thus largely preserving the pentasaccharide sequence responsible for the binding to antithrombin III (AT). Both the transition metal ions influence the overall efficiency of the radical chain processes: Fe²⁺ acting as a catalyst, while Cu²⁺ acts as a reagent. LMWHs, especially those afforded by Cu²⁺, are somewhat unstable to the usual basic workup. However, this lack of stability can be eliminated by a previous NaBH4 reduction. Furthermore, with Cu²⁺, the process is much more reproducible than with Fe²⁺. Therefore, for the process of Fenton type depolymerisation of heparin, the use of Cu(OAc)2 is clearly preferable to the more “classical” FeSO4. The resulting activities and characteristics of these LMWHs are peculiar to these oxidative radical processes. In addition, LMWH provided by H2O2/Cu(OAc)2 in optimised conditions was found to posses anti-Xa and anti-IIa activities comparable to those of LMWHs currently in clinical use.

Footnote: Dedicated to Prof. Pietro Bianchini.

Keywords

Heparin radical - LMWH - Fenton type reaction - transition metal ions - NMR

^* Actual position and address: Freelance, Via Osti 5; 40050 Monte S. Pietro (Bologna) Italy.

• References

• 1 Casu B. Structure and active domains of heparin. In: Chemistry and Biology of Heparin and Heparan Sulfate. Oxford: Elsevier; 2005. pp. 1-28.
• 2 Casu B, Torri G. Structural characterization of low molecular weight heparins. Semin Thromb Hemost 1999; 25: 17-25.
• 3 Linhardt JR, Gunay NS. Production and chemical processing of low molecular weight heparins. Semin Thromb Hemost 1999; 25: 5-16.
• 4 Bisio A, Guglieri S, Frigerio M. et al. Controlled g-ray irradiation of heparin generates oligosaccharides enriched in highly sulfated sequences. Carbohydr Polym 2004; 55: 101-112.
• 5 Vismara E, Pierini M, Guglieri S. et al. Structural modification induced in heparin by a Fenton-type depolymerization process. Semin Thromb Hemost 2007; 33: 466-477.
• 6 Fareed J, Ma Q, Florian M. et al. Differentiation of low molecular-weight heparins: impact on the future of the management of thrombosis. Semin Thromb Hemost 2004; 30 (Suppl. 01) 89-104.
• 7 Edwards JO, Curci R. Fenton type activation and chemistry of hydroxyl radical. In: Catalytic oxidations with hydrogen peroxide as oxidant. Strukul G. ed London: Kluwer Academic Publishers; 1992. pp. 97-153.
• 8 De La Mare HE, Kochi JK, Rust FF. The oxidation and reduction of free radicals by Me salts. J Am Chem Soc 1963; 85: 1437-1449.
• 9 Payne GB, Deming PH, Williams PH. Reaction of hydrogen peroxide. Alkali-catalyzed epoxidation and oxidation using a nitrile as co-reactant. J Org Chem 1961; 26: 659-663.
• 10 Mascellani G, Liverani L, Bianchini P. et al. Structure and contribution to the heparin cofactor II-mediated inhibition of thrombin of naturally oversulphated sequence of dermatan sulphate. Biochem J 1993; 296: 639-648.
• 11 Yates EA, Santini F, De Cristofano B. et al. Effect of substitution pattern on ¹H, ¹³C NMR chemical shifts and ¹JCH coupling constants in heparin derivatives. Carbohydr Res 2000; 329: 239-247.
• 12 Guerrini M, Naggi A, Guglieri S. et al. Complex glycosaminoglycans: profiling substitution patterns by two-dimensional nuclear magnetic resonance spectroscopy. Anal Biochem 2005; 337: 35-47.
• 13 Rota C, Liverani L, Spelta F. et al. Free radical generation during chemical depolymerisation of heparin. Anal Biochem 2005; 344: 193-203.
• 14 Torri G, Guerrini M. Quantitative 2D NMR analysis of glycosaminoglycans. In: NMR Spectroscopy in Pharmaceutical Analysis. Integra; India: Elsevier 2008. Part III Chapter 04 12–5; 407.
• 15 Lindahl U, Thunberg L, Bäckström G. et al. The antithrombin-binding sequence of heparin. Biochem Soc Trans 1981; 9: 499-551.
• 16 Casu B, Oreste P, Torri G. et al. The structure of heparin oligosaccharide fragments with high anti-(factor Xa) activity containing the minimal antithrombin III-binding sequence. Chemical and ¹³C-NMR studies. Biochem J 1981; 197: 599-609.
• 17 Guerrini M, Guglieri S, Casu B. et al. Antithrombin binding octasaccharides: role of extensions of the active pentasaccharide sequence on the specificity and strength of interaction. Evidence for very high affinity induced by an unusual glucuronic acid residue. J Biol Chem 2008; 39: 26662-26675.
• 18 Abraham RJ, Loftus P. Proton and Carbon-13 NMR Spectroscopy. An integrated approach. London: Heyden & Son Ltd; 1978
• 19 Fransson L, Malmström A, Sjöberg I. et al. Periodate oxidation and alkaline degradation of heparin -related glycans. Carbohydr Res 1980; 80: 131-145.
• 20 Walenga JM, Jeske WP, Fareed J. Biochemical and Pharmacological Rationale for Synthetic Heparin Pentasaccharides. In Chemistry and Biology of Heparin and Heparan Sulfate. Oxford: Elsevier; 2005. pp. 143-178.