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

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