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Low-molecular weight heparins (LMWHs), as compared with unfractionated heparin (UFH), present superior bioavailability, much longer plasma half-life, and lower incidence of side effects. For these reasons, over the past two decades LMWHs have become the drugs of choice for the treatment of deep venous thrombosis, pulmonary embolism, arterial thrombosis, and unstable angina. Furthermore, their use in acute ischemic stroke is currently under study. LMWHs are obtained by UFH depolymerization, which can be performed using various methods, including nitrous acid depolymerization, cleavage by beta-elimination of benzyl ester, enzymatic depolymerization, and peroxyl radical-dependent depolymerization. This article addresses the chemical depolymerization, obtained by free radical attack (mainly hydroxyl radical), of heparin. The electron spin resonance (ESR) spectroscopy, coupled to the spin trapping technique, was employed to study this reaction. Free radical-mediated heparin depolymerization was performed under different chemical conditions. The final products of the reactions were purified and classified on the basis of their molecular weight and other characteristics. The level of heparin fragmentation was different depending on the type of depolymerization reaction used. Moreover, the level of reproducibility and the resulting radical species were different for every type of reaction performed.

Heparin plays an important role in anticoagulation and several other biological processes. Cleavage of heparin by nitrous acid results in a reactive 2,5-anhydromannose (Am) which can be used to selectively insert primary and secondary amines by reductive amination. Low-molecular-mass heparin (LMMH) was bound to 4-(2-aminoethylphenol) as shown by nuclear magnetic resonance spectroscopy (NMR), high-performance size-exclusion chromatography (HPSEC), polyacrylamide gel electrophoresis (PAGE), and ultraviolet/visible (uv/vis) spectroscopy. 1H NMR spectra revealed an average sequence of (IdoA2SO3-GlcNSO36SO3)9-IdoA2SO3-Am-tyramine and a 50% binding rate of tyramine to LMMH. LMMH-Tyr had an anticoagulant activity of 108 antifactor Xa activity (aXa) U/mg and 42 antifactor IIa activity (aIIa) U/mg. The compound was neutralized by protamine. The N-alkylamine derivative was adopted to label LMMH with iodine-125 by oxidation with chloramine T. Fluorescein-5-isothiocyanate (Fitc) was used to label LMMH-Tyr with fluorescence. NMR, HPSEC, PAGE, and uv/vis spectroscopy demonstrated the binding of Fitc to LMMH-Tyr. 1H NMR spectra indicated that about 80% of the LMMH-Tyr was labeled at the secondary amino group. The fluorescent compound exhibited 70 aXa and 5 aIIa U/mg and was neutralized by protamine. The selectively bound labeled heparin derivatives are "endpoint attached" and have intact anticoagulant activity.