SYNTHESIS OF A N'-ALKYLAMINE ANTICOAGULANT ACTIVE LOW-MOLECULAR-MASS HEPARIN FOR RADIOACTIVE AND FLUORESCENT LABELING

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. H-1 NMR spectra revealed an average sequence of (IdoA2SO(3)-GlcNSO3 6SO(3))(9)-IdoAaSO(3)-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. H-1 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. (C) 1994 Academic Press, Inc.