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 b iological processes. Cleavage of heparin by nitrous acid results in a reactive 2,5-anhydromannose (Am) which can be used to selectively inse rt primary and secondary amines by reductive amination. Low-molecular- mass heparin (LMMH) was bound to 4-(2-aminoethylphenol) as shown by nu clear magnetic resonance spectroscopy (NMR), high-performance size-exc lusion chromatography (HPSEC), polyacrylamide gel electrophoresis (PAG E), and ultraviolet/visible (uv/vis) spectroscopy. H-1 NMR spectra rev ealed 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 wi th iodine-125 by oxidation with chloramine T. Fluorescein-5-isothiocya nate (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 lab eled at the secondary amino group. The fluorescent compound exhibited 70 aXa and 5 aIIa U/mg and was neutralized by protamine. The selective ly bound labeled heparin derivatives are ''endpoint attached'' and hav e intact anticoagulant activity. (C) 1994 Academic Press, Inc.