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. 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.
Malsch
R
R
First Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Mannheim, Germany.
Guerrini
M
M
Torri
G
G
Löhr
G
G
Casu
B
B
Harenberg
J
J
D000925:Anticoagulants / Q000138:chemical synthesis*; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006495:Heparin, Low-Molecular-Weight / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000494:pharmacology; D007457:Iodine Radioisotopes; D007553:Isotope Labeling / Q000379:methods*; D008969:Molecular Sequence Data; D013056:Spectrophotometry, Ultraviolet; D014439:Tyramine / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000494:pharmacology
D000925:Anticoagulants; D005456:Fluorescent Dyes; D006495:Heparin, Low-Molecular-Weight; D007457:Iodine Radioisotopes; D016650:Fluorescein-5-isothiocyanate; D014439:Tyramine