Malsch R, Guerrini M, Torri G, Löhr G, Casu B, Harenberg J
First Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Mannheim, Germany.
Anal Biochem. 1994 Mar;217(2):255-64. doi: 10.1006/abio.1994.1117.
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.
肝素在抗凝及其他多种生物学过程中发挥着重要作用。亚硝酸对肝素的切割会产生一种活性2,5-脱水甘露糖（Am），它可通过还原胺化反应用于选择性插入伯胺和仲胺。通过核磁共振光谱（NMR）、高效尺寸排阻色谱（HPSEC）、聚丙烯酰胺凝胶电泳（PAGE）和紫外/可见（uv/vis）光谱表明，低分子质量肝素（LMMH）与4-(2-氨基乙基苯酚)结合。1H NMR光谱显示其平均序列为(艾杜糖醛酸2-磺酸-氨基葡萄糖6-磺酸)9-艾杜糖醛酸2-磺酸-Am-酪胺，且酪胺与LMMH的结合率为50%。LMMH-酪胺的抗凝活性为108抗Xa因子活性（aXa）U/mg和42抗IIa因子活性（aIIa）U/mg。该化合物可被鱼精蛋白中和。采用N-烷基胺衍生物通过氯胺T氧化用碘-125标记LMMH。用异硫氰酸荧光素（Fitc）对LMMH-酪胺进行荧光标记。NMR、HPSEC、PAGE和uv/vis光谱证明了Fitc与LMMH-酪胺的结合。1H NMR光谱表明约80%的LMMH-酪胺在仲氨基处被标记。该荧光化合物表现出70 aXa和5 aIIa U/mg的活性，且可被鱼精蛋白中和。选择性结合的标记肝素衍生物是“末端连接”的，并且具有完整的抗凝活性。
