6-生物素氨基己酸-七叶亭合成及其生物活性评价

Synthesis and Bioactivity Evaluation of 6-biotinamidohexanoic Acid-esculetin

[目的]优化6-生物素氨基己酸-七叶亭(6-biotinamidohexanoic acid-esculetin,Bio-AHA-ESC)制备工艺,并初步评价其生物学活性。[方法]以1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐[1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,EDCI]和N,N’-二环己基碳化二亚胺(N,N’-dicyclohexyl carbodiimide,DCC)为偶联催化剂,通过考察反应温度、时间、EDCI用量、4-二甲氨基吡啶(4-dimethylaminopyridine,DMAP)用量和七叶亭(esculetin,ESC)与6-生物素氨基己酸(6-biotinamidohexanoic acid,Bio-AHA)投料比,优化合成制备工艺;采用半制备液相色谱法对产物进行分离、纯化,并通过紫外光谱、高分辨质谱和核磁氢谱进行结构鉴定。最后对其氧化应激损伤保护作用和抗炎活性进行初步评价。[结果] EDCI是较为理想的催化剂,优化反应条件为:ESC与Bio-AHA摩尔比为2:1,DMAP添加量为EDCI摩尔数的十分之一,干燥N,N-二甲基甲酰胺(N,N-dimethylformamide,DMF)溶解,室温搅拌反应6 h。经鉴定产物为Bio-AHA-6-位羟基衍生化ESC,该产物在20.00~80.00μmol·L^(-1)剂量范围内均可剂量依赖性抑制过氧化氢诱导的人肝癌细胞HepG2损伤和脂多糖(lipopolysaccharide,LPS)诱导的小鼠单核巨噬细胞RAW264.7白细胞介素-6(interleukin-6,IL-6)分泌,尤其在80.00μmol·L^(-1)时其与ESC生物学活性差异无统计学意义(P>0.05)。[结论]本研究建立了一种温和、高效、专一,而且易于纯化的Bio-AHA-ESC制备方法,所得产物较好保持了ESC生物活性,为下一步深入探究中药活性单体ESC结合靶蛋白奠定了良好基础。

[Objective] To optimize the preparation process of 6-biotinamidohexanoic acid-esculetin(Bio-AHA-ESC) and evaluate its biological activities. [Methods] Using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(EDCI) and N,N’-dicyclohexyl carbodiimide(DCC) as coupling catalysts, the synthesis process was optimized by investigating the reaction temperature, time, EDCI and4-dimethylaminopyridine(DMAP) addition, as well as the ratio of esculetin(ESC) to 6-biotinamidohexanoic acid(Bio-AHA). The biotinlated product was separated and purified by semi-preparative liquid chromatography, and its structure was identified by ultraviolet-visible spectroscopy(UV-VIS), ultra performance liquid chromatography tandem-mass spectrometry(UPLC-MS) and hydrogen spectrum of nuclear magnetic resonance(1H NMR). Finally, its biological activity was evaluated by hydrogen peroxide induced oxidative stress injury model of human hepatoellular carcinomas HepG2 cells and lipopolysaccharide(LPS) induced inflammation model of RAW264.7 cells. [Results]EDCI is an ideal catalyst. The optimized reaction conditions are as follows: The molar ratio of ESC to Bio-AHA is 2:1, the addition amount of DMAP is one tenth of the moles of EDCI, dissolves with dried N,N-dimethylformamide(DMF) and stirs at room temperature for 6 h. The product was identified as Bio-AHA-6-hydroxyl derived ESC by UV-VIS, UPLC-MS and1H NMR. Bioactivity analysis showed that BioAHA-ESC could protect the injury of HepG2 cells induced by hydrogen peroxide and inhibit the secretion of interleukin-6(IL-6) induced by LPS in RAW264.7 cells in a dose-dependent manner, especially at a high dose of 80.00 μmol·L^(-1), the biological activities of BioAHA-ESC were comparable to that of ESC. [Conclusion] In present work, a mild, efficient, specific and easy to purify Bio-AHA-ESC preparation method was established. The obtained Bio-AHA-ESC maintained the biological activity of ESC, which laid a good foundation for further exploring the binding target protein of ESC.