离子液体中酶催化黄酮苷定向水解与糖基选择性酰化及其合成应用

This project is to investigate the enzyme-catalyzed oriented hydrolysis of flavonoid glycosides and selective acylation of sugar moiety in ionic liquids as well as their synthetic application, using the most abundant natural flavonoid glycosides naringin, hesperdin and rutin as the starting materials. (1) Studies on the enzymatic oriented hydrolysis of naringin, hesperidin and rutin in ionic liquids, catalysed by α-L-rhamnosidase to produce flavonoid monoglycosides, isoquercitrin and L-rhamnose. (2) The regioselective acylation of sugar moiety of flavonoid monoglycosides in ionic liquids by a variety of acyl groups, such as galloyl, caffeoyl, feruloyl, p-coumaroyl, acetyl or long chain fatty acyl, using lipase as biocatalyst in ionic liquids will be investigated, three series of naturally occurring with important biological properties acylated flavonoid glycosides and their derivatives will be synthesized. (3) The catalytic activity and model of α-L-rhamnosidase and lipase in different ionic liquids will be measured, various influence flactors on the conversion yield and regioselective of flavonoid glycosides including the kind of enzymes, the concentration of substrates, water content, reaction temperature, reaction time, the recycling use of enzymes and the isolation of products will be investigated in detail, the optimum processing conditions will be determined. (4) The antioxidant capacity and α-glucosidase inhibition activity of all synthesized acylated flavonoid glycosides will be evaluated , and their structure-activity relationship will be further investigated, with the aim of developing new flavonoids functional molecule having higher medicinal value and economic value.

本项目以丰富的天然黄酮类资源柚皮苷、橙皮苷和芦丁为原料，进行离子液体中酶催化黄酮苷定向水解与糖基选择性酰化及其合成应用研究。（1）离子液体中柚皮苷、橙皮苷或芦丁分别通过α-L-鼠李糖苷酶催化定向水解，制备黄酮类单糖苷、异槲皮苷和鼠李糖等医药和生物化工产品；（2）离子液体中黄酮类单糖苷经脂肪酶催化的选择性酰化，合成系列具有重要生物活性的糖基上连有没食子酰、咖啡酰、阿魏酰、香豆酰、乙酰或各种长链脂肪酰基的天然产物及其它黄酮苷衍生物。（3）探讨α-L-鼠李糖苷酶和脂肪酶在不同离子液体中的催化性能和反应动力学，考察酶种类、底物浓度、温度、体系含水量和反应时间对产率和选择性的影响以及酶循环使用和产物分离等问题，获得最佳合成条件；（4）对合成的酰基黄酮苷进行抗氧化和α-葡萄糖苷酶抑制活性及构效关系研究，以期获得具有较高药用或经济价值的新型黄酮类功能分子。

本项目以来源丰富的天然二氢黄酮类资源柚皮苷和橙皮苷为原料，对黄酮类天然产物及其新型Mannich碱衍生物的合成和生物活性进行了系列研究。（1）分别将柚皮苷和橙皮苷进行微波协助的酸催化水解、O-甲基化或O-异戊烯基化、碱催化的开环反应，或脱氢、溴代、微波协助的芳香族亲核取代反应、过氧丙酮（DMDO）氧化和选择性脱甲基化等反应步骤，半合成了卡瓦胡椒素A和雷必诺亭等天然黄酮类和多甲氧基黄酮类化合物。该合成方法具有工业应用价值。 （2）以间苯三酚为原料，分别经F-C酰化、醇醛缩合、环化、过氧丙酮（DMDO）氧化、O-异戊烯基化和微波协助的Claisen重排等反应步骤，完成了具有重要生物活性的卡瓦胡椒素B和8-异戊烯基黄酮淫羊藿素等黄酮类天然产物新方法全合成。（3）将所合成的黄酮类化合物分别与甲醛、二级胺在酸性醇溶剂中发生Mannich反应，合成了32种未见文献报道的新型黄酮Mannich碱衍生物，并通过乙酰胆碱酯酶（AChE）抑制活性测试，筛选出了11种具有很高AChE抑制活性的黄酮类功能分子。通过药理作用研究表明其中2种化合物具有进一步开发为治疗早老性痴呆症疾病阿尔次海默症（Alzheimer’s）药物的潜力。（4）对脂肪酶催化的香豆素糖苷选择性酰化反应进行了研究。以香豆素糖苷为底物，脂肪酶为催化剂，长链脂肪酸为酰基供体，在有机溶剂的反应介质中，以50-65%的产率合成了9种新型香豆素糖苷长链脂肪酸酯。酰化反应区域选择性地发生在糖基的6´-位羟基上。优化的反应条件为：使用固定化的lipase B（Novozym 435），酶用量10mg/ml，以丙酮和吡啶混合物作溶剂（含水量小于1%），酰基供体和香豆素糖苷的摩尔比为10:1，反应温度为50-60℃，反应时间为96h。