DMATS家族酶催化黄酮定向异戊烯基化的方法研究及其合成应用

Prenylflavonoids (or prenylated flavonoids) are an important class of natural products with special bioactivities. However, the diversity and productivity of the naturally occurring prenylflavonoids are limited. Their chemical synthesis is also difficult due to lack of general strategy to introduce the prenyl groups following the regioselectivity in the alkylation steps. Development of efficient methods to synthesize prenylflavonoids thus become very important. The DMATS superfamily of prenyltransferases are soluble enzymes with wide substrate promiscuities. Their catalytic activities can be easily improved by directed mutagenesis and they showed great potential to be biocatalysts. The previous research by the applier had disclosed the potential to use members from the DMATS superfamily for prenylation of flavonoids and several prenylflavonoids were successfully synthesized by using the 7-DMATS. Based on these results, intensive research is proposed to study the catalytic properties of DMATSs on prenylation of a large variety of flavonoids to figure out the efficient conversions. Site-directed mutagenesis will be used to modify those enzymes with low catalytic efficiencies. Mutants with improved catalytic efficiency, broader substrate specificity and higher regioselectivity will be screened out. Eventually, the appliers will develop highly effective enzymatic approaches to catalyze the prenylation of natural flavonoids, which can be used for production of various prenylflavonoids and will contribute to their potential application as prodrugs.

异戊烯基黄酮是一类有独特生物活性的重要天然产物。因为其分布范围狭窄、多样性有限，化学合成选择性差且效率低，限制了异戊烯基黄酮作为药物的开发。因此，针对异戊烯基黄酮高效定向合成法的构建至关重要。DMATS家族的异戊烯基转移酶是可溶性蛋白，有显著的底物杂泛性，可通过位点突变改善催化性能，具有成为生物催化剂的巨大潜力。申请者研究发现多个DMATS家族的异戊烯基转移酶能催化黄酮的异戊烯基化反应，并利用7-DMATS酶催化快速定向合成了一系列异戊烯基黄酮。在此基础上，本项目将深入研究DMATS家族异戊烯基转移酶对多种黄酮的催化作用，阐明不同反应的催化活性和选择性，筛选出高效的异戊烯基化反应，并利用定点突变技术对低效反应进行优化，拓展底物选择性，提高反应的特异性和产率，从而构建高效催化黄酮异戊烯基衍生化反应的方法，实现多类型衍生物的高收率、高选择性制备，为异戊烯基黄酮药物开发奠定物质基础。

异戊烯基黄酮是一类有独特生物活性的重要天然产物，特征为在黄酮的骨架上有异戊烯基侧链取代。异戊烯基取代与其生物活性之间存在显著关联，往往可以提高药物对靶点蛋白的亲和力，从而提高生物利用度和成药性。然而，异戊烯基黄酮分布范围狭窄、多样性有限，化学合成选择性差且效率低，限制了异戊烯基黄酮作为药物的开发。因此，针对异戊烯基黄酮高效定向合成法的构建至关重要。利用具有底物杂泛性的异源异戊烯基转移酶对黄酮进行异戊烯基化修饰为异戊烯基黄酮的制备提供了新的方向和策略。本项目围绕DMATS家族酶催化黄酮的异戊烯基化开展了一系列研究，首次报道了能催化双黄酮和花青素异戊烯基取代反应的异戊烯基转移酶和他们在对应化合物的生物制备方面的应用。运用异戊烯基转移酶，制备了一系列5-O''-或C3'''-异戊烯基取代的双黄酮类化合物和一系列7-O-异戊烯基取代的花青素类化合物。在此基础上，本项目利用定点突变技术对CdpC3PT催化的双黄酮异戊烯基取代反应进行优化，鉴定了参与双黄酮底物识别的关键氨基酸。F253是改变供体选择性的关键残基，该突变体可改变CdpC3PT对异戊烯基供体的选择性，而不改变异戊烯基取代位点。另一个氨基酸W181的突变能显著提高野生型和F253突变体的反应活性。本项目首次筛选出催化双黄酮和花青素类化合物异戊烯基取代的生物催化反应，并利用定点突变技术改变了底物选择性，同时对低效反应进行优化，提高了反应的特异性和产率，实现了这类化合物的绿色制备，为后续药物开发奠定了物质基础。