利用分子间Diels-Alder反应酶实现D-A类型天然产物的精准高效合成

桑科植物中富含endo和exo构型D-A类型天然产物,由于其良好的生物活性和合成上的挑战性，这类天然产物近年来受到合成化学家不断地关注。目前这类天然产物主要是通过手性路易斯酸促进的不对称D-A反应来实现其仿生合成，然而由于该方法无法高效精准控制D-A反应过程中的立体选择性，使得这类天然产物不能以光学纯的形式高效获取。在我们前期的研究中，我们在桑树中找到了首个单功能分子间D-A反应酶MaDA，并初步探讨了其在endo类型天然产物化学酶法合成中的应用。在本研究中，申请人将对桑树中MaDA同源蛋白进行外源表达和体外酶活测试，从而发现桑科植物中新颖的exo选择性D-A反应酶以及其他endo选择性D-A反应酶。在此基础上，我们将探索这两类D-A反应酶的底物适用范围，并将他们应用于D-A类型天然产物及其类似物的精准高效合成，从而展示其作为生物催化剂在不对称D-A反应中的应用潜力。

Moraceae plants are rich in both endo and exo D-A type natural products. Due to their good biological activities and the synthetic challenges, this kind of natural products has been received more and more attention from synthetic chemists in recent years. By far, the asymmetric synthesis of these natural products is mainly achieved by asymmetric D-A reaction promoted by chiral Lewis acid. However, due to the inability to precisely control stereoselectivity, enantio-pure natural products can’t be obtained efficiently via this strategy. In our previous work, we have identified the first standalone intermolecular Diels-Alderase MaDA from mulberry, and preliminarily discussed its application in the chemo-enzymatic synthesis of D-A type natural products with endo configuration. In this study, the applicant will carry out heterogeneous protein expression and in vitro enzyme activity test of MaDA homologous protein from mulberry, with the goal to discover novel exo-selective Diels-Alderases and other endo selective Diels-Alderases in mulberry plants. With these two types of Diels-Alderases in hand, we will explore their substrate scopes and apply them to the precise and efficient synthesis of D-A type natural products and their analogues, showcasing their synthetic values as biocatalysts in asymmetric D-A reaction.