Synthesis of Biologically Active Molecular Scaffolds via Enzyme-Catalyzed Asymmetric Carbene Transfer

 描述（由申请人提供）：酶是合成化学的强大工具，不仅因为它们可以催化具有特殊反应性水平的反应，而且还因为它们可以使用定向进化进行调整，以在小分子催化剂难以处理的情况下实现高选择性。酶工程也使血红素依赖性单加氧酶细胞色素P450 BM 3能够催化重氮酯和温和亲核试剂之间的卡宾转移反应，这是一种自然界中完全不存在的反应类型。该建议的重点是通过使用定向的方法来扩展该方法的综合效用。 P450 BM 3的进化，以开发卡宾转移催化剂，该催化剂可以合成用现有方法难以获得的生物学上重要的分子支架。 具体目标是：（1）发展P450 BM 3变体，通过不对称卡宾插入N-H和O-H键合成对映体富集的二芳基和三芳基甲烷;（2）将底物范围扩展到包括富电子芳烃，并将该方法应用于3，3-二取代吲哚和α，α-二取代甘氨酸衍生物的不对称合成。二芳基甲烷和三芳基甲烷基序在许多合成药物候选物中普遍存在，但难以不对称合成，部分原因是手性中心周围的区域具有局部对称性，这使得小分子催化剂难以进行对映选择。然而，使用酶是有利的，因为酶的活性位点产生与底物的整个结构相互作用的手性环境。在第二个目标中，卡宾的应用 转移到富电子芳烃构成了酶催化的C-C键形成的新方法，其通常用醛缩酶完成。吲哚和甘氨酸衍生物，我们建议用这种方法，在许多生物活性的天然和合成化合物中发现，因此有持续的需求，新的方法来合成这些结构不对称。我们将用各种技术（如X射线晶体学）表征该项目产生的酶，以阐明选择性的机制，并协助将来开发用于其他反应类型的酶催化剂。

 DESCRIPTION (provided by applicant): Enzymes are powerful tools for synthetic chemistry not only because they can catalyze reactions with exceptional levels of reactivity, but also because they can be tuned using directed evolution to achieve high selectivity in situations that would be intractable for small-molecule catalysts. Enzyme engineering has also enabled the heme-dependent monooxygenase cytochrome P450BM3, to catalyze carbene transfer reactions between diazoesters and mild nucleophiles, a reaction type that is completely absent in nature. The focus of this proposal is to expand the synthetic utility of this methodology by using directed evolution of P450BM3 to develop carbene transfer catalysts that can synthesize biologically important molecular scaffolds that are difficult to access with existing methods. The specific aims are: (1) to evolve P450BM3 variants to synthesize enantioenriched di- and triarylmethanes via asymmetric carbene insertion into N-H and O-H bonds; and (2) to extend the substrate scope to include electron-rich arenes and apply this methodology to the asymmetric synthesis of 3,3-disubstituted indole and α,α-disubstituted glycine derivatives. The di- and triarylmethane motif is prevalent in numerous synthetic drug candidates, but is difficult t synthesize asymmetrically, partly because the area surrounding the chiral center has local symmetry that makes enantioselection difficult for small-molecule catalysts. The use of enzymes is advantageous, however, because an enzyme's active site creates a chiral environment that interacts with the entire structure of the substrate. In the second aim, the application of carbene transfer to electron-rich arenes constitutes a novel approach to enzyme-catalyzed C-C bond formation, which is usually accomplished with aldolases. The indole and glycine derivatives that we propose to make with this method are found in numerous bioactive natural and synthetic compounds, and thus there is continued demand for new methods to synthesize these structures asymmetrically. We will characterize the enzymes that result from this project with a variety of techniques, such as X-ray crystallography, in order to elucidate the mechanisms of selectivity and assist in the future development of enzyme catalysts for other reaction types.