Directed Evolution of Halogenases for Small Molecule Functionalization

项目摘要 卤代有机化合物被广泛用作建筑材料、合成中间体和终点 将产品用于制药和农用化学品。这些化合物的用途，包括它们的 生物活性是由卤素赋予它们的反应性和物理性质引起的 换人。卤化的重要性和与当前卤化方法相关的限制 促使我们开发新的卤化催化剂。这项提议概述了黄素依赖症的演变 一系列合成用卤代酶(FDHS)和Fe(II)依赖的α-酮戊二酸卤代酶(FeDHs) 涉及选择性卤化和相关(即假卤素)原子转移反应的方法。 在项目结束期间完成的工作包括第一批外佣指导进化的例子 改善这些酶的底物范围、选择性和反应性，以用于实际的合成应用。 自那以后，结构、动力学和计算方法被用来提供严格的表征 改造的酶，并为后续的工程工作提供信息。在拟议的项目期内，更多 这些酶的高级合成应用，包括缺乏电子的底物的卤化， 用于铅多样化的顺序卤化/交叉偶合，以及对映选择性卤化 被追捕。在一个重要的新方向上，FeDHs也将被进化成催化非定向的位点选择性SP3C-H 卤化、叠氮化和其他原子转移反应。这些外佣和食环署的工作将涉及新的 加快识别和优化的基因组挖掘和定向进化方法学的进展 用于非天然催化、C-H键功能化和合成应用的酶。

Project Summary Halogenated organic compounds are used extensively as building blocks, synthetic intermediates, and end use products for pharmaceutical and agrochemical applications. The utility of these compounds, including their biological activity, arises from the reactivity and physical properties uniquely conferred to them by halogen substitution. The importance of halogenation and limitations associated with current halogenation methods prompted us to develop new halogenation catalysts. This proposal outlines the evolution of flavin dependent halogenases (FDHs) and Fe(II) α-ketoglutarate dependent halogenases (FeDHs) for a range synthetic methods involving selective halogenation and related (i.e. pseudohalogen) atom transfer reactions. Work completed in the concluding project period included the first first examples of FDH directed evolution to improve the substrate scope, selectivity, and reactivity of these enzymes for practical synthetic applications. Structural, kinetic, and computational methods have since been used to provide rigorous characterization of the engineered enzymes and to inform subsequent engineering efforts. In the proposed project period, more advanced synthetic applications of these enzymes, including halogenation of electron deficient substrates, sequential halogenation/cross-coupling for lead diversification, and enantioselective halogenation will be pursued. In a major new direction, FeDHs will also be evolved to catalyze non-directed site-selective sp3 C-H halogenation, azidation, and other atom transfer reactions. These FDH and FeDH efforts will involve new advances in genome mining and directed evolution methodology to expedite the identification and optimization of enzymes for non-native catalysis, C-H bond functionalization, and synthetic applications.