Directed Evolution of Halogenases for Small Molecule Functionalization

项目摘要 卤代有机化合物被广泛用作结构单元、合成中间体和最终产物。 用于制药和农业化学应用的产品。这些化合物的用途，包括它们的 生物活性，源于卤素赋予它们的独特反应性和物理性质 替代。卤化的重要性和现有卤化方法的局限性 促使我们开发新的卤化催化剂。该提案概述了黄素依赖性 卤化酶（FDH）和Fe（II）α-酮戊二酸依赖性卤化酶（FeDH）用于一系列合成 涉及选择性卤化和相关（即拟卤素）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定向进化的例子 以改进这些酶的底物范围、选择性和反应性，用于实际合成应用。 结构、动力学和计算方法已经被用来提供严格的表征。 工程酶，并告知随后的工程努力。在项目实施期间，更多 这些酶的高级合成应用，包括缺电子底物的卤化， 顺序卤化/交叉偶联的铅多样化，和对映选择性卤化将 追求。在一个主要的新方向，FeDH也将被进化为催化非定向位点选择性sp3 C-H 卤化、叠氮化和其它原子转移反应。这些外佣和家庭佣工的工作将涉及新的 基因组挖掘和定向进化方法的进步，以加快识别和优化 用于非天然催化、C-H键官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.