Directed Evolution of Halogenases for Small Molecule Functionalization

用于小分子功能化的卤化酶的定向进化

• 批准号: 10183266
• 负责人: JARED C LEWIS
• 金额: $ 31.7万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-15 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10183266
• 关键词: Acids; Address; Agrochemicals; Alkenes; Amines; Biological; Catalysis; Chlorides; Collaborations; Complex; Computer Simulation; Computing Methodologies; Coupling; Development; Directed Molecular Evolution; Disease; Electrons; Engineering; Environment; Enzyme Reactivation; Enzymes; Evolution; Flavins; Genome; Goals; Halogens; Hydrogen Bonding; Kinetics; Lead; Letters; Light; Methodology; Methods; Mining; Mission; Molecular; Organic Synthesis; Oxidants; Pharmacologic Substance; Positron; Preparation; Protein Engineering; Proteins; Reaction; Reagent; Reporting; Research; Seminal; Site; Source; Structure; Techniques; United States National Institutes of Health; Work; X-Ray Crystallography; alpha ketoglutarate; catalyst; chemical synthesis; combat; design; enol; halogenation; improved; molecular recognition; nitration; non-Native; novel strategies; physical property; simulation; small molecule; tool

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.

项目概要 卤代有机化合物主要用作结构单元、合成中间体和最终产品 这些化合物的用途，包括它们的用途。 生物活性，源于卤素独特赋予它们的反应性和物理特性 卤化的重要性以及与当前卤化方法相关的局限性。 促使我们开发新的卤化催化剂。该提案概述了黄素依赖性的演变。 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。 项目结束期间完成的工作包括 FDH 定向演进的第一个示例 提高这些酶在实际合成应用中的底物范围、选择性和反应性。 此后，结构、动力学和计算方法已被用来提供严格的表征 工程酶并为后续工程工作提供信息 在拟议的项目期间，更多内容。 这些酶的先进合成应用，包括缺电子底物的卤化， 顺序卤化/交叉偶联以实现铅多样化，并且对映选择性卤化将 在一个重要的新方向上，FeDH 也将进化为催化非定向位点选择性 sp3 C-H。 这些 FDH 和 FeDH 的工作将涉及新的卤化、叠氮化和其他原子转移反应。 基因组挖掘和定向进化方法的进展，以加快识别和优化 用于非天然催化、C-H 键功能化和合成应用的酶。