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: 8944011
• 负责人: JARED C LEWIS
• 金额: $ 29.43万
• 依托单位: UNIVERSITY OF CHICAGO
• 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 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8944011
• 关键词: Acids; Agrochemicals; Air; Alkenes; Amines; Anions; Benzene; Biological; Biological Factors; Catalysis; Chemicals; Computing Methodologies; Coupling; Data; Development; Disease; Eating; Engineering; Environment; Enzymes; Evolution; Flavins; General Practitioners; Goals; Grant; Halogens; Hand; Hydrogen Bonding; Indoles; Kinetics; Lead; Metals; Methods; Mission; Molecular; Mutagenesis; Organic Synthesis; Oxidants; Pharmacologic Substance; Positron; Positron-Emission Tomography; Preparation; Proteins; Protocols documentation; Reaction; Reagent; Relative (related person); Research; Roentgen Rays; Shapes; Site; Source; Specialist; Staging; Structure; Structure-Activity Relationship; Tryptophan; United States National Institutes of Health; Variant; catalyst; chemical synthesis; chlorination; combat; design; directed evolution; enol; halogenation; improved; molecular recognition; physical property; predictive modeling; public health relevance; research study; screening; small molecule; tool

 DESCRIPTION (provided by applicant): 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) for a range synthetic methods involving selective halogenation. Our preliminary results include the first examples of FDH directed evolution via iterative mutagenesis, and we will use the methods we have developed to further improve the substrate scope, selectivity, and reactivity of these enzymes for practical synthetic applications. This will entail generating FDH variants and screening those variants for activity on substrates with progressively increased sizes, altered shapes, and reduced nucleophilicities, relative to the native substrate, tryptophan. Structural, kinetic, and computational methods will then be used to provide rigorous characterization of the engineered enzymes. These data will inform subsequent engineering efforts. Specifically, FDHs will be engineered to override substrate reactivity using molecular recognition and to tune the biological activity of arene and olefin-containing substrates via late-stage halogenation and tandem halogenation/cross-coupling. Synthesis of PET probes using FDH catalyzed incorporation of positron emitting halogens into these compounds will also be demonstrated. Together, these experiments will significantly advance modern synthetic halogenation methods and improve our ability to engineer enzymes for other C-H bond functionalization reactions.

 描述（由申请人提供）：卤化有机化合物主要用作药物和农业化学应用的结构单元、合成中间体和最终产品，这些化合物的效用（包括其生物活性）源于其所赋予的反应性和物理独特性质。卤化的重要性和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卤化酶的演变。 （FDH）用于涉及选择性卤化的一系列合成方法，我们的初步结果包括通过迭代诱变进行 FDH 定向进化的第一个例子，我们将使用我们开发的方法进一步提高这些酶的底物范围、选择性和反应性。对于实际的合成应用，这将需要生成FDH变体并筛选这些变体对相对于天然底物色氨酸逐渐增加的尺寸、改变的形状和降低的亲核性的活性。然后将使用动力学和计算方法来提供工程酶的严格表征，这些数据将为后续的工程工作提供信息。使用 FDH 催化将正电子发射卤素掺入这些化合物中，通过后期卤化和串联卤化/交叉偶联合成 PET 探针。这些实验将共同显着推进现代合成卤化方法，并提高我们为其他 C-H 键功能化反应设计酶的能力。