Directed Evolution of Metal-Substituted Carbonic Anhydrases for Catalysis

项目摘要/摘要 独立之路奖申请中所描述的研究的目标是提供 在蛋白质工程方面受过培训并有开发人造金属酶经验的候选人 催化新的或改进的合成反应以制备具有生物活性的化合物。候选人利维 斯坦利博士目前是NRSA/NIGMS博士后研究员，近期目标是完成 额外一到两年的指导性研究，以建立蛋白质工程领域的研究技能 并将这些新技术应用于人工金属酶的开发，使其结合了广泛的反应性 具有酶能力的过渡金属络合物精细控制合成的选择性 变形。斯坦利博士的长期职业目标包括将自己确立为一名独立科学家 在终身教职的学术职位上，并通过 开发产生生物活性化合物的催化反应和构建此类化合物的基础。 斯坦利博士通过他目前在《教授》杂志上的研究建立了过渡金属催化方面的专业知识。 约翰·哈特威格在伊利诺伊大学的实验室。职业发展计划和研究战略将 允许候选人将他在过渡金属催化方面的知识与目前蛋白质的研究方法结合起来 工程学。该方案的指导阶段将与约翰教授合作进行 过渡金属催化领域的领军人物哈特威格和过渡金属催化领域的专家赵惠民教授 蛋白质工程。候选人的联合导师在伊利诺伊大学的成熟实验室 提供一个理想的环境，以便在各自的专业领域进行研究。这个 在指导阶段提出的研究将允许候选人为发现创建一个新的平台 以及开发新的人造金属酶，催化对合成化学家有用的反应。 独立阶段的拟议研究将集中在这些新的人工应用上 金属酶在区域选择性、化学选择性和立体选择性C-H功能化中的催化作用， 环丙烷化和氮杂环化反应。 拟议的研究计划概括为四个具体目标。第一个目标是关注 具有C-H官能化催化活性的金属取代碳酸酐酶的鉴定 反应。第二个目标是开发产生、表达、纯化、 以及突变的碳酸氢酶文库中金属的取代。第三个目标旨在通过以下方式改进 具有C-H官能化活性的金属取代碳酸酐酶的定向进化 反应。前两个目标和第三个目标的开始将是被辅导者的主要焦点(K99) 阶段，并将为候选人提供进行蛋白质工程和 在计划的独立阶段进行定向进化。第四个目标是针对 金属取代碳酸氢酶的不同进化以鉴定导致不同 区域选择性和化学选择性来自共同的起点。关于完成的研究 目标三和目标四将在该计划的独立(R00)阶段进行。结果是 这些研究将建立一个新的平台来产生和改进人造金属酶，并将为 为扩大由对合成有用的金属酶催化的转化的广度奠定基础 化学家。

Project Summary/Abstract The objectives of the research described in this Pathway to Independence Award application are to provide the candidate with training and experience in protein engineering to develop artificial metalloenzymes that catalyze new or improved synthetic reactions to prepare biologically active compounds. The candidate, Levi Stanley, Ph. D., is currently an NRSA/NIGMS postdoctoral fellow with the immediate goal of completing an additional one to two years of mentored research to establish research skills in the field of protein engineering and to apply these new skills to the development of artificial metalloenzymes that combine the broad reactivity of transition metal complexes with the ability of enzymes to exquisitely control the selectivity of synthetic transformations. Dr. Stanley's long-term career goals include establishing himself as an independent scientist in a tenure-track academic position and contributing to the improvement of human health through the development of catalytic reactions that generate bioactive compounds and building blocks to such compounds. Dr. Stanley has established expertise in transition metal catalysis through his current research in Professor John Hartwig's laboratory at the University of Illinois. The career development plan and research strategy will allow the candidate to combine his knowledge of transition metal catalysis with current methods for protein engineering. The mentored phase of the program will be carried out in collaboration with Professor John Hartwig, a leader in the field of transition metal catalysis, and Professor Huimin Zhao, an expert in the field of protein engineering. The established laboratories of the candidate's co-mentors at the University of Illinois provide an ideal environment to conduct research at the interface of their respective areas of expertise. The proposed research in the mentored phase will allow the candidate to create a novel platform for the discovery and development of new artificial metalloenzymes that catalyze reactions that are useful to synthetic chemists. The independent phase of the proposed research will focus on the application of these new artificial metalloenzymes as catalysts in regioselective, chemoselective, and stereoselective C-H functionalization, cyclopropanation, and aziridination reactions. The proposed research program is outlined in four specific aims. The first aim is focused on the identification of metal-substituted carbonic anhydrases that exhibit activity as catalysts for C-H functionalization reactions. The second aim is directed toward developing methods for the generation, expression, purification, and substitution of the metal in mutant carbonic anhydrase libraries. The third aim is designed to improve by directed evolution the activity of metal-substituted carbonic anhydrases that are active for C-H functionalization reactions. The first two aims and the beginning of the third aim will be the primary focus of the mentored (K99) phase and will provide the background necessary for the candidate to conduct protein engineering and directed evolution during the independent phase of the program. The fourth aim is directed toward the divergent evolution of metal-substituted carbonic anhydrases to identify mutants that lead to different regioselectivities and chemoselectivities from a common starting point. Research toward the completion of aims three and four will be conducted during the independent (R00) phase of this program. The results of these studies will establish a new platform to generate and improve artificial metalloenzymes and will lay the groundwork to expand the breadth of transformations catalyzed by metalloenzymes that are useful to synthetic chemists.