Directed Evolution of Metal-Substituted Carbonic Anhydrases: Applications as Cat

金属取代碳酸酐酶的定向进化：作为 Cat 的应用

基本信息

批准号：
8028609
负责人：
Levi Michael Stanley
金额：
$ 8.22万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-01-01 至 2011-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8028609
关键词：
Active Sites Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly Amino Acids Area Award Azides Catalysis Chemistry Collaborations Data Development Development Plans Dialysis procedure Doctor of Philosophy Engineering Environment Enzymes Evolution Excision Exhibits Felis catus Foundations Generations Genes Goals Health Histidine Human Hydrogen Bonding Illinois Ions Isoenzymes Knowledge Laboratories Lead Libraries Mediating Medical Mentors Metals Methods Mutagenesis Mutation National Institute of General Medical Sciences National Research Service Awards Organism Pathway interactions Phase Positioning Attribute Postdoctoral Fellow Procedures Protein Engineering Protocols documentation Reaction Reporting Research Roentgen Rays Science Scientist Screening procedure Site Sulfonamides Synthesis Chemistry System Training Transition Elements Universities Zinc amidation analog base carbene carbonate dehydratase career career development catalyst cycloaddition design diazo compound directed evolution experience high throughput screening human disease improved metal complex metalloenzyme mutant nitrene novel professor programs research and development scaffold skills

项目摘要

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Synthetic chemistry provides a foundation for modern medical science, and the development of new catalysts and synthetic reactions lies at the forefront of chemistry targeted at generating biologically active materials. The proposed research program is focused on the study and development of new artificial metalloenzymes that combine the broad reactivity of transition metal complexes with the exquisite control of enzymes. These new metalloenzymes will be engineered to promote synthetic reactions that will ultimately facilitate the discovery of medicinal agents for treating human disease.

描述（由申请人提供）：本独立奖申请途径中描述的研究的目标是为候选人提供蛋白质工程方面的培训和经验，以开发人工金属酶，从而催化新的或改进的合成反应以制备生物活性化合物。候选人Levi Stanley，Ph。D。目前是NRSA/NIGMS博士后研究员，其直接的目标是完成额外的一到两年的指导研究，以在蛋白质工程领域建立研究技能，并将这些新技能应用于人工金属酶的开发，从而结合过转化金属的整体反应性，使其与启用的能力相结合。斯坦利博士的长期职业目标包括将自己确立为独立科学家的统治轨道学位，并通过发展催化反应的发展来改善人类健康，从而产生生物活性化合物和对这些化合物的建筑块。斯坦利博士通过他目前在伊利诺伊大学的约翰·哈特维格（John Hartwig）教授的实验室中的研究来建立过渡金属催化的专业知识。职业发展计划和研究策略将使候选人可以将他的过渡金属催化知识与当前的蛋白质工程方法相结合。该计划的指导阶段将与过渡金属催化领域的领导者约翰·哈特维格（John Hartwig）教授以及蛋白质工程领域的专家Huimin Zhao教授合作进行。伊利诺伊大学候选人联合收取者的既定实验室为在其各自专业领域的界面进行研究提供了理想的环境。在指导阶段提出的研究将使候选人能够创建一个新的平台，以发现和开发新的人工金属酶，从而催化对合成化学家有用的反应。拟议的研究的独立阶段将重点介绍这些新的人工金属酶作为区域选择性，化学选择性和立体选择性C-H官能化，环丙烷化和副丙烯酸反应的催化剂的应用。提出的研究计划以四个具体目标概述。第一个目的是鉴定金属取代的碳赤霉酶，这些碳酸酐酶作为C-H官能化反应的催化剂表现出活性。第二个目的是针对突变碳酸酐酶文库中金属的产生，表达，纯化和取代的方法。第三个目标旨在通过定向进化来改善金属取代的碳赤霉酶的活性，该碳氧化酶为C-H功能化反应活跃。前两个目标和第三个目标的开始将是指导阶段（K99）阶段的主要重点，并将为候选人提供在计划的独立阶段进行蛋白质工程和定向进化所必需的背景。第四个目标是针对金属取代的碳酸酐酶的分歧演化，以鉴定导致与公共起点不同的区域选择性和化学启示性的突变体。在该计划的独立（R00）阶段将进行第三和第四个目标的研究。这些研究的结果将建立一个新的平台，以生成和改善人工金属酶，并将奠定基础，以扩大对合成化学家有用的金属酶催化的转化的广度。公共卫生相关性：合成化学为现代医学科学提供了基础，而新的催化剂和合成反应的发展位于旨在生成生物活性材料的化学方面的最前沿。拟议的研究计划的重点是研究和开发新的人工金属酶，这些金属酶结合了过渡金属复合物的广泛反应性和精美的酶控制。这些新的金属酶将经过设计，以促进合成反应，最终将促进发现治疗人类疾病的药用剂。