Development and Applications of Photoinducible Bioorthogonal Chemistry

光诱导生物正交化学的发展及应用

• 批准号: 7793428
• 负责人: Qing Lin
• 金额: $ 29.71万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7793428
• 关键词: Address; Alkenes; Amber; Amino Acids; Biological; Biological Assay; Biology; Biomedical Research; Buffers; Cells; Chemicals; Chemistry; Codon Nucleotides; Complex; Culture Media; Development; Dimerization; Engineering; Epidermal Growth Factor Receptor; Escherichia coli; Goals; Hela Cells; In Vitro; Label; Life; Ligation; Lipids; MDCK cell; Mammalian Cell; Measures; Mediating; Membrane; Membrane Proteins; Methionine; Methods; Modeling; Modification; Molecular; Monitor; Organism; Phosphorylation; Post-Translational Protein Processing; Protein Dynamics; Proteins; Reaction; Research; Role; Site; Specificity; Structure; System; Techniques; Tetrazoles; Transcriptional Activation; Tyrosine Phosphorylation; Tyrosine Phosphorylation Site; abstracting; analog; base; cycloaddition; design; enhanced green fluorescent protein; in vivo; insight; intein; mutant; novel; nucleocytoplasmic transport; prevent; protein function; public health relevance; research study; tandem mass spectrometry; tool; Address; Alkenes; Amber; Amino Acids; Biological; Biological Assay; Biology; Biomedical Research; Buffers; Cells; Chemicals; Chemistry; Codon Nucleotides; Complex; Culture Media; Development; Dimerization; Engineering; Epidermal Growth Factor Receptor; Escherichia coli; Goals; Hela Cells; In Vitro; Label; Life; Ligation; Lipids; MDCK cell; Mammalian Cell; Measures; Mediating; Membrane; Membrane Proteins; Methionine; Methods; Modeling; Modification; Molecular; Monitor; Organism; Phosphorylation; Post-Translational Protein Processing; Protein Dynamics; Proteins; Reaction; Research; Role; Site; Specificity; Structure; System; Techniques; Tetrazoles; Transcriptional Activation; Tyrosine Phosphorylation; Tyrosine Phosphorylation Site; abstracting; analog; base; cycloaddition; design; enhanced green fluorescent protein; in vivo; insight; intein; mutant; novel; nucleocytoplasmic transport; prevent; protein function; public health relevance; research study; tandem mass spectrometry; tool

DESCRIPTION (provided by applicant): Development and Applications of Photoinducible Bioorthogonal Chemistry ABSTRACT Bioorthogonal chemistry has emerged as a powerful tool in probing biomolecular structure and function in living systems. Combining with recent developments in introducing novel chemical reactivity into biomolecules site- selectively in vivo, bioorthogonal chemistry offers an unprecedented opportunity to monitor and expand biomolecular function in living systems. Our long term goal is to develop a toolbox of photoinducible bioorthogonal reactions and apply them to study protein function in living systems. The bioorthogonal reactions we are developing build from our chemical insights into unusual heterocycles which are thermodynamically stable, and yet undergo rapid photoinduced ring openings to generate the highly reactive intermediates. These intermediates then react selectively with their cognate, externally introduced partners in living systems. In the Preliminary Studies, we show the first photoinducible bioorthogonal reaction between diaryltetrazoles and alkenes, and its application in the site-specific modification of proteins both in biological buffer and in living E. coli cells. In this project, we propose to significantly expand the scope and the utility of this reaction toolbox by: 1) identifying tetrazoles with enhanced reactivity toward unactivated alkenes; 2) developing a photoinducible diarylazirine-based bioorthogonal reaction; 3) developing a general strategy for functionalizing newly synthesized proteins in living cells; and 4) probing protein posttranslational modifications such as lipidation and phosphorylation in living cells. We hope these new developments will enable functional study of proteins in vivo with exquisite specificity at the molecular level and operational simplicity at the system level. Our specific aims are the follows: (1) To optimize the reactivity of tetrazoles and develop a diarylazirine- based photoinducible bioorthogonal reaction. Substituent effect based on a "push-pull" hypothesis will be explored to achieve the selective and enhanced reactivity toward unactivated alkenes. (2) To develop a general strategy for labeling newly synthesized proteins in mammalian cells through co-translational alkene incorporation followed by selective functionalization with the tetrazole-based chemistry. Experiments are proposed to examine the co-translational activities of several activated alkene amino acids and their subsequent functionalization by the tetrazole compounds. (3) To apply the tetrazole-based bioorthogonal chemistry to model Ras lipidation in living cells and probe the role of lipid structures on Ras membrane targeting dynamics, specificity, and function. Both the intein-mediated chemical ligation and the amber codon suppression methods will be employed in constructing the tetrazole-encoded N-Ras mutant for this study. (4) To apply the tetrazole-based bioorthogonal chemistry to mimic STAT-1 tyrosine phosphorylation by incorporating a tetrazole amino acid at the tyrosine phosphorylation site (Tyr-701) using both native chemical ligation and amber codon suppression techniques. We will examine the effect of chemical phosphorylation on the engineered STAT-1 dimerization, nuclear transport, and transcriptional activation in living cells. PUBLIC HEALTH RELEVANCE: The development of chemical tools for the study of complex and dynamic biological problems represents a central challenge in chemical biology. As a new class of chemical tools, the bioorthogonal reactions have significantly advanced our understanding of biomolecular structure, function, and dynamics in living systems, however, various limitations of currently available bioorthogonal reactions prevent their wider applications in the biomedical research. This proposal addresses the development of a class of photoinducible bioorthogonal reactions with many desirable reaction attributes, and their applications in functionalizing newly synthesized proteins as well as the study of the dynamics of protein posttranslational modifications such as lipidation and phosphorylation in living cells.

描述（由申请人提供）：光诱导生物正交化学的开发和应用摘要生物正交化学已成为探测生物分子结构和活性系统功能的强大工具。结合最新的发展化学反应性在体内有选择性地将新型化学反应性引入生物分子位点，生物正交化学为监测和扩展生物分子功能在生物系统中提供了前所未有的机会。我们的长期目标是开发一个可光诱导的生物正交反应的工具箱，并将其应用于生命系统中的蛋白质功能。我们正在从化学见解到热力学稳定的异常杂环，但经历了快速的光诱导环开口以产生高反应性的中间体，我们正在发展的生物正交反应。然后，这些中间体与它们的同源，外部引入的伴侣在生活系统中有选择性反应。在初步研究中，我们显示了日记二唑和烯烃之间的第一个可诱导的生物正交反应，及其在生物缓冲液和生物大肠杆菌细胞中的蛋白质特异性修饰中的应用。在这个项目中，我们建议通过以下方式显着扩大此反应工具箱的范围和实用性，以下是识别对未激活烷烃反应性增强的四唑； 2）开发基于光光诱导的二二乙醇的生物正交反应； 3）制定一般策略，以使活细胞中新合成的蛋白质功能化； 4）探测蛋白质后翻译后修饰，例如活细胞中的脂化和磷酸化。我们希望这些新的发展能够在分子水平上具有精致的特异性和系统级别的操作简单性来对蛋白质的体内进行功能研究。我们的具体目的是以下内容：（1）优化四唑的反应性并发展基于二维拉齐瑞碱的光诱导生物正交反应。将探索基于“推拉”假设的取代基效应，以实现对未激活烷烃的选择性和增强的反应性。 （2）制定一种通用策略，用于通过共同翻译烯烃掺入将哺乳动物细胞中新合成的蛋白标记，然后与基于四唑的化学选择性官能化。提出了实验来检查几种活化的烯烃氨基酸的共译活性及其随后通过四唑化合物的功能化。 （3）将基于四唑的生物核化学应用于活细胞中的RAS脂质进行建模，并探测脂质结构对RAS膜靶向动力学，特异性和功能的作用。在本研究中，将采用内部介导的化学连接和琥珀色密码子抑制方法来构建四唑编码的N-RAS突变体。 （4）通过使用天然化学结扎和琥珀色codon抑制作用的酪氨酸磷酸化位点（Tyr-701）在酪氨酸磷酸化位点（Tyr-701）掺入四唑氨基酸，将基于四唑的生物核化学应用于模仿STAT-1酪氨酸磷酸化。我们将研究化学磷酸化对活细胞中工程STAT-1二聚化，核转运和转录激活的影响。 公共卫生相关性：用于研究复杂和动态生物学问题的化学工具的开发代表了化学生物学的核心挑战。作为一种新的化学工具，生物正交反应显着提高了我们对生物分子结构，功能和活性动态的理解，但是，当前可用的生物正交反应的各种局限性阻止了其在生物医学研究中的更广泛应用。该提案介绍了具有许多理想反应属性的一类可光诱导的生物正交反应的发展，以及它们在功能化新合成蛋白的功能中的应用，以及研究蛋白质后型后修饰的动力学，例如脂质化和磷酸化细胞中的蛋白质。