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: 8055023
• 负责人: Qing Lin
• 金额: $ 29.42万
• 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/8055023
• 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; High Pressure Liquid Chromatography; 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; Role; Site; Specificity; Structure; System; Techniques; Tetrazoles; Transcriptional Activation; Tyrosine Phosphorylation; Tyrosine Phosphorylation Site; 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命系统中生物分子结构和功能的强大工具。结合体内位点选择性地将新型化学反应性引入生物分子的最新进展，生物正交化学为监测和扩展生命系统中的生物分子功能提供了前所未有的机会。我们的长期目标是开发光诱导生物正交反应的工具箱，并将其应用于研究生命系统中的蛋白质功能。我们正在开发的生物正交反应是基于我们对不寻常杂环的化学见解，这些杂环在热力学上是稳定的，但会经历快速的光诱导开环，产生高反应性中间体。然后，这些中间体选择性地与生命系统中的同源、外部引入的伙伴发生反应。在初步研究中，我们展示了二芳基四唑和烯烃之间的第一个光诱导生物正交反应，及其在生物缓冲液和活大肠杆菌细胞中蛋白质的位点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展该反应工具箱的范围和实用性：1）识别对未活化烯烃具有增强反应性的四唑； 2) 开发基于二芳基氮丙啶的光诱导生物正交反应； 3）制定在活细胞中功能化新合成蛋白质的总体策略； 4) 探测活细胞中的蛋白质翻译后修饰，例如脂化和磷酸化。我们希望这些新进展将使体内蛋白质的功能研究成为可能，在分子水平上具有精确的特异性，在系统水平上具有操作简单性。我们的具体目标如下：（1）优化四唑的反应活性并开发基于二芳基氮丙啶的光诱导生物正交反应。将探索基于“推拉”假设的取代效应，以实现对未活化烯烃的选择性和增强的反应性。 (2) 通过共翻译烯烃掺入，然后用基于四唑的化学物质进行选择性功能化，制定标记哺乳动物细胞中新合成蛋白质的一般策略。提出了实验来检查几种活化的烯烃氨基酸的共翻译活性以及它们随后被四唑化合物官能化。 (3) 应用基于四唑的生物正交化学来模拟活细胞中的 Ras 脂质化，并探讨脂质结构对 Ras 膜靶向动力学、特异性和功能的作用。本研究将采用内含肽介导的化学连接和琥珀密码子抑制方法构建四唑编码的 N-Ras 突变体。 (4) 应用基于四唑的生物正交化学通过使用天然化学连接和琥珀密码子抑制技术在酪氨酸磷酸化位点 (Tyr-701) 处掺入四唑氨基酸来模拟 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化 STAT-1 二聚化、核运输和转录激活的影响。 公共卫生相关性：用于研究复杂和动态生物问题的化学工具的开发是化学生物学的一个核心挑战。作为一类新型化学工具，生物正交反应极大地促进了我们对生命系统中生物分子结构、功能和动力学的理解，然而，现有生物正交反应的各种局限性阻碍了其在生物医学研究中的更广泛应用。该提案致力于开发一类具有许多理想反应属性的光诱导生物正交反应，及其在新合成蛋白质功能化中的应用，以及活细胞中蛋白质翻译后修饰（例如脂化和磷酸化）动力学的研究。