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(申请人提供)：光诱导生物正交化学的发展和应用摘要生物正交化学已成为探索生命系统中生物分子结构和功能的有力工具。结合在活体内将新的化学反应引入到生物分子中的新进展，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一个光诱导生物正交反应工具箱，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建立在我们对不寻常的杂环的化学洞察力的基础上，这些杂环在热力学上是稳定的，但通过快速的光诱导开环来产生高活性的中间体。然后，这些中间体有选择地与它们的同类、外部引入的伙伴在生活系统中反应。在初步研究中，我们展示了第一个光诱导的二芳基四氮唑和烯烃之间的生物正交反应，以及它在生物缓冲液和活的大肠杆菌细胞中对蛋白质的定点修饰的应用。在这个项目中，我们建议通过以下方式显著扩展这个反应工具箱的范围和用途：1)鉴定对未活化的烯烃具有更高反应活性的四唑类化合物；2)开发基于光诱导的二芳基氮杂环的生物正交反应；3)开发在活细胞中使新合成的蛋白质功能化的一般策略；以及4)探索活细胞中蛋白质的翻译后修饰，如脂肪和磷酸化。我们希望这些新的发展将使蛋白质在体内的功能研究能够在分子水平上具有精致的特异性，在系统水平上操作简单。我们的具体目标如下：(1)优化四唑类化合物的反应活性，发展一种基于二芳基氮杂环丙烷的光诱导生物正交反应。基于“推-拉”假说的取代基效应将被用来实现对未活化的烯烃的选择性和增强的反应性。(2)开发一种通用的策略，通过共翻译的烯烃掺入，然后用四唑类化学选择性官能化来标记哺乳动物细胞中新合成的蛋白质。通过实验考察了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类化合物官能化的情况。(3)应用四唑类生物正交化学方法模拟活体细胞中RAS的脂化反应，探讨脂质结构对RAS膜靶向动力学、特异性和功能的影响。本研究将采用内含素介导的化学连接和琥珀密码子抑制两种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模拟STAT-1酪氨酸磷酸化，利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)引入四唑氨基酸。我们将研究化学磷酸化对活细胞中工程STAT-1二聚化、核运输和转录激活的影响。 公共卫生相关性：开发用于研究复杂和动态生物学问题的化学工具是化学生物学中的一个中心挑战。生物正交反应作为一类新的化学工具，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种种局限性阻碍了其在生物医学研究中的广泛应用。这项建议致力于发展一类具有许多理想反应属性的光诱导生物正交反应，以及它们在新合成蛋白质功能化以及活细胞中蛋白质翻译后修饰(如脂肪和磷酸化)动力学研究中的应用。