Development and Applications of Photoinducible Bioorthogonal Chemistry

光诱导生物正交化学的发展及应用 摘要 生物正交化学已成为探索生物分子结构和功能的有力工具 系统。结合将新的化学反应性引入生物分子位置的最新进展- 在体内选择性地，生物正交化学提供了一个前所未有的机会来监测和扩展 生命系统中的生物分子功能。我们的长期目标是开发一种光诱导工具箱 生物正交反应，并将其应用于研究生命系统中的蛋白质功能。生物正交表 我们正在开发的反应是从我们的化学洞察力建立起来的，这些不寻常的杂环是 热力学稳定，但会经历快速的光致开环以产生高活性 中间体。然后，这些中间体有选择地与它们的同类、外部介绍的合作伙伴在 生命系统。在初步研究中，我们展示了第一个光诱导的生物正交反应 二芳基四氮唑和烯烃及其在蛋白质定点修饰中的应用 缓冲液和活的大肠杆菌细胞中。在这个项目中，我们建议显著扩大 该反应工具箱通过：1)鉴定对未活化的烯烃具有增强的反应性的四氮唑； 开发一种基于光诱导的双芳氮杂环丙烷生物正交反应；3)开发一种通用的策略 在活细胞中使新合成的蛋白质功能化；以及4)探测蛋白质翻译后修饰 例如活细胞中的脂化和磷酸化。我们希望这些新的发展将使功能 蛋白质在体内的研究具有分子水平上的高度特异性和系统操作的简便性 水平。我们的具体目标是：(1)优化四唑类化合物的反应活性，开发一种双芳氮杂环-氮杂环丙烷。 基于光诱导的生物正交反应。基于“推拉”假说的取代基效应将是 探索实现对未活化的烯烃的选择性和增强的反应性。(2)发展一种 通过共翻译烯烃标记哺乳动物细胞中新合成蛋白质的一般策略 与四氮唑为基础的化学合成，然后是选择性官能化。实验是 建议研究几种活化的烯烃氨基酸及其共翻译活性 随后由四唑类化合物进行官能化。(3)四唑生物正交法的应用 用化学方法模拟活细胞中RAS的脂化作用并探讨RAS膜上脂质结构的作用 目标动态、特异性和功能。内含素介导的化学连接和琥珀密码子 本研究将采用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模拟STAT-1酪氨酸磷酸化 使用两种天然化学物质在酪氨酸磷酸化位点(Tyr-701)引入四唑氨基酸 连接和琥珀密码子抑制技术。我们将研究化学磷酸化对 活细胞中STAT-1的工程二聚化、核运输和转录激活。

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.