Development and Applications of Photoinducible Bioorthogonal Chemistry

光诱导生物正交化学的发展与应用 摘要 生物正交化学已成为研究生物分子结构和功能的有力工具 系统.结合最近在生物分子中引入新的化学反应性的进展， 选择性地在体内，生物正交化学提供了前所未有的机会，监测和扩大 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的 生物正交反应，并将其应用于研究蛋白质在生命系统中的功能。生物正交 我们正在开发的反应是基于我们对不寻常的杂环的化学见解， 化学稳定，但仍经历快速光诱导开环以产生高反应性的 中间体的这些中间体然后选择性地与它们的同源的、外部引入的配偶体反应， 生活系统在初步研究中，我们展示了第一个光诱导生物正交反应之间的 二芳基四唑和烯烃，及其在蛋白质的定点修饰中的应用， buffer和活E. coli细胞。在这个项目中，我们建议大大扩大 该反应工具箱通过：1）鉴定对未活化烯烃具有增强反应性的四唑; 2） 开发基于光诱导的二芳基精氨酸的生物正交反应; 3）开发用于以下的一般策略： 在活细胞中功能化新合成的蛋白质;和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 在分子水平上以精确的特异性和系统的操作简单性研究体内蛋白质 水平我们的具体目标如下：（1）优化四唑的反应性，开发二芳基月桂酸酯- 基于光诱导生物正交反应。基于“推拉”假设的替代效应将是 探索以实现对未活化烯烃的选择性和增强的反应性。(2)开发一个 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 引入，然后用基于四唑的化学进行选择性官能化。实验 建议检查几种活化的烯烃氨基酸的共翻译活性及其 随后通过四唑化合物官能化。(3)应用基于四唑的生物正交 化学模拟活细胞中Ras脂化并探测Ras膜上脂质结构的作用 靶向动力学、特异性和功能。内含肽介导的化学连接和琥珀密码子 抑制方法将用于构建用于本研究的四唑编码的N-Ras突变体。（四） 应用基于四唑的生物正交化学模拟STAT-1酪氨酸磷酸化， 在酪氨酸磷酸化位点（Tyr-701）处掺入四唑氨基酸， 连接和琥珀密码子抑制技术。我们将研究化学磷酸化对 工程STAT-1二聚化，核运输，和活细胞中的转录激活。

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.