Development and Applications of Photoinducible Bioorthogonal Chemistry

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。结合最近在生物分子中引入新的化学反应活性的研究进展，生物正交化学为监测和扩展生物分子在生命系统中的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见解之上，这些杂环是热力学稳定的，但是经过快速的光诱导开环来产生高活性的中间体。然后，这些中间体选择性地与它们在生命系统中外部引入的同源伙伴发生反应。在初步研究中，我们首次在二芳基四唑和烯烃之间进行了光诱导的生物正交反应，并将其应用于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。在这个项目中，我们建议通过以下方式显著扩大这个反应工具箱的范围和效用：1)鉴定对非活化烯烃具有增强反应性的四唑；2)建立光诱导的二氮拉嗪基生物正交反应；3)制定活细胞中新合成蛋白质功能化的总体策略；4)探索活细胞中的蛋白质翻译后修饰，如脂化和磷酸化。我们希望这些新的发展将使体内蛋白质的功能研究在分子水平上具有精致的特异性，在系统水平上具有操作简单性。我们的具体目标是：(1)优化四唑类化合物的反应活性，建立一种基于二芳拉津的光诱导生物正交反应。基于“推拉”假说的取代基效应将被探索，以实现对非活化烯烃的选择性和增强反应性。(2)通过共翻译烯烃结合，然后用四唑基化学选择性功能化，开发一种标记哺乳动物细胞中新合成蛋白质的一般策略。提出了实验来检验几种活化的烯烃氨基酸的共翻译活性及其随后被四唑化合物功能化。(3)应用基于四氮唑的生物正交化学方法模拟Ras在活细胞中的脂化过程，探讨脂质结构在Ras膜靶向动力学、特异性和功能中的作用。本研究将采用内部介导的化学连接和琥珀色密码子抑制方法构建四唑编码的N-Ras突变体。(4)利用天然化学连接和琥珀色密码子抑制技术，在酪氨酸磷酸化位点（Tyr-701）加入四氮唑氨基酸，应用基于四氮唑的生物正交化学模拟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、核转运和转录激活的影响。

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.