Development and Applications of Photoinducible Bioorthogonal Chemistry

光诱导生物正交化学的发展和应用摘要生物正交化学已经成为探测生命系统中生物分子结构和功能的有力工具。结合在体内将新的化学反应性位点选择性地引入生物分子中的最新发展，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个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从我们对不寻常的杂环的化学见解出发的，这些杂环是化学稳定的，但会经历快速的光诱导开环以产生高度反应性的中间体。这些中间体然后选择性地与它们的同源物反应，外部引入的伙伴在生命系统中。在初步研究中，我们首次报道了二芳基四唑与烯烃之间的光诱导生物正交反应，以及它在生物缓冲液和活大肠杆菌中蛋白质定点修饰中的应用。coli细胞。在这个项目中，我们提出了显着扩大的范围和实用性，这个反应工具箱：1）确定四唑与未活化的烯烃增强的反应活性; 2）开发一个光诱导的二芳基精氨酸为基础的生物正交反应; 3）开发一个通用的战略功能化新合成的蛋白质在活细胞中;和4）探测活细胞中的蛋白质翻译后修饰，例如脂化和磷酸化。我们希望这些新的发展将使蛋白质的功能研究在体内与精致的特异性在分子水平和操作简单的系统水平。具体目标如下：（1）优化四唑类化合物的反应活性，发展基于二芳基吲哚的光诱导生物正交反应。基于“推拉”假设的取代基效应将被探索以实现对未活化烯烃的选择性和增强的反应性。(2)开发一种通用的策略，用于标记哺乳动物细胞中新合成的蛋白质，通过共翻译烯烃掺入，然后用基于四唑的化学进行选择性功能化。实验提出了几个活化的烯烃氨基酸的共翻译活性和随后的功能化的四唑化合物。(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，并探索脂质结构对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法和琥珀密码子抑制法构建四唑编码的N-Ras突变体。(4)采用天然化学连接和琥珀密码子抑制技术，通过在酪氨酸磷酸化位点（Tyr-701）掺入四唑氨基酸，应用基于四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STAT-1二聚化、核转运和转录激活的影响。 公共卫生相关性：开发化学工具来研究复杂和动态的生物学问题是化学生物学的一个核心挑战。生物正交反应作为一类新的化学工具，极大地促进了人们对生物分子结构、功能和动力学的理解，但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广泛应用。该建议解决了一类光诱导生物正交反应的发展与许多理想的反应属性，和它们的应用功能化新合成的蛋白质，以及蛋白质的翻译后修饰，如脂质化和磷酸化在活细胞中的动力学研究。

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.