新型生物正交反应的研究及应用

The vast complexity of cellular system presents significant challenges for the studies of biomolecules in the native environments. For the past decade, bioorthogonal ligations have been emerging as promising chemical tools to probe the biomolecules in living systems. While powerful, to date only a handful of reactions have been utilized as true bioorthogonal ligations, due to the stringent requirements such as high reactivity and selectivity in a physiological environment, and non-toxic and cell-permeable reagents, reaction product must be stable as well as fast reaction kinetics. In our previous study, we have developed a novel bioorthogonal reaction based on the click hetero-Diels-Alder cycloaddition of o-quinolinone quinone methide (oQQM) and thiol vinylether (TV). This ligation is efficient and robust under physiological environment and is fully compatible with the widely used strain-promoted azide-alkyne cycloaddition. In addition, we have demonstrated that the ligation is a promising bioorthogonal reaction that can be used for site-specific labeling of proteins as well as imaging of bioactive small molecules inside live cells. However, the reaction kinetics of the first generation ligation was not very satisfying. Therefore, in this proposal study, we would like to systematically investigate how to improve the reaction rate of the click reaction for the development of the second generation bioorthogonal ligation. We also plan to further apply the new ligation method in subsequent chemical biology studies, like: 1) dual labeling of proteins and organelles in vitro and in vivo respectively; 2) probing the crucial post-translational modifications of functional proteins.

由于细胞内环境的复杂性，在生理条件下研究生物大分子的功能和生命过程具有很大的挑战性。基于此科学问题，近十年来，化学生物学家开发出生物正交反应并且已经广泛应用于研究各种复杂的生物学问题。近期我们课题组报道了一类基于邻亚甲基喹啉醌和烯基硫醚的点击化学反应所实现的具有原创性的生物正交反应，并成功地运用该反应实现了选择性标记蛋白和对生物活性分子进行活细胞内成像。然而，该生物正交反应急需进一步优化的是反应速率。因此，本课题将围绕对邻亚甲基喹啉醌和烯基硫醚点击化学做系统地改进和优化，以实现更快速和更高效的新一代生物正交反应的开发。此外，本课题还计划进一步将优化好的新一代生物正交反应运用于研究一些复杂的生物学问题，例如：对于复杂生物体系的双标记研究，以及对于重要功能蛋白质的翻译后修饰研究等。

在该面上项目的资助下，申请人课题组开发出一类基于邻亚甲基喹啉醌和烯基硫醚的点击化学反应所实现的具有原创性的生物兼容反应（TQ-ligation），并成功地运用该反应实现了选择性标记蛋白和对生物活性分子进行活细胞内成像。通过围绕对邻亚甲基喹啉醌和烯基硫醚点击化学做系统地改进和优化，进而实现了更快速和更高效的第二代TQ-ligation。此外，申请人还进一步拓展了生物兼容反应的应用范围，开发出新一代化学交联结合质谱技术，并且用于该技术系统地研究了复杂蛋白质结构和揭示了新的蛋白-蛋白相互作用网络。通过以上科学研究，开发出一系列新颖、高效的化学生物学工具，为生物医学研究提供了新的技术手段。