生物体系中的生物化学反应（包括键的形成和断裂）通常是在酶催化条件下实现的，然而在非酶的生理条件下，开发有效的用于对生物分子进行化学修饰的反应是一个巨大挑战。近年来，研究者们开发出了一些实用的生物正交反应，这些反应在生物分子的修饰和功能调控等方面发挥了重要作用。然而，由于生物体系的复杂性，在众多的生物正交反应中，同时具备良好生物兼容性和化学选择性并能够适用于不同生物体系的正交反应非常有限。在一个生物反应体系中，能够同时实现生物正交连接和剪切的方法更为稀少。因此，开发新型、高效的生物正交连接和剪切方法是化学生物学领域中的一项重要任务。本文围绕生物正交连接和剪切反应展开研究，取得了如下成果： 发展了一种高效的氯代喹喔啉衍生物与邻二硫酚的生物正交连接和剪切新方法。该方法能够在生理条件下，通过氯代喹喔啉（CQ）与邻二硫酚（DT）的双亲核取代反应得到四环苯并[5,6][1,4]二硫代[2,3-b]喹喔啉产物，同时释放出一些有价值的功能分子。该生物正交反应通过两个容易获得的非天然正交基团自发进行，具有高度的化学选择性和良好的生物兼容性。更重要的是，反应具有良好的动力学性质，25 ?C下，在磷酸盐缓冲溶液（pH 7.4）和乙腈（1:1）的混合溶液中，二级速率常数k2可达到1.3 M-1s-1。此外，反应产物四环苯并[5,6][1,4]二硫代[2,3-b]喹喔啉结构在生理条件下非常稳定，并且在紫外光照射下，该结构能够释放出较强的荧光，这是探针开发和筛选应用的一个关键特性，这种性质使得在对生物分子进行荧光标记时，避免了二次荧光团的引入而对生物分子活性产生影响。首先在模型肽和蛋白质上验证了该CQ-DT反应作为生物正交连接方法的可行性。随后，又设计、合成了三个带有生物素的可剪切链，通过CQ-DT反应成功地将其用于从蛋白质-生物素缀合物中控制蛋白质的释放，利用产物的内置荧光特性，同时实现了对蛋白质的荧光标记。因此，氯代喹喔啉和邻二硫酚的生物正交反应可用于蛋白质的生物正交连接，生物正交剪切以及蛋白质所带标签的替换，为生物分子的修饰和功能调控等应用提供了一种全新的方法。
The biochemical reactions including the formation and cleavage of bonds in the life systems are performed under the catalysis of enzymes. However, it is a great challenge to develop the efficient chemical reactions to modify biomolecules under non-enzymatic physiological conditions. In recent years, some bioorthogonal reactions have been developed, and they play important roles in chemical modification and function regulation of biomolecules. However, the bioorthogonal reactions with good biological compatibility and chemical selectivity are limited in the complex biological systems, and the reactions suitable for both the biorthogonal ligations and cleavages are very rare in a biological system. Therefore, the development of new and highly efficient bioorthogonal reactions is of great importance in chemical biology field. In this dissertation, some bioorthogonal ligations and cleavages were investigated, and the corresponding results were acquired as follows: A novel and highly efficient bioorthogonal ligation and cleavage method has been developed via reactions of chloroquinoxaline (CQ) derivatives and ortho-dithiophenols (DT). Double nucleophilic substitutions of ortho-dithiophenols to chloroquinoxalines provide conjugates containing tetracyclic benzo[5,6][1,4]dithiino[2,3-b]quinoxaline together with release of the other functional molecules. The tetracyclic benzo[5,6][1,4]dithiino[2,3-b]quinoxaline derivatives show strong fluorescence under irradiation of ultraviolet light. The built-in fluorescence avoids active alteration of biomolecules for secondary derivatization with a fluorophore, which is a key property in development and screening of probes. The feasibility of the CQ-DT reactions as the verstile method was confirmed in protein modification with a model protein as the example. Three cleavable linkers were designed and successfully applied in release of the biologically active molecules containing biotin from the protein conjugates. The CQ-DT bioorthogonal reactions can be used as the biorthogonal ligations, bioorthogonal cleavages, and trans-tagging of proteins, and show some advantages including readily accessible unnatural orthogonal groups, appealing reaction kinetics (a second-order rate constant up to 1.3 M-1s-1 in PBS/MeCN), excellent biocompatibility of orthogonal groups, and high stability of conjugates. Therefore, our developed bioorthogonal reactions can be widely applied in chemical modification and function regulation of biomolecules.
