组蛋白(Histone)去泛素化酶(hDUB)是表观遗传调控的关键去修饰酶，已成为极具潜力的药物发现靶标。共价捕获催化中心的活性探针(ABP: Activity-based probe)作为研究去泛素化酶的核心工具已得到广泛应用。然而，特异性靶向hDUB需求基于泛素修饰组蛋白的ABP(UbH-ABP)，而其制备仍是蛋白质化学合成领域的重要挑战。因此，本项目聚焦于UbH-ABP的化学合成：1.基于多肽酰肼连接和硒代氨基酸的选择性消除反应，发展化学全合成方法。2.应用新设计的含有掩蔽麦氏受体的双功能连接臂，开发半合成方法。3.设计和合成新结构类型的UbH-ABP。4.以UbH-ABP为工具分析hDUB的底物特异性及其复合物的冷冻电镜结构。该项目有望形成独特的分子工具以促进hDUB的机制和药理干预研究，并在蛋白质化学合成方面产生重要的技术创新。

Histone deubiquitinases (hDUBs) are key enzymes in epigenetics and have emerged as attractive targets for pharmacological intervention. Activity-based probes (ABPs) covalently reacting with the active center have been widely used as powerful tools for studying deubiquitinases. However, interrogating the specificity of hDUBs requires ubiquitinated histone based ABPs(UbH-ABPs), whose preparation is still challenging in the field of chemical protein synthesis. Therefore, this project will focus on the chemical synthesis of UbH-ABPs. Firstly, we plan to establish the efficient total chemical synthesis of these probes based on the ligation of peptide hydrazides and the selective elimination of seleno-amino acids. Secondly, using newly designed bifunctional handles containing masked Michael acceptors, methods for semi-synthesis will be developed. Thirdly, a new type of ABP will be designed and synthesized. Finally, using UbH-ABPs as tools to investigate the substrate specificity of hDUBs and solving Cryo-EM structure of hDUB complex. This project is expected to produce unique tools enabling the mechanism and pharmacological intervention studies of hDUBs, as well as important technological innovations in chemical protein synthesis.