Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195 (USA).
Chembiochem. 2014 Jun 16;15(9):1263-7. doi: 10.1002/cbic.201402135. Epub 2014 May 18.
The reversible post-translational modification of eukaryotic proteins by ubiquitin regulates key cellular processes including protein degradation and gene transcription. Studies of the mechanistic roles for protein ubiquitylation require quantities of homogenously modified substrates that are typically inaccessible from natural sources or by enzymatic ubiquitylation in vitro. Therefore, we developed a facile and scalable methodology for site-specific chemical ubiquitylation. Our semisynthetic strategy utilized a temporary ligation auxiliary, 2-(aminooxy)ethanethiol, to direct ubiquitylation to specific lysine residues in peptide substrates. Mild reductive removal of the auxiliary after ligation yielded ubiquitylated peptides with the native isopeptide linkage. Alternatively, retention of the ligation auxiliary yielded protease-resistant analogues of ubiquitylated peptides. Importantly, our strategy was fully compatible with the presence of protein thiol groups, as demonstrated by the synthesis of peptides modified by the human small ubiquitin-related modifier 3 protein.
真核蛋白质的翻译后可逆修饰由泛素调节，包括蛋白质降解和基因转录等关键细胞过程。研究蛋白质泛素化的机制作用需要大量均匀修饰的底物，这些底物通常无法从天然来源获得，也无法通过体外酶促泛素化获得。因此，我们开发了一种简便且可扩展的定点化学泛素化方法。我们的半合成策略利用了一种临时连接辅助剂 2-(氨氧基)乙硫醇，将泛素化引导到肽底物中的特定赖氨酸残基上。连接后温和还原去除辅助剂可得到具有天然异肽键的泛素化肽。或者，保留连接辅助剂可得到蛋白酶抗性的泛素化肽类似物。重要的是，我们的策略与蛋白质巯基的存在完全兼容，这一点通过用人类小泛素相关修饰蛋白 3 修饰的肽的合成得到了证明。
