Abstract
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.