A Picolyl‐Based Cys Caging/Uncaging Strategy Facilitates Protein Synthesis

Abstract Endowed with a reactive thiol group, cysteine (Cys) provides a versatile handle for site‐specific bioconjugation and serves as a cornerstone of chemical protein synthesis, particularly in native chemical ligation (NCL). Extensions such as expressed protein ligation (EPL)‐desulfurization have significantly broadened access to challenging proteins. However, they require orthogonal caging/uncaging protecting groups to enable selective desulfurization in the presence of native cysteines, a process that is crucial for synthetic applications. Photolabile protecting groups (PPGs), which are cleaved via irradiation, offer a simpler and less disruptive approach to protein assembly compared to traditional thiol protecting groups. However, current commercially available PPGs are not compatible with orthogonal protection and EPL‐desulfurization. To address this challenge, we developed a novel and simple picolyl‐based PPG for Cys caging/uncaging, which enables rapid orthogonal caging of thiols and their subsequent uncaging via pH and wavelength control. Notably, the picolyl group undergoes photoorthogonal activation in the presence of a nitrobenzyl group. The efficient synthesis of interleukin‐4 (IL‐4) via one‐pot iterative ligation and tumor necrosis factor‐alpha (TNF‐α) via EPL‐desulfurization further highlights how this strategy significantly advances the synthesis of complex proteins.