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

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.