Site-specific protein functionalization by late-stage reversible cysteine masking

Abstract Site-selective functionalization strategies are essential for generating complex proteins for basic research and biomedical applications. However, achieving precise transformations across protein sequences remains a significant challenge and often requires protein engineering with orthogonal handles or sequence interventions to achieve selectivity. Here, we report a modular strategy for site-selective protein functionalization that enables the selective targeting of desired Cys residues through late-stage reversible thiol caging. This strategy involves splitting the protein sequence into two segments and transiently masking native Cys residues to enable chemoselective ligation, thereby restoring the full-length protein with a single reactive Cys for late-stage functionalization. A subsequent decaging step regenerates the native Cys residues, yielding the desired site-specifically modified protein while preserving the native sequence. We demonstrate the versatility of this strategy through multiple transformations, including Cys arylation, alkylation, and elimination, and demonstrate the power of this approach by selective modification of 30 examples in high resolution. Furthermore, we apply this approach to edit Cys-rich zinc fingers, leading to the discovery of advanced analogs with enhanced DNA-binding activity. The versatility and modularity of the developed reversible thiol caging approach establish a powerful platform for site-selective protein modification, providing rapid access to structurally diverse proteins for fundamental research and therapeutic development.