Programmable Site-Selectivity: pH-Modulated Triazine–Thiol Exchange for Site- and Chemoselective Cysteine Labeling

The chemical modification of peptides is a powerful method to enhance their pharmacological properties, including membrane permeability, metabolic stability, and binding affinity. Over recent decades, advances in chemoselective modifications have enabled the construction of well-defined peptide scaffolds with uniform and precise molecular architectures. However, beyond chemoselectivity, achieving true site-selectivity by differentiating between identical amino acids at distinct positions within complex peptide scaffolds remains a key challenge. So far, site-selectivity of cysteine labeling has been largely restricted to N-terminal cysteines. Herein, a programmable strategy for site-selective cysteine modifications is reported, ultimately enabling precise control over the location of cysteine functionalization within peptides. This is accomplished by employing a triazine–thiol exchange, a dynamic covalent reaction with pH-adjustable site-selectivity. It is shown that under acidic conditions internal cysteines are modified while preserving the N-terminal cysteine functionality. Conversely, at neutral pH, site-selective modification of N-terminal cysteines is achieved. The modification of N-terminal cysteines using triazine–thiol exchange proceeds via an S–N shift, which converts the dynamic linkage into an irreversible modification. Density functional theory computations reveal that the site-selectivity originates from modulation of the formed intermediate, providing insights for future mechanism-based designs of site-selective peptide chemistries. The here presented methodology allows chemists to gain control over site-selectivity and unlock new possibilities for precision peptide engineering.