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.