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

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 labelling has been largely restricted to N-terminal cysteines. Here, we report a programmable strategy for site-selective cysteine modifications, 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. We show 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. Molecular dynamics simulations and 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.