Protein Labeling and Intramolecular Cross-Arylation via Rational Tuning of Bis-Halogenated Pyridiniums

Efficient and highly selective protein arylation labeling and cross-linking under mild and biocompatible conditions remain significantly underexplored. Such reactions form nucleophile-sp 2 carbon bonds, enabling functionalization of biomacromolecules or rigid short-chain cross-linking. This work achieves efficient labeling and cross-linking of primary amine-containing amino acids (lysine and N-terminus) containing peptides and proteins through rational modulation of substituent electronic effects and leaving groups in pyridinium platforms. Concurrent DFT simulations clearly demonstrate the stabilizing effect of electron-withdrawing substituents on transition states in aromatic nucleophilic substitution (S N Ar) pathways. Furthermore, probes designed based on this principle enable efficient labeling on a global proteome-wide scale. Finally, using bovine serum albumin (BSA) as exemplar model, we demonstrate that bis-halogenated pyridinium-based cross-linker (∼5 Å) achieves intramolecular lysine–cysteine cross-arylation under mild conditions (pH 7.4).