Click, Lock & Dye: a chromogenic handle for selective cysteine modification

The addition of a sulfhydryl group to water-soluble N-alkyl(o-nitrostyryl)pyridinium ions (NSPs) followed by fast and irreversible cyclization and aromatization results in a stable S-Csp2-bond. The reaction sequence, termed Click & Lock, engages accessible cysteine residues under the formation of N-hydroxy indole pyridinium ions. The accompanying red-shift of > 70 nm to around 385 nm enables convenient monitoring of the labeling yield by UV-VIS spectroscopy at extinction coefficients of ≥ 2 × 104 M-1cm-1. The versatility of the linker is demonstrated in the stapling of peptides and the derivatization of proteins, including the modification of reduced Trastuzumab with Val-Cit-PAB-MMAE. The high stability of the linker, fast reaction rates (kapp up to 4.4 M-1s-1 at 20°C), high selectivity, and the bathochromic properties of the Click & Lock reaction provide an appealing alternative to existing methods for cysteine conjugation.