NSPs: chromogenic linkers for fast, selective, and irreversible 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–C sp${}^2$-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 × 10${}^4$ M${}^{-1}$ cm${}^{-1}$ . 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 in human plasma, fast reaction rates (k${}_{app}$ up to 4.4 M${}^{-1}$ s${}^{-1}$ at 20 °C), high selectivity for cysteine, favorable solubility of the electrophilic moiety and the bathochromic properties of the Click & Lock reaction provide an appealing alternative to existing methods for cysteine conjugation.