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 sp2-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−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 (kapp 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.
This article is Open Access
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