Biomolecule labeling in living systems is crucial for understanding biological processes and discovering therapeutic targets. A variety of labeling warheads have been developed for multiple biological applications, including proteomics, bioimaging, sequencing, and drug development. Quinone methides (QMs), a class of highly reactive Michael receptors, have recently emerged as prominent warheads for on-demand biomolecule labeling. Their highly flexible functionality and tunability allow for diverse biological applications, but remain poorly explored at present. In this regard, we designed, synthesized, and evaluated a series of new QM probes with a trifluoromethyl group at the benzyl position and substituents on the aromatic ring to manipulate their chemical properties for biomolecule labeling. The engineered QM warhead efficiently labeled proteins both in vitro and under living cell conditions, with significantly enhanced activity compared to previous QM warheads. We further analyzed the labeling efficacy with the assistance of density functional theory (DFT) calculations, which revealed that the QM generation process, rather than the reactivity of QM, contributes more predominantly to the labeling efficacy. Noteworthy, twelve nucleophilic residues on the BSA were labeled by the probe, including Cys, Asp, Glu, His, Lys, Asn, Gln, Arg, Ser, Thr, Trp and Tyr. Given their high efficiency and tunability, these new QM warheads may hold great promise for a broad range of applications, especially spatiotemporal proteomic profiling for in-depth biological studies.

Tryptophan (Trp) plays a critical role in the regulation of protein structure, interactions and functions through its π system and indole N–H group. A generalizable method for blocking and rescuing Trp interactions would enable the gain-of-function manipulation of various Trp-containing proteins in vivo, but generating such a platform remains challenging. Here we develop a genetically encoded N1-vinyl-caged Trp capable of rapid and bioorthogonal decaging through an optimized inverse electron-demand Diels–Alder reaction, allowing site-specific activation of Trp on a protein of interest in living cells. This chemical activation of a genetically encoded caged-tryptophan (Trp-CAGE) strategy enables precise activation of the Trp of interest underlying diverse important molecular interactions. We demonstrate the utility of Trp-CAGE across various protein families, such as catalase-peroxidases and kinases, as translation initiators and posttranslational modification readers, allowing the modulation of epigenetic signalling in a temporally controlled manner. Coupled with computer-aided prediction, our strategy paves the way for bioorthogonal Trp activation on more than 28,000 candidate proteins within their native cellular settings.