Catalyst-Free Late-Stage Functionalization to Assemble α-acyloxyenamide Electrophiles for Selectively Profiling Conserved Lysine Residues

14 December 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Covalent probes coupled with chemical proteomics represent a powerful method for investigating small molecule and protein interactions. However, the creation of a reactive warhead within various ligands to form covalent probes has been a major obstacle. Herein, we report a convenient and robust process to assemble a unique electrophile, an α-acyloxyenamide, through a one-step late-stage coupling reaction. This procedure demonstrates remarkable tolerance towards other functional groups and facilitates ligand-directed labeling in proteins of interest. The reactive group has been successfully incorporated into a clinical drug targeting the EGFR L858R mutant, erlotinib, and a pan-kinase inhibitor. The resulting probes have been shown to be able to covalently engage a lysine residue proximal to the ATP-binding pocket of the EGFR L858R mutant. A series of active sites, and Mg2+, ATP-binding sites of kinases, such as K33 of CDK1, CDK2, CDK5; K278 of PAK2 and K270 of KPYM were detected. This is the first report of engaging these conserved catalytic lysine residues in kinases with covalent inhibition. Further application of this methodology to natural products has demonstrated its success in profiling ligandable conserved lysine residues in whole proteome. These findings offer insights for the development of new targeted covalent inhibitors (TCIs) and pave the way for future advancements in drug discovery and chemical proteomics.


electrophilic warhead
α-acyloxyenamide Electrophiles
Conserved Lysine Residues
Covalent probes
targeted covalent inhibitors


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