Biological and Medicinal Chemistry

α-Methylene-β-Lactone Probe for Measuring Live-Cell Reactions of Small Molecules



The novel use of the α-methylene-β-lactone (MeLac) moiety as a warhead of multiple electrophilic sites is reported. In this study, we demonstrate that a MeLac-alkyne is a competent covalent probe and reacts with diverse proteins in live cells. Proteomics analysis of affinity-enriched samples identifies probe-reacted proteins, resolves their modified peptides/residues, and thus characterizes probe-protein reactions. Unique methods are developed to evaluate confidence in the identification of the reacted proteins and modified peptides. Tandem mass spectra of the peptides reveal that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael addition or acyl addition. A peptide-centric proteomics platform, using MeLac-alkyne as the measurement probe, successfully analyzes the Orlistat selectivity in live HT-29 cells. MeLac is a versatile warhead demonstrating enormous potential to expedite the development of covalent probes and inhibitors in interrogating protein (re)activity. MeLac-empowered platforms in chemical proteomics are widely adaptable for measuring the live-cell action of reactive molecules.

Version notes

Ver2.0/Modifications of Figures 1 and 2


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Supplementary material

Thumbnail image of MeLac_UConn_XYao_Appn_2020Apr.pdf
MeLac UConn XYao Appn 2020Apr
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MeLac UConn XYao preprint 2020Apr ver2
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MeLac UConn XYao preprint SI 2020Apr ver2