α-Halothioamide warheads with enhanced cysteine reactivity and specificity for covalent protein labelling

Abstract

Abstract Covalent labelling is a promising modality relying on electrophilic warheads that form covalent bonds with their targets. Here we present the oxygen-to-sulfur exchange strategy, that transforms traditional carboxamides to thioamides, yielding electrophilic warheads, including chlorothioacetamides and fluorothioacetamides, with enhanced cysteine reactivity, while retaining aqueous stability and selectivity. We demonstrate their utility in targeted covalent inhibitor development targeting Janus kinase 3 and Bruton’s tyrosin kinase, when installed on relevant scaffolds and also in the development of antibody-drug conjugates. Next, alkyne-tagged α-halothioamide probes are evaluated by quantitative chemoproteomics. These studies reveal that the chlorothioacetamide probe preferentially labelled a distinct subset of the proteome, which results in Cys-targeted covalent phosphodiesterase 6δ labelling with functional impact. We discuss that the oxygen-to-sulfur exchange strategy offers alternative cysteine-specific thioamide-derived warheads and enables precise and even late-stage modulation of covalent reactivity, highlighting their promise for covalent probe design and applications in medicinal chemistry and chemical biology.