Bioorthogonally activated reactive species for target identification

A target identification platform derived from the bioorthogonal activation of reactive species is described. We explore the reactivity of halogenated enamine N-oxides and report that the previously undisclosed α,γ-halogenated enamine N-oxides can be reduced biooorthogonally by diboron reagents to produce highly electrophilic α,β-unsaturated haloiminium ions suitable for labeling a range of amino acid residues on proteins in a 1,2- or 1,4-fashion. Affinity labeling reagents bearing this motif enable ligand-directed protein modification and afford highly sensitive and selective target identification in unbiased chemoproteomics experiments. Target identification is supported in both cell lysate and live cells. [Display omitted] •Reagent-driven platform for target identification using bioorthogonal activation•Access to highly reactive haloiminium ions that label proteins effectively•Ligand-directed protein modification is operable in cells and cell lysate•High sensitivity and low background labeling complements existing photochemical tools Target identification is an important step in the drug discovery process that involves identifying the target of a pharmacologically active small molecule or the collection of off-target proteins that contribute to the overall risk profile of a drug candidate. In phenotype-driven drug discovery, target identification is often a rate-limiting yet essential step that precedes structure-guided drug development. Affinity-based label transfer strategies have been widely adopted for target identification and involve the induced covalent capture of a protein target directed by the ligand. Although photoaffinity labeling (PAL) is frequently used, more effective tools are desired. Here, we introduce a chemically activated reactive species platform that leverages bioorthogonal chemistry together with the unique chemistry of enamine N-oxides to enable target identification of bioactive small molecules with a level of sensitivity and specificity that compares favorably to existing methods. A platform for target identification using bioorthogonally activated reactive species (BARS) has been developed. The rapid, chemically induced reduction of halogenated enamine N-oxides is employed to generate highly electrophilic haloiminium ions capable of labeling proteins in a ligand-dependent manner. Affinity labeling reagents leveraging this chemistry were applied in immunoblot-based target validation and unbiased chemoproteomics experiments to d