To investigate whether the covalent ligands showed selective labeling of BRD4(1) L94C over BRD4(2), the in-cell labeling assay was repeated using HEK293T cells transiently expressing either the wildtype tandem bromodomain [BRD4(1,2) WT ] or the tandem bromodomain bearing the L94C mutation within the first domain [BRD4(1,2) L94C ]. Compound 42 was observed to selectively label BRD4(1,2) L94C , which was observed as a fluorescent band corresponding to ~80 kDa, while no covalent engagement with BRD4(1,2) WT was observed (Figure and). This result confirms that the labeling results from a selective covalent interaction with the L94C-containing first bromodomain of BRD4(1,2) L94C (Figure ). ) suggesting that subtle variations in the reactivity or vector of the electrophile are responsible for the difference in selectivity observed. The electron-withdrawing nature of the isoxazole ring likely increases the electrophilicity of the acrylamide in 30, while the electron-donating nature of the phenyl ring substituents in 42 have the opposite effect. This idea is supported by the less complete labeling of purified BRD4(1) L94C by 42, compared to 30 (Table ). The lower reactivity of the electrophile means that compound 42 does not react covalently with proteins that it interacts with transiently. The intrinsic non-covalent affinity of 42 for BRD4(1) L94C is required to provide sufficient protein residency time to allow a covalent reaction to occur.