Bicyclobutane\nCarboxylic Amide as a Cysteine-Directed\nStrained Electrophile for Selective Targeting of Proteins

Expanding\nthe repertoire of electrophiles with unique reactivity\nfeatures would facilitate the development of covalent inhibitors with\ndesirable reactivity profiles. We herein introduce bicyclo[1.1.0]­butane\n(BCB) carboxylic amide as a new class of thiol-reactive electrophiles\nfor selective and irreversible inhibition of targeted proteins. We\nfirst streamlined the synthetic routes to generate a variety of BCB\namides. The strain-driven nucleophilic addition to BCB amides proceeded\nchemoselectively with cysteine thiols under neutral aqueous conditions,\nthe rate of which was significantly slower than that of acrylamide.\nThis reactivity profile of BCB amide was successfully exploited to\ndevelop covalent ligands targeting Bruton’s tyrosine kinase\n(BTK). By tuning BCB amide reactivity and optimizing its disposition\non the ligand, we obtained a selective covalent inhibitor of BTK.\nThe in-gel activity-based protein profiling and mass spectrometry-based\nchemical proteomics revealed that the selected BCB amide had a higher\ntarget selectivity for BTK in human cells than did a Michael acceptor\nprobe. Further chemical proteomic study revealed that BTK probes bearing\ndifferent classes of electrophiles exhibited distinct off-target profiles.\nThis result suggests that incorporation of BCB amide as a cysteine-directed\nelectrophile could expand the capability to develop covalent inhibitors\nwith the desired proteome reactivity profile.