Proteolysis‐Assisted Cyclization Facilitates Site‐Centric Target Deconvolution of Isothiocyanates

Isothiocyanates (ITCs) are a unique class of electrophilic natural products that exert biological effects by reacting with proteinous cysteines to generate thionoacyl adducts. However, the identification of ITCs’ target sites is still an unmet task due to the high lability of such adducts. Here, we report an unexpected chemistry through which the ITC‐protein adduct forms a stable N‐terminal dihydrothiazole peptide adduct during proteolysis. This proteolysis‐assisted cyclization (PAC) reaction can be harnessed for developing affinity‐based and activity‐based chemoproteomic methods to site‐specifically profile targets of ITCs. Applying these methods not only adds further complexity to the known polypharmacological landscape of sulforaphane but also expands the ligandable cysteinome with site‐level resolution through a 55‐member ITC library. Given the promising chemopreventive and therapeutic effects of ITCs, the PAC‐based chemoproteomic platform may lay the groundwork for elucidating their mechanisms of action and ultimately diversifying cysteine targetability for drug discovery.