Dual-Performing\nVinyltetrazine for Rapid, Selective\nBioconjugation and Functionalization of Cysteine Proteins

Although methods for Cys-specific bioconjugation and\nfunctionalization\nof proteins have been developed and widely utilized in biomolecule\nengineering and therapeutic development, reagents for this purpose\nare generally designed to accomplish bioconjugation only. Consequently,\nadditional clickable groups must be attached to these reagents to\naccomplish functionalization. Herein, we describe a new, simple, dual-performing\nbioconjugation–functionalization reagent, VMeTz, which possesses\nan electron-withdrawing tetrazine (Tz) substituted vinyl (V) moiety\nto serve as both a Michael receptor for selective conjugation with\nCys and a site for click with TCO derivatives to introduce functionality.\nCritically, VMeTz contains a methyl group that prevents the formation\nof multiple Tz-containing Cys-adducts. Reactions of VMeTz with Cys-containing\npeptides and proteins both in vitro and in live cells produce single\nstable Michael adducts with high selectivity. Moreover, the Cys-VMeTz\npeptide and protein conjugates undergo facile click reactions with\nTCO-functionalized reagents for labeling and protein profiling. Furthermore,\nVMeTz selectively activates and delivers the TCO-caged toxic substances\nDox and PROTAC ARV-771 to cancer cells to produce therapeutic effects\nthat are comparable to those of the parent drugs. Collectively, the\nstudies demonstrate that VMeTz is a useful reagent for therapeutically\nsignificant Cys-specific protein bioconjugation and functionalization.