The Unique Properties and Click Capacity of the Tetrazine Group Enable Tetrazine Allyl Acetate to Serve as a Versatile Bis-Thiol Bioconjugation and Functionalization Agent

The inverse electron demand Diels–Alder (IEDDA) click reaction of tetrazine (Tz) with trans-cyclooctene (TCO), which takes place at an exceptionally high rate, has excellent orthogonality and biocompatibility. As a result, the unrivaled capacity of this process enables it to serve as a viable chemical tool for protein modification even in humans. However, the utility of this click reaction is impeded by the limited number of practical methods for the incorporation of the Tz moiety into proteins. Herein, we describe the simple dual-functional Tz-substituted allyl acetate reagent, TzAA, that targets bis-thiol moieties for direct, site-selective installation of a Tz group into native peptides and proteins. In addition to functioning as an IEDDA-based click reaction center, the Tz moiety in TzAA activates double Michael cascade reactions of the allylic acetate moiety with bis-thiols or from disulfide derivatives. We demonstrated the versatility and generality of bioconjugation reactions of TzAA that produce well-defined peptide and protein conjugates without compromising biological activities. The Tz-conjugates serve as a versatile handle for facile click reactions to introduce various functionalities. Moreover, the reagent is validated as a versatile linker for the synthesis of stapled peptides with protecting-group-free peptides. As shown, the Tz-derived cyclic peptides offer unrivaled capacity for selective targeting and delivery of TCO-caged prodrugs with reduced side effects. Collectively, the results of this investigation demonstrate that the probe with its unrivaled dual bioconjugation and click capacity, operational simplicity, and high labeling efficiency will offer a powerful chemical tool for direct, site-selective peptide/protein modification.