Bioorthogonal Tetrazine Carbamate Cleavage by Highly Reactive <i>trans</i>-Cyclooctene

The high rate of the ‘click-to-release’ reaction between an allylic substituted <i>trans</i>-cyclooctene linker and a tetrazine activator has enabled exceptional control over chemical and biological processes. Here we report the development of a new bioorthogonal cleavage reaction based on <i>trans</i>-cyclooctene and tetrazine, which allows the use of highly reactive <i>trans</i>-cyclooctenes, leading to 3 orders of magnitude higher click rates compared to the parent reaction, and 4 to 6 orders higher than other cleavage reactions. In this new pyridazine elimination mechanism, wherein the roles are reversed, a <i>trans</i>-cyclooctene activator reacts with a tetrazine linker that is substituted with a methylene-linked carbamate, leading to a 1,4-elimination of the carbamate and liberation of a secondary amine. Through a series of mechanistic studies, we identified the 2,5-dihydropyridazine tautomer as the releasing species and found factors that govern its formation and subsequent fragmentation. The bioorthogonal utility was demonstrated by the selective cleavage of a tetrazine-linked antibody–drug conjugate by <i>trans</i>-cyclooctenes, affording efficient drug liberation in plasma and cell culture. Finally, the parent and the new reaction were compared at low concentration, showing that the use of a highly reactive <i>trans</i>-cyclooctene as the activator leads to a complete cycloaddition reaction with the antibody–drug conjugate in seconds vs hours for the parent system. Although the subsequent release from the IEDDA adduct is slower, we believe that this new reaction may allow markedly reduced click-to-release reagent doses <i>in vitro</i> and <i>in vivo</i> and could expand the application scope to conditions wherein the <i>trans</i>-cyclooctene has limited stability.