Bioorthogonal Tetrazine Carbamate Cleavage by Highly Reactive Trans-Cyclooctene

<p>The high reaction rate of the 'click-to-release' reaction between allylic substituted <i>trans</i>-cyclooctene and tetrazine 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 with up to 3 orders of magnitude higher reactivity compared to the parent reaction, and 4 to 6 orders higher than other cleavage reactions. In this new pyridazine elimination mechanism, wherein the roles a reversed, a <i>trans</i>-cyclooctene activator reacts with a tetrazine that is substituted with a methylene-linked carbamate, leading to an 1,4-elimination of the carbamate and liberation of an 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 activator leads to a complete reaction with antibody-drug conjugate in seconds <i>vs</i>. hours for the parent system. 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. </p>