Computation-Guided Discovery of Diazole Monosubstituted Tetrazines as Optimal Bioorthogonal Tools

Monosubstituted tetrazines are important bioorthogonal reactive tools due to their rapid ligation with <i>trans</i>-cyclooctene. However, their application is limited by the reactivity-stability paradox in biological environments. In this study, we demonstrated that steric effects are crucial in resolving this paradox through theoretical methods and developed a simple synthetic route to validate our computational findings, leading to the discovery of 1,3-azole-4-yl and 1,2-azole-3-yl monosubstituted tetrazines as superior bioorthogonal tools. These new tetrazines surpass previous tetrazines in terms of high reactivities and elevated stabilities. The most stable tetrazine exhibits a reasonable stability (71% remaining after 24 h incubation in cell culture medium) and an exceptionally high reactivity (<i>k</i>₂ > 10⁴ M^-1 s^-1 toward <i>trans</i>-cyclooctene). Due to its good stability in biological systems, a noncanonical amino acid containing such a tetrazine side chain was genetically encoded into proteins site-specifically via an expanded genetic code. The encoded protein can be efficiently labeled using cyclopropane-fused <i>trans</i>-cyclooctene dyes in living mammalian cells with an ultrafast reaction rate exceeding 10⁷ M^-1 s^-1, making it one of the fastest protein labeling reactions reported to date. Additionally, we showed its superiority through <i>in vivo</i> reactions in living mice, achieving an efficient local anchoring of proteins. These tetrazines are expected to be optimal bioorthogonal reactive tools within living systems.