Computation-Guided\nDiscovery of Diazole Monosubstituted\nTetrazines as Optimal Bioorthogonal Tools

Monosubstituted tetrazines are important bioorthogonal\nreactive\ntools due to their rapid ligation with <i>trans</i>-cyclooctene.\nHowever, their application is limited by the reactivity–stability\nparadox in biological environments. In this study, we demonstrated\nthat steric effects are crucial in resolving this paradox through\ntheoretical methods and developed a simple synthetic route to validate\nour computational findings, leading to the discovery of 1,3-azole-4-yl\nand 1,2-azole-3-yl monosubstituted tetrazines as superior bioorthogonal\ntools. These new tetrazines surpass previous tetrazines in terms of\nhigh reactivities and elevated stabilities. The most stable tetrazine\nexhibits a reasonable stability (71% remaining after 24 h incubation\nin cell culture medium) and an exceptionally high reactivity (<i>k</i><sub>2</sub> > 10<sup>4</sup> M<sup>–1</sup> s<sup>–1</sup> toward <i>trans</i>-cyclooctene). Due to\nits good stability in biological systems, a noncanonical amino acid\ncontaining such a tetrazine side chain was genetically encoded into\nproteins site-specifically via an expanded genetic code. The encoded\nprotein can be efficiently labeled using cyclopropane-fused <i>trans</i>-cyclooctene dyes in living mammalian cells with an\nultrafast reaction rate exceeding 10<sup>7</sup> M<sup>–1</sup> s<sup>–1</sup>, making it one of the fastest protein labeling\nreactions reported to date. Additionally, we showed its superiority\nthrough <i>in vivo</i> reactions in living mice, achieving\nan efficient local anchoring of proteins. These tetrazines are expected\nto be optimal bioorthogonal reactive tools within living systems.