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

Monosubstituted tetrazines are important bioorthogonal
reactive
tools due to their rapid ligation with trans-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 (k2 > 104 M–1 s–1 toward trans-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 trans-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 in vivo reactions in living mice, achieving
an efficient local anchoring of proteins. These tetrazines are expected
to be optimal bioorthogonal reactive tools within living systems.