Conformationally strained trans-cyclooctene with improved stability and excellent reactivity in tetrazine ligation

Computation has guided the design of conformationally-strained dioxolane-fused <i>trans</i>-cyclooctene (d-TCO) derivatives that display excellent reactivity in the tetrazine ligation. A water soluble derivative of 3,6-dipyridyl-<i>s</i>-tetrazine reacts with d-TCO with a second order rate <i>k</i>₂ 366,000 (+/- 15,000) M^-1s^-1 at 25 °C in pure water. Furthermore, d-TCO derivatives can be prepared easily, are accessed through diastereoselective synthesis, and are typically crystalline bench-stable solids that are stable in aqueous solution, blood serum, or in the presence of thiols in buffered solution. GFP with a genetically encoded tetrazine-containing amino acid was site-specifically labelled <i>in vivo</i> by a d-TCO derivative. The fastest bioorthogonal reaction reported to date [<i>k</i>₂ 3,300,000 (+/- 40,000) M^-1s^-1 in H₂O at 25 °C] is described herein with a cyclopropane-fused <i>trans</i>-cyclooctene. d-TCO derivatives display rates within an order of magnitude of these fastest <i>trans</i>-cyclooctene reagents, and also display enhanced stability and aqueous solubility.