Conformationally Strained trans-Cyclooctene with Improved Stability and Excellent Reactivity in Tetrazine Ligation

Abstract

Computation has guided the design of conformationally-strained dioxolane-fused trans-cyclooctene (d-TCO) derivatives that display excellent reactivity in the tetrazine ligation. A water soluble derivative of 3,6-dipyridyl-s-tetrazine reacts with d-TCO with a second order rate k₂ 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 in vivo by a d-TCO derivative. The fastest bioorthogonal reaction reported to date [k₂ 3,300,000 (+/- 40,000) M^-1s^-1 in H₂O at 25 °C] is described herein with a cyclopropane-fused trans-cyclooctene. d-TCO derivatives display rates within an order of magnitude of these fastest trans-cyclooctene reagents, and also display enhanced stability and aqueous solubility.

Scheme 1

(a) Conformation analysis of ground states show that the half- chair conformation of TCO is higher in energy by 5.6–5.9 kcal/mol relative to the lowest energy crown conformation. (b) As predicted computationally, the conformationally constrained s-TCO (1b) combines with 3,6-diphenyl-s-tetrazine in with a rate that is 160 times faster than trans-cyclooctene itself. (c) For GFP with a genetically encoded tetrazine-containing amino acid, s-TCO enables rapid fluorogenic labelling inside living bacteria.

Scheme 2

Transition state calculations (level) for 3,6-diphenyl-s-tetrazine with (a) trans-cyclooctene and (b) dioxolane-fused trans-cyclooctene 3a. The cis-ring fusion of 3a confines the 8-membered ring to a strained half-chair conformation, resulting in a significantly lower transition state barrier for the cycloaddition.

Scheme 3

Synthesis of d-TCO derivatives

Scheme 4

Comparison of second order rate constants

Scheme 5

Characterization of tetrazine d-TCO reaction on GFP (a) in vitro labelling rates of d-TCOs with tetrazine unnatural amino acid derived GFP 2 (b) Q-TOF mass spectrum from the in vivo labelling of syn-3a and 2. Near quantitative conversion of 2 with syn-3a demonstrating specific conversion to 16a (expected 28130 Da; observed 28130 ± 1 Da). Each sample did show a small peak at −131 ±1 Da indicating minor amounts of peptidase-based removal of N-terminal methionines and +22 sodium adducts.