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.

Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells

“…We focused on bioorthogonal IEDAC reactions because of their fast reaction rates (up to 10 6 M −1 s −1 ) 66,67 and excellent biocompatibility. In this regard, we chose UAAs bearing strained alkyne or alkene functionalities (Figure 2A), including 1,3-disubstituted cyclopropene-lysine (CpK), 68 bicyclo[6.1.0]nonyne-lysine (BCNK), 54,69 trans -cyclooct-4-ene-lysine (4’-TCOK), 51,54 and axial trans -cyclooct-2-ene-lysine (2’-aTCOK), 55,70,71 all of which can be site-specifically incorporated into proteins expressed in mammalian cells using variants of the pyrrolysyl-tRNA synthetase (PylRS)/Pyl-tRNA CUA pair and react with tetrazines chemoselectively.…”

Enhancing Reactivity for Bioorthogonal Pretargeting by Unmasking Antibody-Conjugated trans-Cyclooctenes

“…Future work could explore whether similar masking effects occur for different hydrophobic reactants such as DBCO, norbornene, and methyl-cyclopropene, alternative linkers directly distal to the TCO such as those containing bulky phenyl groups, 16 or new bicyclic TCO derivatives that have faster reaction rates, greater stability, and, for the dioxolane-fused TCO, potentially greater hydrophilicity. 46,47 Expanding these investigations to different types of proteins or biological molecules, including site-specific methods that involve genetic encoding, 48-50 would also be of interest.…”

Rapid Bioorthogonal Chemistry Turn-on through Enzymatic or Long Wavelength Photocatalytic Activation of Tetrazine Ligation

“…31 With strained trans -cyclooctenes, we have measured bimolecular rate constants as high as 3.3 × 10 6 M −1 s −1 in reactions with tetrazines (Tz), representing the fastest bioorthogonal reactions reported to date. 32,33 Tetrazines are typically synthesized through the chemical oxidation of dihydrotetrazine (DHTz) precursors, 34 and the electroactivity of tetrazines is well established. 35 Recently, Devaraj has demonstrated that electrochemistry can be used to control the redox state of pendant DHTz/Tz groups on the surface of microelectrodes, allowing selective bioconjugation to oxidized electrode surfaces.…”