Described is a bioorthogonal reaction that proceeds with unusually fast reaction rates without need for catalysis: the cycloaddition of s-tetrazine and trans-cyclooctene derivatives. The reactions tolerate a broad range of functionality, and proceed in high yield in organic solvents, water, cell media or in cell lysate. The rate of the ligation between trans-cyclooctene and 3,6-di-(2-pyridyl)-stetrazine is very rapid (k 2 2000 M −1 s −1 ). This fast reactivity enables protein modification at low concentration.Described herein is a bioorthogonal reaction that proceeds with unusually fast reaction rates without need for catalysis: the cycloaddition of s-tetrazine and trans-cyclooctene derivatives. The reactions tolerate a broad range of functionality, and proceed in high yield in organic solvents, water, cell media or in cell lysate. The rate of the ligation between trans-cyclooctene and 3,6-di-(2-pyridyl)-s-tetrazine is very rapid (k 2 2000 M −1 s −1 ). This fast reactivity enables protein modification at low concentration.Bioorthogonal reactions-unnatural transformations that are unaffected by biological functionality-are broadly useful tools with applications that span synthesis, chemical biology, and materials science. 1 The utility of bioorthogonal reactivity has been augmented by recent developments in post-translational strategies for incorporating bioorthogonal functionality into proteins. 2 Bioorthogonal reactions must be exceptionally fast in order to be useful at the low concentrations relevant to many biological applications. Recently, Bertozzi and coworkers described strain-driven click reactions that take advantage of the intrinsic reactivity of cyclooctyne toward organic azides, 3,4 and work by Bertozzi 4a-d and by Boons 4e has shown that click reactions of cyclooctyne derivatives are fast (up to k 2 2.3 M −1 s −1 ). 4 Importantly, this method avoids Cu-catalysts, which are cytotoxic, and the enhanced reactivity enables applications for dynamic in vivo imaging. 4 Recently, Lin and coworkers elegantly described bioconjugation based on a photoinducible 1,3-dipolar cycloaddition reaction that proceeds with fast rates (k 2 11 M −1 s −1 ) with acrylamide. 5 Faster ligation chemistry will allow for the E-mail: jmfox@udel.edu. Unlike normal-electron-demand Diels-Alder chemistry, 6 inverse-electron demand DielsAlder reactions have not previously been applied to bioconjugation. Tetrazines are voracious dienes for inverse-electron-demand Diels-Alder reactions, and N 2 is produced as the only byproduct upon subsequent retro-[4+2] cycloaddition. 7 In 1990, Sauer described the kinetics of electron deficient tetrazines (Scheme 1, structure 1, where R=CO 2 Me or CF 3 ) with a number of dienophiles, and quantitatively demonstrated that their reactions with strained alkenes are exceptionally fast. 8 The most reactive dienophile is trans-cyclooctene, which is 7 orders of magnitude more reactive than cis-cyclooctene toward these tetrazines. 8 In protic solvents, the 4,5-dihydropyridizine 2 rapidly rearranges t...

Rapid, site-specific labeling of proteins with diverse probes remains an outstanding challenge for chemical biologists. Enzyme-mediated labeling approaches may be rapid but use protein or peptide fusions that introduce perturbations into the protein under study and may limit the sites that can be labeled, while many “bioorthogonal” reactions for which a component can be genetically encoded are too slow to effect quantitative site-specific labeling of proteins on a time scale that is useful for studying many biological processes. We report a fluorogenic reaction between bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) and tetrazines that is 3–7 orders of magnitude faster than many bioorthogonal reactions. Unlike the reactions of strained alkenes, including trans-cyclooctenes and norbornenes, with tetrazines, the BCN–tetrazine reaction gives a single product of defined stereochemistry. We have discovered aminoacyl-tRNA synthetase/tRNA pairs for the efficient site-specific incorporation of a BCN-containing amino acid, 1, and a trans-cyclooctene-containing amino acid 2 (which also reacts extremely rapidly with tetrazines) into proteins expressed in Escherichia coli and mammalian cells. We demonstrate the rapid fluorogenic labeling of proteins containing 1 and 2 in vitro, in E. coli, and in live mammalian cells. These approaches may be extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on labeling and imaging studies.

Computation was used to design a trans-cyclooctene derivative that displays enhanced reactivity in the tetrazine-trans-cycloctene ligation. The optimized derivative is a (E)-bicyclo[6.1.0]non-4-ene with a cis-ring fusion, in which the eight-membered ring is forced to adopt a highly strained ‘half-chair’ conformation. Toward 3,6-dipyridyl-s-tetrazine in MeOH at 25 °C, the strained derivative is 19 and 27 times more reactive than the parent trans-cyclooctene and 4E-cyclooct-4-enol, respectively. Toward 3,6-diphenyl-s-tetrazine in MeOH at 25 °C, the strained derivative is 160 times more reactive than the parent trans-cyclooctene.

Bioorthogonal ligation methods with improved reaction rates and less obtrusive components are needed for site-specifically labeling proteins without catalysts. Currently no general method exists for in vivo site-specific labeling of proteins that combines fast reaction rate with stable, nontoxic and chemoselective reagents. To overcome these limitations we have developed a tetrazine-containing amino acid, 1, that is stable inside living cells. We have site-specifically genetically encoded this unique amino acid in response to an amber codon allowing a single 1 to be placed at any location in a protein. We have demonstrated that protein containing 1 can be ligated to a conformationally strained trans-cyclooctene, 2, in vitro and in vivo with reaction rates significantly faster than most commonly used labeling methods.

A radiolabeling method for bioconjugation based on the Diels-Alder reaction between 3,6-diaryl-s-tetrazines and an 18F-labeled trans-cyclooctene is described. The reaction proceeds with exceptionally fast rates, making it an effective conjugation method within seconds at low micromolar concentrations.