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.

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.

Bulky, electron-rich phosphine ligands with a biphenyl backbone, when combined with Pd(OAc)2, give highly active catalysts for the α-arylation of ketones. The ligand 2-methyl-2‘-dicyclohexylphosphinobiphenyl is particularly effective, and with 0.1−1.0 mol % Pd, a large variety of aryl halides and ketones react efficiently and with high selectivity. For two types of substrates, the ligands BINAP and Xantphos are more effective than the biphenyl-based ligands. It is also shown that K3PO4 can be used as the base in these reactions, and that base-sensitive functional groups are better tolerated if this is used instead of NaO t Bu or NaHMDS. In some cases, α-aryl ketones can be produced without adding a ligand to the reaction. Although the substrate scope of the ligandless conditions is limited, some combinations react in high yield, and in one case, 100 000 turnovers were obtained. The results of experiments on the Pd-catalyzed arylation of diethyl malonate, cyclic 1,3-diketones, and nitroalkanes are also reported.