Joseph M. Fox

Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels−Alder Reactivity

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 cell lysate. The rate of the ligation between trans-cyclooctene and 3,6-di-(2-pyridyl)-s-tetrazine is very rapid (k2 2000 M-1 s-1). This fast reactivity enables protein modification at low concentration.

Genetic Encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels-Alder reactions.

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.

Diels-Alder cycloaddition for fluorophore targeting to specific proteins inside living cells.

The inverse-electron-demand Diels-Alder cycloaddition between trans-cyclooctenes and tetrazines is biocompatible and exceptionally fast. We utilized this chemistry for site-specific fluorescence labeling of proteins on the cell surface and inside living mammalian cells by a two-step protocol. Escherichia coli lipoic acid ligase site-specifically ligates a trans-cyclooctene derivative onto a protein of interest in the first step, followed by chemoselective derivatization with a tetrazine-fluorophore conjugate in the second step. On the cell surface, this labeling was fluorogenic and highly sensitive. Inside the cell, we achieved specific labeling of cytoskeletal proteins with green and red fluorophores. By incorporating the Diels-Alder cycloaddition, we have broadened the panel of fluorophores that can be targeted by lipoic acid ligase.

Genetically Encoded Tetrazine Amino Acid Directs Rapid Site-Specific In Vivo Bioorthogonal Ligation with trans-Cyclooctenes

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 in vitro and in vivo with reaction rates significantly faster than most commonly used labeling methods.

Tetrazine–trans-cyclooctene ligation for the rapid construction of 18F labeled probes

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

Rhodium(II)-catalyzed enantioselective C-H functionalization of indoles.

A catalytic, enantioselective method for the C-H functionalization of indoles by diazo compounds has been achieved. With catalytic amounts of Rh(2)(S-NTTL)(4), the putative Rh-carbene intermediates from α-alkyl-α-diazoesters react with indoles at C(3) to provide α-alkyl-α-indolylacetates in high yield and enantioselectivity. From DFT calculations, a mechanism is proposed that involves a Rh-ylide intermediate with oxocarbenium character.

A Photochemical Synthesis of Functionalized trans-Cyclooctenes Driven by Metal Complexation

Described is a scalable procedure for driving photochemical sytheses of trans-cyclooctene derivatives through metal complexation. During photoirradiation, reaction mixtures are continuously pumped through a column of a AgNO3-impregnated silica gel. The trans-cyclooctene derivative is selectively retained by the AgNO3-impregnated silica, but the cis-isomer elutes from the column back to the reaction flask, where it is photoisomerized and recirculated through the column. The method provides access to a range of usefully functionalized derivatives of trans-cyclooctene, including a derivative of 5-aza-trans-cyclooctene that underwent transannular cyclization upon treatment with bromine. The alkene stereochemistry is transferred with high fidelity to the hexahydropyrrolizine framework in the transannular cyclization.

The Use of Catalytic Amounts of CuCl and Other Improvements in the Benzyne Route to Biphenyl-Based Phosphine Ligands

Biphenyl-based phosphine ligands can be prepared on a significantly larger scale than previously possible as a result of the following discoveries and improvements to the original experimental procedure: the finding that CuCl catalyzes the coupling of hindered dialkylchlorophosphines with Grignard reagents; the development of conditions that permit ClPCy2 to be prepared and utilized in situ; the development of a more reliable large-scale preparation of 2-dimethylaminophenylmagnesium halide.

Development and Evaluation of 18F-TTCO-Cys40-Exendin-4: A PET Probe for Imaging Transplanted Islets

Because islet transplantation has become a promising treatment option for patients with type 1 diabetes, a noninvasive imaging method is greatly needed to monitor these islets over time. Here, we developed an 18F-labeled exendin-4 in high specific activity for islet imaging by targeting the glucagonlike peptide-1 receptor (GLP-1R). Methods: Tetrazine ligation was used to radiolabel exendin-4 with 18F. The receptor binding of 19/18F-tetrazine trans-cyclooctene (TTCO)-Cys40-exendin-4 was evaluated in vitro with INS-1 cell and in vivo on INS-1 tumor (GLP-1R positive) and islet transplantation models. Results: 18F-TTCO-Cys40-exendin-4 was obtained in high specific activity and could specifically bind to GLP-1R in vitro and in vivo. Unlike the radiometal-labeled exendin-4, 18F-TTCO-Cys40-exendin-4 has much lower kidney uptake. 18F-TTCO-Cys40-exendin-4 demonstrated its great potential for transplanted islet imaging: the liver uptake value derived from small-animal PET images correlated well with the transplanted β-cell mass determined by immunostaining. Autoradiography showed that the localizations of radioactive signal indeed corresponded to the distribution of islet grafts in the liver of islet-transplanted mice. Conclusion: 18F-TTCO-Cys40-exendin-4 demonstrated specific binding to GLP-1R. This PET probe provides a method to noninvasively image intraportally transplanted islets.

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

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.