Fast and sensitive pretargeted labeling of cancer cells through a tetrazine/trans-cyclooctene cycloaddition.

There is considerable interest in the use of bioorthogonal covalent chemistry, such as “click” reactions, to label small molecules located on live or fixed cells. Such labeling has been used for the visualization of glycans, activity-based protein profiling, the site-specific tagging of proteins, the detection of DNA and RNA synthesis, investigation of the fate of small molecules in plants, and the detection of posttranslational modification in proteins. Most reported applications rely on either the copper-catalyzed azide–alkyne cycloaddition, which is limited to in vitro application owing to the cytotoxicity of copper, or the elegant strain-promoted azide–alkyne cycloaddition, which is suitable for live-cell and in vivo application but is hindered by relatively slow kinetics and the often difficult synthesis of cyclooctyne derivatives. New bioorthogonal reactions that do not require a catalyst and show rapid kinetics are therefore of interest for different molecular-imaging applications at the cellular level. Herein we demonstrate the use of an inverse-electron-demand Diels– Alder cycloaddition between a serum-stable 1,2,4,5-tetrazine and a highly strained trans-cyclooctene to covalently label live cells. We applied this reaction to the pretargeted labeling of epidermal growth factor receptor (EGFR) tagged with cetuximab (Erbitux) on A549 cancer cells. We found that the tetrazine cycloaddition to trans-cyclooctene-labeled cells is fast and can be amplified by increasing the loading of the dienophile on the antibody. This highly sensitive targeting strategy can be used to label proteins by treatment with a secondary agent at nanomolar concentrations for short durations of time. Recently, we and others explored strain-promoted inverse-electron-demand Diels–Alder cycloaddition reactions of 1,2,4,5-tetrazines for bioconjugation. We showed that the cycloaddition of a tetrazine with a norbornene can be applied to the pretargeted imaging of live breast cancer cells. However, the rate of cycloaddition of the tetrazine with norbornene was 1.6m!1 s!1 in serum at 20 8C. This rate is comparable to previously reported rates for optimized azide– cyclooctyne cycloaddition reactions and requires micromolar concentrations for sufficient labeling. On the basis of previously reported rate constants, we decided to investigate the coupling of tetrazines with more-strained dienophiles. Higher rate constants would enable faster and more efficient labeling. Thus, less of the labeling agent would be required, and the background signal would be decreased. Fox and co-workers recently reported the use of a highly strained trans-cyclooctene for bioconjugation. Although the rates reported were impressive, the tetrazine that yielded the fastest rate has limited stability to nucleophiles and aqueous media, with significant degradation observed after several hours. In contrast, we reported the use of the novel asymmetric tetrazine 1, which is very stable in water as well as in whole serum: a prerequisite for in vivo applications. We hypothesized that tetrazine 1 would react with trans-cyclooctene significantly faster than the previously reported norbornene and this would greatly improve the sensitivity of cell labeling by tetrazine cycloaddition. With this goal in mind, we synthesized the trans-cyclooctene dienophile 2 in two steps from a commercially available cyclooctene epoxide. The trans-cyclooctene reacted readily with tetrazine 1 to form isomeric dihydropyrazine conjugation products in greater than 95% yield (Figure 1a; see also the Supporting Information). The trans-cyclooctenol 2 can be converted into the reactive succinimidyl carbonate, and the carbonate can be conjugated to amine-containing biomolecules, such as monoclonal antibodies, through the formation of a carbamate linkage. To determine the secondorder rate constant for the reaction of the tetrazine with the trans-cyclooctene, we modified surface arrays of trans-cyclooctene-functionalized antibodies with a fluorescent tetrazine probe and monitored the fluorescence signal over time (see Figure S3a in the Supporting Information). From these data, we determined a second-order rate constant of 6000" 200m!1 s!1 at 37 8C (see Figure S3b in the Supporting Information). This rate constant is several orders of magnitude higher than the previously reported value for the cycloaddition of tetrazine 1with a norbornene derivative, as well as the previously reported rate constants for bioorthogonal click reactions used to label live cells covalently. To demonstrate the utility of the reaction of a tetrazine with a trans-cyclooctene for live-cell imaging, we chose to label EGFR expressed on A549 lung cancer cells with the anti-EGFR monoclonal antibody cetuximab. The concept of pretargeting is illustrated in Figure 1b. The multistep labeling of monoclonal antibodies is of interest as a result of the long blood half-life of antibodies. This property leads to poor target-to-background ratios when the antibodies are labeled directly with imaging agents or cytotoxins. A small[*] Dr. N. K. Devaraj, R. Upadhyay, Dr. J. B. Haun, Dr. S. A. Hilderbrand, Prof. R. Weissleder Center for Systems Biology, Massachusetts General Hospital Richard B. Simches Research Center 185 Cambridge Street, Suite 5.210, Boston, MA 02114 (USA) Fax: (+1)617-643-6133 E-mail: [email protected] [email protected] Homepage: http://csb.mgh.harvard.edu/ [**] We thank Dr. Ned Keliher for helpful advice. This research was supported in part by NIH grants U01-HL080731 and T32-CA79443. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.200903233. Angewandte Chemie