Advances in Tetrazine Bioorthogonal Chemistry Driven by the Synthesis of Novel Tetrazines and Dienophiles

Bioorthogonal chemistry has found increased application in living systems over the past decade. In particular, tetrazine bioorthogonal chemistry has become a powerful tool for imaging, detection, and diagnostic purposes, as reflected in the increased number of examples reported in the literature. The popularity of tetrazine ligations are likely due to rapid and tunable kinetics, the existence of high quality fluorogenic probes, and the selectivity of reaction. In this Account, we summarize our recent efforts to advance tetrazine bioorthogonal chemistry through improvements in synthetic methodology, with an emphasis on developing new routes to tetrazines and expanding the range of useful dienophiles. These efforts have removed specific barriers that previously limited tetrazine ligations and have broadened their potential applications. Among other advances, this Account describes how our group discovered new methodology for tetrazine synthesis by developing a Lewis acid-promoted, one-pot method for generating diverse symmetric and asymmetric alkyl tetrazines with functional substituents in satisfactory yields. We attached these tetrazines to microelectrodes and succeeded in controlling tetrazine ligation by changing the redox state of the reactants. Using this electrochemical control process, we were able to modify an electrode surface with redox probes and enzymes in a site-selective fashion. This Account also describes how our group improved the ability of tetrazines to act as fluorogenic probes by developing a novel elimination-Heck cascade reaction to synthesize alkenyl tetrazine derivatives. In this approach, tetrazine was conjugated to fluorophores to produce strongly quenched probes that, after bioorthogonal reaction, are "turned on" to enhance fluorescence, in many cases by >100-fold. These probes have allowed no-wash fluorescence imaging in living cells and intact animals. Finally, this Account reviews our efforts to expand the range of dienophile substrates to make tetrazine bioorthogonal chemistry compatible with specific biochemical and biomedical applications. We found that methylcyclopropene is sufficiently stable and reactive in the biological milieu to act as an efficient dienophile. The small size of the reactive tag minimizes steric hindrance, allowing cyclopropene to serve as a metabolic reporter group to reveal biological dynamics and function. We also used norbornadiene derivatives as strained dienophiles to undergo tetrazine-mediated transfer (TMT) reactions involving tetrazine ligation followed by a retro-Diels-Alder process. This TMT reaction generates a pair of nonligating products. Using nucleic acid-templated chemistry, we have combined the TMT reaction with our fluorogenic tetrazine probes to detect endogenous oncogenic microRNA at picomolar concentrations. In a further display of dienophile versatility, we used a novel vinyl ether to cage a near-infrared fluorophore in a nonfluorescent form. Then we opened the cage in a "click to release" tetrazine bioorthogonal reaction, restoring the fluorescent form of the fluorophore. Combining this label with a corresponding nucleic acid probe allowed fluorogenic detection of target mRNA. In summary, this Account describes improvements in tetrazine and dienophile synthesis and application to advance tetrazine bioorthogonal chemistry. These advances have further enabled application of tetrazine ligation chemistry, not only in fundamental research but also in diagnostic studies.