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, 51 (5), 1249-1259

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

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

Haoxing Wu et al. Acc Chem Res.

Abstract

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.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
(A) Optimized reaction condition of one-pot tetrazine synthesis. (B) Representative substrate scope. Reaction yield shown under each product. (C) Synthesis of monosubstituted tetrazine.
Figure 2.
Figure 2.
(A) In situ elimination–Heck cascade reaction. (B) Representative substrate scope. The number of bromide equivalents, reaction time, and yield are shown under each product.
Figure 3.
Figure 3.
(A–C) Fluorogenic reaction of 22–24 with dienophiles. (D) Live-cell imaging of LS174T cells using fluorogenic probe 22. Left: cells were pretargeted with TCO-decorated A33 antibodies. Right: cells were treated with unmodified antibodies. (E) Structure of cyclopropene 25.
Figure 4.
Figure 4.
(A) Phenoxide anion and its resonance structure within various fluorophore scaffolds. (B) Schematic of fluorogenic probe design based on a decaging reaction. (C) Fluorogenic decaging reaction of probe 26 with tetrazine 27. (D) Fluorescence spectra of 26 and its corresponding uncaged cyanine compound 28. (E) Absorption spectra of 26 and 28. (F) Caged fluorogenic probes with different cassettes.
Figure 5.
Figure 5.
(A) Schematic of mRNA imaging using probe-decorated antisense oligonucleotides. (B) Fluorogenic probes covering the visible and near-IR light spectrum based on tetrazine bioorthogonal chemistry.
Figure 6.
Figure 6.
(A) Synthetic of cyclopropene derivatives. (B) Kinetic of cyclopropene–tetrazine reactions.
Figure 7.
Figure 7.
(A) Rate constants of the reaction involving cyclopropene or TCO with selected tetrazines. (B) M06–2X/6–31G(d)-optimized transition-state structures for the tetrazine ligation and M06–2X/6–311+G(d,p)//6–31G(d)-computed activation free energies in water and relative rate constants. (C) Chemical structure of cyclopropene tags.
Figure 8.
Figure 8.
(A). Schematic of templated fluorogenic tetrazine ligations. (B) Sequences of the template strand and corresponding DNA probes. (C) Rates of the templated reaction for different probes.
Figure 9.
Figure 9.
(A) Schematic of templated fluorogenic reactions with turnover-driven signal amplification. (B) Proposed mechanism of the TMT reaction. (C) Reaction of tetrazine-BODIPY with azabenzonorbornadiene. (D) Fluorescence from 100 nM d21′-Tz and 200 nM d21′-ABN with variable d21 concentrations. (E) Distinguish the target mir21 from two single-mismatch variants. (F) Sequences of probes and templates. (G) Normalized fluorescence from different cell lysates. The SKBR3 (–) contained probe lacking reactive groups (10-fold excess over mir21′-Tz and mir21′ABN) as a competitive inhibitor. (H) Detection of miRNAs in living human cancer cells.
Figure 10.
Figure 10.
(A) Chemical structures of thiolated tetrazine probes. (B) Tetrazine redox behavior. (C) Sequential cyclic voltammograms of a mixed SAM during reacting with 1 μM TCO-PEG3-amine. (D) Electrochemically controlled tetrazine ligation at a SAM.

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