2.1. Typical Pinner and “Pinner-Like” Reactions

The first synthetic route to tetrazine, reported by Adolf Pinner, involves the condensation reaction of imidoesters with hydrazine to afford dihydrotetrazine intermediates (Scheme 1a) [15]. The final tetrazines were prepared by the subsequent oxidation of the dihydrotetrazine intermediates. Based on this study, alternative, one-pot syntheses of tetrazines using nitriles and hydrazine (Scheme 1b), commonly known as “Pinner-like” reactions, were reported [16]. Although beneficial, the practical applications of these approaches are restricted because the preparation of unsymmetrical 3,6-disubstituted tetrazines using two different nitriles results in the formation of inevitable byproducts such as symmetrical tetrazines. Moreover, they are not suitable for the synthesis of alkyl tetrazines from aliphatic nitriles [13]. To overcome these limitations, Devaraj et al. developed a metal-catalyzed reaction for the synthesis of a series of alkyl tetrazines, where Zn(OTf)₂ and Ni(OTf)₂ act as Lewis acids to promote the addition of hydrazine to inactivate alkyl nitriles (Scheme 1c) [17]. This approach has significantly improved access to alkyl and unsymmetrical tetrazines. Additionally, monosubstituted, unsymmetrical tetrazines can be synthesized in high yields via the condensation of a formamidine salt and nitrile in the presence of a Ni or Zn catalyst. However, this method is limited by the use of reactive and hazardous anhydrous hydrazine. Hydrazine is a good reductant and a good nucleophile; thus, the reactions it is employed in usually offer poor substrate scopes and harsh conditions [14]. To overcome these limitations, organocatalytic approaches for the preparation of unsymmetrical tetrazines were developed by Wu et al. in 2019 (Scheme 1d) [18]. It was suggested that a thiol-containing organocatalyst could promote the formation of unsymmetrical tetrazines at room temperature from hydrazine hydrate and nitriles bearing reactive functional groups. These mild reaction conditions expanded the access to gram-scale synthesis and imparted a broad substrate scope.

Scheme 1. Synthetic approaches to tetrazines through classical Pinner and “Pinner-like” reactions. (a) Pinner reaction. (b) “Pinner-like reaction. (c) Metal-catalyzed Pinner-like reaction. (d) Thiol-catalyzed “Pinner-like” reactions. (e) Stepwise reaction for preparation of unsymmetrical tetrazines. (f) Sulfur-mediated “Pinner-like” reactions.

Additionally, several studies have been conducted to address unmet needs. For example, applications of N,N′-diacylhydrazines for the synthesis of unsymmetrical tetrazines have been reported (Scheme 1e) [19]. N,N′-Diacylhydrazines were prepared by the sequential introduction of acyl groups into hydrazine. They were first converted to 1,2-dichloromethylene hydrazines by treatment with PCl₅, followed by the condensation of hydrazine and subsequent oxidation to yield unsymmetrical tetrazines with aliphatic substituents. Sulfur-mediated “Pinner-like” reactions generating reactive nucleophile NH₂NHSH were generally used to synthesize aromatic tetrazines [20]. Recently, Audebert et al. demonstrated that dichloromethane (DCM) could be used as an alternative reagent for the formamidine salt when preparing monosubstituted unsymmetrical tetrazines via sulfur-mediated “Pinner-like” reactions (Scheme 1f) [21].

2.2. Tetrazine Synthesis Based on C–C Bond Formation

Despite notable advances in “Pinner-like” reactions, it is challenging to develop synthetic methods for unsymmetrical 3,6-disubstituted tetrazines with a broad substrate scope and high utility. While previous studies mostly focused on the synthesis of tetrazines through the condensation of nitriles and hydrazine, the feasibility of metal-catalyzed C–C bond formation reactions has rendered versatile tetrazine intermediates as attractive targets for the synthesis of unsymmetrical disubstituted tetrazines. Kotschy et al. reported a new method for the preparation of alkynyl tetrazines through Sonogashira or Negishi cross-coupling reactions with chlorotetrazine intermediates (Scheme 2a) [22]. The coupling partners, 3-amino-6-chlorotetrazine intermediates, were synthesized by nucleophilic aromatic substitution, where one chlorine atom of 3,6-dichlorotetrazine was replaced with different amine nucleophiles. Although the substrate scope of tetrazine intermediates is limited due to decomposition, this method has significant potential for the synthesis of new tetrazines via C–C bond formation. As a result, significant efforts have been devoted to the application of other metal-catalyzed cross-coupling reactions and the development of tetrazine intermediates. Recently, Lindsley et al. reported the use of a Suzuki cross-coupling reaction to introduce aryl, heteroaryl, and vinyl groups onto 3-amino-6-chlorotetrazines (Scheme 2b) [23].

Scheme 2. Synthetic approaches to tetrazines through metal-catalyzed C–C bond formations. (a) Sonogashira or Negishi cross-coupling reaction of chlorotetrazine. (b) Suzuki cross-coupling reaction of chlorotetrazine. (c) Liebeskind–Srogl cross-coupling reaction of thioether tetrazines. (d) Ag-mediated Liebeskind–Srogl cross-coupling reaction of b-Tetrazine. (e) Liebeskind–Srogl cross-coupling reaction or reduction of 3-substituted-6-thiomethyltetrazines. (f) Elimination/Heck cross-coupling cascade reaction.

Thioether tetrazines are one of the most useful intermediates for the synthesis of unsymmetrical disubstituted tetrazines. Suzenet et al. reported the Liebeskind–Srogl cross-coupling reaction of disubstituted thioether tetrazines with various boronic acids and organostannanes to yield tetrazines with aryl, heteroaryl, and vinyl substituents (Scheme 2c) [24]. Similar to 3-amino-6-chlorotetrazines, 3-amino-6-methylthiotetrazines were easily obtained from 3,6-bis(methylthio)-1,2,4,5-tetrazine through nucleophilic displacement. This approach has sparked great interest in the development of other valuable thioether tetrazine intermediates. In 2019, Fox et al. demonstrated that 3-thioalkyl-6-methyltetrazines, especially 3-((p-biphenyl-4-ylmethyl)thio)-6-methyltetrazine (b-tetrazine), could serve as a useful intermediate for the synthesis of 3-aryl-6-methylterazines through Ag-mediated Liebeskind–Srogl cross-coupling reactions (Scheme 2d) [25]. To prepare b-tetrazine on a decagram scale, thiocarbohydrazide was subjected to S-alkylation with 4-bromomethylbiphenyl, followed by the condensation of the resultant salt with triethylorthoacetate and subsequent Cu(OAc)₂-catalyzed air oxidation. Compared to those of the typical, copper(I)-mediated Liebeskind–Srogl cross-coupling reaction, the synthetic scope and utility of this Ag-mediated reaction were significantly broader. Furthermore, this new Liebeskind–Srogl cross-coupling protocol is an outstanding synthetic method for the preparation of unsymmetrical tetrazines. In 2020, the same group also synthesized 3-substituted-6-thiomethyltetrazines intermediates bearing various alkyl or aryl groups (Scheme 2e) [26]. This versatile synthetic route begins with the conversion of (3-methyloxetan-3-yl)methyl carboxylic esters into oxabicyclo [2.2.2]octyl (OBO) orthoester intermediates in the presence of BF₃∙OEt₂. These compounds then undergo further condensation reactions with S-methylisothiocarbohydrazide hydroiodide and subsequent oxidation to afford the 3-substituted-6-thiomethyltetrazines intermediates. These intermediates can be converted into unsymmetrical tetrazines through the Liebeskind–Srogl cross-coupling reaction, as well as into monosubstituted tetrazines through catalytic thioether reduction. It is worth mentioning that these synthetic methods provide convenient access to various unsymmetrical, aliphatic, aromatic, and heterocycle-substituted tetrazines.

Furthermore, Devaraj et al. prepared a novel mesylate tetrazine precursor that could react with aryl halides through cascades of eliminations and Heck cross-couplings reaction, affording various (E)-3-substituted-6-alkenyltetrazines (Scheme 2f) [27]. Using this method, tetrazine derivatives with different π-conjugation lengths can be easily prepared.

2.3. Conjugation of Tetrazine to Fluorophores

In addition to the synthetic endeavors to address the challenges associated with tetrazine, various studies have been conducted on the conjugation of tetrazine to fluorophores for applications in the field of bioorthogonal chemistry. As mentioned previously, 3-thioalkyltetrazine can be converted into various unsymmetrical 3,6-disubstituted tetrazines. Thus, Fox et al. synthesized 3-BODIPY-6-methyltetrazine (BODIPY = 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) modifying a BODIPY core with b-tetrazine and phenyl boronic acid through an Ag-mediated Liebeskind–Srogl coupling reaction (Scheme 3a) [25]. Additionally, they demonstrated the application of (3-methyloxetan-3-yl)methyl carboxylic esters with BODIPY for the synthesis of a BODIPY dye with mono-or di-substituted tetrazines (Scheme 3b) [26]. Wombacher et al. utilized Stille cross-coupling to prepare 3-fluorescein-6-methyltetrazine (Scheme 3c) [28]. They prepared another tetrazine intermediate, 3-bromo-6-methyltetrazine, from 3-(methylthio)-6-methyltetrazine through nucleophilic addition of hydrazine followed by bromination. Park et al. utilized Pd-mediated C–H activation for the incorporation of the tetrazine moiety into the Seoul-Fluor (SF) scaffold to synthesize SF_Tz01 (Scheme 3d) [29]. SF_Tz02 could also be synthesized through a Zn-catalyzed “Pinner-like” reaction (Scheme 3d). In addition, Oregon-Green and tetramethylrhodamine (TMR) derivatives with π-conjugated tetrazines were prepared via cascades of eliminations and Heck cross-couplings reaction using a mesylate tetrazine precursor (Scheme 3e) [27].

Scheme 3. Synthetic examples of tetrazines conjugated with various fluorophores. (a) Ag-mediated Liebeskind–Srogl cross-coupling reaction for modification of BODIPY core. (b) Liebeskind–Srogl cross-coupling reaction or reduction for synthesis of a BODIPY dye with mono-or di-substituted tetrazine. (c) Stille cross-coupling reaction for preparation of 3-fluorescein-6-methyltetrazine. (d) Pd-mediated C–H activation or Zn-catalyzed “Pinner-like” reaction for synthesis of Seoul-Fluor (SF) scaffold with tetrazine. (e) Elimination/Heck cross-coupling cascade reaction for preparation of Oregon-Green and tetramethylrhodamine (TMR) derivatives with π-conjugated tetrazine.

3. Development of Fluorogenic Tetrazine Fluorophores

Tetrazine has been used as a fluorescence quencher. Depending on the design strategy, tetrazine can non-radiatively release molecular energy via different mechanisms, thereby quenching the fluorescence. After the iEDDA reaction, pathways for the non-radiative release of energy are no longer available; therefore, fluorescence is restored. Consequently, fluorophores conjugated with tetrazine could be used as bioorthogonal fluorogenic probes for wash-free molecular imaging. Fluorophores modified by various tetrazines and four different types of quenching mechanisms have been used to develop tetrazine-modified fluorophores. These include electronic energy transfer (EET) (e.g., Förster resonance energy transfer (FRET), Dexter, and through-bond energy transfer (TBET)), monochromophoric design, and the formation of new fluorophores after an iEDDA reaction. EET-type fluorogenic probes typically employ the energy of an excited molecule. FRET is one type of EET that takes place between the excited states of donor molecules and the ground states of acceptor molecules via non-radiative dipole–dipole interactions. For this process, the distance between the donor and acceptor is important, and the emission spectrum of the donor must overlap with the absorption of the acceptor. Due to the uniqueness of this process, there are many applications employing FRET phenomena for the development of molecular sensors and investigations into bioimaging probes [30]. In contrast to FRET, Dexter-type EET [31] does not require spectral overlap between the donor and acceptor molecules. Instead, electron hopping occurs from the donor to the acceptor molecule; therefore, the wavefunctions of both the donor and acceptor must overlap. In general, Dexter-type EET requires a shorter distance between the donor and acceptor molecules than that required by FRET-type EET. TBET [32,33] is the EET between a donor and acceptor that occurs through an electronically conjugated, rigid π-system linker, which twists the donor and acceptor groups out of co-planarity. It is still difficult to explain the exact mechanism of TBET or to differentiate TBET from FRET; however, the fast energy transfer rate of TBET, which can be suppressed at low temperatures, is a key characteristic of this process that could be used to examine the mechanism behind its occurrence. The basic principle of EET-type strategies is based on the non-radiative decay of fluorophore energy, where the energy is transferred to a fluorescence quencher (e.g., tetrazine) from the fluorophore; thus, it is important to optimize the efficiency of this energy transfer. In contrast to EET-type quenching, the monochromophoric-design strategy is based on the dark-state quenching of tetrazine. Before the iEDDA reaction, the optically forbidden n→π* transition dominates the S₀–S₁ transition of the molecule, which becomes a π→π* transition after the reaction. The main advantage of the monochromophoric-design strategy is that the unique mechanism it employs allows for the development of a wider range of fluorogenic probes than that offered by EET-type quenching strategies. On the other hand, several studies have reported the formation of new fluorophores after an iEDDA reaction. Because there is no fluorescent molecule before the reaction, this strategy provides a high turn-on ratio. 

3.1. FRET-Type EET Strategy

The most convenient way to modify a fluorophore is to conjugate the fluorophore by amide coupling with the amine form of tetrazine [34]. This approach is based on FRET-type fluorescence quenching, wherein energy is transferred from the excited fluorophore to the tetrazine units. The transferred energy is released by the tetrazine in a non-radiative manner. The first example of the fluorescence quenching effect of tetrazine was reported by Weissleder et al. in 2010 [35]. They conjugated 3-H-6-phenyl-1,2,4,5-tetrazine (H-Tet) derivatives with BODIPY-FL, Oregon Green 488, BODIPY TMR-X, and Vivotag-680 dyes and found a 15- to 20-fold enhancement in fluorescence in Phosphate Buffered Saline (PBS) for green- and red-emitting tetrazine dyes (Figure 1). However, there was no quenching observed for near-IR-emitting dyes with this molecular design.

Figure 1. Synthesis of a tetrazine-functionalized fluorophore via Förster resonance energy transfer (FRET). (a) Schematic representation of the reaction between trans-cyclooctenol and tetrazine-BODIPY-FL (BODIPY = 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) via an inverse electron demand Diels–Alder (iEDDA) reaction. (b) and (c) Fluorogenic properties of tetrazine–BODIPY-FL. Comparison of the image (b) and emission spectrum (c) of tetrazine–BODIPY-FL (left cuvette and black line, respectively) and the corresponding dihydropyrazine product (right cuvette and dashed blue line, respectively). Reproduced with permission from [36]. Copyright (2018) American Chemical Society.

In this study, the probe was used to visualize tubulin filaments using Taxol

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3.2. Dexter-Type, Electron-Exchange EET Strategy

Because the FRET-based strategies for fluorescence quenching have higher efficiency with regard to the spectral overlap of the donor and acceptor fluorophores, their quenching efficiency is wavelength-dependent. Since tetrazine has an absorption maximum of approximately 530 nm, the quenching efficiency of tetrazine decreases with fluorophores exhibiting a red shift. To overcome this limitation, Dexter-type electron exchange strategies have been examined, which involve the direct incorporation of tetrazines into fluorophores to decrease the distance between the donor (fluorophore) and acceptor (tetrazine) molecules. For instance, Wombacher et al. reported green- to far-red-emitting fluorogenic tetrazine probes, which were prepared by the direct conjugation of tetrazine with xanthene fluorophores via C–C bond-forming reactions [28]. As this molecular design provides a short distance between tetrazine and the fluorophore, this strategy allowed for the development of xanthene–tetrazine derivatives that exhibited fluorescence over the broad visible range of emission, from green to far-red (Figure 2). This could not be achieved with previous FRET-type molecular-design strategies.

T

Figure 3. Molecular-design strategy for Dexter-type fluorogenic tetrazine probe. (a) Chemical structures of tetrazine isomers and unquenched derivatives of rhodamine dyes. (b) Density functional theory (DFT)-optimized structures of regioisomers and expected intermolecular distances between their tetrazine units and xanthene core. Reproduced from [39].

 Density functional theory (DFT) calculations suggested that the regioisomers with tetrazine connected at different positions have different proximities between the fluorophore and tetrazine, resulting in better quenching efficiency (Figure 3b). Dexter-type electron exchange was confirmed by recording the femtosecond transient absorption spectra. ortho-Tetrazine substituents provided higher fluorescence quenching efficiency than those of meta- or para-tetrazine substituents, with a 95-fold increase in fluorescence turn-on. In this study, HD653 (em = 676 nm) exhibited a 50-fold increase in fluorescence turn-on. Furthermore, the probe was used for multicolor, no-wash imaging of the target-of-interest (TOI) protein. By modifying the chemical structure, the cell permeability of this tetrazine probe was controlled and it was successfully used for fluorogenic bioorthogonal imaging of the TOI protein labeled with Bicyclononyne-containing, unnatural amino acids. In addition, the self-blinking tetrazine probe allowed for the stimulated emission depletion (STED) imaging of the TOI protein and organelles, including UAA-labeled vimentin, H2A protein, mitochondria, and actin filaments [39,40].

W geissledner et al. reporated a fluorogenic tetrazine-functionalized fluorophore based on the TBET mechanism [41]. By direscent product. Through a Heck cross-coupling reaction, symmetric and asymmectly conjugatingric tetrazine with BODIPY, they achieved a highly fluorogenic probe whose fluorescence turn-on magnitude was on the order of 10³. Thderivatives bearing various substituents were synthesized. Interestingly, they found that the iEDDA reaction products of testrazine super-dark probes are important for the fluorescence imaging of less abundant proteins or for super-resolution imaging [5]. They derivatives and TCOa exhibited varying photophysical properties. The products exhibited a broad range of emissions (478–605 nm) and valso srynthesized tetrazine-functionalized BODIPY derivativeing turn-on ratios (Figure 4a)9- to 91-fold enhance the spatial donor–acceptor proximity and ement). By conjugating TPP and Taxol^® to ptetrovide predictable donorazine (TPP–Tet and Taxol^®–accTeptor transition-dipole orientations, which could enable access to alternative modes oft, respectively), the modified tetrazines were successfully applied to bioorthogonal fluorescence quenchogenic bioimaging.

In this study, the conjugation of a phenyl pendent to a tetramethyl BODIPY fluorophore exhibited 1600-fold fluorescence enhancement after an iEDDA reaction (Figure 4a,b). The authors claimed that their molecular design resulted in a parallel alignment of the tetrazine absorption dipole and BODIPY emission dipole, thereby maximizing the TBET (Figure 4c). Using phalloidin-TCO, they successfully visualized actin filaments via fluorescence with a high signal-to-noise (S/N) ratio. Soon after, Weissleder et al. reported a fluorogenic bioorthogonal probe based on a coumarin fluorophore [42]. Employing their TBET strategy, they modified the coumarin fluorophore to synthesize hyperemissive ligation-initiated orthogonal sensing (HELIOS) probes having a phenyl pendent with tetrazine at the meta-position of the coumarin core (Figure 5).

Figure 5. Coumarin-tetrazine probes for fluorogenic imaging. (a) Schematic representation of the orientation of the transition dipole between coumarin and tetrazine. (b) No-wash, bio-orthogonal fluorogenic imaging of actin filament with phalloidin-TCO. COS-1 cells were sequentially incubated with phalloidin-TCO and DRAQ5. After brief rinsing, cells were imaged upon the addition of the coumarin–tetrazine probe. Reproduced from [42] with permission from Wiley-VCH.

Depending on the coumarin structure, the HELIOS probe can have a varying blue-green spectrum (from 455 to 502 nm) with a high turn-on ratio (2500- to 11,000-fold enhancement). This study again highlighted that the transition dipole between the fluorophore and tetrazine is important for efficient fluorescence quenching (Figure 5a). The HELIOS probe was used to visualize the actin cytoskeleton to investigate the imaging potential of the probe. By sequential addition of phalloidin-TCO and HELIOS probes, vivid fluorogenic images of the actin filament with excellent S/N ratios were successfully obtained (Figure 5b).

3.4. Monochromophoric Design

Figure 6. Development of a monochromophore-based, tetrazine-functionalized fluorophore. (a) Upper panel: Normalized emission spectrum of SF_Tz (SF = Seoul-Fluor) probes after reaction with TCO. Lower panel: Photograph of the SF_Tz probes before (left cuvette) and after (right cuvette) reaction with TCO; irradiated under 365-nm UV light. (b) Molecular orbital distribution, vertical transition energy, and oscillator strength (f) of SF_Tz02 (left) and the corresponding iEDDA reaction product (right) obtained by Time-Dependent-DFT calculations (CAM-B3LYP/6-31G*) of the corresponding first excited-state optimized structures. Reproduced with permission from [29]. Copyright (2018) American Chemical Society.

They confirmed full integration between tetrazine and a model, SF fluorophore system [43] by acquiring the absorption spectra. In addition, they found that the oscillator strength (f) of the structure significantly increased after the iEDDA reaction. Based on this observation, they proposed that the iEDDA reaction changed the major contributor of the S₀–S₁ transition of the probes from the optically forbidden n→π* transition to the optically active π→π* transition, thereby promoting the fluorescence emission process (Figure 6b). Considering the increased interest in mitochondrial biology, a new tetrazine fluorophore was used to visualize the mitochondria (Figure 7). Triphenylphosphonium (TPP)–TCO was synthesized and incubated with HeLa cells. After treatment with SF_Tz02* or SF_Tz08* (the more soluble forms of SF_Tz02 and SF_Tz08, respectively), crisp fluorescent mitochondrial images with exceptional contrast were obtained. They further confirmed the specificity of the probe through a co-staining experiment with a conventional fluorescent mitochondrial probe, MitoTracker Deep Red.  

Figure 7. No-wash fluorogenic imaging of mitochondria with SF_Tz probes. HeLa cells were treated with dimethylsulfoxide (a) or triphenylphosphonium (TPP)−TCO (10 μM; b, c, e, g, and h) with MitoTracker Deep Red (f−h). After brief washing with phosphate buffered saline, SF_Tz02* (a, b, c, e, g, and h) was used to treat the cells, and the images were immediately observed without washing. (d) Plot intensity values of pixels along the red line in (c) were analyzed with the ImageJ program. ROI = region of interest. Reproduced with permission from [29]. Copyright (2018) American Chemical Society.

 3.5. Formation of New Fluorophore

Owing to their structure, tetrazines are intrinsically unstable under biological conditions. Therefore, this instability could restore the fluorescence signal of a tetrazine-modified fluorophore without the iEDDA reaction. To address this, Vrabel et al. reported a new concept involving the formation of fluorescent products upon the iEDDA reaction between tetrazine and a particular TCO (Figure 8) [44]. It was found that the reaction between 3,6-di(2′-pyridyl)-5-tetrazine (DiPyTet) and the axial TCO isomer (TCOa) generated a fluorescent product. Through a Heck cross-coupling reaction, symmetric and asymmetric tetrazine derivatives bearing various substituents were synthesized. Interestingly, they found that the iEDDA reaction products of tetrazine derivatives and TCOa exhibited varying photophysical properties. The products exhibited a broad range of emissions (478–605 nm) and varying turn-on ratios (9- to 91-fold enhancement). By conjugating TPP and Taxol^® to tetrazine (TPP–Tet and Taxol^®–Tet, respectively), the modified tetrazines were successfully applied to bioorthogonal fluorogenic bioimaging.

 

  Recently, Varbel et al. reported an extended approach of their strategy. Compared to previous studies, wherein TCOa was used for fluorescent product formation, in this study, they developed new tetrazine derivatives that could afford fluorescent products from both TCOa and equatorial-TCO (TCOe) [45]. Similar to their previous study, they synthesized alkenyl tetrazine derivatives with different substituents. Interestingly, many of the tetrazine derivatives afforded fluorescent products after undergoing iEDDA reactions with TCOa, and most of the 4,5-dihydropyridazine products exhibited red-shifted emissions (626 to 643 nm) and reasonable turn-on ratios (3- to 18-fold enhancement). Furthermore, they conjugated tetrazine with TPP and concanavalin A (ConA-TCO) and used the resultant tetrazine derivative for bio-orthogonal fluorogenic imaging.