Tetrazines: A World of New Molecules Emerging from Old Chemistry

Tetrazines with branched alkoxy substituents are liquids at ambient temperature that despite the high chromophore density retain the bright orange fluorescence that is characteristic of this exceptional fluorophore. Here, we study the photophysical properties of a series of alkoxy-tetrazines in solution and as neat liquids. We also correlate the size of the alkoxy substituents with the viscosity of the liquids. We show using time-resolved spectroscopy that intersystem crossing is an important decay pathway competing with fluorescence, and that its rate is higher for 3,6-dialkoxy derivatives than for 3-chloro-6-alkoxytetrazines, explaining the higher fluorescence quantum yields for the latter. Quantum chemical calculations suggest that the difference in rate is due to the activation energy required to distort the tetrazine core such that the $n{\pi }^{*}$$S_1$ and the higher-lying $\pi {\pi }^{*}$$T_2$ states cross, at which point the spin-orbit coupling exceeding 10 cm${}^{-1}$ allows for efficient intersystem crossing to occur. Femtosecond time-resolved anisotropy studies in solution allow us to measure a positive relationship between the alkoxy chain lengths and their rotational correlation times, and studies in the neat liquids show a fast decay of the anisotropy consistent with fast exciton migration in the neat liquid films. Full article

The carbon-carbon cross-coupling of phenyl s-tetrazine (Tz) units at their ortho-phenyl positions allows the formation of constrained bis(tetrazines) with original tweezer structures. In these compounds, the face-to-face positioning of the central tetrazine cores is reinforced by π-stacking of the electron-poor nitrogen-containing heteroaromatic moieties. The resulting tetra-aromatic structure can be used as a weak coordinating ligand with cationic silver. This coordination generates a set of bis(tetrazine)-silver(I) coordination complexes tolerating a large variety of counter anions of various geometries, namely, PF₆⁻, BF₄⁻, SbF₆⁻, ClO₄⁻, NTf₂⁻, and OTf⁻. These compounds were characterized in the solid state by single-crystal X-ray diffraction (XRD) and diffuse reflectance spectroscopy, and in solution by ¹H-NMR, mass spectrometry, electroanalysis, and UV-visible absorption spectrophotometry. The X-ray crystal structure of complexes {[Ag(3)][PF₆]}_∞ (4) and {[Ag(3)][SbF₆]}_∞ (6), where 3 is 3,3′-[(1,1′-biphenyl)-2,2′-diyl]-6,6′-bis(phenyl)-1,2,4,5-tetrazine, revealed the formation of 1D polymeric chains, characterized by an evolution to a large opening of the original tweezer and a coordination of silver(I) via two chelating nitrogen atom and some C=C π-interactions. Electrochemical and UV spectroscopic properties of the original tweezer and of the corresponding silver complexes are reported and compared. ¹H-NMR titrations with AgNTf₂ allowed the determination of the stoichiometry and apparent stability of two solution species, namely [Ag(3)]⁺ and [Ag(3)₂]²⁺, that formed in CDCl₃/CD₃OD 2:1 v/v mixtures. Full article

Four new compounds of formulas [Cu(hfac)₂(L)] (1), [Ni(hfac)₂(L)] (2), [{Cu(hfac)₂}₂(µ-L)]·2CH₃OH (3) and [{Ni(hfac)₂}₂(µ-L)]·2CH₃CN (4) [Hhfac = hexafluoroacetylacetone and L = 3,6-bis(picolylamino)-1,2,4,5-tetrazine] have been prepared and their structures determined by X-ray diffraction on single crystals. Compounds 1 and 2 are isostructural mononuclear complexes where the metal ions [copper(II) (1) and nickel(II) (2)] are six-coordinated in distorted octahedral MN₂O₄ surroundings which are built by two bidentate hfac ligands plus another bidentate L molecule. This last ligand coordinates to the metal ions through the nitrogen atoms of the picolylamine fragment. Compounds 3 and 4 are centrosymmetric homodinuclear compounds where two bidentate hfac units are the bidentate capping ligands at each metal center and a bis-bidentate L molecule acts as a bridge. The values of the intramolecular metal···metal separation are 7.97 (3) and 7.82 Å (4). Static (dc) magnetic susceptibility measurements were carried out for polycrystalline samples 1–4 in the temperature range 1.9–300 K. Curie law behaviors were observed for 1 and 2, the downturn of χ_MT in the low temperature region for 2 being due to the zero-field splitting of the nickel(II) ion. Very weak [J = −0.247(2) cm⁻¹] and relatively weak intramolecular antiferromagnetic interactions [J = −4.86(2) cm⁻¹] occurred in 3 and 4, respectively (the spin Hamiltonian being defined as H = −JS₁·S₂). Simple symmetry considerations about the overlap between the magnetic orbitals across the extended bis-bidentate L bridge in 3 and 4 account for their magnetic properties. Full article

Herein, we tune the redox potential of 3,6-diphenyl-1,2,4,5-tetrazine (DPT) by introducing various electron-donating/withdrawing groups (methoxy, t-butyl, H, F, and trifluoromethyl) into its two peripheral benzene rings for use as electrode material in a Li-ion cell. By both the theoretical DFT calculations and the practical cyclic voltammetry (CV) measurements, it is shown that the redox potentials (E_1/2) of the 1,2,4,5-tetrazines (s-tetrazines) have a strong correlation with the Hammett constant of the substituents. In Li-ion coin cells, the discharge voltages of the s-tetrazine electrodes are successfully tuned depending on the electron-donating/withdrawing capabilities of the substituents. Furthermore, it is found that the heterogeneous electron transfer rate (k₀) of the s-tetrazine molecules and Li-ion diffusivity (D_Li) in the s-tetrazine electrodes are much faster than conventional electrode active materials. Full article

Pretargeted nuclear imaging for the diagnosis of various cancers is an emerging and fast developing field. The tetrazine ligation is currently considered the most promising reaction in this respect. Monoclonal antibodies are often the preferred choice as pretargeting vector due to their outstanding targeting properties. In this work, we evaluated the performance of [⁶⁴Cu]Cu-NOTA-PEG₇-H-Tz using a setup we previously used for [¹¹¹In]In-DOTA-PEG₁₁-BisPy-Tz, thereby allowing for comparison of the performance of these two promising pretargeting imaging agents. The evaluation included a comparison of the physicochemical properties of the compounds and their performance in an ex vivo blocking assay. Finally, [⁶⁴Cu]Cu-NOTA-PEG₇-H-Tz was evaluated in a pretargeted imaging study and compared to [¹¹¹In]In-DOTA-PEG₁₁-BisPy-Tz. Despite minor differences, this study indicated that both evaluated tetrazines are equally suited for pretargeted imaging. Full article

Novel bioorthogonal tools enable the development of new biomedical applications. Here we report the concise synthesis of a series of aryl-functionalized cyclobutene analogues using commercially available starting materials. Our study demonstrates that cyclobutene acts as a small, strained dienophile to generate stable substrates suitable for bioorthogonal tetrazine ligation. Full article

Various bioorthogonal chemistries have been used for fluorescent imaging owing to the advantageous reactions they employ. Recent advances in bioorthogonal chemistry have revolutionized labeling strategies for fluorescence imaging, with inverse electron demand Diels–Alder (iEDDA) reactions in particular attracting recent attention owing to their fast kinetics and excellent specificity. One of the most interesting features of the iEDDA labeling strategy is that tetrazine-functionalized dyes are known to act as fluorogenic probes. In this review, we will focus on the synthesis, molecular-design strategies, and bioimaging applications of tetrazine-functionalized fluorogenic probes. Traditional Pinner reaction and “Pinner-like” reactions for tetrazine synthesis are discussed here, as well as metal-catalyzed C–C bond formations with convenient tetrazine intermediates and the fabrication of tetrazine-conjugated fluorophores. In addition, four different quenching mechanisms for tetrazine-modified fluorophores are presented. Full article