Bioorthogonal chemistries are not affected by and minimally perturbing to biological systems, which can be used to label diverse kinds of biomolecules in cells and other complex environments [1]. Since the early successes of Bertozzi group carrying a modified Staudinger reaction on cell surface twenty years ago, a variety of bioorthogonal chemistries have been developed and applied in not only numerous chemical biology studies [2–4] but also synthetic chemistry and materials science [5,6]. Among the extensively studied bioorthogonal chemistry, the tetrazine bioorthogonal reactions, including inverse-electron-demand Diels–Alder (iEDDA) reaction with dienophiles and [4 + 1] cycloaddition with isonitriles, have attracted more and more attention due to their unique fluorescence properties [7]. The iEDDA reaction between tetrazines and strained dienophiles are widely used for protein, lipid and glycan labeling because of fast reaction kinetics and good fluorescence quenching efficiency of tetrazine moiety [8].

On the other side, tetrazine was also adapted as electron acceptor in some optoelectronic materials due to its unique electron-deficient property. Several groups have systematically investigated a series of thiophene coupled acceptors, and found that tetrazine-embedded oligothiophene demonstrated the lowest unoccupied molecular orbital (LUMO) level among twenty usual electron-deficient units [9–11]. Especially, these tetrazine derivatives are electroactive both in oxidation and reduction, they were incorporated into conjugated backbone of some polymer for solar cell [12]. Furthermore, it can also be used as precursor of other electroactive unit [13].

In the past 20 years, our group [14,15] and Marder et al. [16–18] have found that many dimesitylboryl ended quadrupolar oligothiophene compounds exhibit large two-photon absorption cross-sections (σ₂) and emit bright two-photon excited fluorescence (TPEF). Especially, Marder's group reported the σ₂ value of 4590 GM on a tetracationic quadrupolar chromophore with two triarylboranes linked by 5, 5′-di(thien-2-yl)−3, 6-diketopyrrolopyrrole and successfully applied realized TPEF imaging of lysosomes in living cells [19]. The systemic comparison of these TPEF emissive dimesitylboryl-ended quadrupolar oligothiophenes clearly indicated the structure of π-bridges play significant roles [15–19].

In contrast to the previously adapted electron-donating core, we herein embedded electron-accepting tetrazine into a quadrupolar hybrid-oligothiophenes (BTz in Scheme 1). The synthesis, structure, and photophysical properties and its Diels–Alder (D-A) reactivity toward strained dienophiles, such as TCO-OH, cis-cyclooctene, 5-norbornen-2-ol and bicyclo[6.1.0]non-4-yn-9-ylmethanol, were also examined. Especially, the first crystal structure of the cycloaddition product of tetrazine-cyclooctene was obtained, which confirm the dehydrogenation of direct D-A produced dihydropyridazine derivative to an aromatized pyridazine compound under ambient conditions.

As outlined in Scheme 1, the title compound BTz is a dimesitylboryl-ended quadrupolar oligothiophene derivative with tetrazine as core, which could be readily prepared through common procedure for similar compounds as reported [15,17]. Firstly, the dibrominated precursor (BrTz) was prepared starting from 5-cycano-2-bromine-thiophene using hydrazine hydrate as a reductive agent. Then BrTz was lithiated and then quenched with dimesitylfluoroborane to finally afford BTz. Although the direct iEDDA reaction of BTz with (4E)-TCO-OH was not carried out at preparative scale, we did isolate two compounds from the reaction product of cis-cyclooctene with BrTz and BTz, respectively, which were characterized as BrOC and BOC. Especially, single crystals of BrTz, BTz, BrOC and BOC suitable for X-ray diffraction were successfully grown by slow evaporation of their solution of DCM. To the best of our knowledge, this is the first crystallographic proof of iEDDA product between cyclooctene with tetrazine derivatives.

As presented in Fig. 1 and Table 1, both BrTz (Fig. S1 in Supporting information) and BTz adapt central symmetric configurations, with S atoms of thiophene moieties pointing to opposite directions. The lengths of the central N=N bond of BrTz and BTz are 1.320 Å, which is slightly shorter than typical N–N single bond but longer than that of isolated N=N bond, indicating the aromaticity of tetrazine. However, the planarity of thiophene-tetrazine-thiophene backbone in BrTz and BTz are much better than other reported tetrazine derivatives [20]. The dihedral angle between tetrazine and thiophene are only 2.26° for BrTz and 3.43° for BTz, respectively.

Importantly, the structural information of BrOC and BOC clearly indicated that no hydrogen could be reasonably added on the central C4N2 rings and they are all pyridazine derivatives rather than theoretically predicted dihydropyridazine as direct product of D-A reactions. All the bond lengths in the central C4N2 ring clearly indicated the aromatic character. Among them, the length of bridged C–C bond between pyridazine and cyclooctene moiety in BOC is 1.399 Å, which indicates that these three C···C bonds are between single and double bonds. The cyclooctene moiety adapts a half-chair conformation. Although the thiophene-pyridazine-thiophene backbone still keep nearly planar conformation, the dihedral angle between pyridazine-thiophene obviously increased to 19.72°. Interestingly, the two thiophene unit take a cis-conformation, that is, with S atoms of thiophene moieties pointing to same directions.

The ultraviolet–visible (UV–vis) absorption and emission properties of BTz and BOC were summarized in Table S4 (Supporting information). Generally, similar with the reported quadrupolar dimesitylboryl-endded oligothiophenes [15], the absorption and fluorescence emission spectra of BTz and BOC in the common solvents show negligible solvent effects. However, the spectra patterns of BTz and BOC are distinctly different (Fig. S3 in Supporting information). BTz displays a strong absorption band around 410 nm and maintains the well-resolved vibronic structures, while the main absorption band of BOC is degenerated to a broad structure-less peak and blue-shifted to around 370 nm.

To track the tetrazine bioorthogonal reaction process and avoid possible distractions of oxygen in air, BTz and (4E)-TCO-OH were mixed and degassed in a quartz pool and the absorption and emission behavior were recorded in real-time. It seems that tetrazine very effectively quenched the fluorescence of BTz, but the ligation with (4E)-TCO-OH will rapidly turn on the fluorescence (Fig. 2). Liu et al. has proposed two kinds of quenching mechanism of tetrazine-functionalized labels as "energy transfer to a dark state (ETDS)" and "internal conversion to a dark state (ICDS)" [21]. In our BTz, the tetrazine moiety is directly ''embedded'' to the oligothiophene fluorophore, resulting in an integrated π-conjugation. The time dependent density functional theory (TD-DFT) quantum chemical calculations show that the n–π* transition of the tetrazine fragment will produced low-lying dark states (S₁, f = 0.003; S₂, f = 0.004) during the vertical excitation (Table S5 in Supporting information). So, we believe the fluorescence quenching mechanism of BTz could be attribute to the "internal conversion to a dark state". The subsequent iEDDA reaction destroyed the tetrazine part, eliminated the corresponding dark state and turned on the bright fluorescence [21].

Especially, the emission peak intensity enhanced by more than 100 times and peak position remarkably shifted to 510 nm. When cis-cyclooctene was used as dienophile, the emission spectra of the reactant mixture changed very slowly, even with the aid of irradiation of UV-lamp [22]. However, the emission spectra of BTz+cis-cyclooctene is very similar with that of BTz+(4E)-TCO-OH, which indicated the formation of similar intermediate dihydropyridazine derivative. In addition, the existence of dihydropyridazine derivative were also verified by in-situ high-resolution mass spectrometry (MS) spectra (Figs. S9–S11 in Supporting information).

Beside the reaction of tetrazine with alkene, we also studied the D-A reaction of tetrazine with alkyne. The in-situ reaction of BTz and bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) was also performed in a degassed quartz pool. As shown in Fig. 3, both BOC and BTz+BCN present strong emission band at 380–520 nm with similar vibration structures, which should result from the formation of aromatized pyridazine core. The quantum yield of BOC was determined as 3.05% in toluene, which is much higher than that of BTz (Φ = 0.20%).

The reaction kinetics of BTz with dienophiles was also examined. The second-order rate constant for BTz+(4E)-TCO-OH at 25 ℃ is k₂ 0.3 L mol^-1 s^-1 in toluene, which is obviously slower than that of reported phenyl or pyridinyl substituted s-tetrazine [22]. According to the crystal structures of BTz and BOC, it seems one thienyl of BTz need to be rotated before it matches the D-A reaction In addition, Houk et al. had found that the reactivity of substituted tetrazines correlate with the electron-withdrawing abilities of the substituents [23]. In BTz molecule, thiophene group is a weak electron donor in BTz, which will increase the LUMO+1 energy and decreases the activity of the D-A reaction. So, relatively slow reaction kinetic of BTz could be attribute to the rotation barrier and electronic donating properties of the adjacent thienyl groups. Furthermore, the TPEF spectra of in situ BTz+(4E)-TCO-OH indicated BTz could be precursor of TPEF emitter (Fig. 4).

In order to understand the spectroscopic results in depth, TD-DFT calculation at B3LYP/6–31G(d) basis set with Gaussian 09 [24] were carried out on BTz, BOC and possible intermediate dihydropyridazine derivative BOC-H. As shown in Fig. 5, both highest occupied molecular orbital (HOMO) and LUMO energy levels of BOC are higher than those of BTz. But since the enhancement of LUMO is bigger than HOMO, so the HOMO-LUMO gap of BOC (3.77 eV) is obvious larger than that of BTz (3.54 eV). Furthermore, both the HOMO and LUMO of BOC is lower than that of BOC-H, which is also consistent with the experimental results.

In summary, a dimesitylboryl-ended oligothiophene with tetrazine as core (BTz) was synthesized and its bioorthogonal chemistry toward cyclooctene was comprehensively studied by spectral technique and crystal structures of isolated product compounds. The first crystal structure of isolated bioorthogonal product clearly confirmed that a dehydrogenation occurred under ambient conditions after bioorthogonal iEDDA reaction between tetrazine and cyclooctene. We believe this finding will prove useful in designing new ICDS type of tetrazine-based bioorthogonal probes.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by National Natural Science Foundation of China (Nos. 52150222, 21672130 and 52073163), the Shenzhen Science and Technology Research and Development Funds (No. JCYJ20190806155409104), and the State Key Laboratory of Crystal Materials. We acknowledge Prof. Dr. Cuihua Zhao of Shandong University for her valuable suggestions.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2023.108325.