Tetrazine metallation boosts rate and regioselectivity of inverse electron demand DielsAlder (iEDDA) addition of dienophiles

The inverse electron demand Diels–Alder (iEDDA) reaction between 1,2,4,5-tetrazines and olefins is a substrate controlled click-reaction and does not require the addition of a catalyst (CuAAC) or light (thiol–ene). The modification of the 1,(4)positions on the tetrazine can be synthetically arduous but rational design of the tetrazine diene and dienophile has resulted in very fast iEDDA reaction rates, where rates 410 M s 1 have been reported. These factors have made the iEDDA addition a useful reaction in several applications, chiefly among them in biological labelling experiments. The iEDDA addition is, however, not regioselective and it produces a mixture of regioisomers, e.g., 1,4and 1,5-isomers (Scheme 1). Transition metal(s) are known to coordinate tetrazines and these complexes can exhibit ligand non-innocence. Coordinated tetrazines show anodically shifted reduction potentials due to metal back-bonding, i.e., the tetrazine moiety is more electrophilic. The metal back-bonding would also lower the activation energy of the addition of a dienophile to the tetrazine diene, however, the often transoid bridging geometry prevents the approach of a dienophile to the tetrazine. Unlike the majority of the reported symmetric tetrazines, 2-pyridyltetrazine (TzPy), isoelectronic and isostructural to 2,20-bipyridine, can be used as bidentate ligand, and the tetrazine diene is free to add dienophiles. For example, addition of cyclooctyne to the fluorescent iridium complex [Ir(PhPy)2(TzPy)] + (PhPy = 2-phenylpyridine) have been described and the rate of the iEDDA addition was between 2.5 and 60 times faster than with the free TzPy ligand. The addition of cyclooctyne to [Ir(PhPy)2(TzPy)] + generates the aromatic 1,2-diazine and thus no stereochemical information was generated from this reaction. Herein we report the synthesis and rate of addition of three dienophiles to the metallotetrazine [ReCl(CO)3(TzPy)] [1], see ESI,† for synthetic and kinetic details. The ReCl(CO)3 moiety was chosen to coordinate TzPy because tricarbonylrhenium(I) complexes with pyridine donor ligands have found uses as imaging reagents in cells and they have also shown cytotoxic activity for cancer treatment. The water soluble variants [Re(OH2)(CO)3(L^L)] + (L^L = bidentate amine donor ligands) have also been described by replacing the Cl-ligand for the aquo ligand. Additionally, tricarbonylrhenium(I) coordinated to a bidentate ligand, e.g., 2,20-bipyridine, are electrocatalysts for CO2 reduction. 16 The tetrazine moiety in [1] can add dienophiles and the rate of addition of vinylferrocene (ViFc), styrene (Ci), and transcyclooctene (TCO) to [1] were measured and are reported in Table 1. Different dienophiles were also tested for their ability

to the tetrazine. Tetrazine coordiation lowers the DS ‡ contribution to DG ‡ for iEDDA addition.
The inverse electron demand Diels-Alder (iEDDA) reaction between 1,2,4,5-tetrazines and olefins is a substrate controlled click-reaction 1,2 and does not require the addition of a catalyst (CuAAC) 3 or light (thiol-ene). 4,5 The modification of the 1,(4)positions on the tetrazine can be synthetically arduous but rational design of the tetrazine diene and dienophile has resulted in very fast iEDDA reaction rates, where rates 410 6 M s À1 have been reported. 6,7 These factors have made the iEDDA addition a useful reaction in several applications, chiefly among them in biological labelling experiments. 1,5,8 The iEDDA addition is, however, not regioselective 9 and it produces a mixture of regioisomers, e.g., 1,4-and 1,5-isomers (Scheme 1).
Transition metal(s) are known to coordinate tetrazines and these complexes can exhibit ligand non-innocence. 10,11 Coordinated tetrazines show anodically shifted reduction potentials due to metal back-bonding, i.e., the tetrazine moiety is more electrophilic. 10,12 The metal back-bonding would also lower the activation energy of the addition of a dienophile to the tetrazine diene, however, the often transoid bridging geometry prevents the approach of a dienophile to the tetrazine. 10,11 Unlike the majority of the reported symmetric tetrazines, 10 2-pyridyltetrazine (TzPy), isoelectronic and isostructural to 2,2 0 -bipyridine, can be used as bidentate ligand, and the tetrazine diene is free to add dienophiles. For example, addition of cyclooctyne to the fluorescent iridium complex [Ir(PhPy) 2 (TzPy)] + (PhPy = 2-phenylpyridine) have been described and the rate of the iEDDA addition was between 2.5 and 60 times faster than with the free TzPy ligand. 13 The addition of cyclooctyne to [Ir(PhPy) 2 (TzPy)] + generates the aromatic 1,2-diazine and thus no stereochemical information was generated from this reaction.
Herein we report the synthesis and rate of addition of three dienophiles to the metallotetrazine [ReCl(CO) 3 (TzPy)] [1], see ESI, † for synthetic and kinetic details. The ReCl(CO) 3 moiety was chosen to coordinate TzPy because tricarbonylrhenium(I) complexes with pyridine donor ligands have found uses as imaging reagents in cells and they have also shown cytotoxic activity for cancer treatment. 14 The water soluble variants [Re(OH 2 )(CO) 3 (L^L)] + (L^L = bidentate amine donor ligands) have also been described by replacing the Cl-ligand for the aquo ligand. 15 Additionally, tricarbonylrhenium(I) coordinated to a bidentate ligand, e.g., 2,2 0 -bipyridine, are electrocatalysts for CO 2 reduction. 16 The tetrazine moiety in [1] can add dienophiles and the rate of addition of vinylferrocene (ViFc), styrene (Ci), and transcyclooctene (TCO) to [1] were measured and are reported in Table 1. Different dienophiles were also tested for their ability Scheme 1 Addition of dienophile to [1]. to add to [1], such as vinyl and allyl functionality (e.g. vinyltrimethoxysilane and allyltrimethylsilane), a bulky olefin (e.g. quinine) and phenylacetylene to [1] was also demonstrated (see ESI †) indicating coordination of TzPy does not inhibit addition of electron rich olefins. The addition of Ci and TCO to [1], respectively, showed enhanced rates compared to the reported rates between the symmetric 3,6-di(pyridin-2-yl)-1,2,4,5-tetrazine (Py 2 Tz) and the self-similar dieneophiles. 7,17,18 The work herein was performed in 1,2-dichloroethane (DCE) due to the poor solubility of [1] in H 2 O, although [1] does show moderate solubility in MeOH, the comparison is used to demonstrate the enhanced rate compared to the reported k 2 values.
The rate of the addition of ViFc, Ci, or TCO to [1] was measured using time-resolved variable-temperature UV vis spectroscopy in C 2 H 4 Cl 2 , respectively and the rates and thermodynamic values are reported in Table 1. The rate for [1] + ViFc k 2 = 2.80 AE 0.1 M À1 s À1 at 22 1C was 160 times faster than the control reaction TzPy + ViFc k 2 = 0.0180 M À1 s À1 at 22 1C. The Eyring analysis 19 of [1] + ViFc found DH ‡ = 22.6 kJ mol À1 and DS ‡ = À150 J mol À1 K À1 , with DG ‡ (25 1C) = 68 kJ mol À1 . The Eyring analysis of TzPy + ViFc showed a small increase in the DH ‡ = 27 kJ mol À1 , however the DS ‡ = À192 J mol À1 K À1 contributed more to the DG ‡ (25 1C) = 84 kJ mol À1 . Coordination of TzPy lowers the DDG ‡ = 16 kJ, and the DDG ‡ (calc.) = 14 kJ was in good agreement with experimental value (see ESI †). The discrepancy between DFT and experimental values can be attributed to solvent effects.
TzPy shows resonance stabilization of d + at the C4 of the tetrazine, and coordination of TzPy enhances this resonance structure due to back-bonding of the d À on the ReCl(CO) 3 moiety (Fig. 2). The addition of ViFc to TzPy shows a larger contribution of DS ‡ to the transition state DG ‡ . One rational is that the molecular structure of TzPy (see Fig. S27, ESI †) is planar while the TzPy in [1] (Fig. 1) tilts towards the Cl-ligand. This distortion may approximate the dien-dienophile transition state (Scheme 2), another contribution could be that backbonding affords a weakening of the double bonds in the tetrazine. Additionally, coordination of TzPy restricts its motion, which may also contribute to a lower transition state energy. The effect as to why coordination of the tetrazine lowers the DG ‡ is currently under investigation.
ViFc is an electron rich dienophile (d À on a-carbon, fulvene resonance with d + on Fe atom), 20 therefore the addition of the unactivated styrene (Ci) to [1] was also studied. The rate of addition of Ci to [1], k 2 = 6.03 AE 0.02 Â 10 À2 M À1 s À1 was nearly 20 times faster than the addition of Ci to Py 2 Tz (k 2 = 3.0 Â 10 À3 M À1 s À1 18 ). The Eyring analysis of the addition of Ci to [1] found a larger DH ‡ = 55 kJ mol À1 was more than double the DH ‡ for the addition of ViFc to [1]. The lower contribution of DS ‡ = À125 J mol À1 K Àl to the transition state (Table 1) can be attributed to the size of the Ph verse Fc. The rate of the addition of TCO to [1] was k 2 = 4.06 AE 0.52 Â 10 5 M À1 s À1 which is 200 time faster than the addition of TCO to Py 2 Tz. 7 The DH ‡ = 26 kJ mol À1 is on the order of the addition of ViFc to [1], however there is a significantly lower contribution of the DS ‡ = À50 J mol À1 K À1 to DG ‡ = 41 kJ mol À1 , as should be expected with the strained TCO dienophile. The difference in rates between the addition of ViFc and TCO respectively to [1], shows  that coordination lowers the entropic barrier (DS ‡ ) as the enthalpic barriers (DH ‡ ) are nearly isoenergetic ( Table 1). The addition of dienophiles to the tetrazine diene is generally unselective (Scheme 2), and the 1,3-prototropic isomerization is rapid which prevents the 4,5-dihydropyridazine (Scheme 2, intermediate 2) from being isolated. 11 The Alder-Stein principle (the relative stereochemistry of dienes and dienophiles is conserved), and Alder's endo-rule (the endo-adduct is the kinetically preferred product), apply in the iEDDA addition, but stereochemical information is often lost due to rapid isomerization and rearomatisation of the 1,4-dhp to the 1,2-pyrazine, especially under aerobic conditions (Scheme 2).
The 4,5-dhp isomer of [1Fc] is, to the best of our knowledge, the first molecular structure of this intermediate. Based on the structure of [1Fc] the endo approach of the dienophile to [1] at CO-face of the molecule (Chart 1, Fig. 1) is favorable. The 1 H NMR 21 of the reaction product [1Fc] showed a mixture of two major 4,5-dhp products in a ratio of 80 : 12, and a minor product (B8%) that appears to be the 1,4-dhp isomer due to slow 1,3-prototropic isomerization. The major 4,5-dhp product is the same found in the crystal state, however, based purely on these data the dienophile's exo approach at the Cl-face cannot be differentiated from the endo approach. However, DFT calculations found that exo approach of any dienophile to [1] at either Cl-or CO-face produced unrealistic activation energy (450 kcal mol À1 ) and was therefore not considered. The endo approach of ViFc to [1] was found to be favorable at the CO-face versus the Cl-face of [1] based on DFT calculations (Fig. S5, ESI †).
The crystal structure of [1TCO] and [1Ci] revealed that the 1,3-prototropic isomerization had occurred. The 1 H NMR of the reaction mixture for [1Ci] confirmed the 1,4-dhp isomer in a ratio of 46 : 46 with 8% other products (Fig. S22, ESI †). According to DFT analysis the endo approach of Ci to [1] is nearly isoenergetic at both the faces, however, only the product of the endo CO-face addition was found in the crystal state (Fig. 3). Analysis of the reaction mixture from [1] + TCO also showed two similar products in a ratio of 71 : 29, and the 1,4dhp isomer was assigned based on 1 H NMR analysis, the molecular structure, and DFT calculations (Fig. S25, ESI †). These data show that the endo approach of dienophiles is favored to occur and only the 1,4-additon and not the 1,5addition are observed.
We report the increased rate of the iEDDA addition of three dienophiles to the metallotetrazine [1]. The combination of the strong endo effect and the back-bonding from the tetrazine to the metal is thought to increase the rate of this reaction. Coordination of the rhenium(I) moiety to the tetrazine lowers the DS ‡ , while the nature of the dienophile shows the larger influence on the contribution of DH ‡ to the transition state DG ‡ . The metallotetrazine [1] also allows for the facial approach of the dienophile to be prejudiced, albeit the Cl-and CO-face of [1] only imparts a small influence. Currently we are exploring this complex for its biological activity, and for immobilization of the complex onto solid supports to generate new electrocatalysts.
M. Schnierle and this project funded by Deutsche Forschungsgesellschaft SFB1333 C2. Calculations supported by the state of Baden-Württemberg through bwHPC and the German Research Foundation (DFG) through Grant INST 40/467-1 FUGG for access to the Justus cluster. The authors gratefully acknowledge Dr Wolfgang Frey and Dr Ingo Hartenbach for advice and crystallographic measurements.

Conflicts of interest
There are no conflicts to declare.