Regioselective inverse Diels-Alder reaction of unsymmetrical tetrazines with aldehydes and ketones

The amine-catalyzed inverse electron demand Diels-Alder reaction of unsymmetrical tetrazines with aldehydes and ketones was investigated. We found that only one regioisomer could be observed when pyrrolidine was applied as the catalyst. Using this reaction, fluorogenic dyes could be developed with a tetrazine unit as the “triggering group”


Introduction
2][3][4] The reaction goes through two steps: an inverse-electron-demand Diels-Alder reaction (iDA) and a retro Diels-Alder (rDA) reaction, which results in the loss of a nitrogen molecule to produce pyridazine as the final product. 5][18][19] To perform the reaction under mild conditions, strained or electron-rich dienophiles are needed to react with tetrazines. 20On the other hand, although enamines and enolates are two major types of electron rich dienophiles, their low stability greatly compromises their potential applications in organic synthesis.To circumvent this, Wang and coworkers used proline as a catalyst to promote direct iDA reactions of ketones with tetrazines via the in situ formation of the enamine intermediate (Scheme 1). 21However, only symmetric tetrazines were tested in that study.In this paper, we report the regioselectivity of the direct iDA reaction of aldehydes and ketones with unsymmetrical tetrazines as well as the potential application of this protocol in bioimaging.

Results and Discussion
To investigate the reactivity and regioselectivity of the secondary amine-catalyzed iDA reaction of unsymmetrical tetrazines with aldehydes and ketones, a selection of secondary amines (A-D) was screened as catalysts for the reaction of 3-methyl-6-phenyl-1,2,4,5-tetrazine (1) with hexanal (2).All reactions proceeded smoothly at room temperature to produce a single cycloadduct 3, in 45-95% yield.The use of pyrrolidine and morpholine in acetonitrile or dichloromethane furnished the product in 89-95% yield within 5 minutes (Entries 1, 2, 4); whereas using diethylamine took 15 minutes (Entry 5).When we replaced the secondary amines with proline, similar yields were observed in DMSO (Entry 9-10), but no products were observed when other solvents were used, even with heating.The use of acetonitrile and water (1:1) resulted in a 45% yield (Entry 3), and a significant amount of unidentified by-products.As control reactions, by mixing the tetrazine with secondary amines in the absence of aldehydes or ketones, we observed no product formation, but a gradual degradation of the starting material over 24 hours.This also could be confirmed by NMR, and by observing the disappearance of the purple color of the tetrazine.Using pyrrolidine as the catalyst, we studied the reactivity and regioselectivity of a number of aldehydes and ketones with the same asymmetric tetrazine 1 (Table 2).NMR analysis of the crude products taken after each reaction unambiguously confirmed the formation of a sole isomeric product in each case (Table 2).Upon purification, single crystal analysis of some of the products were obtained confirm their regioselectivity.Figure 1 shows the crystal structure of the product obtained by the reaction of the tetrazine with 3-phenylpropionaldehyde (Table 2, Entry 6).The regioselectivity of the products formed was consistent with what one would expect according to the zwitterionic models of alignment of dienophiles with unsymmetrical dienes during the Diels-Alder reaction. 22In this case, the transition state of the intermediate favors placement of the secondary amine and the phenyl substituent of the tetrazine on the same side (Scheme 2).2).
We further explored the possibility of using this reaction to modulate the fluorescence of dyes by making a fluorogenic naphthalimide-tetrazine dye.As shown in Scheme 3, the dye was prepared by addition of dichlorotetrazine to the amine terminal of 9 to obtain 10, which subsequently underwent diethylamine-catalyzed iDA reaction with 3-phenylpropionadehyde to furnish a non-aromatized adduct 11.The elimation of the amine to produce pyradizine was not observed in this instant, indicating the relative stability of the dihydropyridazine of compound 11 over those of the analogues tetrazines in Table 2. Compound 10 was almost completely nonfluorescent due to the quenching effect of the tetrazine motif.However, upon an iDA reaction with an aldehydes or a ketone, e.g.phenylpropionaldehyde, a highly fluoresced 11 could be produced.Photophysical studies of the dyes showed that relative to the quenched fluorophore 10, the turn-on ratio after reacting with the aldehyde is 635 fold in dichloromethane (Table 3).This is a considerable improvement over reported fluorophore-tetrazine pairs joined by a flexible linker, which yielded turn-on ratios of only 15-20 folds after reaction with dienophiles. 11,13,23The only reported fluorophore-tetrazine pairs with turn-on ratios greater than we report here had tetrazine directly linked to phenyl rings appended to the fluorophores. 14,24heme 3. Synthesis of fluorogenic naphthalimide-tetrazine dye.

Conclusions
We have established the regioselectivity of a secondary amine-catalyzed iDA reaction with unsymmetrical tetrazines such as 3-methyl-6-phenyl-1,2,4,5-tetrazine, and applied this unique reaction to tuning fluorogenic dyes having a tetrazine motif.We envisage that this method will find utility in imaging of the intracellular distribution of target molecules.Investigations are therefore ongoing to apply this method to target small molecules inside living cells.

Table 1 .
Regioselectivity of secondary amine-catalyzed iDA reaction of hexanaldehyde with an unsymmetrical tetrazine

Table 3 .
Photophysical properties of the NT-tetrazine before and after iDA reaction with propionaldehyde