Stable, Reactive, and Orthogonal Tetrazines: Dispersion Forces Promote the Cycloaddition with Isonitriles

Abstract

Bioorthogonal reactions are of great value in the life sciences. The isocyano group is a structurally compact bioorthogonal functional group that reacts with tetrazines under physiological conditions. Here we report that bulky tetrazine substituents accelerate this cycloaddition. Computational studies suggest that dispersion forces between the isocyano group and the tetrazine substituents in the transition state contribute to the atypical structure-activity relationship. Stable asymmetric tetrazines that react with isonitriles at rate constants as high as 57 M −1 s −1 were accessible by combining bulky and electron-withdrawing substituents. Sterically encumbered tetrazines react selectively with isonitriles in the presence of strained alkenes/alkynes, which allows for the orthogonal labeling of three proteins. The established principles will open new opportunities for developing tetrazine reactants with improved characteristics for diverse labeling and release applications with isonitriles.

Isonitrile-responsive and bioorthogonally removable tetrazine protecting groups

“…This effect may in part be because of a slightly slowed bimolecular reaction rate (Table S1 †), which agrees with the lack of dispersion forces between the tert-butyl group and the incoming isocyano group. 38 These results show that the substituents on both the tetrazyl ring and the methylene position are important to achieve prompt and high-yielding release. These insights provide guidance for further improvement of probe performance in future studies.…”

“…2c), which we expected not to release pNA because tertiary isonitriles form stable 4H-pyrazol-4-imine conjugates. 36,38,42 Indeed, pNA-release was undetectable in experiments with t-OcNC conrming that TzMe-removal follows the designed release principles. We were interested whether electrondonating groups adjacent to the isocyano functionality would accelerate the inverse-electron demand cycloaddition step.…”

“…The bimolecular reaction rates of tetrazines and isonitriles were in the range of those observed in previous studies (Table S1 †). 37,38 Under these conditions, (DMSO : PBS pH 7.4, 4 : 1, v/v at T ¼ 37 C) the rate constants of the reactions with 4a ranged from k 2 ¼ 0.05-0.38 M À1 s À1 . The water content strongly inuences the kinetics of the cycloaddition step, and based on previous studies, 34,38 we extrapolate the reaction to be about 10-fold faster in purely aqueous solutions.…”

“…37,38 Under these conditions, (DMSO : PBS pH 7.4, 4 : 1, v/v at T ¼ 37 C) the rate constants of the reactions with 4a ranged from k 2 ¼ 0.05-0.38 M À1 s À1 . The water content strongly inuences the kinetics of the cycloaddition step, and based on previous studies, 34,38 we extrapolate the reaction to be about 10-fold faster in purely aqueous solutions. These initial results indicate that the TMSgroup promotes pNA release as the faster bimolecular rate of TMS-MeNC compared to n-BuNC is insufficient to explain the rapid elimination of pNA for the former.…”

“…1). 37,38 Given the prominent role and favorable properties of tetrazine-based cycloadditions in dissociative bioorthogonal chemistry, it would be valuable to have tetrazine-based protecting groups that release a payload upon reaction with some of these dienophiles. An example of such a molecule was disclosed by Wang et al as demonstrated by a tetrazine-based prodrug that was activated through a reaction with a cyclooctyne modied with a hydroxyl group at the propargylic position.…”