Intramolecular tetrazine-acryloyl cycloaddition: chemistry and applications

An unprecedented intramolecular [4 + 2] tetrazine-olefin cycloaddition with alpha,beta-unsaturated substrates was discovered. The reaction produces unique coumarin-dihydropyridazine heterocycles that exhibited strong fluorescence with large Stokes shifts and excellent photo- and pH-stability. This property can be used for reaction analysis. The rate of cycloaddition was found to be solvent dependent and was determined using experimental data with a kinetic modeling software (COPASI) as well as DFT calculations (k(1) = 0.64 +/- 0.019 s(-1) and 4.1 s(-1), respectively). The effects of steric and electronic properties of both the tetrazine and alpha,beta-unsaturated carbonyl on the reaction were studied and followed the known trends characteristic of the intermolecular reaction. Based on these results, we developed a "release-then-click" strategy for the ROS triggered release of methylselenenic acid (MeSeOH) and a fluorescent tracer. This strategy was demonstrated in HeLa cells via fluorescence imaging.