Proximity-Induced Bioorthogonal Chemistry Using Inverse Electron Demand Diels-Alder Reaction

Bioorthogonal chemistry techniques enable the selective and targeted manipulation of living systems. In order to yield universally applicable techniques, it is of great importance for bioorthogonal reactions to take place rapidly, selectively, and with the formation of only benign side products. One of the reactions that match these criteria well is the inverse electron demand Diels-Alder reaction (DA_inv) between tetrazines and strained dienophiles. However, even this prime technique comes with the disadvantage of its reactants having limited stability under physiological conditions. In our protocol, an unreactive and therefore stable DA_inv diene/dienophile pair reacts rapidly using DNA hybridization as secondary rate-accelerating process. Due to the fluorogenicity of the presented tetrazine rhodamine conjugate, this method enables the selective screening and evaluation of reactant pairs for proximity-mediated bioorthogonal chemistry.