Two‐Factor Fluorogenicity of Tetrazine‐Modified Cyanine‐Styryl Dyes for Bioorthogonal Labelling of DNA

Two green fluorescent tetrazine‐modified cyanine‐styryl dyes were synthesized for bioorthogonal labelling of DNA by means of the Diels‐Alder reaction with inverse electron demand. With DNA as target biopolymer the fluorescence of these dyes is released by two factors: (i) sterically by their interaction with DNA, and (ii) structurally via the conjugated tetrazine as quencher moiety. As a result, the reaction with bicyclononyne‐modified DNA is significantly accelerated up to ≥284,000 M(−1) s(−1), and the fluorescence turn‐on is enhanced up to 560 by the two‐factor fluorogenicity. These dyes are cell permeable even in low concentrations and undergo fluorogenic reactions with BCN‐modified DNA in living HeLa cells. The two‐factor fluorescence release improves the signal‐to‐noise ratio such that washing procedures prior to cell imaging are not needed, which is a great advantage for live cell imaging of DNA and RNA in the future.