Two-Factor Fluorogenic Cyanine-Styryl Dyes with Yellow and Red Fluorescence for Bioorthogonal Labelling of DNA

An orange- and a red-emitting tetrazine-modified cyanine-styryl dyes were synthesized for bioorthogonal labelling of DNA by means of the Diels-Alder reaction with inverse electron demand. Both dyes use the concept of the "two-factor" fluorogenicity for nucleic acids: (i) The dyes are nucleic-acid sensitive by their non-covalent binding to DNA, and (ii) their covalently attached tetrazine moiety quench the fluorescence. As a result, the reaction with bicyclononyne- and spirohexene-modified DNA is significantly accelerated up to k2=280,000 M-1 s-1, and the fluorescence turn-on is enhanced up to 305. Both dyes are cell permeable even in low concentrations and undergo fluorogenic reactions with spirohexene-modified DNA in living HeLa cells. The fluorescence is enhanced in living cells to such an extent that washing procedures before cell imaging are not required. Their large Stokes shifts (up to 0.77 eV) also makes them well suited for imaging because the wavelength ranges for excitation and emission can be best possible separated. Furthermore, the spirohexene-modified nucleosides and DNA extend and improve the toolbox of already existing "clickable" dyes for live cell imaging. High Turn-on and fast kinetics! The inverse electron demand Diels-Alder reactions of the synthesized yellow and red tetrazine-modified dyes with spirohexene-modified nucleosides and DNA provide large Stokes-shifts with high quantum yields. With no washing steps, cell imaging could be performed. We introduce two completely new dyes and spirohexene modfied compounds which extend the toolbox of already existing clickable modifications.image