Selective green-light triggered release of an anticancer agent under physiological conditions from a supramolecular hydrogel.

Two green fluorescent tetrazine-modified cyaninestyryl 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 À 1 s À 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.

A selection of four different 2′-deoxyuridines with three different dienophiles of different sizes was synthesized. Their inverse electron demand Diels–Alder reactivity increases from k 2 = 0.15 × 10–2 M–1 s–1 to k 2 = 105 × 10–2 M–1 s–1 with increasing ring strain of the dienophiles. With a fluorogenic tetrazine-modified cyanine-styryl dye as reactive counterpart the fluorescence turn-on ratios lie in the range of 21–48 suitable for wash-free cellular imaging. The metabolic DNA labeling was visualized by a dot blot on a semiquantitative level and by confocal fluorescence microscopy on a qualitative level. A clear correlation between the steric demand of the dienophiles and the incorporation efficiency of the modified 2′-deoxyuridines into cellular DNA was observed. Even 2′-deoxyuridines with larger dienophiles, such as norbornene and cyclopropene, were incorporated to a detectable level into the nascent genomic DNA. This was achieved by an optimized way of cell culturing. This expands the toolbox of modified nucleosides for metabolic labeling of nucleic acids in general.