The Efficiency of Metabolic Labeling of DNA by Diels–Alder Reactions with Inverse Electron Demand: Correlation with the Size of Modified 2′-Deoxyuridines

[Image: see text] 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.