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

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 x 10(-2) M-1 s(-1) to k(2) = 105 x 10(-2) M-1 s(-1) with increasing ring strain of the dienophiles. With a fluorogenic tetrazinemodified cyanine-styryl dye as reactive counterpart the fluorescence turn-on ratios lie in the range of 21-48 suitable for washfree 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.

Automated summary

Four different 2'-deoxyuridines were synthesized and reacted with three different dienophiles. The reactivity of inverse electron demand Diels-Alder reaction increased with the increasing ring strain of the dienophiles. The fluorescence turn-on ratios of the resulting fluorogenic tetrazinemodified cyanine-styryl dye were suitable for washfree cellular imaging. The incorporation efficiency of the modified 2'-deoxyuridines into cellular DNA was influenced by the steric demand of the dienophiles and an optimized way of cell culturing allowed even larger dienophiles to be incorporated.