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

Abstract

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.

Nucleobase-Functionalized 7-Deazaisoguanine and 7-Deazapurin-2,6-diamine Nucleosides: Halogenation, Cross-Coupling, and Cycloaddition

“…Apart from the studies with clickable nucleosides and click adducts obtained by the CuAAC reaction, the biorthogonal iEDDA reaction is useful to functionalize and visualize biological molecules such as nucleosides, nucleic acids, and proteins. , It utilizes electron-deficient dienes such as tetrazines and electron-rich or strained alkenes as dienophiles. , The reaction goes back to investigations made by Sauer and others . In this context, it was observed on vinyl nucleosides of uridine and 7-deazaadenine that vinyl side chains of nucleosides can function as target sites for iEDDA cycloadditions as electron-rich dienophiles . When electron-deficient symmetric tetrazines are used in cycloadditions, two diastereoisomeric dihydropyridazines are formed. , They represent the initial cycloaddition products, and their formation is fast.…”

Metabolic labelling of DNA in cells by means of the “photoclick” reaction triggered by visible light

“…It is known that the efficiency of metabolic labelling strongly depends on the size of the applied modified 2′-deoxynucleoside. 12,13 The nucleoside 12 has a similar size as the natural thymidine and is therefore well accepted by the cellular enzymes to be metabolically incorporated into DNA. Fixed HeLa cells were first incubated with 12 and labelled using both tetrazoles by means of visible light at 405 nm and 450 nm.…”

“…11 The use of UV light has several principal disadvantages, including safety hazards, cell damage, cell toxicity, and missing chemical selectivity (in the UV range many biocompounds are excited). Shifting the irradiation wavelength into the visible light range would require even larger diaryltetrazoles as DNA modifications, which are very likely not compatible with metabolic labelling 12,13 as a new technique for DNA labelling in cells. 14 Therefore, it looked reasonable to use the complementary approach and apply tetrazoles as reactive units at the fluorophore together with alkene-modified DNA for “photoclick” labellings.…”

Cyclopropenes as Chemical Reporters for Dual Bioorthogonal and Orthogonal Metabolic Labeling of DNA

“…To increase metabolic labeling efficiency by pressuring cells to incorporate the modified 2'-deoxyuridines 1 and 6 we applied our recently published and optimized cell culture conditions. [13] HeLa cells were cultured in minimum…”

“…In comparison to more reactive but larger chemical reporters such as trans-cyclooctene, cyclopropenes offer the best compromise of small size and reactivity. [13] Additionally, cyclopropenes are sufficiently stable as well as reactive in the cellular environment due to their moderate ring strain. [8a,14] Our group has previously established the use of 1-methylcyclopropenyl-modified nucleosides for the labeling of DNA in vitro through IEDDA reaction with fluorogenic tetrazine-modified dyes.…”