Oligonucleotides Functionalized by Fluorescein and Rhodamine Dyes: Michael Addition of Methyl Acrylate to 2'-Deoxypseudouridine

The 2′-deoxypseudouridine (5) was functionalized at N(1) with methyl acrylate by Michael addition. The resulting methyl 2′-deoxypseudouridine-1-propanoate (6) was converted to the phosphoramidite 8 and to the amino-functionalized derivative 9, which was transformed into the fluorescein-labeled phosphoramidites 14 and 16. Fluorescent oligonucleotides were synthesized either from these building blocks or by post-synthetic modification of oligomers containing 2′-deoxypseudouridine subunits. The stability of oligonucleotide duplexes was determined from the melting profiles, measured by UV- or VIS-light absorbance, as well as from the fluorescence emission spectra. While small spacer residues did not affect the thermal stability of the 2′-deoxypseudouridine-containing duplexes, large dye residues led to destabilization.