Nitroxide-derived pyrimidines for noncovalent spin-labeling of nucleic acids

Abstract

To understand the function of nucleic acids in biological systems, their structural information is important. Electron paramagnetic resonance (EPR) spectroscopy has been increasingly used to study spin-labeled nucleic acids. Spin labels are usually incorporated site-specifically through covalent bonding, which can be difficult and time consuming. A new method was previously developed in our lab using noncovalent interactions where a spin label binds to an abasic site in a duplex DNA at low temperatures. However, the spin label (ҫ) that was used has a lengthy synthetic route and limited solubility in aqueous solutions. To search for spin labels with higher affinity for abasic sites and easier synthesis we have incorporated several nitroxides into the 5-position of pyrimidines. Two were made by an azide-alkyne Huisgen-Meldal-Sharpless (3+2) cycloaddition reaction (click reaction) and another two using palladium-catalyzed Sonogashira coupling. Among the click spin-labels, a U-analogue was fully bound at -30 °C while ca. 60% of a C-analogue bound to a duplex DNA containing an abasic site when placed opposite to A and G, respectively. However, 5-alkyne-linked pyrimidine spin-labeled derivatives of U and C bind only ca. 30% and 90% when paired with A and G, respectively. A C-analogue prepared by coupling 4-amino-TEMPO to 4-TPS U-derivative did not bind at all. Combined, our results indicate that stacking interactions contribute significantly to noncovalent binding of spin-labels at abasic sites in duplex DNA. Further work involving conjugation of a polyamine linker and an intercalator to the 1-position of the pyrimidines was initiated