Nitroxide-Derived N-Oxide Phenazines for Noncovalent Spin-Labeling of DNA.

Two o-benzoquinone derivatives of isoindoline were synthesized for use as building blocks to incorporate isoindoline nitroxides into different compounds and materials. These o-quinones were condensed with a number of o-phenylenediamines to form isoindoline-phenazines in high yields. Subsequent oxidation gave phenazine-di-N-oxide isoindoline nitroxides that were evaluated for noncovalent and site-directed spin-labeling of duplex DNA and RNA that contained abasic sites. Although only minor binding was observed for RNA, the unsubstituted phenazine-N,N-dioxide tetramethyl isoindoline nitroxide showed high binding affinity and selectivity towards abasic sites in duplex DNA that contained cytosine as the orphan base.

Reduction Resistant and Rigid Nitroxide Spin-Labels for DNA and RNA.

Electron paramagnetic resonance (EPR) spectroscopy, coupled with site-directed spin labeling (SDSL), is a useful method for studying conformational changes of biomolecules in cells. To employ in-cell EPR using nitroxide-based spin labels, the structure of the nitroxides must confer reduction resistance to withstand the reductive environment within cells. Here, we report the synthesis of two new spin labels, EÇ and EÇm, both of which possess the rigidity and the reduction resistance needed for extracting detailed structural information by EPR spectroscopy. EÇ and EÇm were incorporated into DNA and RNA, respectively, by oligonucleotide synthesis. Both labels were shown to be nonperturbing of the duplex structure. The partial reduction of EÇm during RNA synthesis was circumvented by the protection of the nitroxide as a benzoylated hydroxylamine.