Abstract
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.
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