Journal
EUROPEAN JOURNAL OF ORGANIC CHEMISTRY
Volume 2019, Issue 23, Pages 3799-3805Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/ejoc.201900553
Keywords
EPR spectroscopy; Nitroxides; Oligonucleotides; Site-directed spin labeling
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Funding
- Icelandic Research Fund [141062-051]
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Oligonucleotides containing nitroxide spin labels, used in biophysical studies of nucleic acids, are frequently prepared by chemical synthesis. However, during the synthesis of spin-labeled oligonucleotides, the nitroxides are partially reduced to the corresponding amines. Here we report that a benzoylated hydroxylamine can be used as a protected form of the nitroxide to avoid this reduction. The benzoyl group is stable through the oligonucleotide synthesis and is readily removed under standard oligonucleotide deprotection conditions, yielding a hydroxylamine that is oxidized in situ to the nitroxide. This method was used to incorporate the rigid spin labels c and cm into DNA and RNA oligonucleotides, respectively, including a doubly labeled 36-nucleotide long DNAzyme. Enzymatic digestion of the spin-labeled oligonucleotides and subsequent HPLC analysis showed that the nitroxides were intact. This protecting group strategy facilitates the high-yielding synthesis of spin-labeled DNA and RNA oligonucleotides using the phosphoramidite method.
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