Benzoyl-Protected Hydroxylamines for Improved Chemical Synthesis of Oligonucleotides Containing Nitroxide Spin Labels

Abstract

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 Ç and Çm 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.