EPR detection of an electron scavenging contaminant in irradiated deoxyoligonucleotides: one-electron reduced benzoyl

Our lab investigated damage of DNA due to ionizing radiation using electron paramagnetic resonance (EPR). Through studies focused on one-electron-reduction of synthetic oligodeoxynucleotides containing only thymine and adenine, we discovered the significant presence of a contaminant in all samples. The contaminant was observed to have a reduction potential greater than that of thymine. In addition, the contaminant yielded a sharp EPR singlet when it was one-electron reduced that interfered with the distinctive doublet of one-electron reduced thymine. We determined that the contaminant contained a benzoyl group, a chemical used in to protect the amine group of adenine during oligodeoxynucleotide synthesis. Derivatives of benzoyl and 16 different oligomer sequences were prepared in a LiCl glass and studied using EPR after X-irradiating at 4K. This treatment selectively creates one-electron reduced radicals. Synthetic derivatives were used to develop an EPR benchmark of the benzoyl radical. Using this, along with the known spectra of one-electron reduced nucleobases, we performed component analysis of the EPR signal from each sample. This analysis revealed that 2-9% of adenines, in the commercially synthesized oligomers delivered to us, were left contaminated with benzoyl. We concluded that the presence of benzoyl is a potential source of error in a variety of experiments utilizing synthesized oligodeoxynucleotides.