Nucleobase radicals are the major family of reactive intermediates produced when nucleic acids are exposed to γ-radiolysis. The 5,6-dihydrouridin-5-yl radical (1), the formal product of hydrogen atom addition and a model for hydroxyl radical addition, was independently generated from a ketone precursor via Norrish Type I photocleavage in single and double stranded RNA. Radical 1 produces direct strand breaks at the 5'-adjacent nucleotide and only minor amounts of strand scission are observed at the initial site of radical generation. Strand scission occurs preferentially in double stranded RNA and in the absence of O(2). The dependence of strand scission efficiency from the 5,6-dihydrouridin-5-yl radical (1) on secondary structure under anaerobic conditions suggests that this reactivity may be useful for extracting additional RNA structural information from hydroxyl radical reactions. Varying the identity of the 5'-adjacent nucleotide has little effect on strand scission. Internucleotidyl strand scission occurs via β-elimination of the 3'-phosphate following C2'-hydrogen atom abstraction by 1. The subsequently formed olefin cation radical yields RNA fragments containing 3'-phosphate or 3'-deoxy-2'-ketonucleotide termini from competing deprotonation pathways. The ketonucleotide end group is favored in the presence of low concentrations of thiol, presumably by reducing the cation radical to the enol. Competition studies with thiol show that strand scission from the 5,6-dihydrouridin-5-yl radical (1) is significantly faster than from the 5,6-dihydrouridin-6-yl radical (2) and is consistent with computational studies using the G3B3 approach that predict the latter to be more stable than 1 by 2.8 kcal/mol.
© 2012 American Chemical Society