Oxidative Generation of Guanine Radicals by Carbonate Radicals and Their Reactions with Nitrogen Dioxide to Form Site Specific 5-Guanidino-4-nitroimidazole Lesions in Oligodeoxynucleotides

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Abstract
A simple photochemical approach is described for synthesizing site specific, stable 5-guanidino-4-nitroimidazole (NIm) adducts in single- and double-stranded oligodeoxynucleotides containing single and multiple guanine residues. The DNA sequences employed, 5‘-d(ACC CG1C G*2TC CG*3C G4CC) and 5‘-d(ACC CG1C G*2TC C), were a portion of exon 5 of the p53 tumor suppressor gene, including the codons 157 (G*2) and 158 (G*3) mutation hot spots in the former sequence with four Gs and the codon 157 (G*2) mutation hot spot in the latter sequence with two Gs. The nitration of oligodeoxynucleotides was initiated by the selective photodissociation of persulfate anions to sulfate radicals induced by UV laser pulses (308 nm). In aqueous solutions, of bicarbonate and nitrite anions, the sulfate radicals generate carbonate anion radicals and nitrogen dioxide radicals by one electron oxidation of the respective anions. The guanine residue in the oligodeoxynucleotide is oxidized by the carbonate anion radical to form the neutral guanine radical. While the nitrogen dioxide radicals do not react with any of the intact DNA bases, they readily combine with the guanine radicals at either the C8 or the C5 positions. The C8 addition generates the well-known 8-nitroguanine (8-nitro-G) lesions, whereas the C5 attack produces unstable adducts, which rapidly decompose to NIm lesions. The maximum yields of the nitro products (NIm + 8-nitro-G) were typically in the range of 20−40%, depending on the number of guanine residues in the sequence. The ratio of the NIm to 8-nitro-G lesions gradually decreases from 3.4 in the model compound, 2‘,3‘,5‘-tri-O-acetylguanosine, to 2.1−2.6 in the single-stranded oligodeoxynucleotides and to 0.8−1.1 in the duplexes. The adduct of the 5‘-d(ACC CG1C G*2TC C) oligodeoxynucleotide containing the NIm lesion in codon 157 (G2) was isolated in HPLC-pure form. The integrity of this adduct was established by a detailed analysis of exonuclease digestion ladders by matrix-assisted laser desorption ionization with time-of-flight detection MS techniques.

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