Krivokapić, A., Hole, E. O. and Sagstuen, E. EPR and ENDOR Study of Crystalline Cytosine·HCl Doped with 5-Methylcytosine. Radiation-Induced Radical Formation and Hole Transfer. Radiat. Res. 160, 340–354 (2003).
Radical formation and hole transfer were investigated in crystals of cytosine·HCl (C·HCl) doped with 0–1.1 mol-% 5-methylcytosine·HCl (5MC·HCl). The doping level was determined by NMR spectroscopy. Crystals and polycrystalline samples were X-irradiated at 295 K, 77 K and 12 K and studied with EPR, ENDOR and FSE spectroscopy at these temperatures. At 295 K the dominant radicals were the so-called 3αH radical, formed in 5MC by a net H-abstraction from the methyl group, and the cytosine C6 H-addition (5-yl) radical. At 12 K five radicals were identified. These were the 3αH radical, cytosine reduction and oxidation products, and the cytosine C6 and C5 H-addition (5-yl and 6-yl, respectively) radicals. The spectroscopic parameters for the 3αH radical are very similar to those of a radical observed previously in the crystalline cytosine derivatives cytidine (CR), 2′deoxycytidine hydrochloride (CdR·HCl), 5′dCMP and 3′CMP as well as in the uracil derivative 2-thiouracil (2-TU). It was shown that amounts of the order of tenths of a percent 5MC·HCl doped into crystals of C·HCl give rise to a considerable yield of 3αH radicals after exposure to ionizing radiation both at room temperature and at lower temperatures. This supports a previous suggestion that naturally occurring 5-methylated cytosine impurities may be responsible for the formation of 3αH radicals in the crystalline cytosine derivatives CR, CdR·HCl, 5′dCMP and 3′CMP and suggests that the 3αH radical in these systems is a 5-methylated base-centered radical. The total radical yield in doped C·HCl crystals increased considerably with the doping level, both at low temperatures and at room temperature, implying that the 3αH radical is more stable than the primary cytosine radicals. The relative amounts of the 3αH radical were obtained by using simulated benchmark spectra to reconstruct experimental EPR spectra of doped polycrystalline samples. Evidence is presented suggesting that the enhanced yield of the 3αH radical in doped samples is due to holes originally formed at cytosine bases and transferred to 5-methylcytosine bases in addition to the 3αH radical being less exposed to recombination than other cytosine radicals.