Radical formation in salts of pyrimidines. II. Cytosine. HCl crystals.

Radicals produced by X-irradiation at 77 K and at 300 K of cytosine. HCl crystals have been analysed by electron spin resonance spectroscopy. Four radicals have been identified: the anion radical of the cytosine molecule, the radical resulting from H-addition at position C6, the radical resulting from H-addition at position O2, and finally a radical resulting from addition of a Cl- to nitrogen N3. Hückel molecular orbital calculations are presented, which support the hypothesis according to which in unsaturated pyrimidines the site of hydrogenation or protonation depends on the state of the molecule.     

Radical formation in pyrimidines. IV. 1,3-dimethyluracil, a non-hydrogen-bonding crystal.

Radical formation in single crystals of 1,3-dimethyluracil by X-irradiation has been studied by electron spin resonance at 9.5 GHz. This crystal contains no hydrogen bonds. Only Van der Waals forces are present. Accordingly, after X-irradiation at 300 K, the only radicals observed are those resulting from the excitation path: the H-addition radical at C5 and an H-abstraction radical from a methyl group. Irradiation with light of lambda more than 400 nm induces the transformation of the C5-addition into the C6-addition radical. INDO calculations indicate that the C6-addition radical is protonated at O4. Since this crystal does not contain N-H or O-H bonds, this protonation can only occur through proton-abstraction from a C-H bond of a neighbouring molecule by the carbonyl group. The presence of short contacts between C6 and O4 is taken to suggest that the abstraction occurs at C6.     

Radical formation in salts of pyrimidines. I. 1-methyluracil. HBr crystals.

Radicals produced by X-irradiation at 77 K of 1-methyluracil. HBr crystals have been analysed by electron spin resonance spectroscopy. The results are compared with those obtained on 1-methyluracil crystals. Four radicals have been identified, two of which are present only in 1-methyluracil. HBr crystals: the C6-addition radical and the pyrimidine--anion radical. Warming-up experiments have been performed in order to study the secondary radical reactions. The different mechanisms proposed for the radiolysis of DNA constituents in the solid state are discussed in connection with these findings.     

EPR and ENDOR study of crystalline cytosine x HCl doped with 5-methylcytosine. Radiation-induced radical formation and hole transfer

Radical formation and hole transfer were investigated in crystals of cytosine.HCl (C.HCl) doped with 0-1.1 mol-% 5-methylcytosine x HCl (5MC x 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 3alphaH 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 3alphaH 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 3alphaH radical are very similar to those of a radical observed previously in the crystalline cytosine derivatives cytidine (CR), 2'deoxycytidine hydrochloride (CdR x 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 x HCl doped into crystals of C.HCl give rise to a considerable yield of 3alphaH 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 3alphaH radicals in the crystalline cytosine derivatives CR, CdR.HCl, 5'dCMP and 3'CMP and suggests that the 3alphaH radical in these systems is a 5-methylated base-centered radical. The total radical yield in doped C x HCl crystals increased considerably with the doping level, both at low temperatures and at room temperature, implying that the 3alphaH radical is more stable than the primary cytosine radicals. The relative amounts of the 3alphaH 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 3alphaH radical in doped samples is due to holes originally formed at cytosine bases and transferred to 5-methylcytosine bases in addition to the 3alphaH radical being less exposed to recombination than other cytosine radicals.     

E.s.r. of free radicals in irradiated uracil-beta-D-arabinofuranoside.

Electron-spin-resonance measurements have been made on single crystals of uracil-beta-D-arabinofuranoside, which were irradiated by 4-0 MeV electrons at 77 K. At low temperatures, two radicals have been identified, one attributed to a hydrogen abstraction from 05' in the sugar moiety and the other to a radical anion located on the pyrimidine ring. The former is very unstable and seems to act as a precursor to other unidentified radical species stable at 77K. At room temperature, the main resonance is due to hydrogen addition to C5 and is probably produced by protonation of the anion. This same radical is also produced by X-irradiation at room temperature.     

Free radicals in dicarboxylic acids: an e.s.r. study of gamma-irradiated single crystals of glutaric acid at 77 K

Electron spin resonance techniques were used to study the gamma-radiation-induced free radicals in single crystals of glutaric acid in the temperature range from 77 K to 300 K. Three different radicals are stabilized at 77 K. The decarboxylation radical is the dominant species and the other two radicals are assigned to the anion and to the substituted acetyl sigma-radical. When the temperature of the crystal is raised, these radicals disappear and the previously studied room temperature radicals appear. E.S.R.-data and the results from semi-empirical INDO-MO calculations were compared in order to elucidate the structures of the various radicals.     

Free radicals in pyrimidines:single crystals of dihydro-6-methyl uracil irradiated and observed at 77 K.

Electron-spin-resonance measurements have been made on single crystals of dihydro-6-methyl uracil, which were irradiated by 4-0 MeV electrons at 77 K. At low temperature, four radicals have been identified. Two, the C5 and C6 hydrogen abstraction radicals, were also studied in a previous work. On exposure of U.V., the C6 abstraction radical was found to convert into the C5 abstraction radical. Annealing at room temperature reversed the process. The other radicals identified are that formed after H-atom addition to O4, and that formed after H-atom abstractions from N1, C5, and C6. The latter was not present in deuterated crystals. Both radicals were found to be unstable at intermediate temperatures. Thermoluminescence glow-curves were found to correlate to the formation and the decay of these radicals. Semi-empirical INDO MO-calculations have been performed for identification purposes.     

Free radical formation in X-irradiated crystals of 2'-deoxycytidine hydrochloride. Electron magnetic resonance studies at 10 K

Single crystals of deoxycytidine hydrochloride (CdR.HCl) have been X-irradiated at 10 K with doses up to about 150 kGy and studied using 24 GHz (K-band) EPR, ENDOR and FSE spectroscopy. In this system, the cytosine base is protonated at the N3 position. Nine different radicals were characterized and identified. Three of these are ascribed to three versions of the one-electron reduced species, probably differing in their protonation state. Radicals formed by net hydrogen addition to the cytosine C5 and C6 positions were observed at 10 K. The hydrogen-abstraction radical at the deoxyribose C1' position most probably results from initial oxidation of the base. The remaining radical species are all localized to the sugar moiety, representing products formed by net hydrogen abstraction from three of the five available carbons of the deoxyribose sugar. The lack of base-centered oxidation products as well as the structures of the one-electron reduced species is rationalized by considering the specific proton donor-acceptor properties of this crystalline lattice in comparison with similar systems.     

Free radicals in dicarboxylic acids: an e.s.r. study of radical conversions in gamma-irradiated single crystals of glutaric acid and glutaric-2,2,4,4-d4 acid

The gamma-radiation-induced free radicals in single crystals of glutaric acid and glutaric-2,2,4,4-d4 acid were studied in the temperature range 77-300 K by e.s.r. techniques. At 77 K the decarboxylation radical and the anion are stabilized. At higher temperatures the decarboxylation radical is found to be converted into a hydrogen abstraction radical with an activation energy of 6.3 +/- 0.5 kcal/mole for the non-deuterated crystal. This radical is stable at room temperature. The anion seems be be converted to an unidentified intermediate radical which in turn is converted to the gamma-acyl radical. An analysis of the g-value anisotropy and of the 13C hyperfine splitting variation for this radical in the deuterated crystal is consistent with the assigned radical structure. By heat treatment the alpha-acyl radical is converted to another form of the hydrogen abstraction radical with an activation energy of 9.6 +/- 0.6 kcal/mole in the deuterated crystal. U.V.-light (gamma = 254 nm) transforms one of the room temperature radicals into the other.     

Electron trapping and reactions in rhamnose by ESR and ENDOR.

The main objective for a reinvestigation of rhamnose was to devise a mechanistic link between the trapped electron detected previously and the secondary radicals observed at 77 K and at room temperature. Single crystals of rhamnose were X-irradiated at temperatures between 15 and 300 K and examined using ESR, ENDOR, and field-swept ENDOR techniques. After low-temperature irradiation a C3 H-abstraction radical is formed following the visible light-induced decay of the trapped electron. This species was previously assigned erroneously to a C2 H-abstraction species. At temperatures above 120 K, this radical deprotonates at the C3 hydroxy group. Furthermore, a C2 H-abstraction radical is formed following the thermally induced decay of the trapped electron. The C2 and C3 H-abstraction radicals did not convert into each other. A third radical species formed at low temperatures is a C5 H-abstraction radical. It is unstable above 250 K and decays without any apparent successor. The C2 and C3 H-abstraction radicals are formed thermally and photochemically from the parent trapped electron. The conversions are mediated by hydrogen atoms formed intermediately or by elimination of hydride ions. The thermal decomposition pathway requires further studies, in particular with respect to the possible role of water. Recently, Box et al. analyzed the site of the trapped electron in rhamnose crystals. The present results support the results obtained by these authors (Radiat. Res. 121, 262 (1990)). In particular, trapped electron vs proton distances closely match the conversion mechanisms suggested.     

Radical formation in single crystals of hypoxanthine.HCl.H2O, inosine, and the disodium salt of 5'-inosine-monophosphate.

Radical formation in single crystals of hypoxanthine.HCl.H2O, inosine and Na2-5'-IMP.(7.5 H2O) by X-irradiation has been studied using electron-spin-resonance spectroscopy at 9.5 and 35 GHz. In all crystals both H-addition radicals at position C2 and C8 of the purine ring are found. The coupling constants of these two radicals are different and depend strongly on the protonation state of the base. INDO-calculations indicate that the C8-radical is protonated at O6. In Na2-5'-IMP OH-addition radicals at position C2 of the purine ring are formed. Electron adduct radicals are found in the neutral and the N7-protonated base after X-irradiation at 77 K. In Na2-5'-IMP no electron adduct is formed but a radical which probably is the cation. In hypoxanthine.HCl.H2O a radical could be observed after X-irradiation at 77 K, which results from addition of a Cl- to the nitrogen N1.     

Free radicals from X-irradiated single crystals of uridine-5'-phosphate disodium salt

X-irradiation of single crystals of uridine-5'-phosphate (disodium salt) between 10 and 300 K as well as storage of irradiated crystals at 300 K produces at least seven different radical species. Between 10 and 77 K, the uracil base anion and a secondary alkoxy radical at the ribose-O3'-site are formed. The latter transforms into a C5'-centred alkylphosphate species between 110 and 130 K which in turn decays between 180 and 220 K under formation of a base 5-yl hydrogen addition radical. Irradiation at 300 K additionally produces the base-located 6-yl radical together with a radical tentatively assigned to the doubly protonated base anion. Storage of crystals for several months results in decay of most of these species leaving a radical possibly located at c5' of the ribose. The spectral parameters of these radicals are given and discussed.     

Radioprotective effects of dimethyl sulfoxide in golden hamster embryo cells exposed to gamma rays at 77 K. I. Radical formation as studied by electron spin resonance

Formation of free radicals in golden hamster embryo (GHE) cells in the frozen living state by gamma irradiation has been studied by electron spin resonance spectroscopy at 4.2 and 77 K. The relative yields of H atoms, OH radicals, and organic radicals trapped in the irradiated GHE cells are 12, 72, and 16%, respectively, of total radical yields. When dimethylsulfoxide (DMSO) is added to GHE cells at 77 K, a large quantity of CH2SOCH3 radicals (DMSO radicals) are formed after gamma irradiation. The yields of OH radicals are not affected by the addition of DMSO. When the GHE cell-DMSO mixtures are irradiated with gamma rays at 77 K and then warmed to 111 K, the OH radicals decay, whereas the DMSO radicals do not increase complementarily. Moreover, the decay rates of the OH radicals at 111 K do not depend upon the concentration of DMSO. Thus OH radicals do not react with DMSO during warming of the irradiated sample. When H atoms are produced by gamma irradiation of acid ice at 60 K, the decay rates of the H atoms at 77 K increase with increasing DMSO concentration, indicating that DMSO reacts with H atoms (CH3SOCH3 + H----.CH2SOCH3 + H2) at 77 K by quantum-mechanical tunneling. When the GHE cell-DMSO mixture is irradiated with gamma rays at 77 or 4.2 K in the dark, DMSO ions are produced in addition to DMSO radicals. Therefore it is concluded that DMSO does not scavenge OH radicals, but does capture H atoms, holes and/or electrons in the gamma-irradiated cells, resulting in the remarkable formation of DMSO radicals. This scavenger effect of DMSO may be related to the radioprotection of DMSO against cell killing described in the companion paper (Watanabe et al., Radiat. Res., this issue).     

E.s.r. studies of sulphur-substituted pyrimidines. 2-thio-5-carboxyuracil at 77 K.

Single crystals of 2-thio-5-carboxyuracil were irradiated and studied at 77 K with e.s.r. spectroscopy. Five resonances were observed and related to the sulphur atom in the 2 position of the pyrimidine ring. Three of the resonances have been assigned to three conformations of a radical formed by hydrogen abstraction from N1. The principal values for the nitrogen coupling are 9-7, 0-0 and 0-0 gauss. The g tensor principal values are 2-173, 1-997 and 1-990 for the dominant conformation of this radical. Two other radicals could not be identified unambiguously.     

Track structure in DNA irradiated with heavy ions

The spatial properties of trapped radicals produced in heavy-ion-irradiated solid DNA at 77 K have been probed using pulsed electron paramagnetic double resonance (PELDOR or DEER) techniques. Salmon testes DNA hydrated to 12 water molecules per nucleotide was irradiated with 40Ar ions of energy 100 MeV/nucleon and LET ranging from 300 to 400 keV/microm. Irradiated samples were maintained at cryogenic temperature at all times. PELDOR measurements were made using a refocused echo detection sequence that allows dipolar interaction between trapped radicals to be observed. The EPR spectrum is attributed to electron loss/gain DNA base radicals and neutral carbon-centered radicals that likely arise from sugar damage. We find a radical concentration of 13.5 x 10(18) cm(-3) in the tracks and a track radius of 6.79 nm. The cross section of these tracks is 144 nm2, yielding a lineal radical density of 2.6 radicals/nm. Based on the yields determined previously for particles having calculated LET values of 300-400 keV/microm and our measured lineal density, we obtain an LET of 270 keV/microm, which is in good agreement with the calculated range of values. These measurements of radical density and spatial extent provide the first direct experimental determination of track characteristics in irradiated DNA.     

Free radical formation in single crystals of 2'-deoxyguanosine 5'-monophosphate tetrahydrate disodium salt: an EPR/ENDOR study.

Single crystals of 2'-deoxyguanosine 5'-monophosphate were X-irradiated at 10 K and at 65 K, receiving doses between 4.5 and 200 kGy, and studied using K-band EPR, ENDOR, and field-swept ENDOR (FSE) spectroscopy. Evidence for five base-centered and more than nine sugar-centered radicals was found at 10 K following high radiation doses. The base-centered radicals were the charged anion, the N10-deprotonated cation, the C8 H-addition radical, a C5 H-addition radical, and finally a stable radical so far unidentified but with parameters similar to those expected for the charged cation. The sugar-centered radicals were the H-abstraction radicals centered at C1', C2', C3', and C5', an alkoxy radical centered at O3', a C5'-centered radical in which the C5'-O5' phosphoester bond appears to be ruptured, a radical tentatively assigned to a C4'-centered radical involving a sugar-ring opening, as well as several additional unidentified sugar radicals. Most radicals were formed regardless of radiation doses. All radicals formed following low doses (4.5-9 kGy) were also observed subsequent to high doses (100-200 kGy). The relative amount of some of the radicals was dose dependent, with base radicals dominating at low doses, and a larger relative yield of sugar radicals at high doses. Above 200 K a transformation from a sugar radical into a base radical occurred. Few other radical transformations were observed. In the discussion of primary radicals fromed in DNA, the presence of sugar-centered radicals has been dismissed since they are not apparent in the EPR spectra. The present data illustrate how radicals barely traceable in the EPR spectra may be identified due to strong ENDOR resonances. Also, the observation of a stable radical with parameters similar to those expected for the charge guanine cation is interesting with regard to the nature of the primary radicals stabilized in X-irradiated DNA.     

Radical formation in X-irradiated single crystals of guanine hydrochloride monohydrate. II. ESR and ENDOR in the range 10-77 K

In a study of guanine.HCl.H2O (Gm) single crystals X-irradiated at temperatures between 10 and 77 K, three radical species were found and characterized by ESR and ENDOR spectroscopy. All three are primary products in that they were present immediately following irradiation at T less than 10 K. Radical I, which apparently can exist in two slightly different conformations, was identified as the product of electron gain by the parent molecule and subsequent protonation at O6. Radical I decayed only after warming the crystals beyond 250 K. Radical II was the guanine cation previously reported (D. M. Close, E. Sagstuen, and W. H. Nelson, J. Chem. Phys. 82, 4386 (1985)); however, ENDOR data are reported here which confirm the previous results. The guanine cation in Gm resulted from electron loss from the parent and subsequent deprotonation at N7. It is proposed that Radical III results from OH attack at C8 of the parent molecule, followed by rupture of the C8-N9 bond and ring opening. The OH radicals thought to produce Radical III result from electron loss by the cocrystallized water molecules. The reaction leading to Radical III, unusual in solid-state radiation chemistry, is thought to be mediated by the specific hydrogen bonding network in this crystal.     

E.s.r. of free radicals in aqueous solutions of substituted pyrimidines.

Reactions of OH radicals with methyl and ethyl derivatives of uracil, cytosine and thymine in aqueous solutions have been investigated. Photolysis of H2O2 was used to generate OH radicals and the radicals on the base derivatives were spin-trapped using t-nitrosobutane and identified with the help of e.s.r. spectroscopy. Addition of OH radicals was found to take place predominantly to the C(5)--C(6) double bond of the bases. H-abstraction from the methyl group occurred in the N(1) methyl derivatives of uracil, cytosine and thymine. Radicals formed by H-abstraction from the methyl group were also detected for 3-methyluracil, thymine, 1-methylthymine and 1-ethylthymine. Introduction of a methyl or ethyl group at the N(1) position of uracil, cytosine and thymine causes an increase in the C(6) proton coupling and a decrease in the N(1) splitting for radicals formed by OH addition at the C(5) position.     

ESR and ENDOR study of adenosine single crystals X-irradiated at 10 K

Single crystals of adenosine were X-irradiated at 10 K and investigated between 10 and 300 K using K-band ESR and ENDOR spectroscopy. Two free radicals were analyzed. Radical I exhibits small hyperfine couplings to the C8-H, C2-H, and a N3-H protons, and was identified as the N3 protonated base anion radical. Radical II exhibits small hyperfine couplings to a C8-H and an exocyclic -N10-H proton. It is suggested that this is therefore the N10 deprotonated base cation radical. Enough data were not available to analyze a third primary radical believed to be located on the ribose moiety. Upon warming Radical I decays at ca. 40 K with no apparent successor. Likewise, no successor was identified for Radical II, which decays at ca. 100 K. At ca. 200 K there is ESR evidence for the C2 and C8 H-addition radicals. Their precursors have not been identified.     

Reaction of substituted pyrimidines with photochemically generated t-BuO* radicals

Humans are exposed to various organic peroxides through chemical, pharmaceutical and cosmetic products. On photolysis, these peroxides produce alkoxyl radicals and hydroxyl radicals. The reaction of *OH radicals with DNA and its constituents have been extensively studied, but very little is known about the reactions of alkoxyl radicals with DNA and its constituents. In view of this, the oxidation of pyrimidine bases viz., thymine, uracil, cytosine, 5-bromouracil, 6-methyluracil and 1,3-dimethyluracil by t-BuO* radicals in aqueous solution at pH 7.5 has been carried out. The reaction between pyrimidine and t-BuO* is followed by measuring the absorbance of pyrimidine at the respective lambdamax. The rates of oxidation of pyrimidines are calculated from the plot of absorbance vs time. The rates of oxidation of pyrimidines have been found to increase with increase in [t-BuOOH], [pyrimidine] and light intensity. The quantum yields are calculated from the initial rates of oxidation of pyrimidine and the measured light intensity at 254 nm the wavelength at which t-BuOOH is activated to give radicals. The quantum yields are found to depend on [pyrimidine] as well as on [t-BuOOH] while they are independent of light intensity. The product analysis was carried out on HPLC with UV-visible detector. The corresponding 5,6-dihydroxypyrimidine and isobarbituric acid have been identified by comparing the retention times of the authentic samples. On the basis of experimental results and product analysis, it is suggested that t-BuOOH on photolysis gives t-BuO* radical, which initiates the reaction by adding to C (5) or C (6) position of pyrimidine base, leading to the formation of pyrimidine base radical via hydrolysis. The pyrimidine radical further reacts with t-BuO* radical to give the final product. This study predicts the probable transient pyrimidine radicals.