Free radical formation in X-irradiated anhydrous crystals of inosine studied by EPR and ENDOR spectroscopy.

Single crystals of anhydrous inosine were studied subsequent to exposure to high and low doses of X radiation at 10 K using K-band, EPR, ENDOR, and field-swept-ENDOR (FSE) techniques. Immediately following high radiation doses at 10 K at least eight different radicals, RI-RVIII, were observed. All radicals, except for RVIII, were also observed at low doses, but the relative yields varied with the radiation doses. RI, which decayed with no observable successor at about 65 K, has magnetic characteristics similar to those expected for the hypoxanthine base cation. RII, the dominating radical at low radiation doses, exhibits only one hyperfine coupling amenable for ENDOR analysis. From the nature of this coupling and the EPR and FSE characteristics of the resonance, it is suggested that RII is formed by addition of a neighbor sugar fragment to the C2 position of a hypoxanthine base, forming a C2-O5'-C5' ester bond. RII is unstable and decayed at about 60 K without any detectable successor. RIII and RIV are the C2 and C8 H-addition radicals, respectively. These species are formed in minor amounts after irradiation at low temperatures, and they are the only observable radicals left at room temperature. Two sugar-centered radicals, RV and RVI, are formed by net H-abstraction from the C4' and C5' positions, respectively. These radicals dominate the EPR spectra after high radiation doses at low temperatures. A transformation from RV into RIII, the C2 H-adduct, started at about 80 K. Similarly, a transformation of RVI into RIV started at about 210 K. Several minor species were analyzed. RVII is characterized by an alpha-coupling due to 26% spin density at C8, and RVIII is characterized by 12% pi-spin density at N1. Possible structures for these radicals are discussed.     

Radiation-induced free radicals in solid 5-halouracil derivatives: single crystals of 5-iododeoxyuridine

X-irradiation of single crystals of 5-iododeoxyuridine (IUdR) in the temperature range 8-300 K produces mainly four different radicals which have been studied by electron spin resonance (e.s.r.) and electron nuclear double resonance (ENDOR)-spectroscopy. At low temperatures, a pi-anion is formed which shows predominantly an interaction of the unpaired electron with a proton at carbon C6 of the base (-11.8 G, -23.9 G, -4.6 G). Above 10-20 K, the anion protonates at C6 to yield a RC-I(CH2)-R' radical comprising alpha-iodo and beta-methylene proton hyperfine interactions. The primary oxidation product is an O5'-situated alkoxy radical RCH2O which shows inequivalent beta-proton couplings of about 100 G and 35 G together with a highly anisotropic g-tensor. Upon warming to 265 K, a C2'-located radical on the deoxyribose is formed which is stable at room temperature. A detailed account of its spectral features as obtained by ENDOR exhibits three different alpha-type couplings, two small beta-protons and a dipolar interaction. Other radicals, not reproducibly observed, involve a C5'-hydroxyalkyl radical and a species related to the base cation at low temperatures.     

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.     

Free radical formation in crystals of guanine hydrochloride dihydrate: an ESR and ENDOR study

Radiation-induced free radical formation in single crystals of guanine hydrochloride dihydrate has been studied at temperatures between 20 and 300 K using ESR and ENDOR spectroscopy. At low temperatures three radical species are trapped. Two of these are the C8 H-addition radical R1 previously analysed by Alexander and Gordy (1967) and the O6-protonated anion radical R2. The third species (R4) remains unidentified. Upon annealing at 280 K for an extended period the protonated anion R2 transforms into a new radical R3 which exhibit a well-defined hyperfine pattern but still could not be identified unambiguously. Also radical R4 probably transforms into a new radical (R5) upon such treatment. One proton coupling due to R5 was detected. A scheme of radical reactions incorporating these five radicals is proposed. This scheme also suggests that differences in radical formation between the monohydrate and dihydrate crystals of guanine hydrochloride depends upon differences in the hydrogen bonding network.     

Environmental effects on primary radical formation in guanine: solid-state ESR and ENDOR of guanine hydrobromide monohydrate.

Single crystals of guanine hydrobromide monohydrate, in which the guanine base is protonated at N7, were X-irradiated at 8 and 65 K. Using K-band ESR, ENDOR, and field-swept-ENDOR (FSE) techniques, the crystals were studied between 8 K and room temperature. There was evidence for five different radicals, RI-RV, immediately following irradiation at 8 or 65 K. RI was identified as the O6-protonated anion. It decayed near room temperature with no detectable successor. RII was identified as the N7-deprotonated cation, and decayed near 130 K. RIII is thought to be a ring-opened product formed by C8-N9 bond rupture; upon warming, it decayed at 150 K. RIV is the well-known C8 H-addition radical. These four radicals have been observed previously in the hydrochloride salt of guanine monohydrate. RV is novel, however, with magnetic characteristics consistent with those of the product formed by net OH addition to C5 of the unsaturated C4-C5 bond. It is characterized by four alpha-proton couplings indicating pi-electron spin as follows: 13% at C8; 11% at N7; and 12% at N10. RV decayed between 240 and 255 K with no detectable successor. Upon further warming, very weak resonance lines due to additional, unidentified radicals were observed. A comparison of these results with those obtained from other systems containing N7-protonated guanine bases demonstrates the effect of the environment on the primary radical formation.     

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.     

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.     

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.     

Radiation-induced hydroxyl addition to purine molecules: EPR and ENDOR study of hypoxanthine hydrochloride monohydrate single crystals

Three radical species were detected in an EPR/ENDOR study of X-irradiated hypoxanthine.HCl.H2O single crystals at room temperature: RI was identified as the product of net H addition to C8, RII was identified as the product of net H addition to C2, and RIII was identified as the product of OH addition to C8. The observed set of radicals was the same for room-temperature irradiation as for irradiation at 10 K followed by warming the crystals to room temperature; however, the C2 H-addition and C8 OH-addition radicals were not detectable after storage of the crystals for about 2 months at room temperature. Use of selectively deuterated crystals permitted unique assignment of the observed hyperfine couplings, and results of density functional theory calculations on each of the radical structures were consistent with the experimental results. Comparison of these experimental results with others from previous crystal-based systems and model system computations provides insight into the mechanisms by which the biologically important purine C8 hydroxyl addition products are formed. The evidence from solid systems supports the mechanism of net water addition to one-electron oxidized purine bases and demonstrates the importance of a facial approach between the reactants.     

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.     

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 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 radical formation in crystals of 2'-deoxyguanosine 5'-monophosphate irradiated at 15 K: an ESR study

Radiation-induced radicals in single crystals of 2'-deoxyguanosine 5'-monophosphate (5'-dGMP) at 15 K have been studied by electron spin resonance (ESR) spectroscopy. At low temperatures three radicals were analyzed in detail. The negatively charged pi anion of the guanine base completely dominated the spectra. Weaker resonances were due to an alkoxy radical with the spin density in the C3'-O3' region of the sugar moiety as well as another sugar-centered radical. The anion rapidly decayed upon exposure to uv light at 15 K or by annealing above 25 K. In both cases no successor radical was observed. The second sugar-centered radical decays at 200 K with a concomitant appearance of the resonance from the C8 H-addition radical. By annealing at 295 K the latter resonance was the only one observed. After irradiation at 295 K, however, an additional resonance from a sugar-centered radical, which has been analyzed previously by B. Rakvin and J. N. Herak (Radiat. Res. 88, 240-250 (1981)) was observed. A reinvestigation of this resonance was performed.     

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.     

The structure of the guanine cation: ESR/ENDOR of cyclic guanosine monophosphate single crystals after X irradiation at 10 K.

Following X irradiation of 3',5'-cyclic guanosine monophosphate single crystals at 10 K, several free radicals were trapped and detected by ESR/ENDOR/FSE spectroscopy. The two dominant species both have unpaired spin located on the guanine base. One is the product of net hydrogen atom loss from the exocyclic amino group. The spectroscopic characteristics of this resonance leave this assignment unambiguous. The experimental conditions make it likely that this species was formed by deprotonation of the guanine base cation. The nature of the other species is more uncertain. However, the evidence is consistent with the assignment that it is a net OH adduct to the C4 position of the base. Several species in which the unpaired spin was located on the sugar-phosphate region of the molecule were also observed. The mechanisms for the decay of the primary radicals, also leading to the well-known C8 hydrogen addition radical of the guanine base, are described and discussed.     

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 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.     

Mechanistic aspects of radiation-induced free radical formation in frozen aqueous solutions of DNA constituents: consequences for DNA?

Frozen aqueous solutions of 1 M thymidine-5'-monophosphate were X-irradiated 77 K. The free radicals formed were analyzed by electron spin resonance spectroscopy between 77 K and about 260 K and were shown to result nearly exclusively from electron reaction at 77 K forming the thymine base anion, which converts into the well known 5-thymyl radical upon annealing. Primary oxidation of the substrate was not detectable. A minority species denoted TOH., which appeared at about 200 K, was suggested to result from OH. addition to carbon C6 of the base, perhaps via intermediate oxidation involving H2O2 or from direct reaction of OH. with the base. Another minority species at 77 K up to about 150 K, which was strongly enhanced by H2O2, was shown to be the allyl radical formed by reaction of the OH. with the methyl group. Support for this was given from experiments using BeF2 glasses. The possible spectral features for the cation of dTMP were extracted from aqueous pastes of the Ca2+ salt at 77 K. The mechanistic aspects derived from the results are in conflict with previous assumptions and are discussed for DNA model compounds and DNA.     

Ionization of adenine derivatives: EPR and ENDOR studies of X-irradiated adenine.HCl.1/2H2O and adenosine.HCl.

Following X irradiation of adenine.HCl.H2O at 10 K, evidence for five distinct radical products was present in the EPR/ENDOR. (In both adenine.HCl.1/2H2O and adenosine.HCl, the adenine base is present in a cationic form as it is protonated at N1). From ENDOR data, radical R1, stable at temperatures up to 250 K, was identified as the product of net hydrogen loss from N1. This product, evidently formed by electron loss followed by proton loss, is equivalent to the radical cation of the neutral adenine base. Radical R2, unstable at temperatures above 60 K, was identified as the product of net hydrogen addition to N3, and evidently formed by electron addition followed by proton addition. Radicals R3-R5 could not be identified with certainty. Similar treatment of adenosine.HCl provided evidence for six identifiable radical products. Radical R6, stable to ca. 150 K, was identified as the result of net hydrogen loss from the amino group, and evidently was the product of electron loss followed by proton loss. Radical R7 was tentatively identified as the product of net hydrogen addition to C4 of the adenine base. Radical R8 was found to be the product of net hydrogen addition to C2 of the adenine base, and R9 was the product of net hydrogen addition to C8. Radical R10 was identified as the product of net hydrogen abstraction from C1' of the ribose, and R11 was an alkoxy radical formed from the ribose. With the exception of R11, all products were also found following irradiation at 65 K. Only radical R8 and R9 were stable at room temperature. Most notable is the different deprotonation behavior of the primary electron-loss products (radical R1 vs. R6) and the different protonation behavior of the primary electron-gain products (radical R2 vs. no similar product in adenosine.HCl). The major structural difference in the two crystals is the electrostatic environment of the adenine base. Therefore, this study provides further evidence that environmental influences are important in determining proton transfer processes.