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.     

E.S.R. studies of free radicals derived from reaction of OH with uracil and thymine

Short-lived free radicals produced in N2O-saturated aqueous solutions of uracil and thymine have been studied using the in situ radiolysis steady-state e.s.r. method. Radical formed in alkaline aqueous solutions by OH addition to either positions C(5) or (6) were observed. Mechanisms for the formation of transient species were derived. The spin density distribution of the unpaired electron was calculated by means of the INDO method.     

Free radical generation by ultrasound in aqueous solutions of nucleic acid bases and nucleosides: an ESR and spin-trapping study

Direct evidence for the detection of intermediate radicals of nucleic acid constituents induced by ultrasound in argon-saturated aqueous solution is presented. The method of spin trapping with 3,5-dibromo-4-nitrosobenzene sulphonate, which is a water-soluble, non-volatile, aromatic nitroso spin trap, combined with ESR, was used for the detection of sonochemically induced radicals. Spin adducts were also generated by OH radicals produced by UV photolysis of aqueous solution containing H2O2. ESR spectra observed from these photolysis experiments were identical to those after sonolysis. The ESR spectra of the spin adducts suggest that the major spin-trapped radical of thymine and thymidine was the 5-yl radical, and that of cytosine, cytidine, uracil, and uridine was the 6-yl radical. To compare the radicals induced by sonolysis and photolysis, the decay of the ESR spectra of the thymine and thymidine spin adducts was investigated. The decay curves of thymine and thymidine after sonolysis indicated biphasic decay. However, after photolysis the spin adducts from both compounds showed very little decay. These results suggest that the observed spin adducts in the sonolysis of pyrimidine bases and nucleosides were formed by OH radical and H atom addition to the 5,6 double-bond.     

An e.s.r. and spin-trapping study of the reactions of the SO4- radical with protein and nucleic acid constituents.

Reactions of the SO4- radical, generated by U.V. photolysis of Na2S2O8, were studied in aqueous solutions of amino acids, dipeptides, nucleic acid bases, nucleosides and nucleotides. The transient free radicals so formed were spin-trapped by t-nitrosobutane and identified by e.s.r. spectroscopy. The amino acids primarily undergo oxidative decarboxylation. The pKs of the ammonium groups of the spin-trapped decarboxylated radicals of glycine and alanine in D2O were determined to be 8.3 +/- 0.2. An oxidation product, which is the precursor of the decarboxylated radical, is tentatively identified for alanine, valine and isoleucine. Radicals formed by hydrogen abstraction by SO-4 are identified for leucine, serine, phenylalanine and 4-hydroxyproline. In dipeptides, SO-4 produces decarboxylation of the amino acid located at the carboxylate terminal residue. For gly-ala and ala-ala, radicals generated by hydrogen abstraction from the carboxylate terminal residue alanine were also characterized. Radicals centered on the C(5) carbon were observed for uracil, cytosine and thymine. For nucleosides and nucleotides, radicals situated on the base and/or the sugar moiety were assigned.     

Photoinduced reactions of anthraquinone antitumor agents with peptides and nucleic acid bases: an electron spin resonance and spin trapping study

The photoexcitation (lambda = 313 +/- 10 nm) of adriamycin, daunomycin, and mitoxantrone in the presence of peptides or pyrimidine nucleic acid bases was investigated. In air-saturated and air-free solutions, peptides are decarboxylated by the photoexcited drug molecules. The decarboxylation reactions were shown to occur specifically at the C-terminal amino acid of the peptide. The decarboxylated peptide radicals were spin-trapped using 2-methyl-2-nitrosopropane (MNP) and identified by electron spin resonance (ESR). In air-free solutions, nucleic acid bases are oxidized by the photoexcited drug molecules predominantly generating C(5)-carbon-centered radicals in the pyrimidine rings of uracil, cytosine, and thymine. However, spin adducts of MNP and thymine were also obtained at the N(1) or N(3) positions of the pyrimidine ring. In air-saturated adriamycin and daunomycin solutions, the spin adducts of MNP with uracil or thymine are similar to those obtained following hydroxyl radical reactions with these pyrimidines. This suggests that in the presence of oxygen, the photoexcited adriamycin and daunomycin transfer an electron to oxygen generating the superoxide anion radicals (O2-.), which are precursors of hydroxyl radicals. O2-. was also formed when O2-saturated DNA solutions were photoirradiated (lambda = 313 +/- 10 and 438 +/- 10 nm) in the presence of adriamycin and daunomycin, indicating that the photodegradation of DNA in the presence of these drugs caused by hydroxyl radicals is mediated by dissolved oxygen.     

Radiation-induced crosslinks between thymine and phenylalanine

OH radicals generated by ionizing radiation in aqueous solutions of thymine (T) and phenylalanine (Phe) induce crosslinking between thymine and phenylalanine. The crosslinked products were isolated and characterized by capillary gas chromatography-mass spectrometry. They are formed via OH radical adducts to thymine and phenylalanine and the reaction between dissimilar radicals is greatly favoured (T-Phe:Phe-Phe:T-T = 0.46:0.14:0.05). The reaction mechanism presented may serve as a model for radiation or any free radical-induced crosslinks between DNA and proteins.     

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.     

E.s.r. study of spin-trapped radicals formed during the photolysis of aqueous solutions of acid amides and H2O2.

Free radicals formed by the reactions of OH radicals with amides and their N-methylated derivatives in aqueous solutions have been studied. The OH radicals were produced by U.V.-photolysis of H2O2, and the short-lived amide radicals were converted to more stable nitroxide radicals by addition to a spin-trap, tert-nitrosobutane. The spin-trapped radicals were identified by e.s.r. spectroscopy. For acetamide, chloroacetamide, malonamide, succinamide and propionamide, the observed radicals were formed by H-abstraction from the carbon atoms attached to the carbonyl group. The H atom attached to the carbonyl group was abstracted in formamide. For N-methyl acetamide, N,N-dimethyl acetamide and the corresponding formamide derivatives, H-abstraction occurred only from the N-methyl group. The non-equivalency of the amide protons was observed in the spin-trapped radicals for acetamide, formamide, malonamide, succinamide and propionamide. The identification of the site of OH attack on N-methyl amides is helpful for the study of radical formation in peptides and proteins.     

Effect of non-volatile scavengers of hydroxyl radicals on thymine radical formation induced by gamma-rays and ultrasound

In order to investigate the mechanism of sonolysis of nucleic acid constituents, the yield of thymine radicals generated by 50 kHz ultrasound in Ar-saturated aqueous solution was compared with that formed by gamma-radiolysis in N2O-saturated solutions in the presence of various non-volatile scavengers, which cannot act in the gas phase of the cavitation bubbles. For comparison of thymine radical yields by sonolysis and gamma radiolysis, the method of spin trapping with 3,5-dibromo-4-nitrosobenzenesulphonate (a water-soluble, non-volatile, aromatic nitroso spin trap) combined with ESR was used. The efficiency of OH radical scavenging is expressed by the reciprocal value of C1/2, the scavenger concentration at which the thymine radical yield is decreased by 50 per cent. In gamma radiolysis the scavenging efficiencies of the solutes depend on their rate constants with OH radicals. For sonolysis the C1/2 values were similar to those obtained for gamma radiolysis except for the hydrophobic 5,5-dimethyl-1-pyrroline-N-oxide. These results suggest that thymine radicals induced by ultrasound are produced in the bulk of the solution as well as in the interfacial region.     

E.s.r. of spin-trapped radicals in aqueous solutions of peptides. Reactions of the hydroxyl radical.

The reactions of hydroxyl radicals with 30 dipeptides and several larger peptides were studied in aqueous solutions. The OH radicals were generated by U.V. photolysis of H2O2. The short-lived peptide radicals were spin-trapped using t-nitrosobutane and identified by e.s.r. For dipeptides containing the amino terminal residues glycine, alanine and phenylalanine, abstraction of the hydrogen from the carbon adjacent to the peptide nitrogen was the major process leading to the spin-adducts. Such radicals will be referred to as backbone radicals. Dipeptides with a carbonyl terminal serine residue and also glycylglutamic acid form both backbone and side-chain radicals, with the latter being formed in larger quantities. For dipeptides, side-chain radicals were detected on either the carboxyl or amino terminal residues of both. The effect of pD on the e.s.r. sectrum of the spin-adducts of glycylglycine was studied and the pK of the carboxyl group of this radical was determined to be 2.5. For (Ala)3 and (Ala)n, with an average value of n = 1800, backbone and minor side-chain radicals were observed. For ribonucleases-S-peptide, containing 20 amino acid residues, both backbone and side-chain radicals were detected.     

Yields of radiation-induced main chain scission of poly U in aqueous solution: strand break formation via base radicals

The G values for single-strand breaks G(ssb) in polyuridylic acid (poly U) have been measured by low-angle laser light scattering in aqueous solutions under various conditions (e.g. in the presence of N2O, Ar and t-butanol). In N2O-saturated solutions at room temperature and pH 5.6, the G(ssb) is 2.3. The efficiency of ssb formation was found to be 41 per cent for OH radicals, 19 per cent for H atoms and congruent to zero for e-aq. On the basis of 20 per cent and less than 5 per cent attack on the sugar moiety by OH radicals and H atoms, respectively, the large G(ssb) values obtained cannot be explained solely as resulting from radicals produced by reaction of OH radicals and H atoms on the sugar moiety. It is therefore proposed that base radicals produced by the reaction of OH radicals or H atoms with the uracil moiety can also lead to chain break formation in poly U via radical transfer to the sugar moiety.     

Model studies for the direct effect of high-energy irradiation on DNA. Mechanism of strand break formation induced by laser photoionization of poly U in aqueous solution

Laser flash photolysis of polyuridylic acid (poly U) in anoxic aqueous solutions leads to biphotonic photoionization of the uracil moiety followed by the formation of single strand breaks (ssb). The rate constant for ssb formation (1.0 s-1, obtained from the slow component of conductivity increase at 23 degrees C and pH 6.8) increases with decreasing pH to 235 s-1 at pH 3.5. The activation energy (pre-exponential factor) was measured to be 66 kJ mol-1 (5 X 10(11) s-1) at pH 6.8. Addition of dithiothreitol (DTT) or glutathione (GSH) prevents ssb formation by reacting with a poly U intermediate (rate constant = 1.2 X 10(6) and 0.16 X 10(6) dm3 mol-1 s-1, respectively). Since with OH radicals as initiators very similar data have been obtained for the kinetics of ssb formation and for the reaction with DTT, we conclude that photoionization of the uracil moiety in poly U leads eventually to the same chemical pathway for ssb formation as that induced by OH radicals. Furthermore, we propose that protection by DTT and GSH occurs via H donation to the C-4' radicals of the sugar moiety of DNA and to the C-4' and the C-2' radicals of poly U.     

A possible prebiotic synthesis of thymine: uracil-formaldehyde-formic acid reaction.

When uracil is reacted with formaldehyde and formic acid in dilute aqueous solutions at 100-140 degrees C, 5-hydroxymethyluracil (5-HMU), methylenebiuracil (MBU) and thymine are formed. It has been shown that 5-HMU is an intermediate in the formation of MBU and thymine. In the presence of formic acid, 5-HMU gives MBU, thymine and in some cases uracil. The formation of thymine is generally favoured under acidic conditions, although small amounts of this base could also be obtained when the reactions were carried out under mildly basic conditions. A hydride ion transfer mechanism is suggested for some of these reactions. These results have relevance to the formation of thymine under prebiotic conditions.     

On the radiation chemistry of thymine in aqueous solution

The reactions of thymine in aqueous solution with radiation-induced radicals OH, H, and e-aq were studied under various conditions. Competition studies using scavengers of OH radicals (methanol, ethanol, iodide) or of e-aq and/or H atoms (N2O, H+, O2) led to the conclusion that OH and H radicals destroy the chromophoric group of thymine, but e-aq does not. A trace of O2 proved to be necessary to obtain maximal destruction. Removal of the last traces of O2 resulted in a decrease of the destruction yield, possibly through restitution reactions. It was found that (1) alcohol radicals destroy thymine, even in the presence of O2; (2) the rate constant, k(OH + thymine) = 4.3 X 10(9) M(-1) sec(-1) (from competition with iodide); and (3) k(H + thymine) = 8 X 10(8) M(-1) sec(-1) (from competition with O2 in acid solution).     

E.S.R. of spin-trapped radicals in gamma-irradiated polycrystalline nucleic acid constituents and their halogenated derivatives

Free radicals in gamma-irradiated polycrystalline nucleic acid constituents and their 5-halogenated derivatives have been studied by e.s.r. and spin-trapping. After gamma-irradiation at room temperature, the polycrystalline samples were dissolved in aqueous solutions of t-nitrosobutane (tNB) in the absence or presence of oxygen. For many of the nucleic acid constituents, two types of radicals, -C(5)RH-C(6)H- and -C(5)R-C(6)H2-, formed by H-addition to the double bond [-C(5)R=C(6)H-] of the base, were observed, where R is -CH3 or -H. In addition, radicals formed on the sugar moiety were found for some nucleosides. When oxygen was present in the tNB solution, the relative stability of trapped radicals was changed, and thus the presence of more than one radical species could be established. For halogenated bases, the radical produced by H-abstraction from N(1) was observed, and an additional radical species formed by H-addition to the C(6) position was found for 5-fluorouracil. For halogenated nucleosides, the same spectrum was observed in all compounds except the 5-fluoroderivatives, and was assigned to the radicals produced on the sugar moiety. For 5-fluorodeoxyuridine and 5-fluorouridine, the radical formed by H-addition to the C(6) position of the base was observed. In general, the present results are in good agreement with those of previous single crystal studies, but in the case of halogenated compounds other than the 5-fluoroderivatives, it was not possible to spin-trap the alpha-halo radicals which were the most prominent radicals formed from gamma-irradiation of single crystals at room temperature.     

On the radiation chemistry of some pyrimidine-electron adducts: reaction of pyrimidine negative ions with t-butanol radicals.

The rates of decay of the electron adducts of thymine, thymidylic acid and of uracil at pH approximately 8-5 and at wave-lengths of 340 or 350 nm in the presence of t-butanol as scavenger for OH radicals are not influenced significantly by the addition of 0-1 M NaClO4. It follows that the decay reactions do not take place between negatively-charged species. The best explanation seems to be an efficient reaction of the electron adducts with the t-butanol radicals formed by reaction of OH radicals with the t-butanol.     

Sonolysis, radiolysis, and hydrogen peroxide photolysis of pyrimidine derivatives in aqueous solutions: a spin-trapping study

The sonolysis of aqueous solutions of various dihydropyrimidines and substituted pyrimidines was investigated by ESR and spin trapping with the nonvolatile, water soluble spin trap, 3,5-dibromonitrosobenzene sulfonate (DBNBS) and its deuterated analog to examine the possibility of detecting new radicals specifically generated in the high temperature zones produced by collapsing cavitation bubbles. Similar ESR spectra were obtained from sonolysis of argon-saturated aqueous solutions, from uv photolysis of aqueous solutions containing H2O2, and from gamma radiolysis of nitrous oxide saturated solutions, although sonolysis of aqueous solutions leads to the formation of pyrimidine radicals by H atom as well as OH radical addition to the 5,6 double bond of pyrimidines. No evidence for specific new radicals formed in the high temperature regions induced by cavitation could be found. For the reactions of dihydropyrimidines with hydroxyl radicals additional spin adducts could be detected and identified with the spin trap DBNBS compared to 2-methyl-2-nitrosopropane which was used in previous studies; however, for alkylpyrimidines fewer spin adducts were observed. The use of the deuterated analog of DBNBS is helpful for unambiguous radical structure assignment.     

超氧自由基与嘧啶碱基及其核苷反应的ESR研究

用自旋捕集技术和ＥＳＲ方法,以ＭＮＰ为捕集剂,研究了紫外线辐照核黄素产生超氧自由基等活性氧与嘧啶碱基及其核苷的反应,确定了尿嘧啶,胞嘧啶和胸腺嘧啶及其核苷所产生的自旋加合物自由基的类别,讨论了自由基的形成机制,揭示了超氧自由基与嘧啶碱基及其核苷的反应是不是直接进行的,而是通过羟基自由基来实现的。     

Radiolysis of poly(U) in oxygenated solution

Aqueous N2O/O2-saturated solutions of poly(U) were irradiated at 0 degrees C and the release of unaltered uracil determined. Immediately after irradiation G(uracil release) was 1.5 which increased to a value of 5.3 +/- 0.3 upon heating to 95 degrees C. Thereby all of the organic hydroperoxides (G = 6.8 +/- 0.7) and some of the hydrogen peroxide (G = 1.7 +/- 0.2) was destroyed leaving G(peroxidic material; mainly hydrogen peroxide) = 1.0 +/- 0.7. G(chromophore loss) = 8-11 was measured immediately after irradiation, but no increase was observed upon heating. Addition of iodide destroyed the hydroperoxides and caused immediate base release to rise to G = 4 and further heating brought the value to that observed in the absence of iodide. In contrast, on reducing the hydroperoxides with NaBH4, immediate uracil release rose to only G = 2.8 and no further increase was observed on heating. A major product (G = 2.7) is carbon dioxide. There are also osazone-forming compounds produced (G = 2.7), all of which are originally bound to poly(U). Heating in acid solutions, as is required for this test, releases glycoladehyde-derived osazone (G = 0.8) and further unidentified low molecular weight material (G = 0.9). It is concluded that the primary radicals which cause these lesions are the base OH adduct radicals. In the presence of oxygen these are converted into the corresponding peroxyl radicals which abstract an H atom from the sugar moiety. In the course of this reaction base-hydroperoxides are formed. However, such base hydroperoxides cannot be the only organic hydroperoxides, but some (G congruent to 2.5) sugar-hydroperoxides must be formed as indicated by the increase in base release by the addition of iodide. It is speculated that a sugar-hydroperoxide located at C(3') is reduced by iodide to a carbonyl function at C(3'), a lesion that releases the base, while reduction with NaBH4 reduces it to an alcohol function at C(3') thus preventing base release.     

Free radicals in U.V.-irradiated aqueous solutions of substituted amides: an e.s.r. and spin-trapping study.

The radicals produced by reactions of hydroxyl radicals with alkyl substituted ureas and amides in aqueous solutions have been investigated. Hydroxyl radicals were produced by U.V. photolysis of H2O2 and the short-lived amide and urea radicals were spin-trapped by t-nitrosobutane and identified by e.s.r. For all N-alkyl derivatives of urea and acetamide, and for N,N-dimethyl propionamide and N,N-diethyl formamide, only radicals centred on N-alkyl groups were detected. Radicals situated only on alkyl groups attached to the carbonyl carbon were observed for dimethyl acetamide, trimethyl acetamide and butyramide. However, for N,N-dimethyl butyramide, N, N-diethyl butyramide, N-methyl propionamide and N, N-diethyl propionamide, free radicals were formed which were localized on the alkyl group attached to the amide carbon as well as those attached to nitrogen. The hydrogen atom bound to the carbonyl carbon was abstracted in N-ethyl formamide. Acyl radicals formed by C-N scission due to direct U.V. photolysis of N, N-dimethyl butyramide and N,N-dimethyl propionamide were also detected.