Günter Behrens

Comparison of the reaction of ˙OH and of SO4–˙ radicals with pyrimidine nucleosides. An electron spin resonance study in aqueous solution

Reactions of photolytically generated ˙OH and SO4–˙ radicals with uridine, cytidine, 2′-deoxyuridine (dU) 2′-deoxycytidine (dC), and thymidine have been studied by e.s.r. spectroscopy under anoxic conditions. In the experiments with ˙OH, the spectra of the uracil compounds were dominated by the signals of radicals originating from ˙OH addition at the alkenic double bond of the nucleobase. No spectra were observed for the cytosine derivatives and thymidine. With SO4–˙, base radicals were generated from the deoxyribonucleosides [C(5)-OH-6-yl from dU, C(6)-OH-5-yl from thymidine, and a nitrogen-centred radical from dC] whereas the ribonucleosides lead to two different types of sugar radical. One of them is derived from the 2′-hydroxyalkyl radical by heterolytic elimination of the nucleobase and the other is the 3′-hydroxyalkyl radical which undergoes ring-opening by heterolytic cleavage of the C(4′)-oxygen bond at neutral and alkaline pH. Both the ˙OH and SO4–˙ radicals add to the base moieties in the primary step. The adduct radicals formed with ˙OH from uridine and dU are stable on the millisecond time-scale of the e.s.r. experiment whereas the sulphate adducts are too short-lived to be detected by e.s.r. In the deoxyribose derivatives they either hydrolyse (dU and thymidine) or eliminate SO42– and a proton (dC) whereas in the ribonucleosides they induce intramolecular H abstraction from positions 2′ and 3′ of the sugar residues.

Model Reactions for the Degradation of DNA-4′ Radicals in Aqueous Solution. Fast Hydrolysis of α-Alkoxyalkyl Radicals with a Leaving Group in β-Position Followed by Radical Rearrangement and Elimination Reactions

Abstract α-Alkoxyalkyl radicals with a leaving group L = Cl or OCOCH3 in β-position are produced by H-abstraction from the corresponding saturated substrates by ·OH, SO·4- or (CH3)3CO· radicals. From ESR spectroscopic observations it is concluded that in aqueous solution at pH 5 -9 the following fast hydrolysis reactions take place: The rate constants of these reactions and for the hydrolysis of CH3O-ĊH-CH2Cl are k ≥ 106 s-1, whereas the rate constant for CH3O-ĊH-CH2OCOCH3 was determined to be ≈ 2 × 103 s-1 at room temperature. The radicals with L = Cl cannot be scavenged by O2 which fact leads to a value of k ≥ 2 × 10-7 s-1. α-Alkoxyalkene radical cations are assumed as intermediates in the hydrolysis reactions. The radicals with L = OCOCH3 and the radical CH3O--ĊH-CH2Cl are observable in acetone solution ESR spectroscopically. In aqueous solution at pH below 3 proton catalyzed reactions are observed by ESR spectroscopy: Radicals resulting from H-abstraction at the CH3O-groups of the substrates or at the 5-positions of the cyclic ethers are also observed. The ESR parameters and the pH-ranges of existence of the above radicals are given. Support of the reported reactions comes from quantitative analysis of stable products such as H+, Cl- or CH3OH after 60Co-γ-irradiation of N2O saturated aqueous solutions of the substrates. The behaviour of the radicals is used as a model to describe a modified version of the degradation of DNA-4′ radicals in aqueous solution in the absence of oxygen.

Reaction of SO4–˙ with methylated uracils. An electron spin resonance study in aqueous solution

Radicals obtained by reaction of photolytically generated SO4–˙ with 1-methyluracil (1), 1,3-dimethyluracil (2), 1-methylthymine (3), 1,3-dimethylthymine (4), and 1,3,6-trimethyluracil (5) were studied by e.s.r. spectroscopy in aqueous solutions. In situ photolysis of neutral and acidic solutions containing (1) and persulphate in low concentrations (3mM) resulted in the e.s.r. spectrum of the C(5)-OH adduct radical. E.s.r. spectra obtained from (1) and (2) with high persulphate concentrations (30mM) were assigned to the C(6)-OH adduct radicals. It is proposed that this change in radical population is due to a persulphate-induced chain reaction which results in selective oxidation of the C(5)-OH radicals and simultaneous accumulation of the C(6)-OH adducts generated in side reactions. When the persulphate concentration was raised to 60mM, 5-oxo-6-yl radicals were formed in secondary processes from (1) and (2) besides the OH adducts. In contrast to these results the thymines (3) and (4) yielded only C(6)-OH adduct radicals. Addition of phosphate dianions to the photolysis solutions containing persulphate and the pyrimidine bases (1)–(4) resulted in the e.s.r. spectra of the C(6)-phosphate adduct radicals (pH 6.5–9.5). Identical spectra were obtained by reaction of (1)–(4) with HPO4–˙ radical anions generated by photolysis of Li4P2O8. The results of the experiments with 1,3,6-trimethyluracil (5) were completely different. First, reaction of SO4–˙ with (5), even at low persulphate concentration (3mM), did not lead to an OH adduct but to a 5-oxo-6-yl secondary radical. Secondly it was not possible to generate phosphate adduct radicals from (5) either with a mixture of persulphate and phosphate or with peroxodiphosphate. The spectral parameters of the radicals derived from (1)–(5) are given and possible pathways for the SO4–˙-induced radical formation are discussed.

Reactions of 1,1-dialkoxyalkene radical cations in aqueous solution with OH–, HPO42–, and H2O. Electron spin resonance spectroscopic, pulse conductometric, and product analytical studies

The reactions of H2O, HPO42–, and OH– with eight, 1,1-dialkoxyalkene radical cations have been studied in aqueous solution by e.s.r. spectroscopy and in part by pulse conductometry and product analysis. Hydroxide ion reacts with the radical cations with rate constants ranging from k 2 × 108 for species (5) to 6 × 109 l mol–1 s–1 for (6), producing mainly alkoxycarbonylalkyl radicals. With (1) and with (6), in addition, the formation of 1,1-dialkoxy-2-hydroxyethyl radicals was observed and e.s.r. parameters are given. The HPO42– anion was found to react with four of the radical cations to form phosphato-dianion-substituted radicals, (RO)2(PO42–)C–ĊH2(e.s.r. parameters are given), with k values ranging from 2 × 106 for (1) to 3 × 108 l mol–1 s–1 for (6). Water reacted analogously with rate constants ranging from k 3 × 102 for (1) to 104 s–1 for (27) at 20 °C, the reaction yielding 2,2-dialkoxy-2-hydroxyethyl radicals (e.s.r. parameters are given) and protons. Attack of water occurs virtually only at the dioxygen-substituted carbon as is shown in one example. Product analysis by g.l.c.–m.s. of the higher boiling material from 60Co γ-irradiation of N2O-saturated aqueous solutions of (CH3O)2CH–CH2Cl gave evidence for the formation of dimers and cross-dimers of the radicals ĊH2OCH(OCH3)CH2Cl, CH3OCOĊH2, (CH3O)2CHĊHCl, (CH3O)2ĊHCH2, and (CH3O)2ĊH2OH. Structures and yields of the products agree with the conclusions drawn from e.s.r. spectroscopic and conductometric measurements.

Formation and reaction of peroxyl radicals of polynucleotides and DNA in aqueous solution

Radiation-induced strand breaks of polyuridylic acid [poly(U)] in aqueous solution in the presence of oxygen are known to proceed via peroxyl radicals of the uracil residues. The peroxyl radicals abstract H atoms from sugar moieties, and reaction of the sugar radicals leads to chain breaks. The lifetimes of the peroxyl radicals of poly(U), polyadenylic acid [poly(A)] and of single- and double-stranded DNA were determined by time-resolved e.s.r. spectroscopy and found to be in the range of seconds whereas the peroxyl radicals of low-molecular-weight model compounds have lifetimes of several milliseconds. In poly(U), the rate of decay of the peroxyl radicals was shown to coincide with the rate of strand-break formation, determined from pulse conductivity measurements. This leads to the conclusion that the reaction of peroxyl radicals with the sugar moieties is the rate-limiting step in strand-break formation. The peroxyl radicals of poly(U), poly(A) and DNA react with various thiols with rate constants of ⋍104–105 M−1 s−1. Strand-break formation in poly(U) was found to be prevented by thiols. The consequences of these results for the radioprotection of living cells are discussed.

Formation and structure of 1,1-dialkoxyalkene radical cations in aqueous solution. An in situ electron spin resonance and pulse conductivity study

The radical cations (1), (10), (11a and b), and (12)–(15) have been produced in aqueous solution and have been identified by e.s.r. spectroscopy and conductivity investigation. The open chain radical cations exist in Z,E-configurations. They exhibit two sets of aδH couplings. The larger couplings were assigned to the protons in groups with the Z-configuration. In all cases the radical spin is located mainly at the carbon atoms. Under our conditions radical (13) disappeared pseudo-monomolecularly upon reaction with water (k= 7 × 103 s–1) whereas the other radical cations are longer lived and decayed bimolecularly with diffusion controlled rates. The radical cations were generated from open chain or cyclic acetals bearing Br, Cl, or CH3CO2 groups β to the acetal CH group, e.g.(CH3O)2CH–CH2Cl or (CH2O)2CH–CH2Cl. These substrates were subjected to hydrogen abstraction by OH˙ or SO4˙– radicals or triplet acetone in aqueous solution. Hydrogen abstraction from the acetal CH group led to radicals which undergo fast heterolytic dissociation into radical cations and leaving group anions, e.g.(CH3O)2Ċ–CH2Cl →(CH3O)2C–ĊH2+ Cl–.

Elimination of Ammonium Ion from the a-Hydroxyalkyl Radicals of Serine and Threonine in Aqueous Solution and the Difference in the Reaction Mechanism

Abstract The zwitterionic radicals HO-ĊH-CH(COO-)NH3+ (4a) and HO-Ċ(CH3)-CH(COO-)NH3+ (4b) are the main species produced upon OH· radical attack in aqueous solutions at pH 3-7 at the amino acids serine, HO-CH2-CH(COO-)NH3+, or threonine, HO-CH(CH3)-CH(COO-)NH3+, respectively. Both radicals undergo elimination of NH4+ ion to form the radicals O=CH-ĊH-COO- (7) or CH3-CO-ĊH-COO- (9) respectively. The pKa of the serine-derived cationic radical HO-ĊH-CH(COOH)NH3+ (3a) (3a ⇄ 4a + H+), was determined by ESR spectroscopy to 2.2 ± 0.1 at 276 K. From kinetic data the pKa(OH) of radical 4a (4a ⇄ O-ĊH-CH(COO-)NH3+ (5a) + H+) was calculated to 7.0. The elimination of NH3 takes place from the ketyl radical 5a (type-B mechanism), the rate constant was calculated from kinetic data to 2.4 × 106 s-1 at 290 K. The half-lives of radicals 4a and 4b were measured by time-resolved conductivity changes upon pulse radiolysis, 170 ± 10 μs for 4a and 26 ± 2 μs for 4b, at 290 K and pH 5.8 . With the threonine derived radicals elimination of NH3 takes place at the stage of the α-hydroxyalkyl radical 4b (type-A mechanism). In this series the pKa of the product radical CH3-CO-ĊH-COOH (8) (8 ⇄ 9 + H+), was determined by ERS spectroscopy to 2.7 ± 0.1. The reasons for the observed mechanistic differences (type-A versus type-B decay) are discussed. As further examples for a type-B decay some preliminary data on the elimination of HF from the radicals CF3-Ċ(OH)-CF3 and CF3-ĊH-OH have been added.

Formation and Structure of Radicals from ᴅ-Ribose and 2-Deoxy- ᴅ-ribose by Reactions with SO4·̅ Radicals in Aqueous Solution. An in-situ Electron Spin Resonance Study

Abstract ESR spectroscopy has been used to analyse the conformation of the radicals produced by the reaction of SO4·̅ with ᴅ-ribose (1), and 2-deoxy-ᴅ-ribose (6), at pH 1.3-5. From ribose three different types of radicals formed by H abstraction at C-1, C-2 and C-3 followed by a regio-selective α,β-water elimination have been identified: the 2-deoxy-ribonolacton-2-yl (3), the 1-deoxy-pentopyranos-2-ulos-1-yl (4). and the 4-deoxy-pentopyranos-3-ulos-4-yl (2). Using deoxyribose two radicals of similar type, formed by H abstraction at C-3 and C-4 followed by water elimination, have been observed: the 2,4-dideoxy-3-ulos-4-yl (7) and the 2,3-dideoxy-4-ulos-3-yl (8). In addition, from both sugars an a-hydroxyalkyl radical has been identified based in part on the timing of their conformational motions: the ribos-3-yl (5) (the precursor of 2) and the 2-deoxy-ribos-1-yl (9), respectively. For radical 5 the rate constant k(e) for the water elimination and hence transformation into radical 2 was estimated. From the analysis of selective line broadening the frequencies of conformational changes of radicals 2 and 7 have been estimated. For 7 the frequencies of exchange of the two methylene groups were found to differ by more than 3 orders of magnitude.