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Chem. Res. Toxicol. 2008, 21, 958–965
Generation of Thymine Radicals from 6-Alkoxy-5-bromo-5,6-dihydrothymine Derivatives by Radiolytic Reduction and Photolytic Dehalogenation Mayuko Mori, Takeo Ito, Mitsunobu Kawano, Yukinari Takao, Hiroshi Hatta, and Sei-ichi Nishimoto* Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto UniVersity, Nishikyo-ku, Kyoto 615-8510, Japan ReceiVed December 27, 2007
The reduction of 6-alkoxy-5-bromo-5,6-dihydrothymine derivatives (1a,b) by hydrated electrons (eaq-) generated in the radiolysis of deoxygenated aqueous solution was investigated. As the major products, 1-(6′-alkoxy-5′,6′-dihydrothymin-5′-yl)thymines (6a,b), 5-(hydroxymethyl)uracil (8), 6-alkoxy-5,6-dihydrothymines (9a,b), and thymine (10) were produced in sufficient yields. This product distribution is indicative of the generation of 6-alkoxy-5,6-dihydrothymin-5-yl radicals (2a,b) as primary intermediates that undergo elimination of alkoxide ions (RO-) into thymine radical cations (3) followed by deprotonation at the N1 to form N-centered thymine radicals (4). The transient absorption spectra of the 5-yl radicals 2a-c were observed by means of nanosecond laser flash photolysis of 1a,b and 5-bromo-6-ethoxy-5,6dihydrothymidine (1c) in deoxygenated aqueous solution, in which homolytic C5-Br bond dissociation occurred. In contrast to the reaction characteristics in aqueous solutions, the dimeric products were not obtained in acetonitrile, probably because in-cage hydrogen abstraction from the C5 methyl group by bromine atom leads to formation of methide type intermediates 20. Introduction The action of ionizing radiation on living cells is known to cause mutations and cell death either directly or indirectly (1). The indirect effect of radiation originates from radiolysis of excess cellular water giving rise to water radicals such as hydroxyl radicals (•OH), hydrated electrons (eaq-) and hydrogen atoms (•H) that attack on a target cellular molecule of DNA (reaction 1). On the other hand, the direct effect involves the absorption of radiation energy by cellular DNA, thereby undergoing electron ejection to form a DNA radical cation (DNA•+) (reaction 2) (2, 3). Considerable investigations of identifying the products derived from the oxidative and reductive base modification of DNA bases have been hitherto carried out to get mechanistic insight into the primary processes of various modes of radiation-induced oxidative damage to DNA.
H2O f •OH + H• + eaq-
(1)
DNA f DNA•+ + eAttempts have been made to generate radical intermediates of DNA bases in aqueous model reaction systems involving oxidations by two-photon ionizaion (4–6), photoexcited quinones (7–9), and various oxidants such as Br2•- (10), Ti2+ (10), and SO4•- (11–18). As conventional methods, pulse radiolysis and laser photolysis of aqueous solutions of peroxodisulfate (S2O82-) for generating strongly oxidizing sulfate radical anion (SO4•-) have also been employed to characterize spectroscopic properties and kinetic behaviors of the radical ions of uracil, thymine, and their related pyrimidine derivatives (12, 15, 17–26). In a mechanistic view, the addition of the oxidizing SO4•- to the C5-C6 double bonds of pyrimidines followed by rapid elimina* To whom correspondence should be addressed. Tel: +81(75)383-2501. Fax: +81(75)383-2504. E-mail:
[email protected].
Chart 1. Thymidine Derivatives Generated by γ-Irradiation of Thymidine in Frozen Aqueous Solution
tion of sulfate ion (SO42-) leads to the formation of radical cations as a result of net one-electron oxidation. The resulting pyrimidine radical cations without N1 substituents are extremely short-lived, since rapid deprotonation at the N1 position occurs to form N1-centered radicals (11, 23). On the other hand, the radical cations of N1-substituted pyrimidines undergo OH addition at the C5 to afford 5-hydroxy-5,6-dihydrouracil-6-yl radicals (12, 26) or deprotonation at the C5-methyl group, leading to allyl type radicals. According to previous product studies, the former process affords pyrimidine glycols and isobarbituric acid, while the latter results in 5-(hydroxymethyl)uracils and 5-formyluracils (16). In addition, N1-C5′-linked thymine dimer hydrates (I, Chart 1) have been identified (27) in the near-UV photooxidation of thymine sensitized by a biorelated quinone of menadione (9) or in the anodic electrolysis of various uracil derivatives in aqueous solution (28, 29). Synthetically modified DNA bases have been developed for radiation, chemically or photochemically generating the corresponding radical intermediates in model systems of diluted aqueous solutions (30–32). In light of the reported reactivity (33, 34), C5-brominated thymine derivatives are one-electron reduced by eaq- upon radiolysis in aqueous solution, thereby readily eliminating bromide ions (Br-) to produce thymine C5
10.1021/tx700451u CCC: $40.75 2008 American Chemical Society Published on Web 03/28/2008
Generation of Thymine Radicals
radicals. We investigated herein reactivities of radical intermediates of thymine derivatives by radiolytic one-electron reduction or direct photolysis of 6-alkoxy-5-bromo-5,6-dihydrothymine derivatives (1a-c) in aqueous solution and observed the formation of 5-(hydroxymethyl)uracils most likely via a mechanism involving generation of allyl type radicals from thymine radical cation intermediates. Similar radical cation formation under reductive conditions has been observed in the radiolysis of 8-haloguanines in aqueous solution (35, 36). In this study, considerable attention was paid to the identification of products such as 5-(hydroxymethyl)uracil derivatives and dimeric adducts to understand the reactivity of primary 6-alkoxy-5,6-dihydrothymin-5-yl radicals (2a-c). Photolysis of thymidine bromohydrins has also been investigated for understanding the mechanism of formation of 5-(alkoxymethyl)- or 5-(hydroxymethyl)-2′-deoxyuridines from the precursors, in which generation of a methide type intermediate has been suggested as an alternative pathway (37). Laser flash photolysis of 1a-c was also performed to obtain complimentary information on the formation of key intermediates, such as thymine C5 radicals and radical cations as described below.
Materials and Methods Materials and General Procedures. Thymine (10) and thymidine (19) purchased from Wako Pure Chemical Industries (Osaka, Japan) and 5-(hydroxymethyl)uracil (8) received from Sigma (St. Louis, MO) were used without further purification. 5-(Bromomethyl)uracil (7) was prepared by conventional bromination of 8. Acetonitrile used as a solvent for the photolysis was dried and distilled over P2O5. All other reagents and solvents obtained from Nacalai Tesque (Kyoto, Japan) were of the highest available purities and were used as received. Phosphate buffer solutions (KH2PO4 and Na2HPO4, 10 mM) for radiolysis were prepared with water ion-exchanged by a Corning Mega-Pure System MP-190 (>16 MΩ cm). 6-Alkoxy-5-bromo-5,6-dihydrothymines (1a,b) were prepared by alkoxylation of 5-bromo-6-hydroxy-5,6-dihydrothymine. 5-Bromo6-ethoxy-5,6-dihydrothymidine (1c) was prepared by the reaction of thymidine 19 with bromine and dibutyltin oxide in ethanol, following the method reported by Samuel et al. (38), and purified by preparative HPLC. 1H and 13C NMR spectra were recorded on a JEOL EX-400 MHz spectrometer. High-resolution fast atom bombardment mass spectrometry (FAB-HRMS) was performed on a JEOL JMS-SX102A mass spectrometer, using a glycerol matrix. Steady-State Radiolysis. Typically, solutions (4 mL) of 6-alkoxy5-bromo-5,6-dihydrothymine derivatives 1a-c (0.5 mM) containing an excess amount (100 mM) of 2-methyl-2-propanol or sodium formate in phosphate buffer at pH 7.0 were purged with Ar for 20 min. The solution in a sealed Pyrex glass tube was irradiated with a γ-ray source from 60Co at a dose rate of 0.79 Gy min-1 at room temperature. Steady-State Photolysis. An aqueous solution or dry acetonitrile solutions (2 mL) of 5-bromo-6-ethoxy-5,6-dihydrothymine 1b (3 mM) were purged with Ar prior to photoirradiation. The solution in a sealed Vycor tube was photoirradiated for 20 min under magnetic stirring at room temperature with a low-pressure Hg lamp. Nanosecond Laser Flash Photolysis. Laser flash photolysis experiments were carried out with a Unisoku TSP-601 flash spectrometer. A Continuum Surelite-I Nd:YAG (Q-switched) laser with the fourth harmonic at 266 nm (ca. 50 mJ per 6 ns pulse) was employed for the flash photoirradiation. The probe beam from a Hamamatsu 150 W xenon short arc (CA 263) was guided with an optical fiber scope to be arranged in an orientation perpendicular to the exciting laser beam. The probe beam was monitored with a Hamamatsu R2949 photomultiplier tube through a Hamamatsu S3701–512Q MOS linear image sensor (512 photodiodes). Timing of the exciting pulsed laser, the probe beam, and the detection system was achieved through a Tektronix model TDS 320 double channel oscilloscope that was interfaced to an NEC PC. Aqueous
Chem. Res. Toxicol., Vol. 21, No. 4, 2008 959 solutions of 1a-c (3.0 mM) and dry acetonitrile solution of 1b (2.0 mM) were deaerated by Ar bubbling prior to the laser flash photolysis. Product Analysis. The sample solutions were subjected to HPLC analysis immediately after γ-irradiation or photoirradiation, using a Shimadzu 10A HPLC system equipped with a Rheodyne 7725 sample injector. Aliquots (5 µL) of irradiated solutions were injected onto a reversed-phase column containing C18 chemically bonded silica gel with 5 µm particle size (GL science 5C18, φ 4.6 mm × 250 mm, GL science). The phosphate buffer solution (10 mM, pH 3.0) containing various concentrations of methanol (5–30 vol %) were delivered at a flow rate of 0.6 mL min-1. The eluents were monitored by the UV absorbance at 210 nm. For isolation of products, the radiation-irradiated solutions (up to 800 Gy) or the photoirradiated solutions (for 20 min) were evaporated to a minimum volume and subjected to a preparative HPLC using a Tosoh Preparative HPLC system equipped with a Chromatocorder 12 (System Instruments). Isolation was performed on a reversephase column (GL science 10C18, φ 10 mm × 250 mm) containing C18 chemically bonded silica gel (10 µm particle size), and an aqueous solution containing 5–30 vol % methanol was delivered at a flow rate of 3.0 mL min-1. Aqueous fractions that contained the respective products were then collected and evaporated. The resulting residues were lyophilized and subjected to spectroscopic measurements. 1-(6′-Methoxy-5′,6′-dihydrothymin-5′-yl)thymine (6a). 1H NMR (400 MHz, DMSO): δ 11.26 (br s, 1H), 10.45 (br s, 1H), 8.48 (d, J ) 2.93 Hz, 1H), 7.59 (s, 1H), 5.34 (d, J ) 3.91 Hz, 1H), 3.20 (s, 3H), 1.77 (s, 3H), 1.65 (s, 3H). 13C NMR (100 MHz, DMSO): δ 168.9, 167.8, 155.3, 151.9, 143.9, 102.1, 82.1, 78.0, 55.8, 16.0, 11.3. FAB-HRMS (glycerol matrix) m/z: calcd for C11H15O5N4 [(M + H+)], 283.1043; found, 283.1023. 1-(6′-Ethoxy-5′,6′-dihydrothymin-5′-yl)thymine (6b). 1H NMR (400 MHz, DMSO): δ 11.24 (br s, 1H), 10.41 (br s, 1H), 8.42 (d, J ) 3.42 Hz, 1H), 7.59 (s, 1H), 5.45 (d, J ) 3.42 Hz, 1H), 3.66 (dq, J ) 9.77, 7.08 Hz, 1H), 3.46 (dq, J ) 9.77, 7.08 Hz, 1H), 1.77 (s, 3H), 1.65 (s, 3H), 1.09 (t, J ) 6.84 Hz, 3H). 13C NMR (100 MHz, DMSO): δ 168.9, 167.8, 155.2, 151.9, 143.9, 102.1, 80.4, 78.0, 63.7, 16.0, 14.9, 11.3. FAB-HRMS (glycerol matrix) m/z: calcd for C12H17O5N4 [(M + H+)], 297.1199; found, 297.1172. 6-Methoxy-5,6-dihydrothymine (9a). 1H NMR (400 MHz, DMSO): δ 10.08 (br s, 1H), 8.59 (br s, 1H), 4.34 (t, J ) 3.91 Hz, 1H), 3.22 (s, 3H), 2.81 (dd, J ) 3.91, 6.83 Hz, 1H), 1.05 (d, J ) 6.83 Hz, 3H). 13C NMR (100 MHz, DMSO): δ 172.1, 153.1, 82.2, 54.5, 39.8, 10.2. FAB-HRMS (glycerol matrix) m/z: C6H11O3N2 [(M + H+)], 159.0770; found, 159.0777. 6-Ethoxy-5,6-dihydrothymine (9b). 1H NMR (400 MHz, acetoned6): δ 8.89 (br s, 1H), 7.08 (br s, 1H), 4.61 (t, J ) 4.15 Hz, 1H), 3.68 (dq, J ) 9.52, 7.08 Hz, 1H), 3.40 (dq, J ) 9.28, 6.96 Hz, 1H), 2.78 (dd, J ) 3.90, 6.84 Hz, 1H), 1.14 (d, J ) 6.84 Hz, 3H), 1.08 (t, J ) 7.08 Hz, 3H). 13C NMR (100 MHz, acetone-d6): δ 172.1, 153.1, 80.6, 62.2, 39.5, 14.8, 10.1. FAB-HRMS (glycerol matrix) m/z: calcd for C7H13O3N2 [(M + H+)], 173.0898; found, 173.0912. 6-Ethoxy-5,6-dihydrothymidine (9c). 1H NMR (400 MHz, acetone-d6): δ 9.03 (br s, 1H), 5.96 (dd, J ) 5.78, 2.39 Hz, 1H), 4.96 (d, J ) 2.20 Hz, 1H), 4.40–4.37 (m, 1H), 4.25 (d, J ) 4.04 Hz, 1H), 3.97 (t, J ) 5.41 Hz, 1H), 3.89–3.85 (m, 1H), 3.77–3.54 (m, 5H), 2.79 (s, 1H), 2.70–2.68 (m, 1H), 2.35–2.66 (m, 1H), 1.18 (d, J ) 7.34 Hz, 3H), 1.09 (t, J ) 6.97 Hz, 3H). FAB-HRMS (glycerol matrix) m/z: calcd for C12H21O6N2 [(M + H+)], 289.1400; found, 289.1411. 3-(6′-Ethoxy-5′,6′-dihydrothymid-5′-yl)thymidine (14). 1H NMR (300 MHz, acetone-d6): δ 9.37 (br s, 1H), 8.17 (s, 1H), 6.14 (t, J ) 6.10 Hz, 1H), 5.97 (s, 1H), 5.87 (dd, J ) 5.85, 1.96 Hz, 1H), 4.45–4.41 (m, 1H), 4.16–4.13 (m, 1H), 3.94–3.53 (m, 9H), 2.38–2.32 (m, 1H), 2.17–2.10 (m, 1H), 1.98–1.92 (m, 3H), 1.81 (s, 3H), 1.79 (s, 3H), 1.09 (t, J ) 7.32 Hz, 3H). 13C NMR (75 MHz, acetone-d6): δ 169.64, 167.50, 155.12, 152.37, 143.28, 104.72, 89.02, 87.78, 87.74, 86.73, 84.48, 80.16, 71.87, 71.22, 66.16, 63.16, 62.24, 42.15, 39.99, 15.99, 15.54, 12.34. FAB-HRMS
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Chem. Res. Toxicol., Vol. 21, No. 4, 2008 Scheme 1. Radiation-Induced Reduction of 5-Bromo-6-alkoxy-5,6-dihydrothymine Derivatives
Mori et al. Table 1. Radiolytic One-Electron Reduction of 1a,b in Ar-Saturated Aqueous Solution under Several Conditions substrate
OH radical scavenger
reductant
1a 1a 1b 1b 1b
t-BuOH HCOOt-BuOH t-BuOH HCOO-
none none none TMPD none
G (mol J-1)a -substrate 6 8 5.4 32.7 6.4 3.4 30.6
1.2 1.5 1.1 0.2 1.7
1.0 0 0.9
