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Chem. Res. Toxicol. 2007, 20, 1820–1824
(5′S)- and (5′R)-5′,8-Cyclo-2′-deoxyguanosine: Mechanistic Insights on the 2′-Deoxyguanosin-5′-yl Radical Cyclization Chryssostomos Chatgilialoglu,*,† Rita Bazzanini,† Liliana B. Jimenez,†,‡ and Miguel A. Miranda‡ ISOF, Consiglio Nazionale delle Ricerche, Via P. Gobetti 101, I-40129 Bologna, Italy, and Instituto de Tecnología Química UPV-CSIC, UniVersidad Politécnica de Valencia, AVenido de los Naranjos, s/n E-46022 Valencia, Spain ReceiVed August 5, 2007
The two diastereomeric forms (5′S) and (5′R) of 5′,8-cyclo-2′-deoxyguanosine have been synthesized and fully characterized. They have been used as references for the investigation of γ-irradiation of 2′deoxyguanosine and photolysis of 8-bromo-2′-deoxyguanosine in aqueous solutions. The observed (5′R)/ (5′S) ratio of 8:1 was obtained in both sets of experiments. The mechanism of the cyclization reaction is discussed in some detail, and the diastereomeric outcome is rationalized in terms of favorable hydrogenbonded structures in the pro-(5′R) conformation. Introduction It is well known that DNA has limited chemical stability. Free radicals and ionizing radiation induce damage in the DNA of living cells, resulting in mutation, chromosomal aberration, carcinogenesis, and aging (1, 2). Understanding the chemical nature of radiation- and free-radical-induced DNA lesions and elucidating the mechanisms which regulate this damage are essential for assessment of the possible biological consequences and the mechanism of the enzymatic repair process. The hydroxyl radical reacts with the various hydrogen atoms of the deoxyribose moieties of DNA in the order H5′ > H4′ > H3′ ≈ H2′ ≈ H1′ (3). This order of reactivity parallels the exposure of the deoxyribose hydrogens to solvent. Hydrogen abstraction from the 5′-position of purine nucleosides can lead to the formation of 5′,8-cyclonucleosides, which have an additional base-sugar linkage between the C5′ position of the 2-deoxyribose and the C8 position of the purine (Figure 1) (4–6). They have also been identified in mammalian cellular DNA in vivo, where their levels are enhanced by conditions of oxidative stress (7–10). These lesions represent a unique class of oxidative DNA lesions in that they are repaired by the nucleotide excision repair (NER) pathway but not by base excision repair (BER) or direct repair (11). Both the adenine derivative 1 and the guanine derivative 2 have two diastereoisomeric forms, the (5′S) and (5′R) isomers, which differ in configuration at the C5′ position. Depending on the substrate and the experimental conditions, the ratio of the (5′S) and (5′R) isomers changes substantially. The (5′R) isomers of both purines were found to predominate in γ-irradiated aqueous solutions containing either the simple nucleoside or single-stranded DNA (4), whereas (5′R)/(5′S) ratios of approximately 2 and 0.3 were reported for cyclodeoxyadenosine and cyclodeoxyguanosine, respectively, in double-stranded DNA (5, 6). In our laboratories, we have studied in some detail the synthesis, kinetics, and stereochemistry of various intermediates * To whom correspondence should be addressed. Tel: 39-051-639-8309. Fax: 39-051-639-8349. E-mail:
[email protected]. † Consiglio Nazionale delle Ricerche. ‡ Universidad de Politécnica de Valencia.
of 5′,8-cyclo-2′-deoxyadenosine (12–14). Today the most important reactions related to 2′-deoxyadenosin-5′-yl radical are well understood, whereas the available knowledge about the analogous reactions of 2′-deoxyguanosin-5′-yl radical is limited (15). Here we have extended our previous work with an investigation of the formation of 5′,8-cyclo-2′-deoxyguanosine by either γ-irradiation of 2′-deoxyguanosine or photolysis of 8-bromo-2′-deoxyguanosine in aqueous solutions. Our objectives were to determine the diastereomeric ratio of the cyclic nucleosides formed by the two independent methods and to shed light on the mechanism of their formation as well as on the origin of its stereoselectivity.
Experimental Procedures Starting Materials. 2′-Deoxyguanosine was purchased from Fluka. 8-Bromo-2′-deoxyguanosine (16) and 8-hydroxy-2′-deoxyguanosine were purchased from Berry & Associates. Continuous Radiolysis. The experiments were performed at room temperature (rt, 22 ( 2 °C) on 10 mL samples using 60CoGammacells. N2O-saturated aqueous solutions containing 1.5 mM 2′-deoxyguanosine at natural pH were irradiated with a total dose of 2 kGy at a dose rate of 18 Gy min-1. Steady-State Photolysis. Aqueous solutions (5 mL) containing 8-bromo-2′-deoxyguanosine (1 mM) were irradiated under N2. The irradiation source was a 125 W medium-pressure mercury lamp. HPLC and LC/MS Analyses. Reaction mixtures were analyzed with a Zorbax MS-C18 column (4.6 × 150 mm) eluted in triethylammonium acetate buffer (20 mM, pH 7) with a 0–25% acetonitrile linear gradient over 30 min at a flow rate of 1.0 mL/ min (detection at 250 nm). Products were identified by LC/MS analysis. 5′,8-Cyclo-2′,5′-dideoxyguanosine (4). 1H NMR (400 MHz, DMSO-d6): δ 10.53 (br s, 1H, NH), 6.43 (br s, 2H, NH2), 6.10 (d, J1′,2′′ ) 4.9 Hz, 1H, H1′), 5.33 (d, J3′,OH3′ ) 3.9 Hz, 1H, OH3′, D2O exchange), 4.56 (d, J4′,5′ ) 6.1 Hz, 1H, H4′), 4.25 (m, 1H, H3′), 3.22 (dd, J5′,5′′ ) -17.3 Hz, 1H, H5′), 2.79 (d, 1H, H5′′), 2.39 (dd, J2′,2′′ ) -13.2 Hz, J2′,3′ ) 7.3 Hz, 1H, H2′), 2.02 (dt, J2′′,3′ ) 4.7 Hz, 1H, H2′′). 13C NMR (100 MHz, DMSO-d6): δ 158.1 (CdO), 154.8 (C–NH2), 148.8 (C4), 138.8 (C8), 115.4 (C5), 83.3 (C4′), 82.4 (C1′), 73.2 (C3′), 46.1 (C2′), 29.8 (C5′). MS (ES–): m/z 248 (M – 1). (5′S)-5′,8-Cyclo-2′-deoxyguanosine (6). 1H NMR (400 MHz, DMSO-d6): δ 10.53 (br s, 1H, NH), 6.44 (br s, 2H, NH2), 6.27 (d,
10.1021/tx700282x CCC: $37.00 2007 American Chemical Society Published on Web 11/08/2007
Mechanism of 2′-Deoxyguanosin-5′-yl Radical Cyclization
Chem. Res. Toxicol., Vol. 20, No. 12, 2007 1821
Figure 1. Structures of 5′,8-cyclo-2′-deoxyadenosine (1) and 5′,8-cyclo2′-deoxyguanosine (2).
Scheme 1. Synthetic Paths for the Preparation of 5′,8-Cyclo-2′,5′-dideoxyguanosine 4 and (5′S)-5′,8-Cyclo-2′-deoxyguanosine 6a
Figure 2. HPLC chromatogram obtained from a sample of 2′deoxyguanosine 3 γ-irradiated in N2O-saturated aqueous solution.
angle is ∼90°. It is worth noting that 5′,8-cyclo-2′,5′-dideoxyguanosine 4 is an intermediate of the synthetic sequence shown in Scheme 1. In the synthesis, the intermediate 5 was deprotected at the 3′-OH position with 2 equiv of tetrabutylammonium fluoride (TBAF) in THF (62% yield), and subsequently the free amino group was obtained nearly quantitatively by heating at 50 °C overnight (o.n.) in a saturated methanolic ammonia solution to give the expected product, (5′S)-5′,8-cyclo-2′deoxyguanosine 6. The (5′R) isomer 7 was obtained by photolysis of 8-bromo2′-deoxyguanosine (see below) followed by preparative HPLC. It has been fully characterized.
a Synthetic details: (a) Bu3P (2 equiv), (PhS)2 (2 equiv), DMF, rt, 24 h (79%); (b) hν (λ ) 254 nm), (EtO)3P (10 equiv), t-BuOH, argon, 16 h (53%); (c) TBDMSCl (1.85 equiv), imidazole (4.2 equiv), DMF, o.n. (62%); (d) BzCl (2.5 equiv), pyridine, rt, 2 h (80%); (e) SeO2 (5 equiv), 1,4-dioxane, reflux, o.n. (72%); (f) NaBH4 (2 equiv), CH3OH, rt, 20 min (81%); (g) TBAF (1.85 equiv), THF, rt, o.n. (62%); (h) NH3/MeOH, 50 °C, o.n. (96%).
J5′,OH5′ ) 6.1 Hz, 1H, OH5′, D2O exchange), 6.05 (d, J1′2′′ ) 4.6 Hz, 1H, H1′), 5.33 (d, J3′,OH3′ ) 4.8 Hz, 1H, OH3′, D2O exchange), 4.86 (t, J5′,4′ ) 6.2 Hz, 1H, H5′), 4.65–4.50 (m, 1H, H3′), 4.38 (d, 1H, H4′), 2.31 (dd, J2′,2′′ ) -13.0 Hz, J2′,3′ ) 7.4 Hz, 1H, H2′), 1.97 (dt, J2′′,3′ ) 4.4 Hz, 1H, H2′′). 13C NMR (100 MHz, DMSOd6): δ 156.1 (CdO), 153.5 (C–NH2), 148.0 (C4), 143.5 (C8), 115.6 (C5), 86.0 (C4′), 83.8 (C1′), 67.0 (C3′), 63.8 (C5′), 45.0 (C2′). MS (ES–): m/z 264 (M – 1). Anal. Calcd for C10H11N5O4: C, 45.28; H, 4.18; N, 26.41; O, 24.13. Found: C, 45.35; H, 4.20; N, 26.38; O, 24.15. (5′R)-5′,8-Cyclo-2′-deoxyguanosine (7). 1H NMR (300 MHz, DMSO-d6): δ 10.4 (br s, 1H, NH), 6.4 (s, 2H, NH2), 6.11 (d, J1′,2′′ ) 4.8 Hz, 1H, H1′), 5.96 (d, J5′,OH5′ ) 5.2 Hz, 1H, OH5′, D2O exchange), 5.32 (d, J3′,OH3′ ) 1.6 Hz, 1H, OH3′, D2O exchange), 4.43 (d, J ) 1.2 Hz after D2O exchange, J5′,4′ ) 0.8 Hz, 1H, H5′), 4.35 (s, 1H, H4′), 4.11 (m, 1H, H3′), 2.22 (dd, J2′,2′′ ) -13.4 Hz, J2′,3′ ) 7.4 Hz, 1H, H2′), 1.92 (dt, J2′′,3′ ) 4.6 Hz, 1H, H2′′). 13C NMR (75.5 MHz, DMSO-d6): δ 156.7 (CdO), 153.9 (C–NH2), 148.1 (C4), 141.6 (C8), 115.8 (C5), 89.6 (C4′), 83.6 (C1′), 69.3 (C3′), 64.6 (C5′), 44.1 (C2′). MS (ES–): m/z 264 (M – 1).
Results and Discussion Synthesis of (5′S)- and (5′R)-5′,8-Cyclo-2′-deoxyguanosine. The protected (5′S)-5′,8-cyclo-2′-deoxyguanosine 5 was synthesized in six steps following a slightly modified version of the procedure of Cadet and co-workers (16) (Scheme 1). The (5′S) configuration of 5 was assigned by 1H NMR analysis on the basis of the value of the H4′,H5′ coupling constant J4′,5′. A J4′,5′ value of >6 Hz is diagnostic of the (5′S) configuration, since the 4′,5′ dihedral angle is 30°, while a J4′,5′ value of
