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New Insights into the Reaction Paths of Hydroxyl Radicals with 20-Deoxyguanosine Chryssostomos Chatgilialoglu,*,† Mila D’Angelantonio,† Gabriel Kciuk,‡ and Krzysztof Bobrowski‡ † ‡
ISOF, Consiglio Nazionale delle Ricerche, Via P. Gobetti 101, 40129 Bologna, Italy Institute of Nuclear Chemistry and Technology, Dorodna 16, 03-195 Warsaw, Poland
bS Supporting Information ABSTRACT: The reaction of HO• radical with 20 -deoxyguanosine is intensively studied as a model for DNA damage. Several aspects related to the reaction paths responsible for the most relevant lesions are not well understood. We have reinvestigated the reaction of HO• with 20 -deoxyguanosine by pulse radiolysis and extended our studies to a variety of substituted derivatives. The main path of hydrogen abstraction was confirmed to be from the exocyclic NH2 group, followed by a waterassisted tautomerization. The rate constant (k = 2.3 104 s1) obtained from the spectral changes at 620 nm is influenced by the substituent at the C8 position. When N1H is replaced by N1CH3, the tautomerization does not occur. The spectral changes at 370 nm that correspond to a rate constant of 6.9 105 s1 were assigned to the cyclization of 20 -deoxyguanosin-50 -yl radical with formation of 50 ,8-cyclo-20 -deoxyguanosine as the product. When NEt2 replaces the exocyclic NH2, the spectral changes at all wavelengths follow second-order kinetics, suggesting a “slow” ring-opening of the 8-hydroxyl adduct of 20 -deoxyguanosine.
’ INTRODUCTION One-electron oxidation of guanosine (1a) or 20 -deoxyguanosine (1b) using oxidants like SO4• or CO3• affords the guanyl radicals 2 by electron transfer coupled with deprotonation (Scheme 1).1,2 The analogous reaction with HO• radicals also produces the guanyl radical 2 through a more complex path, although the redox properties are still favorable for an electron transfer, viz., E7(2/1) = 1.29 V3 versus E°(SO4•/ SO42) = 2.43 V, E°(HO•/HO) = 1.90 V, or E°(CO3•/ CO32) = 1.59 V.4 In 2000, Candeias and Steenken revisited the reaction of HO• with guanine derivatives in aqueous solution.5 They reported that the reaction of HO• radicals is partitioned between the base and sugar moieties of 1, by addition at the base moiety (8085%) and by hydrogen abstraction at the (20 -deoxy)ribose unit.5 They concluded that addition to the base occurs at the C4 position (∼65%), which is the precursor of guanyl radicals (2) via a dehydration path, and at the C8 position (∼17%), affording an adduct radical that undergoes a fast ring-opening.5 We have recently revisted this ambident reactivity of guanine moiety toward HO• radicals and observed that the main reaction of the HO• radical with guanine moiety is not the addition at the C4 position but a hydrogen abstraction from the exocyclic NH2 group (1 f 3), followed by the tautomerization 3 f 2 (Scheme 1).6 The reaction products from the hydrogen abstraction at the 2-deoxyribose unit by HO• radicals have been recently identified in the absence or presence of oxygen for 1b.7,8 The two diastereomeric forms (50 S and 50 R) of 50 ,8-cyclo-20 -deoxyguanosine (4) r 2011 American Chemical Society
were obtained by γ-irradiation of a N2O-saturated aqueous solution of 20 -deoxyguanosine, in a (50 R)/(50 S) ratio of 8.3:1 and an overall yield of 810% (Scheme 2).7 These products derive specifically from the C50 radical cyclization; therefore, the hydrogen abstraction from the C50 position is the most important path regarding the sugar moiety of the nucleoside. In the presence of 2.66 104 M O2, substantially different products were observed (Scheme 2). The consumption of 1b was accompanied by the formation of two main products, identified as guanine and the hydrated 50 -aldehyde 5.8 The majority of cyclonucleosides (4) were replaced by hydrated 50 -aldehyde 5 (∼8%), which suggests that the oxygen concentration would be high enough to trap most of the C50 radicals prior to cyclization, whereas guanine (free base) is formed via well-known pathways of C10 , C30 and C40 sugar radicals trapped by oxygen.9 The above considerations at the nucleoside level are strictly connected to DNA lesions. HO• radicals are known to cause chemical modifications to DNA through the formation of strand breaks and nucleobase modifications. The majority of HO• attacks occur at the base moieties, and in this context the reaction with guanine (G) is one of the most important paths. When DNA is exposed to HO• radicals, significant amounts of 8-oxoG (8oxo-7,8-dihydroguanine) and FapyG (2,6-diamino-4-hydroxy-5formamidopyrimidine) lesions are produced from G.10,11 Both lesions give rise to guanine f thymine (T) transversions.11,12 It Received: August 4, 2011 Published: September 22, 2011 2200
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Scheme 3. The H-Atom Addition to 1 Selectively Occurs at C8
’ EXPERIMENTAL PROCEDURES
a
The atom numbering of the guanine moiety is given in red.
Scheme 2. Reaction Products from the Reaction of HO• Radicals with 20 -Deoxyguanosine (1b) in the Absence or Presence of Oxygen
Materials. Compounds 1a, 1b and t-BuOH were purchased from Sigma-Aldrich or Fluka. Compound 8 was commercially available from Berry & Associates (Ann Arbor, MI). Water was purified through a Millipore (Milli-Q) system. Compounds 7,19 11,20 13,20 2121 and 2421 were prepared following known procedures. Pulse Radiolysis. Pulse radiolysis with optical absorption detection was performed either by using a 12 MeV linear accelerator in Bologna, which delivered 20200 ns electron pulses with doses between 5 and 50 Gy, or the 10 MeV LAE 10 electron accelerator in Warsaw, which provided 8 ns pulses with doses between 16 and 20 Gy. All experiments were performed in a flow system using N2O saturated solutions. Radiolytic Production of Transients. Radiolysis of neutral water leads to the reactive species eaq, HO• and H• together with H+ and H2O2 as shown in eq 1. The values in parentheses represent the radiation chemical yields (G) in units of μmol J1.22,23 In N2O-saturated solution (∼0.02 M of N2O), eaq are efficiently transformed into HO• radical via reaction 2 (k2 = 9.1 109 M1 s1),23 affording a G(HO•) = 0.55 μmol J1, i.e., HO• radicals and H• atoms account for 90% and 10%, respectively, of the reactive species. H2 O ' eaq ð0:27Þ, HO• ð0:28Þ, H• ð0:06Þ, Hþ ð0:27Þ, H2 O2 ð0:07Þ
ð1Þ eaq þ N2 O þ H2 O f HO• þ N2 þ HO
has been suggested that the ratio of 8-oxoG/FapyG may be characteristic of the cancerous state of a cell.13,14 It is believed that both 8-oxoG and FapyG are produced via the same intermediate (8-hydroxyl radical adduct), although the detailed mechanism is poorly understood.10 On the other hand, the positioning of the C40 - and C50 -hydrogen atoms on the edge of the minor groove renders them accessible to diffusible species like HO• radicals.15 H50 of DNA is estimated to be the major (55%) site of attack by HO• radicals, in the presence of multiple reactive positions on the sugar moiety.16 DNA C50 radicals are the precursor of unique cyclic basesugar adducts (purine 50 ,8cyclo-20 -deoxynucleosides).17 These tandem-type lesions are observed among the DNA modifications and have also been identified in mammalian cellular DNA in vivo.17,18 Using time-resolved UVvis spectroscopy (pulse radiolysis), we will show that the reaction of HO• radicals with 20 -deoxyguanosine occurs, at least partly, by the following three pathways: (i) H-abstraction from the NH2 group followed tautomerization, (ii) addition at C8 position affording an adduct radical with no evidence of ring-opening in the ms time-scale, and (iii) hydrogen abstraction from the C50 position followed by radical cyclization. Substituent effects on the guanyl tautomerization have been also examined.
ð2Þ
In a typical experiment, the UVvis spectral changes obtained from the pulse irradiation of N2O-saturated solution containing ca. 1 mM of nucleoside are monitored. It is well documented that the reaction of H-atom with guanine derivatives occurs with a rate constant of ca. 5 108 M1 s1 selectively at the C8 position to give the H-adducts 6 (Scheme 3). The experimental UVvis spectra present the main band around 300 nm (∼12,000 M1 cm1) with a shoulder around 380 nm and a smaller one around 460 nm (∼2,500 M1 cm1).24 It is worth underlining that there are no absorptions in the range 500700 nm.
’ RESULTS AND DISCUSSION Reaction of HO• with Guanosine Derivatives. The spectral
changes obtained in the range 500700 nm from the reaction of HO• radicals with guanosine were reported in our earlier work and assigned to the formation of guanyl radical 3a and its tautomerization to 2a (Scheme 1).6 In this work we considered for comparison the 8-substituted guanosines. The spectral changes obtained from the pulse irradiation of a N2O-saturated unbuffered aqueous solutions of 1 mM 8-(2-hydroxypropan-2yl)guanosine (7) or 8-bromoguanosine (8) are shown in Figures 1 and 2. The behavior in both cases was very similar to the analogous reaction of guanosine.6 The optical absorption spectrum taken 1 μs after the pulse (black) showed a sharp band around 305 nm and a very broad one around 610 nm. A rate 2201
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Figure 1. Absorption spectra obtained from the pulse radiolysis of N2O-saturated unbuffered solution containing 1 mM 8-(2-hydroxypropan-2-yl)guanosine (7) at pH 6.6 taken 1 μs (black), 60 μs (red) and 1 ms (blue) after the pulse; dose per pulse = 20 Gy, optical path = 1 cm. Inset: Time dependence of absorption at 630 nm in the same experimental conditions of the spectra; the red line represents the first-order kinetic fit to the data.
constant of ca. 5 109 M1 s1 was obtained for the reaction of HO• radical with each derivative, by measuring the growth of the band at 305 or 610 nm as a function of the concentration of the added nucleoside (see Supporting Information). Broad bands in the range 500700 nm and decay by first-order kinetics at 630 nm (see insets) are shown in Figures 1 and 2. The decay kinetics observed at 305 and 360 nm for each substrate are identical (see Supporting Information). These transient species were assigned to guanyl radical in its iminic form 9, and the firstorder decays to the tautomerization process. In Scheme 4 these transformations are illustrated together with the corresponding paths of guanosine (1a) for comparison. By replacing hydrogen by an alkyl group, the rate constant of tautomerization is similar, whereas a 15-fold increase is observed by replacing H with Br. Due to the steric hindrance of the 8-substituents, the competitive addition of HO• radicals to the C8 position should be negligible (vide infra). When the guanosine was methylated at the N1 position, the scenario changed. Figure 3 (left) shows the optical absorption spectrum taken 1.2 μs after the pulse from the reaction of HO• with 1-methylguanosine (11) (black). Again the spectrum showed a sharp absorption band around 305 nm and a very broad one around 600 nm. A rate constant of 6.7 109 M1 s1 was obtained for the reaction of HO• radical with 11. In contrast with the 8-substituted guanosine, the decay of this transient followed second-order kinetics, as illustrated in the inset of Figure 3 (left). The spectrum taken 1 ms after the pulse (red) showed the disappearance of the band above 400 nm and a residual absorption around 300 nm. The absorption around 610 nm of the spectra in Figure 2 taken ∼1 μs after the pulse is very similar in shape to the reported spectra of 12 obtained by one-electron reduction of 8-bromo-1methylguanosine (13) (Scheme 5).25 Figure 3 (right) shows the comparison of the spectra resulting from the reactions HO• + 11 (black) and eaq + 13 (red). The ε values of the spectrum arising from the reaction with HO• + 11 were calculated using G = 0.61 μmol J1, since both HO• and H• species are scavenged by the 1-methylguanosine. The spectrum of eaq + 13 makes up for 55% of the ε values calculated using G = 0.27 μmol J1 of hydrated electrons. The overlap in the range 500700 nm is excellent,
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Figure 2. Absorption spectra obtained from the pulse radiolysis of N2O-saturated unbuffered solution containing 1 mM 8-bromoguanosine (8) at pH 5.5 taken 1.6 μs (black), 32 μs (red) and 1 ms (blue) after the pulse; dose per pulse = 20 Gy, optical path = 1 cm. Inset: Time dependence of absorption at 630 nm in the same experimental conditions of the spectra; the red line represents the first-order kinetic fit to the data.
Scheme 4. Reaction of HO• with a Variety of 8-Substituted Guanosines and the Corresponding Rate Constants for the Tautomerization Steps in Aqueous Medium
suggesting that 55% of HO• radicals react with 1-methylguanosine by hydrogen abstraction from the exocyclic NH2 group to give 12 (Scheme 5). This radical follows a second-order decay because the N1 position is blocked for tautomerization. Similar analysis for the guanosine case suggested that 65% of HO• radicals react by hydrogen abstraction from the exocyclic NH2.6 The inset in Figure 3 (right) shows the resulting difference of the two spectra, which is a strong band around 300 nm and various smaller broad bands up to 500 nm. These bands are expected to be the sum of various contributions: (i) from the 8-hydroxyl radical adduct (vide infra), (ii) from the H-atom adduct, and (iii) from the sugar-derived radicals. Reaction of HO• with 20 -Deoxyguanosine and Its Derivatives. The spectral changes obtained from pulse irradiation of a N2O-saturated aqueous solution of 1 mM 20 -deoxyguanosine (1b) at natural pH are shown in Figure 4. The optical absorption spectrum taken 4 μs after the pulse (black) showed a sharp band around 305 nm and a very broad one around 600 nm. A rate constant of (5.7 ( 0.4) 109 M1 s1 was obtained for the reaction of HO• radical with 1b, by measuring the growth of the band at 305 or 620 nm as a function of the concentration of the added nucleoside (see Supporting Information). The spectrum taken 800 μs after the pulse (red) showed the disappearance of the band around 600 nm and the decrease of the absorption around 300 nm. By monitoring the absorbance changes at 305 and 620 nm, a first-order rate constant of (2.3 ( 0.1) 104 s1 was obtained. 2202
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Figure 3. Left: Absorption spectra obtained from the pulse radiolysis of N2O-saturated unbuffered solution containing 1 mM 1-methylguanosine (11) at pH 5.9 taken 1.2 μs (black) and 1 ms (red) after the pulse; dose per pulse = 20 Gy, optical path = 1 cm. Inset: Time dependence of absorption at 610 nm; the red line represents the second-order kinetic fits to the data, slope = (1.1 ( 0.3) 106 s1. Right: Absorption spectra obtained from the reaction of HO• with 1-methylguanosine (11) recorded 1.2 μs (black) after the pulse and 55% of the intensity of the absorption spectra obtained from the reaction of eaq with 8-bromo-1-methylguanosine (13) recorded 10 μs (red) after the pulse. Inset: The spectrum resulting from the subtraction of red from black.
Scheme 5. Reactions of HO• and eaq with Guanine and 8-Bromoguanine Derivativesa
a The percentages of HO• attack are estimated from the fitting reported in Figure 3.
Figure 5. Plot of kobs vs dose for the second buildup at 370 nm after pulse radiolysis of N2O-saturated unbuffered solution containing 1 mM 20 -deoxyguanosine at pH = 6.2, optical path = 1 cm. The intercept obtained after interpolation of data represents the rate constant at zero dose, i.e. kc = (6.9 ( 0.8) 105 s1. Inset: Time dependence of absorption at 370 nm, dose = 15 Gy. The red line represents a consecutive buildup and decay first order kinetic fit to the data.
Figure 4. Absorption spectra obtained from the pulse radiolysis of N2O-saturated unbuffered solution containing 1 mM 20 -deoxyguanosine (1b) at pH 5.8 taken 4 (black) and 800 μs (red) after the pulse; dose per pulse = 20 Gy, optical path = 1 cm. Insets: Time dependence of absorption at 305 (upper) and 620 nm (lower) in the same experimental conditions of the spectra; the red lines represent the first-order kinetic fits to the data.
In their paper, Candeias and Steenken mentioned a buildup at 305 nm (k = 2 105 s1) and a decay at 620 nm (k = 6 103 s1).5 In our experiments the spectral changes at 305 and 620 nm corresponded to the same kinetics as described above. We would like to stress that our results are quite different from those previously reported, although we do not have an explanation for
this discrepancy (it is worth noting that in Figure 1 reported by Candeias and Steenken,5 the decay traces at 620 nm do not correspond to the reported spectra because the decay does not reach the zero absorbance as evidenced from the spectra). In addition, in our previous study similar results were obtained for guanosine (1a) under identical conditions.6 The only difference between 1a and 1b is the behavior of the spectral changes around 370 nm. Although in 1a no changes are observed, in 1b there were some new observations. The time profile for the formation of the transient at 370 nm followed first-order kinetics with a rate constant that was independent of the 1b concentration, and slightly increased with dose/pulse (inset of Figure 5). This dose dependence was due to the mixing of first-order growth and second-order decay of the species. By extrapolation to zero dose, a rate constant kc = (6.9 ( 0.8) 105 s1 at 20 °C was obtained (Figure 5). On the basis of the experimental findings, we propose the mechanism illustrated in Scheme 6 for the reaction of HO• radicals with 20 -deoxyguanosine (1b), where the three initial 2203
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Scheme 6. Revised Mechanism of the Reaction of HO• Radicals with 20 -Deoxyguanosine (1b)
paths are shown in red. Hydrogen abstraction from the exocyclic NH2 moiety with formation of guanyl radical 3b accounted for ∼65% of the total reactivity. As discussed in the previous section, this iminic form (3b) can readily undergo a waterassisted tautomerization with a rate constant of 2.3 104 s1 to give the guanyl radical (2b) in its aminic form. Hydrogen abstraction from the sugar moiety was estimated to be ∼18% (Scheme 6) on the basis of the product studies, with half of this percentage occurring at the H50 positions (see Introduction).7,8 We assigned the observed buildup at 370 nm and the rate constant of 6.9 105 s1 to the cyclization of C50 radical with formation of aminyl radical 14 (Scheme 6). For comparison, a rate constant of 1.6 105 s1 was reported for 20 -deoxyadenosin-50 -yl radical in aqueous solution by pulse radiolysis,26 and more importantly, a rate constant ca. 1 106 s1 was estimated for the cyclization of protected 20 -deoxyguanosin-50 -yl radical 19 to 20 in competition kinetics, using the well-known freeradical clock methodology (Scheme 7).27 Based on the reactivity of adenosin-50 -yl radical,28 the absence of a 370 nm buildup in reaction of HO• with 1a can likely derive from (i) a lower percentage of C50 attack and (ii) the slowness of C50 radical cyclization in the ribo series. In Scheme 6, the remaining ∼17% of HO• radicals can add to the C8 position to give the 8-hydroxyl adduct 15.5 So far very little is known about radical 15, neither spectral properties nor its fate is well understood, although it is of great importance being the precursor of 8-oxoG (16) and FapyG (18).11 Candeias and Steenken assigned a rate constant of 2 105 s1 to the ringopening without any real evidence. In order to gain further information on this path we considered the mono- and diethyl substitutions of the NH2 moiety. The latter cannot undergo the main path of HO• radical attack shown in Scheme 6. Rate constants of 6 109 and 7.9 109 M1 s1 were obtained for the reaction of HO• radical with N2-ethyl-20 deoxyguanosine (21) and N2,N2-diethyl-20 -deoxyguanosine (24), respectively (Scheme 8). Figure 6 shows the spectral changes obtained from the pulsed irradiation of N2O saturated aqueous solution (unbuffered) of 1 mM 21. The behavior was very similar to that of 20 -deoxyguanosine (1b), that is, a broad band around 650 nm and decay by first-order kinetics, as shown
Scheme 7. C50 Radical 19 Undergoes Cyclization with a kc ≈ 1 106 s1 in THF at 30 °C (Ref 27)
Scheme 8. Reaction of HO• with N2-Ethyl- and N2,N2-Diethyl-20 -deoxyguanosine
in the inset of Figure 6. This species was assigned to guanyl radical in its iminic form 22, and the decay to the tautomerization process (22 f 23) with a rate constant of (3.6 ( 0.5) 104 s1 (Scheme 8). On the other hand, the optical absorption spectrum obtained from the pulsed irradiation of N2O saturated aqueous solution (unbuffered) of 1 mM 24 is reported in Figure 7, and it is identical to those of the ribo analogues (see Supporting Information). It was gratifying to see that the previously discussed band in the range 500700 nm is absent and that the transient decays by second-order kinetics, without evidence of unimolecular transformation. We suggest that the observed spectrum in Figure 7 represents the overlap of the 2204
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Figure 6. Absorption spectra obtained from the pulse radiolysis of N2O-saturated unbuffered solution containing 1 mM N2-ethyl-20 -deoxyguanosine (21) at pH 6.7 taken 1.2 μs (black), 40 μs (red) and 1 ms (blue) after the pulse; dose per pulse = 20 Gy, optical path = 1 cm. Inset: Time dependence of absorption at 660 nm in the same experimental conditions of the spectra; the red line represents the first-order kinetic fit to the data.
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radicals can be modulated by the appropriate substituents and it is also expected to depend upon the environment, e.g., the local conformation of DNA. Abstraction of a hydrogen atom from 2-deoxyribose produces a carbon-centered radical. Experimental data from the reaction of HO• radicals with simple nucleosides, like 20 deoxyadenosine and 20 -deoxyguanosine, indicated that 2025% of H-atom abstraction occurs at the sugar moiety, 4050% of which takes place at the H50 position. The observed spectral changes around 370 nm in the case of 1b are assigned to the cyclization of 20 -deoxyguanosin-50 -yl radical, in agreement with previous information gathered in our laboratory.7,8,27 The measured rate constant of 6.9 105 s1 is in the expected interval. Our findings are of considerable importance as model for analogous reactions in DNA. (50 R)- and (50 S)-50 ,8-cyclo-20 deoxyguanosine stereoisomers are lesions observed among other DNA modifications when HO• radicals react with DNA in the presence of oxygen, which suggests a fast cyclization of C50 radicals.17,18 Anderson and co-workers reported that the reaction of benzothiazinyl radical (aromatic aminyl radical) with DNA results in the formation of the neutral guanyl radical by hydrogen abstraction from the exocyclic NH2 of the guanine moiety.29 The deprotonation of the exocyclic NH2 of the guanine moiety is also reported in the one-electron oxidation of ds-oligonucleotides rich in GC units.30,31 Methylated guanines at the N2-position in ds-oligonucleotides are thought to be a combination of guanyl radical in the iminic form with methyl radical.32 Our findings can assist further research for a better interpretation of the reaction of HO• with DNA structures.
’ ASSOCIATED CONTENT
bS
Supporting Information. Pulse radiolysis techniques, additional results and spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Figure 7. Absorption spectrum obtained from the pulse radiolysis of N2O-saturated unbuffered solution containing 1 mM N2,N2-diethyl-20 deoxyguanosine (24) at pH 6.6 taken 1 μs after the pulse; dose per pulse = 20 Gy, optical path = 2 cm. Inset: Time dependence of absorption at 320 nm; the red line represents the second-order kinetic fits to the data.
spectra derived from the addition of HO• to the C8 position to give 25 (main path) and from the hydrogen abstraction at the sugar and NEt2 moieties (Scheme 8). These findings suggest that the ring-opening of radicals 25 is a relatively slow process (cf. kro for reaction 15 f 17).
’ CONCLUSIONS The reaction of HO• radicals with 20 -deoxyguanosine (1b) occurs with a rate constant close to 5 109 M1 s1. The most important site of attack (∼80%) involves the base unit and is partitioned between two reaction channels, i.e., hydrogen abstraction from the exocyclic NH2 (main path) and addition at the C8 position (minor path). The initially formed guanyl radical 3b, characterized by a broad band around 610 nm, undergoes a water-assisted tautomerization to give the most stable tautomer 2b, which is also formed directly by one-electron oxidation of guanosine or 20 -deoxyguanosine (cf. Scheme 1). The ring-opening of 8-hydroxyl radical adduct (15 f 17 in Scheme 6) is much slower than previously reported.5 However, the ambident reactivity of guanine moiety toward HO•
Corresponding Author
*E-mail:
[email protected]. Funding Sources
The support by the COST Action CM0603 “Free Radicals in Chemical Biology” is kindly acknowledged.
’ ABBREVIATIONS FapyG, 2,6-diamino-4-hydroxy-5-formamidopyrimidine; G, guanine; 8-oxoG, 8-oxo-7,8-dihydroguanine; T, thymine; UVvis, utravioletvisible. ’ REFERENCES (1) Candeias, L. P., and Steenken, S. (1989) Structure and acid-base properties of one-electron-oxidized deoxyguanosine, guanosine, and 1-methylguanosine. J. Am. Chem. Soc. 111, 1094–1099. (2) Crean, C., Geacintov, N. E., and Shafirovich, V. (2005) Oxidation of guanine and 8-oxo-7,8-dihydroguanine by carbonate radical anions: Insight from oxygen-18 labeling experiments. Angew. Chem., Int. Ed. 44, 5057–5060. (3) Steenken, S., and Jovanovic, S. V. (1997) How easily oxidizable is DNA? One-electron reduction potentials of adenosine and guanosine radicals in aqueous solution. J. Am. Chem. Soc. 119, 617–618. (4) Wardman, P. (1989) Reduction potentials of one-electron couples involving free radicals in aqueous solution. J. Phys. Chem. Ref. Data 14, 1637–1756. 2205
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