Comparison of isoelectronic 8-HO-G and 8-NH2-G derivatives in redox processes

Article abstract of DOI:10.1021/ja9065464

8-Oxo-7,8-dihydroguanine (8-oxo-G) is the major lesion of oxidatively generated DNA damage. Despite two decades of intense study, several fundamental properties remain to be defined. Its isoelectronic 8-aminoguanine (8-NH ₂-G) has also received considerable attention from a biological point of view, although its chemistry involving redox processes remains to be discovered. We investigated the one-electron oxidation and one-electron reduction reactions of 8-oxo-G and 8-NH₂-G derivatives. The reactions of hydrated electrons (e_aq^-) and azide radicals (N ₃^?) with both derivatives were studied by pulse radiolysis techniques, and the transient absorption spectra were assigned to specific tautomers computationally by means of time-dependent DFT (TD-B3LYP/6-311G**//B1B95/6-31+G**) calculations. The protonated electron adducts of 8-NH₂-G and 8-oxo-G showed a substantial difference in their absorption spectra, the unpaired electron being mainly delocalized in the imidazolyl ring and in the six-membered ring, respectively. On the other hand, the deprotonated forms of one-electron oxidation of 8-NH₂-G and 8-oxo-G showed quite similar spectral characteristics. In a parallel study, the one-electron reduction of 8-azidoguanine (8-N₃-G) afforded the same transient of one-electron oxidation of 8-NH₂-G, which represents another example of generation of one-electron oxidized guanine derivatives under reducing conditions. Moreover, the fate of transient species was investigated by radiolytic methods coupled with product studies and allowed self- and cross-termination rate constants associated with these reactions to be estimated.

Full text of DOI:10.1021/ja9065464

Published on Web 10/12/2009
Comparison of Isoelectronic 8-HO-G and 8-NH -G Derivatives
2
in Redox Processes
†
,‡
†
†
†
Panagiotis Kaloudis, Mila D’Angelantonio, Maurizio Guerra, Marie Spadafora,
‡,§
‡
†
Crina Cisma s¸ , Thanasis Gimisis, Quinto G. Mulazzani, and
,
†
Chryssostomos Chatgilialoglu*
ISOF, Consiglio Nazionale delle Ricerche, Via P. Gobetti 101, 40129 Bologna, Italy, and
Department of Chemistry, UniVersity of Athens, 15771 Panepistimiopolis, Athens, Greece
Received August 5, 2009; E-mail: chrys@isof.cnr.it
Abstract: 8-Oxo-7,8-dihydroguanine (8-oxo-G) is the major lesion of oxidatively generated DNA damage.
Despite two decades of intense study, several fundamental properties remain to be defined. Its isoelectronic
8
-aminoguanine (8-NH
its chemistry involving redox processes remains to be discovered. We investigated the one-electron oxidation
and one-electron reduction reactions of 8-oxo-G and 8-NH -G derivatives. The reactions of hydrated
) with both derivatives were studied by pulse radiolysis techniques,
2
-G) has also received considerable attention from a biological point of view, although
2
-
•
electrons (eaq ) and azide radicals (N
3
and the transient absorption spectra were assigned to specific tautomers computationally by means of
time-dependent DFT (TD-B3LYP/6-311G**//B1B95/6-31+G**) calculations. The protonated electron adducts
of 8-NH
being mainly delocalized in the imidazolyl ring and in the six-membered ring, respectively. On the other
hand, the deprotonated forms of one-electron oxidation of 8-NH -G and 8-oxo-G showed quite similar spectral
characteristics. In a parallel study, the one-electron reduction of 8-azidoguanine (8-N -G) afforded the same
transient of one-electron oxidation of 8-NH -G, which represents another example of generation of one-
2
-G and 8-oxo-G showed a substantial difference in their absorption spectra, the unpaired electron
2
3
2
electron oxidized guanine derivatives under reducing conditions. Moreover, the fate of transient species
was investigated by radiolytic methods coupled with product studies and allowed self- and cross-termination
rate constants associated with these reactions to be estimated.
Introduction
Scheme 1. Equilibrium of Two Tautomeric Forms of 8-Oxo-G (1)
12 a
2
and of 8-NH -G (2)
8
-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo, 1), an
oxidation product of 2′-deoxyguanosine (dGuo), is commonly
1
-4
used as a biomarker of oxidative stress.
This damage to the
guanine moiety of DNA can be caused by hydroxyl radicals,
singlet oxygen, or one-electron oxidants. Evidence that there
are ∼2000 8-oxo-G lesions generated per human cell per day
5
6
7
has been reported. NMR studies and calculations have
determined that the 6,8-diketo tautomer 1a is the predominant
species in solution with respect to the 8-hydroxy tautomer 1b
a
The equilibrium is shifted to the left for 1 and to the right for 2 (in
red, the atom numbering of guanine).
8
9
(Scheme 1). One-electron oxidation of 8-oxo-dGuo (as nucleo-
side or incorporated in oligonucleotides ) has been studied in
1
0
some detail. Owing to its unique electronic properties, it has
now been established that 8-oxo-dGuo is very susceptible to
oxidation and far more reactive than dGuo itself toward one-
electron oxidants and singlet oxygen. During the past 10 years
a large number of studies appeared in the literature reporting
the major and minor lesions formed from the oxidation of 8-oxo-
†
Consiglio Nazionale delle Ricerche.
University of Athens.
Present address: Organic Chemistry Department, Babes-Bolyai Uni-
‡
8,9
§
versity, 11 Arany Janos str., 400028 Cluj-Napoca, Romania.
(
(
(
1) Gimisis, T.; Cismas, C. Eur. J. Org. Chem. 2006, 1351–1378.
2) Pratviel, G.; Meunier, B. Chem.sEur. J. 2006, 12, 6018–6030.
3) Neeley, W. L.; Essigmann, J. M. Chem. Res. Toxicol. 2006, 19, 491–
1-4,11
dGuo analogues under a variety of oxidation conditions.
5
05.
4) Cadet, J.; Douki, T.; Ravanat, J.-L. Acc. Chem. Res. 2008, 41, 1075–
083.
5) (a) Beckman, K. B.; Ames, B. N. J. Biol. Chem. 1997, 272, 19633–
9636. (b) Foksinski, M.; Rozalski, R.; Guz, J.; Ruszkowska, B.;
(
(
(8) Steenken, S.; Jovanovic, S. V.; Bietti, M.; Bernhard, K. J. Am. Chem.
Soc. 2000, 122, 2373–2374.
1
(9) Shafirovich, V.; Cadet, J.; Gasparutto, D.; Dourandin, A.; Huang, W.;
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J. G.; Burrows, C. J. J. Am. Chem. Soc. 2009, 131, 89–95.
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1
Sztukowska, P.; Piwowarski, M.; Klungland, A.; Olinski, R. Free
Radical Biol. Med. 2004, 37, 1449–1454.
(
6) Oda, Y.; Uesugi, S.; Ikehara, M.; Nishimura, S.; Kawase, Y.; Ishikawa,
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(
7) Cysewski, P. J. Chem. Soc., Faraday Trans. 1998, 94, 3117–3125.
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10.1021/ja9065464 CCC: $40.75  2009 American Chemical Society
J. AM. CHEM. SOC. 2009, 131, 15895–15902 9 15895

A R T I C L E S
Kaloudis et al.
8
-Amino-2′-deoxyguanosine (8-NH
2
-dGuo) and 8-aminogua-
-Guo, 2) are produced together with the corre-
important intermediate for which spectroscopic and kinetic
information is very scarce.
2
6
nosine (8-NH
2
sponding 8-oxo-guanine derivative, in rat liver treated with
-nitropropane (2-NP), a hepatocarcinogen widely used as an
industrial solvent, a component of coatings, and also found in
2
13a
cigarette smoke. This has been shown to result from amination
of DNA or RNA, respectively, by a metabolite of 2-NP,
hydroxylamine-O-sulfonate, capable of releasing a reactive
+
13b
nitrenium ion (NH
2
). Metal-mediated DNA damage caused
Herein we report detailed chemical radiation studies of com-
pounds 1 and 2 in aqueous solution. In particular, we have
investigated their one-electron oxidation and one-electron reduction
reactions and clarified the major tautomeric forms of the various
intermediates by the combined use of pulse radiolysis and DFT
calculations. In addition, continuous radiolysis coupled with product
studies allowed a better understanding of the fate of the transients.
14
by 2-NP metabolites may also play a role. This is a mutagenic
lesion that has been found to generate GfT and GfC
15
transversions in mammalian cells and to be weakly mutagenic
-
3
16
(
mutation frequency, 10 ) in Escherichia coli. To extenuate
the difference between 1 and 2, it was found that 8-NH -dGTP
is incorporated and extended more efficiently than 8-oxo-dGTP
by both HIV type I and murine leukemia virus reverse
transcriptases and by DNA polymerases R and ꢀ. The
analogous one-electron oxidation of 8-aminoguanine derivatives
2
Results and Discussion
1
7
Protonated One-Electron Reduced 8-Oxo-dGuo (1) and
-
8
-NH
2
-Guo (2) Species. Radiolysis of neutral water leads to eaq
,
2
has not received any attention. The 8-NH -G moiety is also of
particular interest, since it can be considered as a model
•
•
HO , and H as shown in eq 2. The values in parentheses represent
the radiation chemical yields (G) in units of µmol J . The reactions
of eaq with the substrates were studied in O -free solutions
-1
compound of protein-DNA cross-links with the C8 position
-
1
8,19
20
21
of the guanine moiety.
NMR studies and calculations
2
containing 0.25 M t-BuOH. Based on the available kinetic data,
suggest that the amino form 2b rather than the imino 2a form
is the most stable tautomer in aqueous solutions, contrary to
•
we expected that HO is scavenged efficiently (eq 3, k
3
) 6.0 ×
27,28
8
-1 -1
•
1
0 M s ), whereas H is trapped only partially (eq 4, k
10 M s ) under the utilized concentrations of t-BuOH.
4
) 1.7
8
-oxo-G (Scheme 1).
5
-1 -1
×
Another interesting aspect related to 8-oxo-dGuo is the one-
electron reduction process. Up to now this reaction has not
-
•
•
H O ' e (0.27), HO (0.28), H (0.062)
(2)
(3)
(4)
2
aq
received any attention. Nucleosides react with hydrated electrons
-
(
e
aq ) at practically diffusion-controlled rates to give radical
•
•
2
HO + t-BuOH f (CH ) C(OH)CH + H O
3
2
2
anions. Radical anions of purine nucleosides, in particular, are
rapidly protonated by water preferably at the C8 position, since
•
•
2
2
2
H + t-BuOH f (CH ) C(OH)CH + H
3
2
2
the corresponding pK
a
values are very high. For example, it
has been recently reported that the guanine radical anion is
The pseudo-first-order rate constants, kobs, for the reaction of
2
3
protonated at C8. 8-Bromoguanine derivatives are also pro-
tonated at C8 affording the corresponding one-electron oxidized
-
e
aq with 8-oxo-dGuo (1) and 8-NH
2
-Guo (2) were determined
-
by measuring the rate of the optical density decrease of eaq at
2
4,25
guanine.
If an analogous reaction applies to 1, it can be
4
-1
-1 29
7
20 nm (ε ) 1.9 × 10 M cm ) at pH ∼7. From the slope
•
expected to generate the HO radical adduct 3 (Reaction 1), an
of kobs vs nucleoside concentration, the bimolecular rate
9
-1 -1
9
-1 -1
constants (k) of 5.6 × 10 M
s
for 1 and 4.4 × 10 M
s
(
(
(
12) In schemes and figures, R ) 2-deoxyribosyl for 8-oxo-G and R )
ribosyl for 8-NH -G and 8-N -G. In the theoretical part (all tables) R
2-deoxyribosyl.
for 2 are obtained. The optical absorption spectrum recorded at
-
2
3
2
µs after the pulse from the reaction of 1 (1.0 mM) with eaq
)
at natural pH (pH 7.2) is presented in Figure 1 (red circles) in
13) (a) Fiala, E. S.; Nie, G.; Sodum, R.; Conaway, C. C.; Sohn, O. S.
Cancer Lett. 1993, 74, 9–14. (b) Sodum, R. S.; Nie, G.; Fiala, E. S.
Chem. Res. Toxicol. 1993, 6, 269–276.
the range 300-700 nm and shows a broad band that decreases
at longer wavelengths. The time profile of the formation of this
14) Sakano, K.; Oikawa, S.; Murata, M.; Hiraku, Y.; Kojima, N.;
Kawanishi, S. Mutat. Res., Fundam. Mol. Mech. Mutagen. 2001, 479,
-
transient absorbance is identical to the disappearance of eaq
,
denoting a single process. The transient species decays with
1
01–111.
5
-1
(
(
(
(
(
(
15) Tan, X.; Suzuki, N.; Johnson, F.; Grollman, A. P.; Shibutani, S. Nucleic
second-order kinetics, k/ε330 nm ) 8.2 × 10 cm s (right inset).
Acids Res. 1999, 27, 2310–2314.
The optical absorption spectrum recorded at 2 µs after the pulse
16) Venkatarangan, L.; Sivaprasad, A.; Johnson, F.; Basu, A. K. Nucleic
Acids Res. 2001, 29, 1458–1463.
-
from the reaction of 2 (1.0 mM) with eaq at natural pH (pH
17) Kamath-Loeb, A. S.; Hizi, A.; Kasai, H.; Loeb, L. A. J. Biol. Chem.
8.1) is also presented in Figure 1 (blue triangles). The spectrum
1
997, 272, 5892–5898.
of this transient species is more intense than that obtained by
18) Perrier, S.; Hau, J.; Gasparutto, D.; Cadet, J.; Favier, A.; Ravanat,
J.-L. J. Am. Chem. Soc. 2006, 128, 5703–5710.
-
the reaction of 1 with eaq , although its shape is only slightly
different. This transient species decays with second-order
19) Xu, X.; Muller, J. G.; Ye, Y.; Burrows, C. J. J. Am. Chem. Soc. 2008,
5
-1
1
30, 703–709.
kinetics, k/ε370 nm ) 1.8 × 10 cm s (left inset), with a non-
20) Guengerich, F. P.; Mundkowski, R. G.; Voehler, M.; Kadlubar, F. F.
Chem. Res. Toxicol. 1999, 12, 906–916.
negligible plateau.
(
(
(
21) Cysewski, P. Z. Phys. Chem. 2005, 219, 213–234.
22) Steenken, S. Chem. ReV. 1989, 89, 503–520.
(26) Chatgilialoglu, C.; D’Angelantonio, M.; Guerra, M.; Kaloudis, P.;
Mulazzani, Q. G. Angew. Chem., Int. Ed. 2009, 48, 2214–2217.
(27) Buxton, G. V; Greenstock, C. L.; Helman, W. P.; Ross, A. B. J. Phys.
Chem. Ref. Data 1988, 17, 513–886.
23) D’Angelantonio, M.; Russo, M.; Kaloudis, P.; Mulazzani, Q. G.;
Wardman, P.; Guerra, M.; Chatgilialoglu, C. J. Phys. Chem. B 2009,
1
13, 2170–2176.
(
24) Chatgilialoglu, C.; Caminal, C.; Guerra, M.; Mulazzani, Q. G. Angew.
Chem., Int. Ed. 2005, 44, 6030–6032.
(28) Ross, A. B., Mallard, W. G.; Helman, W. P.; Buxton, G. V.; Huie,
R. E.; Neta, P. NDRLNIST Solution Kinetic Database - Ver. 3, Notre
Dame Radiation Laboratory, Notre Dame, IN and NIST Standard
Reference Data, Gaithersburg, MD (1998).
(
25) Chatgilialoglu, C.; Caminal, C.; Altieri, A.; Mulazzani, Q. G.;
Vougioukalakis, G. C.; Gimisis, T.; Guerra, M. J. Am. Chem. Soc.
2
006, 128, 13796–13805.
(29) Hug, G. L. Nat. Stand. Ref. Data Ser., Nat. Bur. Stand. 1981, 69, 6.
1
5896 J. AM. CHEM. SOC. 9 VOL. 131, NO. 43, 2009

8-Oxo-G and 8-NH
2
-G in Redox Processes
A R T I C L E S
Figure 2. SOMO of the radicals 5 (left) and 7 (right) computed at the
B3LYP/6-311G(d,p)//B1B95/6-31+G(d,p) level.
band of moderate intensity at a longer wavelength (349 nm).
Interestingly, tautomer 5 is slightly more stable than tautomer
-
1
6
by 0.6 kcal mol at the PCM/B1B95/6-311+G(2df,p)//
B1B95/6-31+G(d,p) level and more stable than tautomer 4 by
.4 kcal mol . This stability is due to the fact that in 5 the
Figure 1. Absorption spectrum (red circles) obtained after pulse radiolysis
at natural pH (pH 7.2) of 1.0 mM 8-oxo-dGuo (1), 0.25 M t-BuOH, Ar-purged,
-
1
6
unpaired electron is delocalized in the six-member ring (Figure
2, left), whereas in 4 it is localized at C8.
2
µs after the pulse. Absorption spectrum (blue triangles) obtained after pulse
3
3
radiolysis at natural pH (pH 8.1) of 1.0 mM 8-NH -Guo (2), 0.25 M t-BuOH,
2
Ar-purged, 2 µs after the pulse. Insets: Time dependence of absorptions at 330
In Figure 1 is shown that substitution of the oxy group with
the amino/imino group at C8 produces an unexpected change
in the absorption spectrum in that the intensity of the main band
is twice as strong as that in the 8-oxo derivative. Furthermore,
two shoulders are present at approximately 360 and 450 nm.
This could be ascribed to different structures assumed by the
two precursors. Indeed, DFT calculations at the PCM/B1B95/
nm, the solid lines represent the second-order kinetic fit to the data; (right/red)
-1
optical path length of 2 cm, dose 27.8 Gy, G(5) ) 0.33 µmol J , (left/blue)
-1
optical path 2 cm, dose 26.7 Gy, G(7) ) 0.33 µmol J
.
What is the structure of the observed intermediate in the case
of 8-oxo-dGuo? The assignment is not straightforward. As
discussed above the electron adducts should be rapidly protonated.
The hydroxyl radical adduct 3 (Reaction 1) can be excluded
because it is reported to undergo fast ring opening with a rate
6
-311+G(2df,p)//B1B95/6-31+G(d,p) level indicate that the
amino 2b form is more stable than the imino 2a form by 4.8
kcal mol , whereas the oxy 1a form is more stable than the
hydroxy 1b form by 12.6 kcal mol , in accord with NMR
studies
-
1
5
-1 30
constant of 2 × 10 s , whereas the recorded intermediate decays
-
1
by second-order kinetics. Unfortunately an optical absorption
6
,20
7,21
and the previous calculations.
Then, DFT-TD
spectrum of 3 is not available because only the minor path (ca.
•
calculations were carried out on the possible tautomers 7, 8, 9
deriving from protonation of the radical anion of 2b at N7, O6′,
and N3 heteroatoms, respectively.
1
7%) of the HO radical reaction with guanine affords radical 3,
the main reaction (65%) being the hydrogen abstraction from the
26
NH
2
moiety. However, DFT calculations predicted the following
As revealed in Table 2, the computed spectrum for the N7-H
tautomer 7 is in rather good accord with the experimental one.
The main peak is computed to be due to overlap of two strong
transitions at 313 and 331 nm; furthermore two shoulders are
computed at 342 and 420 nm. On the other hand, the computed
spectra for both tautomers 8 and 9 are in disaccord with the
experimental one, since no band of moderate intensity is computed
above 400 nm. In particular, only one band of moderate intensity
is computed for 8 at 327 nm, and two bands of low intensity are
computed for 9 in the 300-350 nm region. Interestingly, tautomer
vertical transitions λ/nm (oscillator strength, f): 294 (0.157), 308
26
(0.010), 366 (0.029), and 433 (0.44).
Time-dependent DFT calculations (TD-B3LYP/6-311G(d,p)//
B1B95/6-31+G(d,p)) were carried out to compute vertical optical
transitions of other possible intermediates. Comparison with the
experimental UV-visible spectrum could allow establishment of
which tautomer of the protonated one-electron reduced 8-oxo-dGuo
radical is responsible for the absorption spectrum recorded after
pulse radiolysis, since this theoretical method was found by us to
23-26,31,32
predict optical transitions in nucleosides with reliability.
7
is computed to be much more stable than both tautomers 8 (by
The computed optical transitions are reported in Table 1.
-1
-1
5
.9 kcal mol ) and 9 (by 6.8 kcal mol ).
As can be seen in Table 1, the absorption spectrum displayed
in Figure 1 is well reproduced by the optical transitions
computed for tautomer 5. DFT-TD calculations predict two main
transitions of moderate intensity at 299 and 315 nm with a
slightly weaker transition at 354 and two weak transitions at
From Figure 2 (right), the SOMO of the radical 7 in which
the unpaired electron is mainly delocalized in the imidazolyl
ring can be seen, in contrast with radical 5 (left) where the
unpaired electron is mainly delocalized in the six-membered
3
3
ring. These findings are in accord with experimental spectra
recorded by pulse radiolysis upon one-electron reduction of
4
33 and 647 nm in good accord with the experimental spectrum
that presents a broad main band of moderate intensity between
00 and 400 nm with weak features above 430 nm. Also the
8
-oxo-G (1) and 8-NH
2
-G (2) derivatives, respectively.
3
To investigate in more detail the fate of the intermediate
radicals, γ-radiolysis coupled with product studies was carried
out. Deaerated samples containing either 1 (1.6 mM) and
computed spectrum of tautomer 4 presents two bands of
moderate intensity in the 300-350 spectral region at 300 and
3
20 nm; however a strong peak is computed at 341 nm in
evident contrast with the experimental spectrum. On the other
hand, the computed spectrum for tautomer 6 is in complete
disaccord with the experimental one, since it presents only one
(33) See Supporting Information.
(
34) The experiments of 1 (R ) deoxyribosyl) in natural pH produce small
amounts of free base (8-oxo-7,8-dihydro-guanine). It is known that
radiolysis of neutral water, besides the transient species (eq 1),
+
(
(
30) Candeias, L. P.; Steenken, S. Chem.sEur. J. 2000, 6, 475–484.
31) Chatgilialoglu, C.; Guerra, M.; Mulazzani, Q. G. J. Am. Chem. Soc.
produces H in amounts that can alter the pH of the irradiated solution.
In an independent experiment, the hydrolysis of 1’s glycosidic bond
was observed even in slightly acidic solutions. On the other hand, the
increased stability of the glycosidic bond of the ribose moiety did not
require buffered solutions as in the case of 2 (R ) ribosyl).
2
003, 125, 3839–3848.
(32) Kaloudis, P.; D’Angelantonio, M.; Guerra, M.; Gimisis, T.; Mulazzani,
Q. G.; Chatgilialoglu, C. J. Phys. Chem. B 2008, 112, 5209–5217.
J. AM. CHEM. SOC. 9 VOL. 131, NO. 43, 2009 15897

A R T I C L E S
Kaloudis et al.
Table 1. Vertical Optical Transitions (Wavelengths λ, Oscillator Strengths f) for the Tautomers of Protonated One-Electron Reduced
a
8-Oxo-dGuo Computed at the TD-B3LYP/6-311G(d,p)//B1B95/6-31+G(d,p) Level
a
Only transitions with f greater than, or equal to, 0.010 are reported.
kGy, as illustrated in Figure 3 for 8-oxo-dGuo (see Supporting
Information for 8-NH -Guo). Under our experimental conditions,
a dose of 2 kGy generates ca. 0.66 mM radicals in the irradiated
2
-
•
solution. Assuming that both eaq and H react with the substrates
cf. eq 2), a consumption of up to 41% or 60% would be
(
expected for the starting nucleoside 1 or 2 after 2 kGy, in
contrast with the experimental results.
Our mechanistic proposal to explain this unexpected finding
involves a cross disproportionation reaction between the nucleosidic
•
neutral radical and the CH
2
C(CH
3
)
2
OH radical, which is formed
•
•
after the reaction between HO or H and t-BuOH (eqs 5 and 6).
As a result, both the starting nucleoside and t-BuOH are regener-
ated. This mechanism accounts for the lack of novel products in
the irradiated solution, even after irradiation with high doses, as
well as the low consumption of the starting nucleoside.
•
(
(
CH ) C(OH)CH + 5 f t-BuOH + 1
(5)
3
2
2
Figure 3. Irradiation of 1.6 mM 8-oxo-dGuo (1) in the presence of 0.25
M t-BuOH at pH ) 7.0 (phosphate buffer) and at a dose rate of ∼10 Gy
•
2
CH ) C(OH)CH + 7 f t-BuOH + 2
(6)
3 2
-
1
min . Inset: concentration of 1 vs dose.
8
Taking into consideration that a rate constant of 6.0 × 10
-
1
-1
t-BuOH (0.25 M) in phosphate buffered solution (pH ) 7.0) or
M
s
)
is reported for the radical-radical recombination of
•
28
2
(1.1 mM) and t-BuOH (0.25 M) at natural pH (pH 8.1) were
(CH
3
2
C(OH)CH
2
in aqueous solutions and assuming a rate
8
irradiated under stationary state conditions with a dose rate of
constant for self-termination of radicals 5 and 7 of 2.0 × 10
ca. 10 Gy min- followed by HPLC analysis. Surprisingly,
the only detectable product in both cases was the initial
nucleoside, with a measurable loss of 1 or 2 of ca. 10% after 2
1
34
M
-1 -1
s , it was possible to fit very well the decays of transients
9
in Figure 1 (insets) using rate constants of 1.4 × 10 and 3.0 ×
8
-1 -1
33
10 M
s
for eqs 5 and 6, respectively.
1
5898 J. AM. CHEM. SOC. 9 VOL. 131, NO. 43, 2009

8-Oxo-G and 8-NH
2
-G in Redox Processes
A R T I C L E S
Table 2. Vertical Optical Transitions (Wavelengths λ, Oscillator Strengths f) for the Tautomers of Protonated One-Electron Reduced
a
2
8-NH -dGuo Computed at the TD-B3LYP/6-311G(d,p)//B1B95/6-31+G(d,p) Level
a
Only transitions with f greater than 0.010 are reported.
Deprotonated One-Electron Oxidized 8-Oxo-dGuo (1) and
-
8
-NH
2
-Guo (2) Species. In the presence of N
2
O, eaq are
7
•
-
efficiently transformed into the O radical (eq 7, k
1
equilibrium with its conjugated base O (eq 8, k
M
) 9.1 ×
9
-1 -1 27,28
•
•
0 M s ).
The HO radical [pK (HO ) ) 11.9] is in
a
•
-
10
8
) 1.2 × 10
s , k-8 ) 1 × 10 s ). The irradiation of N O-saturated
solutions containing 0.1 M NaN
at pH ∼7 causes the formation
-1
-1
8
-1 27
2
3
of the azide radical through the process shown in eq 9 (k )
9
10
-1 -1 27,28
1
.2 × 10
M
s ).
-
•-
e
+ N O f O + N2
(7)
(8)
(9)
aq
2
•
-
•-
HO + HO a O + H O
2
•
-
3
-
•
3
HO + N f HO + N
Figure 4. Absorption spectrum (black line) of one-electron oxidized 8-oxo-
dGuo (1) taken from ref 8. Absorption spectrum (red circles) obtained after
pulse radiolysis at natural pH (pH 8.1) of 1.0 mM 8-NH
NaN , N O-saturated, 2 µs after the pulse. Absorption spectrum (blue
triangles) obtained after pulse radiolysis at natural pH of 1.0 mM 8-N
2
-Guo (2), 0.1 M
The optical absorption spectrum of the one-electron oxidized
•
3
2
8
3
-oxo-dGuo (1) obtained by reaction of N with 1 has been
8
3
-
previously reported by Steenken and co-workers. Figure 4
shows this spectrum (black line) in the 300-700 nm range
which consists of a sharp band centered at 320 nm and a broader
Guo (16), 0.25 M t-BuOH, Ar-purged, 2 µs after the pulse. Insets: Time
dependence of absorption at 420 nm; the solid lines represent the second-
order kinetic fit to the data; (left/red) optical path 2 cm, dose 28.2 Gy,
-1
G(12) ) 0.61 µmol J , (right/blue) optical path 2 cm, dose 26.2 Gy, G(12)
band at 400 nm, assigned by Steenken to radical 10 or/and its
-
1
)
0.27 µmol J .
•
tautomeric form 11. The analogous reaction of N
3
with 8-NH
2
-
Guo (2) afforded a transient species that in the 320-700 nm
range resembles the deprotonated one-electron oxidized 8-oxo-
dGuo species (Figure 4, red circles). By measuring the rate of
Time-dependent DFT calculations (TD-B3LYP/6-311G(d,p)//
B1B95/6-31+G(d,p)) were carried out to compute vertical
optical transitions of two tautomers 10 and 11 and to compare
with the experimental UV-visible spectra. The optical absorp-
tion spectrum of the one-electron oxidized 8-oxo-dGuo displayed
in Figure 4 is well reproduced by the optical transitions
computed for tautomer 10 (Table 3). DFT-TD calculations
predict one strong absorption at 296 nm that is in accord with
the optical density increase at 320 nm at different 8-NH
2
-Guo
9
-1 -1
concentrations, a rate constant of 3.5 × 10 M
s
is obtained
•
33
for the oxidation of 2 by N
3
.
In analogy with 8-oxo-dGuo,
we assign this transient to radical 12 or/and its tautomeric form
3 (Scheme 2).
1
J. AM. CHEM. SOC. 9 VOL. 131, NO. 43, 2009 15899

A R T I C L E S
Kaloudis et al.
Table 3. Vertical Optical Transitions (Wavelengths λ, Oscillator Strengths f) for the Tautomers of Deprotonated One-Electron Oxidized
a
8-Oxo-dGuo Computed at the TD-B3LYP/6-311G(d,p)//B1B95/6-31+G(d,p) Level
Tautomers
λ, nm
f
Transition {spin}
1
0
1
296
0.234
0.031
0.045
0.017
0.018
0.044
0.013
0.010
SOMO(pπ(N7) - pπ(O8′)) f π*(C2-N3) - π*(C6-O6′) {R}
SOMO(pπ(N7) - pπ(O8′)) f π*(N1-C2) + π*(C4-N9) {R}
pπ(out-of-phase N2′, N1, (O6′) f SOMO(pπ(N7) - pπ(O8′)) {ꢀ}
pπ(N7) - pπ(N9) - pπ(O6′) f SOMO(pπ(C5)) {ꢀ}
pπ(N7) f SOMO (pπ(C5)) {ꢀ}
pπ(out-of-phase N2′, N3, N9, O8′) f SOMO(pπ(C5)) {ꢀ}
pπ(out-of-phase N2′, N3, N9, O8′) f SOMO(pπ(C5)) {ꢀ}
pπ(out-of-phase N2′, N1, O6′) f SOMO(pπ(C5)) {ꢀ}
3
3
62
82
1
300
316
443
496
703
a
Only transitions with f greater than 0.010 are reported.
Scheme 2. One-Electron Oxidation of 8-Oxo-dGuo (1) and
increases in the range of 0-1.5 kGy, and analysis of the data
in terms of radiation chemical yield (G) gives G(-1) ) 0.38
•
8
2 3
-NH -Guo (2) by N Radical (the Dot in the Radicals Are Located
on the Atom with Highest Calculated Spin Density)
-
1
33
µmol J when the line is extrapolated to zero dose. Taking
•
•
into account that G(N
3
) + G(H ) ) 0.55 + 0.06 ) 0.61 µmol
-1
35
J , ∼40% of the starting material is regenerated. We suggest
that under our experimental conditions the main reaction of
radical 10 is the disproportionation to give 1 and 14, the latter
being further transformed to the several minor products either
under irradiation or during the workup. By replacing 8-oxo-
dGuo with 8-NH -Guo, similar results were also obtained.
1
1
2
the sharp and intense band at 320 nm and two transitions of
moderate intensity at 362 and 382 nm that account for the broad
band observed experimentally at 400 nm. On the other hand,
the computed absorption spectrum for tautomer 11 is in complete
disagreement with the experimental one, since the computed
transitions have low intensity and the main transition is
computed to occur at a much longer wavelength (443 nm) than
in the experimental spectrum. Furthermore, tautomer 10 is more
stable than tautomer 11 by 3.8 kcal mol- at the PCM/B1B95/
One-Electron Reduced 8-Azidoguanosine (16): An
Unexpected Finding. In a parallel pulse radiolysis study of
-
the reaction of hydrated electrons (eaq ) with 8-N
3
-Guo (16),
we noticed the formation of a single transient whose spectrum
shows a remarkable similarity to that of deprotonated one-
1
2
electron oxidized 8-NH -Guo species (Scheme 3). First of
6
-311+G(2df,p)//B1B95/6-31+G(d,p) level.
10
-1 -1
all, a rate constant of 1.8 × 10
M
s
was determined
Substitution of the oxy group with an amino group at C8
-
33
for the reaction of eaq with 16 at pH ∼7. The optical
produces only a red shift of the broad band by ∼20 nm (at 420
nm, Figure 4). This suggests that, in this case, the two radical
species have the same π electronic structure. Indeed, DFT-TD
calculations carried out on tautomer 12, deriving from depro-
tonation of the amino group at C8 in 2b, reproduce very well
this experimental feature. On the other hand, the computed
spectrum for 8-amino tautomer 13, deriving from deprotonation
of 2b at N1, is in disagreement with the experimental one, since
the main band of moderate intensity is computed at 360 nm
and the intensity of the band at a longer wavelength (423 nm)
is computed to be too low. Furthermore, the imino tautomer 12
is computed to be more stable by 2.3 kcal mol- than the amino
tautomer 13 at the PCM/B1B95/6-311+G(2df,p)//B1B95/6-
-
absorption spectrum obtained from the reaction of eaq with
1
6 (blue triangles) is also shown in Figure 4. This is another
example of “paradoxical” generation of one-electron oxidized
guanine derivatives under reducing conditions, in analogy
to the one-electron oxidized guanosine obtained either by
24,25
oxidationofguanosineorbyreductionof8-bromoguanosine.
We suggest that the initial electron adduct is rapidly
protonated on the azide moiety to give radical 17 that loses
nitrogen, with formation of the observable transient species
assigned to radical 12 and/or its tautomeric form 13 (Scheme
•
3
). Based on product studies (see below), also the H atom
1
Scheme 3. One-Electron Reduction of 8-N -Guo (16) Affords 12,
3
3
1+G(d,p) level.
the Same Transient Species of One-Electron Oxidation of
8
2
-NH -Guo (2)
It is worth mentioning that the spin density (F) distribution
changes substantially going from radical 10 to radical 12: in
the former F is 0.30 on N7 and 0.25 on O8′, whereas in the
3
3
latter F is 0.31 on N7 and 0.43 on N8′.
γ-Radiolysis coupled with product studies was also carried
out with 8-oxo-dGuo (1). N O saturated solutions containing 1
1.5 mM) and NaN (12.5 mM) at natural pH (pH 7.2) were
2
(
3
irradiated under stationary state conditions with a dose rate of
ca. 10 Gy min- followed by HPLC analysis. The consumption
of 1 led to the formation of several minor products (not
identified). The concentrations of 1 decreased as the dose
1
1
5900 J. AM. CHEM. SOC. 9 VOL. 131, NO. 43, 2009

8-Oxo-G and 8-NH
2
-G in Redox Processes
A R T I C L E S
Table 4. Vertical Optical Transitions (Wavelengths λ, Oscillator Strengths f) for the Tautomers of Deprotonated One-Electron Oxidized
a
2
8-NH -dGuo Computed at the TD-B3LYP/6-311G(d,p)//B1B95/6-31+G(d,p) Level
Tautomers
λ, nm
f
Transition {spin}
1
2
3
300
0.190
0.036
0.010
0.065
0.099
0.082
0.020
SOMO (pπ(N7) - pπ(N8′)) f π*(C2-N3) - π*(C6-O6′) {R}
SOMO (pπ(N7) - pπ(N8′)) f π*(N1-C2) + π*(C4-N9) {R}
π(C5-N7) - pπ(Ν9) - pπ(O6′) f SOMO (pπ(N7) - pπ(O8′)) {ꢀ}
pπ(out-of-phase N2′, N1, O6′) f SOMO (pπ(N7) - pπ(O8′)) {ꢀ}
SOMO(pπ(C5) - pπ(N3) - pπ(O6′)) f π*(C8-N8′) {R}
pπ(N7) f SOMO(pπ(C5) - pπ(N3) - pπ(O6′)) {ꢀ}
pπ(out-of-phase N2′, N3, N9, N8′) f SOMO(pπ(C5) - pπ(N3) -
pπ(O6′)) {ꢀ}
370
380
421
1
283
3
3
60
88
4
23
96
0.013
0.010
pπ(out-of-phase N2′, N3, N9, N8′) f SOMO(pπ(C5) - pπ(N3) -
pπ(O6′)) {ꢀ}
6
pπ(out-of-phase N2′, N1, O6′) f SOMO(pπ(C5) - pπ(N3) - pπ(O6′))
{ꢀ}
a
Only transitions with f greater than 0.010 are reported.
Scheme 4. Proposed Mechanism for the Quantitative Formation of
-
2
from the Reaction of eaq with 16 via Radical 12
reacted with 16 to potentially afford the same transient species
(
see below).
3
Stationary state radiolysis of 8-N -Guo (16) under reducing
conditions was performed. Aqueous solutions of 16 (ca. 1.0 mM)
and t-BuOH (0.25 M) at pH ∼7 were prepared. The samples
containing 10 mL of deoxygenated solution were irradiated
under stationary-state conditions with a dose rate of ca. 10 Gy/
min followed by HPLC and LC-MS analysis. The consumption
of 16 (blue peaks, Figure 5) led to the formation of a new
3
Figure 5. Irradiation of 8-N -Guo 16 (1.0 mM) in the presence of 0.25 M
-1
t-BuOH at a dose rate of ∼10 Gy min . The HPLC peaks of 16 are
highlighted in blue, while the peaks for 8-NH -Guo (2) are highlighted in
2
red. The inset shows the chemical radiation yields (G) for the consumption
of 16 and the production of 2 as a function of the irradiation dose.
t-BuOH (0.25 M) was irradiated with ca. 1.5 kGy, the resulting
2
product, which was determined to be 8-NH -Guo 2 (red peaks).
2
mixture was lyophilized, and the residue was dissolved in D O
The concentrations of 16 and 2 decreased and increased,
respectively, as the dose increased in the range of 0-1 kGy.
1
33
and analyzed by H NMR. A new peak at 2.65 ppm was
observed, which corresponds to the CH protons of the oxirane
2
Analysis of the data in terms of radiation chemical yield (G)
3
7
gives G(-16) ) 0.33 and G(2) ) 0.31 µmol J-1 when the lines
ring, in accordance with literature data (Scheme 4). Unfor-
tunately, due to the overwhelming concentration of t-BuOH with
respect to that of 1,1-dimethyloxirane, the peaks of the methyl
groups could not be observed at 1.30 ppm. Further proof was
derived from the acidic hydrolysis of 1,1-dimethyloxirane, which
leads to 2-methyl-1,2-propanediol. A drop of DCl was intro-
duced into the NMR tube, and the spectrum obtained after 15
min showed complete conversion of the first to the latter. Indeed,
the peak at 2.65 ppm is replaced by a new peak at 3.42 ppm,
2
3
5
are extrapolated to zero dose (inset, Figure 5). Taking into
-
•
-1
account that G(eaq ) + G(H ) ) 0.33 µmol J , our results lead
to the conclusion that solvated electrons and hydrogen atoms
react quantitatively with 16 to yield 2.
3
The effective reduction of 8-N -Guo (16) to the corresponding
8
-NH -Guo (2) by hydrated electrons deserves a mechanistic
2
36
consideration. Once the intermediate radical 12 is formed (cf.
Scheme 3), we suggest that a cross-termination reaction between
•
which corresponds to the hydrogen atoms of the CH group in
the nucleoside radical and the CH
2
C(CH
3
)
2
OH radical affords
3
8,39
2
-methyl-1,2-propanediol.
adduct 18 (Scheme 4). Adduct 18 can provide 2 upon either
hydrolysis and/or intramolecular epoxide ring formation leading
to 1,1-dimethyloxirane which subsequently is hydrolyzed to
Conclusions
2
-methyl-2,3-propanediol.
To support the proposed mechanism, we tried to identify 1,1-
Radical anions of DNA bases have held the interest of
chemists for years, and a large body of results exist for
dimethyloxirane in the irradiated mixture. For this purpose, a
.5 mM solution of 8-N -Guo in 5 mL of D O containing
40
nucleosides as simple models. Our work here demonstrates
1
3
2
-
that the reaction of eaq with 8-oxo-dGuo (1a) does not lead,
after protonation, to transient 3, an otherwise important inter-
mediate of the hydroxyl radical addition to guanine, but to a
-
1
(
35) The product yield (mol kg ) divided by the absorbed dose (1 Gy )
-1
1
J kg ) gives the radiation chemical yield.
36) The reduction of 16 to 2 in water by the couple (TMS)
HOCH CH SH under free radical conditions has been recently
(
3
SiH/
2
2
(37) Liu, J.; Houk, K. N.; Dinoi, A.; Fusco, C.; Curci, R. J. Org. Chem.
1998, 63, 8565–8569.
reported, see: Postigo, A.; Kopsov, S.; Ferreri, C.; Chatgilialoglu, C.
Org. Lett. 2007, 9, 5159–5162.
(38) Cook, G. K.; Mayer, J. M. J. Am. Chem. Soc. 1995, 117, 7139–7156.
J. AM. CHEM. SOC. 9 VOL. 131, NO. 43, 2009 15901

A R T I C L E S
Kaloudis et al.
transient species that was characterized as tautomer 5, through
cells with a 2 cm optical path length. Solutions were protected from
the analyzing light by means of a shutter and appropriate cutoff
filters. The bandwidth used throughout the pulse radiolysis experi-
ments was 5 nm. The radiation dose per pulse was monitored by
transient spectroscopy and DFT calculations. Contrary to the
-
above system, eaq addition to isoelectronic 8-NH
2
-Guo (2b)
leads to a transient species that was characterized as tautomer
. Whereas the 8-oxo-dGuo protonated radical anion has the
unpaired electron mainly delocalized in the six-membered
pyrimidine ring, the 8-NH -Guo analogue is mainly delocalized
means of a charge collector placed behind the irradiation cell and
7
-
2 2
calibrated with an N O-saturated solution containing 0.1 M HCO
-4
2
-1
and 0.5 mM methylviologen, using Gε ) 9.66 × 10 m J at
2
45
02 nm. G(X) represents the number of moles of species X
6
in the imidazolyl ring, accentuating the electronic differences
between the two functional groups. On the other hand, the
formed, consumed, or altered per joule of energy absorbed by the
system.
deprotonated one-electron oxidized 8-oxo-dGuo and 8-NH
2
-Guo
Computational Details. Hybrid meta DFT calculations with the
46
47
transients were characterized as tautomers 10 and 12, respec-
B1B95 (Becke88 -Becke95 1-parameter model for thermochem-
48
tively, clarifying, especially in the case of 8-oxo-dGuo, the
istry) functional were carried out using the Gaussian 03 system
49
8
of programs. This HMDFT model was found to give excellent
50,51
ambiguity of previous studies. In this case, both systems have
48,50,51
performanceforthermochemistry
andmoleculargeometries.
the unpaired electron density localized in the imidazolyl ring
An unrestricted wave function was used for radical species.
Optimized geometries and total energies were obtained employing
the valence double-ꢁ basis set supplemented with polarization
functions and augmented with diffuse functions on heavy atoms
(
with the highest calculated spin density residing always on a
N). In the case of the 8-NH -Guo, the same transient was
obtained through eaq reaction with 8-N -Guo, providing yet
another example of generation of one-electron oxidized guanine
2
-
3
(
6-31+G(d,p)). The nature of the ground states (zero imaginary
2
4,25
derivatives under reducing conditions.
frequency) was verified by frequency calculations. Total energies
were corrected for the zero-point vibrational energy (ZPVE)
computed from frequency calculations using a scaling factor of
Experimental Section
50
0
.9735 to account for anharmonicity. The effect of water solution
Materials. 8-Oxo-7,8-dihydro-2′-deoxyguanosine was purchased
from Berry & Associates and used without any further purification.
on the total energies was computed with the polarizable continuum
5
2
model (PCM) carrying out calculations at the optimum B1B95/
6-31+G(d,p) geometries (PCM(solvent)water)/B1B95/6-
11+G(2df,p)//B1B95/6-31+G(d,p)). The time dependent B3LYP
8
-Aminoguanosine and 8-azidoguanosine were synthesized using
41,42
standard procedures.
All other chemicals and solvents were
3
purchased from commercial suppliers at the highest purity available.
method employing a triple-ꢁ basis set (TD-B3LYP/6-311G(d,p)//
B1B95/6-31+G(d,p)) was used to compute optical transitions, since
this hybrid DFT method was found by us to provide reliable optical
transitions in nucleosides (see text). In Tables 1-4 we have reported
the electronic transitions having an oscillator strength f greater than
Continuous Radiolysis. Experiments were performed at room
6
0
temperature (22 ( 2 °C) on 10 mL samples using a Co-
-1
Gammacell, with a dose rate of ca. 10 Gy min . The absorbed
radiation dose was determined with a Fricke chemical dosimeter,
3
+
-1 43
by taking G(Fe ) ) 1.61 µmol J . Reaction mixtures were
0
.010 and a wavelength λ greater than 280 nm.
analyzed by reversed phase HPLC using a Zorbax SB-C18 column
(
(
3
4.6 mm × 150 mm) and eluted in triethylammonium acetate buffer
Acknowledgment. This research was supported in part by the
20 mM, pH 7) with a 0-25% acetonitrile nonlinear gradient over
European Community’s Marie Curie Research Training Network
under Contract MRTN-CT-2003-505086 (CLUSTOXDNA). The
support and sponsorship concerned by COST Action CM0603 on
Free Radicals in Chemical Biology (CHEMBIORADICAL)” are
kindly acknowledged. We thank C. Ferreri for many useful
discussions and A. Altieri for some preliminary experiments with
0 min with a flow rate of 1.0 mL/min (detection at 254 nm). LC/
MS analyses were performed with an Agilent 1100 HPLC system
and an Esquire 3000 Plus Bruker mass spectromer using a Zorbax
C8 column (4.6 mm × 150 mm, 5 µm) with a 0-25% acetonitrile
nonlinear gradient over 30 min at a flow rate of 0.1 mL/min,
detection at λ ) 254 nm.
Pulse Radiolysis. Pulse radiolysis with optical absorption
detection was performed by using a 12 MeV linear accelerator,
which delivered 20-200 ns electron pulses with doses between 5
“
3
8-N -Guo. We thank M. Lavalle, A. Monti, and A. Martelli for
assistance with the pulse radiolysis experiments.
•
•
-
and 50 Gy, by which OH , H , and eaq are generated with 1-20
µM concentrations. The pulse irradiations were performed at room
temperature (22 ( 2 °C) on samples contained in Spectrosil quartz
Supporting Information Available: Product studies, DFT
calculations (spin density at the guanine atoms of radicals 4-13,
SOMO of radicals 10 and 12, absolute energies and optimized
geometries of calculated structures), pulse radiolysis experi-
ments, and complete ref 49. This material is available free of
charge via the Internet at http://pubs.acs.org.
44
(
39) Kinetic support of the mechanism proposed in Scheme 4 can be
obtained by analysis of the kinetic decays of radical 12 (insets in Figure
4
). The transient 12 obtained from the one-electron oxidation of 8-NH -
2
•
Guo by N decays with second-order kinetics (left inset). A value of
3
8
-1 -1
JA9065464
k ) 2.3 × 10 M
s
was obtained as an average from the linear
and nonlinear analysis of the trace and associated with the self-
disproportionation to give 2 and 15. On the other hand, the decay of
(45) Mulazzani, Q. G.; D’Angelantonio, M.; Venturi, M.; Hoffman, M. Z.;
Rodgers, M. A. J. J. Phys. Chem. 1986, 90, 5347–5352.
(46) Becke, A. D. Phys. ReV. A 1988, 38, 3098–3100.
(47) Becke, A. D. J. Chem. Phys. 1996, 104, 1040–1046.
(48) Zhao, Y.; Pu, J.; Lynch, B. J.; Truhlar, D. G. Phys. Chem. Chem.
Phys. 2004, 6, 673–676.
(49) Frisch, M. J. Gaussian 03, ReVision D.02; Gaussian, Inc.; Wallingford,
CT, 2004 (See Supporting Information for the full list of authors).
(50) Zhao, Y.; Lynch, B. J.; Truhlar, D. G. J. Phys. Chem. A 2004, 108,
2715–2719.
(51) Zhao, Y.; Truhlar, D. G. J. Phys. Chem. A 2004, 108, 6908–6918.
(52) Tomasi, J.; Menucci, B.; Cances, E. THEOCHEM 1999, 464, 211–
226.
transient 12 generated by the one-electron reduction of 8-N
3
-Guo (right
8
-1 -1
inset) corresponds to a larger value of k ) 4.4 × 10 M
s , which
33
is associated with the cross-recombination reported in Scheme 4.
(
(
40) von Sonntag, C. Free-Radical-Induced DNA Damage and Its Repair;
Springer-Verlag: Berlin; 2006.
41) Lin, T.-C.; Cheng, J.-C.; Ishiguro, K.; Sartorelli, A. C. J. Med. Chem.
1
985, 28, 1194–1198.
(
(
42) Saneyoshi, M. Chem. Pharm. Bull. 1968, 16, 1616–1619.
43) Spinks, J. W. T.; Woods, R. J. An Introduction to Radiation Chemistry,
3
rd ed.; Wiley: New York, 1990; p 100.
44) Hutton, A.; G; Roffi, G.; Martelli, A. Quad. Area Ric. Emilia Romagna
974, 5, 67–74.
(
1
1
5902 J. AM. CHEM. SOC. 9 VOL. 131, NO. 43, 2009