Photooxiation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by thermally generated triplet-excited ketones from 3-(hydroxymethyl)-3,4,4-trimethyl-1,2-dioxetane and comparison with type I and type II photosensitizers

Article abstract of DOI:10.1021/ja953980+

Calf thymus DNA and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) were photooxidized in the dark by triplet-excited ketones generated in the thermal decomposition of 3-(hydroxymethyl)-3,4,4-trimethyl-1,2-dioxetane (HTMD). The oxidation of DNA led to 8-oxodGuo and the type I photooxidation product 2,2-diamino[2-deoxy-β-D-erythro-pentofuranosyl)-4-amino]-5(2H)-oxazol one (oxazolone). While the yield of oxazolone progressively increased, 8-oxodGuo was substantially consumed in DNA on successive exposure to HTMD. The oxidation of authentic 8-oxodGuo by HTMD and established photosensitizers such as benzophenone (mainly type I) and Rose Bengal (predominantly type II) was studied in detail in regard to the concentration and time dependence and the influence of D₂O versus H₂O. The singlet-oxygen-derived 4R* and 4S* diastereomers of 4-hydroxy-8-oxo-4,8-dihydro-2'-deoxyguanosine (4-HO-8-oxodGuo) and oxazolone were the major products. A substantial kinetic D₂O effect (ca. 10-fold) in the Rose Bengal-photosensitized degradation of 8-oxodGuo unequivocally established that in this case singlet oxygen (type II photooxidation) is involved. However, the efficient formation of oxazolone by benzophenone as a characteristic type I photooxidant, as well as in the HTMD-mediated oxidation (predominantly type I), and the fact that these processes exhibit a negligible D₂O effect provide cogent experimental evidence for an electron or hydrogen atom transfer mechanism (type I photooxidation) in the oxidative degradation of 8-oxodGuo into oxazolone. The unprecedented observation that comparable product ratios of 4-HO-8-oxodGuo and oxazolone were obtained in the 8-oxodGuo oxidations, irrespective of whether Rose Bengal as a typical type II photooxidant or benzophenone as an established type I photooxidant was employed, is presumably due to electron-transfer chemistry of ¹O₂ with the easily oxidized 8-oxodGuo in view of its low oxidation potential. This nicely accounts for the fact that the primary oxidation product 8-oxodGuo, which serves as an important monitor of oxidative genotoxicity, may not accumulate appreciably in the photooxidation of DNA.

Full text of DOI:10.1021/ja953980+

J. Am. Chem. Soc. 1996, 118, 9233-9238
9233
Photooxidation of 8-Oxo-7,8-dihydro-2′-deoxyguanosine by
Thermally Generated Triplet-Excited Ketones from
3-(Hydroxymethyl)-3,4,4-trimethyl-1,2-dioxetane and
Comparison with Type I and Type II Photosensitizers
Waldemar Adam, Chantu R. Saha-Mo1ller, and Andre´ Scho1nberger*
Contribution from the Institute for Organic Chemistry, UniVersity of Wu¨rzburg,
Am Hubland, D-97074 Wu¨rzburg, Germany
ReceiVed NoVember 27, 1995X
Abstract: Calf thymus DNA and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) were photooxidized in the dark
by triplet-excited ketones generated in the thermal decomposition of 3-(hydroxymethyl)-3,4,4-trimethyl-1,2-dioxetane
(HTMD). The oxidation of DNA led to 8-oxodGuo and the type I photooxidation product 2,2-diamino[2-deoxy-
â-D-erythro-pentofuranosyl)-4-amino]-5(2H)-oxazolone (oxazolone). While the yield of oxazolone progressively
increased, 8-oxodGuo was substantially consumed in DNA on successive exposure to HTMD. The oxidation of
authentic 8-oxodGuo by HTMD and established photosensitizers such as benzophenone (mainly type I) and Rose
Bengal (predominantly type II) was studied in detail in regard to the concentration and time dependence and the
influence of D2O Versus H2O. The singlet-oxygen-derived 4R* and 4S* diastereomers of 4-hydroxy-8-oxo-4,8-
dihydro-2′-deoxyguanosine (4-HO-8-oxodGuo) and oxazolone were the major products. A substantial kinetic D2O
effect (ca. 10-fold) in the Rose Bengal-photosensitized degradation of 8-oxodGuo unequivocally established that in
this case singlet oxygen (type II photooxidation) is involved. However, the efficient formation of oxazolone by
benzophenone as a characteristic type I photooxidant, as well as in the HTMD-mediated oxidation (predominantly
type I), and the fact that these processes exhibit a negligible D2O effect provide cogent experimental evidence for
an electron or hydrogen atom transfer mechanism (type I photooxidation) in the oxidative degradation of 8-oxodGuo
into oxazolone. The unprecedented observation that comparable product ratios of 4-HO-8-oxodGuo and oxazolone
were obtained in the 8-oxodGuo oxidations, irrespective of whether Rose Bengal as a typical type II photooxidant
or benzophenone as an established type I photooxidant was employed, is presumably due to electron-transfer chemistry
of 1O2 with the easily oxidized 8-oxodGuo in view of its low oxidation potential. This nicely accounts for the fact
that the primary oxidation product 8-oxodGuo, which serves as important monitor of oxidative genotoxicity, may
not accumulate appreciably in the photooxidation of DNA.
Introduction
tion of singlet oxygen13-17 to the C4-C5 and N7-C8 double
bonds of guanine (Diels-Alder reaction) and rearrangement of
the initially formed endo-peroxide to the C-8 hydroperoxide,
which is expected to be readily reduced by oxidizable compo-
nents in the system.17 The third alternative is the photosensitized
oxidation18-20 to generate the guanine radical cation through
electron transfer from guanine by the excited sensitizer (type I
photooxidation21 ). Only in the DNA matrix is the radical cation
persistent enough10,22 to add water at the C-8 position and form
8-oxodGuo. In aqueous solution at pH 7.0, however, the highly
acidic intermediary guanosine radical cation predominantly
deprotonates to afford a radical, which after addition of
molecular oxygen at the C-5 position yields the characteristic
type I photooxidation products of dGuo.23,24 The latter is, as
in the case of hydroxyl radical attack at guanine, the 2,2-
8-Oxo-7,8-dihydro-2′-deoxyguanosine, for short 8-oxodGuo,
constitutes an important product of oxidative attack on nucleic
acids.1-4 This mutagenic DNA lesion5-9 is generally agreed
to be formed by at least three mechanisms. The first involves
attack of hydroxyl radicals10-12 at the C-8 position of the
guanine and subsequent tautomerization to the more stable
8-keto form. The second mechanism entails [4+2] cycloaddi-
X Abstract published in AdVance ACS Abstracts, September 1, 1996.
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S0002-7863(95)03980-1 CCC: $12.00 © 1996 American Chemical Society

9234 J. Am. Chem. Soc., Vol. 118, No. 39, 1996
Adam et al.
diamino[(2-deoxy-â-D-erythro-pentofuranosyl)-4-amino]-5(2H)-
oxazolone (oxazolone), which is the hydrolysis product of the
corresponding imidazolone precursor. Additionally, the cyclic
3-(hydroxymethyl)-3,4,4-trimethyl-1,2-dioxetane (HTMD) the
yield of 8-oxodGuo decreased significantly. In the reaction of
dGuo with HTMD,38 besides a significant yield of 8-oxodGuo
(up to 1%), also the characteristic singlet oxygen product
4-hydroxy-8-oxo-4,8-dihydro-2′-deoxyguanosine,39,40 for short
4-HO-8-oxodGuo, was detected, in addition to the type I
photooxidation products oxazolone and oxoimidazolidine. It
1
was established that HTMD generates O2 on thermal decom-
position46 and singlet oxygen is involved in the formation of
8-oxodGuo from dGuo (substantial D2O effect). Nevertheless,
comparison with the well-established type I (benzophenone,41
riboflavin20) and predominant type II (Rose Bengal,42 methylene
blue43) photosensitizers revealed that both type I and type II
photooxidation modes operate with comparable efficiency in
the HTMD-induced photooxidation of dGuo.
For several years it has been suggested that 8-oxodGuo is
readily attacked by singlet oxygen to produce the two 4R* and
4S* diastereomers of 4-HO-8-oxodGuo.40 This was recently
confirmed by Sheu and Foote,44 by employing a derivative of
8-oxodGuo soluble in organic solvents, which was photooxi-
dized with tetraphenylporphine (TPP) as sensitizer in the
presence of O2. Low-temperature NMR studies in acetone-d6
revealed that, indeed, singlet oxygen adds to the C4-C5 double
bond of 8-oxodGuo through [2+2] cycloaddition to yield a set
of intermediary diastereomeric dioxetanes, which are trans-
formed in situ to 4-HO-8-oxodGuo through the corresponding
4-hydroperoxides.
Recently, the photosensitized degradation of 8-oxodGuo by
methylene blue was reported,45 in which 4-HO-8-oxodGuo and
imidazolone (the precursor of oxazolone) were efficiently
generated. On the basis of a substantial D2O effect on the
conversion rate of 8-oxodGuo and the pronounced quenching
with NaN3, singlet oxygen was proposed to be responsible for
the formation of both observed oxidation products.
Additional to singlet oxygen chemistry, photoexcited meth-
ylene blue is also capable of electron transfer chemistry with
nucleic acids (45% type I and 55% type II photooxidation
products with acetylated 2′-deoxyguanosine).19,42 Therefore, to
avoid such type I activity, we employed the quite selective
singlet-oxygen-generating photosensitizer Rose Bengal (95%
type II products19,42) to clarify definitively the mechanism of
the photooxidative fate of 8-oxodGuo. For comparison, we
investigated the photooxidation of authentic 8-oxodGuo by the
predominant type I photosensitizer benzophenone,42 which
mainly reacts through electron and/or hydrogen transfer chem-
istry, and by HTMD as a thermal source of triplet-excited
ketones in the dark. Our present results confirm the efficient
photooxidative reactivity of Rose Bengal, benzophenone, and
HTMD toward 8-oxodGuo and suggest that, in addition to the
previously proposed type II photooxidation (1O2), type I
photooxidation is also involved in the degradation of 8-oxodGuo
to afford oxazolone.
nucleoside 2(S)-2,5′-anhydro-1-(2-deoxy-â-D-erythro-pentofura-
nosyl)-5-guanidinylidene-2-hydroxy-4-oxoimidazolidine25
(oxoimidazolidine) is formed through intramolecular attack of
the 5′-hydroxyl functionality on the C-8 position of the
intermediary guanine radical.
As alternatives to the well-established triplet photosensitizers,
1,2-dioxetanes may be used, since they serve as efficient sources
of triplet-excited carbonyl products on thermal activation.26-28
This unique class of four-membered ring peroxides is of
biological interest, since they have been implicated as labile
intermediates in oxidative stress, for the induction of spontane-
ous mutations and in enzymatic oxidations.1,27,29-31 The triplet-
state ketones generated in the thermal decomposition of 1,2-
dioxetanes give rise to photochemical and photobiological
transformations in the dark. They exhibit strong genotoxic
activity in isolated as well as in bacterial and cellular DNA,32-34
with the predominant formation of oxidative base modifications
in isolated DNA.35,36
Our recently published studies37 on the oxidation of calf
thymus DNA by 1,2-dioxetanes revealed that 8-oxodGuo is
efficiently formed, unequivocally through the action of thermally
generated triplet-excited ketones. With a large excess of
(23) Cadet, J.; Berger, M.; Decarroz, C.; Mouret, J.-F.; van Lier, J. E.;
Wagner, R. J. J. Chim. Phys. Phys.-Chim. Biol. 1991, 88, 1021-1042.
(24) Cadet, J.; Berger, M.; Buchko, G. W.; Joshi, P. C.; Raoul, S.;
Ravanat, J.-L. J. Am. Chem. Soc. 1994, 116, 7403-7404.
(25) Buchko, G. W.; Cadet, J.; Ravanat, J.-L.; Labataille, P. Int. J. Radiat.
Biol. 1993, 63, 669-676.
(26) Adam, W.; Heil, M.; Mosandl, T.; Saha-Mo¨ller, C. R. In Organic
Peroxides; Ando, W., Ed.; John Wiley & Sons: New York, 1992; pp 221-
254.
(27) Cilento, G.; Adam, W. Photochem. Photobiol. 1988, 48, 361-368.
(28) Adam, W.; Cilento, G. Angew. Chem., Int. Ed. Engl. 1983, 22, 529-
542.
(29) Smith, K. C. Mutat. Res. 1992, 277, 139-162.
(30) Cadenas, E. Photochem. Photobiol. 1984, 40, 823-830.
(31) Baader, W. J.; Bohne, C.; Cilento, G.; Dunford, H. B. J. Biol.
Chem.1985, 260, 10217-10225.
(38) Scho¨nberger, A. Ph.D. Dissertation, University of Wu¨rzburg,
Germany, February 1996.
(39) Buchko, G. W.; Cadet, J.; Berger, M.; Ravanat, J.-L. Nucleic Acids
Res. 1992, 20, 4847-4851.
(32) Adam, W.; Beinhauer, A.; Mosandl, T.; Saha-Mo¨ller, C. R.; Vargas,
F.; Epe, B.; Mu¨ller, E.; Schiffmann, D.; Wild, D. EnViron. Health Perspect.
1990, 88, 89-97.
(40) Ravanat, J.-L.; Berger, M.; Benard, F.; Langlois, R.; Ouellet, R.;
van Lier, J. E.; Cadet, J. Photochem. Photobiol. 1992, 55, 809-814.
(41) Morin, B.; Cadet, J. Photochem. Photobiol. 1994, 60, 102-109.
(42) Cadet, J.; Decarroz, C.; Wang, S. Y.; Midden, W. R. Isr. J. Chem.
1983, 23, 420-429.
(33) Epe, B.; Mu¨ller, E.; Adam, W.; Saha-Mo¨ller, C. R. Chem.-Biol.
Interact. 1992, 85, 265-281.
(34) Adam, W.; Ahrweiler, M.; Saha-Mo¨ller, C. R.; Sauter, M.; Scho¨n-
berger, A.; Epe, B.; Mu¨ller, E.; Schiffmann, D.; Stopper, H.; Wild, D.
Toxicol. Lett. 1993, 67, 41-55.
(43) Tuite, E. M.; Kelly, J. M. J. Photochem. Photobiol., B 1993, 21,
103-124.
(35) Tchou, J.; Bodepudi, V.; Shibutani, S.; Anthoshechkin, I.; Miller,
J.; Grollman A. P.; Johnson, F. J. Biol. Chem. 1994, 269, 15318-15324.
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(45) Buchko, G. W.; Wagner, J. R.; Cadet, J.; Raoul, S.; Weinfeld, M.
Biochim. Biophys. Acta 1995, 1263, 17-24.
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Biol. Int. 1996, 38, 647-651.

Photooxidation of 7,8-Dihydro-8-oxo-2′-deoxyguanosine
J. Am. Chem. Soc., Vol. 118, No. 39, 1996 9235
Table 1. Product Studies of the Guanine Photooxidation in the
Repetitive Thermal Treatment of Calf Thymus DNA with HTMD
yieldb
product
oxa- balancec
conversion of
guanine (%) oxoGua zolone
8-
batch
conditionsa
(%)
1
2
3
4
5
5 mM HTMD
+ 5 mM HTMD
+ 5 mM HTMD
+ 5 mM HTMD
+ 5 mM HTMD
8
22
34
58
79
4.3
4.8
3.8
2.4
1.9
2.6
4.7
6.2
7.4
8.5
86
43
29
17
14
a 0.1 mg/mL calf thymus DNA in 5 mM phosphate buffer (pH 7.0),
at 37 °C, repetitively treated with 5 mM HTMD in aqueous acetonitrile
(90:10) each for 24 h. b Yield relative to available guanine in DNA
(625 pmol/µg DNA), mean value of three independent runs, standard
deviation (5% of the stated value. c Detected products relative to
converted guanine.
Figure 1. Concentration profile for HTMD-induced oxidation of 8-
oxodGuo: carried out in the dark at 50 °C for 12 h with 100 µM
8-oxodGuo in 5 mM sodium cacodylate buffer (pH 7.0) and 10%
acetonitrile as cosolvent. The values on the ordinate are mean values
of at least three independent runs, error (10% of the stated value:
conversion of 8-oxodGuo (+), absolute yield ([) of 4-HO-8-oxodGuo,
and relative one, based on consumed 8-oxodGuo (0).
Results and Discussion
As listed in Table 1, the thermal treatment of calf thymus
DNA at 37 °C for 24 h in the presence of 5 mM HTMD
afforded, besides substantial amounts of 8-oxoGua (4.3%
relative to guanine in DNA), the oxazolone in 2.6% yield. For
the latter photooxidation product the well-established fluorescence-
labeling HPLC assay with 1,2-naphthoquinone-4-sulfonic acid,
introduced by Ravanat et al.,40 was employed. To assess the
persistence of these guanine oxidation products toward HTMD,
we repetitively treated the oxidized DNA solution with new
HTMD batches at 37 °C. The determination of the guanine
conversion in DNA allowed the evaluation of the product
balance, which we define as the yield of detected guanine
oxidation products relative to the amount of converted guanine.
The second treatment of an already oxidized DNA solution with
5 mM HTMD resulted in 22% conversion of the guanine in
DNA, and the yields of 8-oxoGua and oxazolone both increased
to almost 5% (entry 2, Table 1). Further treatments of the
already oxidized DNA with additional HTMD batches selec-
tively consumed up to 80% of the available guanine, whereas
the other DNA bases remained intact, as confirmed by HPLC/
UV detection. After the fifth batch of HTMD, the maximum
yield (4.8%, entry 2) of the already formed 8-oxoGua was
substantially reduced to 1.9% (entry 5) and the yield of
oxazolone increased continuously to 8.5%. The product balance
dropped from ca. 80% after the first to 14% after the fifth
HTMD treatment.
The results of the repetitive treatment of DNA with a large
excess of HTMD imply that 8-oxodGuo is readily further
oxidized by HTMD and that oxazolone is a likely product of
this degradation. However, since the product balance for
guanine decreased significantly in the repetitive treatment of
DNA with HTMD (Table 1), the formation of oxazolone cannot
account exclusively for the consumption of guanine and/or
8-oxodGuo in DNA. This suggests that other still unknown
oxidation products are generated. Recently, 1-(2-deoxy-â-D-
erythro-pentofuranosyl)cyanuric acid and its relatively unstable
precursor have been identified as major singlet oxygen products
in the oxidation of 8-oxodGuo.54 In fact, the almost linear
increase in the yield of oxazolone on repetitive HTMD
treatment suggests that, at least in DNA, the subsequent
oxidation of 8-oxodGuo contributes only a fraction of the
formed oxazolone.
Figure 2. Time profile for the HTMD-induced oxidation of 8-oxod-
Guo: carried out in the dark at 50 °C for 12 h with 100 µM 8-oxodGuo
in 5 mM sodium cacodylate buffer (pH 7.0) and 4 mM HTMD,
dissolved in acetonitrile (10 vol %). The values on the ordinate are
mean values of at least three independent runs, error (10% of the stated
value: conversion of 8-oxodGuo (+), absolute yield ([) of 4-HO-8-
oxodGuo, and relative one, based on consumed 8-oxodGuo (0).
reaction times to assess the respective concentration and time
profiles for this dioxetane-induced oxidation of 8-oxodGuo
(Figures 1 and 2). Control experiments without HTMD
revealed that authentic 8-oxodGuo and 4-HO-8-oxodGuo
persisted (less than 2% consumption, as determined by HPLC
analysis) under the reaction conditions (50 °C, 15 h, pH 7.0).
44
In agreement with the proposed singlet oxygen mechanism
for the oxidation of 8-oxodGuo, the characteristic type II
photooxygenation products of dGuo,39,40 namely, the 4R* and
4S* diastereomers of 4-HO-8-oxodGuo, were detected in high
yields. At an excess of 40 equiv (4 mM) of HTMD, the
8-oxodGuo was nearly quantitatively (91%) consumed after 9
h at 50 °C (Figure 1) to afford 4-HO-8-oxodGuo in 50% yield.
Remarkable is the observation that the relative yield of 4-HO-
8-oxodGuo (based on converted 8-oxodGuo) was significantly
increased to ca. 90% when a lower HTMD excess (10 equiv.)
was employed (Figure 1). Thus, the substantial decrease of the
relative 4-HO-8-oxodGuo yield at high HTMD excess suggests
the formation of additional oxidation products, such as 1-(2-
deoxy-â-D-erythro-pentofuranosyl)-cyanuric acid54 or yet un-
known products.
To clarify the fate of 8-oxodGuo in the HTMD-induced
oxidation of DNA and to gain mechanistic insight, we examined
the oxidation of authentic 8-oxodGuo with HTMD. For
comparison, characteristic type I (benzophenone) and predomi-
nant type II (Rose Bengal) photosensitizers were employed.
The thermal decomposition of HTMD in the presence of
8-oxodGuo was carried out at different concentrations and
In contrast to the concentration profile (Figure 1), the time
profile (Figure 2) reveals that the relative yield of 4-HO-8-
oxodGuo is independent of time for 2-12 h, even when lower
HTMD concentrations (1 or 2 mM) are applied (data not shown

9236 J. Am. Chem. Soc., Vol. 118, No. 39, 1996
Adam et al.
Table 2. Product Studies of the Oxidation of 8-oxodGuo by
in Figure 2). Additionally, the concentration profile exhibits a
linear increase of 8-oxodGuo consumption with increasing
HTMD concentration (Figure 1), whereas in the time profile
(Figure 2) a nonlinear dependence is observed. Both profiles
establish that the conversion of 8-oxodGuo is proportional to
the yield of the triplet-excited ketones, which are released from
HTMD by its thermal decomposition. In this context it should
be kept in mind that contrary to the photosensitized oxidations,
which operate catalytically by energy transfer to generate time-
invariant concentrations of triplet-excited states, the dioxetane-
derived triplet states are stoichiometrically produced; i.e., the
dioxetane decomposition is irreversible and the thermally
produced triplet states are not recycled. Therefore, the mo-
mentary concentration of triplet-excited ketones depends on the
initial concentration of the dioxetane and its decomposition rate.
The concentration-dependent (Figure 1) and time-independent
(Figure 2) relative yields of 4-HO-8-oxodGuo suggest that the
formation of this photooxidation product is mainly governed
by the steady-state concentration of triplet-excited states gener-
ated from the HTMD Versus the concentration of available
substrate (8-oxodGuo or O2).
Thermal Treatment with HTMD or Photosensitized by
Benzophenone or Rose Bengala
yielde (%)
conversion of
product
8-oxodGuo 4-HO-8- oxa- balanceg
oxidant
(%)
oxodGuo zolonef
(%)
HTMD/∆Tb
51
85
50
15 (29) 21 (41)
22 (26) 40 (47)
13 (25) 24 (47)
70
73
72
benzophenone/hνc
Rose Bengal (2 µM)/hνd
a 100 µM 8-oxodGuo, 5 mM sodium cacodylate buffer, pH 7.0. b 7
mM HTMD, aqueous acetonitrile (90:10), 50 °C, 180 min. c 50 µM
benzophenone, aqueous acetonitrile (98:2), RPR (350 nm), irradiated
at a distance of ca. 5 cm, 20 min, 4 °C. d 2 µM Rose Bengal, 150-W
sodium lamp, irradiated at a distance of ca. 20 cm, 30 min, 4 °C.
e Yields relative to conversion of 8-oxodGuo given in parantheses.
f Detected by the fluorescence-labeling assay with 1,2-naphthoquinone-
4-sulfonic acid (NQS) after alkaline treatment (1 N NaOH) at 65 °C
for 9 min. g Detected products relative to converted 8-oxodGuo.
tion of guanidine (the latter is released after alkaline treatment
from oxazolone). This assay, unfortunately, cannot distinguish
the origin of guanidine,49 i.e., whether it stems from oxazolone
or free guanidine, the latter formed as a side product in the
oxidation of 8-oxodGuo.44 Nonetheless, in analogy with the
previously reported2,40,45 photooxidations of nucleic acid deriva-
tives, we assume that the guanidine in our oxidations of
8-oxodGuo also originates mainly from the oxazolone. In fact,
control experiments revealed that no detectable amounts of
guanidine were observed without alkaline treatment of the
reaction mixture nor after thermal treatment of authentic
8-oxodGuo or 4-HO-8-oxodGuo for 15 h at 50 °C. The yield
of the guanidine-releasing oxazolone in the oxidation of
8-oxodGuo on HTMD treatment and by the photosensitizers
benzophenone and Rose Bengal are listed in Table 2. Thus,
with a large excess of HTMD (entry 1, Table 2), as well as in
the photosensitized oxidation of 8-oxodGuo by benzophenone
or rose bengal (entries 2 and 3, Table 2), high yields (41 and
47% referred to consumed 8-oxodGuo) of oxazolone were
observed, which even exceed the yield (25-29%, Table 2) of
4-HO-8-oxodGuo. The product balance could be greatly
improved (from ca. 26% to ca. 70%) through the quantitative
determination of oxazolone by the HPLC/fluorescence assay
(Table 2). Interesting and unexpected is the result that the ratio
of oxidation products 4-HO-8-oxodGuo and oxazolone is
independent of the employed sensitizer within the experimental
error.
The estimate47 that ca. 90% of the HTMD-derived triplet
states react with 8-oxodGuo, whereas only ca. 40% react with
dGuo,38 emphasizes that 8-oxodGuo is more efficiently oxi-
dized by the thermally generated triplet ketones than dGuo. This
is expected since the oxidation potential of 8-oxodGuo (0.85
V Vs AgCl) is significantly lower than for dGuo (1.28 V Vs
AgCl).17 Therefore, it is not surprising that the yield of
8-oxodGuo in the HTMD-induced oxidation of DNA decreases
significantly at a high excess of dioxetane. Consequently,
8-oxodGuo does not accumulate appreciably in the reaction with
DNA and dGuo because its further oxidation is preferred over
that of unmodified guanine.
For mechanistic elucidation, the photosensitized oxidation of
8-oxodGuo by well-established type I and type II photosensi-
tizers was investigated. As a characteristic type I sensitizer
benzophenone, which mainly acts by hydrogen abstraction,41
was chosen, and Rose Bengal42 was employed as an efficient
type II (1O2) sensitizer. With both sensitizers, quantitative
conversion of 8-oxodGuo was achieved.48 The relative yield
of 4-HO-8-oxodGuo for both sensitizers did not exceed 35 (
5% (referred to consumed 8-oxodGuo), within the experimental
error independent of the sensitizer concentration (5-500 µM
benzophenone, 0.2-2 µM Rose Bengal) and the irradiation time
(10-70 min).
The moderate 4-HO-8-oxodGuo yield (ca. 35% relative to
converted 8-oxodGuo), especially for both photosensitizers
benzophenone (type I) and Rose Bengal (type II), implies that,
besides 4-HO-8-oxodGuo, other products, such as 1-(2-deoxy-
â-D-erythro-pentofuranosyl)cyanuric acid,54 are formed in the
photosensitized or the HTMD-induced oxidation of 8-oxodGuo.
As a significant oxidation product in the methylene blue-
sensitized photooxidation of 8-oxodGuo,45 oxazolone was
detected, which has been rigorously characterized spectrally (1H
NMR, UV, FAB-MS). To assess the formation of oxazolone
in our HTMD-induced or photosensitized (benzophenone, Rose
Bengal) oxidation of 8-oxodGuo, we employed the established40
fluorescence-labeling HPLC assay with 1,2-naphthoquinone-4-
sulfonic acid (NQS), which quantitatively monitors the forma-
To probe for the participation of singlet oxygen in the
HTMD-induced and photosensitized oxidation of 8-oxodGuo,
the effect of deuterium oxide on the conversion of 8-oxodGuo
and the formation of 4-HO-8-oxodGuo was investigated (Table
3). The thermal treatment of 8-oxodGuo with HTMD (entries
1-3, Table 3) exhibits a negligible D2O effect in regard to the
conversion of 8-oxodGuo (ca. 20% decrease in D2O) and the
relative yield of 4-HO-8-oxodGuo (ca. 10% decrease in D2O).
Similarly, only a slight but opposite D2O effect was observed
for the benzophenone-sensitized oxidation of 8-oxodGuo (entry
4, Table 3). In contrast, a pronounced kinetic D2O effect was
found in the Rose-Bengal-sensitized oxidation since 8-oxodGuo
was degraded about 10-fold faster in D2O than in H2O (entry 5
with 6, Table 3). The relative yield of 4-HO-8-oxodGuo,
however, remained constant at 31% in both solvents.
(47) A triplet yield of ca. 5% for HTMD in aqueous solution33 and a
half-life of t1/2 ) 6 h at 50 °C (measured by chemiluminescence) were
employed for this estimate.
(48) Reaction conditions for the quantitative conversion of 8-oxodGuo:
100 µM 8-oxodGuo in 5 mM phosphate buffer, pH 7.0, 4 °C, 500 µM
benzophenone, 70-min irradiation with a 125-W blacklight lamp at a 10-
cm irradiation distance, and 2 µM Rose Bengal after 60-min irradiation
with a 150-W sodium lamp at a distance of 20 cm.
(49) At present, no direct assay is available to monitor the formation of
oxazolone specifically and quantitatively accurate enough. The employed
indirect fluorescence-labeling HPLC assay requires alkaline treatment not
only for the release of guanidine from oxazolone but also for its
condensation with NQS. This prevents a clear distinction to be made between
free and released guanidine.

Photooxidation of 7,8-Dihydro-8-oxo-2′-deoxyguanosine
J. Am. Chem. Soc., Vol. 118, No. 39, 1996 9237
Table 3. Effect of D2O on the Oxidation of 8-oxodGuo in the Thermal Treatment with HTMD and in the Photosensitization by
Benzophenone and Rose Bengala
H2O
D2O
time
(min)
conversione of
8-oxodGuo (%)
yielde,f of
4-HO-8-oxodGuo (%)
conversione of
8-oxodGuo (%)
yielde,f of
4-HO-8-oxodGuo (%)
oxidant
HTMD/∆Tb
90
360
780
30
60
6
22
61
73
58
81
7.7 (35)
20 (33)
27 (37)
24 (41)
25 (31)
21
48
55
42
6.4 (31)
15 (31)
17 (31)
16 (38)
HTMD/∆Tb
HTMD/∆Tb
benzophenone/hνc
Rose Bengal/hνd
Rose Bengal/hνd
86
27 (31)
a 100 µM 8-oxodGuo, 10 mM phosphate buffer (pH, pD 7.0). b 5 mM HTMD, aqueous acetonitrile (90:10), 50 °C. c 400 µM benzophenone,
aqueous acetonitrile (98:2), 125-W blacklight, irradiated at a distance of 10 cm, 4°C. d 2 µM Rose Bengal, 150-W sodium lamp, irradiated at a
distance of 20 cm, 4 °C. e Mean value of triplicate determinations, standard deviation (5% of the stated value. f Yield of 4-HO-8-oxodGuo relative
to conversion of 8-oxodGuo given in parantheses.
Scheme 1. Proposed Type I Photooxidation (Electron
Transfer) Mechanism for the Formation of Oxazolone from
8-oxodGuo
Initial electron transfer from 8-oxodGuo in its enolic form
to the excited sensitizer leads after deprotonation to the reactive
C-5 radical species, which in the presence of molecular oxygen
yields the intermediary C-5 hydroperoxide. This mechanism
is analogous to the one reported for the formation of oxazolone
in the type I photooxidation of dGuo.24 That electron transfer
from 8-oxodGuo to the excited triplet state occurs more easily
than from dGuo is corroborated by the favorable oxidation
potential for the 2′,3′,5′-tris[(tert-butyldimethylsilyl)oxy] deriva-
tive of 8-oxodGuo (0.85 V against AgCl) Versus that of dGuo
(1.28 V).17 Consequently, 8-oxodGuo is readily photooxidized
not only by type II sensitizers (singlet oxygen) to yield 4-HO-
8-oxodGuo, but by type I sensitizers (electron transfer, hydrogen
abstraction) as well to afford oxazolone as a secondary oxidation
product of dGuo.
At first glance, the sensitizer-independent product distribution
in the photooxidation of 8-oxodGuo (Table 2) is puzzling in
view of the established predominant reaction modes of the
employed sensitizers. In particular, the characteristic type II
(1O2) photooxidant Rose Bengal also affords a high yield of
the type I product oxazolone. This may be explained by the
fact that singlet oxygen, in addition to the established [2+2] or
[4+2] cycloaddition, is also prone to engage in electron transfer
with electron-rich substrates, especially in polar aqueous
media.50 Because of the low oxidation potential of 8-oxodGuo,
we suggest that singlet oxygen accepts an electron from the
•-
substrate with formation of the superoxide radical anion (O2
)
and the intermediary radical cation of 8-oxodGuo. Although
this electron transfer is by ca. 10 kcal/mol endergonic,51 if the
process is irreversible and initiates a radical chain oxidation of
8-oxodGuo to oxazolone, it is a viable transformation. Indeed,
it was reported that an electron transfer process takes place to
the extent of 15% between singlet oxygen and sodium azide,
although the latter has an even higher oxidation potential (1.35
V against NHE)52 than 8-oxodGuo (0.85 V against AgCl).
Consequently, the singlet oxygen derived from Rose Bengal
photosensitization reacts with 8-oxodGuo through electron
transfer to afford substantial amounts of oxazolone and thereby
mimics type I photoactivity. This unprecedented reaction mode
of 1O2 accounts for the ca. 10-fold faster oxidation but similar
relative yields of 4-HO-8-oxodGuo in the Rose Bengal-
The fair (ca. 70%) balance and sensitizer-independent ratio
of oxidation products and the D2O effect allow us to draw some
pertinent mechanistic conclusions on these HTMD-induced and
photosensitized 8-oxodGuo oxidations. The present observa-
tions that 8-oxodGuo is efficiently degraded by benzophenone
as photosensitizer, which is a characteristic type I photooxidant,
and the negligible D2O effect in the HTMD-induced and
benzophenone-photosensitized oxidations (Table 3) are in
contrast with a solely singlet-oxygen-mediated mechanism45 for
the oxidative degradation of 8-oxodGuo. On the basis of our
results, we conclude that at least in the benzophenone-
photosensitized and the thermal HTMD-induced oxidations of
8-oxodGuo, the type I process (electron transfer, hydrogen
abstraction) plays a decisive role, additional to the established
singlet oxygen photooxidation (type II) of 8-oxodGuo to 4-HO-
8-oxodGuo. We offer the mechanism in Scheme 1 to account
for the present type I photooxidation of 8-oxodGuo to yield
oxazolone.
(50) Saito, I.; Matsuura, T.; Inoue, K. J. Am. Chem. Soc. 1983, 105,
3200-3206.
(51) ∆G ) 23.06[E(D/D+*) - E(A-*/A)] - e2/ꢀR - ∆E0,0; (E(8-
oxodGuo/8-oxodGuo+*) ) 0.85 V Vs AgCl,17 E(O2-*/O2) ) 0.57 V Vs
SCE,53 e2/ꢀR ) 0.53 kcal/mol, ∆E0,0 ) 22.5 kcal/mol, ∆G ) 9.2 kcal/
mol). Rehm, D.; Weller, A. Isr. J. Chem. 1970, 8, 259-271.
(52) Alfassi, Z. B.; Harriman, A.; Huie, R. E.; Mosseri, S.; Neta, P. J.
Phys. Chem. 1987, 91, 2120-2122.
(53) Sawyer, D. T.; Seo, E. T. Inorg. Chem. 1977, 16, 499-501.
(54) Raoul, S.; Cadet, J. J. Am. Chem. Soc. 1996, 118, 1892-1898.

9238 J. Am. Chem. Soc., Vol. 118, No. 39, 1996
Adam et al.
sensitized oxidations of 8-oxodGuo when D2O instead of H2O
is used as the reaction medium.
sitized oxidation of 8-oxodGuo, singlet oxygen is made
responsible, through electron transfer from the readily oxidizable
8-oxodGuo.
Conclusion
Since 8-oxodGuo is extensively used as a characteristic
marker for oxidative DNA damage, caution must be exercised
when the oxidative reactivity of mutagens is assessed by solely
monitoring the formation of 8-oxodGuo. Furthermore, the
proposed type I mediated oxidation of 8-oxodGuo suggests that
a clear distinction between the type I and type II photooxidation
modes requires a detailed product study to ascertain the
predominant mode of photooxidation for a given photosensitizer.
Our results clearly establish that triplet-excited ketones,
generated thermally from the dioxetane HTMD in the dark,
provide a powerful mechanistic tool to study photobiological
problems, as demonstrated here for the photosensitized oxidation
of 8-oxodGuo. On thermal activation, HTMD efficiently
oxidizes 8-oxodGuo and affords up to ca. 90% relative yield
of the singlet oxygen product 4-HO-8-oxodGuo. The estab-
lished photosensitizers benzophenone and Rose Bengal, which
were examined for comparison, both yield ca. 35% 4-HO-8-
oxodGuo. Additionally, guanidine, which presumably is re-
leased from oxazolone through alkaline hydrolysis, is detected
in the HTMD-induced and directly photosensitized oxidations.
We propose that, in addition to the already established singlet-
oxygen-mediated degradation of 8-oxodGuo,40 a type I photo-
oxidation process (electron transfer or hydrogen abstraction from
the substrate to the triplet excited state) operates, which leads
to oxazolone. For the unexpected high yields of oxazolone
(type I photooxidation product) in the Rose-Bengal-photosen-
Acknowledgment. The generous financial support by the
Deutsche Forschungsgemeinschaft (SFB 172, “Molekulare
Mechanismen kanzerogener Prima¨rvera¨nderungen”) and the
Fonds der Chemischen Industrie is gratefully appreciated.
Supporting Information Available: Text describing the
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