Type I and type II photosensitized oxidative modification of 2'-deoxyguanosine (dGuo) by triplet-exicted ketones generated thermally from the 1,2-dioxetane HTMD

Article abstract of DOI:10.1021/ja9629827

The nucleoside 2'-deoxyguanosine (dGuo) was treated with 3-(hydroxymethyl)-3,4,4-trimethyl-1,2-dioxetane (HTMD), the latter generates efficiently triplet-excited carbonyl products on thermal decomposition in the dark. The type I photooxidation products, 2,2-diamino-[(2-deoxy-β-D-erythro-pentofuranosyl)-4-amino]-5(2H)-oxaz olone (oxazolone) and the cyclic nucleoside 2-(S)-2,5'-anhydro-1-(2-deoxy-β-D-erythro-pentofuranosyl)-5-guanidiny lidene-2-hydroxy-4-oxoimidazolidine (oxoimidazolidine), as well as the type II photooxidation products 4-(R)*- and 4-(S)*-4-hydroxy-8-oxo-4,8-dihydro-2'-deoxyguanosine (4-HO-8-oxodGuo) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), were quantitatively determined by appropriate selective and sensitive HPLC assays. The concentration and time profiles revealed that about 40% of the triplet ketones derived from the thermal decomposition of HTMD led to photooxidation of dGuo. Essentially equal amounts of type I and type II photooxidation products were found, as could be established by comparison with predominant type I (benzophenone, riboflavin) and type II (Rose Bengal, methylene blue) photosensitizers. The participation of singlet oxygen (type II activity) was confirmed by the substantial D₂O effect in the formation of 8-oxodGuo. The results demonstrate that dioxetanes, particularly HTMD, are efficient photooxidants of dGuo on thermal activation in the dark and constitute excellent chemical tools to study photobiological processes without the use of light, in the present case, photogenotoxicity.

Full text of DOI:10.1021/ja9629827

J. Am. Chem. Soc. 1997, 119, 719-723
719
Type I and Type II Photosensitized Oxidative Modification of
2′-Deoxyguanosine (dGuo) by Triplet-Excited Ketones
Generated Thermally from the 1,2-Dioxetane HTMD
Waldemar Adam, Chantu R. Saha-Mo1ller, and Andre´ Scho1nberger*
Contribution from the Institute of Organic Chemistry, UniVersity of Wu¨rzburg, Am Hubland,
D-97074 Wu¨rzburg, Germany
ReceiVed August 26, 1996X
Abstract: The nucleoside 2′-deoxyguanosine (dGuo) was treated with 3-(hydroxymethyl)-3,4,4-trimethyl-1,2-dioxetane
(HTMD), the latter generates efficiently triplet-excited carbonyl products on thermal decomposition in the dark.
The type I photooxidation products, 2,2-diamino-[(2-deoxy-â-D-erythro-pentofuranosyl)-4-amino]-5(2H)-oxazolone
(oxazolone) and the cyclic nucleoside 2-(S)-2,5′-anhydro-1-(2-deoxy-â-D-erythro-pentofuranosyl)-5-guanidinylidene-
2-hydroxy-4-oxoimidazolidine (oxoimidazolidine), as well as the type II photooxidation products 4-(R)*- and 4-(S)*-
4-hydroxy-8-oxo-4,8-dihydro-2′-deoxyguanosine (4-HO-8-oxodGuo) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-
oxodGuo), were quantitatively determined by appropriate selective and sensitive HPLC assays. The concentration
and time profiles revealed that about 40% of the triplet ketones derived from the thermal decomposition of HTMD
led to photooxidation of dGuo. Essentially equal amounts of type I and type II photooxidation products were found,
as could be established by comparison with predominant type I (benzophenone, riboflavin) and type II (Rose Bengal,
methylene blue) photosensitizers. The participation of singlet oxygen (type II activity) was confirmed by the substantial
D2O effect in the formation of 8-oxodGuo. The results demonstrate that dioxetanes, particularly HTMD, are efficient
photooxidants of dGuo on thermal actiVation in the dark and constitute excellent chemical tools to study
photobiological processes without the use of light, in the present case, photogenotoxicity.
Introduction
8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo)20-23 and the
2,2-diamino-[(2-deoxy-â-D-erythro-pentofuranosyl)-4-amino]-
5(2H)-oxazolone (oxazolone), which results from hydrolysis of
the corresponding imidazolone precursor,24 constitute the major
photooxidation products of DNA. While 8-oxodGuo can be
formed either directly through type I (hydrogen atom abstraction
or electron transfer)24c or indirectly through type II (singlet
oxygen, energy transfer)25,26 photooxidation processes, the
oxazolone is derived from a type I photooxidation.5,27,28
Recently, it was reported29 that oxazolone is also produced by
singlet oxygen oxidation of 8-oxodGuo, which is a primary
photooxidation product of dGuo.
Due to the importance of the oxidative degradation of nucleic
acids in mutagenesis, carcinogenesis, and aging,1-4 numerous
chemical and biological investigations have been made on this
subject in the past decade.5-7 These intensive studies have
contributed significantly in understanding the reaction mecha-
nism of nucleic acid oxidations mediated by photosensitizers,8-11
hydroxyl radicals,12,13 singlet oxygen,14,15 and other reactive
oxygen species which are involved in oxidative stress.16,17
The purine base guanine is the predominant target in the
photosensitized oxidation of DNA.18,19 The highly mutagenic
X Abstract published in AdVance ACS Abstracts, January 1, 1997.
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D., Bartsch, H., Eds.; IARC Publications: Lyon, France, 1994; Vol. 125,
pp 245-276.
The photosensitized oxidation of the monomeric nucleoside
2′-deoxyguanosine (dGuo) affords (Scheme 1), in addition to
8-oxodGuo and the oxazolone, the two 4R* and 4S* diastere-
omers of 4-hydroxy-8-oxo-4,8-dihydro-2′-deoxyguanosine
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S0002-7863(96)02982-4 CCC: $14.00 © 1997 American Chemical Society

720 J. Am. Chem. Soc., Vol. 119, No. 4, 1997
Adam et al.
Scheme 1. Photochemical Mechanisms of the Oxidation of 2′-Deoxyguanosine (dGuo) in the Thermal Decomposition of the
1,2-Dioxetane HTMD
(4-HO-8-oxodGuo) as characteristic singlet oxygen products
either by [4 + 2] cycloaddition with dGuo28,30 or by [2 + 2]
cycloaddition with 8-oxodGuo.30 Furthermore, 2-(S)-2,5′-
anhydro-1-(2-deoxy-â-D-erythro-pentofuranosyl)-5-guanidi-
nylidene-2-hydroxy-4-oxoimidazolidine (oxoimidazolidine) was
characterized as a type I photooxidation product of dGuo,31
which results from intramolecular attack of the 5′-hydroxyl
functionality on the C-8 position of the intermediary guanine
radical. In contrast to the photooxidation of guanine in DNA,
the formation of 8-oxodGuo through type I photooxidation is
essentially negligible for the nucleoside dGuo as substrate.
Presumably, in this case the fast deprotonation of the intermedi-
ary, highly acidic guanosine radical cation32 prevents the
addition of water to afford 8-oxodGuo. Therefore, significant
amounts (>0.1%) of 8-oxodGuo are exclusively produced by
the type II process in the photosensitized oxidation of dGuo.5
An important class of photooxidative sensitizers constitutes
the triplet-excited ketones,16,33 which are of biological interest
since they may be generated in cellular systems upon exposure
of endogeneous chromophores to UV irradiation or by dark
reactions [e.g., lipid peroxidation (Russell mechanism) and
enzymatic oxidation].34-36 Alternative to their conventional
photochemical generation, triplet-excited ketones may be con-
veniently produced by the thermal decomposition of 1,2-
dioxetanes,37,38 which are high-energy, four-membered ring
cyclic peroxides. The thermally generated triplet-excited spe-
cies, analogous to those produced photochemically, also operate
as type I or type II photooxidants. The advantageous feature
that their thermal generation circumvents the exposure of
biological systems, particularly cells, directly to UV radiation.
This attractive opportunity of employing 1,2-dioxetanes as
chemical sources for electronically excited ketones paved the
way for extensive studies on the new bioorganic topic “photo-
biology without light” to explore the biological function, in
particular genotoxicity, of such peroxides.39-41 These studies
revealed that, on thermal decomposition, 1,2-dioxetanes ef-
ficiently damage DNA in cell-free and cellular systems.39 The
dioxetane-mediated DNA lesions are mostly sensitive to form-
amidopyrimidine DNA-glycosylase (FPG protein),40 which
strongly indicates that 1,2-dioxetanes preferentially generate
guanine lesions (e.g., 8-oxodGuo) besides AP sites and form-
amidopyrimidine (Fapy) residues.42,43
To evaluate the chemical nature of these dioxetane-induced
DNA modifications, we have recently investigated the oxidation
of calf thymus DNA by 1,2-dioxetanes in the dark.44 It has
been established that 1,2-dioxetanes, in particular those that are
alkyl-substituted, oxidize efficiently and almost exclusively the
guanine bases in DNA to form 8-oxodGuo in high yields.
Triplet-excited states generated by thermal decomposition of
dioxetanes were found to be responsible for the observed
guanine oxidation in DNA.
The mechanistic origin of guanosine photooxidation products
is less clear-cut. For example, although in DNA 8-oxodGuo
can be formed through type I and type II photooxidations, this
particular photooxidation product of the dGuo nucleoside is
derived exclusively through a type II process (1O2). Therefore,
the relative contribution of type I versus type II photooxidation
of dGuo is a more definitive monitor of the photooxidation
mechanism. Consequently, in order to define the predominant
chemical oxidation mode of the triplet-excited species generated
thermally from 3-(hydroxymethyl)-3,4,4-trimethyl-1,2-dioxetane
(HTMD), we have investigated its reaction with the nucleoside
dGuo. The results are compared with those of the photosen-
(30) (a) Sheu, C.; Foote, C. S. J. Am. Chem. Soc. 1993, 115, 10446-
10447. (b) Sheu, C.; Foote, C. S. J. Am. Chem. Soc. 1995, 117, 474-477.
(31) Buchko, G. W.; Cadet, J.; Ravanat, J. L.; Labataille, P. Int. J. Radiat.
Biol. 1993, 63, 669-676.
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F.; Epe, B.; Mu¨ller, E.; Schiffmann, D.; Wild, D. EnViron. Health Perspect.
1990, 88, 89-97.
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Interact. 1992, 85, 265-281.
(32) Steenken, S. Free Radical Res. Commun. 1992, 16, 349-379.
(33) Epe, B.; Henzl, H.; Adam, W.; Saha-Mo¨ller, C. R. Nucleic Acids
Res. 1993, 21, 863-869.
(41) 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.
(34) Cilento, G. Pure Appl. Chem. 1984, 56, 1179-1190.
(35) Cadenas, E. Photochem. Photobiol. 1984, 40, 823-830.
(36) Baader, W. J.; Bohne, C.; Cilento, G.; Dunford, H. B. J. Biol. Chem.
1985, 260, 10217-10225.
(37) Adam, W.; Cilento, G. Angew. Chem., Int. Ed. Engl. 1983, 22, 529-
542.
(42) Tchou, J.; Bodepudi, V.; Shibutani, S.; Anthoshechkin, J.; Miller,
J.; Grollman, A. P.; Johnson, F. J. Biol. Chem. 1994, 269, 15318-15324.
(43) Boiteux, S.; Gajewski, E.; Laval, J.; Dizdaroglou, M. Biochemistry
1992, 31, 106-110.
(44) Adam, W.; Saha-Mo¨ller, C. R.; Scho¨nberger, A.; Berger, M.; Cadet,
J. Photochem. Photobiol. 1995, 62, 231-238.
(38) Cilento, G.; Adam, W. Photochem. Photobiol. 1988, 48, 361-368.

OxidatiVe Modification of 2′-Deoxyguanosine
J. Am. Chem. Soc., Vol. 119, No. 4, 1997 721
Figure 1. Concentration profile for the thermally HTMD-induced
photooxidation of dGuo. In the dark with 0.5 mM dGuo at 50 °C for
15 h in 10 mM sodium cacodylate buffer (pH 7.0) and acetonitrile as
cosolvent (10 vol %), 70% consumption of dioxetane (chemilumines-
cence measurements). Absolute yield derived from the mean values
of three independent runs, error (10% of the stated value, conversion
Figure 2. Time profile for the thermally HTMD-induced photooxi-
dation of dGuo. In the dark at 50 °C with 0.5 mM dGuo in 10 mM
sodium cacodylate buffer (pH 7.0) and 25 mM HTMD dissolved in
acetonitrile (10 vol %). Absolute yield derived from the mean values
of at least three independent runs, error (10% of the stated value,
conversion dGuo (crosses), yields of type II products 8-oxodGuo
100 (filled circles) and 4-HO-8-oxodGuo (filled squares) and type I
products oxazolone and oxoimidazolidine (open triangles).
dGuo (crosses), yields of type II products 8-oxodGuo
100 (filled
circles) and 4-HO-8-oxodGuo (filled squares) and type I products
oxazolone and oxoimidazolidine (open triangles).
characteristic type II photooxidation product, 4-HO-8-oxodGuo,
was formed in up to 20% absolute yield at 25 mM HTMD,
while the type I photooxidation products oxazolone and oxo-
imidazolidine amounted to ca. 18% at the same HTMD
concentration. In these reactions, the product balance (i.e., the
sum of quantified products relative to consumed dGuo) amounted
to ca. 55% and was essentially independent of the HTMD
concentration. The time profile (Figure 2), determined with 25
mM HTMD, revealed a gradual increase of all dGuo oxidation
product yields with reaction time. Also in this case, the product
balance amounted to 55 ( 3% and was independent of reaction
time.
sitized oxidation of dGuo by characteristic type I45 (benzophe-
none and riboflavin) and predominant type II (Rose Bengal and
methylene blue) photosensitizers.46,47
Results
On thermal treatment of dGuo with HTMD at 50 °C, five
major oxidation products of the guanine base were detected by
means of appropriate HPLC assays. These products are the type
II photooxidation products 8-oxodGuo and the two 4R* and
4S* diastereomers of 4-HO-8-oxodGuo, in addition to the type
I photooxidation products oxazolone (also produced by singlet
oxygen oxidation of 8-oxodGuo,29 the primary type II photo-
oxidation product of dGuo) and oxoimidazolidine.5 The latter
two products were assessed together by the indirect fluorescence-
labeling HPLC assay of the alkaline-released guanidine, intro-
duced by Ravanat et al.;28 whereas, 8-oxodGuo and 4-HO-8-
oxodGuo were directly detected, either by electrochemical48,49
or by spectrophotometric HPLC analyses.50 Furthermore, the
amount of decomposed HTMD was monitored by chemilumi-
nescence measurements (t1/2 of 6 ( 1 h at 50 °C).
The concentration and time profiles (Figures 1 and 2) for
the HTMD-induced oxidation of dGuo were determined by using
sodium cacodylate buffer as the reaction medium with 10
vol % acetonitrile as cosolvent. As shown in Figure 1, when
dGuo was thermally treated with HTMD at 50 °C for 15 h, a
linearly dependent degradation of dGuo was observed with
increasing HTMD concentration (about 70% dGuo conversion
at 25 mM, 50 equiv of HTMD). The absolute yield (based on
initial amount of dGuo) of 8-oxodGuo increased continuously
up to ca. 0.4% (in Figures 1 and 2 yields of 100[8-oxodGuo]
are given), but at HTMD concentrations above 2 mM, it dropped
significantly to ca. 0.1% 8-oxodGuo at 25 mM HTMD (i.e.,
the 8-oxodGuo yield went through a maximum). The other
To define the predominant photooxidation mechanism (type
I versus type II) for the oxidation of dGuo by electronically
excited species generated thermally from HTMD, the results
of the HTMD reaction were compared with those of the dGuo
photooxidation by the predominant type I (benzophenone and
riboflavin) and type II (Rose Bengal and methylene blue)
photosensitizers.46,47 For this purpose, the product distributions
of dGuo photooxidation induced by these sensitizers were
investigated over a wide range of sensitizer concentrations and
irradiation times at 20-80% conversion of dGuo (Table 1).
In the type II sensitized photooxidation of dGuo by Rose
Bengal and methylene blue, as well as in the HTMD-induced
oxidation (cf. entries 1, 4, and 5, Table 1), the minor product
8-oxodGuo (>1%) was detected in significant yields (relative
to dGuo conversion), whereas the type I photosensitizers
riboflavin and benzophenone (cf. entries 2 and 3) gave only
negligible amounts of 8-oxodGuo (less than 0.1%). Since the
relative yields (based on converted dGuo) of the major type II
(4-HO-8-oxodGuo) and type I (oxazolone and oxoimidazolidine)
photooxidation products remained within the experimental error
independent on sensitizer concentration and reaction time for
all employed photosensitizers, the type II/type I product ratio
may serve as a mechanistic probe to assess the predominant
photooxidation mode. For example, for predominant type I
photooxidation, this product ratio lies below 1 (entries 2 and 3,
Table 1) while for predominant type II photooxidation it is aboVe
4 (entries 4 and 5). For HTMD the type II/type I product ratio
is 1.3 ( 0.2 (entry 1) and, thus, entails both photooxidation
modes. Also for the product balance, the HTMD oxidation (56
( 2%, entry 1) lies between the type I (44-47%, entries 2 and
3) and type II (73-78%, entries 4 and 5) processes. Further-
(45) For definition of type I and type II photosensitized oxidation, see:
Foote, C. S. Photochem. Photobiol. 1991, 54, 659.
(46) Cadet, J.; Decarroz, C.; Wang, S. Y.; Midden, W. R. Isr. J. Chem.
1983, 23, 420-429.
(47) Buchko, G. W.; Cadet, J. Can. J. Chem. 1992, 70, 1827-1832.
(48) Floyd, R. A.; Watson, J. J.; Wong, P. K.; Altmiller, D. H.; Rickard,
R. C. Free Radical Res. Comms. 1986, 1, 163-172.
(49) Floyd, R. A.; West, M. S.; Eneff, K. L.; Schneider, J. E.; Wong, D.
T.; Tingey, D. T.; Hogsett, W. E. Anal. Biochem. 1990, 188, 155-158.
(50) Ravanat, J.-L.; Douki, T.; Incardona, F.; Cadet, J. J. Liq. Chro-
matogr. 1993, 16, 3185-3202.

722 J. Am. Chem. Soc., Vol. 119, No. 4, 1997
Adam et al.
Table 1. Product Studies of the dGuo Oxidation by the Dioxetane HTMD and by Predominantly Type I (Benzophenone, Riboflavin) and
Type II (Rose Bengal, Methylene Blue) Photosensitizersa
product yields (%)b
type II
4-HO-8-oxodGuo
type I
productsc
entry
oxidant
concn (µM)
8-oxodGuo
product balance
type II/type I
1
2
3
4
5
HTMD/50 °Cd
10000
500-2500
1-10
<1.2
<0.1
<0.1
<2.1
<3.0
31 ( 2
17 ( 3
15 ( 2
65 ( 2
58 ( 3
24 ( 2
27 ( 4
32 ( 2
13 ( 1
15 ( 2
56 ( 2
44 ( 5
47 ( 1
78 ( 2
73 ( 3
1.3 ( 0.2
0.7 ( 0.2
0.5 ( 0.1
5.0 ( 0.9
4.0 ( 0.9
benzophenone/350 nme
riboflavin/564 nmf
Rose Bengal/564 nmf
methylene blue/564 nmf
2-20
2-20
a dGuo (0.5 mM) in 10 mM sodium cacodylate buffer (pH 7.0). b Relative yields based on consumed dGuo, mean values of at least 10 independent
runs at 20-80% conversions of dGuo. c Oxazolone and oxoimidazolidine. d In the dark, 15 h, acetonitrile (10 vol %) as cosolvent. e Blacklight
lamp (125 W), irradiation distance (10 cm), 30-180 min, 4 °C, acetonitrile (1 vol %) as cosolvent. f Sodium lamp (150 W), irradiation distance (20
cm), 30-180 min, 4 °C.
significantly decomposed at this temperature during 15 h)
showed that HTMD does not oxidize dGuo directly. Ap-
proximately 60% of the consumed dGuo is accounted for in
terms of these guanine oxidation products. The remaining
products were not pursued, since some of them most likely result
from multiple oxidative degradation. Indeed, 1-(2-deoxy-â-D-
erythro-pentofuranosyl)cyanuric acid and its precursor 3-(2-
deoxy-â-D-erythro-pentofuranosyl)tetrahydro-2,4,6-trioxo-1,3,5-
triazine-1(2H)-carboximidamide have been recently identified29
as major singlet oxygen products in the subsequent photooxi-
dation of 8-oxodGuo, the primary oxidation product of dGuo.
Generally, photosensitizers with ketone chromophores operate
through low-lying triplet n,π* states and are highly efficient
for electron transfer with substrate or hydrogen abstraction from
the substrate.53 Consequently, they react preferentially through
Figure 3. Effect of D2O on the yield of 8-oxodGuo in the thermally
HTMD-induced photooxidation of dGuo. In the dark at 50 °C for 12
the type I photooxidation mechanism. However, the dioxetane
h with 0.5 mM dGuo in 10 mM sodium cacodylate buffer [pH(D) 7.0]
HTMD as an efficient thermal source of triplet-excited ketones,
and acetonitrile as cosolvent (10 vol %). Absolute yield derived from
yields large amounts of the characteristic type II products
the mean values of at least three independent runs, error (10% of the
8-oxodGuo and 4-HO-8-oxodGuo, indicative of singlet oxy-
stated value.
gen.5,54 It was, therefore, important to compare the product
composition of the dGuo photooxidation by predominant type
more, the relative yield of the type II product 4-HO-8-oxodGuo
I (benzophenone and riboflavin) and type II (methylene blue,
in the HTMD-induced oxidation (ca. 31%) is significantly higher
Rose Bengal) photosensitizers8,46 with those observed in the
(ca. 16%) than that with type I photooxidants and smaller than
thermally-promoted dGuo oxidation by the dioxetane HTMD.
that in the type II case (ca. 60%). In contrast, the yields of the
In this comparison it should be kept in mind that the photo-
type I products in the HTMD-induced oxidation (ca. 24%) are
sensitized oxidations operate catalytically [i.e., the photosensi-
comparable with those of type I sensitizers (ca. 30%) and
tizers are not consumed (except for photobleaching) and are
significantly higher than those in the type II photooxidation (ca.
recycled to afford a steady-state concentration of the triplet-
14%).
excited species]. In contrast, the thermal dioxetane oxidations
To confirm the participation of singlet oxygen, the HTMD-
are stoichiometric in type since on fragmentation of the
mediated oxidation of dGuo was examined in deuterium oxide,
dioxetane, concentration- and time-dependent quantities of
which is known to prolong the lifetime of singlet oxygen about
10-fold.51,52 The results for the HTMD-induced formation of
8-oxodGuo in H2O and D2O are shown in Figure 3. At HTMD
concentrations up to 5 mM (10 equiv), the yield of 8-oxodGuo
is almost 3-fold enhanced in D2O (1.04%) compared to the yield
of the reaction in H2O (0.38%). However, at higher HTMD
concentration (10 mM), the yield of 8-oxodGuo in D2O
decreases slightly to 0.85%, while in H2O it increases insig-
nificantly to 0.45%.
triplet-excited ketones are produced irreversibly. Moreover,
another significant difference is the necessity that, in the thermal
cleavage of the dioxetane to triplet-excited ketone products,
eleVated temperatures must be employed.
The type II/type I product ratio and product balance were
shown to differ characteristically with the photosensitizer
employed (Table 1) and may, therefore, serve as probe for the
predominant photooxidation mode. The type II/type I product
ratio (Table 1, entries 2 and 3) for the characteristic type I
photosensitizers benzophenone and riboflavin (poor singlet
oxygen generators) is quite high (0.7 and 0.5). However, the
product balance (i.e., the sum of the quantified products relative
to consumed dGuo) in the benzophenone- and riboflavin-
sensitized photooxidation of dGuo (Table 1, entries 2 and 3) is
only ca. 45%. Presumably, a significant fraction of the
unidentified photooxidation products may derive from the type
Discussion
Our present investigation reveals that triplet-excited ketones
generated in the thermal decomposition of the dioxetane HTMD
oxidize dGuo efficiently to the established guanine photooxi-
dation products 8-oxodGuo, 4-HO-8-oxodGuo, oxazolone, and
oxoimidazolidine.5 Control experiments at 0 °C (HTMD is not
(51) Rodgers, M. A. J.; Snowden, P. T. J. Am. Chem. Soc. 1982, 104,
5541-5543.
(52) Kearns, D. R. In Singlet Oxygen; Wasserman, H. H., Murray, R.
W., Eds.; Academic Press: New York, San Francisco, London, 1979; pp
115-137.
(53) Rosenthal, I. In Singlet Oxygen; Frimer, A. A., Ed.; CRC Press:
Boca Raton, FL, 1985; Vol 1, Chapter 2, pp 13-38.
(54) Buchko, G. W.; Cadet, J.; Berger, M.; Ravanat, J.-L. Nucleic Acids
Res. 1992, 20, 4847-4851.

OxidatiVe Modification of 2′-Deoxyguanosine
J. Am. Chem. Soc., Vol. 119, No. 4, 1997 723
I process, and since these unidentified products are not ac-
counted for, a high type II/type I product ratio is observed. The
comparison of the thermally HTMD-induced oxidation of dGuo
with that of authentic type I and type II photosensitized
processes establishes, however, that HTMD is neither a typical
type I (riboflavin, benzophenone) nor a characteristic type II
(Rose Bengal, methylene blue) photooxidant; in particular, both
photooxidation modes occur quite efficiently.
Proof of the involvement of singlet oxygen in the HTMD-
induced oxidation of dGuo comes from the substantial effect
of D2O on the formation of the primary type II oxidation product
8-oxodGuo. Unfortunately, singlet oxygen quenchers such as
DABCO, â-carotene, and sodium azide could not be employed
to corroborate the involvement of singlet oxygen in the HTMD
photooxidation because they react with the dioxetane.55-57
However, the up to three times higher yields of 8-oxodGuo in
D2O compared to those in H2O implicate that singlet oxygen is
involved in the dioxetane-mediated photooxidation of dGuo.
Singlet oxygen persists 10-fold longer in D2O,51,52 and photo-
oxidation is more effective in this medium. Moreover, the
observed decrease of the 8-oxodGuo yield in D2O at dioxetane
concentrations higher than 2.5 mM may be rationalized by the
high reactivity of 8-oxodGuo toward further oxidation by singlet
oxygen.58,59
The linear increase of dGuo conversion (Figure 1) with
increasing HTMD concentration (up to 25 mM) suggests that
the HTMD oxidation of dGuo is directly proportional to the
amount of triplet-excited ketones formed by the thermal
decomposition of the dioxetane. At a triplet yield of ca. 5%
for HTMD in aqueous solution40 and a half-life of t1/2 ) 6 h at
50 °C (measured by chemiluminescence decay), it may be
estimated that ca. 40% of the thermally produced triplet-excited
ketones cause the oxidation of dGuo, either through direct (type
I) or indirect (type II) photoreaction. Indeed, this efficiency is
remarkable in view of the numerous competitive physical
quenching processes that deactivate triplet-excited ketones. In
addition to this physical deactivation of the triplet-excited
ketones, it should be noted that successive oxidation reactions
(e.g., the oxidation of 8-oxodGuo to 4-HO-8-oxodGuo)59 also
require triplet states without consuming dGuo. For example,
for singlet oxygen, generated by quenching of triplet-excited
species through energy transfer, the reactivity toward 8-oxodGuo
was estimated to be more than 100 times faster than with
dGuo.58 Thus, high amounts of singlet oxygen will be
consumed by its reaction with 8-oxodGuo without further
consumption of dGuo. In fact, a maximum in the yield of
8-oxodGuo is expected at low HTMD concentration because
with a larger excess of dioxetane the 8-oxodGuo is consumed
by further oxidation, as is confirmed experimentally (Figure 1).
In contrast, the relative yields of the type II and type I
photooxidation products of guanine remain approximately
constant with reaction time. The time profile of the thermally
HTMD-induced photooxidation of dGuo exhibited a nearly
exponential decrease of the dGuo concentration with reaction
time (Figure 2). This reflects that the dGuo substrate is
consumed in parallel with the generation of triplet-excited
ketones, which are formed thermally from the HTMD by first-
order kinetics. As in the concentration profile, the time profile
revealed that about 40% of the thermally generated triplet-
excited ketones react with dGuo.
Conclusion
Our results on the oxidation of dGuo by the thermal treatment
with HTMD reveal that through the action of triplet-excited
ketones derived from the dioxetane, photooxidation products
of the guanine base are formed in relatively high yields.
Comparison with well-established type I and type II photosen-
sitizers clearly points out that HTMD acts through both
photooxidation types on thermal activation. This was convinc-
ingly demonstrated by the type II/type I product ratio, which
serves as a mechanistic probe to assess the predominant
photooxidation mode. The pronounced D2O effect (a 3-fold
increase in 8-oxodGuo product) confirms the participation of
singlet oxygen. The concentration and time profiles for the
HTMD-mediated photooxidation of dGuo establish the direct
proportionality between product yield and availability of triplet
ketones. The approximate 40% quantum yield of triplet-ketone-
promoted dGuo oxidation demonstrates the high efficiency of
the dioxetane HTMD.
We emphasize that dioxetanes, particularly HTMD, constitute
unique chemical sources for the efficient photooxidation of
biological substrates through the action of their thermally
generated triplet-excited ketones. In aqueous media, HTMD
serves as convenient chemical photooxidant for photobiological
studies in the dark, which circumvents the disadvantages of
direct exposure of the biological material, in particular isolated
or cellular DNA, to ultraviolet radiation.
Acknowledgment. The generous financial support by the
Deutsche Forschungsgemeinschaft (SFB 172, Molekulare Mech-
anismen kanzerogener Prima¨rvera¨nderungen) and the Fonds der
Chemischen Industrie is gratefully appreciated.
(55) Adam, W.; Heil, M. J. Am. Chem. Soc. 1992, 114, 5591-5598.
(56) Richardson, W. H.; Hodge, V. F. Tetrahedron Lett. 1971, 10, 749-
751.
(57) Sundquist, A. R.; Hanusch, M.; Stahl, W.; Sies, H. Photochem.
Photobiol. 1993, 57, 785-791.
(58) Sheu, C.; Foote, C. S. J. Am. Chem. Soc. 1995, 117, 6439-6442.
(59) Adam, W.; Saha-Mo¨ller, C. R.; Scho¨nberger, A. J. Am. Chem. Soc.
1996, 118, 9233-9238.
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