2′-deoxyguanosine (DG) oxidation and strand-break formation in DNA by the radicals released in the photolysis of N-tert-butoxy-2-pyridone. Are tert-butoxyl or methyl radicals responsible for the photooxidative damage in aqueous media?

Article abstract of DOI:10.1021/ol016955j

Chemical equation presented The photolysis of pyridone 3b (photo-Fenton reagent) in benzene releases tert-butoxyl radicals, which have been trapped by DMPO and EPR-spectrally identified. In aqueous solution, however, the fragmentation of the tert-butoxyl into methyl radicals prevails and the former radicals are of no direct consequence in the photooxidation of 2′-deoxyguanosine (dG) and pBR 322 DNA. The photooxidative damage of nucleic acids is caused by the oxyl radical species generated from the methyl radicals with oxygen.

Full text of DOI:10.1021/ol016955j

ORGANIC
LETTERS
2002
Vol. 4, No. 2
225-228
2′-Deoxyguanosine (DG) Oxidation and
Strand-Break Formation in DNA by the
Radicals Released in the Photolysis of
N-tert-Butoxy-2-pyridone. Are
tert-Butoxyl or Methyl Radicals
Responsible for the Photooxidative
Damage in Aqueous Media?
,†
Waldemar Adam,* Stefan Marquardt,† Diana Kemmer,‡
Chantu R. Saha-Mo1ller,† and Peter Schreier‡
Institut fu¨r Organische Chemie and Institut fu¨r Lebensmittelchemie,
UniVersita¨t Wu¨rzburg, Am Hubland, D-97074 Wu¨rzburg, Germany
adam@chemie.uni-wuerzburg.de
Received October 24, 2001
ABSTRACT
The photolysis of pyridone 3b (photo-Fenton reagent) in benzene releases tert-butoxyl radicals, which have been trapped by DMPO and
EPR-spectrally identified. In aqueous solution, however, the fragmentation of the tert-butoxyl into methyl radicals prevails and the former
radicals are of no direct consequence in the photooxidation of 2′-deoxyguanosine (dG) and pBR 322 DNA. The photooxidative damage of
nucleic acids is caused by the oxyl radical species generated from the methyl radicals with oxygen.
Alkoxyl radicals, endogenously formed during the autoxi-
dation of polyunsaturated fatty acids, have been suspected
to cause cell damage and, consequently, speculated to be
involved in aging and carcinogenesis.1-3 With the aim of
assessing the reactivity of alkoxyl radicals toward biomol-
ecules such as DNA, photochemical alkoxyl-radical sources
(so-called photo-Fenton reagents) have been developed.4-8
In particular, the pyridinethione 16,7 and the perester 24,5 have
been employed for this purpose. Indeed, we have previously
reported that on photochemical activation of both the
pyridinethione 1 and the perester 2, tert-butoxyl radicals are
(4) Adam, W.; Grimm, G. N.; Saha-Mo¨ller, C. R.; Dall’Acqua, F.; Miolo,
G.; Vedaldi, D. Chem. Res. Toxicol. 1998, 11, 1089-1097.
(5) Mahler, H. C.; Schulz, I.; Adam, W.; Grimm, G. N.; Saha-Mo¨ller,
C. R.; Epe, B. Mutat. Res. 2001, 461, 289-299.
† Institut fu¨r Organische Chemie.
(6) Adam, W.; Grimm, G. N.; Saha-Mo¨ller, C. R. Free Radical Biol.
Med. 1989, 24, 234-238.
‡ Institut fu¨r Lebensmittelchemie.
(1) Sies, H., Ed. OxidatiVe Stress, Oxidants and Antioxidants; Academic
Press: New York, 1991.
(2) Halliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology and
Medicine; Oxford University Press: Oxford, 1989.
(3) Harman, D. J. Gerontol. 1985, 11, 298-300.
(7) Adam, W.; Grimm, G. N.; Marquardt, S.; Saha-Mo¨ller, C. R. J. Am.
Chem. Soc. 1999, 121, 1179-1185.
(8) Adam, W.; Arnold, M. A.; Grimm. G. N.; Saha-Mo¨ller, C. R.;
Dall’Acqua, F.; Miolo, G.; Vedaldi, D. Photochem. Photobiol. 1998, 68,
511-518.
10.1021/ol016955j CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/03/2002

released.4,6 In the case of the perester 2, strand-break
formation and guanine oxidation in DNA and dG have been
attributed to the tert-butoxyl radicals.4 In particular, on short-
time (2-20 min) irradiation of the highly photoactive
pyridinethione 1 the observed strand breaks in pBR 322 DNA
have been assigned to the released alkoxyl radicals.6 In
contrast, the prolonged photolysis of the pyridinethione 1
affords photoproducts, which were shown to act as efficient
photosensitizers for the guanine oxidation in dG and DNA.7
This photosensitized electron-transfer process outweighs the
oxyl-radical-mediated oxidation of nucleic acids and makes
such a photo-Fenton reagent unsuitable for photobiological
investigations.7
Scheme 1
Since photobiological studies with nucleic acids are usually
carried out in aqueous solutions, water was used instead of
Figure 1. EPR spectrum of DMPO spin adducts observed upon
irradiation (300 nm, 10 °C, 5 min) of pyridone 3b in benzene.
It has been established through transient spectroscopy that
pyridone 3a represents an efficient photochemical hydroxyl-
radical source.9 Indeed, we have recently demonstrated that
the pyridone 3a generates photochemically hydroxyl radicals
without any photosensitization of DNA by the photoprod-
ucts.10 Thus, in view of this advantageous photochemical
behavior of the pyridone chromophore, we have examined
the corresponding tert-butoxy derivative 3b to assess whether
the latter would produce tert-butoxyl radicals on photolysis
and, thereby, be suitable for photobiological studies on the
oxidative DNA damage by alkoxyl radicals. Our present
results disclose that the photolysis of the tert-butoxy-
substituted pyridone 3b does release efficiently tert-butoxyl
radicals (the quantum yield of its decomposition φdec was
determined to be 0.17); however, in aqueous media, the latter
cleave predominantly into methyl radicals, which either
directly (no O2) or indirectly (with O2) oxidize 2′-deoxygua-
nosine (dG) and induce strand breaks in DNA.
benzene as solvent for the EPR studies. On photolysis of
the pyridone 3b in water under the exclusion of molecular
oxygen, besides the expected tert-butoxyl radicals, also
methyl radicals were trapped by DMPO (Figure 2). The
The tert-butoxy-substituted pyridone 3b was synthezised
from the hydroxy derivative 3a by addition of tert-butyl
bromide in the presence of Ag2O as catalyst (Scheme 1).
Upon photolysis of a benzene solution of pyridone 3b in
the presence of DMPO, the generated tert-butoxyl radicals
were trapped in form of the DMPO adduct, as confirmed
by EPR spectroscopy.6,11,12 This substantiates that NO bond
cleavage took place to release tert-butoxyl radicals (Figure
1).
Figure 2. EPR spectra observed in the photolysis (300 nm,
20 °C, 10 min) of pyridone 3b (3.00 mM) as a function of DMPO
concentration in aqueous solution under the exclusion of molecular
oxygen.
corresponding coupling constants for both radical adducts
are identical to those reported in the literature.13
It is known that in aqueous solutions tert-butoxyl radicals
rapidly (kâ ) 1.5 × 106 s-1) fragment into methyl radicals
and acetone (Scheme 2).13,14 We observe that when a high
DMPO concentration of 90 mM is employed, mainly tert-
butoxyl radicals are trapped, whereas the methyl-radical
adducts are formed in minor amounts; however, this ratio is
reversed when the DMPO concentration is decreased from
90 mM to 30 mM (Figure 2). Thus, at 30 mM [DMPO]
only traces of the tert- butoxyl radicals are trapped, whereas
the signals of the methyl-radical adduct of DMPO (DMPO-
(9) Aveline, B. M.; Kochevar, I. E.; Redmond, R. W. J. Am. Chem. Soc.
1996, 118, 10124-10133.
(10) Adam, W.; Marquardt, S.; Saha-Mo¨ller, C. R. Photochem. Photobiol.
1999, 70, 287-291.
(11) Janzen, E. G.; Krygsman, P. H.; Lindsay, D. A.; Haire, D. L. J.
Am. Chem. Soc. 1990, 112, 8279-8284.
(12) Janzen, E. G.; Liu, J. I.-P. J. Magn. Reson. 1973, 9, 510-512.
(13) Bors, W.; Michel, C.; Stettmaier, K. J. Chem. Soc., Perkin Trans.
2 1992, 1513-1517.
(14) Erben-Russ, M.; Michel, C.; Bors, W.; Saran, M. J. Phys. Chem.
1987, 91, 2362-2365.
226
Org. Lett., Vol. 4, No. 2, 2002

Scheme 2
Me) dominates in the EPR spectrum. Consequently, upon
photolysis of the pyridone 3b in water, a mixture of tert-
butoxyl and methyl radicals is generated, with their propor-
tion depending on the [DMPO]. At the lower DMPO
concentration, the extent of â cleavage is higher and less
DMPO adduct is formed with the tert-butoxyl radicals.
The photolysis of the pyridone 3b in the presence of
supercoiled pBR 322 DNA in phosphate buffer induced
strand-break formation (35% open-circular DNA), as verified
by gel-electophoretic analysis (Figure 3, lane 1). The
Figure 4. Time dependence of the dG (0.10 mM) conversion upon
its irradiation (300 nm) in the presence of pyridone 3b (0.50 mM)
in phosphate buffer (5 mM, pH 7.0) at 10 °C; the error bars
represent at least two independent runs.
in negligible (<0.1%) amounts (not shown). This is analo-
gous to the oxidative behavior observed for the perester 2,
a photochemical tert-butoxyl-radical source which was
shown to oxidize dG also predominantly to guanidine-
releasing products (GRP).4 Additionally, the time profile
(Figure 4) of the dG oxidation reveals that after complete
decomposition of pyridone 3b (10-15 min) and prolonged
irradiation, no further oxidation of dG takes place. Therefore,
it is concluded that the radicals released in the photolysis of
pyridone 3b are responsible for the observed damage;
sensitization by photoproducts may be ruled out as possible
oxidation process. This favorable result encourages the use
of the pyridone system as suitable photo-Fenton reagent for
biological studies.
Figure 3. Gel-electrophoretic analysis of strand breaks generated
in the photolysis (312 nm, 0 °C, 30 min) of supercoiled pBR 322
DNA (10 mg/L in 0.5 mM KH2PO4 buffer, pH 7.4) in the presence
of pyridone 3b (1.50 mM).
It still needs to be determined whether the observed dG
oxidation and DNA cleavage may be attributed to tert-
butoxyl radicals, methyl radicals, or peroxyl radicals derived
therefrom under aerobic conditions. To confirm the interven-
tion of methyl radicals during the irradiation of the pyridone
3b in the presence of dG, efforts were expended to detect
8-methyl-2′-deoxyguanosine (8-MedG), a known methyl-
radical adduct of dG.16-18 Indeed, in the photolysis of a
mixture of dG and pyridone 3b, under the exclusion of
molecular oxygen, appreciable amounts of 8-MedG (2.5%)
were detected and identified by comparison of the MS, the
UV spectrum, and the retention time (on HPLC column) with
that of the authentic material synthesized according to the
literature procedure.19 Therefore, the fragmentation of the
photolytically generated tert-butoxyl radicals into methyl
radicals occurs in aqueous phosphate buffer, and when dG
is present, but no O2, the 8-MedG adducts are produced.
From the known rate constants for the reaction of tert-butoxyl
radicals with the guanine base14 and for the â fragmentation
of the tert-butoxyl radical into methyl radicals,13 it may be
estimated (Scheme 3) that not more than 0.3% of the
generated tert-butoxyl radicals react with dG (0.10 mM) and,
presence of isopropyl alcohol (8 vol %) as radical scavenger
significantly (3%) suppressed the yield of open-circular DNA
(Figure 3, lane 2). This inhibitory effect establishes that
radicals participate in the photolytic strand-break formation
by pyridone 3b.
To assess whether guanine oxidation is caused by the
photolysis of pyridone 3b, 2′-deoxyguanosine (dG) was
irradiated in the presence of the photochemical radical source
3b. Complete photodecomposition of the latter (Figure 4)
revealed that dG was converted to the extent of about 25%
to yield mainly (15%) the guanidine-releasing products
(GRP)15 such as oxazolone, whereas 8-oxodG was detected
(15) Adam, W.; Kurz, A.; Saha-Mo¨ller, C. R. Chem. Res. Toxicol. 2000,
13, 1199-1207.
(16) Augusto, O. Free Radical Biol. Med. 1993, 15, 329-336.
(17) Kwee, J. K.; Armelin, M. S.; Stefani, H. A.; Augusto, O. Chem.-
Biol. Interact 1998, 116, 61-77.
(18) Hix, S.; Kadiiska, M. B.; Mason, R. P.; Augusto, O. Chem. Res.
Toxicol. 2000, 13, 1056-1064.
(19) Kohda, K.; Tsunomoto, H.; Minoura, Y.; Tanabe, K.; Shibutani, S.
Chem. Res. Toxicol. 1996, 9, 1278-1284.
Org. Lett., Vol. 4, No. 2, 2002
227

Scheme 3a
Scheme 4
On the basis of the present results it is concluded that tert-
butoxyl radicals, despite their higher reactivity compared with
the peroxyl radicals,21,22 do not have a chance to react with
dG and DNA in aqueous media, since the tert-butoxyl
species cleaves too efficiently into methyl radicals. Any
observed damage is ascribed to the latter, either directly by
addition to the guanine base or indirectly through oxidation
by the methylperoxyl radical obtained on O2 trapping of the
methyl radical. Also the methoxyl radicals generated from
the latter by disproportionation may be involved.20 Since
under cellular conditions the concentration of oxygen is
significantly lower than under aerobic aqueous conditions,
the addition of methyl radicals to cellular DNA has been
observed.18 We advocate that future photobiological work
on the oxidative propensity of tert-butoxyl radicals, and for
that matter quite generally for alkoxyl radicals generated
photochemically, thermally or enzymatically, should take into
account the facile â cleavage in aqueous media.
a φdG ) efficiency of the dG oxidation. φâ ) efficiency for the
â cleavage of the tert-butoxyl radical in water.
consequently, more than 99% undergo â cleavage to methyl
radicals. Evidently, the fragmentation pathway (φâ) of the
tert-butoxyl radical into methyl radicals overwhelmingly
dominates over the direct reaction with dG (φdG). Thus, the
tert-butoxyl radicals, initially released in the photolysis of
the pyridone 3b, cannot be responsible for the presently
observed and previously reported4 dG photooxidation. It
must be the methyl radicals that are formed by the tert-
butoxyl-radical fragmentation, either directly by reaction with
the guanine in dG and DNA or indirectly through the radicals
derived from the methyl radical. Since methyl radicals are
scavenged by molecular oxygen at diffusion-controlled rates
to form methylperoxyl radicals, the latter or radicals derived
from them (e.g., the methoxyl radical)20 are the most likely
damaging species (Scheme 4).
Acknowledgment. The generous funding by the Deutsche
Forschungsgemeinschaft (SFB172 “Molekulare Mechanis-
men kanzerogener Prima¨rvera¨nderungen”) and the Fond der
Chemischen Industrie is gratefully appreciated.
Supporting Information Available: Experimental Sec-
tion with the details of the synthesis and the photolysis
studies of the pyridone 3b. This material is available free of
charge via the Internet at http://pubs.acs.org.
(20) Schuchmann, H.-P.; von Sonntag, C. Z. Naturforsch. 1984, 39b,
217-221.
(21) Korcek, S.; Chenier, J. H. B.; Howard, J. A.; Ingold, K. U. Can. J.
Chem. 1972, 50, 2285-2297.
(22) Malatesta, V.; Scaiano, J. C. J. Org. Chem. 1982, 47, 1455-1459.
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