Photoinduced reductive repair of thymine glycol: Implications for excess electron transfer through DNA containing modified bases

Article abstract of DOI:10.1021/ja061304+

Photoinduced reduction of thymine glycol in oligodeoxynucleotides was investigated using either a reduced form of flavin adenine dinucleotide (FADH^-) as an intermolecular electron donor or covalently linked phenothiazine (PTZ) as an intramolecular electron donor. Intermolecular electron donation from photoexcited flavin (*FADH^-) to free thymidine glycol generated thymidine in high yield, along with a small amount of 6-hydroxy-5,6-dihydrothymidine. In the case of photoreduction of 4-mer long single-stranded oligodeoxynucleotides containing thymine glycol by *FADH^-, the restoration yield of thymine was varied depending on the sequence of oligodeoxynucleotides. Time-resolved spectroscopic study on the photo-reduction by laser-excited N,N-dimethylaniline (DMA) suggested elimination of a hydroxyl ion from the radical anion of thymidine glycol with a rate constant of ～10⁴ s^-1 generates 6-hydroxy-5,6-dihydrothymidine (6-HOT^?) as a key intermediate, followed by further reduction of 6-HOT^? to thymidine or 6-hydroxy-5,6-dihydrothymdine (6-HOT). On the other hand, an excess electron injected into double-stranded DNA containing thymine glycol was not trapped at the lesion but was further transported along the duplex. Considering redox properties of the nucleobases and PTZ, competitive excess electron trapping at pyrimidine bases (thymine, T and cytosine, C) which leads to protonation of the radical anion (T^-?, C^-?) or rapid back electron transfer to the radical cation of PTZ (PTZ^-?), is presumably faster than elimination of the hydroxyl ion from the radical anion of thymine glycol in DNA.

Full text of DOI:10.1021/ja061304+

Published on Web 07/29/2006
Photoinduced Reductive Repair of Thymine Glycol:
Implications for Excess Electron Transfer through DNA
Containing Modified Bases
Takeo Ito,* Akiko Kondo, Satoru Terada, and Sei-ichi Nishimoto*
Contribution from the Department of Energy and Hydrocarbon Chemistry, Graduate School of
Engineering, Kyoto UniVersity, Kyoto Daigaku Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
Received February 23, 2006; E-mail: takeoit@scl.kyoto-u.ac.jp; nishimot@scl.kyoto-u.ac.jp
Abstract: Photoinduced reduction of thymine glycol in oligodeoxynucleotides was investigated using either
a reduced form of flavin adenine dinucleotide (FADH-) as an intermolecular electron donor or covalently
linked phenothiazine (PTZ) as an intramolecular electron donor. Intermolecular electron donation from
photoexcited flavin (*FADH-) to free thymidine glycol generated thymidine in high yield, along with a small
amount of 6-hydroxy-5,6-dihydrothymidine. In the case of photoreduction of 4-mer long single-stranded
oligodeoxynucleotides containing thymine glycol by *FADH-, the restoration yield of thymine was varied
depending on the sequence of oligodeoxynucleotides. Time-resolved spectroscopic study on the photo-
reduction by laser-excited N,N-dimethylaniline (DMA) suggested elimination of a hydroxyl ion from the
radical anion of thymidine glycol with a rate constant of ∼104 s-1 generates 6-hydroxy-5,6-dihydrothymidine
(6-HOT•) as a key intermediate, followed by further reduction of 6-HOT• to thymidine or 6-hydroxy-5,6-
dihydrothymdine (6-HOT). On the other hand, an excess electron injected into double-stranded DNA
containing thymine glycol was not trapped at the lesion but was further transported along the duplex.
Considering redox properties of the nucleobases and PTZ, competitive excess electron trapping at pyrimidine
bases (thymine, T and cytosine, C) which leads to protonation of the radical anion (T-•, C-•) or rapid back
electron transfer to the radical cation of PTZ (PTZ+•), is presumably faster than elimination of the hydroxyl
ion from the radical anion of thymine glycol in DNA.
Introduction
between radiation quanta and DNA molecules gives rise to
electron-loss centers and the counterpart electrons at the early
A wide variety of genotoxic agents, such as ionizing radiation,
ultraviolet radiation, and chemical oxidants, induce chemical
modifications of DNA bases.1 5,6-Dihydroxy-5,6-dihydrothy-
mine (thymine glycol) is a major oxidative DNA damage
structure that generates in DNA as a consequence of oxidative
stress.2 Exposure of chromatin DNA of a cultured human cell
to γ-radiation (116 Gy) caused a significant increase of thymine
glycol formation in a yield of ∼6.6 × 105 DNA bases.3 Although
thymine glycol is a lethal lesion in DNA, it is subjected to
cellular repair process in vivo and removed by excision repair
enzymes, such as endonuclease III (Endo III).4 Reactive oxygen
species including the hydroxyl radical (OH•) generated as a
result of water radiolysis accounts for the formation of thymine
glycol in DNA (reaction 1). In addition, direct interaction
stage of the DNA damage reaction induced by ionizing radiation
(reaction 2)
H2O f OH•, H•, eaq
(1)
(2)
-
DNA f DNA+• + e-
Recent studies have shown that the radical cations generated
as such migrate hundreds of angstroms through DNA and cause
oxidation of DNA bases as a result of irreversible trapping by
the nucleobases.5 Due to lower HOMO energy of guanine (G)
than those of the other bases, G sites undergo site-specific
oxidation to form an alkaline-labile 8-oxoguanine structure, and
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10.1021/ja061304+ CCC: $33.50 © 2006 American Chemical Society

Photoinduced Reductive Repair of Thymine Glycol
A R T I C L E S
thereby such oxidative damage from a distance possibly
influences the distribution of oxidative lesions in DNA.6
Experiments have also shown that hole transfer in DNA is
extremely sensitive toward structural changes caused by modi-
fied nucleobases or mismatched base pairs.7 Several mechanisms
for hole migration depending on the energetics of the nucleo-
bases and on the structural characteristics of the system under
investigation were proposed in the past decade. The mechanisms
include single-step superexchange, multistep hopping, phonon-
assisted polaron hopping, and ion-gated hopping.5
A counterpart of the hole transfer process, which has been
referred to as excess electron transfer (EET), has been studied
by electron spin resonance (EPR) of γ-irradiated frozen solutions
of DNA8 and also recently demonstrated by means of direct
injection of electrons into DNA from photoexcited flavins,9
aromatic amines,10 pyrenes,11 phenothiazine12 or other chemi-
cally synthesized electron donors13 linked to the DNA duplexes.
Intensive investigations so far have suggested that excess
electrons hop more efficiently over T-A base pairs than G-C
base pairs and are trapped by electron acceptors, such as a
cyclobutane pyrimidine dimer (CPD)9 or 5-bromo-2′-deoxyuri-
dine (BrdU).10 Comprehensive knowledge about such an
interaction between electrons and DNA is essential for inves-
tigating the roles of proteins which contain redox-active
cofactors. For example, DNA photolyase transfers a single
electron to a CPD site in a DNA duplex upon photoexcitation
of a flavin chromophore (FADH-) in the enzyme and initiates
redox repair of the cyclobutane ring of CPD.14 Recent col-
laborative work by David, Barton, and co-workers has demon-
strated rapid scanning of DNA lesions by electronic long-range
communication between DNA-bound repair enzymes.15 From
a viewpoint of cancer radiotherapy, such a reductive electron
generated in the radiolysis is operative as a reactive species
under hypoxic conditions in solid tumor tissues, and thus it is
highly possible that such a charge transfer in DNA occurs at
the earliest stage of radiation-induced DNA damage processes,
which affects the distribution of the base lesions.
In a series of studies on the chemical reactivity of thymine
glycol, our group has shown previously that thymine glycol is
chemically repaired to the original thymine structure by reduc-
tion with radiolytically generated electrons (eaq-, reaction 1) or
photoexcited aromatic amines. From the results of quantitative
product analysis, it was predicted that thymine glycol undergoes
one-electron reduction to generate a 6-hydroxy-5,6-dihydrothymin-
5-yl radical (6-HOT•), followed by the second one-electron
reduction to thymine.16 So far, there have been no reports of an
enzyme that photochemically catalyzes the reductive repair of
thymine glycol in DNA via the similar redox mechanism, but
interaction between the reductive electron and the thymine
glycol lesion may change the DNA-mediated electron transfer
characteristics. Indeed, it has been recently demonstrated that
redox properties of intervened artificial nucleobases influence
the efficiency of EET in DNA.9e Furthermore, knowledge of
the fundamental properties of damaged DNA bases is important
to develop novel devices for electrochemical detection of DNA
lesions.
We describe herein our first attempt to investigate the redox
reactivity of thymine glycol lesions in DNA and the influence
of such a base modification on the efficiency of EET through
the duplex DNA. As described below, we have prepared a series
of oligodeoxynucleotides containing a single thymine glycol
residue in each sequence by well-established methods and
examined the photoreduction of the single strands by photo-
excited FADH- (*FADH-). In addition, we have synthesized
DNA-phenothiazine (PTZ) conjugates for photochemical injec-
tion of an electron into the duplex DNA containing thymine
glycol and studied EET through the lesion by employing gel
electrophoresis. On the basis of our findings, we discuss the
possibility of reductive repair of thymine glycol in DNA and
implications of DNA mediated EET with respect to DNA base
damage reactions.
(6) For example, see: (a) Seidel, C. A. M.; Schulz, A.; Sauer, H. M. J. Phys.
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Am. Chem. Soc. 1996, 118, 7063-7068. (e) Shafirovich, V.; Dourandin,
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Results
Photoinduced Reductive Repair of Thymine Glycol in
DNA. First of all, we examined the reactivities of free thymidine
glycol and oligodeoxynucleotides containing thymine glycol
toward the photoexcited reduced form of flavin, *FADH- in
anoxic aqueous solution. The mechanistic roles of the flavin
chromophore in DNA photolyase have been well established
by employing varieties of analytical methods14 including laser
flash photolysis.17 The excited state of FADH- has enough
reducing power (Eox* ) -2.84 V against SCE) to initiate single-
electron transfer to the pyrimidine dimers. Flavin derivatives
have been used by Carell and co-workers for photochemical
studies on EET through DNA duplexes, where they incorporated
(9) (a) Schwogler, A.; Burgdorf, L. T.; Carell, T. Angew. Chem., Int. Ed. 2000,
39, 3918-3920. (b) Behrens, C.; Burgdorf, L. T.; Schwogler, A.; Carell,
T. Angew. Chem., Int. Ed. 2002, 41, 1763-1766. (c) Cichon, M. K.; Haas,
C. H.; Grolle, F.; Mees, A.; Carell, T. J. Am. Chem. Soc. 2002, 124, 13984-
13985. (d) Breeger, S.; Hennecke, U.; Carell, T. J. Am. Chem. Soc. 2004,
126, 1302-1303. (e) Manetto, A.; Breeger, S.; Chatgilialoglu, C.; Carell,
T. Angew. Chem., Int. Ed. 2006, 45, 318-321.
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Ito, T.; Rokita, S. E. Angew. Chem., Int. Ed. 2004, 43, 1839-1842. (c)
Ito, T.; Rokita, S. E. J. Am. Chem. Soc. 2004, 126, 15552-15559.
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Eur. J. 2002, 8, 4877-4883. (b) Kaden, P.; Mayer A.; Trifonov, A.; Fiegig,
T.; Wagenknecht, H.-A. Angew. Chem., Int. Ed. 2005, 44, 1636-1639.
(12) Wagner, C.; Wagenknecht, H.-A. Chem.sEur. J. 2005, 11, 1871-1876.
(13) (a) Giese, B.; Carl, B.; Barl, T.; Carell, T.; Behrens, C.; Hennecke, U.;
Schiemann, O.; Feresin, E. Angew. Chem., Int. Ed. 2004, 43, 1848-1851.
(b) Lewis, F. D.; Liu, X.; Miller, S. E.; Hayes, R. T.; Wasielewski, M. R.
J. Am. Chem. Soc. 2002, 124, 11280-11281.
(16) (a) Ide, H.; Otsuki, N.; Nishimoto S.; Kagiya, T. J. Chem. Soc., Perkin
Trans. 2 1985, 1387-1392. (b) Nishimoto, S.; Ide, H.; Otsuki, N.;
Nakamichi, K.; Kagiya, T. J. Chem. Soc., Perkin Trans. 2 1985, 1127-
1134.
(17) (a) Kim, S. T.; Heelis, P. F.; Okamura, T.; Hirata, T.; Mataga, N.; Sancar,
A. Biochemistry 1991, 30, 11262-11270. (b) Kim, S. T.; Volk, M.;
Rousseau, G.; Heelis, P. F.; Sancar, A.; Michelbeyerle, M. E. J. Am. Chem.
Soc. 1994, 116, 3115-3116.
(14) Sancar, A. Chem. ReV. 2003, 103, 2203-2238.
(15) (a) Boon, E. M.; Livingston, A. L.; Chmiel, N. H.; David, S. S.; Barton, J.
K. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 12543-12547. (b) Yavin, E.;
Boal, A. K.; Stemp, E. A.; Boon, E. M.; Livingston, A. L.; O’Shea, V. L.;
David, S. S.; Barton, J. K. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 3546-
3551. (c) Yavin, E.; Boal, A. K.; Lukianova, O. A.; O’Shea, V. L.; David,
S. S.; Barton, J. K. Biochemistry 2005, 44, 8397-8407.
9
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A R T I C L E S
Ito et al.
Figure 1. Photoinduced reduction of thymidine glycol (0.5 mM) by FADH-
(0.2 mM) in the presence of EDTA (20 mM) in Ar-purged phosphate buffer
solution (pH 7.0): (b) decomposition of thymidine glycol, (0) formation
of thymidine, and (2) 6-hydroxy-5,6-dihydrothymidine.
Scheme 1. Photoinduced Reduction of Thymidine Glycol
Figure 2. Typical HPLC profiles of 5′-GATgG-3′ (a) before and (b) after
90-min UV irradiation as eluted with 12% aqueous acetonitrile solution.
Table 1. Photoinduced Reductive Repair of Thymine Glycol in
Single-Stranded Oligodeoxynucleotidesa
sequence of
XX(Tg/T)X
decomposition of
XX(Tg)X/%
yield of
XXTX/%
selectivity/%
both the flavin derivative and a thymine cyclobutane photodimer
into DNA and investigated the electron-transfer process from
*FADH- to the photodimer.9
5′-GG(Tg/T)G-3′
5′-GA(Tg/T)G-3′
5′-GG(Tg/T)A-3′
5′-GA(Tg/T)A-3′
5′-CG(Tg/T)A-3′
62
62
61
85
54
<1
37
41
46
15
<1
60
67
54
28
A deoxygenated aqueous solution of thymidine glycol (0.5
mM) was photoirradiated with a high-pressure Hg arc through
a Pyrex glass filter (> 300 nm)18,19 in the presence of FAD
(0.2 mM) and excess EDTA (20 mM) to generate a reduced
form of FAD (FADH-) in situ.9e,20,21 As analyzed by HPLC,
the photoirradiated solution gave two major products, which
were assigned to thymidine and 6-hydroxy-5,6-dihydrothy-
midines (6-HOT) by coeluting the authentic samples (Scheme
1). Formation of the structures was observed in the photosen-
sitized reduction of thymine glycol by a series of aromatic
amines, and also in the radiation-induced reduction of thymine
glycol, where either hydrated electrons (eaq-) and/or carbon
dioxide radical anions (CO2-•) are generated as reducing
species.16 About 20% of thymidine glycol was decomposed after
6-h photoirradiation, and 91% of the products accounted for
thymidine (Figure 1), which is comparable with the yield of
thymine from thymine glycol as sensitized by N,N,N′,N′-
tetramethyl-p-phenylenediamine (TMPD) (72%).16a,22 The gen-
eration of 6-HOT supports the previous prediction that 5,6-
dihydro-6-hydroxythymidin-5-yl radical (6-HOT•) is an inter-
mediate in the reaction. In addition to the two major products,
a few minor products were observed by the HPLC analysis (data
not shown). Considering the possible pathway for generation
a The single-stranded DNA (66 µM) in phosphate buffer Solution (pH
7) was exposed to UV light in the presence of FAD (0.1 mM) and EDTA
(20 mM) for 90 min at 24 °C. The amounts of decomposed oligodeoxy-
nucleotide and the restoration were quantified based on the absorbance at
260 nm on the HPLC profiles (Figure 2).
of the 5-yl radical, the minor products may include 5-hy-
droxymethyl-2′-deoxyuridine23 and/or the stereoisomers of
6-HOT, but they were not identified in this study.
Next, we prepared 4-mer long oligodeoxynucleotides contain-
ing thymine glycol by chemical oxidation of the corresponding
thymine-containing DNA in the presence of KMnO4 and
examined FADH- sensitized reduction of the sequences under
anaerobic conditions. The Ar-saturated oligodeoxynucleotide (66
µM) solutions were exposed to UV light (λ > 300 nm) in the
presence of FAD (0.1 mM) and EDTA (20 mM) and analyzed
by the use of HPLC (Figure 2). Restoration of thymine from
thymine glycol in the oligodeoxynucleotides after 90-min
irradiation was quantitated with reference to authentic DNA
samples (Table 1). Compared with the reaction of free thymidine
glycol, the selectivity of thymine restoration for each sequence
was relatively low, probably because of some side reactions,
such as reduction of pyrimidine bases. Interesting sequence
dependence was observed as can be seen in Table 1; the solution
of oligodeoxynucleotide containing only G and thymine glycol
(5′-GGTgG-3′) gave a trace of a repaired sequence (5′-GGTG-
3′). On the other hand, G and adenine (A) containing oligode-
oxynucleotides (5′-GATgG-3′, 5′-GGTgA-3′, and 5′-GATgA-
(18) Thymine glycol absorbs light at wavelengths shorter than 300 nm, and
UV light through the cutoff filter selectively generates the excited state of
flavin.
(19) Heyroth, F. F.; Loofbourow, J. R. J. Am. Chem. Soc. 1934, 56, 1728-
1734.
(20) An excess amount of EDTA generates a two-electron reduced form of FAD.
Restoration of thymidine from thymidine glycol was negligible in the
photolysis of FAD without EDTA.
(21) Traber, R.; Kramer, H. E. A.; Hemmerich, P. Biochemistry 1982, 21, 1687-
1693.
(22) Photolysis (λ > 300 nm, 45 min) of thymine glycol (1 mM) in the presence
of TMPD (1 mM) in aqueous buffer solution (pH 7).
(23) Berger, M.; Sarrazin, F.; Cadet, J. J. Chim. Phys. Phys. Chim. Biol. 1993,
90, 853-861.
9
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Photoinduced Reductive Repair of Thymine Glycol
A R T I C L E S
Figure 4. Time courses of transient absorption at (a) 460 and (b) 360 nm
upon 266-nm laser photolysis of DMA (0.2 mM) in the presence of
thymidine glycol (3.0 mM). (c) Time course of transient absorption at 360
nm upon 266-nm laser photolysis of DMA (0.2 mM).
Figure 3. Transient absorption spectra of the intermediates as observed
10 µs and 800 µs after the 266-nm laser photolysis of DMA (0.2 mM) in
phosphate buffer solution (pH 7.0) in the (a) absence or (b) presence of
thymidine glycol (3.0 mM).
previous pulse radiolysis study of 5-bromo-6-hydroxy-5,6-
dihydrothymine.26 Therefore, the intermediate observed 800 µs
after the pulse is assigned to 6-HOT• generated by elimination
of the hydroxyl ion from the radical anion of thymidine glycol.
Due to the well-established oxidizing ability of the 6-hydroxy-
5,6-dihydrothymin-5-yl radical,27 it is expected that structurally
similar 6-HOT• undergoes further one-electron reduction to the
corresponding anion by the ground-state DMA (reaction 3).
3′) showed higher reactivity (37-46%) compared to that of
cytidine (C) containing DNA (5′-CGTgA-3′, 15%). In consid-
eration of reduction potentials of DNA bases and the high
reducing power of *FADH-, short-range EET in the single
strands might be responsible for the difference in the restoration
yield.
Laser Flash Photolysis Study on the Repair Mechanism.
Our previous product studies on the photoreduction of thymine
glycol were indicative that the reductive repair proceeds via
elimination of the hydroxyl ion from the radical anion of
thymine glycol, thereby 6-HOT• is generated as an intermedi-
ate;16 however, the involvement of the 5-yl radical has not been
directly observed yet. In this context, we next performed a time-
resolved spectroscopic study of radical intermediates in the
photoinduced reduction of thymidine glycol. Since FAD has
multiple redox states and the corresponding protonated and
deprotonated structures depending on the pH of the solutions,
spectral overlaps of the flavin chromophores prevented direct
observation of 6-HOT• in the photoreduction of thymidine glycol
by *FADH-. As is well-known, N,N-dimethylaniline (DMA)
is a photoinducible electron donor with a high reducing power
(Eox* ) -3.3 V),24 and the radical cation of DMA (DMA+•)
shows a characteristic transient absorption band at around 460
nm (Figure 3a).25 Laser excitation at 266 nm of an Ar-saturated
aqueous solution of thymidine glycol (3.0 mM) in the presence
of DMA (0.2 mM) at pH 7 resulted in the intense absorption of
DMA+• at 460 nm, as shown in Figure 3b. The radical cation
decays relatively slowly (Figure 4a), and thereafter an absorption
band with λmax ) 360 nm emerged (Figure 4b), as was evidently
observed 800 µs after excitation (Figure 3b). The transient
spectrum of the slower decayed intermediate at around 360 nm
shown in Figure 3b was quite similar to that of the 6-hydroxy-
5,6-dihydrothymin-5-yl radical which was obtained in the
6-HOT• + DMA f 6-HOT- + DMA+•
(3)
If one-electron reduction of 6-HOT• occurs during the DMA-
sensitized reaction, we could observe a slow growing component
of DMA+• along with the rapid buildup due to laser-induced
ionization of DMA. A not so simple decay profile in Figure 4a
might be indicative of involvement of such a process, but
unfortunately, analysis of the decay kinetics of DMA+• based
on the time profile at 460 nm was not successful because of
spectral overlap of the radical cation and the excited state of
DMA, as has been suggested previously.28
The rate constant of electron transfer from photoexcited DMA
(DMA*) to thymidine glycol was thus conveniently estimated
by Stern-Volmer analysis of the fluorescence quenching. Upon
excitation of DMA (0.3 mM) in anoxic aqueous buffer solution
(pH 7) at 300 nm, corresponding fluorescence at around 320-
450 nm was observed, and the intensity of fluorescence of DMA
(λem ) 360 nm) decreased with increasing concentration of
thymidine glycol (0-3.3 mM) in the DMA solution. The Stern-
Volmer treatment of the data yields a linear plot (Supporting
Information) with a slope of kqτ ) 0.11 × 103 M-1, which is
deduced to be kq ) 3.8 × 1010 M-1 s-1 using a reported singlet
lifetime of DMA, τ ) 2.8 × 10-9 s.29 This almost diffusion
(26) Deeble, D. J.; von Sonntag, C. Z. Naturforsch. 1985, 40c, 925-928.
(27) (a) Jovanovic, S. V.; Simic, M. G. J. Am. Chem. Soc. 1986, 108, 5968-
5972. (b) Steenken, S. Chem. ReV. 1989, 89, 503-520.
(24) Scannell, M. P.; Fenick, D. J.; Yeh, S.-R.; Falvey, D. E. J. Am. Chem.
Soc. 1997, 119, 1971-1977.
(25) (a) Habersbergerova´, A.; Janovsky´, I.; Teply´, J. Radiat. Res. ReV. 1968, 1,
109-181. (b) Land, E. J.; Porter, G. Trans. Faraday Soc. 1963, 59, 2027-
2037. (c) Ito, T.; Shinohara, H.; Hatta, H.; Fujita, S.; Nishimoto, S. J. Phys.
Chem. A 2000, 104, 2886-2893
(28) (a) Cadogan, K. D.; Albrecht, A. C. J. Phys. Chem. 1969, 73, 1868-1877.
(b) Land, E. J.; Richards, J. T.; Thomas, J. K. J. Phys. Chem. 1972, 76,
3805-3812.
(29) Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd ed.;
Beriman, I. B., Ed.; Academic Press: New York, 1971; p 473.
9
J. AM. CHEM. SOC. VOL. 128, NO. 33, 2006 10937

A R T I C L E S
Ito et al.
Chart 1. Sequences of Oligodeoxynucleotides Used in This Study
Figure 5. Autoradiogram of a 15% polyacrylamide gel containing 7 M
urea showing cleavage products of 5′-32P-labeled Tg-ODN 1. Duplex DNA
(Tg-ODN 1/PTZ-ODN 1) was photoirradiated for the indicated periods in
the presence (lanes 1-8) or absence (lanes 9-16) of Na2S2O4, then analyzed
either directly (lanes 5-8 and 13-16) or after subsequent treatment with
piperidine (lanes 1-4 and 9-12). Lane 17 is the Maxam-Gilbert A+G
sequencing lane.
Scheme 2. Synthesis of Phenothiazine Containing
Oligodeoxynucleotides
basic solution at a high temperature,31 an oligodeoxynucleotide
containing a repaired thymine residue (13-mer) can be detected
as a slow-moving band, while a fast-moving band corresponding
to a fragment of 9-mer long oligodeoxynucleotide is observed
for the thymine-glycol containing DNA.
Figure 5 shows a gel image of photoirradiated samples of
Tg-ODN 1/PTZ-ODN 1 as analyzed before (lanes 13-16) and
after (lanes 9-12) piperidine treatment. On this gel, however,
increase of the full length oligodeoxynucleotide band (T13) was
not observed, but most of the irradiated oligodeoxynucleotides
were hydrolyzed at the thymidine glycol site (Tg). As was
proposed by the mechanistic studies above, a total of two
electron reductions would be necessary for complete repair of
thymidine glycol. Based on this prediction, photoreaction in the
presence of excess sodium dithionite (Na2S2O4) as a second
electron source was also performed under the same conditions
(lanes 1-8), but no remarkable difference on the cleavage
pattern was observed. Maximum yield of a reductive repair of
thymine glycol was obtained from the single-stranded oligo-
nucleotide 5′-GATgA-3′ as shown above; thus photoreactivity
of a duplex bearing the sequence (Tg-ODN 2/PTZ-ODN 2) was
also examined under the same conditions, but essentially the
same results were obtained (Supporting Information).
In addition, we designed an oligodeoxynucleotide containing
both thymine glycol and BrdU in the sequence (Tg-BrdU-ODN),
hybridized it with the PTZ-containing complimentary oligo-
deoxynucleotide (PTZ-ODN 3), and then investigated photo-
induced EET through the duplex (Chart 1). As has been used
as a chemical probe for EET in DNA,10 BrdU traps an electron
and generates the 2′-deoxyuridyl-5-yl radical by losing the
bromide anion, and then the radical in turn abstracts a hydrogen
atom from the deoxyribose of a 5′-adjacent nucleotide.32
Therefore, EET followed by piperidine treatment results in
strand cleavage at the 5′-adjacent base of BrdU. Figure 6a is
the gel image of a control photoirradiated solution of a duplex
containing double BrdU sites (BrdU-BrdU-ODN, Chart 1),
showing a cleavage band at the 5′-next of the first BrdU site
controlled rate constant is consistent with the observed fast
buildup of the absorption of DMA+• at 460 nm (Figure 4a).
Excess Electron Transfer through Thymine Glycol in
DNA. The experimental data obtained from the photoinduced
reduction of thymine glycol in 4-mer long oligodeoxynucleotides
suggest potential electron hopping between vicinal nucleobases
in the single-stranded DNA. We investigated the effect of
thymine glycol in duplex DNA on the efficiency of DNA-
mediated EET by the use of polyacrylamide gel electrophoresis.
Commercially available thymidine glycol phosphoramidite was
site-specifically incorporated into oligodeoxynucleotides using
a DNA synthesizer (Tg-ODN 1, 2 and Tg-BrdU-ODN, Chart
1). To avoid unnecessary oxidation of nucleotides by the
photoexcited oxidized form of flavin (*FAD), phenothiazine
(PTZ) was employed as a photoinducible electron donor and
was covalently linked to the complimentary strand with a 5′-
amino linker by the standard amine-N-hydroxysuccinimide
coupling (PTZ-ODN 1-3, Scheme 2). In a separate study, we
confirmed that photoexcited PTZ has enough reducing power
(*Eox ) -2.7 V)30 to reductively repair thymidine glycol by
photoirradiating (λ > 300 nm) a deaerated thymidine glycol
solution in the presence of free PTZ (data not shown). 5′-End
32P-radiolabeled Tg-ODN 1 was annealed with the complimen-
tary electron donor strand (PTZ-ODN 1), and the duplex was
exposed to UV light (365 nm) under an anoxic atmosphere at
4 °C. The irradiated samples were then treated with hot
piperidine and subjected to polyacrylamide gel electrophoresis.
Since the thymine glycol structure is intrinsically unstable in a
(30) Excited-state oxidation potential was calculated using *Eox ) Eox - E00
with the oxidation potential of PTZ (Eox ) 0.59 V; Mecklenburg, S. L.;
Peek, B. M.; Schoonover, J. R.; McCafferty, D. G.; Wall, C. G.; Erickson,
B. W.; Meyer, T. J. J. Am. Chem. Soc. 1993, 115, 5479-5495) and zero
excitation energy (E00) evaluated from λ00 () 381 nm) in aqueous solution.
*Eox ) -2.0 V has been adopted for phenothiazine incorporated in DNA
in a recent study by Wagenknecht (see ref 12).
(31) (a) Iida, S.; Hayatsu, H. Biochim. Biophys. Acta 1971, 228, 1-8. (b) Cadet,
J.; Ducolomb, R.; Teoule, R. Tetrahedron 1977, 33, 1603-1607. (c) Cadet,
J.; Teoule, R. J. Chromatogr. 1975, 115, 191-194.
(32) (a) Cook, G. P.; Greenberg, M. M. J. Am. Chem. Soc. 1996, 118, 10025-
10030. (b) Cook, G. P.; Chen, T.; Koppisch, A. T.; Greenberg, M. M. Chem.
Biol. 1999, 6, 451-459. (c) Fujimoto, K.; Ikeda, Y.; Ishihara, S.; Saito, I.
Tetrahedron Lett. 2002, 43, 2243-2245.
9
10938 J. AM. CHEM. SOC. VOL. 128, NO. 33, 2006

Photoinduced Reductive Repair of Thymine Glycol
A R T I C L E S
Scheme 3. Proposed Reaction Mechanism of Thymidine Glycol
Repair by Photoinduced Electron Transfer from Either FAD or
DMA
Figure 6. (a) Autoradiogram of a 15% polyacrylamide gel containing 7
M urea showing cleavage products of 5′-32P-labeled BrdU-BrdU-ODN.
Duplex DNA (BrdU-BrdU-ODN/PTZ-ODN 3) was photoirradiated for the
indicated periods under aerobic (lanes 1-4) or anaerobic atmosphere (lanes
5-8) and then treated with piperidine. Lane 9 is the Maxam-Gilbert A+G
sequencing lane. (b) Autoradiogram of a 15% polyacrylamide gel containing
7 M urea showing cleavage products of 5′-32P-labeled Tg-BrdU-ODN.
Duplex DNA (Tg-BrdU-ODN/PTZ-ODN 3) was photoirradiated for the
indicated periods under aerobic (lanes 1-4) or anaerobic atmosphere (lanes
5-8) and then treated with piperidine. Lane 9 is the Maxam-Gilbert A+G
sequencing lane.
a high reducing power for electron donation to thymidine glycol
but also because it could be a model reaction of thymine glycol
repair by flavin-containing photoenzymes. As in the previous
cases of photoreduction of thymine glycol by aromatic amines,16
efficient restoration of thymidine from thymidine glycol by the
reduction with *FADH- was observed despite structural dif-
ferences between thymine glycol and thymidine glycol. Tran-
sient absorption data obtained by laser flash photolysis indicated
that DMA+• is generated immediately after laser excitation
followed by relatively slow formation of the intermediate 5-yl
radical, 6-HOT•, while the precursor radical anion of thymidine
glycol was not detected by the current flash photolysis system.
These results and our previous findings are inconsistent with
the stepwise two-electron-transfer mechanism, in which the
radical anion of thymidine glycol loses the hydroxyl ion and
then the resulting radical undergoes further reduction, as shown
in Scheme 3. A hypothetical decay profile of 6-HOT• can be
obtained by subtracting the contribution of the transient species
of DMA (Figure 4c) from the observed decay profile at 360
nm in Figure 4b. By analyzing the differential time profile
(Supporting Information), the built-up component (<100 µs)
of 6-HOT• observed at 360 nm gave a first-order rate constant
of ∼3 × 104 s-1, which is much slower than that of debromi-
nation from the BrdU radical anion (k ) ∼109 s-1).35 As
discussed earlier, 6-HOT• is a relatively stable oxidizing radical
as it can oxidize aromatic amines,27 but the source of the second
electron in this reaction is not clear. Taking into account the
long lifetime of 6-HOT• (τ1/2 > 1 ms) obtained from the
differential time profile (Supporting Information), further reduc-
tion by another excited state of the electron donor or dispro-
portionation in 6-HOT• might be responsible.
(C15), not of the second BrdU site (T13). This result suggests
an excess electron injected from PTZ was efficiently trapped
at the proximal BrdU site, which prevented further migration
to the distal BrdU. Oxygen did not have a significant effect on
the electron-transfer efficiency (lanes 1-4), implying electron
transfers inside of the DNA duplex rather than becomes detached
from and reattaches to the DNA. In the case of the thymine-
glycol-containing duplex Tg-BrdU-ODN/PTZ-ODN 3, how-
ever, an excess electron migrated to the distal BrdU site beyond
the thymine glycol site and induced strand scission at T13 (Figure
6b). According to previous studies,33 DNA replication is
inhibited by thymine glycol in the DNA strand, presumably
because hydrogen bonding between thymine glycol and the
complimentary adenine is less effective than that within the
normal A-T base pair. The lower melting temperature of Tg-
BrdU-ODN/PTZ-ODN 3 (61.3 °C) compared to BrdU-BrdU-
ODN/PTZ-ODN 3 (67.5 °C) suggests slight destabilization of
the glycol-containing duplex (Supporting Information), which
is inconsistent with the previous results. At this point, it is not
clear whether thymine glycol is flipped from the center of the
duplex,34 but the result that an excess electron reached the distal
BrdU is noteworthy as an example of EET through a modified
DNA base.
Discussion
Excess Electron Hopping through Thymidine Glycol in
DNA. Several groups have made efforts toward understanding
the mechanism of EET in DNA, but our knowledge about EET
is still limited to elementary principles. Early investigation into
temperature dependent EET in DNA by EPR as well as recent
studies on distance dependent EET have suggested that an excess
electron hops between nucleobases, especially pyrimidines, by
a thermally activated mechanism at ambient temperatures.8-11
Escape to the surrounding aqueous phase or trapping at
pyrimidines to generate dihydropyrimidine derivatives is ex-
Reductive Repair Mechanism of Thymidine Glycol. We
employed herein FADH- as an electron donor for photoreduc-
tion of thymidine glycol not only because the excited state has
(33) For example, see: (a) Hayes, R. C.; Petrullo, L. A.; Huang, H.; Wallace,
S. S.; LeClerc, J. E. J. Mol. Biol. 1988, 201, 239-246. (b) Rouet, P.;
Essigmann, J. M. Cancer Res. 1985, 45, 6113-6118. (c) Ide, H.; Kow, Y.
W.; Wallace, S. S. Nucleic Acids Res. 1985, 13, 8035-8052. (d) Clark, J.
M.; Pattabiraman, N.; Jarvis, W.; Beardsley, G. P. Biochemistry 1987, 26,
5404-5409. (e) Basu, A. K.; Loechler, E. L.; Leadon, S. A.; Essigmann,
J. M. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 7677-7681. (f) Mlasklewicz,
K.; Miller, J.; Ornstein, R.; Osman, R. Biopolymers 1995, 35, 113-124.
(g) Iwai, S. Chem.sEur. J. 2001, 7, 4344-4351.
(34) Kao, J. Y.; Goljer, I.; Phan, T. A.; Bolton, P. H. J. Biol. Chem. 1993, 268,
17787-17793.
(35) Rivera, E.; Schuler, R. H. J. Am. Chem. Soc. 1983, 87, 3966-3971.
9
J. AM. CHEM. SOC. VOL. 128, NO. 33, 2006 10939

A R T I C L E S
Ito et al.
pected as the fate of the excess electron on DNA,1a,36 but this
has not been experimentally demonstrated yet.
Scheme 4. Excess Electron Transfer through Thymine Glycol (Tg)
in DNA
The results of photoinduced reduction obtained above show
that photoexcited FADH- can promote efficient reduction of
thymine glycol in the single-stranded 4-mer long oligodeoxy-
nucleotides. Interesting is the fact that the yield and the
selectivity of repaired thymine are sequence dependent and seem
to be correlated with reduction potentials of the nucleobases
(G, -3.00 V; A, -2.76 V; C, -2.59 V; T, -2.42 V vs SCE)37
in the sequence (Table 1). Due to the high reducing power of
*FADH-, any nucleobases including thymine glycol in the short
single-stranded DNA could undergo direct one-electron reduc-
tion; thereafter the reductive electron possibly hops to a vicinal
nucleobase until it is irreversibly trapped at high reduction
potential sites, or otherwise it may escape to aqueous phase.
While the reduction potential of thymine glycol in DNA is not
available at this point, it was suggested that 5,6-saturated
pyrimidine derivatives have slightly more negative reduction
potentials than those of the original pyrimidines.38 Therefore,
C may most easily undergo one-electron reduction among the
four nucleobases (A, T, G, C) and thymine glycol. The low
yield of 5′-CGTA-3′ from a photoirradiated solution of 5′-
CGTgA-3′ (28%) supports the prediction, where trapping of the
excess electron by C and competitively fast protonation of the
cytosine radical anion prevent further electron transfer to the
thymine glycol residue.8b,10b,12 In fact, kinetic analyses of the
reactions of pyrimidine radical anions (C-•, T-•) in aqueous
solution have demonstrated the quite fast protonation of C-•
completing on a nanosecond time scale or faster39 and the
comparatively slow protonation of T-• (k < 103 s-1).40 On the
other hand, adenine-containing sequences showed high reactivity
among the sequences, presumably because adenine is a temporal
electron mediator as it can transport an excess electron to
thymine glycol or other nucleobases. In contrast, no restoration
of 5′-GGTG-3′ was observed in the photoreduction of the 5′-
GGTgG-3′ sequence; nevertheless the sequence was decom-
posed during the photoirradiation. Previous theoretical calcu-
lations estimated the electron affinity (EA) of hydroxyl radical
adducts of DNA bases, suggesting the 6-hydroxy-5-yl radical
6-HOT• possesses a high EA of >2 eV.41 Thus, one explanation
for the reactivity of 5′-GGTgG-3′ might be that 6-HOT•
generated by direct reduction of thymine glycol underwent
further reduction by the adjacent G sites which have low
ionization potentials (IP, 6-7 eV for GG)6c,d and then the
generated guanine radical cations undergo hydrolysis to afford
final oxidation products, such as 8-oxo-2′-deoxyguanine.42 In
addition, such a radical may give rise to cross-linked lesions
via coupling to 5′-adjacent guanine, as discussed earlier41,42
Further product analysis of the electron-transfer reaction in
single-stranded DNA would be necessary for understanding the
reactivity and the detailed reaction mechanisms.
Carell and co-workers have synthesized varieties of oligo-
deoxynucleotides with the thymine cyclobutane dimer as a
chemical probe of EET through a DNA duplex.9 In the reaction
system, intramolecular electron transfer to a thymine dimer in
DNA causes splitting of the C5-C6 cyclobutane ring with a
relatively slow rate constant of k ) ∼107 s-1, and the single
electron further migrates along the duplex after repairing the
dimer. Thus, the apparent EET efficiency in the system is less
sequence dependent compared to the system using BrdU as the
detection probe of EET.13a Considering this fact, it is not
surprising that we observed efficient excess electron trapping
at BrdU rather than at the thymine glycol site (Figure 6). Given
that the radical anion of thymine glycol in duplex DNA also
loses the hydroxyl ion with a rate constant of ∼104 s-1, most
of the excess electrons injected from the covalently linked PTZ
reach the thymine glycol residue and migrate either further to
BrdU or back to the PTZ radical cation (back electron transfer,
BET) before the glycol radical anion irreversibly loses the
hydroxyl ion (Scheme 4). On the other hand, in the case of
intermolecular electron donation to the 4-mer long single-
stranded DNA by free FADH-, electron recombination between
the neutral FADH radical (FADH•) and the excess electron on
the oligodeoxynucleotide is less expected once charge separation
occurs between them. Under such circumstances, an injected
excess electron would eventually react with thymine glycol
unless it is irreversibly trapped by high electron-affinic cytosines
or it escapes to the solution phase.
While biological implications of EET in DNA are still
uncertain, recent collaborative work by David, Barton, and co-
workers have demonstrated rapid scanning of DNA abasic sites
using DNA-mediated electron transfer between DNA-bound
repair enzymes, such as Escherichia coli MutY and Endo III.15
Inefficient electron transfer through damaged sites prevents
relocation of the repair enzymes containing redox active [4Fe-
4S]2+ cofactor, thereby increasing the local concentration of
repair enzymes in the vicinity of damaged DNA. However our
results are indicative that the thymine glycol structure in DNA
does not work as an efficient excess-electron trap, although
thymine glycol is also a substrate for damage repair by Endo
III.4 It is thus likely that not all of the damaged structures induce
the mode of electron communication for efficient detection of
the substrates, since it seems that DNA-mediated excess
electron-transfer efficiency depends on the redox property of
the intervened nucleobases as shown above. For investigating
such an electron communication between DNA-bound proteins,
(36) (a) Nishimoto, S.; Ide, H.; Nakamichi, K.; Kagiya, T. J. Am. Chem. Soc.
1983, 105, 6740-6741. (b) Ito, T.; Shinohara, H.; Hatta, H.; Nishimoto,
S. J. Org. Chem. 1999, 64, 5100-5108.
(37) Seidel, C. A. M.; Schulz, A.; Sauer, M. H. M. J. Phys. Chem. 1996, 100,
5541-5553.
(38) Scannell, M. P.; Prakash, G.; Falvey, D. E. J. Phys. Chem. A 1997, 101,
4332-4337.
(39) (a) Visscher, K. J.; de Haas, M. P.; Loman, H.; Vojnovic, B.; Warman, J.
M. Int. J. Radial. Biol. 1987, 52, 745-753. (b) Visscher, K. J.; Hom, M.;
Loman, H.; Spoelder, H. J. W.; Verberne, J. B. Radiat. Phys. Chem. 1988,
32, 465-473. (c) Raytchev, M.; Mayer, E.; Amann, N.; Wagenknecht, H.-
A.; Fiebig, T. ChemPhysChem 2004, 5, 706-712.
(40) (a) Novais, H. M.; Steenken, S. J. Am. Chem. Soc. 1986, 108, 1-6. (b)
Steenken, S. Chem. ReV. 1989, 89, 503-520.
(41) Colson, A.-O.; Sevilla, M. D. J. Phys. Chem. 1995, 99, 13033-13037.
(42) (a) Box, H. C.; Budzinski, E. E.; Dawidzik, J. B.; Wallace, J. C.; Iijima,
H. Radiat. Res. 1998, 149, 433-439. (b) Box, H. C.; Dawidzik, J. B.;
Budzinski, E. E Free Radical Biol. Med. 2001, 31, 856-868. (c) Douki,
T.; Rivie`re, J.; Cadet, J. Chem. Res. Toxicol. 2002, 15, 445-454.
9
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Photoinduced Reductive Repair of Thymine Glycol
A R T I C L E S
the oligodeoxynucleotides were typically less than 30% as calculated
based on their absorbance at 260 nm.
further studies on the EET through other types of modified DNA
bases would be necessary.43
Photosensitized Reduction by *FADH-. Typically, solutions of
thymidine glycol (0.1-20 mM) in phosphate buffer containing FAD
(0.1-0.2 mM) were purged with Ar before photoirradiation. For
effective generation of the reduced form of FAD (FADH-) in situ,
EDTA‚2Na (20 mM) was added to the solutions. The solutions in sealed
Pyrex glass tubes were photoirradiated (λex > 300 nm) under magnetic
stirring (1000 rpm) at 24 °C with a high-pressure Hg arc (450 W,
Eikosha 400). In the case of photoreduction of 4-mer long oligonucleo-
tides containing thymine glycol, an Ar-saturated phosphate buffer
solution (2.5 mM, pH 7.0) of FAD (0.1 mM) and EDTA‚2Na (20 mM)
was preirradiated for 30 min, synthesized 4-mer long DNA (66 µM)
was added, and then the solution was exposed to UV light for 90 min.
Analytical HPLC was performed with a Shimadzu 10AS HPLC system
equipped with a Rheodyne 7725 sample injector. Sample solutions were
injected onto a 5 µm C18 reversed-phase column (Wakosil 5C18, φ
4.6 mm × 150 mm, Wako). Phosphate buffer solutions (10 mM, pH
3.0) containing various concentrations of methanol (10-25 vol %) were
delivered as the mobile phase. The column eluents were monitored by
the UV absorbance at 210 or 260 nm.
Conclusions
In this study, we have demonstrated that photoreduction of
thymine glycol in short single-stranded DNA by a photoexcited
reduced form of flavin affords repaired thymine. A laser flash
photolysis study suggested an electron adduct of thymidine
glycol loses the hydroxyl ion to afford a 6-hydroxy-5,6-
dihydrothymidin-5-yl radical as an intermediate, which is further
reduced to thymidine. On the other hand, as investigated with
chemically synthesized duplex DNAs containing phenothiazine
and thymine glycol, it was demonstrated that a photoinjected
excess electron does not induce reductive repair, since elimina-
tion of the hydroxyl ion from the radical anion of thymine glycol
is relatively slow compared to competitive electron trapping at
5-bromouracil or cytosine. In this context, it is less expected
that an oxidative lesion such as thymine glycol undergoes further
reduction to thymine in radiation-exposed hypoxic cells. Another
new finding is that this type of DNA base lesion does not
necessarily prevent excess electron transfer through DNA, while
hole transfer along the DNA duplex is remarkably sensitive to
base-stacking perturbation induced by intervening base lesions.
Nanosecond Laser Flash Photolysis. The laser flash photolysis
experiments were carried out with a Unisoku TSP-601 flash spectrom-
eter with a Continuum Surelite-I Nd:YAG (Q-swithched) laser, as
described previously.46 A fourth harmonic emission at 266 nm (ca. 50
mJ per 6 ns pulse) was employed for the photolysis, and the data
obtained were analyzed on NEC PC-9801 and IBM ThinkCentre
computers. Aqueous solutions of thymidine glycol (3.0 mM) at pH
7.0 containing DMA (0.2 mM) were deaerated by Ar bubbling prior
to the laser flash photolysis experiments.
Experimental Section
Materials. Flavin adenine dinucleotide disodium salt was used as
received from Sigma Chemical. Potassium permanganate, ethylenedi-
aminetetraacetic acid, and N,N-dimethylaniline were purchased from
Nacalai Tesque and used without further purification. Reagents for high-
performance liquid chromatography (HPLC) including solvents, ac-
etonitrile, and methanol (HPLC grade) were used as received from
Wako Pure Chemical Industries. cis-Thymidine glycol and 6-hydroxy-
5,6-dihydrothymidines were synthesized and purified following the
methods reported previously.30 The stereostructure was not further
determined in this study. 10H-Phenothiazine-10-acetic acid was
synthesized by the same method as reported.44 Aqueous solutions for
all experiments were prepared using water purified with Corning Mega-
Pure System MP-190 (>16 M Ωcm).
Fluorescence Quenching. Various concentrations of thymidine
glycol in phosphate buffer (10 mM, pH 7.0) solution containing DMA
(0.3 mM) were purged with Ar for 10 min and sealed in a quartz cuvett
with a Teflon cap. Fluorescence spectra were recorded on a Hitachi
F-2000 fluorescence spectrophotometer (slit width: 1.0 nm, λex ) 300
nm, λem ) 360 nm). The dynamic quenching rate constant kq was
determined by the Stern-Volmer equation: I0/I ) 1 + kqτ0 [thymidine
glycol], where I0 and I are the intensity of the fluorescence at 360 nm,
and τ0 is the lifetime of the excited singlet state of DMA28 in the absence
of thymidine glycol.
Synthesis of Oligodeoxynucleotides Containing Modified Bases.
Modified oligonucleotides were synthesized on a 1.0 µmol scale (500
Å CPG column) using an Applied Biosystems 3400 DNA synthesizer.
cis-Thymidine glycol phosphoramidite and 5-bromo-2′-deoxyuridine
phosphoramidite (Glen Research) were site specifically incorporated
into the oligonucleotides by trityl-off synthesis. Coupling reactions were
allowed to proceed for 2 min (for A, T, G, or C) or 16.7 min (for
BrdU or thymidine glycol) and were subsequently followed by standard
oxidation and capping steps. Upon completion of the oligodeoxynucleo-
tide synthesis, the CPG column was treated with 28% aqueous
ammonium hydroxide for 1 h at room temperature. The resulting
aqueous ammonium hydroxide solutions were consolidated and stored
at room temperature for 24 h. The aqueous ammonium hydroxide
solutions were lyophilized and purified by the use of HPLC (Hitachi
D-7000). For deprotecting TBDMS groups on thymine glycol, the solid
was treated with triethylamine trihydrofluoride (0.5 mL) at 40 °C for
12 h and desalted on an NAP 10 column (Amersham Biosciences)
before HPLC purification. The DNAs were characterized by MALDI-
TOF mass spectrometry; m/z: calcd for Tg-ODN 1 (C126H164N46O81P12)
4014.62, found 4014.12; calcd for Tg-ODN 2 (C128H162N52O78P12)
4048.64, found 4048.32; calcd for Tg-BrdU-ODN (C173H221BrN63O111P17)
5565.42, found 5563.5; calcd for BrdU-BrdU-ODN (C172H216Br2-
N63O109P17) 5596.28, found 5595.34.
Synthesis of 4-mer Long Oligodeoxynucleotides Containing cis-
Thymine Glycol.45 Commercially purchased 15 OD260 oligodeoxy-
nucleotide (Nisshinbo, Tokyo) was stirred in 4.7 mL of K2HPO4 (0.1
M, pH 8.6) buffer solution containing 5.5 mM KMnO4 at 4 °C for 15
min. The reaction was quenched by the addition of allyl alcohol (0.1
mL, Wako), and the mixture was stirred at 4 °C for another 1 h. After
centrifugation (1500 rpm) at room temperature for 5 min, the
supernatant was desalted with a short column of reversed phase (C18
Sep-Pak, Waters). The DNA was eluted with 2 × 5 mL of 50% CH3-
CN/H2O. The single-stranded DNA solution was injected to a reversed-
phase HPLC column (5C18), and the corresponding peak was isolated.
A small portion of the isolated DNA was digested with Phosphodi-
esterase I (Snake Venon, Funakoshi) and Alkaline Phosphatase I
(Nacalai) in Tris (0.1 M)-MgCl2 (20 mM) buffer solution at 37 °C
for 1 h. Samples were injected on a reversed-phase column (Inertsil
ODS-3, GL Science Inc., φ 4.6 mm × 250 mm) and analyzed with a
Hitachi D-7000 HPLC system. The elution of A, C, G, and cis-
thymidine glycol was observed from the thymine-glycol-containing
DNA by comparing the authentic sample solution of them. Yields of
(43) Recent systematic screenings have found modified base structures that
impact electron-transfer efficiency (Boal, A. K.; Barton, J. K. Bioconjugate
Chem. 2005, 16, 312-321).
(44) Tierney, M. T.; Grinstaff, M. W. J. Org. Chem. 2000, 65, 5355-5359.
(45) Jerkovic, B.; Kung, H. C.; Bolton, P. H. Analytical Biochemistry 1998,
255, 90-94.
(46) Ito, T.; Shinohara, H.; Hatta, H.; Fujita, S.; Nishimoto, S. J. Phys. Chem.
A 1999, 103, 8413-8420.
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J. AM. CHEM. SOC. VOL. 128, NO. 33, 2006 10941

A R T I C L E S
Ito et al.
Synthesis of Phenothiazine-10-acetic Acid Succinimidyl Ester.
Phenothiazine-10-acetic acid (329 mg, 1.28 mmol), N-hydroxysuccin-
imide (177 mg, 1.54 mmol, Nacalai), and ethyl-3-(3-dimethylamino-
propyl)carbodiimide (294 mg, 1.54 mmol, Nacalai) were dissolved in
dry DMF under a N2 atmosphere. The solution was stirred at room
temperature overnight. The solution was extracted with ethyl ether (30
mL) and water, washed with water (10 mL × 2), and then dried over
MgSO4. Solvent was removed under reduced pressure. Silica gel column
chromatography (1:0-2:1 hexane/ethyl acetate) gave a white solid
saturated buffer (10 mM phosphate buffer (pH 7.0) with 90 mM NaCl)
by heating to 90 °C, followed by slow cooling to room temperature.
The solution in a 1.5-mL eppendorf tube was exposed to 365 nm UV
light with a FTI-20L transilluminator (Funakoshi, Tokyo) at 4 °C. All
reaction mixtures were precipitated by addition of herring sperm DNA
(1 mg/mL), 3 M sodium acetate (pH 5.2), and ethanol. The precipitated
DNA was dissolved in 10 vol % piperidine, heated at 90 °C for 20
min, and then dried under reduced pressure. The radioactivity of the
samples was assayed using an Aloka 1000 liquid scintillation counter
(Aloka, Tokyo), and the dried DNA pellets were resuspended in loading
buffer (80 vol % formamide, 1 mM EDTA, 0.1% xylene cyanol, and
0.1% bromophenol blue). All reaction mixtures, along with a Maxam-
Gilbert G+A sequencing reaction, were heat-denatured at 90 °C for 3
min and quickly chilled on ice. The samples (1.0 µL, (5-20) × 103
cpm) were loaded onto a 15% polyacrylamide (acrylamide-bisacryl-
amide 19:1) gel containing 7 M urea, electrophoresed at 1900 V for
approximately 80 min, transferred to a cassette, and stored at -80 °C
with Fuji X-ray films (RX-U). Cleavage of the labeled strand was
quantified by autoradiography using ATTO Densitograph software
(version 3.0).
1
(72%). H NMR (CDCl3): 7.17-7.08 (m, 4H), 6.96-6.92 (m, 2H),
6.73 (d, J ) 8.2 Hz, 2H), 4.85 (s, 2H), 2.87 (s, 4H). 13C NMR: 168.64,
165.48, 143.13, 127.49, 126.90, 123.40, 123.35, 114.63, 48.62, 25.54.
HR-FAB MS (matrix: 3-nitrobenzyl alcohol) m/z: (M+) calcd for
C18H14N2O4S 354.0674, found 354.0671.
Synthesis of Oligodeoxynucleotides Containing Phenothiazine.
Oligodeoxynucleotides with an amino-linker C6 at the 5′-termini (ODN
1-3) were commercially synthesized (Sigma) and desalted by a size
exclusion chromatograph column (MicroBioSpin 6, Biorad). To an
acetonitrile solution (200 µL) of N-hydrosuccinimide derivatized
phenothiazine (3.0 mg, 8.4 µmol) was added ODN 1, 2, or 3 (200 µM,
200 µL) and 100 µL of saturated NaHCO3, and the solution was
incubated at 37 °C for 24 h. Monitoring of the coupling reaction and
purification of the conjugate were carried out by the use of HPLC
(Hitachi D-7000) with a 5-50% (over 45 min) gradient of MeCN/100
mM TEAA buffer (pH 5.0). The purified DNAs were characterized by
MALDI-TOF mass spectrometry; m/z: calcd for PTZ-ODN 1
(C146H182N53O77P13S) 4346.04, found 4345.81; calcd for PTZ-ODN 2
(C145H183N48O80P13S) 4313.00, found 4312.39; calcd for PTZ-ODN 3
(C195H242N76O107P18S) 5952.05, found 5951.43.
Acknowledgment. This research was partially supported by
the Ministry of Education, Science, Sports and Culture, Grant-
in-Aid for Young Scientists (B). We also gratefully acknowledge
the Japan Securities Scholarship Foundation and the Japan
Atomic Energy Research Institute for financial support of this
work. We thank Professors Steven E. Rokita, Hiroshi Ide, and
Kazuhito Tanabe for helpful discussions.
Supporting Information Available: Melting curves of oli-
godeoxynucleotide duplexes, Stern-Volmer plot for fluores-
cence quenching of DMA by thymidine glycol, time-dependent
transient absorption of 6-hydroxy-5,6-dihydrothymidin-5-yl
radical, and polyacrylamide gel analysis (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
Photolysis and Polyacrylamide Gel Electrophoresis Analysis of
DNA. The oligonucleotides (20 µL, 400 pmol) were 5′-32P labeled with
4 µL of [γ-32P]ATP (Amersham Bioscience, 10 mCi/mL) using T4
polynucleotide kinase (Nippon Gene) following the manufacture’s
instruction. Labeled DNA samples were recovered by ethanol precipita-
tion, further purified by a 15% polyacrylamide gel, and extracted by
the crush and soak method. Duplex DNA (1 µM) containing the labeled
and the complementary PTZ-containing strand was annealed in a N2-
JA061304+
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10942 J. AM. CHEM. SOC. VOL. 128, NO. 33, 2006