Photoinduced electron injection into DNA by N-cyclopropyl-1- aminonaphthalene

Article abstract of DOI:10.1016/j.jphotochem.2011.01.025

DNA containing either N-cyclopropyl-1-aminonaphthalene (cAN) or N-methyl-1-aminonaphthalene (mAN) as a photoinduced electron donor have been developed to investigate the electron injection and charge recombination processes in DNA duplex. Oxidation potentials of the photo-excited aminonaphthalenes (ANs) were estimated to be approximately -3.0 V vs. SCE, which are high enough to induce one-electron reduction of DNA bases. Photoinduced excess electron transfer in the AN-tethered DNA duplexes has been examined using gel electrophoresis, but the apparent electron-transfer efficiencies were almost the same for both of the cAN- and the mAN-tethered DNA. The marginal effect of cyclopropyl substitution on the efficiency of charge separation suggests that charge recombination in the contact radical ion pair is faster and more efficient than cyclopropane-ring opening.

Full text of DOI:10.1016/j.jphotochem.2011.01.025

Journal of Photochemistry and Photobiology A: Chemistry 219 (2011) 115–121
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Photoinduced electron injection into DNA by N-cyclopropyl-1-aminonaphthalene
Takeo Ito∗, Tsukasa Uchida, Kazuhito Tanabe, Hisatsugu Yamada, Sei-ichi Nishimoto∗
Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
DNA containing either N-cyclopropyl-1-aminonaphthalene (cAN) or N-methyl-1-aminonaphthalene
(mAN) as a photoinduced electron donor have been developed to investigate the electron injection and
charge recombination processes in DNA duplex. Oxidation potentials of the photo-excited aminonaph-
thalenes (ANs) were estimated to be approximately −3.0 V vs. SCE, which are high enough to induce
one-electron reduction of DNA bases. Photoinduced excess electron transfer in the AN-tethered DNA
duplexes has been examined using gel electrophoresis, but the apparent electron-transfer efficiencies
were almost the same for both of the cAN- and the mAN-tethered DNA. The marginal effect of cyclo-
propyl substitution on the efficiency of charge separation suggests that charge recombination in the
contact radical ion pair is faster and more efficient than cyclopropane-ring opening.
Received 30 November 2010
Received in revised form 19 January 2011
Accepted 31 January 2011
Available online 4 February 2011
Keywords:
Photoinduced electron transfer
Aminonaphthalenes
Charge recombination
Radical ions
© 2011 Elsevier B.V. All rights reserved.
5-Bromouracil
DNA
1. Introduction
In the current study, we have developed DNA duplexes contain-
ing N-cyclopropyl-1-aminonaphthalene (cAN) as a photoinduced
It has been demonstrated that DNA undergoes reduction by
hydrated electrons generated in the radiation-induced ionization
of water molecules [1] to afford reductively damaged base struc-
tures such as 5,6-dihydrothymine [2,3]. Thus far, many studies have
been performed to elucidate the mechanism of DNA damage by
radiation-chemically generated hydrated electrons or externally
added photoinduced electron donors [2–6]. Site-specific injection
of electrons into the DNA duplex has been attempted with the aim
of investigating DNA-mediated excess electron transfer reactions;
such site-specific injection can be accomplished by incorporating
photoinduced electron donors into the DNA strand via covalent
linkers [7–27]. In a previous study, we found that electrons injected
from an external free donor can induce the reduction of 5,6-
thymine glycol to thymine, but those injected from a covalently
linked electron donor cannot. This difference can be explained by
the rapid charge recombination of the injected electron and the rad-
ical cation formed from the internal electron donor. On the other
hand, charge recombination between the excess electron and the
unanchored donor radical cation is less likely [7]. Recent studies on
excess electron transfer in DNA have suggested that back electron
transfer occurs rapidly and efficiently, except when deep electron
traps exist in the DNA sequences [21–23,26].
electron donor and investigated the effects of the N-cyclopropyl
substitution on the apparent electron transfer efficiency through
DNA; the radical cation of this donor can undergo spontaneous
ring opening. We expected that the modification on the electron
donor would not retard back electron transfer, if quite fast and effi-
cient charge recombination process are involved in the reactions.
The method using chemical probes to quantify the electron transfer
products is useful for determining relative charge-transfer proper-
ties of DNA duplexes at a time and would provide complementary
data to those obtained from kinetic analyses of the process using
time-resolved spectroscopy techniques. N-Cyclopropyl-modified
DNA bases have been previously used to trap holes migrating along
DNA sequences [28–30].
2. Experimental
2.1. General methods
Reagents were purchased from various commercial sources and
used without additional purification. NMR spectra were recorded
on a JEOL JNM-AL 300 or a JEOL EX 400 spectrometer and chem-
ical shifts are expressed in ppm relative to the residual signals
of chloroform (ı = 7.24 in 1H NMR and ı = 77.0 in 13C NMR). Fast
atom bombardment (FAB) mass spectrometry was performed with
a JEOL JMS-SX 102A mass spectrometer, using nitrobenzyl alco-
hol as a matrix. Electrospray ionization (ESI) mass spectrometry
∗
Corresponding authors. Tel.: +81 75 3837054; fax: +81 75 3832504.
E-mail addresses: takeoit@scl.kyoto-u.ac.jp (T. Ito), nishimot@scl.kyoto-u.ac.jp
(S.-i. Nishimoto).
1010-6030/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotochem.2011.01.025

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T. Ito et al. / Journal of Photochemistry and Photobiology A: Chemistry 219 (2011) 115–121
Scheme 1. Synthesis of N-alkylaminonaphthalene derivatives (cAN, mAN and iAN).
was carried out with a Bruker Daltonics microTOF spectrometer.
DNA synthesis was performed with an Applied Biosystems 3400
DNA synthesizer. Analytical and preparative high-performance liq-
uid chromatographies (HPLCs) were carried out with a Hitachi
D2000 HPLC system equipped with a reversed phase column
(Inertsil ODS-3, GL Science Inc., ␾ 4.6 mm × 250 mm). Fluores-
cence measurement was carried out on a Shimadzu RF-5300PC
spectrofluorophotometer. UV–visible spectra were obtained using
a JASCO V-530 UV/VIS spectrophotometer. Thermal melting of
duplex DNA was observed with a JASCO UV–VIS Spectrophotome-
ter V-530 by monitoring absorbance at 260 nm with a heating
rate of 1 ◦C/min. The temperature at the midpoint of the transi-
tion was defined as the melting temperature (Tm). Solutions of
oligodeoxynucleotides (1 ␮M) in 10 mM sodium phosphate and
200 mM sodium chloride (pH 7.0) were prepared to determine
the melting temperatures. The voltammetric measurements were
made under potentiostatic conditions with a three-electrode sys-
tem consisting of an ALS/CH Instruments 660B electrochemical
analyzer (Austin, TX), a platinum wire counter electrode, a platinum
working electrode, and a saturated calomel reference electrode
(BAS Inc., Tokyo). Aminonaphthalene derivatives (0.5 mM) in phos-
phate buffered saline (10 mM phosphate, 137 mM NaCl, and 2.7 mM
KCl, pH 7.4) containing 1 vol% acetonitrile were used for the AC
voltammetry measurements. The laser flash photolysis experi-
ments were carried out with a Unisoku TSP-601 flash spectrometer.
A Continuum Surelite-I Nd:YAG (Q-switched) laser with a emis-
sion at 355 nm was employed for the photolysis. The probe beam
from a Hamamatsu 150 W Xe short arc (CA 263) was guided with
an optical fiber scope to be arranged in an orientation perpendic-
ular to the exciting laser beam. The probe beam was monitored
with a Hamamatsu R2949 photomultiplier tube through a Hama-
matsu S3701-512Q MOS linear image sensor (512 photodiodes).
Timing of the exciting pulsed laser, the probe beam, and the
detection system was achieved through a Tektronix model TDS
320 double channel oscilloscope that was interfaced to a Dell
PC.
2.2. Synthesis (Scheme 1)
2.2.1. N-Cyclopropyl-1-naphthylamine (cAN) [31]
To a solution of 1-bromonaphthalene (1.47 g, 7.09 mmol) and
cyclopropylamine (537 mg, 9.38 mmol) in 15 mL of anhydrous
toluene were added tris(dibenzylideneacetone)dipalladium(0)
(42.8 mg, 0.046 mmol),
( )-BINAP (58.8 mg, 0.094 mmol) and
sodium t-butoxide (794 mg, 8.26 mmol). The mixture was stirred
under an N2 atmosphere at 80 ◦C for 4 h. The resulting mixture was
then cooled, diluted with chloroform, filtered through Celite and
concentrated under vacuum. The crude product was purified by sil-
ica gel column chromatography (30:1 chloroform/acetone) to give
cAN (1.20 g, 92%) as a yellow oil.
2.2.2. N-(Benzotriazol-1-ylmethyl)-1-naphthylamine (2) [32]
To a solution of 1-naphthylamine (1, 1.77 g, 12.4 mmol) and
benzotriazole (1.56 g, 13.1 mmol) in 40 mL of ethanol was added
37% aqueous formaldehyde (1.0 mL, 12.3 mmol). The solution was
stirred under an N2 atmosphere at room temperature for 1 h. White
solid obtained after evaporation was used for the next reaction
without purification.
2.2.3. N-methyl-1-naphthylamine (mAN) [32]
To a solution of compound 2 (1.77 g, 12.4 mmol) in 40 mL
of tetrahydrofuran was added sodium borohydride (2.31 g,
61.0 mmol) over a 2-h period at 20 ◦C with vigorous stirring. The
mixture was kept well-stirred for another 3 h. Solvent was removed
under reduced pressure. The residue was extracted with ethyl
acetate, washed with aqueous NaHCO3 and brine, dried over MgSO4
and concentrated under vacuum. The crude product was purified
by silica gel column chromatography (10:1 hexane/ethyl acetate)
to give mAN (777 mg, 40%) as a pale pink oil.
2.2.4. N-isopropyl-1-naphthylamine (iAN) [33]
To a solution of 1-naphthylamine (1, 1.44 g, 10.0 mmol) and
sodium cyanoborohydride (629 mg, 10.0 mmol) in 10 mL of ace-
tone was added acetic acid (2.0 ml, 35.0 mmol) over a 5-min period.

T. Ito et al. / Journal of Photochemistry and Photobiology A: Chemistry 219 (2011) 115–121
117
The mixture was stirred at 0 ◦C for 2 h under an N2 atmosphere.
The solvent was removed under reduced pressure. The residue was
neutralized with NaHCO3 and extracted with ethyl acetate, washed
with brine, dried over MgSO4 and concentrated under vacuum. The
crude product was purified by silica gel column chromatography
(10:1 hexane/ethyl acetate) to give iAN (1.75 g, 94%) as a colorless
oil.
54.8, 33.4, 25.5, 8.71; HR-MS (FAB-pos) m/z calcd. for C19H19N2O4:
339.1345; found 339.1340 (M+H+).
2.2.8. Ethyl (N-methyl-N-naphthalen-1-yl-amino)acetate (5)
Starting with the ethyl ester 3 and formaldehyde, the same pro-
cedure as for 4 gave (methyl-naphthalen-1-yl-amino)acetic acid
ethyl ester 5 (yield 94%) as a bright red oil. 1H NMR ((CDCl3) ı 8.23
(dd, 1H, J = 7.56, 0.73 Hz), 7.82 (dd, 1H, J = 9.03, 1.95 Hz), 7.37–7.56
(m, 4H), 7.19 (d, 2H, J = 9.03 Hz), 4.21 (q, 2H, J = 7.08 Hz), 3.93 (s, 2H),
3.04 (s, 3H), 1.26 (t, 3H, J = 7.08 Hz); 13C NMR ı 170.8, 148.7, 134.8,
128.6, 128.3, 125.7, 125.5, 125.4, 123.6, 123.4, 115.9, 60.6, 58.7,
41.7, 14.2; HR-MS (FAB-pos) m/z calcd. for C15H18N1O2: 244.1337;
found 244.1331 (M+H+).
2.2.5. Ethyl (N-naphthalen-1-yl-amino)acetate (3)
A solution of 1-naphthylamine (1, 2.03 g, 14.2 mmol) and ethyl
chloroacetate (1.92 g, 15.7 mmol) in 5 mL of ethanol was stirred at
80 ◦C for 14 h under an N2 atmosphere. The solvent was removed
under reduced pressure. The crude product was purified by silica
gel column chromatography (60:1 chloroform/acetone) to give 3
(1.61 g, 50%) as a red oil. 1H NMR ((CDCl3) ı 7.90–7.93 (m, 1H),
7.79–7.83 (m, 1H), 7.44–7.49 (m, 2H), 7.27–7.37 (m, 2H), 6.47 (dd,
1H, J = 6.34, 0.98 Hz), 5.03 (broad, 1H), 4.22–4.32 (m, 2H), 4.04 (2H),
1.32 (3H); 13C NMR ı 171.0, 142.2, 134.2, 128.5, 126.3, 125.8, 124.9,
123.4, 120.0, 118.1, 104.4, 61.3, 45.9, 14.1; HR-MS (FAB-pos) m/z
calcd. for C14H16N1O2: 230.1181; found 230.1186 (M+H+).
2.2.9. (N-Methyl-N-naphthalen-1-yl-amino)acetic acid
succinimidyl ester (mAN-suc)
Starting with 5, the same procedure as for cAN-suc gave mAN-
suc as a brown oil. 1H NMR ((CDCl3) ı 8.19 (dd, 1H, J = 8.08, 1.47 Hz),
7.83 (dd, 1H, J = 8.52, 1.65 Hz), 7.39–7.61 (m, 5H), 4.26 (s, 2H), 3.08
(s, 3H), 2.70 (s, 4H); 13C NMR ı 169.1, 165.9, 147.5, 134.6, 128.4,
125.9, 125.8, 125.5, 125.4, 125.3, 124.1, 123.1, 55.8, 41.5, 25.3;
HR-MS (FAB-pos) m/z calcd. for C17H17N2O4: 313.1188; found
313.1195 (M+H+).
2.2.6. Ethyl (N-cyclopropyl-N-naphthalen-1-yl-amino)acetate
(4)
To
a solution of compound 3 (646 mg, 2.82 mmol), (1-
ethoxycyclopropoxy)trimethylsilane (1.32 g, 7.56 mmol) and
sodium cyanoborohydride (440 mg, 7.00 mmol) in 1.5 mL of
anhydrous methanol was added acetic acid (1.23 g, 20.4 mmol).
The mixture was stirred at room temperature for 18 h under an
N2 atmosphere [34]. The solvent was removed under reduced
pressure. The residue was extracted with ethyl acetate, washed
with brine, dried over anhydrous Na2SO4 and concentrated under
vacuum. The crude product was purified by silica gel column
chromatography (10:1 hexane/ethyl acetate) to give 4 (485 mg,
64%) as a bright red oil. 1H NMR ((CDCl3) ı 7.87–7.90 (m, 1H),
7.61–7.63 (m, 1H), 7.36–7.41 (m, 2H), 7.21–7.28 (m, 3H), 3.89–3.94
(m, 4H), 2.97 (sept, 1H), 1.00 (t, 3H, J = 7.20 Hz), 0.48–0.52 (m, 2H),
0.36–0.40 (m, 2H); 13C NMR ı 171.1, 147.2, 134.4, 128.9, 128.3,
125.2, 125.4, 125.6, 123.4, 123.2, 119.2, 60.1, 56.9, 33.4, 14.0, 8.5;
HR-MS (FAB-pos) m/z calcd. for C17H20N1O2: 270.1494; found
270.1500 (M+H+).
2.3. Synthesis of aminonaphthalene-tethered
oligodeoxynucleotides
Oligodeoxynucleotide containing an amino-linker at their 5ꢀ-
terminus (ODN 1) was synthesized at a 1 ␮mol scale (500 A CPG
˚
column) on an Applied Biosystems (ABI) 3400 DNA synthesizer
using a standard phosphoramidite chemistry. The C3-amino-
linker phosphoramidite was purchased from Glen Research
(5ꢀ-Aminomodifier C3-TFA) and used following the procedure pro-
vided by the supplier. To the succinimidyl ester derivative (cAN-suc
or mAN-suc, 3.0–7.0 mg) in acetonitrile (200 ␮L) were added ODN
1 bearing the C3-amino linker (200 ␮M, 200 ␮L) and 100 ␮L of
saturated aqueous NaHCO3, and the reaction mixture was incu-
bated at 37 ◦C for 24 h. Purification of the conjugates was carried
out by the use of HPLC. ESI-MS (negative): calcd. for cAN–ODN
1 (C196H242N76O106P18) [(M−4H)4−]: 1479.0; found 1478.8;
calcd. for mAN–ODN 1 (C194H240N76O106P18) [(M − 4H)4−]:
1472.5; found 1472.5.
2.2.7. (N-Cyclopropyl-N-naphthalen-1-yl-amino)acetic acid
succinimidyl ester (cAN-suc)
To a solution of compound 4 (485 mg, 1.80 mmol) in 3.5 mL of
1,4-dioxane was added 1 M aqueous sodium hydroxide (2.5 mL,
2.5 mmol). The solution was stirred at room temperature for
8 h. The solvent was removed under reduced pressure. The
residue was acidized with saturated aqueous citric acid and
extracted with ethyl acetate, washed with brine, dried over
Na2SO4 and concentrated under vacuum. Obtained colorless oil
was used for the next reaction without purification. To the product
in 5 mL of anhydrous dimethyl sulfoxide were added 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide hydrochloride (578 mg,
3.02 mmol), 1-hydroxybenzotriazole (397 mg, 2.93 mmol). After
30-min continuous stirring at 0 ◦C, N-hydroxysuccinimide (362 mg,
3.15 mmol) was added to the solution. The reaction mixture
was stirred at room temperature for another 24 h. The reaction
mixture was quenched by adding water, extracted with ethyl
acetate, washed with saturated aqueous NaHCO3 and brine, dried
over Na2SO4 and concentrated under vacuum. The crude product
was purified by silica gel column chromatography (50:1 chloro-
form/acetone) to give cAN-suc (185 mg, 31% in 2 steps) as a white
solid. 1H NMR ((CDCl3) ı 7.94–7.97 (m, 1H), 7.81–7.84 (m, 1H),
7.39–7.60 (m, 5H), 4.38 (s, 2H), 3.11 (sept, 1H, J = 3.30 Hz), 2.76 (s,
4H), 0.71–0.78 (m, 2H), 0.58–0.60 (m, 2H); 13C NMR ı 168.9, 166.1,
146.1, 134.6, 128.8, 128.5, 125.8, 125.7, 125.6, 124.4, 122.8, 119.7,
2.4. Photolysis and polyacrylamide gel electrophoresis.
Oligodeoxynucleotides were 5ꢀ-32P-labeled with [␥-32P]ATP
(Perkin Elmer, 370 MBq/mL) and T4 polynucleotide kinase (Nip-
pon Gene). The labeled mixtures were subsequently centrifuged
through MicroBio-Spin 6 or 30 columns (Biorad) to remove
excess unincorporated nucleotide. Complementary ODNs (1 ␮M,
respectively) were annealed in phosphate buffer (10 mM sodium
phosphate, 90 mM NaCl, pH 7.0) by heating to 90 ◦C, followed by
slow cooling to room temperature. The samples (20 ␮L) in 1.5-
mL eppendorf tubes were exposed to UV light with a LAX-100
Xe lamp (Asahi Spectra) through a UTVAF-50S-36U glass filter
(Sigma Koki) at 4 ◦C. The DNA samples were precipitated by adding
10 ␮L of herring sperm DNA (1 mg/mL), 5 ␮L of 3 M sodium acetate
(pH 5.2), and 400 ␮L of ethanol, and then chilling at −20 ◦C. The
precipitated DNA were dissolved in 10vol% piperidine, heated at
90 ◦C for 20 min, and then dried under reduced pressure. The
radioactivities of the samples were assayed using an Aloka 1000
liquid scintillation counter (Aloka), and the dried DNA pellets
were resuspended in loading buffer (8 M urea, 40% sucrose, 0.025%
xylene cyanol, 0.025% 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

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T. Ito et al. / Journal of Photochemistry and Photobiology A: Chemistry 219 (2011) 115–121
Fig. 1. UV-absorption (dotted lines) and fluorescence (solid lines, ꢀex = 312 nm)
spectra of cAN, cAN, and iAN in deoxygenated aqueous buffer containing 0.1% ace-
tonitrile.
Fig. 3. Transient absorption spectra of the intermediates as observed after the 355-
nm laser photolysis of cAN (upper) and mAN (lower) (0.1 mM) in the presence of
1,4-dicyanobenzene (1 mM) in Ar-saturated acetonitrile.
(2.0 ␮L, (5 − 10) × 103 cpm) were loaded onto a 20% polyacrylamide
(acrylamide–bisacrylamide 19:1) gel containing 7 M urea, elec-
trophoresed at 1700 V for approximately 60 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). Individual yields were calcu-
lated relative to each total band intensity. The averages of three
independent measurements for each sample were indicated.
voltammetry [35]. Excited state oxidation potential (Eo∗x) can be
calculated using Eo∗x = Eox − E0–0 with the oxidation potential of
1-aminonaphthalene (Eox) and zero excitation energy evaluated
from ꢀ0–0 (=371 nm) in aqueous solution. The excited-state oxida-
tion potentials of ANs were thus calculated to be approximately
−3.0 V, which is sufficiently high for thymidine (Ered = − 2.35 V)
and 2ꢀ-deoxycytidine (Ered = − 2.48 V) reduction [22]. Indeed, fluo-
rescence intensities of ANs monitored in the presence of thymidine
decreased as increasing the concentration of thymidine (data not
shown), which suggests that photo-excited ANs can induce one-
electron reduction of thymidine. It seemed that bulkiness of the
substituent on the amino group have little effect on their reducing
properties, thus we proceeded the following experiments for cAN
and mAN derivatives.
3. Results and discussion
3.1. Photophysical properties of aminonaphthalene derivatives
Before
synthesizing
cyclopropylaminonaphthalene-
tethered DNA, we investigated photophysical properties
of three 1-aminonaphthalene (AN) derivatives in aqueous
solution. N-Cyclopropyl-1-aminonaphthalene cAN, N-methyl-
1-aminonaphthalene mAN, and N-isopropyl-1-aminonaphthalene
iAN were synthesized according to previously reported procedures
[31–33]. Fig. 1 shows the UV-absorption and fluorescence spectra
of the naphthalene chromophores in 0.1% acetonitrile–water.
The spectra of all the derivatives were similar with ꢀmax at
approximately 320 nm (for absorption) and ꢀflmax at approximately
440 nm (for fluorescence). As shown in Fig. 2, the ground-state
oxidation potentials of ANs were determined to be +0.38 V vs.
SCE (cAN), +0.27 V (mAN), and +0.30 V (iAN) by employing AC
Ring opening of the cyclopropyl radical cation of cAN in aqueous
buffer was observed by nanosecond laser flash photolysis (Fig. 3).
Excitation of mAN with a 355-nm Nd:YAG laser in the presence
of 1,4-dicyanobenzene as an electron acceptor resulted in the for-
mation of transient species (ꢀmax; around 580 nm) within 1 ␮s after
the pulse. This absorption spectrum is quite similar to that observed
for the 1-aminonaphthalene radical cation [36] and hence, it can be
assigned to the relatively stable mAN radical cation (mAN+•). On the
other hand, no such broad absorption at 580 nm was detected in the
photolysis of cAN. Dynamic absorption spectra of the ring-opening
products have not been reported so far, but it is proposed that spon-
taneous ring opening of the cyclopropylamine radical cation affords
an iminium carbon radical as an intermediate. Considering that the
spectra obtained after excitation showed no remarkable changes
in the time region of 0.1–100 ␮s, the observed spectra might be
assigned to the iminium carbon radical or more stable final prod-
ucts. It is thus estimated that decomposition of cAN radical cation
(cAN+•) proceeds with a rate constant of k > 107 s−1
.
3.2. Electron injection from photo-excited ANs onto DNA
To synthesize AN-tethered oligodeoxynucleotides, succinimidyl
esters of cAN and mAN were prepared from 1-aminonaphthalene
(Scheme 2). Ethyl ester derivatives (4 and 5) obtained by reductive
amination were derivatized into succinimidyl esters by follow-
ing a standard procedure. The resulting succinimidyl esters were
incubated with oligodeoxynucleotide containing a 5ꢀ-amino linker
(ODN 1, Fig. 4) at ambient temperature to afford cAN- (cAN–ODN
1) or mAN- (mAN–ODN 1) tethered DNA. Complementary strand
containing 5-bromouracil (BrU) as the electron-transfer probe in
Fig. 2. AC voltammographs of (ꢁ) cAN, (ꢂ) mAN, and (×) iAN measured in phos-
phate buffered saline (10 mM phosphate, 137 mM NaCl, and 2.7 mM KCl, pH 7.4)
containing 1 vol% acetonitrile.

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119
Scheme 2. Synthesis of N-alkylaminonaphthalene derivatives (4 and 5).
the sequence (ODN 2) was synthesized using a DNA synthesizer.
The thermal stabilities of the duplexes were evaluated on the basis
of their thermal melting temperatures (Tm). Tm of the unmodi-
fied ODN 1/ODN 2 duplex was 67.5 ◦C, which was close to that
of cAN–ODN 1/ODN 2 (68.6 ◦C) and mAN–ODN 1/ODN 2 (68.4 ◦C).
The stabilization effect by ANs indicates that the naphthalene rings
directly interact with DNA, although binding mode of the chro-
mophores is not clear at this point. It has been reported that
anchored aminonaphthalene partly stabilizes duplex DNA [18,20].
For a quantitative analysis of the excess electron transfer in
DNA, the 5ꢀ-terminus of ODN 2 was 32P-labeled, and subjected to
photochemical analysis. To avoid undesirable photo-excitation of
DNA bases and BrU, UV-light through a glass filter (320–390 nm)
was employed for the photoinduced electron-injection experi-
ment. The duplexes (cAN–ODN 1/ODN 2 and mAN–ODN 1/ODN
2) in a N2 atmosphere were exposed to UV-light, then treated
with piperidine at 90 ◦C. Because oxygen molecules could quench
excess electrons on the DNA duplex, we examined the experiments
under N2-saturated conditions. The base-catalyzed hydrolysis pro-
cess yielded DNA fragments those correspond to the products
obtained when the DNA strand was cleaved at the nucleobase
adjacent to the 5ꢀ-end of BrU (Fig. 5). Electron capture by BrU
affords a uracil 5-yl radical, which in turn abstracts a hydrogen
from the 5ꢀ-adjacent deoxyribose. The resulting intermediates are
considered to be alkali-labile [18,37]. The intensity of the cleav-
age bands increased with the irradiation time, suggesting that
electrons could be injected into the DNA duplexes by photo-
excitation of the AN derivatives. In the absence of either AN or
BrU, strand cleavage at the corresponding sites was negligible
(see Supplementary data, Fig. S1). As can be seen in Fig. 5, the
yields of the electron-transfer fragments from cAN–ODN 1/ODN
2 and mAN–ODN 1/ODN 2 were almost equal (5.4 0.4%/30 min
for cAN–ODN 1/ODN 2, 6.6 0.6%/30 min for mAN–ODN 1/ODN
2). If the rapid cyclopropane-ring-opening of cAN competes with
the charge recombination between the injected electrons and the
AN radical intermediates, the yields of electron-transfer products
should be higher for cAN–ODN 1/ODN 2 than mAN–ODN 1/ODN
Fig. 5. Polyacrylamide gel electrophoresis image of (lanes 1–7) cAN–ODN 1/ODN 2
and (lanes 8–14) mAN–ODN 1/ODN 2. Duplexes (1 ␮M) in NaCl (90 mM) and phos-
phate buffer (10 mM, pH 7.0) were UV-irradiated at 4 ◦C for 0–30 min under an N2
atmosphere, followed by piperidine treatment (90 ◦C, 20 min). The arrow indicates
the fragment bands generated as a result of electron capture by BrU.
2. Thus, our results suggest that very fast back electron transfer is
involved in the reaction pathways.
Photo-excitation of the AN moiety in DNA results in the forma-
tion of a contact radical ion pair of AN and a nucleobase (Scheme 3).
It has been proposed that some of the injected electrons migrate
along the duplex via a thermally activated hopping mechanism
at ambient temperature [9,12,38]. These negative charges may
move back to the radical cation of AN (AN+•), unless they are irre-
versibly trapped by BrU. Release of Br− from the radical anion
of BrU (BrU−•) proceeds with a rate constant of k ∼ 108 s−1 [39],
which might be slower in duplex DNA due to stabilization of the
excess electron by hydrogen bonding interaction between BrU−•
Scheme 3. Photoinduced charge separation and recombination processes in the
DNA conjugates.
Fig. 4. Sequences of oligodeoxynucleotides.

120
T. Ito et al. / Journal of Photochemistry and Photobiology A: Chemistry 219 (2011) 115–121
and the complementary adenine [40]. As has been demonstrated
in a recent research [21–23,26], charge recombination in a contact
ion pair (kCR1) occurs rapidly (within a few picoseconds), while
back electron transfer from a charge separated state (kCR2) may
be slow enough to compete with the ring opening of cAN+•. Rate
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constant for excess electron transfer between each base pair (kET
)
has been estimated to be ∼108 s−1 [10,26]. In view of the marginal
effect of the ring opening in cAN-tethered DNA as shown above,
it is presumable that charge recombination in the contact ion
pair is quite efficient. This is supported by the fact that the dis-
tance between the AN and BrU units has a negligible effect on the
apparent electron transfer efficiency, as has been observed in the
experiments performed using DNA containing a 2-basepairs-longer
bridge sequence (see Supplementary data, Fig. S2). There is some
controversy about quantification of hole migration through DNA
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in DNA, especially when the rate constants for product formation
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4. Conclusion
In summary, we have synthesized cAN-tethered oligodeoxynu-
cleotides to investigate the mechanism of excess electron transfer
in DNA. Almost no effect of cyclopropyl substitution on the forward
electron transfer to BrU suggests that charge recombination in the
contact-ion-pair state is more efficient than the further hopping
of electrons along the duplex. We believe that the results of this
study would inspire the design of new DNA materials that facilitate
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Acknowledgements
The authors thank Professor Steven Rokita for helpful com-
ments. This work was supported by Grant-in-Aid for Young
Scientists (B) and by Grant-in-Aid for the Global COE program,
“International Center for Integrated Research and Advanced Edu-
cation in Material Science” from the Ministry of Education, Culture,
Sports, Science and Technology of Japan. A part of this study was
done under the support of the Radioisotope Research Center, Kyoto
University.
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Appendix A. Supplementary data
cyclopropyldeoxyadenosine suggests
a direct contribution of adenine
bases to hole transport through DNA, J. Am. Chem. Soc. 125 (2003) 10154–
10155.
Supplementary data associated with this article can be found, in
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