Modulated drug release from the stem-and-loop structured oligodeoxynucleotide upon UV-A irradiation in the presence of target DNA

Article abstract of DOI:10.1039/b510608g

o-Nitrobenzyl photochemistry as induced by UV-A irradiation was applied to a photoactivated drug releasing system based on a molecular beacon strategy. A stem-and-loop structured oligodeoxynucleotide (ODN) possessing a photoreactive o-nitrobenzyl chromoph

Full text of DOI:10.1039/b510608g

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A R T I C L E
Modulated drug release from the stem-and-loop structured
oligodeoxynucleotide upon UV-A irradiation in the presence
of target DNA
Kazuhito Tanabe,* Hiroyuki Nakata, Shin Mukai and Sei-ichi Nishimoto*
Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering,
Kyoto University, Kyoto, 615-8510, Japan.
E-mail: tanabeka@scl.kyoto-u.ac.jp, nishimot@scl.kyoto-u.ac.jp; Fax: +81 75 383 2501;
Tel: +81 75 383 2500
Received 26th July 2005, Accepted 31st August 2005
First published as an Advance Article on the web 26th September 2005
o-Nitrobenzyl photochemistry as induced by UV-A irradiation was applied to a photoactivated drug releasing system
based on a molecular beacon strategy. A stem-and-loop structured oligodeoxynucleotide (ODN) possessing a
photoreactive o-nitrobenzyl chromophore at the 3ꢀ-end and 1-aminonaphthalene quencher at the 5ꢀ-end underwent
conformational change into a conventional double strand structure by hybridization with a specified target
DNA. The intrinsic stem-and-loop structure suppressed photoactivated release of benzoic acid as a phantom drug
from the o-nitrobenzyl chromophore because of intramolecular quenching by the 1-aminonaphthalene unit in close
proximity to the chromophore. Formation of the double strand structure in the presence of perfectly matched target
DNA minimized occurrence of intramolecular quenching and thereby enhanced the photoactivated drug release.
aminonaphthalene unit that was located in close proximity to
Introduction
the o-nitrobenzene chromophore in the stem-and-loop structure.
Smart drugs, which manifest a specific activity according to
In contrast, the presence of complementary target DNA could
individual genetic information, are of particular significance for
switch over the stem-and-loop structure of the photoreactive
selective treatment of diseased cells.1 This family of drugs can
ODN to an ordinary double-strand structure by hybridization
mediate effective medical treatment of diseased cells without
with target, thereby resulting in the efficient photochemical drug
inducing side-effects in normal cells. In this view, development
release with decreased extent of intramolecular quenching due to
of such drugs is recognized as the ultimate goal of chemotherapy.
A new strategy for creating these functional drugs involves the
separation of the o-nitrobenzene and 1-aminonaphthalene units.
In addition, we obtained the first evidence that the enhanced
combination of a prescribed DNA hybridization sensor with a
drug release could not occur even in the presence of DNA with
drug releasing system to set up a prodrug, the activation of which
a single base mismatch.
is modulated by sensing its target DNA sequence.2 Recently, an
attempt was made to develop a DNA sequence-specific drug-
Results and discussion
releasing system, using a photoreactive oligodeoxynucleotide
(ODN) possessing phenacyl ester and naphthalene derivatives.3
This photoreactive ODN consisted of a stem-and-loop structure
to modulate the photoactivated drug release by a molecular
beacon strategy.4 Although the photoreaction of the phenacyl
ester attachment for drug release could be effectively modulated
whether or not there was target DNA present, the drawback
to the previous photoreactive ODN was that UV-B irradiation
(290–320 nm) is required for photoactivation and thus induces
direct oxidative stress on normal cells to invoke a strong
cytotoxic and mutagenic effect.5 Furthermore, UV-B radiation
has the demerit of less penetration through the epidermis and
the upper dermis due to shorter wavelength. Therefore, an
alternative photoactivation system with longer wavelengths of
light to achieve deeper penetration through skin and avoid
unfavorable direct radiation effect on normal tissues is essential.
We report here construction of a photoactivated drug re-
leasing system based on a molecular beacon strategy and
o-nitrobenzyl photochemistry,6 using UV-A irradiation (320–
400 nm) that does not cause direct excitation of DNA.7,8 We
synthesized a photoreactive ODN possessing an o-nitrobenzyl
chromophore, which released benzoic acid as a phantom
drug compound upon photoirradiation at 365 nm, and a
1-aminonaphthalene unit with an intramolecular quench-
ing function.9 Upon photoirradiation of the o-nitrobenzyl
chromophore attached to the photoreactive ODN in the
absence of complementary target DNA, the drug release
was effectively suppressed because of quenching by the 1-
The synthesis of photoreactive ODN is outlined in Scheme 1.
The o-nitrobenzyl ester 6 possessing a phantom drug com-
pound of benzoic acid, and succinimidyl ester as connected to
amino-modified DNA was synthesized from a 4-nitrophenol
derivative 1. A primary alcohol 8 prepared from N-methyl-
1-aminonaphthalene 710 was converted to phosphoramidite
9, followed by introduction into DNA by automated DNA
synthesis. Incorporation of the o-nitrobenzyl chromophore into
DNA was achieved by coupling of the amino group in 10 and 6.
The formation of modified ODN was confirmed by MALDI–
TOF mass spectrometry. The structures of the ODNs used in
this study are shown in Fig. 1.
Fig. 1 Structure and sequences of oligodeoxynucleotides (ODNs) used
in this study.
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 8 9 3 – 3 8 9 7
3 8 9 3
©

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Scheme 1 Reagents: (a) 2-bromo-1-tert-butyldimethylsilyloxyethane, K2CO3, DMF, 76%; (b) NaBH4, THF, 74%; (c) benzoyl chloride, Et3N,
CH2Cl2, 74%; (d) TBAF, acetic acid, THF, 94%; (e) N,Nꢀ-disuccinimidyl carbonate, Et3N, CH3CN, 66%; (f) 2-bromoethanol, Et3N, toluene, 88%; (g)
(iPr2N)2PO(CH2)2CN, 1H-tetrazole, acetonitrile, quant.; (h) DNA autosynthesizer, then 28% ammonia; (i) 6, satd NaHCO3 aq., acetonitrile–water
(1 : 1).
To characterize the functionality of 1-aminonaphthalene as an
intramolecular quencher influencing on the photoactivated re-
lease of benzoic acid, we performed photoirradiation at 365 nm
of ODN 1 in 10 mM sodium cacodylate (pH 7.0) for 30 min at
various temperatures in the range 0–60 ◦C. As shown in Fig. 2,
photoactivated drug release was suppressed at temperatures
lower than 20 ◦C, where ODN 1 would favor the stabilized
stem-and-loop structure. In contrast, the elevated temperatures
enhanced the drug releasing efficiency to a substantial amount
up to a five-fold increase at 60 ◦C. These results strongly suggest
that the 1-aminonaphthalene attachment to the 5ꢀ-end of ODN
1 can display an effective intramolecular quenching ability for
the photochemical reaction of the o-nitrobenzyl chromophore in
the stem-and-loop structure stabilized at lower temperatures, in
which the o-nitrobenzyl chromophore remains in close proximity
to the 1-aminonaphthalene attachment. At higher temperatures,
however, such a quenching by 1-aminonaphthalene becomes
minor to induce efficient release of benzoic acid, because the
stem strand dissociates to separate the average distance between
chromophore and quencher.11
To identify the functionality of the loop strand to hybridize
with its target DNA possessing a specified base sequence, we
further compared photoactivated releases of benzoic acid from
ODN 1 at 0 ◦C in the presence and absence of complementary
DNA. The time courses of the photoreactions are shown in
Fig. 3. ODN 1 released benzoic acid about twice as efficiently
in the presence of complementary ODN 2 as in its absence. This
result indicates that hybridization of ODN 1 with ODN 2 into a
duplex structure gives rise to separation of the o-nitrobenzyl
chromophore and 1-aminonaphthalene quencher. A smaller
efficiency of the apparent photoactivated release of benzoic acid
from ODN 1 in the presence of ODN 2 at 0 ◦C (Fig. 3), relative
to the heat denatured ODN 1 at 60 ◦C (Fig. 2), suggests that the
stem-and-loop formation of ODN 1 may still occur to a con-
siderable extent at lower temperatures in competition with the
duplex formation between ODN 1 and ODN 2. In the separate
experiments, we also confirmed that the presence of a single-base
mismatched ODN 3 or a non-complementary ODN 4, instead
of the complementary ODN 2, was ineffective for the enhanced
release of benzoic acid, the efficiency of which was practically
the same as in the photoirradiation of ODN 1 alone. Thus, the
characteristic photoreactivity of ODN 1 with a stem-and-loop
structure could discriminate a single-base mismatched DNA.
Fig. 2 Influence of the reaction temperature on the photoactivated
release of benzoic acid from ODN 1. Irradiation of 10 lM ODN 1 with
365 nm light obtained from transilluminator was carried out in 10 mM
sodium cacodylate (pH 7.0) for 30 min at various temperatures. The
photoreaction was monitored by reversed-phase HPLC.
3 8 9 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 8 9 3 – 3 8 9 7

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tra were measured with JEOL JNM-AL 300 (300 MHz), JEOL
JMN-EX-400 (400 MHz) or JEOL JNM-A500 (500 MHz)
spectrometers. Mass spectra were recorded on a JEOL JMS-
SX102A spectrometer. Matrix-assisted laser desorption ion-
ization time-of-flight (MALDI-TOF) mass spectrometry of
oligonucleotides were obtained on JEOL JMS-ELITE MALDI-
TOF MASS spectrometer with 2ꢀ,3ꢀ,4ꢀ-trihydroxyacetophenone
as matrix. Calf intestine alkaline phosphotase (AP), snake
venom phosphodiesterase (svPDE) and nuclease P1 (P1) were
purchased from PROMEGA, YAMASA and ICN, respectively.
The oligonucleotide was purchased from INVITROGEN. The
reagents for the DNA synthesizer such as A, G, T, C and 3ꢀ-
Amino-Modifier C7 CPG support were purchased from Glen
Research. The purity and concentration of all oligodeoxynu-
cleotides were determined by complete digestion with svPDE,
AP and P1 to 2ꢀ-deoxymononucleotides. Reversed phase HPLC
was performed on an Inertsil ODS-3 column (4.6 × 150 mm)
with Shimadzu 10A or HITACHI D-7000 HPLC system using a
UV detector at 230 or 260 nm. Photoirradiation at 365 nm was
carried out using TFX-40.M. transilluminator.
Fig. 3 Release of benzoic acid upon photoirradiation of ODN 1.
Photoirradaition at 365 nm of 10 lM ODN 1 for 0, 20, 40, 60 and
120 min at 0 ◦C in 10 mM sodium cacodylate (pH 7.0) was carried out in
the presence of 50 lM complementary ODN 2 (ꢀ), 50 lM single-base
mismatched ODN 3 (ꢁ), or 50 lM noncomplementary ODN 4 (᭹).
Release of benzoic acid upon photoirradiation of ODN 1 alone is also
indicated by (ꢂ). The reaction was monitored by HPLC.
5-[2-(tert-Butyldimethylsilyloxy)ethyloxy]-2-nitrobenzaldehyde (2)
Compared with the previous photo-induced drug release
system with functionalized ODN possessing phenacyl ester as
a chromophore,3 the difference in efficiency between the drug
releases in the presence and absence of complementary DNA
is smaller in the present o-nitrobenzyl photoreaction system.
This may be attributed to the evidence that photoreaction of the
o-nitrobenzyl chromophore occurring both in the singlet and
triplet excited states12 was partly quenched by aminonaphtha-
lene quencher via long-range dipole–dipole interaction.13 Thus,
such a long-range quenching could occur even in the double
strand structure of ODN 1 with complementary ODN 2 to
reduce efficiency of drug release in the present system.
To a solution of 5-hydroxy-2-nitrobenzaldehyde 1 (2.01 g,
12.03 mmol) in dry DMF (30 ml) was added 2-bromoethoxy-
tert-butyldimethylsilane 3 (3.0 g, 12.59 mmol) and K2CO3
(1.89 g, 13.67 mmol), and the mixture was stirred at 80 ◦C
for 1.5 h. The resulting mixture was diluted with sat. NH4Cl
and extracted with ethyl acetate. The extract was washed with
brine, dried over anhydrous MgSO4, filtered, and concentrated in
vacuo. The crude product was purified by flash chromatography
(SiO2, 20% ethyl acetate–hexane) to give 2 (2.97 g, 76%) as a
1
yellow oil: H NMR (CDCl3, 400 MHz) d 10.45 (d, 1H, J =
4.0 Hz), 7.31 (dd, 1H, J = 2.4, 4.0 Hz), 7.15 (dd, 1H, J = 8.8,
2.4 Hz), 4.17 (t, 2H, J = 4.6 Hz), 3.98 (t, 2H, J = 4.6 Hz),
0.86 (s, 9H), 0.06 (s, 6H); 13C NMR (CDCl3, 100 MHz) d 188.3,
163.4, 142.1, 134.2, 127.1, 119.0, 113.9, 70.6, 61.6, 25.9, 18.4,
−5.2; FABMS (NBA) m/z 326 [(M + H)+]; HRMS calcd. for
C15H24NO5Si [(M + H)+] 326.1424, found 326.1422.
Conclusion
In summary, we improved a photoactivated drug releasing
system modulated by a molecular beacon strategy. The presented
molecular system consisted of an o-nitrobenzyl chromophore, a
1-aminonaphthalene quencher, and an ODN with a stem-and-
loop structure, the base sequence of which was so arranged that
it can hybridize with a given target DNA. The ODN underwent
conformational change from a stem-and-loop structure into
double strand structure in the presence of complementary DNA
that had a perfect base match, and thereby resulted in efficient
drug release. Although the present system showed a smaller
extent of enhancement of photo-induced drug release in the
presence of target complementary DNA relative to the previous
system, a major advantage is that we could selectively excite the
o-nitrobenzyl chromophore of functionalized ODN by a less
harmful light of UV-A. In addition, nitrobenzyl photochemistry
employed herein has been demonstrated to be applicable to the
development of photoactivated prodrugs releasing actual drugs
such as 5-fluorouracil, phosphoramide mustard and L-leucyl-L-
leucine methyl ester.6,14 In these views the present system would
be a promising candidate for effective medical treatment without
serious side-effects.
5-[2-(tert-Butyldimethylsilyloxy)ethyloxy]-2-nitrobenzyl alcohol (3)
To a solution of 2 (1.63 g, 5.02 mmol) in THF (25 ml) was
added NaBH4 (188 mg, 4.84 mmol), and the mixture was stirred
at 0 ◦C for 2 h. The resulting mixture was diluted with water
and extracted with ethyl acetate. The extract was washed with
brine, dried over anhydrous MgSO4, filtered, and concentrated in
vacuo. The crude product was purified by flash chromatography
(SiO2, 20% ethyl acetate–hexane) to give 3 (1.22 g, 74%) as a
yellow solid: mp 51–52 ◦C; 1H NMR (CDCl3, 400 MHz) d 8.11
(d, 1H, J = 9.2 Hz), 7.21 (d, 1H, J = 2.8 Hz), 6.86 (dd, 1H,
J = 9.2, 2.8 Hz), 4.95 (s, 2H), 4.12 (t, 2H, J = 5.0 Hz), 3.97 (t,
2H, J = 5.0 Hz), 0.87 (s, 9H), 0.06 (s, 6H); 13C NMR (CDCl3,
100 MHz) d 163.5, 140.3, 140.1, 127.8, 114.4, 113.5, 70.0, 62.8,
61.7, 25.9, −5.2; FABMS (NBA) m/z 328 [(M + H)+]; HRMS
calcd. for C15H26NO5Si [(M + H)+] 328.1580, found 328.1588.
5-[2-(tert-Butyldimethylsilyloxy)ethyloxy]-2-nitrobenzyl
benzoate (4)
To a solution of 3 (1.01 g, 3.09 mmol) in CH2Cl2 (20 ml) was
added benzoyl chloride (520 mg, 3.70 mmol) and triethylamine
(374 mg, 3.69 mmol), and the mixture was stirred at 0 ◦C
for 2 h. The resulting mixture was diluted with sat. NaHCO3
and extracted with ethyl acetate. The extract was washed with
brine, dried over anhydrous MgSO4, filtered, and concentrated in
vacuo. The crude product was purified by flash chromatography
(SiO2, 13% ethyl acetate–hexane) to give 4 (983 mg, 74%) as a
yellow solid: mp 78–79 ◦C; 1H NMR (CDCl3, 400 MHz) d 8.21
(d, 1H, J = 9.2 Hz), 8.10 (dd, 2H, J = 8.0, 1.2 Hz), 7.59 (m,
1H), 7.47 (dd, 2H, J = 8.0, 8.0 Hz), 7.13 (d, 1H, J = 2.8 Hz),
Experimental
General methods
Melting points were determined with a Yanagimoto micro melt-
ing point apparatus and are uncorrected. 1H NMR spectra were
measured with JEOL JNM-AL 300 (300 MHz), or JEOL JMN-
EX-400 (400 MHz) spectrometers. The chemical shifts are ex-
pressed in ppm downfield from tetramethylsilane, using residual
chloroform (d = 7.24 in 1H NMR) and residual dimethyl sulfox-
ide (d = 2.49 in 1H NMR) as internal standards. 13C NMR spec-
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 8 9 3 – 3 8 9 7
3 8 9 5

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6.91 (dd, 1H, J = 9.2, 2.8 Hz), 5.79 (s, 2H), 4.09 (t, 2H, J =
tetraisopropylphosphorodiamidite (60 mg, 0.20 mmol), and the
mixture was stirred at ambient temperature for 30 min. After
the reaction, crude product 9 was used for automated DNA
synthesis without further purification.
4.8 Hz), 3.95 (t, 2H, J = 4.8 Hz), 0.85 (s, 9H), 0.04 (s, 6H); 13
C
NMR (CDCl3, 100 MHz) d 165.7, 163.3, 140.1, 135.6, 133.3,
129.7, 129.6, 128.5, 128.0, 114.4, 113.0, 70.0, 63.6, 61.6, 25.9,
18.4, −5.2; FABMS (NBA) m/z 432 [(M + H)+]; HRMS calcd.
for C22H30NO6Si [(M + H)+] 432.1842, found 432.1845.
Synthesis of modified oligodeoxynucleotides (10)
Modified oligodeoxynucleotides possessing amino group at 3ꢀ-
terminal and aminonaphthalene quencher at 5ꢀ-terminal were
prepared by the b-cyanoethylphosphoramidite method on 3ꢀ-
Amino-Modifier C7 controlled pore glass support (1 lmol) by
using an Applied Biosystems Model 392 DNA/RNA synthe-
sizer. After the automated synthesis, oligomers were deprotected
by heating the solutions at 55 ◦C for 12 h. The synthesized
oligomers were purified by reversed phase HPLC, elution with a
solvent mixture of 0.1 M triethylamine acetate (TEAA), pH 7.0,
linear gradient over 60 min from 0% to 30% acetonitrile at a
flow rate 3.0 mL min−1. The synthesized oligodeoxynucleotides
was identified by MALDI-TOF mass (10: calcd. 8162.39, found
8162.59).
5-[2-Hydroxyethyloxy]-2-nitrobenzyl benzoate (5)
To a solution of 4 (621 mg, 1.44 mmol) in THF (25 ml) was added
acetic acid (432 mg, 7.20 mmol) and TBAF (2.88 mL, 1.0 M
in THF, 2.88 mmol), and the mixture was stirred at ambient
temperature for 10 h. The resulting mixture was diluted with
sat. NaHCO3 and extracted with ethyl acetate. The extract was
washed with brine, dried over anhydrous MgSO4, filtered, and
concentrated in vacuo. The crude product was purified by flash
chromatography (SiO2, 3% methanol–CHCl3) to give 5 (429 mg,
94%) as a white solid: mp 75–76 ◦C; 1H NMR (CDCl3, 400 MHz)
d 8.20 (d, 1H, J = 9.2 Hz), 8.10 (dd, 2H, J = 8.4, 0.8 Hz), 7.59
(m, 1H), 7.47 (dd, 2H, J = 8.4, 8.4 Hz), 7.13 (d, 1H, J = 2.4 Hz),
6.91 (dd, 1H, J = 9.2, 2.4 Hz), 5.78 (s, 2H), 4.13 (t, 2H, J =
4.6 Hz), 3.96 (t, 2H, J = 4.4 Hz); 13C NMR (CDCl3, 100 MHz)
d 165.7, 162.8, 140.4, 135.7, 133.4, 129.7, 129.5, 128.5, 128.0,
114.4, 112.8, 69.9, 63.6, 61.0; FABMS (NBA) m/z 318 [(M +
H)+]; HRMS calcd. for C16H16NO6 [(M + H)+] 318.0977, found
318.0985.
Incorporation of nitrobenzyl chromophore into
oligodeoxynucleotides (ODN 1)
To a solution (total volume 20 lL) of 10 was added the solution
of 6 (550 lg, 1.20 lmol) and sat. NaHCO3 (10 lL), and the
mixture was incubated at 25 ◦C for 12 h. After the reaction,
the crude product was purified by reversed phase HPLC to give
ODN 1. The formation of ODN 1 was confirmed by MALDI–
TOF mass (ODN 1: calcd. 8505.68, found 8505.27).
5-[2-(2,5-Dioxopyrrolidinyloxycarbonyloxy)ethyloxy]-2-
nitrobenzyl benzoate (6)
To a solution of 5 (295 mg, 0.93 mmol) in CH3CN (3 ml) was
added N,Nꢀ-disuccinimidyl carbonate (1.19 g, 4.65 mmol) and
triethylamine (471 mg, 4.65 mmol), and the mixture was stirred
at ambient temperature for 24 h. The resulting mixture was
diluted with sat. NaHCO3 and extracted with ethyl acetate. The
extract was washed with brine, dried over anhydrous MgSO4,
filtered, and concentrated in vacuo. The crude product was
purified by flash chromatography (SiO2, 5% acetone–CHCl3)
to give 6 (279 mg, 66%) as a white solid: mp 47–48 ◦C; 1H NMR
(CDCl3, 400 MHz) d 8.23 (d, 1H, J = 9.2 Hz), 8.11 (dd, 2H, J =
8.4, 1.6 Hz), 7.59 (m, 1H), 7.48 (dd, 2H, J = 8.4, 8.4 Hz), 7.17
(d, 1H, J = 2.4 Hz), 6.93 (dd, 1H, J = 9.2, 2.4 Hz), 5.79 (s, 2H),
4.66 (t, 2H, J = 4.4 Hz), 4.31 (t, 2H, J = 4.4 Hz), 2.81 (s, 4H);
13C NMR (CDCl3, 100 MHz) d 168.2, 165.8, 162.1, 151.5, 140.9,
135.8, 133.3, 129.7, 129.5, 128.6, 128.0, 114.5, 113.1, 68.4, 65.6,
63.5, 25.5; FABMS (NBA) m/z 459 [(M + H)+]; HRMS calcd.
for C21H19N2O10 [(M + H)+] 459.1039, found 459.1044.
Photoreactions of ODN 1
Before irradiation, we carried out formation of stem-and-loop
structure or hybridization with target DNA (ODN 2, ODN
3 or ODN 4) of ODN 1, which was achieved by heating
the sample at 90 ◦C for 5 min and slowly cooing to room
temperature. Photoreactions of 10 lM ODN 1 in the presence
or absence of 50 lM target DNA at given temperatures were
carried out in a buffer containing 10 mM sodium cacodylate
(pH 7.0), using 365 nm light from transilluminator. After the
reaction, the released benzoic acid was determined by reversed
phase HPLC (HITACHI D-7000) equipped with Intersil ODS-
3 column (4.6 × 150 mm, GL Sciences) using a UV detector
(L-7455) at 230 nm.
References
2-(N-Methyl-N-1-naphthylamino)ethanol (8)
1 For examples with drugs for selective treatment of diseased cells. See:
(a) G. M. Dubowchik and M. A. Walker, Pharmacol. Ther., 1999,
83, 67–123; (b) S. Kizaka-Kondoh, M. Inoue, H. Harada and M.
Hiraoka, Cancer Sci., 2003, 94, 1021–1028; (c) M. Trepel, W. Arap
and R. Pasqualini, Curr. Opin. Chem. Biol., 2002, 6, 399–404; (d) K.
Tanabe, Y. Makimura, Y. Tachi, A. Imagawa-Sato and S. Nishimoto,
Bioorg. Med. Chem. Lett., 2005, 15, 2321–2324.
2 (a) J. Cai, X. Li and J.-S. Taylor, Org. Lett., 2005, 7, 751–754; (b) Z.
Ma and J.-S. Taylor, Bioorg. Med. Chem., 2001, 9, 2501–2510; (c) Z.
Ma and J.-S. Taylor, Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 11159–
11163.
To a solution of 710 (295 mg, 2.00 mmol) in toluene (3 ml) was
added triethylamine (405 mg, 4.00 mmol) and 2-bromoethanol
(501 mg, 4.01 mmol), and the mixture was refluxed for 2 h. The
resulting mixture was diluted with sat. NH4Cl and extracted
with ethyl acetate. The extract was washed with brine, dried
over anhydrous MgSO4, filtered, and concentrated in vacuo. The
crude product was purified by flash chromatography (SiO2, 20%
ethyl acetate–hex◦ane) to give 8 (353 mg, 88%) as a pale purple
1
solid: mp 53–54 C; H NMR (CDCl3, 300 MHz) d 8.28 (dd,
3 A. Okamoto, K. Tanabe, T. Inasaki and I. Saito, Angew. Chem., Int.
Ed., 2003, 42, 2502–2504.
1H, J = 7.0, 2.0 Hz), 7.82 (dd, 1H, J = 6.9, 2.7 Hz), 7.58 (d, 1H,
J = 8.4 Hz), 7.50–7.36 (3H), 7.17 (dd, 1H, J = 7.4, 1.1 Hz), 3.80
(dt, 2H, J = 5.4, 5.4 Hz), 3.30 (t, 2H, J = 5.4 Hz), 2.87 (s, 3H),
2.41 (t, 1H, J = 5.4 Hz); 13C NMR (CDCl3, 100 MHz) d 149.4,
134.6, 129.3, 128.2, 125.6, 125.4, 125.4, 123.8, 123.3, 116.0, 59.2,
58.0, 42.9; FABMS (NBA) m/z 202 [(M + H)+]; HRMS calcd.
for C13H16NO [(M + H)+] 202.1232, found 202.1225.
4 (a) A. S. Piatek, S. Tyagi, A. C. Pol, A. Telenti, L. P. Miller, F. R.
Kramer and D. Alland, Nat. Biotechnol., 1998, 16, 359–363; (b) S.
Tyagi, D. P. Btaru and F. R. Kramer, Nat. Biotechnol., 1998, 16,
49–53; (c) S. Tyagi and F. R. Kramer, Nat. Biotechnol., 1996, 14,
303–308.
5 M. Ichihashi, M. Ueda, A. Budiyanto, T. Bito, M. Oka, F. Fukunaga,
K. Tsuru and T. Horikawa, Toxicology, 2003, 189, 21–39.
6 H. Morrison, ed. in Biological Applications of Photochemical
Switches, Wiley-Interscience, New York, 1993.
7 B. S. Rosenstein and D. L. Mitchell, Photochem. Photobiol., 1987,
45, 775–780.
8 DNA may be damaged indirectly by reactive oxygen species (ROS)
generated by UV-A irradiation to endogeneous photosensitizer.
N-Methyl-N-[2-(N,N-diisopropylamino-2-
cyanoethoxyphosphinyloxy)ethyl]-1-aminonaphthalene (9)
To a solution of 8 (40 mg, 0.20 mmol) and tetrazole (18 mg,
0.26 mmol) in dry CH3CN was added 2-cyanoethyl-N,N,Nꢀ,Nꢀ-
3 8 9 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 8 9 3 – 3 8 9 7

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9 Triplet and singlet excited energies of nitrobenzene are determined
to be 243 and 372 kJ mol−1, respectively. On the other hand,
these parameters of aminonaphthalene are also reported to be
229 and 348 kJ mol−1, respectively. In view of such photophysical
parameters, aminonaphthalene was expected to act as a quencher
of photoreaction of o-nitrobenzyl chromophore. See: S. L. Murov,
I. Carmichael and G. L. Hug, in Handbook of Photochemistry, 2nd
edn., Marcel Dekker, New York, 1993.
competition, we preferred a rather short stem of 5 mer base pairs, as
a fundamental structure of the molecular beacon.
12 R. W. Yip, Y. X. Wen, D. Gravel, R. Giasson and D. K. Sharma,
J. Phys. Chem., 1991, 95, 6078–6081.
13 G. J. Kavarnos, in Fundamentals of Photoinduced Electron Transfer,
VCH Publishers, Inc., New York, 1993.
14 Recent reports on photoactivated prodrugs, see: (a) S. Watanabe,
M. Sato, S. Sakamoto, K. Yamaguchi and M. Imamura, J. Am.
Chem. Soc., 2000, 122, 12588–12589; (b) Y. Wei, Y. Yan, D. Pei
and B. Gong, Bioorg. Med. Chem. Lett., 1998, 8, 2419–2422; (c) R.
Reinhard and B. F. Schmidt, J. Org. Chem., 1998, 63, 2424–2441;
(d) A. R. Katritzky, Y. Xu, A. V. Vakulenko, A. L. Wilcox and
K. R. Bley, J. Org. Chem., 2003, 68, 9100–9104; (e) Z. Zhang, H.
Hatta, T. Ito and S. Nishimoto, Org. Biomol. Chem., 2005, 3, 592–
596.
10 A. R. Katritzky, M. Black and W. Q. Fan, J. Org. Chem., 1991, 56,
5045–5048.
11 Employment of longer stems may produce a more stable stem-and-
loop structure, thereby inducing more efficient overall quenching of
the photoreaction of the nitrobenzyl chromophore. However, such
a stable stem-and-loop formation is expected to compete with the
duplex formation that is essential for the drug release. In view of this
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 8 9 3 – 3 8 9 7
3 8 9 7