Quencher-free molecular beacon tethering 7-hydroxycoumarin detects targets through protonation/deprotonation

Article abstract of DOI:10.1016/j.bmc.2012.05.052

In this study, we synthesized a simple but efficient quencher-free molecular beacon tethering 7-hydroxycoumarin on d-threoninol based on its pK_a change. The pK_a of 7-hydroxycoumarin in a single strand was determined as 8.8, whereas that intercalated in the duplex was over 10. This large pK_a shift (more than 1.2) upon hybridization could be attributed to the anionic and hydrophobic microenvironment inside the DNA duplex. Because 7-hydroxycoumarin quenches its fluorescence upon protonation, the emission intensity of the duplex at pH 8.5 was 1/15 that of the single strand. We applied this quenching mechanism to the preparation of a quencher-free molecular beacon by introducing the dye into the middle of the stem part. In the absence of the target, the stem region formed a duplex and fluorescence was quenched. However, when the target was added, the molecular beacon opened and the dye was deprotonated. As a result, the emission intensity of the molecular beacon with the target was 10 times higher than that without the target. Accordingly, a quencher-free molecular beacon utilizing the pK _a change was successfully developed.

Full text of DOI:10.1016/j.bmc.2012.05.052

Bioorganic & Medicinal Chemistry 20 (2012) 4310–4315
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Quencher-free molecular beacon tethering 7-hydroxycoumarin detects
targets through protonation/deprotonation
⇑
Hiromu Kashida, Kyohei Yamaguchi, Yuichi Hara, Hiroyuki Asanuma
Graduate School of Engineering, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8603, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we synthesized a simple but efficient quencher-free molecular beacon tethering 7-hydroxy-
Received 23 April 2012
Revised 23 May 2012
Accepted 23 May 2012
Available online 1 June 2012
coumarin on D-threoninol based on its pKa change. The pKa of 7-hydroxycoumarin in a single strand was
determined as 8.8, whereas that intercalated in the duplex was over 10. This large pKa shift (more than
1.2) upon hybridization could be attributed to the anionic and hydrophobic microenvironment inside the
DNA duplex. Because 7-hydroxycoumarin quenches its fluorescence upon protonation, the emission
intensity of the duplex at pH 8.5 was 1/15 that of the single strand. We applied this quenching mecha-
nism to the preparation of a quencher-free molecular beacon by introducing the dye into the middle of
the stem part. In the absence of the target, the stem region formed a duplex and fluorescence was
quenched. However, when the target was added, the molecular beacon opened and the dye was depro-
tonated. As a result, the emission intensity of the molecular beacon with the target was 10 times higher
than that without the target. Accordingly, a quencher-free molecular beacon utilizing the pKa change was
successfully developed.
Keywords:
DNA
Fluorescence
7-Hydroxycoumarin
Molecular beacon
D-Threoninol
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
multiple fluorophores.22 Here, we utilized a new strategy, quench-
ing upon protonation, for the preparation of quencher-free MBs.
A molecular beacon (MB) is a hairpin-shaped oligonucleotide
probe that tethers a fluorophore and a quencher at its termini.1
In the absence of the target, the stem part closes and the fluoro-
phore and quencher interact with each other so that the emission
is quenched. On the other hand, in the presence of the target, the
MB opens and the fluorophore emits fluorescence. As a result,
the target can be detected by the difference of the emission inten-
sity with high sensitivity. To date, MBs have been widely used in
SNP typing, real-time PCR, and mRNA imaging in living cells.2
We have also proposed In-Stem Molecular Beacons, which tether
a fluorophore and a quencher in the middle of the stem region.3,4
However, these conventional MBs require at least two kinds of
dyes, a fluorophore and a quencher, in order to quench emission
in the absence of targets.
Recently, a novel class of molecular beacons that tethers no
quencher but only fluorophore(s) has been reported.5 Such quench-
er-free molecular beacons are advantageous over conventional ones
due to their cost effectiveness and easy handling. In quencher-free
MBs, the emission from the fluorophore(s) should change in the
presence of the target. Several strategies have been reported for
the emission change, such as quenching from nucleobases,6–12
FRET,13–18 excimer/exciplex formation,19–21 and self-quenching of
Previously, we incorporated several pH-sensitive dyes into DNA
D-threoninol and demonstrated that the dyes significantly chan-
on
ged their pKas upon hybridization due to the more anionic environ-
ment in the DNA duplex.23,24 For example, Brooker’s Merocyanine,
which is known to change both absorption and emission maxima
upon protonation, shifted its pKa from 9.5 to 10.1 upon duplex for-
mation.24 Although ratiometric detection of duplex formation was
possible with this dye, its quantum yield was below 0.01, which se-
verely limited the detection sensitivity. To overcome low emission,
we utilized a new fluorophore, 7-hydroxycoumarin (X in Scheme 1),
that has a quantum yield as high as 0.63 under basic conditions, and
quenches its fluorescence upon protonation, to design more sensi-
tive probe.25,26 Seela et al. previously synthesized modified
oligodeoxyribonucleotides (ODNs) tethering 7-hydroxycoumarin
directly to nucleobases by using Click chemistry to locate it at the
groove of the duplex.27–30 In our case,
D-threoninol is used as a linker
to incorporate the dye into the middle of the ODN.31 Previous
NMR structural analyses revealed that dyes are intercalated be-
tween base pairs when they are introduced into DNA on D-threoni-
nol.23,32,33 Therefore, 7-hydroxycoumarin is expected to be located
near the phosphate anions of the complementary strand between
the base pairs. In addition, the hydrophobic environment should
also promote the protonation of the dye, allowing an increase in
pKa of the dye upon hybridization. If the fluorescence is quenched
simply by hybridization, 7-hydroxycoumarin is available for the
preparation of quencher-free MBs. Here, we first investigated
⇑
Corresponding author.
E-mail address: asanuma@mol.nagoya-u.ac.jp (H. Asanuma).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.052

H. Kashida et al. / Bioorg. Med. Chem. 20 (2012) 4310–4315
4311
Scheme 1. Sequences of ODNs synthesized in this study. The chemical structure of 7-hydroxycoumarin (X) is also shown. Stem regions of MBs are underlined. The sequence
of the target (Surv), which is complementary to MBs, is also underlined.
Table 1
the spectroscopic behaviors of 7-hydroxycoumarin-modified ODNs
Effects of pH on the melting temperature (Tm) of CXG/cCXG and absorption maxima of
single-stranded CXG and CXG/cCXG
in order to evaluate their structure, pKa, and quenching efficiency.
Then, we applied the quenching system to the preparation of
quencher-free MBs.
a
b
pH
Tm (°C)
kmax (nm)
CXG
CXG/cCXG
2. Results and discussion
7.0
8.0
8.5
9.0
9.5
43.2
43.1
42.7
40.9
39.2
27.7
330
331
333
369
371
371
334
334
335
335
335
341
2.1. Synthesis of modified ODN with 7-hydroxycoumarin
The synthesis of phosphoramidite monomers tethering
7-hydroxycoumarin was conducted by using standard phospho-
ramidite chemistry (Scheme 2). First, 7-hydroxycoumarin was cou-
10.0
a
Solution conditions: [ODN] = 5.0
Absorption maxima at 0 °C.
l
M, [NaCl] = 100 mM.
b
pled to DMT-protected
D
-threoninol4 (compound 1). Then, the
hydroxyl group of the dye was protected by an acetyl group and
converted to a phosphoramidite monomer (compound 4) for incor-
poration into ODN. After DNA synthesis, the acetyl group was suc-
cessfully deprotected by treatment with ammonium hydroxide
solution. The sequences of modified DNA are shown in Scheme 1.
Before evaluating the pKa of 7-hydroxycoumarin, the stability of
the duplex (CXG/cCXG) was evaluated at a different pH (Table 1).
At pH 7.0, the melting temperature (Tm) of CXG/cCXG was
43.2 °C, which was 4.5 °C lower than that of the native duplex
without the dye (47.7 °C). This destabilization is attributable to
the small stacking area of 7-hydroxycoumarin. As listed in Table
1, the Tm was almost constant as a function of pH from 7 to 9.5,
whereas it decreased to 27.7 °C at pH 10 due to the deprotonation
of the imino protons of thymine and guanine. Accordingly, spectro-
scopic measurements were carried out below 20 °C, where the du-
plex is formed even at pH 10.
Figure 1. UV–Vis spectra of CXG and CXG/cCXG at pH 7.0. Solution conditions are
as follows: [ODN] = 10.0 lM, [NaCl] = 100 mM, pH 7.0 (10 mM phosphate buffer),
0 °C.
Then, we investigated the spectroscopic behaviors of CXG at pH
7.0 where 7-hydroxycoumarin is fully protonated. Single-stranded
CXG showed a single peak at 330 nm attributable to the p–p* tran-
Intercalated dyes are known to show much smaller ICD than
groove-binder dyes.35,36 Taken together, we concluded that 7-
hydroxycoumarin is intercalated between base pairs.
sition of protonated 7-hydroxycoumarin, as shown in Figure 1. On
the other hand, the addition of its complementary strand, cCXG, in-
duced a small bathochromic shift (kmax = 334 nm) and hypochro-
mism of the band (Table 1, Figure 1). These spectral changes are
caused by excitonic interactions between natural nucleobases
and intercalated dyes.34 CD spectra of CXG/cCXG showed symmet-
rical Cotton effects around 260 nm below 20 °C, demonstrating
that the insertion of the X residue did not destroy the B-form du-
plex of natural nucleobases (Fig. 2). In addition, CXG/cCXG exhib-
2.2. Evaluation of pKa change upon hybridization
Next, we investigated the effect of pH on the absorption spectra.
Figure 3A shows the UV–Vis spectra of single-stranded CXG at var-
ious pH. At pH 7.01, a peak maximum was observed at 331 nm,
which disappeared with pH increase, and concurrently a new peak
ited
a small positive induced CD at 300–350 nm at 0 °C.
O
O
OH
(b)
O
O
O
O
O
O
O
O
DMTO
DMTO
DMTO
DMTO
H
N
H
N
H
N
(c)
(a)
NH2
O
O
O
N
OH
OH
OH
O
P
NC
1
2
3
4
O
Scheme 2. Synthesis of phosphoramidite monomers tethering 7-hydroxycoumarin. Reagents and conditions: (a) 7-Hydroxycoumarinyl-4-acetic acid, PyBOP, Et3N, CH2Cl2, rt,
3d, 94%; (b) acetic anhydride, tBuOK, THF, rt, overnight, 68%; (c) (iPr)2NP(Cl)(OCH2CH2CN), Et3N, CH3CN, 0 °C ? rt, 1 h, quant.

4312
H. Kashida et al. / Bioorg. Med. Chem. 20 (2012) 4310–4315
UV–Vis spectra of CXG/cCXG at various pH. In contrast to single-
stranded CXG, the peak at 330 nm remained even at a pH higher
than 10, demonstrating that the pKa of CXG/cCXG is higher than
that of single-stranded CXG. From the titration curve of CXG/cCXG
shown in Figure 4b, pKa was estimated to be over 10.0. Although
the precise pKa could not be determined due to the protonation
of coumarin even at pH 10.0, its pKa in the duplex was at least
1.2 higher than that in the single strand. The increase in pKa upon
duplex formation is much larger than that of merocyanine (9.5–
10.1)24 and Naphthyl Red (5.8–6.5).23 Presumably, the smaller size
of coumarin facilitated its accommodation between base pairs,
resulting in the large increase in pKa within the hydrophobic envi-
ronment around the dye.
The change of pKa by hybridization can be exploited for the
detection of DNA hybridization. Figure 5 shows the absorption
spectra of single-stranded CXG and CXG/cCXG at pH 8.5. At this
pH, CXG/cCXG showed only a single peak at 335 nm assignable
to protonated coumarin. On the contrary, single-stranded CXG
gave a shoulder peak at about 370 nm, demonstrating that couma-
rin was partly deprotonated in the single-stranded state. Because
the fluorescence emission of 7-hydroxycoumarin is quenched upon
protonation, its emission should be lower upon hybridization.
Actually, at pH 8.5, single-stranded CXG showed strong emission
at 467 nm when excited at 365 nm where deprotonated coumarin
has its kmax (see solid line in Figure 6). On the other hand, hybrid-
ization with cCXG significantly lowered its emission (see dotted
line in Fig. 6). As a result, the emission intensity of single-stranded
Figure 2. CD spectra of CXG/cCXG at various temperatures at pH 7.0. Solution
conditions are as follows: [ODN] = 5.0
phosphate buffer).
lM, [NaCl] = 100 mM, pH 7.0 (10 mM
assignable to the deprotonated dye appeared at 371 nm. From the
titration curve monitored at 370 nm (Fig. 3B), the pKa of the single-
stranded CXG was determined as 8.8. Since the pKa of 7-hydroxy-4-
methylcoumarin is 7.8,26 the introduction of 7-hydroxycoumarin
into single-stranded DNA raised the pKa by 1.0 units. This increase
is probably caused by the anionic environment inside the DNA gen-
erated by phosphate anions.
Furthermore, pKa significantly increased when the complemen-
tary strand, cCXG, was added to the solution. Figure 4A shows
Figure 3. (A) UV–Vis spectra of CXG at various pH (from 7.01 to 10.45) and (B) plots of the absorbance of CXG at 370 nm as a function of pH at 20 °C. Solution conditions were
as follows: [ODN] = 5.0 M, [NaCl] = 100 mM, [NaH2PO4/Na2HPO4] = 10 mM.
l
Figure 4. (A) UV–Vis spectra of CXG/cCXG at various pH (from 7.03 to 10.03) and (B) plots of the absorbance of CXG/cCXG at 370 nm as a function of pH at 20 °C. Solution
conditions are as follows: [ODN] = 5.0 M, [NaCl] = 100 mM, [NaH2PO4/Na2HPO4] = 10 mM.
l

H. Kashida et al. / Bioorg. Med. Chem. 20 (2012) 4310–4315
4313
above, UV–Vis and CD spectra showed that 7-hydroxycoumarin
was also located between the base pairs. Accordingly, 7-hydroxy-
coumarin was located in the hydrophobic environment and direc-
ted towards the phosphate anions of the complementary strand,
which enhanced protonation upon hybridization.
2.3. Preparation of a quencher-free molecular beacon by
introducing 7-hydroxycoumarin into the stem region
7-Hydroxycoumarin quenched its fluorescence upon hybridiza-
tion. This quenching system can be utilized for the design of a
molecular beacon (Scheme 3). We introduced 7-hydroxycoumarin
into the stem region of the MB. In the absence of the target, the
stem part should form a duplex, and thus the emission of this
MB is quenched in the closed state because the dye is protonated.
On the other hand, when the complementary target is added, the
MB opens and the dye is in the single-stranded state. Therefore,
the dye is deprotonated and the emission from 7-hydroxycouma-
rin should be observed. Consequently, the emission of 7-hydroxy-
coumarin should turn on in the presence of the target, that is, the
dye positively responds to the target.
Figure 5. UV–Vis spectra of CXG (solid line) and CXG/cCXG (dotted line) at pH 8.5.
Solution conditions were as follows: [ODN] = 10.0 lM, [NaCl] = 100 mM, pH 8.5
(10 mM Tris buffer), 0 °C.
In ‘conventional’ MBs, dyes were introduced into the termini.
However, in our design, it is necessary to introduce the dye into
the middle of the stem region to create a negatively charged and
hydrophobic microenvironment around the dye for efficient
quenching by protonation. We synthesized five MBs that have 8-
mer stem regions with a shared-stem design37 (Scheme 1). We
used the survivin gene as the target DNA, which is highly ex-
pressed in breast cancer (Surv in Scheme 1).38 The melting temper-
atures of all the MBs with and without the target are listed Table S1
in Supplementary data. MB1, MB2, and MB3 have a single dye in
the stem region at different positions while MB4 has two dyes to
raise the emission intensity. MBt is the control MB which has a
tethered the dye at its 50 terminus. In MBt, the dye is located out-
side the DNA duplex even in the closed state. Accordingly, the
emission should be observed even in the absence of the target.
Figure 7A shows the emission spectra of MB1, which gave weak
emission without the target because the dye was protonated in the
closed state (solid line). In contrast, when the target was added,
strong emission was observed from the deprotonated coumarin
at around 465 nm (dotted line). The ratio of the intensities be-
tween the open and closed states (S/B ratio) was 10.0 (Table 2).
This value is comparable to the ratio of the intensity of CXG to that
of CXG/cCXG (15.3). MB2 and MB3, each of which has the dye at
different positions, showed similar S/B ratios to MB1 (8.3 and
7.3, respectively). Thus, MB with 7-hydroxycoumarin in the stem
could detect the target in a positive response as designed without
significant sequence dependence (see Fig. S3 in Supplementary
data for actual data). MB4 with two dyes also showed a similar po-
sitive response to the target, although its S/B ratio was 6.1. This rel-
atively low S/B ratio was caused by low emission intensity (1.3
times higher than MB1) in the open state,39 probably due the
self-quenching of dyes in the single-stranded state.
Figure 6. Fluorescence emission spectra (365 nm excitation) of CXG (solid line) and
CXG/cCXG (dotted line) at pH 8.5. Solution conditions were as follows:
[ODN] = 5.0 lM, [NaCl] = 100 mM, pH 8.5 (10 mM Tris buffer), 0 °C. The excitation
wavelength was 365 nm.
CXG was 15.3 times higher than that of CXG/cCXG. In addition,
strong emission of coumarin was observed in the single-stranded
CXG at pH higher than 7.0 (see Fig. S1 in Supplementary data).
Accordingly, duplex formation was successfully monitored with a
negative response to the target by using coumarin-modified DNA.
We also examined the sequence dependence on the emission spec-
tra (see Fig. S2 in Supplementary data), and found that duplex dis-
sociation could be monitored irrespective of the neighboring bases,
although the ratio of the intensities between the single strand and
duplex slightly depended on the sequence.
According to Seela et al., if the dye is attached to the 5 position
of pyrimidines or the 7 position of purines, it results in localization
at the groove of the duplex.27–30 In this case, the pKa of the dye
should not change because the dye is located in a relatively hydro-
philic environment, even in the duplex. In the studies of Seela et al.,
the difference in emission intensities between single strands and
duplexes was not very large, although they did not determine each
On the other hand, MBt, the control MB, did not show a positive
response to the target (Fig. 7B), because strong emission was ob-
served even in the closed state. Since the dye in MBt was located
pKa directly. In our design, the dye was introduced on
D-threoninol
to facilitate its intercalation between the base pairs. As mentioned
Scheme 3. Schematic illustration of target detection by using an MB with 7-hydroxycoumarin.

4314
H. Kashida et al. / Bioorg. Med. Chem. 20 (2012) 4310–4315
Figure 7. Fluorescence emission spectra (365 nm excitation) of (A) MB1 and (B) MBt with (solid line) and without Surv (dotted line) at pH 8. Solution conditions are as
follows: [MB] = 1.0
lM, [Surv] = 2.0
l
M, [NaCl] = 100 mM, pH 8.0 (10 mM Tris buffer), 0 °C. The excitation wavelength was 365 nm.
Table 2
4. Experimental section
Effects of pH on the melting temperature (Tm) of CXG/cCXG and the absorption
maxima of single-stranded CXG and CXG/cCXG
4.1. General
Sequence
Relative intensitya
S/B ratiob
Without Surv
With Surv
All conventional phosphoramidite monomers, CPG columns, re-
agents for DNA synthesis, and Poly-Pak II cartridges were pur-
chased from Glen Research. Other reagents for the synthesis of
phosphoramidite monomers were purchased from Tokyo Kasei
Co., Ltd, and Sigma–Aldrich.
MB1
MB2
MB3
MB4
MBt
1
10.0
9.3
8.9
13.3
1.8
10.0
8.3
7.3
6.1
0.3
1.1
1.2
2.2
5.6
a
Relative intensity at 465 nm relative to that of MB1 without Surv. Solution
conditions: [MB] = 1.0 lM, [Surv] = 2.0 lM, [NaCl] = 100 mM, pH 8.0 (10 mM Tris
4.1.1. Preparation of compound 2
buffer), 0 °C.
Compound 1 was synthesized according to a previous report.4
7-hydroxycoumarinyl-4-acetic acid (1.33 g, 6.0 mmol) was reacted
with PyBOP (3.67 g, 7.1 mmol) and Et3N (10 ml) in CH2Cl2 (30 ml)
for 10 min. Then, a solution of compound 1 (2.05 g, 5.0 mmol) in
CH2Cl2 (10 ml) was added to the above mixture. After vigorous stir-
ring for 3 days, the organic solution was washed with a saturated
aqueous solution of NaHCO3. The solvent was removed by evapora-
tion, followed by silica gel column chromatography (CHCl3/MeOH/
Et3N = 60:1:2, Rf = 0.2) to afford 2 (2.90 g, yield 94%). 1H NMR
[CDCl3, 500 MHz] d = 7.49 (d, J = 8.5 Hz, 1H), 7.29–7.18 (m, 9H),
6.82–6.78 (m, 4H), 6.73 (m, 2H), 6.59 (m, 1H), 6.19 (s, 1H), 4.01
(m, 1H), 3.92 (m, 1H), 3.78 (s, 6H), 3.69 (s, 2H), 3.35 (dd,
J = 4.5 Hz, 9.5 Hz, 1H), 3.21 (dd, J = 3.5 Hz, 9.5 Hz, 1H), 1.04 (d,
J = 6.5 Hz, 3H). 13C NMR [CDCl3, 126 MHz] d = 169.0, 161.9, 161.1,
158.6, 155.1, 150.0, 144.3, 135.4, 135.3, 129.9, 129.1, 128.0,
127.9, 127.0, 125.8, 113.8, 113.3, 112.6, 111.6, 103.2, 86.7, 67.9,
64.0, 55.2, 54.6, 39.9, 20.1. HRMS(FAB) Calcd for C36H35NO8 (M+)
609.2363. Found: 609.2343.
b
Ratio of the fluorescence intensity of the MB in open and closed state.
outside of the duplex, protonation did not occur. The addition of
Surv did not increase, but actually decreased, the emission of cou-
marin, probably due to quenching by the neighboring base pairs.
Accordingly, incorporation of coumarin into the stem is essential
for the design of an MB for positive response.
3. Conclusions
In conclusion, we have successfully developed a simple but effi-
cient quencher-free molecular beacon, only by introducing a single
7-hydroxycoumarin into the middle of the stem region via D-thr-
eoninol. UV–Vis and CD spectra of 7-hydroxycoumarin-modified
ODN indicated that the dye is intercalated between base pairs.
The pKa of 7-hydroxycoumarin in single-stranded state was 8.8,
whereas that in the duplex was more than 10. Consequently, the
pKa change upon hybridization was over 1.2. This large difference
could be attributed to electrostatic interactions with phosphate an-
ions in the complementary strand. In addition, the hydrophobic
microenvironment between base pairs could also contribute to
the pKa shift.
We incorporated 7-hydroxycoumarin into the stem region of
molecular beacons. In the absence of the target, the emission from
7-hydroxycoumarin was quenched due to the protonation. On the
other hand, strong emission was observed by the addition of the
target. Consequently, the S/B ratio of the molecular beacon was
10.0. In our design of quencher-free MBs based on pKa change,
the incorporation of chromophores into the stem region is essen-
tial. Otherwise, quenching of the dye in the closed state does not
occur as evidenced by MBt.
4.1.2. Preparation of compound 3
Potassium tert-butoxide (0.184 g, 1.64 mmol) was added to a
solution of compound 2 (1.0 g, 1.6 mmol) in dry THF (10 ml) under
nitrogen. Then, acetic anhydride (0.16 ml, 1.6 mmol) was added to
the above mixture. After vigorous stirring overnight, the solvent
was removed by evaporation, followed by silica gel column chro-
matography (CHCl3/MeOH = 60:1, Rf = 0.2) to afford 3 (0.73 g, yield
68%). 1H NMR [CDCl3, 500 MHz] d = 7.67 (d, J = 8.5 Hz, 1H), 7.34–
7.19 (m, 9H), 7.11 (s, 1H), 7.01 (d, J = 8.5 Hz, 1H), 6.81 (m, 4H),
6.44 (m, 1H), 6.38 (s, 1H), 4.05 (m, 1H), 3.94 (m, 1H), 3.77 (s,
6H), 3.68 (s, 2H), 3.35 (dd, J = 4.5 Hz, 9.5 Hz, 1H), 3.27 (dd,
J = 4 Hz, 10 Hz, 1H), 2.87 (br, 1H), 2.32 (s, 3H), 1.05 (d, J = 6.5 Hz,
3H). HRMS(FAB) Calcd for C38H37NO9 (M+) 651.2468. Found:
651.2449.

H. Kashida et al. / Bioorg. Med. Chem. 20 (2012) 4310–4315
4315
4.1.3. Preparation of compound 4
were as follows: [NaCl] = 100 mM, [Na2HPO4/NaH2PO4] = 10 mM.
Since pH titration could not be conducted at a pH higher than 10
due to the dissociation of the duplex, pKa was determined by
assuming the following equation with a curve-fitting program
using KaleidaGraph 3.5 (Synergy Software):
Et3N (0.13 ml, 1.2 mmol) and 2-cyanoethyldiisopropylchloro
phosphoramidite (0.075 ml, 0.34 mmol) were added to a solution
of compound 3 (0.20 g, 0.31 mmol) in CH3CN (5.0 ml) at 0 °C under
nitrogen. After 20 min of vigorous stirring, the solution was stirred
for 1 h at room temperature. Then, an excess of AcOEt was added to
the reaction mixture which was washed with a saturated aqueous
solution of NaHCO3 and of NaCl. After drying over MgSO4, the sol-
vent was removed by evaporation. The oily product 4 was used for
DNA synthesis without further purification. 31P NMR [121 MHz,
ðAAꢀ ꢀ AHA
Þ
A ¼ AHA
þ
aꢀpHÞ
10ðpK
where AHA and AAꢀ denote the absorbances of fully protonated and
deprotonated forms.
CDCl3] d = 148.7, 147.8. HRMS(FAB) Calcd for C47H55N3O10
P
(M+H+) 852.3625. Found: 852.3647.
Acknowledgments
4.2. DNA synthesis
This work was partially supported by a Grant-in-Aid for Scientific
Research (A) (21241031) and a Grant-in-Aid for Young Scientists (B)
(22750149) from the Ministry of Education, Culture, Sports, Science
and Technology, Japan. Partial supports by SENTAN program (JST)
and The Asahi Glass Foundation are also acknowledged.
All of the modified DNAs were synthesized on an automated
DNA synthesizer (ABI-3400 DNA synthesizer, Applied Biosystems)
by using phosphoramidite monomers bearing dye molecules. The
coupling efficiency of the monomers corresponding to the modi-
fied residues was as high as that of the conventional monomers,
as judged from the coloration of the released trityl cation. The acet-
yl group protecting the hydroxyl group of 7-hydroxycoumarin was
deprotected by treatment with an ammonium hydroxide solution
at 55 °C for 8 h. After the recommended workup, they were puri-
fied by reversed phase (RP)-HPLC and were characterized by MAL-
DI-TOFMS (Autoflex, Bruker Daltonics). The MALDI-TOFMS data for
the DNAs were as follows: CXG: Obsd 4015 (Calcd for [CXG+H+]:
4014). MB1: Obsd 9319 (Calcd for [MB1+H+]: 9318). MB2: Obsd
9317 (Calcd for [MB2+H+]: 9318). MB3: Obsd 9319 (Calcd for
[MB3+H+]: 9318). MB4: Obsd 9687 (Calcd for [MB4+H+]: 9687).
MBt: Obsd 9319 (Calcd for [MBt+H+]: 9318).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.052.
References and notes
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100 mM, [DNA] = 5.0 lM. For measurements at pH 7.0, 8.0–9.5, or
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doubled.