Rational design of novel photoinduced electron transfer type fluorescent probes for sodium cation

Article abstract of DOI:10.1016/j.tet.2004.06.108

We have developed novel fluorescence probes for sodium cation based on photoinduced electron transfer (PeT). In this study, we rationally designed new probes and succeeded in achieving fluorescence enhancement upon sodium ion binding by reducing the HOMO

Full text of DOI:10.1016/j.tet.2004.06.108

Tetrahedron 60 (2004) 11067–11073
Rational design of novel photoinduced electron transfer type
fluorescent probes for sodium cation
Suguru Kenmoku,a Yasuteru Urano,a Koujirou Kanda,a Hirotatsu Kojima,a Kazuya Kikuchia,b
and Tetuso Naganoa,
*
aGraduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
bPresto, JST Corporation, 4-8-1 Honcho, Kawaguchi, Saitama 332-0012, Japan
Received 31 January 2004; accepted 22 June 2004
Available online 23 August 2004
Abstract—We have developed novel fluorescence probes for sodium cation based on photoinduced electron transfer (PeT). In this study, we
rationally designed new probes and succeeded in achieving fluorescence enhancement upon sodium ion binding by reducing the HOMO
energy level of the chelator group within the probe molecule. Our new probes show low pH dependency, possibly because of their simple
structures. Our results confirm the value of rational probe design based on PeT.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
vative.7 In this sense, design of these fluorescence probes is
largely empirical, and this limits the design flexibility.
Fluorescein is highly fluorescent in aqueous media and
emits longer wavelength light upon excitation at 490 nm.
For these reasons, various fluorescein derivatives have been
used as fluorescent tags1 or fluorescence probes.2 For
example, many fluorescein-based fluorescence probes are
available for various cations, such as Fluo-3,3 ZnAF-24 and
CoroNa Red5 (Fig. 1). Most of these probes are based on an
aminofluorescein structure containing a selective chelator
for the target cation. Why has this structure been retained in
so many cation probes? One answer is that many chelators
consist of nitrogen atoms.6 However, another and more
important reason is that aminofluorescein is non-fluorescent
and becomes fluorescent upon formation of the amidoderi-
Recently, we showed that the fluorescein molecule is a
directly conjugated electron donor and acceptor system.8
Furthermore, the fluorescence properties of fluorescein
derivatives can be controlled by intramolecular photo-
induced electron transfer (PeT). PeT is a well-known
mechanism through which the fluorescence of a fluorophore
is quenched by electron transfer from the donor to the
acceptor.9 In a molecule in which two groups, a fluorophore
(electron acceptor) and its fluorescence quencher (electron
donor) are located in close proximity and have no ground
state interaction with each other, when the fluorophore is
photochemically excited, a single electron is transferred
Figure 1. Cation probes based on PeT. Solid lines indicate the aminofluorescein or aminorhodamine platform.
Keywords: Photoinduced electron transfer; Rational design; Fluorescence probe; Sodium cation.
* Corresponding author. Tel.: C81358414854; fax: C81358414855.; e-mail: ff26014@mail.ecc.u-tokyo.ac.jp
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.06.108

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S. Kenmoku et al. / Tetrahedron 60 (2004) 11067–11073
Eox, oxidation potential of electron donor; Ered, reduction
potential of electron acceptor; DE00, singlet excitation
energy of fluorophore; wp, work term for charge separation
state.
We very recently found that the carboxyl group of
fluorescein molecule can be replaced with other substituents
such as methyl or methoxyl10 (Fig. 3). For example, the
methyl-substituted derivative called 2-MeTokyoGreen
(2-MeTG) was as fluorescent as the traditional fluorescein
molecule (Fig. 3). We synthesized many derivatives, and
found an excellent relationship10 between the HOMO
energy levels of the benzene moiety (electron donor) and
the quantum efficiency (QE) of TGs: a higher-HOMO-
energy-level benzene moiety diminishes the fluorescence of
xanthene more efficiently (Fig. 3), and the threshold HOMO
energy level for fluorescence off/on switching could be
precisely determined. Those findings enabled us to control
the fluorescence properties of xanthene without an aniline
moiety and gave great flexibility in designing novel probes.
Using these findings, we tried to develop novel cation
probes bearing only O atoms as ligands.
Figure 2. On the left, the HOMO energy level of the electron donor moiety
is too low to allow electron transfer. Consequently, emission from the
singlet-excited fluorophore is not affected. On the right, the HOMO energy
level is enough high to allow electron transfer, so the fluorescence
decreases.
from the electron donor to the excited fluorophore.
Consequently, the fluorophore loses its energy thermally
instead of by emitting fluorescence (Fig. 2).
Indeed, many fluorescence probes for cations as (Fig. 1) are
based on PeT. Furthermore, almost all probes that detect
cations are structurally based on aminofluorescein. The
aniline moiety within aminofluorescein quenches the
fluorescence of the singlet excited xanthene dye. However,
is aminofluorescein necessary for developing novel probes?
Our recent work suggests that the answer to this question is no.
2. Results and discussion
Our report8 revealed that the rate of intramolecular PeT in
the fluorescein molecule follows the Marcus and Rehm–
Weller equations and that fluorescein derivatives have
characteristically small l and V values.
We chose benzo-15-crown-5-ether derivatives as candidates
for the electron donor. We calculated the HOMO energy
levels of o-dimethoxybenzenes as analogues of the benzo-
crown-ether moiety. As shown in Figure 3, 3,4-dimethox-
ybenzoic acid (compound (1), whose HOMO energy level is
K0.220 hartrees, solid line) seems not to be a good electron
donor group; on the other hand, 3,4-dimethoxytoluene ((2),
K0.206 hartrees, dashed line) seems to be a good donor. So
compound (3) should emit strong fluorescence (in other
words, retention of the carboxyl group has prevented the
development of cation probes with a plain benzo-crown-
ether), while compound (4) should emit little fluorescence.
Thus, we synthesized the novel sodium probes, (10a) and
(10b) shown in Scheme 1. In general, the pKa value of the
phenolic hydroxyl group of xanthene is about 611 and that of
2,7-dichloroxanthene is about 4.5, so it is likely that (10b)
will be a better probe than (10a) under acidic conditions.
Marcus equation:
ꢀ
ꢁ
ꢂ
ꢃ
1=2
2
4p3
h2lkBT
KðDG0PeT ClÞ
4lkBT
kPeT
Z
V2 exp
(1)
l, reorganization energy; V, electronic coupling matrix
element between electron donor and acceptor; DG0PeT, free
energy of electron transfer reaction.
Rehm–Weller equation:
DG0PeT Z Eox KEred KDE00 Kwp
(2)
Figure 3. (A) Electron donors and TokyoGreens. (B) Tested analogues for rational design of novel probes. (C) The graph is a plot of the HOMO level of the
benzene moiety (electron donor) versus the QE of TGs. HOMO energy levels were calculated by B3LYP/6-31G. The points represent 2-MeTG, 2,4-diMeTG,
2,5-diMeTG, 2-OMeTG, 2-Me, 4-OMeTG, 2-OMe,5-MeTG, 2,4-diOMeTG, and 2,5-diOMeTG, from left to right. The solid line and dashed line indicate the
calculated HOMO energy levels of compounds (1) and (2), respectively. QE of (3) and (4) can be estimated from the intersection of the respective lines with the
best fit to the points on the graph.

S. Kenmoku et al. / Tetrahedron 60 (2004) 11067–11073
11069
Scheme 1. Synthesis of compounds (10a) and (10b).
Figure 4. Panels A and B are emission spectra of (10a) and (10b) excited at 492 and 506 nm, respectively. Panel C and D are absorbance spectra of (10a) and
(10b), respectively. Each sample was measured in aqueous MOPS buffer, pH 7.0, containing 0.5% DMF as a cosolvent. Probes were added as stock solution in
DMF, finally 3 mM. [NaClO4] was 0, 200, 700, 1000, 1500, 2000 or 3000 mM. In panel A, Ffl([NaClO4]Z3000 mM)Z0.041, Ffl([NaClO4]Z0 mM)Z0.008.
In panel B, Ffl([NaClO4]Z3000 mM)Z0.025, Ffl([NaClO4]Z0 mM)Z0.005.

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S. Kenmoku et al. / Tetrahedron 60 (2004) 11067–11073
Figure 5. Fluorescence intensity and absorbance were normalized to those at pH 8. Each sample was measured in aqueous MOPS buffer of various pH values,
adjusted with trimethylammonium hydroxide, containing 0 or 1000 mM NaClO4, and 0.1% DMF as a cosolvent. The final concentrations of (10b) and Sodium
Green are 3 and 0.5 mM, respectively. Panels A and C are normalized absorbance (open circle) and normalized fluorescence (closed circle) of (10b) in 0 and
1000 mM NaClO4, respectively. Panels B and D are normalized absorbance (open square) and normalized fluorescence (closed square) of Sodium Green in 0
and 1000 mM NaClO4, respectively.
Absorption and emission spectra of (10a) and (10b) are
shown in Figure 4. For both probes, the fluorescence
intensity was enhanced by the addition of sodium
perchlorate while there was little change in the absorption
spectrum, which means that the fluorescence increases of
(10a) and (10b) resulted from increases of QE. The QE of
(10a) increased from 0.008 to 0.041, that is, about 5-fold,
upon binding NaC. The QE of (10b) also increased from
0.005 to 0.025, that is, also 5-fold. Quite similar results were
obtained in the case of NaCl addition, so the photochemical
properties of (10a) and (10b) are independent of the counter
fluorescence is more complex. In panel B, the fluorescence
intensity peaked at pH 5.8 and decreased at lower pH. In
panel D, the fluorescence intensity tended to decrease in
lower pH. These data clearly show that the fluorescence
intensity of Sodium Green is dependent not only on the
concentration of NaC, but also on the pH. Some fluorescent
cation probes consist of one chelator and two fluorophores,
for example Calcium Green-214 and SBFI.15 The main aim
of using two fluorophores in one molecule is to achieve
greater fluorescence enhancement by unstacking of the
fluorophores upon cation binding. Indeed, Sodium Green
has two absorption peaks at 492 and 506 nm, of which the
former is thought to correspond to the molecule with
intramolecular stacking and the latter to the unstacked
molecule. Higher NaC concentration makes Abs492 lower
and Abs506 higher. This fluorescence enhancement mechan-
ism provides a low detection limit, but may cause
unexpected effects, for example undesirable pH depen-
dency. Our probe design is so simple that there is little
possibility of unexpected effects.
anion. These fluorescence enhancements are large com-
¨
12
pared with that of benzos (Boens et al. ), which has a plain
benzo-crown-ether as the chelator. Benzos may become
more or less fluorescent upon binding cations, but PeT-type
probes such as (10a) and (10b) always become more
fluorescent. The Kd values for NaC are about 0.38 and
0.44 M for (10a) and (10b), respectively. Fluorescence of
(10a) and (10b), which are distinct from aminofluorescein,
could thus be controlled through rational design based
on TGs.
We obtained similar results in the case of adding potassium
ion (shown in Fig. 6). We employed potassium chloride as
the potassium salt. The Kd values for KC are 0.29 M for
(10a) and 0.25 M for (10b).
We compared the pH dependency of the fluorescence
intensity and absorbance of (10b) with those of a well-
known NaC probe, Sodium Green.13 In panels A and C in
Figure 5, in the presence of 0 and 1 M NaClO4, (10b)
showed plateau profiles in the range of pH 5–8. On the other
hand, as shown in panels B and D, the absorbance of Sodium
Green decreased drastically below pH 6.5. This pH profile
of Sodium Green is distinct from that of dichlorofluorescein,
whose pKa was reported to be below 5; this indicates the
existence of the intramolecular stacking of the two
fluorophores. The pH dependency of Sodium Green’s
The cation affinity did not differ much between (10a) and
(10b) because the chelator moiety and the xanthene dye are
orthogonal and resonantly independent of each other. The
Kd values described above imply that the affinities of (10a)
and (10b) for KC are higher than those for NaC, and that
full binding to NaC enhances the fluorescence of (10a) and
(10b) about 5-fold while full binding to KC does so by only

S. Kenmoku et al. / Tetrahedron 60 (2004) 11067–11073
11071
Figure 6. Panels A and B are emission spectra of (10a) and (10b) excited at 492 and 506 nm, respectively. Each sample was measured in aqueous MOPS
buffer, pH 7.0, containing 0.5% DMF as a cosolvent. Probes were added as stock solution in DMF, finally 3 mM. [NaClO4] was 0, 200, 700, 1000, 1800 or
2700 mM. In panel A, Ffl([NaClO4]Z2700 mM)Z0.021, Ffl([NaClO4]Z0 mM)Z0.008. In panel B, Ffl([NaClO4]Z2700 mM)Z0.010, Ffl([NaClO4]Z
0 mM)Z0.005.
Table 1. Comparison of NaC addition and KC addition to (10a) and (10b).
Kd (M)
Fmax/Fmin
For NaC
For KC
NaC
KC
Compound (10a)
Compound (10b)
0.38
0.44
0.29
0.25
5.3
4.6
2.4
2.4
Fmax/Fmin, fluorescence saturated with MC/fluorescence in MC-free condition
2–3-fold. Although (10a) and (10b) have higher affinity for
KC than NaC, the electron-withdrawing effect of NaC is
stronger than that of KC, so the oxidation potentials of
sodium-bound benzo-crown-ether are higher than those of
potassium-bound benzo-crown-ether. Consequently, (10a)
and (10b) are more fluorescent in the presence of
NaC than in the presence of the same concentration of
KC (Table 1).
3. Conclusion
By predicting QE from the HOMO energy level of the
electron donor, we succeeded in easily designing reasonable
PeT-type cation probes. Compounds (10a) and (10b)
become more fluorescent upon binding NaC, and (10b)
shows a good pH profile (low pH-dependency). These
rationally designed probes are simple, having one chelator
as the electron donor and one fluorophore, so that stacking
should not be a problem. This strategy can be applied for
designing molecules containing other fluorescent dyes, for
example, BODIPY16 and rhodamine.17 Our strategy should
be useful not only for developing new probes, but also for
improving existing probes because the key factors for
obtaining better probes, for example, the extent of the
change of HOMO energy level upon the binding the target
and the selectivity of chelator itself, can be controlled by
means of minor structural modifications. We intend to
search for good pairs of electron donors and fluorophores
based on the estimated HOMO levels of electron donor
candidates and thereby to develop novel probes for various
biomolecules.
To confirm the change of HOMO energy level of the benzo-
crown moiety as an electron donor upon NaC binding, we
measured Eox of 2,3,5,6,8,9,11,12-octahydro-16-methyl-
1,4,7,10,13-benzopentaoxacyclopentadecin (6b) in aceto-
nitrile containing various concentrations of sodium per-
chlorate (Fig. 7). The oxidation potential of (6b) was shifted
to the more positive with the increase of sodium perchlorate
content. This indicates that the oxidation potential of (6b)
was raised upon binding NaC. The oxidation potential
difference between the NaC-free form and occupied form
was about 0.10 V. Thus, we obtained the evidence that the
oxidation potential of the electron donors within (10a) and
(10b) was raised upon binding NaC.
Figure 7. Cyclic voltammetry of compound (6b) in acetonitrile containing various concentrations of NaClO4. Ionic strength was kept the same for all solutions
with tetrabutylammonium perchlorate.

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S. Kenmoku et al. / Tetrahedron 60 (2004) 11067–11073
4. Experimental
The reaction mixture was washed with 100 ml of satd
ascorbic acid solution in water, and 100 ml of brine. The
organic layer was dried over Na2SO4 and concentrated
under reduced pressure to give 4.1 g of white solid (yield
71%). 1H NMR (300 MHz, CDCl3) d 7.05 (1H, s, aromatic
H), 6.76 (1H, s, aromatic H), 5.16 (1H, s, OH), 5.14 (1H, s,
OH), 2.27 (3H, s, CH3); MS (EI) m/z (%) 204 [MC2], 202
[MC], 18 (100).
4.1. Materials and general instrumentation
General chemicals were of the best grade available, supplied
by Tokyo Chemical Industries, Wako Pure Chemical or
Aldrich Chemical Company, and were used without further
purification. Special chemicals consisted of dimethyl
sulfoxide (DMSO, fluorometric grade, Dojindo) and tetra-
butylammmonium perchlorate (TBAP, electrochemical
grade, dried over P2O5 before use, Fluka). Acetonitrile,
acetone, N,N-dimethylformamide (DMF), tetrahydrofuran
(THF), methanol, and ethanol were used after appropriate
distillation or purification. NMR spectra were recorded on a
4.4.2. 2,3,5,6,8,9,11,12-Octahydro-15-bromo-16-methyl-
1,4,7,10,13-benzopentaoxacyclopentadecin (4a). A mix-
ture of 3-bromo-4-methylcatechol (5 mmol), tetra(ethylene
glycol) di-p-tosylate (5 mmol), CsF (33 mmol), and dry
acetonitrile (100 ml) was refluxed at 90 8C for 22 h.
Acetonitrile was removed under reduced pressure and the
residue was suspended in AcOEt, filtered to remove CsF,
and concentrated. The resulting residue was chromato-
graphed (NH-silica gel, AcOEt/MeOHZ20/1) to give 0.6 g
1
JNM-LA300 (JEOL) instrument at 300 MHz for H NMR.
Mass spectra (MS) were measured with a JMS-DX300
(JEOL), for EI, an MS700 (JEOL) for FAB and a JMS-
T100LC (JEOL) for ESI-TOF. All experiments were carried
out at 298 K, unless otherwise specified.
1
of white solid (yield 34%). H NMR (300 MHz, CDCl3) d
7.01 (1H, s, 17-H), 6.74 (1H, s, 14-H), 4.1 (4H, m, 2, 12-H),
3.9 (4H, m, 3, 11-H), 3.7 (8H, m, 5, 6, 8, 9-H), 2.30 (3H, s,
CH3); MS (EI) m/z (%) 362 [MC2], 360 [M], 228 (100).
4.2. Fluorescence properties and quantum efficiency of
fluorescence
4.4.3. 2,3,5,6,8,9,11,12-Octahydro-15-methyl-1,4,7,10,13-
benzopentaoxacyclopentadecin (6b). The synthesis of
(6b) followed that of (6a).
Steady-state fluorescence spectroscopic studies were per-
formed on an F4500 (Hitachi). UV–visible spectra were
obtained on a UV-1600 (Shimadzu), with 0.1 mol LK1
NaOHaq (pH 13) as the solvent, unless otherwise specified.
Each solution contained up to 0.1% (v/v) DMF as a
cosolvent. For determination of the fluorescence (Ffl),
fluorescein in 0.1 mol LK1 NaOHaq (FflZ0.85) was used as
a fluorescence standard.18 The quantum efficiencies of
fluorescence were obtained with the following equation
4.4.4. 3,30-Dichloro-2,20,4,40-tetrahydroxybenzophenone
(7b). Compound 7b was prepared according to the
literature.19
4.4.5. 3,6-Dihydroxyxanth-9-one (8a). Compound 8a was
prepared according to the literature.20
(F denotes fluorescence intensity at each wavelength and
[F] was calculated by summation).
P
4.4.6. 2,7-Dichloro-3,6-dihydroxyxanth-9-one (8b).
Compound 8b was prepared according to the literature.20
ðsampleÞ
ðstandardÞ
F
Z F
AbsðstandardÞ=AbsðsampleÞ
fl
fl
4.4.7. O,O0-Bis(tert-butyldimethylsilyl)-3,6-dihydroxy-
xanth-9-one (9a). Compound 9a was prepared according
to the literature.21
X
4.3. Cyclic voltammetry
X
ꢀ
½FðsampleÞꢁ= ½FðstandardÞꢁ
4.4.8. O,O0-Bis(tert-butyldimethylsilyl)-2,7-dichloro-3,6-
dihydroxyxanth-9-one (9b). Compound 9b was prepared
according to the literature.21
Cyclic voltammetry was performed on a 600A electro-
chemical analyzer (ALS). A three-electrode arrangement in
a single cell was used for the measurements: a Pt wire as the
auxiliary electrode, a glassy carbon electrode as the working
electrode, and an Ag/AgC electrode as the reference
electrode. The sample solutions contained 1.0!10K3 M
sample and 0.1 M tetrabutylammonium perchlorate (TBAP)
as a supporting electrolyte in acetonitrile, and argon was
bubbled through the solution for 10 min before each
measurement.
4.4.9. 9-(20,30,50,60,80,90,110,120-Octahydro-160-methyl-
10,40,70,100,130-benzopentaoxacyclopentadecin-150-yl)-6-
hydroxy-3H-xanthen-3-one (10a). To a solution of (6a)
(150 mg) in dry 2-methyltetrahydrofuran (15 ml), a portion
of tert-butyllithium in n-pentane (1.54 N, 1 ml) was added
dropwise via a syringe at below K150 8C over 10 min. The
mixture was stirred for 30 min at below K150 8C, then a
solution of (9a) in dry 2-methyltetrahydrofuran (3 ml) was
added dropwise via a syringe over 10 min, and the reaction
mixture was allowed to warm to room temperature over 1 h.
A portion of 2 N HClaq was added, a solution of
tetra(n-butyl)ammonium fluoride in THF was added, and
the reaction mixture was refluxed at 80 8C for 1 h. The
mixture was evaporated under reduced pressure. The
residue was dissolved in CH2Cl2, and the solution was
washed with a portion of 2 N HClaq. The organic layer was
extracted with five portions of 2 N NaOHaq, and then
the aqueous solution was acidified with 2 N HClaq, and
4.4. Preparation
4.4.1. 4-Bromo-5-methylcatechol (3). To a solution of
4-methylcatechol (28 mmol) in CH2Cl2 (100 ml), a mixture
of bromine (31 mmol) and CH2Cl2 (20 ml) was added
dropwise at K80 8C. When a white solid precipitate
appeared, addition was stopped to avoid formation of the
dibromoderivative (about 15 mmol of bromine was added).

S. Kenmoku et al. / Tetrahedron 60 (2004) 11067–11073
11073
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extracted with CH2Cl2. The organic layer was dried over
Na2SO4, and evaporated under reduced pressure. The
resulting residue was chromatographed (silica gel,
CH2Cl2/methanolZ10/2) to give orange-colored solid
1
(yield 8.0%). H NMR (300 MHz, CDCl3) d 7.09 (2H, d,
1, 8-H, JZ1.9 Hz), 7.05 (2H, d, 4, 5-H, JZ9.2 Hz), 6.91
(1H, dd, 2, 7-H, JZ1.9, 9.2 Hz), 6.86 (2H, s, 1, 140-H), 6.66
(1H, s, 1700-H), 4.2 (2H, m, 120-H), 4.1 (2H, m, 20-H), 4.0
(2H, m, 11 -H), 3.9 (2H, m, 30-H), 3.8 (4H, m, 50, 90-H), 3.77
(4H, s, 60, 80-H), 1.95 (3H, s, CH3) %). 13C NMR (300 MHz,
CDCL3) d 19.4, 68.2, 68.8, 69.6, 70.2, 103.1, 114.2, 115.4,
116.0, 121.5, 132.5, 159.0, 207.1; MS (FAB) m/z 515
[MNaC]; HRMS (ESI-Tof) [MNaC] 515.16819, found
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4.4.10. 9-(20,30,50,60,80,90,110,120-Octahydro-160-methyl-
10,40,70,100,130-benzopentaoxacyclopentadecin-150-yl)-
2,7-dichloro-6-hydroxy-3H-xanthen-3-one (10b). The
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1
synthesis of (10b) followed that of (10a) 0(yield 10%). H
NMR (300 MHz, DMSO) d 7.04 (1H, s, 14 -H), 6.84 (1H, s,
1700-H), 6.81 (2H, s, 1,08-H), 6.26 (2H, s, 4, 05-H), 4.2 (2H, m,
12 -H), 4.0 (2H, m,0 2 -H), 3.8 (2H, m, 11 -H), 3.7 (2H, m,
30-H), 3.6 (4H, m, 5 , 90-H), 3.6 (4H, s, 60, 80-H) 1.94 (3H, s,
CH3). 13C NMR (300 MHz, CDCL3) d 18.6, 68.0, 68.4,
68.5, 69.3, 70.1, 103.6, 109.3, 114.4, 115.3, 124.3, 126.9,
127.0, 128.1, 132.9, 146.1, 148.6, 149.8, 156.2; MS (FAB)
m/z 583 [MNaC], 585 ([MC2]NaC); HRMS (ESI-Tof)
[MNaC] 583.09024, found 583.08912 (K1.12 mmu).
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Acknowledgements
10. Urano, Y.; Kanda, K.; Kamiya, M.; Ueno, T.; Hirose, K;
Nagano, T. Submitted for publication.
This study was supported in part by the Advanced and
Innovational Research Program in Life Sciences from the
Ministry of Education, Culture, Sports, Science and
Technology, the Japanese Government, by Takeda Science
Foundation, by Nagase Science and Technology Foun-
dation, by research grants from the Ministry of Education,
Science, Sports and Culture of Japan (Grant Nos. 12771349,
13557209, and 14030023 to Y.U.), and by Kowa Life
Science Foundation to Y.U.
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