Design and synthesis of an enzyme-cleavable sensor molecule for phosphodiesterase activity based on fluorescence resonance energy transfer

Article abstract of DOI:10.1021/ja011251q

Ratiometric measurement is a technique that can provide precise data and even quantitative detection. To carry out ratiometric measurements, it is necessary that the sensor molecule exhibits a large shift in its emission or excitation spectrum after react

Full text of DOI:10.1021/ja011251q

Published on Web 02/01/2002
Design and Synthesis of an Enzyme-Cleavable Sensor
Molecule for Phosphodiesterase Activity Based on
Fluorescence Resonance Energy Transfer
Hideo Takakusa,† Kazuya Kikuchi,† Yasuteru Urano,† Shigeru Sakamoto,‡
Kentaro Yamaguchi,‡ and Tetsuo Nagano*,†
Contribution from the Graduate School of Pharmaceutical Sciences, UniVersity of Tokyo,
7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and Chemical Analysis Center,
Chiba UniVersity, Yayoi-cho, Inage-ku, Chiba 263-8522, Japan
Received May 22, 2001. Revised Manuscript Received November 5, 2001
Abstract: Ratiometric measurement is a technique that can provide precise data and even quantitative
detection. To carry out ratiometric measurements, it is necessary that the sensor molecule exhibits a large
shift in its emission or excitation spectrum after reaction with the target molecule. Fluorescence resonance
energy transfer (FRET) is one mechanism used to obtain a large spectral shift. In this study, our aim was
to develop a ratiometric fluorescent sensor molecule for phosphodiesterase activity based on FRET. We
designed and synthesized CPF4 with a coumarin donor, a fluorescein acceptor, and two phenyl linkers
having the phosphodiester moiety interposed between them. In the emission spectrum of CPF4 in aqueous
buffer excited at 370 nm, the emission of the coumarin donor was strongly quenched and the emission of
the fluorescein acceptor was observed. This emission spectrum demonstrates that energy transfer from
the coumarin donor to the fluorescein acceptor proceeds efficiently. Addition of a phosphodiesterase to an
aqueous solution of CPF4 resulted in an increase in the donor fluorescence and a decrease in the acceptor
fluorescence. CPF4 exhibited a large shift in its emission spectrum after the hydrolysis of the phosphodiester
group by the enzyme. This large shift of the emission spectrum indicates that ratiometric measurements
can be made by using CPF4. The method described in this paper for designing enzyme-cleavable sensor
molecules based on FRET should be readily applicable to other hydrolytic enzymes.
Introduction
molecule exhibits a large shift in its emission or excitation
wavelength after reaction with a target molecule. Fluorescence
In recent years, many fluorescent sensor molecules have been
developed to probe biological phenomena in living cells.1 A
fluorescent sensor is advantageous due to its high sensitivity,
but may be influenced by many factors such as photobleaching,
changes in the sensor molecule concentration, changes of
environment around the sensor molecule (pH, polarity, temper-
ature, and so forth), and stability under illumination. To reduce
the influence of such factors, ratiometric measurements are
utilized. Ratiometric measurements involve observation of
changes in the ratio of the fluorescence intensities of the
excitation or the emission at two wavelengths. This technique
allows more precise measurement and, with some sensor
molecules, quantitative detection is possible. To carry out
ratiometric measurements, it is necessary that the sensor
resonance energy transfer (FRET) is one mechanism used to
obtain a large spectral shift. FRET is an interaction between
the electronic excited states of two fluorophores, in which
excitation energy of a donor is transferred to an acceptor without
emission of a photon.2 Recently, some ratiometric fluorescent
sensor molecules based on FRET have been developed and
widely used, such as the calcium sensor cameleon3 and the
â-lactamase sensor CCF2.4 In this study, our aim is to develop
a ratiometric fluorescent sensor molecule with low molecular
weight for phosphodiesterase activity based on FRET.
Phosphodiesterases catalyze the hydrolysis of phosphodiester
bonds and their substrates are nucleic acids and cyclic nucle-
otides.5 Although phosphodiesterases have many important
cellular roles, there is no effective method to monitor their
activity in real time with high sensitivity. For example,
nucleotide pyrophosphatase/phosphodiesterases6 (NPPs), one
group of phosphodiesterases, have been reported to be implicated
* Corresponding author: (e-mail) tlong@mol.f.u-tokyo.ac.jp; (fax) +81-
3-5841-4855.
† University of Tokyo.
‡ Chiba University.
(1) (a) Kojima, K.; Nakatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino, Y.;
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Kikuchi, K.; Higuchi, T.; Nagano, T. Angew. Chem., Int. Ed. Engl. 1999,
38, 2899-2901. (d) Hirano, T.; Kikuchi, K.; Urano, Y.; Higuchi, T.;
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M.; Tsien, R. Y. Nature 1997, 388, 882-887.
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9
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J. AM. CHEM. SOC. VOL. 124, NO. 8, 2002 1653

A R T I C L E S
Takakusa et al.
Chart 1. The Structures of CPFs
they should form the ground-state dye-to-dye close contact in
an aqueous environment and the fluorescence should be
quenched.11,12 CPF2 with two cyclohexane linkers exhibited
acceptor fluorescence due to FRET by hindering the close
contact of the two fluorophores. So, it is necessary that the
structure of the sensor molecule is such as to prevent dye-to-
dye close contact in an aqueous environment. However, CPF2
was not hydrolyzed by phosphodiesterases due to the structural
features of the molecule. Thus, it is also necessary that the
molecule should be recognized as a substrate by phosphodi-
esterases. None of the first-generation CPFs satisfied both
requirements (no close contact of fluorophores in aqueous
environments and recognition as a substrate by the enzyme)
and worked as a ratiometric fluorescent sensor molecule for
phosphodiesterase activity. In this study, we intended to develop
an enzyme-cleavable sensor molecule for ratiometric measure-
ment by optimizing the linker such as to satisfy both require-
ments.
Results and Discussion
Design and Synthesis of CPF4 and Its Spectroscopic
Properties. There are several reports13 about the features of
the substrate binding site of phosphodiesterase I based on the
specificity and kinetics of some artificial substrates. According
to the reports, phosphodiesterase I catalyzes the hydrolysis of
a wide range of synthetic phosphodiesters in addition to naturally
occurring nucleotide substrates. In particular, the compounds
which contain diphenyl phosphate structure [e.g., bis(4-nitro-
phenyl) phosphate,13a,b 4-nitrophenyl phenyl phosphate,13a
4-nitrophenyl 4-tert-butylphenyl phosphate,13d and bis(4-meth-
ylumbelliferyl) phosphate13e] could be hydrolyzed by phospho-
diesterase I. Thus, it was presumed that the phenyl moiety would
be appropriate as the enzyme cleavable linker and designed
CPF4 with two phenyl linkers. The structure of CPF4 and the
sensor mechanism are shown in Scheme 1. In CPF4, coumarin
as the donor and fluorescein as the acceptor are held in close
proximity, and the absorption spectrum of the acceptor and the
emission spectrum of the donor overlap. Therefore FRET takes
place and the acceptor fluorescence can be observed. After the
hydrolysis of the phosphodiester group by a phosphodiesterase,
FRET no longer takes place and the donor fluorescence can be
observed because the donor and acceptor diffuse away from
one another. Synthesis of CPF4 is described in Scheme 2. The
control compounds 1 and 2 were also synthesized to investigate
the spectroscopic properties of CPF4.
in the regulation of various intra- and extracellular processes,
including cell differentiation and motility, bone and cartilage
mineralization, and signaling by nucleotides and insulin. Some
important proteins categorized in NPPs such as PC-17 and
Autotaxin8 have been the focus of much interest in recent years.
Since NPPs consist of phosphodiesterase I (EC 3.1.4.1), the
probe for phosphodiesterase I activity will be a useful tool for
investigating the role of NPPs. Berkessel and Riedl have
developed fluorescence reporters for phosphodiesterase I activ-
ity, in which a naphthalene moiety acts as the fluorophore and
an azobenzene moiety as the quencher.9 These reporters
fluoresce upon addition of phosphodiesterases, but have limita-
tions for biological application because of their short excitation
wavelength and weak fluorescence. Also, the change of
fluorescence intensity is observed at one wavelength. To obtain
a ratiometric fluorescent sensor molecule for phosphodiesterase
activity, we previously designed and synthesized intramolecular
FRET compounds (CPFs) which have coumarin as a donor,
fluorescein as an acceptor, and phosphodiester as a linker (Chart
1).10 CPF1 with two ethylene linkers and CPF3 with one
ethylene and one cyclohexane linker exhibited fluorescence
quenching due to the dye-to-dye close contact. If the fluoro-
phores are introduced intramolecularly with a flexible linker,
The emission spectrum of CPF4 in an aqueous buffer solution
excited at 370 nm, which is the excitation wavelength of the
coumarin moiety, is shown in Figure 1 (spectrum A). The
emission of the coumarin donor at around 450 nm was strongly
quenched and the emission of the fluorescein acceptor at around
515 nm was observed. This emission spectrum demonstrates
that energy transfer from the coumarin donor to the fluorescein
acceptor can proceed efficiently. The energy transfer efficiency
(6) (a) Bollen, M.; Gijsbers, R.; Ceulemans, H.; Stalmans, W.; Stefan, C. Crit.
ReV. Biochem. Mol. 2000, 35, 393-432. (b) Zimmermann, H.; Braun, N.
Prog. Brain Res. 1999, 120, 371-385. (c) Zimmermann, H. Trends.
Pharmacol. Sci. 1999, 20, 231-236. (d) Goding, J. W. J. Leukoc. Biol.
2000, 67, 285-311.
(11) Takakusa, H.; Kikuchi, K.; Urano, Y.; Higuchi, T.; Nagano, T. Anal. Chem.
2001, 73, 939-942.
(12) Mizukami, S.; Kikuchi, K.; Higuchi, T.; Urano, Y.; Mashima, T.; Tsuruo,
T.; Nagano, T. FEBS Lett. 1999, 453, 356-360.
(13) (a) Landt, M.; Everard, R. A.; Butler, L. G. Biochemistry 1980, 19, 138-
143. (b) Razzell, W. E.; Khorana, H. G. J. Biol. Chem. 1959, 234, 2105-
2113. (c) Burgers, P. M. J.; Eckstein, F. Proc. Natl. Acad. Sci. 1978, 75,
4798-4800. (d) Hengge, A. C.; Tobin, A. E.; Cleland, W. W. J. Am. Chem.
Soc. 1995, 117, 5919-5926. (e) Wright, C. E.; Beratis, N. G. Am. J. Human
Gen. 1980, 32, 60A.
(7) Goding, J. W.; Terkeltaub, R.; Maurice, M.; Deterre, P.; Sali, A.; Belli, S.
I. Immunol. ReV. 1998, 161, 11-26.
(8) (a) Stracke, M. L.; Clair, T.; Liotta, L. A. AdV. Enzyme Regul. 1997, 37,
135-144. (b) Clair, T.; Lee, H. Y.; Liotta, L. A.; Stracke, M. L. J. Biol.
Chem. 1997, 272, 996-1001.
(9) Berkessel, A.; Riedl, R. Angew. Chem., Int. Ed. Engl. 1997, 36, 1481-
1483.
(10) Kawanishi, Y.; Kikuchi, K.; Takakusa, H.; Mizukami, S.; Urano, Y.;
Higuchi, T.; Nagano, T. Angew. Chem., Int. Ed. 2000, 39, 3438-3440.
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Design of Enzyme-Cleavable Sensor Molecules
A R T I C L E S
Scheme 1. The Structure of CPF4 and the Sensor Mechanism
Figure 1. The emission spectra of a 1.0 µM solution of CPF4 in Tris-HCl
buffer (100 mM, pH 8.8) at 37 °C: (A) without enzyme; (B) 1 min; (C) 5
min; (D) 10 min; (E) 15 min; (F) 20 min; (G) 30 min; (H) 45 min after
addition of 0.05 u of phosphodiesterase I (Crotalus adamanteus venom);
(I) a mixture of 1.0 µM 1 and 1.0 µM 2.
Scheme 2. Synthetic Scheme for CPF4
Figure 2. Effect of pH on the fluorescence intensity of CPF4. Closed
circle: fluorescence intensity of 1.0 µM compound 1, λex 370 nm, λem 450
nm. Open circle: fluorescence intensity of 1.0 µM CPF4, λex 370 nm, λem
515 nm. Open triangle: fluorescence ratio of compound 1 and CPF4.
Solvent: 100 mM sodium phosphate buffer.
approximately 48 Å from the fluorescence of compound 1 and
the absorption of compound 2. Thus, these values indicate that
the transfer efficiency should be close to 100% and the
experimentally obtained value for the FRET efficiency of CPF4
(94%) is reasonable. Figure 2 shows the pH profile of
fluorescence intensity of the donor and acceptor. Both the donor
and acceptor fluorescence strikingly decreased below pH 7. The
pKa values of the donor and acceptor emission were calculated
as 5.95 and 6.48, respectively. The pH profile of the fluorescence
ratio of the donor and acceptor emission is also shown. The
fluorescence ratio value was hardly affected by change of pH
in the physiologically possible region.
The absorption spectra of CPF4 and the mixture of com-
pounds 1 and 2 were measured in aqueous environment (Figure
3). It was reported that a large red shift in the absorption
spectrum is observed if the coumarin and fluorescein moieties
form dye-to-dye close contact.7,8 CPF1, which shows fluores-
cence quenching due to dye-to-dye close contact, exhibited a
large red shift in its absorption spectrum (λmax ) 380 nm, 500
nm). No shift in the absorption spectrum of CPF4 (λmax ) 372
was calculated as 94%. According to the Fo¨rster theory,2 the
rate of FRET is inversely proportional to R.6 So, the energy
transfer efficiency is strongly dependent on R when R is near
the Fo¨rster distance (R0), which can be defined as the donor-
acceptor distance at which the transfer efficiency is 50%. The
transfer efficiency abruptly increases to 100% as R decreases
below R0. In CPF4, R was estimated to be shorter than 30 Å by
means of the CPK model and R0 was calculated to be
nm, 495 nm) was observed compared to the control (λmax
)
372 nm, 495 nm). So, the absorption spectrum demonstrates
that the two fluorophores in CPF4 cannot interact with each
other and fluorescence quenching due to dye-to-dye close
contact is not observed.
Enzyme Assay. Addition of a phosphodiesterase, for example
phosphodiesterase I from Crotalus adamanteus venom,14 to an
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A R T I C L E S
Takakusa et al.
Table 1. Summary of CPFs
abs max (nm)
acceptor
FRET effd
(%)
vmax
e
acceptor
quantum effc
CPFs
donor
Kme (M)
(mol/min/mg)
CPF1a
380
372
373
372
372
500
494
497
495
494
0.10
0.83
0.27
0.58
0.69
2.5 × 10-6
1.6 × 10-9
CPF2a
>95
CPF3a
CPF4
94
1.6 × 10-7
1.0 × 10-3
1.8 × 10-9
3.4 × 10-7
controlb
bis(4-nitrophenyl) phosphate
a Reference 10. b A mixture of 1 and 2. c Data were measured in aqueous buffers, pH 7.4 (λex ) 492 nm, λem ) 515 nm). d Data were obtained from the
fluorescence quantum yield of donor in the presence and in the absence of acceptor. e Data were measured in 100 mM Tris-HCl buffer (pH 8.8) at 37 °C.
data are summarized in Table 1. As a standard, Km and Vmax of
bis(4-nitrophenyl) phosphate were also measured under the same
conditions (Table 1). The kinetic parameters (Km and Vmax) of
phosphodiesterase I were reported against the various sub-
strates.13,15 It can be seen that these values vary widely according
to the structures of the substrates [e.g., bis(4-nitrophenyl)
phosphate:13b Km ) 770 µM, Vmax ) 650 nmol/min/mg;
thymidine dinucleotide:13b Km ) 210 µM, Vmax ) 110 µmol/
min/mg; 5′-ATP:15a Km , 200 µM, Vmax ) 4.6 µmol/min/mg;
p-nitrophenyl thymidine 5′-phosphate:15b Km ) 46 µM, Vmax
)
50 µmol/min/mg]. Comparing the experimentally obtained
kinetic parameters of CPF4 with the literature data, Km and Vmax
values of CPF4 are much lower than that for the other substrates
including some native substrates. These observations suggest
that CPF4 have a high affinity for phosphodiesterase I though
the catalytic rate of CPF4 is relatively slow. In addition to
phosphodiesterase I, phosphodiesterases consist of cyclic-
nucleotide phosphodiesterases and nucleic acid phosphodi-
esterases (ribonucleases and deoxyribonucleases). So, we per-
formed the fluorogenic assays for these two enzymes by using
CPF4. Consequently, the fluorescence spectrum of CPF4 was
not affected by the addition of phosphodiesterase 3′,5′-cyclic-
nucleotide (from bovine heart, Sigma) nor nuclease (from
staphylococcus aureus, Sigma). These observations indicate that
CPF4 should be a specific probe for phosphodiesterase I.
Figure 3. The absorption spectra of a 10 µM solution of CPF4 (open circle),
a mixture of 10 µM 1 and 10 µM 2 (closed circle), and a 10 µM solution
of CPF1 (closed square) in HEPES buffer (100 mM, pH 7.4) at 25 °C.
Conclusion
The phenyl linkers were sufficiently rigid to prevent dye-to-
dye close contact and the diphenyl phosphate could be recog-
nized as a substrate by phosphodiesterase I. Thus, we succeeded
in developing a ratiometric fluorescence sensor molecule for
phosphodiesterase I activity by introducing two phenyl linkers
between the donor and acceptor moieties. Since CPF4 allows
more precise and quantitative detection of phosphodiesterase I
activity, it should be useful for studies on the biological
functions of phosphodiesterases. For example, CPF4 will be
applied as a ratiometric probe for the molecules that possess
the phosphodiesterase I activity such as NPPs. The method
described in this paper for designing enzyme-cleavable sensor
molecules based on FRET should be readily applicable to other
hydrolytic enzymes.
Figure 4. Michaelis-Menten plot of CPF4 hydrolysis by phosphodiesterase
I (0.01 u) in 100 mM Tris-HCl buffer (pH 8.8) at 37 °C.
aqueous solution of CPF4 resulted in a rapid increase in the
donor fluorescence at around 460 nm and a rapid decrease in
the acceptor fluorescence at around 515 nm as shown in Figure
1. The emission intensity of CPF4 asymptotically approached
that of an equimolar mixture of 1 and 2 (spectrum I). CPF4
exhibited a large shift in its emission spectrum after the
hydrolysis of the phosphodiester group by the enzyme. The
emission maximum shifted from 515 to 460 nm. This large shift
of the emission spectrum indicates that ratiometric measurements
can be feasible by using CPF4. A plot of initial velocity of
hydrolysis versus substrate concentration is shown in Figure 4.
The values of initial velocity were calculated from the enhance-
ment of the donor emission intensity (λex 370 nm, λem 450 nm).
From this Michaelis-Menten plot, Km and Vmax were calculated
as 160 nM and 1.8 × 10-9 mol/min/mg, respectively. The CPFs
Experimental Section
Instruments. NMR spectra were recorded on a JEOL JNM-LA300
1
instrument at 300 MHz for H NMR and at 75 MHz for 13C NMR.
Mass spectra (MS) were measured with JEOL SX-102A and JEOL
JMS-700T mass spectrometers.
(14) Phosphodiesterase I (from Crotalus adamanteus venom), Sigma. Definition
of activity by the suppliers: 1 unit hydrolyzes 1.0 µmol of bis(p-
nitrophenyl)phosphate per min at pH 8.8 and 37 °C.
(15) (a) Kelly, S. J.; Dardinger, D. E.; Butler, L. G. Biochemistry 1975, 14,
4983-4988. (b) Razzell, W. E. J. Biol. Chem. 1961, 236, 3031-3036.
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Design of Enzyme-Cleavable Sensor Molecules
A R T I C L E S
Synthesis of Bis(4-aminophenyl) Phosphoric Acid (3). Bis(4-
nitrophenyl)phosphoric acid (1.0 g, 2.9 mmol) was dissolved in
ethanol-water (2:1) (30 mL). After the addition of 10% Pd-C (300
mg), the mixture was stirred vigorously under a H2 atmosphere for 3
h. The Pd-C was filtered off and washed with water. The residue after
evaporation of the filtrate was purified by column chromatography with
use of HP-20SS (Mitsubishi Chemical Corporation, Tokyo, Japan) resin,
eluted with H2O. The appropriate fractions, after evaporation to dryness,
gave 600 mg of 3 (yield 73%). 1H NMR (300 MHz, DMSO-d6) δ 5.35
(s, 4H); 6.51 (d, 4H, J ) 8.8 Hz); 6.82 (d, 4H, J ) 8.8 Hz). 13C NMR
(75 MHz, DMSO-d6) δ 119.0; 120.5; 135.3; 148.0. MS (FAB): 181
(M + H+).
Synthesis of 6-Chloro-7-hydroxy-4-(4-hydroxyphenylcarbamoyl-
methyl)coumarin (1). 4-Aminophenol (420 mg, 3.9 mmol) and
6-chloro-7-hydroxycoumarin-4-acetic acid (100 mg, 0.39 mmol) were
dissolved in DMF. EDC-HCl (150 mg, 0.77 mmol) and HOBt (100
mg, 0.74 mmol) were added to the solution and the mixture was stirred
at 0 °C overnight. The reaction mixture was diluted with 2 N HCl and
was extracted with ethyl acetate. The organic phase was washed with
2 N HCl and brine and dried over sodium sulfate. The residue after
the removal of solvent was chromatographed on silica gel, eluted with
dichloromethane to dichloromethane/MeOH ) 19/1. The appropriate
fraction gave 80 mg of 1 (yield 59%). 1H NMR (300 MHz, DMSO-d6)
δ 3.83 (s, 1H); 6.29 (s, 1H); 6.68 (d, 2H, J ) 8.6 Hz); 6.91 (s, 1H);
7.33 (d, 2H, J ) 8.6 Hz); 7.69 (m, 1H); 7.86 (s, 1H); 9.21 (s, 1H);
10.0 (s, H). 13C NMR (75 MHz, DMSO-d6) δ 39.3; 103.4; 112.2; 112.8;
115.5; 116.9; 121.2; 126.3; 130.3; 150.3; 153.2; 153.6; 156.4; 159.7;
165.7. HRMS (EI+): calcd for M+, 345.0404; found, 345.0425.
Synthesis of 2-Aminophenyl 6-Chloro-7-hydroxycoumarin-4-
acetamidophenyl Phosphoric Acid (4). Compound 3 (150 mg, 0.52
mmol) and 6-chloro-7-hydroxycoumarin-4-acetic acid (140 mg, 0.52
mmol), which had been prepared from chlororesorcinol and acetonedi-
carboxylic acid, were dissolved in DMF. Diisopropylethylamine (DIEA)
(0.10 mL, 580 µmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC-HCl) (190 mg, 1.0 mmol), and hydroxybenzo-
triazole (HOBt) (130 mg, 0.96 mmol) were added to the solution and
the mixture was stirred at 0 °C overnight. The reaction mixture was
poured into 2 N HCl (50 mL) and the resulting precipitate was removed
by filtration. The mother liquor was evaporated and the residue was
purified by column chromatography with use of HP-20SS resin, eluted
with H2O to MeOH/H2O ) 1/1. The appropriate fractions, after
Synthesis of 6-(2-Hydoxyphenylcarbamoyl)fluorescein (2). 4-Amino-
phenol (390 mg, 3.6 mmol) and 6-carboxyfluorescein (100 mg, 0.27
mmol) were dissolved in DMF. EDC-HCl (160 mg, 0.84 mmol) and
HOBt (110 mg, 0.81 mmol) were added to the solution and the mixture
was stirred at 0 °C overnight. The reaction mixture was diluted with 2
N HCl and extracted with ethyl acetate. The organic phase was washed
with 2 N HCl and brine, and dried over sodium sulfate. The residue
after the removal of solvent was chromatographed on silica gel, eluted
with dichloromethane/MeOH ) 19/1 to dichloromethane/MeOH ) 9/1.
1
evaporation to dryness, gave 24 mg of 4 (yield 8.9%). H NMR (300
1
The appropriate fraction gave 70 mg of 2 (yield 56%). H NMR (300
MHz, DMSO-d6) δ 3.87 (s, 2H); 6.30 (s, 1H); 6.91 (s, 1H); 6.98 (d,
2H, J ) 8.4 Hz); 7.06 (d, 2H, J ) 9.0 Hz); 7.08 (d, 2H, J ) 8.4 Hz);
7.41 (d, 2H, J ) 9.0 Hz); 7.85 (s, 1H); 10.2 (s, 1H). 13C NMR (75
MHz, DMSO-d6) δ 103.5; 112.3; 113.0; 116.9; 119.9; 120.3; 120.8;
121.8; 125.6; 126.4; 133.6; 149.1; 150.2; 150.8; 153.2; 156.4; 159.8;
166.1. MS (FAB): 517 (M + H+).
MHz, DMSO-d6) δ 6.56 (dd, 2H, J ) 3.0, 8.6 Hz); 6.61 (d, 2H, J )
8.6 Hz); 6.69 (d, 2H, J ) 3.0 Hz); 6.69 (d, 2H, J ) 9.2 Hz); 7.42 (d,
2H, J ) 9.2 Hz); 7.80 (s, 1H); 8.11 (d, 1H, J ) 7.9 Hz); 8.25 (d, 1H,
J ) 7.9 Hz); 9.27 (s, 1H); 10.1 (s, 2H). 13C NMR (75 MHz, DMSO-
d6) δ 83.4; 102.2; 109.1; 112.7; 114.9; 122.6; 122.7; 124.8; 128.2; 129.2;
129.7; 129.9; 141.2; 151.8; 152.6; 154.1; 159.6; 163.1; 168.0. HRMS
(FAB+): calcd for (M + H+), 468.1083; found, 468.1115.
Fluorometric Analysis. A fluorescence spectrophotometer (F4500,
Hitachi, Tokyo, Japan) was used. The slit width was 2.5 nm for both
excitation and emission. The photomultiplier voltage was 950 V. CPF4
was dissolved in DMSO to make a 10 mM stock solution, which was
diluted to the required concentration for measurement.
Synthesis of CPF4. Compound 4 (16 mg, 31 µmol) and 6-carboxy-
fluorescein (23 mg, 62 µmol), which had been prepared according to
the procedure reported by Rossi and Kao,16 were dissolved in DMF.
DIEA (10 µL, 58 µmol), EDC-HCl (19 mg, 0.1 mmol), and HOBt
(14 mg, 0.1 mmol) were added to the solution and the mixture was
stirred at 0 °C overnight. The reaction mixture was poured into 2 N
HCl (5 mL) and the resulting precipitate was collected by filtration.
This precipitate was purified by column chromatography with use of
HP-20SS resin, eluted with H2O. The appropriate fractions, after
evaporation to dryness, gave 2.1 mg of compound CPF4 (yield 7.7%).
1H NMR (300 MHz, DMSO-d6) δ 3.68 (s, 2H); 5.98 (d, 2H, J ) 2.0
Hz); 6.06 (dd, 2H, J ) 2.0, 9.1 Hz); 6.61 (d, 2H, J ) 9.1 Hz); 7.12 (d,
4H, J ) 9.0 Hz); 7.46 (d, 2H, J ) 9.0 Hz); 7.49 (s, 1H); 7.61 (d, 2H,
J ) 9.0 Hz); 7.75 (s, 1H); 8.09 (s, 2H). 13C NMR (75 MHz, D2O) δ
39.9; 105.1; 106.3; 108.5; 108.6; 113.3; 118.5; 121.8; 122.0; 123.7;
124.2; 124.3; 124.5; 125.3; 130.0; 132.3; 132.7; 134.3; 134.4; 135.6;
144.3; 150.1; 150.2; 152.7; 155.9; 158.2; 159.7; 166.2; 168.6; 169.0;
170.5; 175.3; 181.9. HRMS (FAB+): calcd for (M + H+), 875.1045;
found, 875.1080.
Absorption Analysis. A spectrometer (UV-1600, Hitachi, Japan)
was used. All samples were prepared from 10 mM stock solutions in
DMSO.
Acknowledgment. This work was supported in part by the
Ministry of Education, Science, Sports and Culture of Japan
(grant numbers 11794026, 12470475, and 12557217 to T.N.,
11771467 and 12045218 to K.K.), as well as the Mitsubishi
Foundation and the Research Foundation for Opt-Science and
Technology. K.K. gratefully acknowledges financial support
from the Nissan Science Foundation, the Shorai Foundation of
Science and Technology, and the Uehara Memorial Foundation.
(16) Rossi, F. M.; Kao, J. P. Y. Bioconj. Chem. 1997, 8, 495-497.
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