Intramolecular fluorescence resonance energy transfer system with coumarin donor included in β-cyclodextrin

Article abstract of DOI:10.1021/ac001016a

In aqueous solutions, the fluorescence of the intramolecular fluorescence resonance energy-transfer (FRET) system 1 was strongly quenched, because of close contact between the donor and acceptor moieties. FRET occurred, and the acceptor fluorescence was i

Full text of DOI:10.1021/ac001016a

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Anal. Chem. 2001, 73, 939-942
Intramolecular Fluorescence Resonance Energy
Transfer System with Coumarin Donor Included in
â-Cyclodextrin
Hideo Takakusa, Kazuya Kikuchi, Yasuteru Urano, Tsunehiko Higuchi, and Tetsuo Nagano*
Graduate School of Pharmaceutical Sciences, the University of Tokyo,7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
possible. Recently, Tsien and co-workers reported several indica-
tors using FRET for the detection of adenosine 3′,5′-cyclic
monophosphate (cAMP),3 calcium cation,4 and â-lactamase2 activ-
ity, and they employed these probes for assays at physiological
concentrations and for imaging activity changes in living cells.
Peptides bearing fluorescent dyes have been widely used in the
protease assay.5 FRET peptide probes are superior to single dye-
labeled probes for this biological application, because we can
observe the ratio of the fluorescence. However, it is difficult to
obtain such FRET peptide probes, because peptides in aqueous
solutions take conformations such that the donor and acceptor
moieties are in close proximity, and the emissions of the fluoro-
phores are quenched.6 This quenching mechanism can be
explained in terms of ground-state complex formation.7 It has been
reported that the fluorescence quenching of the ground-state dye-
to-dye complex formation is observed in various fluorophore pairs.6
In general, if the fluorophores have the hydrophobic character-
istics, they would form dye-to-dye close contact in aqueous
environment and the fluorescence should be quenched.8,10 For
practical use of peptide-based FRET probes, it is necessary to
employ conformationally constrained oligopeptides such as pro-
In aqueous solutions, the fluorescence of the intramo-
lecular fluorescence resonance energy-transfer (FRET)
system 1 was strongly quenched, because of close contact
between the donor and acceptor moieties. FRET occurred,
and the acceptor fluorescence was increased, by adding
â-cyclodextrin (â-CD) to aqueous solutions of 1 . Spectral
analysis supported the idea that the FRET enhancement
was due to the formation of an inclusion complex of the
coumarin moiety in â-CD, resulting in separation of the
fluorophores. On the basis of this result, we propose that
covalent binding of coumarin to â-CD will provide a FRET
cassette molecule. So, compound 2 bearing â-CD co-
valently was designed and synthesized. Fluorescence
intensity of 2 was enhanced markedly compared to the
intensity of 3 . Applying this FRET system, various FRET
probes that will be useful for ratio imaging and also the
high-throughput screening will be provided.
In recent years, many fluorescent probes1 have been developed
to study biological phenomena in living cells. A fluorescence
resonance energy-transfer (FRET) technique was used in some
fluorescent probes. FRET is an interaction between the electronic
excited states of two fluorophores, in which excitation energy is
transferred from a donor to an acceptor without emission of a
photon. The FRET technique has been applied to probe biological
systems and also for high-throughtput screening of combinatorial
libraries,2 by means of ratiometric measurements. Ratiometric
measurements are methods that observe the changes in the ratio
of the fluorescence intensities at two wavelengths. Using ratio-
metric measurements, it is possible to reduce the influence of
many artifacts due to the change of the probe concentration and
excitation intensity. Therefore, this technique allows more precise
measurements, and with some probes, quantitative detection is
(3) Adams, S. R.; Harootunian, A. T.; Buechler, Y. J.; Taylor, S. S.; Tsien, R. Y.
Nature 1 9 9 1 , 349, 694-697.
(4) Miyawaki, A.; Llopis, J.; Heim, R.; McCaffery, J. M.; Adams, J. A.; Ikura,
M.; Tsien, R. Y. Nature 1 9 9 7 , 388, 882-887.
(5) (a) Matayoshi, E.; Wang, G. T.; Krafft, G. A.; Erickson, J. Science 1 9 9 0 ,
247, 954-958. (b) Garcia-Echeveria, C.; Rich, D. H. FEBS Lett. 1 9 9 2 , 297,
100-102. (c) Gurtu, V.; Kain, S. R.; Zhang, G. Anal. Biochem. 1 9 9 7 , 251,
98-102. (d) Packard, B. Z.; Toptygin, D. D.; Komoriya, A.; Brand, L. Proc.
Natl. Acad. Sci. U.S.A. 1 9 9 6 , 93, 11640-11645.
(6) (a) Mizukami, S.; Kikuchi, K.; Higuchi, T.; Urano, Y.; Mashima, T.; Tsuruo,
T.; Nagano, T. FEBS Lett. 1 9 9 9 , 453, 356-360. (b) Daugherty, D. L.;
Gellman, S. H. J. Am. Chem. Soc. 1 9 9 9 , 121, 4325-4333. (c) Geoghegan,
K. L.; Rosner, P. J.; Hoth, L. R. Bioconjugate Chem. 2 0 0 0 , 11, 71-77. (d)
Wei, A.; Blumenthal, D. K.; Herron, J. N. Anal. Chem. 1 9 9 4 , 66, 1500-
1506. (e) Loura, L. M. S.; Fedorov, A.; Prieto, M. J. Phys. Chem. B 2 0 0 0 ,
104, 6920-6931. (f) Jones, L. J.; Upson, R. H.; Haugland, R. P.; Panchuk-
Voloshina, N.; Zhou, M.; Haugland, R. P. Anal. Biochem. 1 9 9 7 , 251, 144-
152. (g) Packard, B. Z.; Komoriya, A.; Toptygin, D. D.; Brand, L. J. Phys.
Chem. B 1 9 9 7 , 101, 5070-5074. (h) Packard, B. Z.; Komoriya, A.; Toptygin,
D. D.; Brand, L. Proc. Natl. Acad. Sci. U.S.A. 1 9 9 6 , 93, 11640-11645. (i)
Zych, A. J.; Iverson, B. L. J. Am. Chem. Soc. 2 0 0 0 , 122, 8898-8909.
(7) Packard, B. Z.; Toptygin, D. D.; Komoriya, A.; Brand, L. Biophys. Chem.
1 9 9 7 , 67, 167-176.
(8) (a) Valdes-Aguilera, O.; Neckers, D. C. Acc. Chem. Res. 1 9 8 9 , 22, 171-
177. (b) West, W.; Pearce, S. J. Phys. Chem. 1 9 6 5 , 69, 1894-1903.
(9) (a) Li, Y.; Glazer, A. N. Bioconjugate Chem. 1999, 10, 241-245. (b) Imperiali,
B.; Rickert, K. W. Proc. Natl. Acad. Sci. U.S.A. 1 9 9 5 , 92, 97-101.
(10) Kawanishi, Y.; Kikuchi, K.; Takakusa, H.; Mizukami, S.; Urano, Y.; Higuchi,
T.; Nagano, T. Angew. Chem., Int. Ed. 2 0 0 0 , 39, 3438-3440.
* Corresponding author: (e-mail) tlong@mol.f.u-tokyo.ac.jp; (fax) +81-3-5841-
4855.
(1) (a) Kojima, K.; Nakatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino, Y.; Nagoshi,
H.; Hirata, Y.; Nagano, T. Anal. Chem. 1 9 9 8 , 70, 2446-2453. (b) Hirano,
T.; Kikuchi, K.; Urano, Y.; Higuchi, T.; Nagano, T. Angew. Chem., Int. Ed.
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K.; Higuchi, T.; Nagano, T. Angew. Chem., Int. Ed. Engl. 1 9 9 9 , 38, 2899-
2901. (d) Seifert, J. L.; Connor, R. E.; Kushon, S. A.; Wang, M.; Armitage,
B. A. J. Am. Chem. Soc. 1 9 9 9 , 121, 2987-2995. (e) Niikura, K.; Metzger,
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(2) (a) Zlokarnik, G.; Negulescu, P. A.; Knapp, T. E.; Mere, L.; Burres, N.; Feng,
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10.1021/ac001016a CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/26/2001
Analytical Chemistry, Vol. 73, No. 5, March 1, 2001 939

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under this condition, FRET occurred, resulting in the appearance
of acceptor emission.
CDs are torus-shaped cyclic oligosaccharides composed of six,
seven, or eight -glucopyranose units (R,â,γ-CD, respectively). A
D
variety of organic compounds can be included in their central
cavities in aqueous solution.11 It was reported that â-CD formed
inclusion complexes with coumarin derivatives12 as well as with
naphthalene13 and dansyl14 derivatives. In this report, we describe
the spectroscopic analysis of the â-CD inclusion complex with 1
in an aqueous environment. On the basis of the results of the
titration experiment of â-CD, we propose that covalent binding of
coumarin to â-CD will provide a FRET cassette molecule. To
demonstrate the validity of this concept, we have designed and
synthesized 2 bearing â-CD covalently (Figure 1).
EXPERIMENTAL SECTION
Synthetic details are described in Supporting Information.
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. Compound 1 was dissolved in DMSO as a 10 mM
stock solution and then diluted to the corresponding concentration
for measurement.
Circular Dichroism Analysis. A spectropolarimeter (J-600,
Jasco) was used. All samples were prepared from 10 mM stock
solutions in DMSO. The following conditions were used: band-
width, 1.0 nm; slit width, 1.0 nm; autosensitivity, 10 mdeg; time
constant, 1.0 s; step resolution, 0.2 nm; scan speed, 20 nm/ min;
number of scans, 3.
Absorption Analysis. A spectrometer (UV-1600, Hitachi) was
used. All samples were prepared from 10 mM stock solutions in
DMSO.
Figure 1. Structures of compounds 1-4.
RESULTS AND DISCUSSION
line-containing oligopeptides as linkers of the donor and acceptor
dyes.9 It is also shown that it is possible to observe the emission
of the acceptor if the structure of the probe is such as to prevent
close contact of the two fluorophores by restricting the flexibility
of the linker.10 In other words, there are no successful examples
of FRET peptide probes with conformationally flexible oligopep-
tides as linkers. If such FRET systems could be developed, they
would be useful for assay of a wide range of proteolytic activities.
This study was intended to develop a conformationally flexible
FRET system usable in an aqueous environment without quench-
ing of the two dyes.
First, we designed and synthesized the intramolecular FRET
compound 1 bearing the coumarin donor and the fluorescein
acceptor (Figure 1). Ethylene glycol was employed as a flexible
linker. In this system, the excitation energy of the coumarin donor
at 408 nm would be transferred to the fluorescein acceptor, which
would emit green light. As we expected, the fluorescence emission
of 1 was strongly quenched in an aqueous environment, whereas
FRET was observed when 1 was dissolved in methanol. It appears
that these fluorophores did not come into close proximity in
methanol because of its apolar environment compared to water.
We thought that if we could prevent close approach of the
fluorophores by surrounding one of the fluorophores with an
appropriate host molecule, we could obtain a FRET system. So,
in the present study, we added â-CD to aqueous solutions of 1 ;
Enhancement of the Fluorescence of 1 Caused by the
Addition of â-CD. Compound 1 was obtained according to the
reaction scheme described in the Supporting Information. The
fluorescence emission spectra of 1 in aqueous solutions containing
â-CD at various concentrations are shown in Figure 2 a). The
spectra were obtained by irradiating the solutions at 408 nm, which
is the excitation wavelength of the coumarin fluorophore. Without
â-CD, both the donor fluorescence around 445 nm and the
acceptor fluorescence around 515 nm were strongly quenched,
reflecting close contact between the donor and acceptor moieties
in aqueous solutions. However, with increasing concentration of
â-CD, the intensity of the acceptor emission was markedly
enhanced. These results demonstrate that the energy transfer
from the coumarin moiety to the fluorescein moiety can proceed
efficiently after the addition of â-CD.
(11) Peczuh, M. W.; Hamilton, A. D. Chem. Rev. 2 0 0 0 , 100, 2479-2494.
(12) (a) Brett, T. J.; Alexander, J. M.; Clark, J. L.; Ross, C. R., II.; Harbison, G.
S.; Stezowski, J. J. Chem. Commun. 1 9 9 9 , 1275-1276.
(13) (a) Moriwaki, F.; Kaneko, H.; Ueno, A.; Osa, T.; Hamada, F.; Murai, K. Bull.
Chem. Soc. Jpn. 1 9 8 7 , 60, 3619-3623. (b) Sidney Cox, G.; Turro, N. J. J.
Am. Chem. Soc. 1 9 8 4 , 106, 422-424. (c) Hamai, S. Bull. Chem. Soc. Jpn.
1 9 8 2 , 55, 2721-2729.
(14) (a) Wang, Y.; Ikeda, T.; Ikeda, H.; Ueno, A.; Toda, F. Bull. Chem. Soc. Jpn.
1 9 9 4 , 67, 1598-1607. (b) Flink, S.; van Veggel, F. C. J. M.; Reinhoudt, D.
N. Chem. Commun. 1 9 9 9 , 2229-2230. (c) Ueno, A.; Minato, S.; Suzuki, I.;
Fukushima, M.; Ohkubo, M.; Osa, T.; Hamada, F.; Murai, K. Chem. Lett.
1 9 9 0 , 605-608.
940 Analytical Chemistry, Vol. 73, No. 5, March 1, 2001

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Figure 3. Induced circular dichroism spectra and absorption spectra
of 1 (50 and 10 µM, respectively) at various concentrations of â-CD
(0, 1.0, 5.0, 10, 20 mM) (25 °C, sodium phosphate buffer, pH 9.0).
Figure 2. Increase of fluorescence caused by the addition of â-CD
(25 °C, sodium phosphate buffer, pH 9.0): (a) fluorescence spectra
of 1 (1.0 µM) at various concentrations of â-CD (Ex 408 nm); (b)
dependence of fluorescence intensity on the concentration of R,â,
and γ-CD (Ex 408 nm; Em 515 nm).
isosbestic points at 429 and 504 nm. These observations suggest
that the addition of â-CD caused an environmental change around
the donor-acceptor moieties. The reason for the changes in
absorption spectra is described in the Supporting Information.
There were two peaks around 400 and 500 nm in the ICD
spectrum of 1 alone. With increasing â-CD concentration, the
positive ICD signal around 400 nm due to coumarin was increased
dose-dependently, whereas the signal around 500 nm due to
fluorescein showed no change. These results suggest that the
coumarin moiety is included in â-CD and the fluorescein moiety
is not. We also confirmed that coumarin can be fit into the â-CD
cavity by means of a molecular modeling calculation using Spartan
(version 5.0).16 There was nonzero CD signal of 1 in the absence
of â-CD. We measured the CD spectrum of fluorescein (50 µM)
in aqueous solution and there was a negative signal around 500
nm. Because fluorescein (50 µM) had no signal in methanol, the
negative signal in aqueous solution would be due to the intermo-
lecular close contact. And we measured the CD spectrum of 1
(50 µM) in methanol and there was no signal. From these
observations, the nonzero CD signal of 1 in the absence of â-CD
in aqueous solution was also due to the dye-to-dye contact.
Evidence of 1:1 host-guest complex formation (m/ z 1923.7
(â-CD + 1 + Na)+, theoretical m/ z 1923.6) was also obtained by
ESI MASS spectrometry. A minor signal of a 2:1 host-guest
complex (m/ z 1530.2 (2 â-CD + 1 + H + Na)2+, theoretical m/ z
1530.0) was observed at lower â-CD concentrations, and the signal
was enhanced by the addition of excess â-CD. Thus, the stoichi-
ometry of host-guest complex formation is dependent on the
concentration of â-CD.
In titration experiments with solutions of R-CD and γ-CD, the
emission intensity of the acceptor fluorescence was not changed
(Figure 2b). These observations show that the enhanced fluores-
cence in the presence of â-CD is not due to a change in the polarity
of the solution caused by the addition of CDs. Considering the
size of the CD cavity, that is, R-CD small, â-CD intermediate, and
γ-CD large, the size of â-CD should be sufficient to accommodate
one coumarin moiety. So, the enhanced fluorescence should be
due to inclusion complex formation between â-CD and the
coumarin moiety. The dissociation constant of â-CD and 1 was
calculated to be 4.2 mM.
Spectral Analysis of the Inclusion Complex between
Coumarin and â-CD. Since the cyclodextrins are composed of
chiral glucose units, circular dichroism is expected to be induced
at the absorption bands of guest molecules which are included in
the cavity of chiral â-CD.15 The absorption and induced circular
dichroism (ICD) spectra of 1 in aqueous solutions without and
with various concentrations of â-CD are shown in Figure 3. In
the absence of â-CD, the absorption spectrum exhibits two peaks
at 408 nm (donor absorption) and 501 (acceptor absorption). The
coumarin absorption intensity was decreased dose-dependently
by the addition of â-CD to a 10 µM aqueous solution of 1 , while
the absorption of fluorescein was increased. There are two
(15) (a) Harata, K.; Uedaira, H. Bull. Chem. Soc. Jpn. 1 9 7 5 , 48, 375-378. (b)
Shimizu, H.; Kaito, A.; Hatano, M. J. Am. Chem. Soc. 1 9 8 2 , 104, 7059-
7065. (c) Kobayashi, N. J. Chem. Soc., Chem. Commun. 1 9 8 8 , 918-919.
(d) Kodaka, M.; Fukaya, T. Bull. Chem. Soc. Jpn. 1 9 8 9 , 62, 1154-1157.
(16) Hehre, W. J. Wavefunction, Inc.: Irvine, CA, 1997.
Analytical Chemistry, Vol. 73, No. 5, March 1, 2001 941

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the coumarin and fluorescein moieties and thereby enhances the
fluorescence intensity. In an analytical format, this FRET system
will be applied as ratiometric probes for the hydrolytic enzymes.
To obtain such probes, â-CD should be incorporated at the ends
of the substrate peptides by following strategy. The FRET donor
cassette 4 (Figure 1) should be conjugated at the C-terminus of
the peptides and the carboxyfluorescein should be conjugated at
N-terminus by the peptide bonding. In such probes, FRET can
take place and the acceptor fluorescence can be observed before
the hydrolysis by an enzyme. After the hydrolysis, the excitation
energy of the donor cannot be transferred to the acceptor
intermolecularly and the donor fluorescence will be observed. So,
by detecting the ratio of the donor and acceptor fluorescence
intensities, the activities of the hydrolytic enzymes can be
measured.
Figure 4. Fluorescence spectra of 2 and 3 (25 °C, sodium
phosphate buffer, pH 9.0; Ex 408 nm).
The above experimental observations demonstrate that â-CD
inclusion complex formation with the coumarin moiety of 1 results
in disruption of close contact between the coumarin and fluores-
cein moieties in aqueous solution. Consequently, FRET can take
place.
ACKNOWLEDGMENT
We thank Dr. Kentaro Yamaguchi and Mr. Shigeru Sakamoto
for mass spectrometry. This work was supported in part by the
Ministry of Education, Science, Sports and Culture of Japan (Grant
11794026, 12470475, and 12557217 for T.N. and 11771467 and
12045218 for K.K.), by the Mitsubishi Foundation and by the
Research Foundation for Opt-Science and Technology. K.K. is
thankful for financial support from the Nissan Science Foundation,
Shorai Foundation of Science and Technology, and Uehara
Memorial Foundation.
Fluorometric Analysis of 2 . Compound 2 bearing â-CD
covalently and a reference compound 3 (Figure 1) were synthe-
sized according to the reaction scheme in the Supporting Informa-
tion. The fluorescence spectra of 2 and 3 are shown in Figure 4.
The fluorescence intensity of 2 was enhanced markedly compared
to the intensity of 3 . This fluorescence enhancement was
considered to be due to the formation of an inclusion complex
between coumarin and â-CD. To estimate the fraction of coumarin
that is actually bound within the cavity of â-CD of 2 , we added
â-CD to the solution of 2 and observed the changes in the
fluorescence spectra (Supporting Information). In the titration
experiment, it was shown that the fluorescence spectra of 2 were
not changed by the addition of â-CD. If there was a coumarin
molecule out of the cavity of intramolecular â-CD due to the linker
length, the fluorescence will be enhanced by the addition of â-CD.
So, it was suggested that all the coumarin moiety that can be
included in â-CD was in â-CD binding pocket of 2 .
SUPPORTING INFORMATION AVAILABLE
Synthetic details, a discussion concerning the absorption
spectra of 1 , and the titration experiment of â-CD to an aqueous
solution of 2 . This material is available free of charge via the
Internet at http:/ / pubs.acs.org.
Received for review August 24, 2000. Accepted December
18, 2000.
CONCLUSION
From these results, we suggest that covalent binding of
coumarin to â-CD facilitates disruption of close contact between
AC001016A
942 Analytical Chemistry, Vol. 73, No. 5, March 1, 2001