Fluorescent cyclodextrins for molecule sensing: Fluorescent properties, NMR characterization, and inclusion phenomena of N-dansylleucine-modified cyclodextrins

Article abstract of DOI:10.1021/ja960183i

Fluorophore-amino acid-cyclodextrin (CD) triad systems, N-dansyl-L-leucine-modified and N-dansyl-D-leucine-modified β-CD (1, 2) and N-dansyl-L-leucine-modified and N-dansyl-D-leucine-modified γ-CD (3, 4), were synthesized and characterized as fluorescent indicators of molecular recognition. Fluorescence decay analyses of these CD derivatives indicated that there exist two lifetime components, being in equilibrium with each other in aqueous solution, with the dansyl moiety included in its own cavity (self-inclusion) for the larger lifetime component while located outside the cavity for the shorter lifetime one. The structural analyses of β-CD derivatives 1 and 2 undertaken by combined use of 1D and 2D NMR spectra indicate that the dansyl moiety of 2 is more deeply included in the CD cavity than that of 1. The leucine residue between the dansyl and the CD moieties of these hosts was more effective in enhancing the binding abilities for various guests when compared with the glycine residue of the corresponding hosts. The difference in the enantiomeric configuration of the leucine residue caused the difference in the binding constants with larger values for 1 than 2 and with the opposite trend for the γ-CD derivatives 3 and 4. Upon guest addition, the fluorescence intensities of 1 and 2 decreased, reflecting the exclusion of the dansyl moiety from inside to outside of the β-CD cavity, while the fluorescence intensities of the γ-CD derivatives 3 and 4 depended on the guest as shown by the increase induced by cyclohexanol and the decrease by (-)-borneol and other larger guests. These guest-responsive variations of the fluorescence intensity enabled these hosts to be used as effective fluorescent indicators of molecular recognition.

Full text of DOI:10.1021/ja960183i

10980
J. Am. Chem. Soc. 1996, 118, 10980-10988
Fluorescent Cyclodextrins for Molecule Sensing: Fluorescent
Properties, NMR Characterization, and Inclusion Phenomena of
N-Dansylleucine-Modified Cyclodextrins
†
‡
Hiroshi Ikeda, Michiei Nakamura, Nobuyuki Ise, Naomi Oguma,
§
Asao Nakamura, Tsukasa Ikeda, Fujio Toda, and Akihiko Ueno*
Contribution from the Department of Bioengineering, Faculty of Bioscience and Biotechnology,
Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226, Japan, and
Dainichiseika Color and Chemicals Mfg. Co. Ltd., 9-4 Horinouchi 1-chome, Adachi-ku,
Tokyo 123, Japan
X
ReceiVed January 19, 1996
Abstract: Fluorophore-amino acid-cyclodextrin (CD) triad systems, N-dansyl-L-leucine-modified and N-dansyl-
D-leucine-modified â-CD (1, 2) and N-dansyl-L-leucine-modified and N-dansyl-D-leucine-modified γ-CD (3, 4), were
synthesized and characterized as fluorescent indicators of molecular recognition. Fluorescence decay analyses of
these CD derivatives indicated that there exist two lifetime components, being in equilibrium with each other in
aqueous solution, with the dansyl moiety included in its own cavity (self-inclusion) for the larger lifetime component
while located outside the cavity for the shorter lifetime one. The structural analyses of â-CD derivatives 1 and 2
undertaken by combined use of 1D and 2D NMR spectra indicate that the dansyl moiety of 2 is more deeply included
in the CD cavity than that of 1. The leucine residue between the dansyl and the CD moieties of these hosts was
more effective in enhancing the binding abilities for various guests when compared with the glycine residue of the
corresponding hosts. The difference in the enantiomeric configuration of the leucine residue caused the difference
in the binding constants with larger values for 1 than 2 and with the opposite trend for the γ-CD derivatives 3 and
4
. Upon guest addition, the fluorescence intensities of 1 and 2 decreased, reflecting the exclusion of the dansyl
moiety from inside to outside of the â-CD cavity, while the fluorescence intensities of the γ-CD derivatives 3 and
depended on the guest as shown by the increase induced by cyclohexanol and the decrease by (-)-borneol and
4
other larger guests. These guest-responsive variations of the fluorescence intensity enabled these hosts to be used
as effective fluorescent indicators of molecular recognition.
Introduction
changing fluorescence and absorption intensities upon accom-
modation of a guest molecule, and these spectral variations
were indicated to occur associated with the induced-fit confor-
mational changes of the hosts, usually excluding the chro-
mophore moiety from inside to outside the CD cavity upon guest
binding.
Cyclodextrins (CDs) are cyclic oligosaccharides, mostly
consisting of six, seven, and eight glucose units for R-, â-, and
γ-CD, respectively. They have the remarkable property of
including various organic molecules in their central cavity in
aqueous solution and have been used as molecular vessels for
reactions, binding sites of enzyme models, solubilizers of water-
insoluble substances, and molecular capsules for stabilizing
unstable chemicals.1 Although CDs themselves are spectro-
scopically inert, we have recently shown that they can be
So far most of these systems have been confined to CD-
chromophore diad systems, and here we report on a new series
of CD-amino acid-chromophore triad systems, where CD is
â- or γ-CD, amino acid is D- or L-leucine, and chromophore is
dansyl. The leucine residue inserted between CD and dansyl
has a hydrophobic isobutyl side chain, so the moiety is expected
to affect the binding and molecular recognition abilities of the
hosts. It is also interesting how the stereochemical difference
between D- and L-leucine units is reflected in the molecular
recognition abilities in these triad systems. We also used
dansylglycine-modified â- and γ-CD for comparison.
-4
converted into spectroscopically active compounds by modifica-
tion with appropriate chromophores.5
-17
On this basis, it was
shown that many chromophore-modified CDs are capable of
*
To whom correspondence should be addressed.
Present address: Dainichiseika Color and Chemicals Mfg. Co. Ltd.
Dainichiseika Color and Chemicals Mfg. Co. Ltd.
†
‡
§
Present address: Tokyo Polytechnic College, 2-32-1, Ogawanisi,
Kodaira 187, Japan.
X
Abstract published in AdVance ACS Abstracts, October 15, 1996.
(1) Bender, M. L.; Komiyama, M. Cyclodextrin Chemistry; Springer-
(10) Minato, S.; Osa, T.; Morita, M.; Nakamura, A.; Ikeda, H.; Toda,
F.; Ueno, A. Photochem. Photobiol. 1991, 54, 593-597.
(11) Hamasaki, K.; Ikeda, H.; Nakamura, A.; Ueno, A.; Toda, F.; Suzuki,
I.; Osa, T. J. Am. Chem. Soc. 1993, 115, 5035-5040.
(12) Hamasaki, K.; Ueno, A.; Toda, F.; Suzuki, I.; Osa, T. Bull. Chem.
Soc. Jpn. 1994, 67, 516-523.
Verlag: Berlin, 1978.
2) Szejtli, S. Cyclodextrin Technology; Kluwer Academic Publishers:
Dordrecht, 1988.
(
(3) Cyclodextrins; Toda, F., Ueno, A., Eds.; Sangyo-Tosho: Tokyo, 1995.
(4) Wenz, G. Angew. Chem., Int. Ed. Engl. 1994, 33, 803-822.
(5) Ueno, A. In Fluorescent Chemosensors for Ion and Molecule
(13) Ueno, A.; Minato, S.; Suzuki, I.; Fukushima, M.; Ohkubo, M.; Osa,
T.; Hamada, F.; Murai, K. Chem. Lett. 1990, 605-608.
(14) Hamada, F.; Kondo, Y.; Ito, R.; Suzuki, I.; Osa, T.; Ueno, A. J.
Incl. Phenom. 1993, 15, 273-279.
Recognition; Czarnik, A. W., Ed.; ACS Symposium Series 538; American
Chemical Society: Washington, DC, 1993; pp 74-84.
(
6) Ueno, A.; Kuwabara, T.; Nakamura, A.; Toda, F. Nature 1992, 356,
1
1
6
36-137.
(15) Wang, Y.; Ikeda, T.; Ikeda, H.; Ueno, A.; Toda, F. Bull. Chem.
Soc. Jpn. 1994, 67, 1598-1607.
(7) Kuwabara, T.; Nakamura, A.; Ueno, A.; Toda, F. J. Phys. Chem.
994, 98, 6297-6303.
(16) Nakamura, M.; Ikeda, T.; Nakamura, A.; Ikeda, H.; Ueno, A.; Toda,
F. Chem. Lett. 1995, 343-344.
(
8) Ueno, A.; Suzuki, I.; Osa, T. J. Am. Chem. Soc. 1989, 111, 6391-
397.
9) Ueno, A.; Suzuki, I.; Osa, T. Anal. Chem. 1990, 62, 2461-2466.
(17) Nakamura, M.; Ikeda, A.; Ise, N.; Ikeda, T.; Ikeda, H.; Toda, F.;
Ueno, A. J. Chem. Soc., Chem. Commun, 1995, 721-722.
(
S0002-7863(96)00183-7 CCC: $12.00 © 1996 American Chemical Society

Fluorescent Cyclodextrins for Molecule Sensing
J. Am. Chem. Soc., Vol. 118, No. 45, 1996 10981
Chart 1
Experimental Section
General Methods. Thin-layer chromatography (TLC) was carried
out with silica gel 60 F254 (Merck Co.). Absorbance and fluorescence
spectra were recorded on a Shimadzu UV-3100 spectrometer and a
Hitachi 850 fluorescence spectrometer, respectively. Fluorescence
decay was measured by a time-correlated single-photon counting
method on a Horiba NAES-550 system. A self-oscillating flash lamp
filled with H was used as a light source. The excitation beam was
2
passed through the filters UV34, U340, and U350 (Hoya), and the
emission beam was passed through the filters L42 (Hoya) and Y46
4 2
(Toshiba) and an aqueous solution of NiSO ‚6H O (500 g/L) in a 1-cm
pathlength cell. The accumulated data in a memory of the system were
transferred to and analyzed on a desktop computer (Hewlett-Packard
HP9000 Series Model 330). Lifetime was obtained by the deconvo-
lution with a nonlinear least-square fitting procedure. 1D and 2D NMR
2
spectra were recorded in D O at 25 °C on Varian VXR-500S and
UNITY plus-400 spectrometers operating at 499.843 and 399.973 MHz,
1
respectively, for H. All the NMR spectra were measured by using
pulse sequences and standard procedures offered by Varian. HDO (δ
)
4.70) was used as an internal standard, and 3-(trimethylsilyl)propionic
-
6
Figure 1. Fluorescence spectra of 1-7 (2 × 10 M) in aqueous
acid-d sodium salt (TSP, δ ) 0) was used as an external standard.
4
solution; excitation wavelength was 370 nm.
Mass spectrometry was performed on a Hitachi M-1200H mass
spectrometer. Optical rotation was measured with a JASCO DIP-1000
digital polarimeter at room temperature. Elemental analyses were
performed by the Analytical Division in Research Laboratory of
Resources Utilization of Tokyo Institute of Technology.
Materials. â- And γ-cyclodextrin were kindly gifted from Nihon
Shokuhin Kako Co., Ltd. All chemicals were reagent grade and were
used without further purification unless otherwise noted. Distilled water
and dimethyl sulfoxide as solvents for spectroscopy were special grade
for fluorometry (Uvasol) from Kanto Chemicals. Deuterium oxide,
with an isotopic purity of 99.95%, was purchased from Merck Co.
Synthesis of N-Dansyl-D-leucine. To a solution of D-leucine (0.75
g, 5.7 mmol) in 10 mL of 1 N NaOH aqueous solution was added a
solution of dansyl chloride (2.7 g, 10.0 mmol) in 10 mL of acetone.
The resulting mixture was stirred at room temperature for 4 h. After
evaporation of acetone, insoluble materials were removed with filtration.
The filtrate was made acidic to Congo red with 1 N HCl aqueous
solution to obtain a crude product as a solid. Column chromatography
on silica gel (eluted with methanol/chloroform ) 1/9) afforded the
desired product as a pale yellow solid (30% yield). TLC: R
CHCl /MeOH ) 4/1). Anal. Calcd for C18 :C, 59.32; H,
.64; N, 7.69, S, 8.80. Found: C, 58.57; H, 6.54; N, 7.79; S, 8.55.
R] : 37.4° (c 0.1, methanol) ([R] ) -37.7° for N-dansyl-L-leucine).
H NMR (D O): δ 0.01 (d, J ) 6.6, 3H, H-δ), 0.51 (d, J ) 6.6 Hz,
H, H-δ′), 0.98 (m, 1H, H-γ), 1.16 (ddd, J ) 4.4, 9.5, and 14.1 Hz,
H, H-â), 1.28 (ddd, J ) 4.4, 10.0 and 14.1 Hz, 1H, H-â′), 2.92 (s,
H, NMe ), 3.33 (dd, J ) 4.4 and 10.0 Hz, 1H, H-R), 7.47(d, J ) 7.8
f
) 0.34
(
6
3
24 2 4 1
H N O S
[
D
D
-6
Figure 2. Fluorescence spectra of 1 (2 × 10 M) in aqueous solution
1
2
in the presence of various concentrations of 1-adamantanol; excitation
wavelength was 370 nm.
3
1
6
2
lcarbodiimide (0.45 g, 2.2 mmol) and 1-hydroxybenzotriazole (0.30 g,
2.2 mmol) were added. The resulting mixture was stirred at room
temperature for 2 days. After insoluble materials were removed with
filtration, the filtrate was poured into acetone, and the precipitate was
collected and dried in Vacuo. The crude product was purified by
column chromatography on high porous polystyrene gel, DIAION HP-
20 (eluted with water/methanol ) 100/0 to 50/50). The eluent was
concentrated to give the desired product as a pale yellow solid (50%
Hz, 1H, H-6′), 7.67 (m, 2H, H-3′ and H-7′), 8.25 (dd, J ) 1.1 and 7.4
Hz, 1H, H-2′), 8.37 (d, J ) 8.5 Hz, 1H, H-8′), 8.43 (d, J ) 8.4 Hz,
1
H, H-4′).
Synthesis of 6-(N-Dansyl-L-leucylamino)-6-deoxy-â-cyclodextrin
(
1). To a solution of 6-amino-6-deoxy-â-cyclodextrin11 (2.1 g, 1.9
mmol) in 10 mL of DMF was added N-dansyl-L-leucine (0.73 g, 2.0
mmol). After the solution was cooled below 0 °C, N,N-dicyclohexy-

10982 J. Am. Chem. Soc., Vol. 118, No. 45, 1996
Ikeda et al.
Chart 2
yield). TLC: R
f
) 0.60 (n-BuOH/EtOH/H
2
O ) 5/4/3). Anal. Calcd
for C60
H O N
93 37 3
S‚3H O: C, 47.0; H, 6.50; N, 2.74; S, 2.09. Found:
2
C, 47.3; H, 6.52; N, 2.76; S, 2.05. MS (ESI): m/e 1478 (calcd for (M
-
-
H) , 1478).
Synthesis of 6-(N-Dansyl-D-leucylamino)-6-deoxy-â-cyclodextrin
(
2). This compound was prepared by the condensation of 6-amino-6-
deoxy-â-cyclodextrin and N-dansyl-D-leucine by the same method as
for 1 (40% yield). TLC R ) 0.61 (n-BuOH/EtOH/H O ) 5/4/3). Anal.
Calcd for C60 S‚4H O: C, 46.4; H, 6.56; N, 2.71; S, 2.07.
f
2
H
93
O
37
N
3
2
Found: C, 46.5; H, 6.52; N, 2.84; S, 2.47. MS (ESI): m/e 1478 (calcd
-
for (M - H) , 1478).
Synthesis of 6-(N-Dansyl-L-leucylamino)-6-deoxy-γ-cyclodextrin
(
3). This compound was prepared by the condensation of 6-amino-6-
1
1
deoxy-γ-cyclodextrin and N-dansyl-L-leucine by the same method as
for 1 (45% yield). TLC: R ) 0.52 (n-BuOH/EtOH/H O ) 5/4/3).
Anal. Calcd for C66 S‚4H O: C, 46.2; H, 6.52; N, 2.45; S,
f
2
H O
103 42
N
3
2
1
(
.87. Found: C, 46.2; H, 6.27; N, 2.39; S, 1.90. MS (ESI): m/e 1640
-
calcd for (M - H) , 1640).
Synthesis of 6-(N-Dansyl-D-leucylamino)-6-deoxy-γ-cyclodextrin
4). This compound was prepared by the condensation of 6-amino-6-
(
deoxy-γ-cyclodextrin and N-dansyl-D-leucine by the same method as
for 1 (60% yield). TLC: R ) 0.52 (n-BuOH/EtOH/H O ) 5/4/3).
Anal. Calcd for C66 S‚8H O: C, 44.4; H, 6.71; N, 2.35; S,
-
6
Figure 3. Fluorescence spectra of 4 (2 × 10 M) in aqueous solution
in the presence of various concentrations of cyclohexanol; excitation
wavelength was 370 nm.
f
2
H O
103 42
N
3
2
1
.79. Found: C, 44.0; H, 6.24; N, 2.28; S, 1.86. MS (ESI): m/e 1640
-
(
calcd for (M - H) , 1640).
ment or most deeply included in the CD cavity. On this basis,
the order of the maximum intensities, 2 > 1 > 5 > 4 > 3 > 6,
may be regarded roughly as the order of the hydrophobic nature
around the dansyl moiety of the fluorescent CDs, consequently
suggesting that the â-CD cavity is more appropriate than the
γ-CD one in including the appending dansyl moiety.
The maximum wavelengths of the fluorescence spectra of 1,
2, 3, 4, 5, 6, and 7 are 543, 540, 550, 553, 546, 556, and 571
nm, respectively, reflecting the polarity of the environment
around the dansyl moiety in each CD derivative with shorter
maximum wavelength in less polar environment. This result
also suggests that the dansyl moiety of 2 is located in the most
hydrophobic environment.
Results and Discussion
Fluorescence Spectra. Figure 1 shows the fluorescence
spectra of N-dansyl-L-leucine-modified â-CD (1), N-dansyl-D-
leucine-modified â-CD (2), N-dansyl-L-leucine-modified γ-CD
(
3), N-dansyl-D-leucine-modified γ-CD (4), N-dansylglycine-
13
14
modified â-CD (5), N-dansylglycine-modified γ-CD (6), and
N-dansyl-L-leucine (7) in aqueous solution. The fluorescence
intensities of the dansyl-modified CDs are much larger than
that of the reference compound 7, and among the modified CDs,
2
exhibits the largest fluorescence intensity. Since the fluo-
rescence intensity of the dansyl moiety is sensitive to a micro-
environment, being stronger in a hydrophobic environment than
this result suggests that the
dansyl moiety of 2 is located in the most hydrophobic environ-
(
19) Li, Y.-H.; Chan, L.-M.; Tyer, L.; Moody, R. T.; Himel, C. M.;
in a hydrophilic environment,1
8-21
Hercules, D. M. J. Am. Chem. Soc. 1975, 97, 3118-3126.
(20) Bridge, J. B.; Johnson, P. Eur. Polym. J. 1973, 9, 1327-1346.
(21) Hoenes, G.; Hauser, M.; Pfleiderer, G. Photochem. Photobiol. 1986,
(18) Chen, R. F. Arch. Biochem. Biophys. 1967, 120, 609-620.
43, 133-137.

Fluorescent Cyclodextrins for Molecule Sensing
J. Am. Chem. Soc., Vol. 118, No. 45, 1996 10983
Table 1. Fluorescence Lifetimes of 1-7 Alone and in the
a
Presence of Guest in Aqueous Solution
host
guest
τ
1/ns
A
1
τ
2
/ns
A
2
ø2
1
2
3
4
5
6
7
1
2
5
3
4
6
3
4
6
5.7
6.9
7.3
7.9
5.9
8.2
3.5
5.5
5.9
4.4
5.5
5.7
6.0
7.6
7.3
7.3
0.33
0.23
0.46
0.78
0.29
0.79
17.7
17.8
13.1
13.5
15.5
13.4
0.67
0.77
0.54
0.22
0.71
0.21
1.32
1.38
1.13
1.06
1.24
1.11
1.28
1.38
1.07
1.39
1.23
1.03
1.20
1.22
0.95
1.04
13
13
13
16
16
16
14
14
14
0.99
0.97
0.92
0.57
0.86
0.53
0.40
0.45
0.41
19.0
17.9
15.3
13.7
13.2
12.3
14.8
14.1
13.3
0.01
0.03
0.08
0.43
0.14
0.47
0.60
0.55
0.59
Figure 4. (I) Conformational equilibria in aqueous solution and guest-
induced conformational change of â-CD derivatives (1, 2, and 5) and
γ-CD derivatives (3, 4, and 6). In the case of γ-CD derivatives, this
type of conformational change occurs for larger guests. (II) Confor-
mational change of γ-CD derivatives (3, 4, and 6) occurring associated
with accommodation of a smaller guest.
a
-5
-3
[
1-7] ) 2 × 10 M, [1-adamantanol (13)] ) 5 × 10 M,
-3 -2
[
(-)-borneol (16)] ) 3 × 10 M, [cyclohexanol (14)] ) 5 × 10 M.
Decay curves were fitted to the equation: I(t) ) A exp(-t/t ) + A
). ø is the parameter for the goodness of the fit.
1
1
2
2
exp(-t/t
2
Upon addition of 1-adamantanol as a guest, the fluorescence
intensities of 1, 2, and 5 decreased as shown in Figure 2. Similar
phenomena were observed for all the guests used in this work
exponential function, and the results are summarized in Table
1
. These results indicate that there are two kinds of observable
(
Chart 2), when the dansyl-modified â-CD derivatives were used
conformational isomers. The longer and shorter lifetime species
as the hosts. This guest-induced fluorescence variation suggests
that the hosts exclude the dansyl moiety from their hydrophobic
cavities to the bulk water environment associated with the guest
accommodation. The degrees of the fluorescence intensity
variation were evaluated by a sensitivity parameter expressed
as ∆I/I0, where ∆I ) I0 - I and I0 and I are the fluorescence
intensities in the absence and presence of the guest, respectively,
measured at the maximum emission wavelength of the host
alone. The variation of the ∆I/I0 value of 1 with increasing
concentration of 1-adamantanol was fitted to a Benesi-
may be ones with the dansyl moiety inside and outside the
15,32
cavity, respectively.
These two species would be in
equilibrium as shown in parentheses in Figure 4. Table 1 shows
fractions A1 and A2 of the two species and reveals that 2 has
the largest fraction of the longer lifetime (A2, 0.77). It means
that the proportion of the self-inclusion form of 2 is largest
among all the fluorescent hosts, being consistent with the largest
fluorescence intensity of 2. The data in Table 1 also reveal
that the fractions of the longer lifetime forms of the â-CD
derivatives 1, 2, and 5 are larger than those of the γ-CD
derivatives 3, 4, and 6, consistent with the previous argument
that the self-inclusion forms of the â-CD derivatives are more
stable than those of the γ-CD derivatives. The data indicate
that the cavity of γ-CD is too large to form stable self-inclusion
complexes. This is substantiated particularly by the data of 4
and 6 that the shorter lifetime forms become the major
components with A1 values of 0.78 and 0.79 for 4 and 6,
respectively. Therefore, the dansyl moiety of 4 and 6 is located
predominantly in a polar environment, being in contact with
water. However, it is noted that the dansyl moiety in the shorter
lifetime form is still surrounded by a less polar environment
than in pure bulk water, because the shorter lifetimes of 4
and 6 are still larger than the lifetime of 7 in bulk water
solution.
22
Hildebrand type equation for 1/1 host/guest complex formation
11-17
similarly to the fluorophore-appended CDs already reported.
In the cases of γ-CD derivatives 3, 4, and 6, addition of a
guest caused increase or decrease of the fluorescence intensity,
depending on the kind of the guest. For example, their
fluorescence intensities decreased upon addition of (-)-borneol,
while increased upon addition of cyclohexanol (Figure 3). Both
guest-induced variations were fitted to the equation for the 1/1
host/guest complex formation. These results indicate that 3, 4,
and 6 form inclusion complexes of 1/1 stoichiometry with
different structures, depending on the kind of the guest. The
∆
I/I0 values for other guests are discussed in a later section.
Fluorescence Lifetimes. In aqueous solution, the pendant
groups of modified CDs are flexible and not fixed in many cases,
usually being located inside the cavity in one conformation and
Upon addition of 1-adamantanol as a guest, the fractions of
the shorter lifetime form of 1, 2, and 5 increased, while the
fractions of the longer lifetime forms of the hosts decreased
outside the cavity in another.1
1,15
Although the conformational
interconversion occurs too rapidly to be followed by NMR
spectroscopy, the analysis of the fluorescence decay of the
fluorophore pendant may provide useful information with respect
to the conformational features, because of the fact that fluo-
rescence lifetimes of many fluorophores are in the range of a
measurable time scale (nanoseconds) for the conformations. This
is particularly true for the fluorescent CDs with a fluorophore
pendant, which is sensitive to environment and has different
lifetimes when located inside the cavity and in bulk water
solution.1
(Table 1). Obviously, the locational change of the dansyl moiety
from inside to outside the cavity occurs in association with the
guest accommodation as shown by I in Figure 4, and this
conformational change of the hosts is reflected in the change
in lifetime distribution. The fact that the fluorescence decay
curves of the hosts are still double exponential indicates that
the fluorescence lifetimes of species B and C in I of Figure 4
are similar to each other and not resolved by our lifetime
measurement system.
On the other hand, the fractions of the longer lifetime
component of 3 and 4 increased upon addition of cyclohexanol
but decreased upon addition of (-)-borneol (Table 1). In the
case of 6, however, the addition of (-)-borneol increased the
5,32
All the fluorescence decays of the dansyl moiety of the
fluorescent CDs could be analyzed by a simple double-
(
22) Benesi, H. A.; Hildebrand, J. H. J. Am. Chem. Soc. 1949, 71, 2703-
2
707.

10984 J. Am. Chem. Soc., Vol. 118, No. 45, 1996
Ikeda et al.
1
Figure 5. 500 MHz H NMR spectra of 1 (a-1) alone, (a-2) in the presence of 1-adamantanol, 2 (b-1) alone, (b-2) in the presence of 1-adamantanol,
-
3
and (c) 7 in D
2
O at 25 °C in the region of (A) aromatic protons and (B) δ protons of leucine: [1] ) [2] ) [7] ) [1-adamantanol] ) 1 × 10 M.
fraction of the longer lifetime component. The results sug-
gest that the dansyl moiety of 3 and 4 is included together
with cyclohexanol in the cavity, whereas it is excluded by
reported elsewhere, and the only important results are shown
in this paper.
The H resonances for H-A5 (the proton at the C5 position
1
(
-)-borneol (II in Figure 4). On the same basis, it is likely
in the glucose unit A), H-E3, and H-E5 of 1 and H-A3, H-A5,
H-D3, H-D5, H-E3, H-E5, and H-G5 of 2, these protons being
located at the inside of the cavities, are shifted upfield. These
shifts would be attributed to the anisotropic ring current effects
from the dansyl moiety, and the result indicates that the dansyl
moiety is included in the CD cavities. Figure 5A shows the
that the dansyl moiety of 6 is included together with the guest
in both cases of cyclohexanol and (-)-borneol.
Previous reports substantiated that γ-CD, which has a larger
2
3-26
cavity than â-CD, can include two small guest molecules.
Since the cavity of γ-CD is too large to form a stable
intramolecular complex with the dansyl moiety, a small guest
1
dansyl region of the H NMR spectra of 1, 2, and 7, and Figure
such as cyclohexanol may act as a spacer to form stable
5B shows the regions of the δ protons of the leucine moiety of
the H NMR spectra of these compounds. The resonance
complexes.5
,12,14,27
Therefore, it is reasonable that both the
1
dansyl moiety and cyclohexanol are included in the γ-CD cavity
in 3, 4, and 6. However, the inclusion of the dansyl moiety in
the γ-CD cavity in 3 and 4 is not enhanced by (-)-borneol,
which is much larger than cyclohexanol. This result suggests
that there is a steric hindrance for such co-inclusion due to the
presence of the isobutyl side chain of their leucine residue.
Conformational Analyses of 1 and 2 by NMR Spectros-
copy. The dansyl moiety of the fluorescent CDs is flexible
rather than fixed in aqueous solution, and the observed NMR
spectra are the averages of the spectra of the species in various
conformational states weighted by the fractions of the conform-
ers. This situation is due to the fact that the interconversion
rates are fast on the NMR time scale. The analysis of the
fluorescence decay of the dansyl moiety of the CD derivatives
indicates that there are two kinds of observable conformational
isomers, which are in equilibrium. The dansyl moieties of 1
and 2 are located in the cavity in one of the states and are located
out of the cavity in another, and the former is the major state.
Therefore, the NMR spectra of 1 and 2 may be expected to
provide information about their structures in the self-inclusion
state.
patterns of 1 and 2 are different from each other, both being
also different from the pattern of 7. These spectra suggest that
the dansyl moiety of 1 and 2 is included in the cavities, and the
positions of the dansyl moiety in the cavities of 1 and 2 are
different from each other.
The orientation of the dansyl moiety of 1 and 2 can be
estimated from the NOE correlations between the dansyl protons
and the CD protons. The NOE correlation can be observed by
the ROESY spectrum, in the case when a proton of the dansyl
moiety and a proton of the CD moiety are closer than 4 Å
3
3,34
through space.
Therefore, if the dansyl moiety is included
in the CD cavity, the NOE correlations between the protons of
the dansyl moiety and the protons (H-3, H-5, or H-6) of the
CD moiety should be observed, and consequently, on this basis,
it is possible to estimate the orientation of the dansyl moiety in
the CD cavity using the assigned NOE correlations.
Figures 6 and 7 show the ROESY spectra with assignments.
The NOE correlations were observed between the protons of
CD and the protons of the naphthyl and methyl parts of the
dansyl moiety. A weak NOE correlation was observed between
the protons of CD and the δ protons of the leucine moiety. The
flexible movement of the δ protons would make the NOE
weaker.
1
The full assignments of the H NMR spectra of 1 and 2 are
1
required to elucidate their structures. Although the H NMR
spectra of 1 and 2 are complex, it is possible to assign almost
(
28) Deschenaux, R.; Harding, M. M.; Ruch, T. J. Chem. Soc., Perkin
all of their resonances by combined use of various 1D and 2D
Trans. 2, 1993, 1251-1258.
(29) Ikeda, H.; Du, Y.-q.; Nakamura, A.; Toda, F. Chem. Lett. 1991,
2
8-31
NMR techniques.
Their detailed NMR analyses will be
1
495-1498.
(23) Ueno, A.; Takahashi, K.; Osa, T. J. Chem. Soc., Chem. Commun.
(30) Ikeda, H.; Moon, H.-t.; Du, Y.-q.; Toda, F. Supramol. Chem. 1993,
1
980, 921-922.
1, 337-342.
(
24) Kano, K.; Takenoshita, I.; Ogawa, T. Chem. Lett. 1982, 321-324.
25) Yorozu, T.; Hoshino, M.; Imamura, M. J. Phys. Chem. 1982, 86,
(31) Djeda ¨ı ni-Pilard, F.; Azaroual-Bellanger, N.; Gosnat, M.; Vernet, D.;
Perly, B. J. Chem. Soc., Perkin Trans. 2 1995, 723-730.
(32) Dunbar, R. A.; Bright, F. V. Supramol. Chem. 1994, 3, 93-99.
(33) Neuhaus, D.; Williamson, M. The Nuclear OVerhauser Effect in
Structural and Conformational Analysis; VCH Publishers: New York, 1989.
(34) Bothner-By, A. A.; Stephens, R. L.; Lee, J.-m.; Warren, C. D.;
Jeanloz, R. W. J. Am. Chem. Soc. 1984, 106, 811-813.
(
4426-4429.
26) Turro, N. J.; Okubo, T.; Weed, G. C. Photochem. Photobiol. 1982,
35, 325-329.
27) Ueno, A.; Tomita, Y.; Osa, T. J. Chem. Soc., Chem. Commun. 1983,
976-977.
(
(

Fluorescent Cyclodextrins for Molecule Sensing
J. Am. Chem. Soc., Vol. 118, No. 45, 1996 10985
2
Figure 6. 500 MHz ROESY spectrum of 1 in D O at 25 °C with mixing time of 300 ms, indicating the cross peaks between the protons of the
dansyl-L-leucine moiety and the protons of the â-CD moiety.
Figure 8 shows the structures of 1 and 2 estimated from the
difference in the stability of the self-inclusion complexes
NOE data and the degree of the anisotropic ring current effect
from the dansyl moiety in the H NMR spectra of the CD
between 1 and 2 would be reflected in their binding abilities.
1
1
Upon addition of 1-adamantanol, H NMR spectra of 1 and
1
protons. The dansyl moiety of 2 is included in the cavity more
deeply than that of 1. These structures can elucidate the
difference of the resonance of the δ protons of the leucine
between those of 1 and 2. The resonances of the δ protons of
the leucine moiety of 1 are more dispersed and are shifted more
upfield than that of 2. This difference is caused by the
difference in the position of the dansyl moiety and indicates
that the δ protons of 1 are more influenced by the anisotropic
ring current effects of the dansyl moiety than those of 2. The
deeper inclusion of the dansyl moiety of 2 into its own cavity
would make the more stable self-inclusion complex, and the
2 were changed as shown in Figure 5. The H resonances of
the dansyl moiety in the presence of the guest are different from
those of the hosts alone and are similar to that of 7. The
resonances of the δ protons of the leucine moiety of 1 and 2
were also changed in a similar way upon addition of the guest.
These changes suggest that the dansyl moiety is excluded from
the cavity upon addition of the guest. In the formed inclusion
complexes of 1 and 2, the dansyl moiety causes a larger
anisotropic ring current effect on the δ protons of the leucine
moiety, and the resonances of the δ protons of the leucine
moiety are more dispersed.

10986 J. Am. Chem. Soc., Vol. 118, No. 45, 1996
Ikeda et al.
Figure 7. 500 MHz ROESY spectrum of 2 in D
2
O at 25 °C with mixing time of 300 ms, indicating the cross peaks between the protons of the
dansyl-D-leucine moiety and the protons of the â-CD moiety.
The resonances of CD protons were also changed upon
addition of 1-adamantanol. The upfield shifts of the protons
(1-6) for various guests were obtained from their guest-induced
variations of the sensitivity parameter expressed as ∆I/I0 (Table
2). All the variations were fitted with the Benesi-Hildebrand
type equation for the 1/1 host/guest complex formation as
described in the previous section. Binding constants of 3, 4,
and 6 for some guests were too small to be determined.
The order of the binding abilities of the â-CD derivatives
for all the guests is 5 < 2 < 1. This result indicates that the
isobutyl side chain of the leucine moiety increases the hydro-
phobic nature of the environment around the guest, thus resulting
in the larger binding abilities. It is interesting that the binding
constants of 1 are over two times larger than those of 2 for
most of the guests. One extreme result is that the binding
constant of 1 for cholic acid (11) is 27.3-fold larger than that
(H-3, H-5 or H-6) of CD, arising from the ring current effects
of the dansyl moiety, were not observed in the presence of the
guest. The resonances for the anomeric protons of 1 and 2 in
the absence of the guest were dispersed in the range from 4.7
to 5.2 ppm, showing distinctly separated peaks for all glucose
units. Upon addition of the globular guest, 1-adamantanol, these
spectral dispersions were collapsed and the resonances were
almost overlapped around 5.0 ppm, This spectral change also
indicates the exclusion of the dansyl moiety from the cavities
of 1 and 2.
Binding Constants of the Fluorescent CDs (1-6) for
Various Guests. The binding constants of the fluorescent CDs

Fluorescent Cyclodextrins for Molecule Sensing
J. Am. Chem. Soc., Vol. 118, No. 45, 1996 10987
Figure 8. Structures of 1 and 2 estimated from the NMR data. The energy was not minimized.
Table 2. Binding Constants of 1-6 for the Guests Shown in
larger than that of 2, respectively. The difference in guest
a
Chart 2
binding between â- and γ-CD derivatives indicates that the
fitness of the cavity size for the guest is another significant factor
in the stabilization of the inclusion complexes.
K/M-
1
guest
1
2
3
4
5
6
Sensitivities and Selectivities of Fluorescent CDs in
Molecule Sensing. Compounds 1-4 can be used as fluorescent
indicators of molecular recognition, since their guest-induced
variations in the fluorescence intensities depend on the kind of
the guest as described above. From this viewpoint, we examined
the sensitivities and molecular recognition abilities of the
fluorescent CDs for the guests shown in Chart 2 using the ∆I/
I0 value as a sensing parameter and the results are shown in
Figures 9 and 10. Here, since ∆I is equated as I0 - I, the ∆I/I0
value becomes positive when the fluorescence intensity de-
creases upon guest addition.
8
9
77100 41000 6250
21900 11800 5620
24600
16100
9420
1960
b
370
43.5
b
1430
852
352
b
37600
10200
1030
4110
2660
658
b
b
b
29.3
76.2
b
10
11
12
13
14
15
16
17
18
19
20
2660
1650
59300 20400
54200 19900
1520 5600
588 1320
60.4
b
116
9630
8910
540
1700
4020
1230
1010
1740
6110
26000
8470
3690
2860
1650
860
2450
9230
2160
1830
1470
1230
27.1
b
358
138
436
b
b
116
185
26.5
83.3
68.4
b
b
Figure 9 shows that the fluorescence intensities of the â-CD
derivatives 1 and 2 (2 µM) always decreased upon addition of
the guests (A, 10 µM; B, 100 µM) and 1 and 2 exhibit similar
trends in the guest dependency of the ∆I/I0 value, although the
a
The binding constants for guests 8-12 were measured in phosphate
buffer (I ) 0.01, pH 7.0) at 25 °C, and the binding constants for the
other guests were measured in aqueous solution at 25 °C. The values
are too small to be accurately determined.
b
∆
I/I0 value of 1 is larger than that of 2 in any case. Four
of 5, but that of 2 is only 9.7-fold larger than that of 5. It is
worth noting that the large difference in the binding ability is
caused only by the difference in the enantiomeric configuration
of the amino acid residue. This fact substantiates the unique
feature of this system that not only the hydrophobicity of the
isobutyl side chain but also the enantiomeric configuration of
the amino acid residue is an important factor to determine the
binding abilities of the hosts.
steroidal compounds such as ursodeoxycholic acid (8), cheno-
deoxycholic acid (9), deoxycholic acid (10), and cholic acid
(
1
11) were detected by 1 and 2 with the sensitivity order 11 <
0 < 9 < 8, and this order is the same as the order of their
binding constants. These compounds have the same steroidal
framework, and 8 and 9 are isomers with the difference of
stereochemistry of the hydroxyl group at C-7. Compound 10
is the regioisomer of 8 and 9 with one hydroxyl group at C-12
in place of the C-7 of 8 and 9. Compound 11 has one more
hydroxyl group than 8, 9, and 10. These differences were
reflected in the different sensitivity values of these steroids and
accordingly in the sensitivity order as indicated above. 1-Ada-
mantanecarboxylic acid (12) and 1-adamantanol (13) are known
as good guests for â-CD, and here, they give large ∆I/I0 values
comparable with those of steroidal compounds. The ∆I/I0 values
for smaller guests, cyclohexanol (14) and cyclooctanol (15) were
not so large, but the difference of the ring size is reflected to
some extent in the larger values for 15. (-)-Borneol (16) and
(+)-camphor (17) have the same framework although with
different functional groups, a hydroxyl group for 16 and a ketone
group for 17 at the same position. This difference was detected
by 1 and 2 with higher sensitivity values for 16 than those for
17. Nerol (19) and geraniol (20) are geometrical isomers and
were detected with similar low sensitivities. A cyclic mono-
terpene, (-)-menthol (18), was detected with a larger sensitivity
than the open chain monoterpenes 19 and 20. The results of a
series of monoterpenes 16-20 clearly indicate that the order
The order of the binding abilities of the γ-CD derivatives is
< 3 < 4 for most of the guests. It implies that the binding
6
constants of the D-leucine-modified γ-CD (4) are larger than
those of the L-leucine-modified γ-CD (3) with one exception
for (-)-menthol (18). It is rather surprising that the effect of
the leucine chirality of the γ-CD derivatives is opposite to that
of the â-CD derivatives in guest binding. The enantiomeric
configuration of the amino acid residue is a dominant factor
for determining the positions of the isobutyl side chain and the
dansyl moiety in the complexes, and the above result indicates
that the L- and D-configurations are more favorable for forming
stable inclusion complexes with guests in the case of the â-
and γ-CD derivatives, respectively. The binding constants of
the γ-CD derivatives are smaller than those of the â-CD
derivatives in most cases, but one can see an exception for
deoxycholic acid (10) as shown by the fact that the binding
constants of the γ-CD derivatives 3 and 4 are 2.1- and 6.2-fold
larger for 10, respectively, than those of the corresponding â-CD
derivatives 1 and 2. Another exception is seen in the binding
constant of 4 for 9 and 11, the value being 1.4- and 3.3-fold

10988 J. Am. Chem. Soc., Vol. 118, No. 45, 1996
Ikeda et al.
) of 1 and 2 for various guests: [1-2] ) 2 × 10 M, [guest] ) (A) 10-5 M, (B) 10-4 M. λex ) 370 nm.
-6
Figure 9. Sensitivity parameters (∆I/I
em ) 543 nm for 1 and 540 nm for 2.
0
λ
) of 3 and 4 for various guests: [3-4] ) 2 × 10 M, [guest] ) (A) 10-4 M, (B) 10-3 M. λex ) 370 nm.
-6
Figure 10. Sensitivity parameters (∆I/I
em ) 550 nm for 3 and 553 nm for 4.
0
λ
of the sensitivity for the terpenes is acyclic < monocyclic <
bicyclic terpenes.
enantiomeric isomers might be improved. The construction of
more effective molecule sensing systems are now underway.
Figure 10 shows the variations of the ∆I/I0 value of the γ-CD
derivatives 3 and 4 (2 µM) induced by guest addition (A, 0.1
mM; B, 1 mM). The guest dependency is similar for 3 and 4
with exceptions for 15 and 19, although the ∆I/I0 value of 4 is
larger than that of 3, indicating that the D-leucine-modified γ-CD
Conclusion
A new series of CD-amino acid-chromophore triad systems,
where CD was â- or γ-CD, amino acid was D- or L-leucine,
and chromophore was dansyl, were prepared for molecule
sensing, and their abilities as fluorescent indicators of molecular
recognition were studied. The insertion of the leucine residue
between CD and the dansyl residue improved the binding ability
of the hosts when compared with the glycine residue of the
corresponding hosts, and the difference in the enantiomeric
configuration of leucine caused the difference in the sensitivity
of molecular recognition. The fluorescence intensities of â-CD
derivatives always decreased upon guest addition, but the guest-
induced variation of the fluorescence intensities of the γ-CD
derivatives depended on the guest as shown by the increase by
cyclohexanol and the decrease by (-)-borneol and other larger
guests. The results demonstrate that the presence of a hydro-
phobic side chain of amino acid in the fluorescent CDs improves
their molecule sensing abilities and the guest binding, and
molecule recognition behavior of these sensors are delicately
affected by the chirality of the amino acid.
4
is more sensitive to the guest than the L-leucine-modified
γ-CD 3. It is noted that this chirality dependency is opposite
to that of the corresponding â-CD derivatives 1 and 2, and this
result agrees with the opposite chirality dependency described
for the binding data. Detailed examination of Figure 10 reveals
that the steroidal compounds were selectively detected with the
same sensitivity order 11 < 10 < 9 < 8 as that detected by the
fluorescent â-CDs 1 and 2, and the ∆I/I0 values for 1-adaman-
tanecarboxylic acid (12), 1-adamantanol (13), and (-)-borneol
(16) are rather depressed. Another remarkable result is that the
negative ∆I/I0 values are seen for cyclohexanol (14), cyclooc-
tanol (15), (-)-menthol (18), nerol (19), and geraniol (20). Since
these guest compounds belong to the category of smaller guests
among the guests examined here, it is reasonable that the
complexation proceeds according to the case II in Figure 4.
Molecule sensing abilities of the fluorescent CDs were
examined for some enantiomeric isomers such as camphor or
menthol. However, their molecular recognition abilities for
enantiomeric isomers were not so large, probably due to the
symmetric shape of the cavity and the flexibility of the dansyl
moiety. If the fluorescent CDs have a coordination group for
specific interaction with the guests, the sensing abilities for
Acknowledgment. We are grateful to Nihon Shokuhin Kako
Co. Ltd. for providing us with cyclodextrins. This work was
supported by a Grant-in-Aid for Science Research from the
Ministry of Education, Science, Sports, and Culture of Japan.
JA960183I