Boronic acid fluorophore/β-cyclodextrin complex sensors for selective sugar recognition in water

Article abstract of DOI:10.1021/ac001363k

A novel boronic acid fluorophore 1/β-cyclodextrin (β-CyD) complex sensor for sugar recognition in water has been designed. The probe 1 bearing pyrene moiety as a fluorescent signal transducer exhibits no fluorescence emission, due to its aggregation in water containing 2% DMSO; however, the addition of β-CyD to this solution largely changes UV-vis and fluorescence spectra of 1 by forming an inclusion complex with β-CyD, and an efficient fluorescence emission response of 1/β-CyD complex upon sugar binding is found to be obtained at pH 7.5. The pH-fluorescence profile of the 1/β-CyD complex reveals that the boronate ester formation with fructose induces the apparent pK_a shift from 7.95 ± 0.03 in the absence of fructose to 6.06 ± 0.03 in the presence of 30 mM fructose, resulting in the fluorescence emission response under the neutral condition. The spectral properties of 1 in 95% methanol:5% water (v/v), as well as the fluorescence quenching study of 1-methylpyrene with 4-methoxycarbonylphenyl-boronic acid 2, demonstrate that the response mechanism is based on the photoinduced electron transfer (PET) from the pyrene donor to the acid form of phenylboronic acid acceptor in 1, and thus, the proton dissociation of phenylboronic acid induced by sugar binding inhibits the PET system while increasing the fluorescence intensity of the pyrene moiety. To evaluate the binding ability and selectivity of the 1/β-CyD complex for monosaccharides in water, the response equilibria have been derived. The 1:1 binding constants of the 1/β-CyD complex obtained from the equilibrium analysis are in the order: D-fructose (2515 ± 134 M^-1) ? L-arabinose (269 ± 28 M^-1) > D-galactose (197 ± 28 M^-1) > D-glucose (79 ± 33 M^-1), which is consistent with the binding selectivity of phenylboronic acid.

Full text of DOI:10.1021/ac001363k

Anal. Chem. 2001, 73, 1530-1536
Bo ro n ic Ac id Flu o ro p h o re /â-Cyc lo d e x t rin Co m p le x
S e n s o rs fo r S e le c t ive S u g a r Re c o g n it io n in Wa t e r
†
‡
,‡
§
§
Ai-J un Tong, Akiyo Ya m a uc hi, Ta ka s hi Ha ya s hita ,* Zhi-Yi Zha ng, Bra dle y D. Sm ith, a nd
,
‡
Norio Te ra m a e *
Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980-8578, Japan, and
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556
A novel boronic acid fluorophore 1/ â-cyclodextrin (â-CyD)
complex sensor for sugar recognition in water has been
designed. The probe 1 bearing pyrene moiety as a
fluorescent signal transducer exhibits no fluorescence
emission, due to its aggregation in water containing 2 %
DMSO; however, the addition of â-CyD to this solution
largely changes UV-vis and fluorescence spectra of 1 by
forming an inclusion complex with â-CyD, and an efficient
fluorescence emission response of 1/ â-CyD complex upon
sugar binding is found to be obtained at pH 7 .5 . The pH-
fluorescence profile of the 1 / â-CyD complex reveals that
the boronate ester formation with fructose induces the
fundamental roles in controlling an individual’s birth, differentia-
tion and immunity. Because of these important properties, there
is a need to develop methods for in-situ sugar sensing in aqueous
1
solution. One approach that has recently attracted strong interest
is the use of boronic acid derivatives as fluorescent sugar
sensors.2
-10
For example, Czarnik and co-workers reported the
properties of anthrylboronic acid 3, which senses sugars in neutral
2
aqueous solution via a fluorescence quenching process. Similarly,
Aoyama and co-workers synthesized indolylboronic acid 4 , which
recognizes fructose and oligosaccharides at pH 9 with chain length
selectivity due to the CH-π interaction in addition to boronate ester
3
formation. A major design effort has been made by Shinkai et al.
to develop sensors for precise and selective sugar recognition.5
A notable design uses a boronic acid unit connected to a
fluorescent tertiary amine, as shown in 5.6 Sugar binding results
in fluorescence enhancement due to a strengthening of the
boron-nitrogen Lewis acid-base interaction which suppresses
electron-transfer quenching. This photoinduced electron transfer
(PET) system senses sugars in neutral aqueous methanol solution
with fluorescence intensity increases.
Molecular recognition by multicomponent assemblies of host
molecules appears to be another promising way to construct novel
molecular sensors.11 Self-assembled systems often exhibit different
recognition properties as compared to the original host molecule.12
apparent pK shift from 7 .9 5 ( 0 .0 3 in the absence of
a
fructose to 6 .0 6 ( 0 .0 3 in the presence of 3 0 mM
fructose, resulting in the fluorescence emission response
under the neutral condition. The spectral properties of 1
in 9 5 % methanol:5 % water (v/ v), as well as the fluores-
cence quenching study of 1 -methylpyrene with 4 -meth-
oxycarbonylphenyl-boronic acid 2 , demonstrate that the
response mechanism is based on the photoinduced
electron transfer (P ET) from the pyrene donor to the acid
form of phenylboronic acid acceptor in 1 , and thus, the
proton dissociation of phenylboronic acid induced by
sugar binding inhibits the P ET system while increasing
the fluorescence intensity of the pyrene moiety. To evalu-
ate the binding ability and selectivity of the 1 / â-CyD
complex for monosaccharides in water, the response
equilibria have been derived. The 1 :1 binding constants
of the 1 / â-CyD complex obtained from the equilibrium
-10
(2) Yoon J.; Czarnik A. W. J. Am. Chem. Soc. 1 9 9 2 , 114, 5874-5875. Czarnik,
A. W. In Fluorescent Chemosensors for Ion and Molecule Recognition; Czarnik,
A. W., Ed.; ACS Symposium Series 538; ACS Books: Washington, D.C.,
1992; pp 104-129.
(3) Nagai, Y.; Kobayashi, K.; Toi, H.; Aoyama, Y. Bull. Chem. Soc. Jpn. 1 9 9 3 ,
-
1
66, 2965-2971.
analysis are in the order:
D
-fructose (2 5 1 5 ( 1 3 4 M
)
(
(
4) Adhikiri, D. P.; Heagy, M. D. Tetrahedron Lett. 1 9 9 9 , 40, 7893-7896.
5) Shinkai, S. In Chemosensors of Ion and Molecule Recognition; NATO ASI
Series C492; Kluwer: London, 1996; pp 37-59. James, T. D.; Sandanayake,
K. R. A. S.; Shinkai, S. Supramol. Chem. 1 9 9 5 , 6, 141-157. James, T. D.;
Linnane, P.; Shinkai, S. Chem. Commun. 1 9 9 6 , 281-288. James, T. D.;
Sandanayake, K. R. A. S.; Shinkai, S. Angew. Chem., Int. Ed. Engl. 1 9 9 6 ,
35, 1910-1922. Sandanayake, K. R. A. S.; James, T. D.; Shinkai, S. Pure
Appl. Chem. 1 9 9 6 , 68, 1207-1212.
1
.
M
L
-arabinose (2 6 9 ( 2 8 M- ) >
D
-galactose (1 9 7 ( 2 8
) > -glucose (7 9 ( 3 3 M ), which is consistent with
-1
-1
D
the binding selectivity of phenylboronic acid.
Sugars are important biological molecules that are essential
in processes such as nutrition, metabolism, and cell structure.1
Sugars are also physiologically active substances, and they play
(
(
6) James, T. D.; Sandanayake, K. R. A. S.; Iguchi, R.; Shinkai, S. J. Am. Chem.
Soc. 1 9 9 5 , 117, 8982-8987.
7) James, T. D.; Shinmori, H.; Takeuchi, M.; Shinkai, S. Chem. Commun. 1996,
705-706.
†
Present address: Department of Chemistry, Tsinghua University, Beijing
1
00084, PR China.
(8) James, T. D.; Shinmori, H.; Shinkai, S. Chem. Commun. 1 9 9 7 , 71-72.
(9) Cooper, C. R.; James, T. D. Chem. Commun. 1 9 9 7 , 1419-1420. Cooper, C.
R.; James, T. D. Chem. Lett. 1 9 9 8 , 883-884.
‡
Tohoku University.
§
University of Notre Dame.
(
1) Davis, A. P.; Wareham, R. S. Angew. Chem., Int. Ed. Engl. 1 9 9 9 , 38, 2978-
996. Henning, T. P.; Cunningham, D. C. In Commercial Biosensors:
(10) Kijima, H.; Takeuchi, M.; Robertson, A.; Shinkai, S.; Cooper, C. R.; James,
2
T. D. Chem. Commun. 1 9 9 9 , 2011-2012.
Applications to Clinical, Bioprocess, and Environmental Samples; Ramsay,
G., Ed.; Wiley: New York, 1998.
(11) Lehn, J.-M. Supramolecular Chemistry: Concepts and Perspectives; VCH:
Weinheim, 1995.
1530 Analytical Chemistry, Vol. 73, No. 7, April 1, 2001
10.1021/ac001363k CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/06/2001

For example, we recently used a crown ether fluorophore/ γ-
cyclodextrin (γ-CyD) complex to selectively sense potassium ions
distilled and deionized by a Milli-Q Labo system (Millipore) before
use. Other chemicals used were of reagent grade and were used
as received.
Synthesis of 4-(4-(1-Pyrenyl)butoxy)carbonylphenylboronic
Acid (1 ). To a mixture of 4-(1-pyrenyl)butanol (80 mg, 0.29
mmol), 2-(4-carboxyphenyl)-1,3,2-dioxaborinane (60 mg, 0.29
mmol), and 4-(dimethylamino)pyridine (18 mg, 0.15 mmol) in dry
1
3
in water. Dimerization of the probe inside the γ-CyD provides a
highly selective binding site for the potassium ion. This finding
has led us to design additional multicomponent probe/ CyD
complex sensors for ion and molecule recognition in water.
The objective of this study is to develop a highly sensitive and
selective recognition system for sugars in aqueous solution by
using the inclusion complex of newly designed fluorescent probe,
CH
1-(3-dimethylamino)propyl-3-ethylcarbodiimide hydrochloride (87
mg, 0.44 mmol) in dry CH Cl / DMF (10:1 v/ v, 2 mL) at 0 °C.
2 2
Cl / DMF (10:1 v/ v, 15 mL) was slowly added a solution of
1
, with â-CyD. Probe 1 contains a fluorescent pyrenyl group and
2
2
a sugar-binding arylboronic acid moiety.1 The hydrophobic cavity
of â-CyD can incorporate probe 1 and the assembled probe 1 / â-
CyD complex acts as a sugar sensor. We find that the 1 / â-CyD
complex exhibits an increased fluorescence emission response
upon sugar binding in water.
4
The reaction mixture was stirred at 0 °C for 2 h and at room
temperature for 2 days. The solvent was evaporated in vacuo and
the residue was taken up with CH
solution was washed with 10% aqueous HCl (20 mL), water (20
mL), 4% aqueous Na CO (20 mL), 5% aqueous HCl (20 mL), and
water (20 mL), was dried over MgSO , and was evaporated in
vacuo. The residue was chromatographed on silica gel sequentially
with EtOAc/ hexane (3:1, v/ v) and MeOH/ CHCl (1:1, v/ v) as
2 2
Cl (20 mL). The organic
2
3
4
3
eluents to give 73 mg (60%) of the desired product as a white
solid. mp 194-197 °C. MS (+ FAB in glycerol) m/ z 478 for
1
boronic acid/ glycerol adduct. H NMR (300 MHz; DMSO-d
6
) δ
1
8
.9 (m), 3.38 (peak + water impurity), 4.36 (t), 7.88 (d), 7.97 (d),
.04 (t), 8.12 (d), 8.19 (t), 8.23-8.25 (m), 8.27-8.28 (m), 8.37 (d).
C NMR (75 MHz; DMSO-d ) δ 27.97, 28.25, 32.24, 64.23, 123.55,
6
13
1
1
1
1
24.23, 124.34, 124.90, 125.06, 126.27, 126.61, 127.31, 127.56,
27.61, 128.00, 128.16, 129.37, 130.48, 130.99, 131.08, 134.31,
36.78. FT-IR (KBr pellet) cm- 3036, 2925, 1714; 1400, 1362, 1268,
115, 1015, 949, 834, 707.
1
Synthesis of 4 -Methoxycarbonylphenylboronic Acid (2 ).
-Carboxyphenylboronic acid (1.00 g, 6.03 mmol) was dissolved
4
2 4
in methanol (25 mL) and 5 drops of concentrated H SO was
added. The mixture was stirred at reflux temperature for 1 day.
After removal of the solvents in vacuo, the residue was dissolved
in EtOAc (20 mL), and the solution was extracted with 5% aqueous
2 3 4
Na CO and water. The EtOAc layer was dried over MgSO , and
the solvent was removed in vacuo. The resulting residue was
purified by recrystallization from toluene to give 0.166 g (15%) of
1
the desired product as a white solid. H NMR (DMSO-d
6
plus a
EXPERIMENTAL SECTION
drop of D
CH ). Anal. Calcd for C
Found: C, 55.74; H, 4.99.
2
O, 270 MHz) δ 7.83-7.94 (m, 4 H, ArH), 3.83 (s, 3 H,
Apparatus. Fluorescence spectra were measured with a
BO (-0.4 H
4
2
O):16 C, 55.61; H, 4.78.
3
8
H
9
JASCO FP-770 spectrofluorometer at 25 °C; the excitation and
emission bandwidths were 5.0 and 3.0 nm, respectively. The scan
speed was 50.0 nm/ min. All fluorescence spectra were recorded
under an aerated condition. UV-vis spectra were measured by a
Hitachi U-3000 UV-vis spectrophotometer (Hitachi Ltd.) by using
RESULTS AND DISCUSSION
Spectral P roperties of 1 in Aqueous DMSO Solution.
Probe 1 was prepared in straightforward fashion by condensing
1
a 5-cm quartz cell at 25 °C. H NMR spectra were obtained using
2
-(4-carboxyphenyl)-1,3-dioxaborinane with 4-(1-pyrenyl)butanol
a JEOL-GSX270 (270 MHz; JEOL DATUM).
followed by deprotection of the boronic acid. The absorption and
fluorescence spectra of 1 were recorded in aqueous solutions with
different DMSO contents. Figure 1a shows the UV-vis spectra
of 1 in 2% DMSO solution (spectrum 1). The absorption peaks
are quite broad when compared to those in 25% DMSO solution
Reagents.
(+)-glucose were special grade reagents obtained from Wako
Pure Chemical Industries, Ltd. The first reagent grade of â-CyD
Wako Pure Chemical Industries, Ltd) was recrystallized twice
L(+)-Arabinose,
D
(-)-fructose,
D
(+)-galactose, and
D
(
from water. 2-(4-Carboxyphenyl)-1,3,2-dioxaborinane was prepared
by a slightly modified procedure;15 benzene (reaction time, 12 h)
was used instead of toluene (reaction time, 2 h). Water was doubly
(
spectrum 2), indicating that 1 aggregates in water. The addition
of â-CyD to a 2% DMSO solution of probe 1 dramatically changes
the absorption and fluorescence spectra. The absorption spectrum
of 1 in 2% DMSO solution containing 5.0 mM â-CyD (spectrum
3 in Figure 1a) becomes similar to that in 25% DMSO solution.
(
(
12) Lawrence, D. S.; Jiang, T.; Levett, M. Chem. Rev. 1 9 9 5 , 95, 2229-2260.
13) Yamauchi, A.; Hayashita, T.; Nishizawa, S.; Watanabe, M.; Teramae, N. J.
Am. Chem. Soc. 1 9 9 9 , 121, 2319-2320. Yamauchi, A.; Hayashita, T.; Kato,
A.; Nishizawa, S.; Watanabe, M.; Teramae, N. Anal. Chem. 2 0 0 0 , 72, 5841-
(15) Matsubara, H.; Seto, K.; Tahara, T.; Takahashi, S. Bull. Chem. Soc. Jpn.
1 9 8 9 , 62, 3896-3901.
5
846.
(
14) Lorand, J. P.; Edwards, J. O. J. Org. Chem. 1 9 5 9 , 24, 769-774.
(16) Products are the mixture of compound 2 and its anhydride. See ref 3.
Analytical Chemistry, Vol. 73, No. 7, April 1, 2001 1531

Fig u re 1 . Spectral properties of 1 in aqueous DMSO solution. (a)
UV-vis spectra: optical path length, 5 cm. (b) Fluorescence spec-
tra: excitation wavelength, 328.0 nm; excitation bandwidth, 5 nm;
emission bandwidth, 3 nm. [1] ) 1.05 µM in (1) 2% DMSO/98% water
Fig u re 2 . (a) Fluorescence spectra of the 1/â-CyD complex upon
the addition of fructose: [1] ) 1.05 µM and [fructose] ) 0-30.0 mM
in 2% DMSO/98% water (v/v) containing 5.0 mM â-CyD; excitation
wavelength, 328.0 nm; excitation bandwidth, 5 nm; emission band-
width, 3 nm; pH ) 7.5, adjusted by 0.015 M phosphate buffer (I )
(v/v), (2) 25% DMSO/75% water (v/v), and (3) 2% DMSO/98% water
(v/v) containing 5.0 mM â-CyD. pH ) 7.5, adjusted by 0.015 M
0.08 M with NaCl). (b) Dependence of I377.5 on the concentration of
phosphate buffer (I ) 0.08 M with NaCl).
fructose: [1] ) 1.05 µM in (1) 2% DMSO/98% water (v/v), (2) 25%
DMSO/75% water (v/v), and (3) 2% DMSO/98% water (v/v) containing
5
0
.0 mM â-CyD; pH ) 7.5, adjusted by 0.015 M phosphate buffer (I )
.08 M with NaCl).
This indicates the presence of monomeric 1 due to the formation
of an inclusion complex with â-CyD. Figure 1b shows the
fluorescence spectra of 1 in the same solvent mixtures. In 2%
DMSO solution, the aggregated 1 exhibits no emission, due to a
self-quenching. In contrast, probe 1 in 25% DMSO solution shows
fluorescence at 370-430 nm with a vibronic structure, which can
be ascribed to the monomer emission of a pyrene moiety. It is
interesting to note that the fluorescence intensity of 1 is dramati-
cally enhanced in the presence of â-CyD (spectrum 3 in Figure
1b). This is attributed to an enhancement in fluorescence quantum
yield for 1 , because it forms an inclusion complex with â-CyD,
which restricts the molecular motion of 1 and reduces the
radiationless transition process.
Fructose Recognition of the 1 / â-CyD Complex in Water.
The effect of added fructose on the emission intensity of 1 was
monitored at pH 7.5 in 2% DMSO solution, 25% DMSO solution,
and 2% DMSO solution containing 5.0 mM of â-CyD. Figure 2a
shows a typical fluorescence response for the 1 / â-CyD complex
in the presence of fructose. The fluorescence emission of the 1 / â-
CyD complex in 2% DMSO solution intensifies as the fructose
concentration is raised from 0 to 30 mM (curve 3 in Figure 2b).
In contrast, no fluorescence response is noted for 1 in 2% DMSO
solution (curve 1 in Figure 2b), and in 25% DMSO solution, probe
Fig u re 3 . Dependence of I377.5 as a function of pH: [1] ) 1.05 µM
in 2% DMSO/98% water (v/v) containing 5.0 mM â-CyD: (a) [fructose]
)
0.0 mM; (b) [fructose] ) 30.0 mM.
The pH-dependent changes in the fluorescence intensity of the
/ â-CyD complex in the absence (a) and presence (b) of 30 mM
1
fructose are presented in Figure 3. The solid lines show the
theoretical curves calculated from eq 1,
1
exhibits a reasonable increase in monomer fluorescence
Ka
intensity (curve 2). Thus, highly sensitive fructose recognition in
water is achieved by the 1 / â-CyD complex sensor.
To clarify the response function, the fluorescence behavior of
the 1 / â-CyD complex was examined at different pH conditions.
â[L]
t
φ
HL + φ
L
[
(
+
)
H ]
I )
(1)
Ka
1
+
+
[H ]
1532 Analytical Chemistry, Vol. 73, No. 7, April 1, 2001

S c h e m e 1 . P ro t o n Dis s o c ia t io n a n d S u g a r Bin d in g Eq u ilib ria o f 1 in Aq u e o u s S o lu t io n
L
where φHL and φ are the fluorescence quantum yields for HL
and L- species of 1 , respectively, and â is a constant that is
proportional to the intensity of the excitation light and the molar
extinction coefficient of 1 . K
sociation constant of 1 . As can be seen from Figure 3, the addition
of 30 mM fructose decreases the apparent pK value of 1 from
.95 ( 0.03 to 6.06 ( 0.03. This pK shift enables 1 to recognize
a
represents the boronic acid dis-
a
7
a
fructose at neutral pH. The sugar recognition process is illustrated
in Scheme 1. The fluorescence quantum yield for 1 apparently
increases when 1 is converted to its tetrahedral boronate form.
Thus, the increased amount of tetrahedral boronate induced by
sugar binding causes an enhancement in the emission intensity.
It is notable that the ratio of quantum yield between the acidic
Fig u re 4 . UV-vis and fluorescence spectra of 1 in 95% methanol/
5% water (v/v): [1] ) 1.05 µM when (1) [BTA] ) 0.0 mM and (2)
[BTA] ) 1.0 mM; BTA, benzyltrimethylammonium hydroxide.
L
and basic forms of 1 (φ / φHL) reaches 15.2. This pH-dependent
fluorescence profile is opposite to that observed with the previ-
ously reported fluorescence quenching probes.2 In addition,
Shinkai and co-workers have developed probes exhibiting in-
creased emission response to sugars, but their fluorescence
intensities become reduced with increasing pH, which is different
from the profile shown in Figure 3. This demonstrates a clear
difference in the response mechanism of the 1 / â-CyD complex.
Response Mechanism of the 1 / â-CyD Complex in Water.
The low fluorescence intensity for 1 when its boronic acid group
is trigonal suggests that the arylboronic acid can act as an electron
acceptor from the excited-state pyrene donor.17 To test this idea,
we examined the UV-vis and fluorescence spectra of 1 in 95%
methanol/ 5% water (v/ v). The fluorescence quantum yield of 1
is greatly enhanced by the addition of 1.0 mM benzyltrimethyl-
ammonium hydroxide (Figure 4). No changes are observed in
the UV-vis spectra (dotted lines in Figure 4), which indicates
that no ground state interaction is taking place. This result strongly
supports a PET pathway from the pyrene donor to the acid form
of the arylboronic acid acceptor.17 The 95% methanol is relatively
hydrophobic and no UV-vis spectral change is noted, which is
evidence against a sensing mechanism based on changes in
solubility or aggregation.5
-4
To obtain further evidence, we studied 1-methylpyrene and
4-methoxycarbonylphenylboronic acid (2 ) as donor and acceptor
models of the intramolecular pyrene/ arylboronic acid system in
1 . A fluorescence quenching study was carried out in 95%
methanol/ 5% water (v/ v). The UV-vis and fluorescence spectra
-
5
of 1-methylpyrene (5 × 10 M) and increasing amounts of the
acidic form of 2 (0-20 mM) are depicted in Figure 5a. Similar to
the result in Figure 4, the fluorescence of 1-methylpyrene is
significantly quenched by addition of the acid form of 2 without
changing its UV-vis spectra; however, when 40 mM benzyltri-
methylammonium hydroxide is added, which converts 2 to its
tetrahedral boronate form, no quenching behavior is observed.
The Stern-Volmer plot for this system in the absence (condition
1) and presence (condition 2) of base is shown in Figure 5b. A
clear straight line is obtained for condition 1, and the Stern-
-1
Volmer constant (KSV) is determined to be 110 M from its slope
analysis. On the assumption that the fluorescence lifetime of
18
1-methylpyrene is ∼100 ns, the quenching rate constant is
9
-1
s
-1, which is in the expected range for
estimated to be ∼10 M
a diffusion-controlled quenching by 2 . Thus, the main mechanism
(
17) Kavarnos, G. J. Fundamentals of Photoinduced Electron Transfer; Wiley-
(18) Snare, M. J.; Thistlethwaite, P. J.; Ghiggino, K. P. J. Am. Chem. Soc. 1 9 8 3 ,
105, 3328-3332.
VCH: New York, 1993.
Analytical Chemistry, Vol. 73, No. 7, April 1, 2001 1533

S c h e m e 2 . Re s p o n s e Me c h a n is m o f t h e 1 /â-CyD Co m p le x fo r S u g a r Bin d in g
for fluorescence quenching appears to be electron transfer from
the excited 1-methylpyrene to the acid form of 2 , which acts as
electron acceptor.
These results strongly support intramolecular PET from the
pyrene donor in 1 to the trigonal form of its arylboronic acid
acceptor. Sugar binding converts the boronic acid to tetrahedral
boronate, which decreases the amount of PET quenching and
increases the monomer fluorescence intensity (Scheme 2).
Overall, the 1 / â-CyD complex provides a new mechanism for
1
9
sugar sensing in water.
Binding Equilibrium and Recognition Selectivity of 1 / â-
CyD Complex for Sugars. According to the equilibria shown in
Scheme 1, the different equilibrium constants can be expressed
by the following equations:20
+
-
[
H ][L ]
K )
(2)
(3)
(4)
(5)
a
[
HL]
+
-
[
H ][LS ]
K ′ )
a
[
HLS]
-
[
LS ]
KLS
)
)
[
L-][S]
HLS]
HL][S]
Fig u re 5 . (a) UV-vis and fluorescence spectra of 1-methylpyrene
upon the addition of 2 in 95% methanol/5% water (v/v): [1-meth-
ylpyrene] ) 50.0 µM and [2] ) 0-20 mM; (b) Stern-Volmer plots,
[
KHLS
[
(1) [BTA] ) 0.0 mM and (2) [BTA] ) 40.0 mM.
a a
Because K ′ is equal to K KLS/ KHLS, at least three main equilibrium
be seen from eqs 1 and 6, the apparent acid dissociation constant
app) is defined as
constants must be taken into consideration. From eqs 2-5, the
fluorescence intensity of 1 / â-CyD is expressed as
(K
a
1
+ K [S]
LS
K (1 + K [S])
app
a
LS
Ka ) Ka
(9)
â[L] φ′ + φ′L
t
HL
+
1 + KHLS[S]
(
[S]))
[
H ](1 + K
HLS
I )
(6)
K (1 + K [S])
Ka
1 + K [S]
LS
a
LS
1
+
∆pK ) -log
) log
(10)
a
app
+
1
+ KHLS[S]
[
H ](1 + KHLS[S])
Ka
φ
+ φHLSKHLS[S]
HL
φ′HL
)
(7)
(8)
When the KHLS value is comparable to the KLS value, eqs 9
and 10 imply that no pK shift can be observed upon the addition
1
+ KHLS[S]
a
φ + φ K [S]
L
LS LS
(
19) The glucose-selective fluorescent probe reported by Eggert et al. seems to
φ′ )
L
1
+ K [S]
respond using the same mechanism as described here, although they do
not discuss the details. See: Eggert, H.; Frederiksen, J.; Morin, C.; Norrild,
J. C. J. Org. Chem. 1 9 9 9 , 64, 3846-3852.
LS
(
20) Hayashita, T.; Teramae, N.; Kuboyama, T.; Nakamura, S.; Yamamoto, H.;
Nakamura, H. J. Inclusion Phenom. Mol. Recognit. Chem. 1 9 9 8 , 32, 251-
265.
where φHLS and φLS are the fluorescence quantum yields for HLS
and LS- species of the 1 / â-CyD complex, respectively. As can
1534 Analytical Chemistry, Vol. 73, No. 7, April 1, 2001

Ta b le 1 . Bin d in g Co n s t a n t s o f Va rio u s Flu o re s c e n t P ro b e s fo r Mo n o s a c c h a rid e s
Binding constants (KLS/ M-1)
probes
D-fructose
D-galactose
276
L-arabinose
391
D-glucose
ref.
phenylboronic acid
4370
110
14
4
5
1
6300
1000
2515 ( 134
71
63
79 ( 33
3
6
(in 33% MeOH)
/ â-CyD complex
158
269 ( 28
197 ( 28
this work
of sugars; however, it is known, at least in dilute aqueous solution,
that the trigonal boronate esters (HLS) are very unstable, and
-
only anionic boronate complexes (LS ) are formed to a detectable
extent.14,21 Thus, in Scheme 1, the formation of HLS complex is
negligible in water (1 . KHLS[S]). In addition, because the
fluorescence intensity of the 1 / â-CyD complex in the presence
of fructose reaches the same level of fluorescence intensity as
the sugar-free 1 / â-CyD complex at pH 10, we can assume that
φ
L
is mostly equal to φLS. Thus, eq 6 is more simply expressed as
Ka
â[L] φ + φL
(1 + K [S])
LS
t
HL
+
(
)
[
H ]
I )
(11)
Ka
Fig u re 6 . Effect of sugar concentration on the fluorescent response
of the 1/â-CyD complex: [1] ) 1.05 µM in 2% DMSO/98% water
1
+
(1 + K [S])
+
LS
[
H ]
(v/v) containing 5.0 mM â-CyD; excitation wavelength, 328.0 nm;
excitation bandwidth, 5 nm; emission bandwidth, 3 nm; pH ) 7.5,
adjusted by 0.015 M phosphate buffer (I ) 0.08 M with NaCl); (1)
D-fructose; (2) L-arabinose; (3) D-galactose; and (4) D-glucose.
Equation 11 clearly indicates that the binding constant (KLS) can
be determined by nonlinear curve fitting analysis of fluorescence
intensity (I) as a function of sugar concentration ([S]) at the fixed
pH condition. The determination of pK
alternative method to obtain the KLS value upon the basis of eq
2.
a a
shift (∆pK ) is the
the order
D
-fructose .
L
-arabinose >
D
-galactose >
-fructose calculated
value in eq 12 is 2554 M-1, which coincides well
a
D-glucose. The
binding constant of 1 / â-CyD complex for
from the ∆pK
with the KLS value of 2515 M-1 in Table 1.
D
1
∆
pK ) log(1 + K [S])
(12)
a
LS
It should be noted that the Shinkai group produced efficient
glucose sensors, as well as probes for chiral sugar recognition,
by incorporating two boronic acid units as a sugar recognition
site with a tertiary amine PET system.5,23,24 In comparison, the
advantages of the self-assembled 1 / â-CyD complex sensor shown
here are (1) the high probe solubility in water, and (2) the high
fluorescent quantum yield due to an inclusion complex formation.
In addition, the combination of boronic acid probes with the CyD
derivatives bearing various functional groups25 may provide
multipoint sugar recognition. The evolutional development of
additional probe/ CyD complex sensors is actively underway in
our laboratory.
It should be noted that a Benesi-Hildebrand plot22 is only
applicable at a constant pH, with the reservation of 1 , KLS[S].
In this condition, the apparent experimental binding constant
K
LSapp) can be expressed as eq 13.
(
Ka
app
LS
K
) KLS
(13)
+
[
H ]
Binding of the monosaccharides
L-arabinose,
D
-galactose,
D
-glucose, and -fructose with the 1 / â-CyD complex was exam-
D
ined in water containing 2% DMSO. Figure 6 shows the fluores-
cence intensity changes of the 1 / â-CyD complex as a function of
sugar concentration at pH 7.5. The observed results are fitted well
with eq 11 (solid lines), and the 1:1 binding constants that are
calculated from the nonlinear program are summarized in Table
CONCLUSION
This study demonstrates that a boronic acid fluorophore 1 / â-
CyD complex binds sugars and produces increased fluorescence
emission in water. The fluorescence quantum yield is greatly
enhanced by forming an inclusion complex of sugar-1 / â-CyD.
A pH-fluorescence profile for the 1 / â-CyD complex reveals that
the fluorescence intensity increases upon formation of the bor-
, together with the reported data of phenylboronic acid,14
1
3
indolylboronic acid 4 , and the tertiary-amine-supported fluores-
6
cent probe 5 . As is seen from Table 1, the monosaccharides
binding selectivity of the 1 / â-CyD complex is essentially the same
as phenylboronic acid; thus, the binding constants decrease in
(
(
23) Bielecki, M.; Eggert, H.; Norrild, J. C. J. Chem. Soc., Perkin Trans. 2 1 9 9 9 ,
449-455.
24) James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. Nature 1 9 9 5 , 374, 345-
(
(
21) Smith, B. D.; Gardiner, S. J.; Munro, T. A.; Paugam, M. F.; Riggs, J. A. J.
Inclusion Phenom. Mol. Recognit. Chem. 1 9 9 8 , 32, 121-131.
22) Connors, K. A. Binding Constants; Wiley: New York, 1987; pp 147-187.
347.
(25) Suzuki, I.; Obata, K.; Anzai, J.; Ikeda, H.; Ueno, A. J. Chem. Soc., Perkin
Trans. 2, 2 0 0 0 , 1705-1710.
Analytical Chemistry, Vol. 73, No. 7, April 1, 2001 1535

onate conjugate base (pK
fructose, the apparent pK
increased fluorescence at neutral pH. The fluorescence emission
response of the 1 / â-CyD complex upon sugar binding appears
to be due to suppression of the photoinduced electron transfer
a
) 7.95). Upon the addition of 30 mM
decreases to 6.06, which results in
ACKNOWLEDGMENT
We acknowledge the Japan Society for The Promotion of
Science (JSPS) for funding the invitation fellowship programs for
research in Japan to Prof. A.-J. Tong. This research was supported
by a Grant-in-Aid for Scientific Research (12554029) from the
Ministry of Education, Science, Sports, and Culture, Japan, and
the National Science Foundation, U.S.A.
a
(
PET) from a pyrene donor to a trigonal arylboronic acid acceptor.
The 1:1 binding constants of the 1 / â-CyD complex decrease in
-
1
the order:
D
-fructose (2515 ( 134 M ) .
L
-arabinose (269 ( 28
M-1) >
-galactose (197 ( 28 M-1) >
-1
Received for review November 21, 2000. Accepted
January 15, 2001.
D
D
-glucose (79 ( 33 M ),
which is consistent with the known binding selectivity of phenyl-
boronic acid.
AC001363K
1536 Analytical Chemistry, Vol. 73, No. 7, April 1, 2001