Fluorescence turn-on sensing of lectins with mannose-substituted tetraphenylethenes based on aggregation-induced emission

Article abstract of DOI:10.1002/asia.200900430

The synthesis of mannosesubstituted tetraphenylethenes (TPEs) and their aggregation-induced emission (AIE) behavior, induced by interactions with concanavalin A (Con A), are reported. A mixture of the mannose-TPE conjugates and Con A in a buffer solution displays an intense blue emission on agglutination within a few seconds, which serves as a turn-on fluorescent sensor for lectins. The sensing is also selective: the conjugates act as a sensor for Con A, but do not sense a galactose-binding lectin, PNA. Con A-recognition is not affected even in the presence of other proteins in a mixture. The conjugates also exhibit high sensitivity to detect Con A. An increased sensitivity of the conjugates results if mannopyranoside substituents are linked to the TPE-core unit with a flexible chain and/or when the number of mannose residues increases.

Full text of DOI:10.1002/asia.200900430

DOI: 10.1002/asia.200900430
Fluorescence Turn-On Sensing of Lectins with Mannose-Substituted
Tetraphenylethenes Based on Aggregation-Induced Emission
Takanobu Sanji,* Kentaro Shiraishi, Mitsutaka Nakamura, and Masato Tanaka*[a]
Abstract: The synthesis of mannose-
substituted tetraphenylethenes (TPEs)
and their aggregation-induced emission
(AIE) behavior, induced by interac-
tions with concanavalin A (Con A),
are reported. A mixture of the man-
nose-TPE conjugates and Con A in a
buffer solution displays an intense blue
emission on agglutination within a few
seconds, which serves as a “turn-on”
fluorescent sensor for lectins. The sens-
ing is also selective: the conjugates act
as a sensor for Con A, but do not sense
a galactose-binding lectin, PNA. Con
A-recognition is not affected even in
the presence of other proteins in a mix-
ture. The conjugates also exhibit high
sensitivity to detect Con A. An in-
creased sensitivity of the conjugates re-
sults if mannopyranoside substituents
are linked to the TPE-core unit with a
flexible chain and/or when the number
of mannose residues increases.
Keywords: aggregation
·
biosen-
sors · fluorescence · glycoconju-
gates · proteins
Introduction
rotations of the molecules.[9] Based on the state (aggrega-
tion)-dependent fluorescence, AIE-active molecules can be
used as a convenient tool for the sensing of biomolecular
species, in which they aggregate by interactions, resulting in
their display of an intense emission. For example, a phosp-
hole oxide, which is AIE-active,[10] was used as the sensory
element, and hence sugar–phosphole oxide conjugates act as
a fluorescence turn-on sensor for lectins. While the conju-
gates displayed significant binding, it was clearly not suffi-
ciently sensitive for trace detection of lectins. To advance
fluorescence turn-on sensing with AIE-active materials as a
detection tool, a modification is required to improve the
sensitivity to analytes. Here, we describe an alternative fluo-
rescence turn-on sensing of lectins with mannose-substituted
tetraphenylethenes (TPEs) 1 based on the AIE feature
(Scheme 1). It may not be suitable to call the compounds
“sensors”, as they are just optical probes.
The development of sensitive, convenient, and precise pro-
tein-sensing methods is of great importance in the fields of
biological, medical, and environmental science because pro-
tein–carbohydrate interactions mediate a wide variety of
biological events, such as cell-cell recognition, cell adhesion,
differentiation, trafficking, signaling between cells, cellular
metastasis, and viral or bacterial infections.[1] A variety of
optical-sensing methods for carbohydrate-binding proteins,
lectins, bacteria, and viruses have been reported.[2,3] For in-
stance, fluorescent p-conjugated polymers for lectins and
bacteria in biosensing applications,[4] which usually work in
fluorescence-quenching mode, turn-off upon interactions
with biological analytes.
Recently, we[5] and others[6,7] have demonstrated that ag-
gregation-induced emission (AIE)-active materials, first re-
ported by Tang and co-workers,[8] have a potential utility in
biosensor fields. AIE-active molecules show no emission in
solution but an intense emission when aggregated or in the
solid state because of the restriction of the intramolecular
Results and Discussion
Design
Our design for lectin sensing is schematically illustrated in
Figure 1. TPE, which shows no emission in solution but an
intense blue emission upon aggregation, is AIE active,[6] and
was our choice of a basic fluorescent sensory element. This
is because TPE has key advantages because of its emission
efficiency, facile synthesis, accessible structural modification
for specific utility, and stability among AIE-active materials.
[a] Dr. T. Sanji, K. Shiraishi, M. Nakamura, Prof. M. Tanaka
Chemical Resources Laboratory
Tokyo Institute of Technology
4259-R1-13 Nagatsuta, Midori-ku, Yokohama 226-8503 (Japan)
E-mail: sanji@res.titech.ac.jp
m.tanaka@res.titech.ac.jp
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.200900430.
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FULL PAPERS
crease of the number of sugar units installed to bind more
efficiently multivalent Con A.
Synthesis
Scheme 2 shows the synthetic route for the mannose-modi-
fied TPE 1a and 1b. An a-d-mannopyranoside derivative
1a was synthesized by the reaction of 4-hydroxy-substituted
Scheme 1. Chemical structures of mannose-substituted tetraphenyl-
ACHTUNGTRENNUNGethenes (TPEs), 1a–c.
Figure 1. Schematic representation of a “turn-on” fluorescent sensor for
Con A using a sugar-modified tetraphenylethene (TPE) based on an ag-
gregation-induced emission (AIE).
Scheme 2. Synthesis of 1a and 1b.
On the other hand, concanavalin A (Con A), which is a
well-studied lectin from Canavalia ensiformis (Jack bean),
was used as a model protein to study protein–carbohydrate
interactions.[11] Con A exists as a tetramer at neutral pH,
and is known to selectively recognize a-mannopyranoside
and a-glucopyranoside at its four binding sites located about
6.5 nm apart on one side of the protein.
For sensing of Con A, a-d-mannopyranoside units were
incorporated into TPE.[12] In this study, two possible princi-
ples to enhance the sensitivity toward Con A were exam-
ined; 1) elongation of the linker between the sugar and the
AIE molecules to endow the interaction with flexibility and
to decrease the entropic cost for the interaction, and 2) in-
TPE 3 and acetyl-protected d-mannopyranoside 4 followed
by deprotection. The NMR analysis showed that the incor-
porated mannopyranosides involve the a-isomer. TPE with
a-mannopyranoside linked by a diethylene-glycol unit, 1b,
was prepared by the reaction of 4 and diethylene glycol-
tethered TPE 5 followed by deprotection.
As shown in Scheme 3, TPE with eight a-mannopyrano-
side units linked by diethylene-glycol chains 1c was also syn-
thesized from tetrakis(3,5-dihydroxyphenyl)ethene 7 as a
starting material that was easily obtained by the McMurry
coupling of 3,3’,5,5’-tetramethoxybenzophenone 9 followed
by demethylation.
The products were fully characterized by spectroscopic
methods. Importantly, the sugar-modified TPEs 1a–c dis-
solve in water completely, which is usually required for bio-
sensing in aqueous media.
Abstract in Japanese:
Fluorescence Response toward Lectins
Sensing Studies of 1a to Con A
In the sensing studies, the interaction of 1 with Con A was
used as an example. As designed, an aqueous solution of 1a
was practically nonluminescent, with a quantum yield of
1.5% (relative to quinine sulfate as a standard). When Con
818
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Fluorescence Turn-On Sensing of Lectins
Scheme 3. Synthesis of 1c.
A was added to an aqueous solution of 1a in a buffer solu-
tion (10 mm Tris-HCl, 1 mgmLꢀ1 CaCl2, 1 mgmLꢀ1 MnCl2,
pH 7.6), the mixture exhibited an intense emission under ir-
radiation with UV light (Figure 2a and b), in which the
quantum yield was 10%. Note that when Con A was in a
great excess relative to the concentration of 1a, a further in-
crease in the Con A concentration did not result in a signifi-
cant change in the fluorescence intensity (Figure S2 in the
Supporting Information). The increase in the fluorescence
intensity was rapid, reaching a final value within a few sec-
onds (Figure 2c). The final intensity at 460 nm represented
an approximately 25-fold increase relative to the original
value. As can be seen in Figure 3a, the increase in the emis-
sion intensity of 1a upon addition of Con A was easily con-
firmed by the naked eye.
The dramatic enhancement of the fluorescence intensity
upon addition of Con A is ra-
tionalized in terms of aggrega-
tion triggered by multivalent
complexation of Con A with
1a, appending a-mannopyra-
noside units at its periphery,
which in turn induces enhance-
ment of the emission, as illus-
trated in Figure 1. Laser scat-
tering studies showed aggre-
gate formation with an average
diameter of 10.3 mm (see Fig-
ure S3 in the Supporting Infor-
mation), demonstrating that a
few thousands of Con A as-
semble into the aggregate
through the multivalent inter-
action with 1a.
The fluorescence intensity as
a function of the concentration
of Con A allows the determi-
nation of the binding constant
(Ka) to be 2.5ꢁ105 mꢀ1 (Fig-
ure 2d and Table 1), which is
Figure 2. a) Change in fluorescence spectra of 1a (7.2 mm) upon addition of Con A in a buffer solution (lex
=
much higher than that of
methyl mannose.[13] This is be-
cause the clustered carbohy-
drate enhances the binding
constant of generally weak in-
320 nm). b) Fluorescence intensity at 460 nm (F) relative to the value in its absence (F0) upon addition of Con
A (&) or PNA (*) in a buffer solution (lex =320 nm, [1a]=7.2 mm). c) Changes in fluorescence intensity,
monitored at 460 nm, on addition of Con A (2.0 mm) to 1a (7.2 mm) in a buffer solution (lex =320 nm).
d) Scatchard plot of 1a upon addition of Con A as shown in (a). All measurements were performed in a
buffer solution (10 mm Tris-HCl, 1 mgmLꢀ1 CaCl2, 1 mgmLꢀ1 MnCl2, pH 7.6).
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T. Sanji, M. Tanaka et al.
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has verified the selectivity to mannose-binding lectins; it
showed no increase in the fluorescence of 1a (Figures 2b
and 4). Figure 3b demonstrates that no fluorescence re-
Figure 3. A photograph of optical changes of fluorescence of 1a (7.7 mm)
on addition of a) Con A and b) PNA (0.0, 0.05, 0.10, 0.20, 0.50, 1.0, and
2.0 mm, from left to right) in
a buffer solution (10 mm Tris-HCl,
1 mgmLꢀ1 CaCl2, 1 mgmLꢀ1 MnCl2, pH 7.6) under irradiation with UV
light of 365 nm.
Table 1. Binding constant (Ka) and limit of detection (LOD) of 1a–c
based on the change in the fluorescence intensity as a function of Con A
content.
Compound
Ka [mꢀ1
]
LOD [nm]
1a
1b
1c
2.5ꢁ105
7.9ꢁ105
5.3ꢁ106
100
50
20
Figure 4. a) Change in fluorescence spectra (lex =320 nm) of 1a (7.7 mm)
upon addition of Con A (2.0 mm), a mixture of Con A (3.0 mm) and PNA
(3.0 mm), BSA (2.0 mm), BS-I (2.0 mm), PNA (2.0 mm), Tg (2.0 mm), and
WGA (2.0 mm) in a buffer solution. b) Fluorescence-response patterns (F/
F0, the fluorescence intensity (F) relative to the initial fluorescence inten-
sity of the sensor element (F0), monitored at 460 nm) of 1a against the
protein analytes. All measurements were performed in a buffer solution
(10 mm Tris-HCl, 1 mgmLꢀ1 CaCl2, 1 mgmLꢀ1 MnCl2, pH 7.6).
teraction between carbohydrate and lectin.[14] In addition,
the fluorescence titration shows that the optimal signal is
reached when the mole ratio of Con A to 1a is approxi-
mately 1:1 (Figure 2b). As Cloninger and co-workers report-
ed that the sugar-conjugated dendrimers show a size de-
pendence when binding to lectins,[15] TPE conjugate 1a is
too small to bind to multiple binding sites on tetrameric
Con A, in which only one sugar of the conjugate can bind at
a time, promoting extensive aggregation.
sponse was also observed during the addition of PNA. 1a
does not sense PNA.
An excess amount of mannan, which is a polysaccharide
of mannose, as an inhibitor was added to the mixture of 1a
and Con A (see Figure S4 in the Supporting Information).
The solution returned to being nonluminescent, indicating
that disaggregation of the 1a–Con A aggregate had taken
place. This observation further supports that the intense
fluorescence of 1a upon addition of Con A is attributed to
the complexation with Con A.
Furthermore, the selectivity of 1a for Con A was con-
firmed by treatment of various proteins, such as bovine
serum albumin (BSA, protein), bandeiraea simplicifolia
(BS-I, galactose-binding lectin), thyroglobulin (Tg, glycopro-
tein), and wheat germ agglutinin (WGA, N-acetyl-b-d-glu-
cosaminyl residue-binding lectin). As shown in Figure 4,
only the addition of Con A exhibited the fluorescence emis-
sion of 1a. Figure 5 clearly shows the different optical image
of 1a in the presence of Con A. Thus, mannose-substituted
TPE 1a only responds to Con A, but not to others. This is
because the mannopyranoside units on 1a specifically recog-
nize Con A.
Selective Detection of 1a for Con A and Other Proteins
A control experiment using 1a and peanut agglutinin
(PNA), a galactose-binding lectin from Arachis hypogaea,[16]
820
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Fluorescence Turn-On Sensing of Lectins
mixture instantly displayed an intense emission, as shown in
Figure 4. The fluorescence pattern is almost the same as that
observed in the case of Con A, as shown in Figure 4b.
Responsive Sensitivity of 1a–c to Con A
TPE 1b and 1c behaved similarly, and have higher sensitivi-
ties in the sensing study. Again, 1b and 1c were practically
nonluminescent in solution. Upon addition of Con A, the
fluorescence of 1b and 1c was turned on, as shown in Fig-
ures 6 and 7. The emission maximum and the intensity were
different from that of 1a. This is probably because the bind-
ing mode is different from that of the Con A–1a complex.
The binding constant was estimated at 7.9ꢁ105 mꢀ1 for 1b
and 5.3ꢁ106 mꢀ1 for 1c (Table 1), indicative of 1b and 1c
possessing higher affinities for Con A, as compared with 1a.
This is presumably because the sugars of the conjugates can
be bound many at a time to promote extensive aggregation
and precipitation. TPE 1b has the di(ethylene glycol) chain
as the flexible linker between the sugar and the TPE core,
resulting in an affinity for Con A that is three-fold higher
than 1a. TPE 1c, on the other hand, has eight mannose resi-
Figure 5. A photograph of a mixture of 1a (7.7 mm) and Con A, BSA,
BS-I, PNA, Tg, and WGA (2.0 mm, from left to right) in a buffer solution
(10 mm Tris-HCl, 1 mgmLꢀ1 CaCl2, 1 mgmLꢀ1 MnCl2, pH 7.6) under irra-
diation with UV light of 365 nm.
Most importantly, mannose-modified 1a has the ability to
sense Con A even in the presence of other proteins in a
mixture. For example, when 1a was mixed with an equiva-
lent amount of Con A and PNA in a buffer solution, the
Figure 6. a) Change in fluorescence spectra of 1b (7.1 mm), and b) the
fluorescence intensity at 470 nm (F) relative to the value in its absence
(F0) upon addition of Con A in a buffer solution (lex =320 nm). The mea-
surement was performed in a buffer solution (10 mm Tris-HCl, 1 mgmLꢀ1
CaCl2, 1 mgmLꢀ1 MnCl2, pH 7.6).
Figure 7. a) Change in fluorescence spectra of 1c (7.3 mm), and b) the
fluorescence intensity (F) relative to the value in its absence (F0) at
470 nm upon addition of Con A in a buffer solution (lex =320 nm). The
measurement was performed in
a buffer solution (10 mm Tris-HCl,
1 mgmLꢀ1 CaCl2, 1 mgmLꢀ1 MnCl2, pH 7.6).
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T. Sanji, M. Tanaka et al.
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Materials
dues linked with the di(ethylene glycol) chain, which makes
1c exhibit an even greater affinity than 1b. The typical limit
of detection (LOD) of 1a–c, evaluated from a ratio of
signal/noise (S/N) higher than 3,[17] was estimated at 100, 50,
and 20 nm, respectively (Table 1). The increased affinities
lead to a decrease in the LODs of 1b and 1c toward Con A.
As designed, the increased sensitivity of the conjugates re-
sults if mannopyranoside substituents are linked to the
TPE-core unit with a flexible chain and/or when the number
of mannose residues increases. Thus, 1 show high sensitivity
and are noticeably more sensitive to previously reported
sensors.[18]
All manipulations involving air and moisture-sensitive compounds were
carried out under atmosphere of dry nitrogen. All solvents and reagents
were of reagent quality, purchased from commercial sources and used
without further purification, unless otherwise noted below. Toluene,
Et2O, and THF were dried and distilled from sodium/benzophenone. Di-
chloromethane was dried and distilled from calcium hydride. 4,4’-Dime-
thoxybenzophenone was purchased from Aldrich. 3,3’,5,5’-Tetramethoxy-
benzophenone[20] and tetra(4-hydroxyphenyl)ethene 3[21] were prepared
by the reported procedures. Concanavalin A (Con A), mannan, peanut
agglutinin (PNA), bovine serum albumin (BSA), bandeiraea simplicifolia
(BS-I), thyroglobulin (Tg), and wheat germ agglutinin (WGA) were pur-
chased from Sigma–Aldrich.
Synthesis
2a: To a mixture of 3 (200 mg, 0.50 mmol), 4 (1.21 g, 2.46 mmol), and
MS4A in dichloromethane (15 mL) was added BF3·Et2O (0.76 mL,
6.0 mmol). The mixture was stirred at room temperature under N2 atmos-
phere for 16 h and then poured into saturated aqueous solution of
NaHCO3. The organic layer was washed with saturated aqueous solutions
of NaHCO3, H2O, and NaCl and dried over anhydrous Na2SO4. After fil-
tration and removal of the solvent, the residue was chromatographed
over silica gel (dichloromethane/acetone=20:1) to give 2a (28 mg,
Conclusions
In conclusion, we report the synthesis of mannose-substitut-
ed TPEs, 1a–c, with sensitivity toward Con A. When Con A
was added to 1a–c, they specifically formed aggregates, ex-
hibiting an intense blue emission. The sensing affords rapid,
sensitive, specific, and real-time detection of Con A. Sensi-
tivity of 1a–c increases if the mannopyranoside substituent
is linked to the TPE-core unit with a flexible chain and/or
when the number of mannose residues increases. This sensor
assay is feasible for lectins that are multivalent or multiva-
lently presented. Further experiments are required to dem-
onstrate the practical use of the fluorescence turn-on assay.
In particular, the assay must be tested in a more complex
environment, that is, biologically relevant fluids and mix-
tures of proteins, for their ability to detect species present at
very low concentrations. However, this study introduces a
principle to design sensitive “turn-on” fluorescent sensors
for potential applications in the fields of chemical, biologi-
cal, medical, and environmental science. Light-up sensors
with fluorescence turn-on responses as signal readout pos-
sess reduced false-positive signal and increased sensitivity,
offering a dark background for easy and accurate measure-
ment of trace analytes.[19] Further study is currently in prog-
ress.
0.016 mmol, 3% yield) as
a white solid. 2a: m.p.: 114.1–118.48C;
1H NMR (300 MHz, CDCl3): d=2.02 (m, 24H), 2.04 (s, 12H), 2.17 (s,
12H), 4.04–4.12 (m, 8H), 4.25–4.31 (m, 4H), 5.31–5.54 (m, 16H), 6.82 (d,
8H, J=8.7 Hz), 6.91 ppm (d, 8H, J=8.7 Hz); 13C NMR (75 MHz,
CDCl3): d=20.6, 20.8, 62.0, 65.9, 68.8, 69.1, 69.4, 95.9, 115.9, 132.5, 138.5,
138.8, 154.2, 169.7, 169.86, 169.93, 170.4 ppm; HRMS (FAB, matrix: 2-ni-
trobenzyl alcohol): m/z (%) calcd for C82H92O40: 1716.5165 [M]+; found:
1716.5171.
1a: A mixture of 2a (25 mg, 0.015 mmol) and sodium methoxide (3 mg,
0.05 mmol) in methanol (2 mL)/THF (2 mL) was stirred at room temper-
ature for 60 min. The mixture was made acid with a cation exchange
resin (DOWEX 50WX8-100) and filtered. The filtrate was evaporated to
give 1a (13 mg, 0.012 mmol, 87% yield) as a pale yellow solid. 1a: m.p.:
255.2–258.98C; 1H NMR (300 MHz, D2O): d=3.52–3.58 (m, 8H), 3.67–
3.70 (m, 4H), 3.77–3.83 (m, 4H), 3.93–4.00 (m, 8H), 5.34 (s, 4H), 6.61–
6.67 (m, 8H), 6.71–6.75 ppm (m, 8H); 13C NMR (75 MHz, D2O): d=
61.3, 67.1, 71.1, 71.6, 74.2, 99.2, 117.0, 133.5, 139.0, 139.7, 155.3 ppm;
HRMS (FAB, matrix: glycerol): m/z (%) calcd for C50H61O24: 1045.3553
[M+H]+; found: 1045.3533.
5: A mixture of 3 (0.40 g, 1.00 mmol), 2-chloroethoxyethanol (1.25 g,
10.0 mmol), K2CO3 (4.16 g, 30.1 mmol), and potassium iodide (0.21 g,
1.27 mmol) in DMF (100 mL) was stirred at 1008C for 8 h under N2 at-
mosphere. After removal of solvent, ethyl acetate was added to the mix-
ture. The organic layer was washed with saturated aqueous solutions of
NaHCO3, H2O, and NaCl and dried over anhydrous Na2SO4. After filtra-
tion and removal of the solvent, the residue was chromatographed over
silica gel (dichloromethane/acetone=1:3) to give 5 (0.35 g, 0.47 mmol,
47% yield) as a white solid. 5: m.p.: 138.6–140.08C; 1H NMR (300 MHz,
CDCl3): d=2.25–2.29 (m, 4H), 3.63–3.66 (m, 8H), 3.72–3.75 (m, 8H),
3.81–3.84 (m, 8H), 4.06–4.87 (m, 8H), 6.65 (d, 8H, J=8.4 Hz), 6.91 ppm
(d, 8H, J=8.4 Hz); 13C NMR (75 MHz, CDCl3): d=61.8, 67.2, 69.7, 72.6,
113.7, 132.5, 137.1, 138.5, 156.9 ppm; elemental analysis: calcd (%) for
C42H52O12: C 67.36, H 7.00; found: C 67.30, H 6.97.
Experimental Section
Measurements
1H and 13C NMR spectra were recorded using a Bruker AVANCE DPX
300 FT-NMR spectrometer at 300 and 75.4 MHz, respectively. H and 13C
1
chemical shifts were referenced to solvent residues. GC-MS was run
using a Shimadzu GC-MS 17A/QP-5000 mass spectrometer. High resolu-
tion and FAB mass spectra were obtained with a JEOL JMS-700 mass
spectrometer at an ionization potential of 70 eV. Matrix-assisted laser de-
sorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)
was performed on a Shimadzu-Kratos AXINA-GFR plus. Preparative
GPC was performed on a JAI LC-908 equipped with JAIGEL 1H and
2H columns with toluene as an eluent. Melting points were measured on
a Micro melting point apparatus IA9100. UV/Vis spectra were recorded
using an Agilent 8453 diode array spectrophotometer. Fluorescence spec-
tra were recorded using a Hitachi F 4500 spectrophotometer. Particle
size was recorded using a Horiba laser scattering particle size distribution
analyzer LA-950.
2b: To a mixture of 6 (0.23 g, 0.30 mmol), 4 (0.73 g, 1.48 mmol), and
MS4ꢂ in dichloromethane (15 mL) was added BF3·Et2O (0.45 mL,
3.6 mmol). The mixture was stirred at room temperature for 20 h under
N2 atmosphere and poured into saturated aqueous solution of NaHCO3.
The organic layer was washed with saturated aqueous solutions of
NaHCO3, H2O, and NaCl and dried over anhydrous Na2SO4. After filtra-
tion and removal of the solvent, the residue was chromatographed over
silica gel (dichloromethane/acetone=10:1) to give 2b (0.31 g, 0.15 mmol,
50% yield) as a pale yellow solid. 2b: m.p.: 57.3–62.08C; 1H NMR
(300 MHz, CDCl3): d=1.95–1.96 (m, 24H), 2.05 (s, 12H), 2.10 (s, 12H),
3.68–3.78 (m, 24H), 4.02–4.07 (m, 16H), 4.21–4.25 (m, 4H), 4.85 (s, 4H),
5.24–5.31 (m, 12H), 6.60 (d, 8H, J=7.2 Hz), 6.85 ppm (d, 8H, J=
822
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Chem. Asian J. 2010, 5, 817 – 824

Fluorescence Turn-On Sensing of Lectins
7.2 Hz); 13C NMR (75 MHz, CDCl3): d=20.5, 20.6, 20.7, 62.3, 66.0, 67.0,
67.2, 68.3, 68.9, 69.4, 69.7, 70.0, 97.6, 113.5, 132.4, 136.9, 138.2, 156.8,
169.6, 169.7, 169.8, 170.4 ppm; elemental analysis: calcd (%) for
C98H124O48: C 56.86, H 6.04; found: C 57.15, H 5.96.
3.64–3.73 (m, 40H), 3.78–3.85 (m, 24H), 4.05–4.08 (m, 16H), 4.24–4.29
(m, 8H), 4.85 (s, 8H), 5.25–5.33 (m, 24H), 6.17 (s, 8H), 6.24 ppm (s,
4H); 13C NMR (75 MHz, CDCl3): d=20.61, 20.64, 20.7, 20.8, 62.3, 66.0,
67.2, 67.2, 68.3, 69.0, 69.4, 69.6, 70.0, 97.7, 100.6, 109.8, 140.6, 144.8, 159.0,
169.7, 169.8, 169.9, 170.6 ppm; elemental analysis: calcd (%) for
C170H228O96: C 53.63, H 6.04; found: C 53.84, H 5.92.
1b: A mixture of 2b (24 mg, 0.011 mmol) and sodium methoxide (2.0 mg,
0.04 mmol) in methanol (2 mL) was stirred at room temperature for
1c: A mixture of 2c (41 mg, 0.011 mmol) and sodium methoxide (4 mg,
0.07 mmol) in methanol (3 mL) was stirred at room temperature for 2 h.
The mixture was made acid with a cation exchange resin (DOWEX
50WX8-100) and filtered. The filtrate was evaporated to give 1c (24 mg,
9.2ꢁ10ꢀ3 mmol, 83% yield) as a pale yellow solid. 1c: m.p.: 83.7–87.68C;
1H NMR (300 MHz, CD3OD): d=3.59–3.72 (m, 72H), 3.81–3.93 (m,
40H), 4.82 (s, 8H), 6.28 (s, 8H), 6.37 ppm (s, 4H); 13C NMR (75 MHz,
CD3OD): d=62.9, 67.8, 68.6, 68.7, 70.6, 71.5, 72.1, 72.5, 74.6, 101.8, 102.1,
111.0, 142.3, 146.5, 160.8 ppm; MS (MALDI-TOF, matrix: 2,5-dihydroxy-
benzoic acid): m/z (%) calcd for C106H164O64Na: 2483.95 [M+Na]+;
found: 2484.26.
80 min. The mixture was made acid with
a cation exchange resin
(DOWEX 50WX8-100) and filtered. The filtrate was evaporated to give
1b (14 mg, 0.009 mmol, 88% yield) as a pale yellow solid. 1b: m.p.: 86.6–
89.38C; 1H NMR (300 MHz, CD3OD): d=3.59–3.73 (m, 28H), 3.81–3.87
(m, 20H), 4.02–4.05 (m, 8H), 4.81 (s, 4H), 6.68 (d, 8H, J=8.4 Hz),
6.89 ppm (d, 8H, J=8.4 Hz); 13C NMR (75 MHz, CD3OD): d=62.9, 67.8,
68.5, 68.6, 70.8, 71.5, 72.1, 72.5, 74.6, 101.8, 114.8, 133.7, 138.3, 140.0,
158.7 ppm; HRMS (FAB, matrix: glycerol): m/z (%) calcd for C66H92O32
1396.5572 [M]+; found: 1396.5605.
:
8: To a suspension of zinc powder (4.46 g, 68 mmol) in THF (100 mL)
was added TiCl4 (IV) (6.5 g, 34 mmol) at ꢀ108C. The mixture was
warmed slowly to room temperature and then stirred at 708C for 2 h.
3,3’,5,5’-Tetramethoxybenzophenone 9 (3.6 g, 12 mmol) in THF (50 mL)
was added to the mixture at 08C. The mixture was stirred at 708C for
5 h. After the addition of an aqueous solution of K2CO3 (100 mL, 10%),
the organic layer was extracted with ethyl acetate (200 mL), washed with
saturated aqueous solution of NaCl, and dried over MgSO4, subsequently.
After filtration and removal of the solvent, the residue was chromato-
graphed over silica gel (dichloromethane/hexane=2:1) to give 8 (1.5 g,
2.5 mmol, 43% yield) as a white solid. 8: m.p.: 210.0–210.88C; 1H NMR
(300 MHz, CDCl3): d=3.65 (s, 24H), 6.21–6.25 ppm (m, 12H); 13C NMR
(75 MHz, CDCl3): d=55.3, 99.4, 109.0, 140.9, 145.0, 160.0 ppm; HRMS
(FAB, matrix: 2-nitrobenzyl alcohol): m/z (%) calcd for C34H36O8:
572.2410 [M]+; found: 572.2407; elemental analysis: calcd (%) for
C34H36O8: C 71.31, H 6.34; found: C 71.20, H 6.04.
Sample Preparation for Sensing Studies
A typical example is as follows: A 10 mL stock solution of 1a (1.05 mg)
in a buffer solution (10 mm Tris-HCl, 1 mgmLꢀ1 CaCl2, 1 mgmLꢀ1 MnCl2,
pH 7.6) was prepared. To a 1 mL aliquot of the stock solution, was added
Con A at room temperature, and then diluted to 5 mL with the buffer so-
lution. The mixture was stirred for 10 min, and then subjected to the fluo-
rescence measurements (lex =320 nm). The concentration of 1a and Con
A were [1a]=7.8 mm, [Con A]=0.1, 0.2, 0.8, 1.2, 1.6, 2.0, 3.0, 4.0, 6.0, and
16.0 mm, respectively.
Binding Constant (Ka)
The binding constant (Ka) was estimated from the Scatchard plot using
the following equation:
7: To 8 (1.25 g, 2.18 mmol) in dichloromethane (50 mL) was added a di-
chloromethane solution (1m) of boron tribromide (34.9 mL, 34.9 mmol)
at ꢀ788C, and then the mixture was stirred at room temperature for
18 h. The mixture was poured into ice water (200 mL) and extracted with
ethyl acetate (300 mL). The organic layer was washed with saturated
aqueous solution of NaCl and dried over MgSO4. After filtration and re-
moval of the solvent, the residue was chromatographed over silica gel
½ConAꢁ ꢂ Fo=DF ¼ ½ConAꢁ ꢂ F0=DFmax þ Ka ꢂ F0=DFmax
ð1Þ
in which, [Con A] is the concentrations of the Con A, F0, and F are the
initial fluorescence intensity and the fluorescence intensity of 1a–c, re-
spectively.[22]
(dichloromethane/acetone=1:1) to give
7
(0.792 g, 1.72 mmol, 79%
yield) as
a
white solid. 7: m.p.: >3008C; 1H NMR (300 MHz,
[D6]acetone): d=6.07 (s, 8H), 6.12 (s, 4H), 8.04 ppm (s, 8H); 13C NMR
(75 MHz, [D6]acetone): d=101.7, 110.0, 140.5, 146.6, 158.3 ppm; HRMS
(FAB, matrix: 2-nitrobenzyl alcohol): m/z (%) calcd for C26H21O8:
461.1236 [M+H]+; found: 461.1230.
Acknowledgements
We thank Horiba Co. for the laser scattering measurement. This work
was supported by Special Education and Research Expenses (Post-Silicon
Materials and Devices Research Alliance) and the G-COE program
from the Ministry of Education, Culture, Sports, Science, and Technology
of Japan. K.S. also thanks the Office of Industry Liaison at Tokyo Insti-
tute of Technology for financial support.
6: A mixture of 7 (0.38 g, 0.82 mmol), 2-chloroethoxyethanol (2.04 g,
16.4 mmol), K2CO3 (6.83 g, 49.4 mmol), potassium iodide (0.33 g,
2.02 mmol) in DMF (100 mL) was stirred at 1008C for 13 h. After evapo-
ration of the solvent, dichloromethane (200 mL) was added to the resi-
due. The organic layer was washed with saturated aqueous solutions of
NH4Cl and NaCl, and dried over Na2SO4. After filtration and removal of
the solvent, the residue was chromatographed over silica gel (chloro-
form/methanol=5:1) to give 6 (0.42 g, 0.36 mmol, 44% yield) as a pale
yellow oil. 6: 1H NMR (300 MHz, CDCl3): d=3.17–3.20 (m, 8H), 3.54–
3.55 (m, 16H), 3.62–3.71 (m, 16H), 3.84–3.91 (m, 16H), 6.21 (s, 8H),
6.26 ppm (s, 4H); 13C NMR (75 MHz, CDCl3): d=61.5, 67.2, 69.2, 72.6,
100.9, 110.0, 140.7, 144.8, 159.0 ppm; HRMS (FAB, matrix: 2-nitrobenzyl
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2c: To a mixture of 6 (330 mg, 0.28 mmol), 4 (1.43 g, 2.90 mmol), and
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Chem. Asian J. 2010, 5, 817 – 824
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
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Received: September 8, 2009
Published online: February 8, 2010
824
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2010, 5, 817 – 824