Building fluorescent sensors by template polymerization: The preparation of a fluorescent sensor for d-Fructose

Article abstract of DOI:10.1021/ol9908732

(Matrix presented) The application of molecular imprinting in making fluorescent sensors has been hampered by the lack of suitable fluorescent tags, which would respond to the binding event with significant fluorescence intensity changes. We have designed

Full text of DOI:10.1021/ol9908732

ORGANIC
LETTERS
1999
Vol. 1, No. 8
1209-1212
Building Fluorescent Sensors by
Template Polymerization: The
Preparation of a Fluorescent Sensor for
D-Fructose
Wei Wang, Shouhai Gao, and Binghe Wang*
Department of Chemistry, North Carolina State UniVersity,
Raleigh, North Carolina 27695-8204
binghe_wang@ncsu.edu
Received July 28, 1999
ABSTRACT
The application of molecular imprinting in making fluorescent sensors has been hampered by the lack of suitable fluorescent tags, which
would respond to the binding event with significant fluorescence intensity changes. We have designed and synthesized a fluorescent monomer
which allows for the preparation of fluorescent sensors of cis diols using molecular imprinting methods. This monomer was used for the
preparation of sensitive fluorescent sensors for D-fructose.
Custom-made fluorescent sensors for organic molecules have
a wide range of application potentials.1-4 Traditionally, such
sensors have been prepared through de noVo design and
synthesis. For example, many very sensitive fluorescent
sensors have been designed for peptides,5 metal ions,6-10
saccharides,11-13 and others.12,14-18 Recently, molecular
imprinting or template polymerization has gained much
attention as a convenient method for the construction of
binding sites for different analytes. Such an approach does
not require the prior knowledge of the 3-dimensional
structure of the analyte and the de noVo construction of the
complementary binding site.
Molecular imprinting is a technique first demonstrated in
the late 1940s by Dickey.19 The preparation of imprinted
polymers involves (1) prearrangement of the print molecule
(template) and the functional monomers at low temperature
so that complementary intermolecular interactions among
functional groups can develop, (2) polymerization of the
monomers under conditions that cause minimal disturbance
to the print molecule-monomer interactions, and (3) extrac-
tion of the print molecules from the polymers, which leaves
behind “receptor sites” that are complementary to the
templates or print molecules in terms of size, shape, and
(1) de Silva, A. P.; Gunaratne, N.; Gunnlaugsson, T.; Huxley, A. J. M.;
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10.1021/ol9908732 CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/28/1999

Scheme 1a
a (a) TBSCl/imidazole/DMF, 91%; (b) (i) NH2Me/MeOH, 4-5 h, (ii) NaBH4/MeOH, 2 h, 89%; (c) 6, K2CO3/MeCN, reflux, 42%; (d)
TBAF/THF, 74%; (e) methacrylic anhydride, DMAP/TEA/CH2Cl2, 70%.
functional group orientations. This technique has been used
for the preparation of selective recognition sites for a wide
variety of molecules.20-23 Naturally, such polymeric receptors
also have the potential to be developed as fluorescent
sensors.22,24-28 Conceivably, polymeric receptors could be
constructed with a fluorescent tag built into them so that
binding and dissociation of the analyte would change the
fluorescence emission sufficiently for the binding event to
be monitored conveniently. However, such efforts have been
hampered by the lack of appropriate fluorescent tags which
could respond to the binding event with a significant
fluorescence intensity change unless certain chromophores
are present in the template molecules, which could quench
the fluorescence of the fluorescent tag. Recently, we have
reported a method of making fluorescent sensors using
molecular imprinting methods through the use of an external
fluorescence quencher to manifest the fluorescence intensity
change upon binding of the target compound.29
imprinted polymers. The fluorescence intensity of the an-
thracene moiety of the functional monomer (1) is expected
to increase with the formation of an ester with a cis diol
structure (8, Scheme 2) because of the increased acidity of
the boron atom after the ester formation, which increases
the tendency for the nitrogen lone pair electrons to be donated
to the open shell of boron. Because the lone pair electrons
of the nitrogen can quench the fluoresence of the anthracene
moiety through photoelectron transfer, this complex (8)
formation will take away the quenching mechanism and,
therefore, increase the fluorescence intensity. The utility of
such a functional monomer (1) was tested by preparing a
polymeric fluorescent sensor for D-fructose, which is known
to bind to two boronic acid moieties.40 As designed, the bind-
ing of fructose to the resulting fluorescent polymers showed
significant fluorescence intensity changes (Figure 1).
In this report, we describe our efforts in the development
of polymeric sensors which respond to the binding event with
a significant fluorescence intensity change without the use
of an external quencher. The key to the project was the design
and synthesis of a fluorescent tag which is intrinsically
sensitive to the binding event and the incorporation of this
fluorescent tag into the imprinted polymers. There are ample
literature precedents showing that boronic acid derivatives
can bind to cis diols tightly through ester formation30-32 and
boronic acid-containing monomers can be used for the
preparation of selective binding sites for saccharides through
molecular imprinting.33-35 Furthermore, boronic acid moi-
eties, when attached to certain fluorescent molecules, have
been shown to significantly affect the fluorescence intensity
of such fluorescent tags upon ester formation with cis
diols.12,15,36-38 One specific example is the conjugate of an
anthracene moiety and a benzylamine moiety with boronic
acid attached at the ortho position.39 Such a conjugate was
known to show significant fluorescence intensity changes
upon ester formation with cis diols. By taking advantage of
such known properties, we designed and synthesized an
anthracene-boronic acid conjugate (1) with a methacrylate
moiety attached to it to allow for its incorporation into
Figure 1. Typical set of fluorescence emission spectra of D-fructose
imprinted polymer at different concentrations of D-fructose (λex 370
nm).
Functional monomer 1 was synthesized in five steps
starting from 10-(hydroxymethyl)-9-anthraldehyde (2)41
(Scheme 1). Hydroxy aldehyde 2, upon treatment with tert-
1210
Org. Lett., Vol. 1, No. 8, 1999

butyldimethylsilyl chloride (TBSCl) in the presence of
imidazole in DMF at room temperature, yielded the silyl
ether aldehyde in 91% yield.42 Treatment of the silyl ether
aldehyde with methylamine in methanol followed by reduc-
tion with sodium borohydride afforded silyl ether amine 3
in 89% yield. The reaction of amine 3 with 2,2-dimethyl-
propane-1,3-diyl[o-(bromomethyl)phenyl]boronate (6)39 in
the presence of potassium carbonate in acetonitrile gave
amine boronate intermediate 4 in 42% yield. The cleavage
of the silyl protecting group with tetrabutylammonium
fluoride (TBAF) in THF at room temperature followed by
an aqueous workup gave 5 in 74% yield. Reaction of 5 with
methacrylic anhydride in anhydrous THF in the presence of
DMAP (4-(dimethylamino)pyridine) afforded methacrylic
ester 1 in 70% yield.
diastereomers. This mixture of diastereomers 8 was used
directly for the next step, polymerization, without purifica-
tion. 2-Hydroxyethyl methacrylate was used as a comonomer
and EGDM (ethyleneglycol dimethacrylate) was used as the
cross linker. Then polymerization was carried out by
following procedures previously reported with a ratio of
complex 8, 2-hydroxyethyl methacrylate, and EGDM of 1:4:
50 at 65 °C under nitrogen using AIBN as the free radical
initiator.29 Then the polymer was ground and the template
molecules were extracted by washing with MeOH twice for
1.5-2 h, a mixture of 0.1 N HCl and MeOH (1/1, v/v) three
times for 3-5 h, and MeOH 10 times for a total of 22-24
h. After drying in a vacuum oven at 35 °C overnight, the
polymer particles were sieved and then used for the binding
studies.
The template-directed polymerization was carried out using
D-fructose (7) as the print molecule. D-Fructose was chosen
as the test compound because it was known to bind tightly
with the boronic acid moiety in a 1:2 ratio.35 The D-fructose-
boronic acid complex was preformed so as to maximize the
complementary interactions at the binding sites. Therefore,
D-fructose-boronic acid complex 8 was prepared by mixing
D-fructose (7) with 1 in a 1:2 ratio in a mixture of dioxane
and pyridine (9:1) (Scheme 2). Distillation of water from
Briefly, for the binding studies, about 10 mg of the
polymer particles was suspended in 4.0 mL of D-fructose
solutions at different concentrations in 50% MeOH/H2O (v/
v) with 0.05 M phosphate as the buffer at pH 7.4. The
suspension was vortex-mixed for 3.0 h. Then about 3.5 mL
of the suspension was transferred into a cuvette for fluores-
cence measurements. The emission spectra was recorded
from 380 to 650 nm immediately after the solution was
stirred with an INSTECT stirrer for 30 s. The excitation
wavelength was set at 370 nm. A typical set of spectra is
shown in Figure 1.
Without the addition of D-fructose, the fluorescence
intensity (I) was low (Figures 1 and 2). However, with the
addition of D-fructose, the fluorescence intensity was en-
hanced significantly in a concentration-dependent fashion.
Fluorescence intensity changes were observed at high µM
to high mM fructose concentrations (Figures 1 and 2). The
fluorescence binding studies were conducted in triplicate.
Because boronic acid was known to bind to any com-
pounds with a cis diol structural moiety, the fluorescence
intensity change in response to D-fructose itself, as shown
in Figures 1 and 2, did not necessarily indicate that the
Scheme 2
the reaction mixture yielded complex 8, which was confirmed
with FAB-MS (1015, M + 1). Due to the creation of four
new chiral centers, 8 was expected to be a mixture of many
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1997; pp 13-26.
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H. Nature Biotechnology 1997, 15, 354-357.
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Figure 2. Fluorescence intensity changes of D-fructose imprinted
polymers vs concentration of sugars ((, D-fructose; 2, D-mannose;
9, D-glucose; b, control polymer) (λex 370 nm, λem 426 nm).
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Org. Lett., Vol. 1, No. 8, 1999
1211

imprinting process created specific polymeric receptors for
D-fructose. Furthermore, very often the boronic acid moiety
shows preferential binding for fructose12,39,40 and additional
structural constrains are needed to change this preference.43-46
Therefore, several control experiments were conducted. First,
the binding of the polymeric receptors with D-glucose and
D-mannose was also examined. It was found that the
fluorescence intensity changes of the imprinted polymers
upon addition of glucose and mannose at the same concen-
trations were far smaller than that of D-fructose (Figure 2).
Second, the effect of D-fructose, D-glucose, and D-mannose
on the fluorescence intensity of the fluorescent monomer 1
at a concentration of 10-5 M, a concentration comparable to
that of the fluorescent moiety in studying the imprinted
polymers, was also examined. It was found that fluorescent
monomer 1 also exhibited preferential recognition of D-
fructose (Figure 3), however, to a lesser degree than that of
Table 1. Comparison of Monomer 1 and the Imprinted
Polymer in Their Preference for D-Fructose over D-Glucose.
(∆I/I0)fru/(∆I/I0)glu
concn (mM)
monomer
polymer
a
1.0
5.0
10.0
50.0
100.0
7.7
4.1
2.7
1.4
1.2
∞
8.8
5.7
2.7
3.0
a No intensity change was observed at 1.0 mM of D-glucose.
minimal fluorescence intensity changes upon addition of
D-fructose (Figure 2). All of these indeed indicated that the
imprinting process helped in the creation of binding sites
which preferentially recognized the template molecules.
Our studies demonstrated that indeed sensitive fluorescent
sensors can be prepared using molecular imprinting methods
for compounds that do not necessarily have chromophores
or quench fluorescence themselves. This can be achieved
with properly designed recognition moieties. The fluorescent
monomer 1 developed in this study could also be used for
the preparation of fluorescent sensors of other sugars and
biologically important catecholamines, which also have a cis
diol structural moiety.
Acknowledgment. Financial support from the National
Institutes of Health (DK55062) is gratefully acknowledged.
Wei Wang acknowledges a Glaxo graduate fellowship.
OL9908732
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Figure 3. Fluorescence intensity changes of monomer 1 vs
concentration of sugars ((, D-fructose; 2, D-mannose; 9, D-glucose)
(λex 370 nm, λem 426 nm).
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