Building fluorescent sensors for carbohydrates using template-directed polymerizations

Article abstract of DOI:10.1006/bioo.2001.1219

The ability to custom-make fluorescent sensors for different analytes could have a tremendous impact in a variety of areas. Template-directed polymerization or molecular imprinting seems to be a promising approach for the preparation of high-affinity and

Full text of DOI:10.1006/bioo.2001.1219

Bioorganic Chemistry 29, 308–320 (2001)
doi:10.1006/bioo.2001.1219, available online at http://www.idealibrary.com on
Building Fluorescent Sensors for Carbohydrates Using
Template-Directed Polymerizations1
Shouhai Gao, Wei Wang, and Binghe Wang2
Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204
Received May 3, 2001
The ability to custom-make fluorescent sensors for different analytes could have a tremendous
impact in a variety of areas. Template-directed polymerization or molecular imprinting seems
to be a promising approach for the preparation of high-affinity and specific binding sites for
different template molecules. However, the application of molecular imprinting in the prepara-
tion of 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 (1) that allows for the preparation of fluorescent
sensors of cis diols using molecular imprinting methods. This monomer has been used for the
preparation of imprinted polymers as sensitive fluorescent sensors for
polymers prepared showed significant fluorescence intensity enhancement upon binding with
the template carbohydrate.
D-fructose. The imprinted
᭧ 2001 Academic Press
Key Words: fluorescence; chemical sensor; boronic acid; molecular imprinting; polymeri-
zation; saccharides.
INTRODUCTION
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. Recently, molecular imprinting or template polymeriza-
tion, first demonstrated in the late 1940s by Dickey (5), 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 three-dimensional
structure of the analyte and the de novo design of the complementary binding site.
This technique has been used for the preparation of selective recognition sites for a
wide variety of molecules (6–9). For example, molecular imprinting has been used
for the preparation of HPLC stationary phases (10,11), antibody mimics (12–14), and
sensors (15,16). The preparation of imprinted polymers usually involves: (i)
1 Part of this work has been communicated: Wang, W., Gao, S., and Wang, B. (1999) Org. Lett.
1, 1209–1212.
2 To whom correspondence should be addressed. Fax: (919) 515-3757. E-mail: binghe wang@
ncsu.edu.
308
0045-2068/01 $35.00
Copyright ᭧ 2001 by Academic Press
All rights of reproduction in any form reserved.

CARBOHYDRATES USING TEMPLATE-DIRECTED POLYMERIZATIONS
309
prearrangement of the print molecule (template) and the functional monomers at
low temperature so that complementary intermolecular interactions among functional
groups can develop; (ii) polymerization of the monomers under conditions that cause
minimal disturbance to the print molecule–monomer interactions; and (iii) extraction
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
functional group orientations. Naturally, such polymeric receptors also have the poten-
tial to be developed as fluorescent sensors (17–29). 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 that could respond to the binding
event with a significant fluorescence intensity change unless certain chromophores
are present in the template molecule, which could quench the fluorescence of the
fluorescent tag (26,27). Recently, we have reported a method of making fluorescent
sensors using molecular imprinting methods through the use of an external fluores-
cence quencher to manifest the fluorescence intensity change upon binding of the
target compound (22). Ideally, one would like to develop a fluorescent sensing system
that is intrinsically sensitive to the binding event without the use of an external
quencher. To this end, we have been interested in the synthesis of fluorescent sensors
for carbohydrates because of their importance as biomarkers in different biological
systems (30–34). Herein, we report our efforts in searching for ways to prepare
fluorescent sensors for carbohydrates using molecular imprinting methods.
RESULTS AND DISCUSSION
The Design. One of the key elements for the success of this project is the design
of a functional monomer that has intrinsically strong intermolecular interactions with
certain structural elements of carbohydrates and such interactions result in changes
in fluorescence intensity. There are ample literature precedents showing that boronic
acid derivatives can bind to cis diols tightly through ester formation (35–37) and
boronic acid-containing monomers can be used for the preparation of selective binding
sites for saccharides through molecular imprinting (38–40). Furthermore, boronic
acid moieties, 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 (41–43). One specific example is the conjugate of an anthra-
cene moiety and a benzylamine moiety with boronic acid attached at the ortho position
(44). 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 have designed an anthracene–boronic acid conjugate (1) with a methacrylate
moiety attached to it to allow for its incorporation into imprinted polymers. The
fluorescence intensity of the anthracene moiety of the functional monomer (1) is
expected to increase with the formation of an ester with a cis diol structure 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 fluorescence
of the anthracene moiety through photoelectron transfer (PET), this complex formation

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GAO, WANG, AND WANG
FIG. 1. The binding of the carbohydrate with the boronic acid-containing monomer 1.
will take away the quenching mechanism and, therefore, increase the fluorescence
intensity (Fig. 1).
Synthesis of the monomer. The functional monomer (1) was synthesized in five
steps starting from 10-(hydroxymethyl)-9-anthraldehyde (2) (46) (Scheme 1). Reaction
of the hydroxy aldehyde (2) with tert-butyldimethylsilyl chloride (TBSCl) in the
presence of imidazole in DMF at room temperature yielded the silyl ether aldehyde
in 91% yield. Treatment of the silyl ether aldehyde with methylamine in methanol
followed by reduction with sodium borohydride afforded the silyl ether amine 3 in
89% yield. The reaction of amine 3 with 2,2-dimethylpropane-1,3-diyl[o-(bromometh-
yl)phenyl]-boronate (6) (44) in the presence of potassium carbonate in acetonitrile
SCHEME 1. Synthesis of the functional monomer 1. (a) TBSCl/imidazole/DMF, 91%; (b) (i) NH2Me/
MeOH, 6 h, (ii) NaBH4/MeOH, 2 h, 89%; (c) 6, K2CO3/MeCN, reflux, 42%; (d) TBAF/THF, 74%;
(e) methacrylic anhydride, DMAP/TEA/CH2Cl2, 70%.

CARBOHYDRATES USING TEMPLATE-DIRECTED POLYMERIZATIONS
311
gave the amine boronate intermediate 4 in 42% yield. The cleavage of the silyl
protecting group with tetrabutylammonium fluoride (TBAF) in THF at room tempera-
ture followed by an aqueous work-up gave 5 in 74% yield. Reaction of 5 with
methacrylic anhydride in dry CH2Cl2 in the presence of DMAP (4-dimethylaminopyri-
dine) afforded the methacrylic ester (1) in 70% yield.
Imprinted polymer preparations. The utility of the monomer (1) prepared was
tested through template-directed polymerization using
molecule. -Fructose was chosen as the test compound because it was known to bind
tightly with a boronic acid moiety in a 1:2 ratio (40). The -fructose-boronic acid
complex (8) was preformed so as to maximize the complementary interactions at the
binding sites. Therefore, the -fructose-boronic acid complex 8 was prepared by
mixing -fructose (7) with 1 in a 1:2 ratio in a mixture of dioxane and pyridine (9:1)
D-fructose (7) as the print
D
D
D
D
(Scheme 2). Distillation of water from the reaction mixture yielded the complex 8,
which was confirmed with FAB-MS (1015, M+ ϩ H). Due to the creation of four
new chiral centers, 8 was expected to be a mixture of many diastereomers. This
mixture of diastereomers 8 was used directly for the next step polymerization with-
out purification.
Polymerizations. For the imprinted polymer preparation, 2-hydroxyethyl methacry-
late (HEMA) was used as a comonomer to afford other possible interaction sites with
the carbohydrate. For the imprinted polymers to maintain certain three-dimensional
structures, it is important that the polymers have a high degree of cross-linking.
Ethyleneglycol dimethacrylate (EGDM) was used as the cross linker. For the free-
radical initiated polymerization reactions, we have tested both AIBN-initiated poly-
merization and transition metal-catalyzed atom transfer radical polymerization
(ATRP). AIBN-initiated polymerization has traditionally been used for the preparation
of imprinted polymers. However, such bulk polymerization methods often suffer from
the drawbacks of poor control of the macromolecular structures and the lack of
homogeneity in the polymers prepared. Therefore, there has been a great deal of
interest in developing new methods that can be used for preparing imprinted polymers
(47–51). In recent years, controlled/“living” radical polymerization, using transition
metal-catalyzed atom transfer radical polymerization (ATRP), has been developed for
making new polymeric materials (52). Several transition metal systems have been
SCHEME 2. Synthesis of the boronate–D-fructose complex 8.

312
GAO, WANG, AND WANG
used in ATRP (53–59). ATRP was said to have better control of the final average
molecular weight of the polymer by varying the initial monomer to initiator ratio
while maintaining a narrow molecular weight distribution. Therefore, we were also
interested in examining how the use of ATRP would affect of the overall outcome
of molecular imprinting.
Again, for the polymerization HEMA was used as a comonomer and EGDM was
used as the cross-linker. The AIBN-initiated polymerization was carried out using a
procedure similar to one previously reported (22). The ATRP was carried out using
copper chloride (CuCl) as the catalyst, 2,2Ј-bypyridine (bpy) as the ligand, and ethyl
2-bromoisobutyrate (BriB) as the initiator in DMF (60). The polymers prepared were
ground, washed, and dried before they were used for the evaluation of their binding
to the target carbohydrate.
Fluorescent binding studies of the imprinted polymers. 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 of phosphate as the
buffer at pH 7.4. The emission spectra were recorded from 380 nm to 550 nm with
an excitation wavelength of 370 nm. The fluorescence binding studies were conducted
in triplicate.
As designed, all imprinted polymers showed significant fluorescence intensity
changes upon the addition of the print molecule, D-fructose, and the fluorescence
intensity increase is concentration-dependent. A typical set of fluorescence spectra
is shown in Fig. 2. Figure 3 shows the concentration-depended fluorescence intensity
changes of the polymer prepared using AIBN-initiated polymerization (P-a) and Fig.
FIG. 2. A typical set of fluorescence spectra of
D-fructose-imprinted polymer (P-b) at different
concentrations of -fructose (␭ex, 370 nm; ␭em, 426 nm).
D

CARBOHYDRATES USING TEMPLATE-DIRECTED POLYMERIZATIONS
313
FIG. 3. Fluorescence intensity changes of
sugars (␭ex, 370 nm; ␭em, 426 nm).
D-fructose-imprinted polymer (P-a) vs concentration of
4 shows the concentration-depended fluorescence intensity changes of the polymer
prepared using atom transfer radical polymerization (P-b).
Because boronic acid was known to bind to any compounds with a cis diol structural
moiety, the fluorescence intensity changes in response to
in Figs. 2–4, do not necessarily indicate that the imprinting process created specific
polymeric receptors for -fructose. Furthermore, very often the boronic acid moiety
D-fructose itself, as shown
D
shows preferential binding for fructose (44,45,61) and additional structural constrains
are needed to change this preference (62–67). Therefore, several control experiments
were conducted. First, the binding of the polymeric receptors with D-glucose was
also examined. It was found that the fluorescence intensity changes of the imprinted
polymers upon addition of glucose at the same concentrations were far smaller than
that of D-fructose (Figs. 3 and 4). Second, the effect of D-fructose and D-glucose on
the fluorescence intensity of the fluorescent monomer 1 at a concentration of 10Ϫ5
M, a concentration comparable to that of the fluorescent moiety in studying the
imprinted polymers, was also examined. It was found that the fluorescent monomer
1 also exhibited preferential recognition of D-fructose (Table 1), however, to a lesser
degree than that of the imprinted polymers (P-a and P-b). The ratio of (⌬I/I0)fru/(⌬I/
I0)glu, indicating this preference, was found to be about two-fold greater for the AIBN-
initiated polymer (P-a) than for the monomer 1 (Table 1 and Fig. 5). The polymer
prepared using ATRP showed comparable or greater preference for
1 and Fig. 5), the template molecule. Furthermore, the control polymer prepared in the
absence of -fructose showed minimal fluorescence intensity changes upon addition of
D-fructose (Table
D

314
GAO, WANG, AND WANG
FIG. 4. Fluorescence intensity changes of D-fructose-imprinted polymer (P-b) vs concentration of
sugars (␭ex, 370 nm; ␭em, 426 nm).
D-fructose (Figs. 3, 4, and 5). All of these indeed indicate that the imprinting process
helped in the creation of binding sites which preferentially recognized the template
molecules. Both the AIBN-initiated and atom transfer radical polymerizations can be
used for the creation of such imprinted polymers with specific recognition sites for
the template carbohydrates.
TABLE 1
Comparison of the Monomer 1 and the Imprinted Polymers in Their Preference for
over -Glucose
D-Fructose
D
(⌬I/I0)fru/(;gDI/I0)glu
Concentration
(mM)
Monomer 1
P-aa
P-bb
P-cc
1.0
5.0
10.0
50.0
100.0
7.72 Ϯ 0.54
4.16 Ϯ 0.64
2.75 Ϯ 0.47
1.43 Ϯ 0.24
1.25 Ϯ 0.07
ϱd
33.1 Ϯ 2.71
17.3 Ϯ 1.56
11.6 Ϯ 0.75
5.96 Ϯ 0.63
4.52 Ϯ 0.49
3.16 Ϯ 0.98
2.22 Ϯ 0.52
1.94 Ϯ 0.49
1.85 Ϯ 0.70
1.74 Ϯ 0.42
8.85 Ϯ 1.24
5.74 Ϯ 0.63
3.23 Ϯ 0.25
2.88 Ϯ 0.33
a The imprinted polymer prepared using AIBN initiated free radical polymerization.
b The imprinted polymer prepared using atom transfer radical polymerization (ATRP).
c Control polymer.
d No fluorescence intensity change at 1.0 mM of
D-glucose.

CARBOHYDRATES USING TEMPLATE-DIRECTED POLYMERIZATIONS
315
FIG. 5. The preference of the
D-fructose imprinted polymers and the monomer 1 for
D-fructose over
D-glucose (P-a, the imprinted polymer prepared using AIBN-initiated free radical polymerization; P-b,
the imprinted polymer prepared using ATRP; P-c, the control polymer).
CONCLUSION
Our studies demonstrated that (i) the fluorescent monomer (1) designed can indeed
be used for the preparation of fluorescent imprinted polymers, (ii) the imprinted
polymers created with both atom transfer radical polymerization and AIBN-initiated
free radical polymerization were able to preferentially recognize the template molecule,
and (iii) the imprinted polymer prepared with ATRP seems to have a better preference
for the template molecule than the fluorescent monomer and the polymer prepared
using AIBN-initiated free radical polymerization. The fluorescent monomer 1 devel-
oped 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.
EXPERIMENTAL
General. All 1H and 13C NMR spectra were recorded at 300 and 75 MHz, respec-
tively, with tetramethylsilane as the internal standard. Column chromatography was
performed using silica gel (200–400 mesh) from Aldrich and neutral activated Brock-
mann I aluminum oxide (ϳ150 mesh) from EM Science. Elemental analyses were
performed by Atlantic Microlab Inc. (Norcross, GA). Mass spectral analyses were
conducted by North Carolina State University Mass Spectrometry Laboratory. IR

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GAO, WANG, AND WANG
spectra were recorded on a Perkin-Elmer 1600 series spectrometer using KBr as
dispersant. Commercially available starting materials and reagents were purchased
from Aldrich. Tetrahydrofuran (THF) was distilled from Na and benzophenone. Aceto-
nitrile (CH3CN) and dichloromethane (CH2Cl2) were distilled from CaH2. All pH
values were determined with an Accumet 1003 handheld pH/mV/ion meter (Fisher
Scientific). A Shimadzu RF-5301 PC fluorometer was used for the fluorescent studies.
The excitation wavelength was set at 370 nm. An INSTECT Model 1060 stirrer was
used for the stirring of the samples during the fluorescent studies and a Thermolyn
vortex mixer (Type 16700) was used for equilibrating the polymer–analyte mixtures.
[10-(tert-Butyldimethylsilanyloxymethyl)-anthracen-9-ylmethyl]-methylamine
(3). Compound 2 (46) (2.0 g, 8.47 mmol), TBSCl (1.66 g, 11.0 mmol, 1.3 equiv.),
and imidazole (0.75 g, 11.0 mmol, 1.3 equiv.) were dissolved in anhydrous DMF
(40 mL) and stirred for 14 h. After solvent removal under reduced pressure, the
residue was dissolved in ethyl acetate (300 mL) and then washed with water (100
mL), 0.5 N of hydrochloric acid (100 mL), and saturated brine (100 mL). The organic
layer was separated and dried over anhydrous MgSO4 and then evaporated to give a
yellow solid. The crude product was purified by silica gel chromatography using
ethyl acetate/hexanes (1:50) as the eluent to give 2.71 g (91%) of the TBDMS
1
protected aldehyde as a yellow solid. H NMR (CDCl3) 0.14 (s, 6H), 0.90 (s, 9H),
5.64 (s, 2H), 7.59–7.67 (m, 4H), 8.48 (d, 2H, J ϭ 8.6 Hz), 8.89 (d, 2H, J ϭ 8.6 Hz),
11.49 (s, 1H). 13C NMR (CDCl3) Ϫ4.79, 18.57, 26.08, 58.15, 124.16, 125.49, 126.13,
126.42, 128.36, 129.96, 131.64, 139.78, 193.89. FAB-MS m/z: 350.17 (M+). IR (KBr,
cmϪ1): 1670. Anal. Calcd for C22H26O2Si: C, 75.38; H, 7.48. Found: C, 75.08; H, 7.51.
The TBDMS-protected aldehyde (2.10 g, 6.00 mmol) obtained in the previous
reaction was dissolved in a 2 M solution of methylamine in methanol (80 mL) and
the resulting mixture was stirred at RT for 6 h. Then 0.684 g (18.0 mmol, 3.0 equiv.)
of sodium borohydride was added and the reaction mixture was stirred at RT for 2 h.
Then the solvent was removed under reduced pressure and the residue was dissolved
in ethyl acetate (300 mL). The organic solution was washed with water (3 ϫ 200
mL) and dried over anhydrous MgSO4. Solvent evaporation gave a yellow solid,
which was purified on a silica gel column (methylene chloride then methanol as the
1
solvents) to afford 1.95 g (89%) of 3 as a yellow solid. H-NMR (CDCl3) 0.12 (s,
6H), 0.91 (s, 9H), 2.67 (s, 3H), 4.69 (s, 2H), 5.65 (s, 2H), 7.51–7.54 (m, 4H),
8.37–8.42 (m, 4H). 13C-NMR (CDCl3) Ϫ4.78, 18.60, 26.15, 37.23, 48.12, 58.20,
124.81, 125.44, 125.50, 125.76, 130.36, 132.15. FAB-MS m/z: 365.3 (M+). Anal.
Calcd for C23H31NOSi: C, 75.56; H, 8.55; N, 3.83. Found: C, 75.54; H, 8.58; N, 3.82.
[10-(tert-Butyldimethyl-silanyloxymethyl)-anthracen-9-ylmethyl]-[2-(5,5-
dimethyl-[1,3,2]dioxaborinan-2-yl)-benzyl]-methylamine (4). Compound 3 (0.20 g,
0.548 mmol), 0.186 g (0.657 mmol, 1.2 equiv.) of 2-(2-bromomethyl-phenyl)-5,5-
dimethyl-[1,3,2]dioxaborinane (6) (44), and 0.091 g (0.657 mmol, 1.2 equiv.) of
potassium carbonate were gently refluxed in dry acetonitrile (50 mL) under argon
atmosphere for 20 h. The insoluble material was filtered off and the filtrate was
evaporated to give a yellow oil, which solidified when treated with ethyl acetate. The
solid was collected by filtration. Recrystallization of the solid with CH2Cl2/EtOAc
1
(1:1) gave 130 mg (42%) of 4 as a yellow crystalline solid. H NMR (CDCl3) 0.15
(s, 6H), 0.89 (s, 6H), 0.91 (s, 9H), 2.74 (d, 3H, J ϭ 5.0 Hz), 3.52 (d, 2H, J ϭ 10.8

CARBOHYDRATES USING TEMPLATE-DIRECTED POLYMERIZATIONS
317
Hz), 3.67 (d, 2H, J ϭ 10.8 Hz), 4.61 (m, 1H), 5.15 (m, 1H), 5.46 (m, 1H), 5.67
(s, 2H), 5.76 (m, 1H), 7.43–7.67 (m, 7H), 7.97 (t, 2H, J ϭ 8.1 Hz), 8.49 (d, 3H,
J ϭ 8.7 Hz). 13C NMR (CDCl3) Ϫ4.7, 18.2, 21.8, 26.1, 31.7, 40.2, 50.5, 58.2, 60.0,
72.5, 121.0, 124.3, 124.6, 125.9, 126.1, 127.9, 129.6, 130.2, 131.6, 131.8, 133.3,
134.7, 136.2, 136.7. FAB-HRMS m/z Calcd for C35H46BNO3Si (M+ ϩ H): 568.3418.
Found: 568.3451.
9-Hydroxymethyl-10-[[N-methyl-N-(o-boronobenzyl)amino]methyl]anthracene
(5). To a solution of compound 4 (1.0 g, 2.00 mmol) in dry THF (20 mL) was added
tetrabutylammonium fluoride (1.57 g, 6.00 mmol, 3.0 equiv.). After stirring at RT
for 3 h, the solvent was removed and the residue was dissolved in methylene chloride
(100 mL), washed with water (3 ϫ 100 mL), and dried over anhydrous MgSO4.
Solvent evaporation gave a yellow oil, which was purified on a neutral aluminum
oxide (ϳ150 mesh) column with CH2Cl2/MeOH (100:1) as the solvent to give 0.57
1
g (74%) of 5 as a slightly yellow solid. H-NMR (CD3OD) 2.28 (s, 3H), 4.30
(s, 2H), 4.83 (s, 2H), 5.44 (s, 2H), 7.27–7.73 (m, 8H), 8.06 (d, 2H, J ϭ 8.5 Hz),
8.43 (d, 2H, J ϭ 8.4 Hz). 13C-NMR (CD3OD) 40.43, 50.15, 57.25, 64.45, 125.29,
125.59, 126.56, 126.98, 127.83, 128.10, 129.06, 131.38, 131.66, 132.75, 135.44,
136.14. IR (KBr, cmϪ1) 3436, 1444, 1341. Anal. Calcd for C24H24BNO3 и 1/2 H2O:
C, 73.11; H, 6.39; N, 3.55. Found: C, 72.63; H, 6.17; N, 3.71.
9-Hydroxymethyl-10-[[N-methyl-N-(o-boronobenzyl)amino]methyl]anthracene-9-
methacrylate (1). Compound 5 (340 mg, 0.883 mmol), 408 mg (2.64 mmol, 3.0
equiv.) of methacrylic anhydride, and 323 mg (2.64 mmol, 3.0 equiv.) of DMAP
(dimethylaminopyridine) were dissolved in dry methylene chloride (50 mL). The
reaction mixture was stirred at RT for 20 h. Then the reaction mixture was washed
with 5% aqueous solution of sodium bicarbonate (80 mL), water (2 ϫ 100 mL), and
saturated brine (100 mL) and dried over anhydrous MgSO4. Then solvent evaporation
gave a yellow oil, which was purified on a neutral aluminum oxide (ϳ150 mesh)
column with CH2Cl2/MeOH (100:1) to give 280 mg (70%) of 1 as a yellow solid.
1H NMR(CD3OD) 1.86 (s, 3H), 2.36 (s, 3H), 4.30 (s, 2H), 4.90 (s, 2H), 5.53 (s, 1H),
5.97 (s, 1H), 6.11 (s, 2H), 7.26–7.37 (m, 3H), 7.50–7.60 (m, 4H), 7.68 (m, 1H), 8.18
(d, 2H, J ϭ 8.4 Hz), 8.38 (d, 2H, J ϭ 8.4 Hz). 13C NMR (CD3OD) 18.51, 42.25,
50.98, 65.80, 124.28, 124.74, 124.84, 125.48, 125.64, 126.26, 127.63, 127.79, 127.96,
129.94, 130.13, 130.65, 130.94, 131.16, 136.04, 136.25, 141.44, 167.72. ESI-MS
(MeOH) m/z: 482.2 (M+ Ϫ 2 H2O ϩ 2 CH3OH ϩ H). IR (KBr, cmϪ1) 3433, 1717,
1447, 1292, 1150. Anal. Calcd for C28H28BNO4: C, 74.18; H, 6.23; N, 3.09. Found:
C, 74.17; H, 6.65; N, 3.72.
Preparation of the
D-fructose–boronate complex (8). A mixture of D-fructose
(18 mg, 0.1 mmol) and boronic acid monomer 1 (91 mg, 0.2 mmol) in 9.0 mL of
dry dioxane and 1.0 mL of dry pyridine was stirred at 45–50ЊC for 26 h and then
65–70ЊC for 1 h. Then the solvents and water were removed under reduced pressure
during 1.5 h to afford a yellow oil. The residue was further dried under an oil pump
overnight. The complex was used directly without purification for the next step
polymerization. FAB-MS m/z: 1015 (M+ ϩ H).
Preparation of the
polymerization (P-a). To a solution of the
0.1 mmol) in 2.5 mL of dry THF and 1.5 mL of CH3CN were added HEMA (42 mg,
D-fructose imprinted polymer using AIBN-initiated free radical
D-fructose–boronate complex 8 (101 mg,

318
GAO, WANG, AND WANG
0.4 mmol) and EGDM (980 mg, 5.0 mmol). The mixture was purged with argon for
5 min, and then AIBN was added. The reaction flask was capped with a glass stopper
and then placed in an oil bath. The polymerization was carried out at 65ЊC with
stirring for 45 h and then 90ЊC for 4 h. The rigid polymer was ground and then
washed with MeOH (2 ϫ 35 mL) for 1.5–2 h, 0.1 N HCl aqueous solution with
MeOH (1/1, v/v) (3 ϫ 35 mL) for 3–5 h, and MeOH (10 ϫ 35 mL) for 22–24 h
with a fritted glass filter. The washed polymer was dried at 35ЊC under reduced
pressure overnight to afford 880 mg (79%) of the product. The polymer particles
were sieved with a 100-mesh sieve. The polymer particles smaller than 100 mesh
were used for the binding studies.
Preparation of the D-fructose imprinted polymer using atom transfer radical poly-
merization (P-b). Copper chloride (CuCl) (12 mg, 0.12 mmol) and 2,2Ј- bypyridine
(bpy) (39 mg, 0.24 mmol) were mixed in a round flask, it was degassed by vacuum
for 1 h and back filled argon for three times. To this flask was added the solution of
HEMA (3.12 g, 24 mmol), EGDM (4.75 g, 24 mmol) and the D-fructose–boronate
complex 8 (122 mg, 0.12 mmol) in 5 mL of anhydrous DMF, which was degassed
previously by bubbling argon for 1 h. Then 72 ␮L of ethyl 2-bromoisobutyrate (BriB)
was added, the polymerization was proceeded at 50ЊC for 12 h. The polymer formed
was ground to fine particles and then washed with MeOH (2 ϫ 50 mL) for 1.5–2 h,
0.1 N HCl aqueous solution with MeOH (1/1, v/v) (3 ϫ 50 mL) for 3–5 h, and
MeOH (10 ϫ 50 mL) for 22–24 h with a fritted glass filter. The washed polymer
particles were dried at 35ЊC under reduced pressure overnight to afford 6.52 g (82%)
of the product. The polymer particles were sieved with a 100-mesh sieve. The polymer
particles smaller than 100 mesh were used for the binding studies.
Preparation of the control polymer. The control polymer was prepared following
the same procedure for the preparation of the
absence of the imprinted molecule, -fructose.
D-fructose-imprinted polymers in the
D
Fluorescent binding studies. For each fluorescence measurement, 10 mg of the
polymer particles was added to a 15-mL test tube, followed by the addition of 4.0 mL
of D-fructose solution at different concentrations in 50% MeOH/H2O (v/v) phosphate
(0.05 M) buffer at pH 7.4. The suspension was mixed by using a Thermolyn vortex
mixer for 3 h. Then about 3.5 mL of the suspension was transferred into a cuvette
for fluorescence measurement. The emission spectra were recorded from 380 to 550
nm immediately after the solution was stirred with an INSTECT stirrer for 30 s. The
excitation wavelength was set at 370 nm with both the excitation and the emission
slits of 1.5 nm.
ACKNOWLEDGMENTS
Financial support from the National Institutes of Health (DK55062 and CA88343) is gratefully acknowl-
edged. Wei Wang acknowledges a Glaxo graduate fellowship.
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