Selectivity control by chemical modification of the recognition sites in two-point binding molecularly imprinted polymer

Article abstract of DOI:10.1021/ma0496089

We demonstrated the possibility of modifying the selectivity of a two-point binding imprinted polymer by chemical modification of the binding sites inside the cavities. We used a thermally reversible bond for the preparation of the monomer-template complex, which allowed us to remove the template easily by means of a simple thermal reaction and to simultaneously introduce various functional groups into the cavity. A phenylmaleimide having an azidocarbonyl group was reacted with diethylstilbestrol (DES, template) to yield a monomer, where the template was linked to two polymerizable maleimido groups via a thermally reversible urethane bond. The polymerization of the monomer was carried out in the presence of ethylene glycol dimethacrylate (EGDMA) by the initiation with 2,2-azobis(isobutyronitrile) (AIBN) at 54°C in DMF. The polymers were refluxed in 1,4-dioxane in the presence of a nucleophile such as water, methanol, or aniline. In this extraction step, the template molecules were removed from the polymer matrix, and simultaneously the isocyanato groups, which were generated by the thermal cleavage of the urethane bond, were converted to amino, urethane, or urea groups through their reaction with water, methanol, or aniline, respectively. The specific recognition ability of the imprinted polymers for the template and its structural analogues was dependent on the space between the two binding points as well as on the nature of the functional group. This method is especially propitious for developing artificial receptors for molecules lacking strongly interactive groups.

Full text of DOI:10.1021/ma0496089

5544
Macromolecules 2004, 37, 5544-5549
Selectivity Control by Chemical Modification of the Recognition Sites in
Two-Point Binding Molecularly Imprinted Polymer
Ka n gw on Lee,† Ch a n g Do Ki,† Ha su ck Kim ,‡ a n d J i You n g Ch a n g*,†
Hyperstructured Organic Materials Research Center, School of Materials Science and Engineering,
and School of Chemistry, Seoul National University, Seoul 151-744, Korea
Received February 27, 2004; Revised Manuscript Received May 13, 2004
ABSTRACT: We demonstrated the possibility of modifying the selectivity of a two-point binding imprinted
polymer by chemical modification of the binding sites inside the cavities. We used a thermally reversible
bond for the preparation of the monomer-template complex, which allowed us to remove the template
easily by means of a simple thermal reaction and to simultaneously introduce various functional groups
into the cavity. A phenylmaleimide having an azidocarbonyl group was reacted with diethylstilbestrol
(DES, template) to yield a monomer, where the template was linked to two polymerizable maleimido
groups via a thermally reversible urethane bond. The polymerization of the monomer was carried out in
the presence of ethylene glycol dimethacrylate (EGDMA) by the initiation with 2,2-azobis(isobutyronitrile)
(AIBN) at 54 °C in DMF. The polymers were refluxed in 1,4-dioxane in the presence of a nucleophile
such as water, methanol, or aniline. In this extraction step, the template molecules were removed from
the polymer matrix, and simultaneously the isocyanato groups, which were generated by the thermal
cleavage of the urethane bond, were converted to amino, urethane, or urea groups through their reaction
with water, methanol, or aniline, respectively. The specific recognition ability of the imprinted polymers
for the template and its structural analogues was dependent on the space between the two binding points
as well as on the nature of the functional group. This method is especially propitious for developing
artificial receptors for molecules lacking strongly interactive groups.
In tr od u ction
reported of imprinted polymers recognizing molecules
other than the template.8 Whitcombe and co-workers
prepared an imprinted polymer showing high selectivity
for a molecule that could not normally form a stable
complex with a monomer.8a-c They used a functionalized
template which was structurally analogous to this
molecule. The template was linked to a monomer
through a sacrificial spacer. After polymerization, the
template and the spacer were removed to create a
recognition site for the poorly functionalized molecule.
Recently, Zimmerman and co-workers reported on a
monomolecularly imprinted dendrimer which showed
better selectivity for its structural analogues than the
porphyrin imprinted originally.8d
Several studies have shown that the major factors
governing the recognition ability of an imprinted poly-
mer are the shape of the cavities and the orientation of
the functional groups situated inside them, with the
latter considered to be predominant.1a For this reason,
multipoint binding imprinted polymer shows superior
performance to the one-point binding one. The arrange-
ment of the functional groups inside the cavities is
determined by the formation of a template-monomer
complex and its subsequent polymerization. Since the
functional groups generated by the removal of the
template are directly attached to the polymer matrix,
chemical modification under mild conditions does not
change their orientation significantly, but it does have
an effect on the amount of space available inside the
cavities.
Molecular imprinting constitutes a valuable method
of preparing polymeric materials with specific binding
properties, which have potential uses in applications
such as chemical sensors, microreactors mimicking
enzymes, stationary phases for high-performance chro-
matography, catalysts, and membranes for separating
toxic chemicals.1-7 The most conspicuous merit of mo-
lecular imprinting is that structurally three-dimensional
recognition sites can be introduced into a polymer
matrix with ease and low cost when compared with the
complicated process of biological system for antigen and
antibody. In the molecular imprinting process, a target
molecule (template) is first complexed with a functional
monomer and then frozen into a matrix by polymeriza-
tion. The formation of a stable complex before polym-
erization is crucial for the imprinted polymer to have
high rebinding ability. So most templates reported so
far have functional groups that react or interact strongly
with a monomer.
In this work, we demonstrated the possibility of
modifying the selectivity of an imprinted polymer by
chemical modification of the binding sites inside the
cavities. We used a thermally reversible urethane bond
for the preparation of the monomer-template complex,
which allowed us to remove the template easily by
means of a simple thermal reaction and to simulta-
neously introduce various functional groups into the
cavity. This method is especially propitious for develop-
ing artificial receptors for molecules lacking strongly
interactive groups. Only a few examples have been
It is known that the urethane bond formed between
an isocyanate and a phenol is stable at room tempera-
ture, but the reversible cleavage occurs at elevated
temperatures.9-12 Diethylstilbestrol (DES) having two
terminal phenol groups was chosen as a template. A
monomer with an isocyanato group can be attached to
DES on both sides by means of a thermally reversible
† Hyperstructured Organic Materials Research Center, School
of Materials Science and Engineering.
‡ School of Chemistry.
* Corresponding author: Tel +82-2-880-7190, Fax +82-2-885-
1748, e-mail jichang@snu.ac.kr.
10.1021/ma0496089 CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/23/2004

Macromolecules, Vol. 37, No. 15, 2004
Two-Point Binding Molecularly Imprinted Polymer 5545
Sch em e 1
Sch em e 2
urethane bond. The resulting monomer-template com-
plex presents two point binding sites13 into the polymer
matrix after extraction of template molecules. Besides,
the choice of DES as a template was motivated by
environmental necessity of its detection. DES is a
synthetic nonsteroidal estrogen, which is known for its
ability to treat prostate cancer but which is suspected
of being an endocrine disrupting chemical.14
Resu lts a n d Discu ssion
Mon om er Syn th esis. Monomer 2 was prepared
according to Scheme 1. Phenylmaleimide 1 having an
azidocarbonyl group was converted to an isocyanate
through Curtuis rearrangement by heating at 110 °C
in toluene. Diethylstilbestrol was reacted with the
isocyanate to yield monomer 2, where the template was
linked to two polymerizable groups via a thermally
reversible urethane bond. The thermal cleavage of the
1
urethane bond was investigated by H NMR spectros-
copy. Monomer 2 was dissolved in DMSO-d6 containing
moisture, and its 1H NMR spectra were measured at
various temperatures (Figure 1). The aromatic ring
proton peaks appeared at 7.64, 7.31, and 7.29 ppm and
the NH peak at 10.40 ppm at room temperature. The
vinyl proton peak of a maleimido group showed up at
7.17 ppm. After increasing the sample temperature to
110 °C, the peak corresponding to the urethane group
at 10.40 ppm completely disappeared, and the aromatic
ring proton peaks corresponding to free DES appeared
at 6.75 and 6.98 ppm. A peak corresponding to an amino
group also showed up at 4.9 ppm, resulting from the
reaction of an isocyanato group with the water present
in DMSO-d6 (Scheme 2).
Syn th esis of Im p r in ted P olym er . The polymeri-
zation of monomer 2 was carried out in the presence of
ethylene glycol dimethacrylate (EGDMA)15 by the ini-
tiation with 2,2-azobis(isobutyronitrile) (AIBN) at 54 °C
in DMF (Scheme 3).
To remove the template molecules from the polymer
matrix, the polymer was refluxed in 1,4-dioxane in the
presence of a nucleophile such as water, methanol, or
aniline. In this extraction step, the template molecules
were removed from the polymer matrix, and simulta-
neously the isocyanato groups, which were generated
by the thermal cleavage of the urethane bond, were
converted to amino, urethane, or urea groups through
their reaction with water, methanol, or aniline, respec-
tively. Extraction was monitored by UV-vis spectros-
copy and continued until the absorption intensity for
dissociated DES in the solvent reached the constant
(Figure 2). The control polymer was also prepared
following the same procedure as that used for the
imprinted polymers, except that N-(4-acylazidophenyl)-
maleimide (1) was used as a monomer in place of
compound 2.
F igu r e 1. 1H NMR spectra (DMSO-d6 + moisture) of com-
pound 2 obtained (a) at 25 °C and (b) at 110 °C.

5546 Lee et al.
Macromolecules, Vol. 37, No. 15, 2004
Sch em e 3
Solid -Sta te 13C CP -MAS NMR An a lysis. The re-
Recogn ition Test for DES. The ability of polymer
4 to recognize the template was investigated. In the
rebinding test, polymer particles were added to the
solution of the template in 10% (v/v) tetrahydrofuran-
chloroform at various concentrations. Imprinted poly-
mer 4 had two binding points in a cavity. The amount
of an analyte in a sample solution was decided on the
basis of the capacity of imprinted polymers, calculated
theoretically.16 After incubating for 24 h at room tem-
perature, the polymer particles were isolated by filtra-
tion. The amount of template absorbed by the polymer
was determined by measuring the residual analyte in
the filtrate by reverse phase HPLC. Figure 4 shows the
amount of template (DES) bound to polymer 4 and to
the control polymer according to the sample concentra-
tion. The imprinted polymer exhibited a much higher
recognition ability than the control polymer. The supe-
rior rebinding ability of the imprinted polymer, com-
pared to the control polymer, proved that the target
molecules were not simply adsorbed at the polymer
surface but were trapped in the cavities through hy-
drogen bonding.
moval of the template and the resultant generation of
the recognition site were investigated by solid-state 13
C
NMR spectroscopy. In Figure 3, all of the strong peaks
were assigned to the carbons of the cross-linker, by
comparing them with the spectrum obtained from the
polymerization of EGDMA only. After extraction in the
presence of water, the peak at 153 ppm corresponding
to the carbonyl carbon of the urethane group disap-
peared along with the peaks at 14 and 30 ppm for the
CH3 and CH2 groups of the DES moieties, respectively,
indicating that most of the template molecules were
removed (Figure 3b). This spectrum coincided exactly
with the spectrum of the control polymer. Figure 3c
shows the spectrum of the imprinted polymer prepared
by the extraction in the presence of methanol. The
carbonyl group peak of the newly formed urethane bond
and the methoxy carbon peak appeared at 154 and 52
ppm, respectively. In the spectrum of the imprinted
polymer obtained by the reaction with aniline (Figure
3d), the peak for the urea carbonyl group showed up at
154 ppm, and the intensities of the phenyl carbon peaks
increased.
Selectivity Test. We also investigated the specific
recognition ability of the imprinted polymers for the
template and its structural analogues (Figure 5). Mo-
lecular modeling revealed that the space between the
binding points in the cavity decreased in the order of
an amino, a methylurethane, and a phenylurea group,
while the relative direction of the two functional groups
in the cavity was essentially unchanged. The rebinding
test was carried out in the same manner as described
above. The polymers were incubated into a solution (1
mM) of an analyte in 10% (v/v) tetrahydrofuran-
chloroform. Except for A6 and A7 in Figure 5, all of the
analogues possessed functional groups capable of hy-
drogen bonding. As expected, the imprinted polymer (4),
containing amino groups inside the cavities, showed the
highest recognition ability for the template (DES) and
the lowest recognition ability for A6 and A7. The lower
response for A1-A5 was attributed to their smaller
sizes than that of the template. They would plausibly
F igu r e 2. Extraction of template molecule from DES-bound
polymer monitored by UV-vis spectroscopy (λmax ) 243 nm).

Macromolecules, Vol. 37, No. 15, 2004
Two-Point Binding Molecularly Imprinted Polymer 5547
F igu r e 3. Solid-state 13C NMR spectra of (a) polymer 3 before removal of the template molecules, (b) polymer 4, (c) polymer 5,
and (d) polymer 6. The asterisk (/) denotes a spinning sideband.
template. Since A2 had two hydroxyl groups as the
template, the selectivity change was likely due to the
space contraction between the two binding points. More
striking results came from polymer 6, wherein phenyl-
urea groups were introduced into the cavity. This
polymer showed the highest recognition ability for trans-
stilbene (A7), which contains two phenyl groups. The
π-π interaction between the phenyl urea group and
trans-stilbene seemed to play a key role, leading to a
two-point binding.17 Molecular modeling showed that in
the cavity of the polymer the urea group was screened
by the bulky phenyl group, and accordingly, its ability
of hydrogen bonding with an analyte would be ob-
structed.
F igu r e 4. Amount of bound DES (a) by polymer 4 and (b) by
the control polymer according to the sample concentration.
Con clu sion s
We demonstrated how to control the selectivity of two-
enter into the cavity with little steric hindrance but
would not be able to form stable two-point binding.
Our major concern in this test was to determine
whether the selectivity of the imprinted polymers could
be controlled by tailoring the nature of the functional
groups as well as by adjusting the space between them
in the cavity. In this regard, Figure 6 clearly shows that
the approach we were taking was on target. When
methylurethane groups were introduced into the cavity,
the imprinted polymer (5) showed the highest affinity
for 4,4′-biphenol (A2), which has a smaller size than the
point binding imprinted polymers. In our approach,
after molding the overall shape of the imprint cavity
by means of the template molecule, the affinity of the
cavity was tailored by adjusting the space between the
two binding points as well as by changing the nature of
the functional group. We utilized a thermally reversible
urethane bond in the template-monomer complexation,
which allowed us to readily introduce various functional
groups into the cavity, during the process of template
removal after polymerization. The rebinding test results
were very encouraging for the development of an
F igu r e 5. Template molecule (DES) and its analogues.

5548 Lee et al.
Macromolecules, Vol. 37, No. 15, 2004
DMSO-d6) δ (ppm): 10.40 (s, 2H, NH), 7.64, 7.31 (dd, 8H, J ≈
9.0, 99 Hz, phenyl protons), 7.29 (s, 8H, phenyl protons), 7.17
1
(s, 4H, vinyl protons), 2.16 (q, 4H, CH2), 0.78 (t, 6H, CH3). H
NMR (300 MHz, THF-d8) δ (ppm): 9.39 (s, 2H, NH), 7.61, 7.32
(dd, 8H, J ≈ 8.9, 87 Hz, phenyl protons), 7.23 (s, 8H, phenyl
protons), 6.91 (s, 4H, vinyl protons), 2.20 (q, 4H, CH2), 0.81 (t,
6H, CH3). 13C NMR (500 MHz, THF-d8) δ (ppm): 169.74 (Cd
O of maleimide), 151.79 (CdO of urethane), 151.20, 139.56,
139.32, 138.63, 134.47, 129.71, 127.29, 126.83, 121.45, 118.57,
28.20 (CH2 in DES), 12.18 (CH3 in DES). Anal. Calcd for
C
40H32N4O8: C, 68.96; H, 4.63; N, 8.04. Found: C, 69.24; H,
4.85; N, 7.78.
P r ep a r a tion of Im p r in ted P olym er 4. A mixture of
monomer 2 (1.50 g, 2.15 mmol), EGDMA (13.60 g, 68.61 mmol),
and DMF (20 mL) was charged to a polymerization tube (50
mL), and AIBN (0.17 g, 0.73 mol % with respect to polymer-
izable double bonds) was added. After three freeze-thaw cycles
under N2, the tube was sealed and placed in an oil bath at 54
°C for 24 h. The cross-linked polymer was washed with THF
and dried in vacuo at room temperature for 48 h. The polymer
(3) was ground with a mechanical mortar and pestle. The
polymer particles were refluxed in 1,4-dioxane/water (7/1 v/v).
The process of extraction was monitored by UV spectroscopy.
The absorption intensity at 243 nm for the dissociated DES
in the solution reached a constant after 24 h.19 The polymer
particles were isolated by filtration, washed with 1,4-dioxane,
THF, methylene chloride, and acetone, and dried in vacuo at
room temperature for a week.
P r ep a r a tion of Con tr ol P olym er . The control polymer
was synthesized in the same manner for the preparation of
the imprinted polymer (4), except that N-(4-azidocarbonyl-
phenyl)maleimide (1) was used instead of monomer 2. Azido
groups in the polymers were converted to amino groups when
the polymers were refluxed in 1,4-dioxane/water (7/1 v/v).
P r ep a r a tion of Im p r in ted P olym er 5. DES bound poly-
mer 3 (3.00 g) was added to a solution of 1,4-dioxane (50 mL)
and methanol (10 mL). The mixture was refluxed for 24 h. The
resulting mixture was isolated by filtration, washed with 1,4-
dioxane, THF, methylene chloride, and acetone, and dried in
vacuo at room temperature for a week.
P r ep a r a tion of Im p r in ted P olym er 6. DES bound poly-
mer 3 (3.00 g) was added to a solution of 1,4-dioxane (50 mL)
and aniline (10 mL). The mixture was refluxed for 24 h. The
polymer was isolated by filtration, washed with DMSO, DMF,
1,4-dioxane, THF, methylene chloride, and acetone, and dried
in vacuo at room temperature for a week.
Rebin d in g Test for DES. Imprinted polymer 4 (0.1 g) or
the control polymer was added to a solution of DES in 10%
(v/v) tetrahydrofuran-chloroform (7.4 mL) at various concen-
trations (1, 2, and 3 mM). After incubating for 24 h at 25 °C,
the polymer particles were isolated by filtration and washed
with chloroform and THF. The filtrate was concentrated to
dryness by evaporation of the solvent before HPLC analysis.
Selectivity Test. An imprinted polymer or the control
polymer (0.1 g) was added to a solution (1 mM) of an analyte
listed in Figure 5 in 10% (v/v) tetrahydrofuran-chloroform (7.4
mL). After incubating for 24 h at 25 °C, the polymer particles
were isolated by filtration and washed with chloroform and
THF. The filtrate was concentrated to dryness by evaporation
of the solvent before HPLC analysis.
F igu r e 6. Biding selectivity of polymers 4-6 and the control
polymer. In all experiments, 0.1 g of the polymers was added
to 7.4 mL of the sample solutions (1 mM) in chloroform/THF
(9/1 v/v).
artificial receptor for an unfunctionalized molecule.
Further studies are in progress along these lines.
Exp er im en ta l Section
Ma ter ia ls a n d Meth od s. Maleic anhydride, p-aminoben-
zoic acid, sodium acetate, sodium azide, 2,2-azobis(isobu-
tyronitrile) (AIBN), dibutyltin dilaurate (DBTDL), and ethyl-
ene glycol dimethacrylate (EGDMA) were obtained from
Aldrich. AIBN was purified by recrystallization from methanol.
EGDMA was dissolved in diethyl ether, washed with 1 M
aqueous NaOH three times, and dried over anhydrous MgSO4.
After filtration, it was purified by distillation under reduced
pressure. (E)-Diethylstilbestrol (DES) was purchased from
Sigma. Bisphenol A, 4,4-biphenol, 4-hydroxybenzoic acid,
4-aminobenzoic acid, hydroquinone, p-xylene, and trans-stil-
bene were obtained from Aldrich. Acetic anhydride, toluene,
and triethylamine were purchased from J unsei Chemical
(J apan). Chloroform, methanol, water, and tetrahydrofuran
for HPLC analysis were purchased from J .T. Baker.
The 1H and 13C NMR spectra were obtained on Bruker
Avance DPX-300 (300 MHz) spectrometer and Bruker Avance
DPX-500 (500 MHz). FT-IR spectra were recorded on a Perkin-
Elmer Spectrum 2000 equipped with a temperature controller.
The solid-state 13C NMR spectra were obtained on Unity/
Inova200 solid-state NMR 50 MHz spectrometer equipped with
a CP-MAS probe (Varian). Samples were spun in air at
approximately 5 kHz. Extraction of template molecules was
monitored by a SCINCO S-3150 UV-vis spectrophotometer.
Reverse phase HPLC analysis was carried out using a M930
solvent delivery system, a M720 UV-vis detector (YOUNG
LIN Instrument Co., Ltd., Korea), a MetaSil 5u ODS column
from Metachem (Torrance, Canada) with methanol for DES,
bisphenol A, 4,4-biphenol, hydroquinone, p-xylene, and trans-
stilbene, or water/methanol for 4-hydroxybenzoic acid and
4-aminobenzoic acid, as an eluent at a rate of 1.0 mL/min at
room temperature. For each analysis 20 µL of sample was
injected.
N-[4-(N′-Diet h ylst ilb est r oxyca r b on yla m in o)p h en yl]-
m a leim id e (2). Toluene was dried by azeotropic distillation
with a Dean-Stark trap for 24 h. N-(4-Azidocarbonylphenyl)-
maleimide (1) (3.00 g, 12.39 mmol), prepared following the
procedures in the literature,18 was dissolved in toluene (70
mL). After refluxing for 3 h, diethylstilbestrol (DES 1.67 g,
6.22 mmol) and dibutyltin dilaurate (DBTDL, 1 mL) were
added to the solution. The solution was refluxed for 5 h. The
resulting precipitates were isolated by filtration and purified
by column chromatography on silica gel (ethyl acetate:hexane
) 5:4 v/v). Yield: 1.63 g (38%).
Ack n ow led gm en t. This work was supported by the
HOMRC and MOST (National R&D Project).
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(16) The capacity of an imprinted polymer was calculated on the
assumption that all monomer-template complexes produced
the imprinted sites. For example, capacity of imprinted
polymer 4 ) [sample polymer (g)] ÷ (mol wt of 4-aminophen-
ylmaleimide × 2 + mol wt of EGDMA × 32).
(17) (a) Dickert, F. L.; Besenbo¨ck, H.; Tortschanoff, M. Adv. Mater.
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(18) (a) Oishi, T.; Fujimoto, M. J . Polym. Sci., Polym. Chem. Ed.
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(19) Duffy, D. J .; Das, K.; Hsu, S. L.; Penelle, J .; Rotello, V. M.;
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