Molecularly imprinted polymers with signaling function based on the UV-Vis spectral change by diastereoselective binding events

Article abstract of DOI:10.1246/bcsj.78.356

Signaling cinchonidine-imprinted polymers were prepared using a polymerizable iron(III) porphyrin and/or methacrylic acid as functional monomer(s), and evaluated by chromatographic tests, Scatchard analysis, and spectroscopic analysis. An imprinted polyme

Full text of DOI:10.1246/bcsj.78.356

356 Bull. Chem. Soc. Jpn., 78, 356–360 (2005)
ꢀ 2005 The Chemical Society of Japan
Molecularly Imprinted Polymers with Signaling Function Based on
the UV–Vis Spectral Change by Diastereoselective Binding Events
ꢀ;1;2
Toshifumi Takeuchi,
Akinobu Seko,3 and Takashi Mukawa1
1Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501
2PRESTO, Japan Science and Technology Agency (JST), Kawaguchi 332-0012
3Graduate School of Information Sciences, Hiroshima City University, Asaminami-ku, Hiroshima 731-3194
Received July 20, 2004; E-mail: takeuchi@scitec.kobe-u.ac.jp
Signaling cinchonidine-imprinted polymers were prepared using a polymerizable iron(III) porphyrin and/or meth-
acrylic acid as functional monomer(s), and evaluated by chromatographic tests, Scatchard analysis, and spectroscopic
analysis. An imprinted polymer prepared with an equimolar amount of the iron(III) porphyrin monomer to cinchonidine
and six equivalents of methacrylic acid gave the strongest binding (Ka ¼ 5:7 ꢁ 105
M
ꢂ1) and highest selectivity
(ꢀ ¼ 14:6) for cinchonidine against its diastereomer, cinchonine. Thus, it was concluded that the iron(III) porphyrin
and methacrylic acid residues worked cooperatively at the imprinted binding site. There is also the added advantage that
the porphyrin provides a signaling function proportional to the chiral binding event of cinchonidine. A linear relationship
was observed between the UV–vis absorbance of the imprinted polymer and the logarithm of cinchonidine concentra-
tions, indicating that the polymer may be used as a selective cinchonidine sensing material. Moreover, various metal-
loporphyrins can be used as functionalized monomers in order to prepare functional MIPs for a wide range of target
compounds capable of coordination with such metalloporphyrins, even if the target compounds have no characteristic
UV–vis absorption or fluorescence.
Molecularly imprinted polymers (MIPs) are attractive mate-
rials due to the elimination of complicated molecular designs,
ease of syntheses, effective molecular recognition, and high se-
lectivity for target molecules.1–7 MIPs are formed by com-
plexes comprised of a target molecule with a functional mono-
mer(s) that are fixed by polymerization with appropriate cross-
linking agent(s). Removal of the target molecule leaves cavi-
ties complementary to the target molecule produced in the
resulting polymer matrices. MIPs have excellent stability due
to the high level of crosslinking, and can be used as artificial
receptors without the restrictions that bio-macromolecules pos-
sess.
Although molecular imprinting was first reported in 1931,8
the number of papers regarding MIPs has only rapidly increas-
ed in the last decade. Two major applications of MIPs have
been reported: as stationary phases for liquid chromatography,
and as sorbents for solid-phase extraction and binding assays.
In recent years, MIPs have been investigated for the incorpo-
ration of functions in addition to molecular recognition, which
will allow MIPs to play an important role in the construction of
tailor-made sensors toward desired molecules.9–15
cence sensors based on fluorescence quenching; however, the
fluorescence of porphyrins is limited to zinc(II) and magne-
sium(II), so that the use of metalloporphyrins does not always
work as a fluorescent probe due to the lack of fluorescence by
other metalloporphyrins.
Metalloporphyrins have characteristic UV–vis spectra
that are independent of the coordinated metal ions, in which
specific Soret band and Q bands in long-wavelength regions
can be utilized for sensing based on the coordination of
axial ligands. Herein, we report on signaling CD imprinted
polymers, using a combination of two types of monomers,
chloro[5,10,15,20-tetrakis(4-methacryloyloxyphenyl)porphyri-
nato]iron(III) (Scheme 1) as a signaling monomer and meth-
acrylic acid (MAA) as a stereo-determining monomer.
Experimental
Chemicals. Cinchonidine (CD) and cinchonine (CN), chloro-
form, pyridine, methacrylic acid, and ethylene dimethacrylate
(EDMA) were purchased from Wako Pure Chemical Industry
(Osaka, Japan). Other reagents and solvents were obtained from
commercial sources and used without further purification.
We have reported that MIPs having binding sites with zinc-
(II) porphyrins residues exhibit fluorescence quenching by the
selective and strong binding of 9-ethyladenine16 and cinchoni-
dine (CD).17 The porphyrins can coordinate to various metal
ions to give metalloporphyrins18 that exhibit various functions
that play important roles in functionalized MIPs. The chemical
and physical properties of metalloporphyrins strongly depend
on the kinds and valences of coordinated metal ions. The
zinc(II) porphyrin-based MIPs have been utilized as fluores-
Preparation of the Iron(III) Porphyrin Monomer.
5,10,15,20-Tetrakis(4-hydroxyphenyl)-21H,23H-porphinato iron-
(III) chloride 1 was prepared using a mixture of 5,10,15,20-tetra-
kis(4-hydroxyphenyl)-21H,23H-porphine (0.5 g, 0.7 mmol), iron-
(II) chloride tetrahydrate (3 g, 15 mmol), and 2,6-lutidine (3 mL,
26 mmol) in DMF, which was stirred at 95 ꢃC for 2 h under nitro-
gen. The solution was cooled to room temperature, then added to
5% hydrochloric acid, and the resulting precipitate was collected
and washed with water. The crude iron complex was purified by
Published on the web February 11, 2005; DOI 10.1246/bcsj.78.356

T. Takeuchi et al.
Bull. Chem. Soc. Jpn., 78, No. 2 (2005)
357
silica-gel chromatography (ethyl acetate/hexane, 2:1, v/v) to
yield 1 as a violet solid (0.3 g, 0.4 mmol, 59%). UV/vis (CH2Cl2)
ꢁmax (relative intensity) 418 nm (1.000), 512 nm (0.058), 572
nm (0.044), 609 nm (0.024), 648 nm (0.016). Subsequently, 1
was treated with methacryloyl chloride to obtain chloro[5,10,
lution was stirred for 8 h, washed with water (50 mL ꢁ 3), and
then dried over MgSO4. After removing diethyl ether, the residue
was purified by silica-gel chromatography (ethyl acetate/hexane,
4:1, v/v). The obtained solid was dissolved in benzene, and the
solution was reprecipitated with hexane to yield 2 as a violet solid
(0.3 g, 0.3 mmol, 75%). UV/vis (CH2Cl2) ꢁmax (relative intensi-
ty) 419 nm (1.000), 518 nm (0.053), 553 nm (0.045), 593 nm
(0.030), 651 nm (0.033).
Polymer Preparation. Compositions of pre-polymerization
mixtures for the synthesis of CD-imprinted polymers (IP) and
pyridine-imprinted polymers (RP) as reference polymers are listed
in Table 1. The pre-polymerization mixture was placed in a glass
tube and purged with nitrꢃogen. The resulting mixture was poly-
merized by heating at 50 C for 15 h in the dark.
15,20-tetrakis(4-methacryloyloxyphenyl)porphyrinato]iron(III)
2
(Scheme 1). A solution of methacryloyl chloride (0.78 mL, 8
mmol) in diethyl ether (18 mL) was added dropwise to a solution
of 1 (0.3 g, 0.4 mmol) and triethylamine (1.12 mL, 8 mmol) in di-
ethyl ether (24 mL) under a nitrogen atmosphere. The resulting so-
O
O
Chromatographic Study. The obtained polymer was ground
and sieved to yield polymer particles of 32–63 mm. The polymer
particles were slurried with CHCl3/MeCN (1:1, v/v) and packed
into a stainless steel column (100 mm ꢁ 4.6 mm i.d.). The packed
particles were washed with methanol/acetic acid (7:3, v/v) to
remove the template and unreacted compounds. A mixture of
CH2Cl2/acetic acid (99:1, v/v) was used as an eluent at 1.0
mL/min. The template and related compounds (Scheme 1), name-
ly CD, CN, pyridine, and quinoline, were injected independently
in triplicate. The retention factors were calculated by the equation,
k0 ¼ ðtR ꢂ t0Þ=t0, where tR is the retention time of a sample and t0
is the retention time of a marker (acetone). 0Separation f0actors
were calculated by the equation, ꢀ ¼ k0CD=k CN, where k CD is
the retention factor for CD and k0CN is the retention factor for
CN, respectively. Chromatographic studies were conducted with
a Gilson HPLC system consisting of a 234 auto injector (sample:
5.0 mM, 10 mL), 305+306 pumps and a 117 UV detector (280
nm).
O
O
N
N
N
N
Fe(III)
O
O
Cl
O
O
The iron(III) porphyrin monomer 2
H
H
Scatchard Analysis. Polymer particles (size: less than 32 mm)
were washed with methanol/acetic acid (7:3, v/v). The polymers
(3.0 mg) were suspended into CH2Cl2 solutions containing vari-
ous amounts of CD (1.5 mL, 5.0 to 2000 mM) in vials. After the
polymer suspensions were rotated for 15 h at 25 ꢃC, an aliquot
(1 mL) of each CD-incubated suspension was taken and dried in
vacuo. The residue was dissolved in CH3CN (1 mL) and the con-
centration of free CD ([CD]), was determined with the Gilson
HPLC with a reverse-phase column (TSK gel ODS-80Ts, TOSOH
Corporation, Japan). The eluent was 0.1 M ammonium acetate
buffer (pH 6.0)/CH3CN (1:1, v/v). Each vial was determined in
N
N
HO
HO
H
H
N
N
Cinchonidine (CD)
Cinchonine (CN)
Scheme 1.
Table 1. Composition of Pre-polymerization Mixture (mmol)
Polymers
Cinchonidine
Pyridine
2
MAA
IP(P1)
IP(M4)
IP(M6)
IP(P1M2)
IP(P1M4)
IP(P1M6)
RP(P1)
RP(M4)
RP(M6)
RP(P1M2)
RP(P1M4)
RP(P1M6)
0.22
0.22
0.22
0.22
0.22
0.22
—
—
—
—
—
—
—
—
—
—
—
0.22
0.22
0.22
0.22
0.22
0.22
0.22
—
—
0.22
0.22
0.22
0.22
—
—
0.88
1.32
0.44
0.88
1.32
—
0.88
1.32
0.44
0.88
1.32
—
0.22
0.22
0.22
—
EDMA (ethylene dimethacrylate) 14.7 mmol, ADVN (2,20-azobis(2,4-dimethylvaleroni-
trile)) 0.06 mmol, CHCl3 7.75 mL.

358 Bull. Chem. Soc. Jpn., 78, No. 2 (2005)
Diastereoselective Signaling MIPs
triplicate. The amount of CD bound to polymers (B) was calculat-
ed by subtracting [CD] from the initial CD concentration. Scatch-
ard analysis was based on the equation, B=[CD] ¼ ðBmax ꢂ BÞKa,
where Ka is an association constant and Bmax is the apparent max-
imum number of binding sites, was performed from the binding
data.
Spectroscopic Analysis. Polymer particles (size: less than 32
mm, 2.0 mg) were placed into a vial tube, and various concentra-
tions of a CD or CN solution ꢃin CH2Cl2 (2 mL, 0 to 1000 mM)
were incubated for 15 h at 25 C. The suspensions were transfer-
red to a quartz cell and UV–vis spectra were measured with a
JascoV-560 UV/VIS spectrophotometer (Japan) equipped with
an integral sphere (ISV-469).
100.0
80.0
60.0
40.0
20.0
0.0
Results and Discussion
CD-imprinted polymers were prepared with 2, MAA, or a
combination of both 2 and MAA (Fig. 1). After preparing
the imprinted polymers and corresponding reference polymers,
chromatographic tests were performed using stainless steel
columns packed with these polymers. The retention factors
(k0) in the imprinted and reference polymers for CD, CN, pyri-
dine, and quinoline are listed in Fig. 2. Pyridine was used as
the template instead of CD for preparing reference polymers,
because radical polymerization did not proceed without a li-
gand capable of coordinating to 2. The retention factors of
CD in the CD-imprinted polymers were significantly higher
compared to those on the reference polymer. These results re-
veal that the imprinting of CD effectively proceeded and the
binding sites suitable for CD were induced in the imprinted
polymer matrices during the imprinting process. Retention
times of the corresponding diastereomer CN on the CD-im-
printed polymers were weak, indicating that the binding sites
are complementary to the structure of CD in terms of the steric
and functional topography.
Fig. 2. Retention factors (k0) on the polymers prepared. A
mixture of CH2Cl2/acetic acid (99:1, v/v) was used as
the eluent at 1.0 mL/min, and the detection was carried
out at 280 nm. Retention factors (k0) were calculated by
an equation, k0 ¼ ðtR ꢂ t0Þ=t0, where tR is the retention
time of a sample and t0 is the retention time of a marker.
CD: cinchonidine, CN: cinchonine, QN: quinoline, PY:
pyridine.
Table 2. Separation Factors for CD and CN Given by the
Imprinted Polymers Prepared
Separat0ion factor
Polymers
ꢀ ¼ k CD=k0CN
IP(P1)
IP(M4)
IP(M6)
IP(P1M2)
IP(P1M4)
IP(P1M6)
1
4.7
7.8
4.8
8.3
14.6
IP(P1) showed less retention and no diastereoselectivity.
This polymer was prepared with equimolar amounts of CD
and 2, where CD may form a 1:1 complex with 2 in a pre-poly-
k0CD and k0CN represent retention factors for CD and CN
shown in Fig. 2.
merization mixture. In this case, the polymer would bind CD at
one point, resulting in a low affinity (Fig. 2). On the other
hand, IP(M4) and IP(M6), which were prepared using only
MAA as the functional monomer, showed strong binding to
CD, and the separation factors increased when more MAA
was used (Table 2). Because the use of MAA induced the dia-
stereoselectivity to the polymers, MAA may be referred to as a
‘‘stereo-determining monomer’’.
N
N
HO
O
H
O
O
H
N
N
N
When both MAA and 2 were used as functional monomers,
the resulting imprinted polymers, IP(P1M2), IP(P1M4), and
IP(P1M6), strongly retained CD, and the retention factors for
CD increased with the amount of MAA used. The poor reten-
tion by IP(P1) was greatly improved by cooperative work of
MAA and 2 in the binding site and this cooperativity enhanced
the affinity of CD. The increase in the retention factor was ac-
companied by an increase in the separation factor as MAA was
increased (Table 2). In the pre-polymerization stage, the for-
mation of a ternary complex of CD, 2, and MAA was possible,
N
FeIII
N
N
Fig. 1. Schematic representation of molecular imprinting of
CD with the cooperative use of MAA and 2 as the func-
tional monomers.

T. Takeuchi et al.
Bull. Chem. Soc. Jpn., 78, No. 2 (2005)
359
Table 3. Association Constants (Ka) and Maximum Num-
bers of Binding Sites (Bmax
because bases such as imidazole and pyridine are known to co-
ordinate to iron(III) porphyrins.18 Thus, the quinoline-nitrogen
of CD would coordinate to the iron(III) center and the hydroxy
group of CD would interact with the carboxyl group of MAA
by hydrogen bonding. After polymerization, these interactions
would be memorized in the polymer matrices, resulting in
binding sites that are complementary to the stereochemistry
of CD (Fig. 1). The results presented here reveal that the
MAA residues in IP(P1M6) were more suitably assembled
for chiral recognition in a binding site than those in IP(M6),
due to the fixation of CD by coordination bonding by the
iron(III) porphyrin during the polymerization. As a result,
IP(P1M6) could recognize the stereochemistry of CD more
precisely (k0CD and ꢀ in IP(P1M6): 85.9 and 140.6), compared
to the corresponding MAA-based polymers (k CD and ꢀ in
IP(M6): 54.1 and 7.8).
Particles of the imprinted polymers were suspended in
known concentrations of CD solutions to perform batch re-
binding tests, and Scatchard analyses were performed from
the obtained binding isotherms. A typical binding isotherm
and a Scatchard plot are shown in Fig. 3. All of the Scatchard
plots indicated the typical shape, reported in most cases for
non-covalent imprinting systems; two straight lines were fit
corresponding to the high and low affinity sites. According
to the Scatchard equation, the association constant (Ka) and
the maximum number of binding sites (Bmax) were estimated
from the inclinations and the intercepts of the lines in the
low concentration range (Table 3).
)
Polymers
Ka/Mꢂ1
Bmax/mmol gꢂ1
IP(P1)
IP(M4)
IP(M6)
IP(P1M2)
IP(P1M4)
IP(P1M6)
7:0 ꢁ 104
2:4 ꢁ 105
3:7 ꢁ 105
1:1 ꢁ 105
3:6 ꢁ 105
5:7 ꢁ 105
37
43
42
33
41
37
The values of Ka and Bmax were determined from the Scatch-
ard plots: B=[CD] ¼ ðBmax ꢂ BÞKa.
The values of Ka for the higher affinity sites (in the low con-
centration range) increased with the amount of MAA used as
the functional monomer. The order of the Ka values was con-
sistent with the results in chromatographic tests, meaning that
regardless of the equilibrium state (batch re-binding tests), or
non-equilibrium state (chromatographic tests), IP(P1M6) pre-
sented the highest affinity for CD (Ka ¼ 5:7 ꢁ 105 Mꢂ1). Be-
cause equilibria of the hydrogen-bonding complex between
CD and MAA exist, the use of six equivalents of MAA was
effective for the formation of high affinity binding sites in
the resulting polymer network owing to a displacement of
the equilibria toward complex association; however, more
MAA may result in non-specific binding. The maximum num-
bers of higher affinity sites (Bmax) were almost identical in all
polymers, indicating that the binding sites corresponding to the
high affinity sites in each polymer were generated on the basis
of CD added.
We observed absorbance changes of the imprinted polymers
suspended in known concentrations of a CD solution by using
an integral sphere-equipped spectrophotometer. The UV–vis
spectral change given by the iron(III) porphyrin residues of
IP(P1M6) suspended in CD or CN solutions (0–1000 mM in
CH2Cl2) are shown in Fig. 4. Beyond 400 nm, the visible ab-
sorbance is only derived from the iron(III) porphyrin residues
in the polymer network, because CD and CN are optically
transparent in this region, and the absorbance at 572 nm (Q-
band) of IP(P1M6) and IP(P1) was plotted against concentra-
tions of CD or CN incubated (Fig. 5). The absorbance of
IP(P1M6) showed a larger increase with the concentrations
of CD incubated than with those of CN incubated. In contrast,
the absorbance changes showed an identical manner in IP(P1).
Therefore, in the case of IP(P1M6), the iron(III) porphyrin res-
idue could be located with MAA as a reporter of the binding
events in the high affinity binding sites and provide the signal-
ing function as well as stabilizing the complex of CD and
MAA to develop the chiral selectivity.
The present study demonstrates that two functional mono-
mers that have different functional groups can be suitably as-
sembled in the polymer matrix using the template molecule.
This means that one can construct cavities in synthetic poly-
mers with a desirable assembly of multiple functional mono-
mers; for example, an adjacent assembly of a polymerizable
Lewis acid as a catalytic site and a hydrogen bonding mono-
mer for binding site construction using an appropriate template
molecule may lead to the preparation of a new type of artificial
enzymes.
120
(A)
100
80
60
40
20
0
0
1000
2000
3000
Initial CD concentration/µM
3.0
2.5
2.0
1.5
1.0
0.5
0
(B)
0
50
100
B / µmol g-1
150
Fig. 3. Typical binding isotherm (A) and Scatchard plot (B)
of IP(P1).

360 Bull. Chem. Soc. Jpn., 78, No. 2 (2005)
Diastereoselective Signaling MIPs
polymer displayed changes in the presence of CD, based on the
coordination of the quinoline nitrogen to the iron(III) porphy-
rin. The relationship between the absorbance and the logarith-
mic concentrations of the incubated CD was linear, indicating
that the binding events could be quantitatively read out by the
spectral change. Since many compounds to be analyzed have
no characteristic absorbance or fluorescence, the present read-
out system for the binding events based on the UV–vis absorp-
tion change of metalloporphyrins enables us to perform specif-
ic detection for many target compounds. The characteristic
UV–vis spectra of metalloporphyrins, namely Soret band and
Q bands, are independent of the kind of central metal ions;
therefore, various metal ions other than iron(III) can be applied
to the formation of metalloporphyrins, which can be used as
functionalized monomers in order to prepare functional MIPs
for a wide range of compounds capable of binding the metal-
loporphyrins.
3
2.5
2
1.5
1
0.5
0
250
350
450
550
650
750
Wavelength / nm
Fig. 4. UV–vis spectra of IP(P1M6) with various concen-
trations of CD (from bottom to top: 0, 10, 50, 100, 250,
500, and 1000 mM in CH2Cl2, respectively).
The authors acknowledge financial support from the Minis-
try of Education, Culture, Sports, Science and Technology.
0.35
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