Effect of porogen on the catalytic activity of molecular imprinted polymers

Article abstract of DOI:10.14233/ajchem.2013.13261

A synthetic polymer catalyst was designed and synthesized for enhancing the rate of the hydrolysis reaction of Z-L-phenylalanine-PNP ester. Transition state analogues for the ester hydrolysis reaction pathways were used as templates for the synthesis of molecular imprinted N-methacryloyl L-histidine(MAH)-ethylene glycol dimethacrylate copolymers. Catalytic studies revealed that the imprinted polymers were selective for the transition state analogue corresponding to the template used in the polymer synthesis. Substrate hydrolysis was measured under pseudo first order reaction conditions (rate constant k). The rates of transition state analogue imprinted polymer catalyzed reactions were compared with those carried out in acetonitrile-Tris HCl buffer solution of pH 7.25 at room temperature as well as to the rate of reaction in the presence of non-imprinted polymer. In order to provide comparison between effect of a thermodynamically stable and an unstable solvent on the morphology of the polymer matrix and its catalytic activity the polymerization was carried out using DMSO and chloroform as the porogen. The catalytic activity of the transition state analogue imprinted polymer is found to be substrate specific, solvent and pH dependent. The molecular imprinted polymer synthesized in DMSO showed high selectivity supporting that the nature of the porogen has an effect on the hydrolysis by transition state analogue imprinted catalysts.

Full text of DOI:10.14233/ajchem.2013.13261

Asian Journal of Chemistry; Vol. 25, No. 4 (2013), 1985-1990
http://dx.doi.org/10.14233/ajchem.2013.13261
Effect of Porogen on the Catalytic Activity of Molecular Imprinted Polymers
*
K.P. DEEPTHI and BEENA MATHEW
School of Chemical Sciences, Mahatma Gandhi University, Kottayam-686 560, India
*Corresponding author: E-mail: beenam4@gmail.com
(Received: 15 December 2011;
Accepted: 10 October 2012)
AJC-12263
A synthetic polymer catalyst was designed and synthesized for enhancing the rate of the hydrolysis reaction of Z-L-phenylalanine-PNP
ester. Transition state analogues for the ester hydrolysis reaction pathways were used as templates for the synthesis of molecular imprinted
N-methacryloyl L-histidine(MAH)-ethylene glycol dimethacrylate copolymers. Catalytic studies revealed that the imprinted polymers
were selective for the transition state analogue corresponding to the template used in the polymer synthesis. Substrate hydrolysis was
measured under pseudo first order reaction conditions (rate constant k). The rates of transition state analogue imprinted polymer catalyzed
reactions were compared with those carried out in acetonitrile-Tris HCl buffer solution of pH 7.25 at room temperature as well as to the
rate of reaction in the presence of non-imprinted polymer. In order to provide comparison between effect of a thermodynamically stable
and an unstable solvent on the morphology of the polymer matrix and its catalytic activity the polymerization was carried out using
DMSO and chloroform as the porogen. The catalytic activity of the transition state analogue imprinted polymer is found to be substrate
specific, solvent and pH dependent. The molecular imprinted polymer synthesized in DMSO showed high selectivity supporting that the
nature of the porogen has an effect on the hydrolysis by transition state analogue imprinted catalysts.
Key Words: Molecular imprinting, Transition state analogue, Amino acid ester, Hydrolysis, Porogen, Enantioselectivity.
stabilizing the transition state and/or to impart higher selectivity
by favouring the formation of one of the possible transition
states12-14. The developing understanding of the mechanisms by
which enzymic catalysts function has been a major source of
inspiration for researchers in molecular imprinted polymer
(MIP) catalysis and a number of active imprinted polymer
INTRODUCTION
Molecular imprinting is an emerging technology leading
to highly stable synthetic polymers with predetermined selec-
tivity and applications in the field of protein recognition1,2,
biosensor development3-5, enantiomer separation6,7 and mole-
cular catalysis8,9. The principle of molecular imprinting consists
of polymerizing and crosslinking functional monomers,
previously positioned by low energy or covalent interactions
around a guest or template molecule so as to freeze the imprint.
Following extraction of the template molecule, the remaining
three dimensional networks presents pores with geometry and
positioning of the functional groups complementary to those
of the template. This enables it to specifically recognize the
template molecule10,11. In the area of molecular catalysis,
imprinted polymers seem to be particularly attractive with
cavities designed to bind to the transition state of a given
reaction should function as a selective catalyst for that reaction.
In order to prepare molecular imprinted catalyst, molecules
that mimic the transition state of a given reaction are used as
templates. Furthermore, the system must be designed in such
a way that a reactive moiety or a convenient precursor thereof
is placed in the cavity in the course of the imprinting procedure.
The resulting functionalized cavities are then potentially able
to accelerate the rate of a reaction taking place inside them by
catalysts have now been prepared15-18
.
Even though there are a lot of work has been reported on
the transition state analogue imprinted molecular imprinted
polymer catalyzed ester hydrolysis, the dependence of the
nature of porogen used during polymerization have been less
studied19-22. The selectivity and efficiency of molecular
imprinted polymers depend on the nature of the solvent used
for the synthesis of the molecular imprinted polymers23. The
properties of molecular imprinted polymers like swellability
and porosity are often correlated with the solvent conditions
employed during polymerization and there are two effects of
the polymerization solvent, referred to as the porogen on the
formation of molecular imprinted polymers24-26. The first is
the formation of prepolymerization complex. For weaker non-
covalent interactions such as ionic or hydrogen bonds non-
polar solvents are preferred in order to drive the template and
functional monomer towards complex formation. The second
effect of the polymerization solvent is on the formation of the

1986 Deepthi et al.
Asian J. Chem.
porous structure of the polymer, which is the reason that it is
referred to as porogen. The type of porogen used during
the polymerization has a strong influence on the morphology
of the imprinted polymer and its catalytic activity. In the
present study in order to provide comparison between effect
of a thermodynamically stable and an unstable solvent on the
morphology of the polymer matrix and its catalytic activity
the polymerization was carried out using chloroform and
DMSO as the porogen.
and strirred under nitrogen atmosphere for 1 h at room tempe-
rature. Ethylene glycol dimethacrylate (2.75 cm3, 14.95 mmol)
and 50 mg initiator AIBN were added and polymerized at 60
ºC for 24 h. The process for the preparation of non-imprinted
polymers was the same as that of the molecular imprinted
polymers except that the imprinted transition state analogue
molecules were not added during polymerization. The polymer
(P-1) formed was collected by filtration, washed with acetone,
Soxhlet extracted with chloroform for complete removal of
transition state analogue and dried in vaccum. The same
procedure was followed by using DMSO as porogen instead
of chloroform resulted in polymer (P-2).
EXPERIMENTAL
Solid state 13C NMR spectra were recorded on AMX-400
NMR spectrometer. 1H NMR spectra were taken using Bruker
Avance DPX-300MHz FT-NMR spectrometer in CDCl3.
31P-NMR spectra was taken on AC200 NMR spectrometer.
Infrared spectra were recorded on a Shimadzu FTIR-8400S
spectrophotometer. Kinetic determinations were performed
using a Shimadzu UV-visible 2450 spectrophotometer
equipped with a thermostated cell. SEM analyses were
performed on JEOL JSM 6390 SEM analyzer.
Catalytic activity measurement of imprinted and non-
imprinted polymers: Z-L-phenylalanine p-nitrophenyl ester
was chosen as the substrate to evaluate the hydrolytic activity
of the imprinted and non-imprinted polymers P-1 and P-2 in a
9:1 (v/v) solution of tris-HCl buffer and acetonitrile at 25 ºC.
A 10 mg each molecular imprinted polymer and non-imprinted
polymers of polymers P-1 and P-2 was placed in a sealed test
tube, to which a 2 mL acetonitrile-tris HCl buffer (pH = 7.25)
added and allowed to equilibrate for 30 min. Acetonitrile
solution (0.2 mL) containing 3.42 × 10-3 mmol Z-L-phenyl-
alanine p-nitrophenyl ester as a substrate [functional host
molecule/substrate = 1:0.36 (molar ratio)]. The hydrolysis
activity of the polymers was determined by measuring the
hydrolytic product (p-nitrophenol) produced in the reaction
mixture. The produced p-nitrophenol was detected at 400 nm
with UV-VIS spectrophotometer. The degree of self-hydrolysis
of the substrate was also measured without the functional host
molecule under the same conditions. The rate of hydrolysis
was determined by measuring the concentration of p-
nitrophenol released at definite time intervals. Rates were
determined by calculating the slope of ln(Aα-At) (where Aα is
the measured absorbance at infinity and At is the absorbance
at time t) vs. time, after correcting for background hydrolysis
without polymer and dividing by the molar concentration of
imidazole present.
Synthesis of monomer: The monomer N-methacryloyl
L-histidine was prepared from methacryloyl chloride, L-histidine
monohydrochloride and NaOH following the reported proce-
dure20. FTIR (cm-1): 1612 (HC=CH), 1651 (amide carbonyl),
1
1
1705 (acid carbonyl). H NMR (CDCl3) δ ppm: 7.6 (s, H,
imidazole NH), 7.37 (s, 1H, imidazole CH), 6.76 (s, 1H,
imidazole CH), 3.03, 2.56 (s, CH2), 4.29 (d, 1H, C=CH), 1.83
(s, 3H, CH3).
Synthesis of transition state analogue (phenyl 1-
benzyloxycarbonyl amino-4-methoxybenzyl phosphonate):
Triphenyl phosphite (13.2 mmol), p-methoxy benzaldehyde
(19.8 mmol), benzyl carbamate (13.2 mmol) and glacial
acetic acid (2 mL) were stirred for 4 h at 100 ºC in an oil bath.
The diphenyl phosphonate formed was hydrolyzed with NaOH
(0.4 N) at room temperature for 2 days (Fig. 1). It was acidified
with conc. HCl and the product formed was purified by column
chromatography using 9:1 chloroform-methanol mixture.
FTIR (KBr, νmax, cm-1): 1300 (P=O stretching), 945 (P-OH
stretching), 1250 (P-O-benzyl stretching). 1H NMR (CDCl3)
δ ppm: 1.73 (s, H, OH), 3.77 (s, 3H, OCH3), 5.49 (d, 2H,
CH2), 6.73-7.42 (m, 13H, aromatic CH). 31P NMR δ ppm: 24.99
P(O)(OPhe)(OH),16.50 (P-O-P angle).
RESULTS AND DISCUSSION
Spectral analysis of transition state analogue imprinted
and non-imprinted polymers: FTIR spectroscopy and solid
state 13C NMR spectroscopy were used to characterize the
polymer structure. From the FTIR spectrum of molecular
imprinted polymer and non-imprinted polymers, the peak at
1712 cm-1 represents the C=O double-bond stretching vibration
in the carboxylic acid groups of poly N-methacryloyl L-histi-
dine. The peak at 1549 cm-1 indicates the N-H deformation
vibration in the amide groups and a peak at 3417 cm-1 corres-
ponds to NH streching frequency of amide. The small peaks
at 1387 and 1367 cm-1 indicated the stretching vibration of
methyl group protons.
The 13C NMR spectrum (Fig. 2) give the information about
the chemical structure of polymer backbone. The peak at 178.7
ppm is due to the ester carbonyl carbon of EGDMA. The small
peak at 113.1 ppm corresponds to the -CH carbon of imidazole
ring. The peaks at 63.9 ppm and 26.9 ppm are due to the -CH2
carbon atom of ethylene-glycol dimethacrylate copolymer and
peak at 20.3 ppm was appeared by the -CH3 group.
O
O
OH
O
CH3COOH/1000C
+
P(OC6H5)3
+
P
N
O
O
N
H
NaOH
2
O
O
OMe
OMe
Fig. 1. Synthesis of transition state analogue
Preparation of N-protected phenylalanine esters: N-
protected phenylalanine esters were prepared with coupling
between the corresponding protected amino acids and
p-nitrophenol in presence of DCC27.
Synthesis of transition state analogue imprinted and
non-imprinted polymers: The transition state analogue (0.319
g, 0.748 mmol) and monomer N-methacryloyl L-histidine
(0.329 g, 1.495 mmol) were dissolved in chloroform (30 mL)

Vol. 25, No. 4 (2013)
Effect of Porogen on the Catalytic Activity of Molecular Imprinted Polymers 1987
Morphological studies: The morphology of the transition
state analogue imprinted and non-imprinted polymers prepared
in two solvents was assessed by SEM analysis (Fig. 3). The
images of SEM analysis reveals a smooth surface for molecular
imprinted polymer and non-imprinted polymers of polymer
prepared in chloroform (P-2) comparable to that prepared in
DMSO (P-1). This nonporous morphology is very different
from the porous morphologies that have been typically observed
in molecular imprinted polymers that were prepared in the
solvent DMSO. The roughness of the surface should be consi-
dered as a factor providing an increase in the surface area. It is
a strong evidence for the importance of the use of a thermo-
dynamically stable solvent during the preparation of an efficient
imprinted polymer catalyst.
in the dry state and solvent can be used to access the pore
network. By measuring the swelling ratio of the polymers in
different solvents an estimate can be made of the porous
structure. The swelling ratios of imprinted polymer and non-
imprinted polymer of P-1 and P-2 are tabulated in Table-1.
Porogenic solvents play an important role in the formation of
the porous structure of molecular imprinted polymer, which
known as macroporous polymers. Swelling ratio of the
imprinted polymer was found to be higher than that of the
non-imprinted polymer because of the porous structure of the
molecular imprinted polymer. The studies showed that as the
polarity of solvent increases the swelling of both imprinted
polymer and non-imprinted polymer increased. Different
swelling properties of different solvents may play a role in
determining shape and distance parameters that are locked into
the imprinted polymer. The swelling ratio of polymer P-1
prepared in DMSO and P-2 prepared in chloroform show
considerable difference. The highest swelling ratio exhibited
by polymer P-1 is indicative of highly porous structure of
polymer formed using thermodynamically more stable
solvent DMSO than chloroform.
TABLE-1
SWELLING RATIO OF POLYMERS P-1AND P-2
Swelling ratio
Polymer Chloroform Acetonitrile Methanol DMSO Water
MIP
NIP
MIP
NIP
8.29
2.20
0.96
0.52
3.84
1.91
1.87
0.55
5.99
5.90
2.56
1.43
16.30
15.59
9.40
14.59
13.99
9.76
P-1
P-2
8.34
7.38
Catalytic activities of imprinted polymer and non-
Fig. 2. 13C NMR spectrum of transition state analogue imprinted polymer
imprinted polymer: The catalytic hydrolysis of molecular
imprinted polymer is higher than non-imprinted polymer and
there is a striking difference in the hydrolysis of esters by
imprinted polymer depending upon the porogen used for the
polymerization (Fig. 4). The data indicate that the catalytic
rate for hydrolysis of Z-L-Phe-PNP by transition state
analogue imprinted polymer prepared in DMSO (P-1) is very
high as compared to the corresponding polymer prepared in
chloroform (P-2). The transition state analogue imprinted
polymer P-1 exhibited catalytic activity -55 % of the hydrolysis
of Z-L-phenylalanine p-nitrophenyl ester while transition state
analogue imprinted polymer P-2 showed less than 10 %
hydrolysis of the ester. It is known that the nature and level of
porogenic solvents determines the strength of non-covalent
interactions and influences polymer morphology which,
obviously, directly affects the performance of molecular
imprinted polymer. The rate of hydrolysis catalyzed by tran-
sition state analogue imprinted polymer is higher than that of
the non-imprinted polymer and it represented the imprinting
effect.
Fig. 3. SEM analysis of (a) transition state analogue imprinted and (b)
non-imprinted polymers in DMSO (P1) and (c) transition state
analogue imprinted and (d) non-imprinted polymers prepared in
chloroform (P2)
Substrate selectivity of transition state analogue
imprinted polymers: In order to study the substrate selectivity
of transition state analogue imprinted polymers catalytic
hydrolysis of structurally related esters t-Boc-L-Phe-PNP and
N-Phth-L-Phe-PNP were also carried out. The polymer
imprinted with transition state analogue exhibited the highest
activity with the rate constant ratio of kMIP/kuncat = 2.33 for the
Swelling studies: Swelling studies of the imprinted
polymer and non-imprinted polymer is an important tool to
differentiate the morphological difference between the
polymers. Through the molecular imprinting procedure
macroporous polymers obtained are permanently porous even

1988 Deepthi et al.
Asian J. Chem.
TABLE-2
KINETIC PARAMETERS FOR THE HYDROLYSIS OF VARIOUS N-PROTECTED L-Phe-PNP
ESTER BY MIP AND NIP OF POLYMERS P1 AND P2
Polymer
Substrate
103kuncat
0.56
103kNIP
4.97
103kMIP
7.76
kMIP/kuncat
13.86
5.88
102kcat
81.77
36.56
12.96
11.04
6.99
Z-L-Phe-PNP
P1
P2
t-Boc-L-Phe-PNP
N-Phth-L-Phe-PNP
Z-L-Phe-PNP
0.59
2.24
3.47
0.41
0.99
1.23
3.15
0.45
0.62
1.05
2.33
t-Boc-L-Phe-PNP
N-Phth-L-Phe-PNP
0.41
0.515
0.456
0.664
0.476
1.62
0.34
1.4
5.02
TABLE-3
KINETIC PARAMETERS FOR THE HYDROLYSIS OF Z-(L)/(D)-Phe-PNP BY
EGDMA CROSSLINKED MIP AND NIP OF POLYMERS P-1 AND P-2
Polymer
P-1
Substrate
103kuncat
0.56
103kNIP
4.97
103kMIP
7.76
kMIP/kuncat
13.86
5.06
102kMIP/[Im]
81.77
kL/kD
4.51
Z-L-Phe-PNP
Z-D-Phe-PNP
Z-L-Phe-PNP
Z-D-Phe-PNP
0.34
1.15
1.72
18.12
0.45
0.62
1.05
2.33
11.04
P-2
1.81
0.44
0.46
0.58
1.32
6.11
Z-L-Phe-PNP hydrolysis, but it indicated the lower activity
for the hydrolysis of other amino acid p-nitrophenyl esters
possessing small sized t-Boc-L-Phe-PNP and more sterically
hindered N-Phth-L-Phe-PNP (Table-2). Hence, the imprinted
polymer catalyst was capable of exhibiting efficient selectivity
in its esterolytic catalysis. The polymer catalyst recognized
the structure of imprinted molecule and selectively catalyzed
the substrate that has a similar structure of the imprinted
transition state analogue molecule than the non-imprinted
molecule. The transition state analogue imprinted polymer
prepared in DMSO (P-1) retains higher substrate selectivity
than that prepared in chloroform (P-2).
of p-nitrophenyl Z-D-phenylalanine. This result indicates that
the transition state analogue imprinted polymer exhibits
efficient enantioselectivity. The ratio kL/kD for imprinted
polymer made with DMSO as porogen (P-1) is 4.51 and for
the polymer synthesized in chloroform as porogen (P-2) is
only 1.81. This explains the role of porogen in creating
the macroporous structure in the polymer matrix and thus
enhancing catalytic activity and enantioselectivity.
Optimization of conditions of catalytic hydrolysis: For
optimizing the conditions of catalytic hydrolysis the effect of
parameters like substrate concentration, solvent and pH on
catalytic hydrolysis were carried out.
a) Effect of substrate concentration: The catalytic
hydrolysis of the transition state analogue imprinted and
non-imprinted polymers were carried out using different
molar concentrations of Z-L-phenylalanine p-nitrophenyl ester.
The studies with different concentrations by polymers P-1 and
P-2 showed highest rate of hydrolysis for 1.14 mM concen-
tration of ester (Fig. 5). The rate of hydrolysis was found to be
increased with increase in concentration upto 1.14 mM and
beyond this concentration catalytic activity decreased due to
substrate inhibition.
60
Blank
NIP Chloroform
MIP Chloroform
NIP DMSO
MIP DMSO
50
40
30
20
10
0
200
NIP(P1)
0
20
40
60
80
100
120
140
MIP(P1)
Time (h)
150
NIP(P2)
Fig. 4. Comparison of catalytic activity with time by TSA imprinted and
non-imprinted polymers prepared in chloroform (P2), DMSO (P1)
and uncatalyzed reaction
MIP(P2)
100
Enantioselectivity of transition state analogue imprinted
polymer: The enantioselectivity studies of the transition state
analogue imprinted polymers prepared in two solvent systems
were carried out using p-nitrophenyl Z-L-phenylalanine and
p-nitrophenyl Z-D-phenylalanine. The results of the
enantioselectivity studies of the transition state analogue
imprinted polymer catalysts for the hydrolysis of p-nitrophenyl
Z-L-phenylalanine and p-nitrophenyl Z-D-phenylalanine are
given in Table-3. The imprinted polymer catalyzed the
hydrolysis of p-nitrophenyl Z-L-phenylalanine more than that
50
0
0.6735
1.14
3.42
10.2
17.05
[S] mmol
Fig. 5. Effect of substrate concentration on the hydrolysis of Z-(L)/(D)-
Phe-PNP by EGDMA-crosslinked molecular imprinted polymer and
non-imprinted polymer of polymers P-1 and P-2

Vol. 25, No. 4 (2013)
Effect of Porogen on the Catalytic Activity of Molecular Imprinted Polymers 1989
TABLE-4
CATALYTIC ACTIVITY OF MIP AND NIP PREPARED OF POLYMERS P-1 AND P-2
WITH DIFFERENT RATIOS OF SOLVENT-BUFFER SYSTEM
Polymer
P-1
Acetonitrile-Tris HCl ratio
103kuncat
0.56
103kNIP
4.97
1.54
1.15
0.62
0.72
1.26
103kMIP
7.76
1.67
1.19
1.05
1.37
1.52
kMIP/kuncat
13.86
1.99
102kMIP/[Im]
81.77
1:9
3:7
5:5
1:9
3:7
5:5
0.84
17.56
0.96
1.24
12.54
0.45
2.33
11.04
P-2
0.48
2.85
14.44
0.47
3.23
16.02
b) Effect of pH on the catalytic hydrolysis: The pH
effect on the activities of imprinted and non-imprinted polymers
P-1 and P-2 for ester hydrolysis was studied in the pH range
6.0 and 7.25 in acetonitrile and tris HCl buffer and the results
are presented in Fig. 6. The catalytic activity of imprinted polymer
prepared in DMSO (P-1) is higher than those prepared in chloro-
form (P-2). The pH profile follows the same manner in the
polymers P-1 and P-2 that it exhibits an optimum at pH 7.25.
The shrinkage or expansion of the polymer matrix took place
on varying the pH of the reaction medium due to changes in
the number of protonated imidazole residues and it will also
resulted in decreased catalytic activity.
catalytic activity. The polymer prepared in chloroform resulted
in more rigid polymer matrix with low swelling for rebinding
the template.
Conclusion
The molecular-imprinting technique provide new catalytic
systems with tremendous performances for reactions for which
enzymes cannot be efficient at all and under the reaction
conditions where enzyme systems cannot be applied. We have
successfully demonstrated that a transition state analogue
imprinted polymeric catalysts can be prepared in a bulk
polymerization protocol utilizing N-methacryloyl L-histidine
as functional monomer, DMSO and chloroform as porogen.
The morphological studies of the imprinted polymer and non-
imprinted polymer synthesized in DMSO exhibited highly
porous morphology while those prepared in chloroform
exhibited nonporous structure. Results of catalytic studies of
polymer prepared with DMSO displayed the highest catalytic
activity, substrate selectivity and enantioselectivity. The drastic
behaviour of the imprinted polymers could be attributed to
the different kinetics of polymerization in the two systems.
The high catalytic activity, efficiency and selectivity together
with the strong chemical, mechanical and thermal stability give
the catalysts a real advantage compared to catalytic antibodies
and also provide an alternative compared to natural enzymes.
8
NIP (P1)
7
MIP (P1)
6
NIP (P2)
MIP (P2)
5
4
3
2
ACKNOWLEDGEMENTS
1
0
The authors gratefully acknowledged the financial support
of Department of Science and Technology, Government of
India by awarding the Bio-inorganic Pre-doctoral Fellowship
to K.P. Deepthi.
5.5
6
6.5
7
7.5
8
pH
Fig. 6. Effect of pH on catalytic hydrolysis by molecular imprinted polymer
and non-imprinted polymer of polymers P-1 and P-2
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