Amidase activity of phosphonate TSA-built polymer catalysts derived from organic monomers in the amidolysis of amino acid p-nitroanilides

Article abstract of DOI:10.1016/j.apcata.2016.09.020

Highly crosslinked transition state analogue imprinted macromatric polymer catalysts having imidazole, carboxyl and hydroxyl functional groups in the catalytic sites were synthesized as chymotrypsin mimics using achiral organic monomers 4-vinylimidazole, methacrylic acid, allyl alcohol and phenyl-1-(N-benzyloxycarbonylamino)-2-(phenyl)ethyl phosphonate (transition state analogue of ester and amide hydrolytic reactions) as the template. The catalytic properties of the enzyme mimics were investigated in the amidolytic reactions of L-amino acid p-nitroanilides and correlated to the amidase activities of the catalysts derived from chiral methacryloyl-L-amino acid monomers methacryloyl-L-histidine, methacryloyl-L-aspartic acid and methacryloyl-L-serine. A two-fold enhancement in rate acceleration, substrate specificity, substrate shape-selectivity and stereoselectivity was observed for polymers made up of flexible amino acid monomers compared to the copolymers of organic monomers. The pre-polymerization complex of TSA with methacryloyl-L-amino acid monomers fabricates specific 3D-memory cavity preferentially of L-enantiomer of the TSA in the polymer matrix while the achiral organic monomers designs both L- and D- cavities The effect of crosslink density on the catalytic efficiencies of the polymer catalysts was also investigated. Replacement of allyl alcohol by vinylpyridine afforded catalyst with better enzymatic activity.

Full text of DOI:10.1016/j.apcata.2016.09.020

Applied Catalysis A: General 528 (2016) 93–103
Contents lists available at ScienceDirect
Applied Catalysis A: General
journal homepage: www.elsevier.com/locate/apcata
Feature Article
Amidase activity of phosphonate TSA-built polymer catalysts derived
from organic monomers in the amidolysis of amino acid
p-nitroanilides
Divya Mathewa, Benny Thomasa,b, K.S. Devakya,∗
a School of Chemical Sciences, Mahatma Gandhi University, Kottayam, 686 560, India
b Department of Chemistry, St. Berchmans College, Changanacherry, 686 101, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Highly crosslinked transition state analogue imprinted macromatric polymer catalysts having imida-
zole, carboxyl and hydroxyl functional groups in the catalytic sites were synthesized as chymotrypsin
mimics using achiral organic monomers 4-vinylimidazole, methacrylic acid, allyl alcohol and phenyl-1-
(N-benzyloxycarbonylamino)-2-(phenyl)ethyl phosphonate (transition state analogue of ester and amide
hydrolytic reactions) as the template. The catalytic properties of the enzyme mimics were investigated
in the amidolytic reactions of l-amino acid p-nitroanilides and correlated to the amidase activities
of the catalysts derived from chiral methacryloyl-l-amino acid monomers methacryloyl-l-histidine,
methacryloyl-l-aspartic acid and methacryloyl-l-serine. A two-fold enhancement in rate accelera-
tion, substrate specificity, substrate shape-selectivity and stereoselectivity was observed for polymers
made up of flexible amino acid monomers compared to the copolymers of organic monomers. The
pre-polymerization complex of TSA with methacryloyl-l-amino acid monomers fabricates specific 3D-
memory cavity preferentially of l-enantiomer of the TSA in the polymer matrix while the achiral organic
monomers designs both L- and D- cavities The effect of crosslink density on the catalytic efficiencies
of the polymer catalysts was also investigated. Replacement of allyl alcohol by vinylpyridine afforded
catalyst with better enzymatic activity.
Received 25 July 2016
Received in revised form
26 September 2016
Accepted 30 September 2016
Available online 1 October 2016
Keywords:
Molecularly imprinted macromatric
polymer catalyst
Transition state analogue
Imprinting efficiency
Enzyme-mimetic catalytic amidolysis
© 2016 Published by Elsevier B.V.
1. Introduction
have reported the amidolysis of phenylalanine p-nitroanilides
using phosphonate TSA imprinted copolymers of amino acid
Molecularly imprinted polymer catalysts (MIPs) have received
considerable attention due to their thermal and pH stability and
increased shelf life where natural enzymes may lose activity [1–4].
The proteolytic enzyme chymotrypsin continues to be a good
model of MIP catalysts. Imidazole group of histidine residue is
the essential catalytic group in the active site of the hydrolase
protein chymotrypsin which cleaves the phenylalanine, trypto-
phan and tyrosine residues in peptide chains. Based on the theory
of transition state stabilization for ester hydrolysis, phosphonic
monoester TSA is generally used as a template molecule in molec-
ular imprinting [6–12]. Even though there are many reports on
MIP catalyzed esterolytic reactions, amide or peptide hydroly-
sis reactions using artificial enzyme mimics are less studied. We
monomers-methacryloyl-l-histidine, methacryloyl-l-aspartic acid
and methacryloyl-l-serine [13]. Phosphonate analogue imprinted
polymers with 4-vinylimidazole [14–16], methacrylic acid [9,17]
and 4-vinylpyridine [18] have been used for esterolytic reac-
tions. The present work describes the design and synthesis
of catalytically active phenyl-1-(N-benzyloxycarbonylamino)-2-
(phenyl)ethyl phosphonate (TSA). The individual and combined
effects of 4-vinylimidazole, methacrylic acid, allyl alcohol and 4-
vinylpyridine were studied in detail.
2. Experimental
2.1. Materials and methods
Dicyclohexylcarbodiimide (DCC), ethylene glycol dimethacry-
late (EGDMA) and 4-vinylpyridine (VP) were purchased from Sigma
Aldrich, USA. Z-l-Phenylalanine, methacrylic acid (MAA), allyl
alcohol (AA), benzylcarbamate, triphenyl phosphite and pheny-
∗
Corresponding author at: School of Chemical Sciences, Mahatma Gandhi Uni-
versity, Priyadarshini Hills, P.O Kottayam, 686 560, Kerala, India.
E-mail address: ksdevakyscs@yahoo.com (K.S. Devaky).
http://dx.doi.org/10.1016/j.apcata.2016.09.020
0926-860X/© 2016 Published by Elsevier B.V.

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D. Mathew et al. / Applied Catalysis A: General 528 (2016) 93–103
lacetaldehyde were purchased from SRL, Mumbai. All chemicals
used, other than listed above were from local suppliers, which
were purified prior to use by following the standard procedures.
IR spectra were recorded on a Shimadzu FT-IR-8400S spectropho-
tometer. Kinetic studies were performed using Shimadzu UV 2450
spectrophotometer. JEOL JSM6390 SEM analyzer was used for
SEM analysis. 1H NMR spectra were taken using Bruker Advance
DPX–300 MHz FT-NMR spectrometer in CDCl3. BET analyzer used
was Thermo Fisher Scientific Surface Analyzer V-230 50/60 Hz.
Scheme 1. Synthesis of the phosphonate TSA.
2.2. Synthesis of TSA
(Phenyl-1-(N-benzyloxycarbonylamino)-2-(phenyl)ethyl
phosphonate)
The transition state analogue was synthesized by refluxing
4.1 mL, 13.2 mmol triphenyl phosphite, 2.0 g, 13.2 mmol benzyl car-
bamate, 2.38 mL, 19.8 mmol phenylacetaldehyde and 2.0 mL glacial
acetic acid for 4 h at 100 ◦C in an oil bath. FTIR:−1301 cm−1(P
O
stretching), 946 cm−1(P-OH stretching) and 1252 cm−1 (P-O-
benzyl stretching) [13].
Fig. 1. Structure of methacrylated amino acid monomers and their organic ana-
logues.
2.3. Synthesis of monomer 4-vinylimidazole
5%. Since the heterogeneity of the polymer catalysts and solvation
problems, the results show some deviations in reproducibility.
The functional monomer 4-vinyimidazole (VIm) was prepared
from urocanic acid as per the reported procedure [14].
2.4. Synthesis of TSA imprinted enzyme mimics and
non-imprinted control polymers
3. Results and discussion
3.1. Synthesis of TSA-built and non-built polymer catalysts
The polymer catalyst P1a was prepared by radical initiated bulk
polymerization of 752 mg, 8.0 mmol, of monomer 4-vinylimidazole
(VIm) and 396 mg, 2.0 mmol, of the crosslinking agent EGDMA in
presence of 1.8 g, 4.0 mmol TSA in 40 mL DMSO for 6 h at 80 ◦ C. The
template was completely leached out by washing with methanol
and then subjected to Soxhlet extraction with chloroform. The
polymer obtained was collected and dried over vacuum. Enzyme
mimics P1b-P3e were also synthesized as per the same procedure
by varying the molar ratios of monomer and crosslinker. The corre-
sponding non-imprinted control polymers CPs were also prepared
by the same procedure in the absence of TSA.
The TSA, which has more structural resemblance with the sub-
strate Z-l-Phe-PNA was synthesized using triphenyl phosphite,
benzyl carbamate and phenylacetaldehyde (Scheme 1). The TSA
synthesized possesses N-benzyloxycarbonyl (Z) protecting group
of the substrate and the “specificity determinant”- C6H5CH2 group
− complementary to the hydrophobic pocket of chymotrypsin CT
[19,20]. The TSA was characterized by FTIR and NMR spectroscopic
techniques.
The methacrylated l-amino acid monomers − methacryloyl-
l-histidine (MALH), methacryloyl-l-aspartic acid (MALA) and
methacryloyl-l-serine (MALS) − were synthesized by Schotten-
Bauman reaction [5] and the organic monomer 4-vinylimidazole
was prepared from urocanic acid [14].
2.5. Synthesis of the substrates phenylalanine p-nitroanilides
The substrate Z-l-phenylalanine p-nitroanilide was synthe-
sized by DCC coupling of a suspension of 299 mg, 1 mmol of
Z-l-phenylalanine and 138 mg, 1 mmol of p-nitroaniline in 30 mL
ethyl acetate. The dicyclohexylurea (DCU) formed was filtered off
and the filtrate was evaporated in vacuum. The residue obtained
was recrystallized from hot ethanol containing 1% acetic acid.
The TSA imprinted polymers were prepared by radical initiated
bulk polymerization method using the functional monomers, tem-
plate TSA and the crosslinker EGDMA as per the reported procedure.
The structure of the methacrylated l-amino acid monomers and
their organic analogues used are shown in Fig. 1 and the details of
polymer synthesis are given in Table 1.
The monofunctional mimics (P1a-P1c), bifunctional mimics
(P2a-P2c) and trifunctional mimics (P3a-P3d) were synthesized
using the functional monomers 4-VIm/MAA/AA with EGDMA. In all
enzyme mimics −mono/bi/trifunctional mimics, the concentration
of the print molecule and total concentration of monomers is taken
fixed as 1:2. Since the allyl alcohol containing polymer catalysts
were found to be less efficient in amidolytic reactions, we prepared
trifunctional mimic P3e by replacing allyl alcohol by vinylpyridine.
The corresponding non-imprinted control polymers (CPs) were
also synthesized in the same molar ratios of monomers and the
crosslinker without the TSA molecule. The total functional group
contents were estimated by titration method as per the reported
procedures.
2.6. Amidolysis of phenylalanine p-nitroanilides using TSA
imprinted and non-imprinted enzyme mimics
In a reagent bottle, to a suspension of 10 mg, 0.012 mmol of the
polymer catalyst P1a in 5 mL acetonitrile-Tris HCl buffer (1:9 by
volume, pH 7.75), 503 mg, 1.2 mmol of the substrate Z-Phe-PNA in
50 mL acetonitrile was added. The reaction mixture was placed in
a water bath shaker at 45 ◦ C and shaken gently. Amidolysis of Z-
Phe-PNA was followed spectrophotometrically by monitoring the
absorbance of released p-nitroaniline at 374 nm. A blank reaction
was also carried out in the absence of the enzyme mimic. From
the absorbance data, the rate constants and percentage amidoly-
sis were evaluated. Amidase activity of the mimics P1b-P3d was
evaluated in a similar manner.
The trifunctional mimic (C3) comprising of amino acid
monomers was synthesized in presence of EGDMA crosslinker
(90%) and TSA. The total functional group content (TFGC) was esti-
mated using ninhydrin reagent [5].
All the experiments were repeated 4 times and the average of
the results were reported here. The error of the results lies within

D. Mathew et al. / Applied Catalysis A: General 528 (2016) 93–103
95
Table 1
Details of the synthesis of polymer catalysts.
Polymer Catalysts
FunctionalMonomers
%Crosslinker
TFGC
P1a
P1b
P1c
P2a
P2b
P2c
P3a
P3b
P3c
P3d
P3e
4-VIm
MAA
AA
20%
20%
20%
20%
20%
20%
90%
10%
20%
30%
20%
1.187
1.156
1.144
1.021
1.078
1.072
0.643
1.084
1.065
1.037
1.072
Monofunctional
4-VIm/MAA
4-VIm/AA
MAA/AA
4-VIm/MAA/AA
4-VIm/MAA/AA
4-VIm/MAA/AA
4-VIm/MAA/AA
4-VIm/MAA/4-VP
Bifunctional
Trifunctional
Table 2
Details of BET surface area analysis and TSA rebinding studies.
the initial and equilibrium concentration, V (L) is the volume of
the TSA solution and M (g) is the weight of the imprinted polymer.
The polymers could rebind the TSA molecules effectively and the
amounts of TSA rebound evaluated were given in Table 2. The two
values obtained were in good agreement.
The morphological differences of imprinted and non-imprinted
polymer catalysts were assessed by SEM analysis. The SEM picture
of C3 and P3e is full of cavities by the removal of the imprinted phos-
phonate TSA. But, the surface of the corresponding non-imprinted
control polymers is almost smooth without cavities (Fig. 2).
Enzyme
mimics
Surface area
(m2/g)
Pore volume
(cm3/g)
[TSA rebound]
mmol/g
C3
P3c
P3e
311.55
267.16
285.37
0.898
0.770
0.823
0.184
0.358
0.364
Table 3
Catalytic amidolysis and effect of the extent of EGDMA crosslinking.
Kinetic parameters/Catalysts
Trifunctional Mimics
3.2. Evaluation of the kinetics of catalytic amidolysis
P3a
P3b
P3c
P3d
C3
kacc
kim
St (h)
6.78
1.02
nd
21.62
1.43
50
35.42
2.12
24
23.56
1.37
70
75.38
4.22
22
Amidolysis proceeds through
a higher energy tetrahedral
oxyanion intermediate and the “shape-selective-stabilization”
of the unstable intermediate in the memorized cavity fabricated
by the print molecule leads to the rate enhancement with the
enzyme mimics. The key acceleration processes of catalytic ami-
dolysis are substrate binding process, stabilization of the T.S of
the amidolytic reaction and finally release of the product. The
amidolysis of Z-L-Phe-PNA was found to be too slow in aqueous
medium (0.16 × 10−8 min−1) and in Tris HCl buffer at room tem-
perature (0.98 × 10−5 min−1).The amidolysis was carried out in 1:9
acetonitrile–Tris HCl buffer of pH 7.75 at 45 ◦ C which is found to
be the optimum temperature and the kinetics of amidolysis with
catalysts was evaluated spectrophotometrically at 374 nm by mon-
itoring the amount of p-nitroaniline released. A blank experiment
was also carried out under similar conditions without the catalysts.
The pseudo-first order rate constants were obtained from the fol-
St–time for saturation, nd– not observed saturation even after several days, all kinetic parameters within the limit 5%.
Proficient pre-organization is essential for successful imprint-
ing [21–24]; from the rac-TSA synthesized, the achiral organic
monomers (4-VIm, MAA and AA) having the functional groups
present in active site of chymotrypsin −imidazole, carboxyl
and hydroxyl- formed TSA-monomer pre-polymerization complex
with both L- and D-TSA. A suitable crosslinker with sufficient
crosslink density freezes the stable, H-bonded TSA-monomer
pre-polymerization complex into rigid polymer matrix and after
polymerization, removal of TSA leaves behind its ‘3D- imprints’
lined with catalytic entities. The ‘specificity determinant’ C6H5CH2
group of TSA, designs the hydrophobic pocket in the poly-
mer matrix. The pre-polymerization complexes (PPC) formed are
depicted in Scheme 2. The PPC of methacrylated l-amino acid
monomers (a) and that of their organic analogues-vinylimidazole,
methacrylic acid and allyl alcohol (b) are held together by H-
bonding interactions. The chiral l-amino acid monomers form
PPC selectively with L-TSA and fabricate catalytic cavities with
l-configuration in the polymer matrix. But, the achiral organic
monomers form PPC with TSA having both L- and D- configurations.
The ␲-stacking interaction of pyridine moiety with the specificity
determinant C6H5CH2 group of TSA makes the PPC (c) more stable.
The structural difference between the imprinted polymers
become more obvious upon assessment of the data received from
BET surface area analysis (Table 2). The higher surface area of
imprinted polymers is indicative of distribution of pores (cavities)
and higher binding capacity. The pore volume measurements show
that C3 has a maximum pore volume of 0.898 cm3/g [25].
ꢀ
ꢁ
A
max
max
−A
lowing equation: ln
= kt where Amax is the absorbance of
A
t
p-nitroaniline at infinite time, At is the absorbance of p-nitroaniline
at time t and k is the pseudo-first-order rate constant. At 45 ◦ C, in
1:9 MeCN–Tris HCl buffer of pH 7.75 the rate constant for the blank
is found to be 0.52 × 10−4 min−1. The rate acceleration kacc val-
ues of the polymer catalysts were evaluated in terms of the ratio
kobs./kuncat.and the imprinting efficiency kim of the polymer cata-
lysts is expressed in terms ofkMIP/kCP. The possible mechanism for
the amidolysis catalyzed by C3 is depicted in Scheme 3.
In our previous study of the amidolysis of Z-L-Phe-PNA
using trifunctional imprinted polymer catalyst (C3) comprises of
methacrylated l-amino acid monomers; it was found that the
90% EGDMA crosslinking is essential to preserve the 3D-geometry
of the imprint and to facilitate the uptake of the substrate Z-L-
Phe-PNA molecule [13]. But, the rate acceleration exhibited by
trifunctional mimic P3a comprises of the organic analogues of
amino acid monomers polymerized with 90% EGDMA crosslinks
is only 6.78, compared to the kacc value 75.38 for C3, 90% EGDMA
crosslinked trifunctional mimic made up of methacrylated l-amino
acid monomers. Further, the imprinting efficiency values kim evalu-
ated are 1.02 (P3a) and 4.22 (C3) indicating poor catalytic efficiency
of P3a. Moreover P3a did not exhibit any saturation in its amidase
The amount of bound TSA was quantitatively eluted by Soxhlet
extraction using chloroform and the TSA in the eluate was esti-
mated spectrophotometrically at 245 nm. The specific rebinding of
the TSA molecules by the imprinted polymers C3, P3c and P3e were
carried and the am−oCunt of TSA rebound was determined using the
C
V
)
(
e
0
equation, Qe
=
where C0 (mmol/g) and Ce (mmol/g) are
M

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D. Mathew et al. / Applied Catalysis A: General 528 (2016) 93–103
Scheme 2. Pre-polymerization complexes.
Fig. 2. SEM pictures a) P3c, b) P3e and c) C3.
Scheme 3. Catalytic amidolysis of Z-l-Phe-PNA using C3.

D. Mathew et al. / Applied Catalysis A: General 528 (2016) 93–103
97
Table 4
Co-operative effect of functional monomers and catalytic amidolysis.
Catalysts with l-amino acid monomers
Catalysts with organic monomers
Enzyme mimic
kacc
kim
St (h)
Enzyme mimic
kacc
kim
St (h)
C1a
C1b
C1c
C2a
C2b
C2c
C3
29.62
12.50
15.00
41.53
45.77
15.96
75.38
2.37
1.12
1.08
2.60
2.74
1.15
4.22
50
P1a
P1b
P1c
P2a
P2b
P2c
P3c
10.46
6.52
6.34
17.78
14.08
7.66
1.05
1.03
1.02
1.06
1.04
1.01
2.12
70
nd
nd
45
45
nd
24
120
120
40
35
120
22
35.68
Allkineticparameters within the limit 5%.
Table 5
Co-operative effect of functional monomers in the trifunctional mimics in the cat-
alytic amidolysis-a comparison.
Enzyme mimic
kacc
kim
St (h)
C3
P3c
P3e
75.38
35.68
44.60
4.22
2.12
2.46
22
24
20
Allkineticparameters within the limit 5%.
polymer matrix of P-type polymer catalysts may be composed of
both L- and D-cavities.
3.3. Co-operative effect of functional monomers on amidolysis
The number, strength and orientation of the functional groups
and their interactions with the print molecule are the key fac-
tors in determining the shape-selective binding of the substrate
molecules. The individual and combined effects of imidazole,
carboxyl and hydroxyl groups of methacrylated l-amino acid
monomers in the chymotrypsin mimics on the amidolysis of Z-l-
Phe-PNA were demonstrated in detail in our previous paper and
it was observed that only the mimics containing imidazole moi-
eties exhibit chymotrypsin like behaviour towards Z-l-Phe-PNA.
The same effect was observed for polymer catalysts with organic
monomers (Table 4).
Fig. 3. % Amidolysis vs time.
activity even after several days. The lack of well-defined substrate
recognition sites in the matrix of P3a as a consequence of higher
rigidity provided by 90% EGDMA crosslinks may be the reason for
its poor catalytic activity.
Among the monofunctional catalysts, the highest rate accel-
eration kacc value (10.46) is observed for P1a polymer with
imidazole moieties. The bifunctional mimic P2a with imidazole
and carboxylic groups exhibited enhanced [kcat/kuncat] value of
17.78 and P2b with imidazole and hydroxyl residues showed
[kcat/kuncat]value of 14.08. But bifunctional mimic P2c with car-
boxyl and hydroxyl residues is found to be catalytically poor with
kacc value of 7.66. The trifunctional polymer catalyst P3c showed
the maximum kacc value 35.68. The polymer catalysts formed from
organic monomers showed lower kacc values compared to the cat-
Since the functional groups in the organic monomers VIm, MAA
and AA are less flexible compared to the amino acid monomers
MALH, MALA and MALS, the super crosslinked networks of EGDMA
makes the polymer highly rigid to impede the release of the print
molecule and uptake of the substrate molecule and catalysis was
not observed in the hydrolytic reaction (Fig. 3).
There are some reports in which imprinted catalysts have been
prepared with low degree of crosslinking [26,27,32]. Hence in the
case of trifunctional mimics of organic monomers, crosslink den-
sity in the range of 10–30% (P3b-P3d) was used to freeze the
catalytic functions intact and to preserve the exact geometry of
the imprint. The amidolysis was carried out in the framework of
Michaelis-Menten kinetics using polymers P3b-P3d with 10, 20
and 30% EGDMA crosslinks and the results are given in Table 3.
The rate acceleration value exhibited by P3b was observed to be
21.62. The trifunctional mimic P3c with 20% EGDMA crosslinks
was observed to be the best polymer catalyst with kacc, kim and
St values 35.42, 2.12 and one day respectively. On further increase
in crosslink density to 30% decelerates the rate acceleration (23.56)
and imprinting efficiency (1.37). The P3c polymer showed 5.2 times
rate acceleration than P3a polymer; but, showed 2.1 times lower
rate acceleration compared to the trifunctional mimic C3 com-
prises of amino acid monomers. The methacrylated l-amino acid
monomers selectively forms TSA-monomer pre-polymerization
complex with L-TSA over D-TSA and leaves out memory cavities
with l-configuration and selectively binds the substrate Z-L-Phe-
PNA. But, due to the achiral nature of organic monomers, the
alysts derived from amino acid monomers. The ratios kCac1ca/kP1a
,
acc
kCac2ca/kPac2ca, kCac2cb/kP2b and kCac3cc/kP3c are 2.83, 2.34, 3.25 and 2.11
acc
acc
respectively. Further the ratio kCac1cc/kPac1cc and kCac2cc/kaPc2cc are 2.37 and
2.08. These results reveal that the catalytic property contributed by
the hydroxyl group of allyl alcohol is low in the amidolytic reaction
compared to serine.
The catalytic amidolysis of the substrate Z-l-Phe-PNA using P3c
comprising of rigid organic monomers is depicted in Scheme 4.
Since the polymer catalysts with allyl alcohol showed poor
nucleophilicity compared to serine in the amidolytic reaction, we
synthesized a trifunctional polymer catalyst P3e by replacing allyl
alcohol by vinylpyridine. It is reported that pyridine units provide
better nucleophilic sites in chymotrypsin mimics for esterolytic
reactions [18]. The mimic P3e exhibited a kaccvalue of 44.60. The
ratio kPac3ce/kP3c was found to be 1.25. The selective binding of sub-
acc
strate in the catalytic cavity of P3e is expected to be facilitated
by the ꢀ stacking interactions of pyridine moieties. The proton

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D. Mathew et al. / Applied Catalysis A: General 528 (2016) 93–103
Scheme 4. Amidolysis of Z-l-Phe-PNA using enzyme mimic P3c.
Scheme 5. Amidolysis of Z-l-Phe-PNA using enzyme mimic P3e.
shuttle between pyridine and carboxylic functions at the active
sites increases the nucleophilicity of imidazole units. Moreover,
P3e attains saturation within a shorter time period (20 h) than P3c
(24 h). The proposed mechanism is depicted in Scheme 5.
A comparison of the amidase activities of the trifunctional mim-
ics P3c and P3e derived from achiral organic monomers and C3
made from chiral amino acid monomers is listed in Table 5. Thus,
for polymers comprises of amino acid monomers, the rate enhance-
ment is observed only with higher extreme crosslink density. But,
for the polymers of organic monomers, a rate enhancement is
observed, in the lower range of crosslink density (20%).
3.4. Effect of pH on the amidase activities of the trifunctional
enzyme mimics
Amidolysis of Z-l-Phe-PNA was carried out in the pH range
6.0–8. The polymers P3c, P3e and C3 follow the same pH profile
that it exhibits an optimum rate at pH 7.75 (Fig. 4). The mechanism

D. Mathew et al. / Applied Catalysis A: General 528 (2016) 93–103
99
Fig. 4. Effect of pH on catalytic amidolysis.
Fig. 6. Effect of temperature on saturation time.
Fig. 5. Effect of temperature on rate acceleration.
Fig. 7. Michaelis- Menten plots.
shows that the catalytic activity of the enzyme mimics arises due
to the cooperative effect of neutral imidazole groups and most of
the imidazole units are non-protonated at the optimum pH 7.75.
As the pH of the reaction medium decreases protonation of imi-
dazole nitrogen decreases the nucleophilicity. At higher pH of the
reaction medium, the rate of uncatalyzed reaction increased due
to the increased concentration of hydroxyl ions. At higher pH of
the reaction medium, the polymer bound negatively charged car-
boxylic acid groups provides electrostatic exclusion of nucleophilic
OH- from polymer-substrate complex resulting in the inhibition
[9]. At higher pH of the reaction medium, the rate of uncatalyzed
reaction increased due to the increased concentration of hydroxyl
ions.
3.6. Michaelis- Menten kinetics of polymers
The Michaelis-Menten kinetic studies were carried using the
polymer catalysts P1b-P3e to evaluate their substrate affinity
towards Z-l-Phe-PNA in the enzyme-mimetic catalytic amidolysis
and a comparison was made with the polymer catalysts made up
of l-amino acid monomers [1,5,28,29]. Different enzyme-substrate
concentrations were taken (1:25 to 1:150). The Michaelis- Menten
plots for the trifunctional polymer catalysts P3b-P3e are shown in
Fig. 7.
The competence of the polymer catalyst to mimic natural chy-
motrypsin has been evaluated in terms of substrate affinity of the
polymer catalysts from the double reciprocal plots for the ami-
dolytic reaction (Fig. 8).
The enzyme characteristics- Km, Vmax/Km and kcat/Km- evalu-
ated are given in Table 6. Both P3c and P3e are found to be obeying
Michaelis- Menten kinetics with Km values 1.419 and 1.151 mmol
respectively. The ratio KPm3c/KmP3e was observed to be 1.23 strongly
supporting the ␲-stacking interactions of pyridine moiety. The
mimic C3 exhibited more pronounced initial burst kinetics and sat-
uration than P3c and P3e. The ratios KPm3c/KmC3 and KmP3e/KmC3 were
found to be 4.26 and 5.26. Specific stereochemical “memory” of
l-configuration due to the presence of l-amino acid monomers is
responsible for the higher substrate specificity of the mimic C3.
3.5. Effect of temperature on the catalytic activities of the
trifunctional polymer catalysts
To investigate the optimum temperature for catalytic amidoly-
sis, the reaction was carried out at different temperatures (30, 35,
40, 45 and 50 ◦ C) using the mimics P3c, P3e and C3 and the results
are given in Fig. 5. It is reported that, as temperature increases, the
catalytic sites become more accessible and exhibit higher catalytic
efficiency [3]. The time taken for saturation in amidase activity of
the polymer catalyst is also found to be decreasing as temperature
increases (Fig. 6).

100
D. Mathew et al. / Applied Catalysis A: General 528 (2016) 93–103
Fig. 9. Enantioselective binding of substrate by imprinted polymer.
enantioselectivity towards L-substrates. If an optically active
template is used as TSA for imprinting, the polymer catalysts cause
enantiomers to react at different rates. Chymotrypsin exhibits
stereochemical specificity that they catalyze the reactions of l-
amino acid esters or amides. It has been reported that the selective
stabilization of one particular complex conformation should have
a superficial influence on the enantioselectivity of the enzyme
mimic (Fig. 9).
Fig. 8. Lineweaver-Burk plots.
It is worth emphasizing that the chymotrypsin mimic C3, a
trifunctional polymer catalyst of l-amino acid monomers, essen-
tially incorporates the L-Phe-PNA primarily over the D-Phe-PNA
to form the catalyst–substrate complex with KDm/KmL = 10.59
and hydrolyzes the L-Phe-PNA competently with kLacc/kaDcc = 7.56.
Thus, the enzyme mimic C3 have memory cavities with the shape
of L-TSA, and show proficient substrate-stereospecific amidolysis
of Z-L-Phe-PNA through shape-selective binding of the substrate
into its recognition sites. The KDm/KLm and kaLcc/kaDcc values of P3c
(1.03, 1.02) and P3e (1.05 and 1.03) indicate that these polymers
possess little enantioselectivity. This result probably came from the
presence of both Land D-cavities in the polymer matrix.
Table 6
Michaelis-Menten kinetics for the copolymers of amino acid monomers.
Trifunctionalpolymer catalystsKm(mmol)103Vmax/Km(min−1)kcat /Km(mmol/min)
C3
0.27
26.74
0.829
4.956
1.427
6.718
4.934
0.153
0.914
0.263
1.239
P3b
P3c
P3d
P3e
2.516
1.419
2.890
1.151
Allkinetic parameters within the limit 5%.
Concerning the substrate-binding affinity K−m1 of the polymer
catalysts in the catalyst-substrate complex formation, the negative
activation free energy ꢁG#binding = −RTlnK−m1 indicates the non-
barrier of the substrate-binding process. The ꢁG#binding of C3 for
Z-L-Phe-PNA and Z-D-Phe-PNA were observed to be −5.17 kcal/mol
and −3.68 kcal/mol respectively. The ꢁG#binding values of the poly-
mer catalysts are given in Table 7. Relatively lower ꢁG#binding of C3
for Z-L-Phe-PNA probably reflected the formation of well-defined
three dimensional memory cavities with l-configuration for the
incorporation of the substrate.
Scheme 6. Simplified reaction process of amidolysis with TSA imprinted polymer
catalyst.
The polymer catalysts P3b and P3d from organic monomers do
not obey Michaelis- Menten kinetics and behave like uncatalyzed
reaction. The polymer P3b having lower EGDMA crosslinking (10%)
is not capable of preserving the exact geometry of the imprint and
P3d with higher crosslink density (30%) is having highly rigid poly-
mer network reducing the accessibility of the catalytic sites to the
substrate molecule.
The substrate affinity of C3 is reflected mainly in the acti-
vation free energy of the shape-selective binding of substrate.
ꢂ
ꢃ
The ꢁ ꢁG#binding facilitated binding of Z-L-Phe-PNA as com-
pared with the D-form in the range of −1.49 (C3) to 0.031 (P3e)
and 0.019 kcal mol−1 (P3c). Predominant reaction for the L-form
of the substrate over the D-form was also reflected in the dif-
ꢂ
ꢃ
ference of activation free energy of ꢁ ꢁG#
for the reaction
C.S
[
]
ꢂ
ꢃ
process; ꢁ ꢁG#
values observed were −1.27, −0.012 and
C.S
[
]
3.7. Enantioselective amidolysis
−0.019 kcal mol−1 for the polymer catalysts C3, P3c and P3e. As
a result, the substrate stereospecificity of the highly competent
C3 catalyst is reflected mostly by the activation free energy of the
stereospecific substrate-binding in the recognition site in the poly-
mer catalyst and to a certain extent by the stereospecific reaction
in the catalyst–substrate complex.
The substrate stereospecificity of the mimics P3c and P3e
were investigated in the amidolysis of Z-L/D-Phe PNAs [9,17,30].
The substrate stereospecificity is evaluated in terms ofkLacc/kaDcc
,
ꢂ
ꢃ
ꢂ
ꢃ
KDm/KLm, ꢁ ꢁGb#inding and ꢁ ꢁG#
and compared with the
mimic C3 [26,27]. The activation free energy ꢁ ꢁG#binding of
the shape-selective binding of substrate Z-L-Phe-PNA is evalu-
C.S
[
]
ꢂ
ꢃ
Even though 20% EGDMA crosslinked polymers P3c and P3e
obey Michaelis-Menten kinetics, the lack of substrate stereose-
lectivity can be explained as due to the incorporation of both D-
and L-TSA molecules along with the achiral monomers in the PPC.
The chirality of the print molecule along with chiral monomers
bestowed enhanced enantioselectivity on C3. The CT mimic C3 is
found to be 9.64 times stereoselective than P3e due to the pres-
ence of stereoselective L-cavity furnished by L-TSA-l-Monomer
pre-polymerization complex during TSA imprinting, which selec-
tively binds L-PNA over D-PNA.
ꢄ
ꢅ
D
m
L
K
ated as −RTln
and the difference of activation free energy
K
m
ꢄ
ꢅ
ꢂ
ꢃ
L
k
k
ꢁ
ꢁG#[C.S] as −RTln
in the frame work of Michaelis-
acc
D
acc
Menten kinetics (Scheme 6).
Since
the
racemic
phosphonate
TSA, phenyl-1-(N-
benzyloxycarbonylamino)-2-(phenyl)ethyl phosphonate was
used as the print molecule for the synthesis of all the mimics,
only C3 with methacryloyl-l-amino acid residues can exhibit

D. Mathew et al. / Applied Catalysis A: General 528 (2016) 93–103
101
Table 7
Kinetic parameters supporting substrate stereospecificity of the polymer catalysts.
ꢂ
ꢃ
ꢂ
ꢃ
Polymercatalysts
kLacc /kDacc
KDm/KLm
ꢁG#binding (kcal/mol)
ꢁ
ꢁG#binding
ꢁ
ꢁG#
C.S
[
]
L-PNA
D-PNA
C3
P3c
P3e
7.56
1.02
1.03
10.59
1.03
1.05
−5.17
−4.13
−4.26
−3.68
−4.09
−4.23
−1.49
−1.27
−0.019
−0.031
−0.012
−0.019
Allkineticparameters within the limit 5%.
Table 8
Table 9
Influence of chiral amino acid monomers and achiral organic monomers on substrate
specificity of polymer catalysts.
Influence of chiral amino acid monomers and achiral organic monomers on substrate
shape-selectivity of polymer catalysts.
Kineticparameter
C3
P3c
P3e
Selectivity ratio
P3c
P3e
C3
kZacc /kBoc
2.56
4.21
8.04
3.18
4.83
8.85
4.56
8.21
14.24
Phe
acc
Tyr
acc
Trp
acc
Ala
k
123.78
108.58
24.86
60.54
51.09
11.35
9.15
66.54
55.39
14.45
9.65
acc
kZacc /kFmoc
k
acc
k
kZacc /kNphth
acc
k
19.43
All kinetic parameters within the limit 5%.
acc
Allkineticparameters within the limit 5%.
3.8. Substrate specific amidolysis of P3c and P3e
The substrate-specificity ‘the discrimination of a substrate over
other competing molecules’ of the TSA-built polymer catalysts P3c
and P3e towards chymotrypsin specific and non-specific amino
acids was investigated [19,20,31,32]. The mimics showed higher
specificity towards Z-L-Phe-PNA and Z-L-Tyr-PNA. But the speci-
ficity towards Phe is slightly higher than Tyr (Table 8). However,
the mimics P3c and P3e exhibited almost half the specificity of
C3. The mimics showed reduced specificity towards Trp, which is a
chymotrypsin specific amino acid. The rate acceleration kTarcpc values
Trp
acc
observed are 24.86 (C3), 11.35 (P3c) and 14.45 (P3e). The lower k
values are due to the incompatibility of the indole ring in the cat-
alytic cavity. The substrate Ala-PNA was found to be non-specific to
the mimics. Like natural chymotrypsin, the mimics are also found
to be non-specific towards Ala-PNA.
Fig. 10. TSA inhibition on the amidase activity of C3, P3c and P3e.
3.9. Substrate shape-selective amidolysis
The substrate-recognition ability of TSA imprinted polymer
catalysts P3c and P3e was investigated using phenylala-
nine p-nitroanilides with various N˛-protecting groups
like benzyloxylcarbonyl(Z), N-t-butyloxycarbonyl (Boc), 9-
fluorenylmethoxycarbonyl (Fmoc) and N-phthaloyl (Nphth) [27].
The mimics kept the structure of the imprinted TSA with ben-
zyloxycarbonyl group in memory and recognized the structure
of Z-L-Phe-PNA and shape-selectively accommodated it in the
catalytic cavity. The substrate selectivity is observed in the order
Z > Boc > Fmoc > Nphth-L- Phe-PNAs. The capacity of the mimic
to recognize the structure of the protecting group was evaluated
3.10.TSA inhibition study
A typical property of enzymes is the inhibition by certain
molecules. A transition-state analogue imprinted polymer should
show competitive inhibition by the template [6,33,34]. The TSA
inhibition studies were carried out in the amidolytic reaction in
presence of the imprinted phosphonate TSA using the polymer cat-
alysts P3c, P3e and C3.
As shown in Fig. 10, as the concentration of the template TSA
increases, the amidase activity of the imprinted polymers was
found to be fairly inhibited and then finally gets stopped. This
results may be explained as due to “the shape-selective rebind-
ing” of the imprinted TSA over the substrate Z-l-Phe-PNA. A better
rebinding within shorter time is observed for C3 due to the exact
geometry of the imprinted TSA in the polymer matrix. This would
be a further proof for catalysis to occur inside the imprinted cavity.
as shape-selectivity, in terms of the ratio kZacc/kaBcocc, kaZcc/kFmoc
acc
and kaZcc/kaNcpchth. The shape-selectivity ratios were evaluated;
compared with the substrate shape-selective stabilization power
of C3. The kacc values calculated for the mimic P3c were found to
be 60.54 (Z-L-Phe-PNA), 23.65 (t-Boc-L-Phe-PNA), 14.38 (Fmoc-
L-Phe-PNA) and 7.53 (Nphth-L- Phe-PNA). For the mimic P3e the
corresponding values are 66.54, 20.92, 13.78 and 7.52. The sub-
strate shape-selectivity ratios exhibited by the polymer catalysts
are recorded in Table 9. The maximum substrate-shape selectivity
is observed between Z and Nphth-L-Phe-PNAs and the selectivity
kZacc/kaNcpchth value was 8.04 for P3c and 8.85 for P3e. But, for the
3.10. Comparison with natural chymotrypsin
The kinetics of amidolysis of Z-l-Phe-PNA catalyzed by trifunc-
tional mimics P3c and P3e synthesized from organic analogues
of amino acid monomers was compared with the amidolysis cat-
alyzed by natural chymotrypsin, CT and the mimic C3 made up of
methacrylated amino acid monomers (Fig. 11).
mimic C3, the selectivity kZacc/kaNcpchth value was 14.24.

102
D. Mathew et al. / Applied Catalysis A: General 528 (2016) 93–103
more in the case of polymers derived from organic monomers.
The results show that the cavities in the catalysts P3c and P3e get
deformed easily than C3.
4. Conclusion
Polymer catalysts with the imprints of phenyl-1-(N-
benzyloxycarbonylamino)-2-(phenyl)ethyl phosphonate were
made up of achiral organic monomers and l- amino acid monomers
to mimic natural chymotrypsin. The polymers derived from l-
amino acid monomers are found to be substrate stereospecific.
But the catalysts synthesized from organic monomers pos-
sess both L-and D-cavities in the polymer matrix and hence
exhibited no enantioselectivity. Imidazole group of histidine
residue/vinylimidazole monomer is the essential catalytic group
in the active site of the mimics as in the case of hydrolase protein
chymotrypsin. Allyl alcohol showed poor nucleophilicity in the
amidolytic reaction. The pyridine moiety is capable of exerting
H-bonding interaction with carboxyl group of methacrylic acid
and ꢀ- stacking interaction with aromatic side chain of amino acid
residue of the substrate p-nitroanilide. For the polymers derived
from organic monomers, the rate enhancement and enzyme-like
behaviour are observed, in the lower range of crosslink density
(20 mol%). The thermal stability and recycling ability of these
mimics were lower than the trifunctional mimic from amino acid
monomers, but higher than natural chymotrypsin.
Fig. 11. % Amidolysis using natural chymotrypsin CT and trifunctional mimics P3c,
P3e and C3.
Acknowledgements
The authors gratefully acknowledge the support from Coun-
cil of Scientific and Industrial Research (CSIR), India for awarding
junior and senior research fellowships to Divya Mathew. We are
also thankful to IIRBS, Mahatma Gandhi University, Kottayam, Ker-
ala, India for providing facilities for spectral analysis and School of
Biosciences, Mahatma Gandhi University for providing facilities for
incubation studies.
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