Hemo-acrylic polymers as catalyst for the oxidative dehalogenation of 2,4,6-trichlorophenol. Chloroperoxidase's mimic imprinting effects

Article abstract of DOI:10.1016/j.molcata.2011.11.014

Acrylic polymers with catalytic activity for the oxidative degradation of 2,4,6-trichlorophenol (TCP) were developed. In order to mimic the active site of chloroperoxidase (CPO), chloro-iron(III)-protoporphyrin IX was used as the catalytic centre, and methacrylamide (MA) and 4-vinylpyridine (VPY) were used as the monomers that build up the active sites. Taking as basis 3:1 (w/w) acid:basic aminoacidic composition of CPO, three MA:VPY combinations were tested: one keeping the same ratio (3:1) i.e. 25% VPY in the functional monomer mixture, one with lower content of the basic monomer (9:1) i.e. 10% VPY, and one with higher concentration of it (1:1) i.e. 50% VPY. Polymers synthesized with the lowest VPY content exhibited the highest catalytic efficiency, which was improved by the creation of specific TCP binding sites through molecular imprinting technology. In these way, synthetic enzymes with useful properties for analytical and bioremediation applications were obtained.

Full text of DOI:10.1016/j.molcata.2011.11.014

Journal of Molecular Catalysis A: Chemical 353–354 (2012) 117–121
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Journal of Molecular Catalysis A: Chemical
journal homepage: www.elsevier.com/locate/molcata
Hemo-acrylic polymers as catalyst for the oxidative dehalogenation of
2,4,6-trichlorophenol. Chloroperoxidase’s mimic imprinting effects
Goretti Díaz-Díaz, M. Carmen Blanco-López, M. Jesús Lobo-Castan˜ón, Arturo J. Miranda-Ordieres,
Paulino Tun˜ón-Blanco∗
Departamento de Química Física y Analítica, Universidad de Oviedo, Julián Clavería, 8, 33006 Oviedo, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2011
Received in revised form
30 September 2011
Accepted 13 November 2011
Available online 22 November 2011
Acrylic polymers with catalytic activity for the oxidative degradation of 2,4,6-trichlorophenol (TCP) were
developed. In order to mimic the active site of chloroperoxidase (CPO), chloro-iron(III)-protoporphyrin
IX was used as the catalytic centre, and methacrylamide (MA) and 4-vinylpyridine (VPY) were used as the
monomers that build up the active sites. Taking as basis 3:1 (w/w) acid:basic aminoacidic composition
of CPO, three MA:VPY combinations were tested: one keeping the same ratio (3:1) i.e. 25% VPY in the
functional monomer mixture, one with lower content of the basic monomer (9:1) i.e. 10% VPY, and one
with higher concentration of it (1:1) i.e. 50% VPY. Polymers synthesized with the lowest VPY content
exhibited the highest catalytic efficiency, which was improved by the creation of specific TCP binding
sites through molecular imprinting technology. In these way, synthetic enzymes with useful properties
for analytical and bioremediation applications were obtained.
Keywords:
Heterogeneous catalyst
Chloroperoxidase mimic
2,4,6-Trichlorophenol dechlorination
Molecularly imprinted polymer
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
to microorganisms, and therefore a biological pre-treatment is
required. Depending on the enzyme, the reaction conditions and
Chlorophenols constitute a group of priority pollutants listed
by the US Environmental Protection Agency (EPA) and the Euro-
pean Union (EU) in the Clean Water Act and the Decision
2455/2001/EC, respectively. They have been widely employed
in different manufacturing processes, and they also result from
pulp bleaching, drinking water dechlorination and incineration
processes. Chlorophenols can be found in ground and wastew-
aters [1], and their toxicity and persistence in the environment
increase with the degree of chlorine substitution. Therefore, 2,4,6-
trichlorophenol (TCP) is one of the most harmful chlorophenols due
to the pulmonary lesions that it causes.
When chlorophenols reach soils or natural water reservoirs,
they are degraded by microorganisms, which live in muds,
sludges and sewages. This natural degradation consists in reductive
dehalogenation in anaerobic conditions or in oxidative path-
ways. However, the majority of chlorophenols are poisonous
the chlorophenol characteristics, the sole use of enzymes could
lead to the complete degradation of the pollutant, and the sub-
sequent treatment with microorganisms could be avoided [2,3].
Peroxidases have been commonly used for this purpose [4,5]. As
an alternative solution there is a recent interest in developing arti-
ficial catalysts mimicking these natural enzymes. These synthetic
receptors can work in harsh conditions (pH, temperature), which
provides advantages over natural enzymes. As a first approach
to develop catalysts that mimic the degradation ability of natu-
ral enzymes over chlorophenols, water soluble iron(III)-porphyrins
have been used as homogeneous catalysts in batch assays [6,7]. A
step forward is the immobilization of metalloporphyrins on differ-
ent solid supports that has been found efficient not only to ensure
catalyst recovery but also to prevent loss of activity through cata-
lyst aggregation [8–12]. Although these systems allow the catalytic
degradation of chlorophenols, they lack of selectivity. In this con-
text, the generation of recognition and catalytic sites in polymeric
macromolecular synthetic receptors by molecular imprinting tech-
nology have emerged as an attractive approach in order to mimic
the catalytic ability of enzymes [13,14]. These artificial receptors
consisted on organic polymers synthesized from a suitable combi-
nation of monomers and cross-linkers, where the catalytic centre
is included. Metalloporphyrins have been used as catalytic centres,
and the resulting artificial catalysts exhibit both hydrophobic bind-
ing pockets and polar recognition groups, capable of recognizing
polar functional groups under aqueous conditions [15–17].
Abbreviations:
TCP, 2,4,6-trichlorophenol; CPO, chloroperoxidase; MA,
methacrylamide; VPY, 4-vinylpyridine; EPA, US Environmental Protection
Agency; EU, European Union; 4-C-3-MP, 4-chloro-3-methylphenol; 2,4-DCP, 2,4-
dichlorophenol; 2,4-DNP, 2,4-dinitrophenol; DCQ, 2,6-dichloro-1,4-benzoquinone;
AIBN, 2,2ꢀ-azo-bis-(isobutyronitrile); DMSO, dimethylsulfoxide; EGDMA, ethileneg-
lycoldimethacrylate; HPLC, High Performance Liquid Chromatography; ODS,
octadecylsilane.
∗
Corresponding author. Tel.: +34 985103487; fax: +34 985103125.
E-mail address: ptb@uniovi.es (P. Tun˜ón-Blanco).
1381-1169/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcata.2011.11.014

118
G. Díaz-Díaz et al. / Journal of Molecular Catalysis A: Chemical 353–354 (2012) 117–121
Taking chloroperoxidase as a model enzyme, the objective of
2.3. Procedures
this work was the synthesis of a polymer that resembles CPO and
mimics its catalytic activity towards the oxidative dehalogena-
tion of TCP, which is one of the most substituted chlorophenols.
To achieve this aim, we have reproduced the catalytic centre of
CPO with Fe(III)-protoporphyrin IX, which has been covalently
included as a co-monomer in the polymer backbone through its two
vinyl groups. In our previous works, we have used either a neutral
(methacrylamide, MA) [15,18] or a basic (4-vinylpyridine, VPY) [15]
functional monomer to prepare the catalysts. But in order to mimic
the aminoacidic residues of CPO, both neutral and basic monomers
are needed. Therefore, in this work we propose the use of mixtures
of the two monomers for preparing the polymers. Different MA:VPY
compositions have been tested in order to study the effect of these
two monomers in the catalytic behaviour of the synthetic catalyst.
In addition, we have tried to improve the properties of the catalysts
through an imprinting process, with the ultimate goal of obtaining
a catalytic synthetic receptor with analytical and biotechnological
applications such as development of sensors for the detection of
the pollutant or decontamination applications. Equilibrium experi-
ments and kinetic evaluation were carried out through batch assays
in order to assess the catalytic efficiency and selectivity of these
polymers towards the oxidative dehalogenation of TCP.
The preparation of the polymers was carried out as follows:
chlorohemin (5 ␮mol), MA and VPY (25 ␮mol total amount, with
different ratios of MA to VPY as explained below), and EGDMA
(250 ␮mol) were placed in a vial, and DMSO (7.5 mL) was employed
as porogenic solvent to achieve the solubilization of hemin. The vial
was sealed and the mixture was purged with nitrogen for 15 min.
Then AIBN (240 mg) was quickly added and the mixture was purged
again for 5 min. The polymerization reaction was carried out in an
oven at 65 ◦C for 24 h. As a first approach, we have selected a propor-
tion of MA to VPY in the pre-polymerization mixture of 3:1, which
matches the aminoacidic content of the natural CPO, 16% (w/w)
of neutral aminoacids and 6% of basic aminoacids over the whole
protein [19]. In addition, two more MA:VPY ratios of 1:1 and 9:1
were tested. These proportions correspond to VPY molar content
of 25, 50 and 10% VPY with respect to MA. The resultant polymer
was subjected to Soxhlet extraction using methanol with 15% (v/v)
acetic acid in order to remove the non-polymerized hemin and then
washed with methanol to remove the acid. Finally, the polymers
were crushed and sieved to obtain particles sized below 25 ␮m
that were used in the batch experiments. Molecularly imprinted
polymers (MIPs) were synthesized in the same way, adding TCP
(5 ␮mol) in the pre-polymerization mixture. TCP was subsequently
extracted from the polymer by Soxhlet, which was carried out until
no TCP was detected by cyclic voltammetry.
2. Experimental
The iron present in the polymers was determined by ICP-MS
measuring the released iron after lixiviation of the polymers with
hydrogen peroxide 15% (w/v) in acidic medium (2 M HClO4). 20 mg
of each polymer were suspended in 1 mL of HClO4 2 M, H2O2 15%
(w/v) was added, and the mixture was stirred at 600 rpm for 14 h.
Before ICP-MS analysis, the mixture was centrifuged at 10,000 rpm,
and iron was determined in the supernatant. The lixiviation was
carried out again for 15 min in the same conditions, and significant
iron quantities were only detected in the first supernatant.
The polymers were morphologically characterized by scanning
electron microscopy (SEM) on a MEB JEOL-6100 instrument. SEM
specimens were prepared by placing a little amount of the polymer
in a support and coating it with gold under vacuum.
Kinetics of the oxidative transformation of TCP with the differ-
ent polymers as catalyst was studied through batch experiments in
acetate 0.01 M pH 5.0, with 10% DMSO in order to improve the wet-
tability of the polymers. The concentration of polymer and H2O2
was fixed at 0.5 g L−1 and 10−3 M, respectively, measuring the initial
reaction rate to increasing concentrations of TCP.
2.1. Reagents
Chlorohemin (iron(III)-protoporphyrin IX) was purchased from
Frontier Scientific (UK). 2,4,6-Trichlorophenol (TCP), 4-chloro-
3-methylphenol (4-C-3MP), ␣,␣ꢀ-azoisobutyronitrile (AIBN) and
dimethylsulfoxide (DMSO) were purchased from Fluka. Ethy-
lene glycol dimethacrylate (EGDMA), acetic acid and perchlo-
ric acid were purchased from Merck. Methacrylamide (MA),
4-vinylpiridine (VPY), 2,6-dichloro-1,4-benzoquinone (DCQ) 2-
chloro-1,4-benzoquinone and 3-methyl-1,4-benzoquinone were
purchased from Aldrich. Hydrogen peroxide was obtained from
Prolabo. Methanol and acetonitrile were purchased from J.T. Baker.
All chemicals were of analytical grade and used as received
except for EGDMA, whose inhibitors were removed by successive
liquid–liquid extractions. Buffer solutions were prepared with high
purity water produced by a Milli-Q purification system (Millipore),
and stock standard solutions of phenols were used to daily prepare
working standard solutions by suitable dilution in a 0.01 M acetate
buffer.
The binding of TCP to the polymers was studied by equilibrium
experiments, carried out in 0.01 M acetate buffer with 10% of DMSO,
and varying the TCP concentrations from 2.5 × 10−5 to 5 × 10−4 M
with a fixed concentration of polymer (125 ␮g mL−1) for 12 h in a
tilting mixer. The amount of free TCP was determined by HPLC-
UV/VIS.
2.2. Apparatus
Selectivity studies were performed in two different ways: firstly,
TCP kinetics was carried out at a fixed concentration of the inter-
ferent (10−4 M), and secondly, the interferent itself was used as
substrate of the catalytic reaction, and the resulting product was
quantified by HPLC-UV/VIS.
An HPLC instrument (Shimadzu 20A) made up of a gradient sys-
tem fitted with a SPD-20MA diode array detector and a Rheodyne
7725i rotating valve with a 20 ␮L loop was used. A precolumn (TR-
C-160 with a ODS cartridge) was coupled to the analytical column
(150 mm Pinnacle C18 column, 5 ␮m particle diameter and 4.6 mm
I.D.) supplied by Teknokroma. The chromatographic separation was
performed with a mobile phase consisting of 0.05 M acetate buffer
pH 3.5 (solvent A) and acetonitrile (solvent B). The gradient elution
program was as follows: the percentage of solvent B was set at 50%
B for the first 6 min, increased to 90% B in 1 min and then kept con-
stant until 9 min. The flow rate was 1 mL min−1. Data analysis was
carried out with Shimadzu LC Solution software. The detector was
set at 280 nm to detect both TCP and the products of the catalytic
reaction in a single chromatogram.
3. Results and discussion
3.1. Morphological characterization of the polymers
The morphology of the polymers was studied by SEM experi-
ments. In all cases, polymer particles exhibited an irregular shape
and a dual distribution of sizes. However, the size differences
between the two particle populations were more remarkable for

G. Díaz-Díaz et al. / Journal of Molecular Catalysis A: Chemical 353–354 (2012) 117–121
119
50
40
30
20
10
0
catalyzed by CPO [20] and by polymers synthesized with just MA
(n = 1.6 0.4) or VPY (n = 2.7 0.5) [15]. The lower n values corre-
spond to the polymers synthesized with 25% VPY while the rest
exhibit Hill parameters close to 2, which indicates that there could
be up to two TCP molecules bound at the catalytic centre of the
polymer. However, the vmax values resemble the ones obtained for
VPY-based polymers (1.35 0.05 × 10−5 M min−1), and this param-
eter takes its maximum values when 25% VPY polymers catalyze
the oxidative dehalogenation of TCP. An additional parameter
useful to describe the efficiency of a biomimetic catalyst is the
catalytic constant (kcat) or turnover number that can be calcu-
lated as the ratio between vmax and the concentration of catalyst.
The turnover number for the different polymers was obtained
assuming that the concentration of the active sites is equiva-
lent to the amount of chlorohemin present in the polymers. The
catalytic constants displayed similar values to kcat obtained for MA-
based polymers (0.45 0.04 min−1), which were slightly higher
than the obtained for VPY-based polymers (0.34 0.01 min−1) [15].
The pseudo-Michaelis constant Ks* indicates that these polymers
exhibit affinities similar to those of the polymers prepared with
the basic functional monomer VPY (7.8 0.5 × 10−5 M) [15].
Finally, the global effect of kcat and Ks* was evaluated as the
ratio kcat/Ks* (catalytic efficiency) [14]. The catalytic efficiency is
similar to the VPY-based catalyst and one order of magnitude
lower than the MA-based one [15]. kcat/Ks* takes it lowest value
for the 50% VPY polymer whereas similar values were shown for
the polymers synthesized with 10 or 25% VPY, which indicates
that the polymers containing the lower VPY percentages mimicked
more efficiently the CPO catalytic dehalogenation activity. It should
be taken into account that the aminoacidic composition of these
polymers resembled the basic aminoacidic percentage found in
chloroperoxidase.
% VPY
10%
25%
50%
0
2 × 10-4
6 × 10-4
10-3
[TCP] / M
Fig. 1. Initial reaction rate at different TCP concentrations with the polymers as
catalysts. Reaction conditions: 0.5 g L−1, polymer, 10−3 M H2O2 in 0.01 M acetate
buffer with 10% DMSO, reaction time: 2 min.
the 10% VPY polymers (5 2 and 26 4 ␮m) compared to the ones
synthesized with 25 and 50% VPY (10 3 and 31 4 ␮m), which
did not exhibit significant differences between them. This fact
indicated that the particle size increased with the VPY content.
Comparing these results with those of the polymers synthesized
with a unique functional monomer [15], 10% VPY polymers are
formed by particles with intermediate sizes between the polymers
synthesized with MA (10 3 and 20 3 ␮m) or VPY (10 3 and
31 4 ␮m), while 25 and 50% VPY ones resembled the ones syn-
thesized with VPY.
3.2. Oxidative dehalogenation of TCP with polymers as catalysts
3.3. Oxidative dehalogenation of TCP with molecularly imprinted
polymers
The oxidative degradation of TCP was carried out with the syn-
thesized polymers as catalysts. The first step was to confirm that
they exhibit dehaloperoxidase activity, giving 2,6-dichloro-1,4-
benzoquinone (DCQ) as the main reaction product, which matches
with that obtained when either CPO [20–22] or polymers synthe-
sized with only MA [15,18] or only VPY [15].
The reaction–progress curve was obtained by mixing each poly-
mer (10, 25 and 50% VPY) as catalysts with the substrate (TCP
10−5 M) and the oxidant (H2O2 10−3 M), and measuring the DCQ
generated over a period of 25 min at 30 s intervals. The initial veloc-
ity region of the catalytic reaction was below 5 min. Initial rates,
obtained by measuring the concentration of the generated DCQ at a
fixed reaction time of 2 min, were measured as a function of the TCP
concentration to generate the saturation curve (Fig. 1). These data
do not obey the Michaleis–Menten equation but yield sigmoidal
profiles that were fit to Hill equation by an iterative procedure fol-
lowing Marquardt–Levenberg non-linear least squares algorithm
using Origin 8.1 software. A similar behaviour was obtained for the
natural enzyme, CPO, catalyzing the same process [18].
In order to improve the catalytic properties of the polymers, spe-
cific binding sites for the substrate, TCP, were introduced during
their synthesis. Molecular imprinting technology was used for this
purpose [14], and TCP was included in the pre-polymerization mix-
ture, giving 10, 25 and 50% VPY-MIPs. These imprinted polymers
exhibit similar morphology when compared to their respective
non-imprinted counterparts. They were also made up of spheri-
cal substructures, whose rapprochement created pores that were
larger in the MIP as compared to the NIP, which indicates that the
accessibility of TCP to the recognition cavities will be favored. The
oxidative dehalogenation of TCP was carried out with MIPs as cat-
alysts, and the parameters n, vmax and Ks*, which were compiled
in Table 2, follow the same trend as those of the non-imprinted
polymers discussed previously. However, the catalytic efficiency
reaches its minimum value for 25% VPY-MIP. This fact could indi-
cate the higher ability of this polymer to specifically bind TCP
and catalyze its oxidative dehalogenation, due to the presence of
specific template binding sites created during the polymerization
process. The imprinting factor (IF) also reflects this fact, and it can be
estimated as the ratio MIP/NIP of the respective catalytic efficien-
cies. According to this parameter, polymers synthesized with 10%
VPY exhibit a marked imprinting effect. Therefore, based on the
The Hill equation provides the values for the parameters n,
vmax and the pseudo-Michaelis constant (Ks*), which are shown in
Table 1. The Hill parameter (n) indicates the presence of coopera-
tive effects (n > 1) [23]. We had previously observed this behaviour
(n = 1.7 0.2) when we studied the oxidative dehalogenation of TCP
Table 1
Kinetic parameters for the polymers synthesized in this work.
Polymer
n
vmax × 10−5 (M min−1
)
kcat (min−1
)
Ks* × 10−4 (M)
kcat/Ks* × 103 (M−1 min−1
)
R2
10% VPY
25% VPY
50% VPY
1.8 0.6
1.3 0.2
2.0 0.4
1.5 0.1
2.1 0.2
2.1 0.2
0.42 0.03
0.55 0.05
0.42 0.04
1.1 0.2
1.5 0.2
1.4 0.2
3.8 0.7
3.6 0.6
3.0 0.5
0.973
0.993
0.968

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G. Díaz-Díaz et al. / Journal of Molecular Catalysis A: Chemical 353–354 (2012) 117–121
Table 2
Kinetic parameters of the molecularly imprinted polymers (MIPs).
Polymer
n
vmax × 10−5 (M min−1
)
kcat (min−1
)
Ks* × 10−4 (M)
kcat/Ks* × 103 (M−1 min−1
)
R2
IF
10% VPY-MIP
25% VPY-MIP
50% VPY-MIP
1.9 0.2
1.2 0.2
1.9 0.3
1.26 0.04
2.2 0.2
1.78 0.06
0.54 0.02
0.57 0.05
0.45 0.02
1.22 0.08
1.8 0.3
1.18 0.08
4.5 0.3
3.2 0.6
3.8 0.3
0.998
0.992
0.993
1.2
0.9
1.3
Table 3
Table 5
Binding parameters of the polymers synthesized with 10% VPY: Kk min−k max (affin-
ity constant), Nk min−k max (average number of binding sites) and m (heterogeneity
index).
Initial rate of the oxidative dehalogenation of DCP and 4C-3MP with 10% VPY-MICs
as catalysts.
Interferent
DCP
Polymer
vo × 10−5 (M min−1
)
Polymer
m
Nk min−k max (␮mol g−1
)
Kk min−k max (␮mol g−1
)
R2
10% VPY-MIP
10% VPY-NIP
1.4 0.4 (6)
1.9 0.5 (6)
10% VPY-MIP 0.84
793
6.3 × 104
0.968
0.916
10% VPY-NIP
0.78 1197
5.0 × 104
10% VPY-MIP
10% VPY-NIP
1.2 0.3 (7)
1.4 0.5 (6)
4C-3MP
DCP, 2,4-dichlorophenol; 4C-3-MP, 4-chloro-3-methylphenol.
criteria of the catalytic efficiency, the 10% VPY-MIC seems to
present the best characteristics to mimic CPO in catalyzing the
oxidative dehalogenation of TCP. Nevertheless, the imprinting
effect was also demonstrated by substrate binding and selectivity
studies.
Hill equation was used to analyse the kinetic data obtained
for increasing amounts of TCP and a fixed concentration (10−4 M)
of each interferent, and these results were displayed in Table 4.
When 2,4-DCP and 4-C-3-MP were in the reaction media, the Hill
index decreases until no cooperative effects took place, whereas
a substantial increase of n is observed with 2,4-DNP. No signifi-
cant changes were observed in vmax and kcat with respect to the
absence of interferents, and the pseudo-Michaelis constant dou-
bled its value with 2,4-DCP and 4-C-3-MP, which indicated that
the affinity of the catalyst to TCP decreased in the presence of
these compounds. Besides, the catalytic efficiencies obtained for
TCP with 2,4-DCP and 4-C-3-MP in the reaction media decreased
respect to TCP alone. These facts indicated that these two com-
pounds significantly interfere with TCP, and they both could
compete for the active sites of MIPs. Finally, kcat/Ks* increased
in the presence of 2,4-DNP with a more marked increase when
MIPs acted as catalysts. This implied that this compound acti-
vates the oxidative dehalogenation of TCP under the described
conditions.
To confirm the interference of 2,4-DCP and 4-C-3-MP in TCP
reaction, these compounds were taken as substrates instead of
TCP, and their main reaction products (2-chloro-1,6-benzoquinone
and 3-methyl-1,6-benzoquinone, respectively) were monitored
by HPLC-UV/VIS. Unexpectedly, at the concentration of polymer
assayed these compounds exhibited constant substrate conversion
rate, which is independent of the substrate concentration in the
range 10−6–10−3 M. This fact was observed for 2,4-DCP or 4-C-3-
MP substrates with MIPs and NIPs as catalysts. Therefore a mean vo
was obtained in each case, as shown in Table 5.
3.4. Binding properties
The binding parameters of the polymer synthesized with 10%
VPY were obtained from equilibrium experiments, using the Fre-
undlich isotherm to fit the data (Table 3). This model gives a good
mathematical approximation of the binding characteristics of non-
covalently imprinted polymers, and it is widely used to describe
the heterogeneity of MIPs in the subsaturation region [24,25]. The
heterogeneity index (m) is similar for both imprinted and non-
imprinted polymers, although a slightly increase was observed for
the MIPs. It could be due to the presence of specific TCP binding sites
apart from the unspecific ones present in both types of polymers.
The binding parameters were estimated in the (Kmin, Kmax) inter-
vals (1.5 × 104, 5.4 × 105) and (1.7 × 104, 2.0 × 105) for 10% VPY-MIP
and 10% VPY-NIP, respectively. The average number of binding sites
(Nk min−k max) was lower for the imprinted polymer compared to the
control one. However, 10% VPY-MIP exhibited more affinity for TCP
than 10% VPY-NIP regarding the affinity constant (Kk min−k max). This
could be related to the presence of specific TCP recognition sites,
which had been created during the imprinting process.
3.5. Selectivity
For the selectivity studies we have selected 2,4-dichlorophenol
(2,4-DCP) and 4-chloro-3-methylphenol (4-C-3-MP), two struc-
turally similar compounds that could compete against TCP for the
active sites of the polymers, and undergo oxidative dehalogenation
since they have a chlorine atom in the para position. Addition-
ally, we considered 2,4-dinitrophenol (2,4-DNP), which can block
the binding/catalytic sites although it cannot undergo the catalytic
reaction.
These facts seem to indicate that 2,4-DCP and 4-C-3-MP have
competed with TCP, and they acted as inhibitors of TCP oxida-
tive dehalogenation. However, these interferents were bound to
the MIPs in different sites from the specific ones created for TCP
during the imprinting process, although these binding sites were
also hemin-containing because the catalytic reaction could also
take place. Nevertheless, their presence did not totally limit the
Table 4
Kinetic parameters for TCP with 10% VPY-MICs as catalyst and a fixed concentration (10−4 M) of interferent.
Interferent
DCP
Polymer
n
vmax × 10−5 (M min−1
)
kcat (min−1
)
Ks* × 10−4 (M)
kcat/Ks* × 103 (M−1 min−1
)
MIP
NIP
1.0 0.2
2.0 0.2
1.3 0.3
1.2 0.3
0.6 0.2
0.35 0.08
3
1
1.9 0.8
2.9 0.7
1.19 0.07
MIP
NIP
1.0 0.3
1.3 0.4
1.1 0.3
1.4 0.2
0.5 0.2
0.39 0.06
2
1
2
3
1
1
4C-3MP
DNP
1.2 0.4
MIP
NIP
2.7 0.7
2.2 0.6
1.2 0.5
1.32 0.07
0.52 0.02
0.37 0.02
0.83 0.08
0.9 0.1
6.2 0.6
4.1 0.5
DCP, 2,4-dichlorophenol; 4C-3-MP, 4-chloro-3-methylphenol; DNP, 2,4-dinitrophenol.

G. Díaz-Díaz et al. / Journal of Molecular Catalysis A: Chemical 353–354 (2012) 117–121
121
oxidative dehalogenation of TCP, and even more, the oxidative
dehalogenation of TCP was the preferred route, over those of the
interferents.
Científica Aplicada y la Tecnología (COF 09-20 FICYT) for a postdoc-
toral contract.
References
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monomers (methacrylamide, MA; 4-vinylpyridine, VPY, respec-
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efficiency. Substrate selectivity was introduced by the use of molec-
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This research was financially supported by the Spanish Govern-
ment, Ministerio de Ciencia y Tecnología (CTQ2008-02429/BQU)
and the European Social Fund (ESF). G. Díaz-Díaz also thanks the
Spanish Government Ministerio de Educación y Ciencia and the
ESF for a FPI Fellow grant, and the Principado de Asturias Govern-
ment Fundación para el Fomento en Asturias de la Investigación