Atrazine transformation using synthetic enzymes prepared by molecular imprinting.

Article abstract of DOI:10.1039/b407002j

Atrazine imprinted synthetic polymers were prepared using a combination of methacrylic acid and 2-sulfoethyl methacrylate that bound and converted atrazine and other 6-chlorotriazine herbicides to less toxic compounds. A chloride at the 6-position of the triazines was converted to a methoxy group by the polymer in a methanol-containing solvent, where the imprinting effects enhanced the catalytic activity. Competitive inhibition of the atrazine methanolysis was observed in the presence of a structurally related binder, ametryn, suggesting that the catalytic reaction proceeded in the binding sites generated by the molecular imprinting process.

Full text of DOI:10.1039/b407002j

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A R T I C L E
Atrazine transformation using synthetic enzymes prepared by
molecular imprinting†
Toshifumi Takeuchi,*a,b Satoshi Ugata,c Shuichi Masuda,c Jun Matsuic and
Masayoshi Takaseb
a
Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai-cho,
Nada-ku, Kobe, 657-8501, Japan. E-mail: takeuchi@scitec.kobe-u.ac.jp;
Fax: +81-78-803-6158; Tel: +81-78-803-6158
PRESTO, Japan Science and Technology Agency (JST), Kawaguchi, 332-0012, Japan
b
c
Faculty of Information Sciences, Hiroshima City University, Hiroshima, 731-3194, Japan
Received 10th May 2004, Accepted 23rd July 2004
First published as an Advance Article on the web 17th August 2004
Atrazine imprinted synthetic polymers were prepared using a combination of methacrylic acid and 2-sulfoethyl
methacrylate that bound and converted atrazine and other 6-chlorotriazine herbicides to less toxic compounds. A chloride
at the 6-position of the triazines was converted to a methoxy group by the polymer in a methanol-containing solvent,
where the imprinting effects enhanced the catalytic activity. Competitive inhibition of the atrazine methanolysis was
observed in the presence of a structurally related binder, ametryn, suggesting that the catalytic reaction proceeded in the
binding sites generated by the molecular imprinting process.
and 6 equiv. of MAA and imprinted against atrazine (IP(S2M6)).7
Introduction
Chemical structures of the triazine herbicides and other pesticides
used in this study are shown in Fig. 1. Similar to previously reported
atrazine-imprinted polymers,3 only triazine herbicides were strongly
adsorbed and other pesticides were hardly retained (Fig. 2). These
results clearly showed that the polymers prepared in this study have
the selectivity for triazine herbicides.
The design and synthesis of synthetic materials with specific
binding sites and catalytic functions are an area of increasing
current interest.1 Selective recognition and subsequent chemical
reactions within binding pockets are instructive as well as of
interest not only to supramolecular chemists but also to synthetic
chemists. In particular, synthetic enzymes that selectively convert
triazine herbicides into to less or non-toxic compounds would be
useful catalytic systems as triazine herbicides are acknowledged
widespread environmental pollutants.2
To date we3 and others4 have reported atrazine binding polymers
prepared by molecular imprinting for the purpose of detection,
separation, and purification.5 The technique is now known as a
means to prepare tailor-made synthetic polymers that are highly
selective for target substances. In our recent study on employing
automated polymer preparation/evaluation systems for molecu-
larly imprinted polymers,3a we found that the use of 2-sulfoethyl
methacrylate (SEMA) provides atrazine transformation polymers.
Herein we show the selective binding and subsequent transforma-
tion of 6-chlorotriazine herbicides, using imprinted polymers that
were prepared with methacrylic acid (MAA) as the binding site-
constructing monomer and SEMA as the catalytic monomer. The
resulting imprinted polymer was demonstrated to act as a novel
synthetic enzyme with catalytic ability.
Results and discussion
Scheme 1 Schematic illustration of the imprinted polymer preparation.
To prepare polymers with catalytic activity for triazine herbicides,
atrazine was used as a template molecule with two functional mono-
mers: MAA and SEMA. The choice of MAA and SEMA as func-
tional monomers was based on their abilities to form complementary
hydrogen bonds with atrazine and to act as an acid catalyst for the
nucleophilic aromatic substitution of atrazine with MeOH.3a,6 The
former constructs binding sites and the latter provides catalytic sites
(Scheme 1). For the characterization of binding sites induced by the
imprinting process, binding behavior of the imprinted polymers was
examined by injecting triazine herbicides and other pesticides onto
a column packed with the polymer prepared using 2 equiv. of SEMA
In order to estimate the affinity of the binding sites for triazine
herbicides, a binding isotherm was drawn using ametryn which
is structurally similar to atrazine and is more resistant to acid-
catalyzed methanolysis. A Scatchard plot from the binding data
(ESI†) showed a non-linear profile, suggesting that the binding
sites are heterogeneous with respect to the affinity for ametryn. A
dissociation constant could be estimated to be 6.5 × 10−6 M from
the low concentration range (ESI†). Although this figure is not
precisely calculated because of the non-linear profile, it gives an
idea for the possessing affinity.
Transformation of atrazine to the less toxic derivative atraton
(2-ethylamino-4-isopropylamino-6-methoxy-1,3,5-triazine)2 was
demonstrated by incubation of the polymers with atrazine in the
mixed solvent CHCl3–MeOH, 9:1, v/v (Fig. 3). Aproposed mecha-
nism is the protonation of the atrazine by a sulfonyl group of SEMA
† Electronic supplementary information (ESI) available: (1) Binding
isotherm and Scatchard plot for ametryn in IP(S2M6). (2) Transformation
of simazine and propazine by IP(S2M6). (3) GC–MS of a transformation
reaction mixture with IP(S2M6). See http://www.rsc.org/suppdata/ob/b4/
b407002j/
T h i s j o u r n a l i s
©
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 5 6 3 – 2 5 6 6
2 5 6 3

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within the binding pockets, followed by the nucleophilic substitu-
tion at the 6-position by methanol yielding atraton (Scheme 2).2
The obtained atraton was identified by comparing with the authentic
sample on GC-MS (ESI†).
Since atrazine is essentially transformed under acidic or basic
conditions,2 reaction kinetics were examined to evaluate the effects
of changing imprinting conditions on the catalytic activity.
IP(S2M6) showed higher activity than the polymer prepared with
1 equiv. of SEMAand 7 equiv. of MAAagainst the amount of initial
atrazine (IP(S1M7)). The differences between the imprinted and
reference polymers was larger in IP(S2M6) than IP(S1M7) (Fig. 4).
The activity of the corresponding reference polymers (RP(S2M6)
and RP(S1M7)) was lower than that of the imprinted polymers,
revealing that the binding sites formed during the imprinting
process appear to enhance the catalytic activity. It is obvious that
more SEMA residues provide higher activity but too many SEMA
residues may lead to non-specific binding and to less selective
catalytic reactions, therefore IP(S2M6) was used for the following
experiments.
Fig. 1 Chemical structures of the triazine herbicides and pesticides used
in this study.
Fig. 4 Transformation velocity in the imprinted and reference polymers:
IP(S2M6) (), RP(S2M6) (), IP(S1M7) () and RP(S1M7) ().
Attempts were made to evaluate the rate enhancements in the
polymer by comparing transformation velocities of atrazine, i.e. the
velocity of atraton production in imprinted polymers against that in
free SEMA solutions. Firstly, effects of substrate concentrations on
the reaction velocity were investigated in the imprinted polymer. As
seen in Fig. 5, saturation kinetics was obtained, suggesting that the
atrazine transformation reaction proceeds within discrete binding
sites within the polymer. From a Lineweaver–Burk plot using the
kinetic data, we estimated a velocity constant of this reaction;
a maximal velocity Vmax of 1.0 × 10−7 M min−1 and a Michaelis
constant Km of 5.2 × 10−4 M. A kcat value was also estimated
to be 1.0 × 10−6 M min−1 (Fig. 5). According to the elemental
analysis,8 the polymer contained 0.87% of S, meaning that there
was 0.815 mol of SEMA residues in 3 mg of the polymer and the
concentration of total catalytic sites can be estimated as 0.54 mM in
the 1.5 mL incubation solutions. It should be noted that this figure is
probably an overestimation because not all SEMA residues will be
incorporated into a catalytic site, and there is no way to distinguish
SEMA in the catalytic sites from randomly located SEMA in the
polymer. However, the maximum number of binding/catalytic
sites should not exceed the amount of atrazine added in the
polymerization process, which was half the SEMA concentration.
Secondly, the transformation was carried out in homogeneous
solutions as a reference, using free SEMA solutions and V′max, K′m,
and k′cat were also estimated to be 2.4 × 10−7 M min−1, 3.1 × 10−4 M,
Fig. 2 Retention factors in IP(S2M6); IP(S2M6) was ground and wet-
sieved (methanol) to obtain polymer particles with a size of 32–63 m, then
packed into a stainless column (4.6 × 100 mm, i.d). The eluent used was
CH2Cl2–CH3CN, 3:1, v/v (1.0 mL min−1) and the detection was carried out
at 254 nm.
Fig. 3 Atrazine transformation by IP(S2M6). The polymer particles
were incubated with atrazine in CHCl3–MeOH (1.5 mL, 9:1, v/v) at 25 °C.
After 24 h, the suspension was filtered (0.2 L) and the filtrate was dried
in vacuo. The residues were dissolved in CH3CN (1.0 mL) and analyzed
(Waters HPLC system) with a reversed phase column (Supelco LC-8-DB)
and an eluent of CH3CN–0.1 M ammonium acetate buffer (47:53, v/v, pH
6.0, 1.0 mL min−1).
Fig. 5 Effects of substrate concentrations on the atraton production
(a) and Lineweaver–Burk plot (b) in IP(S2M6): Vmax = 1.0 × 10−7 M min−1,
Km = 5.2 × 10−4 M,[E]total = 5.4 × 10−4 M,k2 = Vmax/[E]total = 1.9 × 10−3 min−1,
kcat = k2Km = 1.0 × 10−6 M min−1. [E]: IP(S2M6) conc.
Scheme 2 A possible mechanism of atrazine transformation with MeOH
activated by SEMA residues.
2 5 6 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 5 6 3 – 2 5 6 6

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and 1.3 × 10−7 M min−1, respectively (Fig. 6). Thus, a ratio of the
two kcat, i.e. imprinting effects for the transformation, was evaluated
to be at least 7.6. Unfortunately, the increment by the imprinting
effect was not as high as previously reported MIP catalyzed reac-
tions,5 this may be partly due to the influence of improperly located
SEMA residues and the overestimation of SEMA in the polymer
involved in the catalytic reaction. Product inhibition may also occur
because atraton is structurally similar to atrazine, contributing to the
low kcat value.
values were underestimated because the number of catalytic sites
was overestimated. The low rate accelerations may be caused by
randomly positioned SEMAthat arises from the use of non-covalent
imprinting strategy, in which an excess of functional monomer is
used to shift the equilibrium in favor of complex formation with the
template molecule. We found that the product inhibition occurred
at a high substrate concentration range. This may be due to the
structure similarity of the substrates and the products. Therefore
the construction of more precise binding/catalytic sites will be
necessary to improve this system, and polymers that organize the
catalytically active site more precisely using combined use of
covalent and non-covalent imprinting systems are currently being
studied in our group.
Experimental
Materials
Atrazine was kindly donated by Nissan Chemical Industries, Ltd.
(Tokyo, Japan). Propazine, ametryn, prometrine, terbutylazine,
cyanazine, simazine, propazine, asulam, thiuram, propyzamide,
iprodion, methacrylic acid and 2,2′-azobis(isobutyronitrile) were
purchased from Chemical Industry (Tokyo, Japan). 2-Sulfoethyl
methacrylate(SEMA)waspurchasedfromPolysciences(Warrington,
PA). Ethylene glycol dimethacrylate (EGDMA) and other solvents
were obtained from Katayama Chemical (Osaka, Japan).
Fig. 6 Effects of substrate concentrations on atraton production (a) and
Lineweaver–Burk plot (b) in free SEMA: Vmax = 2.4 × 10−7 M min−1,
Km = 3.1 × 10−4 M, [E]total = 5.9 × 10−4 M, k2 = Vmax/[E]total = 4.1 × 10−4 min−1,
kcat = k2Km = 1.3 × 10−7 M min−1.
To confirm that the reaction proceeds in the binding sites of the
imprinted polymers, inhibition experiments were conducted using
an inactive compound. Ametryn was ideal for this purpose because
it has almost the same structure as atrazine except for the methylthio
group at the 6-position. Inhibition of the reaction by ametryn would
indicate that it was competing for the binding sites in the atrazine-
imprinted polymers, and the nucleophilic substitution with MeOH
would not occur even if in the presence of the SEMA catalytic side
chains. The reaction velocities were measured for three different
atrazine concentrations at a variety of inhibitor concentrations. The
activity was inhibited with increasing ametryn concentration, and a
Dixon plot showed that ametryn was a competitive inhibitor. Also
calculated from these plots was the inhibition constant Ki, indicating
that the dissociation constant for the enzyme-inhibitor complex
was 4.7 × 10−7 M (Fig. 7). Thus, it was confirmed that atrazine
methanolysis reactions proceeds within the binding sites that were
constructed during the molecular imprinting process.
Preparation of atrazine imprinted polymers using SEMA and
MAA as functional monomers
Atrazine (1.67 mmol), functional monomers (SEMA 1.67 mmol/
MAA 11.69 mmol or SEMA 3.34 mmol/MAA 10.02 mmol) and
EGDMA (47.2 mmol) were mixed in 25 mL of chloroform. After
the polymerization was initiated by photo-irradiation using a radi-
cal initiator, 2,2′-azobis(isobutyronitrile) (120 mg) for 12 h at 5 °C,
the obtained polymer was crushed, washed to remove atrazine first
with the mixed solvent (100 mM H2SO4 aqueous solution–MeOH,
1/9, v/v) and then with MeOH. The resulting polymers were further
ground and wet-sieved in methanol to obtain polymer particles
with the size of 32–63 m. Corresponding reference polymers were
prepared with the same recipe without atrazine.
Selectivity and kinetic experiments
For the selectivity experiments, the particles were packed into a
stainless steel column (4.6 mm × 100 mm i.d.). A Waters HPLC
system consisting of pumps (model 626), an auto-sampler (model
717 plus, sample size: 20 l), a detector (model 996, 254 nm) and
an eluent of dichloromethane–acetonitrile, 3:1, v/v (1.0 mL min−1)
was used.
In the kinetic experiments, the polymer particles (3 mg) were
incubated with atrazine (0, 50, 100, 200, 400 or 500 M) in
methanol–chloroform (1.5 mL, 1:9, v/v) at 25 °C. At appropriate
intervals, the suspensions were filtered (0.2 m) and the filtrates
were dried in vacuo. The residues were dissolved in acetonitrile
(1 mL) and analyzed by the Waters system with a reversed phase
column (Supelco LC-8-DB) and an eluent of acetonitrile–0.1 M
ammonium acetate buffer (47:53, v/v, pH 6.0, 1 mL min−1). For
the Lineweaver–Burk and Dixon plots, the velocities of atraton
production for the first 3 h were measured against each substrate
concentration based on the amount of atraton in the supernatants as
quantified by HPLC.
Fig. 7 Inhibition of the transformation activity by ametryn. Atrazine
(200 (), 300 () and 400 () M) and ametryn (0–100 M) were
incubated with the polymers (3 mg in methanol–chloroform, 1:9, v/v,
1.5 mL) at 25 °C. The Ki value was estimated to be 4.7 × 10−7 M.
Other triazine herbicides that also contained a Cl leaving group
at the 6-position, such as simazine and propazine, showed similar
reactivity in the presence of the imprinted polymer (ESI†). This
demonstrates that the binding sites produced from the molecular
imprinting of atrazine showed selectivity not only in binding but
also in catalytic activity.
As a reference, free SEMA (590 M) was used instead of the
polymer for the kinetics of free SEMA in solution. The amount
was adjusted to be the theoretical amount of SEMA in 3 mg of the
particles.
Conclusion
Inhibition experiments
We have succeeded in the preparation of synthetic polymers with
binding and catalytic sites for triazine herbicides with Cl at the 6-
position using molecular imprinting. As compared to homogeneous
SEMA, this synthetic enzyme exhibited higher catalytic activities.
At the present stage, the activity seems to be low even though the kcat
The polymer particles (3 mg) were incubated with atrazine
(200, 300 and 400 M) and ametryn (an inhibitor, 0–100 M) in
methanol–chloroform, 1:9, v/v (1.5 mL) at 25 °C. At appropriate
intervals, the suspensions were filtered (0.2 m) and the filtrates
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 5 6 3 – 2 5 6 6
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were dried in vacuo. The residues were dissolved in acetonitrile
(1 mL) and analyzed by the Waters HPLC system using a reversed
phase column (Supelco LC-8-DB) and an eluent of acetonitrile–
0.1 M ammonium acetate buffer (47:53, v/v, pH 6.0, 1 mL min−1).
4
5
Acknowledgements
This work was supported partly by a Grant-in-aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science
and Technology of Japan.
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