Atrazine transforming polymer prepared by molecular imprinting with post-imprinting process

Article abstract of DOI:10.1039/b612407k

Molecularly imprinted polymers bearing atrazine transforming activity were prepared by using newly designed templates that are atrazine analogues attached with an allyl or a styryl group via a disulfide bond at the 6-position, methacrylic acid as a functional monomer and styrene/divinylbenzene as crosslinkers. After polymerization, the disulfide bond was reduced to remove the atrazine moiety from the polymer matrix, followed by oxidation of the remaining thiol group to generate sulfonic acid (post-imprinting treatment), so that both a methacrylic acid residue and a sulfonic acid residue existed in an atrazine-imprinted cavity. The polymers indicated the selective binding of triazine herbicides and catalytic activity for methanolysis at the 6-position of atrazine, yielding a low toxic atraton. The Royal Society of Chemistry.

Full text of DOI:10.1039/b612407k

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Atrazine transforming polymer prepared by molecular imprinting with
post-imprinting process†
Takehisa Yane, Hideyuki Shinmori and Toshifumi Takeuchi*
Received 29th August 2006, Accepted 20th October 2006
First published as an Advance Article on the web 8th November 2006
DOI: 10.1039/b612407k
Molecularly imprinted polymers bearing atrazine transforming activity were prepared by using newly
designed templates that are atrazine analogues attached with an allyl or a styryl group via a disulfide
bond at the 6-position, methacrylic acid as a functional monomer and styrene/divinylbenzene as
crosslinkers. After polymerization, the disulfide bond was reduced to remove the atrazine moiety from
the polymer matrix, followed by oxidation of the remaining thiol group to generate sulfonic acid
(post-imprinting treatment), so that both a methacrylic acid residue and a sulfonic acid residue existed
in an atrazine-imprinted cavity. The polymers indicated the selective binding of triazine herbicides and
catalytic activity for methanolysis at the 6-position of atrazine, yielding a low toxic atraton.
employed, where 6-Cl is replaced by OCH3 to yield atraton (6-
Introduction
methoxy-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine).
Molecular imprinting has come to be regarded as one of the
Atrazine has two amino groups and the three nitrogen atoms
potentially promising and convenient methods for the preparation
of a triazine in its structure and they can be utilized to form hy-
drogen bonding with carboxylic acid. By using hydrogen bonding,
this technique, a molecular template that is often a target molecule
atrazine-imprinted polymers have been successfully prepared, in
of synthetic polymers having specific binding/catalytic sites.1 In
which specific binding was shown for atrazine.5 Thus we utilized
this non-covalent atrazine imprinting system as a way to construct
carboxylic acid-based binding cavities for the recognition of
atrazine. As a catalytic functional group inside the recognition
cavities, we intended to introduce a strongly acidic functional
group such as sulfonic acid at a desirable position inside the specific
binding sites, preferably around the 6-Cl position of atrazine in its
bound position, because the nucleophilic reaction should proceed
at the 6-position of atrazine.
For this purpose, novel atrazine analogue template molecules
attached to a polymerizable group via a disulfide bond at the
6-position were designed. By using these templates, molecularly
imprinted polymers bearing an atrazine transforming activity
were prepared, involving covalent and non-covalent molecular
imprinting (Scheme 1). This strategy can introduce minimum
amounts of sulfonic acid at appropriate positions and this may
lead to enhanced specificity due to low non-specific binding.
itself or its analogue is added to a prepolymerization mixture
containing crosslinker(s) and other monomers. After polymeriza-
tion, the template molecule is removed from the obtained polymer,
yielding three dimensional binding cavities specific for the target
molecule.
Many molecularly imprinted catalysts (MIC) for various reac-
tions have been prepared by using various template molecules such
as transition state analogues, substrates and products.2 For the
preparation of MIC, both covalent3 and non-covalent4 molecular
imprinting have been used, in which the most important issue is
how we can insert catalytic functional groups into the specific
binding sites, since randomly located catalysts may cause non-
specific catalytic reactions.
Covalent molecular imprinting, in which polymerizable tem-
plates are used, can provide fairly homogeneous binding sites,
because the remaining functional groups in the resulting polymers
are only located inside the binding sites after the cleavage of
templates. Therefore covalent molecular imprinting would be more
favorable for the introduction of desirable functional groups into
target-selective binding sites than non-covalent techniques in order
to develop catalytic activities.
Herein we propose a technique for preparing substrate-selective
catalysts by the combined use of covalent and non-covalent
molecular imprinting coupled with a post-imprinting chemical
modification (post-imprinting process). As a model catalytic
reaction, methanolysis assisted by acids of a herbicide, atrazine
(6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine), was
Results and discussion
Firstly, we designed Template 1 (T1: allyl 4-ethylamino-6-
isopropylamino-1,3,5-triazine-2-yl disulfide) that is an atrazine
analogue attached to an allyl group via a disulfide bond at the
6-position, in order to obtain MIC bearing carboxylic acid-based
atrazine imprinted cavities with a sulfonic acid group located
inside the cavity. The imprinted polymers were prepared by radical
polymerization of a monomer mixture containing T1, methacrylic
acid (MAA) as a functional monomer for the construction
of atrazine binding sites, and divinylbenzene and styrene as
crosslinkers in chloroform. After polymerization, the disulfide
bonds originating from T1 were reduced by NaBH4 to remove the
atrazine moiety, yielding the atrazine binding cavities. In the final
step, the remaining thiol groups in the polymers were transformed
Graduate School of Science and Technology, Kobe University, Kobe 657-
8501, Japan. E-mail: takeuchi@gold.kobe-u.ac.jp; Fax: +81-788036158;
Tel: +81-788036158
† Electronic supplementary information (ESI) available: Michaelis–
Menten plots of IP(T1) and IP(T2), Lineweaver–Burk plot of IP(T2),
selectivity of catalysis by IP(T2). See DOI: 10.1039/b612407k
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Scheme 1 Illustration of the preparation of atrazine transforming polymers using the post-imprinting process.
into sulfonic acids by oxidation with hydrogen peroxide (post-
imprinting process).6 After all the processes were completed, the
imprinted polymer IP(T1) had binding/catalytic sites containing
both MAA and sulfonic acid residues. The conversion of T1 in
the obtained polymer was about 20% (215 lmol per g polymer) as
estimated by the S-content determined by elemental analysis.7
Binding properties of IP(T1) were investigated by batch binding
tests, where the imprinted polymers were incubated with triazine
herbicides and other pesticides (100 lM) in chloroform for 24 h
at 25 ◦C. Under the conditions, no catalytic reaction proceeded
because no nucleophilic reagent existed. IP(T1) showed strong
adsorption for triazine herbicides (atrazine, atraton, ametryn, and
cyanazine), while other pesticides (thiuram, propyzamide, and
iprodion) were hardly adsorbed (Fig. 1). These results clearly
indicate that atrazine imprinting was successfully achieved in
IP(T1). The binding of cyanazine, that has a bulky substituent, was
weaker than others, suggesting that the polymers cannot recognize
the difference between Cl and OCH3 substitutes at the 6-position,
however they can recognize larger side chains even if the base
structure is the same.
The transformation of atrazine (methanolysis of atrazine to
atraton) by IP(T1) was examined by incubation with atrazine in
CHCl3–MeOH (9 : 1, v/v) at 40 ◦C. Transformation velocities of
atrazine in the presence of IP(T1) were estimated by monitoring
the atraton production with time. Upon the investigation of
the effects of atrazine concentrations on the reaction velocities,
saturation behaviors were observed and a linear Lineweaver–Burk
plot was obtained (Fig. 2), suggesting that the reactions may
proceed with Michaelis–Menten kinetics under the conditions
employed. Therefore, similar to enzymatic catalysis, the substrate
bound to the binding site in the imprinted polymers should react
with a nucleophilic reagent, methanol. Kinetic parameters were
calculated to be as follows; Vmax: 2.1 × 10−7 M min−1, Km: 1.6 ×
10−4 M, kcat: 4.9 × 10−4 min−1, whereas the total concentration
of catalytic sites was estimated from the result of elemental
Fig. 1 Binding selectivity of IP(T1). Selectivity is shown by binding
amounts for 100 lM substrate solution.
analysis (4.3 × 10−4 M). At high substrate concentrations, product
inhibition may occur. To avoid this, other nucleophilic reagents
could be helpful if chemical properties of products can be
significantly changed after the nucleophilic reaction.
To prove that the catalytic reaction proceeded inside the binding
sites of the imprinted polymers, competition experiments were
carried out using an inactive triazine herbicide, ametryn, for
methanolysis with two different atrazine concentrations. The
reaction velocities diminished with the increase of ametryn
concentrations. This means that atrazine is transformed in the
binding sites that can be bound by ametryn, i.e. the catalytic site is
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Fig. 2 Lineweaver–Burk plots on atraton production catalyzed by IP(T1)
at 40◦ C; Vmax = 2.1 × 10−7 M min−1, Km = 1.6 × 10−4 M.
Fig. 4 Proportions of transformed substrates catalyzed by IP(T1) for
250 lM substrate solutions after 12 h incubation at 40 ◦C.
located inside the cavity constructed during the imprinting process.
The inhibition constant Ki was estimated to be 2.6 × 10−6 M from
a Dixon plot (Fig. 3). The figure is smaller than for the previously
reported MIC prepared by non-covalent molecular imprinting
using 2-sulfoethyl methacrylate as a catalytic monomer (4.7 ×
10−5M),8 indicating that the homology of binding/catalytic sites
could be improved by the proposed combined method involving
covalent and non-covalent imprinting.
conversion rate was almost 100% (256 lmol per g polymer), as
estimated by the S-content in elemental analysis,9 and the binding
specificity observed was higher than that of IP(T1) (Fig. 5).
Fig. 3 Inhibition of the transformation activity by ametryn. Atrazine (ꢀ:
160 lM, ᭹: 320 lM) and ametryn were incubated with IP(T1).
The specificity of the catalytic activity for various triazine
herbicides was evaluated by the ratio of the amount of product to
the initial amount of substrate after 12 h (Fig. 4). IP(T1) exhibited
catalytic activities for all the triazine substrates and atrazine se-
lectivity was observed. This also proved that the atrazine-selective
binding sites catalyze the transformation of triazine herbicides.
Because the co-polymerization efficiency of T1 with other
monomers was not good (20%), we designed Template 2 (T2:
4-vinylbenzyl (4-ethylamino-6-isopropylamino-1,3,5-triazine-2-yl
disulfide). It has a styryl group instead of an allyl group that
should easily co-polymerize with styrene/divinylbenzene. For the
preparation, a smaller amount of T2 was used because T2 may give
a high conversion rate and further, higher content of crosslinker
would give more highly selective binding sites. As expected,
when an imprinted polymer was prepared using T2 (IP(T2)), the
Fig. 5 Binding selectivity of IP(T2). Selectivity is shown by binding
amounts for 100 lM substrate solutions.
Table 1 summarizes kinetic parameters of IP(T1) and IP(T2)
(detailed data are given in the ESI†). The value of Vmax for IP(T2)
was higher than that for IP(T1), meaning that more catalytic sites
exist per unit weight of IP(T2) than in IP(T1). Methanolysis of
atrazine was catalyzed by the sulfonic acid residues in the binding
sites and with the concentration of catalytic sites estimated by the
S-contents in the polymers, kcat values of IP(T1) and IP(T2) were
calculated to be 4.9 × 10−4 and 1.2 × 10−3 min−1, respectively; this
is a measure of the catalytic activity. These results reveal that the
imprinting process using T2 can provide higher catalytic ability,
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Table 1 Kinetic parameters of the imprinted polymers for the catalytic
succinimide (810 mg, 1.98 mmol) in benzene (45 ml) was stirred at
70 ◦C for 12 h. Afterwards hexane was added to the mixture,
which was filtered to extract precipitate and the filtrate was
evaporated. The crude product was purified by silica gel column
chromatography (dichloromethane–methanol = 200 : 1) to yield
T1 (215 mg, 44%); dH (300 MHz; CDCl3; Me4Si) 1.25 (9H, m,
atrazine transformation
IP(T1)
IP(T2)
Vmax (10−7 M min−1
Km (10−4 M)
)
2.1
1.6
4.9
6.0
5.7
12
kcat (10−4 min−1
)
=
CH3), 3.49 (4H, m, NCH2 and SCH2), 5.15 (2H, m, CH2) and
IP(T1): [E]total = 4.3 × 10−4 M; IP(T2): [E]total = 5.1 × 10−4 M.
=
5.90 (1H, m, CH).
4-Vinylbenzyl 4-ethylamino-6-isopropylamino-1,3,5-triazin-2-yl
disulfide (T2). A solution of 2 (296 mg, 1.40 mmol), di(4-
vinylbenzyl)disulfide (2.70 g, 11.2 mmol) and triethylamine
(3.5 ml) in benzene (150 ml) was stirred at 80 ◦C for 48 h. The
crude product was purified by silica gel column chromatography
(dichloromethane–ethyl acetate = 10 : 1) to yield Template 2
(150 mg, 30%); dH (300 MHz; CDCl3; Me4Si) 1.23 (9H, m, CH3),
3.45 (2H, m, NCH2), 4.15 (3H, m, NCH and SCH2), 5.19 and 5.70
i.e. the population of properly working sites may be higher. The
Km of IP(T1) was smaller than that of IP(T2). Thus, relatively
more stable substrate–catalyst complexes may form in the less
sterically hindered binding sites of IP(T1) because of the absence
of a phenylene group. Since the catalytic activity was affected by
the conversion rate of the templates used and the steric hindrance
of the linkers between the disulfide and vinyl groups, more careful
design of the template molecules would lead to the development
of higher catalytic activity.
=
=
(2H, d, CH2), 6.70 (1H, m, CH) and 7.33 (4H, m, ArH).
Preparation of IP(T1) and IP(T2)
Conclusions
IP(T1). T1 (720 mg, 2.52 mmol), the functional monomer
MAA (214 ll, 2.52 mmol), divinylbenzene (1.08 ll, 7.56 mmol)
and styrene (533 ll, 3.78 mmol) as crosslinkers were mixed in
1.64 ll of chloroform. Polymerization was initiated by photo-
irradiation using a ra◦dical initiator, 2,2ꢀ-azobis(isobutyronitrile)
(60 mg) for 12 h at 5 C, and the solution was heated for 4 h at
60 ◦C to remove the solvent. The resulting polymer was ground to
obtain polymer particles. The polymer particles (2.0 g) and sodium
borohydride (2.0 g, 20 eq. with respect to the disulfide bond)
were suspended in methanol (80 ml). The mixture was stirred.
This cleavage procedure was repeated a further two times. After
reduction, the polymer particles were washed with methanol–
water (9 : 1) and methanol at room temperature. Post-imprinting
treatment to convert of thiol groups in the polymers to sulfonic
acid groups was performed as follows. The polymer particles were
suspended in H2O2–acetic acid (80 ml, 1 : 1, v/v) and stirred at
room temperature for 12 h. This post-imprinting treatment was
repeated a further two times. After post-imprinting treatment, the
polymer particles were stirred in 100 mM sulfuric acid–methanol
(1 : 1) for 8 h at room temperature, then were washed with
methanol.
We proposed new MIC preparation methods involving the
combination of covalent and non-covalent molecular imprinting
processes with post-imprinting chemical modification for the
introduction of one catalytic site per one specific binding cavity.
The resulting imprinted polymer has a catalytic activity for the
transformation of atrazine to atraton with Michaelis–Menten
kinetics-like behavior. This method will provide a new way to
introduce catalytic functional groups into molecularly imprinted
cavities, resulting in more homogeneous catalytic sites.
Experimental
Preparation of T1 and T2
4-Ethylamino-6-isopropylamino-2-(4-methoxybenzylsulfanyl)-
1,3,5-triazine (1). A solution of atrazine (2.15 g, 10 mmol), 4-
methoxy-a-toluenthiol (1.4 ml, 10 mmol), sodium-tert-butoxide
(1.92 g, 20 mmol), tetrakis (triphenylphosphine)palladium(0)
(1.15 g, 1.0 mmol) in DMF (200 ml) was stirred at 100 ◦C for
20 h under nitrogen. The resulting mixture was concentrated and
extracted with CHCl3. The mixture was purified by silica gel
column chromatography (dichloromethane–methanol = 100 : 1)
to yield 1 (2.26 g, 68%); dH (300 MHz; CDCl3; Me4Si) 1.23 (9H,
m, CH3), 3.45 (2H, m, NCH2), 3.80 (3H, s, OCH3), 4.38 (3H, m,
NCH and SCH2), 6.78 (2H, d, ArH), 7.21 (2H, s, NH) and 7.20
(2H, d, Ar–H).
IP(T2). T2 (181 mg, 0.50 mmol), the functional monomer
MAA (42.5 ll, 0.50 mmol), divinylbenzene (1.67 ml, 10 mmol)
and styrene (573 ll, 5.0 mmol) as crosslinkers were mixed in 1.6 ml
of chloroform. Polymerization was initiated by photo-irradiation
using a radical initiator, 2,2ꢀ-azobis(isobutyronitrile) (59 mg) for
20 h at 5 ◦C, and the solution was heated for 4 h at 60 ◦C to remove
the solvent. The resulting polymer was ground to obtain polymer
particles. The polymer particles (1.34 g) and sodium borohydride
(380 mg, 20 eq. with respect to the disulfide bond) were suspended
in methanol (100 ml). The mixture was stirred. This cleavage
procedure was repeated a further three times. After reduction,
the polymer particles were washed with methanol–water (9 : 1)
and methanol at room temperature. Post-imprinting treatment to
convert the thiol groups in the polymers to sulfonic acid groups
was performed as follows. The polymer particles were suspended
in H2O2–acetic acid (100 ml, 1 : 1, v/v) and stirred at room
temperature for 8 h. This post-imprinting treatment was repeated
4-Ethylamino-6-isopropylamino-1,3,5-triazine-2-thiol (2).
A
solution of 1 (1.97 g, 5.9 mmol) in TFA (20 ml) was stirred at room
temperature for 6 h. The resulting mixture was diluted with CHCl3
and was washed with NaHCO3 solution. The crude product was
purified by silica gel column chromatography (chloroform–ethyl
acetate = 1 : 1) to yield 2 (1.29 g, 100%); dH (300 MHz; CDCl3;
Me4Si) 1.25 (9H, m, CH3), 3.48 (2H, m, NCH2), 4.20 (1H, m,
NCH), 8.70 (1H, s, NH) and 8.99 (1H, s, NH).
Allyl 4-ethylamino-6-isopropylamino-1,3,5-triazin-2-yl disulfide
(T1). A solution of 2 (422 mg, 1.98 mmol) and N-allylsulfanyl
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a further two times. After post-imprinting treatment, the polymer
particles were stirred in 1.0 N hydrochloric acid–methanol (1 : 1)
for 12 h at room temperature, then were washed with methanol.
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Binding selectivity of imprinted polymers
The polymer particles (3.0 mg) were incubated with atrazine,
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an eluent of acetonitrile–10 mM ammonium acetate buffer (50 :
50 v/v, pH 6.0, 0.8 ml min−1).
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Catalytic activities for various triazine substrates
7 Elemental analysis data of IP(T1)(wt%): C, 78.77; H, 7.52; N, 0.37; S,
0.69.
The polymer particles (3.0 mg) were incubated with atrazine,
simazine, propazine and cyanazine (250 lM at 40 ◦C) in methanol–
chloroform (1.5 ml, 1 : 9, v/v) for 12 h. After incubation, the
suspensions were filtered and the filtrates (1.0 ml) were dried in
vacuo. The residues were dissolved in acetonitrile (1.0 ml) and
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9 Elemental analysis data of IP(T2)(wt%): C, 84.80; H, 7.7.93; N, 0.26; S,
0.82.
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