Dopamine selective molecularly imprinted polymers via post-imprinting modification

Article abstract of DOI:10.1039/b514432a

A novel synthetic dopamine receptor bearing bidentate binding sites were prepared by covalent imprinting using a disulfide linkage which is cleaved and oxidized to a non-covalent sulfoxide recognition group. The used templates have dopamine-like structure

Full text of DOI:10.1039/b514432a

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Dopamine selective molecularly imprinted polymers via post-imprinting
modification
Toshifumi Takeuchi,*a,b Nobuo Murase,a Hideshi Maki,a Takashi Mukawaa and Hideyuki Shinmoria
Received 11th October 2005, Accepted 5th December 2005
First published as an Advance Article on the web 5th January 2006
DOI: 10.1039/b514432a
A novel synthetic dopamine receptor bearing bidentate binding sites were prepared by covalent
imprinting using a disulfide linkage which is cleaved and oxidized to a non-covalent sulfoxide
recognition group. The used templates have dopamine-like structures connected to an allyl moiety via a
disulfide and to a 4-vinylphenyl group via a cyclic boronic diester. After the polymerization, the ester
bonds were hydrolyzed and the disulfide bond was reduced to remove the template moiety from the
polymer matrix, followed by the oxidation to transform the thiol residues into sulfonic acid (post
imprinted process). The imprinted polymer adsorbed dopamine selectively in aqueous solution with the
two-point interaction, i.e. the formation of cyclic boronic diester and electrostatic interaction with the
sulfonic acid residue.
previous reported catecholamine-imprinted polymers prepared by
Introduction
non-covalent imprinting using organic3 and inorganic materials.4
Molecular imprinting has been widely recognized as a technique
Because an excess of functional monomers, usually used in non-
for the construction of materials containing binding sites that can
covalent imprinting, may generate nonspecific binding sites upon
recognize a given target molecule.1 The most attractive aspect of
being randomly grafted into the polymer matrix, we employed a
this technique is the use of molecular templates to assemble func-
covalent molecular imprinting strategy for the introduction of
tional monomers around them into complementary orientations,
minimum amounts of boronic acid and sulfonic acid into the
forming tailor-made binding sites for the target molecules within
polymer matrices (Fig. 1, step 1).
a synthetic polymer matrix. The resulting imprinted polymers
show specific binding behaviors for the target molecules. Template
analog–functional monomer complexes were fashioned using
covalent and non-covalent interactions in which all or part of the
template analog is removed after polymerization, yielding a three
dimensional cavity. In this manner, not only molecular recognition
but also catalytic and signalling functionality can be easily intro-
duced into the synthetic polymers, using appropriately designed
template molecules.2 Unlike low-molecular weight synthetic recep-
tors, in which proper design of the binding site is the most crucial
aspect, binding sites of imprinted polymers are automatically
constructed according to the template molecules designed.
Here, we report on a novel synthetic dopamine receptor bearing
two-point binding sites prepared by a covalent imprinting system
using disulfide templates followed by a post-imprinting treatment,
Fig. 1 Schematic diagram of dopamine imprinting with the post-
imprinting oxidation.
in which the original functional groups are chemically transformed
into other functional groups that can form strong non-covalent
interactions.
Results and discussion
The target molecule, dopamine, has 3,4-dihydroxyphenyl and
We designed a template molecules 5-[2-(allyldithio)ethyl]-2-(4-
amino groups in its structure that were utilized to form a
vinylphenyl)benzo[1,3,2]dioxaborole, (Template 1) that has a
cyclic diester with a boronic acid and non-covalent electrostatic
dopamine-like core connected to an allyl moiety via a disulfide
group and to a cyclic boronic diester via 4-vinyl phenylboronic
interactions with acidic functional groups such as sulfonic acid.
Therefore, we designed a molecularly imprinted polymer contain-
acid. For the introduction of the boronic acid moiety, we employed
ing both a boronic acid and a sulfonic acid in the binding site by
a conventional imprinting method as previously reported by Wulff
using covalent molecular imprinting. This polymer differs from
et al. for imprinting carbohydrates.5 Disulfides are easily cleaved
from polymer matrices under reducing conditions (Fig. 1, step
aGraduate School of Science and Technology, Kobe University, Kobe, 657-
2)6 and the cyclic boronic diesters can be hydrolyzed under
8501, Japan. E-mail: takeuchi@scitec.kobe-u.ac.jp; Fax: +81-788036158;
acidic conditions (Fig. 1, step 3). The cleavage would create a
cavity complementary to dopamine and the imprinting process is
completed at this point.7
Tel: +81-788036158
bPRESTO, Japan Science and Technology Agency (JST), Kawaguchi, 332-
0012, Japan
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The corresponding reduced polymer contains the thiol residues
located in the proper position in the binding site that can interact
with the amino group of dopamine, after being transformed
into a sulfonic acid by oxidation with H2O2 (post-imprinting
treatment, Fig. 1, step 4).6 Finally, after completing all of the
treatments, phenylboronic acid and sulfonic acid residues are
assembled suitably for the binding of dopamine, meaning that the
dopamine binding sites can be generated, in which dopamine will
be captured with two-point binding. A reference template, 5-[2-
(allyldithio)ethyl]-2-(phenyl)benzo[1,3,2]dioxaborole (Template 2)
was prepared, in order to verify the formation of binding sites
capable of two-point binding in the imprinted polymers prepared
using Template 1.
Fig.
IP(T2–SO3H).
2
pH Dependence of the retention in IP(T1-SO3H) and
The two imprinted polymers IP(T1) and IP(T2) were prepared
by co-polymerizing styrene, divinylbenzene and either Template 1
or Template 2. A blank polymer (BP) was also prepared without
any template. After all the post-imprinting chemical modifications
were completed, the binding sites in the polymer prepared using
Template 1 (IP(T1–SO3H)) consist of both boronic acid and
sulfonic acid residues, and the polymer prepared using Template
2 (IP(T2–SO3H) has binding sites with only sulfonic acid residues.
Styrene/divinylbenzene based polymers were used as the matrix
because it was resistant to the redox reaction conditions that were
employed. The numbers of thiol groups in the polymers (IP(T1-
SH) and IP(T2–SH)) after the cleavage by the NaBH4 treatment
were estimated to be 175 lmol g−1 for the both polymers, which
corresponds to about 70% of the templates used in the imprinting
process. Following oxidation of the thiol groups into sulfonic acid
residues6 the loading of sulfonic acid residues in the polymers
were 120 lmol g−1 IP(T1–SO3H) and 110 lmol g−1 IP(T2–SO3H),
respectively, meaning that about 70 to 80% of the thiol residues
were converted to sulfonic acids and the overall yields of the
binding sites were about 50%.
In order to examine effects of the post-imprinting treatment,
the binding of dopamine was investigated by HPLC method using
columns packed with IP(T2–SO3H) and IP(T2–SH), which have
no boronic acid residues. The retention factors were 13.08 0.24
for IP(T2–SO3H) and 0.03 0.01 for IP(T2–SH) (n = 3) at pH 9.5.
The binding of dopamine became dramatically strengthened after
the post-imprinting treatment. This reveals that the sulfonic acid
residues in the binding sites of IP(T2–SO3H) can form strong
electrostatic interactions with the positively charged amino group
of dopamine under the testing conditions. From these results,
the effectiveness of the post-imprinting treatment was clearly
demonstrated by the enhancement in affinity of the post-modified
polymer.
effective formation of cyclic boronic diester in alkaline solutions.9
Dopamine and catechol were strongly bound by IP(T1–SO3H)
especially at higher pHs. In contrast, tyramine that has no
dihydroxyl group showed a similar ion-exchange profile to that
of IP(T2–SO3H). At pH 11.0, the binding of dopamine for IP(T1–
SO3H) was the strongest among the tested compounds, and the kꢀ
value of dopamine was approximately given by adding up those
of tyramine (to a sulfonic acid binder) and catechol (to a boronic
acid binder), suggesting that dopamine could be bound via two-
point binding to the boronic acid and sulfonic acid residues. The
blank polymer prepared without the templates showed almost
no binding under all the conditions employed. Therefore, the
imprinting effect clearly appears to be dominant, demonstrating
that the template molecules worked well to generate binding sites
selective for dopamine.
The relative selectivity of IP(T1–SO3H) and IP(T2–SO3H) at
pH 11.0 is summarized in Table 1. In IP(T1–SO3H), dopamine
showed the strongest binding because it has cis-diol and amino
group. Tyramine has a primary amine but no cis-diol and
catechol has no amine, and therefore, these two compounds had
lower retention factors than dopamine. The compounds having
carboxylic acid such as 3,4-dihydroxyphenylacetic acid (DOPAC)
and homovanillic acid (HVA) showed lower affinity; of the two,
Table 1 Relative selectivitya of IP(T1–SO3H) and IP(T2–SO3H)
With IP(T2–SO3H), dopamine and tyramine showed the
strongest binding at pH 9.5. pKa values of both amino and
phenolic hydroxyl groups are around 10,8 therefore, at pH 9.5,
the compounds may exist as zwitter ions and the cooperation
of ion-exchange and hydrophobic effects could contribute to the
stronger binding. At pH 6.8 and 11, the binding may occur via a
simple ion-exchange process (Fig. 2). Catechol has no functional
group capable of forming an electrostatic interaction with the
sulfonic acid residues, therefore, the binding was weak and the
pH dependence was not clearly observed.
Sample
IP(T1–SO3H)
IP(T2–SO3H)
Dopamine
Tyramine
Catechol
DOPAC
HVA
Epinephrine
Norepinephrine
1.00
0.54
0.45
0.36
0.08
0.05
0.35
1.00
1.02
0.05
0.02
0.00
0.12
0.05
The introduction of boronic acid residues to the imprinted
binding sites using Template 1 (IP(T1–SO3H)) provided an
enhancement in binding affinity at higher pHs, because of the
a The relative selectivity is expressed as ratios of retention factors of the
tested samples to a retention factor of dopamine at pH 11.0.
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DOPAC was better retained because it possesses a catechol
structure. In IP(T2–SO3H), negatively charged compounds having
no amino groups such as catechol, DOPAC and HVA showed
almost no affinity due to the electrostatic repulsion. Finally, it was
notable that IP(T1–SO3H) showed higher affinity for dopamine
than IP(T2–SO3H).
sodium sulfate and the solvent was removed. The residue was
purified by a silica-gel column chromatography (ethyl acetate–n-
hexane = 1 : 2) to give 2 (4.56 g, 86%); dH (300 MHz; CDCl3;
Me4Si) 2.33 (3H, s, CH3), 2.73 (2H, t, CH2), 3.06 (2H, t, CH2),
6.19 (2H, br s, 2 × OH) and 6.63–6.81 (3H, m, Ar–H).
1,2-Dihydroxy-4-(2-mercaptoethyl)benzene 3. Under a nitro-
gen atmosphere, 2 (4.24 g, 20.0 mmol) was dissolved in 0.2 mol L−1
aqueous sodium hydroxide–ethanol (80 mL, 1 : 1, v/v) and the
resulting solution was stirred for 5 h at room temperature. After
neutralization with 1 mol L−1 hydrochloric acid, the resulting
mixture was extracted with ethyl acetate (50 mL × 5). The
combined extracts were dried over anhydrous sodium sulfate and
the solvent was removed. The residue was purified by a silica-gel
column chromatography (ethyl acetate–n-hexane = 1 : 2) to give
3 (3.11 g, 92%); dH (300 MHz; CDCl3; Me4Si) 1.41 (1H, t, SH),
2.72–2.83 (4H, m, 2 × CH2), 5.83 (2H, br s, 2 × OH) and 6.61–6.82
(3H, m, Ar–H).
Although epinephrine and norepinephrine have also the both
functional groups, the binding was weaker. Epinephrine showed
especially low affinity to IP(T1–SO3H). The basicity of the
secondary amine of epinephrine is higher than that of nore-
pinephrine, thus epinephrine should be more strongly bound to
IP(T1–SO3H) than norepinephrine, but the opposite trend was
observed. In IP(T2–SO3H), epinephrine was more retained than
norepinephrine, as expected. This trend can be explained by the
binding sites in IP(T2–SO3H) being large enough to fit these
compounds and can bind them by an ion-exchange mechanism,
since it was generated by using Template 2, which generates a larger
cavity than Template 1 due to the presence of unpolymerizable
boronic acid moiety in Template 2. In contract, norepinephrine
was retained longer than epinephrine by IP(T1–SO3H), suggesting
that epinephrine does not fit as well into the imprinted cavity gen-
erated by Template 1 as norepinephrine has a less bulky primary
amine. These results suggest that the selectivity is affected not only
by the functional groups but also by the size of imprinted cavities.
4-[2-(Allyldithio)ethyl]catechol 4. To a mixture of 3 (3.00 g,
17.6 mmol) and diallyldisulfide (25.7 g, 176 mmol) was added
dropwise 5 mL of triethylamine and the resulting mixture was
stirred for 8 h at 60 ◦C under a nitrogen atmosphere. The mixture
was cooled to room temperature and then n-hexane was added.
The resulting precipitate was collected and purified by a silica-gel
column chromatography (ethyl acetate–n-hexane = 1 : 2) to give
4 (1.96 g, 46%); dH (300 MHz; CDCl3; Me4Si) 2.72–2.94 (4H, m,
Conclusions
=
CH2CH2), 3.37 (2H, d, CH2), 5.15–5.25 (2H, m, CH CH2), 5.56
The polymers with the post-imprinting treatment showed en-
hanced affinity without loss of the imprinting effect in aqueous
solution. The present results prove that the proposed imprinting
system involving the post imprinting oxidation can generate
binding cavities as intended with two functional groups positioned
in the binding site and work cooperatively. Tailoring the binding
sites after constructing preferable molecularly imprinted binding
sites by using organic chemistry shown here would open a new
strategy to design more desirable molecular recognition and/or
catalytic materials.
=
(2H, br s, 2 × OH), 5.83–5.94 (1H, m, CH CH2) and 6.63–6.83
(3H, m, Ar–H).
5-[2-(Allyldithio)ethyl]-2-(4-vinylphenyl)benzo[1,3,2]dioxaborole
(Template 1). A solution of 4-vinylphenylboronic acid (1.48 g,
10.0 mmol) in toluene (80 mL) was refluxed for 3 h and then the
solvent was removed. A part of the resulting anhydride (351 mg,
0.90 mmol) and 4 (654 mg, 2.7 mmol) was dissolved in toluene
(80 mL) and the mixture was refluxed for 3 h. The resulting
insoluble precipitate was removed by filtration and the filtrate
was evaporated to give Template 1 (774 mg, 81%); dH (300 MHz;
CDCl3; Me4Si) 2.69–2.91 (4H, m, CH2CH2), 3.36 (2H, d, CH2),
Experimental
=
=
5.14–5.28 (2H, m, CH CH2), 5.35 (1H, d, CH CH2), 5.83–5.94
=
=
(2H, m, CH CH2 and CH CH2), 6.63–6.83 (4H, m, Ar–H and
Preparation of Templates 1 and 2
=
CH CH2), 7.51 (2H, d, Ar–H) and 8.15 (2H, d, Ar–H).
S-[2-(3,4-Dimethoxyphenyl)ethyl]thioacetate 1. A mixture of
3,4-dimethoxystyrene (5.00 g, 30.5 mmol) and thioacetic acid
(2.62 mL, 36.6 mmol) was stirred for 7 h at room temperature
while irradiating with a 500 W bulb. The resulting mixture was
purified by a silica-gel column chromatography (ethyl acetate–n-
hexane = 1 : 3) to give 1 (6.14 g, 84%); dH (300 MHz; CDCl3;
Me4Si) 2.34 (3H, s, CH3), 2.80 (2H, t, CH2), 3.11 (2H, t, CH2),
3.86 (3H, s, OCH3), 3.88 (3H, s, OCH3) and 6.74–6.81 (3H, m,
Ar–H).
5-[2-(Allyldithio)ethyl]-2-(phenyl)benzo[1,3,2]dioxaborole (Tem-
plate 2). A solution of phenylboronic acid (1.22 g, 10.0 mmol)
in toluene (80 mL) was refluxed for 3 h and then the solvent was
removed. A part of the resulting anhydride (281 mg, 0.90 mmol)
and 4 (654 mg, 2.7 mmol) was dissolved in toluene (150 mL) and
the mixture was refluxed for 3 h. The resulting insoluble precipitate
was removed by filtration and the filtrate was evaporated to give
Template 2; dH (300 MHz; CDCl3; Me4Si) 2.63–2.89 (4H, m,
=
CH2CH2), 3.28–3.35 (2H, d, CH2), 5.09–5.26 (2H, m, CH CH2),
S-[2-(3,4-Dihydroxyphenyl)ethyl]thioacetate 2. To a solution
of 1 (6.00 g, 25.0 mmol) in CH2Cl2 (80 mL) was added dropwise
a solution of boron tribromide (4.10 mL, 42.4 mmol) in CH2Cl2
=
5.78–5.94 (1H, m, CH CH2), 6.60–6.78 (3H, m, Ar–H) and 7.30–
8.20 (5H, m, Ar–H).
◦
(20 mL) at −78 C. Then the resulting mixture was refluxed for
Preparation of IP(T1) and IP(T2)
7 h and was further stirred for 24 h at room temperature. To
the mixture was added 10 mL of water. After separating the
CH2Cl2 layer, the aqueous layer was washed with diethyl ether
(80 mL × 4). The combined organic layer was dried over anhydrous
Either Template 1 or Template 2 (2 mmol) was dissolved in
chloroform (5 mL) with divinylbenzene (50 mmol), styrene
(10 mmol) and 2,2ꢀ-azobis(isobutyronitrile) (500 mg). The mixture
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was purged with nitrogen gas for 5 min. The glass tube was sealed
and placed under UV light (XX-15L, UVP, Upland, CA) for 24 h
at 5 ◦C, followed by heating for 3 h at 80 ◦C. A blank polymer
(BP) was prepared without the template molecules.
carried out at 254 nm. The sample size was 10 lL. Retention
factors were calculated using the equation kꢀ = (tR − t0)/t0, where
tR is the retention time of the solutes and t0 is the retention time
of potassium nitrate used as a void marker. The measurement of
each sample was performed in triplicate.
Removal of the template molecule to yield IP(T1–SH) and
IP(T2–SH)
Acknowledgements
The obtained polymers were crushed roughly, then the particles
were suspended in methanol (100 mL) with NaBH4 (20 mmol).
The mixture was stirred for 12 h to cleave the disulfide bond of the
template and the NaBH4 treatment was carried out three times.
The particles were then treated with a diluted HCl solution
containing 50% (v/v) methanol to hydrolyze the boronic acid ester
of the template. The particles were washed twice with 50% (v/v)
methanolic aqueous solution, then washed with methanol. Finally
the particles obtained were dried in vacuo.
The authors thank Prof. Ken Shimizu (University of South
Calorina) for his helpful discussion.
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Post-imprinting treatment to yield IP(T1–SO3H) and
IP(T2–SO3H)
Polymer particles (200 mg) were suspended in acetic acid con-
taining ca. 15% hydrogen peroxide, and stirred for 12 h. After
the filtration, the particles were washed twice with 50% (v/v)
methanolic aqueous solution containing 50 mM sulfonic acid,
then washed twice with methanol.
The particles (200 mg) were treated with 1 M sodium chloride
(100 mL) and pH in the supernatant was measured. This treatment
was carried out three times and an amount of hydrogen released
from sulfonic acid residues by ion exchange was calculated.
7 Whitcombe et al. reported a covalent molecular imprinting system
using 4-vinylphenyl carbonate ester to make non-covalent recognition
sites for cholesterol. In this case, no post-imprinting treatment was
performed. See M. J. Whitcombe, M. E. Rodriguez, P. Villar and E. N.
Vulfson, J. Am. Chem. Soc., 1995, 117, 7105.
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HPLC conditions
An HPLC used consisted of two pumps (Gilson model 305
and 306), an auto-injector (Gilson model 234) and a UV/VIS
detector (Gilson model 119). The eluents used (1 mL min−1)
were acetonitrile–10 mM phosphate buffer pH 11.0 (1 : 1 v/v),
acetonitrile–10 mM glycine buffer pH 9.5 (1 : 1 v/v) or acetonitrile–
10 mM phosphate buffer pH 6.8 (1 : 1 v/v). The detection was
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568 | Org. Biomol. Chem., 2006, 4, 565–568
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The Royal Society of Chemistry 2006
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