Molecular and Ionic Recognition with Imprinted Polymers - American

Chapter 9

Noncovalent Molecular Imprinting of a Synthetic Polymer with the Herbicide 2,4-Dichlorophenoxyacetic Acid in the Presence of Polar Protic Solvents

Downloaded by PENNSYLVANIA STATE UNIV on July 31, 2012 | http://pubs.acs.org Publication Date: May 7, 1998 | doi: 10.1021/bk-1998-0703.ch009

Karsten Haupt Department of Pure and Applied Biochemistry Chemical Center, Lund University, P.O. Box 124, S-22100 Lund, Sweden Non-covalent molecular imprinting of a synthetic polymer with the herbicide 2,4-dichlorophenoxyacetic acid has been achieved in the presence of the polar solvents methanol and water. Formation of the prearranged complex results from hydrophobic and ionic interactions between the template and the functional monomer 4-vinylpyridine. The polymer obtained binds the original template with an appreciable selectivity over structurally related compounds. The potential use of micrometer-sized imprinted polymer particles as the recognition element in a radioligand binding assay for 2,4-dichlorophenoxyacetic acid is demonstrated. Molecular imprinting is becoming increasingly recognized as a powerful technique for the preparation of synthetic polymers containing tailor-made recognition sites for certain target molecules (7). The imprinting process is performed by copolymerizing functional and crosslinking monomers in the presence of a molecular template. After elution of the template, complementary binding sites are revealed within the polymer network that allow rebinding of the template with a high specificity, sometimes comparable to that of antibodies (2, 3). The artificial receptors so obtained may be used in applications that demand specific ligand binding, such as the analytical and preparative separations of closely related compounds (4, 5), solid phase extraction (6), directed synthesis and catalysis (7-9), as recognition elements in sensors (10, 77) and immunoassay-type binding assays (2, 72). There exist two conceptionally different approaches for molecular imprinting in synthetic polymers. In the "covalent approach" that has been developed by Wulff and others, the functional monomers are covalently coupled to the template prior to polymerization (13). These bonds have to be cleaved to liberate the template and reveal the binding sites, and are subsequently reformed during rebinding of the target molecule. The second approach, which has primarily been developed by the Mosbach group (7), is generally called the "non-covalent approach". It relies on a pre-arrangement of functional monomers with the template prior to polymerization via non-covalent bonds. In some cases, a "hybrid approach" has been adopted wherein the polymerization is performed with the functional monomer(s) covalently bound to the template and, after cleavage of the template from the polymer, rebinding

©1998 American Chemical Society

In Molecular and Ionic Recognition with Imprinted Polymers; Bartsch, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

135


136 takes place by non-covalent interactions. An interesting example of this approach has recently been reported by Whitcombe et al. (14). Also of note is another imprinting protocol which is somewhat different from the covalent and non-covalent approaches in that it involves the use of metal coordination (15). Among these different methods for the preparation of molecularly imprinted polymers, the non-covalent approach is probably the most flexible in terms of the choice of functional monomers and possible template molecules and has therefore been most widely adopted. However, it does have some limitations. The non covalent bonds formed during pre-arrangement, e.g. hydrogen bonds or other electrostatic interactions, are relatively weak. Therefore conditions must be chosen to shift the equilibrium towards complex formation. As a result non-covalent imprinting has been mostly performed in apolar organic solvents, since in the presence of polar solvents, and especially water, the prearranged complex is destabilized. On the other hand, hydrophobic interactions are strong in water, and ionic bonds might also add to the stability of the complex in an aqueous environment. The aim of the present work was therefore to investigate whether specific non-covalent molecular imprints can be obtained in the presence of high concentrations of water using a combination of the hydrophobic effect and ionic interactions. The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was selected as the model template owing to its hydrophobic aromatic ring and its ionizable carboxyl group. Moreover, molecularly imprinted polymers specific for phenoxyacid herbicides could be attractive as artificial receptors in environmental analysis. Experimental Ethyleneglycol dimethacrylate (EDMA) and 4-vinylpyridine (4-VP) were from Merck (Darmstadt, Germany). 2,4-Dichlorophenoxyacetic acid (2,4-D), 2,4dichlorophenoxybutyric acid (2,4-DB), 2,4-dichlorophenoxyacetic acid methyl ester (2,4-D-OMe), 4-chlorophenoxyacetic acid (CPOAc), 4-chlorophenylacetic acid (CPAc), phenoxyacetic acid (POAc), phenoxyethanol (POEtOH) and naphthoxyacetic acid (NOAc) were from Sigma (St. Louis, M O , USA). 2,2'azobis(2,4-dimethylvaleronitrile) (ABDV) was from Wako (Osaka, Japan). A l l other chemicals were of analytical grade and solvents were of HPLC quality. Preparation of Polymers. Compositions of the polymerization mixtures are summarized in Table I. The ratio of crosslinker : functional monomer : template was 20:4:1. Template, monomers, and polymerization initiator (ABDV) were weighed into glass test tubes and mixed with the solvent. The solutions were then sonicated, sparged with nitrogen for 2 minutes and placed in a thermostated water bath at 45°C for 4 hours, followed by 2 h at 60°C. The resultant hard bulk polymers were ground in a mechanical mortar and wet-sieved in acetone through a 25-pm sieve. The particles were washed by incubation in methanol/acetic acid (7:3) (2x), acetonitrile/acetic acid (9:1) (2x), acetonitrile (lx), methanol (2x) for 2 hours each time, followed by centrifugation. The particles were then resuspended in acetone and allowed to settle for 4 hours. The ones that remained in suspension (fines) were collected and the procedure was repeated 4 times. The solvent was removed by centrifugation and the particles were dried in vacuo. The fine particles obtained in this way had an average diameter of 1 pm and were used in all further experiments. Radioligand Binding Assays. The polymer particles were suspended in the incubation solvent and appropriate volumes were added into 1.5-ml polypropylene test tubes, followed by the radioligand C-2,4-D (0.26 nmol, specific activity 15.7 mCi/mmol), varying amounts of a solution of a competing ligand if appropriate, and solvent to give a total volume of 1.0 ml. The samples were incubated on a rocking table for 2 hours. After centrifugation, 700 pi of supernatant was withdrawn and measured by liquid scintillation counting. 14


137


Table I. Composition of the different imprinted and control polymers Polymer

EDMA

4-VP

Template

Solvent

1 (imprinted)

20 mmol

4 mmol

1 mmol 2,4-D

4 ml methanol + 1 ml H 0

0.31 mmol

2 (control)

20 mmol


0.31 mmol

3 (control)

20 mmol


0.31 mmol

ABDV

2

4 mmol

0

2

4 mmol

1 mmol toluene + 1 mmol acetic acid

2

Results Preparation of the Polymers. The imprinted polymer (1, Table I) was prepared by copolymerization of a crosslinking monomer (EDMA) with a functional monomer (4VP) in the presence of 2,4-D as the template. Given that the goal was to prepare and use the imprinted polymers in water-containing solvents, complex formation had to rely on hydrophobic and ionic interactions which, unlike hydrogen bonding, are not, or at least are to a lesser extent, disturbed in the presence of water. 4-VP was chosen as the functional monomer to allow for ionic interaction with the carboxyl group of the template as well as for the hydrophobicity of its aromatic ring. The imprinting was performed in methanol/H 0 (4:1), as the crosslinking monomer E D M A and 2,4-D were only poorly soluble in pure water. After polymerization, a faint blue color was observed in the bulk polymer. The nonimprinted control polymer (2) was uncolored. However, a control polymer prepared in presence of toluene and acetic acid (3) was also slightly blue. The color disappeared in all cases during washing of the polymers. In contrast to other reports on binding assays with imprinted polymers (2, 7, 77), in the present work we used fine particles with a diameter of about one micrometer. These particles are normally discarded. We found that micrometer-sized particles and smaller had the same binding characteristics for the target molecule as the 25 pm particles normally used. Furthermore, not only were the incubation times reduced due to shorter diffusion distances, but fines were found to be more practical for binding assays as they stayed in suspension longer and were easier to pipette. Moreover, if the template is expensive, it is undesirable to lose a considerable amount of polymer by discarding the fines. 2

14

Rebinding of C-2,4-D. Initially, all three polymers were tested for rebinding of C-2,4-D in the original imprinting solvent. The imprinted polymer (1) could rebind the radiolabeled template in methanol/H 0 (4:1). Only 200 pg polymer was needed to adsorb ca. 50% of the added radioligand. The control polymers 2 and 3 showed only very low binding of the radioligand. As the polymers were intended for use in aqueous buffer, the conditions for aqueous binding assays had to be optimized. There have been some reports in the literature where polymers imprinted in organic solvents have been used in aqueous buffer for binding assays (72,16). Normally low percentages of a water-miscible organic solvent, such as ethanol or acetonitrile, are added to the buffer to increase the wettability of the polymer and to prevent adsorption of hydrophobic templates on test tubes, pipette tips, etc. In initial binding assays, we found that the addition of small amounts of a non-ionic surfactant (0.02-0.1% Triton-X-100) had the same effect, but also improved the mixing of the polymer particles in the test tubes. Therefore, we employed 0.1% Triton X-100 instead of ethanol or acetonitrile in all our binding assays. I4

2


138


14

Also of importance is the pH of the buffer used. Figure 1 shows binding of C-2,4-D to polymers I and 2 in different buffers from pH 3 to pH 9. Binding to polymer I was approximately constant between pH 3 and 7, and then decreased with further increase in pH. The local minimum at pH 5 can be attributed to the fact that at this pH both the pyridine and the 2,4-D are partially charged, which renders them less hydrophobic. Binding should now be due mostly to ionic interactions where the buffer ions can effectively act as competitors. The non-imprinted control polymer 2 showed highest binding at pH 3 which rapidly decreased to remain at a constant low level above pH 6. A l l subsequent experiments were performed in phosphate buffer at pH 7 where binding to the control polymer was minimized. As can be seen from Figure 2a, polymer 1 was able to rebind radiolabeled 2,4-D in 20 m M phosphate buffer at pH 7 and 150 pg/ml of polymer was needed to bind 50% of the added radioligand. At this polymer concentration, very low binding to the corresponding blank polymer was observed. It has to be added that only with polymers imprinted in methanol/H 0 (4:1) and having 4-VP as the functional monomer Was appreciable rebinding capacity observed. Polymers prepared with methacrylic acid as the functional monomer did not specifically rebind C-2,4-D, either in buffer at different pH or in toluene or acetonitrile. Polymers prepared with 4-VP as the functional monomer but in dry toluene as the imprinting solvent could specifically rebind C-2,4-D, but with a 13 times lower capacity than polymer 1. The corresponding control polymer exhibited a significant amount of non-specific binding (not illustrated). 2

l4

14

14

Competitive Binding Assays. Figure 2b shows the competition of C-2,4-D binding to polymers 1-3 by unlabelled 2,4-D. A typical sigmoid competition curve similar to those observed in competitive immunoassays was obtained for the imprinted polymer L The useful concentration range for detection of 2,4-D is from 30 ng/ml (135 nM) to 10 pg/ml (45 pM).

80

14

Figure 1. Binding of C-2,4-D to polymers 1 (•) and 2 (O) as a function of pH. Conditions: 150 pg/1 ml assay; 20 mM buffer, 0.1% Triton X-100.


139


100

Polymer concentration (jug/ml)

0.001

0.01

0.1

1

10

100

[2,4-D] (pg/ml) Figure 2. (a) Binding of radioligand relative to polymer concentration and (b) radioligand displacement curves with unlabelled 2,4-D as competitor at 150 pg polymer/1 ml assay for polymer I (•), polymer 2 (O) and polymer 3 (O). Conditions: 20 m M sodium phosphate buffer pH 7, 0.1% Triton X-100.


140 Cross-reactivity Studies. To assess the specificity of the imprinted polymer, competition of C-2,4-D binding by structurally related compounds was studied. By comparing the concentration that yields 50% inhibition of C-2,4-D binding (IC value) for the different competitors to that of 2,4-D, the cross-reactivities of the related compounds can be estimated. The structures of the different competitors and the corresponding cross-reactivities are shown in Figure 3. A l l evaluated compounds exhibited lower binding to the polymer than the original template. The lowest crossreactivities were obtained with compounds not having a charged group (2,4-D-OMe, POEtOH), whereas 2,4-dichlorophenoxybutyric acid showed almost the same binding as 2,4-D. To assess whether the added surfactant Triton X-100 had an influence on the binding of the different compounds to the polymer, binding was also studied in the same buffer containing 10% ethanol instead of the surfactant. The cross-reactivities obtained were very similar to or identical with those measured in the presence of surfactant.


50

Discussion Molecular imprinting of polymers using only non-covalent interactions between the template and the functional monomers has until now been limited to apolar environments. In some cases however, the use in aqueous buffer of polymers initially imprinted in non-polar solvents has been demonstrated to be feasible. It has been suggested that the interaction between the molecule of interest and the polymer is governed by different molecular forces in non-polar solvents and in aqueous buffer (72). This would mean that the interactions that are predominant in the latter case, mainly hydrophobic interactions and ionic bonds, are strong enough to allow for complex formation in a polar environment. The herbicide 2,4-dichlorophenoxyacetic acid seemed to be a good model compound if the imprinting itself should also be done in presence of water, as it contains an aromatic ring and an ionizable carboxyl group. On the other hand, we presumed that there would only be a small chance of success for imprinting of 2,4-D in non-polar solvents such as toluene, as 2,4-D has too few possible electrostatic interaction points. The use of the solvent system methanol/water instead of pure water in our experiments was dictated by the low solubility of the crosslinker and the template in pure water. We believe, however, that other templates, used in conjunction with water-soluble crosslinkers may work even better at much higher water concentrations. From a comparison of polymer 1 (imprinted with 2,4-D) and polymer 2 (control polymer, no template) in rebinding of the template, it can be concluded that polymer 1 is clearly templated. The observation of a faint blue color during polymer formation in the case of the imprinted polymer could be an indication of a charge transfer interaction between the aromatic rings of 2,4-D and 4-vinylpyridine. The formation of a 7C-complex (77) between the two aromatic rings may also be possible. This may contribute to the stability of the prearranged complex mainly formed through ionic interactions and the hydrophobic effect in the polar environment. Hydrophobic interactions are strong in water and should thus make the biggest contribution to adsorption. However, they are generally non-specific. Therefore it was of interest to investigate the specificity of polymer I by use of related compounds as competitors for C-2,4-D-binding in radioligand binding assays. Given the conditions during imprinting and rebinding as well as the structure of the template, the cross-reactivities obtained between the different competitors and 2,4-D are surprisingly low. Moreover, these data provide additional information about the contribution of different forces to the interaction. Comparing 2,4-D and 2,4-D-OMe, it is obvious that cancelling out the effect of the negative charge greatly reduces binding (7% cross-reactivity, Figure 3). Removing one (CPOAc) or two (POAc) chlorine atoms from the ring also progressively weakens the affinity (24 and 2 % cross-reactivity, respectively). In the case of POEtOH which has no chlorine and no charged group, binding is completely suppressed. On the other hand, NOAc which 14


141


CL

Compound

Cross-reactivity (%)

2,4-D

100

XOOH

DB

95

CI ° \ ^

C

0

0

C

H

3

XOOH

>

V k

|f'*^r ^ COOH

KJ

CV

o o

2,4-D-OMe

CPOAc

24

CPAc

10

XOOH POAc O .CH OH v

2

^COOH

POEtOH

Molecular and Ionic Recognition with Imprinted Polymers - American

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