Post-Cross-Linked Molecular Imprinting with Functional Polymers as a

Sep 27, 2017 - These cavities could be easily tuned to recognize the target molecules by changing the template using a common FP as a universal buildi...
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Post-Cross-Linked Molecular Imprinting with Functional Polymers as a Universal Building Block for Artificial Polymeric Receptors Yukiya Kitayama,* Kazuki Yoshikawa, and Toshifumi Takeuchi* Graduate School of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan S Supporting Information *

ABSTRACT: A post-cross-linked molecular imprinting (PCMI) technique utilizing a functional polymer (FP) with interacting and post-cross-linking groups was developed to create molecularly imprinted polymeric (MIP) receptors. Molecular recognition cavities were formed in the cross-linked polymer matrix by a posteriori cross-linking of the FP with template molecules using photoirradiation. These cavities could be easily tuned to recognize the target molecules by changing the template using a common FP as a universal building block. Thus, precise chiral recognition cavities were successfully created using PC-MI and optimizing the molar ratio of the functional groups between the FP and the target molecules, which suppressed the nonspecific binding of the offtarget molecules. Furthermore, the morphology of the MIPs could be changed from bulk to particles. This study provides a facile and efficient synthetic route for MIPs with tailor-made properties. Thus, PC-MI can be utilized to create molecular recognition elements for purification, hygiene control, disease diagnosis, and sensors.

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INTRODUCTION In nature, antibodies and enzymes work as molecular recognition elements and have been utilized for bio-based applications, such as purification, proteomics, diagnostics, sensors, and pharmaceutical agents.1−5 They are biosynthesized by folding genetically translated polypeptides into thermodynamically stable tertiary and quaternary structures,6 and the folding occurs via inter- and intramolecular interactions between the polypeptide backbone and the amino acid residues. Part of the side chains can be cross-linked via a disulfide linkage between two cysteine residues to form a more rigid structure. Thus, the formed proteins can have specific recognition cavities that complement the size and shape of the target molecules with multiple weak interactions such as hydrogen bonding, electrostatic, hydrophobic, and van der Waals interactions.7,8 These examples from nature suggest that the three-dimensionally regulated polymers formed via the folding process of prematured polymers can demonstrate sophisticated properties such as precise recognition. The artificial molecular recognition materials based on synthetic polymers are attracting significant attention for their high stability and easy functionalization, which allows for the use of a wide range of monomeric species as building blocks. Sequence-controlled polymerizations such as living anionic polymerization and reversible deactivation radical polymerization are precise and have enormous potential for creating three-dimensionally regulated polymers as a bottom-up approach from a wide range of monomer building blocks.6−11 However, the bottom-up approach still faces great challenges. For example, this approach requires complex procedures and © XXXX American Chemical Society

must presume a tertiary structure for the obtained polymers from the polymer sequence. Molecular imprinting (MI) is a promising template-assisted strategy for creating polymeric three-dimensionally regulated recognition materials for specific target molecules. The templates induce the assembly of functional groups that interact with the surrounding templates and create recognition cavities of complementary sizes and shapes to that of the target molecules with integrated interaction sites placed at appropriate positions in the recognition cavities of the artificial polymer matrix.12−29 Conventional MI based on radical polymerization uses functional monomers that are either covalently conjugated to or via noncovalent interactions with the template molecules.30,31 There are several problematic issues with the conventional MI technique, namely: (i) Side reactions with radical species: template molecules bearing radical scavenging groups such as phenol and thiol may be partially denatured by radical-related side reactions during the radical polymerization. (ii) Weakening intermolecular interactions under polymerization conditions: the temperature during the polymerization is normally higher than that during complex formation via noncovalent imprinting, and it affects the intermolecular interactions such as hydrogen bonding. The previously reported prepolymer-based MI technique requires radical polymerization for cross-linking to take place, which also requires these challenges to be overcome.32−34 Received: June 10, 2017 Revised: September 9, 2017

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DOI: 10.1021/acs.macromol.7b01233 Macromolecules XXXX, XXX, XXX−XXX

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technique, radical-related side reactions with the template molecules can be avoided because the radical polymerization step is separated from the molecular imprinting process. In the latter process, the photoirradiation-initiated molecular imprinting is performed either at or below room temperature, enabling more stable interactions between the template molecules and FP. Further, the morphology can be easily changed from the bulk to the particle state. MIPs capable of recognizing different target molecules can be easily synthesized by changing only the template molecules by the same procedure using a common FP as a universal building block. Therefore, the PC-MI technique involving the polymerization-free molecular imprinting process has an enormous potential as an alternate method of conventional MI for producing three-dimensionally regulated polymer-based molecular recognition materials.

Inspired by the folding-based strategy in nature, herein, a new methodology of post-cross-linked molecular imprinting (PC-MI) has been developed as a tailor-made synthetic route for three-dimensionally regulated polymers bearing molecular recognition capability. This approach uses a presynthesized linear functional polymer (FP) bearing groups that can be postcross-linked and interact with the target molecules. First, a thermodynamically stable complex is formed between the presynthesized FP and the target molecule. Subsequently, the a posteriori cross-linking of the complexes via specific stimuli and eventual template removal forms the molecularly imprinted polymers (MIPs) (Scheme 1). By utilizing the PC-MI Scheme 1. Post-Cross-Linked Molecular Imprinting (PCMI)

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RESULTS AND DISCUSSION PC-MI Using a Designed FP. The cinnamoyl group was selected as a post-cross-linking group,35,36 and cinnamoyloxyethyl methacrylate (CEMA) was prepared by the coupling reaction between 2-hydroxyethyl methacrylate and cinnamoyl chloride. UV−vis measurements showed that CEMA has a UV absorption at 280 nm (Figure S1). For the synthesis of the FPs, methacrylic acid (MAA) and methyl methacrylate (MMA) were selected as the monomer bearing interaction sites and a comonomer, respectively. After solution polymerization of CEMA, MAA, and MMA, the linear poly(MAA-MMA-CEMA) was obtained as the FP (number-average molecule weight, Mn = 7700 (Mw/Mn: 2.0)). The 1H NMR spectrum of the FP

Figure 1. (a) Binding isotherms of BPA-MIP and NIP with BPA. (b) Chemical structures of BPA, BPB, 4-cumylphenol, 17β-estradiol, and atrazine. Selectivity tests for (c) BPA-MIP and (d) NIP with BPA and reference compounds at a concentration of 25 μM. B

DOI: 10.1021/acs.macromol.7b01233 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules Scheme 2. PC-MI for the Preparation of BPA-MIP, Atrazine-MIP, and (S)-Ketoprofen-MIP

also prepared by a similar procedure without using the template molecules, and it was found that the presence of BPA in the FP film did not affect the yields of the cross-linked polymer (approximately 70% for BPA-MIP and 70% for NIP). The BPA binding experiments for the BPA-MIPs were demonstrated by quantifying the BPA concentration in the supernatant of the incubated DCM solution, where the functional groups (carboxyl groups) in the molecularly imprinted cavities interacted with the target molecules via hydrogen bonding. At all BPA concentrations, the amount of BPA binding with BPA-MIP was found to be greater than that for NIP, and the dissociation constant (Kd) of BPA-MIP was estimated to be 156 μM, which is approximately 5 times smaller than that for NIP (794 μM) (Figure 1 and Figure S7). This result indicated that the PC-MI process positively affects the BPA binding performance. The selectivity was also checked by using bisphenol B (BPB) and 4-cumylphenol as a structural analogue (Figure 1). In addition, 17β-estradiol and atrazine were selected as other types of endocrine disruptors. The highest binding amount was observed for BPA; i.e., the selectivity factors of BPA-MIP at 25 μM were −0.13, 0.41, −0.18, and 0.27 for BPB, 4-cumylphenol, 17β-estradiol, and atrazine, respectively (Figure 1). However, significant nonspecific binding toward the reference compounds was observed by using NIP (Figure 1). These results indicate that molecularly imprinted three-dimensional cavities bearing BPA recognition capabilities are created via PC-MI using an appropriate FP. Expansion of Target Molecules (Amino and Carboxylic Compounds). The proposed PC-MI methodology is advantageous as it is possible to create various molecular recognition cavities by changing only the template molecules. The synthetic procedure is the same and involves using a common FP as a universal building block. The crucial point is that the carboxyl groups of the FP must be able to interact with various functional groups such as phenols, amino, and carboxyl groups. Therefore, in next step, we demonstrated the use of PC-MI technique toward atrazine and (S)-ketoprofen bearing different interaction groups (amino and carboxyl groups) by using a suitable FP (Scheme 2). Atrazine is an estrogen-like endocrine disruptor. In previous studies, MIPs targeting atrazine have been widely reported in

clarified that the molar ratios of CEMA, MAA, and MMA were 20, 4, and 5, respectively (Figure S2). The cinnamoyl group was not denatured in the FP after the polymerization, and the maximum absorption wavelength was still observed at 280 nm (Figure S3). In order to check the photoinduced cross-linking (λ = 254 nm) property, the time dependence of the cinnamoylderived absorbance at 280 nm of the FP film formed on a BK7 optical glass substrate was investigated by UV−vis measurements. It was found that the absorbance gradually decreased as the photoirradiation time increased (Figure S3). In addition, the FT-IR peak intensity at 1636 cm−1 arising from the CC bond of the cinnamoyl group clearly decreased after photoirradiation, suggesting that the FP has a posteriori photoinduced cross-linking capability (Figure S4). Bisphenol A (BPA), an endocrine disruptor, was selected as the first target molecule bearing phenol groups. The interaction between FP and BPA was investigated by a 1H NMR titration. The proton peak derived from the phenolic groups of BPA clearly shifted to downfield with broadening after mixing with FP (Figure S5). The peak shift with broadening indicates that the phenol groups interact with the carboxyl groups of the FP via hydrogen bonding. The apparent dissociation constant (Kd) between FP and BPA (2.60 mM) is 6.2 times smaller than that between MAA and BPA (16.2 mM) (Figure S6). The high affinity toward the target BPA may be a result of the multiple interactions of carboxyl groups and the π−π interactions of the cinnamoyl groups. A small peak shift for the BPA aromatic protons also was observed (Figure S5). The BPA-imprinted polymers (BPA-MIPs) were prepared by using the PC-MI technique as follows. FP and BPA were mixed in dichloromethane (DCM) to form the complex. The photoinduced post-cross-linking took place after the FP film interacted with BPA via DCM evaporation, and the obtained cross-linked polymers were crushed to 99% (Figure S15)) have a spherical morphology with a number-average particle size of 960 nm (coefficient of variation (CV) = 17%) (Figure 7). The obtained spherical BPA-MIP particles were examined in terms of their affinity and selectivity toward the target molecules. BPA-MIP particles showed a greater binding affinity toward the target BPA compared to the NIP particles (the number-average particle size is 830 nm (CV = 23%)) (Figure 7 and Figure S16). The selectivity could only be confirmed for the BPA-MIP particles using BPB, and atrazine as reference compounds; the selectivity factors of the BPA-MIP particles for BPB and atrazine were determined to be

0.27 and 0.12, respectively (Figure 7). These results indicate that the PC-MI technique was successfully applied for the synthesis of spherical BPA-MIP particles.

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CONCLUSIONS PC-MI was developed by using a FP with various target molecules bearing different interaction sites (phenol, amine, and carboxyl groups) and chiralities. The molecular recognition capabilities were confirmed by the binding experiments of the corresponding target molecules and selectivity tests with structural analogues. The shape control of the MIPs from bulk to spherical particles was also demonstrated. Furthermore, the nonspecific binding of the off-target compounds was successfully suppressed, and recognition cavities with high affinities and selectivities were formed by selecting an optimum G

DOI: 10.1021/acs.macromol.7b01233 Macromolecules XXXX, XXX, XXX−XXX

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molar ratio of the interaction sites between the FP and the target molecules. The template-assisted construction approach is a powerful strategy for creating three-dimensional, regulated, polymer-based recognition cavities for a wide range of applications such as separation, sensing, and drug delivery.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.7b01233. Detailed experimental section, UV−vis spectra of CEMA and FP, 1H NMR and FT-IR spectra of FP, 1H NMR titration of FP or MAA with the target molecules for the estimation of binding constants, selectivity tests for NIP (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Y.K.). *E-mail: [email protected] (T.T.). ORCID

Yukiya Kitayama: 0000-0002-7418-301X Toshifumi Takeuchi: 0000-0002-5641-2333 Funding

This work was partially supported by JSPS KAKENHI Grant 17K05997. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS BPA bisphenol A BPB bisphenol B CEMA cinnamoyloxyethyl methacrylate DCM dichloromethane FP functional polymer MAA methacrylic acid MI molecular imprinting MIP molecularly imprinted polymer MMA methyl methacrylate NIP nonimprinted polymer PC-MI post-cross-linked molecular imprinting.

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DOI: 10.1021/acs.macromol.7b01233 Macromolecules XXXX, XXX, XXX−XXX