Regioselective Molecularly Imprinted Reaction Field for [4+ 4

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A Regioselective Molecularly Imprinted Reaction Field for the [4+4] Photocyclodimerization of 2-Anthracenecarboxylic Acid Satoshi Nakai, Hirobumi Sunayama, Yukiya Kitayama, Masaki Nishijima, Takehiko Wada, Yoshihisa Inoue, and Toshifumi Takeuchi Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b04104 • Publication Date (Web): 08 Feb 2017 Downloaded from http://pubs.acs.org on February 9, 2017

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A Regioselective Molecularly Imprinted Reaction Field for the [4+4] Photocyclodimerization of 2-Anthracenecarboxylic Acid

Satoshi Nakai,†, Hirobumi Sunayama,†,‡ Yukiya Kitayama,† Masaki Nishijima,§ Takehiko Wada,∥ Yoshihisa Inoue,⊥ and Toshifumi Takeuchi*,†

†

Graduate School of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501,

Japan §

∥

Office for University-Industry Collaboration, Osaka University, Yamada-oka, Suita 565-0871, Japan Institute of Multidisciplinary Research for Advanced Materials (IMRAM), Tohoku University, 2-1-1,

Katahira, Aoba-ku, Sendai 980-8577, Japan ⊥

Department of Applied Chemistry, Osaka University, Yamada-oka, Suita 565-0871, Japan

1 ACS Paragon Plus Environment

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ABSTRACT Molecularly imprinted cavities have functioned as a regioselective reaction field for the [4+4] photocyclodimerization of 2-anthracenecarboxylic acid (2-AC). Molecularly imprinted polymers (MIPs) were prepared by the precipitation polymerization of N-methacryloyl-4-aminobenzamidine as a functional monomer to form a complex with template 2-AC and ethylene glycol dimethacrylate as a crosslinking monomer. The 2-AC-imprinted cavities thus constructed preferentially bound 2-AC with an

affinity

greater

than

that

towards

structurally

related

9-anthracenecarboxylic

acid,

2-aminoanthracene, and unsubstituted anthracene. Moreover, from the four possible regioisomeric cyclodimers, they mediated the [4+4] photocyclodimerization of 2-AC specifically to the anti-head-to-tail (HT) isomer. This indicates that the imprinted cavities accommodate two 2-AC molecules

in

an

anti-HT

manner,

thereby

facilitating

photocyclodimerization.


the

subsequent

regioselective

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1. Introduction Molecular imprinting is a well-established methodology for constructing recognition cavities tailor-made for versatile target molecules in an artificial polymer matrix.1-10 This methodology, categorized as a template polymerization technique, employs the crosslinking polymerization of a functional monomer covalently or non-covalently bound to a target molecule or its derivative (used as a template molecule). Subsequent extraction of the template from the polymer matrix results in the formation of imprinted cavities that are complementary to the template molecule in size and shape. The molecularly imprinted polymers (MIPs) afforded exhibit highly sensitive and selective recognition capabilities. Hence, MIPs have found applications in a wide range of research areas, for example, as separation media, sensors, and pharmaceutical agents. 11-15 This technique has also been applied to the construction of reaction fields. Several groups have reported enzyme-inspired MIPs prepared by using this strategy.16-23 Template molecules that mimic the transition state of a desired reaction have been used in molecular imprinting to endow the imprinted cavities with the ability to catalyze the target reaction through stabilization of the transition state.24 In our previous study, toxic atrazine, a triazine herbicide, was successfully converted to the less toxic atraton by methanolysis in the presence of MIPs.25 Furthermore, high-performance catalytic MIPs have been prepared via post-imprinting modifications. 26 Anthracene undergoes photoinduced [4+4] cyclodimerization.27 The photocyclodimerization of 2-anthracenecarboxylic acid (2-AC) leads to four regioisomeric cyclodimers, i.e., anti-head-to-tail 3 ACS Paragon Plus Environment

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(anti-HT), syn-head-to-tail (syn-HT), anti-head-to-head (anti-HH), and syn-head-to-head (syn-HH) isomers (Scheme 1). Therefore, the photocyclodimerization of 2-AC has been used as a model system for evaluating how and to what extent a potentially regioselective reaction field can modify the original reactivity and selectivity to afford a specific regioisomer via its interaction with natural and artificial supramolecular hosts such as proteins, cyclodextrins, liquid crystals, and organogels.28-33 In this study, we found that molecularly imprinted cavities prepared by using 2-AC as a template molecule do not merely serve as a recognition site for 2-AC but unprecedentedly function as a regioselective reaction field that facilitates the [4+4] photocyclodimerization of 2-AC to a specific cyclodimer. Thus, the imprinted cavities selectively recognize not only 2-AC but also the anti-HT dimer, exhibiting the ability to simultaneously bind two 2-AC molecules in a specific orientation. This result prompted us to examine the regioselective photocyclodimerization of 2-AC mediated by the MIP that eventually enabled us to preferentially produce one of the four regioisomeric cyclodimers of 2-AC. The present method provides a new convenient tool for constructing a regioselective reaction field in MIPs by using the substrate itself, rather than the final product, as the template molecule.


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Scheme 1. [4+4] Photocyclodimerization of 2-anthracenecarboxylic acid (2-AC) COOH COOH

+ COOH

hν

HOOC

HOOC

anti-HT

syn-HT

HOOC

2-AC

HOOC

+ HOOC

HOOC

syn-HH

anti-HH

2. Results and Discussion N-Methacryloyl-4-aminobenzamidine (MABA) hydrochloride was synthesized as a functional monomer for molecular imprinting by 2-AC. This was achieved by the coupling reaction of p-aminobenzamidine with methacryloyl chloride. The amidine and carboxylic acid were expected to form a stoichiometric complex.33-34 The 1:1 complex of 2-AC with MABA (template complex employed in molecular imprinting) was precipitated as a white solid [in the ethyl acetate (AcOEt) phase] by mixing an aqueous sodium hydroxide (NaOH) solution of 2-AC with an AcOEt solution of MABA. The successful synthesis of the template complex was confirmed by 1H-NMR, where the integrated areas of the proton signals derived from the anthracene moiety were in good agreement with the expected theoretical values (Figure S1). In addition, the amidine proton peak (δ 9.2 ppm) disappeared, implying that the complex formation occurred via electrostatic interactions and/or hydrogen bonding between the amidine and carboxy groups. The MIP particles were prepared by precipitation polymerization using the template-MABA complex,

ethylene

glycol

dimethacrylate

(EGDM)

as


a

cross-linking

agent,

and

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2,2’-azobis(4-methoxy-2,4-dimethylvaleronitrile) (V-70) as an initiator. The reaction was carried out in methanol at 30 °C. The interaction between the amidine and carboxy groups in methanol has been reported in a previous study.35 Scanning electron microscopy (SEM) confirmed the particulate morphology of the synthesized polymers (Figure S2). The estimated number-average particle size (dn) was ~353 nm. After the removal of the unreacted monomers by extraction with methanol, the template 2-AC molecules were washed out from the polymer particles by centrifugation with a methanol solution comprising 10% acetic acid. This process was repeated for another five times to afford template-free MIP particles for molecular recognition and photochemical examinations. To confirm the removal of the template molecule, the supernatant afforded after each centrifugal washing was subjected to fluorescence spectrophotometry. Strong 2-AC fluorescence was detected in the early washing solutions; the accumulated intensity reached a plateau after the fifth washing where negligible fluorescence was observed (Figure S3). This indicates that the 2-AC molecules were successfully removed from the MIP particles. The binding behavior of the MIP particles was examined in a 4:1 acetonitrile/water mixture to verify the successful formation of the 2-AC-specific binding cavities by the molecular imprinting procedures employed. For comparison, 9-anthracenecarboxylic acid (9-AC), 2-aminoanthracene (2-AA), and unsubstituted anthracene (AN), compounds that was structurally related to the target guest 2-AC, were also employed as reference guests. As illustrated in Figure 1a, neither 2-AA nor AN exhibited any adsorption to the MIP particles due to the lack of an effective interacting site that is 6 ACS Paragon Plus Environment

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complementary to the hydrogen bonding motif in the amidine moiety. Moreover, non-specific binding, based on the π-π stacking and hydrophobic interactions between the aromatic rings, did not appear to operate. In contrast, 2-AC and 9-AC, possessing a carboxyl group at different positions, adsorbed onto the MIP particles smoothly to exhibit distinctly different binding isotherms (Figure 1a). The affinity was appreciably larger for 2-AC than for 9-AC. From the Scatchard analyses (Figure S4), the dissociation constant (Kd) for 2-AC was determined as 8.1 µM, a factor of 3.6 smaller than that of 9-AC (29 µM). The selectivity factor, the ratio of bound 9-AC to bound 2-AC, was employed as an index of the selective binding capability and was calculated as ~0.53 for the MIP at 50 µM (Figure 1b), where the bound amounts of 2-AC and 9-AC were 27.4 nmol/mg and 14.5 nmol/mg, respectively. These results confirm that the MIP particles bear the selective recognition cavities for 2-AC. On the other hand, the non-imprinted polymer (NIP) particles, prepared by the same protocol used for the MIP but without the addition of 2-AC, possessed no recognition cavities but only interaction sites (amidine groups). These particles exhibited comparable binding abilities for 2-AC (34.3 nmol/mg) and 9-AC (35.2 nmol/mg) with a selectivity factor of ~1.0 for 9-AC, although the amount of bound 2-AC was slightly higher than that of the MIP particles. This indicates that adsorption of 2-AC onto the NIP should be considered as non-specific binding (Figure 1c). These results can be rationalized by assuming that 2-AC and 9-AC can interact with the amidine group in both the MIP and NIP, however, the MIP is more efficient in stabilizing the shape-matched 2-AC guest included in its cavity. This implies that the molecular imprinting protocol employed was successful in creating specific recognition 7 ACS Paragon Plus Environment

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cavities for the target guest 2-AC.

Bound amount (nmol/mg)

40

(a) 2-AC

30

9-AC 2-AA

20

AN

10 0 0

20

40

60

80

100

-10 Concentration (μM) 1.4 1.2

1.4

(b) MIP

1.2

1

Selectivity factor

Selectivity factor

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0.8 0.6 0.4 0.2

(c) NIP

1 0.8 0.6 0.4 0.2

0 2-AC

0

9-AC

2-AC

9-AC

Figure 1. (a) Binding isotherms of 2-anthracenecarboxylic acid (2-AC), 9-anthracenecarboxylic acid (9-AC), 2-aminoanthracene (2-AA), and anthracene (AN) toward the molecularly imprinted polymer (MIP) particles, and selectivity factors of 9-AC relative to 2-AC upon adsorption to (b) MIP and (c) non-imprinted polymer (NIP) particles at 50 µM; each value is an average of three independent runs.


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We further investigated the binding behavior of the regioisomeric 2-AC dimers (Scheme 1) with the MIP particles. Thus, a mixture of anti-HT, syn-HT, anti-HH, and syn-HH dimers was incubated with the MIP particles under the same conditions employed in the above experiments. To calculate the relative affinities of the 2-AC dimers to the MIP particles, the concentrations of each dimer before and after incubation (C0 and C, respectively) were determined by HPLC. Surprisingly, only the anti-HT dimer was appreciably bound to the MIP particles, while the other three dimers displayed no sign of adsorption (Figure 2); the bound amount of anti-HT dimer was determined as 17.6 nmol/mg at an initial concentration of 61 µM. The exclusive binding of the anti-HT dimer indicates that the MIP particles possess imprinted cavities that are nicely fitted to this specific 2-AC dimer, despite the use of monomeric 2-AC as a template for molecular imprinting. Although the exact reason for the specific binding of anti-HT dimer is not clear, it is likely that 2-AC does not function as an isolated monomer in the imprinting process. Instead, it forms a stacked dimer through π-π interactions between two 2-AC molecules that are oriented anti-HT to each other due to the steric repulsion of the carboxyl groups. This facilitates the subsequent regioselective photocyclodimerization. To confirm the dimerization of the 2-AC monomers via π-π stacking during MIP preparation, UV-Vis spectra were measured at various concentrations of 2-AC in methanol. The absorption peaks clearly displayed a longer wavelength shift with peak divisions (Figure S5), suggesting that the 2-AC molecules interact with each other in methanol, thereby facilitating the subsequent regioselective photocyclodimerization.


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0.15

0.1 (C0-C)/C0

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0.05

0 anti-HT

syn-HT

anti-HH

syn-HH

-0.05

Figure 2. Affinities of the four regioisomeric 2-anthracenecarboxylic acid (AC) dimers toward the molecularly imprinted polymer (MIP) particles, evaluated as the adsorbed amount (C0 – C) relative to the initial concentration (C0) in a 1:4 H2O-CH3CN mixture; average of three independent runs; C is the dimer concentration after incubation.

The discovery of the highly preferential binding of the anti-HT dimer prompted us to use the MIP as a reaction field for regioselective photocyclodimerization of 2-AC. THF solutions of 2-AC with and without the added MIP particles were photoirradiated (>320 nm) for 2 h under identical conditions. The irradiated solutions were filtered to remove the MIP particles and the resultant filtrates were analyzed by HPLC. The effects of the MIP on the product distribution were examined by comparing the molar fractions of the 2-AC dimers afforded in the presence and absence of the MIP particles. The change in molar fraction (∆F) caused by the addition of the MIP was calculated for each dimer by 10 ACS Paragon Plus Environment

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using equation 1.

∆F =

AMIP A0 − ∑ AMIP ∑ A0

(1)

where AMIP and A0 are the HPLC peak areas of each 2-AC dimer produced in the presence and absence of MIP particles, respectively and ΣAMIP and ΣA0 are their respective sums. The total conversion of 2-AC by the photoirradiation process in the presence of the MIP (~67%) was slightly higher than that observed when the MIP was absent (64%). This may be due to the locally enhanced 2-AC concentration in the imprinted cavities. Notably, the HT/HH as well as the anti/syn ratios were enhanced in the presence of the MIP. In particular, the molar fraction of the anti-HT dimer significantly increased (that of the syn-HT dimer increased to a lesser extent) in the presence of the MIP particles at the expense of the anti- and syn-HH dimers (Figure 3). These results reveal that the MIP cavities prepared by using 2-AC as a template can accommodate not one but two 2-AC molecules preferentially in an anti-HT manner and more crucially act as a regioselective reaction field to facilitate the subsequent photocyclodimerization of the included 2-ACs to the specific dimer. The initial amount of 2-AC added (15 µmol) was in large excess when compared to the number of maximum 2-AC binding cavities in the MIP (35.1 nmol/mg-MIP) calculated by Scatchard analysis (Figure S4). Thus, competitive replacement may occur between 2-AC and the produced dimers. This prevents the dimers from occupying the cavity for a long period of time and consequently facilitates the progress of the 11 ACS Paragon Plus Environment

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photoreaction. The regioselective synthesis should be prominent by further optimizing various conditions such as the photoirradiation time, 2-AC/MIP molar ratio, and MIP synthetic conditions. 0.04 0.03 0.02 0.01 ΔF

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-2E-16 -0.01

anti-HT

syn-HT

anti-HH

syn-HH

-0.02 -0.03

Figure 3. A comparison of the molar fraction changes (∆F) for the anti-head-to-tail (HT), syn-HT, anti-head-to-head (HH), and syn-HH dimers caused upon photocyclodimerization in the presence of the molecularly imprinted polymer (MIP) particles; average of three independent runs.


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3. Conclusions Molecular imprinting has long been employed as a convenient tool for constructing a cavity complementary in shape and functional group to the template molecule incorporated in the crosslinking polymerization of functional monomers. Consequently, the void cavity prepared by this technique functions as an efficient recognition site for specific guests with dimensions and functional groups comparable to the original template. In this study, using MABA as a functional monomer and 2-AC as a template molecule, we have succeeded to create a dually and regiospecifically functionalized cavity that is twice as large as the single-functionalized template used; this has not been reported to date. The three-dimensionally well-organized cavity thus prepared preorganizes two 2-AC molecules in a regiospecific manner to facilitate the subsequent regioselective photocyclodimerization to the anti-HT dimer. This method leads to a new convenient tool for constructing a regioselective reaction field in MIPs, whereby the 2-AC substrate is employed instead of a 2-AC dimer as the template molecule. Thus, the tedious and time-consuming process to produce the 2-AC dimer is avoided. The scope of this new MIP methodology is not restricted to photocyclodimerization reactions but is potentially expandable to other bimolecular (photo)reaction systems. This includes a reactive donor-acceptor pair that forms a charge-transfer (CT) complex in the ground state and undergoes photochemical or thermal reactions upon irradiation or heating. In such a system, the CT complex is expected to function as a supramolecular template for creating a large imprinted cavity suitable for inclusion and subsequent bimolecular (photo)reaction of the specific donor and acceptor pair. 13 ACS Paragon Plus Environment

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4. Experimental Section 4-1. Materials p-Aminobenzamidine dihydrochloride, N, N-dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), and trifluoroacetic acid (TFA) were purchased from Nacalai Tesque Co. (Kyoto, Japan). Methacryloyl chloride, 2-AC, 9-AC, and AN were purchased from Tokyo Chemical Industries (Tokyo, Japan). NaOH, pyridine, methanol, ethanol, AcOEt, dichloromethane (DCM), EGDM, V-70, acetic acid, and 2-AA were purchased from Wako Co. Ltd. (Osaka, Japan). Acetonitrile was purchased from SIGMA-Aldrich (USA). 4-2. Characterization The particle sizes and morphologies were observed under a VE-9800 scanning electron microscope (KEYENCE, Osaka, Japan). The 1H-NMR spectra were measured using FT-NMR apparatus (300 MHz, JNM-LA300 FT NMR system, JEOL Ltd., Tokyo, Japan). Fluorescence spectra were measured using an F-2500 fluorescence spectrophotometer (Hitachi, Japan). UV-Vis spectral measurements were performed using a V-560 spectrophotometer (JASCO Ltd., Tokyo, Japan). 4-3. Synthesis of MABA The monomer has been reported by Haupt et al.36 Pyridine (0.75 mL, 9.3 mmol) and p-aminobenzamidine dihydrochloride (212.5 mg, 1 mmol) were dissolved in dry DMA (3 mL). Methacryloyl chloride (200 µL, 2 mmol) dissolved in dry DMA (0.5 mL) was added to the mixture at 0 °C under N2 atmosphere. After the reaction was allowed to stand at room temperature for 2 h, the 14 ACS Paragon Plus Environment

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solvent was evaporated in vacuo at 40 °C. The resulting orange oil was washed with a 10:1 (v/v) DCM/methanol mixture to afford the product as a white solid. Yield: 170 mg (71%). 1H-NMR (DMSO-d6, 300 MHz, ppm) δ 10.24 (s, 1H), 9.22 (s, 2H), 8.80 (s, 2H), 7.91 (m, 4H), 5.88 (s, 1H), 5.61 (s, 1H), 1.96 (s, 3H).

4-4. Synthesis of the MABA-Template Complex MABA (29.7 mg, 0.12 mmol) dissolved in AcOEt (100 mL) was added to 2-AC (60.6 mg, 0.24 mmol) dissolved in aqueous NaOH solution (100 mM, 40 mL) to form the crude product (as a precipitate in the AcOEt phase). The precipitate was collected by filtration, washed with AcOEt and dried in vacuo to afford the product as a white solid. Yield: 56.3 mg (97.6%). 1H-NMR (DMSO-d6, 300 MHz, ppm) δ 10.25 (s, 1H, amide), 8.61–7.49 (m, 13H, benzene), 8.61–7.49 (m, 14H, benzene), 5.88 (s, 1H, CH2), 5.61 (s, 1H, CH2), 1.97 (s, 3H, CH3). 4-5. UV-Vis Measurements of Various Concentrations of 2-AC 2-AC/methanol solutions in different concentrations (0, 2.88, 5.76, 14.4, 28.8, 72, 144, 360, and 720 µM) were prepared for UV-Vis measurements at 25 °C. 4-6. Synthesis of the MIP and NIP Particles The MIP particles were synthesized by precipitation polymerization. The template-monomer complex (12.5 mg, 0.027 mmol), EGDM (0.10 mL, 0.54 mmol), and V-70 (3.39 mg, 1.1 µmol) were dissolved in methanol (7.5 mL). The polymerization reaction was carried out at 30 °C for 24 h with constant stirring 15 ACS Paragon Plus Environment

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(450 rpm). The polymerized sample was washed three times with methanol to remove the free template molecules and unreacted monomers. The afforded polymers were mixed with methanol containing 10% acetic acid (4 mL) for 45 min. The polymer dispersion was completely transferred to the centrifuge tube with the aid of the same mixed solvent (total solution volume: 7 mL) and centrifuged at 18900 rpm for 15 min. To measure the 2-AC concentration, the supernatant (6 mL) was corrected and the fluorescence intensities (excitation wavelength: 365 nm) were determined after six-fold dilution of the 10% acetic acid solution. The centrifugal washing process was repeated for six times. The same procedure, without the addition of the template, was employed for NIP preparation. 4-7. Binding and Selectivity Tests for 2-AC The 2-AC solution was prepared from 2-AC dissolved in an acetonitrile/water mixture (80/20 vol/vol, 1–100 µM). After the MIP particles (1 mg) were added to the solution, incubation was carried out at r.t. for 20 min. The supernatant was corrected by filtration. The amount of bound 2-AC was measured by HPLC [Waters ACQUITYTM fitted with an ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 mm × 150 mm), temperature: 40 °C, injection volume: 10 µL, eluent: acetonitrile (0.1% TFA):water ratio = 50:50 (vol/vol), flow rate: 0.6 mL/min, detector: UV (254 nm)]. The binding capability of the NIP particles was also estimated using the same protocol. Selectivity tests (using the same protocol as that used for 2-AC) were also carried out with AN, 2-AA, and 9-AC as reference compounds.


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4-8. Binding Tests for the 2-AC Dimers The binding tests for the 2-AC dimer mixture dissolved in 80% acetonitrile solution (0.25 mM, 2.5 mL) were carried out using MIP particles (1 mg). The mixture comprised four regioisomeric 2-AC dimers (HPLC area percentage: syn-HH: 24.6%, anti-HH: 16.8%, syn-HT: 34.5%, and anti-HT: 24.2%). The amount of each bound 2-AC dimer was measured by HPLC [Waters ACQUITYTM fitted with an ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 mm × 50 mm), temperature: 40 °C, injection volume: 10 µL, eluent: acetonitrile/water ratio = 80:20 (vol/vol), flow rate: 0.2 mL/min, detector: UV (254 nm)]. Because of the solubility of the AC dimers, an acetonitrile-water mixture was employed as the solvent in the binding experiments. 4-9. Photocyclodimerization of 2-AC with the MIP Particles 2-AC dissolved in THF (5 mM, 3 mL) was mixed with the MIP (1 mg) in a 1 × 1 cm quartz cell. The MIP-dispersed 2-AC solution was photoirradiated (>320 nm) for 2 h using an ultrahigh-pressure mercury lamp (SX-UI500HQ, Ushio Inc., Japan) fitted with a UV band-pass filter (UTVAF-50S-34U, 251–398 nm, Sigma Koki Co., Ltd., Japan; distance from the light source to the sample = 40 cm). After filtration to remove the MIP particles, the product was analyzed using HPLC [Waters ACQUITYTM fitted with an ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 mm × 50 mm), temperature: 40 °C, injection volume: 10 µL, eluent: acetonitrile-water ratio = 80:20, flow rate: 0.2 mL/min, detector: UV (254 nm)]. To investigate whether the imprinted cavities in the MIP particles work as a reaction field, ∆A was calculated using equation 1. The conversion of 2-AC was estimated from the peak area of the 17 ACS Paragon Plus Environment

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2-AC dimers and 2-AC monomer afforded from HPLC analysis after 2 h photoirradiation and from the ε values of the 2-AC dimer (88,000 M-1cm-1) and 2-AC monomer (13,000 M-1cm-1) at 254 nm.30

ASSOCIATED CONTENT Supporting Information 1

H-NMR spectrum of the template molecule, SEM image of the MIP particles, template removal test

using fluorescence spectrophotometry, Scatchard plots, and UV-Vis spectra of various concentrations of 2-AC can be viewed in the Supporting Information.

AUTHOR INFORMATION Corresponding Author * [email protected] ORCID Toshifumi Takeuchi: 0000-0002-5641-2333 Present Addresses ‡

Faculty of Pharmacy, Yasuda Women’s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima

731-0153, Japan Notes The authors declare no competing financial interest. 18 ACS Paragon Plus Environment

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ACKNOWLEDGMENTS This work was partially performed under the Cooperative Research Program of Network Joint Research Center for Materials and Devices from the Japan Society for the Promotion of Science (JSPS). The authors would like to thank SYSTEM INSTRUMENTS Co., Ltd. (Tokyo, Japan) for their financial support.

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Regioselective Molecularly Imprinted Reaction Field for [4+ 4

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