Phase Inversion Molecular Imprinting by Using Template Copolymers

These copolymers were synthesized in the presence of the THO template at ..... Molecularly Imprinted Membranes Having Amphiphilic Scaffold Media for T...
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Phase Inversion Molecular Imprinting by Using Template Copolymers for High Substrate Recognition Takaomi Kobayashi,* Takahiro Fukaya, Masanori Abe, and Nobuyuki Fujii Department of Chemistry, Nagaoka University of Technology, Kamitomioka, Nagaoka, Niigata 940-2188, Japan Received May 3, 2001. In Final Form: January 2, 2002 Phase inversion molecular imprinting of theophylline (THO) was performed using acrylonitrile (AN) copolymers with acrylic acid (AA) (P(AN-co-AA)) and methacrylic acid (MA) (P(AN-co-MA). These copolymers were synthesized in the presence of the THO template at different template concentrations with [THO]/ [COOH] ) 0.4, 1, and 2. Then, polymer solutions were casted and phase-inversed in water to prepare THO-imprinted copolymers. After template extraction, heterogeneous binding experiments of THO and its analogues caffeine, hydroxyethyl THO, uracil, theobromine, lumazine, and purine were performed. It was found that binding amounts of THO depended on [THO]/[COOH]. At [THO]/[COOH] ) 1, THOimprinted copolymers bound effectively to the THO substrate. Comparison was made by 1H NMR experiments for a monomer solution with THO and copolymers prepared with and without template polymerization. Evidence showed that the template interacted with the COOH proton in the monomer solution via hydrogen bonding and that template copolymerizations caused a conformation change of copolymer segments. Resultant template copolymers had selective recognition to THO, and effective imprinting of template copolymers with MA segments was observed.

Introduction The molecular imprinting technique has been found to be an effective means of encoding template molecule information in bulk material on a molecular scale.1,2 This procedure involves incorporation of small amounts of template molecules in a polymerization medium. After polymerization, template molecules are removed from the rigid polymer. Thus, template polymerization builds up functionalized volumetric sizes of the template molecule in the polymer network.3-6 Most molecular-imprinted polymers are applied mainly for chromatographic uses because only rigid gel matrixes are suitable for those applications. More recently, we developed a new molecular-imprinting technique for porous membrane material preparation using poly(acrylonitrile-co-acrylic acid) (P(AN-co-AA)) (Chart 1).7,8 As shown in Figure 1, template molecule information was encoded by the copolymer phase inversion process.9-12 As is advantageous for methods without the polymerization step in the imprinting process, polymer solidification was applied. Here, the polymer solution with the template was used. The copolymer was (1) Mosbach, K. Trends Biochem. Sci. 1994, 19, 9. (2) Wulff, G. Molecular Interaction in Bioseparations; Ngo, T., Ed.; Plenum Press: New York, 1993; p 363. (3) Andersson, I. L.; Ekberg, B.; Mosbach, K. Molecular Interaction in Bioseparations; Ngo, T., Ed.; Plenum Press: New York, 1993; p 383. (4) Sellergren, B.; Shea, K. J. J. Chromatogr., A 1993, 654, 17. (5) Sellergren, B.; Ekberg, B.; Mosbach, K. J. Chromatogr. 1985, 347, 1. (6) Vlatakis, G.; Andersson, L. I.; Muller, R.; Mosbach, K. Nature 1993, 361, 645. (7) Kobayashi, T.; Wang, H. Y.; Fujii, N. Chem. Lett. 1995, 927. (8) (a) Wang, H. Y.; Kobayashi, T.; Fujii, N. Langmuir 1996, 12, 4850. (b) Wang, H. Y.; Kobayashi, T.; Fujii, N. Langmuir 1997, 13, 5396. (9) Mulder, M. Basic Principles of Membrane Technology; Kluwer Academic: The Netherlands, 1991; pp 83-86. (10) Loeb, S.; Milstein, F. Paper presented before the First European Symposium on Fresh Water from the Sea, Athens, May 31-June 3, 1962. (11) Loeb, S.; Milstein, F. Summary Report No. 2369; Aerojet-General Corp.: Azusa, CA, Oct 1962. (12) Kobayashi, T.; Nagai, T.; Ono, M.; Wang, H. Y.; Fujii, N. Eur. Polym. J. 1996, 3, 1187.

Figure 1. Illustration of phase inversion imprinting. Chart 1

coagulated in nonsolvent in the presence of the template. Hence, the resultant polymer contains the volumetric size of the template after template extraction. In the case of acrylonitrile (AN) copolymer, AN residues work as solidified residues; acrylic acid (AA) residues having the carboxylic acid function interact with template molecules

10.1021/la0106586 CCC: $22.00 © 2002 American Chemical Society Published on Web 02/16/2002

Phase Inversion Molecular Imprinting Chart 2

to fix the template volumetric size in the copolymer.13 We reported that resulting imprinted copolymers selectively recognize the template theophylline (THO) (Chart 2).13,14 In this case, we used copolymer without template polymerization for phase inversion imprinting of THO. To obtain high substrate recognition by phase inversion imprinting polymers, the present paper describes P(ANco-AA) and P(AN-co-MA) copolymers (MA, methacrylic acid) (Chart 1) prepared in the presence of the THO template. Then, the resultant template-polymer solution was used for phase inversion imprinting of THO. To incorporate specific template sites into the template polymer, we sought to study the effect of template polymerization on recognition of the phase inversion imprinted polymer. We placed emphasis on binding properties of THO to THO-imprinted polymers which were obtained at different [THO]/[COOH] ratios in a monomer solution. Experimental Methods Materials and Copolymerization. All experiment reagents were of reagent grade unless otherwise described. The THO, CAF, and other substrates were products of Tokyo Kasei and used without purification. Water used in experiments was distilled and then purified by passing it through ion-exchange resin before use. Chart 1 shows the copolymer chemical structure (y denotes the fraction of AA or MA segments having a COOH unit). Template polymers, P(AN-co-AA)THO and P(AN-co-MA)THO, were prepared in the presence of THO by the modified method previously reported.15 As shown in Table 1, copolymerization of AN and AA or MA was carried out in the presence of THO in dimethyl sulfoxide (DMSO) solution (300 mL). The monomer feed of AN and AA or MA was fixed as 85 and 15 mol % based on total monomer with 0.1 wt % 2,2′-azobis(2 methyl propionitrile), and THO concentration relative to AA or MA concentration was changed with [THO]/[COOH] ) 0.4, 1, and 2. For example, copolymerization of P(AN-co-AA)THO at [THO]/[COOH] ) 1 was performed as follows: In a reaction vessel of 500 mL capacity, 0.572 mol of purified AN, 0.106 mol of AA, 0.106 mol of THO, and 1.3 mmol of AIBN were soluble in 110.5 g of DMSO. Copolymerization was carried out at 60 °C for 6 h in a nitrogen atmosphere. The viscous polymerization contents mixture was used for phase inversion imprinting without further polymer purification. Table 1 lists properties of the resultant copolymers, P(AN-co-AA)THO, P(AN-co-MA)THO, and PANTHO. The subscript THO was for template copolymerization. The P(AN-co-AA) and P(AN-co-MA) without subscript THO were copolymerized without the template according to previous reports7,15 and used for phase inversion imprinting after preparation of the DMSO solution with the template. Phase Inversion Imprinting. Phase inversion imprinting of P(AN-co-AA)THO, P(AN-co-MA)THO, and PANTHO was carried out by using the copolymer-DMSO solution with the template. Figure 1 illustrates the imprint process involving polymer phase inversion. The viscous solution containing the copolymer and THO template was cast on a flat glass plate (200 × 200 cm2) with about 100 µm thickness. To solidify the PAN copolymer with (13) Bartsch, R. A.; Maeda, M. Molecular and ionic recognition with imprinted polymers; ACS Symposium Series 703; American Chemical Society: Washington, DC, 1998. (14) Kobayashi, T.; Wang, H. Y.; Fujii, N. Anal. Chim. Acta 1998, 365, 81. (15) Oak, M. S.; Kobayashi, T.; Wang, H. Y.; Fukaya, T.; Fujii, N. J. Membr. Sci. 1997, 123, 185.

Langmuir, Vol. 18, No. 7, 2002 2867 THO, water was selected as a coagulation medium. After casting the polymer solution on the glass plate, the polymer solution was coagulated in water at 30 °C. Then, the copolymer solidified was kept overnight in water in order to remove DMSO and template. The resulting copolymer was washed with 0.1 wt % acetic acid aqueous solution for complete template extraction and then rinsed with a large excess of water. As a reference, P(AN-co-AA) and P(AN-co-MA) without template polymerization were used for phase inversion imprinting. The procedure was done according to previous reports.7,8,14 The DMSO solution containing 10 wt % of P(AN-co-AA) or P(AN-co-MA) was prepared at 50 °C, and equivalent THO to AA or MA residues was added to the solution and kept overnight at the temperature. The polymer-template solution was cast on the plate. Then the solution was coagulated in water. The solidified copolymer was washed with an excess of water to remove DMSO and THO. It was then washed with 0.1 wt % acetic acid aqueous solution. The imprinted copolymer was rinsed with a large excess of water and used for binding experiments. After template extraction, copolymerization was checked by measuring copolymer viscosity, FT-IR spectra by FT-IR 8200 (Shimadzu), and 1H NMR spectra by JNM GX400 FT-NMR in d6-DMSO at 60 °C. Viscosity of the copolymer was measured at 30 °C using an Ubbelohde viscometer to evaluate intrinsic viscosity, [η] (cm3/g). The resultant copolymer molecular weight (Mw) was estimated from [η] according to the literature using the viscometric method in dimethylformamide solution.15 Table 1 lists results of viscosity and mole fraction of copolymers estimated by the 1H NMR spectrometer. The fraction of AA or MA units in the copolymer was calculated from the integrals of total aliphatic protons for polyacrylonitrile (PAN) and AA or MA segments. For all copolymers, y fraction values approximated 12.7-14.9 mol %. The result of 1H NMR measurements was also used to confirm template extraction of copolymers by the disappearance of methyl protons of THO at higher (3.50 ppm) and lower (3.63 ppm) magnetic fields. To study substrate uptake of THO and its analogous molecules, heterogeneous batch experiments were carried out in 3.6 µM concentration. The THO-imprinted copolymer with weight W (g) was equilibrated in the THO aqueous solution at 30 °C. Then, the THO or CAF concentration of bulk solution was estimated at various times by a UV-visible detector ((UV 8000) monitored at 270 nm) with a TSKgel-ODS column. The value of the substrate binding to the THO-imprinted membrane, [St] (µmol/g polymer), was calculated by following eq 1:

[St] ) (Cb - Ca)V/W

(1)

where Cb and Ca are the mole concentration (µM) of THO or another substrate before and after equilibrium time (h), respectively: V (L) is the volume of bulk equilibrium solution.

Results and Discussion Template Copolymerization and Phase Inversion Imprinting. Table 1 lists monomer feed and properties of resultant copolymers prepared with various template concentrations. In the table, influence of template feeds on the template polymerization of P(AN-co-AA)THO, P(AN-co-MA)THO, and PANTHO was seen. Here, concentration of THO and AA or MA was changed in the range of [THO]/[COOH] ) 0.4, 1, and 2. The resultant copolymers, P(AN-co-AA)THO and P(AN-co-MA)THO, had almost similar y contents of the COOH segments in the range of y ) 0.124-0.149. Also, Mw data suggest that THO addition to copolymerization was almost the same in each copolymer with AA segments for P(AN-co-AA)THO and P(ANco-AA) and with MA segments for P(AN-co-MA)THO and P(AN-co-MA). However, Mw’s of copolymers with the MA system were somewhat lower than those with the AA system. This may be attributed to the difference in polymerization ability of AA and MA. Furthermore, we observed scanning electron micrograph (SEM) morphology for polymers solidified. Figure

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Table 1. Copolymerization of AN with AA and MA in the Absence and Presence of the THO Template monomer feed copolymer

AN

AA

P(AN-co-AA)THO

0.572 0.572 0.572 0.572 0.572 0.572 0.572 0.572 0.572

0.106 0.106 0.106

P(AN-co-MA)THO PANTHO P(AN-co-AA) P(AN-co-MA)

MA

0.106 0.106 0.106 0.106 0.106

THO

yield (%)

[η]

Mw

y (mol %)

1.0 2.8 4.7 1.0 2.8 4.7 2.8

77.7 70.8 79.5 41.0 51.4 27.9 67.6 77.7 78.0

2.30 2.64 2.50 1.23 1.19 1.16 1.86 2.30 1.30

212 000 254 000 238 000 92 000 88 000 87 000 159 000 212 000 96 000

14.4 14.7 13.0 13.3 14.9 12.5 14.5 13.5

Figure 3. THO amounts taken into the THO-imprinted copolymers of (a) P(AN-co-AA) and (b) P(AN-co-MA) with 3.6 µM concentration of THO for equilibrium experiments. Open symbols show the copolymer without template polymerization.

Figure 2. SEM photographs of (a) cross section and (b) surface of P(AN-co-MA)THO.

2 shows SEM images of (a) a cross section and (b) the surface for P(AN-co-MA)THO. The copolymer looked like a porous membrane structure. We confirmed that both P(AN-co-AA)THO and P(AN-co-MA)THO showed quite similar morphologies, as we previously reported.8,14 The resultant copolymers have asymmetric porous voids and about 44 µm membrane thickness with macrovoids and a spongelike layer in the cross section. THO Binding to THO-Imprinted Copolymers. Figure 3 shows the time change of THO binding amounts, [St]THO, for (a) P(AN-co-AA)THO and (b) P(AN-co-MA)THO. In Figure 3a, amounts of THO binding were increased over time. After about 10 h, the value of [St]THO reached

a constant value of binding amounts. This means that imprinted sites were saturated by THO binding after 10 h of equilibrium. That is, the constant value showed total binding amounts of THO in the imprinted polymer. Here, the value was defined as saturation binding amounts of THO, [S]THO. At [THO]/[COOH] ) 1, which means equivalent THO concentration to AA in polymerization, the value of [S]THO was 1.4 µmol/g polymer, higher than both the [S]THO ) 0.36 and 0.4 µmol/g polymer for [THO]/ [COOH] ) 0.4 and 2, respectively. Open symbol data in the figure were for the P(AN-co-AA) value, which was prepared without THO and was phase-inversed in the presence of THO in water. The [S]THO value for P(ANco-AA) was 0.265 µmol/g polymer, somewhat lower than that of [THO]/[COOH] ) 0.4 and 2. The result means that AA segments in the copolymer acted as nonspecific binding sites to THO recognition. This was supported by binding data of PANTHO; no THO binding to the polymer without AA segments was observed. On the other hand, Figure 3b presents binding of THO for P(AN-co-MA)THO. As in the case of P(AN-co-AA)THO, THO binding to P(AN-co-MA)THO was efficient at [COOH]/ [THO] ) 1, and the resulting [S]THO was 1.2 µmol/g polymer. Figure 4 summarizes the value of [S]THO observed

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Figure 4. Plots of saturated binding amounts of THO versus [THO]/[COOH] for (b) P(AN-co-AA)THO and (9) P(AN-co-MA)THO. Open symbols are for (O) P(AN-co-AA) and (0) P(AN-co-MA). Chart 3

at different copolymers and PANs. As seen, no binding of THO was observed in both PANs with and without template polymerization. The [S]THO of P(AN-co-AA) and P(AN-co-MA) prepared at [THO]/[COOH] ) 1 was shown by open symbols and was 0.75 and 0.62 µmol/g polymer, respectively. The value observed at [THO]/[COOH] ) 2 was somewhat higher than that of [THO]/[COOH] ) 1. Note that [S]THO of P(AN-co-AA)THO and P(AN-co-MA)THO had a different dependence on [THO]/[COOH]. The tendency of [S]THO for both P(AN-co-AA)THO and P(ANco-MA)THO showed a maximum value at [THO]/[COOH] ) 1. Also, a comparison between PANTHO and THOimprinted copolymers P(AN-co-AA)THO and P(AN-coMA)THO indicates that COOH segments of the copolymer have a very important role to fix the template molecule into the imprinted volumetric space of PAN segments. Since solidification of the PAN segments builds up imprinted volumetric space during the phase inversion process (Figure 1), the interaction site between THO and the copolymer is necessarily needed to have high recognition of the substrate. In cases of copolymers prepared without template polymerization, complexation between the template and the copolymer is performed in the viscous polymer solution. Hence, it may be more difficult to form hydrogen bonding between the template and the interaction site of the copolymer because of the difficulty of copolymer chain orientation. As a result, imprinting efficiency was lower than that observed in the template copolymer. These results suggest that the COOH group of AA or MA in template polymerization interacts easily with the THO molecule via hydrogen bond interaction (Chart 3). Consequently, the template copolymer could easily encode the shape of the template by phase inversion imprinting. Analysis of Template Copolymerization by NMR. It is meaningful to analyze interaction of the template monomer in the monomer-DMSO medium. We applied NMR measurements for the d6-DMSO medium containing AN, AA or MA, and THO. Figure 5 shows 270 MHz 1H NMR spectra of (a) THO and (b) AN-MA in d6-DMSO.

Figure 5. 1H NMR spectra (270 MHz) of (a) THO, (b) AN-MA, and (c) AN-MA-THO at [THO]/[COOH] ) 0.4 and (d) [THO]/ [COOH] ) 1 in d6-DMSO measured at 25 °C.

As shown in Figure 5a, methyl groups of THO resulted in the chemical shift near 3.50 and 3.63 ppm. The lower field resonance peak at 8.30 ppm was attributed to the proton on the 7-nitrogen of the xanthine ring. In Figure 5b, multiplet peaks appeared near 5.8-6.6 ppm for the CH and CH2 groups for AN and the CH2 group for the methacryl group of MA. The resonance peak of CH3 for MA was observed at 2.0 ppm. Furthermore, when the template was absent in AN-MA d6-DMSO solution, the characteristic peak of the proton of the COOH group of MA was seen as a singlet near the lower resonant field of 12.68 ppm. When template THO was present in the solution, the peak was slightly shifted to the lower magnetic field of (c) 12.7 ppm at [THO]/COOH] ) 0.4 and (d) 12.76 ppm at [THO]/[COOH] ) 1. A downfield shift of the H proton signal resonance for the COOH group of MA is a general characteristic of protons participating in hydrogen bond formation.17 It is also apparent that the proton peak of COOH broadened with increased THO concentration, especially for [THO]/[COOH] ) 1. Chemical shift and broadening strongly suggest that the H proton of the COOH group interacts with THO by hydrogen bonding. In addition, the characteristic proton peak at 8.30 ppm for the THO proton was somewhat slightly shifted to the higher side of 8.34 ppm at [THO]/[COOH] ) 1, when both MA and THO were present in d6-DMSO with AN. This is because of the decrease of electron density around the proton of THO by hydrogen bond interaction (Chart 3). In the case of the AN-AA-THO system, a similar observation of NMR spectra was obtained in d6-DMSO. The resonance peak of COOH of AA appeared near 12.73 (16) Onyon, P. F. J. Phys. Soc. 1959, 37, 315. (17) (a) Lancelot, G. J. Am. Chem. Soc. 1977, 99, 7037. (b) Silverstein, R. M.; Bassler, G. C.; Morrill, T. C. Spectrometric Identification of Organic Compounds, 4th ed.; John Wiley & Sons: New York, 1991; p 183.

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Kobayashi et al. Chart 4. Chemical Structure of THO Analogues

Figure 6. 1H NMR spectra (400 MHz) of (a) copolymers with AA segments and (b) MA segments in d6-DMSO measured at 60 °C; samples with template extraction were used.

ppm in the absence of THO. Then, the shift of the resonance peak to 12.75 and 12.76 ppm was observed at [THO]/ COOH] ) 0.4 and 1, respectively. Broadening of the singlet peak of the carboxylic acid proton was also found in the spectra, especially for [THO]/[COOH] ) 1. A similar shift of the H proton on the 7 position nitrogen of THO was found in the AN-AA-THO system at [THO]/ [COOH] ) 1. Moreover, to examine the effect of the polymer chain conformation on the template polymer copolymerization, a comparison of 400 MHz 1H NMR spectra was made. The NMR spectra in d6-DMSO were measured after molecules were extracted and then the sample was dried. As shown in Figure 6a for the copolymer with AA segments, resonant peaks near 1.7-2.3 ppm came from methylene (CH2) protons of the copolymer backbone. The CH2 protons of PAN segments and AA segments separated to two signals appeared near 1.95 and 1.8 ppm, respectively. The resonance signal for the CH proton of the copolymer overlapped with 2.7-3.4 ppm peaks for water (3.2 ppm). Here, the sharp peak at 2.5 ppm was from H protons of the DMSO solvent. Signals of AA segment CH protons were observed near 2.7 and 2.82 ppm for P(AN-co-AA) and P(AN-co-AA)THO. The CH signal of template polymerization was slightly shifted to the lower magnetic field of 2.82 ppm relative to P(AN-co-AA). That is, the appearance of the CH proton signal was strongly influenced by the induced magnetic field of the AA segment dO bond. This difference in P(AN-co-AA)THO and P(AN-co-AA) implies that template copolymerization caused different conformations of resultant copolymers.

The NMR spectra of copolymers with MA segments were similarly compared between P(AN-co-MA)THO and P(ANco-MA) prepared at [THO]/[COOH] ) 1 (Figure 6b). Resonance peaks near 1.2-1.5 ppm were attributed to methyl protons of MA, and those at 1.8-2.4 ppm were for the CH2 group of PAN segments in the copolymer. The CH protons near 2.9-3.2 ppm were assigned as PAN segments in the copolymer because MA segments have no CH group in the chemical structure. Apparently, the CH signal near 2.8 ppm was shifted to a higher resonant area rather than that of AN segments. A shift of the CH proton of AA or MA segments in the copolymer means that the proton of the CH backbone is exposed to a shield field of the CdO bonds of MA segments. Resonance peaks of methyl groups obtained in P(AN-co-MA)THO were separated to 1.2, 1.35, and 1.42 ppm, while peaks observed for P(AN-co-MA) had two signals near 1.2 and 1.4 ppm. In addition, CH2 resonance peaks of PAN segments near 2-2.4 ppm also showed two resonance peaks at 2.05 and 2.2 ppm with equivalent resonance intensity for P(ANco-MA). However, the resonance intensity of CH2 for P(ANco-MA)THO was not equivalent in the template copolymer. The difference in resonance intensity is evidence of a polymer backbone difference caused by template copolymerization. Namely, PAN segments in the copolymer were tailor-made in the presence of the template. On the other hand, the resonant areas of the CH2 and CH peaks of PANTHO were 3.2 and 2.1 ppm. However, there was no difference in PANTHO and PAN, which polymerized without the THO template. Therefore, the NMR data comparison of P(AN-co-MA)THO and PANTHO indicated that the tailormade structure appeared in only template copolymerization. Thus, the microstructure tailor-made for the template apparently influenced phase inversion imprinting and resulted in high THO binding by THO-imprinted volumetric space of the copolymer. Substrate Selectivity of THO-Imprinted Copolymers. To confirm the template polymer effect on THO recognition by P(AN-co-AA)THO and P(AN-co-MA)THO, we further compared results of THO binding with those of other xanthine derivatives having a different chemical structure (Chart 4). Here, CAF is structurally close to the THO template and has a methyl group on the 7-nitrogen atom instead of the H group. Figure 7a shows time profiles of [St]CAF for the CAF substrate in cases of P(AN-co-AA)THO and P(AN-co-MA)THO. For further infor-

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Figure 7. Time profiles of binding amounts of (a) CAF and (b) uracil to THO-imprinted copolymers.

binding to the THO-imprinted copolymers was much less than that for THO, while CAF binding to copolymers was higher than that for HETHO. Hence, to bind to the THOimprinted sites of the copolymers for HETHO having a hydroxyl group was not easier than for CAF having a methyl group on the N-atom. However, THB, lumazine, and purine showed no binding to both THO-imprinted copolymers. On the other hand, for uracil, the saturation binding amount to P(AN-co-AA)THO was 0.78 µmol/g polymer, higher than that of HETHO. However, the uracil value was smaller than for THO. Relative to P(AN-coAA)THO, P(AN-co-MA)THO showed low binding of uracil, in which the saturation binding amount of uracil was 0.15 µmol/g polymer, much smaller than for P(AN-co-AA)THO. The result of uracil binding indicates that the THOimprinted copolymer with AA shows a loose recognition relative to that of P(AN-co-MA)THO. This may be due to the presence of methacryl methyl groups being more efficient in the tailor-made structure of the THO template. Furthermore, we examined dynamics of the substrate binding to imprinted copolymers over time. In the figures showing the relationship between [St] and time (Figures 3 and 7), all data points could be fitted with singleexponential curves as shown by solid lines in the figures. The relaxation time τ for each component was determined by using eq 2.18 Figure 8. Saturation binding amounts of various substrates to THO-imprinted copolymers.

mation about recognition of THO-imprinted copolymers, we examined binding of 2-hydroxyethy-l-theophylline (HETHO), theobromine (THB), lumazine, purine, and uracil (Chart 4). Here, HETHO has a hydroxyethyl group instead of the H group of THO. THB, lumazine, and purine are xanthine derivatives. Uracil has H groups on the N-atom and two carbonyl groups in the chemical structure like THO, while the framework structure is different than that of THO. As shown by Figure 7b, the value of binding amounts for uracil increased with increase of time. To compare these binding results, we evaluated amounts of saturated binding, [S]s, for each binding experiment. Figure 8 summarizes saturated binding amounts of each substrate to THO-imprinted copolymers. The HETHO

[St] ) [S]s(1 - exp(-τ/t))

(2)

Here, [St] represents substrate binding amounts measured at each time and [S]s shows saturation binding amounts of the substrate. Values obtained are listed in Table 2. The difference in relaxation time obtained by binding experiments would be attributed mainly to accessibility of the substrate to the imprinted polymer binding site, which encoded the volumetric size of the THO template. Values of τ for both THO-imprinted copolymers imply that THO took a similar time to be accessible to imprint sites of P(AN-co-AA)THO and P(AN-co-MA)THO. In the case of (18) (a) Cox, B. G. Modern liquid-phase kinetics; Oxford Science: New York, 1994; p 61. (b) Barrow, G. Physical Chemistry, 6th ed.; WCB/ McGraw-Hill: Boston, 1996; p 744.

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Table 2. Relaxation Time τ (h) Obtained by Curve Fitting for Substrate Binding to the THO-Imprinted Copolymers substrate

P(AN-co-AA)THO

P(AN-co-MA)THO

THO CAF HETHO uracil

3.2 6.3 8.3 (1.2)a 2.8

3.2 5.0 8.3 (1.2)a 3.5

a

The number in the parentheses was an error of the fitting.

CAF, these took a long time to bind CAF into imprinted sites. This may be due to the methyl group on the CAF substrate. That is, hydrogen bonding between the imprint copolymer and THO may be the effective capture force of the substrate to the imprint sites. In the case of HETHO, binding amounts were much less to both copolymers and values of τ became larger than that of CAF. However, uracil, which has a smaller molecular size than THO, CAF, or HETHO, showed higher binding and a shorter binding time for P(AN-co-AA)THO. This may be attributed to the small size of uracil relative to THO. For P(AN-co-MA)THO, the value of τ was almost identical to that of THO, while the binding amount was very low. This means that uracil accessibility was lowered by high recognition sites for the template. The hydrogen-bonding direction may be regulated by effective encoding in the case of P(AN-co-MA)THO, although uracil’s molecular size was lower than that of

THO. In conclusion, THO-imprinted copolymers show sharp recognition on THO and other substrates for the copolymer with MA segments. This may be due to the effect of the methyl group of the methacryl group on template polymerization and the phase inversion imprinting process. Conclusions. THO-imprinted copolymers prepared from template copolymerization of AN with AA or MA with a THO template significantly increased recognition ability to the THO at [COOH]/[THO] ) 1. This result suggested that AA or MA segments bound the substrate via hydrogen bonding. Proton NMR experiments supported that the hydrogen-bonding interaction was present in polymerization and the resultant copolymer conformation was tailor-made for the template. Selectivity of THOimprinted copolymers was confirmed by using CAF, HETHO, uracil, and other substrates. Results of uptake experiments for THO and its analogues showed that THOimprinted sites in the imprinted copolymer with MA could bind effectively to THO with high selectivity. Acknowledgment. This research was partly supported by Grants-in-Aid for Scientific Research (C) (12650764 and 13027227) of the Ministry of Education, Science, Sports and Culture, Japan. LA0106586