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Chiral-recognition polymer prepared by surface molecular imprinting technique
Colloids and Surfaces A: Physicochemical and Engineering Aspects 169 (2000) 259 – 269 www.elsevier.nl/locate/colsurfa
Chiral-recognition polymer prepared by surface molecular imprinting technique Masahiro Yoshida a, Yasuo Hatate a, Kazuya Uezu b, Masahiro Goto b,*, Shintaro Furusaki b a
Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Kagoshima Uni6ersity, Korimoto, Kagoshima 890 -0065, Japan b Department of Chemical Systems and Engineering, Graduate School of Engineering, Kyushu Uni6ersity, Hakozaki, Fukuoka 812 -8581, Japan
Abstract A highly enantioselective polymer was prepared by the surface molecular imprinting technique for the separation of optically active tryptophan methyl ester. A synthetic host molecule (phenyl phosphonic acid monododecyl ester) was proved to be effective for recognizing the chirality of amino acid esters. The L- or D-tryptophan methyl ester (TrpOMe)-imprinted polymer containing the functional host molecules revealed high enantioselectivity toward the corresponding imprinted isomer. While, the racemic-TrpOMe-imprinted and unimprinted polymers did not show the enantioselectivity at all. These results mean that the complementary binding sites such as ‘template-fit pockets’, in which the position and the alignment of the functional group in the functional host molecule are optimally adjusted for binding the corresponding imprinted isomer, are a principal factor to recognize the target molecule. These enantioselectivities were quantitatively supported by high binding constants for the corresponding imprinted isomer. To verify the recognition mechanism of the imprinted polymer, FT-IR and 1H-NMR measurement and computational modeling were conducted. Based on the results obtained, it was concluded that the enantiomeric selectivity is endowed by the electrostatic and hydrogen bonding interactions between the functional molecule and the target tryptophan methyl ester along with the chiral space formed on the polymer surface. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Molecular imprinting; Surface molecular imprinting; Molecular recognition; Enantioselectivity; Amino acid
1. Introduction
* Corresponding author. Tel.: +81-92-6423576; fax: + 8192-6423575. E-mail address: [email protected] (M. Goto)
The complicated phenomena in various biological systems are led by simple chemical reactions with precise molecular recognition. Enzymes, antibodies, or receptors are typical molecular recognition elements in a biological system. A highly specific molecular recognition in these bio-
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molecules is created by well-organized three-dimensional structures possessing both binding and catalytic sites for a target molecule. The complicated bio-molecules are of course superior to synthetic compounds in this point, and have been widely interested in various fields. However, because most of them are proteins, many difficulties in practical use exist due to their sensitive properties such as instability against high temperature, organic solvents, serious pH conditions, etc. Synthetic approaches are often desirable, and presently one of the most challenging themes for chemists is to create an organic compound possessing a specific molecular recognition ability mimicking bio-molecules [1 – 3]. However, the multi-step procedures are required for the preparation of these organic host compounds and sometimes cause a low yield. Furthermore, the specificity of synthesized materials is not always high, although novel receptor-like materials are emerging rapidly. Another promising approach to design a synthetic receptor is a molecular imprinting technique [4–6]. Several decades ago, Wulff [4] introduced a new methodology for preparing a specific receptor sites in a cross-linked polymer. The process called imprinting polymerization involves formation of a complex of a template molecule and a functional monomer, followed by polymerization in the presence of cross-linking agent. During the polymerization, the geometry of the self-assembled imprint molecule-functional monomer complexes is enclosed into the growing polymer matrix. Removal of the imprint molecules leaves behind cavities possessing a shape and an arrangement of the functional groups corresponding to the structure of the imprint molecule. The polymers prepared by the molecular imprinting technique have attracted much attention as interesting separation tools. One of the attractive applications is optical resolution of chiral materials [7 – 12]: direct enantio-separation of drugs [13,14], and regio- and enantio-separation of sugar or sugar derivatives [15,16]. In addition, antibody and enzyme analogues prepared by the molecular imprinting technique have been studied extensively [17 – 22]. This technique is a conceptually simple and straightforward method to apply to a wide variety of target
molecules, however, it still has some fundamental drawbacks unresolved yet, i.e. inapplicability to water-soluble substances which are important in the biological field. In recent years, to overcome these drawbacks, a novel molecular imprinting technique called ‘surface molecular imprinting technique’ was proposed [23–33]. The concept of the surface molecular imprinting technique is illustrated in Fig. 1. The molecularly imprinted polymer is prepared by polymerizing water-in-oil (W/O) emulsions. In this novel technique, the organic-aqueous interface in W/O emulsions is utilized as a recognition field toward a target molecule. The target molecule forms a complex with the functional host molecule, and the orientation of the functional host molecule is fixed at the oil-water interface. This provides, after polymerization, the complementary recognition sites to the imprint molecule at the inner cavity surfaces of the imprinted bulk polymer. The bulk polymer obtained is ground to appropriate particles in order to interact with the target molecules in an aqueous solution. This technique also promises a rapid and reversible complexation of target molecules with the imprinted polymer. In this study, we demonstrate that amino acidimprinted polymers, which include phenyl phosphonic acid monododecyl ester (abbreviated as n-DDP), exhibit a high enantioselectivity toward an imprinted isomer. Furthermore, we discuss the recognition mechanism of the imprinted polymer for the target molecule with computational modeling. We selected L- or D- tryptophan methyl ester (abbreviated with L-, D- TrpOMe) as the target molecule. The lowest energy structure for the complex between the functional molecule and the target TrpOMe at the interface is analyzed by molecular dynamics (MD) calculation. The enantioselective performance of the L-, D- or racemicTrpOMe-imprinted polymer was evaluated by a competitive adsorption test using analogues of the target TrpOMe. The template effect was also characterized by comparing the adsorption behavior of the imprinted polymers to that of the control adsorbent prepared without the imprint molecules. The specific interaction between TrpOMe and n-DDP was investigated in detail by
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Fig. 1. Schematic illustration of the surface molecular imprinting technique.
FT-IR and 1H-NMR measurement to verify the imprinting phenomena. Finally, we quantitatively discuss the template effect by evaluating their binding constants on the basis of the modified Scatchard analysis.
2. Materials and methods
2.1. Reagents and apparatus Functional host molecule n-DDP was synthesized according to the procedures reported in previous works [29]. N-D-glucono dioleyl glutamate (abbreviated with 2C18D9GE) used as an emulsion stabilizer was synthesized in our previous study [34]. Amino acids (D,L-TrpOMe, D,LPheOMe, and D,L-Trp) were purchased from Sigma. Divinylbenzene (abbreviated as DVB, Wako Pure Chemical Industries) was employed after treatment with silica gel to remove an inhibitor. Fig. 2 shows the structures of (a) Tr-
Fig. 2. Structures of (a) imprint molecule: TrpOMe and (b) functional host molecule: n-DDP.
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pOMe and (b) n-DDP. Other reagents were of commercially available special grades. Particle size analysis was performed by a microtrac optical analyzer (Model 7995-10 SRA, Nikkiso), and the microstructure of particles was observed by using an ABT-32 type microscopy (Akashi Beam Technology).
2.2. Preparation of the L -, D -, or racemic-TrpOMe-imprinted polymer Enantioselective polymers were prepared by the surface molecular imprinting technique utilizing W/O emulsions. A 40-ml DVB containing 60 mM n-DDP and 5 mM 2C18D9GE was mixed with 20 ml toluene. Then, a 30-ml aqueous solution containing 10 mM L-, D-, or racemic-TrpOMe, which pH was adjusted to 4.5 with a 100-mM phosphate buffer solution, was added to the above organic phase. The mixture was sonicated for 4 min to obtain stable W/O emulsions. After the addition of 0.36 g (1.4×10 − 3 mol, 0.01 wt.% for DVB) powder initiator (2,2%-azobis (2,4%-dimethylvaleronitrile), a product of Wako Pure Chemical Industries), the mixture was polymerized at 308 K for 2 h under a flow of nitrogen. The obtained bulk polymer was dried under vacuum and ground into particles of an appropriate size. The particles were washed with 1 M hydrochloric acid to remove the imprinted L-, D-, or racemic-TrpOMe and then filtered off. This procedure was repeated several times until the imprint molecule in the filtrate cannot be detected by a UV spectrometer (V-570 UV/VIS/NR Spectrophotometer, Japan Spectroscopic). Finally, the polymer was dried in vacuo for several days. An unimprinted polymer and a polymer without applying functional host molecule (abbreviated as n-DDP-free polymer) as a reference were similarly prepared without the imprint molecule or the functional host molecule.
2.3. Competiti6e adsorption experiments on L -, or racemic-TrpOMe-imprinted polymer
D-
The batchwise adsorption experiments were conducted for the newly prepared polymers. The polymers (0.05 g) were added to a 5-ml aqueous
solution containing amino acids and placed in a sealed test tube (10 ml volume). The pH was adjusted to a desired value between 1.5 and 7.5 with 100 mM KH2PO4-K2HPO4 and 100 mM HNO3. The mixture was shaken in a thermostated water bath at 308 K for 24 h. The polymers were then filtered off through a polyethylene membrane (Sumplep LCR25-LG, Nippon Millipore). The amount of each amino acid adsorbed to the polymers was calculated on the basis of their residual amount in the filtrate. The concentration of amino acids was analyzed by a HPLC system (Japan Spectroscopic) with a reversed phase column (TSKgel ODS-80Ts column, 4.6×250 mm, Toyo Soda Co., Ltd.). The elution (100 mM CH3COOHCH3COONa (pH 4.5)/acetonitrile = 4/1, v/v) was spectorophotometrically monitored at 278.8 nm and 1.0 ml/min except for PheOMe, whose elution was monitored at 258 nm and 0.5 ml/min. The adsorption tests were carried out at least three times and the data were plotted with the average values. The experimental errors were less than 8%.
2.4. FT-IR and 1H-NMR measurement The interactions between the target L-TrpOMe and n-DDP were investigated in detail by FT-IR and 1H-NMR spectra studies. The measurement was carried out using FT-IR 8300 (Shimadzu) and AC 250 P (Bruker) at 303 K. The FT-IR study was performed by the KBr method. 1H-NMR samples were measured in C6D6 containing 15 vol.% DMSO-d6 ( 250 MHz, Tetramethylsilane (TMS) was used as an internal standard). The following values were used for analysis. 1. FT-IR spectra 1.1. L-TrpOMe 3287 cm − 1: N-H stretching vibration on the indole ring, 1747 cm − 1: CO stretching vibration 1.2. n-DDP-L-TrpOMe complex 3217 cm − 1: N-H stretching vibration on the indole ring, 1751 cm − 1: CO stretching vibration 2. 1H-NMR spectra 2.1. L-TrpOMe
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Fig. 3. SEM photograph of the TrpOMe-imprinted polymers.
d = 11.45, s, NH (indole ring); d= 7.0 – 8.0, m, CH (indole ring); d= 6.43, bs, NH+ (amino group); 3 d= 4.39, t, CH2 (methylene group); d= 3.67, m, CH (methine group); d= 3.47, s, COH3 (methoxyl group) 2.2. n-DDP-L-TrpOMe complex d= 11.28, s, NH (indole ring); d= 8.60, bs, NH+ 3 (amino group); d= 7.0 – 8.0, m, CH (indole ring and phenyl group); d =3.35, s, COH3 (methoxyl group)
2.5. Computational calculation for optimum structure of the complex between amino acid ester and the functional host molecule The molecular mechanic (MM) and molecular dynamic (MD) calculations were performed for the TrpOMe-n-DDP-complex by molecular modeling software, HyperChem Release 5.1 (Hypercube, Canada). In the calculation, the force field parameter set of MM2 was employed [35]. Before calculating the optimized structure of the complex at the oil-water interface, two preliminary calculations were conducted: (1) MM calculation to obtain the lowest energy structure of the complex in a vacuum; and (2) a toluene-water box to calculate the complex at the interface. Firstly the lowest energy structure of the complex in vacuum was calculated by the MM method. The toluene-water
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biphase box (30 A, × 30 A, × 60 A, ) was built from two adjacent cubic boxes (30 A, × 30 A, × 30 A, ) of pure toluene and water. The number of toluene and water molecules was set to be 172 and 856 by taking into account each molecular volume, respectively. Finally the TrpOMe-n-DDP-complex optimized in vacuum was placed at the interface by replacing seven toluene molecules that is almost equivalent to the complex volume. The MD simulation was performed in the (N, V, T) ensemble after MM calculation in the biphase system, which was required to avoid a large strain energy raised when being directly calculated by the MD method. The time step was adjusted to be 1 fs, and the temperature was controlled at 300 K by coupling to a thermal bath with a relaxation time of 0.1 ps.
2.6. Determination of binding constants between L -, D -, or racemic-TrpOMe and the imprinted polymers Binding constants of imprinted polymers were evaluated with a batchwise method. A 0.05-g polymer was immersed in a sealed test tube (10 ml volume). Then, a 5-ml aqueous solution, which is buffered with 100 mM KH2PO4K2HPO4 containing L- or D-TrpOMe adjusted to a desired concentration between 0.05 and 5 mM was added. The mixture was shaken at room temperature for 24 h. The polymers were then filtered off through the polyethylene membrane. The concentration of L- or D-TrpOMe in the filtrate was analyzed by a HPLC system. The binding constants were calculated by a modified Scatchard equation [36,37].
3. Results and discussions Highly cross-linked TrpOMe-imprinted polymers were prepared by the surface molecular imprinting technique with W/O emulsions. All polymers were obtained at more than 80% yields. After polymerization, the bulk polymer was ground into particles, whose volume-averaged diameters were ca. 40 mm. Fig. 3 shows a typical view of the TrpOMe-imprinted polymer prepared from W/O emulsions by scanning electron mi-
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croscopy. Substantial traces of aqueous phases in the emulsions are observed in the polymer. The recognition sites for the TrpOMe isomer are con-
Fig. 5. The pH dependence of the adsorption of L- and D-PheOMe or L- and D-Trp on the unimprinted polymer.
Fig. 4. The pH dependence of the adsorption of L- and or L- and D-Trp on the imprinted polymers: (a) the L-TrpOMe-imprinted polymer; (b) the D-TrpOMe-imprinted polymer; or (c) the Racemic-TrpOMe-imprinted polymer. D-TrpOMe
ceptually produced on the surfaces of the inner cavities in the polymer. A number of micropores facilitate diffusion of the target TrpOMe into the polymer. Fig. 4(a–c) exhibits the pH dependence for the adsorption of L- and D-TrpOMe or L- and D-Trp on the L-TrpOMe-imprinted, D-TrpOMe-imprinted, or racemic-imprinted polymer. The percentage of adsorption increased with increasing pH in the feed solution. This result suggests that the proton-dissociation from the functional host molecule plays a predominant role when the imprinted polymers rebind the amino acid esters. The L-TrpOMe-imprinted polymer showed a high selectivity toward L-TrpOMe over D-TrpOMe in the whole pH range investigated (Fig. 4(a)). Similarly the D-TrpOMe-imprinted polymer has a high affinity to D-TrpOMe imprinted at the preparation (Fig. 4(b)). Although the racemic-TrpOMeimprinted polymer also has high potential for the adsorption, it afforded no evidence of enantioselective adsorption (Fig. 4(c)). That is, the imprinted polymer (L- or D-TrpOMe-imprinted polymer) exhibits much higher template effect toward the corresponding imprinted tryptophan methyl ester than its isomer, while a reference polymer prepared with the racemic molecule does not reveal any selectivity toward the enantiomers, because the non-specific recognition sites are produced on the polymer surface. Fig. 5 shows the
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pH dependence for the adsorption of L- and D-TrpOMe or L- and D-Trp on the unimprinted polymer. The unimprinted polymer prepared without the imprint molecule was not able to recognize the chirality of amino acid derivatives. In the unimprinted polymer, the functional host molecules are randomly distributed on the polymer surfaces. The unimprinted polymer, however, had relatively high adsorption ability compared to that of the imprinted polymers, because the number of adsorption sites, which are non-specific sites, is comparable to that of the imprinted polymers. Fig. 6 exhibits the pH dependence for the adsorption of L- and D-TrpOMe or L- and D-Trp on the n-DDP-free-polymer. The polymer prepared without the host molecules did not adsorb tryptophan methyl ester at all. This result demonstrates that the interaction between the functional host molecule and the substrate should have a crucial effect for creating the adsorption ability. Tryptophan is scarcely adsorbed on all polymers (Fig. 4(a–c), Fig. 5, Fig. 6), because amino acids form intra-/inter-molecular ionic complexes between amino and carboxyl groups or because the electrostatic repulsion between the carboxyl anion in tryptophan and the negatively charged functional molecule inhibits the adsorption. This result indicates that the electrostatic interaction between the phosphonic acid moiety in the functional host molecule and the amino group in the
Fig. 6. The pH dependence of the adsorption of D- and L-TrpOMe or L- and D-Trp on the n-DDP-free polymer.
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substrate is required to succeed in creating the enantiomeric selectivity. In the case of adsorption of the phenylalanine methyl ester, which has a similar structure to the imprinted tryptophan methyl ester, on the imprinted polymer (L- or D-TrpOMe-imprinted polymer), we did not observe enantioselectivity. Hence, we found that the shape and/or space constructed on the polymer surface, which is complementary to the imprinted tryptophan methyl ester, should be a vital factor to recognize the molecular chirality. It is important to elucidate the recognition mechanism of the imprinted polymer in a wide application of the surface molecular imprinting technique. We investigated in detail the interaction between the imprint molecule (L-tryptophan methyl ester) and the functional host molecule (n-DDP) at the pre-polymerization stage by FTIR and 1H-NMR measurement. The characteristic peaks observed for the n-DDP-L-TrpOMe complex and L-TrpOMe itself were used to discuss the recognition morphology. Based on the results of the FT-IR spectra, the N-H stretching vibration peak derived from the indole ring appeared at 3287 cm − 1 on L-TrpOMe, and a similar peak on the n-DDP-L-TrpOMe complex shifted to 3217 cm − 1. This result suggests that the hydrogen bonding interaction between the PO in the phosphonic part of n-DDP and the proton of the indole ring causes the lower shift when forming the complex. On the other hand, the sharp CO stretching vibration peaks derived from L-TrpOMe itself and the n-DDP-L-TrpOMe complex appeared at 1747 and 1751 cm − 1, respectively. It can be assumed that the hydrogen bonding is not formed between the CO group in L-TrpOMe and the P-OH in the phosphonic group in n-DDP. The indole proton in L-TrpOMe was found at 11.45 ppm, and the peak on the complex had shifted to 11.28 ppm. This upfield chemical shift can be rationalized by the NH–OP interactions in forming the complex between n-DDP and L-TrpOMe. Additionally, the resonance peak of the cationic amino group protons on the complex remarkably shifted to the lower magnetic field compared to that of the L-TrpOMe (from 6.43 to 8.60 ppm). It is considered that the electrostatic interaction between n-DDP and L-TrpOMe
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Fig. 7. Schematic illustration of the surface of the TrpOMe-imprinted polymers.
causes a remarkable low-field chemical shift. The down- or up-field chemical shifts are due to the shielding and anti-shielding effect of nearby phosphonic groups on the protons influenced by hydrogen bonding and electrostatic interactions [38]. Based on these remarks one general point becomes very clear: the enantioselective recognition morphology on the L- (D-) TrpOMe-imprinted polymer was accomplished by three important factors as shown in Fig. 7: (i) the memorized space produced on the surface of the imprinted polymer in such a way that it can fit a desirable formation around L- (D-) TrpOMe when it interacts with the template; (ii) the electrostatic interaction between the functional host molecule and the substrate in a higher pH range which allows the proton dissociation from the phosphonic group; and (iii) the hydrogen bonding interactions between the functional host molecule and the indole ring. We calculated the lowest energy structure of the complex in the toluene-water box (Fig. 8) by the
MD method, and the snap shot at 1 ps was shown in Fig. 9(b). In the complex as shown in Fig. 9(b), the right-side host molecule forms the hydrogen bond with the nitrogen atom in the indole ring. While the left-side molecule interacts with the amino group in tryptophan through the ionicbond formation. By comparing it to the overview for the lowest energy structure of the complex in a vacuum in Fig. 9(a), it is clearly found that the indole ring in tryptophan locates along the interface and a little relaxes in the toluene-water box compared to that in a vacuum. The imprinted tryptophan is removed by washing with a week acidic solution and the complementary space to the target tryptophan will be memorized on the polymer surface. Finally, we quantitatively characterized the template effect in the L-, D-, or racemic-TrpOMeimprinted polymers by assessing the binding constant (Ka). The binding constant can be evaluated on the basis of the slope and intercept by the modified Scatchard plot. Table 1 summarized the
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Fig. 8. Toluene-water biphase boxes optimized by molecular dynamic calculation (30 A, ×30 A, ×60 A, ) Fig. 9. (a) The lowest energy structure of complex in a vacuum before MD calculation. Blue, carbon; grey, hydrogen; purple, nitrogen; red, oxygen, and yellow, phosphorus. (b) The lowest energy structure of complex at oil – water interface after MD calculation (at 1 ps). The colors mean the same atoms as that in (a).
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results of the binding constants for the TrpOMeimprinted polymers: (a) the L-TrpOMe-imprinted polymer; (b) the D-TrpOMe-imprinted polymer; and (c) the racemic-imprinted polymer. The binding constant becomes an indicator to represent the adsorption affinity of recognition sites for the target tryptophan methyl ester. The L- or D-imprinted polymers exhibited high binding abilities to the corresponding imprinted guest molecules, while the racemic-TrpOMe-imprinted polymer had a similar binding ability to both isomers. To discuss the enantioselectivity, we defined the separation factor as follows: a =KaL (D)/KaD (L). The separation factors in the L-, D-, and racemic-TrpOMe-imprinted polymers were 1.49, 1.40, or 1.02, respectively (Table 1). It should be concluded that the surface imprinting technique is very useful to create enantioselective sites, which can recognize the chirality of an optically active molecule on the polymer surface. We believe that the use of the surface molecular imprinting technique for the preparation of recognition polymers will find various applications in a variety of chemical fields.
molecule exhibited high enantioselectivity toward the corresponding imprint isomer. However, the racemic-TrpOMe-imprinted and unimprinted polymers did not show enantioselectivity. In addition, the n-DDP-free polymer as a reference has no adsorption ability to tryptophan methyl ester. The enantioselectivity was supported by high binding constants of corresponding imprinted substrates, which are suitable for the recognition sites constructed on the surfaces of the imprinted polymers. FT-IR and 1H-NMR studies provided useful information about the recognition mechanism of the imprinted polymers. Furthermore, computational modeling clearly showed the interaction mode between the target amino acid ester and the functional molecule at the liquid–liquid interface. Based on the results obtained, we found that three critical factors are required for recognizing the chirality of optically active materials: (1) a complementary recognition site for the target molecule; (2) hydrogen bonding formation; and (3) electrostatic interaction between a target guest molecule and a functional host molecule.
Acknowledgements 4. Conclusions The D- or L-TrpOMe-imprinted polymers containing an organophosphorus functional host Table 1 Binding constants of L- or K-TrpOMe on the TrpOMe-imprinted polymers: (a) the L-TrpOMe-imprinted polymer; (b) the D-TrpOMe-imprinted polymer; or (c) the racemic TrpOMe-imprinted polymer Binding constant Ka (M−1)
Separation factor a (= KaL (D)/KaD (L))
(a) L-TrpOMe D-TrpOMe
2.95×103 1.98×103
1.49
(b) D-TrpOMe L-TrpOMe
4.81×103 3.43×103
1.40
( c) L-TrpOMe D-TrpOMe
3.84×103 3.78×103
1.02
Substrate
We are grateful for the Grant-in-Aid for Scientific Research (No. 09750842) from the Ministry of Education, Science, Sports and Culture of Japan. One of the authors (M. Yoshida) was supported by a Research Fellowship from the Japan Society for the Promotion of Science (JSPS) for Young Scientists.
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Chiral-recognition polymer prepared by surface molecular imprinting technique Masahiro Yoshida a, Yasuo Hatate a, Kazuya Uezu b, Masahiro Goto b,*, Shintaro Furusaki b a
Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Kagoshima Uni6ersity, Korimoto, Kagoshima 890 -0065, Japan b Department of Chemical Systems and Engineering, Graduate School of Engineering, Kyushu Uni6ersity, Hakozaki, Fukuoka 812 -8581, Japan
Abstract A highly enantioselective polymer was prepared by the surface molecular imprinting technique for the separation of optically active tryptophan methyl ester. A synthetic host molecule (phenyl phosphonic acid monododecyl ester) was proved to be effective for recognizing the chirality of amino acid esters. The L- or D-tryptophan methyl ester (TrpOMe)-imprinted polymer containing the functional host molecules revealed high enantioselectivity toward the corresponding imprinted isomer. While, the racemic-TrpOMe-imprinted and unimprinted polymers did not show the enantioselectivity at all. These results mean that the complementary binding sites such as ‘template-fit pockets’, in which the position and the alignment of the functional group in the functional host molecule are optimally adjusted for binding the corresponding imprinted isomer, are a principal factor to recognize the target molecule. These enantioselectivities were quantitatively supported by high binding constants for the corresponding imprinted isomer. To verify the recognition mechanism of the imprinted polymer, FT-IR and 1H-NMR measurement and computational modeling were conducted. Based on the results obtained, it was concluded that the enantiomeric selectivity is endowed by the electrostatic and hydrogen bonding interactions between the functional molecule and the target tryptophan methyl ester along with the chiral space formed on the polymer surface. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Molecular imprinting; Surface molecular imprinting; Molecular recognition; Enantioselectivity; Amino acid
1. Introduction
* Corresponding author. Tel.: +81-92-6423576; fax: + 8192-6423575. E-mail address: [email protected] (M. Goto)
The complicated phenomena in various biological systems are led by simple chemical reactions with precise molecular recognition. Enzymes, antibodies, or receptors are typical molecular recognition elements in a biological system. A highly specific molecular recognition in these bio-
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molecules is created by well-organized three-dimensional structures possessing both binding and catalytic sites for a target molecule. The complicated bio-molecules are of course superior to synthetic compounds in this point, and have been widely interested in various fields. However, because most of them are proteins, many difficulties in practical use exist due to their sensitive properties such as instability against high temperature, organic solvents, serious pH conditions, etc. Synthetic approaches are often desirable, and presently one of the most challenging themes for chemists is to create an organic compound possessing a specific molecular recognition ability mimicking bio-molecules [1 – 3]. However, the multi-step procedures are required for the preparation of these organic host compounds and sometimes cause a low yield. Furthermore, the specificity of synthesized materials is not always high, although novel receptor-like materials are emerging rapidly. Another promising approach to design a synthetic receptor is a molecular imprinting technique [4–6]. Several decades ago, Wulff [4] introduced a new methodology for preparing a specific receptor sites in a cross-linked polymer. The process called imprinting polymerization involves formation of a complex of a template molecule and a functional monomer, followed by polymerization in the presence of cross-linking agent. During the polymerization, the geometry of the self-assembled imprint molecule-functional monomer complexes is enclosed into the growing polymer matrix. Removal of the imprint molecules leaves behind cavities possessing a shape and an arrangement of the functional groups corresponding to the structure of the imprint molecule. The polymers prepared by the molecular imprinting technique have attracted much attention as interesting separation tools. One of the attractive applications is optical resolution of chiral materials [7 – 12]: direct enantio-separation of drugs [13,14], and regio- and enantio-separation of sugar or sugar derivatives [15,16]. In addition, antibody and enzyme analogues prepared by the molecular imprinting technique have been studied extensively [17 – 22]. This technique is a conceptually simple and straightforward method to apply to a wide variety of target
molecules, however, it still has some fundamental drawbacks unresolved yet, i.e. inapplicability to water-soluble substances which are important in the biological field. In recent years, to overcome these drawbacks, a novel molecular imprinting technique called ‘surface molecular imprinting technique’ was proposed [23–33]. The concept of the surface molecular imprinting technique is illustrated in Fig. 1. The molecularly imprinted polymer is prepared by polymerizing water-in-oil (W/O) emulsions. In this novel technique, the organic-aqueous interface in W/O emulsions is utilized as a recognition field toward a target molecule. The target molecule forms a complex with the functional host molecule, and the orientation of the functional host molecule is fixed at the oil-water interface. This provides, after polymerization, the complementary recognition sites to the imprint molecule at the inner cavity surfaces of the imprinted bulk polymer. The bulk polymer obtained is ground to appropriate particles in order to interact with the target molecules in an aqueous solution. This technique also promises a rapid and reversible complexation of target molecules with the imprinted polymer. In this study, we demonstrate that amino acidimprinted polymers, which include phenyl phosphonic acid monododecyl ester (abbreviated as n-DDP), exhibit a high enantioselectivity toward an imprinted isomer. Furthermore, we discuss the recognition mechanism of the imprinted polymer for the target molecule with computational modeling. We selected L- or D- tryptophan methyl ester (abbreviated with L-, D- TrpOMe) as the target molecule. The lowest energy structure for the complex between the functional molecule and the target TrpOMe at the interface is analyzed by molecular dynamics (MD) calculation. The enantioselective performance of the L-, D- or racemicTrpOMe-imprinted polymer was evaluated by a competitive adsorption test using analogues of the target TrpOMe. The template effect was also characterized by comparing the adsorption behavior of the imprinted polymers to that of the control adsorbent prepared without the imprint molecules. The specific interaction between TrpOMe and n-DDP was investigated in detail by
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Fig. 1. Schematic illustration of the surface molecular imprinting technique.
FT-IR and 1H-NMR measurement to verify the imprinting phenomena. Finally, we quantitatively discuss the template effect by evaluating their binding constants on the basis of the modified Scatchard analysis.
2. Materials and methods
2.1. Reagents and apparatus Functional host molecule n-DDP was synthesized according to the procedures reported in previous works [29]. N-D-glucono dioleyl glutamate (abbreviated with 2C18D9GE) used as an emulsion stabilizer was synthesized in our previous study [34]. Amino acids (D,L-TrpOMe, D,LPheOMe, and D,L-Trp) were purchased from Sigma. Divinylbenzene (abbreviated as DVB, Wako Pure Chemical Industries) was employed after treatment with silica gel to remove an inhibitor. Fig. 2 shows the structures of (a) Tr-
Fig. 2. Structures of (a) imprint molecule: TrpOMe and (b) functional host molecule: n-DDP.
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pOMe and (b) n-DDP. Other reagents were of commercially available special grades. Particle size analysis was performed by a microtrac optical analyzer (Model 7995-10 SRA, Nikkiso), and the microstructure of particles was observed by using an ABT-32 type microscopy (Akashi Beam Technology).
2.2. Preparation of the L -, D -, or racemic-TrpOMe-imprinted polymer Enantioselective polymers were prepared by the surface molecular imprinting technique utilizing W/O emulsions. A 40-ml DVB containing 60 mM n-DDP and 5 mM 2C18D9GE was mixed with 20 ml toluene. Then, a 30-ml aqueous solution containing 10 mM L-, D-, or racemic-TrpOMe, which pH was adjusted to 4.5 with a 100-mM phosphate buffer solution, was added to the above organic phase. The mixture was sonicated for 4 min to obtain stable W/O emulsions. After the addition of 0.36 g (1.4×10 − 3 mol, 0.01 wt.% for DVB) powder initiator (2,2%-azobis (2,4%-dimethylvaleronitrile), a product of Wako Pure Chemical Industries), the mixture was polymerized at 308 K for 2 h under a flow of nitrogen. The obtained bulk polymer was dried under vacuum and ground into particles of an appropriate size. The particles were washed with 1 M hydrochloric acid to remove the imprinted L-, D-, or racemic-TrpOMe and then filtered off. This procedure was repeated several times until the imprint molecule in the filtrate cannot be detected by a UV spectrometer (V-570 UV/VIS/NR Spectrophotometer, Japan Spectroscopic). Finally, the polymer was dried in vacuo for several days. An unimprinted polymer and a polymer without applying functional host molecule (abbreviated as n-DDP-free polymer) as a reference were similarly prepared without the imprint molecule or the functional host molecule.
2.3. Competiti6e adsorption experiments on L -, or racemic-TrpOMe-imprinted polymer
D-
The batchwise adsorption experiments were conducted for the newly prepared polymers. The polymers (0.05 g) were added to a 5-ml aqueous
solution containing amino acids and placed in a sealed test tube (10 ml volume). The pH was adjusted to a desired value between 1.5 and 7.5 with 100 mM KH2PO4-K2HPO4 and 100 mM HNO3. The mixture was shaken in a thermostated water bath at 308 K for 24 h. The polymers were then filtered off through a polyethylene membrane (Sumplep LCR25-LG, Nippon Millipore). The amount of each amino acid adsorbed to the polymers was calculated on the basis of their residual amount in the filtrate. The concentration of amino acids was analyzed by a HPLC system (Japan Spectroscopic) with a reversed phase column (TSKgel ODS-80Ts column, 4.6×250 mm, Toyo Soda Co., Ltd.). The elution (100 mM CH3COOHCH3COONa (pH 4.5)/acetonitrile = 4/1, v/v) was spectorophotometrically monitored at 278.8 nm and 1.0 ml/min except for PheOMe, whose elution was monitored at 258 nm and 0.5 ml/min. The adsorption tests were carried out at least three times and the data were plotted with the average values. The experimental errors were less than 8%.
2.4. FT-IR and 1H-NMR measurement The interactions between the target L-TrpOMe and n-DDP were investigated in detail by FT-IR and 1H-NMR spectra studies. The measurement was carried out using FT-IR 8300 (Shimadzu) and AC 250 P (Bruker) at 303 K. The FT-IR study was performed by the KBr method. 1H-NMR samples were measured in C6D6 containing 15 vol.% DMSO-d6 ( 250 MHz, Tetramethylsilane (TMS) was used as an internal standard). The following values were used for analysis. 1. FT-IR spectra 1.1. L-TrpOMe 3287 cm − 1: N-H stretching vibration on the indole ring, 1747 cm − 1: CO stretching vibration 1.2. n-DDP-L-TrpOMe complex 3217 cm − 1: N-H stretching vibration on the indole ring, 1751 cm − 1: CO stretching vibration 2. 1H-NMR spectra 2.1. L-TrpOMe
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Fig. 3. SEM photograph of the TrpOMe-imprinted polymers.
d = 11.45, s, NH (indole ring); d= 7.0 – 8.0, m, CH (indole ring); d= 6.43, bs, NH+ (amino group); 3 d= 4.39, t, CH2 (methylene group); d= 3.67, m, CH (methine group); d= 3.47, s, COH3 (methoxyl group) 2.2. n-DDP-L-TrpOMe complex d= 11.28, s, NH (indole ring); d= 8.60, bs, NH+ 3 (amino group); d= 7.0 – 8.0, m, CH (indole ring and phenyl group); d =3.35, s, COH3 (methoxyl group)
2.5. Computational calculation for optimum structure of the complex between amino acid ester and the functional host molecule The molecular mechanic (MM) and molecular dynamic (MD) calculations were performed for the TrpOMe-n-DDP-complex by molecular modeling software, HyperChem Release 5.1 (Hypercube, Canada). In the calculation, the force field parameter set of MM2 was employed [35]. Before calculating the optimized structure of the complex at the oil-water interface, two preliminary calculations were conducted: (1) MM calculation to obtain the lowest energy structure of the complex in a vacuum; and (2) a toluene-water box to calculate the complex at the interface. Firstly the lowest energy structure of the complex in vacuum was calculated by the MM method. The toluene-water
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biphase box (30 A, × 30 A, × 60 A, ) was built from two adjacent cubic boxes (30 A, × 30 A, × 30 A, ) of pure toluene and water. The number of toluene and water molecules was set to be 172 and 856 by taking into account each molecular volume, respectively. Finally the TrpOMe-n-DDP-complex optimized in vacuum was placed at the interface by replacing seven toluene molecules that is almost equivalent to the complex volume. The MD simulation was performed in the (N, V, T) ensemble after MM calculation in the biphase system, which was required to avoid a large strain energy raised when being directly calculated by the MD method. The time step was adjusted to be 1 fs, and the temperature was controlled at 300 K by coupling to a thermal bath with a relaxation time of 0.1 ps.
2.6. Determination of binding constants between L -, D -, or racemic-TrpOMe and the imprinted polymers Binding constants of imprinted polymers were evaluated with a batchwise method. A 0.05-g polymer was immersed in a sealed test tube (10 ml volume). Then, a 5-ml aqueous solution, which is buffered with 100 mM KH2PO4K2HPO4 containing L- or D-TrpOMe adjusted to a desired concentration between 0.05 and 5 mM was added. The mixture was shaken at room temperature for 24 h. The polymers were then filtered off through the polyethylene membrane. The concentration of L- or D-TrpOMe in the filtrate was analyzed by a HPLC system. The binding constants were calculated by a modified Scatchard equation [36,37].
3. Results and discussions Highly cross-linked TrpOMe-imprinted polymers were prepared by the surface molecular imprinting technique with W/O emulsions. All polymers were obtained at more than 80% yields. After polymerization, the bulk polymer was ground into particles, whose volume-averaged diameters were ca. 40 mm. Fig. 3 shows a typical view of the TrpOMe-imprinted polymer prepared from W/O emulsions by scanning electron mi-
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croscopy. Substantial traces of aqueous phases in the emulsions are observed in the polymer. The recognition sites for the TrpOMe isomer are con-
Fig. 5. The pH dependence of the adsorption of L- and D-PheOMe or L- and D-Trp on the unimprinted polymer.
Fig. 4. The pH dependence of the adsorption of L- and or L- and D-Trp on the imprinted polymers: (a) the L-TrpOMe-imprinted polymer; (b) the D-TrpOMe-imprinted polymer; or (c) the Racemic-TrpOMe-imprinted polymer. D-TrpOMe
ceptually produced on the surfaces of the inner cavities in the polymer. A number of micropores facilitate diffusion of the target TrpOMe into the polymer. Fig. 4(a–c) exhibits the pH dependence for the adsorption of L- and D-TrpOMe or L- and D-Trp on the L-TrpOMe-imprinted, D-TrpOMe-imprinted, or racemic-imprinted polymer. The percentage of adsorption increased with increasing pH in the feed solution. This result suggests that the proton-dissociation from the functional host molecule plays a predominant role when the imprinted polymers rebind the amino acid esters. The L-TrpOMe-imprinted polymer showed a high selectivity toward L-TrpOMe over D-TrpOMe in the whole pH range investigated (Fig. 4(a)). Similarly the D-TrpOMe-imprinted polymer has a high affinity to D-TrpOMe imprinted at the preparation (Fig. 4(b)). Although the racemic-TrpOMeimprinted polymer also has high potential for the adsorption, it afforded no evidence of enantioselective adsorption (Fig. 4(c)). That is, the imprinted polymer (L- or D-TrpOMe-imprinted polymer) exhibits much higher template effect toward the corresponding imprinted tryptophan methyl ester than its isomer, while a reference polymer prepared with the racemic molecule does not reveal any selectivity toward the enantiomers, because the non-specific recognition sites are produced on the polymer surface. Fig. 5 shows the
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pH dependence for the adsorption of L- and D-TrpOMe or L- and D-Trp on the unimprinted polymer. The unimprinted polymer prepared without the imprint molecule was not able to recognize the chirality of amino acid derivatives. In the unimprinted polymer, the functional host molecules are randomly distributed on the polymer surfaces. The unimprinted polymer, however, had relatively high adsorption ability compared to that of the imprinted polymers, because the number of adsorption sites, which are non-specific sites, is comparable to that of the imprinted polymers. Fig. 6 exhibits the pH dependence for the adsorption of L- and D-TrpOMe or L- and D-Trp on the n-DDP-free-polymer. The polymer prepared without the host molecules did not adsorb tryptophan methyl ester at all. This result demonstrates that the interaction between the functional host molecule and the substrate should have a crucial effect for creating the adsorption ability. Tryptophan is scarcely adsorbed on all polymers (Fig. 4(a–c), Fig. 5, Fig. 6), because amino acids form intra-/inter-molecular ionic complexes between amino and carboxyl groups or because the electrostatic repulsion between the carboxyl anion in tryptophan and the negatively charged functional molecule inhibits the adsorption. This result indicates that the electrostatic interaction between the phosphonic acid moiety in the functional host molecule and the amino group in the
Fig. 6. The pH dependence of the adsorption of D- and L-TrpOMe or L- and D-Trp on the n-DDP-free polymer.
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substrate is required to succeed in creating the enantiomeric selectivity. In the case of adsorption of the phenylalanine methyl ester, which has a similar structure to the imprinted tryptophan methyl ester, on the imprinted polymer (L- or D-TrpOMe-imprinted polymer), we did not observe enantioselectivity. Hence, we found that the shape and/or space constructed on the polymer surface, which is complementary to the imprinted tryptophan methyl ester, should be a vital factor to recognize the molecular chirality. It is important to elucidate the recognition mechanism of the imprinted polymer in a wide application of the surface molecular imprinting technique. We investigated in detail the interaction between the imprint molecule (L-tryptophan methyl ester) and the functional host molecule (n-DDP) at the pre-polymerization stage by FTIR and 1H-NMR measurement. The characteristic peaks observed for the n-DDP-L-TrpOMe complex and L-TrpOMe itself were used to discuss the recognition morphology. Based on the results of the FT-IR spectra, the N-H stretching vibration peak derived from the indole ring appeared at 3287 cm − 1 on L-TrpOMe, and a similar peak on the n-DDP-L-TrpOMe complex shifted to 3217 cm − 1. This result suggests that the hydrogen bonding interaction between the PO in the phosphonic part of n-DDP and the proton of the indole ring causes the lower shift when forming the complex. On the other hand, the sharp CO stretching vibration peaks derived from L-TrpOMe itself and the n-DDP-L-TrpOMe complex appeared at 1747 and 1751 cm − 1, respectively. It can be assumed that the hydrogen bonding is not formed between the CO group in L-TrpOMe and the P-OH in the phosphonic group in n-DDP. The indole proton in L-TrpOMe was found at 11.45 ppm, and the peak on the complex had shifted to 11.28 ppm. This upfield chemical shift can be rationalized by the NH–OP interactions in forming the complex between n-DDP and L-TrpOMe. Additionally, the resonance peak of the cationic amino group protons on the complex remarkably shifted to the lower magnetic field compared to that of the L-TrpOMe (from 6.43 to 8.60 ppm). It is considered that the electrostatic interaction between n-DDP and L-TrpOMe
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Fig. 7. Schematic illustration of the surface of the TrpOMe-imprinted polymers.
causes a remarkable low-field chemical shift. The down- or up-field chemical shifts are due to the shielding and anti-shielding effect of nearby phosphonic groups on the protons influenced by hydrogen bonding and electrostatic interactions [38]. Based on these remarks one general point becomes very clear: the enantioselective recognition morphology on the L- (D-) TrpOMe-imprinted polymer was accomplished by three important factors as shown in Fig. 7: (i) the memorized space produced on the surface of the imprinted polymer in such a way that it can fit a desirable formation around L- (D-) TrpOMe when it interacts with the template; (ii) the electrostatic interaction between the functional host molecule and the substrate in a higher pH range which allows the proton dissociation from the phosphonic group; and (iii) the hydrogen bonding interactions between the functional host molecule and the indole ring. We calculated the lowest energy structure of the complex in the toluene-water box (Fig. 8) by the
MD method, and the snap shot at 1 ps was shown in Fig. 9(b). In the complex as shown in Fig. 9(b), the right-side host molecule forms the hydrogen bond with the nitrogen atom in the indole ring. While the left-side molecule interacts with the amino group in tryptophan through the ionicbond formation. By comparing it to the overview for the lowest energy structure of the complex in a vacuum in Fig. 9(a), it is clearly found that the indole ring in tryptophan locates along the interface and a little relaxes in the toluene-water box compared to that in a vacuum. The imprinted tryptophan is removed by washing with a week acidic solution and the complementary space to the target tryptophan will be memorized on the polymer surface. Finally, we quantitatively characterized the template effect in the L-, D-, or racemic-TrpOMeimprinted polymers by assessing the binding constant (Ka). The binding constant can be evaluated on the basis of the slope and intercept by the modified Scatchard plot. Table 1 summarized the
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Fig. 8. Toluene-water biphase boxes optimized by molecular dynamic calculation (30 A, ×30 A, ×60 A, ) Fig. 9. (a) The lowest energy structure of complex in a vacuum before MD calculation. Blue, carbon; grey, hydrogen; purple, nitrogen; red, oxygen, and yellow, phosphorus. (b) The lowest energy structure of complex at oil – water interface after MD calculation (at 1 ps). The colors mean the same atoms as that in (a).
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results of the binding constants for the TrpOMeimprinted polymers: (a) the L-TrpOMe-imprinted polymer; (b) the D-TrpOMe-imprinted polymer; and (c) the racemic-imprinted polymer. The binding constant becomes an indicator to represent the adsorption affinity of recognition sites for the target tryptophan methyl ester. The L- or D-imprinted polymers exhibited high binding abilities to the corresponding imprinted guest molecules, while the racemic-TrpOMe-imprinted polymer had a similar binding ability to both isomers. To discuss the enantioselectivity, we defined the separation factor as follows: a =KaL (D)/KaD (L). The separation factors in the L-, D-, and racemic-TrpOMe-imprinted polymers were 1.49, 1.40, or 1.02, respectively (Table 1). It should be concluded that the surface imprinting technique is very useful to create enantioselective sites, which can recognize the chirality of an optically active molecule on the polymer surface. We believe that the use of the surface molecular imprinting technique for the preparation of recognition polymers will find various applications in a variety of chemical fields.
molecule exhibited high enantioselectivity toward the corresponding imprint isomer. However, the racemic-TrpOMe-imprinted and unimprinted polymers did not show enantioselectivity. In addition, the n-DDP-free polymer as a reference has no adsorption ability to tryptophan methyl ester. The enantioselectivity was supported by high binding constants of corresponding imprinted substrates, which are suitable for the recognition sites constructed on the surfaces of the imprinted polymers. FT-IR and 1H-NMR studies provided useful information about the recognition mechanism of the imprinted polymers. Furthermore, computational modeling clearly showed the interaction mode between the target amino acid ester and the functional molecule at the liquid–liquid interface. Based on the results obtained, we found that three critical factors are required for recognizing the chirality of optically active materials: (1) a complementary recognition site for the target molecule; (2) hydrogen bonding formation; and (3) electrostatic interaction between a target guest molecule and a functional host molecule.
Acknowledgements 4. Conclusions The D- or L-TrpOMe-imprinted polymers containing an organophosphorus functional host Table 1 Binding constants of L- or K-TrpOMe on the TrpOMe-imprinted polymers: (a) the L-TrpOMe-imprinted polymer; (b) the D-TrpOMe-imprinted polymer; or (c) the racemic TrpOMe-imprinted polymer Binding constant Ka (M−1)
Separation factor a (= KaL (D)/KaD (L))
(a) L-TrpOMe D-TrpOMe
2.95×103 1.98×103
1.49
(b) D-TrpOMe L-TrpOMe
4.81×103 3.43×103
1.40
( c) L-TrpOMe D-TrpOMe
3.84×103 3.78×103
1.02
Substrate
We are grateful for the Grant-in-Aid for Scientific Research (No. 09750842) from the Ministry of Education, Science, Sports and Culture of Japan. One of the authors (M. Yoshida) was supported by a Research Fellowship from the Japan Society for the Promotion of Science (JSPS) for Young Scientists.
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