Fresenius J Anal Chem (2000) 368 : 461–465

© Springer-Verlag 2000

O R I G I N A L PA P E R

Hongsheng Guo · Xiwen He

Study of the binding characteristics of molecular imprinted polymer selective for cefalexin in aqueous media

Received: 23 March 2000 / Revised: 16 May 2000 / Accepted: 22 May 2000

Abstract A molecularly imprinted polymer was prepared using cefalexin as the template molecule and 2-(trifluoromethyl)acrylic acid as the functional monomer. The bulk polymer was ground, sieved and investigated in an equilibrium binding experiment to evaluate the binding characteristics of the cefalexin-imprinted polymer for a better understanding of the mechanisms of recognition in molecularly imprinted polymers. Scatchard analysis showed that two classes of binding sites were formed in the imprinted polymer under the concentration studied. The dissociation constants were estimated to be 0.14 mmol/L and 2.38 mmol/L. The polymer gave much higher binding capacity for cefalexin than the non-imprinted polymer with the same chemical composition. The selectivity was evaluated by distribution coefficients of cefalexin and other structurally similar compounds. The results showed that the imprinted polymer exhibited high affinity for cefalexin among the tested compounds.

1. Introduction Molecular imprinting is a rapidly developing technique for preparing polymeric materials that are capable of high molecular recognition. This method usually involves crosslinking of the functional monomers in the presence of template molecules by radical polymerization, then removing the target molecules. The imprinted polymers selectively bind again with the template molecules. Molecular imprinting has been successfully applied to chiral separations of amino acid derivatives [1], drugs [2] and sugar derivatives [3], for specific recognition of steroids [4], proteins and protein analogues [5, 6]. Recent work [7, 8] by Mosbach’s group compared the selectivities and cross-reactivities of molecular imprinted polymers

Hongsheng Guo · Xiwen He () Department of Chemistry, Nankai University, Tianjin 300071, P.R.China e-mail: [email protected]

(MIPs) and antibodies for theophylline and diazepam and found them to be equal, confirming the utility of this method. Cefalexin (CFL) is an effective antibiotic with broad spectrum activity against gram-positive cocci and rods and gram-negative cocci [9]. Up to now, a CFL-imprinted polymer is not reported because of the bad solubility of CFL in both non-polar solvents and polar solvents. In most cases, molecular recognition in the MIPs was performed only in non-aqueous organic solvents or aqueous organic solvents with water contents less than 50% [10, 11], although the recognition in aqueous solution would be very desirable because the biological recognition is mainly occurring in aqueous systems. It is quite important to prepare MIPs capable of recognition in aqueous solutions in order to mimic bio-molecular reaction. We describe here the preparation of synthetic polymer receptors for CFL by a molecular imprinting technique using 2-(trifluoromethyl)acrylic acid as functional monomer in methanol and water as solvent to study the binding characteristics of the imprinted-polymer by Scatchard analysis and substrate-selectivity experiment.

2. Experimental 2.1. Materials and instruments. Cefalexin (CFL), cefadroxil, amoxicillin, ampicillin were purchased from the National Institute For The Control of Pharmaceutical and Biological Products (People’s Republic of China). 2-(Trifluoromethyl)acrylic acid (TFMAA) and 4-vinylpyridine (4-VP) were purchased from Aldrich Chem. Co. Ethylene glycol dimethacrylate (EGDM) was prepared from ethylene glycol and methacrylic acid. 2,2′-Azobisisobutyronitrile (AIBN) was purchased from Nankai University Special Reagent Factory. Other chemicals were analytical grade. A Shimadzu UV-240 double-beam spectrophotometer, Varian Unity-Plus 400 NMR spectrometer and a SHZ-82 constant temperature bath oscillator (China) were used. 2.2. Preparation of the cefalexin-imprinted polymer(P(TCFL)) using 2-(trifluoromethyl)acrylic acid as the functional monomer. For the preparation of CFL-imprinted polymer, 365.4 mg (1 mmol) template CFL and 0.5602 g (4 mmol) functional monomer TFMAA were dissolved in 5 mL methanol in a 50 mL glass ampoule, 3.9644 g (20 mmol) cross-linker EGDM and 50 mg AIBN as ini-

462 tiator were added. After nitrogen gas bubbling into the solution for 5 min, the ampoule was sealed under vacuum, and the mixture kept in a thermo-bath at 60 °C for 24 h. The resulting bulk rigid polymers were ground to pass through a 75 µm sieve. Fine particles were removed by decantation in methanol. The resulting particles were washed with 20% aqueous acetic acid solution until the template could no longer be detected with spectrophotometer. Then the particles were washed with distilled water to remove residual acetic acid and dried to constant weight under vacuum at 60 °C. A reference non-imprinted polymer (P(TO)) was prepared using the same procedure without addition of the template.

tometer at appropriate wavelength. The amount of substrates bound to the polymer, Q, was calculated by subtracting the concentration of free substrate from the initial substrate concentration. The average data of triplicate independent results were used for the following investigation.

2.3. Binding experiments. The sized and washed polymer particles (20.0 mg) were placed in a 10 mL conical flask and mixed with 2.0 mL of a known concentration of selected substrate in water. The conical flask was oscillated in a constant temperature bath oscillator at 25 °C for 16 h. The mixture was transferred into a centrifuge tube and centrifuged for 5 min, and the concentration of free substrate in the solution was determined using a spectropho-

2.5. 13CNMR determination. A solution of CFL (20 mmol/L) and a solution of CFL (20 mmol/L) with TFMAA (20 mmol/L) were prepared using water as solvent. The measurements of the chemical shift of the C1, C2, and C3 of CFL were carried out at room temperature.

Fig. 1 Scheme of the molecular imprinting procedure

2.4. Spectrophotometric analysis. A series of solutions were prepared with a fixed concentration of CFL (0.06 mmol/L) and various amounts of TFMAA in water. The absorption spectra of these solutions with corresponding TFMAA solutions as references were determined.

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3. Results and discussion 3.1. Polymer preparation Cefalexin was chosen as the imprinted-template and 2-(trifluoromethyl)acrylic acid as the functional monomer. 2-(Trifluoromethyl)acrylic acid was first used by Jun Matsui to prepare nicotine-imprinted polymers [12]. This monomer is more acidic than methacrylic acid, a common but potentially functional monomer, owing to the electron-withdrawing effect of the trifluoromethyl group. It can be expected that TFMAA can interact with CFL more strongly than MAA by ionic interaction between –COOH group of TFMAA and the –NH2 group of CFL. TFMAA

Fig. 2 Structure of CFL

Fig. 3 UV adsorption spectra of CFL and TFMAA system in water. Concentration of CFL: 0–0.06 mmol/L; concentration of TFMAA(mmol/L): 1–0.06, 2–0.12, 3–0.24, 4–0.32; corresponding pure TFMAA solution as references

is also a strong hydrogen bond donor. The results showed that 4 mmol of TFMAA enables 1 mmol CFL to be dissolved in 5 mL methanol, thus making it possible to prepare the CFL-imprinted polymer (scheme of the molecular imprinting procedure is shown in Fig. 1). 3.2. Interaction between CFL and TFMAA in aqueous media The studies of the interaction between CFL and TFMAA in water are important to understand the imprinting and recognition phenomena. Since the cross-linker and initiator would be much less important for the interaction of template and functional monomer, the 13C NMR study was performed with CFL and TFMAA in water. In this system, the amino group of CFL presumably interact with the carboxyl group of TFMAA. As expected, the addition of TFMAA to the CFL solution resulted in high-field shifts of the peak derived from the C1, C2 and C3 of CFL (the structure of CFL is shown in Fig. 2). The peak of C1 shifted from 58.562 ppm to 57.417 ppm, the peak of C2 shifted from 128.291 ppm to 122.379 ppm, and the peak of C3 shifted from 131.679 ppm to 131.203 ppm because the –NH2 of CFL changed to be –+NH3. The effect of TFMAA on UV-spectra of CFL is shown in Fig. 3. At a fixed concentration of CFL in water, different concentration of TFMAA was added. Figure 3 shows that when the concentration of TFMAA increased, the maximum absorption wavelength of CFL shifted to longer wavelengths. And the maximum absorbances Amax at λmax decrease with the addition of TFMAA. The facts indicate that CFL and TFMAA can interact with each other by ionic interaction in water.

Fig. 4 Binding isotherm of P(TCFL). Q: amount of CFL bound to 20.0 mg of imprinted-polymers; V = 2.00 mL; Adsorption time: 16 h

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3.3. Binding characteristics of P(TCFL) In the binding study of MIPs, it has been found that two classes of binding sites often existed. In order to investigate the binding performance of the P(TCFL), its binding isotherm was determined in the range of 0.1–4.5 mmol/L as initial concentration of CFL (Fig. 4). In this range, the obtained binding data were plotted according to the Scatchard equation [13] to estimate the binding parameters of P(TCFL): Q/[CFL] = (Qmax – Q)/KD

(1)

where KD is an equilibrium dissociation constant and Qmax an apparent maximum number of binding sites. The scatchard plot (Fig. 5) was not linear indicating that the

binding sites in P(TCFL) are heterogeneous with respect to the affinity for CFL. Because there are two distinct sections within the plot which can be regarded as straight lines, it reveals that two classes of binding sites were produced in P(TCFL). The equilibrium dissociation constant KD1 and the apparent maximum number Qmax1 of the higher affinity binding sites can be calculated to be 0.14 mmol/L and 29.9 µmol g–1 of dry polymer from the slope and the intercept of its Scatchard plot. By the same treatment, KD2 and Qmax2 of the lower affinity binding sites were found to be 2.38 mmol/L and 130.1 µmol g–1. 3.4. Substrate-selectivity of CFL-imprinted polymer P(TCFL) The substrate selectivity of P(TCFL) was carried out using a series of structurally related β-lactams cefadroxil, amocillin, ampicillin as substrates in aqueous media (their structures are shown in Fig. 6). The amount of substrates bound to P(TCFL) and P(TO) were determined by the equilibrium binding method. The distribution coefficients of the selected substrates between solution and polymer were calculated and are listed in Table 1. The distribution coefficient KD is defined as: KD = Cp/Cs

Fig. 5 Scatchard plots to estimate the binding nature of P(TCFL). Q is the amount of CFL bound to 20.0 mg of P(TCFL)

Fig. 6 Structures of the substrates

(2)

where CP = concentration of substrate on the polymer (in µmol g–1) and CS = concentration of substrate in the solution (in µmol mL–1). Table 1 shows that P(TCFL) exhibits the highest selectivity for CFL compared to all the other substrates tested. P(TCFL) exhibited higher KD values for cefadroxil than for amoxicillin and ampicillin because of the slight difference between the structure of cefalexin and cefadroxil (there is a –OH in cefadroxil whereas there is no in cefalexin). P(TO) exhibit low values of KD for all the substrates. All the evidences mentioned above indicate that the imprinting method creates a micro-environment based on shape selection and position of functional groups that

465 Table 1 KD of tested substrates on P(TCFL) and P(TO) under equilibrium binding conditions Substrates

P(TCFL)

P(TO)

Cefalexin Cefadroxil Amoxicillin Ampicillin

32.10 27.19 13.61 14.68

15.11 16.04 15.55 15.45

g–1;

polymer: 20.0 mg. Unit: mL [initial substrate] = 2.0 mmol/L; V = 2.00 mL; adsorption time: 16 h.

recognizes best CFL template molecule. The obvious difference of binding selectivity between P(TCFL) and P(TO) mainly results from the carboxyl functional groups within the microcavities created in the P(TCFL) matrix by imprinting, although P(O) has the same chemical composition as P(TCFL), it does not have the recognition sites complementary in both shape and functional groups to those of CFL and the arrangement of carboxyl groups in P(O) are random. So, it would only relatively poorly bind the test substrates by weak non-specific adsorption and does not show any selectivity for them.

4. Conclusions The CFL-imprinted polymer was prepared by molecular imprinting, and P(TCFL) has higher affinity in water than P(TO). The development of such molecular imprinted polymers, which are operational in aqueous solution, may open new applications in the fields of life sciences.

Acknowledgements The authors are grateful to project 29775011 supported by National Natural Science Foundation of China and to project 003602211 supported by Natural Science Foundation of Tianjin.

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