Binding study on 5,5-diphenylhydantoin imprinted polymerconstructed by utilizing an amide functional group1

Jie Zhoua, Xiwen Heb,*, Yijun Lib

aDepartment of Chemistry, Shandong Agricultural University, Taian, Shandong 271018, ChinabDepartment of Chemistry, Nankai University, Tianjin 300071, China

Received 6 November 1998; received in revised form 16 March 1999; accepted 20 March 1999

Abstract

The molecular imprinting technique was applied for the preparation of a polymer selective for an acidic drug, 5,5-

diphenylhydantoin in a polar solvent using acrylamide as the hydrogen-bonding functional monomer. The binding

characteristics of the imprinted polymer were evaluated by batch methods. Scatchard analysis showed that two classes of

binding sites were formed in the imprinted polymer. Their dissociation constants were estimated to be 9.05 mmol/l and

1.87 mmol/l, respectively, by utilizing the model of multiple independent classes of binding sites. These results were more

reasonable than those obtained by the Scatchard equation. Factors that in¯uenced rebinding of the imprinted polymer

including pH, template/monomer ratio and functional monomers were explored. By contrast, when methacrylic acid was used

as functional monomer, the molecular imprinted polymers made in tetrahydrofuran exhibited only very weak binding capacity

for the template molecule. Finally, the substrate selectivity of imprinted polymer was investigated. # 1999 Elsevier Science

B.V. All rights reserved.

Keywords: Molecular imprinting; Substrate selectivity; 5,5-diphenylhydantoin; Binding sites; Dissociation constants

1. Introduction

The development of synthetic receptors that recog-

nize a target molecule at the molecular level is an

important area in chemistry today. On the basis of the

increasing understanding of supramolecular interac-

tions (hydrogen bonding, ionic interaction, van der

Waals interactions, the hydrophobic effect, metal che-

lation, etc.) between substrate±enzyme, antigen±anti-

body and ligand±receptor, several well-known

synthetic recognition systems have been reported

[1] and newly synthesized receptors are emerging

very rapidly [2].

Molecular imprinting is now a well established

technique for the preparation of such arti®cial recep-

tors and has recently been reviewed [3±6]. The process

begins with the desired target molecule called tem-

plate, which serves two functions. The ®rst is as a

space-®lling three-dimensional object around which a

complementary polymer cavity can be formed. The

second is to organize complementary interaction

between groups on the template and functional mono-

Analytica Chimica Acta 394 (1999) 353±359

*Corresponding author. Fax: +86-22-23502458; e-mail:

chemczl@sun.nankai.edu.cn1Project 29775011 supported by National Natural Science

Foundation of China.

0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.

PII: S 0 0 0 3 - 2 6 7 0 ( 9 9 ) 0 0 2 9 8 - 6

mers during polymerization. Organization of polymer-

izable functional monomers by the template can be

achieved by either covalent bonds and/or noncovalent

forces, that is, supramolecular interactions. The poly-

merization reaction mixture consists of the template,

functional monomers and a large excess of cross-

linker. An appropriate volume of inert solvent and

free radical initiator make up the remainder of the

polymerization solution. Thermal or photochemical

initiated polymerization gives a highly cross-linked

insoluble network polymer. The template can be

washed from the polymer matrix, while the functional

monomers remain covalently bound to the polymer

itself. Left in the polymer matrix are three-dimen-

sional cavities that are complementary in both shape

and chemical functionality to that of the template. The

high degree of cross-linking enables the microcavities

to maintain their shape after removal of the template,

and thus the functional groups are held in an optimal

con®guration for rebinding the template, allowing the

receptor to `recognize' the original substrate. Thus,

many molecularly imprinted polymers (MIPs) have

been prepared and utilized mainly as af®nity chroma-

tography media [7±10]. MIP-based binding assays

have been also developed in which MIPs are success-

fully used as antibody mimics [11,12].

The molecular imprinting technique has been

expanded to the ®eld of analytical chemistry of drugs

[13,14]. The used functional monomer is mostly

methacrylic acid (MAA) because the carboxyl group

is the most commonly hydrogen-bonding and acidic

functional group in molecular imprinting. However,

the hydrogen-bonding ability of this functional group

is not very strong in polar solvents and can only form

strong ionic interactions with basic functional groups.

So it is dif®cult to use MAA in molecular imprinting

for acidic drugs insoluble in apolar solvents. Here, we

®rst describe the preparation of a synthetic polymer

receptor for an effective anticonvulsant acidic drug,

5,5-diphenylhydantoin (DPH) using acrylamide as the

hydrogen-bonding functional monomer in a polar

solvent, tetrahydrofuran (THF). The imprinting pro-

cess is shown in Fig. 1. The detailed binding mechan-

ism of the MIP is ®rst examined by using the multisite

binding model in the molecular imprinting technique,

Some factors that in¯uence rebinding of the DPH-

imprinted polymer including pH, template/monomer

ratio and functional monomers are explored. Finally,

the binding selectivity of the MIP for DPH and its

methyl derivatives is also discussed

2. Experimental

2.1. Materials and instruments

DPH was obtained from Aldrich. Acrylamide,

MAA, methanol, acetic acid, acetonitrile, iodo-

mathane, dimethyl sulphate and THF were purchased

from Tianjin No. 2 Chemical Reagent Factory. MAA

was puri®ed by distillation prior to use. Ethylene

glycol dimethacrylate (EGDMA) was prepared from

ethylene glycol and methacrylic acid. 2,20-azobisiso-

butyronitrile (AIBN) was from Nankai University

Special Reagent Factory. Acetonitrile was chromato-

graphic grade. Other chemicals were analytical grade.

A Shimadsu UV-240 double-beam spectrophoto-

meter, a FT-NMR Model FX90 (JEOL) and a Model

SHZ-82 constant temperature bath oscillator (China)

were used.

2.2. Synthesis of Methyl Derivatives of DPH

3-Methyl-5,5-diphenylhydantoin (MDPH): A 2 ml

volume of iodomethane was added with magnetic

stirring to a solution of 2.50 g of DPH in 100 ml of

0.011 mol/l NaOH. The reaction was allowed to pro-

ceed for 3 h at 258C and the white precipitate was

collected by ®ltration and recrystallized from EtOH.

Yield: 67%, mp: 215±2168C (Lit. 2178C), 1H NMR

(CDCl3): � � 3.07 ppm, (N3-CH3); 7.40 ppm (Ar-H);

8.2l ppm (N1-H).

1,3-Dimethyl-5,5-diphenylhydantoin (DMDHP): A

50 ml volume of dimethyl sulphate was added drop-

wise with magnetic stirring to a solution of 1.25 g of

DPH in 250 ml of 2 mol/l NaOH and was prepared

according to the procedure of the preparation of

MDPH. Yield: 73%, mp: 196±1978C (Lit. 1978C),1H NMR (CDCl3): � � 2.80 ppm (N1-CH3); 3.12 ppm

(N3-CH3); 7.38 (Ar-H).

2.3. Polymer preparation

Polymers were prepared using acrylamide or MAA

as functional monomer and EGDMA as the cross-

linker, DPH as the template. The procedure for the

354 J. Zhou et al. / Analytica Chimica Acta 394 (1999) 353±359

synthesis of the standard polymer P2 is as follows.

DPH (1 mmol) was dissolved in THF (10 ml) in a

50 ml glass ampoule. EGDMA (20 mmol), acryl-

amide (4 mmol) and AIBN (50 mg) were added. After

nitrogen gas sparged into the solution for 5 min, the

ampoule was sealed under vacuum, and the mixture

was kept in a shaker bath at 608C for 24 h. The

resultant bulk rigid polymer was ground to pass

through a 75 mm sieve. Fine particles were removed

by repeated sedimentation in acetone. The resulting

particles were placed in a home-made extraction

apparatus [15] and washed at a ¯ow rate of 1.0 ml/

min under continuous stirring condition with 10%

acetic acid methanolic solution until the DPH could

no longer be detected at 240 nm in the eluent. Then the

particles were washed with methanol to remove resi-

dual acetic acid and dried to constant weight under

vacuum at 608C. The polymerization conditions for all

other materials were shown in Table 1.

Fig. 1. Schematic illustration of the preparation of DPH-imprinted polymer.

Table 1

Effect of DPH/monomer molar ratio on affinity of acrylamide MIPa

MIPs DPH/monomer

molar ratio

Q�imp

(mmol/g)

�Q*

(mmol/g)

P1 1 : 2 18.4 5.1

P2 1 : 4 35.0 16.6

P3 1 : 8 43.4 13.2

a Polymers 20.0 mg, [initial DPH] � 1.0 mmol/l, V � 2.0 ml,

t � 258C, Adsorption time � 12 h, solvent: acetonitrile,

Q* � amount of adsorbed DPH.

J. Zhou et al. / Analytica Chimica Acta 394 (1999) 353±359 355

2.4. Binding experiments

The sized and washed polymer particles (20.0 mg)

were placed in a 10 ml conical ¯ask and mixed with

2.0 ml of a known concentration of DPH acetonitrile

solution. The conical ¯ask was oscillated in a constant

temperature bath oscillator at 258C for 12 h. The

mixture was transferred into a centrifuge tube and

centrifuged at 4000 rpm for 5 min. The concentration

of free DPH in the solutions was determined by

measuring the absorbance at 240 nm. The amount

of DPH bound to the polymer Q was calculated by

subtracting the concentration of free DPH from the

initial DPH concentration. We de®ne the imprinting

factor as �Q � Qimp ÿ Qnon, where Qimp and Qnon are

the amounts of bound DPH on the imprinted and

nonimprinted polymers. Thus, Q is a measure of the

af®nity of polymers for DPH, while �Q is a measure

of the effect of the imprinting process. The average

data of triplicate independent results were used for the

following discussion.

3. Results and discussion

3.1. Template/monomer molar ratio

The molecular imprinting of DPH using acrylamide

as functional monomer was performed essentially by a

well-known procedure described previously [16].

Because different template molecules have different

functional groups and different degrees of functiona-

lization [17], a constant ratio of functional monomer to

template is required to obtain a high-af®nity MIP for a

particular template. Thus, in order to ®nd the optimum

conditions for DPH template, we synthesized imprint-

ing polymers P1, P2 and P3 at a constant template/

cross-linker ratio (1 : 20) and corresponding nonim-

printing polymers. The value of Q of the polymers for

DPH was determined by the equilibrium binding

method and �Q was obtained (Table 1). Table 1

shows that Q increases with the acrylamide content

and �Q of P2 is the highest among those of P1, P2, and

P3. This is that the more selective binding sites and

more nonselective adsorption in the imprinted poly-

mers are produced as the acrylamide content increases

The selective binding sites increases faster when the

DPH/monomer molar ratios change from 1 : 2 to 1 : 4.

So �Q is enhanced. While the DPH/monomer molar

ratios vary from 1 : 4 to 1 : 8, on the contrary, the

nonselective adsorption increases faster and �Q

decreases. Based on this, we have chosen P2 as a

standard DPH-MIP to investigate the binding char-

acteristics of the acrylamide imprinting polymer for

DPH.

3.2. Effect of functional monomer

MAA is known as a common functional monomer

and has been extensively used in the preparation of

MIPs [18]. We replaced acrylamide with MAA to

prepare DPH-MIP in polar solvent(THF) with the

same template/monomer/cross-linker molar ratio as

P2. Q and �Q of the polymer for DPH were 7.1 mmol/

g and 4.5 mmol/g under the same conditions. The

properties of the polymer were vastly inferior to those

of P2. This result was in agreement with that of MIPs

made in a polar solvent using carboxylic functional

monomers and print molecules [19]. Theoretically, the

difference between amide and carboxylic group MIPs

can be explained. (i) The dielectric constant and dipole

moment of the amide group is higher than that of the

carboxylic group. For example, acetic acid has a

dielectric constant of 6.20 and a dipole moment of

1.70 D, while for acetamide these values are 67.6 and

3.76 D [20]. (ii) In a peptide, the amide oxygen has

0.42e (e � 1.602 � 10ÿ19 C) negative charge and the

hydrogen 0.20e positive charge [21]. These constants

indicate that the amide functional group may be

capable of forming stronger hydrogen bonds than

the carboxylic group in polar solvents or water. In

addition, DPH is a weakly acidic drug, it can not form

ionic interactions with MAA. So the acrylamide

imprinting polymer yields much more highly selective

binding for DPH than the MAA imprinting polymer

prepared in the polar solvent THF.

3.3. Determination of binding parameters of DPH-

imprinted polymer

In the binding study of MIPs, it has been found that

two classes of binding sites often existed [18]. The

binding parameters of MIPs were mainly estimated by

Scatchard analysis [18]. At ®rst, we investigated the

binding performance of P2. The equilibrium binding

experiments were carried out by varying the concen-

356 J. Zhou et al. / Analytica Chimica Acta 394 (1999) 353±359

tration of DPH from 50 mmol/l to 4.0 mmol/l in acet-

onitrile in the presence of a ®xed amount of P2. The

obtained data were plotted according to the Scatchard

equation [18] to estimate the binding parameters of P2.

As shown in Fig. 2, the Scatchard plot was not linear

indicating that the binding sites in P2 are heteroge-

neous in respect to the af®nity for DPH. 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 P2. The equilibrium

dissociation constant Kd1 and the apparent maximum

number Qmax1 of the higher af®nity binding sites can

be calculated to be 2.l mmol/l and 17.2 mmol/g of dry

polymer from the slope and the intercept of its Scatch-

ard plot. By the same treatment, Kd2 and Qmax2 of the

lower af®nity bonding sites were found to be

1.56 mmol/l and 104.0 mmol/g. However, Scatchard

analysis does not consider the contribution of the

lower af®nity binding sites to the binding capacity

of MIPs at low concentrations of substrates in the

determination of higher af®nity binding parameters.

Similarly, at a high concentration of substrates, bind-

ing capacity of higher af®nity binding sites is ignored

in the determination of lower af®nity binding para-

meters. So the binding parameter values obtained by

Scatchard analysis are less accurate in these systems.

In order to overcome the insuf®ciency of Scatchard

analysis, we ®rst treated our observations according to

the model of many independent classes of binding

sites [22].

Because of the presence of two classes of binding

sites in P2, the equilibrium binding equation can be

written as:

Q � Qmax1�DPH�Kd1 � �DPH� �

Qmax2�DPH�Kd2 � �DPH� (1)

where Q is the amount of DPH bound to P2. [DPH] is

the free concentration in solution, Qmax1 and Qmax2 are

the appearent maximum numbers of the higher and

lower af®nity binding sites, respectively, and Kd1 and

Kd2 are the equilibrium dissociation constants of the

binding sites. Using the binding parameter values

obtained by Scatchard analysis as a set of initial

parameter estimates, we ®tted the experimental points

by Eq. (1). The obtained ®tting curve is in good

agreement with the experimental points as shown in

Fig. 3. Kd1, Kd2, Qmax1 and Qmax2 obtained by the ®nal

parameter estimates were 9.05 mmol/l, 1.87 mmol/l,

l0.0 mmol/g, and 94.6 mmol/g, respectively. Because

the treatment is not limited by the substrate concen-

tration used in the experiments, we think that the

values obtained by the model are more reasonable.

3.4. Effect of pH on characters of DPH-imprinted

polymer

Since biological recognition mainly occurs in aqu-

eous buffer systems and is a function of pH, it is quite

important to make MIPs capable of recognition in

water in order to mimic biomolecules. Unlike the

carboxylic group the amide group is not ionizable,

which could be advantageous for molecular recogni-

tion in water. Using KH2PO4±K2HPO4 (aq)/acetoni-

trile (3/7) as an aqueous buffer solvent system, a

correlation between binding and pH of adsorbed

solution is seen in Fig. 4 for the DPH-imprinted

polymer. The pH was altered by adjusting the balance

of mono- and dibasic phosphate salts(or adding HCl to

KH2PO4 solution for pH values lower than 4.2), while

the total concentration of phosphate salts was held

constant at 0.05 mol/l.

Fig. 2. Scatchard plots to estimate the binding nature of P2. Q is

the amount of DPH bound to 20.0 mg of P2.

J. Zhou et al. / Analytica Chimica Acta 394 (1999) 353±359 357

The binding of DPH on the DPH-imprinted polymer

is strongly in¯uenced by the pH in the solutions as

shown in Fig. 4. Enhanced binding was obtained in the

pH 4.7±6.9 range. This can be accounted for by the

protonation of the amide group and ionization of the

N-3 nitrogen atom of the DPH molecule. When the pH

is lower than 4.7, the amide group of P2 can bind a

proton to form a positively charged group. When the

pH is higher than 6.9, a proton of N-3 in DPH

molecule can be lost to form a negatively charged

DPH molecule. These results lead to the weak selec-

tive hydrogen-bonding interaction between DPH and

P2 and they lower the binding capacity of P2 for DPH.

In particular, this is more obvious in basic solutions.

This suggests that the binding of the DPH-imprinted

polymer for DPH may be controlled by hydrogen-

bonding interaction, which plays a crucial role in

biological recognition systems and in determining

the structure of protein and nucleic acids. In looking

at the nonimprinted polymer, its binding capacity for

DPH is distinctly lower than P2 and there is little

dependence of binding on pH. The reason for this may

be that, although the optimum binding conditions of

the polymer can be controlled by the pH of the

external solvent system, the selective binding is con-

trolled by the imprinting process. The selectivity of the

MIPs is due to the shape-selective cavity built into the

polymer matrix and the preorganization of functional

groups complementary to the template molecule.

3.5. Substrate-selectivity of DPH-imprinted polymer

The substrate selectivity of P2 was studied using

DPH and its methyl derivatives, MDPH and DMDPH,

as substrates in acetontrile and an aqueous buffer

solvent system, KH2PO4±K2HPO4 (pH 6.9)/acetoni-

trile (3/7). Their amounts bound to P2 and nonim-

printed polymer were determined by batch methods

(Table 2).

Table 2 shows that the amount of DPH bound to P2

was 3 to 4 times more than for a nonimprinted polymer

in both acetonitrile and aqueous buffer solvent system.

This indicates that the binding ability is introduced

into the polymer by the molecular imprinting techni-

que. This template effect induced a good binding

performance for DPH. As can be seen, P2 showed

very low af®nity to the DPH methyl derivatives

MDPH and DMDPH. The cause for this can be that,

Fig. 3. The fitting curve obtained by Eq. (1). Q is the amount of

DPH bound to 20.0 mg of P2.

Fig. 4. Effect of pH on the binding of P2 in buffer solvent system.

Solid square: DPH-imprinted polymer (P2); open square: non-

imprinted polymer.

358 J. Zhou et al. / Analytica Chimica Acta 394 (1999) 353±359

in spite of their similar structures to DPH, MDPH has

an active hydrogen on N-1, which can form a hydro-

gen bond as a proton donor whereas DMDPH has not

the active hydrogen in its molecule. In addition, the

molecular structures of MDPH and DMDPH can not

be complementary to the shape of the cavities in P2

which is disadvantageous to the molecules entering

the cavities. This can result in the formation of very

weak or no hydrogen-bonding interactions between

the DPH methyl derivatives and the amide groups of

P2. In conclusion, good hydrogen-bonding speci®c

recognition sites for acidic drugs can be created within

the synthetic polymer P2 in polar solvents using an

amide functional group. This study may further

enlarge the application of molecularly imprinted

polymers to the separation and determination of trace

drugs.

Acknowledgements

The authors are grateful to National Natural Science

Foundation of China for ®nancial support.

References

[1] G. Wenz, Angew. Chem. Int. Ed. Engl. 33 (1994) 803.

[2] M. Famulok, J. Am. Chem. Soc. 116 (1994) 1698.

[3] G. Wulff, Trends in Biotechnol. 11 (1993) 85.

[4] S. Vidyasankar, F.H. Arnold, Curr. Opin. Biotechnol. 6 (1995)

218.

[5] G. Wulff, Angew. Chem. Int. Ed. Engl. 34 (1995) 1812.

[6] J. Zhou, X.W. He, J. Instr. Anal. 17 (1998) 87.

[7] I.A. Nicholls, A. RamstroÈm, K. Mosbach, J. Chromatogr. A

691 (1995) 349.

[8] O. RamstroÈm, I.A. Nicholls, K. Mosbach, Tetrahedron:

Asymmetry 5 (1994) 649.

[9] L. Fischer, R. MuÈller, B. Ekberg, K. Mosbach, J. Am. Chem.

Soc. 113 (1991) 167.

[10] J. Matsui, O. Doblhoffer-Dier, T. Takeuchi, Chem. Lett.

(1995) 489.

[11] G. Vlatakis, L.I. Andersson, R. MuÈller, K. Mosbach, Nature

361 (1993) 645.

[12] M.T. Muldoon, L.H. Stanker, J. Agric. Food Chem. 43 (1995)

1424.

[13] J. Mathew-Krotz, K.J. Shea, J. Am. Chem. Soc. 118 (1996)

8154.

[14] M. Senholdt, M. Siemann, K. Mosbach, L.I. Andersson, Anal.

Lett. 30 (1997) 1809.

[15] G. Wulff, W. Vesper, R. Grobe-Linsler, A. Sarhan, Makromol.

Chem. 178 (1977) 2799.

[16] M. Kempe, K. Mosbach, J. Chromatogr. 664 (1994) 276.

[17] M. Kempe, K. Mosbach, J. Chromatogr. A 691 (1995)

317.

[18] J. Matsui, Y. Miyoshi, O. Doblhoff-Dier, T. Takeuchi, Anal.

Chem. 67 (1995) 4404.

[19] O. RamstroÈm, L.I. Andersson, K. Mosbach, J. Org. Chem. 58

(1993) 7562.

[20] D.R. Lide, CRC Handbook of Chemistry and Physics, CRC

Press, Boca Raton, 1994, pp. 6±155, pp. 9±42.

[21] T.E. Creiqhton, Protein's Structure and Molecular Properties,

W.H. Freem, New York, 1993, p. 45.

[22] A.F. Heny, Anal. Biochem. 48 (1972) 317.

Table 2

Binding amounts (mmol/g) of tested substrates on P2 and Pnon by

batch methoda

Substrates Acetonitrile KH2PO4±K2HPO4/MeCN

P2 Pnon P2 Pnon

DPH 51.3 18.4 44.5 12.5

MDPH 9.0 7.9 5.2 4.8

DMDPH 2.4 6.3 3.2 3.8

a Polymer 20.0 mg, [initial substrate] � 2.0 mmol/l, V � 2.0 ml,

adsorption time � 12 h, t � 258C.

J. Zhou et al. / Analytica Chimica Acta 394 (1999) 353±359 359