Biomacromolecules 2002, 3, 1353-1358
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Enzyme-Based Molecular Imprinting with Metals Aihua Cui, Amarjit Singh, and David L. Kaplan* Department of Chemical & Biological Engineering and Bioengineering Center, Tufts University, 4 Colby Street, Medford, Massachusetts 02155 Received July 13, 2002
Aromatic monomers with various functional groups were utilized in horseradish peroxidase-catalyzed polymerization reactions with metal ions Cu(II), Ni(II), and Fe(III) as imprinting templates. The approach described combines molecular imprinting with enzymatic free radical coupling. Selectivity in metal ion affinity between the various polymer products was assessed and found to depend on the metal used in the imprinting process using aniline, tyramine, and phenol as monomers. Selectivity in binding metals was found when polymers imprinted with copper, nickel, or iron were screened against the three metals, with preference for the metal used in the imprinting step. A model for the structural features of the putative imprinted polymers is proposed based on electron paramagnetic resonance, NMR, and IR analysis. Specific potential benefits to this imprinting method include reactivity with a wide range of aromatic monomers to provide more diverse options for molecular recognition with the target analyte and thus polymer products with higher selectivity, mild reaction conditions for the enzyme polymerization step to enable imprinting against labile substrates, imprinted polymeric products that contain conjugated backbones that could be suitable for electronics-based biosensor applications, and a potential for combinatorial selection to further enhance specificity. Introduction The technique of template polymerization, also known as molecular imprinting, has been used as a route to generate selective matrices for the detection and purification of compounds.1 Polymers were originally prepared by interactive preorganization of a functionalized monomer with a substrate bearing complementary binding sites (the template), followed by polymerization with or without an excess of cross-linking agent. Removal of the template yielded a functional polymer matrix with cavities and spatial arrangements of functional groups corresponding to the template molecule, such that the polymer exhibited selectivity for rebinding the original template. Reversible covalent bonds for monomer-template interactions2,3 and noncovalent (electrostatic and hydrogen bonding) interactions4 have been used to prepare imprinted polymers. These types of imprinting techniques are conceptually attractive; however, they tend to be limited in utility because of the few choices of monomers, and thus chemistries, that can be used in the imprinting step. Often this limitation is due to the constraints of the physical and chemical processes required during the polymerization step. Typically, preorganization of the target molecule with the monomers prior to polymerization is a critical step in the preparation of these materials, and interactions between monomer and template should be as specific as possible during polymerization. Reversible covalent bonds, while providing strong interactions, exhibit slow kinetics during rebinding and often necessitate severe conditions for desorption. Imprinted polymers are known to have enzyme-like catalytic properties and have been used as versatile tools in synthetic transformations including
enantioselective protection-deprotection, transesterifications, selective ester hydrolysis, and catalytic turnover.5-7 In these studies schematic diagrams and binding site interactions responsible for the catalytic transformations have been depicted although no detailed structural validation has been published.6-12 Interactions between biological molecules and metal ions tend to be highly specific and usually reversible under mild reaction conditions.13 Therefore, metal coordination would be a useful binding mode to use in the preparation of specific templated polymers. With metal coordination as a reversible basis for specific interactions, a diverse set of monomers would be required to enhance selectivity in the imprinting process. Polymerization under mild conditions would also be useful in cases where sensitive biological molecules may be used as templates. We selected peroxidase-based free radical coupling reactions for the imprinting system in the present work. These reactions can be carried out with a wide range of aromatic monomers under ambient reaction conditions, thereby satisfying both the selectivity and biological sensitivity criteria.14-16 A variety of polymers can be generated with this approach with a surprising degree of control over the process, depending on the reaction environment (e.g., aqueous, organic solvent, interfacial), monomers, and reaction conditions (pH, solvent, surfactants).17-21 An additional benefit to this approach is that the products (polyaromatics) are highly conjugated in structure and thereby potentially suitable directly as a conductive matrix.22 Thus, imprinting, synthesis, and signal transduction can be integrated in one process. The method studied in the present work combines metal ion imprinting with enzymatic free radical organic coupling
10.1021/bm025615y CCC: $22.00 © 2002 American Chemical Society Published on Web 08/28/2002
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reactions. The method (1) takes advantage of the variety of monomer functional group chemistries that can be utilized in these reactions, (2) utilizes the relatively mild reaction conditions for the enzyme polymerization, (3) generates imprinted polymeric matrices containing conjugated backbones that could be suitable for electronics-based biosensor applications, and (4) makes use of a combinatorial selection strategy to enhance selectivity. The objective of this study was to determine the feasibility of this enzyme-based imprinting approach using metal ions as the target analyte. A variety of aromatic monomers were utilized in reactions with Cu(II), Ni(II), and Fe(III) as template targets. Horseradish peroxidase was the enzyme used to carry out the polymerization process in the presence of hydrogen peroxide. The results suggest that this approach may be a useful addition to the suite of imprinting methods due to the simplicity, selectivity, and mild reaction conditions. Materials and Methods Materials. Horseradish peroxidase (HRP) (type II, 150200 units/mg of solid), hydrogen peroxide (HPLC grade, 30% w/w), and HEPES buffer were purchased from Sigma Chemical Company, St. Louis, MO. Phenolic and aromatic amine compounds were obtained from Aldrich Chemical Co., Milwaukee, WI. Compounds studied included aniline, phenol, thiophenol, tyramine, p-hydroxybenzoic acid, p-hydroxybenzyl alcohol, p-hydroxyphenethyl alcohol, tyrosine, 4-hydroxythiophenol, 4-phenylphenol, p-aminophenethyl alcohol, p-hydroxyphenylacetic acid, and p-hydroxyphenylpropioinic acid. Salts NiCl2‚6H2O, FeCl3‚6H2O, and CuCl2 were reagent grade. Assay of Metal Ion Inhibition on HRP Activity. To establish a baseline of enzyme activity, it was necessary to determine the influence of the metal ions used in the imprinting reactions on the activity of the enzyme, since it had been reported that metal ions inhibited the activity of HRP.20 The polymerization reactions were compared with the addition of different concentrations of the metal ions, and reaction rates were calculated from the yields of polymer with time. Yields of polymer were measured by spectrophotometry after solubilizing the polymer in THF followed by assay at 600 nm. In addition, enzyme activity was assayed with pyrogallol under standard conditions where one unit of enzyme activity resulted in the formation of 1.0 mg of purpurogallin from pyrogallol in 20 s at pH 6.0 at 20 °C. Preparation of Metal Ion Imprinted Polymers. The scheme for the preparation of a metal-imprinted polymer is shown in Figure 1 with copper. Ni(II) and Fe(III) were also imprinted in a similar fashion. The reactions were first explored in a noncombinatorial fashion to ensure the validity of the enzyme-imprinting concept. Reactions were carried out in 10 mL polystyrene tubes in water, with buffer at room temperature. HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) was used at a concentration of 0.05 mol/L at pH 7.5. The monomer (aromatic) and metal ions were dissolved in the buffer at specific molar ratios (e.g., 4:1 for Cu(II) and Ni(II) and 6:1 for Fe(III)) for the imprinting step. After 30 min, the horseradish peroxidase was added slowly
Figure 1. Schematic representation of imprinting and polymerization reactions carried out with aniline, metal ions (Cu2+), horseradish peroxidase (HRP), and hydrogen peroxide in aqueous buffer.
to the solution (with the monomer and metal ions) with gentle mixing. The catalytic reaction was initiated by the addition of hydrogen peroxide. The final 10 mL of reaction mixture contained 0.15 mol/L monomer, 0.04 mol/L Cu(II) or Ni(II) or 0.025 mol/L Fe(III), 0.5 mg/L horseradish peroxidase, and 0.2 mol/L hydrogen peroxide. The reactions were run for 24 h. Polymer products were isolated by centrifugation at 5000g at room temperature for 10 min, and the solid product was washed with water to remove residual buffer, HRP, unreacted monomers, and noncomplexed metal ions. After being washed, the polymer was treated with 0.01 mol/L hydrochloric acid to extract Cu(II) or the other metals from the matrices. The polymer was finally dried in a vacuum oven for subsequent analysis. In a partial combinatorial approach, a variety of monomers was also screened using a microtiter plate format. The 96-well plates had a volume of 300 µL for each well, and similar procedures were used as described above, only scaled to the smaller volume. Reactions were also carried out in organic solvents to compare polymer formation and selectivity with the aqueous systems. Metal Binding. To study the binding capacity and selectivity of the imprinted polymer, a 10 mL solution of 0.1 mol/L metal ions was added (Cu(II), Ni(II), or Fe(III)) to 100 mg of the polymer. The pH was adjusted to a value between 2 and 6 with 5.0 × 10-2 mol/L acetic acid/sodium acetate and 1.0 mol/L nitric acid. The mixture was shaken at room temperature for 5 h. The polymer was then washed and centrifuged to remove unreacted metal ions. The extracts and the original solution were combined, and metal ion concentration adsorbed to the polymer was calculated from the decrease in metal ion concentration in solution. Metal ion concentration was analyzed using a TJA SH-1000 flame GF atomic adsorption spectrophotometer (Billerica, MA). Metal ion binding capacity is reported either as mM metal/g of polymer or as a percentage (wt/wt).
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Figure 2. The influence of Cu(II) concentration on the reaction of horseradish peroxidase and hydrogen peroxide with aniline in terms of yield of polymer product during 24 h. Average of two runs.
Characterization. FTIR spectra of samples were recorded on a Nicolet Magna-760 (Nicolet Instrument Inc., Madison, WI) instrument at a resolution of 2 cm-1 and averages of 64-100 spectra/scan (for enhanced signal) were obtained in the wavenumber range 400-4000 cm-1. Spectra of samples were recorded from KBr pellets, prepared by mixing the samples with KBr in 1:100 (wt/wt) ratio. 1H NMR and 13 C NMR spectra were recorded using a Bruker DPX 300 spectrometer. Chemical shifts in parts per million (ppm) were referenced relative to tetramethylsilane (TMS) as internal reference. Molecular weight was determined on a Waters GPC module I instrument (Millipore, Milford, MA) with columns with a molecular weight separation range of 100 to >200 million. Polymers were dissolved at 3 mg/mL in DMF. To prevent aggregation, 1% (w/v) of LiBr was added to solutions. All samples were filtered twice through 0.2 µm filters prior to injection. Electron spin resonance (EPR) spectra were recorded on a Bruker model EMX EPR spectrophotometer controlled by compatible IBM PC software. All EPR studies were run with solid polymers at room temperature or in liquid nitrogen. Results and Discussion Initially, inhibition of peroxidase activity by metal ions was determined. Figure 2 shows the reaction rates of aniline with the enzyme and hydrogen peroxide in the presence of Cu(II). The results demonstrated that increasing levels of the metal, especially at initial stages of the reaction, resulted in greater inhibition. However, the differences in polymer yield after 24 h were minimal. The impact of other metals on the enzyme activity was lower than that of copper. On the basis of these results, metal ion concentrations were selected based on the following considerations: the coordination number of Cu(II) and Ni(II) is 4; a molar ratio of aromatic monomer to Cu(II) or Ni(II) was selected to be 4:1 in the imprinting reactions, while for Fe(III) the coordination number is 6, so that the molar ratio selected was 6:1. All reactions were run for 24 h to minimize impact on polymer yield based on the results in Figure 2. The structures of monomers used in the imprinting reactions provided a diversity in chemistry to optimize selectivity in terms of molecular recognition while still serving as suitable reactants for the enzymatic free-radical
Figure 3. Chemical structure of the monomers used for imprinting reactions.
coupling step (Figure 3). Polymers formed via imprinting with metal ions are designated PI (polymer imprinted), while the control reference polymer prepared in the absence of metal ion in the imprinting step is referred to as PC (polymer control). The polymerization reactions were first carried out in aqueous solution. The molecular weights of the imprinting reaction products were in the oligomeric range, trimers and tetramers. These findings were in agreement with the prior literature on horseradish peroxidase reactions with aromatic monomers in aqueous systems and reflect the limits of solubility of the products in water.14 Reactions conducted in organic solvents would significantly raise the molecular weight; however, for imprinting from aqueous solutions this was felt to be less representative of structural features to optimize interactions between monomers and metals prior to polymerization. To provide comparisons between choice of solvent and imprinting conditions, reactions were run with (a) metal ion and monomer in aqueous solution for the imprinting reaction, (b) metal ion and monomer in organic solvent for the imprinting reaction, and (c) first running the imprinting reaction in aqueous solution and then dissolving the reaction products in organic solvent and running the polymerization reaction again to increase molecular weight. In these initial experiments the metal binding capacity (millimoles of metal per gram of polymer) for copper by the polyaniline synthesized under these three different conditions was 12.5, 6.9, and 9.1 for the reactions in water during imprinting and polymerization, dioxane, and water for both imprinting and polymerization, and imprinting in water followed by polymerization in dioxane, respectively. These differences in copper binding capacity suggested that the first step (imprinting) in water helps to form the coordination between Cu(II) and
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Table 1. Selectivity of Imprinted Polymers-Oligomer Exposed to Different Metal Ion Solutions Assessed Separatelya Cu(II) binding capacity
Ni(II) binding capacity
Fe(III) binding capacity
entry
sample
%
mmol/g
%
mmol/g
%
mmol/g
1 2 3 4 5 6 7 8 9 10 11 12
polyphenol PC polyphenol PI with Cu(II) polyphenol PI with Ni(II) polyphenol PI with Fe(III) polyaniline PC polyaniline PI with Cu(II) polyaniline PI with Ni(II) polyaniline PI with Fe(III) polytyramine PC polytyramine PI with Cu(II) polytyramine PI with Ni(II) polytyramine PI with Fe(III)
0 0 3.47 6.13 3.10 12.80 10.92 3.97 4.50 16.20 6.13 4.85
0 0 0.373 0.659 0.333 1.376 1.174 0.427 0.328 1.182 0.659 0.521
0 0 7.80 8.21 6.20 6.42 17.10 3.02 2.60 7.12 12.90 4.04
0 0 0.83 0.874 0.667 0.683 2.054 0.321 0.19 0.757 0.942 0.430
8.20 3.31 5.07 16.80 0 2.63 3.29 4.50 6.60 6.98 4.73 9.90
0.872 0.352 0.539 1.787 0 0.208 0.350 0.484 0.481 0.742 0.503 0.723
a PI ) imprinted oligomer, PC ) nonimprinted oligomer or control polymer. Each metal was rebound to the oligomer separately in these experiments. As an example “Polyphenol PI” describes polyphenol prepared by imprinting with copper with the monomer phenol, and after extraction of the imprinted copper, then evaluated for rebinding capacity (mmol of copper per gram of polyphenol and as a percentage) against copper metal ions. Averages from N ) 5 are presented; standard deviations were e0.05 mmol/g.
Table 2. Selectivity of Imprinted Polymers-Oligomer Exposed to a Mixture of Different Metal Ions in One Solutiona Cu(II) binding capacity
Ni(II) binding capacity
Fe(III) binding capacity
entry
sample
%
mmol
%
mmol
%
mmol
1 2 3 4 5 6 7 8 9 10 11 12
polyphenol PC polyphenol PI with Cu(II) polyphenol PI with Ni(II) polyphenol PI with Fe(III) polyaniline PC polyaniline PI with Cu(II) polyaniline PI with Ni(II) polyaniline PI with Fe(III) polytyramine PC polytyramine PI with Cu(II) polytyramine PI with Ni(II) polytyramine PI with Fe(III)
0 0 0.49 1.22 3.10 10.41 6.04 1.13 1.56 7.89 2.21 1.14
0 0 0.052 0.130 0.237 1.107 0.642 0.120 0.166 0.839 0.235 0.121
0 0 4.90 3.12 6.20 1.27 13.78 0.68 1.34 1.95 6.50 1.27
0 0 0.521 0.332 0.428 0.135 1.465 0.072 0.142 0.207 0.692 0.135
8.21 3.11 2.54 13.88 0 0.33 1.01 3.01 2.38 0.35 0.72 5.02
0.873 0.331 0.270 1.475 0 0.035 0.107 0.320 0.253 0.037 0.076 0.531
a PI ) imprinted oligomer, PC ) nonimprinted oligomer or control polymer. Each metal ion was rebound to the oligomers as a mixture in these experiments with exposure level for each metal ion in the miture of 0.1 mol/L at pH 6.5. Averages from N ) 5 are presented; standard deviations were e0.05 mmol/g.
aniline monomer, and the oligomers obtained in aqueous systems were sufficient to detect the differences in binding affinity. The molecular weights of the polymers were increased in the reactions carried out with organic solvent; however, the specificity of the imprinted polymer was lower. The third set of conditions may reflect competition between polymerization and coordination. On the basis of the initial screening described above, reactions were conducted using reaction a, all aqueous imprinting and polymerizations. The results in Tables 1 and 2 provide comparisons between binding of three metals against three different aromatic monomers when each metal was used separately during the imprinting process. In Table 1 the polymers were studied for binding capacity against each of the three metals separately after imprinting and after extraction of the imprinted metal ions. In Table 2, mixtures of metals were studied during the rebinding process. The results showed that Cu(II) and Ni(II) were preferentially complexed with polymers having -NH2 functional groups (polyaniline and polytyramine) while Fe(III) complexed with higher capacity to polymers with -OH groups (e.g., polyphenol). Similar results for binding capacity were obtained when the same strategy was used in microtiter plate volumes (data not shown), supporting this approach in future combinatorial
strategies. On the basis of these data, the imprinted polymers did exhibit some selectivity toward the metal ion to which they were originally imprinted. For example, in Table 1 (entry 6), the polyaniline imprinted with copper had more than twice the binding capacity for rebinding copper than the other two metals. Significantly, the polymers imprinted with the metal ions had higher binding capacities than the corresponding control polymers in which imprinting was not utilized. In Table 2 the data suggest that in some instances the control polymers exhibited some selectivity, as expected, based on the differences in functional group chemistry, as discussed above. For example, polytyramine was more selective for iron than copper and nickel. However, the imprinted polymers demonstrated enhanced selectivity in comparison to the controls. For example, imprinted polytyramine had a higher binding capacity for copper than for nickel. Within the context of these initial experiments, significant selectivity was demonstrated and provides a basis for the further study of this approach to optimize of selectivity. The binding capacity of the imprinted polymers, as expected, was dependent on pH (Figures 4). The results for copper binding are illustrated in Figure 4 while similar results were found for the other metals (data not shown). The
Enzyme-Based Molecular Imprinting
Figure 4. pH dependence of Cu(II) binding capacity of Cu(II)imprinted polyaniline. Averages from N ) 5 are presented. Standard deviations were e0.05 mmol/g.
binding capacity for the polyaniline PI was higher than that for PC over the entire pH range, and the binding capacity of PI and PC decreased as the pH decreased, as expected. Figure 5 illustrates the differences in selectivity of the oligomers to the binding of copper. At a Cu(II) solution concentration of 10 ppm (10 mg/L), binding capacity was still observed, especially for the polyaniline PI. According to the literature, chromatography techniques can be used for the detection and separation of 1-10 ppm of most of metal ions.23-25 Therefore, the levels of selectivity observed with these imprinted systems are in a similar range without optimization and when considering only homopolymers in the imprinting. Transition metal ions can be embedded in a variety of compounds whose structure and dynamic properties modulate their paramagnetic properties through the symmetry and strength of the ligand field and relaxation properties.26 Electron paramagnetic resonance (EPR) can be used to detect subtle changes in the microenvironment of a paramagnetic center. EPR spectra were recorded (data not shown) for (a) polyaniline imprinted with Cu(II) but without extraction of the metal with HCl, (b) polyaniline imprinted with Cu(II) after extraction by HC1 and then rebinding Cu(II), and (c) polyaniline PC after binding Cu(II) but not imprinted. There was no hyperfine structure in the spectra. To try to understand the interactions between the paramagnetic copper centers and the aromatic functional groups, we purposely decreased the copper concentration in the ligand environment and ran EPR in liquid nitrogen (T ) 77 K). For example, polyaniline PI
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before extraction by HCl but imprinted with only 1% copper during the polymerization reaction was characterized; however, again no hyperfine structure was observed. A number of reports have detailed experimental and theoretical studies to explain why smaller or non-EPR parallel hyperfine splitting is observed in some metal ion complexes.27,28 Copper complexes are found in metalloproteins and were characterized by an intense blue color and a large value of optical absorption molecular extinction coefficient around 600 nm, which usually showed small hyperfine splitting. These complexes are usually axially symmetric. Shadle et al.27 also showed that this small hyperfine splitting in EPR spectra may reflect the high degree of covalency of the Cu(II)-ligand bond; moreover, they also demonstrated that different site symmetries of the ligands around the Cu(II) center may also result in different levels of hyperfine splitting, for example, D4h CuCl22- exhibits normal EPR hyperfine splitting while D2d CuCl22- shows smaller hyperfine splitting. On the basis of these previous reports, we suggest that the metal ion-ligand in our imprinted samples may be highly bonded so that they exhibit some level of covalency or the ligands are symmetric around the metal ions, e.g., tetrahedral for Cu(II) and Ni(II) and hexahedral for Fe(III), and this symmetry may result in smaller hyperfine splitting. The peak for polyaniline PI before extraction was greater than the peak after extraction and rebinding of the metal ion. This result suggests that not all of the binding sites that are formed during the polymerization reaction can be rebound, are not accessible, after removal and rebinding of the metal. Nevertheless, the peak for polyaniline PI after rebinding the metal ion was still greater than that of the polyaniline PC. NMR and FTIR were used to characterize the structures of the imprinted and polymerized oligomers. The chloroformsoluble fraction of imprinted polyaniline generated absorption bands in regions 3500-3100 cm-1 and 3100-2800 cm-1 to represent N-H stretching and C-H stretching for the polyaniline benzene and quinoid rings, respectively.29 The main absorption peaks at 3233, 3312, and 3442 cm-1 can be assigned to N-H stretching of B-NH-B and QdNH (where B ) benzene ring and Q ) quinoid ring). The bands
Figure 5. Selectivity and binding capacity of oligomers imprinted individually against the three metals followed by assessments of binding capacity for Cu(II).
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Acknowledgment. We thank Decheng Ma for helpful contributions to the work. References and Notes
Figure 6. Hypothetical representation of imprinting sites in polyaniline where M ) metal ion. Dotted lines between M and the nitrogen atoms in polyaniline represent potential electrostatic interactions, and the dotted line between phenyl rings denotes potential interchain links.
at 3055, 2959, 2922, and 2860 cm-1 may be due to C-H stretching of protons on the benzene ring. N-H deformation and CdN stretching of the polyaniline appear in the region 1600-1450 cm-1. Of the two bands at 1581 and 1500 cm-1, the former is from the quinoid ring and the latter from the benzene ring.29-32 The bands for Q and B are shifted to a lower frequency since in our experiments the samples were extracted with hydrochloric acid to remove the metal ions. HCl doping shifts the bands to a lower frequency by 10 cm-1.31 An additional peak appeared at 1555 cm-1 and may be due to an interchain bond. At 1400-1200 cm-1 the C-N stretch for aromatic amines was detected. The main absorption for C-H stretch on aromatic rings appears at 824, 754, and 692 cm-1. The IR spectrum for the chloroform insoluble fraction of the polyaniline indicated absorbance at 1495 cm-1, characteristic of the benzene ring. In the 1H NMR three singlets appeared at δ 4.8, 5.6, and 6.0 and a broad peak at δ 8.5. The peaks at δ 4.8, 6.0, and 8.5 disappeared upon deuterium exchange in DMSO and can be assigned to the NH protons of polyaniline.33 Small remnants of these peaks were observed after deuterium exchange in CDCl3 even after vigorous and extended stirring in D2O, perhaps due to immiscibility of CDCl3 and D2O. The peak at δ 4.8 did not disappear upon D2O exchange. Aromatic protons appeared at δ 6.9-8.0. In 13C NMR the peaks for the carbon of polyaniline appeared between δ 120.8 and 154.3.31,34-36 The peaks from δ 120.8 to 131.1 were from the benzene ring carbons and from δ 138.2 to 154.3 for the quinoid ring carbons. Additional peaks at δ 0.8-1.5 in 1H NMR and in 13C NMR at δ 29.8, 91.6, 97.4, and 181.5 may be due to artifacts from the HEPES buffer used in the experiments. The results from IR, 1H NMR, and 13C NMR confirm that the polymers contain both benzene and quinoid rings. Matrixassisted laser desortption ionization time-of-flight experiments show a regular loss of fragments with a molecular mass of 91, which corresponds to the molecular weight of the quinoid and benzene rings. Interchain links are a possible scenario, and a drawing of a possible structure is presented in Figure 6 based on the analytical data. It should be recognized that the model offered in this figure is speculative as the analytical results for such complex polymer systems are limited in definitive evaluation, as is the case with all prior reports of imprinting as mentioned earlier.6-12 The functional evidence presented in the present paper based on metal binding suggests an imprinting mechanism.
(1) Wulff, G. Polymeric Reagents and Catalysts; Ford, W. T., Ed.; ACS Symposium Series 308; American Chemical Society: Washington, DC, 1986; pp 186-230. (2) Wulff, G. In Biomimetic Polymers; Gebelein, C. C., Ed.; Plenum Press: New York, 1990; pp 1-14 and earlier references cited therein. (3) Shea, K. J.; Stoddard, G. J.; Shavelle, D. M.; Wakui, F.; Choate, R. M. Macromolecules 1990, 23, 4497-4507 and earlier references therein. (4) Fischer, L.; Muller, R.; Ekberg, B.; Mosbach, K. J. Am. Chem. Soc. 1991, 113, 9358-9360 and earlier references therein. (5) Sellergren, B.; Karmalkar, R. N.; Shea, K. J. J. Org. Chem. 2000, 65, 4009-4027. (6) Whitcombe, M. J.; Alexander, C.; Vulfson, E. N. Synlett 2000, 911923. (7) Wulff, G. Chem. ReV. 2002, 102, 1-27. (8) Wulff, G. TIBTECH 1993, 11, 85-87. (9) Wulff, G.; Gross, T.; Schonfeld, R. Angew. Chem., Int. Ed. Engl. 1997, 36, 1962-1964. (10) Yu, Congr.; Mosbach, K. J. Org. Chem. 1997, 62, 4057-4064. (11) Kim, J.; Ahn, K.; Wulff, G. Macromol. Chem. Phys. 2001, 202, 1105-1108. (12) Mosbach, K.; Yu, Y.; Andersch, J.; Ye, L. J. Am. Chem. Soc. 2001, 123, 12420-12421. (13) Spiro, T. G., Ed. Metal Ions in Biology; Wiley: New York, 1983; Vol. 3. (14) Klibanov, A. M.; Alberti, B. N.; Morris, E. D.; Felshin, L. M. J. Appl. Biochem. 1980, 2, 414. (15) Akkara, J. A.; Senecal, K. J.; Kaplan, D. L. J. Polym. Sci., Part A: Polym. Chem. 1991, 29, 1561-1574. (16) Dordick, J. S.; Marletta, M. A.; Klibanov, A. M. Biotechnol. Bioeng. 1987, 30, 31-36. (17) Rao, A. M.; John, V. T.; Gonzalez, R. D.; Akkara, J. A.; Kaplan, D. L. Biotechnol. Bioeng. 1993, 41, 531-540. (18) Bruno, F. F.; Akkara, J. A.; Kaplan, D. L.; Sekher, P.; Marx, K. A.; Tripathy, S. K. Ind. Eng. Chem. Res. 1995, 34, 4009-4015. (19) Eggins, B. R. Biosensors, An Introduction; Wiley-Teubner: Chichester, 1996. (20) Xu, Y. P.; Huang, G. L.; Yu, Y. T. Biotechnol. Bioeng. 1995, 47, 117-119. (21) Bonn, G.; Reiffenstuhl, S.; Jandik, P. J. Chromatogr. 1990, 499, 669676. (22) Parthasarthy, N.; Buffle, J. Anal. Chim. Acta 1991, 254, 1-7. (23) Wang, Z.; Li, J.; Van Loon, J. C.; Barafoot, R. R. Anal. Chim. Acta 1991, 252, 205-210. (24) Laintz, K. E, Yu, J. J.; Wai, C. M. Anal. Chem. 1992, 64, 311-315. (25) Nesterenko, P. N.; Amirova, G. B. Z.; Bolshova, T. A. Anal. Chim. Acta 1994, 285, 161-168. (26) Brill, A. S. Transition Metals in Biochemistry; Springer-Verlag: New York, 1997. (27) Shadle, S. E.; Penner-Hahn, J. E.; Schugar, H. J.; Hedman, B.; Hodgson, K. O, Solomon, E. I J. Am. Chem. Soc. 1993, 115, 767776. (28) Bizzani, A. R.; Canniistraro, S. Mol. Phys. 1995, 85, 913-929. (29) Tang, J.; Jing, X.; Wang, B.; Wang, F. Synth. Met. 1988, 24, 231238. (30) Furukawa, Y.; Ueda, F.; Hyodo, Y.; Harada, I.; Nakajima, T.; Kawagoe, T. Macromolecules 1988, 21, 1297-1305. (31) Cao, Y.; Li, S. Z.; Xue, Z. J.; Guo, D. Synth. Met. 1986, 16, 305315. (32) Wudl, F.; Angus, R. O.; Lu, F. L.; Allemand, P. M.; Vachon, D. J.; Nowak, M.; Liu, Z. X.; Heeger, A. J. J. Am. Chem. Soc. 1987, 109, 3677-3684. (33) Chen, W.; Jenekhe, S. A. Macromolecules 1992, 25, 5919-5926. (34) Kenwright, A. M.; Feast, W. J.; Adams, P.; Milton, A. J.; Monkman, A. P.; Say, B. J. Polymer 1992, 33, 4292-4298. (35) Ni, S.; Tang, J.; Wang, F.; Shen, L. Polymer 1992, 33, 3607-3610. (36) Kenwright, A. M.; Feast, W. J.; Adams, P.; Milton, A. J.; Monkman, A. P.; Say, B. J. Synth. Met. 1993, 55, 666-671.
BM025615Y
