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Oxidative Polymerization of a Substituted Phenol with Ion-Paired Horseradish Peroxidase in an Organic Solvent Philipp S. Angerer, Andre´ Studer, Bernard Witholt, and Zhi Li* Institute of Biotechnology, ETH-Hoenggerberg, CH-8093 Zurich, Switzerland Received January 14, 2005 Revised Manuscript Received June 10, 2005
Introduction. Enzyme catalysis is an environmentally friendly and useful alternative to chemical catalysis. Moreover, enzymes can catalyze reactions that are difficult to perform using conventional methods. For example, chemical polymerization of phenol requires the use of formaldehyde for the condensation, whereas horseradish peroxidase (HRP) is able to catalyze the oxidative polymerization of phenol giving a new class of polyaromatics1-3 that is structurally different from those synthesized chemically. Since such polyphenols are potentially electrically conductive materials, fluorescent polymers, or substitutions of phenol-formaldehyde resins, HRP-catalyzed polymerization has increasingly received attention. One problem in such reactions is that the aliphatic phenol substrates are poorly soluble in aqueous solution. To increase the substrate solubility, systems involving organic solvents were developed. This includes the use of a mixture of water and water miscible solvents such as 1,4-dioxane,4-6 biphasic systems such as water-isooctane,7 and reversed micelles resembling stable, water-in-oil microemulsions.8,9 Such nontraditional reaction media also allowed the control of polymer morphology:3 polymerization in mono- and biphasic solvent systems gave aromatic polymers with no specific geometry or orientation, polymerization in a reversed micelle system formed microspherical particles, and polymerization of amphiphilic aromatic monomers that are preordered and oriented in a Langmuir trough gave products consisting of two-dimensional monolayer.10 Enzymatic reactions can be performed in organic solvents containing a small amount of water. For instance, lyophilized HRP suspended in organic solvent was reported to catalyze the oxidation of phenol.11 On the other hand, enzymes can be modified to be soluble in organic solvent by forming surfactant ion-paired complex. Paradkar and Dordick reported the successful preparation of ion-paired R-chymotrypsin with the anionic surfactant aerosol OT (AOT) and demonstrated high catalytic activities for such enzyme preparation in isooctane.12 This method was recently applied by Bindhu and Abraham to prepare ion-paired HRP in isooctane.13 However, the potential of using such system for the polymerization of phenol has not yet been explored. Here we report a modified procedure for preparing ionpaired HRP in isooctane, the use of tert-butyl hydroperoxide as the oxidant for the enzymatic polymerization, and the first example of polymerization of phenol with ion-paired HRP in organic solvent. * To whom correspondence should be addressed: Fax +41-16331051; e-mail
[email protected].
Results and Discussion. a. Preparation of IonPaired HRP in Organic Solvent. Commercially available lyophilized HRP was used for the ion-pairing experiments. It was found to contain a major HRP component with a molecular weight of 44 kDa and a pI of 5.2. The Bradford protein determination assay showed 37% of protein in the lyophilized powder. A general methodology for ion-pairing enzyme with AOT has been developed by Paradkar and Dordick.12 It consists of dissolving the enzyme in buffer, mixing with equal volume of isooctane containing AOT, and separating the isooctane phase containing ion-paired enzyme. This method was recently used for the preparation of ionpaired HRP.13 However, repeating the reported procedure (at pH ) 6.6)13 with our HRP afforded only 2% of proteins in isooctane, possibly due to the low pI of our HRP. Decreasing the pH of the buffer significantly increased the extraction efficiency: ion-pairing of HRP (0.5 mg/mL) with AOT (10 mM) in acetate buffer containing CaCl2 (8 mM) at pH of 3.0 followed by extraction with isooctane allowed for an extraction efficiency of 52%. Further decreasing of pH was not attempted due to the risk of denaturing the enzyme.14 The extraction efficiency was then improved by changing the concentration of AOT. An extraction efficiency of 94% was achieved by use of 20 mM AOT, while only 20% efficiency was obtained with 5 mM AOT. To avoid high concentrations of AOT in the final ion-paired enzyme system, 15 mM AOT was used in the standard procedure giving an extraction efficiency of 80%. We found that HRP could also be enriched by sequential extractions at different pH: extraction at pH of 4.4 gave an organic phase containing only other proteins, determined by SDS PAGE, and further extraction at pH of 3.0 allowed for the enrichment of the ion-paired HRP in isooctane. To investigate our ion-paired HRP system, the concentrations of water, AOT, and HRP in isooctane were determined by Karl Fischer titration, Rhodamine 6G dye assay, and UV absorption at 402 nm, respectively. Such a system was found to contain 42 mM water, 9.4 mM AOT, and 3.2 µM HRP, whereas pure isooctane contained only 0.3 mM water. A similar extraction without the use of HRP gave the isooctane phase with 43 mM water and 10 mM AOT. These data suggested the existence of reversed micelles in the HRP-containing organic phase. As water to surfactant molar ratio W0 in this system is about 4.2, the radius of the micelle can be estimated as r ) 0.15 × 4.2 ) 0.63 nm.15 This size is far too small to take up HRP, since a radius of 2.6 nm is needed for the encapsulation of HPR.15 Therefore, HRP is not incorporated into the reversed micelle but dissolved in isooctane through ion-paired form. Ethyl acetate is known to disrupt the structure of reversed micelles,12 and a similar extraction experiment with isooctane containing 10% (v/v) ethyl acetate gave the same amount of HRP in organic phase. This suggested again that the reversed micelles formed in the absence of ethyl acetate did not encapsulate HRP. On the basis of these findings, a schematic diagram of ion-paired HRP in isooctane prepared by our method is proposed in Scheme 1. Some AOT molecules are bound to enzyme by ion-pairing, while some AOT and water molecules form reversed micelles containing no
10.1021/ma050082h CCC: $30.25 © 2005 American Chemical Society Published on Web 06/29/2005
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Scheme 1. Polymerization of 4-Hexyloxyphenol with Ion-Paired HRP and tBuOOH in Isooctane
Figure 1. UV spectra of mixtures of ion-paired HRP (100 µg/ mL), tBuOOH, and 4-hexyloxyphenol in isooctane. (A) HRP, no substrate, no tBuOOH. (B) HRP, no substrate, 10 min after the addition of tBuOOH (2.5 mM). (C) HRP, 4-hexyloxyphenol (1 mM), no tBuOOH. (D) HRP, 4-hexyloxyphenol (1 mM), 40 min after the addition of tBuOOH (2.5 mM).
Table 1. Polymerization of 4-Hexyloxyphenol with and Ion-Paired HRP (100 µg/mL, 2.27 µM) in Isooctane for 17 h
tBuOOH
substrate (mM)
tBuOOH
(mM)
substrate converted (mM)
turnover numbera (µmol/µmol)
10 10 10 10 2 5 8
10 15 20 30 10 10 10
3.7 6.8 7.1 7.2 1.8 4.6 6.2
1600 3000 3100 3200 790 2000 2700
a
µmol substrate converted/µmol ion-paired HRP.
HRP. The total water amount in such ion-paired HRP system is only 0.08% v/v, which provides a rather anhydrous environment. b. Polymerization of 4-Hexyloxyphenol with IonPaired HRP in Organic Solvent and with tertButyl Hydroperoxide as Oxidant. 4-Hexyloxyphenol was chosen as substrate for the enzymatic polymerization (Scheme 1). The long alkyl chain in the substrate provides good solubility in isooctane and could give rise to better thermoplastic properties of the resulting polymer. Thus far, hydrogen peroxide is the only used oxidant for the polymerization of phenols in aqueous, mixed solvent, two-phase, and reversed micelle systems. Since our system contains only 0.08% v/v water and the ion-paired HRP is dissolved in isooctane, the watersoluble H2O2 is not the ideal oxidant. On the other hand, tert-butyl hydroperoxide is soluble in isooctane, thus being examined as the oxidant for ion-paired HRPcatalyzed polymerization. The polymerization of 4-hexyloxyphenol was successfully performed in isooctane containing ion-paired HRP and tBuOOH. The enzyme concentration was fixed at 100 µg/mL, and different concentrations of the substrate and the oxidant were examined for the optimal polymerization. The reaction was followed by UV absorption at 250 nm as well as by HPLC analysis. The results after 17 h polymerization are given in Table 1. A turnover number of ca. 3000 µmol substrate converted/ µmol HRP was achieved. Good results are obtained when the ratio of substrate and tBuOOH is between
1:1.2 and 1:3.0. Further increase of the amount of oxidant did not contribute significantly to the enzymatic reaction. It was known that excess hydrogen peroxide impaired the catalytic activity by decomposing the heme and modifying the apo-HRP structure.16 For efficient polymerization, hydrogen peroxide had to be added in small portions at different time points.6 To examine the possible inhibition of enzyme by excess tBuOOH, polymerization of 5 mM substrate was carried out with 25 µg/mL (0.57 µM) enzyme and 50 mM tBuOOH for 17 h. The oxidant was added in one portion at the beginning in one case and in several small portions at different time points in another case. No difference in the polymerization was observed between the two cases, and 2.8 mM substrate was converted to the product corresponding to a turnover number of 5000 µmol substrate converted/µmol HRP. These results indicated that the organic peroxide tBuOOH has less poisoning effect than hydrogen peroxide on the oxidative polymerization. The poisoning effect of tBuOOH on ion-paired HRP itself was investigated by UV spectroscopy. As shown in Figure 1, incubation of ion-paired HRP (100 µg/mL) with 2.5 mM tBuOOH for 10 min resulted in 80% decrease of UV absorption at 402 nm, indicating the decomposition of the heme. However, a similar experiment in the presence of 1 mM 4-hexyloxyphenol for 40 min gave nearly no change in the UV absorption at 402 nm. This suggested that the poisoning effect of tBuOOH on the enzyme can be efficiently suppressed by the presence of substrate. c. Preparative Polymerization of 4-Hexyloxyphenol. Polymerization of 7.4 mM 4-hexyloxyphenol was carried out on a 36 mL scale with 20 mM tBuOOH and 100 µg/mL ion-paired HRP for 20 h. Substrate was totally converted, and the turnover number for HRP amounted to 3300. The average specific activity within the first 2 h reached 0.25 U/mg protein (U ) µmol/min). The reaction mixture was concentrated at 70 °C under vacuum and subjected to purification. A general problem in the polymerization with AOT-containing systems is the removal of AOT after the reaction. We found that this problem can be easily solved by use of ion-exchange resin Dowex 1X8: while AOT was absorbed on to the resin, the polymer product was not. After purification with Dowex 1X8, 32.6 mg (63%) of product was obtained. The product was characterized by GPC, IR, 1H NMR, 13C NMR, and UV. GPC analysis showed two peaks with
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Scheme 2. Postulated Quinonoid Structures in the Polymerization Producta
v/v water, providing good solubility for hydrophobic phenols and the corresponding oligomers/polymers. Application of this system gives rise to the first example of polymerization of phenol with ion-paired HRP in an organic solvent, with a turnover number of 5000 µmol substrate converted/µmol HRP. For the first time, tertbutyl hydroperoxide has been used as the oxidant for HRP-catalyzed polymerization of phenol, and it has proven to be less toxic and rather efficient for the enzymatic reaction. Different from HRP-catalyzed polymerizations in other systems, polymerization of 4-hexyloxyphenol with ion-paired HRP in organic solvent has resulted in a product mainly containing quinonoid segments.
a Such segments either may be presented as end group or may be presented within the chain.
Acknowledgment. We thank Dr. P. Neuenschwander and Ms. G. Marti at ETH Zurich for the help in GPC measurement and Karl Fischer titration, respectively.
a molecular weight of 1100 and 650. This suggests that the product is a mixture of oligomers mainly consisting of 3 and 6 monomer units. This result is comparable with that using HRP in the reversed micelle system.8,9 Although the enzymatic generation of the radicals in our system is rather efficient, the radical polymerization in isooctane may not be efficient since radicals may transfer to isooctane itself. Thus, using other organic solvents such as n-octane might result in a higher molecular weight of the final product. In the IR spectra, the absorption of the OH group at 3338 cm-1 nearly disappeared, indicating that the poly(phenylene) structure is not the major one in the product; the existence of new absorptions at 1682 and 1654 cm-1 suggested the formation of quinonoid structures (Scheme 2), and the absence of a characteristic band for aromatic ether (Ar-O-Ar) around 1270-1280 cm-1 excludes the formation of oxo-coupled product. In the 13C NMR spectrum, two signals at 188 and 182 ppm were observed which correspond to the keto carbons of the quinonoids; comparing with monomer, no new signals were found between 100 and 0 ppm, which excludes the possibility of forming a Pumerer’s ketone through β-addition.9 In the 1H NMR spectrum, the absorptions at 5.6-5.8 and 7.2-7.4 ppm are assigned to the olefin proton of the quinonoid segments. Thus, the polymerization product contains mainly the quinonoid segments. This is different from the polymerization with HPR in other systems such as water/dioxane mixture and reversed micelles. The electrically conductive and fluorescent properties of the new type of conjugated aromatic oligomer are currently under investigation. Conclusion. An enzymatic system containing ionpaired HRP in isooctane has been prepared by a modified procedure. This system contains less than 0.1%
Supporting Information Available: Experimental details; IR, 1H NMR, and 13C NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
References and Notes (1) Gross, R. A.; Kumar, A.; Kalra, B. Chem. Rev. 2001, 101, 2097. (2) Kobayashi, S.; Uyama, H.; Kimura, S. Chem. Rev. 2001, 101, 3793. (3) Akkara, J. A.; Ayyagari, M. S. R.; Bruno, F. F. Trends Biotechnol. 1999, 17, 67. (4) Dordick, J. S.; Marletta, M. A.; Klibanov, A. M. Biotechnol. Bioeng. 1987, 30, 31. (5) Akkara, J. A.; Senecal, K. J.; Kaplan, D. L. J. Polym. Sci., Part A: Polym. Chem. 1991, 29, 1561. (6) Ikeda, R.; Sugihara, J.; Uyama, H.; Kobayashi, S. Macromolecules 1996, 29, 8702. (7) Ayyagari, M.; Akkara, J. A.; Kaplan, D. L. Acta Polym. 1996, 47, 193. (8) Ayyagari, M. S.; Marx, K. A.; Tripathy, S. K.; Akkara, J. A.; Kaplan, D. L. Macromolecules 1995, 28, 5192. (9) Premachandran, R. S.; Banerjee, S.; Wu, X. K.; John, V. T.; McPherson, G. L.; Akkara, J.; Ayyagari, M.; Kaplan, D. Macromolecules 1996, 29, 6452. (10) Bruno, F. F.; Akkara, J. A.; Samuelson, L. A.; Kaplan, D. L.; Mandal, B. K.; Marx, K. A.; Kumar, J.; Tripathy, S. K. Langmuir 1995, 11, 889. (11) Dai L.; Klibanov, A. M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 9475. (12) Paradkar, V. M.; Dordick, J. S. Biotechnol. Bioeng. 1994, 43, 529. (13) Bindhu, L. V.; Abraham, T. E. Biochem. Eng. J. 2003, 15, 47. (14) Chattopadhyay, K.; Mazumdar, S. Biochemistry 2000, 39, 263. (15) Motlekar, N. A.; Bhagwat, S. S. J. Chem. Technol. Biotechnol. 2001, 76, 643. (16) Akita, M.; Tsutsumi, D.; Kobayashi, M.; Kise, H. Biosci. Biotechnol. Biochem. 2001, 65, 1581.
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