January/February 2004
Published by the American Chemical Society
Volume 5, Number 1
© Copyright 2004 by the American Chemical Society
Communications Horseradish Peroxidase-Catalyzed Polymerization of Cardanol in the Presence of Redox Mediators Keehoon Won, Yong Hwan Kim,* Eun Suk An, Yeon Soo Lee, and Bong Keun Song Korea Research Institute of Chemical Technology, 100 Jang-Dong, Yuseong-Gu, Daejeon 305-343, South Korea Received August 30, 2003; Revised Manuscript Received October 16, 2003
Horseradish peroxidase-catalyzed polymerization of cardanol in aqueous organic solvent was investigated in the presence of a redox mediator. Cardanol is a phenol derivative from a renewable resource mainly having a C15 unsaturated hydrocarbon chain with mostly 1-3 double bonds at a meta position. Unlike soybean peroxidase (SBP), it has been shown that horseradish peroxidase (HRP) is not able to perform oxidative polymerization of phenol derivatives having a bulky meta substituent such as cardanol. For the first time, redox mediators have been applied to enable horseradish peroxidase to polymerize cardanol. Veratryl alcohol, N-ethyl phenothiazine, and phenothiazine-10-propionic acid were tested as a mediator. It is surprising that the horseradish peroxidase-catalyzed polymerization of cardanol took place in the presence of N-ethyl phenothiazine or phenothiazine-10-propionic acid. However, veratryl alcohol showed no effect. FT-IR and GPC analysis of the product revealed that the structure and properties of polycardanol formed by HRP with a mediator were similar to those by SBP. This is the first work to apply a redox mediator to enzyme-catalyzed oxidative polymerization. Our new finding that oxidative polymerization of a poor substrate, which the enzyme is not active with, can take place in the presence of an appropriate mediator will present more opportunities for the application of enzyme-catalyzed polymerization. Recently, there has been strong demand for the synthesis of polymers from renewable resources instead of petroleumbased raw materials. This contributes to promote global sustainability without depletion of scarce resources. Cashew nut shell liquid (CNSL) is a side-product from mechanical processing (hot-bath process) for the edible use of the cashew nut of Anacardium occidentale. Every year a huge amount of CNSL is formed, as CNSL is nearly a third of the total nut weight. Cardanol, a main component of CNSL, is a phenol derivative having a meta substituent of a C15 unsaturated hydrocarbon chain mainly with 1-3 double bonds.1 Cardanol may be used as antioxidants and stabilizers and has potential utilization in various ways such as resins, * To whom correspondence
[email protected].
should
be
addressed.
E-mail:
friction lining materials, and surface coatings. Phenolic resins from cardanol and formaldehyde are industrially produced as the prepolymer of coating materials with high gloss surface mainly for indoor use.2 Recently, we have reported a new application of polycardanol.3 Polymerization catalyzed by enzymes has drawn much attention as a new methodology of polymer syntheses. It is expected that enzymatic polymerization provides new polymeric materials, which are difficult to be obtained by conventional methods. Peroxidase is famous for inducing the oxidative polymerization of phenol derivatives under mild reaction conditions.4-6 Major advantages of the enzymatic polymerization of phenol derivatives are as follows: (i) enzyme-catalyzed polymerization of phenols proceeds under mild conditions without the use of toxic reagents such as formaldehyde; (ii) enzymes catalyze the polymerization of
10.1021/bm034325u CCC: $27.50 © 2004 American Chemical Society Published on Web 11/21/2003
2
Biomacromolecules, Vol. 5, No. 1, 2004
a variety of phenol monomers; (iii) phenol derivatives possessing more than two reactive groups can be chemoselectively polymerized; (iv) the structure and solubility of the polymer can be controlled by changing the reaction conditions.7,8 For oxidative polymerization of cardanol, soybean peroxidase (SBP) was successfully used in aqueous organic solvents. However, horseradish peroxidase (HRP) was not able to catalyze cardanol polymerization under the same conditions.3,9 Interestingly, it was found that HRP did not polymerize cardanol, although it showed the higher peroxidase activity than SBP throughout the reaction period. Excessive HRP failed in cardanol polymerization.3 Our preliminary experiments also showed that HRP was not able to polymerize cardanol even though we tried various organic solvents including dioxane as a reaction medium (data not shown). The reason HRP is not able to polymerize cardanol unlike SBP is not obvious but may be explained by the difference of substrate selectivity between HRP and SBP. Kobayashi’s group showed that HRP had a high catalytic activity toward phenols with a small meta-substituent, whereas SBP had a preference for those with a large one. The polymer formation was not observed in the HRPcatalyzed polymerization of m-isopropyl and m-tert-butylphenols.10 Therefore, it is generally accepted that HRP is not able to polymerize phenols with a bulky group at the meta position. It is well-known that a low-molecular-weight compound called a mediator can mediate oxidation reaction between a substrate and an enzyme and thus enhance the reaction rate. For example, lignin peroxidase alone is not practically able to oxidize 4-methoxybenzyl alcohol and 2-hydroxy-2-(4methoxyphenyl)acetic acid, but these oxidations take place with good efficiency in the presence of veratryl alcohol.11 Redox mediators also allowed laccases to oxidize nonphenolic compounds, thereby vastly expanding the range of substrates.12 In this work, some redox mediators have been introduced in order to perform HRP-catalyzed polymerization of cardanol, which has been shown to be impossible. To the best of our knowledge, this is the first study to apply mediators to oxidative polymerization catalyzed by enzymes. Cardanol polymerization by HRP was carried out using hydrogen peroxide in 2-propanol/phosphate buffer (pH 7) (50:50 vol %) at room temperature under air.13 N-ethyl phenothiazine (EP), phenothiazine-10-propionic acid (PPA), and veratryl alcohol (VA) were used as a redox mediator. In Figure 1, the reaction scheme and molecular structures of the used mediators are shown. PPA was synthesized from phenothiazine, because it was not commercially available unlike EP and VA.14 Phenothiazine-10-propionic acid has been shown to be an excellent mediator, which shows very good performance in the dye-transfer inhibition system and in the decolorization process.15,16 Veratryl alcohol, a secondary metabolite of the fungus P. chrysosporium, is well-known to enhance lignin peroxidase-catalyzed oxidation of poor substrates.11,17 In Figure 2 is depicted the effect of the added mediators on the polymer yield. As shown in our previous work, HRP-
Communications
Figure 1. Reaction scheme of cardanol polymerization catalyzed by peroxidase. Redox mediators: (A) N-ethyl phenothiazine, (B) phenothiazine-10-propionic acid, and (C) veratryl alcohol.
Figure 2. Dependence of the polycardanol yield on the mediator concentration. (b) N-Ethyl phenothiazine. (9) Phenothiazine-10propionic acid.
catalyzed polymerization of cardanol did not occur at all when any mediator was not added. However, when N-ethyl phenothiazine (1000 µM) was added, oily water-insoluble polymeric materials were surprisingly formed. The polymer was purified by reprecipitation using ethyl acetate (good solvent) and methanol (poor solvent). Isolated polycardanol was viscous, dark yellow oil and the yield was as high as 40%. Similarly, polycardanol was also formed in the presence of PPA, which showed the better performance at a much lower concentration (37 µM) than EP. However, veratryl alcohol failed in inducing cardanol polymerization even at a concentration of 2000 µM. The reason polycardanol is formed in the presence of EP or PPA is not perfectly clear, but it is most likely that EP and PPA act as a mediator for electron transfer between HRP and cardanol as demonstrated by other groups (Figure 3).11,17,18 A redox mediator is oxidized by HRP, which in
Biomacromolecules, Vol. 5, No. 1, 2004 3
Communications
Figure 3. Scheme representing the proposed role of mediators for peroxidase-catalyzed oxidation of cardanol.
turn acts as a charge-transfer mediator, oxidizing a poor substrate, cardanol. Thus, the oxidized cardanol radicals start to polymerize. Redox mediators have different mediation efficiency, and two major factors to determine the efficiency were suggested in the literature: a redox potential of a mediator and interaction with enzyme.19 A redox potential of a mediator is an important factor in determining the mediation efficiency. The redox potential is measured in units of volts (V) and determined by measuring the potential difference between an inert indicator electrode in contact with the solution and a stable reference electrode connected to the solution by a salt bridge. The standard hydrogen electrode (SHE) is the reference from which all standard redox potentials are determined and has been assigned an arbitrary half cell potential of 0.0 V. In our case, redox potentials of PPA, EP, and VA are 0.71, 0.81, and 1.36 (V vs SHE), respectively.19,20 When a series of structurally related substrates were tested, it was found that the mediation efficiency first increased and then decreased on increasing the redox potential value. Baciocchi et al. explained that this trend was because a mediator must have a redox potential sufficiently low to allow its oxidation by the enzyme, but at the same time, it should be high enough in order to promote the oxidation of substrates. Even though VA can act as a good mediator for lignin peroxidase, which is more potent than HRP,21 its redox potential may be too high for HRP to oxidize. This might be why VA did not work. The other important factor is the interaction with the enzyme expressed as kinetic effectiveness (kcat/Km). Even though the oxidation potential remained almost constant, the mediation efficiency increased as the kinetic effectiveness became higher.19 As shown in Figure 2, the higher concentration of N-ethyl phenothiazine did not accompany a significant increase in yield. In the case of phenothiazine-10-propionic acid, the polymer yield seems to be slightly decreased at the higher concentration. In other reports, a decrease in the oxidation rate was observed at high mediator concentration when enzyme-catalyzed oxidation was conducted with mediators.12,16,17 This phenomenon was explained by enzyme inactivation by excessive mediator radicals formed by interaction with enzyme.16 FT-IR spectra, which were recorded on a Perkin-Elmer FT-IR 2000, confirmed the polymer structure synthesized in the presence of the mediators. In Figure 4 are shown FTIR spectra of (A) cardanol, polycardanol formed by HRP coupled with (B) N-ethyl phenothiazine, and with (C) phenothiazine-10-propionic acid. Broad peaks at 3400 cm-1 are due to the vibration of the O-H linkage of phenolic group. Figure 4, parts B and C, shows three characteristic peaks at 1239, 1189, and 1155 cm-1 ascribed to the vibrations of the C(Ar)-O-C (Ar) and/or C(Ar)-OH linkages. These
Figure 4. FT-IR spectra of (A) cardanol, polycardanol formed by HRP coupled with (B) N-ethyl phenothiazine (2000 µM) and with (C) phenothiazine-10-propionic acid (74 µM). Table 1. Oxidative Polymerization of Cardanol by Peroxidases without or with Phenothiazine-10-propionic Acid (74 µM) peroxidase
amount (mg)
mediator
yield (%)
Mn
Mw/Mn
HRP HRP SBP SBP
40 40 20 20
without with without with
0 64 48 54
3900 4200 3500
1.5 1.6 1.7
results reveal that polycardanol formed in the presence of the mediators consisted of a mixture of phenylene and oxyphenylene units. This structure is very similar to that of polycardanol formed by SBP.9 Even though cardanol has two groups subject to polymerization (phenolic moiety and unsaturated hydrocarbon group), only the phenolic moiety was polymerized during SBP-catalyzed polymerization.3,9 In Figure 4A, the spectrum of cardanol shows a characteristic peak at 3010 cm-1 due to C-H vibration of the unsaturated hydrocarbon moiety. As shown in the FT-IR spectra of Figure 4, parts B and C, peaks at 3010 cm-1 remained almost unchanged, indicating no reaction of the unsaturated groups during the polymerization. It will be very interesting to compare the molecular weight and polydispersity of polymer formed by peroxidase with mediators to those without mediators. However, it is not feasible with HRP because HRP-catalyzed polymerization does not take place without a mediator. Therefore, SBPcatalyzed polymerization of cardanol was carried out in the absence or presence of a mediator under the same conditions. Phenothiazine-10-propionic acid (74 µM), which had showed the highest efficiency of the three tested mediators for HRP, was used as a mediator for SBP. The molecular weight and polydispersity were determined by gel permeation chromatography (GPC).22 As shown in Table 1, the molecular weight and polydispersity of polycardanol by HRP with PPA were 3900 and 1.5, respectively, which were similar to those by SBP without a mediator. More interestingly, it appeared that PPA had little effect on SBP-catalyzed polymerization of cardanol. Table 1 indicates that yield, molecular weight, and polydispersity with PPA were similar to those without PPA when SBP was used as a catalyst. From this result, we can
4
Biomacromolecules, Vol. 5, No. 1, 2004
expect that mediation efficiency of a mediator depends on the peroxidase nature, and thus, an appropriate mediator should be chosen for successful applications in enzymatic polymerization. In conclusion, redox mediators have been introduced to oxidative enzymatic polymerization in this preliminary work. For the first time, we have found here that simple addition of N-ethyl phenothiazine or phenothiazine-10-propionic acid to the reaction mixture allowed HRP to be capable of cardanol polymerization, which has been known to be impossible with HRP. Furthermore, the FT-IR spectra and GPC data showed that polycardanol formed in the HRPmediator system was similar to that in the SBP system. Our new finding that oxidative polymerization of a poor substrate, which HRP is not active with, can take place in the presence of appropriate mediators will expand the applicability of enzyme-catalyzed polymerization significantly. However, to apply redox mediators to enzymatic polymerization efficiently, a more detailed picture of a peroxidase-mediator system and the rationale for mediators are needed. Further studies on redox mediators are in progress in our laboratory. Acknowledgment. The authors thank Samsung Engineering Co. and Ministry of Commerce, Industry and Energy for their partial financial support for this study. References and Notes (1) Mahanwar, P. A.; Kale, D. D. J. Appl. Polym. Sci. 1996, 61, 21072111. (2) Ikeda, R.; Tanaka, H.; Uyama, H.; Kobayashi, S. Macromol. Rapid Commun. 2000, 21, 496-499. (3) Kim, Y. H.; An, E. S.; Song, B. K.; Kim, D. S.; Chelikani, R. Biotech. Lett. 2003, 25, 1521-1524. (4) Akkara, J. A.; Ayyagari, M. S. R.; Bruno, F. F. Trends Biotechnol. 1999, 17, 67-73. (5) Dordick, J. S.; Marletta, M. A.; Klibanov, A. M. Biotechnol. Bioeng. 1987, 30, 31-36. (6) Uyama, H.; Kobayashi, S. J. Mol. Catal. B Enzymatic 2002, 19-20, 117-127. (7) Ikeda, R.; Tanaka, H.; Uyama, H.; Kobayashi, S. Polymer 2002, 43, 3475-3481.
Communications (8) Tonami, H.; Uyama, H.; Kobayashi, S. Biomacromolecules 2000, 1, 149-151. (9) Ikeda, R.; Tanaka, H.; Uyama, H.; Kobayashi, S. Polym. J. 2000, 32, 589-593. (10) Tonami, H.; Uyama, H.; Kobayashi, S.; Kubota, M. Macromol. Chem. Phys. 1999, 200, 2365-2371. (11) Harvey, P. J.; Schoemaker, H. E.; Palmer, J. M. FEBS Lett. 1986, 195, 242-246. (12) Bourbonnais, R.; Paice, M. G. FEBS Lett. 1990, 267, 99-102. (13) Unless otherwise stated, the polymerization was carried out using the following procedure: Under open air conditions, cardanol (0.6 g, 2.0 mmol) and HRP (40 mg) in 12.5 mL of 2-propanol and 12.5 mL of pH 7 phosphate buffer (0.1 M) were placed in the absence or presence of a mediator in a 50 mL flask. Hydrogen peroxide (15%, 300 µL, 1.0 mmol) was continuously added by a perfusion pump for 5 h at room temperature under mild stirring conditions. After 24 h, the reaction mixture was concentrated under a reduced pressure. Ethyl acetate (20 mL) was added to the residue, and the organic top layer was separated, followed by removal of the solvent under a reduced pressure. Methanol was added to the oily residue to remove unreacted cardanol. The methanol-insoluble material was separated by centrifugation and dried in a vacuum to give polycardanol yield. (14) The Michael reaction of phenothiazine with methyl acrylate and subsequent hydrolysis of methyl phenothiazine-10-propionate gave phenothiazine-10-propionic acid of yellow solid. 1H NMR (300 MHz, CD3OD) spectra are δ2.78 (t, 2H, J ) 7.5 Hz), 4.19 (t, 3H, J ) 7.5 Hz), 6.90-6.95 (m, 4H), 7.10-7.19 (m, 4H). 1H NMR was recorded on a 300 MHz Bruker AM-300. (15) Nakayama, T.; Amachi, T. J. Mol. Catal. B Enzymatic 1999, 6, 185198. (16) Soares, G. M. B.; Pessoa de Amorim, M. T.; Costa-Ferreira, M. J. Biotechnol. 2001, 89, 123-129. (17) ten Have, R.; de Thouars, R. G.; Swarts, H. J.; Field, J. A. Eur. J. Biochem. 1999, 265, 1008-1014. (18) Goodwin, D. C.; Grover, T. A.; Aust, S. D. Biochemistry 1997, 36, 139-147. (19) Baciocchi, E.; Gerini, M. F.; Lanzalunga, O.; Mancinelli, S. Tetrahedron 2002, 58, 8087-8093. (20) Kulys, J.; Krikstopaitis, K.; Ziemys, A. JBIC, J. Biol. Inorg. Chem. 2000, 5, 333-340. (21) Kersten, P. J.; Kalyanaraman, B.; Hammel, K. E.; Reinhammer, B.; Kirk, T. K. Biochem. J. 1990, 268, 475-480. (22) Gel permeation chromatography (GPC) analysis was carried out using Waters 2690 HPLC with a refractive index detector under the following conditions: PL4 mixed BB columns (TOSOH, Japan) and THF effluent at a flow rate of 1.0 mL/min. The calibration curves for GPC analysis were obtained using polystyrene standards.
BM034325U
