Chapter 24
Peroxidase-Mediated Free Radical Polymerization of Vinyl Monomers B. Kalra and R. A. Gross* NSF-I/UCRC for Biocatalysis and Bioprocessing of Macromolecules, Polytechnic University, 6 Metrotech Center, Brooklyn, NY 11201
Peroxidase-mediated polymerizations of methyl methacrylate, acrylamide, and sodium acrylate were studied. Horseradish peroxidase (HRP), hydrogen peroxide, and 2,4-pentanedione functioned as the oxido-reductase, oxidant, and reducing substrate, respectively, for the free radical polymerizations. The polymerizations were conducted in solution (aqueous and water/co-solvent mixtures) and emulsions (oil/water with and without surfactant, concentrated). The poly(acrylamide)s and poly(acrylate)s formed were atactic. However, poly(methylmethacrylate) with high syndiotactic was prepared as a result of the ability of enzymes to function effectively at ambient temperature and below.
© 2003 American Chemical Society
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Introduction The ability of enzymes to provide an advantage over traditional catalysts for monomer and polymer synthesis is gaining in recognition (7). Although enzyme catalysis has been known for well over a century (2), there use has been largely restricted to aqueous systems. However, over the past 15 years, non-aqueous enzymology has continued to grow in importance (3-5) In some cases, non aqueous enzyme-catalysis has provided synthetic routes to chemical transformations that are difficult, or even impossible, with conventional chemical catalysts. Important examples of reactions catalyzed by enzymes in organic media include: i) the lipase-catalyzed synthesis of optically active polyesters (6), ii) lipase-catalyzed interesterification of triglycerides and fatty acids (7), «0 the regioselective oxidation of phenols by phenol oxidase (8), iv) peroxidasemediated reactions that yield polymers with useful electro-optical properties such as polyphenols and polyaromatic amines (9JO), and polysaccharide modification to regulate their properties (11). Horseradish peroxidase (HRP) is an oxido-reductase that acts on hydrogen peroxide and/or alkyl peroxide as an oxidant (12) and on several reducing substrates such as phenol, hydroquinone, pyrogallol, catechol, aniline and paminobenzoate (13). The oxidative coupling of a variety of substrates such as phenols and aromatic amines catalyzed by HRP in the presence of hydrogen peroxide have been reported in aqueous (14), nonaqueous (5-5, 8-10, 15-19) and interfacial systems (20). Recently, HRP has been used to catalyzefree-radicalpolymerizations of commodity vinyl monomers. The potential of such reactions with vinyl monomers including methyl methacrylate, acrylamide, 2-hydroxyethyl methacrylate, and acrylic acid was first recognized and reported by Derango et al. (21). In a related study, Kobayashi and coworkers (22) reported the HRPcatalyzed polymerization of phenylethyl methacrylate. Later, HRP-mediated free-radical polymerizaton of acrylamide was shown to take place in the presence of β-ketones as initiators (23,24). Our group investigated HRPmediated methyl methacrylalte polymerization in a mixture of water and watermiscible solvents at ambient temperature (25, 26). This chapter provides an overview of recent contributions by our laboratory towards enzyme-mediated vinyl monomer polymerizations. These studies addressed how various reaction parameters can be adjusted to regulate: i) the HRP-mediated polymerization of water-soluble acrylamide and sodium acrylate in aqueous medium (27), if) polymerizations of water-insoluble methyl methacrylate, and Hi) polymerizations of acrylamide in concentrated emulsions (27). In addition, we have carried out experiments to probe whether the mechanism of the polymerization involved a direct electron transferfromthe oxoiron(IV) π-radical cation to monomer, or whether chain initiation takes place through the radical species generated by 2,4-pentanedione.
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Experimental
Materials. Horseradish peroxidase (Type II, activity 235 purpullogallin units/mg), Horserdish peroxidase (Type I, activity 100 purpullogallin units/mg), Soybean peroxidase (90 purpullogallin units/mg), Arythomyces ramosus peroxidase [(75 purpullogallin units/mg), Acrylamide, sodium acrylate, dioctyl sulfosuccinate sodium salt, cetyl trimethyl ammonium bromide, sorbitane monooleate, hydrogen peroxide (30% (wt/vol)] were all obtained from the Sigma Chemical Company. Methanol, dioxane, acetone, tetrahydrofiiran and dimethyl formamide were all of analytical grade and were used as received. Methyl methacrylate (MMA), obtained from the Aldrich Chemical Co., wasfractionallydistilled over calcium hydride under reduced pressure with a nitrogen atmosphere. 2,4Pentanedione from Aldrich was distilled prior to use.
Instrumentation The NMR data were recorded on a Bruker DPX300 and Bruker AMX500. The chemical shifts in parts-per-million (ppm) for proton ( H) NMR spectra were referenced relative to tetramethylsilane (TMS, O.OOppm) as the internal reference. The stereochemistry of the polymer backbone was calculated by observing the NMR signals (28) due to the backbone methyl groups: syndiotactic triad, 0.81 ppm; atactic triad, 0.97 ppm; isotactic triad, 1.14 ppm. The standard deviation for the syn dyadfractionswas calculated by taking the mean for three replicates of the NMR integration values. The distribution of repeat unit sequences that differ in stereochemistry was analyzed for poly(acrylamide) by observing the NMR signals due to the methine carbon region in the C-NMR. The number average molecular weights (M ) of the polymer samples (PMMA) were determined by gel permeation chromatography (GPC) using a Waters HPLC system equipped with model 510 pump, Waters model 717 autosampler, model 410 refractive index detector, and model T-50/T-60 detector from Viscotek Corporation with 500, ΙΟ , 10 and 10 Â ultrastyragel columns in series. Trisec GPC software Version 3 was used for calculations. Chloroform was used as the eluent at a flow rate of 1.0 mL/min. Sample concentrations of 0.2 percent wt/vol and injection volumes of 100 μΐ, were used. Molecular weights were determined based on conventional calibration curve generated by polystyrene standards of low dispersity (Aldrich Chemical Company). Since the [
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300 PMMAs formed in this work were not completely soluble in chloroform, the molecular weight values reported are relevant to only part of the product. The weight average molecular weights (M ) of the poly(acrylamide) and poly(sodium acrylate) were determined by gel permeation chromatography (GPC). Studies by GPC were carried out using a Waters, Inc. Model 510 pump, two Shodex KB 80m and one Shodex K B 802.5 columns, and a waters 410 differential refraetometer. The software used for molecular weight calculations was millennium chromatography manager version 2.15. Sodium dihydrogen phosphate, 20 mM, pH 7.0, was used as the eluent. Analyses were carried out at 35 °C, flow rate 1 mL/min and with injection volumes of 10 \\L. Polyethylene glycol standards with narrow polydispersity were used to generate a calibration curve. Differential Scanning Calorimetry (DSC) was performed on TA instrumental model 2920 DSC. The temperature program used was to increase the sample temperaturefromroom temperature to 150 °C, cool to 30 °C and then to reheat to 150 °C. The heating rate was 20 °C min' and the purge gas was helium. The cooling was performed using TA instruments refrigerated cooling systems using an equilibration step that gives rapid cooling. The glass transition temperature (T ) was determined by the midpoint at half-height. The T results were takenfromthe second heating scan. w
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Enzyme-catalyzed polymerization of MMA in binary solvents Methyl methacrylate (MMA) (5.6 mmol) was added to a solution of distilled water (0.7 mL) and organic solvent (0.3 mL) in a dual inlet ampule under nitrogen atmosphere. An example of a typical reaction is the successive addition under a nitrogen atmosphere of 0.2 mL of HRP (80 mg/mL, 16 mg of enzyme), hydrogen peroxide (0.092 mmol) and 2,4-pentanedione (0.136 mmol). The reaction mixture was maintained under nitrogen with stirring at room temperature for a predetermined time period. Then, the reaction mixture was poured into a large excess of methanol. The precipitate obtained was separated by filtration, washed with methanol and dried (in vaccuo, 50 °C, 30 mm Hg, 24 hours).
Polymerization of acrylamide in aqueous medium Acrylamide (2.92 mmol in 4 mL water) in a dual inlet ampule was purged with nitrogen for 10 min. Into the above solution, HRP (8 mg in 0.2 mL water), hydrogen peroxide (0.046 mmol) and 2,4-pentanedione (0.068 mmol) were successively injected while stirring. The reactions were carried out at room
301 temperature for a predetermined time period while maintaining both stirring and a nitrogen atmosphere. After the predetermined time, the reaction mixture was poured into an excess of methanol. The resulting precipitate was filtered off, washed with methanol and dried in vacuo (50 °C, 30mm Hg, 24 h). The enzyme was soluble in methanol and thus removedfromthe polymer.
Polymerization of acrylamide in concentrated emulsion Toluene (42 pL) and sorbitane monooleate (17.2 μΙ,) were degassed in a dual inlet ampule for 10 min. Acrylamide (2.92 mmol) dissolved in water (0.45 mL) was added to the surfactant solution with stirring. To the above was added HRP (8 mg in 0.3 mL water), hydrogen peroxide (0.046 mmol) and 2,4pentanedione (0.068 mmol) while vigorously stirring. The polymerization was carried out under a nitrogen stream for different time periods. The polyacrylamide was isolated by precipitation in methanol and then dried in a vacuum oven (50 °C, 30mm Hg, 24 h).
Polymerization of sodium acrylate in aqueous medium Sodium acrylate (4 mmol) was added to a solution of distilled water (1.5 mL) in a dual inlet ampule under nitrogen atmosphere. An example of a typical reaction is the successive addition under a nitrogen atmosphere of HRP (11.4 mg in 0.5 mL water), hydrogen peroxide (0.064 mmol) and 2,4-pentanedione (0.097 mmol). The reaction mixture was maintained under nitrogen with stirring at room temperature for a predetermined time period. Then, the reaction mixture was poured into a large excess of methanol. The precipitate obtained was separated byfiltration,washed with methanol and dried (in vacuo, 50 °C, 30 mm Hg, 24 h).
Results and Discussion HRP-mediated polymerizations were conducted with 2,4-pentanedione as the reducing substrate and hydrogen peroxide as the oxidant (stoichiometric ratio 1.5:1). The acrylate to 2,4-pentanedione ratio was maintained at about 42-44:1. M M A polymerization was conducted in a mixture of water and a water-miscible co-solvent so that MMA is solubilized. Co-solvents (dioxane, acetone, THF and DMF) were all useful for the HRP-mediated polymerization of MMA.
302 Effect of co-solvents The room-temperature polymerization of MMA was catalyzed by HRP in the presence of hydrogen peroxide and 2,4-pentanedione in binary solvent mixtures consisting of water and different co-solvents. The polymer formed, based on analyses by high field proton (*H) NMR, was predominantly syndiotactic. When the binary mixture was water:DMF (3:1), the polymerization was very slow (
