Horseradish Peroxidase Mediated Free Radical Polymerization of Methyl Methacrylate Bhanu Kalra and Richard A. Gross* Polytechnic University, NSF Center for Biocatalysis and Bioprocessing, Six Metrotech Center, Brooklyn, New York 11201 Received June 15, 2000; Revised Manuscript Received July 7, 2000 This paper reports the free radical polymerization of methyl methacrylate (MMA) catalyzed by horseradish peroxidase (HRP). A novel method was developed whereby MMA polymerization can be carried out at ambient temperatures in the presence of low concentrations of hydrogen peroxide and 2,4-pentanedione in a mixture of water and a water-miscible solvent. Polymers of MMA formed were highly stereoregular with predominantly syndiotactic sequences (syn-dyad fractions from 0.82 to 0.87). Analyses of the chloroform- soluble fraction of syndio-PMMA products by GPC showed that they have number-average molecular weights, M n , that range from 7500 to 75 000. By using 25% v/v of the cosolvents dioxane, tetrahydrofuran, acetone, and dimethylformamide, 85, 45, 7 and 2% product yields, respectively, resulted after 24 h. Increasing the proportion of dioxane to water from 1:3 to 1:1 and 3:1 resulted in a decrease in polymer yield from 45 to 38 and 7%, respectively. Increase in the enzyme concentration from 70 to 80 and 90 mg/mL resulted in increased reaction kinetics. By adjustment of the molar ratio of 2,4-pentanedione to hydrogen peroxide between 1.30:1.0 and 1.45:1.0, the product yields and M n values were increased. On the basis of the catalytic properties of HRP and studies herein, we believe that the keto-enoxy radicals from 2,4-pentanedione are the first radical species generated. Then, initiation may take place through this radical or by the radical transfer to another molecule. Introduction Enzymes have proven to be powerful catalysts for the polymerization of a wide variety of monomers and mac- romonomers. 1 Furthermore, enzymes represent a family of environmentally friendly catalysts. Though enzyme catalysis has been known for well over a century, 2 it has been largely restricted to aqueous systems until the relatively recent development of nonaqueous enzymology. 3-5 Nonaqueous enzymology has had a powerful impact on organic synthetic methods. In some cases, nonaqueous 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 the following: (i) the lipase-catalyzed synthesis of optically active polyesters, 6 (ii) lipase-catalyzed interesterification of triglycerides and fatty acids, 7 (iii) the regioselective oxidation of phenols by phenol oxidase, 8 (iv) peroxidase-mediated reactions that yield poly- mers with useful electrooptical properties such as polyphe- nols and polyaromatic amines, 9,10 (v) and the modification of polysaccharides to enhance their processability and to regulate their biodegradability. 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 p-aminoben- zoate. 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, 3-5,8-10,15-19 and interfacial systems. 20 The potential of using HRP and other oxidases to catalyze the free radical polymerization of vinyl monomers was first reported by Derango et al. 21 The polymers were formed in the presence of a large excess of oxidant (monomer:oxidant, 1.66:1.0 v/v, for, e.g., 1.41:1.0 mol/mol for 2-hydroxyethyl methacrylate). Unfortunately, these workers gave only qualitative descriptors to describe whether a polymer was formed without further information on the polymer structure. Kobayashi and co-workers 22 reported the HRP-catalyzed polymerization of phenylethyl methacrylate. Similar to Derango et al., 21 Kobayashi and co-workers 22 also published the formation of polymer using large quantities of oxidant (equimolar with respect to the monomer). More recently, HRP-mediated free radical polymerization of acrylamide in water was reported. 23,24 These polymerizations took place when -diketones were used as initiators and the molar ratio of hydrogen peroxide to monomer was 1 to 66. 23,24 That -diketones would react under such conditions is related to their weakly bonded R-hydrogens. Indeed, it had previously been shown that cyclic -ketones, such as 5,5-dimethyl-1,3- cyclohexanedione, are substrates for chloroperoxidases which belong to the same subclass of enzymes as HRP (E.C. 1.11.a and 1.11.1.7, respectively). 25 By analogy to phenol, it was assumed that the enolic tautomeric form of 2,4-pentanedione is a key intermediate in the catalytic pathway. One objective of this work was to explore the potential of enzymes to regulate the stereochemical configuration of * Corresponding author. 501 Biomacromolecules 2000, 1, 501-505 10.1021/bm005576v CCC: $19.00 © 2000 American Chemical Society Published on Web 08/03/2000