Enzymatic Template Synthesis of Polyphenol

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approach to the polymerization of phenol in the presence of several polyelectrolyte templates. The synthesis, characterization and properties of these new polyphenolic complexes will be discussed. Experimental Section Horseradish peroxidase (EC 1.11.1.7) was purchased from Sigma Chemical Co. (St. Louis, MO) as a salt free powder. The specific activity was of 240 purpurogallin units/mg solid. Phenol, polystyrene sulfonated (SPS, Mw 1.0M and 70K g/mol), dodecyl benzene sulfonate (DBSA), hydrogen peroxide (30% solution), phosphate buffer, and all the solvents (reagent grade or better) were purchased from Aldrich (Milwaukee, WI). Lignin sulfonate (Lignosol SFX-65) was obtained from Lignotech USA (Rothschild, WI). Enzymatic polymerization was carried out in a 10 mL of aqueous phosphate buffer (10 mM, pH 7.0), phenol (71.3 mM) and equimolar concentrations (with respect to the phenol) of SPS (based on the repeat unit) and

hydrogen peroxide (no more than 20 mM added every 5 minutes). The HRP concentration was 0.1-0.15 mg/mL and was added prior to the hydrogen peroxide. The reactions were carried out for 30 minutes at room temperature and the final products were dialyzed using Centricon concentrators (10,000 cut off, Amicon Inc., Beverly, MA). The samples were dried under vacuum at 50'C and stored until further analysis. The percent yield was typically 95% or higher. Control samples, using denatured enzyme, were prepared following the same procedure. Leaving in buffered water at 100°C for 30 minutes denatured the enzyme. The denatured HRP was then tested using purpurogallin, and was found to be inactive. Spectral characterization of the polymers and controls was performed with a Perkin-Elmer Lambda-9-UV-Vis-Near IR spectra-photometer (Norwalk, CT). FTIR spectra, of the samples deposited on ZnSe, were collected on a Perkin Elmer FTIR 1720X. Thermal gravimetric analysis (TGA) and differential scanning calorimetry (DSC) analysis were conducted using a TA instrument 2950 and a DSC instrument 2910 (New Castle, DE) respectively. The TGA and the DSC measurements were carried out under nitrogen at a rate of 10°C/min. Static light scattering (SLS) measurements were performed on SPS, SPS-Phenol (control), SPS-Polyphenol polymer and DBSA-polyphenol using a Brookhaven instrument (Model SG-7B Rigaku Denki, Japan). A stock solution of SPS was prepared by dissolving an appropriate amount of polymer in 0.01 M of a filtered, pH 7.0 sodium phosphate buffered

solution. The stock solution was sonicated for one hour at 25°C and then re-dialyzed for 24 hours. 0.1M of NaCl was added to the solution and filtered multiple times using a 0.45 Rim filter. Similar procedures were followed for SPS-Phenol prior to and after polymerization. However, SPS-Polyphenol was dissolved in a 50:50 DMSO/water solvent mixture. A series of concentrations required for the measurements was prepared by diluting the stock solution with filtered buffer solution added directly into the scattering cell. Similar procedures were used for the DBSA/polyphenol system. SLS mea