Polymerization of 4´-Hydroxyphenyl α-Glucoside and 4´-Hydroxyphenyl β-Glucoside Catalyzed by Horseradish Peroxidase

Abstract

Polymerization processes of 4´-hydroxyphenyl α-glucoside (α-Arb) and 4´-hydroxyphenyl β-glucoside (arbutin, β-Arb) catalyzed by horseradish peroxidase (HRP) were analyzed in detail. A calculated model based on the coupling of the free radicals formed from the substrates with every degree of polymerization reproduced successfully most of the experimental results: An increase of the polymer with a decrease of the monomer and a temporal accumulation of the dimer responding to the addition of H₂O₂. Reaction controlled at neutral pH improved the polymer yields by preventing partial hydrolysis of the glucose residues. A sequential addition of divided portions of H₂O₂ also increased the yields considerably compared to the single addition, although the yields with too low enzyme activities could not be restored. The main chain structure of the α-Arb polymer was the same as that of the β-Arb polymer which had linkages at the 3´- and 5´-positions of hydroquinone moieties. The polymers showed molecular weight distribution at 1-25 kDa in gel permeation chromatography, when maltooligosaccharides and pullulan were used as standards. Mass spectrometric analysis of deglycosylated polymers suggested that the α-Arb polymer contained smaller components more than the β-Arb polymer. Peroxidases from horseradish, soybean and Arthromyces polymerized β-Arb faster than α-Arb. HRP polymerized the two glucosides faster than the other two peroxidases. Oxidation potentials were determined to be 821 and 835 mV for α-Arb and β-Arb, respectively, by cyclic voltammetry, suggesting almost the same electrochemical reactivity. A higher reactivity of β-Arb in the enzymatic polymerization was thus attributed not to electrochemical reactivity of the substrates, but to stereoselectivity of the enzymes.