Enzymatic dehydrogenative polymerization of monolignol dimers

Abstract

The structure and biosynthesis of lignin are not yet fully understood, especially the step following the initial dimerization of monolignol. Liquid chromatograph mass spectrometer (LC-MS) was used to analyze the consumption rates of monolignol dimers formed by b-O-4, b-5, and b-b couplings between coniferyl alcohols in efforts to understand the activity of monolignol dimers in enzymatic dehydrogenative polymerization. We investigated the reaction kinetics in single-component and mixedcomponent reaction systems containing one and two species of the dimers, respectively. A difference was observed between the consumption rates of the three dimers we tested, and the consumption rate of one dimer in the singlecomponent reaction was different from that in a mixedcomponent reaction. In qualitative LC-MS analyses, coniferyl alcohol oligomers were detected in the reaction products. Some monolignol tetramers were formed by 5-5 and 5-O-4 coupling between the dimers. The results of this work suggested that monolignol dimers with b-5 and b-b linkages could function as radical mediators in enzymecatalyzed polymerization. 12.0 Hz, H-c1), 3.70 (1H, dd, J = 3.2, 12.0 Hz, H-c2), 3.81 (3H, s, 3-OCH 3 ), 3.85 (3H, s, 3 0 -OCH 3 , erythro isomer), 3.89 (3H, s, 3 0 -OCH 3 , threo isomer), 4.22 (1H, m, H-b), 4.31 (2H, dd, J = 5.4, 1.6 Hz, H-c 0 ), 4.90 (1H, br d, J = 5.2, 6.0 Hz, H-a, threo isomer), 4.90 (1H, br d, J = 5.2, 6.0 Hz, H-a, erythro isomer), 6.28 (1H, dt, J = 15.8, 5.4 Hz, H-b 0 , erythro isomer), 6.30 (1H, dt, J = 15.8, 5.4 Hz, H-b 0 , threo isomer), 6.52 (1H, dt, J = 15.8, 1.6 Hz, H-a 0 , erythro isomer), 6.54 (1H, dt, J = 15.8, 1.6 Hz, H-a 0 , threo isomer), 6.77 (1H, d, J = 8.0 Hz, H-5, erythro isomer), 6.78 (1H, d, J = 8.0 Hz, H-5, threo isomer), 6.86 (1H, d, J = 2.0 Hz, H-2, threo isomer), 6.88-6.90 (1H, m, H-6), 6.93 (1H, d, J = 6.0 Hz, H-5 0 , erythro isomer), 7.05 (1H, d, J = 2.0 Hz, H-2, erythro isomer), 7.09 (1H, d, J = 1.6 Hz, H-2 0 ), 7.12 (1H, d, J = 6.0 Hz, H-5 0 , threo isomer); 13 C NMR d:

Difference in enzymatic dehydrogenative polymerization of dilignols using horseradish peroxidase and crude enzyme obtained from Japanese cypress (Chamaecyparis obtusa)

“…In our previous studies [13,14], when HRP was used for the enzymatic dehydrogenative polymerization of dilignol, the consumption rate of I was the lowest among three dilignols. However, the consumption rate of I increased when another type of dilignol was mixed, i.e., when using a mixed-component system of I + II and I + III, while the consumption rates of II and III decreased.…”

“…Compounds I, II, and III were synthesized according to the method in our previous study [13,14]. Briefly, after the oxidation of coniferyl alcohol by silver (I) oxide, these three compounds were isolated by flash column chromatography (Agilent 971-FP, Agilent Technologies) using a silica gel column (Super Flash SF, Agilent Technologies).…”

“…The procedure for estimating the enzyme activity was based on our previous report [13], and the activity was determined from the oxidation rate of guaiacol. The activities of HRP and the crude enzyme obtained from cypress were 387.2 and 5.24 U/mg, respectively.…”

“…Enzymatic dehydrogenative polymerization was carried out by a modification of our previously reported method [13]. In this study, we employed two systems: a singlecomponent system and mixed-component system.…”

“…In addition, a radical transfer system between coniferyl alcohol and sinapyl alcohol [5][6][7][8], p-coumarate and sinapyl alcohol [9,10], superoxide radical in the presence of calcium and coniferyl alcohol [11], and manganese oxalate and monolignols [12] were reported. Previously, we investigated the enzymatic oxidation of dilignols using horseradish peroxidase (HRP) and proposed a radical transfer system, where one dilignol radical transfers its radical site to another dilignol [13,14].…”