Coniferyl aldehyde dimers in dehydrogenative polymerization: model of abnormal lignin formation in cinnamyl alcohol dehydrogenase-deficient plants

The enzymatically dehydrogenative polymerization of coniferyl aldehyde and coniferyl alcohol was studied to understand lignins in cinnamyl alcohol dehydrogenase (CAD)-downregulated plants. The sample dimers were prepared by polymerization under three reaction systems (coniferyl alcohol, coniferyl aldehyde, and their combination) with horseradish peroxidase/H₂O₂ under the conditions of limited reaction time. In addition, the residual amount of substrate in each reaction was determined at specified time intervals. In the reaction system of coniferyl aldehyde, the 5-5-type dimer was formed in preference to- and-5 dimers; in the reaction system of coniferyl alcohol the-5 dimer was preferentially formed. Furthermore, it was revealed when quantifying dimers among reaction systems that the total dimer formation capability of coniferyl alcohol clearly surpassed that of coniferyl aldehyde. However, the dimers cross-coupled with coniferyl alcohol and coniferyl aldehyde were formed in amounts not accounted for by the difference seen in dimer formation abilities with the two substrates.Part of this paper was presented at the 49th Annual Meeting of the Japan Wood Research Society, Tokyo, April 1999     

Stereochemistry and biosynthesis of 8-O-4′ neolignans in Eucommia ulmoides: diastereoselective formation of guaiacylglycerol-8-O-4′-(sinapyl alcohol) ether

Stereochemistry and biosynthesis of guaiacylglycerol-8-O-4′-(sinapyl alcohol) ether (GGSE), an 8-O-4′ neolignan, which consists of coniferyl and sinapyl alcohol moieties, in Eucommia ulmoides were investigated. Four 8-O-4′ neolignans, GGSE, syringylglycerol-8-O-4′-(coniferyl alcohol) ether (SGCE), guaiacylglycerol-8-O-4′-(coniferyl alcohol) ether (GGCE), and syringylglycerol-8-O-4′-(sinapyl alcohol) ether (SGSE), were synthesized. Their erythro and threo diastereomers were separated through acetonide derivatives, intermediates of the synthesis, and identified by means of nuclear magnetic resonance (NMR) spectroscopy. All of the erythro-acetonide derivatives have larger coupling constants (ca 9 Hz) for the C₇-H resonances than those of the threo ones (1.5–2 Hz). In the case of the four 8-O-4′ neolignans, the C₇-H coupling constants of the threo-isomers are not smaller than those of the erythro ones. GGSE isolated previously from this plant was identified as the erythro isomer by comparison of the ¹³C-NMR data with synthetic erythro-GGSE and threo-GGSE and the other 8-O-4′ neolignans mentioned as above. Administration of a mixture of [8-¹⁴C]coniferyl alcohol and [8-¹⁴C]sinapyl alcohol to excised shoots of E. ulmoides was carried out and the incorporation of ¹⁴C into erythro-[¹⁴C]GGSE was found to be higher than that in threo-[¹⁴C]GGSE. The occurrence of diastereoselective formation of erythro-GGSE by cross coupling of coniferyl and sinapyl alcohols is suggested.Part of this paper was presented at the 47th Lignin Symposium, Fukuoka, October 2002 and the 53rd Annual Meeting of the Japan Wood Research Society, Fukuoka, April 2003     

Polymerisation of added coniferyl alcohol by inherent xylem peroxidases and its effect on fungal decay resistance of Norway spruce

It is known that active peroxidase isozymes exist in mature wood of Norway spruce (Picea abies L. Karst.) and that they remain active for years and are found even in the heartwood (in Scots pine), where all cellular activity has ceased. This peroxidase activity was utilised in the impregnation of wood blocks with a natural monolignol, coniferyl alcohol and hydrogen peroxide. The hypothesis was that the internal wood peroxidases would oxidise the added monolignol and bind it stably into the cell wall matrix, which could hinder fungal decay. Since coniferyl alcohol is not very soluble in water, the impregnation was done under vacuum with an acetone–water solution containing 10% coniferyl alcohol and 0.4 mM H₂O₂ at room temperature (ca. 0.02 g of coniferyl alcohol was added to 1 g of wood). After impregnation, dimers of coniferyl alcohol and free coniferyl alcohol were found in acetone extracts with GC–MS analysis. Penetration of coniferyl alcohol and non-extractable reaction products were studied from the wood blocks with FTIR PAS technique. The wood samples treated were also subjected to a fungal decay test with Coriolus versicolor. This treatment hindered fungal decay in a 60-day experiment and led to a dry weight loss of 8.8% in comparison with 19.9% in the control. The reactions of coniferyl alcohol and H₂O₂ in the presence of peroxidases are discussed as well as the use of monolignols to increase wood decay resistance.     

Solvent effects on the electronic state of monolignol radicals as predicted by molecular orbital calculations

The spin and charge densities in three monolignol radicals were computed using the UB3LYP/6-31G* method of molecular orbital calculation. As well, the effects of solvents were simulated by using an SCI-PCM model. It was confirmed that an unpaired electron was localized at C₁, C₃, C₅, C₈, and O₄ for all monolignol radicals. In solvents, the spin density decreased at O₄ with increasing solvent polarity, but increased at C₈. The atomic charges at all reactive atoms had a negative value and were obviously strengthened at O₄ with increasing solvent polarity. These tendencies support the experimental results for radical coupling reactions of monolignols in various solvents; that is, that 8-O₄′ linkages are produced much more often than 8-8′ linkages in nonpolar solvents.     

Reactivity of lignin in supercritical methanol studied with various lignin model compounds

Behavior of lignin in supercritical methanol (250–270°C, 24–27 MPa) was studied by using lignin model compounds at the tin bath temperature of 270°C with a batch-type reaction vessel. Guaiacol and veratrole were selected as a guaiacyl type of aromatic ring in lignin, while 2,6-dimethoxyphenol and 1,2,3-trimethoxybenzene as a syringyl one. In addition, biphenyl and β-O-4 types of dimeric lignin model compounds were, respectively, studied as condensed and ether linkages between C₆-C₃ phenyl propane units. As a result, both guaiacyl and syringyl types of aromatic rings were very stable, and the biphenyl type was comparatively stable under supercritical conditions of methanol. However, β-ether linkage in the phenolic β-O-4 model compound was cleaved rapidly into guaiacol and coniferyl alcohol, which was further converted to its γ-methyl ether. Non-phenolic β-O-4 model compound was, on the other hand, converted initially into its α-methyl ether and degraded further to produce guaiacol. These lines of evidence imply that in lignin macromolecules, the new phenolic residues are continuously formed and depolymerized repeatedly in supercritical methanol into the lower molecular products, mainly by the cleavage of the dominant β-ether structure in lignin.     

The monomer composition controls the Σ<Emphasis Type="Italic">β-O</Emphasis>-4/Σ<Emphasis Type="Italic">O</Emphasis>-4 end monomer ratio of the linear lignin fraction

Lignins are cell wall phenolic heteropolymers that result from the oxidative coupling of three monolignols bearing p-coumaryl (H), coniferyl (G), and sinapyl (S) units, in a reaction mediated by peroxidases. Here, we report the existence of a relationship between the Σβ-O-4/ΣO-4 end monomer ratio of the linear lignin fraction, released through the specific cleavage of the alkyl ether linkages by thioacidolysis, and the G/S ratio of lignins, when this was estimated in differentially evolved vascular land plants. Most importantly, in the case of angiosperms, Gnetales, and lycopods, the Σβ-O-4/ΣO-4 end monomer ratio was apparently predictable from the proportions at which the G and S units were mixed. In the case of G lignins (present in basal gymnosperms and ferns), the Σβ-O-4/ΣO-4 end monomer ratio decayed exponentially to increase the O-4-linked dihydroconiferyl alcohol (DHCA) content. The results obtained suggest that the Σβ-O-4/ΣO-4 end monomer ratio of the linear lignin fraction depends intimately on the lignin monomer composition, and, therefore, on the chemical nature of the radicals derived from three monolignols (coniferyl, dihydroconiferyl, and sinapyl alcohols), whose gain have been finely tuned during land plant evolution.     

Synthesis of isoacteoside, a dihydroxyphenylethyl glycoside

The total chemical synthesis of isoacteoside (1), 2-(3,4-dihydroxyphenyl)ethyl 6-O-caffeoyl-3-O-(-l-rhamnopyranosyl)--d-glucopyranoside, is described. An acteoside acetate with benzyl groups at the catechols (3: 2-(3,4-dibenzyloxyphenyl)ethyl 2,6-di-O-acetyl-4-O-[3,4-bis(O-benzyl)caffeoyl]-3-O-(-l-rhamnopyranosyl)--d-glucopyranoside) was treated with a solution of methy-lamine in methanol (MeNH₂ in MeOH) to perform both deacetylation and caffeoyl migration, affording an isoacteoside derivative with benzyl groups at the catechols4b: 2-(3,4-dibenzyloxyphenyl)ethyl 6-O-[3,4-bis(O-benzyl) caffeoyl] -3-O-(-l-rhamnopyranosyl)--d-glucopyranoside —in 34% yield. Debenzylation of4b was successfully accomplished by catalytic transfer hydrogenation using 1,4-cyclohexadiene to give the target compound isoacteoside (1) in 54% yield.¹H and¹³C nuclear magnetic resonance spectral data of the synthesized isoacteoside (1) were identical with those of the natural isoacteoside isolated fromPaulownia tomentosa (Thumb.) Steud.Part of this research was presented at the 51st Annual Meeting of the Japan Wood Research Society, Tokyo, April 2001     

Sinapyl alcohol-specific peroxidase isoenzyme catalyzes the formation of the dehydrogenative polymer from sinapyl alcohol

Two peroxidases, CWPO-A and CWPO-C, were isolated from the cell walls of poplar (Populus alba L.) callus culture. The cationic CWPO-C showed a strong preference for sinapyl alcohol over coniferyl alcohol as substrate. Thus, the monolignol utilization of CWPO-C is unique compared with other peroxidases, including anionic CWPO-A and horseradish peroxidase (HRP). CWPO-C polymerized oligomeric sinapyl alcohol (S-oligo) and sinapyl alcohol, producing a polymer of greater molecular weight. In contrast, HRP, which is specific to coniferyl alcohol, produced sinapyl alcohol dimers, rather than catalyzing polymerization. Adding coniferyl alcohol as a radical mediator in the HRP-mediated reaction did not result in S-oligo polymerization. This report shows that CWPO-C is an isoenzyme specific to sinapyl alcohol that polymerizes oligomeric lignols. Its catalytic activity toward oligomeric lignols may be related to the lignification of angiosperm woody plant cell walls.Part of this study was presented at the 45th Lignin Symposium, Matsuyama, October 2000     

Effect of coniferyl alcohol addition on removal of chlorophenols from water effluent by fungal laccase

The effect of coniferyl alcohol on removal of chlorinated phenols from a water environment byRhizoctonia praticola andCerrena unicolor laccases was studied. At optimal conditions in which 7 mM coniferyl alcohol and laccase were added to chlorinated phenols over 20h, about 34% of the radioactivity of 4-chlorophenol, 57% of 2,4-dichlorophenol, 66% of 2,4,5-trichlorophenol, and 85% of pentachlorophenol were removed from the supernatants, compared to the level without laccase activity. After 12-h incubation periods at the optimal concentrations of coniferyl alcohol and laccase (added simultaneously), the fast first phase of chlorophenol removal was complete in 1 h, and eventually coniferyl alcohol enhanced the removal of 4-chlorophenol by 40%, 2,4-dichlorophenol by 54%, 2,4,5-trichlorophenol by 60%, and pentachlorophenol by 76%.     

Study on chemical constitution of benzene and alcohol extractives ofTraceutron Sinense Olive

The wood powder ofTetracentron Sinense Olive was extracted with the benzene and alcohol (2:1 v/v). Then, the extractive is analyzed in Gc and Gc-Ms. Twenty chemical constitutions of the extractive are separated and identified, which are as follows: calamenene; 2.6,10-trimethyldihexyl; 3-methoxybenzaldehyde; Methyl-4-methox-_ybenzoate; 4-methoxyphenylacetone; 3.4-dimethoxybenzaldehyde; 3.4-dimethoxyhypnone; methyl-3.4-dimethoxybenzoate; Methyl-4-methoxyphenylacrylate; 4,4′-dimethoxycabonyl-diphenylethane; Methyl-3, 4,5-erimethoxybenzoate; Methyl-3.4-dimethoxyphenylacrylate; Methyl-myristate; Methyl-palmate; Methyl-zoomarate; Methyl-stearate; Methyl-oleate; Methyl-arachidate; Methyl-behenate; Dimethylazelate.     

Role of caffeic glucoside esters in defense-repair processing of trees II: Synthesis of 2-(3,4-dihydroxyphenyl)-ethyl 3-α-L-rhamnopyranosylβ-D-glucopyranoside

The reference compound (9), with a partial structure of acteoside, was synthesized to elucidate the relation between structural features and the precipitation or solubility of the oxidation products of acteoside: 2-(3,4-dihydroxyphenyl)-ethyl 3-O-(-L-rhamnopyranosyl)--D-glucopyranoside (9). The glycosyl acceptor 2-O-benzoyl-3-O-(2,3,4-tri-O-acetyl--L-rhamnopyranosyl)-4,6-O-benzylidene--D-glucopyranosyl trichloroacetimidate (7) was prepared from allyl 2-O-benzoyl-3-O-(2,3,4-tri-O-acetyl--L-rhamnopyranosyl)-4,6-O-benzylidene--D-glucopyranoside (4) via isomerization of the allyl group with an iridium complex to the 1-propenyl group and its hydrolysis with HgCl₂lHgO, followed by treatment with CCl₃CN and DBU in a 65.5% overall yield. The glycosyl acceptor 3,4-diacetoxyphenethyl alcohol (16) was prepared from homoveratric acid via demethylation with 57% HI and its acetylation with Ac₂O and 85% H₃PO₄, followed by selective reduction of the carboxyl group to the alcohol with a borane-tetrahydrofurane complex in a 61% overall yield. The glycosylation of7 with16 in dichloromethane promoted by BF₃-Et₂O gave a 74.3% yield of8. Hydrolysis of8 with 90% CF₃COOH gave the debenzylidenated product, which was treated with NaOMe to afford a 32% overall yield of the desired compound9. This compound9 was identical with the natural specimen.This study was presented in part at the 46th annual meeting of the Japan Wood Research Society, Kumamoto, April 3–5, 1996     

Structural unit of xylans from sugi (<Emphasis Type="Italic">Cryptomeria japonica</Emphasis>) and hinoki (<Emphasis Type="Italic">Chamaecyparis obtusa</Emphasis>)

Arabinoglucuronoxylans (AGXs) isolated from the holocellulose of sugi (Cryptomeria japonica) and hinoki (Chamaecyparis obtusa) contained one 4-O-methyl-d-glucopyranosyluronic acid (4-O-Me-d-GlcAp) residue per 6.2 d-xylopyranose (d-Xylp) residues and one 4-O-Me-d-GlcAp residue per 3.8 d-Xylp residues. These AGXs were subjected to partial acid hydrolysis. Analyses by size exclusion chromatography and electrospray-ionization mass spectroscopy of the neutral sugar fractions in the hydrolysates showed the presence of xylooligosaccharides having a degree of polymerization of 2-8 in addition to d-Xyl, suggesting that the AGXs from sugi and hinoki contained unsubstituted chains consisting of at least eight d-Xyl residues. The acidic sugars in the hydrolysates were separated into two series of aldouronic acids composed of 4-O-Me-d-GlcAp and d-Xylp by ion-exchange chromatography. The first series included aldouronic acids from aldobiouronic acid (4-O-Me-d-GlcAp-Xyl) to aldopentaouronic acids (4-O-Me-d-GlcAp-Xyl₄). The second series were aldouronic acids composed of two 4-O-Me-d-GlcAp residues and 2-4 d-Xyl residues. In these acidic sugars, the uronic acid side chains were located on two contiguous d-Xyl residues. These facts indicated that AGXs from sugi and hinoki had a structural unit containing two 4-O-Me-d-GlcAp residues on two contiguous d-Xyl residues as well as AGXs from spruce and larch.     

Substituent effects of 3,5-disubstituted p-coumaryl alcohols on their oxidation using horseradish peroxidase–H2O2 as the oxidant

The consumption rates of three monolignols (p-coumaryl, coniferyl, and sinapyl alcohols) and eight analogues using horseradish peroxidase (HRP)–H₂O₂ as an oxidant were measured and compared with the anodic peak potentials thereof measured with cyclic voltammetry. 3-Monosubstituted p-coumaryl alcohols, i.e., 3-methoxy-, 3-ethoxy-, 3-n-propoxy-, and 3-n-butoxy-p-coumaryl alcohols, had faster reaction rates than p-coumaryl alcohol. This is most probably due to the electron-donating effect of alkoxyl groups. However, the reaction rates gradually decreased with an increase in the molecular weight of the alkoxyl groups. Furthermore, t-butoxyl group, which is a very bulky substituent, caused an extreme reduction in the reaction rate, even though its electron-donating effect was almost the same as that of other alkoxyl groups. The reaction rates of 3,5-disubstituted p-coumaryl alcohols, especially 3,5-dimethyl-p-coumaryl alcohol, were very low compared with 3-monosubstituted p-coumaryl alcohols. These results suggest that there are three main factors of hindrance during the approach of monolignols to the active site of HRP. First, from the results of 3-monoalkoxy-p-coumaryl alcohols, it was suggested that the volume of substituents could decrease their oxidation rates. Second, from the results of 3,5-disubstituted p-coumaryl alcohols, it was suggested that local steric hindrance by the amino residues quite near the heme decreased the oxidation rates. Third, from the results of the substrates with hydrophobic substituents at their 3,5-positions, we suggested that hydrophilicity near heme would decrease their oxidation rates.     

Biosynthesis of a syringyl 8-O-4′ neolignan in Eucommia ulmoides: formation of syringylglycerol-8-O-4′-(sinapyl alcohol) ether from sinapyl alcohol

To investigate the biosynthesis and stereochemistry of syringylglycerol-8-O-4′-(sinapyl alcohol) ether (SGSE), a syringyl 8-O-4′ neolignan, feeding experiments and enzyme assays using Eucommia ulmoides were carried out. Diastereoselective formation of erythro-SGSE was found. When [8-¹⁴C]sinapyl alcohol was administered to excised shoots of E. ulmoides, ¹⁴C was incorporated into free SGSE and SGSE glucosides. In stems, incorporation into (+)-erythro-[¹⁴C]SGSE (0.037%) with 9.1% enantiomeric excess (% e.e.) was found; incorporation into the threo isomer was not detectable. Erythro-[¹⁴C]SGSE glucosides (0.047%) dominated over threo forms (0.007%) with 74.0% diastereomeric excess (% d.e.); both diastereomers were levorotatory with 32.0% e.e. and 18.3% e.e., respectively. In leaves, higher incorporation into (−)-erythro-[¹⁴C]SGSE (0.500%, 15.9% e.e.) than into the threo isomer (0.206%, 7.4% e.e.) was observed (41.6% d.e.). (−)-Erythro-[¹⁴C]SGSE glucosides (1.692%, 25.0% e.e.) were produced at higher rates than threo isomers (0.177%, 16.4% e.e.) with 81.0% d.e. In incubations of a mixture of [8-¹⁴C]sinapyl and [8-¹⁴C]coniferyl alcohols with an insoluble enzyme preparation from stems of E. ulmoides, erythro-SGSE was preferentially produced. The highest % d.e. (82.8) was observed at 60 min with the (+)-erythro isomer (21.4% e.e.) and the (−)-threo form (4.3% e.e.).Part of this report was presented at the 52nd Annual Meeting of the Japan Wood Research Society, Gifu, April 2002, and the 47th Lignin Symposium, Fukuoka, October 2002     

Reaction behavior of lignin in supercritical methanol as studied with lignin model compounds

 The reaction behavior and kinetics of lignin model compounds were studied in supercritical methanol with a batch-type supercritical biomass conversion system. Guaiacol, veratrole, 2,6-dimethoxyphenol, and 1,2,3-trimethoxybenzene were used as model compounds for aromatic rings in lignin. In addition, 5-5, β-1, β-O-4, and α-O-4 types of dimeric lignin model compounds were used as representatives of linkages in lignin. As a result, aromatic rings and 5-5 (biphenyl)-type structures were stable in supercritical methanol, and the β-1 linkage was not cleaved in the β-1-type structure but converted rapidly to stilbene. On the other hand, β-ether and α-ether linkages of β-O-4 and α-O-4 lignin model compounds were cleaved rapidly, and these compounds decomposed to some monomeric compounds. Phenolic compounds were found to be more reactive than nonphenolic compounds. These results indicate that cleavages of ether linkages mainly contribute to the depolymerization of lignin, whereas condensed linkages such as the 5-5 and β-1 types are not cleaved in supercritical methanol. Therefore, it is suggested that the supercritical methanol treatment effectively depolymerizes lignin into the lower-molecular-weight products as a methanol-soluble portion mainly by cleavage of the β-ether structure, which is the dominant linkage in lignin. Received: December 19, 2001 / Accepted: April 30, 2002 Acknowledgments This research has been done under the research program for the development of technologies for establishing an ecosystem based on recycling in rural villages for the twenty-first century from the Ministry of Agriculture, Forestry and Fisheries, Japan; by a Grant-in-Aid for Scientific Research (B)(2) (no.12460144, 2001.4–2003.3) from the Ministry of Education, Culture, Sports, Science and Technology, Japan; and under the research program from Kansai Research Foundation for Technology Promotion, Japan. The authors thank them for their financial support. This study was presented in part at the 45th Lignin Symposium, Ehime, Japan, October 2000 and the 52nd Annual Meeting of the Japan Wood Research Society, Gifu, Japan, April 2002 Correspondence to:S. Saka     

Synthesis of dihydroxyphenacyl glycosides for biological and medicinal study: β-oxoacteoside from Paulownia tomentosa

The protected structure of -oxoacteoside (tomentoside A), 2-oxo-2-(3,4-dihydroxyphenyl)ethyl 3-O-(2,3,4-tri-O-acetyl--l-rhamnopyranosyl)-4-O-caffeoyl--d-glucopyranoside 14 was synthesized in 14% overall yield in 11 steps, starting from d-glucose for biological and medicinal studies of phenylpropanoid glycosides. The first step was the preparation of a 3-O-rhamnopyranosyl disaccharide sugar core 2 from a suitably protected rhamnosyl trichloroacetimidate 10 and glucose derivative (diacetone-d-glucose 1) in 71% yield. To the glucose moiety of this sugar core, several protection/deprotection procedures were performed sequentially to obtain a fully acetylated sugar core 7 with a 4-OH group on the glucose moiety, in 57% yield in five steps. Thereafter, to the 4-OH group of the glucose moiety, selective 4-O-caffeoylation was achieved by proton-transfer esterification with 3,4-di-O-allylcaffeic acid 16 to give the caffeoyl disaccharide 11 in 97% yield. Then, it was converted to trichloroacetimidate 13 for a glycosylation donor in 90% in two steps. Finally, anomeric glycosylation was conducted with 2-oxo-2-(3,4-di-allyloxyphenyl)ethyl alcohol 19 with catalytic amounts of BF₃·Et₂O to give 2-oxo-2-(3,4-di-allyloxyphenyl)ethyl 2,6-di-O-acetyl-3-O-(2,3,4-tri-O-acetyl--l-rhamnopyranosyl)-4-O-(3,4-di-allyloxycaffeoyl)--d-glucopyranoside 14 in 60% yield. Deprotected intermediates of compounds 2, 11, 14, and 19 which were obtained in high yield would be useful for biological and medicinal studies of phenylpropanoid glycosides.Part of this study was presented at the 52nd Annual Meeting of the Japan Wood Research Society, Gifu, April, 2002     

Inhibition ofTrichoderma cellulase activity by a stilbene glucoside fromPicea glehnii bark

The stilbene glucoside isorhapontin (5,4-dihydroxy-3-methoxystilbene-3--D-glucoside) is the major constituent of the ethyl acetate extracts fromPicea glehnii bark. Isorhapontin inhibited the hydrolytic activity ofTrichoderma cellobiohydrolase I (CBH I) for both bacterial microcrystalline cellulose and the soluble cellooligosaccharide celloheptaitol. The inhibitory effect for celloheptaitol, however, was more drastic than that for bacterial microcrystalline cellulose. The hydrolytic activity of the CBH I core domain for celloheptaitol was also inhibited by isorhapontin to a similar extent, suggesting that the interaction between isorhapontin and the core domain of CBH I is the reason for this phenomenon. The inhibition of CBH I activity by isorhapontin showed mixed noncompetitive and uncompetitive types in a concentration of the inhibitor of less than 125M. TheK _i andK _i values were estimated to be 57.2 and 33.3M, respectively. Whereas isorhapontin strongly inhibited CBH I activity, its aglycone isorhapontigenin (3-methoxy-3,5,4-trihydroxystilbene) showed almost no inhibition. Consequently, both the stilbenic and the-glucosidic structures in isorhapontin are essential for the inhibitory effect on CBH I activity. Isorhapontin also inhibited the activity ofTrichoderma endoglucanase I for celloheptaitol, whereas almost no effect was observed for the activities of both endoglucanases II and III.An outline of this work was presented at the 49th Annual Meeting of the Japan Wood Research Society, Tokyo, April 1999     

Decomposition behavior of woody biomass in water-added supercritical methanol

The chemical conversion of Japanese beech (Fagus crenata Blume) in water-added supercritical methanol was studied for a wide range of water content using a batch-type reaction vessel to obtain chemicals from lignocellulosics. It was consequently found that addition of water enhanced the decomposition of wood cell wall components; cellulose, hemicelluloses, and lignin. In cases of high water content, however, it resulted in low solubility of lignin-derived products causing an increase in the mass of the residue. The water content was thus optimized to be around 10% (v/v) for the decomposition of wood. Concomitantly, the yields and selectivity of the chemicals from wood could be regulated by the addition of water, especially for the lignin-derived products. As a result, the monomeric compounds of lignin, coniferyl alcohol and sinapyl alcohol, were recovered as their γ-methyl ethers in the presence of water in higher yields than those obtained without addition of water.     

MALDI-TOF/MS analyses of decomposition behavior of beech xylan as treated by semi-flow hot-compressed water

Japanese beech (Fagus crenata) was treated with semi-flow hot-compressed water at various temperatures of 150–230 °C under 10 MPa. The obtained various products were then analyzed with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS). In a temperature range of 150 °C up to 210 °C, however, no hydrolyzed products were found, and at 210 °C/10 MPa, O-acetyl-4-O-methylglucuronoxylo-oligosaccharides (X _n Ac _m MG _i ) and O-acetyl-xylo-oligosaccharides (X _n Ac _m ) were obtained, indicating the first cleavage of native xylan β-1,4-glycosidic linkages followed by a cleavage of α-1,2-glycosidic linkage in 4-O-methylglucuronic acid (MG) residue at mainly 220–230 °C under 10 MPa. At subsequent stage of 230 °C/10 MPa, X _n Ac _m were predominantly recovered. As the treatment was prolonged at 230 °C, X _n Ac _m were reduced, but remained to some extent, indicating that the acetyl group which is hydrolyzed to be acetic acid is more resistant than MG residue. In such a stage of treatment, cellulose started to hydrolyze to cello-oligosaccharides. These lines of evidence can clearly indicate the hydrolysis pathway of native O-acetyl-4-O-methylglucuronoxylan as treated by hot-compressed water. Thus, xylo-oligosaccharides recovered in a very early stage of the semi-flow hot-compressed water treatment preserve native O-acetyl-4-O-methylglucuronoxylan.     

5α-Reductase inhibitory compounds produced by polymerization of resveratrol with horseradish peroxidase

To produce 5-reductase inhibitory compounds, resveratrol was enzymatically oxidized in a horseradish peroxidase (HRP)/H₂O₂ system. Ethyl acetate extract of the oxidation products showed strong 5-reductase inhibitory activity with 10%–15% organic solvents in the system, whereas without organic solvent little inhibitory activity was exhibited. The optimum pH of enzymatic oxidation for acquisition of the inhibitory activity was 4.5. The inhibitory compounds were isolated and identified as resveratroltrans-dehydrodimer and resveratrolcis-dehydrodimer by comparing with published nuclear magnetic resonance data. The two resveratrol dehydrodimers have stronger inhibitory activity than natural resveratrol dimers and trimers found inShorea species.