Shingo Kawai

NMR analysis of lignins in CAD-deficient plants. Part 1. Incorporation of hydroxycinnamaldehydes and hydroxybenzaldehydes into lignins

Peroxidase/H2O2-mediated radical coupling of 4-hydroxycinnamaldehydes produces 8-O-4-, 8-5-, and 8-8-coupled dehydrodimers as has been documented earlier, as well as the 5-5-coupled dehydrodimer. The 8-5-dehydrodimer is however produced kinetically in its cyclic phenylcoumaran form at neutral pH. Synthetic polymers produced from mixtures of hydroxycinnamaldehydes and normal monolignols provide the next level of complexity. Spectral data from dimers, oligomers, and synthetic polymers have allowed a more substantive assignment of aldehyde components in lignins isolated from a CAD-deficient pine mutant and an antisense-CAD-downregulated transgenic tobacco. CAD-deficient pine lignin shows enhanced levels of the typical benzaldehyde and cinnamaldehyde end-groups, along with evidence for two types of 8-O-4-coupled coniferaldehyde units. The CAD-downregulated tobacco also has higher levels of hydroxycinnamaldehyde and hydroxybenzaldehyde (mainly syringaldehyde) incorporation, but the analogous two types of 8-O-4-coupled products are the dominant features. 8-8-Coupled units are also clearly evident. There is clear evidence for coupling of hydroxycinnamaldehydes to each other and then incorporation into the lignin, as well as for the incorporation of hydroxycinnamaldehyde monomers into the growing lignin polymer. Coniferaldehyde and sinapaldehyde (as well as vanillin and syringaldehyde) co-polymerize with the traditional monolignols into lignins and do so at enhanced levels when CAD-deficiency has an impact on the normal monolignol production. The implication is that, particularly in angiosperms, the aldehydes behave like the traditional monolignols and should probably be regarded as authentic lignin monomers in normal and CAD-deficient plants.

Degradation mechanisms of a nonphenolic β-O-4 lignin model dimer by Trametes versicolor laccase in the presence of 1-hydroxybenzotriazole

Abstract Past studies have shown that Trametes versicolor laccase catalyzed Cα-Cβ cleavage, Cα-oxidation, β-ether cleavage and aromatic ring cleavage of a nonphenolic β- O -4 lignin model dimer, 1,3-dihydroxy-2-(2,6-dimethoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl)propane (I), in the presence of 1-hydroxybenzotriazole. In this study, we investigated the detailed degradation mechanism for these pathways of substrate I by laccase/1-hydroxybenzotriazole couple, based on incorporation experiments from H 2 18 O and 18 O 2 . Oxidation of substrate I in H 2 18 O resulted in the incorporation of 18 O into three aromatic ring cleavage products and a β-ether cleavage product. These results were identical with those for lignin peroxidase. We inferred that these cleavages of substrate I proceed via the β-aryl cation radical intermediate of I. However, the Cα-Cβ cleavage degradation product of I by the laccase/1-hydroxybenzotriazole couple was 4-ethoxy-3-methoxybenzoic acid, whereas it was 4-ethoxy-3-methoxybenzaldehyde for lignin peroxidase. It is clear that one atom of 18 O from 18 O 2 was incorporated into 4-ethoxy-3-methoxybenzoic acid during the oxidation of substrate I. To confirm the reaction intermediate, we carried out the chemical reaction of 2-(2,6-dimethoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl)- 3-hydroxypropanone with alkaline hydrogen peroxide, since the expected hydroperoxide intermediate is quite similar to that formed from the reaction between the benzylic radical of I and O 2 . The result showed that the main degradation product was 4-ethoxy-3-methoxybenzoic acid. Thus, we propose a novel Cα-Cβ cleavage via a kind of Baeyer-Villiger reaction for nonphenolic β- O -4 lignin model dimer by laccase/1-hydroxybenzotriazole couple.

Aromatic ring cleavage of a non-phenolic β-O-4 lignin model dimer by laccase of Trametes versicolor in the presence of 1-hydroxybenzotriazole

The novel cleavage products, 2,3‐dihydroxy‐1‐(4‐ethoxy‐3‐methoxyphenyl)‐1‐formyloxypropane (II) and 1‐(4‐ethoxy‐3‐methoxyphenyl)‐1,2,3‐trihydroxypropane‐2,3‐cyclic carbonate (III) were identified as products of a non‐phenolic β‐O‐4 lignin model dimer, 1,3‐dihydroxy‐2‐(2,6‐dimethoxylphenoxy)‐1‐(4‐ethoxy‐3‐methoxyphenyl)propane (I), by a Trametes versicolor laccase in the presence of 1‐hydroxybenzotriazole (1‐HBT). An isotopic experiment with a 13C‐labeled lignin model dimer, 1,3‐dihydroxy‐2‐(2,6‐[U‐ring‐13C]dimethoxyphenoxy)‐1‐(4‐ethoxy‐3‐methoxyphenyl)propane (I‐13C) indicated that the formyl and carbonate carbons of products II and III were derived from the β‐phenoxy group of β‐O‐4 lignin model dimer I as aromatic ring cleavage fragments. These results show that the laccase‐1‐HBT couple could catalyze the aromatic ring cleavage of non‐phenolic β‐O‐4 lignin model dimer in addition to the β‐ether cleavage, Cα‐Cβ cleavage, and Cα‐oxidation.

Substrate specificity of the α-l-arabinofuranosidase from Rhizomucor pusillus HHT-1

Abstract The α- l -arabinofuranosidase (AF) from the fungus Rhizomucor pusillus HHT-1 released arabinose at appreciable rates from (1→5)-α- l -arabinofuranooligosaccharides, sugar beet arabinan and debranched arabinan. This enzyme preferentially hydrolyzed the terminal arabinofuranosyl residue [α-(1→5)-linked] of the arabinan backbone rather than the arabinosyl side chain [α-(1→3)-linked residues]. The enzyme-hydrolyzed arabinan reacted at and debranched the arabinan almost at the same rate, and the degree of conversion for both cases was 65%. Methylation analysis of arabinan showed that the arabinosyl-linkage proportions were 2:2:2:1, respectively, for (1→5)-Araf, T-Araf, (1→3, 5)-Araf and (1→3)-Araf, while the ratios for the AF-digested arabinan shifted to 3:1:2:1. Enzyme digestion resulted in an increase in the proportion of (1→5)-linked arabinose and a decrease in the proportion of terminal arabinose indicated this AF cleaved the terminal arabinosyl residue of the arabinan back bone [α-(1→5)-linked residues]. Peak assignments in the 13C NMR spectra also confirmed this linkage composition of four kinds of arabinose residues. Both 1H and 13C NMR spectra are dominated by signals of the α-anomeric configuration of the arabinofuranosyl moieties. No signals were recorded for arabinopyranosyl moieties in the NMR spectra. Methylation and NMR analysis of native and AF-digested arabinan revealed that this α- l -arabinofuranosidase can only hydrolyse α- l -arabinofuranosyl residues of arabinan.

Simple method for synthesizing phenolic β-O -4 dilignols

A modified synthetic method for phenolicβ-O-4 lignin substructure model dimers was developed involving protection of the phenolic hydroxyl group of acetophenons with benzoyl chloride, bromination with 4-dimethylaminopyridiniumbromide perbromide, condensation with phenols in the presence of 18-crown-6-ether, condensation with paraformaldehyde, reduction with NaBH4 and debenzoylation. This method results in shorter reaction times and increasing yields without the application of strict anhydrous and drastic conditions or chloric solvents. This alternative route could be applied to theβ-O-4 dilignol syntheses of four combinations of guaiacyl and syringyl derivatives.

Photodiscoloration of western hemlock (Tsuga heterophylla) sapwood III Early stage of photodiscoloration reaction with lignans

The reaction during the early stage of photodiscoloration of constituents in western hemlock [Tsuga heterophylla (Raf. Sarg., Pinaceae] sapwood was investigated with chemical methods. The main photodiscoloring constituents, hydroxymatairesinol, allohydroxymatairesinol, α-conidendrin, and oxomatairesinol, were used as substrates for light-irradiation experiments in vitro. The structures of photodiscoloration reaction products were elucidated by isolation and instrumental analyses and/or co-high-performance liquid chromatography analyses with authentic specimens. The experiment was undertaken to distinguish each series of liquid phases using chloroform, water (both including a trace of methanol), and methanol, and the solid phase. The reaction products allohydroxymatairesi (2), oxomatairesinol (3), α-conidendrin (4), allo-7′-methoxymatairesinol (5), 7′-methoxymatairesinol (6), and vanillin (7) were isolated or detected in the reaction mixture of a hydroxymatairesinol system. The reaction products hydroxymatairesinol (1), 3, 4, 5, 6, and 7 were confirmed in the reaction system of allohydroxymatairesinol, which was an epimer of hydroxymatairesinol. Product 3 was confirmed from the α-conidendrin system, and reaction product 7 was confirmed from oxomatairesinol. The photodiscoloration reaction of western hemlock sapwood could be initiated by the formation of phenoxy radicals from the respective constituents. The reaction was then presumed to progress via formation of a quinonemethide intermediate in many of them. It was suggested that the reactive species, such as phenoxy radical or quinonemethide intermediate, formed by lightirradiation might be converted to quinone derivatives and colored oligomers. Products 1, 2, 3, 4, and 7, formed from substrates such as hydroxymatairesinol, allohydroxymatairesinol, α-conidendrin, and oxomatairesinol, were the same as the original metabolic constituents of western hemlock. Therefore it was concluded that the photodiscoloration of western hemlock depends not on the quantitative level of a few respective metabolites but, rather, on the coexistence of many metabolites.

Identification of Thuja occidentalis lignans and its biosynthetic relationship

Abstract Five lignans, (8R,8′R)-(−)-matairesinol, (8R,8′R)-(−)-thujaplicatin methyl ether, (8S,8′S)-(−)-wikstromol, 8-hydroxy-thujaplicatin methyl ether and epi-pinoresinol were isolated from Thuja occidentalis branch xylem, besides the previously identified (8R,8′R)-(−)-4-O-demethylyatein. Chiral HPLC analyses of matairesinol, thujaplicatin methyl ether, wikstromol and 4-O-demethylyatein indicated that these compounds were optically pure. A neolignan, dihydrodehydrodiconiferyl alcohol, was isolated from the acid-hydrolyzed extracts of T. occidentalis leaves. The lignans pinoresinol and secoisolariciresinol were also identified by GC–MS analysis of the extracts of xylem and leaves, respectively. Feeding experiments of [9- 2 H 2, OC2H3]coniferyl alcohol into T. occidentalis young shoots showed that two molecules of coniferyl alcohol were incorporated into pinoresinol and lariciresinol.

Norlignan from the knot resin of Araucaria angustifolia

Abstract A new norlignan, 2,3-bis-( p -hydroxyphenyl)-2-cyclopentene-1-one, was isolated from the knot resin powder of Araucaria angustifolia . The structure was elucidated by spectroscopic chemical analyses. In addition 4,4′- dihydroxychalcone, cryptoresinol (norlignan), four known lignans and one known norlignan, were isolated for the first time from the resin. A qualitative and quantitative comparison of these compounds in knot, heartwood and sapwood of A. angustifolia was performed from the viewpoint of plant chemistry. Detectable amounts of the new compound were present in both knot and heartwood.

Sesquilignans and lignans from Tsuga heterophylla

Abstract Two epimeric sesquilignans and two lignans were isolated from the sapwood of Tsuga heterophylla (western hemlock, Pinaceae). By spectroscopic analyses their structures were deduced to be (8 R ,8′ R ,7′ R ,8″ S ,7″ R )-7′-hydroxylappaol E and (8 R ,8′ R ,7′ R ,8″ S ,7″ S )- epi -7′-hydroxylappaol E, and (8′ R ,7′ S )-8-hydroxy-α-conidendrin and (8′ R ,7′ S )-8-hydroxy-α-conidendric acid methyl ester. Their presence in several samples of western hemlock sapwood was confirmed by quantitative analyses.

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 toβ-β andβ-5 dimers; in the reaction system of coniferyl alcohol theβ-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.