Takao Kishimoto

Influence of syringyl to guaiacyl ratio on the structure of natural and synthetic lignins.

Several kinds of natural woods and isolated lignins with various syringyl to guaiacyl (S/G) ratios were subjected to thioacidolysis followed by Raney nickel desulfuration to elucidate the relationships between the S/G ratio and the interunit linkage types of lignin. Furthermore, enzymatic dehydrogenation polymers (DHP) were produced by the Zutropf (gradual monolignol addition) method from mixtures of various ratios of coniferyl alcohol and sinapyl alcohol. The analysis of DHPs and natural wood lignins exhibited basically a similar tendency. The existence of both syringyl and guaiacyl units is effective for producing higher amounts of beta-O-4 and 4-O-5 structures, but it lowers the total amount of cinnamyl alcohol and aldehyde end groups. The relative frequency of the beta-beta structure increased, whereas that of beta-5 and 5-5 structures decreased with increasing syringyl units.

Easy synthesis of β-O-4 type lignin related polymers

The β-O-4 structure is the most abundant substructure in lignin. Lignin related polymers composed of only the β-O-4 structure were prepared using simple aromatic compounds as starting materials. Acetophenone derivatives were brominated, polymerized in the presence of K2CO3 and reduced with NaBH4 to give the lignin related polymers. These are linear polymers which resemble natural lignins in their structures, although they do not have a γ-hydroxymethyl group. The number average degree of polymerization (DPn) was determined with peracetate of the polymers by gel permeation chromatography. The DPn of guaiacyl type polymers ranged from 15.2–21.4, where the value for the syringyl type was 11.3 and for the p-hydroxyphenyl type 16.9. The Guaiacyl type polymer was very soluble in usual lignin solvents such as 1, 4-dioxane–water (96 : 4, v/v) and DMSO, but only slightly soluble in acetone–water (9 : 1, v/v).

Heteronuclear single-quantum coherence nuclear magnetic resonance (HSQC NMR) characterization of acetylated fir (Abies sachallnensis MAST) wood regenerated from ionic liquid.

An ionic liquid, 1-butyl-3-methylimidazolium chloride ([Bmim]Cl), was used to dissolve Japanese fir (Abies sachallnensis MAST) wood. Milled woods prepared by planetary ball-milling for 8 h dissolved completely in [Bmim]Cl at 100 °C in 2 h. The dissolved woods were then subjected to in situ acetylation, and the fully acetylated woods were regenerated from [Bmim]Cl. (1)H-(13)C correlation heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) experiments were successfully conducted with the acetylated woods in dimethyl sulfoxide (DMSO)-d(6). The acetylated lignin and polysaccharide signals dispersed reasonably well on the 2D spectra. Characterization of the NMR signals for the whole cell-wall components, including lignin, cellulose, and hemicelluloses, was achieved by comparison with isolated lignin and commercial cellulose and hemicelluloses (arabinoxylan, galactomannan, and glucomannan). The procedure used here is applicable for the characterization of cell-wall components in various plant biomasses.

Chemical synthesis of β-O-4 type artificial lignin

An artificial lignin polymer containing only the β-O-4 substructure was synthesized. The procedure consists of two key steps: 1) polycondensation of a brominated monomer by aromatic Williamson reaction; and 2) subsequent reduction of the carbonyl polymer. 13C-NMR and HMQC spectra of the polymer were consistent with β-O-4 substructures in milled wood lignin isolated from Japanese fir wood. The weight average degree of polymerization (DPw) ranged from 19.5 to 30.6, which is comparable to enzymatically synthesized artificial lignin from p-hydroxycinnamyl alcohols (dehydrogenation polymer, DHP) and some isolated lignins. Using this new lignin model polymer, it will now be possible to reinvestigate the properties and reactivity of the main lignin structure in terms of its polymeric character.

Delignification Mechanism during High-Boiling Solvent Pulping.Part 1. Reaction of Guaiacylglycerol-β-Guaiacyl Ether

Summary A phenolic β-O-4 type lignin model compound, guaiacylglycerol-β-guaiacyl ether (1) was treated with 70 wt% aq 1,3-butanediol solution at 160–200°C to investigate the delignification mechanism under HBS (high-boiling solvent) pulping conditions. The following compounds were identified from the reaction products by use of GC-MS: guaiacol (2), coniferyl alcohol (3), γ-etherified coniferyl alcohols (4) and α-etherified guaiacylglycerol-β-guaiacyl ethers (5), but acidolysis products, such as Hibberts ketones were not detected. These results strongly suggest that the phenolic β-O-4 linkage was cleaved homolytically under HBS pulping conditions. The cleavage of β-ether exhibited a pseudo first-order rate behavior. The pseudo first-order rate constants were as follows: k = 0.94 × 10−2 min−1 at 160 °C; k = 1.97 × 10−2 min−1 at 170°C; k = 3.22 × 10−2 min−1 at 180 °C; k = 9.76 x 10−2 min−1 at 200 °C. The activation energy was 98.3 kJmol−1. The formation of higher molecular weight compounds was confirmed by GPC. It is highly probable that the oligomeric products were derived from the recombination of phenoxy radicals formed by homolysis of the β-aryl ether.

Synthesis of β-O-4-type artificial lignin polymers and their analysis by NMR spectroscopy

We describe the synthesis and NMR spectroscopic analysis of three artificial lignin polymers containing only the beta-O-4 substructure: syringyl-type homopolymer, p-hydroxyphenyl-type homopolymer and guaiacyl/syringyl-type heteropolymer. Using gel permeation chromatography, the weight-average degree of polymerization (DP(w)) of the three polymers was determined as 19.2, 38.6, and 13.9, respectively. The polymers were prepared based on the synthetic methodology of guaiacyl-type homopolymer, and were fully characterized using (1)H-, (13)C-, and (1)H-(13)C NMR spectroscopy of the acetylated and non-acetylated forms. The spectra of guaiacyl/syringyl-type heteropolymers were in good agreement with those of the beta-O-4 substructure of milled wood lignin obtained from the hardwood of Japanese white birch.

Dissolution and acetylation of ball-milled birch (Betula platyphylla) and bamboo (Phyllostachys nigra) in the ionic liquid [Bmim]Cl for HSQC NMR analysis

Abstract A method for nuclear magnetic resonance (NMR) characterization of whole cell wall components, including lignin, cellulose and hemicelluloses, was recently developed in our laboratory. The method described for fir (Abies sachalinensis) as a softwood consists of ball-milling of cell wall, dissolution in an ionic liquid 1-butyl-3-methylimidazolium chloride ([Bmim]Cl), in situ acetylation, recovery of the material from the solution, and characterization of the product by 1H-13C correlation heteronuclear single quantum coherence (HSQC) NMR spectroscopy in dimethyl sulfoxide (DMSO)-d6. In the present paper, the performance of the method should be tested for a hardwood and a bamboo. Thus, Japanese white birch (Betula platyphylla) and hachiku bamboo (Phyllostachys nigra) have been investigated. Finely ball-milled birch and bamboo materials were completely dissolved in [Bmim]Cl at 100°C without severe chemical modification of the cell wall components. The dissolved cell walls were then subjected to in situ acetylation, and the ball-milled and fully acetylated cell walls were recovered from [Bmim]Cl. Longer ball-milling time was required for birch and bamboo cell walls, because of the lower solubility of acetylated birch and bamboo materials in DMSO-d6compared to the acetylated fir material. However, HSQC NMR experiments were successfully conducted, and the acetylated whole cell wall components in the birch and bamboo could be fully characterized. This method is applicable for the analysis of cell wall components of various plant biomasses without previous isolation. Further studies are necessary to improve the method.

The reactions of lignin model compounds with hydrogen peroxide at low pH

Summary In peroxymonosulfuric acid bleaching, the presence of hydrogen peroxide is dependent on the reaction conditions and the conversion ratios used to generate the peroxy acid. Substantial amounts of hydrogen peroxide may be present in the reaction system under certain conditions. An understanding of the reactions of hydrogen peroxide under these conditions would be beneficial. Therefore, several simple lignin model compounds were reacted with acidic hydrogen peroxide, pH 1-3, at 70°C. In all cases the phenolic lignin model compounds reacted much faster than their non-phenolic counterparts. In fact, the extent of reaction was very much dependent on the structure of the lignin model compound. The α-hydroxyl compounds, 4-(1-Hydroxy-ethyl)-2-methoxy-phenol and 1-(3,4-Dimethoxy-phenyl)-ethanol, reacted faster than the corresponding α-carbonyl compounds with both reacting much faster than the aromatic compounds, with simple alkyl substituents. A new reaction mechanism for α-hydroxyl compounds is proposed, in which benzyl carbocation formation is followed by nucleophilic addition of hydrogen peroxide. Unlike the mechanisms proposed in the past, no evidence of aromatic hydroxylation via perhydronium ion was observed. The reactivities were very pH dependent, in that higher reactivity was associated with lower pH. Decreasing pH further increased the amount of condensation products identified, such that condensation was competitive with degradation. These condensation reactions were also present under the Caros acid bleaching conditions at pH below 2. However, under all conditions the reactivity of acidic peroxide was found to be much less than that of peroxymonosulfuric acid.

A novel xylogenic suspension culture model for exploring lignification in Phyllostachys bamboo

BackgroundSome prominent cultured plant cell lines, such as the BY-2 cell line of tobacco (Nicotiana tabacum cv. ‘Bright Yellow 2’) and the T87 cell line of Arabidopsis (Arabidopsis thaliana L. Heynh., ecotype Columbia) are used as model plant cells. These suspension cell culture systems are highly applicable for investigating various aspects of plant cell biology. However, no such prominent cultured cell lines exist in bamboo species.ResultsWe standardized a novel xylogenic suspension culture model in order to unveil the process of lignification in living bamboo cells. Initial signs of lignin deposition were able to be observed by a positive phloroglucinol-HCl reaction at day 3 to 5 under lignification conditions (LG), i.e., modified half-strength Murashige and Skoog medium (m1/2MS) containing 10 μM 6-benzyladenine (BA) and 3% sucrose. Two types of xylogenic differentiation, both fiber-like elements (FLEs) with cell wall thickening and tracheary elements (TEs) with formation of perforations in the cell wall, were observed under these conditions. The suspension cells rapidly formed secondary cell wall components that were highly lignified, making up approximately 25% of the cells on a dry weight basis within 2 weeks. Detailed features involved in cell growth, differentiation and death during lignification were characterized by laser scanning microscopic imaging. Changes in transcript levels of xylogenesis-related genes were assessed by RT-PCR, which showed that the transcription of key genes like PAL1, C4H, CCoAOMT, and CCR was induced at day 4 under LG conditions. Furthermore, interunit linkage of lignins was compared between mature bamboo culms and xylogenic suspension cells by heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The presence of the most common interunit linkages, including β-aryl ether (β-O-4), phenylcoumaran (β-5) and resinol (β-β) structures was identified in the bamboo cultured cell lignin (BCCL) by HSQC NMR. In addition to these common features of lignin, several differences in lignin substructures were also found between the BCCL and the bamboo milled wood lignin (BMWL).ConclusionsOur xylogenic suspension culture model could be used for detailed characterization of physiological and molecular biological events in living bamboo cells.

Synthesis of Some Phenylpropanoid Monoglycerides via the Mitsunobu Protocol

Natural monoglycerides of cinnamic, ferulic and p-coumaric acids were synthesized in good to high overall yields from isopropylidene glycerol via the Mitsunobu reaction and further deprotection of the corresponding acetonides with Amberlyst 15. The method avoids the need of protection of the phenolic hydroxyls. During the Mitsunobu esterifications a strong influence of the acid strength on the efficacy and outcome of the reaction was observed.