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Bamboo-cultured cells (BCCs) were produced under three phytohormone conditions. BCC lignin was then isolated and characterized by heteronuclear single-quantum coherence-nuclear magnetic resonance (HSQC-NMR) analysis. HSQC-NMR analysis revealed that all three BCC lignin samples were composed of guaiacyl (G), syringyl (S), oxidized syringyl (S′), and p-hydroxyphenyl (H) units. p-Coumaric acid (pCA) and ferulic acid (FA) were identified as well. Main lignin substructures, including β-O-4, β-5, and β-β, were also detected. However, β-O-4/α-O-4, spirodienone, dibenzodioxocin, or tricin structures were absent in the BCC lignin. The BCC lignin contained higher proportions of H, FA, and β-5 structures, but less proportions of S, S′, and β-O-4 structures than the mature bamboo lignin. The removal of auxin 2,4-dichlorophenoxyacetic acid (2,4-D) from the subculture medium promoted G unit formations. Nevertheless, it suppressed H and pCA unit formations. Cytokinin 6-benzyladenine (BA) promoted H and β-β structure formations as well but suppressed β-O-4 formations than in the mature bamboo and BCC lignin produced under phytohormone free conditions.

A 6.2 m high immature bamboo (Phyllostachys nigra) was divided into seven fractions. The bamboo cell walls and lignin samples from young to old were characterized by 1H-13C correlation heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy both qualitatively and semiquantitatively. Mature bamboo and bamboo shoot samples were used as comparison references. HSQC-NMR analysis proved that cellulose and arabinoxylan have already deposited in bamboo shoot, and cellulose amount increased during growth. Lignin side chain linkage formation started from β-ether (β-O-4), then phenylcoumaran (β-5), and finally resinol (β-β). Ferulic acid and p-coumaric acid (pCA) were formed at the earlier stages in the immature bamboo, and the pCA proportion decreased throughout the lignification process. We propose that the bamboo lignification process is distinct from both woody and other herbaceous plants, where syringyl units deposited at the early stage and polymerized with the β-O-4 linkage. Then guaiacyl units formed gradually, and finally, p-hydroxyphenyl units formed.

Lignin acylated with acetate and/or p-coumarate is common in many herbaceous plants. Herein, the biomimetic oxidation of γ-acylated monolignols with Ag2O was studied to understand the effect of γ-acyl groups on monolignol polymerization. The oxidation of sinapyl acetate gave γ-acylated and α-acylated β-O-4 dimers in 71 and 9.5% yields, respectively. The oxidation of sinapyl p-coumarate produced γ-acylated β-O-4 and γ-acylated tetralin β-β dimers in 53 and 16% yields, respectively. Only the sinapyl alcohol moiety in sinapyl p-coumarate reacted, and the p-coumarate moiety remained unchanged, suggesting that p-coumaric acid is not incorporated into the lignin backbone in the acylated lignins. All of the γ-acylated monolignols used in this study produced the γ-acylated β-O-4 dimers, which suggests that the γ-acylated monolignols act as lignin monomers. The relatively high yields of the β-O-4 dimers indicate that Ag2O oxidation of the monolignols can be used as an easy method for synthesizing the β-O-4 dimer model compounds.

Recently, conversion of lignocellulose into useful substances has attracted increasing attention. In our previous investigations, microcrystalline cellulose was successfully converted to methyl glucopyranosides (MeGlc) by the combined use of ionic liquid (IL) and microwave irradiation under moderate reaction conditions. In this study, lignocelluloses, including softwood, hardwood, and rice straw, were directly converted to methyl glycopyranosides (MG), including MeGlc, methyl mannopyranosides (MeMan), and methyl xylopyranosides (MeXyl) using acid-catalyzed methanolysis under microwave irradiation in ILs. Lignocellulose ball-milling was quite effective as a crucial process of increasing the yield of MG. Under the optimized reaction conditions, the molar yield of MeGlc reached 40% from softwood, which was a comparable yield from microcrystalline cellulose. MeXyl was also obtained in a 48% yield. These results showed that the combination of the dissolution of ball-milled lignocellulose in IL and the microwave-assisted methanolysis was an effective method of converting lignocellulose into a high-value-added substance.

Conversion of lignocellulose into useful chemicals is an important research topic in the area of biomass utilization. In this study, microcrystalline cellulose (MC) was dissolved in a mixed-solvent system containing the ionic liquid (IL) 1-allyl-3-methylimidazolium chloride ([Amim]Cl) and N-methyl-pyrrolidone (NMP), and the cellulose was directly converted into methyl glucoside (MG) by acid-catalyzed methanolysis aided by microwave irradiation (μWIr). Under moderate reaction temperature and pressure, and in the presence of acetyl chloride/methanol (in situ formed HCl) as an acid catalyst, MG was obtained in a 42% yield. In contrast, in the absence of either IL or μWIr, the MG yield was only 5 or 21%, respectively. Both μWIr and the dissolution of cellulose in IL were quite effective for the conversion of cellulose into MG.

Lignin acylated with acetate and/or

A new approach for the enzymatic synthesis of methyl β-d-glucoside was proposed, based on commercially available cellulase and cellulose pretreated with phosphonium-based amino acid ionic liquid/cosolvent. The pretreatments were quite effective and methyl β-d-glucoside was successfully synthesized with 40% yield from cellulose (Avicel) pretreated with tetrabutylphosphonium glycine/dimethyl sulfoxide (DMSO), whereas the yield was only 1.4% from untreated cellulose. Preparative-scale synthesis from 1 g cellulose with a reduced amount of cellulase was also conducted, achieving a 33% isolated yield. Results of additional studies with cellobiose and glucose as substrates have been interpreted as cellulose being first enzymatically hydrolyzed to cellobiose, which then reacted with methanol to produce methyl β-d-glucoside by transglycosylation.

The potential of halogen-free and imidazolium-free phosphonium-based amino acid ionic liquids (AAILs) has been investigated as new solvents for cellulose pretreatment for the subsequent enzymatic hydrolysis of cellulose. AAILs alone did not dissolve cellulose (Avicel), even at 120°C. However, when polar solvents such as dimethylsulfoxide (DMSO) were added as cosolvents, AAILs became an acceptable solvent for cellulose at 30°C. The solubility of cellulose in tetrabutylphosphonium glycine ([TBP][Gly])/cosolvent reached 15%. The enzymatic hydrolysis of cellulose was dramatically enhanced by pretreatment with AAIL/cosolvent, and the glucose yield reached 100% when the novel AAIL tetrabutylphosphonium N,N-dimethylglycine ([TBP][DMGly]) was used in combination with DMSO as cosolvent. The enzymatic conversion of cellulose to glucose in 6% and 13% [TBP][DMGly]/DMSO buffer solutions reached 98% and 79%, respectively. The decrease in cellulase activity owing to residual [TBP][DMGly]/DMSO was not significant. Hence, it is possible to conduct the dissolution and enzymatic hydrolysis of cellulose in a one-batch process in a phosphonium-based AAIL/cosolvent system.

To improve the solubility of cellulose at lower temperatures, several polar organic solvents were examined as cosolvents in imidazolium ionic liquid (IL). All tested cosolvents increased the solubilization efficiency of ILs at lower temperatures. Among these, N-methylimidazole, N-methyl-2-pyrrolidone, and dimethyl sulfoxide (DMSO) were notably efficient; in case of Avicel the solubility was increased, with 12–15% cellulose dissolution in 1-allyl-3-methylimidazolium chloride ([Amim]Cl) at 30°C. IR spectra of the regenerated celluloses from IL/cosolvent systems showed characteristic features of cellulose II and/or amorphous cellulose. Thermogravimetric analyses showed significantly higher thermal stability of regenerated cellulose from [Amim]Cl/DMSO compared with that without DMSO. Moreover, dimethylacetamide (DMAc) increased the solubility of filter paper pulp in 1-ethyl-3-methylimidazolium acetate ([Emim]OAc), with 12% pulp dissolution at 30°C. No decrease in the degree of polymerization (DP) of cellulose was observed with [Emim]OAc/DMAc, whereas 8–9% DP decrease was observed with [Emim]OAc, even at 30°C. These results indicate that some cosolvents including DMSO and DMAc increase solubilization efficiency and have superior cellulose-protective effects during enhanced dissolution in ILs.

Tea catechins such as epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate afford many metabolites, including γ-valerolactone derivatives, through metabolic pathways in the human body. Two of these γ-valerolactone metabolite analogues with a 2′-hydroxyphenyl group were synthesized as optically active authentic standards.

Recently, a method was presented for the dissolution and nuclear magnetic resonance analysis of cell wall components in lignocellulosic biomass, which involves cell wall ball-milling, dissolution in ionic liquids (ILs), in situ acetylation, and the recovery of acetylated materials. However, the dissolution in ILs and the relatively long ball-milling times may partially degrade the plant cell wall components. In the present study, the molecular weight (MW) distributions of acetylated biomasses from fir (Abies sachalinensis), birch (Betula platyphylla), and bamboo (Phyllostachys nigra) recovered from IL systems were examined by size exclusion chromatography. The effects of IL types, cosolvents, dissolution temperatures and times, and ball-milling times were evaluated. The MW of acetylated fir woods recovered from 1-allyl-3-methylimidazolium chloride at 30–80°C or from 1-butyl-3-methylimidazolium chloride at 100°C for 2 h were similar to those materials that were recovered from the N-methylimidazole/dimethyl sulfoxide system. In contrast, a significant decrease in MW was observed with 1-ethyl-3-methylimidazolium acetate ([Emim]OAc) even at 30°C. The degradation of cell wall components in [Emim]OAc was reduced to some extent in the presence of N,N-dimethylacetamide or pyridine. The MW decreased gradually with increased ball-milling time.

Nuclear magnetic resonance (NMR) analysis of entire plant cell walls without isolating their components gained importance over the last decade. Recently, a dissolution method and an NMR analysis of entire plant cell walls were reported, in which finely ball-milled lignocellulosic biomass was dissolved at 100°C in the ionic liquid (IL) 1-butyl-3-methylimidazolium chloride. In the present study, several ILs were examined with or without co-solvent to find milder dissolution conditions to mitigate possible degradation processes. Co-solvents, including N,N-dimethylacetamide, pyridine, and dimethyl sulfoxide, increased the solubilization efficiency of ILs. For example, ball-milled fir wood was completely dissolved in 1-allyl-3-methylimidazolium chloride ([Amim]Cl) at 30°C in the presence of these co-solvents. The heteronuclear single-quantum coherence NMR spectra of acetylated fir, birch, and bamboo cell walls, which were recovered from [Amim]Cl/pyridine (1:1, w/w) solution, had a high analytical power.

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.

Several lignin model polymers and their derivatives comprised exclusively of β-O-4 or 8-O-4' interunitary linkages were synthesized to better understand the relation between the thermal mobility of lignin, in particular, thermal fusibility and its chemical structure; an area of critical importance with respect to the biorefining of woody biomass and the future forest products industry. The phenylethane (C6-C2)-type lignin model (polymer 1) exhibited thermal fusibility, transforming into the rubbery/liquid phase upon exposure to increasing temperature, whereas the phenylpropane (C6-C3)-type model (polymer 2) did not, forming a char at higher temperature. However, modifying the Cγ or 9-carbon in polymer 2 to the corresponding ethyl ester or acetate derivative imparted thermal fusibility into this previously infusible polymer. FT-IR analyses confirmed differences in hydrogen bonding between the two model lignins. Both polymers had weak intramolecular hydrogen bonds, but polymer 2 exhibited stronger intermolecular hydrogen bonding involving the Cγ-hydroxyl group. This intermolecular interaction is responsible for suppressing the thermal mobility of the C6-C3-type model, resulting in the observed infusibility and charring at high temperatures. In fact, the Cγ-hydroxyl group and the corresponding intermolecular hydrogen bonding interactions likely play a dominant role in the infusibility of most native lignins.

The lignin syringyl to guaiacyl (S/G) ratio, the composition of monomeric units, is an important parameter that is widely used to obtain information on the complex polymer structure of lignin present in plant cell walls. We assessed the reliability of time-of-flight secondary ion mass spectrometry (TOF-SIMS) for determining the S/G ratio using monomer, dimer and polymer model compounds of lignin. In the case of the monomer and dimer, the S/G ratios were much higher than the actual molar S/G ratios. In contrast, for the aryl ether 8-O-4'-linked polymers, the S/G ratios estimated were similar to the actual S/G molar ratios, suggesting that the ionization efficiencies of the characteristic ions of S and G units detected from the synthetic lignin polymer compounds were similar. The results suggest that TOF-SIMS is useful for rapid and convenient determination of the lignin S/G ratio in plants.

Catechins and epicatechins are extremely useful compounds in the context of biological activities. These compounds afford many metabolites, including γ-valerolactone derivatives, through metabolic pathways in the human body. Several of these γ-valerolactone metabolites were synthesized as optically pure authentic standards.

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.

Dimeric lignin model compounds with non-phenolic and phenolic moieties and a β -O-4 type artificial lignin polymer were subjected to thioacidolysis to evaluate the reaction efficiency of thioacidolysis for cleavage of β -O-4 interunitary linkage. The obtained yields of thioethylated monomeric products from the dimeric lignin model compounds reached nearly 100% under the conventional condition, whereas that from the artificial lignin polymer was as low as 74%. Neither prolonged reaction time nor increased concentration of ethanethiol (the thioacidolysis reagent) enhanced the resulting monomer yields from the polymer (69–79%). Thioacidolysis of the lignin model compounds followed by HPSEC analysis also showed the dimeric model compounds were degraded almost quantitatively, but that the artificial lignin polymer was not. Thioacidolysis followed by desulfurization gave at least one dimeric product resulting from incomplete β -O-4 cleavage at significant yield. These results suggested the conventional...

リグニンは，樹木の細胞壁中に存在する非常に複雑なバイオポリマーである。これまでにリグニンの部分構造のみを取り出したさまざまなリグニンモデル化合物が合成されている。それらは，木材研究のいろいろな分野に応用され，重要な役割を果たしてきた。本論文では，リグニンモデル化合物の合成に関する最近の成果に注目しながら，リグニンモデル化合物の主な合成方法とその木材研究への応用例について概説する。さらに，筆者らが合成に成功したβ-O-4 型人工リグニンポリマーについても，併せて簡単に紹介する。

We are interested in developing chemically modified linear and cyclic polyglycerols and their esters that have a single polymerization degree and fine structure. Triglyceryl di-, tri-, and tetra-fatty acid esters were synthesized from common substrate as new prominent gelators. The triglyceryl esters were capable of gelling up cooking oils. A comparison of the gelation ability of structurally related compounds clarified that the introduction of alkyl chains of suitable length is required for effective gelation.

Labile (+)-6-tuliposide B, an antimicrobial compound produced by tulip, was synthesized in nine steps from d -glucose via the Baylis–Hillman reaction of 2-(tert-butyldimethylsilyloxy)-acetaldehyde with 6-O-acryloyl-1-O-(2-trimethylsilylethyl)-β- d -glucopyranoside, followed by a mild deprotection procedure using TFA in CH2Cl2.

Guaiacyl, syringyl, and p-hydroxyphenyl-type bromoacetophenone derivatives were synthesized as the starting materials for β-O-4 type artificial lignin polymers. They were prepared from 4-hydroxyacetophenone instead of 4-hydroxybenzaldehyde, which was used in the previous investigations. The new route required 4 reaction steps. Overall yields of the monomers were 60, 79, and 68%, respectively.

Human immunodeficiency virus type 1 (HIV-1) is a cytopathic retrovirus and the primary etiological agent of acquired immunodeficiency syndrome (AIDS) and related disorders. In cells chronically infected with HIV-1, nuclear factor-kappaB (NF-kappaB) activation by external stimuli such as tumor necrosis factor alpha (TNFalpha) augments replication of HIV-1. NF-kappaB involves in many diseases such as inflammation, cancer, and Crohn's disease. In this paper, we exhibit that (i) HIV-1gene expression was inhibited by lignin, (ii) fraction of small molecular mass in HBS lignin (less than 0.5kDa) had stronger inhibitory effects than large molecular mass (more than 1.3kDa), (iii) lignin also inhibited activation of NF-kappaB induced by TNFalpha, (iv) among six lignin dimer-like compounds, compound 6 containing beta-5 bond has more potent inhibitory activity than compounds 1, 2, 3, 4 and 5, which contain beta-1, beta-O-4, 5-5, or beta-beta structural units. These results suggested that small molecules of lignin inhibit HIV-1 replication through suppression of HIV-1 transcription from LTR including activation via NF-kappaB. Low molecular lignin may be a beneficial material or drug leads as a new class for AIDS and NF-kappaB-related diseases.

We present two case studies developed in our laboratory that involve the establishment of efficient plant cell tissue and organ culture (PCTOC) models to explore functional differentiation of cells responsible for cell wall and fiber formation in Phyllostachys and Bambusa bamboo plants and their application. First, we standardized a novel xylogenic suspension culture model in order to understand the process of xylogenic cell differentiation during lignification in living Phyllostachys nigra (Pn). The Pn cells rapidly formed secondary cell wall components that were highly lignified, making up approximately 25 % of the dry weight of the cells under the xylogenic differentiation condition (1/2 MS medium supplemented with 10 μM BA). Two types of xylogenic differentiation were observed-fiber-like elements (FLEs) with cell wall thickening and tracheary elements (TEs). We systematically maintained in vitro node culture stocks of two prominent Bambusa bamboo species: B. multiplex (Bm), which shows a normal ‘hollow’ culm, and B. glaucescens f. houraikomachi (Bg), which has a thick-walled ‘solid’ culm. As node portions have apical and intercalary meristems, they could directly be used as explant sources for the establishment of callus and organ cultures without sterilization. When node portions were cultured on an optimized proliferation medium (MSp680 medium supplemented with 10 μM picloram), active callus induction and organ differentiation were seen. Although calli usually proliferate as irregular tissue masses and vary widely in texture, it is still possible to generate different cell lines such as whitish and greenish callus, and bunches of adventitious roots, under the same medium conditions by carefully separating these cultures from the explant. The growth patterns of these two PCTOC model cultures during maintenance and functional cell differentiation cultures are discussed in this chapter.

Bacterial cellulose (BC)-producing bacterium, Gluconacetobacter xylinus (ATCC53582), was found to move along linear microgrooves of a stripe-patterned cellulosic scaffold. On the basis of this finding, fabrication of honeycomb-patterned BC was attempted by controlling the bacterial movement using a agarose film scaffold with honeycomb-patterned grooves (concave type). The patterned agarose film was prepared by three steps. The first was transcription of a honeycomb-patterned polycaprolactone film template with polydimethyl siloxane. When the bacteria were cultured on the scaffold under atmospheric conditions, only bacterial proliferation was observed. Honeycomb-patterned BC was obtained when cultured under a humid CO 2 atmosphere. Electron diffraction and polarized microscopic observation showed that the patterned BC comprised of the well defined cellulose Iα microfibrils. As another attempt to fabricate honeycomb-patterned BC, the bacteria were cultured on the patterned cellulose and agarose film with convex type of honeycomb. This culture yielded no honeycomb-patterned BC. Therefore, concave type honeycomb scaffold is more suitable to fabricate honeycomb-patterned BC.

Interactions of hemicelluloses, xylan and glucomannan, with monolignols, coniferyl alcohol and sinapyl alcohol, and their glucosides, coniferin and syringin, were investigated by surface plasmon resonance analysis. Hemicelluloses with different aldehyde contents, prepared by sodium periodate oxidation, were immobilized onto carboxymethyl–dextran–coated gold plates by reductive amination. The interaction of the hemicelluloses with monolignols was evaluated as weight gain of the hemicelluloses at neutral pH. Both hemicelluloses adsorbed the monolignols in much larger amounts than the corresponding glucosides. The adsorption tendency was independent of aldehyde content. These results suggest the hemicelluloses have higher affinities to the aglycons than the glucosides. Thus, the interactions of the hemicelluloses with monolignols are primarily the result of hydrophobic interactions. However, binding constants were not estimated because of the low solubility of the monolignols in the aqueous buffered...

A lignin-carbohydrate complex (LCC) fraction isolated from the residue of milled wood lignin extraction of birch was found to form self-aggregates in water. The aggregates had relatively strong hydrophobic domains, comparable to the hydrophobicity of 80% ethanol. Dynamic light scattering analysis showed that the aggregates were transformed into small particles by sonication with a number-average diameter of 43.7±9.6 nm, and were stable in water. The surface pressure (π)-surface area (A) profile showed that the LCC also formed a monolayer on the water surface. Based on these results, we suggest that the LCC is amphiphilic in nature, despite its insolubility in low-polarity organic solvents.

High mass resolution ToF-SIMS spectra by Au + primary ion bombardment were used to investigate exact structures of characteristic ions of lignin in plant cell walls. Previous study using Ga primary ion bombardment showed the characteristic peaks of guaiacyl lignin at m / z 137 ([C 8 H 9 O 2 ] + ) and 151 ([C 8 H 7 O 3 ] + and [C 9 H 11 O 2 ] + ), but it was unclear whether the peak at m / z 151 in the spectrum of lignin in situ in plant cell walls is actually a double-component, [C 8 H 7 O 3 ] + (151.0394) and [C 9 H 11 O 2 ] + (151.0758). This report achieved a higher mass resolution with lignin samples, showing that the peak at m / z 151 is dominated by the C 6 –C 1 benzoyl ion, [C 8 H 7 O 3 ] + , not the C 6 –C 2 ion, [C 9 H 11 O 2 ] + .

The lower critical solution temperature (LCST) of hydroxypropylcellulose bearing lignin (HPC-L) prepared from unbleached pulp depends on the amount of residual lignin. An HPC-L gel having thermal properties reflective of original HPC-L was prepared using ethyleneglycol diglycidylether as a crosslinker, as previously reported [Uraki et al. (2004) Carbohydr. Polym. 58:123–130], and the volume transition temperature was detected as an endothermic peak by differential scanning calorimetry (DSC). The adsorption and release behavior of the guest molecules to/from this gel was then examined. When the adsorption of cationic and anionic guests was compared, cationic methylene blue (MB) was adsorbed in larger amounts than anionic methyl orange (MO). In addition, MB adsorption into the HPC-L gel was greater than MB adsorption into the HPC gel prepared from commercially available HPC. This suggests that residual lignin affects the adsorption of organic dyes. Significant differences were not observed with respect to the release of MB from HPC-L at 38 °C and lower temperatures. In the adsorption of surfactants, marked adsorption at around the critical micelle concentration of the ionic surfactants and gel swelling were observed. Such swelling did not occur in the aqueous nonionic surfactant solution, although the nonionic surfactant was adsorbed into the gel. Gel swelling may have been caused by the electrostatic repulsion of the ionic surfactants adsorbed onto the polymer chains within the gel structure.

Lignin in plant cell walls is a complex, irregular polymer built from phenylpropanoid C6-C3 units that are connected via various C-C and C-O linkages. A recent study using time-of-flight secondary ion mass spectrometry (ToF-SIMS) with Ga primary ion bombardment showed that lignin polymers can be characterized by specific positive ions possessing a substituted aromatic ring (so-called guaiacyl or syringyl rings), which are the basic building units of lignin. To study the relationship between the characteristic ions of lignin and the common interunit linkages, various lignin dimer model compounds were investigated using ToF-SIMS. The resulting dimer spectra showed that the characteristic ions with a guaiacyl ring at m/z 137 and 151 result from rupture of most common interunit linkages, not only 8-O-4' linkages, which are the most abundant in lignin, but also 8-1', 8-5', and 8-8'. There was no evidence of rupture of 5-5' linkages. These results show that ToF-SIMS offers a new tool for the direct analysis of the depolymerized fragments of lignin polymers. The mechanisms for the fragmentation of lignin dimer models in ToF-SIMS were proposed that allow ToF-SIMS fragmentation rules to be deduced. Adduct ions such as [M + 13]+ ([M + CH]+) were also produced in fragmentation of the dimers and are thought to arise from the combination of the molecules with their stable fragments.

Hydroxypropylcellulose bearing lignin (HPC-L) prepared from softwood by the atmospheric acetic acid pulping followed by derivatization showed the lower critical solution temperature (LCST) of approximate 38 °C in 2% aqueous solution, which were 5° lower than that of pure HPC with a similar molecular mass to HPC-L. Two types, urethane and epoxy, of chemical gels were prepared from HPC-L and HPC using hexamethylene diisocyanate and polyethylene glycol diglycidyl ether as crosslinkers, respectively. The urethane-type gels from both HPC's have undergone shrinking at less than 20 °C. This thermoresponse of the gels did not reflect LCST of the sources. Such low temperature shrinking was brought about by the hydrophobicity of crosslinker. On the other hand, the epoxy-type gels showed a very similar volume transition temperature (VTT) upon shrinking to LCST; the epoxy-type HPC-L gel showed VTT at 38 °C, while the corresponding HPC gel had VTT at 46 °C. This difference in VTT as well as LCST was attributable to hydrophobicity of lignin dangled to HPC. Thus, the body temperature-responsive gel can be prepared from HPC-L with the water-soluble crosslinker.

A novel method for analysis of benzylic ether type lignin–carbohydrate bonds has been developed by using model compounds. Four diastereomers of model compound 3-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-3-(methyl β-d-glucopyranoside-6-O-yl)-1-propanol (GGMGP), were ozonized in acetic acid/water/methanol 16 : 3 : 1 for 1 h at 0°C. The product from ozonation of each diastereomer was saponified and the corresponding α-etherified tetronic acid (TAMGP) was isolated using ion exchange chromatography. Minor amounts of methyl β-d-glucopyranoside (MGP) and small amounts of a gluconic acid etherified with tetronic acid (TAGLCA), tetronic acid, gluconic acid, and glyceric acid were detected in the product mixture of ozonated benzylic ether type model compounds. The results suggest that a benzyl ether bond between lignin and carbohydrate is rather stable during the ozone treatment. Acid treatments with sulfuric acid or trifluoroacetic acid of the derived TAMGP led to cleavage of the glucosidic bond but only a small amount of products (tetronic acid and glucose) resulting from cleavage of the α-ether bond were formed. The successful chemical treatments were used for studies of benzylic ether bonds in Japanese red pine. The results suggest the presence of benzylic ether bonds to polysaccharides in the wood.

Summary Atmospheric acetic acid pulp from Japanese white birch was bleached with 1–5% ozone with and without intermediate mild alkaline extraction. Concentrated acetic acid was a more effective bleaching media than water. Mild alkaline extraction of ozone-prebleached pulp was found to be very effective in delignification of the pulp and did not have any undesirable effects on pulp properties. A sufficiently delignified acetic acid pulp was obtained with multi-stage ozone bleaching in Z1Z2Z3 or Z1EZ2EZ3 bleaching sequence with a total ozone consumption of 2.17% on oven-dry pulp. The pulp from a Z1EZ2EZ3 sequence still contained 3.51% acetyl groups, which was 75% of the original amount of acetyl groups in unbleached acetic acid pulp.

Nonphenolic β-aryl ether model compounds, veratrylglycerol-β-guaiacyl ether (1) and its α-ethyl ether (2) were treated in 70% aqueous 1,4-butanediol solutions at 180°C. The reactivity of compound 2 was higher than that of compound 1. Benzyl ether formation with 1,4-butanediol was observed. This suggests the high reactivity of lignin–carbohydrate complex with benzyl ether type under HBS pulping conditions. On the other hand, guaiacol was not detected in the reaction mixture. This indicates that β-aryl ether in nonphenolic structures is inert to HBS pulping conditions. Addition of glucose and acetic acid enhanced the benzyl ether formation with 1,4-butanediol, but not cleavage of β-aryl ethers.

A phenolic β-O-4 type lignin model compound, guaiacylglycerol-β-guaiacyl ether was treated with 70 wt% aqueous 1,3-butanediol solution in the presence of glucose at 160–200°C to investigate the effect of reducing sugars on the degradation of lignin during high-boiling solvent (HBS) pulping. Addition of glucose increased the formation of guaiacol, coniferyl alcohol, and its γ-ethers, and decreased the formation of radical coupling compounds dramatically. These results suggest that reducing sugars may stabilize phenoxy radicals formed by homolysis of phenolic β-ethers. The kinetic studies also revealed that the disappearance of the β-ether model compound was enhanced substantially by the presence of glucose, which suggests that in addition to homolysis of the β-ether, a reducing sugar-assisted β-ether cleavage may be involved under the conditions used.

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 Caro's 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.

To explore further potential applications of acetic acid pulp, an investigation was conducted to develop a direct method for producing cellulose acetate from wood in combination with atmospheric acetic acid pulping. The process consists of delignification, totally chlorine-free bleaching, and esterification, with the concentrated acetic acid aqueous solution being used as only solvent throughout the process. The acetic acid pulp with kappa number of 30 and ISO brightness of 16 was bleached with 5% ozone on pulp to kappa number of 1.4 and brightness of 61. The resulting bleached pulp was further bleached with peracetic acid to kappa number of less than 1.0 and brightness of 68. The final bleached acetic acid pulp was acetylated with acetic anhydride in the concentrated acetic acid for 45 min to produce cellulose acetate with an apparent degree of substitution (DS) of 2.54. Although the product was lower grade compared with commercially available cellulose diacetate because it was prepared from the chemical pulp but not dissolving pulp, the product was almost soluble in acetone. Eventually, the DS of the acetone-soluble fraction was 2.62. The acetone solubility might be attributed to the original acetic acid pulp that had been partially acetylated during the pulping.

Biomimetic oxidations of sinapyl alcohol and sinapyl acetate were carried out with Ag2O to better understand the high frequency of β-O-4 structures in highly acylated natural lignins. The major products from the Ag2O oxidation of sinapyl alcohol were sinapyl aldehyde (14% yield), β-O-4-coupled dimer (32% yield), and β-β-coupled dimer (3% yield). In contrast, the Ag2O oxidation of sinapyl acetate produced β-O-4-coupled dimer in 66% yield. Oligomeric products with predominantly β-O-4 structures were also obtained in 18% yield. The yield of the β-O-4-coupled products from sinapyl acetate was much higher than that from sinapyl alcohol. Computational calculations based on density functional theory showed that the negative charge at Cβ was significantly reduced by the γ-acetyl group. The computational calculations suggest that the Coulombic repulsion between Cβ and O4 in sinapyl acetate radicals was significantly reduced by the γ-acetyl group, contributing to the preferential formation of β-O-4 structures from sinapyl acetate.

Summary The water-soluble fraction (WS) from waste liquor of atmospheric acetic acid pulping was analyzed in order to explore its potential utilization as a carbon source for bacterial cellulose (BC) production. The unpurified WS that contained colored compounds had appreciable antibacterial activity and was therefore not used as a carbon source for BC production with the bacteria Acetobacter xylinum ATCC 10245 and 53582. However, the bacteria produced BC from purified WS of both fir and birch after activated carbon treatment. The yields of BC were much lower compared to those using glucose as a typical carbon source in the Hestrin-Schramm (HS) medium. In order to improve the cellulose production, monosaccharides derived from hemicellulose sugars in WS were modified. The WS from softwood was reduced by catalytic reduction, i.e. mannose, the major component of softwood monosaccharides derived from hemicelluloses, was converted to mannitol. In the case of hardwood WS, xylose was isomerized with glucose isomerase to xylulose. The yield of BC was considerably enhanced by both modifications of the WS. The yield of BC using the modified WS from the pulping of fir was superior to that using glucose as carbon source.

Methylβ-d-xylopyranoside was allowed to react withβ-O-4-type quinone methide without a catalyst to elucidate the reactivities of secondary hydroxyl groups at the C2, C3, and C4 positions. Benzyl ether-type lignin-carbohydrate complex (LCC) compounds linked at the C2 and C4 positions were predominant, at a ratio of 2∶3. However, the reactivity of the hydroxyl group at the C3 position was quite low. These results strongly suggest that the reactivity of the C2 hydroxyl group in xylan toward quinone methide intermediate is higher than that of the C3 hydroxyl group during biosynthesis of LCCs.

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 Hibbert's 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.

In order to develop a new pulping process with non-pollution and saving energy, softwood was subjected to pulping with 60-90% HBSs, such as 1, 4-butanediol, 1, 3-butanediol and tetrahydrofurfuryl alcohol, containing 0-10% AcOH at 200-220°C. Todomatsu (A. Sachalinensis Mast.). ezomatsu (P. Glehnii Mast.), karamatsu (L. leptolepis Gordon) and sugi (Cryptomeria japonica D. Don) were easily pulped with aqueous HBS of 70% such as butanediol-1, 4 containing 5% AcOH at 220°C for 1-2h. AcOH promoted the delignification reaction. HBS such as tetrahydrofurfuryl alcohol. butanediol-1, 3 and proplylene glycol were used with satisfactory results for HBS pulping. RHBS, which was obtained by separation of water insolubles such as HBS lignin and evaporation of water from waste liquor, was able to be reused at least five times as a HBS pulping solvent. The higher yield of HBS lignin than the amount of wood lignin is indicates that modified sugar compounds are recovered with the lignin as water insolubles. Pulp showed the strength properties equivalent to those of kraft pulp except tear index.

Methyl 4-O-ethyl-β-D-glucopyranoside was treated with ozone both in anhydrous CH 2 Cl 2 and in aqueous solutions, to investigate the participation of radical species in the degradation of the polysaccharide during ozone bleaching. The reactivity of C in the model compound during ozonation was compared with that during oxidation with Fenton's reagent. It was found that ozone itself brings about glycosidic bond cleavage with a high degree of selectivity. The degradation of the model compound during ozonation in distilled water is due in part to radical species, and in part to ozone itself. The contribution of radical species to the degradation in distilled water is estimated to be about 40-70 %. Initial high pH and Fe 2+ ions enhanced the degradation of the model compound, and the contribution of radical species increased. However, the degradation of the model compound with radical species generated by Fe 2+ ions can be inhibited by adjusting to pH 2 without the removal of Fe 2+ ions. Vanillyl alcohol also enhanced the degradation of the model compound during ozonation. Radical reactions initiated by vanillyl alcohol participate in the degradation of the model compound even at pH 2. although the additional degradation of the model compound was inhibited by the low pH. Inhibition of the degradation of carbohydrates by radical species is one of the keys to prevent the viscosity drop in ozone bleaching of pulp.

Methyl 4-O-ethyl-β-D-glucopyranoside was used as a model for cellulose, and treated with hydrogen peroxide in the presence of Fe 2+ ions (Fenton's reagent) at room temperature for 2h, to investigate the free-radical reactions of polysaccharides during non-chlorine bleaching. Reaction products were analyzed by gas chromatography. The following products were determined: methyl β-D-glucoside, 4-O-ethyl-D-gluconic acid, gluconic acid. methyl 4-O-ethyl-glucuronoside, methyl glucuronoside, methyl 4-O-ethyl-2-keto-glucoside, methyl 4-O-ethyl-3-keto-glucoside, methyl 4-O-ethyl-6-aldehyde-glucoside. methyl 6-aldehyde-glucoside. 4-O-ethyl-glucose and glucose. The degradation of the model compound and the relative reactivities of the carbon atoms at Cl-C6 positions in the model compound were affected very much by the initial pH and oxygen content. Higher initial pH inhibited the degradation of the model compound. The reactivity at Cl, that is, glycosidic bond cleavage decreased with an increase of initial pH. Oxygen also inhibited drastically the degradation of model compound compared with that under nitrogen. The glycosidic bond cleavage was also inhibited by oxygen. The model compound was recovered unchanged in the presence of Mn 2+ or Cu 2+ instead of Fe 2+ .

Exosite 2 of human thrombin contributes to two opposing pathways, the anticoagulant pathway and the platelet aggregation pathway. We reasoned that an exosite 2 directed allosteric thrombin inhibitor should simultaneously induce anticoagulant and antiplatelet effects. To assess this, we synthesized SbO4L based on the sulfated tyrosine-containing sequence of GPIbα. SbO4L was synthesized in three simple steps in high yield and found to be a highly selective, direct inhibitor of thrombin. Michelis–Menten kinetic studies indicated a noncompetitive mechanism of inhibition. Competitive inhibition studies suggested ideal competition with heparin and glycoprotein Ibα, as predicted. Studies with site-directed mutants of thrombin indicated that SbO4L binds to Arg233, Lys235, and Lys236 of exosite 2. SbO4L prevented thrombin-mediated platelet activation and aggregation as expected on the basis of competition with GPIbα. SbO4L presents a novel paradigm of simultaneous dual anticoagulant and antiplatelet effects achieved through the GPIbα binding site of thrombin.