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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.

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。

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.

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.

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.

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.

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.

Evidence for the presence of lignin-carbohydrate bonds of the benzylic ether type in wood and pulps may be obtained by use of ozonation treatment to selectively degrade the lignin. It was found that the benzyl ether bond in 3-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxy-phenoxy)-3-(methyl-β-d-glucopyranosid-6-O-yl)-1-propanol was rather stable during ozonation in acetic acid-water-methanol 16∶3∶1 at 0°C. The corresponding acid, 3,4-dihydroxy-2-(methyl-β-d-glucopyranosid-6-O-yl)-butanoic acid, was found to be the major reaction product. The use of ozonation followed by acid hydrolysis in connection with studies of lignincarbohydrate linkages is briefly discussed.

High incorporation of D-[U-13C] glucose and L-[indole-2-13C] tryptophan into indocarbazostatin B (2) was observed in a biosynthetic study using a mutant strain, Streptomyces sp. MUV-7-8. The original strain, Streptomyces sp. TA-0403 produced a small amount of indocarbazostatin (1) and indocarbazostatin B (2), which displayed potent biological activities. To facilitate biosynthetic studies, we selected high indocarbazostatin producing mutant strains. The first mutants, Streptomyces sp. MUV-6-83 and MUV-6-17, produced indocarbazostatins C (3) and D (4) as well as 1 and 2. When the production medium was supplemented with D-tryptophan, the MUV-6-17 mutant produced K252c (5), whereas when L-tryptophan was added, it produced K252d (6). On further UV treatment of the mutant strain MUV-6-83, we finally obtained a new mutant producer, Streptomyces sp. MUV-7-8, that produced 2 as a major metabolite with higher productivity. This mutant producer enabled us to do a feeding experiment of the envisioned precursors, glucose and tryptophan.

The reaction of bicyclic terpene alcohols, nopol(2-pinene-10-methanol) Cl J and isoborneol C 2, with paraformaldehyde (PFA) in the presence of acetic anhydride and sodium acetate was investigated, and three novel adducts were isolated by distillation and gas chromatography, respectively. Their structures were assigned on the basis of transesterification, their physical constants, IR, NMR and mass spectra as follows; nopyl acetoacetate C/ a, nopyl dioxymethyleneacetoacetate C/ b, nopyl trioxymethyleneacetoacetate 11 cD, isobornyl acetoacetate C2C a, isobornyl dioxymethyleneacetoacetate C2C b and isobornyltrioxymethyleneacetoacetate C2C c. The reaction of PFA with C 1 yields 11 a, C/ bp and CI c in a ratio of 71: 20: 9, and the reaction of PFA with C2C yields C2C a, C2C b and C2C c in a ratio of 80: 15: 5.

The adducts of homoalloocimenol (1) with methyl acrylate (2) was qllgweq to reac. t, . apd the T ddli6-fsMMwere separated by gas chromatography, and identified as. methyl. tr2 .2: mA ethy.1-1 tus wpt @confirmed on the basis of NMR evidence.iiM=fg-Tb6e n found that the addition reaction gave ortho adducts only and that the adduct have weak woody odor.

Camphene (1) reacts with chloroacetic acid (2) or its polymer, polyacetoxamer; α-chloro, ω-carboxy in the presence of potassium bromide at reflux temperature to give the 2,2-dimethyl-exo-3-hydroxynorbornane-endo-3-propanoic acid lactone and the 2,2-dimethyl-endo-3-hydroxynorbornane-exo-3-propanoic acid lactone in a 50% yield. The synthesis of 2-(8-camphenyl)ethanol from 1 was simplified by utilizing this reaction.

Background Some 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. Results We 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). Conclusions Our xylogenic suspension culture model could be used for detailed characterization of physiological and molecular biological events in living bamboo cells.