Masahiro Hamada

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.

Biosynthesis of Drug Glucuronide Metabolites in the Budding Yeast Saccharomyces cerevisiae

Glucuronidation is one of the most common pathways in mammals for detoxification and elimination of hydrophobic xenobiotic compounds, including many drugs. Metabolites, however, can form active or toxic compounds, such as acyl glucuronides, and their safety assessment is often needed. The absence of efficient means for in vitro synthesis of correct glucuronide metabolites frequently limits such toxicological analyses. To overcome this hurdle we have developed a new approach, the essence of which is a coexpression system containing a human, or another mammalian UDP-glucuronosyltransferases (UGTs), as well as UDP-glucose-6-dehydrogenase (UGDH), within the budding yeast, Saccharomyces cerevisiae. The system was first tested using resting yeast cells coexpressing UGDH and human UGT1A6, 7-hydroxycoumarin as the substrate, in a reaction medium containing 8% glucose, serving as a source of UDP-glucuronic acid. Glucuronides were readily formed and recovered from the medium. Subsequently, by selecting suitable mammalian UGT enzyme for the coexpression system we could obtain the desired glucuronides of various compounds, including molecules with multiple conjugation sites and acyl glucuronides of several carboxylic acid containing drugs, namely, mefenamic acid, flufenamic acid, and zomepirac. In conclusion, a new and flexible yeast system with mammalian UGTs has been developed that exhibits a capacity for efficient production of various glucuronides, including acyl glucuronides.

Synthesis of Cyclic Polyglycerols

Authentic standards of cyclic polyglycerol, with well-defined structures and degrees of polymerization from 3 to 6 cyclic oligomers, have been efficiently synthesized. The intramolecular cyclization conditions gave cyclic glycerols more effectively than intermolecular coupling conditions.

Superior cellulose-protective effects of cosolvent during enhanced dissolution in imidazolium ionic liquid

Abstract 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.

Biomimetic Oxidative Coupling of Sinapyl Acetate by Silver Oxide: Preferential Formation of β-O-4 Type Structures

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.

Molecular weight distributions of acetylated lignocellulosic biomasses recovered from an ionic liquid system.

Abstract 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.

Novel cellulose pretreatment solvent: phosphonium-based amino acid ionic liquid/cosolvent for enhanced enzymatic hydrolysis

Abstract 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.

Role of 2,3-cis Structure of (−) -Epicatechin-3,5-O-digallate in Inhibition of HeLa S3 Cell Proliferation

Flavan-3-ol, which is primarily found in tea, is able to inhibit the proliferation of the human cancer cell line HeLa S3; in this study, we investigate the importance of the 2,3-cis structure in this inhibition. We synthesized six (−)-epicatechin and (+)-catechin analogs modified with a galloyl moiety at either the 3-hydroxyl, 5-hydroxyl, or 3,5-dihydroxyl positions. We then investigated their biological activity, DPPH radical scavenging activity and inhibitory activity on HeLa S3 cell proliferation. Among the six compounds, (−)-epicatechin-3,5-O-digallate showed the strongest inhibitory activity on HeLa S3 cell proliferation, whereas (+)-catechin-3,5-O-digallate was not active. In addition, there is no relation among the cell proliferation inhibitory activity and DPPH radical scavenging activity. Furthermore non-specific BSA binding ability of synthesized compounds was demonstrated. Improved photoaffinity beads method revealed that there is no difference between (−)-epicatechin-3,5-O-digallate and (+)-catechin-3,5-O-digallate on the non-specific BSA absorption. These data indicated that the 2,3-cis structure of flavan-3-ol is essential for the inhibition of HeLa S3 cell proliferation

Lymphatic metabolites of quercetin after intestinal administration of quercetin-3-glucoside and its aglycone in rats

Quercetin is a major flavonoid, present as its glycosidic forms in plant foods. In this study, quercetin-3-glucoside (Q3G) was administered intraduodenally to thoracic lymph-cannulated rats, and its lymphatic transport was investigated. The resulting lymphatic and plasma metabolites were identified with LC-MS/MS and compared with those after administration of quercetin aglycone. The total concentration of quercetin metabolites in the lymph was about four times lower than that in the plasma, and quercetin and its methylated form isorhamnetin were detected as their glucuronides, sulfates and diglucuronides both in the lymph and the plasma after Q3G and quercetin administrations. The lymph levels of the glucuronides after Q3G administration were lower than those after quercetin administration, whereas those in the plasma showed the opposite pattern. Both the lymph and plasma levels of the sulfates after Q3G administration were lower than those after quercetin administration. Some of the intestinal metabolites like quercetin monoglucuronides were transported directly into the lymph and the hepatic metabolites like the diglucuronides were eventually transferred from the plasma into the lymph. These results indicate that the absorbed Q3G is partly transported into the intestinal lymph as quercetin metabolites. Deglycosylation in the enterocyte is also suggested to affect the subsequent metabolic pathways.

A synergetic effect of ionic liquid and microwave irradiation on the acid-catalyzed direct conversion of cellulose into methyl glucopyranoside

Abstract 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.