Researcher Profile and Settings

Master

Affiliation (Master)

• Research Faculty of Agriculture　Fundamental AgriScience Research　Forest Science

Affiliation (Master)

• Research Faculty of Agriculture　Fundamental AgriScience Research　Forest Science

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Profile and Settings

Profile and Settings

• Name (Japanese)

  Shigetomi

• Name (Kana)

  Kengo

• Name

  201301098183942078

Alternate Names

http://kaken.nii.ac.jp/d/r/20547202.ja.html

Achievement

Research Interests

• lignin   tuliposide   muconic acid   Organic synthesis   

Research Areas

• Nanotechnology/Materials / Structural/physical organic chemistry
• Life sciences / Bioorganic chemistry
• Life sciences / Wood science

Research Experience

• 2016/10 - Today 北海道大学大学院農学研究院 基盤研究部門森林科学分野 講師
• 2011/10 - 2016/09 北海道大学 (連合)農学研究科(研究院) 助教
• 2009/04 - 2011/09 Hokkaido university Postdoctoral fellow

Awards

• 2021/12 日本農芸化学会北海道支部 日本農芸化学会支部奨励賞
   構造活性相関による天然有機化合物の機能解明
• 2009 日本農芸化学会北海道支部 学生奨励賞
   

  受賞者: 重冨顕吾
• 2005/03 日本農芸化学会, Frontiersシンポジウム Frontiersシンポジウム 西沢賞
   

  受賞者: 重冨顕吾
• 2004/11 日本木材学会北海道支部 北の木材科学賞
   

  受賞者: 重冨顕吾

Published Papers

• UV irradiation during fluorescence measurements affects the structures and fluorescence properties of isolated lignins in solution

  Keiichiro Takeuchi, Shiori Suzuki, Kengo Shigetomi, Yasumitsu Uraki

  Journal of Wood Chemistry and Technology 1 - 11 0277-3813 2024/09/12
• Swelling of lignin-based gel in salt-containing organic solvents and its application as gel electrolyte

  Fumiya Honda, Shogo Taira, Shiori Suzuki, Kazuhiro Shikinaka, Kengo Shigetomi, Yasumitsu Uraki

  Holzforschung 0018-3830 2023/08/31 [Refereed][Not invited]
   

  Abstract A lignin-based gel prepared by the chemical crosslinking of hardwood acetic acid lignin (AL) with poly(ethylene glycol) diglycidyl ether has been reported to shrink in water and organic solvents but swell specifically in aqueous binary solutions. In this study, the AL-based gel was also found to swell in lithium-salt-containing organic solvents, namely, liquid electrolytes. The uptake of salt solutions reached five times the dry weight of the gel. The ionic conductivity of the gel swollen with 1 M LiBF₄ in propylene carbonate or a mixed solution (1:1, v/v) of ethylene carbonate and dimethyl carbonate exceeded 1 mS cm⁻¹ at room temperature (25 °C), suggesting that this gel can be applied as a gel electrolyte for lithium-ion batteries (LIBs). A prototype LIB was assembled with the AL-based gel electrolyte and LiCoO₂/graphite-based electrodes and exhibited low bulk and charge transfer resistances of 4.1 and 9.7 Ω, respectively. Moreover, its initial specific capacity reached 104 mAh g⁻¹ at a current density of 28 mA g⁻¹, which is comparable to that of a reference LIB assembled using a commercial polyethylene separator. These results indicate the significant potential of this lignin-based gel for application in energy storage devices.
• Assembly of electric double-layer capacitors with hardwood kraft lignin-based electrodes and separator together with ionic liquid electrolyte

  Nutthira Pakkang, Shiori Suzuki, Kengo Shigetomi, Yasumitsu Uraki

  HOLZFORSCHUNG 0018-3830 2022/12 

  This study aimed to assemble a high-performance electric double-layer capacitor (EDLC) using a hardwood kraft lignin (HKL)-based separator and HKL-based electrodes, which were fabricated from a nonwoven mat of electrospun HKL fibers. The separator was prepared by the thermostabilization of the mat derived from a mixed dope of HKL, hexamethylenetetramine, and polyethylene glycol (1.66/0.50/0.09, w/w) for electrospinning. Although a mat-type HKL-based electrode containing conductive carbon black (CB) has been reported to be suitable for a commercial cellulosic separator, this electrode was found to be unsuitable for the HKL-based separator because of its rough surface and poor contact with the separator interface. Hence, a powder-type electrode with a smooth surface was fabricated by grinding the mat, followed by casting with a carboxymethyl cellulose aqueous solution, and its EDLC possessed high energy (49 Wh kg(-1)) and power densities (151 kW kg(-1)). Moreover, to provide a simple process for electrode fabrication, another mat-type electrode was fabricated by adding CB to the mixed dope, followed by electrospinning, carbonization, and steam activation. The resultant EDLC exhibited excellent electrochemical performance with energy (58 Wh kg(-1)) and power densities (55 kW kg(-1)).
• Synthesis of highly deuterated coniferyl alcohol for silencing of NMR signals in the resulting dehydrogenative polymer

  Kengo Shigetomi, Mana Tokizawa, Yasumitsu Uraki

  Journal of Wood Science 68 (1) 1435-0211 2022/12 [Refereed]
   

  Abstract To establish a facile methodology for the elucidation of the lignin chain-growth mechanism, the preparation of monolignol that does not show NMR signals in the dehydrogenative polymer (DHP) was attempted. As a monolignol of which aliphatic moieties were deuterated, coniferyl alcohol-d₇ was successfully synthesized from protocatechualdehyde and malonic acid via the modified Knoevenagel–Doebner reaction and the Luche reduction. The process achieved high to excellent deuteration efficiencies at the aimed positions (i.e., methoxy: > 99%D, α: > 99%D, β: 92%D, and γ: 98%D). DHP was prepared solely from coniferyl alcohol-d₇, and its NMR spectra were compared with those from coniferyl alcohol. The results indicated that: (1) the deuterium atoms at methoxy group, α- and β-positions were highly retained even in the DHP, and their signals were effectively suppressed; (2) a part of the deuterium at γ-position was replaced with H through the reaction; (3) meanwhile, the formation of γ-CH₂ was negligible. This study demonstrated that coniferyl alcohol-d₇ could “silence” the majority of the signals even when converted to DHP. Highly deuterated monolignols can be a unique molecular tool that can differentiate the signals of interest from those derived from monolignols.
• Effects of hemicelluloses on dehydrogenative polymerization of monolignols with cationic cell wall-bound peroxidase

  Yan Lyu, Shiori Suzuki, Hiroki Nagano, Kengo Shigetomi, Yutaka Tamai, Yuji Tsutsumi, Yasumitsu Uraki

  Carbohydrate Polymers 301 120305 - 120305 0144-8617 2022/11/07 [Refereed]
  
• Stretched lignin/polyacrylonitrile blended carbon nanofiber as high conductive electrode in electric double layer capacitor

  Manish Kumar, Shogo Taira, Nutthira Pakkang, Kengo Shigetomi, Yasumitsu Uraki

  Advances in Natural Sciences: Nanoscience and Nanotechnology 13 (2) 2022/06 [Refereed][Not invited]
   

  In this study, lignin-based conducting carbon nanofiber mat was prepared by electrospinning followed by a thermal treatment. Lignin is a sustainable carbon precursor. Polyacrylonitrile (PAN) acts as a binder polymer, which increases the viscosity of the lignin solution using dimethylformamide solvent and helps in the formation of a stable nanofiber. The mixture solution was electrospun, followed by stabilisation and carbonisation to yield carbon nanofibers (CNFs). A fixed amount of external load was provided to the lignin fiber mat during the stabilisation procedure and then carbonised to yield stretched carbon nanofibers (S-CNFs). On stretching the mat, surface conductivity was enhanced by 3 times, and the surface area by 1.3 times compared to that of non-stretched CNFs. Finally, the electric double layer capacitor (EDLC) was assembled with the resulting (CNFs and S-CNFs) nanofiber mat using 6 M of KOH aqueous solution. S-CNFs mat exhibits a specific capacitance of 266 F g−1, which was higher than that of CNFs, i.e. 258 F g−1 at a scan rate of 5 mVs−1
• A NEW TEMPLATE OF MITSUNOBU ACYLATE CLEAVABLE IN NON-ALKALINE CONDITIONS

  Yoshimichi Sakakibara, Yusuke Sai, Yasumitsu Uraki, Makoto Ubukata, Kengo Shigetomi

  Heterocycles 104 (1) 140 - 158 0385-5414 2021/10 [Refereed]
   

  The Mitsunobu inversion is one of the reliable methods for stereospecific substitution of chiral alcohols, but its deacylation step has limited the substrate scope. Here, we propose a new template of the Mitsunobu acylate that can be deacylated in non-alkaline treatments. The 3,4-dihydroxy-2-methylenebutanoate was selected as a template structure, and its acetonide- or bisTBS derivatives were synthesized. The latter especially showed excellent inversion efficiency (up to >99% ee) and good elimination performance for a series of secondary alcohols in near-neutral conditions. The results demonstrated the applicability of the new template for the substrates labile in alkaline conditions, such as alpha-hydroxyesters.
• Influences of polysaccharides in wood cell walls on lignification in vitro

  Yan Lyu, Tatsuya Matsumoto, Shogo Taira, Kazumasa Ijiri, Arata Yoshinaga, Kengo Shigetomi, Yasumitsu Uraki

  Cellulose 28 (15) 9907 - 9917 0969-0239 2021/10 [Refereed][Not invited]
   

  To elucidate the effects of polysaccharides, cellulose, water-soluble xylan (WXY), galactoglucomannan (GGM) and xyloglucan (XG) on lignification in vitro, artificial polysaccharide matrices were prepared from a combination of cellulose and hemicelluloses, and dehydrogenation polymer (DHP) was synthesized from coniferyl alcohol in the presence of the matrices by using horseradish peroxidase (HRP). Prior to DHP formation, interactions between cellulose and hemicelluloses were investigated with equilibrium adsorptions of the hemicelluloses on bacterial cellulose (BC) films and with quartz crystal microbalance with dissipation technique (QCM-D) to determine their adsorption on cellulose nanofibers (CNFs). Both analyses showed that the order of adsorption amounts was XG > GGM > WXY. The QCM-D experiments also suggested that HRP strongly interacted with cellulose rather than hemicelluloses. The amount of DHP generated in the XG-BC matrix was the largest among the prepared matrices, and XG facilitated the formation of 5–5′ interunitary linkages. Thus, XG must be involved in the lignification in primary wood cell wall. On the other hand, the amount of DHP in the GGM-BC matrix was the smallest, indicating that GGM hampered lignification. Graphic abstract: [Figure not available: see fulltext.]
• Characterization of a lignin-based gel responsive to aqueous binary solvents and pH

  Shogo Taira, Yuriko Usukura, Mitsukuni Nishida, Kengo Shigetomi, Yasumitsu Uraki

  Holzforschung 75 (8) 765 - 772 0018-3830 2021/08/01 [Refereed][Not invited]
   

  A lignin-based gel (AL-PE gel) was obtained from hardwood acetic acid lignin (AL) and poly(ethylene glycol) diglycidyl ether (PE) as a cross-linker at a high AL concentration, while the reaction at a lower AL concentration yielded an amphipathic derivative (am-AL-PE). The gel has been reported to swell in aqueous ethanol but shrink in pure water and ethanol. In the present work, swelling behaviors in other aqueous binary solvents and the swelling mechanism were investigated to explore novel lignin-based functional materials, such as stimuli-and/or environment-responsive gels. The AL-PE gel swelled in aqueous methanol, isopropanol, acetone, and tetrahydrofuran, and the order of swelling in the solvents was consistent with that of the am-AL-PE. Spin-spin relaxation time (T2) measurements with 1H NMR analysis of the gel in aqueous acetone revealed that gel swelling was closely related to an increasing T2 of acetone bound to the gel network. The nature of the lignin moiety in the gel also enabled a pH response, and the amphipathic nature of the gel provided it with a function as an absorbent for cationic surfactants. The results of this study can contribute to the valorization of lignin as a main component for solvent sensors and environmental purification materials.
• Direct deuteration of hinokitiol and its mechanistic study.

  Yoshimichi Sakakibara, Kota Osada, Yasumitsu Uraki, Makoto Ubukata, Kengo Shigetomi

  Bioscience, biotechnology, and biochemistry 85 (2) 215 - 222 2021/02/18 [Refereed][Not invited]
   

  Hinokitiol has a broad antibacterial activity against bacteria and fungi. While its biosynthetic pathway has been intensively studied, its dynamics in natural environments, such as biodegradation pathway, remain unclear. In this study, the authors report a direct deuterium labeling of hinokitiol as a traceable molecular probe to serve those studies. Hinokitiol was subjected to the H2-Pd/C-D2O conditions and deuterated hinokitiol was obtained with excellent deuteration efficiencies and in moderate yield. The 1H and 2H NMR spectra indicated that all ring- and aliphatic hydrogens except that on C-6 were substituted by deuterium. According to the substrate scope and computational chemistry, deuteration on tropolone ring was suggested to proceed via D+-mediated process, and which was supported by the results of the experiment with trifluoroacetic acid and Pd(TPP)4. On the other hand, the deuteration on aliphatic group was predicted to be catalyzed by Pd(II) species.
• Preparation of kraft lignin-based activated carbon fiber electrodes for electric double layer capacitors using an ionic liquid electrolyte

  Nutthira Pakkang, Manish Kumar, Shogo Taira, Keiichi Koda, Kengo Shigetomi, Yasumitsu Uraki

  Holzforschung 74 (6) 577 - 588 0018-3830 2020/06/25 [Refereed][Not invited]
   

  Abstract This article demonstrates the development of activated carbon fiber electrodes produced from hardwood kraft lignin (HKL) to fabricate electric double layer capacitors (EDLCs) with high energy and power densities using an ionic liquid (IL) electrolyte. A mixture solution of HKL, polyethylene glycol as a sacrificial polymer, and hexamethylenetetramine as a crosslinker in dimethylformamide/acetic acid (6/4) was electrospun, and the obtained fibers were easily thermostabilized, followed by carbonization and steam activation to yield activated carbon fibers (ACFs). The electrochemical performance of EDLCs assembled with the ACFs, 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIBF₄) as an IL electrolyte and a cellulosic separator was insufficient due to the low conductivity of the electrode. The conductivity of the electrode was improved successfully by spraying conductive carbon black (CB) onto the fibers mat during electrospinning. The CB containing electrodes with improved conductivity gave the resulting EDLCs a higher electrochemical performance, with an energy density of 91.5 Wh kg⁻¹ and a power density of 76.2 kW kg⁻¹.
• A branched structure provides kraft lignins a denser morphology and a high molar mass for a given hydrodynamic radius

  Linping Wang, Kengo Shigetomi, Keiichi Koda, Aori Gele, Yasumitsu Uraki

  HOLZFORSCHUNG 74 (6) 551 - 558 0018-3830 2020/06 [Refereed][Not invited]
   

  Size exclusion chromatography with multi-angle laser light-scattering detectors was applied for acetylated softwood and hardwood kraft lignins (Ac-SKL and Ac-HKL) and 8-O-4' type of linear polymeric lignin model (Ac-M-8O4') to compare their swelling behaviors. The plot of molar mass (MM) vs. retention time for Ac-M-8O4' was similar to that of polystyrene, which revealed that Ac-M8O4' exhibited swelling behavior that was similar to that of polystyrene. However, the MM values of both Ac-KLs were larger than those of polystyrene standards at any retention time. This difference indicated that both Ac-KLs had a more compact structure than those of polystyrene and Ac-M-8O4'. One hypothesis is that the larger MM of both Ac-KLs stems from their branched structures. To verify this hypothesis, the frequency of 5-5' interunit linkage in lignin samples was determined by H-1 NMR after nitrobenzene oxidation. A linear relationship between MM and 5-5' abundance was observed in the high MM region.
• Comparison of Dehydrogenation Polymers by Commercial Enzymes, Laccase from Rhus vernicifera and Horseradish Peroxidase

  Tatsuya Matsumoto, Keiichi Koda, Kengo Shigetomi, Manish Kumar, Yasumitsu Uraki

  Lignin 1 20 - 28 2020/04 [Refereed][Not invited]
  
• Isolation, structure determination and structure–activity relationship of anti-toxoplasma triterpenoids from Quercus crispula Blume outer bark

  Marina Endo, Kengo Shigetomi, Shinya Mitsuhashi, Makoto Igarashi, Makoto Ubukata

  Journal of Wood Science 65 (1) 1435-0211 2019/12 [Refereed][Not invited]
   

  Toxoplasma gondii is an intracellular protozoan parasite of which infection can result in serious symptoms for fetuses or people who have weakened immune system. In our effort to discover novel anti-toxoplasma substances from tree barks, only outer bark extract from Quercus crispula Blume (mizunara) was revealed to show potent anti-toxoplasma activity. Isolation of the active principles was performed to identify three pentacyclic triterpenoids, namely 29-norlupane-3,20-dione, oleanolic acid acetate and ursolic acid acetate. These structures were determined by combining a series of spectral data, computational simulation and synthetic approach. All isolated compounds exhibited notable activities at IC50 of 6.8-24.4M and high selectivities against the parasite. The structure-activity relationship study conducted to probe key structure(s) indicated that the lack of free hydroxyl group at 3-position highly contributes to both the titer of activity and the selectivity. Moreover, skeleton and functionalities of E-rings were also suggested to affect to the activity. The present study demonstrated not only that the extract from Q. crispula Blume could be a promising source of toxoplasmacidal agent, but also that related extractive triterpenoids can be modified to furnish anti-toxoplasma activity.
• Novel adenosine-derived inhibitors of Cryptosporidium parvum inosine 5'-monophosphate dehydrogenase.

  Kengo Shigetomi, Albertus Eka Yudistira Sarwono, Satoshi Ichikawa, Makoto Ubukata

  The Journal of antibiotics 72 (12) 934 - 942 0021-8820 2019/12 [Refereed][Not invited]
   

  We have found cyclophane-type adenosine derivatives having p-quinone amide moieties (1 and 2) as weak inhibitors of Cryptosporidium parvum inosine 5'-monophosphate dehydrogenase (CpIMPDH) from the Hokkaido University Chemical Library via the luciferase-based high-throughput screening. To obtain more potent inhibitors, we synthesized four new derivatives free from cyclophane rings (3-6). The N-H derivatives 3 and 5 showed more potent activities (24.4 and 11.1 μM, respectively) in the presence of dithiothreitol (DTT), whereas the N-methyl derivative 4 indicated more potent activity (2.1 μM) without DTT. Conformational analysis of compounds 3 and 4 suggested that N-H amide 3 binds to IMP-binding site in the DTT mediated manner.
• Repurposing existing drugs: identification of irreversible IMPDH inhibitors by high-throughput screening.

  Albertus Eka Yudistira Sarwono, Shinya Mitsuhashi, Mohammad Hazzaz Bin Kabir, Kengo Shigetomi, Tadashi Okada, Fumina Ohsaka, Satoko Otsuguro, Katsumi Maenaka, Makoto Igarashi, Kentaro Kato, Makoto Ubukata

  Journal of enzyme inhibition and medicinal chemistry 34 (1) 171 - 178 1475-6366 2019/12 [Refereed][Not invited]
   

  Inosine 5'-monophosphate dehydrogenase (IMPDH) is an essential enzyme for the production of guanine nucleotides. Disruption of IMPDH activity has been explored as a therapeutic strategy for numerous purposes, such as for anticancer, immunosuppression, antiviral, and antimicrobial therapy. In the present study, we established a luciferase-based high-throughput screening system to identify IMPDH inhibitors from our chemical library of known bioactive small molecules. The screening of 1400 compounds resulted in the discovery of three irreversible inhibitors: disulfiram, bronopol, and ebselen. Each compound has a distinct chemical moiety that differs from other reported IMPDH inhibitors. Further evaluation revealed that these compounds are potent inhibitors of IMPDHs with kon values of 0.7 × 104 to 9.3 × 104 M-1·s-1. Both disulfiram and bronopol exerted similar degree of inhibition to protozoan and mammalian IMPDHs. Ebselen showed an intriguing difference in mode of inhibition for different IMPDHs, with reversible and irreversible inhibition to each Cryptosporidium parvum IMPDH and human IMPDH type II, respectively. In the preliminary efficacy experiment against cryptosporidiosis in severe combined immunodeficiency (SCID) mouse, a decrease in the number of oocyst shed was observed upon the oral administration of disulfiram and bronopol, providing an early clinical proof-of-concept for further utilization of these compounds as IMPDH inhibitors.
• Novel autophagy modulators: Design and synthesis of (+)-epogymnolactam analogues and structure-activity relationship.

  Kazuki Ueda, Yuji Okado, Kengo Shigetomi, Makoto Ubukata

  Bioorganic & medicinal chemistry 26 (18) 5159 - 5168 0968-0896 2018/10/01 [Refereed][Not invited]
   

  (+)-Epogymnolactam (1) was discovered as a novel autophagy inducer from a culture of Gymnopus sp. in our laboratory. To determine structure-activity relationships among (+)-epogymnolactam analogues comparing with cerulenin (2), we synthesized 5 analogues including (-)-epogymnolactam (3) having each different functional group, and 3 analogues with different side-chain lengths. Five analogues, 3, 4, 5, 6, and 7 did not significantly increase the ratio of LC3-II to LC3-I as an autophagy marker in NIH3T3 cells. These results suggest that presence and stereochemistry of (2R,3S)-epoxy group and cyclic syn-form (1b) of 1 are important for the activity as autophagy inducer. Hexyl analogue (8) as well as 1 having butyl side-chain dose-dependently increased the ratio of LC3-II to LC3-I, whereas octyl analogue (9) and 2 rather decreased the ratio. Decyl analogue (10) did not give a change in the ratio. Although 8 seemed to be an excellent autophagy inducer, it dose-dependently increased SQSTM1 (p62) as in the case of 2, whereas 1 showed a slight dose-dependent decrease of p62 as an index of autophagic protein degradation. These observations suggest that 8 is an autophagy modulator with different molecular target from 1 or 2.
• Inhibitory activities of tree bark extracts against AGEs formation and their correlation with phenolic components

  Kengo Shigetomi, Naoki Okada, Takashi Kumaki, Shinya Mitsuhashi, Makoto Ubukata

  Wood Science and Technology 52 (3) 873 - 886 0043-7719 2018/05/01 [Refereed][Not invited]
   

  Advanced glycation end-products (AGEs) are one of the pivotal factors that cause a variety of diabetes complications. To explore a new class of natural resources that can prevent AGEs formation, the inhibitory activities of tree bark extracts against AGEs formation were evaluated. Most of the crude extracts of inner or outer bark showed equal or superior inhibitory activities to aminoguanidine, a conventional AGEs formation inhibitor. The quantification of phenolic components of each bark extract revealed that proanthocyanidins were one of the principal factors for inhibition, whereas flavonoids, such as flavones/flavonols and flavanones/dihydroflavonols were not a major factor. This study focused on the outer bark extract from Betula platyphylla which showed exceptionally high activity despite its low contents of total phenolics and proanthocyanidins. The bioassay-guided isolation revealed that betulin caffeate and its two derivatives were active principles in extract of B. platyphylla. These compounds exhibited up to ninefold higher activity than methyl caffeate, which suggested that betulin moiety plays a synergistic role in the activity. These results demonstrated the potency of bark extract as the screening source of AGEs inhibitors and also provided a new insight for the exploration of anti-AGEs substances from natural resources.
• Identification and characterization of guanosine 5'-monophosphate reductase of Trypanosoma congolense as a drug target.

  Albertus Eka Yudistira Sarwono, Keisuke Suganuma, Shinya Mitsuhashi, Tadashi Okada, Simon Peter Musinguzi, Kengo Shigetomi, Noboru Inoue, Makoto Ubukata

  Parasitology international 66 (5) 537 - 544 1383-5769 2017/10 [Refereed][Not invited]
   

  Trypanosoma congolense is one of the most prevalent pathogens which causes trypanosomosis in African animals, resulting in a significant economic loss. In its life cycle, T. congolense is incapable of synthesizing purine nucleotides via a de novo pathway, and thus relies on a salvage pathway to survive. In this study, we identified a gene from T. congolense, TcIL3000_5_1940, as a guanosine 5'-monophosphate reductase (GMPR), an enzyme that modulates the concentration of intracellular guanosine in the pathogen. The recombinant protein was expressed in Escherichia coli, and the gene product was enzymatically confirmed as a unique GMPR, designated as rTcGMPR. This enzyme was constitutively expressed in glycosomes at all of the parasite's developmental stages similar to other purine nucleotide metabolic enzymes. Mycophenolic acid (MPA) was found to inhibit rTcGMPR activity. Hence, it is a potential lead compound for the design of trypanocidal agents, specifically GMPR inhibitor.
• Quercetin inhibits advanced glycation end product formation via chelating metal ions, trapping methylglyoxal, and trapping reactive oxygen species

  Mohammad Nazrul Islam Bhuiyan, Shinya Mitsuhashi, Kengo Shigetomi, Makoto Ubukata

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 81 (5) 882 - 890 0916-8451 2017/05 [Refereed][Not invited]
   

  Physiological concentration of Mg2+, Cu2+, and Zn2+ accelerated AGE formation only in glucose-mediated conditions, which was effectively inhibited by chelating ligands. Only quercetin (10) inhibited MGO-mediated AGE formation as well as glucose- and ribose-mediated AGE formation among 10 polyphenols (1-10) tested. We performed an additional structure-activity relationship (SAR) study on flavanols (10, 11, 12, 13, and 14). Morin (12) and kaempherol (14) showed inhibitory activity against MGO-mediated AGE formation, whereas rutin (11) and fisetin (13) did not. These observations indicate that 3,5,7,4-tetrahydroxy and 4-keto groups of 10 are important to yield newly revised mono-MGO adducts (16 and 17) and di-MGO adduct (18) having cyclic hemiacetals, while 3-hydroxy group is not essential. We propose here a comprehensive inhibitory mechanism of 10 against AGE formation including chelation effect, trapping of MGO, and trapping of reactive oxygen species (ROS), which leads to oxidative degradation of 18 to 3,4-dihydroxybenzoic acid (15) and other fragments.
• GROWTH MECHANISM OF UNCULTURED ACTINOBACTERIAL STRAIN LEUCOBACTER sp ASN212 BY ZINC COPROPORPHYRIN

  Ryogo Takai, Kengo Shigetomi, Yoichi Kamagata, Makoto Ubukata

  HETEROCYCLES 95 (1) 145 - 151 0385-5414 2017/01 [Refereed][Not invited]
   

  We propose HemH as a primary target of Zn coproporphyrin, which stimulates the growth of uncultured Leucobacter sp. ASN212, based on the structure-activity relationship for transition-metal complexes of coproporphyrin.
• Zincmethylphyrins and coproporphyrins, novel growth factors released by Sphingopyxis sp., enable laboratory cultivation of previously uncultured Leucobacter sp. through interspecies mutualism.

  Mohammad Nazrul Islam Bhuiyan, Ryogo Takai, Shinya Mitsuhashi, Kengo Shigetomi, Yasuhiro Tanaka, Yoichi Kamagata, Makoto Ubukata

  The Journal of antibiotics 69 (2) 97 - 103 0021-8820 2016/02 [Refereed][Not invited]
   

  We have identified coproporphyrins including structurally new zincmethylphyrins I and III as growth factors A-F for the previously uncultured bacterial strain, Leucobacter sp. ASN212, from a supernatant of 210 l of Sphingopyxis sp. GF9 culture. Growth factors A-F induced significant growth of strain ASN212 at the concentrations of picomolar to nanomolar which would otherwise be unculturable in liquid medium or on agar plate. More interestingly, we found that the growth factors functioned as self-toxic compounds for the growth-factor producing strain GF9 at the picomolar to nanomolar levels. As a variety of bacteria could potentially produce coproporphyrins, our findings suggest that these compounds function as a novel class of signal molecules across a boundary at phylum level in the complex bacterial communities.
• Isolation, structure elucidation, and structure-activity relationship of (+)-epogymnolactam

  Okado Yuji, Ueda Kazuki, Shindo Chihaya, Mitsuhashi Shinya, Shigetomi Kengo, Makoto Ubukata

  Symposium on the Chemistry of Natural Products, symposium papers 天然有機化合物討論会実行委員会 58 Poster44  2016 

  Autophagy is one of the major eukaryotic intracellular degradation systems in which damaged organelles and protein aggregates are eliminated in order to maintain cytoplasmic homeostasis. This degradation pathway is involved in various human disorders, including neurodegenerative diseases, cancers and infectious diseases. We screened wild mushrooms growing in Hokkaido for novel autophagy inducers, which would help understanding of a regulation of autophagy in human diseases and provide new insight into the development of autophagy-targeted drugs. Among tested mushrooms, a cluture broth of Gymnopus sp. showed remarkable autophagy inducing activity. Subsequent bioassay-guided fractionation afforded a new active compound, which exists as a mixture of three tautomers, a keto-form and two hydroxylactam-forms in various NMR solvents. The compound was named as (+)-epogymnolactam (1) after dominant tautomers. Its structure was elucidated by such instrumental analyses as HR-ESI-MS, NMR and specific optical rotation. To evaluate biological properties of 1, we conducted the first total synthesis of 1. Optically pure 1 was synthesized in 8 steps from cis-4-benzyloxy-2-buten-1-ol. The high enantiopurity was successfully obtained by a tandem strategy, Sharpless epoxidation and lipase kinetic resolution. Interestingly, whereas synthetic 1 exhibited autophagy-inducing activity, a structural analog of 1, (+)-cerulenin exhibited inhibitory actibity against autophagy. To explore further sturucture-activity relationship, we have conducted synthetic studies of various other analogs of 1. The syntheses of (‒)-epogymnolactam (ent-1), O-methyl analog (1a) and N,N-dimethyl analog (1b) were already achieved. The syntheses of a series of side-chain analogs at C-4 position and SAR are currently underway.
• First total synthesis of (+)-epogymnolactam, a novel autophagy inducer.

  Yuji Okado, Kengo Shigetomi, Shinya Mitsuhashi, Makoto Ubukata

  The Journal of antibiotics 68 (12) 721 - 4 0021-8820 2015/12 [Refereed][Not invited]
   

  A novel autophagy inducer, (+)-epogymnolactam (1), was first synthesized from cis-4-benzyloxy-2-butene-1-ol (2) in eight steps. A reliable preparation of optically pure epoxy alcohol (+)-3 from monobenzyl derivative (2) was established by a tandem strategy, Sharpless epoxidation/lipase kinetic resolution.
• (+)-Epogymnolactam, a novel autophagy inducer from mycelial culture of Gymnopus sp.

  Shinya Mitsuhashi, Chihaya Shindo, Kengo Shigetomi, Toshizumi Miyamoto, Makoto Ubukata

  Phytochemistry 114 163 - 7 0031-9422 2015/06 [Refereed][Not invited]
   

  Mushrooms, including Ganoderma lucidum, have been used as a potential source of therapeutic compounds, and an autophagy inducer would be useful for treatment of diverse diseases in human. Reported here is a full account of screening, isolation, structural determination, and biological activity of an autophagy inducer, (+)-epogymnolactam (1) from a mycelial culture of a Gymnopus sp. strain. Its structure was elucidated by HR-ESI-MS, NMR, and its plus sign by specific rotation. It exists as a tautomeric mixture of 1a and 1b in MeOH. The major tautomer of 1 is (1R,5S)-4-butyl-4-hydroxy-3-aza-6-oxa-bicyclo[3.1.0]hexan-2-one (1a), and the minor tautomeric form is (2R,3S)-3-pentanoyloxirane-2-carboxamide (1b).
• Discovery of N-(2,3,5-triazoyl)mycophenolic amide and mycophenolic epoxyketone as novel inhibitors of human IMPDH.

  Kazuhiro Sunohara, Shinya Mitsuhashi, Kengo Shigetomi, Makoto Ubukata

  Bioorganic & medicinal chemistry letters 23 (18) 5140 - 4 0960-894X 2013/09/15 [Refereed][Not invited]
   

  Syntheses of ten derivatives of mycophenolic acid (MPA) at C-6' position, and structure-activity relationship study among these derivatives, MPA and mycophenolic hydroxamic acid (MPHA) led to discovery of N-(2,3,5-triazolyl)mycophenolic amide 4, (7'S) mycophenolic epoxyketone 9 and (7'R) mycophenolic epoxyketone 10 having potent inhibitory activity against human inosine-5'-monophosphate dehydrogenase (IMPDH) type I and II as well as antiproliferative activity on human leukemia K562 cells. Compounds 4, 9, and 10 showed induction activity of erythroid differentiation in K562 cells. Inhibitory effects of 4 and 10 against IMPDH were attenuated by supplemental guanosine in K562 cells. In contrast, attenuation effect by supplemental guanosine was not significant in the case of 9. Compound 9 weakly inhibited the enzyme activity of HDAC in the nuclear lysate of K562 cells at 10 μM. These observations suggest that the primary target of 4, 9, and 10 is IMPDH, whereas compound 9 partially inhibits a certain type of HDAC.
• MurA as a primary target of tulipalin B and 6-tuliposide B.

  Kengo Shigetomi, Sanne Harder Olesen, Yan Yang, Shinya Mitsuhashi, Ernst Schönbrunn, Makoto Ubukata

  Bioscience, biotechnology, and biochemistry 77 (12) 2517 - 9 0916-8451 2013 [Refereed][Not invited]
   

  (-)-Tulipalin B and (+)-6-tuliposide B were confirmed to inhibit MurA in vitro. However, contrary to fosfomycin, these compounds showed potent inhibitory activities against MurA overexpressing Escherichia coli, especially in the presence of UDP-GlcNAc. These observations suggest that these compounds induced bacterial cell death not through a MurA malfunction, but in such a MurA-mediated indirect manner as the inhibition of other Mur enzymes.
• Maillard reaction inhibitors produced by Paecilomyces sp.

  Daxin Li, Kengo Shigetomi, Shinya Mitsuhashi, Makoto Ubukata

  Bioscience, biotechnology, and biochemistry 77 (12) 2499 - 501 0916-8451 2013 [Refereed][Not invited]
   

  Maillard reaction inhibitors could be useful therapeutics for diabetes and other age-related diseases. We isolated for the first time 4-O-demethylsilvaticol (1) and (-)-mitorubrin (2) as Maillard reaction inhibitors from Paecilomyces sp. 3193B. Among the isolated inhibitors, 2 showed most potent inhibitory effect by an SDS-PAGE assay on cross-linked protein formation and by a fluorescent assay on AGE formation.
• Cleavage of α-dicarbonyl compounds by terpene hydroperoxide.

  Ryu-ichiro Nagamatsu, Shinya Mitsuhashi, Kengo Shigetomi, Makoto Ubukata

  Bioscience, biotechnology, and biochemistry 76 (10) 1904 - 8 0916-8451 2012 [Refereed][Not invited]
   

  The highly reactive α-dicarbonyl compounds, glyoxal, methylglyoxal (MGO), and 3-deoxyglucosone, react with the amino groups of proteins to form advanced glycation end-products (AGEs) which have been implicated in diabetic complications, aging, and Alzheimer's disease. We found that a test sample of terpinen-4-ol (T4) containing hydroperoxides showed cleaving activity toward an α-dicarbonyl compound, but that the freshly isolated pure sample did not. Prepared terpinen-4-ol hydroperoxide (T4-H) also efficiently cleaved the C-C bond of the α-dicarbonyl compounds via Baeyer-Villiger-like rearrangement and subsequent hydrolysis of an acid anhydride moiety in the rearranged product to give carboxylic acids. Other terpene hydroperoxides, as well as T4-H, showed significant cleaving activities, and all these hydroperoxides protected RNase A from the lowering of enzyme activity induced by MGO. The cleaving mechanism via Baeyer-Villiger-like rearrangement was confirmed by time-interval NMR measurements of the reaction mixture of the symmetrical α-dicarbonyl compound, diacetyl with T4-H.
• Absolute configurations of (-)-hirsutanol A and (-)-hirsutanol C produced by Gloeostereum incarnatum.

  Ryo Asai, Shinya Mitsuhashi, Kengo Shigetomi, Toshizumi Miyamoto, Makoto Ubukata

  The Journal of antibiotics 64 (10) 693 - 696 0021-8820 2011/10 [Refereed][Not invited]
  
• Asymmetric total synthesis of 6-Tuliposide B and its biological activities against tulip pathogenic fungi.

  Kengo Shigetomi, Shoko Omoto, Yasuo Kato, Makoto Ubukata

  Bioscience, biotechnology, and biochemistry 75 (4) 718 - 22 0916-8451 2011 [Refereed][Not invited]
   

  The structure-activity relationship was investigated to evaluate the antifungal activities of tuliposides and tulipalins against tulip pathogenic fungi. 6-Tuliposide B was effectively synthesized via the asymmetric Baylis-Hillman reaction. Tuliposides and tulipalins showed antifungal activities against most of the strains tested at high concentrations (2.5 mM), while Botrytis tulipae was resistant to tuliposides. Tulipalin formation was involved in the antifungal activity, tulipalin A showed higher inhibitory activity than 6-tuliposide B and tulipalin B. Both the tuliposides and tulipalins showed pigment-inducing activity against Gibberella zeae and inhibitory activity against Fusarium oxysporum f. sp tulipae. These activities were induced at a much lower concentration (0.05 mM) than the antifungal MIC values.
• The antibacterial properties of 6-tuliposide B. Synthesis of 6-tuliposide B analogues and structure-activity relationship.

  Kengo Shigetomi, Kazuaki Shoji, Shinya Mitsuhashi, Makoto Ubukata

  Phytochemistry 71 (2-3) 312 - 24 0031-9422 2010/02 [Refereed][Not invited]
   

  6-Tuliposide B is a secondary metabolite occurring specifically in tulip anthers. Recently, a potent antibacterial activity of 6-tuliposide B has been reported. However, its molecular target has not yet been established, nor its action mechanism. To shed light on such issues, 6-tuliposide B and tulipalin B analogues were synthesized and a structure-activity relationship (SAR) was examined using a broad panel of bacterial strains. As the results of SAR among a total of 25 compounds, only tulipalin B and the compounds having 3',4'-dihydroxy-2'-methylenebutanoate (DHMB) moieties showed any significant antibacterial activity. Moreover, the 3'R analogues of these compounds displayed essentially the same activities as 6-tuliposide B and the structure of the 3'R-DMBA moiety was the same as that of the proposed active moiety of cnicin. These results suggest that 6-tuliposide B has the same action mechanism as proposed for cnicin and bacterial MurA is one of the major molecular targets of 6-tuliposide B.
• Purification and characterization of a tuliposide-converting enzyme from bulbs of Tulipa gesneriana.

  Yasuo Kato, Kazuaki Shoji, Makoto Ubukata, Kengo Shigetomi, Yukio Sato, Noriyuki Nakajima, Shinjiro Ogita

  Bioscience, biotechnology, and biochemistry 73 (8) 1895 - 7 0916-8451 2009/08 [Refereed][Not invited]
   

  An enzyme that catalyzes the stoichiometric conversion of 6-tuliposide into tulipalin was purified and characterized from bulbs of Tulipa gesneriana. The enzyme appeared to be a dimer, the relative molecular mass (Mr) of each subunit being 34,900; it had maximum activity and stability at neutral pH and moderate temperature. The enzyme preferentially acted on such glucose esters as 6-tuliposides, and to a lesser extent on p-nitrophenylacetate.
• First total synthesis of 6-tuliposide B

  Kengo Shigetomi, Takao Kishimoto, Kazuaki Shoji, Makoto Ubukata

  TETRAHEDRON-ASYMMETRY 19 (12) 1444 - 1449 0957-4166 2008/06 [Refereed][Not invited]
   

  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)-beta-D-glucopyranoside, followed by a mild deprotection procedure using TFA in CH(2)Cl(2). (C) 2008 Elsevier Ltd. All rights reserved.
• Synthesis of tulipalin B and 1-O-methyl-6-tuliposide B

  Kengo Shigetomi, Takao Kishimoto, Kazuaki Shoji, Makoto Ubukata

  HETEROCYCLES 69 (1) 63 - + 0385-5414 2006/12 [Refereed][Not invited]
   

  Toward the total synthesis of 6-tuliposide B, facile synthesis of tulipalin B and 1-O-methyl-6-tuliposide B (Methyl 6-O-((S)-3',4'-dihydroxy-2'-methylenebutanoyl)-beta-D-glucopyranoside) has been achieved.

MISC

• Artifactual lipid coatings on intervessel pit membranes in dried xylem tissues of some angiosperms

  Shohei Yamagishi, Kengo Shigetomi, Syunya Fujiyasu, Dan Aoki, Tetsuro Uno, Kazuhiko Fukushima, Yuzou Sano  IAWA Journal  6-  (3)  1  -19  2021/05  [Refereed][Not invited]
   

  Intervessel pit membranes are recognized as key structures for influencing water flow/embolism resistance. The mechanisms remain largely unclear owing to difficulties in examining them intact in nature. This study investigates ethanol-extractable pit membrane incrustations (PMIs), which were previously reported in certain angiosperms and may affect their water conduction. The presence of PMIs was determined for 40 angiosperms by field-emission scanning electron microscopy (FE-SEM). Candidate components of PMIs were determined by chemical analyses of wood extracts, and their distributions in the xylem were examined by time-of-flight secondary ion mass spectrometry (TOF-SIMS). Cryo-TOF-SIMS and cryo-FE-SEM were also performed to clarify the native distribution of PMIs. PMIs were observed in 11 species. Some of them were categorized as fat trees, which are known to store abundant lipids. Tilia japonica sapwood displaying PMIs contained large amounts of lipids, which were distributed in the dried xylem tissue, consistent with the distribution of the PMIs. In the frozen samples of T. japonica, however, the distributions were restricted to the parenchyma. In conclusion, PMIs consist of an artifactual coating of lipids originated from the parenchyma in dried samples at room temperature. Researchers performing surface analyses of plant cell walls should take strong precautions against such self-coating by these intrinsic chemicals.
• Hinokitiolの直接重水素標識とその反応機構

  榊原義道, 長田晃汰, 浦木康光, 生方信, 重冨顕吾  日本木材学会大会研究発表要旨集(完全版)(CD-ROM)  70th-  2020
• Coproporphyrins, novel interphylum growth factors released by Sphingopyxis sp., enable laboratory cultivation of previously uncultured Leucobacter sp.

  Ryogo Takai, Nazrul Bhuiyan, Kengo Shigetomi, Shinya Mitsuhashi, Yoichi Kamagata, Makoto Ubukata  ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY  257-  2019/03
• 難培養放線菌Leucobacter sp. ASN212株が要求する増殖因子の探索 : 難培養微生物を培養可能にする新たな増殖因子の発見

  TAKAI Ryogo, SHIGETOMI Kengo, UBUKATA Makoto  KAGAKU TO SEIBUTSU  57-  (3)  143  -145  2019  [Refereed][Not invited]
  
• 白色腐朽菌はリグニンを“食べる”のか?

  永井翔龍, 重冨顕吾, 生方信  日本木材学会北海道支部講演集(Web)  (51)  2019
• 白色腐朽菌における低分子芳香族化合物の資化に関する研究

  永井翔龍, 重冨顕吾, 生方信  日本木材学会大会研究発表要旨集(完全版)(CD-ROM)  69th-  2019
• ヒノキチオール耐性菌における生分解性の検証

  長田晃汰, 重冨顕吾, 生方信  日本木材学会大会研究発表要旨集(完全版)(CD-ROM)  69th-  2019
• ミズナラ外樹皮由来の抗トキソプラズマ活性化合物の単離・構造決定

  遠藤麻理奈, 重冨顕吾, 五十嵐慎, 生方信  日本木材学会大会研究発表要旨集(完全版)(CD-ROM)  68th-  2018
• キラルなβ-O-4リグニンポリマーの合成研究

  鈴木薫平, 重冨顕吾, 生方信  日本木材学会大会研究発表要旨集(完全版)(CD-ROM)  68th-  2018
• リグニン分解酵素生産誘導化合物の探索へ向けたペルオキシダーゼ特異的検出法の確立

  落合崇浩, 重冨顕吾, 生方信  日本木材学会大会研究発表要旨集(完全版)(CD-ROM)  68th-  2018
• 難培養放線菌Leucobactersp.212株のZn coproporphyrin類による増殖機構の探索

  高井亮吾, BHUIYAN Mohammad Nazrul Islam, 重冨顕吾, 鎌形洋一, 生方信  日本農芸化学会大会講演要旨集(Web)  2017-  2017
• 白色腐朽菌による低分子芳香族資化に関する研究

  永井翔龍, 重冨顕吾, 生方信  日本木材学会北海道支部講演集(Web)  (49)  2017
• 新規オートファジー誘導物質(+)-エポジムノラクタムの構造活性相関

  岡戸祐治, 上田一貴, 重冨顕吾, 生方信  日本農芸化学会大会講演要旨集(Web)  2017-  2017
• ミズナラ外樹皮由来の抗トキソプラズマ活性物質の単離・構造決定

  遠藤麻理奈, 重冨顕吾, 三橋進也, 五十嵐慎, 生方信  日本農芸化学会大会講演要旨集(Web)  2016-  2016
• (+)-Epogymnolactamの類縁体合成ならびにオートファジー誘導活性評価

  上田一貴, 岡戸祐治, 重冨顕吾, 生方信  日本農芸化学会大会講演要旨集(Web)  2016-  2016
• Leucobacter属ASN212株に対する増殖活性因子としてのporphyrin類金属錯体の構造活性相関研究

  高井亮吾, ISLAM Bhuiyan Mohammad Nazrul, 重冨顕吾, 鎌形洋一, 生方信  日本農芸化学会大会講演要旨集(Web)  2016-  2016
• キラルなβ-O-4リグニンポリマーの合成に関する検討

  鈴木薫平, 重冨顕吾, 生方信  日本木材学会北海道支部講演集(Web)  (48)  2016
• 計算化学によるリグニン局所構造の解明-リグニン分子内でhole transferは起きうるか-

  重冨顕吾, 生方信  日本木材学会北海道支部講演集(Web)  (48)  2016
• オートファジー誘導物質(+)-epogymnolactamの類縁体合成および活性評価

  上田一貴, 岡戸祐治, 重冨顕吾, 生方信  日本木材学会北海道支部講演集(Web)  (48)  2016
• リグニン分解系酵素の発現にプロテインホスファターゼは関与するか?

  落合崇浩, 重冨顕吾, 生方信  日本木材学会北海道支部講演集(Web)  (48)  2016
• 新規オートファジー誘導物質(+)-エポジムノラクタムの単離・構造決定及び構造活性相関

  岡戸祐治, 上田一貴, 新藤千波耶, 三橋進也, 重冨顕吾, 生方信  天然有機化合物討論会講演要旨集(Web)  58th-  2016
• ミズナラ外樹皮由来のトキソプラズマ症治療薬シードの探索

  遠藤麻理奈, 重冨顕吾, 三橋進也, 五十嵐慎, 生方信  日本木材学会北海道支部講演集(Web)  (47)  P-1 (WEB ONLY)  2015/11  [Not refereed][Not invited]
  
• Sphingomonas sp.の異種バクテリアに対する増殖因子の探索

  高井亮吾, 重冨顕吾, BHUIYAN Mohammad, Nazrul Islam, 三橋進也, 鎌形洋一, 生方信  日本農芸化学会大会講演要旨集(Web)  2015-  3E23P18 (WEB ONLY)  2015/03/05  [Not refereed][Not invited]
  
• (-)-Epogymnolactamの合成研究

  上田一貴, 岡戸祐治, 重冨顕吾, 生方信  日本木材学会北海道支部講演集(Web)  (47)  2015
• Gymnopus sp.由来の(+)‐Epogymnolactamの全合成

  岡戸祐治, 重冨顕吾, 三橋進也, 生方信  日本木材学会北海道支部講演集(Web)  (46)  O-4 (WEB ONLY)  2014/11  [Not refereed][Not invited]
  
• Gymnopus sp.油来の(+)‐Epogymnolactamの全合成

  岡戸祐治, 重冨顕吾, 三橋進也, 生方信  日本農芸化学会北海道支部講演会講演要旨  2014-  23  2014/09/22  [Not refereed][Not invited]
  
• Tautomycetinの合成研究―C6‐C11セグメントの構築―

  井上周也, 重冨顕吾, 生方信  日本農芸化学会北海道支部講演会講演要旨  2014-  23  2014/09/22  [Not refereed][Not invited]
  
• ルシフェラーゼを利用したT.congolense HTS系の確立と道産木材樹皮からの新規抗トリパノソーマ化合物探索

  菅沼啓輔, 三橋進也, PUTTIK Allamanda, SARWONO Yudistir, 岡田只士, 重冨顕吾, 河津信一郎, 生方信, 井上昇  日本獣医学会学術集会講演要旨集  157th-  362  2014/08/11  [Not refereed][Not invited]
  
• シラカンバ外樹皮からのAGEs形成阻害物質の探索

  岡田直己, 重冨顕吾, 三橋進也, 生方信  日本農芸化学会大会講演要旨集(Web)  2014-  4A06A15 (WEB ONLY)  2014/03/05  [Not refereed][Not invited]
  
• Sphingomonas sp.GF9株の生産するCatellibacterium nectariphilum AST4^T株生育因子の単離

  三橋進也, BHUIYAN Mohammad, Nazrul Islam, 重冨顕吾, 高井亮吾, 鎌形洋一, 生方信  日本農芸化学会大会講演要旨集(Web)  2014-  2A09P13 (WEB ONLY)  2014/03/05  [Not refereed][Not invited]
  
• 新規オートファジー誘導物質(+)‐エポジムノラクタムの構造と機能

  新藤千波耶, 三橋進也, 重冨顕吾, 生方信  日本農芸化学会大会講演要旨集(Web)  2014-  2A06P17 (WEB ONLY)  2014/03/05  [Not refereed][Not invited]
  
• Gymnopus sp.由来の(+)‐Epogymnolactamの合成研究

  岡戸祐治, 重冨顕吾, 生方信  日本木材学会大会研究発表要旨集(完全版)(CD-ROM)  64th-  ROMBUNNO.M14-05-1600  2014/03/03  [Not refereed][Not invited]
  
• シラカンバ外樹皮からの終末糖化産物(AGEs)形成阻害成分の探索

  岡田直己, 久間木孝史, 三橋進也, 重冨顕吾, 生方信  日本木材学会北海道支部講演集(Web)  (45)  P-7 (WEB ONLY)  2013/11  [Not refereed][Not invited]
  
• Tautomycetinの合成研究(6)

  重冨顕吾, 今村龍太郎, 生方信  日本農芸化学会大会講演要旨集(Web)  2013-  3C25P09 (WEB ONLY)  2013/03/05  [Not refereed][Not invited]
  
• 4-Hydroxybutanoate鎖を応用した光延反応アシロキシドナーの開発検討

  佐井勇介, 重冨顕吾, 生方信  日本農芸化学会北海道支部講演会講演要旨  2013-  2013
• 4-Hydroxybutanoate鎖を応用した光延反応アシロキシドナーの開発検討

  佐井勇介, 重冨顕吾, 生方信  日本農芸化学会大会講演要旨集(Web)  2013-  2013
• 樹皮のフェノール性抽出成分とAGEs形成阻害活性との相関に関する研究

  久間木孝史, 三橋進也, 重冨顕吾, 生方信  日本木材学会大会研究発表要旨集(完全版)(CD-ROM)  63rd-  ROMBUNNO.M28-P-PM06  2013  [Not refereed][Not invited]
  
• ミコフェノール酸6’位誘導体化による新規HDAC阻害剤開発の検討

  春原和宏, 重冨顕吾, 三橋進也, 生方信  日本農芸化学会北海道支部講演会講演要旨  2012-  18  2012/11/01  [Not refereed][Not invited]
  
• AGE形成阻害剤のスクリーニング系に関する研究

  三橋進也, LI Daxin, 重冨顕吾, 生方信  日本農芸化学会大会講演要旨集(Web)  2012-  3A06P19 (WEB ONLY)  2012/03/05  [Not refereed][Not invited]
  
• ミコフェノール酸6’位誘導体化による新規HDAC阻害剤開発の検討

  春原和宏, 重冨顕吾, 三橋進也, 生方信  日本農芸化学会大会講演要旨集(Web)  2012-  4A07A03 (WEB ONLY)  2012/03/05  [Not refereed][Not invited]
  
• Tautomycetinの合成研究(5)

  今村龍太郎, 重冨顕吾, 生方信  日本農芸化学会大会講演要旨集(Web)  2012-  2A07P13 (WEB ONLY)  2012/03/05  [Not refereed][Not invited]
  
• P-37 Synthetic Study of Tautomycetin(Poster Presentation)

  Imamura Ryutaro, Shigetomi Kengo, Ubukata Makoto  Symposium on the Chemistry of Natural Products, symposium papers  54-  375  -380  2012  

  [Background] In 1989, Isono and co-workers reported the isolation of tautomycetin (TC, 1), which had potent activity against Sclerotina sclerotirum, from a culture of soil acitinomycete, Streptmyces griseochromogenes. Later, the antibiotic was found to induce the morphological change (bleb formation) of human leukemia cells K562, and affect to the production of secondary metabolites in Penicillium urtcae. It was revealed that TC is a specific inhibitor of serine/threonine protein phosphatase 1 (PP1). Here, we report synthetic study of four segments of TC and its analog demethyl phenyl tautomycetin. [Optimization of boron aldol reaction] In the course of our synthetic study of TC, we planed to use borone aldol reaction as a key reaction. First we attempted to synthesize model compounds (8 and 9) for optimization of aldol reaction conditions to obtain the aldol adducts with high yield and diastereometric excess (Scheme 2). The desired aldol adduct (17) was obtained in 96%yield and 90%de by DIPOTf mediated enolization for 30min at -78℃ and then for lh at 0℃ (Table2, entry2). [Synthesis of each segment] Segment A: We completed synthesis of Segment A from acetylene dicarboxylic acid dimethyl ester (18) in 8 steps including two key reactions, regioselective nucleophilic addition reaction and asymmetric reduction by (+)-DIPCl (70%ee) (Scheme 3). Segment B and C: Segment B and C were effectively led from the key compound (25) that resembles the model compound (8) of aldol reaction (Scheme 4). Segment D: Chiral dicarboxylic acid was converted into bromide (30) in good yield. And Weinreb amide (34) as another fragment was synthesized from methyl chloroformate (31) in 3steps and 45% overall yield (Scheme 5). Segment D': Bromide(37) was easily obtained from commercially available acetate(35). The organolithium compound generated from 37 was condensed with benzaldehyde to give alcohol (38). TES protection of 38 followed by ozonolysis afforded Segment D' (7)(Scheme 6). Thus we have developed synthetic routes to four segments of TC. The coupling of each segments are under way using Mistunobu reaction and Honor-Emmons reaction.
• Growth promoting compound for Catellibacterium nectariphilum produced by Sphingomonas sp. (2)

  重冨顕吾, BOEY Andrew, 三橋進也, 鎌形洋一, 生方信, 田中靖浩, 玉木秀幸  日本木材学会大会研究発表要旨集(完全版)(CD-ROM)  62nd-  ROMBUNNO.M15-P-PM02  2012  [Not refereed][Not invited]
  
• Tautomycetinの合成研究(3)

  今村龍太郎, 重冨顕吾, 佐藤文彦, 生方信  日本農芸化学会大会講演要旨集  2011-  33  2011/03/05  [Not refereed][Not invited]
  
• ミコフェノール酸6’位誘導体化による新規HDAC阻害剤開発の検討

  春原和宏, 重冨顕吾, 三橋進也, 生方信  日本農芸化学会大会講演要旨集  2011-  22  2011/03/05  [Not refereed][Not invited]
  
• Tuliposide類・tulipalin類の抗菌活性並びに抗真菌活性

  重冨顕吾, 三橋進也, 加藤康夫, 生方信  日本農芸化学会大会講演要旨集  2011-  180  2011/03/05  [Not refereed][Not invited]
  
• 土壌放線菌からの新規プロテインホスファターゼ阻害物質の探索

  柳田進太郎, 三橋進也, 重冨顕吾, 生方信  日本木材学会北海道支部講演集(Web)  (42)  P-4 (WEB ONLY)  2010/11  [Not refereed][Not invited]
  
• 6‐Tuliposide Bの全合成並びに構造活性相関研究

  重冨顕吾, 三橋進也, 加藤康夫, 生方信  日本農薬学会大会講演要旨集  35th-  54  2010/04/21  [Not refereed][Not invited]
  
• Tautomycetinの合成研究(2)

  重冨顕吾, 佐藤文彦, 生方信  日本農芸化学会大会講演要旨集  2010-  145  2010/03/05  [Not refereed][Not invited]
  
• チューリップ組織由来のチューリッポシドB変換酵素

  黒田穂, 庄司和明, 生方信, 重冨顕吾, 野村泰治, 荻田信二郎, 加藤康夫  日本農芸化学会大会講演要旨集  2010-  2010
• プロテインホスファターゼ1型(PP1)阻害剤トウトマイセチンの分子機構

  三橋進也, LI Ying, 佐藤文彦, 重冨顕吾, 生方信  天然有機化合物討論会講演要旨集  51st-  (51)  659  -663  2009/09/01  [Not refereed][Not invited]
   

  Protein phosphatase type1 (PP1), together with protein phosphatase 2A (PP2A), is a major serine/threonine protein phosphatase (PP). Preiously, we reported that tautomycetin (TC) is a specific inhibitor of PP1 using cell extracts and purified enzymes. Then we demonstrated that treatment of COS-7 cells with 5μM TC for 5h inhibited endogenous PP1 by more than 90% without affecting PP2A activity, showing that TC preferentially inhibits PP1 in the cells. Using of tautomycetin, we found that inhibition of PP1 in COS-7 cells by TC specifically suppresses activation of ERK without affecting those of JNK and p38. The TC-mediated inhibition of PP1 also suppressed activation of Raf-1 and B-Raf. Furthermore, a physical interaction between PP1C and Raf-1 was observed. We were also interested in role of PP1 in TNF-α-induced signaling. We show that TC specifically inhibits activation of NF-κB among the three TNF-α effectors (NF-κB, JNK and caspase). Specifically, TC-treatment suppressed activation of IKK. Co-immunoprecipitation experiment showed that PP1C physically interacted with IKK complex. Pull-down analysis using recombinant GST-TNF-α, showed that PP1C was recruited to TNFR1 together with IKK complex, RIP and TAK1 upon stimulus. Ours is the first demonstration that PP1 positively regulates the TNF-α-induced NF-κB pathway at the level of IKK activation. Constitutively activation of Raf/ERK and/or NF-κB pathway was observed in several tumor cells, and it is thought EGF/Raf inhibitor and NF-κB inhibitor could be used for cancer therapy. Thus, TC might be a potential therapeutic reagent for cancer.
• 6‐Tuliposide Bの類縁体合成と構造活性相関研究

  重冨顕吾, 三橋進也, 荘司和明, 生方信  日本農芸化学会大会講演要旨集  2009-  24  2009/03/05  [Not refereed][Not invited]
  
• 6‐Tuliposide Bの不斉合成と構造―抗真菌活性相関

  大本笑子, 重冨顕吾, 加藤康夫, 生方信  日本農芸化学会大会講演要旨集  2009-  24  2009/03/05  [Not refereed][Not invited]
  
• チューリップ花弁,葯中におけるチューリッポシド変換酵素

  加藤康夫, 生方信, 重冨顕吾, 荘司和明, 荻田信二郎, 佐藤幸生, 中島範行  日本農芸化学会大会講演要旨集  2009-  2009
• PP1阻害剤Tautomycetinの合成研究

  佐藤文彦, 大本笑子, 重冨顕吾, 生方信  万有生命科学振興国際交流財団札幌シンポジウム  21st-  2009
• 6‐Tuliposide B並びに類縁体の合成と生物活性

  重冨顕吾, 三橋進也, 荘司和明, 生方信  万有生命科学振興国際交流財団札幌シンポジウム  20th-  48  2008/07/05  [Not refereed][Not invited]
  
• 抗菌活性物質6‐tuliposide Bの全合成

  重冨顕吾, 岸本崇生, 荘司和明, 生方信  日本農芸化学会大会講演要旨集  2008-  17  2008/03/05  [Not refereed][Not invited]
  
• 6‐Tuliposide Bの不斉合成研究

  大本笑子, 重冨顕吾, 生方信  日本農芸化学会北海道支部・日本土壌肥料学会北海道支部・日本生物工学会北日本支部・日本応用糖質科学会北海道支部・北海道農芸化学協会合同学術講演会講演要旨  2008-  25  2008  [Not refereed][Not invited]
  
• P-64 Total Synthesis of 6-Tuliposide B

  Shigetomi Kengo, Kishimoto Takao, Shoji Kazuaki, Ubukata Makoto  天然有機化合物討論会講演要旨集  (49)  667  -672  2007/08/24  [Not refereed][Not invited]
   

  6-Tuliposide B, a secondary metabolite of tulip cultivars was recently revealed to have antimicrobial activity and be specifically distributed in anthers. These observations suggest that the production of 6-tuliposide B is a novel defense strategy in tulips to protect pollens from bacterial pollution1. To investigate the molecular property and biological activity of this natural product, we undertook total synthesis of 6-tuliposide B (1) using Baylis-Hillman (BH) reaction as a key step. 6-Tuliposide B was synthesized via BH reaction of tert-butyldimethylsilyloxy-acetaldehyde (2) and D-glucose-derived sugaracrylate (10c), followed by TFA treatment. In BH reaction, triethylsilyl-protected sugaracrylate (3) provided no BH adduct even after 20 days probably because of its low affinity with catalyst. On the other hand, sugaracrylate (10c) with free hydroxyl group afforded a BH adduct (13c) after 40 hours in 32 % yield with 44 %d.e.. It was considered that remote asymmetric induction in this reaction was originated from interaction between catalyst and chiral sugar moiety having free hydroxy group.
• 抗菌活性物質6-tuliposide Bの合成研究(4)

  重冨顕吾, 岸本崇生, 荘司和明, 生方信  日本農芸化学会大会講演要旨集  2007-  2007
• Synthetic study of antimicrobial substance, 6-tuliposide B

  重冨顕吾, 岸本崇生, 荘司和明, 生方信  薬学雑誌  126-  (Suppl.5)  196  -199  2006/11/01  [Not refereed][Not invited]
  
• チューリッポシドBの合成研究(3)

  重冨顕吾, 岸本崇生, 荘司和明, 下坪訓次, 生方信  日本農芸化学会大会講演要旨集  2006-  16  2006/03/05  [Not refereed][Not invited]
  
• 風倒木の有効利用―北大ポプラ並木のパルプ化を事例として―

  重冨顕吾, 中村淳, 岸本崇生, 浦木康光, 生方信  日本木材学会北海道支部講演集  (37)  17  -20  2005/10/21  [Not refereed][Not invited]
  
• チューリッポシドBの合成研究(2)

  重冨顕吾, 岸本崇生, 荘司和明, 下坪訓次, 生方信  日本農芸化学会大会講演要旨集  2005-  20  2005/03/05  [Not refereed][Not invited]
  
• チューリッポシドBの合成研究

  重冨顕吾, 岸本崇生, 荘司和明, 佐伯嘉彦, 生方信  日本農芸化学会大会講演要旨集  2004-  276  2004/03/05  [Not refereed][Not invited]
  
• Baylis-Hillman反応を用いた生理活性物質の合成研究(3)

  重冨顕吾, 岸本崇生, 生方信, 荘司和明, 下坪訓次  日本木材学会北海道支部講演集  (36)  2004

Research Projects

• Clarification of propagation reaction for lignin formation by using artificial polysaccharide matrix

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)

  Date (from‐to) : 2021/04 -2026/03 

  Author : 浦木 康光, 堤 祐司, 玉井 裕, 綿岡 勲, 鈴木 栞, 重冨 顕吾

   

  当該年度は、3つの課題について検討を行い、以下の成果を得た。 1. rCWPO-Cの高純度化と多量発現：組換体大腸菌によるタンパク質の発現と精製工程にいて、TAPS化とリフォールディングの間に遠心分離を2回の行う操作を加えた。この結果、精製rCWPO-Cの収率を維持したまま、ペルオキシダーゼの精製度を示すRZ値が約1.5倍に増加し、比活性も約1.5倍の560μmol/min/mgまで向上した。 2.細胞壁構成多糖類間および多糖類-ペルオキシダーゼ間のの相互作用解析：木材のヘミセルロース（キシラン、グルコマンナン、キシログルカン）とセルロースとの相互作用を、平衡吸着実験と速度論的解析が可能なQCM-Dを用いて行った。両測定とも、キシログルカンがセルロースに最も吸着することが明らかとなった。また、Horseradish peroxidase(HRP)と多糖類との相互作用解析では、HRPはセルロースに対し、高い親和性を示すことも分かった。さらに、多糖類が脱水素重合体(DHP)の形成に及ぼす影響も調べ、キシログルカンがDHPを多量生成させ、加えて、5-5’縮合結合の形成も促進することが示された。この結果より、一次細胞壁が縮合構造に富む理由が解明できた。 3.高分子リグニンの蛍光共鳴エネルギー移動（FRET)の測定：FRET測定の最初の取り組みとして、固体試料を対象とした蛍光顕微鏡でFRETが示されているリグニンと蛍光試薬ローダミンとの相互作用を、溶液中で検出することから検討した。使用したリグニンは広葉樹のクラフトリグニンである。この測定の結果、プロピレングリコールを溶媒とする溶液中で、FRETを観測することができた。モノリグノールとクラフトリグニンとのFRETの検出を試みたが、どちらも蛍光強度が弱かったために、明確なFRET検出には至っていない。
• 細胞壁成分分布の網羅的解析から迫る樹木の姿勢制御機構

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  Date (from‐to) : 2021/04 -2025/03 

  Author : 吉永 新, 岸本 崇生, 重冨 顕吾, 上高原 浩, 飛松 裕基, 津山 濯, 粟野 達也

   

  あて材形成の比較対象となる針葉樹のヒノキおよび、シリンギルリグニンの割合が高い広葉樹のシラカシの正常材について、リグニン中の結合様式に特異的に反応する4種類のモノクローナル抗体を用いて免疫蛍光標識した。得られた画像を解析して、細胞壁各部位での標識強度を比較する方法を確立した。連続切片の自家蛍光像からリグニン全体の分布も評価し、細胞壁の木化過程と結合様式の変化について考察した。その結果、ヒノキ仮道管二次壁では、8-8'型、8-O-4'型、5-5'型構造は未木化の段階で存在し、木化が進むと8-5'型構造も形成されること、コーナー部の複合細胞間層では未木化の段階で8-8’型構造が存在し、木化が進むと8-5型、8-O-4'型構造も形成されることが明らかになった。一方、シラカシの道管要素と周囲仮道管の二次壁では、5-5'型構造が木化初期に形成され、木化の進行に伴って8-O-4'型構造の割合が増加すること、木部繊維の二次壁では未木化段階で8-O-4'型、5-5‘型構造が二次壁外側に存在し、木化が進むと、8-O-4'型、5-5’型構造が内側に形成され、木化完了段階では二次壁全体で均一に形成されることが明らかになった。 ポプラの引張あて材より、滑走式ミクロトームを用いて横断切片を作製し、超音波処理によりG層を集める方法を検討し、ごく微量に道管要素の断片も混入するものの、純度よくG層を単離する方法を確立し、約200mgのG層を得た。単離されたG層を蛍光顕微鏡で観察したところ、ごく弱い自家蛍光がG層全体に観察された。 H型5-5’型構造を含む2量体モデル化合物を合成した。このモデル化合物はR4年度に新しいモノクローナル抗体作製のための抗原として使用する予定である。
• Roles of Cell Walls of Trees at Subzero Temperatures

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2021/04 -2024/03 

  Author : 荒川 圭太, 重冨 顕吾, 鈴木 伸吾

   

  多くの植物植物は、氷点下温度にさらされると細胞外の水から凍結が始まり、それにともなって細胞は脱水を生じる。このような挙動は細胞外凍結と呼ばれ、樹木では緑葉や樹皮の細胞などが細胞外凍結する。そこでカツラの樹皮から細胞壁画分を調製して調べると、水を凍結へと導く氷核形成を促す活性（氷核活性）が検出された。そこで本研究では、カツラ樹皮から検出された氷核活性の原因物質を特定し、細胞外凍結における役割について調べることを目的とした。初年度は、カツラ樹皮由来の氷核活性成分の特定とその性質について調べることを主な目標とした。 氷核活性成分の調製には、冬季のカツラ樹皮から抽出した細胞壁画分を用い、既知の氷核活性物質と比較しながら活性成分の分画を進めた。細胞壁から水で抽出できた活性画分を用い、熱処理や酸処理、アルカリ処理などの活性への影響について調べたところ、既知の氷核物質であるシュウ酸カルシウムや氷核タンパク質とは性質は異なることが示唆された。これらの処理に加え、限外濾過を利用した簡易的なサイズ分画も併用し、活性成分の部分精製を進めて性質を調べるとともに、スケールアップして活性画分を回収して構造解析にも用いた。 これまでの実験によって知り得た活性成分の性質を鑑み、二次元NMR解析などで構造解析を進めたところ、活性成分は多糖類であることが示唆され、その主たる構成糖などの知見が得られた。氷核活性成分の機能解析については、活性の濃度依存性や季節変動の有無、抗氷核ポリフェノール類による活性阻害の有無について検証などを進めている。また、既知の氷核物質との活性比較を試みたが、それぞれの研究によって活性測定条件が異なることもあり、構造特性や活性の比較を論ずるには慎重な判断を要することを改めて認識した。２年目も研究計画に沿って引き続き氷核活性成分の構造や活性の特性に注目して実験を進めていく予定である。
• A study on mechanisms of barrier function in various types of outer bark of trees

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2020/04 -2023/03 

  Author : 佐野 雄三, 玉井 裕, 重冨 顕吾

   

  生立木から採取した自然状態の外樹皮組織における菌糸体の存否・分布と組織の状態を顕微鏡的に詳しく観察するとともに、モデル培養系を使って樹皮組織に対する菌類の忌避性評価・成長阻害活性評価を行った。主な成果は以下の通りである。 ＜生立木の外樹皮における菌糸体の存否・分布＞広葉樹21 種を対象に、外樹皮が十分に発達した生立木より採取された全樹皮を含む組織の液浸標本を用いて、走査電子顕微鏡により樹皮組織の解剖学的特徴と菌糸体の分布を観察した。樹皮型に拘らず、内樹皮の生きた師部組織には菌糸体は認められなかった。一方、外樹皮における分布には、樹皮型による違いが認められた。リチドームが発達する樹種では菌糸体が内方深くまで分布している傾向が明らかで、外樹皮組織中の最内の周皮まで侵入しているケースも認められた。これに対して、外樹皮が単一の周皮で構成される樹種では、菌糸体は亀裂やコルク組織の部分的な剥離部から深く侵入していたが、無傷の健全部ではごく表層にとどまっていた。 ＜樹皮片に対する菌類の忌避性評価＞6～8月に北海道大学研究林にて計12種（広葉樹9種、針葉樹3種）の生立木より樹皮サンプルを採取した。外樹皮と内樹皮に分け、小片（3×3 cm）に切り分け凍結乾燥（24 h）後、γ線滅菌（15 kGy）を行った。これをPDA 培地上に置き、カワラタケの接種片とともに培養した。12種中1種（ナナカマド）に強い抗菌活性が認められた。樹皮組織片とカワラタケを分断する溝を培地中央に設けても、同様の活性が認められた。この結果から、ナナカマドの樹皮組織は菌類の成長を強く阻害する揮発性物質が含むことが明らかであり、化学的な恒常的防御機構解明の一つのモデルとなりえることが示された。本研究で考案した培養系は、高い抗菌活性を持つ樹皮を備える樹種を選抜するのに有効な手法であるといえる。
• Mineralization and biosynthesis of aromatic compounds by white rot fungi

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2019/04 -2022/03 

  Author : Shigetomi Kengo

   

  White rot fungi, a kind of mushroom, are the only organisms that completely degrade lignin, the aromatic polymer that constitutes wood. For this reason, white rot fungi are sometimes described as lignin "eaters" (i.e., mineralizers), but it is not clear whether they actually eat lignin. In this study, we found that white rot fungi can mineralize veratryl alcohol, a low-molecular-weight aromatic compound that they produce, by converting it into muconic acids. This suggests that lignin can also be partially "eaten" through the same process. It was also suggested that the formation process of muconic acid is different from that of bacteria, which have been known previously.
• Investigation of lignin formed in the mimetics of wood cell wall and clarification of lignification-related compounds

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)

  Date (from‐to) : 2018/04 -2021/03 

  Author : Uraki Yasumitsu

   

  This study clearly demonstrated the effect of hemicelluloses on ligninfication through lignin formation in the artificial polysaccharide matrices; xylan contributed to lignin formation and generation of 8-O-4' interunitary linkage, which was a major linkage in native lignin, while glucomannan inhibited lignin formation. In addition, xyloglucan, which located only at primary wall of tree, was found to enhance the frequency of 5-5' interunitary linkage. From the analysis of solution structure for isolated lignins by using size-exclusion chromatograph equipped with multi-angle laser light scattering detector, 5-5' linkage was found to be closely related to the densed structure, especially much compact structure of lignin fraction with larger molar mass compared to that with small molar mass.
• Regulation mechanisms of cavitation at bordered pits as estimated by analyses of the structures

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2016/04 -2019/03 

  Author : Sano Yuzou, Yamagishi Shohei, Umebayashi Toshihiro, Aoki Dan

   

  In an attempt to elucidate the regulation mechanisms of water flow and cavitation at bordered pits in trees, we revealed the fine structures of intervascular pit membranes and the presence or absence of gel-like incrustations on the pit membranes for a total of 28 species. The porosity of the pit membranes were largely varied among taxa. The gel-like incrustations were estimated as fatty acids. We also compared the sample preparation techniques for electron microscopy to find the intact structures of such delicate bordered pit membranes, and showed that a few traditional and novel methods were very useful for the purpose.
• Action mechanism and physiological function of coproporphyrins, novel bacterial growth factors

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

  Date (from‐to) : 2016/04 -2019/03 

  Author : Shigetomi Kengo

   

  In this research, the action mechanism and physiological effectivities of coproporphyrins, novel bacterial growth factors were investigated. The results revealed that coproporphyrins function as the exogenous biosynthetic intermediate in the incomplete heme biosynthesis of Leucobacter sp. strain ASN212. Moreover, the growth factors were proved to stimulate a part of strains belonging to Firmicutes and gamma-Proteobacteria in bacterial flora.
• Lignification in polysaccharide matrix mimicking wood cell wall and clarification of lignin structure -control factor

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)

  Date (from‐to) : 2014/06 -2018/03 

  Author : Uraki Yasumitsu

   

  An aim of this study is to elucidate lignin formation mechanism (lignification), especially influence of hemicelloses, with polysaccharide matrix mimicking wood cell wall structure. Polysaccharide matrix was successfully prepared by the deposition of hemicelluloses, xylan and glucomannan, onto bacterial cellulose. Dehydrogenative polymerization of coniferyl alcohol, which was one of monolignols, was attempted in the presence of such polysaccharide matrix. As a result, the polymerization was accelerated by xylan deposition, but was inhibited by glucomannan. In addition, the structure of the resultant polymerized product (Dehydronation Polymer: DHP) was investigated on a size-exclusion chromatography equipped with a laser light-scattering detector by the comparison with solution structure of a non-branched lignin model compound that is comprised of only 8-O-4’ linkage. This investigation revealed that DHP had a significant frequency of branched structure.
• Structure and function of the novel autophagy inducer, (+)-epogymnolactam

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2014/04 -2017/03 

  Author : Ubukata Makoto

   

  To determine the structural requirement of (+)-epogymnolactam (1) for its autophagy-inducing activity, we first synthesized 1 in 8 steps. We next synthesized enantiomer of 1 ((-)-Epo), deepoxy derivative (Deepoxy), linear analog (N,N-Dimethyl), cyclic analog (O-Methyl), analog having C6 side chain (C6), analog having C8 side chain (C8). Structure-activity relationship in these compounds, synthetic intermediate (Amide 8), cerulean (Cer), and 1 indicated the importance of (2R,3S)-epoxy group, lactam structure, length of side chain, and presence or absence of double bond for their biological activities. Cer inhibited autophagy in NIH 3T3 cells, and C6 and C8 analogs induced autophagy, whereas these two analogs inhibited the degradation of p62. These observations suggest that each molecular target for 1, Cer, and C6 or C8 should be different. The present study would contribute to the development of chemotherapeutic agents for the treatment of various incurable diseases.
• Phylogeny and ecophysiology of fungi possessing ligninolytic activity under snow cover.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2013/04 -2016/03 

  Author : MIYAMOTO Toshizumi, KODA Keiichi, SHIGETOMI Kengo

   

  We isolated litter-decomposing fungi capable of producing active extracellular phenoloxidases at 0 °C from decomposing litter under snow cover in the forests. Molecular phylogenetic analysis and identification of extracellular enzymes of these fungi were conducted. It was revealed that the fungi involved in lignin decomposition under snow cover were consisted of various taxonomic groups inhabiting in litter of various plant species. These fungi were isolated from beginning of snow cover period to snow melting season. Lac, LiP, and MnP active at 4°C were produced from the mycelia of these fungi. A new method to determine lignin content of decomposing leaf litter was also developed. Loss of lignin in litter during the snow cover period was monitored by the new method. Lignin was lost at higher rate than previously reported.
• The evaluation of anaerobic muconates metabolism and ethanol fermentation

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

  Date (from‐to) : 2012/04 -2015/03 

  Author : SHIGETOMI Kengo, UBUKATA Makoto, MITSUHASHI Shinya

   

  Muconates are the key intermediates in biodegradation of natural or unnatural aromatic compounds (e.e. lignin, plastics and dyes), which means that the exploration of novel fermentation process of muconates can give a new insight for utilization of waste aromatic compounds, such as ethanol production. The present study focused on the anaerobic mineralization of muconates as a sole carbon source. The anaerobical enrichment culture of soil samples by using cis,cis-muconic acid as a sole carbon source gave 4 microbial strains which can mineralize this compounds in anaerobic condition. The precursor compound of 2-hydroxymuconic semialdehyde, the other type of muconate, could be successfully synthesized.
• Focused proteomics of phosphorylated proteins using tautomycetin and its application

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)

  Date (from‐to) : 2007 -2009 

  Author : UBUKATA Makoto, MITSUHASHI Shinya, SHIGETOMI Kenngo

   

  We have conducted focused proteomics of phosphorylated proteins by using tautomycetin. Protein phosphatase 1 (PP1) was found to act as a kind of regulatory enzyme for signal transduction in mammalian cells. For example, it positively regulates TNFα/NF-κB pathway as well as Raf. In addition, we isolated ATM30, a substrate of both PP1 and ATM/ATR kinase. ATM30 was found to be a ribosomal protein RPS3A(a) by MALDI-TOF/MS analysis.