Researcher Profile and Settings

Master

Affiliation (Master)

• Faculty of Pharmaceutical Sciences　Molecular Pharmaceutical Sciences　Chemistry and Medicinal Chemistry

Affiliation (Master)

• Faculty of Pharmaceutical Sciences　Molecular Pharmaceutical Sciences　Chemistry and Medicinal Chemistry

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Profile and Settings

Degree

• Ph.D.（University of Tokyo）

Profile and Settings

• Name (Japanese)

  Wakimoto

• Name (Kana)

  Toshiyuki

• Name

  201501022605283747

Alternate Names

https://www.pharm.hokudai.ac.jp/tennen/
https://www.pharm.hokudai.ac.jp/tennen/en/

Achievement

Research Interests

• チョコガタイシカイメン   蛍光プローブ   タンパク質脱リン酸化酵素   ミドリイガイ   海綿   カリクリン   フラン脂肪酸   抗炎症活性   Calyculin A   二次代謝酵素   機能制御   酵素工学   海洋生物資源   物質生産   生合成   

Research Areas

• Life sciences / Environmental and pharmaceutical development resources
• Life sciences / Bioorganic chemistry
• Life sciences / Marine/Aquatic life sciences

Research Experience

• 2015 - Today 北海道大学大学院 薬学研究院 教授
• 2013 - 2015 東京大学大学院 薬学系研究科 准教授
• 2010 - 2013 東京大学大学院 薬学系研究科 講師
• 2009 - 2010 University of Shizuoka School of Pharmaceutical Sciences
• 2007 - 2009 University of Shizuoka School of Pharmaceutical Sciences
• 2003 - 2007 University of Shizuoka School of Pharmaceutical Sciences
• 2001 - 2003 日本学術振興会海外特別研究員 ピッツバーグ大学化学科 博士研究員

Education

•        - 2001  東京大学大学院農学生命科学研究科
•        - 1996  東京大学農学部

Awards

• 2021/03 日本薬学会 学術振興賞
   

  受賞者: 脇本 敏幸
• 2014/09 日本生薬学会 学術貢献賞
   

  受賞者: 脇本 敏幸
• 2013/09 長瀬科学技術振興財団 長瀬研究奨励賞
   

  受賞者: 脇本 敏幸
• 2012/09 日本生薬学会 学術奨励賞
   

  受賞者: 脇本 敏幸
• 2008/05 新規素材探索研究会 最優秀ポスター賞
   

  受賞者: 脇本 敏幸

Published Papers

• Doubly Homologated Tyrosine-Containing Peptides from the Cyanobacterium Microcystis aeruginosa NIES-4285 and Their Biosynthesis

  Chin-Soon Phan, Zhengyi Ling, Jakia Jerin Mehjabin, Kenichi Matsuda, Nurcahyo Iman Prakoso, Taiki Umezawa, Toshiyuki Wakimoto, Tatsufumi Okino

  Journal of Natural Products 0163-3864 2024/10/30
• Synthesis and Cytotoxicity of Cyclic Octapeptide Surugamides with Varied <i>N</i>-Acyl Moieties

  Kenichi Matsuda, Shinya Niikura, Rintaro Ichihara, Kei Fujita, Anna M. Strasser, Rokusuke Yoshikawa, Jiro Yasuda, Yoshiki Hiramatsu, Hironori Hayashi, Eiichi N. Kodama, Toshiyuki Wakimoto

  Chemical and Pharmaceutical Bulletin 72 (9) 826 - 830 0009-2363 2024/09/20
• Penicillin-binding protein-type thioesterases: An emerging family of non-ribosomal peptide cyclases with biocatalytic potentials

  Kenichi Matsuda, Toshiyuki Wakimoto

  Current Opinion in Chemical Biology 2024/06 [Refereed][Not invited]
  
• Chemoenzymatic tandem cyclization for the facile synthesis of bicyclic peptides

  Masakazu Kobayashi, Naho Onozawa, Kenichi Matsuda, Toshiyuki Wakimoto

  Communications Chemistry 7 (1) 2024/03/28 

  Abstract Bicyclic peptides exhibit improved metabolic stabilities and target specificities when compared to their linear or mono-cyclic counterparts; however, efficient and straightforward synthesis remains challenging due to their intricate architectures. Here, we present a highly selective and operationally simple one-pot chemoenzymatic tandem cyclization approach to synthesize bicyclic peptides with small to medium ring sizes. Penicillin-binding protein-type thioesterases (PBP-type TEs) efficiently cyclized azide/alkyne-containing peptides in a head-to-tail manner. Successive copper (I)-catalyzed azide-alkyne cycloaddition generated bicyclic peptides in one-pot, thus omitting the purification of monocyclic intermediates. This chemoenzymatic strategy enabled the facile synthesis of bicyclic peptides bearing hexa-, octa-, and undecapeptidyl head-to-tail cyclic scaffolds.
• Bacterial hydrazine biosynthetic pathways featuring cupin/methionyl tRNA synthetase-like enzymes.

  Kenichi Matsuda, Toshiyuki Wakimoto

  Chembiochem : a European journal of chemical biology e202300874  2024/03/08 

  Nitrogen-Nitrogen (N-N) bond-containing functional groups in natural products and synthetic drugs play significant roles in exerting biological activities. The mechanisms of N-N bond formation in natural organic molecules have garnered increasing attention over the decades. Recent advances have illuminated various enzymatic and nonenzymatic strategies, and our understanding of natural N-N bond construction is rapidly expanding. A group of didomain proteins with zinc-binding cupin/methionyl-tRNA synthetase (MetRS)-like domains, also known as hydrazine synthetases, generates amino acid-based hydrazines, which serve as key biosynthetic precursors of diverse N-N bond-containing functionalities such as hydrazone, diazo, triazene, pyrazole, and pyridazinone groups. In this review, we summarize the current knowledge on hydrazine synthetase mechanisms and the various pathways employing this unique bond-forming machinery.
• Phylogeny‐guided Characterization of Bacterial Hydrazine Biosynthesis Mediated by Cupin/methionyl tRNA Synthetase‐like Enzymes

  Kenichi Matsuda, Yuto Nakahara, Atina Rizkiya Choirunnisa, Kuga Arima, Toshiyuki Wakimoto

  ChemBioChem 1439-4227 2024/02/25 

  Cupin/methionyl‐tRNA synthetase (MetRS)‐like didomain enzymes catalyze nitrogen‐nitrogen (N–N) bond formation between Nw‐hydroxylamines and amino acids to generate hydrazines, key biosynthetic intermediates of various natural products containing N–N bonds. While the combination of these two building blocks leads to the creation of diverse hydrazine products, the full extent of their structural diversity remains largely unknown. To explore this, we herein conducted phylogeny‐guided genome‐mining of related hydrazine biosynthetic pathways consisting of two enzymes: flavin‐dependent Nw‐hydroxylating monooxygenases (NMOs) that produce Nw‐hydroxylamine precursors and cupin/MetRS‐like enzymes that couple the Nw‐hydroxylamines with amino acids via N–N bonds. A phylogenetic analysis identified the largely unexplored sequence spaces of these enzyme families. The biochemical characterization of NMOs demonstrated their capabilities to produce various Nw‐hydroxylamines, including those previously not known as precursors of N–N bonds. Furthermore, the characterization of cupin/MetRS‐like enzymes identified five new hydrazine products with novel combinations of building blocks, including one containing non‐amino acid building blocks: 1,3‐diaminopropane and putrescine. This study substantially expanded the variety of N–N bond forming pathways mediated by cupin/MetRS‐like enzymes.
• Actinobacterial chalkophores: the biosynthesis of hazimycins

  Kenichi Matsuda, Hiroto Maruyama, Kumiko Imachi, Haruo Ikeda, Toshiyuki Wakimoto

  The Journal of Antibiotics 2024/02/20 [Refereed][Not invited]
  
• FlkO, a penicillin-binding protein-type thioesterase in cyclofaulknamycin biosynthesis

  Kenichi Matsuda, Rintaro Ichihara, Toshiyuki Wakimoto

  Organic & Biomolecular Chemistry 1477-0520 2024 

  In vitro studies of FlkO, a new penicillin-binding protein thioesterase, demonstrated its peptide-cyclizing activity and substrate scope.
• Animal-associated marine Acidobacteria with a rich natural-product repertoire

  Stefan Leopold-Messer, Clara Chepkirui, Mathijs F.J. Mabesoone, Joshua Meyer, Lucas Paoli, Shinichi Sunagawa, Agustinus R. Uria, Toshiyuki Wakimoto, Jörn Piel

  Chem 2451-9294 2023/12
• Parallel loss of sex in field populations of a brown alga sheds light on the mechanisms underlying the emergence of asexuality

  Masakazu Hoshino, Guillaume Cossard, Fabian B. Haas, Emma I. Kane, Kazuhiro Kogame, Takahiro Jomori, Toshiyuki Wakimoto, Susana M. Coelho

  2023/09/12 

  Abstract Sexual reproduction is widespread among eukaryotes, but asexual lineages have repeatedly arisen from sexual ancestors across a wide range of taxa. Despite extensive research on the evolution of asexuality from sexual ancestors, the molecular changes underpinning the switch to asexual reproduction remain elusive, particularly in organisms with haploid sexual systems such as bryophytes, and red and brown algae in which males and females are haploid and multicellular. Here, we investigate independent events in which asexuality has emerged from sexual ancestor lineages in species of the brown algalScytosiphon, we examine the proximate and evolutionary mechanisms involved, and test the importance of sexual conflict on gene expression changes following loss of sex. We find that individuals from asexual, female-only (‘Amazon’) populations lose their ability to produce and sex pheromone and, consequently, are unable to attract and fuse with male gametes, whereas they gain the ability to trigger parthenogenic (asexual) development from large, unfertilized eggs. This independent and convergent decline in pheromone production and optimization of asexual traits is accompanied by convergent changes in gene expression, including de-feminization and masculinization of the Amazon gamete transcriptomes. These data are consistent with the idea that decay of female functions, rather than relaxation of sexual antagonism, is the dominant force at play during the emergence of asexuality in haploid sexual systems. Moreover, we identify a locus on an autosomal protein-coding gene that is associated with the switch to asexuality. We propose that the sex chromosome, together with this autosomal locus, may underlie the switch to obligate asexuality in the Amazon populations.
• Membrane‐Vesicle‐Mediated Interbacterial Communication Activates Silent Secondary Metabolite Production

  Aya Yoshimura, Rio Saeki, Ryusuke Nakada, Shota Tomimoto, Takahiro Jomori, Keisuke Suganuma, Toshiyuki Wakimoto

  Angewandte Chemie International Edition 1433-7851 2023/07/14
• Carrier Protein Mediated Formation of the Dihydropyridazinone Ring in Actinopyridazinone Biosynthesis

  Kuga Arima, Satoko Akiyama, Kazuo Shin-ya, Kenichi Matsuda, Toshiyuki Wakimoto

  Angewandte Chemie International Edition 1433-7851 2023/05/17
• Streamlined Chemoenzymatic Synthesis of Cyclic Peptides by Non-ribosomal Peptide Cyclases

  Masakazu Kobayashi, Kei Fujita, Kenichi Matsuda, Toshiyuki Wakimoto

  Journal of the American Chemical Society 2023/01/13 [Refereed][Not invited]
  
• Chemo-Enzymatic Synthesis of Non-ribosomal Macrolactams by a Penicillin-Binding Protein-Type Thioesterase.

  Masakazu Kobayashi, Kei Fujita, Kenichi Matsuda, Toshiyuki Wakimoto

  Methods in molecular biology (Clifton, N.J.) 2670 127 - 144 2023 

  Penicillin-binding protein-type thioesterases (PBP-type TEs) are an emerging family of non-ribosomal peptide cyclases. PBP-type TEs exhibit distinct substrate scopes from the well-exploited ribosomal peptide cyclases and traditional non-ribosomal peptide cyclases. Their unique properties, as well as their stand-alone nature, highlight PBP-type TEs as valuable candidates for development as biocatalysts for peptide macrocyclization. Here in this chapter, we describe the scheme for the chemoenzymatic synthesis of non-ribosomal macrolactam by SurE, a representative member of PBP-type TEs.
• New Theonellapeptolides from Indonesian Marine Sponge Theonella swinhoei as Anti-austerity Agents

  Jabal Haedar, Agustinus Uria, Subehan Lallo, Dya Fita Dibwe, Toshiyuki Wakimoto

  Marine Drugs 20 (11) 661 - 661 2022/10/25 

  We reported three new members of the theonellapeptolide family from theonellapeptolide II series, namely theonellapeptolides IIb (1), IIa (2), IIc (3), and three known members—IId (4), IIe (5), and Id (6)—from Kodingarengan marine sponge Theonella swinhoei collected in Makassar, Indonesia. The structures of tridecadepsipeptides 1–3, including the absolute configurations of their amino acids, were determined by the integrated NMR and tandem MS analyses followed by Marfey’s analysis. To the best of our knowledge, 1 and 2 are the first theonellapeptolide-type compounds to have a valine residue with D configuration at residue position 6. The isolated theonellapeptolide-type compounds 1–6 showed selective cytotoxic activity against human pancreatic MIA PaCa-2 cancer cells in a nutrient-deprived medium. Among them, the most potent preferential cytotoxicity was observed in new theonellapeptolide IIc (3) and known IId (4), IIe (5), and Id (6).
• New azodyrecins identified by a genome mining-directed reactivity-based screening

  Atina Rizkiya Choirunnisa, Kuga Arima, Yo Abe, Noritaka Kagaya, Kei Kudo, Hikaru Suenaga, Junko Hashimoto, Manabu Fujie, Noriyuki Satoh, Kazuo Shin-ya, Kenichi Matsuda, Toshiyuki Wakimoto

  Beilstein Journal of Organic Chemistry 18 1017 - 1025 2022/08/10 

  Only a few azoxy natural products have been identified despite their intriguing biological activities. Azodyrecins D–G, four new analogs of aliphatic azoxides, were identified from two Streptomyces species by a reactivity-based screening that targets azoxy bonds. A biological activity evaluation demonstrated that the double bond in the alkyl side chain is important for the cytotoxicity of azodyrecins. An in vitro assay elucidated the tailoring step of azodyrecin biosynthesis, which is mediated by the S-adenosylmethionine (SAM)-dependent methyltransferase Ady1. This study paves the way for the targeted isolation of aliphatic azoxy natural products through a genome-mining approach and further investigations of their biosynthetic mechanisms.
• A Natural Dihydropyridazinone Scaffold Generated from a Unique Substrate for a Hydrazine-Forming Enzyme

  Kenichi Matsuda, Kuga Arima, Satoko Akiyama, Yuito Yamada, Yo Abe, Hikaru Suenaga, Junko Hashimoto, Kazuo Shin-ya, Makoto Nishiyama, Toshiyuki Wakimoto

  Journal of the American Chemical Society 0002-7863 2022/06/30
• Insights into Phosphatase-activated Chemical Defense in a Marine Sponge Holobiont

  Takahiro Jomori, Kenichi Matsuda, Yoko Egami, Ikuro Abe, Akira Takai, Toshiyuki Wakimoto

  RSC Chemical Biology 2 (6) 1600 - 1607 2021/10 [Refereed]
   

  Marine sponges often contain potent cytotoxic compounds, which in turn evokes the principle question of how marine sponges avoid self-toxicity. In a marine sponge Discodermia calyx, the highly toxic calyculin...
• Geographical parthenogenesis in the brown alga Scytosiphon lomentaria (Scytosiphonaceae): Sexuals in warm waters and parthenogens in cold waters

  Masakazu Hoshino, Shimpei F. Hiruta, Maria Emilia Croce, Mitsunobu Kamiya, Takahiro Jomori, Toshiyuki Wakimoto, Kazuhiro Kogame

  Molecular Ecology 30 (22) 5814 - 5830 0962-1083 2021/09/06 [Refereed]
   

  Geographical parthenogenesis, a phenomenon where parthenogens and their close sexual relatives inhabit distinct geographical areas, has been considered an interesting topic in evolutionary biology. Reports of geographical parthenogenesis from land and freshwater are numerous, but this occurrence has been rarely reported from the sea. Brown algae are mostly marine and are thought to include numerous obligate parthenogens; still, little is known about the distribution, origin and evolution of parthenogens in this group. Here we report a novel pattern of geographical parthenogenesis in the isogamous brown alga Scytosiphon lomentaria. Sex ratio investigation demonstrated that, in Japan, sexual populations grew in the coast along warm ocean currents, whereas female-dominant parthenogenetic populations grew mainly in the coast along a cold ocean current. In the two localities where sexual and parthenogenetic populations were parapatric, parthenogens grew in more wave-exposed areas than sexuals. Population genetic and phylogenetic analyses, including those based on genome-wide single nucleotide polymorphism data, indicated that parthenogens have initially evolved at least twice and subsequent hybridizations between the parthenogens and sexuals have generated multiple new parthenogenetic lineages. The origin of the initial parthenogens is not clear, except that it would not be interspecies hybridization. Interestingly, we found that the production of sex pheromones, which attract male gametes, has been independently lost in the initial two parthenogenetic lineages. This parallel loss of the sexual trait may represent the direct origin of parthenogens, or the regressive evolution of a useless trait under asexuality.
• Argicyclamides A–C Unveil Enzymatic Basis for Guanidine Bis-prenylation

  Chin-Soon Phan, Kenichi Matsuda, Nandani Balloo, Kei Fujita, Toshiyuki Wakimoto, Tatsufumi Okino

  Journal of the American Chemical Society 0002-7863 2021/06/28 [Refereed]
   

  Guanidine prenylation is an outstanding modification in alkaloid and peptide biosynthesis, but its enzymatic basis has remained elusive. We report the isolation of argicyclamides, a new class of cyanobactins with unique mono- and bis-prenylations on guanidine moieties, from Microcystis aeruginosa NIES-88. The genetic basis of argicyclamide biosynthesis was established by the heterologous expression and in vitro characterization of biosynthetic enzymes including AgcF, a new guanidine prenyltransferase. This study provides important insight into the biosynthesis of prenylated guanidines and offers a new toolkit for peptide modification.
• PenA, a penicillin-binding protein-type thioesterase specialized for small peptide cyclization.

  Kenichi Matsuda, Kei Fujita, Toshiyuki Wakimoto

  Journal of Industrial Microbiology & Biotechnology 48 (3-4) 2021/06/04 [Refereed]
   

  Penicillin-binding protein-type thioesterases (PBP-type TEs) are a recently identified group of peptide cyclases that catalyze head-to-tail macrolactamization of nonribosomal peptides. PenA, a new member of this group, is involved in the biosyntheses of cyclic pentapeptides. In this study, we demonstrated the enzymatic activity of PenA in vitro, and analyzed its substrate scope with a series of synthetic substrates. A comparison of the reaction profiles between PenA and SurE, a representative PBP-type TE, showed that PenA is more specialized for small peptide cyclization. A computational model provided a possible structural rationale for the altered specificity for substrate chain lengths.
• Ascidian-associated photosymbionts from Manado, Indonesia: secondary metabolites, bioactivity simulation, and biosynthetic insight

  Inneke F. M. Rumengan, Vera I. Y. Roring, Jabal R. Haedar, Mayse S. Siby, Aldian H. Luntungan, Beivy J. Kolondam, Agustinus R. Uria, Toshiyuki Wakimoto

  Symbiosis 0334-5114 2021/04/22 [Refereed]
  
• Total Synthesis and Structural Revision of Kasumigamide, and Identification of a New Analogue

  Takefumi Kuranaga, Kenichi Matsuda, Masachika Takaoka, Chisato Tachikawa, Ayae Sano, Kosei Itoh, Ayumu Enomoto, Kei Fujita, Ikuro Abe, Toshiyuki Wakimoto

  ChemBioChem 21 (23) 3329 - 3332 1439-4227 2020/12 [Refereed]
   

  © 2020 Wiley-VCH GmbH Kasumigamide is an antialgal hybrid peptide–polyketide isolated from the freshwater cyanobacterium Microcystis aeruginosa (NIES-87). The biosynthetic gene cluster was identified from not only the cyanobacterium but also Candidatus “Entotheonella”, associated with the Japanese marine sponge Discodermia calyx. Therefore, kasumigamide is considered to play a key role in microbial ecology, regardless of the terrestrial and marine habitats. We now report synthetic studies on this intriguing natural product that have led to a structural revision and the first total synthesis. During this study, a new analogue, deoxykasumigamide, was also isolated and structurally validated. This study confirmed the presence of the unusual pathway in the biosynthesis of a hybrid peptide–polyketide natural product.
• Heterochiral coupling in non-ribosomal peptide macrolactamization

  Kenichi Matsuda, Rui Zhai, Takahiro Mori, Mazakazu Kobayashi, Ayae Sano, Ikuro Abe, Wakimoto Toshiyuki

  Nature Catalysis 32 2020/05 [Refereed][Not invited]
  
• Polyketides Biosynthesis in Marine Sponges of the Family Theonellidae

  Agustinus Robert Uria, Toshiyuki Wakimoto

  Topics in Heterocyclic Chemistry 389 - 413 1861-9282 2020 [Refereed]
  
• Crystal Structures of Flavone C-Glycosides from Oolong Tea Leaves: Chafuroside A Dihydrate and Chafuroside B Monohydrate.

  Yasunori Iwao, Hitoshi Ishida, Shin-Ichiro Kimura, Toshiyuki Wakimoto, Hiromu Kondo, Shigeru Itai, Shuji Noguchi

  Chemical & Pharmaceutical Bulletin 67 (9) 935 - 939 2019 [Refereed]
   

  Chafuroside A and chafuroside B are flavone C-glycosides isolated from oolong tea leaves. They have a number of beneficial pharmacological activities related to antiinflammation at various concentrations. However, no crystallographic study of chafurosides has yet been reported. In the present study, the crystal structures of chafuroside A and chafuroside B were investigated using single-crystal X-ray diffraction. The asymmetric unit of the chafuroside A crystal consists of one chafuroside A and two water molecules, and that of chafuroside B contains one chafuroside B and one water molecule. The flavone moiety of chafuroside A is curved, i.e., the angle between the best-fit planes of the chromene and phenyl rings is 18.9°, whereas the chafuroside B flavone moiety is relatively flat. A comparison of the curvatures of the flavone moieties of various C-glycosides showed that the curvature of chafuroside A is significantly larger than those of the others. This structural feature might contribute to the differences between the strengths of the pharmacological activities of chafurosides A and B.
• The Revised Structure of the Cyclic Octapeptide Surugamide A

  Kenichi Matsuda, Takefumi Kuranaga, Ayae Sano, Akihiro Ninomiya, Kentaro Takada, Toshiyuki Wakimoto

  Chemical & Pharmaceutical Bulletin 67 (5) 476 - 480 2019 [Refereed][Not invited]
   

  Surugamides are a group of non-ribosomal peptides isolated from marine-derived Streptomyces. Surugamide A (1) and its closely related derivatives, surugamides B-E (2-5), are D-amino acid containing cyclic octapeptides with cathepsin B inhibitory activity. The D-isoleucine (Ile), the nonproteinogenic amino acid residue embedded in 1, is less common in natural peptides because a rare Cβ-epimerization is required for its biosynthesis. Taking advantage of the synthetic route of 2 previously established by our group, we synthesized the cyclic octapeptide 1 containing D-Ile by solid phase peptide synthesis. The structure of 1 actually contains D-allo-Ile in place of D-Ile, which was corroborated by chemical syntheses and chromatographic comparisons.
• A new cyclase family catalyzing head-to-tail macrolactamization of non-ribosomal peptides

  Kenichi Matsuda, Takefumi Kuranaga, Toshiyuki Wakimoto

  J. Synth. Org. Chem. Jpn. 77 1106 - 1115 2019 [Refereed][Not invited]
  
• Scrobiculosides A and B from the Deep-Sea Sponge Pachastrella scrobiculosa

  Takahiro Jomori, Shuji Shiroyama, Yuji Ise, Hisanori Kohtsuka, Kenichi Matsuda, Takefumi Kuranaga, Toshiyuki Wakimoto

  J. Nat. Med. 73 (4) 814 - 819 2019 [Refereed][Not invited]
   

  Two new steroidal saponins, scrobiculosides A and B, were isolated from the deep-sea sponge Pachastrella scrobiculosa, collected at a depth of 200 m off Miura Peninsula, Japan. The aglycones of scrobiculosides A and B feature a vinylic cyclopropane and a ∆24,25 exomethylene on the side chains, respectively. Both saponins have a common sugar moiety composed of β-D-galactopyranosyl-(1 → 2)-6-acetyl-β-D-glucopyranoside, with the exception of an acetyl group on C6″ in scrobiculoside A. Scrobiculoside A exhibited cytotoxicity against HL-60 and P388 cells, with IC50 values of 52 and 61 μM, respectively.
• SurE is a trans-acting thioesterase cyclizing two distinct non-ribosomal peptides.

  Matsuda, K, Kobayashi, M, Kuranaga, T, Takada, K, Ikeda, H, Matsunaga, S, Wakimoto, T

  Org. Biomol. Chem. 17 1058 - 1061 2019/01 [Refereed][Not invited]
  
• Palmitoyl lactic acid induces adipogenesis and a brown fat-like phenotype in 3T3-L1 preadipocytes.

  Yuka Unno, Hirona Yamamoto, Shuto Takatsuki, Yoshinori Sato, Takefumi Kuranaga, Kazunaga Yazawa, Yasuo Ono, Toshiyuki Wakimoto

  Biochimica et Biophysica Acta. Molecular and Cell Biology of Lipids 1863 (7) 772 - 782 2018/07 [Refereed][Not invited]
   

  Brown adipose tissue is specialized to generate heat by dissipating chemical energy and may provide novel strategies for obesity treatment in humans. Recently, advances in understanding the pharmacological and dietary agents that contribute to the browning of white adipose tissue have been made to alleviate obesity by promoting energy expenditure. Krill oil is widely used as a health supplement in humans. In this study, the components from krill oil that promote adipogenesis of 3T3-L1 cells were screened to reveal palmitoyl lactic acid (PLA) as a promoter of adipogenesis. The PLA-induced adipocytes contained large number of small lipid droplets. Moreover, similar to the peroxisome proliferator-activated receptor (PPAR)γ agonists, pioglitazone and rosiglitazone, PLA significantly enhances adipogenesis in the presence of dexamethasone compared with PLA alone. Treatment with PLA causes a brown fat-like phenotype in 3T3-L1 cells by enhanced expression of various brown/beige cell-specific genes, such as PR domain containing 16 (Prdm16) and peroxisome proliferative activated receptor, gamma, coactivator 1 alpha (Pgc1a), as well as adiponectin gene. The expression profile of the brown/beige cell-specific genes induced by PLA was similar to that of the PPARγ agonist in 3T3-L1 cells. Our findings suggest that PLA induces a brown fat-like phenotype and, thus, likely has therapeutic potential in treating obesity.
• Discovery of Unprecedented Hydrazine-Forming Machinery in Bacteria.

  Matsuda K, Tomita T, Shin-Ya K, Wakimoto T, Kuzuyama T, Nishiyama M

  Journal of the American Chemical Society 140 (29) 9083 - 9086 0002-7863 2018/07 [Refereed][Not invited]
   

  Recent studies described several different routes that facilitate nitrogen-nitrogen bond formation in natural product biosynthesis. We report herein the identification of unprecedented machinery for hydrazine formation involved in the biosynthesis of s56-p1, a dipeptide natural product with a unique hydrazone unit. The gene cassette comprising this machinery is widespread across several bacterial phyla, highlighting the overlooked potential of bacteria to synthesize hydrazine.
• Biosynthetic Insights of Calyculin- and Misakinolide-Type Compounds in “Candidatus Entotheonella sp.”

  Agustinus R. Uria, Jörn Piel, Toshiyuki Wakimoto

  Methods in Enzymology 604 287 - 330 1557-7988 2018/01/01 [Refereed][Not invited]
   

  Microbial symbionts are recognized as the important sources of numerous sponge-derived metabolites with potent biological activities. The limitation to cultivate the majority of potential symbionts has hampered attempts to explore and exploit their natural products for further development toward medical applications. Metagenomics-guided approaches have enabled cloning of natural product biosynthesis genes from uncultured microbial symbionts. Subsequent activation of biosynthesis genes in easily culturable bacteria could lead to the sustainable production of rare sponge-derived compounds. In this chapter, we highlight metagenomic strategies to reveal natural product biosynthetic pathways in sponge metagenomes based on the calyculin and misakinolide polyketides. Techniques to identify the compound producer are briefly discussed. We further describe examples of functional studies of the biosynthetic pathways of these two compound types with a special emphasis on the general experimental protocols for the activity assays of key proteins involved in their biosynthesis.
• Diastereoselective total synthesis and structural confirmation of surugamide F

  Takefumi Kuranaga, Atsuki Fukuba, Akihiro Ninomiya, Kentaro Takada, Shigeki Matsunaga, Toshiyuki Wakimoto

  Chemical and Pharmaceutical Bulletin 66 (6) 637 - 641 1347-5223 2018 [Refereed][Not invited]
   

  Surugamide F is a linear decapeptide (1) isolated along with the cyclic octapeptides surugamides A–E (2–6), from a marine-derived Streptomyces species. The linear peptide 1 is produced by two nonribosomal peptide synthetases (NRPSs) encoded in adjacent open reading frames, which are further flanked by an additional pair of NRPS genes responsible for the biosyntheses of the cyclic peptides 2–6. While the cyclic peptides 2–6 were identified to be cathepsin B inhibitors, the biological activity of the new metabolite 1 still remained unclear. In order to elucidate its unique biosynthetic pathway and biological activity in detail, we planned to develop an efficient synthetic route toward 1. Here we report the diastereoselective total synthesis of 1, utilizing 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis. During this study, we found that the structural correction of 1 was required, due to the mislabeling of the commercially obtained 3-amino-2-methylpropionic acid, and the true structure of 1 was corroborated by the chemical synthesis and chromatographic comparison.
• Total Synthesis of the Non-Ribosomal Peptide Surugamide B and Identification of a New Offloading Cyclase Family

  Takefumi Kuranaga, Kenichi Matsuda, Ayae Sano, Masakazu Kobayashi, Akihiro Ninomiya, Kentaro Takada, Shigeki Matsunaga, Toshiyuki Wakimoto

  Angew. Chem. Int. Ed. 57 (30) 9447 - 9451 2018 [Refereed][Not invited]
   

  The cathepsin B inhibitor surugamide B (2), along with structurally related derivatives (A and C-E), has previously been isolated from the marine actinomycete Streptomyces sp. JAMM992. The biosynthetic genes are unexpectedly part of a cluster of four non-ribosomal peptide synthetase (NRPS) genes, two of which are responsible for the biosynthesis of the additional linear decapeptide surugamide F. However, the thioesterase domain required for the later stage of the biosynthesis of the cyclic peptides surugamides A-E is not present in any module architecture of the surugamide NRPSs. Herein, we report the first total synthesis of surugamide B (2) through the macrocyclization at the biomimetic position, which not only alleviated the Cα epimerization in the macrolactamization process, but also efficiently provided 2 in 34 % yield for 18 steps. Furthermore, both the chemical and enzymatic studies with the biosynthetic precursor mimics revealed that the stand-alone enzyme SurE, which belongs to the penicillin-binding protein family, is responsible for macrocyclization of the tethered octapeptidyl intermediate.
• Protein phosphatases 1 and 2A and their naturally occurring inhibitors: current topics in smooth muscle physiology and chemical biology

  Akira Takai, Masumi Eto, Katsuya Hirano, Kosuke Takeya, Toshiyuki Wakimoto, Masaru Watanabe

  Journal of Physiological Sciences 68 (1) 1880-6546 2018/01/01 [Refereed][Not invited]
   

  Protein phosphatases 1 and 2A (PP1 and PP2A) are the most ubiquitous and abundant serine/threonine phosphatases in eukaryotic cells. They play fundamental roles in the regulation of various cellular functions. This review focuses on recent advances in the functional studies of these enzymes in the field of smooth muscle physiology. Many naturally occurring protein phosphatase inhibitors with different relative PP1/PP2A affinities have been discovered and are widely used as powerful research tools. Current topics in the chemical biology of PP1/PP2A inhibitors are introduced and discussed, highlighting the identification of the gene cluster responsible for the biosynthesis of calyculin A in a symbiont microorganism of a marine sponge.
• Toward the Dark Matter of Natural Products

  Toshiyuki Wakimoto

  Chemical Record 17 (11) 1124 - 1134 1527-8999 2017/11 [Refereed][Not invited]
   

  Considering the dynamic features of natural products, our access toward exploring the entire diversity of natural products has been quite limited. It is challenging to assess the diversity of natural products by using conventional analytical methods, even with tandem chromatographic techniques, such as LC-MS and GC-MS. This viewpoint is supported by the sequencing analyses of microbial genomes, which have unveiled the potential of secondary metabolite production far exceeding the number of isolated molecules. Recent advancements in metabolomics, in concert with genomics analyses, have further extended the natural product diversity, prompting growing awareness of the existence of reactive or short-lived natural molecules. This personal account introduces some examples of the discoveries of hitherto elusive natural products, due to physico-chemical or biological reasons, and highlights the significance of the dark matter of natural products.
• Identification of Pyridinium with Three Indole Moieties as an Antimicrobial Agent

  Masahiro Okada, Tomotoshi Sugita, Chin Piow Wong, Toshiyuki Wakimoto, Ikuro Abe

  Journal of Natural Products 80 (4) 1205 - 1209 0163-3864 2017/04 [Refereed][Not invited]
   

  A novel pyridinium with three indole moieties, tricepyridinium, was obtained from the culture of an Escherichia coli clone incorporating metagenomic libraries from the marine sponge Discodermia calyx. For the important structural elements of tricepyridinium to be investigated for antibacterial activity, tricepyridinium and its analogues were chemically synthesized. Tricepyridinium had antimicrobial activity, but not against E. coli, and cytotoxicity against P388 cells. Additional bioassays with its synthetic analogues revealed that the intriguing combination of the indole moieties, most likely derived from three tryptamines, as well as the pyridinium moiety were chiefly responsible for its potent biological activities.
• Total Synthesis of Theonellapeptolide Id

  Takefumi Kuranaga, Ayumu Enomoto, Hui Tan, Kazuto Fujita, Toshiyuki Wakirnoto

  Organic Letters 19 (6) 1366 - 1369 1523-7060 2017/03 [Refereed][Not invited]
   

  Theonellapeptolide Id is a tridecapeptide containing a 37-membered lactone, originally isolated from the marine sponge Theonella swinhoei. In addition to moderate cytotoxicity, immunosuppressive activity had been reported for this natural cyclodepsipeptide. However, the synthetic material to verify its unique biological activity has not been available thus far. In this study, the first total synthesis of theonellapeptolide Id has been performed by solid phase peptide synthesis, and the biological activity has been confirmed in comparison with cyclosporin A.
• Characterization of Giant Modular PKSs Provides Insight into Genetic Mechanism for Structural Diversification of Aminopolyol Polyketides

  Lihan Zhang, Takuya Hashimoto, Bin Qin, Junko Hashimoto, Ikuko Kozone, Teppei Kawahara, Masahiro Okada, Takayoshi Awakawa, Takuya Ito, Yoshinori Asakawa, Masashi Ueki, Shunji Takahashi, Hiroyuki Osada, Toshiyuki Wakimoto, Haruo Ikeda, Kazuo Shin-ya, Ikuro Abe

  Angew. Chem. Int. Ed. 56 (7) 1740 - 1745 1433-7851 2017/02 [Refereed][Not invited]
   

  Polyketides form many clinically valuable compounds. However, manipulation of their biosynthesis remains highly challenging. An understanding of gene cluster evolution provides a rationale for reprogramming of the biosynthetic machinery. Herein, we report characterization of giant modular polyketide synthases (PKSs) responsible for the production of aminopolyol polyketides. Heterologous expression of over 150 kbp polyketide gene clusters successfully afforded their products, whose stereochemistry was established by taking advantage of bioinformatic analysis. Furthermore, phylogenetic analysis of highly homologous but functionally diverse domains from the giant PKSs demonstrated the evolutionary mechanism for structural diversification of polyketides. The gene clusters characterized herein, together with their evolutionary insights, are promising genetic building blocks for denovo production of unnatural polyketides.
• Characterization of Giant Modular PKSs Provides Insight into Genetic Mechanism for Structural Diversification of Aminopolyol Polyketides

  Lihan Zhang, Takuya Hashimoto, Bin Qin, Junko Hashimoto, Ikuko Kozone, Teppei Kawahara, Masahiro Okada, Takayoshi Awakawa, Takuya Ito, Yoshinori Asakawa, Masashi Ueki, Shunji Takahashi, Hiroyuki Osada, Toshiyuki Wakimoto, Haruo Ikeda, Kazuo Shin‐ya, Ikuro Abe

  Angewandte Chemie 129 (7) 1766 - 1771 0044-8249 2017/01/11 

  Abstract Polyketides form many clinically valuable compounds. However, manipulation of their biosynthesis remains highly challenging. An understanding of gene cluster evolution provides a rationale for reprogramming of the biosynthetic machinery. Herein, we report characterization of giant modular polyketide synthases (PKSs) responsible for the production of aminopolyol polyketides. Heterologous expression of over 150 kbp polyketide gene clusters successfully afforded their products, whose stereochemistry was established by taking advantage of bioinformatic analysis. Furthermore, phylogenetic analysis of highly homologous but functionally diverse domains from the giant PKSs demonstrated the evolutionary mechanism for structural diversification of polyketides. The gene clusters characterized herein, together with their evolutionary insights, are promising genetic building blocks for de novo production of unnatural polyketides.
• Corrigendum to: Biosynthetic Gene Cluster for Surugamide A Encompasses an Unrelated Decapeptide, Surugamide F (ChemBioChem, (2016), 17, 18, (1709-1712), 10.1002/cbic.201600350)

  Ninomiya A, Katsuyama Y, Kuranaga T, Miyazaki M, Nogi Y, Okada S, Wakimoto T, Ohnishi Y, Matsunaga S, Takada K

  ChemBioChem 18 (17) 2017 [Refereed][Not invited]
  
• Concise Synthesis of Anserine: Efficient Solvent Tuning in Asymmetric Hydrogenation Reaction

  Megumi Yamashita, Keita Shimizu, Yasuaki Koizumi, Toshiyuki Wakimoto, Yoshitaka Hamashima, Tomohiro Asakawa, Makoto Inai, Toshiyuki Kan

  Synlett 27 (19) 2734 - 2736 0936-5214 2016/12/01 [Refereed][Not invited]
   

  © Georg Thieme Verlag Stuttgart.New York-Synlett 2016. A concise synthesis of anserine and related compounds was accomplished by Et-DuPhos-Rh-catalyzed asymmetric hydrogenation of dehydrohistidine derivatives in 2,2,2-trifluoroethanol, which played a key role in improving the yield and selectivity.
• Metagenomic Analysis of the Sponge Discodermia Reveals the Production of the Cyanobacterial Natural Product Kasumigamide by 'Entotheonella'

  Yu Nakashima, Yoko Egami, Miki Kimura, Toshiyuki Wakimoto, Ikuro Abe

  PLOS ONE 11 (10) e0164468  1932-6203 2016/10 [Refereed][Not invited]
   

  Sponge metagenomes are a useful platform to mine cryptic biosynthetic gene clusters responsible for production of natural products involved in the sponge-microbe association. Since numerous sponge-derived bioactive metabolites are biosynthesized by the symbiotic bacteria, this strategy may concurrently reveal sponge-symbiont produced compounds. Accordingly, a metagenomic analysis of the Japanese marine sponge Discodermia calyx has resulted in the identification of a hybrid type I polyketide synthase-nonribosomal peptide synthetase gene (kas). Bioinformatic analysis of the gene product suggested its involvement in the biosynthesis of kasumigamide, a tetrapeptide originally isolated from freshwater free-living cyanobacterium Microcystis aeruginosa NIES-87. Subsequent investigation of the sponge metabolic profile revealed the presence of kasumigamide in the sponge extract. The kasumigamide producing bacterium was identified as an 'Entotheonella' sp. Moreover, an in silico analysis of kas gene homologs uncovered the presence of kas family genes in two additional bacteria from different phyla. The production of kasumigamide by distantly related multiple bacterial strains implicates horizontal gene transfer and raises the potential for a wider distribution across other bacterial groups.
• Biosynthetic Gene Cluster for Surugamide A Encompasses an Unrelated Decapeptide, Surugamide F

  Akihiro Ninomiya, Yohei Katsuyama, Takefumi Kuranaga, Masayuki Miyazaki, Yuichi Nogi, Shigeru Okada, Toshiyuki Wakimoto, Yasuo Ohnishi, Shigeki Matsunaga, Kentaro Takada

  ChemBioChem 17 (18) 1709 - 1712 1439-7633 2016/09/15 [Refereed][Not invited]
   

  Genome mining is a powerful method for finding novel secondary metabolites. In our study on the biosynthetic gene cluster for the cyclic octapeptides surugamides A–E (inhibitors of cathepsin B), we found a putative gene cluster consisting of four successive non-ribosomal peptide synthetase (NRPS) genes, surA, surB, surC, and surD. Prediction of amino acid sequence based on the NRPSs and gene inactivation revealed that surugamides A–E are produced by two NRPS genes, surA and surD, which were separated by two NRPS genes, surB and surC. The latter genes are responsible for the biosynthesis of an unrelated peptide, surugamide F. The pattern of intercalation observed in the sur genes is unprecedented. The structure of surugamide F, a linear decapeptide containing one 3-amino-2-methylpropionic acid (AMPA) residue, was determined by spectroscopic methods and was confirmed by solid-phase peptide synthesis.
• Sulfoureido Lipopeptides from the Marine Sponge Discodermia kiiensis

  Karen Co Tan, Toshiyuki Wakimoto, Ikuro Abe

  Journal of Natural Products 79 (9) 2418 - 2422 0163-3864 2016/09 [Refereed][Not invited]
   

  New N-sulfoureidylated lipopeptides, sulfolipodiscamides A-C (1-3), were isolated by gel filtration chromatography of the n-butanol fraction of the marine sponge Discodermia kiiensis. By extensive NMR. analyses and high-resolution mass spectrometry, the structures of 1-3 were elucidated as having an unprecedented N-sulfoureidyl group on the D-citrulline residue, a distinct feature that was not found in the structurally related lipodiscamides A-C (4-6), derived from the ether fraction of the same sponge. Furthermore, the absolute configurations of 1-3 were confirmed by comparisons of the HPLC retention times of the hydrolytic products and the corresponding authentic lipodiscamides. Interestingly, sulfolipodiscamide A displayed a 2.3-fold increase in cytotoxicity against murine leukemia (P388) cells, compared to the unconjugated parent compound.
• Structural Diversification of Lyngbyatoxin A by Host-Dependent Heterologous Expression of the tleABC Biosynthetic Gene Cluster

  Lihan Zhang, Shotaro Hoshino, Takayoshi Awakawa, Toshiyuki Wakimoto, Ikuro Abe

  ChemBioChem 17 (15) 1407 - 1411 1439-4227 2016/08 [Refereed][Not invited]
   

  Natural products have enormous structural diversity, yet little is known about how such diversity is achieved in nature. Here we report the structural diversification of a cyanotoxin-lyngbyatoxin A-and its biosynthetic intermediates by heterologous expression of the Streptomyces-derived tleABC biosynthetic gene cluster in three different Streptomyces hosts: S. lividans, S. albus, and S. avermitilis. Notably, the isolated lyngbyatoxin derivatives, including four new natural products, were biosynthesized by crosstalk between the heterologous tleABC gene cluster and the endogenous host enzymes. The simple strategy described here has expanded the structural diversity of lyngbyatoxinA and its biosynthetic intermediates, and provides opportunities for investigation of the currently underestimated hidden biosynthetic crosstalk.
• Synthesis of chemical-biology tools enabling in vivo imaging and analysis of epigallocatechin gallate

  Tomohiro Asakawa, Atsushi Yoshida, Yasuo Hirooka, Takashi Suzuki, Kunihiko Itoh, Kosuke Shimizu, Naoto Oku, Takumi Furuta, Toshiyuki Wakimoto, Makoto Inai, Toshiyuki Kan

  Heterocycles 93 (1) 218 - 242 0385-5414 2016/04 [Refereed][Not invited]
   

  (-)-Epigallocatechin gallate (EGCg) has multiple bioactivities, and imaging/analytical tools are required for drug development studies. Here we present full details of our synthetic studies aimed at providing building blocks for development of such tools, including a concise synthesis of model compound 5,7-dideoxyEGCg (DOEGCg, 2) and an asymmetric synthesis of 6-(5-aminopenty1)-5,7-deoxyepigallocatechin gallate (APDOEGCg, 4), which contains a reactive terminal amino group. To demonstrate its utility, APDOEGCg (4) was efficiently converted to a fluorescent probe 53 by linking it to a fluorescein derivative, Tokyo Green, via the amino group. We confirmed that 53 is suitable for in vivo imaging studies. We also prepared an immunogen 56 by conjugation of 4 to human serum albumin carrier protein via a glutaraldehyde linker, and we used 56 to raise anti-EGCg antiserum in mice. The fluorescent probe and antiserum should be useful tools for biochemical investigations of the localization and target sites of EGCg. APDOEGCg should also be available for developing other novel tools for biochemical studies of catechins.
• Syntheses of methylated catechins and theaflavins using 2-nitrobenzenesulfonyl group to protect and deactivate phenol

  Tomohiro Asakawa, Yusuke Kawabe, Atsushi Yoshida, Yoshiyuki Aihara, Tamiko Manabe, Yoshitsugu Hirose, Asuka Sakurada, Makoto Inai, Yoshitaka Hamashima, Takumi Furuta, Toshiyuki Wakimoto, Toshiyuki Kan

  Journal of Antibiotics 69 (4) 299 - 312 0021-8820 2016/04 [Refereed][Not invited]
   

  An efficient and versatile synthetic method for labile polyphenols was established using 2-nitrobenzenesulfonate (Ns) as a protecting group for phenol. This methodology provides regio- and stereoselective access to a range of methylated catechins, such as methylated epigallocatechin gallates, that are not readily available from natural sources. In addition, biomimetic synthesis of theaflavins from catechins was accomplished using Ns protection to minimize undesired side reactions of electron-rich aromatic rings during oxidation, enabling construction of the complex benzotropolone core in a single-step oxidative coupling reaction. Availability of these compounds will aid detailed structure biological activity relationship studies of catechins.
• 複合培養法を用いた新規macrolactam類の単離構造決定

  星野 翔太郎, 岡田 正弘, 張 恵平, 林 文晶, 脇本 敏幸, 尾仲 宏康, 阿部 郁朗

  日本薬学会年会要旨集 (公社)日本薬学会 136年会 (2) 112 - 112 0918-9823 2016/03
• An Unusual Chimeric Diterpene Synthase from Emericella variecolor and Its Functional Conversion into a Sesterterpene Synthase by Domain Swapping

  Bin Qin, Yudai Matsuda, Takahiro Mori, Masahiro Okada, Zhiyang Quan, Takaaki Mitsuhashi, Toshiyuki Wakimoto, Ikuro Abe

  Angew. Chem. Int. Ed. 55 (5) 1658 - 1661 1433-7851 2016/01 [Refereed][Not invited]
   

  Di- and sesterterpene synthases produce C-20 and C-25 isoprenoid scaffolds from geranylgeranyl pyrophosphate (GGPP) and geranylfarnesyl pyrophosphate (GFPP), respectively. By genome mining of the fungus Emericella variecolor, we identified a multitasking chimeric terpene synthase, EvVS, which has terpene cyclase (TC) and prenyltransferase (PT) domains. Heterologous gene expression in Aspergillus oryzae led to the isolation of variediene (1), a novel tricyclic diterpene hydrocarbon. Intriguingly, in vitro reaction with the enzyme afforded the new macrocyclic sesterterpene 2 as a minor product from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). The TC domain thus produces the diterpene 1 and the sesterterpene 2 from GGPP and GFPP, respectively. Notably, a domain swap of the PT domain of EvVS with that of another chimeric sesterterpene synthase, EvSS, successfully resulted in the production of 2 in vivo as well. Cyclization mechanisms for the production of these two compounds are proposed.
• Calyculin: Nature's way of making the sponge-derived cytotoxin

  Toshiyuki Wakimoto, Yoko Egami, Ikuro Abe

  Natural Product Reports 33 (6) 751 - 760 0265-0568 2016 [Refereed][Not invited]
   

  Calyculin A is a major cytotoxic compound isolated from the Japanese marine sponge Discodermia calyx. Its potent cytotoxicity is attributable to the specific inhibition of protein phosphatases 1 and 2A, as in the case of okadaic acid and the microcystins. Its chemical structure is well-designed not only for enzyme inhibition but also for higher membrane permeability in order to impart its potent cytotoxicity. The biosynthetic gene cluster of this densely functionalized polyketide and nonribosomal peptide hybrid molecule was recently identified from the sponge-microbe association. The producer organism and the dynamic bioconversion process were also revealed. In this highlight, we focus on the recent studies addressing nature's design and biogenesis of the sponge-derived cytotoxin, calyculin A.
• Niizalactams A-C, Multicyclic Macrolactams Isolated from Combined Culture of Streptomyces with Mycolic Acid-Containing Bacterium

  Shotaro Hoshino, Masahiro Okada, Toshiyuki Wakirnoto, Huiping Zhang, Fumiaki Hayashi, Hiroyasu Onaka, Ikuro Abe

  Journal of Natural Products 78 (12) 3011 - 3017 0163-3864 2015/12 [Refereed][Not invited]
   

  A terrestrial bacterium, Streptomyces sp. NZ-6, produced niizalactams A-C (1-3), unprecedented di- and tricyclic macrolactams, by coculturing with the mycolic acid-containing bacterium Tsukamurella pulmonis TP-B0596. Their complete structures, including absolute configurations, were elucidated on, the basis of spectroscopic data and chemical derivatization. Their unique skeletons are proposed to be biosynthesized from a common 26-membered macrolactam intermediate by S(N)2 cyclization or an intramolecular Diels-Alder reaction.
• Dietziamides, novel tetramic acid dimers from Dietzia timorensis MZ-3 with antioxidative activity

  Shotaro Hoshino, Toshiyuki Wakimoto, Huiping Zhang, Fumiaki Hayashi, Masahiro Okada, Ikuro Abe

  Bioorganic & Medicinal Chemistry Letters 25 (18) 3953 - 3955 0960-894X 2015/09 [Refereed][Not invited]
   

  Dietziamides A and B, two novel tetramic acid dimers, were isolated from the rare actinomycetes Dietzia timorensis MZ-3 in the course of our HPLC-diode array screening of our collection of terrestrial actinomycetes. The spectroscopic analysis revealed the chemical structures of the first secondary metabolites characterized in the genus Dietzia. Dietziamides A and B showed moderate DPPH (1,1-diphenyl-2-picryl-hydrazyl) radical scavenging activities. (C) 2015 Elsevier Ltd. All rights reserved.
• Structural Basis for beta-Carboline Alkaloid Production by the Microbial Homodimeric Enzyme McbB

  Takahiro Mori, Shotaro Hoshino, Shusaku Sahashi, Toshiyuki Wakimoto, Takashi Matsui, Hiroyuki Morita, Ikuro Abe

  CHEMISTRY & BIOLOGY 22 (7) 898 - 906 1074-5521 2015/07 [Refereed][Not invited]
   

  The beta-carboline (beta C) alkaloids occur throughout nature and exhibit diverse biological activities. In contrast to beta C alkaloid synthesis in plants, the biosynthesis in microorganisms remains poorly understood. The recently reported McbB from Marinactinospora thermotolerans is a novel enzyme proposed to catalyze the Pictet-Spengler (PS) reaction of L-tryptophan and oxaloacetaldehyde to produce the beta C scaffold of marinacarbolines. In this study, we solved the crystal structure of McbB complexed with L-tryptophan at 2.48 angstrom resolution, which revealed the novel protein folding of McbB and the totally different structure from those of other PS condensation catalyzing enzymes, such as strictosidine synthase and norcoclaurine synthase from plants. Structural analysis and site-directed mutagenesis confirmed that the previously proposed catalytic Glu97 at the active-site center functions as an acid and base catalyst. Remarkably, the structure-based mutants R72A and H87A, with expanded active-site cavities, newly accepted bulky phenylglyoxal as the aldehyde substrate, to produce 1-benzoyl-3-carboxy-beta-carboline.
• Arcyriaflavin E, a new cytotoxic indolocarbazole alkaloid isolated by combined-culture of mycolic acid-containing bacteria and Streptomyces cinnamoneus NBRC 13823

  Shotaro Hoshino, Lihan Zhang, Takayoshi Awakawa, Toshiyuki Wakimoto, Hiroyasu Onaka, Ikuro Abe

  Journal of Antibiotics 68 (5) 342 - 344 0021-8820 2015/05 [Refereed][Not invited]
  
• Uncovering the Unusual D-Ring Construction in Terretonin Biosynthesis by Collaboration of a Multifunctional Cytochrome P450 and a Unique Isomerase

  Yudai Matsuda, Taiki Iwabuchi, Toshiyuki Wakimoto, Takayoshi Awakawa, Ikuro Abe

  Journal of the American Chemical Society 137 (9) 3393 - 3401 0002-7863 2015/03 [Refereed][Not invited]
   

  Terretonin (1) is a fungal meroterpenoid isolated from Aspergillus terreus, and possesses a highly oxygenated and unique tetracyclic structure. Although the biosynthetic gene cluster for 1 has been identified and the biosynthesis has recently been studied by heterologous reconstitution and targeted-gene deletion experiments, the last few steps of the terretonin pathway after terrenoid (6) have yet to be elucidated. Notably, the mechanism for the D-ring expansion to afford the terretonin scaffold has been a long-standing mystery to solve. Here we report the characterization of three enzymes that convert 6 into 1, as well as the complete biosynthetic pathway of 1. In the proposed terretonin pathway, the cytochrome P450 Trt6 catalyzes three successive oxidations to transform 6 into an unstable intermediate, which then undergoes the D-ring expansion and unusual rearrangement of the methoxy group to afford the core skeleton of 1. This unprecedented rearrangement is catalyzed by a novel isomerase Trt14. Finally, the nonheme iron-dependent dioxygenase Trt7 accomplishes the last two oxidation reactions steps to complete the biosynthesis.
• Chojalactones A-C, Cytotoxic Butanolides Isolated from Streptomyces sp Cultivated with Mycolic Acid Containing Bacterium

  Shotaro Hoshino, Toshiyuki Wakimoto, Hiroyasu Onaka, Ikuro Abe

  Organic Letters 17 (6) 1501 - 1504 1523-7060 2015/03 [Refereed][Not invited]
   

  The soil-derived bacterium, Streptomyces sp. CJ-5, was cocultured with the mycolic acid-containing bacterium Tsukamurella pulmonis TP-B0596. The combined culture method significantly enhanced the production of the secondary metabolites in Streptomyces sp. CJ-5, leading to the isolation of three novel butanolide chojalactones A-C (1-3), with unusual gamma-butyrolactone scaffolds. The complete structures, including the absolute configurations of 1-3, were determined based on spectroscopic data and total syntheses. In methylthiazole tetrazolium (MTT) assays, 1 and 2 showed moderate cytotoxicity against P388 cells.
• Cytotoxic cyclic peptides from the marine sponges

  Toshiyuki Wakimoto, Karen Co Tan, Hiroki Tajima, Ikuro Abe

  Handbook of Anticancer Drugs from Marine Origin 113 - 144 2015/01/01 [Refereed][Not invited]
   

  To date, a significant number of cyclic peptides Cyclic peptide have been isolated from the marine sponges. Their structures often contain non-proteinogenic amino acids, some of which are derived from the biosynthetic pathway mixed with polyketides synthase. Halogenation, N-formylation, and racemaization to D-isomers were also frequently observed. Here we review the structural features of cytotoxic cyclic peptides from marine sponges. The cyclic peptides and depsipeptides were classified into different cyclization ways. The recent progress on the studies of their mode of action and biosynthesis was also included.
• Production of indole antibiotics induced by exogenous gene derived from sponge metagenomes

  Yuya Takeshige, Yoko Egami, Toshiyuki Wakimoto, Ikuro Abe

  Molecular Biosystems 11 (5) 1290 - 1294 1742-206X 2015 [Refereed][Not invited]
   

  Sponge metagenomes are accessible genetic sources containing genes and gene clusters responsible for the biosynthesis of sponge-derived bioactive natural products. In this study, we obtained the clone pDC112, producing turbomycin A and 2,2-di(3-indolyl)-3-indolone, based on the functional screening of the metagenome library derived from the marine sponge Discodermia calyx. The subcloning experiment identified ORF 25, which is homologous to inosine 50-monophosphate dehydrogenase and required for the production of 2,2-di(3-indolyl)-3-indolone in Escherichia coli.
• Phosphocalyculin C as a pyrophosphate protoxin of calyculin C in the marine sponge Discodermia calyx

  Yoko Egami, Toshiyuki Wakimoto, Ikuro Abe

  Bioorganic & Medicinal Chemistry Letters 24 (22) 5150 - 5153 0960-894X 2014/11 [Refereed][Not invited]
   

  Calyculin C, a minor derivative of the calyculins, has an additional methyl group on C32 of calyculin A. A recent biosynthetic study of calyculins revealed that an end product of calyculin biosynthesis is the pyrophosphate form, phosphocalyculin A. However, the pyrophosphate counterpart derived from calyculin C had not been reported. We isolated phosphocalyculin C as a minor pyrophosphate derivative, by a detailed investigation of an extract from the sponge Discodermia calyx. The treatment of phosphocalyculin C with the D. calyx cell-free extract significantly enhanced its cytotoxicity, providing molecular evidence for its role as the protoxin of calyculin C. (C) 2014 Elsevier Ltd. All rights reserved.
• Complete Biosynthetic Pathway of Anditomin: Nature's Sophisticated Synthetic Route to a Complex Fungal Meroterpenoid

  Yudai Matsuda, Toshiyuki Wakimoto, Takahiro Mori, Takayoshi Awakawa, Ikuro Abe

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 136 (43) 15326 - 15336 0002-7863 2014/10 [Refereed][Not invited]
   

  Anditomin and its precursors, andilesins, are fungal meroterpenoids isolated from Aspergillus variecolor and have unique, highly oxygenated chemical structures with a complex bridged-ring system. Previous isotope-feeding studies revealed their origins as 3,5-dimethylorsellinic acid and farnesyl pyrophosphate and suggested the possible involvement of a Diels-Alder reaction to afford the congested bicyclo[2.2.2]octane core structure of andilesins. Here we report the first identification of the biosynthetic gene cluster of anditomin and the determination of the complete biosynthetic pathway by characterizing the functions of 12 dedicated enzymes. The anditomin pathway actually does not employ a Diels-Alder reaction, but involves the nonheme iron-dependent dioxygenase AndA to synthesize the bridged-ring by an unprecedented skeletal reconstruction. Another dioxygenase, AndF, is also responsible for the structural complexification, generating the end product anditomin by an oxidative rearrangement.
• Calyculin biogenesis from a pyrophosphate protoxin produced by a sponge symbiont

  Toshiyuki Wakimoto, Yoko Egami, Yu Nakashima, Yukihiko Wakimoto, Takahiro Mori, Takayoshi Awakawa, Takuya Ito, Hiromichi Kenmoku, Yoshinori Asakawa, Joern Piel, Ikuro Abe

  Nature Chemical Biology 10 (8) 648 - U193 1552-4450 2014/08 [Refereed][Not invited]
   

  The Japanese marine sponge Discodermia calyx contains a major cytotoxic compound, calyculin A, which exhibits selective inhibition of protein phosphatases 1 and 2A. It has long been used as a chemical tool to evaluate intracellular signal transduction regulated by reversible protein phosphorylation. We describe the identification of the biosynthetic gene cluster of calyculin A by a metagenome mining approach. Single-cell analysis revealed that the gene cluster originates in the symbiont bacterium 'Candidatus Entotheonella' sp. A phosphotransferase encoded in the gene cluster deactivated calyculin A to produce a newly discovered diphosphate, which was actually the biosynthetic end product. The diphosphate had been previously overlooked because of the enzymatic dephosphorylation that occurred in response to sponge tissue disruption. Our work presents what is to our knowledge the first evidence for the biosynthetic process of calyculin A along with a notable phosphorylation-dephosphorylation mechanism to regulate toxicity, suggesting activated chemical defense in the most primitive of all multicellular animals.
• A Methyltransferase Initiates Terpene Cyclization in Teleocidin B Biosynthesis

  Takayoshi Awakawa, Lihan Zhang, Toshiyuki Wakimoto, Shotaro Hoshino, Takahiro Mori, Takuya Ito, Jun Ishikawa, Martin E. Tanner, Ikuro Abe

  Journal of the American Chemical Society 136 (28) 9910 - 9913 0002-7863 2014/07 [Refereed][Not invited]
   

  Teleocidin B is an indole terpenoid isolated from Streptomyces. Due to its unique chemical structure and ability to activate protein kinase C, it has attracted interest in the areas of organic chemistry and cell biology. Here, we report the identification of genes encoding enzymes for teleocidin B biosynthesis, including non-ribosomal peptide synthetase (tleA), P-450 monooxygenase (tleB), prenyltransferase (tleC), and methyltransferase (tleD). The tleD gene, which is located outside of the tleABC cluster on the chromosome, was identified by transcriptional analysis and heterologous expression. Remarkably, TleD not only installs a methyl group on the geranyl moiety of the precursor but also facilitates the nucleophilic attack from the electron-rich indole to the resultant cation, to form the indole-fused six-membered ring. This is the first demonstration of a cation, generated from methylation, triggering successive terpenoid ring closure.
• Three Acyltetronic Acid Derivatives: Noncanonical Cryptic Polyketides from Aspergillus niger Identified by Genome Mining

  Xiao-Long Yang, Takayoshi Awakawa, Toshiyuki Wakimoto, Ikuro Abe

  ChemBioChem 15 (11) 1578 - 1583 1439-4227 2014/07 [Refereed][Not invited]
  
• ミコール酸含有微生物Tsukamurella pulmonisとの複合培養による、放線菌Streptomyces属由来新規生物活性物質の探索

  星野 翔太郎, 張 驪駻, 淡川 孝義, 脇本 敏幸, 尾仲 宏康, 阿部 郁朗

  日本放線菌学会大会講演要旨集 日本放線菌学会 29回 48 - 48 2014/06
• Lipodiscamides A-C, New Cytotoxic Lipopeptides from Discodermia kiiensis

  Karen Co Tan, Toshiyuki Wakimoto, Ikuro Abe

  Organic Letters 16 (12) 3256 - 3259 1523-7060 2014/06 [Refereed][Not invited]
   

  Lipodiscamides A-C, three new lipodepsipeptides, were characterized from the marine sponge Discodermia kiiensis. These structurally rare cyclic lipodepsipeptides were found to possess an unprecedented dilactone macrocycle and, thus, represent a new family of lipopeptides. They are the only lipopeptides bearing 4S-hydroxy-trans-2-enoate, and non-canonical amino acids, L-3-ureidoalanine (Uda), E-dehydronorvaline (Denor), and D-citrulline (Cit). MTT assays against P388 and HeLa cells revealed the moderate cytotoxicity of all three compounds.
• An environmental bacterial taxon with a large and distinct metabolic repertoire (vol 506, pg 58, 2014)

  Micheal C. Wilson, Tetsushi Mori, Christian Rueckert, Agustinus R. Uria, Maximilian J. Helf, Kentaro Takada, Christine Gernert, Ursula A. E. Steffens, Nina Heycke, Susanne Schmitt, Christian Rinke, Eric J. N. Helfrich, Alexander O. Brachmann, Cristian Gurgui, Toshiyuki Wakimoto, Matthias Kracht, Max Cruesemann, Ute Hentschel, Ikuro Abe, Shigeki Matsunaga, Joern Kalinowski, Haruko Takeyama, Joern Piel

  Nature 507 (7491) 0028-0836 2014/03 [Refereed][Not invited]
  
• Enantioselective Synthesis of SB-203207

  Yasuo Hirooka, Kazutada Ikeuchi, Yuichiro Kawamoto, Yusuke Akao, Takumi Furuta, Tomohiro Asakawa, Makoto Inai, Toshiyuki Wakimoto, Tohru Fukuyama, Toshiyuki Kan

  Organic Letters 16 (6) 1646 - 1649 1523-7060 2014/03 [Refereed][Not invited]
   

  Total synthesis of SB-203207 (1) was achieved, beginning with a desymmetrical C-H insertion reaction of a diazoester bearing our recently developed chiral auxiliary. Utilizing the optically active bicyclo[3.3.0]octane ring, four stereogenic centers were efficiently constructed in sequence. Finally, mild oxidation of 27 to carboxylic acid via a cyanohydrin intermediate and hydrolysis of cyanide to carboxyamide in the presence of the labile enamide group completed an efficient total synthesis of 1.
• An environmental bacterial taxon with a large and distinct metabolic repertoire

  Micheal C. Wilson, Tetsushi Mori, Christian Rueckert, Agustinus R. Uria, Maximilian J. Helf, Kentaro Takada, Christine Gernert, Ursula A. E. Steffens, Nina Heycke, Susanne Schmitt, Christian Rinke, Eric J. N. Helfrich, Alexander O. Brachmann, Cristian Gurgui, Toshiyuki Wakimoto, Matthias Kracht, Max Cruesemann, Ute Hentschel, Ikuro Abe, Shigeki Matsunaga, Joern Kalinowski, Haruko Takeyama, Joern Piel

  Nature 506 (7486) 58 - + 0028-0836 2014/02 [Refereed][Not invited]
   

  Cultivated bacteria such as actinomycetes are a highly useful source of biomedically important natural products. However, such 'talented' producers represent only a minute fraction of the entire, mostly uncultivated, prokaryotic diversity. The uncultured majority is generally perceived as a large, untapped resource of new drug candidates, but so far it is unknown whether taxa containing talented bacteria indeed exist. Here we report the single-cell- and metagenomics-based discovery of such producers. Two phylotypes of the candidate genus 'Entotheonella' with genomes of greater than 9 megabases and multiple, distinct biosynthetic gene clusters co-inhabit the chemically and microbially rich marine sponge Theonella swinhoei. Almost all bioactive polyketides and peptides known from this animal were attributed to a single phylotype. 'Entotheonella' spp. are widely distributed in sponges and belong to an environmental taxon proposed here as candidate phylum 'Tectomicrobia'. The pronounced bioactivities and chemical uniqueness of 'Entotheonella' compounds provide significant opportunities for ecological studies and drug discovery.
• Erratum: An environmental bacterial taxon with a large and distinct metabolic repertoire (Nature (2014) 506 (58-62) DOI: 10.1038/nature12959))

  Micheal C. Wilson, Tetsushi Mori, Christian Rückert, Agustinus R. Uria, Maximilian J. Helf, Kentaro Takada, Christine Gernert, Ursula A E Steffens, Nina Heycke, Susanne Schmitt, Christian Rinke, Eric J N Helfrich, Alexander O. Brachmann, Cristian Gurgui, Toshiyuki Wakimoto, Matthias Kracht, Max Crüsemann, Ute Hentschel, Ikuro Abe, Shigeki Matsunaga, Jörn Kalinowski, Haruko Takeyama, Jörn Piel

  Nature 507 262  0028-0836 2014/01/01 [Not refereed][Not invited]
  
• Revised Structure of Cyclolithistide A, a Cyclic Depsipeptide from the Marine Sponge Discodermia japonica

  Hiroki Tajima, Toshiyuki Wakimoto, Kentaro Takada, Yuji Ise, Ikuro Abe

  Journal of Natural Products 77 (1) 154 - 158 0163-3864 2014/01 [Refereed][Not invited]
   

  A cyclic peptide was isolated from the deep-sea marine sponge Discodermia japonica, and its NMR spectroscopic data were identical to those reported for cyclolithistide A, a known antifungal depsipeptide. However, the interresidue HMBC correlations suggested that the amino acid sequence was different from that of the original structure. Moreover, chiral-phase GC-MS, combined with,Marfey's analysis, indicated that the absolute configurations of three amino acids were also antipodal. Here, we propose the revised structure of cyclolithistide A and address the configuration of the previously unassigned 4-amino-3,5-dihydroxyhexanoic acid (Adha) moiety.
• Pyranonigrin E: A PKS-NRPS Hybrid Metabolite from Aspergillus niger Identified by Genome Mining

  Takayoshi Awakawa, Xiao-Long Yang, Toshiyuki Wakimoto, Ikuro Abe

  ChemBioChem 14 (16) 2095 - 2099 1439-4227 2013/11 [Refereed][Not invited]
   

  Induced production of PKS-NRPS metabolites: The genome mining approach is useful for obtaining new compounds. We activated a dormant PKS-NRPS gene cluster in Aspergillus niger ATCC 1015 by expressing its dedicated transcriptional activator (PynR). As a result, the transformant expressing pynR produced a new pyranonigrin compound that we have named pyranonigirin E. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
• Induced production of novel prenyldepside and coumarins in endophytic fungi Pestalotiopsis acaciae

  Xiao-Long Yang, Takayoshi Awakawa, Toshiyuki Wakimoto, Ikuro Abe

  Tetrahedron Letters 54 (43) 5814 - 5817 0040-4039 2013/10 [Refereed][Not invited]
   

  Concomitant supplementation of a histone deacetylase inhibitor, suberoylanilide hydroxamic acid, and a DNA methyltransferase inhibitor, 5-azacytidine, to the culture medium of a plant endophytic fungus, Pestalotiopsis acaciae, dramatically altered its metabolic profiles. As a result, three novel aromatic compounds, 2'-hydroxy-6'-hydroxymethyl-4'-methylphenyl-2,6-dihydroxy-3-(2-isopentenyl)benzoate (1), 4,6-dihydroxy-7-hydroxymethyl-3-methylcoumarin (2) and 4,6-dihydroxy-3,7-dimethylcoumarin (3), were isolated, along with five known polyketides, endocrocin (4), pestalotiollide B (5), pestalotiopyrone G (6), scirpyrone A (7) and 7-hydroxy-2-(2-hydroxypropyl)-5-methylchromone (8). (c) 2013 Elsevier Ltd. All rights reserved.
• Ergot alkaloid from the sea slug Pleurobranchus forskalii

  Toshiyuki Wakimoto, Karen Co Tan, Ikuro Abe

  Toxicon 72 1 - 4 0041-0101 2013/09 [Refereed][Not invited]
   

  The sea slug Pleura bronchus forskalii is a carnivorous scavenger that is widely distributed in shallow subtidal areas. Very few investigations of the chemical components of this gastropod have been reported. In this study we performed a comprehensive analysis of an extract of the marine mollusc, R forskalii, collected off Ishigaki Island, Japan. As a result, an alkaloid was isolated from the chloroform extract. Remarkably, the structure elucidation based on the spectral data revealed that it was an ergot alkaloid peptide, ergosinine. Various ergot alkaloids have previously been isolated mainly from terrestrial higher plants or fungi. This is the first report of the isolation of an ergopeptine from marine life, and thus the known geographical extent of ergot alkaloids now includes both terrestrial and aquatic organisms. (C) 2013 Elsevier Ltd. All rights reserved.
• Induced biosyntheses of a novel butyrophenone and two aromatic polyketides in the plant pathogen Stagonospora nodorum

  Xiao Long Yang, Takayoshi Awakawa, Toshiyuki Wakimoto, Ikuro Abe

  Natural Products and Bioprospecting 3 (4) 141 - 144 2192-2195 2013/08/01 

  © 2013, The Author(s). Fungal aromatic compounds comprise an important and structurally diverse group of secondary metabolites. Several genome sequencing projects revealed many putative biosynthetic gene clusters of fungal aromatic compounds, but many of these genes seem to be silent under typical laboratory culture conditions. To gain access to this untapped reservoir of natural products, we utilized chemical epigenetic modifiers to induce the expression of dormant biosynthetic genes. As a result, the concomitant supplementation of the histone deacetylase inhibitors suberoylanilide hydroxamic acid (500 μM) and nicotinamide (50 μM) to the culture medium of a fungal pathogen, Stagonospora nodorum, resulted in the isolation of three aromatic compounds (1–3), including a novel natural butyrophenone, (+)-4′-methoxy-(2S)-methylbutyrophenone (1), and two known polyketides, alternariol (2) and (−)-(3R)-mellein methyl ether (3). [Figure not available: see fulltext.].
• Cycloforskamide, a cytotoxic macrocyclic peptide from the sea slug Pleurobranchus forskalii

  Karen Co Tan, Toshiyuki Wakimoto, Kentaro Takada, Takashi Ohtsuki, Nahoko Uchiyama, Yukihiro Goda, Ikuro Abe

  Journal of Natural Products 76 (7) 1388 - 1391 0163-3864 2013/07/26 [Refereed][Not invited]
   

  A macrocylic dodecapeptide, cycloforskamide, was isolated from the sea slug Pleurobranchus forskalii, collected off Ishigaki Island, Japan. Its planar structure was deduced by extensive NMR analyses and was further confirmed by MS/MS fragmentation analyses. Finally, the absolute configuration was determined by total hydrolysis and chiral-phase gas chromatographic analysis. This novel dodecapeptide contains three d-amino acids and three thiazoline heterocycles and exhibits cytotoxicity against murine leukemia P388 cells, with an IC 50 of 5.8 μM. © 2013 The American Chemical Society and American Society of Pharmacognosy.
• Induced production of the novel glycolipid ustilagic acid C in the plant pathogen Ustilago maydis

  Xiao-Long Yang, Takayoshi Awakawa, Toshiyuki Wakimoto, Ikuro Abe

  Tetrahedron Letters 54 (28) 3655 - 3657 0040-4039 2013/07/10 [Refereed][Not invited]
   

  Glycolipids have attracted interest from the pharmaceutical, cosmetic, and food industries, due to their unique chemical properties and interesting biological activities. The phytopathogenic fungus Ustilago maydis, a well-known producer of glycolipids, reportedly secretes large amounts of the glycolipid biosurfactant ustilagic acid under special culture conditions. Thus, we utilized chemical epigenetic modifiers, DNA methyltransferase (DNMT) inhibitors and/or histone deacetylase (HDAC) inhibitors, to induce the expression of silent biosynthetic pathways for isolating novel glycolipids produced by this fungus. HPLC analyses revealed that supplementations of the DNMT inhibitor 5-azacytidine and the HDAC inhibitor SBHA to the liquid culture of U. maydis induced the production of the novel glycolipid ustilagic acid C (1), along with the known ustilagic acid B (2). Glycolipids 1 and 2 displayed weak antifungal activities against Aspergillus terreus and Candida albicans. © 2013 Elsevier Ltd. All rights reserved.
• Indole-porphyrin hybrids produced by metagenomics

  Xiao-Long Yang, Toshiyuki Wakimoto, Yuya Takeshige, Rui He, Yoko Egami, Takayoshi Awakawa, Ikuro Abe

  Bioorganic & Medicinal Chemistry Letters 23 (13) 3810 - 3813 0960-894X 2013/07 [Refereed][Not invited]
   

  New indole-porphyrin hybrid molecules were isolated from Escherichia coli harboring metagenomic DNA from the Japanese marine sponge Discodermia calyx. The indole was appended to the reactive vinyl substituent of the harderoporphyrin chromophore, encoded by the insert DNA. Thus, the chimeric pathway between the heterologously expressed porphyrins and the endogenous indole generated new indole-conjugated chiral porphyrins in E. coli. (c) 2013 Elsevier Ltd. All rights reserved.
• Spiro-Ring Formation is Catalyzed by a Multifunctional Dioxygenase in Austinol Biosynthesis

  Yudai Matsuda, Takayoshi Awakawa, Toshiyuki Wakimoto, Ikuro Abe

  Journal of the American Chemical Society 135 (30) 10962 - 10965 0002-7863 2013/07 [Refereed][Not invited]
   

  Austinol, a fungal meroterpenoid derived from 3,5-dimethylorsellinic acid, has a unique chemical structure with a remarkable spiro-lactone ring system. Despite the recent identification of its biosynthetic gene cluster and targeted gene-deletion experiments, the process for the conversion of protoaustinoid A (2), the first tetracyclic biosynthetic intermediate, to the spirolactone preaustinoid A3 (7) has remained enigmatic. Here we report the mechanistic details of the enzyme-catalyzed, stereospecific spiro-lactone ring-forming reaction, which is catalyzed by a non-heme iron-dependent dioxygenase, AusE, along with two flavin monooxygenases, the 5'-hydroxylase AusB and the Baeyer-Villiger monooxygenase AusC. Remarkably, AusE is a multifunctional dioxygenase that is responsible for the iterative oxidation steps, including the oxidative spiro-ring-forming reaction, to produce the austinol scaffold.
• Epigenetic modifier-induced biosynthesis of novel fusaric acid derivatives in endophytic fungi from Datura stramonium L.

  Han Jing Chen, Takayoshi Awakawa, Jie Yin Sun, Toshiyuki Wakimoto, Ikuro Abe

  Natural Products and Bioprospecting 3 (1) 20 - 23 2192-2195 2013/02/01 

  © 2013, The Author(s). Abstract: The treatment of fungi with DNA methyltransferase (DNMT) and/or histone deacetylase (HDAC) inhibitors is a promising way to activate secondary metabolite biosynthetic pathways that are dormant under normal conditions. In this study, we included an HDAC inhibitor, suberoylanilide hydroxamic acid (SBHA), in the culture medium of endophytic fungi isolated from the medicinal plant Datura stramonium L. The production of two compounds was induced in the culture supplemented with SBHA, and their structures were determined to be the fusaric acid derivatives 5-butyl-6-oxo-1,6-dihydropyridine-2-carboxylic acid and 5-(but-9-enyl)-6-oxo-1,6-dihydropyridine-2-carboxylic acid. The result confirmed that the use of chemical epigenetic modifiers is an effective technique for promoting the expression of silent biosynthetic pathways to produce unique secondary metabolites. Graphical abstract: [Figure not available: see fulltext.].
• Allos-hemicalyculin A, a photochemically converted calyculin from the marine sponge Discodermia calyx

  Miki Kimura, Toshiyuki Wakimoto, Ikuro Abe

  Tetrahedron Letters 54 (1) 114 - 116 0040-4039 2013/01 [Refereed][Not invited]
   

  Allos-hemicalyculin A (1), a new derivative of calyculin A, was isolated from the marine sponge Discodermia calyx collected off Shikine-jima Island, Japan. The structure of 1, including the absolute configurations, was elucidated by spectroscopic analyses and photochemical degradation experiments. Consequently, its structure was identical to the distal end of the peptide side chain of calyculin A (2), previously isolated from D. calyx and generated by photochemical oxidative cleavage of the oxazole moiety. In stark contrast to the potent cytotoxicity of 2, 1 is no longer cytotoxic, in agreement with the previously reported structure-activity relationship data. Here we describe the isolation and structural elucidation of 1. (C) 2012 Elsevier Ltd. All rights reserved.
• Cyclodipeptides from metagenomic library of a Japanese marine sponge

  Rui He, Bochu Wang, Toshiyuki Wakimoto, Manyuan Wang, Liancai Zhu, Ikuro Abe

  Journal of the Brazilian Chemical Society 24 (12) 1926 - 1932 0103-5053 2013 [Refereed][Not invited]
   

  Culture-independent metagenomics is an attractive and promising approach to explore unique bioactive small molecules from marine sponges harboring uncultured symbiotic microbes. Therefore, we conducted functional screening of the metagenomic library constructed from the Japanese marine sponge Discodermia calyx. Bioassay-guided fractionation of plate culture extract of antibacterial clone pDC113 afforded eleven cyclodipeptides: Cyclo(L-Thr-L-Leu) (1), Cyclo(L-Val-d-Pro) (2), Cyclo(L-Ile-d-Pro) (3), Cyclo(L-Leu-L-Pro) (4), Cyclo(L-Val-L-Leu) (5), Cyclo(L-Leu-L-Ile) (6), Cyclo(L-Leu-L-Leu) (7), Cyclo(L-Phe-L-Tyr) (8), Cyclo(L-Trp-L-Pro) (9), Cyclo(L-Val-L-Trp) (10) and Cyclo(L-Ile-L-Trp) (11). To the best of our knowledge, these are first cyclodepeptides isolated from metagenomic library. Sequence analysis suggested that isolated cyclodipeptides were not synthesized by nonribosomal peptide synthetases and there was no significant indication of cyclodipeptide synthetases. © 2013 Sociedade Brasileira de Química.
• Stereocontrolled Total Synthesis of (-)-FR901483

  Shigeru Ieda, Akitaka Masuda, Mami Kariyama, Toshiyuki Wakimoto, Tomohiro Asakawa, Tohru Fukuyama, Toshiyuki Kan

  Heterocycles 86 (2) 1071 - 1092 0385-5414 2012/12 [Refereed][Not invited]
   

  The total synthesis of a potent immunosuppressant (-)-FR901483 is accomplished. The skeleton itself is constructed by the Ugi 4CC reaction, subsequent intramolecular Dieckmann condensation, and a diastereoselective intramolecular aldol reaction. However, the remarkable feature is the stereoselective incorporation of the p-methoxybenzyl and methylamino groups within the tricyclic core skeleton.
• Heterologously expressed beta-hydroxyl fatty acids from a metagenomic library of a marine sponge

  Rui He, Toshiyuki Wakimoto, Yoko Egami, Hiromichi Kenmoku, Takuya Ito, Yoshinori Asakawa, Ikuro Abe

  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 22 (24) 7322 - 7325 0960-894X 2012/12 [Refereed][Not invited]
   

  Functional screening based on the antibacterial activity of a metagenomic library of the Japanese marine sponge, Discodermia calyx, afforded three beta-hydroxyl fatty acids: 3-hydroxypalmitic acid, 3-hydroxylauric acid and 3-hydroxymyristic acid, heterologously expressed in an antibacterial clone, pDC113. 3-Hydroxy-palmitic acid showed moderate antibacterial activity against Bacillus cereus and Candida albicans. A sequence analysis of the insert DNA revealed 23 putative ORFs, with most sharing homology to bacterial fatty acid synthase II and lipid A biosynthesis enzymes. The other ORFs were probably transmembrane proteins involved in lipid A biosynthesis. Although lipid A was not detected under our experimental conditions, the production of b-hydroxyl fatty acids as components of lipid A were enhanced in pDC113. (C) 2012 Elsevier Ltd. All rights reserved.
• Whiskey Congeners Suppress LPS/IFN gamma-Induced NO Production in Murine Macrophage RAW 264 Cells by Inducing Heme Oxygenase-1 Expression

  Tomohiro Itoh, Masashi Ando, Yasuyuki Tsukamasa, Toshiyuki Wakimoto, Haruo Nukaya

  JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 60 (51) 12491 - 12500 0021-8561 2012/12 [Refereed][Not invited]
   

  Whiskey includes many nonvolatile substances (whiskey congeners; Whc) that seep from the oak cask during the maturation process. To date, many functions of Whc have reported, such as antiallergy and antimelanogenesis. This study examined the effect of Whc on LPS/IFN gamma-induced nitric oxide (NO) production in murine macrophage RAW 264 cells. Whc suppressed LPS/IFN gamma-induced NO production in a concentration-dependent manner. To determine the active compounds in Whc, the effect of 10 major compounds isolated from Whc on LPS/IFN gamma-induced NO production was examined. Coniferylaldehyde (CA) and sinapylaldehyde (SiA) strongly suppressed LPS/IFN gamma-induced NO production. Pretreatment with Whc, CA, and SiA induced heme oxygenase-1 (HO-1) expression. The expression of HO-1 by Whc, CA, and SiA pretreatment was due to activation of Nrf2/ARE signaling via the elevation of intracellular reactive oxygen species. To investigate the in vivo effects of Whc, Whc was administered to mice with antitype II collagen antibody-induced arthritis, and we the arthritis score and hind paw volume were measured. Administration of Whc remarkably suppressed the arthritis score and hind paw volume. Taken together, these findings suggest that Whc is beneficial for the treatment of inflammatory disease.
• Labile natural products

  Toshiyuki Wakimoto, Ikuro Abe

  MedChemComm 3 (8) 866 - 870 2040-2503 2012/08 [Refereed][Not invited]
   

  The existence of labile natural products in the highly complex endogenous compositions of plants, animals, and even foodstuffs is expected, but the isolation of such unstable compounds remains difficult. We present a review of the current successes in isolating labile natural products.
• Terretonin Biosynthesis Requires Methylation as Essential Step for Cyclization

  Yudai Matsuda, Takayoshi Awakawa, Takayuki Itoh, Toshiyuki Wakimoto, Tetsuo Kushiro, Isao Fujii, Yutaka Ebizuka, Ikuro Abe

  ChemBioChem 13 (12) 1738 - 1741 1439-4227 2012/08 [Refereed][Not invited]
  
• A heptaketide naphthaldehyde produced by a polyketide synthase from Nectria haematococca

  Takayoshi Awakawa, Takuya Kaji, Toshiyuki Wakimoto, Ikuro Abe

  Bioorganic & Medicinal Chemistry Letters 22 (13) 4338 - 4340 0960-894X 2012/07 [Refereed][Not invited]
   

  Bostrycoidin and fusarubin are biologically active fungal polyketides produced by Nectria haematococca. This azaanthraquinone and naphthoquinone are thought to be biosynthesized via formation of a C-14 heptaketide aldehyde as a common key intermediate. A BLAST search against the genome of N. haematococca revealed one candidate gene (NECHADRAFT_101778, NhPKS1), which encodes a multi-domain polyketide synthase (PKS) with a thiol reductase (TR) domain that would facilitate the reductive release of the intermediate to produce a free aldehyde. To investigate the possible involvement of NhPKS1 in the biosynthesis of bostrycoidin and fusarubin, NhPKS1 was heterologously expressed in Aspergillus oryzae, and shown to produce a heptaketide 3-acetonyl-1,6,8-trihydroxy-2-naphthaldehyde as a single product. Thus, NhPKS1 catalyzes a C-2/C-11 and C-4/C-9 aldol-type cyclization of a linear intermediate followed by a subsequent reductive product release to yield the naphthaldehyde. The results indicate NhPKS1 is the enzyme involved in the biosynthesis of bostrycoidin and fusarubin. (C) 2012 Elsevier Ltd. All rights reserved.
• Prenylation of a Nonaromatic Carbon of Indolylbutenone by a Fungal Indole Prenyltransferase

  Jing Chen, Hiroyuki Morita, Toshiyuki Wakimoto, Takahiro Mori, Hiroshi Noguchi, Ikuro Abe

  Organic Letters 14 (12) 3080 - 3083 1523-7060 2012/06 [Refereed][Not invited]
   

  FtmPT1 from Aspergillus fumigatus is a fungal indole prenyltransferase (PT) that normally catalyzes the regiospecific prenylation of brevianamide F (cyclo-L-Trp-L-Pro) at the C-2 position of the indole ring with dimethylallyl diphosphate (DMAPP). Interestingly, FtmPT1 exhibited remarkable substrate tolerance and accepted (E)-4-(1H-indol-3-yl)but-3-en-2-one (1) as a substrate to produce an unnatural novel alpha-prenylindolylbutenone (la). This is the first demonstration of the prenylation of a nonaromatic carbon of the acceptor substrate by a fungal indole PT.
• Calyxamides A and B, Cytotoxic Cyclic Peptides from the Marine Sponge Discodermia calyx

  Miki Kimura, Toshiyuki Wakimoto, Yoko Egami, Karen Co Tan, Yuji Ise, Ikuro Abe

  Journal of Natural Products 75 (2) 290 - 294 0163-3864 2012/02 [Refereed][Not invited]
   

  Cyclic peptides containing 5-hydroxytryptophan and thiazole moieties were isolated from the marine sponge Discodermia calyx collected near Shikine-jima Island, Japan. The structures of calyxamides A (1) and B (2), including the absolute configurations of all amino acids, were elucidated by spectroscopic analyses and degradation experiments. The structures are similar to keramamides F and G, previously isolated from Theonella sp. The analysis of the 16S rDNA sequences obtained from the metagenomic DNA of D. calyx revealed the presence of Canclidatus Entotheonella sp., an unculturable fi-proteobacterium inhabiting the Theonella genus and implicated in the biosynthesis of bioactive peptides.
• Porphyrins from a metagenomic library of the marine sponge Discodermia calyx

  Rui He, Toshiyuki Wakimoto, Yuya Takeshige, Yoko Egami, Hiromichi Kenmoku, Takuya Ito, Bochu Wang, Yoshinori Asakawa, Ikuro Abe

  Molecular Biosystems 8 (9) 2334 - 2338 1742-206X 2012 [Refereed][Not invited]
   

  Marine sponges harbouring uncultured symbiotic bacteria are important sources of biologically active compounds. Since they would be interesting resources to explore unknown functional genes by means of a metagenomic approach, we constructed a metagenomic library of the Japanese marine sponge Discodermia calyx. The functional screening afforded the two clones producing porphyrins as red pigments. The isolation and structural elucidation of the red pigments revealed that the major red pigment was Zn-coproporphyrin III. The sequence data of the clones identified genes encoding glutamyl-tRNA reductase along with other ORFs related to porphyrin biosynthesis.
• Engineering of Plant Type III Polyketide Synthases

  Toshiyuki Wakimoto, Hiroyuki Morita, Ikuro Abe

  Natural Product Biosynthesis by Microorganisms and Plants, Part A 515 337 - 358 0076-6879 2012 [Refereed][Not invited]
   

  Members of the chalcone synthase superfamily of type III polyketide synthases (PKSs) catalyze iterative condensations of CoA thioesters to produce a variety of polyketide scaffolds with remarkable structural diversity and biological activities. The homodimeric type III PKSs share a common three-dimensional overall fold with a conserved Cys-His-Asn catalytic triad; notably, only a slight modification of the active site dramatically expands the catalytic repertoire of the enzymes. In addition, the enzymes exhibit extremely promiscuous substrate specificities, and accept a variety of nonphysiological substrates, making the type III PKSs an excellent platform for the further production of unnatural, novel polyketide scaffolds with promising biological activities. This chapter summarizes recent advances in the engineering of plant type III PKS enzymes in our laboratories, using approaches combining structure-based enzyme engineering and precursor-directed biosynthesis with rationally designed substrate analogs.
• Stereocontrolled Total Synthesis of (+)-UCS1025A

  Kenji Uchida, Takahiro Ogawa, Yoshinori Yasuda, Hiraku Mimura, Teppei Fujimoto, Tohru Fukuyama, Toshiyuki Wakimoto, Tomohiro Asakawa, Yoshitaka Hamashima, Toshiyuki Kan

  Angew. Chem. Int. Ed. 51 (51) 12850 - 12853 1433-7851 2012 [Refereed][Not invited]
   

  Under control: A stereocontrolled total synthesis of (+)-UCS1025A, a potent telomerase inhibitor, was achieved. The synthesis features an intramolecular Diels-Alder reaction, a tandem Staudinger/aza-Wittig reaction, and stereoselective construction of the hemiaminal moiety facilitated by neighboring-group participation. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
• Furan fatty acid as an anti-inflammatory component from the green-lipped mussel Perna canaliculus

  Toshiyuki Wakimoto, Hikaru Kondo, Hirohiko Nii, Kaori Kimura, Yoko Egami, Yusuke Oka, Masae Yoshida, Eri Kida, Yiping Ye, Saeko Akahoshi, Tomohiro Asakaw, Koichi Matsumura, Hitoshi Ishida, Haruo Nukaya, Kuniro Tsuji, Toshiyuki Kan, Ikuro Abe

  Proc. Natl. Acad. Sci. USA 108 (42) 17533 - 17537 0027-8424 2011/10 [Refereed][Not invited]
   

  A lipid extract of Perna canaliculus (New Zealand green-lipped mussel) has reportedly displayed anti-inflammatory effects in animal models and in human controlled studies. However, the anti-inflammatory lipid components have not been investigated in detail due to the instability of the lipid extract, which has made the identification of the distinct active components a formidable task. Considering the instability of the active component, we carefully fractionated a lipid extract of Perna canaliculus (Lyprinol) and detected furan fatty acids (F-acids). These naturally but rarely detected fatty acids show potent radical-scavenging ability and are essential constituents of plants and algae. Based on these data, it has been proposed that F-acids could be potential antioxidants, which may contribute to the protective properties of fish and fish oil diets against chronic inflammatory diseases. However, to date, in vivo data to support the hypothesis have not been obtained, presumably due to the limited availability of F-acids. To confirm the in vivo anti-inflammatory effect of F-acids in comparison with that of eicosapentaenoic acid (EPA), we developed a semisynthetic preparation and examined its anti-inflammatory activity in a rat model of adjuvant-induced arthritis. Indeed, the F-acid ethyl ester exhibited more potent anti-inflammatory activity than that of the EPA ethyl ester. We report on the in vivo activity of F-acids, confirming that the lipid extract of the green-lipped mussel includes an unstable fatty acid that is more effective than EPA.
• 16 Enzymatic Synthesis of Unnatural Novel Biologically Active Compounds by Engineered Plant Polyketide Synthases(Oral Presentation)

  Morita Hiroyuki, Mori Takahiro, Yamashita Makoto, Wakimoto Toshiyuki, Abe Ikuro

  Symposium on the Chemistry of Natural Products, symposium papers 天然有機化合物討論会 (53) 91 - 96 2011/09/02 

  HsPKS1 from Huperzia serrata is a type III polyketide synthase (PKS) with remarkable substrate tolerance and catalytic potential. Here we present the synthesis of unnatural novel polyketide-alkaloid hybrid molecules by exploiting the enzyme reaction using precursor-directed and structure-based approaches. HsPKS1 produced novel pyridoisoindole (or benzopyridoisoindole) with the 6.5.6-fused (or 6.6.5.6-fused) ring system by the condensation of 2-carbamoylbenzoyl-CoA (or 3-carbamoyl-2-naphthoyl-CoA), a synthetic nitrogen-containing non-physiological starter substrate, with two molecules of malonyl-CoA. The structure-based S348G mutant not only extended the product chain length, but also altered the cyclization mechanism to produce a biologically active, ring-expanded 6.7.6-fused dibenzoazepine, by the condensation of 2-carbamoylbenzoyl-CoA with three malonyl-CoAs. Thus, the basic nitrogen atom and the structure-based mutagenesis enabled additional C-C and C-N bond formation to generate the novel polyketide-alkaloid scaffold. Benzalacetone synthase (BAS) from Rheum palmatum is a structurally simple, plant-specific type III PKS, which catalyzes the one-step decarboxylative condensation of malonyl-CoA with 4-coumaroyl-CoA. The type III PKS exhibits unusually broad substrate specificity and notable catalytic versatility. Here we report that R. palmatum BAS efficiently produces a series of unnatural, novel tetramic acid derivatives by the condensation of malonyl-CoA with aminoacyl-CoA thioesters, chemically synthesized from L- and D-amino acids. Remarkably, the novel tetramic acid dimer from D-phenylalanoyl-CoA, showed moderate antiproliferative activity against murine leukemia P388 cells.
• Synthesis of unnatural alkaloid scaffolds by exploiting plant polyketide synthase

  Hiroyuki Morita, Makoto Yamashita, She-Po Shi, Toshiyuki Wakimoto, Shin Kondo, Ryohei Kato, Shigetoshi Sugio, Toshiyuki Kohno, Ikuro Abe

  Proc. Natl. Acad. Sci. USA 108 (33) 13504 - 13509 0027-8424 2011/08 [Refereed][Not invited]
   

  HsPKS1 from Huperzia serrata is a type III polyketide synthase (PKS) with remarkable substrate tolerance and catalytic potential. Here we present the synthesis of unnatural unique polyketide-alkaloid hybrid molecules by exploiting the enzyme reaction using precursor-directed and structure-based approaches. HsPKS1 produced novel pyridoisoindole (or benzopyridoisoindole) with the 6.5.6-fused (or 6.6.5.6-fused) ring system by the condensation of 2-carbamoylbenzoyl-CoA (or 3-carbamoyl-2-naphthoyl-CoA), a synthetic nitrogen-containing nonphysiological starter substrate, with two molecules of malonyl-CoA. The structure-based S348G mutant not only extended the product chain length but also altered the cyclization mechanism to produce a biologically active, ring-expanded 6.7.6-fused dibenzoazepine, by the condensation of 2-carbamoylbenzoyl-CoA with three malonyl-CoAs. Thus, the basic nitrogen atom and the structure-based mutagenesis enabled additional C-C and C-N bond formation to generate the novel polyketide-alkaloid scaffold.
• Enantioselective Total Synthesis of Aperidine

  Toshiyuki Wakimoto, Kakeru Miyata, Hitoshi Ohuchi, Tomohiro Asakawa, Haruo Nukaya, Yoshihide Suwa, Toshiyuki Kan

  Organic Letters 13 (10) 2789 - 2791 1523-7060 2011/05 [Refereed][Not invited]
   

  An efficient total synthesis of aperidine was accomplished using a Rh-catalyzed C-H insertion of a cis-dihydrobenzofuran ring. To circumvent the facile epimerization of the cis-dihydrobenzofuran ring, we designed and prepared the C-H insertion precursor diazoamide by Raines' protocol. Finally, the efficient incorporation of a guanidine group and mild deprotection conditions yielded this labile natural product.
• Cytotoxic Tetramic Acid Derivative Produced by a Plant Type-III Polyketide Synthase

  Toshiyuki Wakimoto, Takahiro Mori, Hiroyuki Morita, Ikuro Abe

  Journal of the American Chemical Society 133 (13) 4746 - 4749 0002-7863 2011/04 [Refereed][Not invited]
   

  The tetramic acid (2,4-pyrrolidinedione) scaffold has been recognized as an important structural feature because of its mycotoxic, antibacterial, antiviral, and antioxidant activities. This important class of natural products is reportedly produced by the type-I polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) hybrid megaenzyme systems. In contrast, the benzalacetone synthase (BAS) from Rheum palmatum is a structurally simple, plant-specific type-III PKS that catalyzes the one-step decarboxylative condensation of malonyl-CoA with 4-coumaroyl-CoA. The type-III PKS exhibits unusually broad substrate specificity and notable catalytic versatility. Here we report that R. palmatum BAS efficiently produces a series of unnatural, novel tetramic acid derivatives by the condensation of malonyl-CoA with aminoacyl-CoA thioesters chemically synthesized from L- and D-amino acids. Remarkably, the novel tetramic acid dimer ID-S formed from D-phenylalanoyl-CoA showed moderate antiproliferative activity against murine leukemia P388 cells.
• Stereocontrolled Synthesis of (+)-Methoxyphenylkainic Acid and (+)-Phenylkainic Acid

  Takumi Higashi, Yoichiro Isobe, Hitoshi Ouchi, Hiroto Suzuki, Yuko Okazaki, Tomohiro Asakawa, Takumi Furuta, Toshiyuki Wakimoto, Toshiyuki Kan

  Organic Letters 13 (5) 1089 - 1091 1523-7060 2011/03 [Refereed][Not invited]
   

  The efficient total syntheses of (+)-methoxyphenylkainic acid (3) and (+)-phenylkainic acid (4) were achieved using a rhodium carbenoid-mediated intermolecular C-H insertion reaction. Complete stereoselective construction of the kainoid skeleton was accomplished by utilizing the stereochemistry at the C-4 position as a pivotal stereogenic center.
• Proof of the Existence of an Unstable Amino Acid: Pleurocybellaziridine in Pleurocybella porrigens

  Toshiyuki Wakimoto, Tomohiro Asakawa, Saeko Akahoshi, Tomohiro Suzuki, Kaoru Nagai, Hirokazu Kawagishi, Toshiyuki Kan

  Angew. Chem. Int. Ed. 50 (5) 1168 - 1170 1433-7851 2011 [Refereed][Not invited]
   

  Angel's wing mushroom, Pleurocybella porrigens, caused fatal acute encephalopathy in Japan in 2004. The structures of cytotoxic amino acids previously isolated from the mushroom motivated a study to prove the existence of an aziridine amino acid, pleurocybellaziridine (1), found in the mushroom. The ester forms of synthetic 1 were used to confirm that the fruiting bodies contained high amounts of 1. Furthermore, 1 showed significant toxicity towards rat oligodendrocytes. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
• Concise synthesis of catechin probes enabling analysis and imaging of EGCg

  Atsushi Yoshida, Yasuo Hirooka, Yusuke Sugata, Mariko Nitta, Tamiko Manabe, Shunsuke Ido, Kouki Murakami, Repon Kumer Saha, Takashi Suzuki, Motohiro Ohshima, Akira Yoshida, Kunihiko Itoh, Kosuke Shimizu, Naoto Oku, Takumi Furuta, Tomohiro Asakawa, Toshiyuki Wakimoto, Toshiyuki Kan

  Chemical Communications 47 (6) 1794 - 1796 1359-7345 2011 [Refereed][Not invited]
   

  A concise synthesis of APDOEGCg (3) was accomplished. Due to the reactivity of its amine group, the compound could be easily converted to the fluorescein probe 21 and immunogen probe 22 efficiently. We then demonstrated the usefulness of the probes for imaging studies and the generation of antibodies.
• PET imaging of nobiletin based on a practical total synthesis

  Tomohiro Asakawa, Aiki Hiza, Miho Nakayama, Makoto Inai, Dai Oyama, Hiroyuki Koide, Kosuke Shimizu, Toshiyuki Wakimoto, Norihiro Harada, Hideo Tsukada, Naoto Oku, Toshiyuki Kan

  Chemical Communications 47 (10) 2868 - 2870 1359-7345 2011 [Refereed][Not invited]
   

  A practical synthesis of nobiletin, a polymethoxylated citrus flavone, was accomplished by utilizing our novel flavone synthesis. Synthetic nobiletin was labelled by selective demethylation and rapid incorporation of C-11 atom. Positron emission tomography images successfully visualized the brain distribution, which may provide therapeutic benefits in the treatment of Alzheimer's disease.
• Proof of the Existence of an Unstable Amino Acid: Pleurocybellaziridine in Pleurocybella porrigens

  Toshiyuki Wakimoto, Tomohiro Asakawa, Saeko Akahoshi, Tomohiro Suzuki, Kaoru Nagai, Hirokazu Kawagishi, Toshiyuki Kan

  Angewandte Chemie Wiley 123 (5) 1200 - 1202 0044-8249 2010/12/23 [Not refereed]
  
• Search for toxic compounds from the mushroom Pleurocybella porrigens

  Suzuki Tomohiro, Fujita Motohiro, Amano Yuko, Asakawa Tomohiro, Akahoshi Saeko, Wakimoto Toshiyuki, Kobayashi Yuka, Morita Tatsuya, Arai Nobutaka, Nagai Kaoru, Tanaka Shigeyasu, Hirai Hirofumi, Kan Toshiyuki, Kawagishi Hirokazu

  Symposium on the Chemistry of Natural Products, symposium papers 天然有機化合物討論会 (52) 655 - 660 2010/09/01 

  The mushroom Pleurocybela porrigens (Angel's wings in English; Sugihiratake in Japanese) is widespread and common throughout temperate regions of the world. It has been eaten for a lng time all over the world. However, in autumn 2004 in Japan, fifty-five people got poisoned by eating this mushroom, and seventeen peoplel among them died of acute encephalopaty. There had been no report regarding toxocity of the fruiting bodies until the incident. Under these circumstances, we tried to isolate teh principle(s) of the disease. Purification of a glycoprotein showing lethal activity against mouse The mushroom was extracted with water and boiling water. After repeated chromatography of the water-soluble fractions, a glycoprotein was purified. The substance showed lethal toxocity toward mice at a dose of 24mg/kg (i.p.). Purification, characterization, and cDNA cloning of a lectin (PPL) PPL was purified from this mushroom. The results of SDS-PAGE gel filtration and MALDI-TOF-mass of PPL indicated that its molecular mass was 56kDa, and it was composed of four 14kDa subunits with no disulfide bonds. The complete amino acid sequence was determined by amino acid sequencing. The cDNA of PPL was cloned from RNA extracted from the mushroom. The open reading frame of the cDNA of the protein consisted of 411 bp encoding 137 amino acids. Intravenous (i.v.) (50mg/kg) or intraperitoneal (i.p.) (150mg/kg) administration of PPL to mice did not show any toxocity. However, i.v. (9mg/kg) administration of the protein to rats exhibited lethal toxocity. Purification of unusual amino acids showing cytotoxicity against mouse cerebrum glial cells Six amino acid derivatives (1-6) including three novel ones (1-3) were isolated from the mushroom. These compounds were cytotoxic to mouse glial cells. The structural novelty and analogy of the amino acids is such that each acid has the β-hydroxyvaline unit adducted to endogenous molecules, which inspired us to conclude the occurrence of an aziridine-amino acid (7) as the common precursor of the six compounds. We synthesized this compound and proved its occurrence in this mushroom. Compound 7 showed toxocity against rat CG4-16 oligodendrocyte cells; 7 significantly reduced the cell viability at concentrations up to 10μg/ml (87μM). Mechanism of the acute encephalopathy We found that a mixture of the letnal glycoprotein and PPL showed protease activity and disrupted the blood-brain barrier (BBB) in mice. We speculated that compound 7 or its derivatives caused demyelinating symptom after disruption of BBB. Verfication of the hypothesis is now on progress.
• Structure Function Studies on Coumaroyl-CoA Ligase

  Wanibuchi Kiyofumi, Mori Takahiro, Niwano Tomoko, Zhang Gang, Wakimoto Toshiyuki, Morita Hiroyuki, Abe Ikuro

  Symposium on the Chemistry of Natural Products, symposium papers 天然有機化合物討論会 (52) 55 - 60 2010/09/01 

  4-Coumareate:coenzyme A ligase (4CL) plays a key role in phenylpropanoid metabilism, providing precursors for a variety of plant secondary metabolites such as flavonoids and lignin. The enzyme catalyzes formation of 4-coumaroyl-CoA by initial activation of 4-coumaric acid with ATP to form an acyl-AMP intermediate, which is followed by thioester bond formation with CoASH and the concomitant release of AMP. The activation of the carboxylate substrate with ATP to form the acyl-AMP intermediate is a common mechanism for other adenylate-forming enzymes, including fatty acyl-CoA synthetase, the adenylation domains of the modular non-ribosomal peptide synthetase, and firefly luciferase (the ANL superfamily enzymes). Here we report that 4CL from Arabiaopsis thaliana (At4CL2) shows remarkable substrate promiscuity and novel catalytic functions. First, it was demonstrated that, in the absence of CoASH, the enzyme catalyzed amide bond forming reactions by condensation of 4-coumaric acid and the amino group fo D- and L- amino acids. Second, the enzyme generated a variety of biologically active amides including homoserine lactones and capsaicin by coupling the corresponding acids and amines. Finally, unexpectedly, structure-based 4CL site-directed mutants, Y253F and Q345A, catalyzed formation of dipeptides from a series of amino acids in the presence of ATP. Thus, the CoA ligase was functionally converted to dipeptide synthetase by the single amino acid substitutions in the substrate binding pocket.
• Structural characteristics of green tea catechins for formation of protein carbonyl in human serum albumin

  Takeshi Ishii, Taiki Mori, Tatsuya Ichikawa, Maiko Kaku, Koji Kusaka, Yoshinori Uekusa, Mitsugu Akagawa, Yoshiyuki Aihara, Takumi Furuta, Toshiyuki Wakimoto, Toshiyuki Kan, Tsutomu Nakayama

  Bioorganic and Medicinal Chemistry 18 (14) 4892 - 4896 0968-0896 2010/07 [Refereed][Not invited]
   

  Catechins are polyphenolic antioxidants found in green tea leaves. Recent studies have reported that various polyphenolic compounds, including catechins, cause protein carbonyl formation in proteins via their pro-oxidant actions. In this study, we evaluate the formation of protein carbonyl in human serum albumin (HSA) by tea catechins and investigate the relationship between catechin chemical structure and its pro-oxidant property. To assess the formation of protein carbonyl in HSA, HSA was incubated with four individual catechins under physiological conditions to generate biotin-LC-hydrazide labeled protein carbonyls. Comparison of catechins using Western blotting revealed that the formation of protein carbonyl in HSA was higher for pyrogallol-type catechins than the corresponding catechol-type catechins. In addition, the formation of protein carbonyl was also found to be higher for the catechins having a galloyl group than the corresponding catechins lacking a galloyl group. The importance of the pyrogallol structural motif in the B-ring and the galloyl group was confirmed using methylated catechins and phenolic acids. These results indicate that the most important structural element contributing to the formation of protein carbonyl in HSA by tea catechins is the pyrogallol structural motif in the B-ring, followed by the galloyl group. The oxidation stability and binding affinity of tea catechins with proteins are responsible for the formation of protein carbonyl, and consequently the difference in these properties of each catechin may contribute to the magnitude of their biological activities. (C) 2010 Elsevier Ltd. All rights reserved.
• Binding affinity of tea catechins for HSA: Characterization by high-performance affinity chromatography with immobilized albumin column

  Takeshi Ishii, Kanako Minoda, Min-Jung Bae, Taiki Mori, Yoshinori Uekusa, Tatsuya Ichikawa, Yoshiyuki Aihara, Takumi Furuta, Toshiyuki Wakimoto, Toshiyuki Kan, Tsutomu Nakayama

  Molecular Nutrition & Food Research 54 (6) 816 - 822 1613-4125 2010/06 [Refereed][Not invited]
   

  Catechins are the major polyphenols in green tea leaves. Recent studies have suggested that the catechins form complexes with HSA for transport in human blood, and their binding affinity for albumin is believed to modulate their bioavailability. In this study, the binding affinities of catechins and their analogs were evaluated and the relationship between the chemical structure of each catechin and its binding property were investigated. Comparing these catechins by HPLC analysis with the HSA column, we showed that galloylated catechins have higher binding affinities with HSA than non-galloylated catechins. In addition, pyrogallol-type catechins have a high affinity compared to catechol-type catechins. Furthermore, the binding affinity of the catechin with 2,3-trans structure was higher than those of the catechin with 2,3-cis structure. The importance of the hydroxyl group on the galloyl group and B-ring was confirmed using methylated catechins. These results indicate that the most important structural element contributing to HSA binding of tea catechins is the galloyl group, followed by the number of hydroxyl groups on the B-ring and the galloyl group or the configuration at C-2. Our findings provide fundamental information on the relationship between the chemical structure of tea catechins and its biological activity.
• Inhibitory Effects of Whisky Congeners on IgE-Mediated Degranulation in Rat Basophilic Leukemia RBL-2H3 Cells and Passive Cutaneous Anaphylaxis Reaction in Mice

  Tomohiro Itoh, Mariko Tsukane, Minako Koike, Chizu Nakamura, Kenji Ohguchi, Masafumi Ito, Yukihiro Akao, Sehchi Koshimizu, Yoshinori Nozawa, Toshiyuki Wakimoto, Haruo Nukaya, Yoshihide Suwa

  Journal of Agricultural and Food Chemistry 58 (12) 7149 - 7157 0021-8561 2010/06 [Refereed][Not invited]
   

  Whisky is matured in oak casks. Many nonvolatile substances (whisky congeners, WC) seep from the oak cask during the maturing process. In this study, three antiallergic agents (syringaldehyde, SA; lyoniresinol, Lyo; and ellagic acid, EA) were isolated from WC. Treatment with SA, Lyo, and EA reduced the elevation of intracellular free Ca(2+) concentration (([Ca(2+)]i)) and intracellular ROS production caused by Fc epsilon RI activation. The inhibitions of the elevation of [Ca(2+)]i and intracellular ROS production by SA and Lyo were mainly due to the suppression of the NADPH oxidase activity and scavenging of the produced radical, respectively. On the other hand, EA inactivated spleen tyrosine kinase and led to the inhibition of the elevation of [Ca(2+)]i and intracellular ROS production. Furthermore, it was found that WC strongly inhibited IgE binding to the Fc epsilon RI alpha chain, whereas SA, Lyo, and EA did not indicate this inhibitory effect. These results suggest that WC inhibits allergic reactions through multiple mechanisms. To disclose the in vivo effects of WC, SA, Lyo, and EA, these compounds were administered to type I allergic model mice, and the passive cutaneous anaphylaxis (PCA) reaction was measured. These compounds remarkably suppressed the PCA reaction. Taken together, these findings suggest that WC seemed to be beneficial to ameliorate allergic reactions.
• Modified Julia-Kocienski Reaction Promoted by Means of m-NPT (Nitrophenyltetrazole) Sulfone

  Yuki Sakai, Kazutada Ikeuchi, Yuji Yamada, Toshiyuki Wakimoto, Toshiyuki Kan

  Synlett (5) 827 - 829 0936-5214 2010/03 [Refereed][Not invited]
   

  m-Nitrophenyltetrazole sulfone (2) was employed in the Julia-Kocienski reaction. The olefination reaction between 2 and carbonyl compounds proceeded smoothly under Masamune-Roush conditions (DBU and LiCl). These conditions were also applicable to our catechin derivative synthesis. Furthermore, phenolic mesylate was also tolerated in this mild reaction.
• Solid-Supported Synthesis of Artificial Phospholipids

  Takumi Furuta, Hitoshi Onuki, Masayoshi Mochizuki, Mai Ito, Makoto Inai, Toshiyuki Wakimoto, Toshiyuki Kan

  Synlett (20) 3373 - 3377 0936-5214 2009/12 [Refereed][Not invited]
   

  A concise solid-supported synthesis of artificial phospholipids was developed. Functionalized phospholipids were prepared by introduction of a head group onto a solid-supported phospholipid framework in good overall yield and purity.
• Structure-activity relationship study on alpha(1) adrenergic receptor antagonists from beer

  Toshiyuki Wakimoto, Makoto Nitta, Kana Kasahara, Taketo Chiba, Ye Yiping, Kuniro Tsuji, Toshiyuki Kan, Haruo Nukaya, Masaji Ishiguro, Minako Koike, Yoshiaki Yokoo, Yoshihide Suwa

  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 19 (20) 5905 - 5908 0960-894X 2009/10 [Refereed][Not invited]
   

  Hordatine A and aperidine have been previously isolated from beer as active ingredients, which bind to muscarinic M-3 receptor. In addition, these compounds have exhibited antagonist activity against the alpha(1A) adrenoceptor. Although the relative structures of these two molecules have previously been determined, the absolute stereochemistry was unclear. Hence, to elucidate the absolute stereochemistry of natural hordatine A, we synthesized each enantiomer of hordatine A and aperidine from optically pure dehydrodi-p-coumaric acid. Several additional related compounds were also synthesized for structure-activity relationship studies. Chiral column HPLC analysis demonstrated that the absolute stereochemistry of natural hordatine A is (2S,3S), while based on the isomerization mechanism, the stereochemistry of aperidine is (2R,3S). The alpha(1A) adrenoceptor binding activity of (2R,3R)-hordatine A is the most potent among the enantiomeric pairs of hordatines and aperidines. Furthermore, the related, synthetic compound, (2R,3R)-methyl benzofurancarboxylate exhibits antagonist activity against the alpha(1A) adrenoceptor at a lower concentration than that of hordatine A. (C) 2009 Elsevier Ltd. All rights reserved.
• Formation Mechanism of Chafuroside A and B, Novel Anti-inflammatory Flavone C-Glycosides in Oolong Tealeaves

  Ishida Hitoshi, Wakimoto Toshiyuki, Nukaya Haruo

  Symposium on the Chemistry of Natural Products, symposium papers 天然有機化合物討論会 (51) 521 - 526 2009/09/01 

  A procedure was developed for quantitative determination of chafuroside A1, a flavone C-glycoside with potent anti-inflammatory activity, and its regioisomer chafuroside B2, as well as isovitexin and vitexin, by selected reaction monitoring liquid chromatography-tandem mass spectrometry analysis. This method was successfully applied to commercial leaves of green tea, houji tea, oolong tea and black tea. High levels of chafuroside A and chafutoside B were found in oolong tea leaves that had been heated at over 140℃. Next, their precursors, prechafuroside A and prechafuroside B, were isolated frommethanol extract of oolong tea leaves prepared from Shizu 7132, Camellia sinensis (L.) O. Kuntze, by partiton with n-butanol and H_2O and chromatography on Diaion SP-825, Sephadex LH-20, and ODS C-18, guided by assay of chafuroside formation. Prechafuroside A and prechafutoside B gave chafuroside A and Chafuroside B, respectively, in good yields when heated at 160℃ for 0.5h. Hydrolysis of prechafuroside A and prechafuroside B with pyridine and dioxane quantitatively afforded isovitexin and vitexin, respectively. Based on these results and physicochemical data (MS, UV and NMR), prechafuroside A and prechafuroside B were concluded to be new flavone C-glycoside sulfates, isovitexin-2"-sulfate 5 and vitexin-2"-sulfate 6, respectively.
• Regioselective synthesis of methylated epigallocatechin gallate via nitrobenzenesulfonyl (Ns) protecting group

  Yoshiyuki Aihara, Atsusi Yoshida, Takumi Furuta, Toshiyuki Wakimoto, Toshifumi Akizawa, Motomi Konishi, Toshiyuki Kan

  Bioorganic and Medicinal Chemistry Letters 19 (15) 4171 - 4174 0960-894X 2009/08 [Refereed][Not invited]
   

  Regioselective synthesis of methylated epigallocatechin gallate from epigallocatechin was accomplished using a 2-nitrobenzenesulfonyl (Ns) group as a protecting group for phenols. This methodology provided several methylated catechins, which are naturally scarce catechin derivatives. (C) 2009 Elsevier Ltd. All rights reserved.
• Quantitation of Chafurosides A and B in Tea Leaves and Isolation of Prechafurosides A and B from Oolong Tea Leaves

  Hitoshi Ishida, Toshiyuki Wakimoto, Yukiko Kitao, Shimako Tanaka, Toshio Miyase, Haruo Nukaya

  Journal of Agricultural and Food Chemistry 57 (15) 6779 - 6786 0021-8561 2009/08 [Refereed][Not invited]
   

  A procedure was developed for the quantitative determination of chafuroside A, a flavone C glycoside with potent anti-inflammatory activity, and its regioisomer chafuroside B, as well as isovitexin and vitexin, by selected reaction monitoring liquid chromatography-tandem mass spectrometry (SRM LC-MS/MS) analysis. This method was successfully applied to commercial leaves of green tea, houji tea, oolong tea, and black tea. High levels of chafurosides A and B were found in oolong tea leaves that had been heated at >140 degrees C. Next, their precursors, prechafurosides A and B, were isolated from methanol extract of oolong tea leaves prepared from Shizu 7132, Camellia sinensis (L.) O. Kuntze, by partition with n-butanol and H(2)O and chromatography on Diaion SP-825, Sephadex LH-20, and ODS C-18, guided by assay of chafuroside formation. Prechafurosides A and B gave chafurosides A and B, respectively, in good yields when heated at 160 degrees C for 0.5 h. Solvolysis of prechafurosides A and B with pyridine and dioxane quantitatively afforded isovitexin and vitexin, respectively. On the basis of these results and physicochemical data (MS, UV, and NMR), prechafurosides A and B were concluded to be new flavone C-glycoside sulfates, isovitexin-2 ''-sulfate and vitexin-2 ''-sulfate, respectively.
• Concise Synthesis of Chafurosides A and B

  Takumi Furuta, Miho Nakayama, Hirotaka Suzuki, Hiroko Tajimi, Makoto Inai, Haruo Nukaya, Toshiyuki Wakimoto, Toshiyuki Kan

  Organic Letters 11 (11) 2233 - 2236 1523-7060 2009/06 [Refereed][Not invited]
   

  The regioselective synthesis of chafurosides A (1) and B (2) from the same methyl ketone 5 was accomplished using a novel protecting group strategy. Both flavone rings were constructed from beta-diketone intermediate 4, which was readily obtained by condensation of an acyl donor and ketone 5. Construction of the dihydrofuran ring was achieved via an intramolecular Mitsunobu reaction.
• Stereocontrolled total synthesis of (-)-myriocin

  Makoto Inai, Toshihiro Goto, Takumi Furuta, Toshiyuki Wakimoto, Toshiyuki Kan

  Tetrahedron: Asymmetry 19 (24) 2771 - 2773 0957-4166 2008/12 [Refereed][Not invited]
   

  The stereocontrolled total synthesis of (-)-myriocin 1 is reported. Optically active epoxide 9 was converted from symmetrical cyclohexadiene 8, utilizing an enzymatic kinetic resolution. The three sequential stereogenic centers of 1 were constructed by a regioselective epoxide-opening reaction and a Hofmann rearrangement. Elongation of the side chain was efficiently accomplished by the Julia-Kocienski reaction. Published by Elsevier Ltd.
• Total Synthesis of (-)-Serotobenine

  Yasuaki Koizumi, Hideki Kobayashi, Toshiyuki Wakimoto, Takumi Furuta, Tohru Fukuyama, Toshiyuki Kan

  Journal of the American Chemical Society 130 (50) 16854 - + 0002-7863 2008/12 [Refereed][Not invited]
   

  The efficient total synthesis of (-)-serotobenine (1) has been achieved by constructing an optically active dihydrobenzofuran ring via a rhodium carbenoid mediated intramolecular C-H insertion reaction, which was developed by our group. Then the possibility of racemization of 1 was investigated using optically active synthetic 1.
• Isolation and identification of bioactive principles in beer

  Haruo Nukaya, Toshiyuki Wakimoto, Yoshiaki Yokoo, Yoshihide Suwa

  Yakugaku Zasshi 127 55 - 57 0031-6903 2007 [Refereed][Not invited]
   

  It has long known that alcoholic beverages stimulate gastric acid secretion. In the 1980s, controlled studies on the action of pure ethanol and alcoholic beverages on gastric acid secretion and gastrin release in humans have confirmed that beer and wine, not pure ethanol, are potent stimulants of gastric acid secretion and gastrin release and endogenous gastrin is the mediator of the stimulation of gastric acid secretion. Moreover, it is known that beer accelerates gastrointestinal motility in humans. However, the active components in beer has not yet been identified. In this study, we isolated and identified N-methyltyramine as a gastrin release inducer in beer and 2 active compounds, hordatine A, a kind of phytoalexin in barley and cis-isomer, bind to the muscarine M-3 receptor.
• Structural determination of two active compounds that bind to the muscarinic M-3 receptor in beer

  Nahoko Yamaji, Yoshiaki Yokoo, Takashi Iwashita, Asuka Nemoto, Minako Koike, Yoshihide Suwa, Toshiyuki Wakimoto, Kuniro Tsuji, Haruo Nukaya

  Alcoholism: Clinical and Experimental Research 31 9S - 14S 0145-6008 2007/01 [Refereed][Not invited]
   

  Background: It is known that beer accelerates gastrointestinal motility in humans. Our previous studies showed that beer congener stimulates gastrointestinal motility by directly stimulating the muscarinic M-3 receptor. Further, we isolated 2 active compounds (compounds A and B) from beer by liquid chromatography. The objective of the present study was to identify the 2 active compounds that bind to the muscarinic M-3 receptor in beer. Methods: Structural analyses of the active compounds were performed by fast atom bombardment mass spectra, H-1-nuclear magnetic resonance (NMR), and C-13-NMR spectroscopy. Active compounds were chemically synthesized from p-coumaric acid and agmatine as starting materials. Binding activity to the muscarinic M-3 receptor was used to confirm the activity of the synthetic compounds. Results: It was identified that 2 active compounds had the same structural characteristics: stereoisomers (cis-isomer and trans-isomer), molecular weight=550 and molecular formula=C28H38N8O4. Trans-isomer (compound B) was identified as the known substance hordatine A, a kind of phytoalexin in barley, and cis-isomer (compound A) was found to be a novel compound (tentatively referred to as aperidine). Both naturally present and chemically synthesized aperidine (compound A) and hordatine A (compound B) were demonstrated to have potent binding activities to the muscarinic M-3 receptor. Conclusions: The 2 active compounds isolated from beer, namely aperidine (compound A) and hordatine A (compound B), have structurally and functionally been identified as active entities of binding to the muscarinic M-3 receptor.
• Concise total synthesis of flavone C-glycoside having potent anti-inflammatory activity

  T Furuta, T Kimura, S Kondo, H Mihara, T Wakimoto, H Nukaya, K Tsuji, K Tanaka

  Tetrahedron 60 (42) 9375 - 9379 0040-4020 2004/10 [Refereed][Not invited]
   

  The total synthesis of anti-inflammatory active flavone C-glycoside isolated from oolong tea extract is achieved. Introducing a C-glucosyl moiety to an aryl system and constructing a fused tetracyclic ring characteristic to this natural product were conducted based on the O-to-C rearrangement of sugar moiety and the successive intramolecular Mitsunobu reaction, respectively. This concise and efficient synthetic pathway is applicable to the large-scale synthesis of target flavone and for constructing a large library of related compounds. (C) 2004 Elsevier Ltd. All rights reserved.
• Erratum: Crystal structure of the complex between calyculin A and the catalytic subunit of protein phosphatase 1 (Structure (May 2002) 10 (715-724))

  Akiko Kita, Akiko Kita, Shigeki Matsunaga, Shigeki Matsunaga, Akira Takai, Akira Takai, Hirotaka Kataiwa, Hirotaka Kataiwa, Toshiyuki Wakimoto, Toshiyuki Wakimoto, Nobuhiro Fusetani, Nobuhiro Fusetani, Minoru Isobe, Minoru Isobe, Kunio Miki, Kunio Miki

  Structure 10 1149  0969-2126 2002/08/29 [Not refereed][Not invited]
  
• Crystal structure of the complex between calyculin A and the catalytic subunit of protein phosphatase 1

  A Kita, S Matsunaga, A Takai, H Kataiwa, T Wakimoto, N Fusetani, M Isobe, K Miki

  Structure 10 (5) 715 - 724 0969-2126 2002/05 [Refereed][Not invited]
   

  The crystal structure of the catalytic subunit of the protein phosphatase 1 (PP1), PP1gamma, in complex with a marine toxin, calyculin A, was determined at 2.0 Angstrom resolution. The metal binding site contains the phosphate group of calyculin A and forms a tight network via the hydrophilic interactions between PP1 and calyculin A. Calyculin A is located in two of the three grooves, namely, in the hydrophobic groove and the acidic groove on the molecular surface. This is the first observation to note that the inhibitor adopts not a pseuclocyclic conformation but an extended conformation in order to form a complex with the protein. The amino acid terminus of calyculin A contributes, in a limited manner, to the binding to PP1gamma, which is consistent with findings from the studies of dose-inhibition analysis.
• Insight into binding of calyculin A to protein phosphatase 1: Isolation of hemicalyculin A and chemical transformation of calyculin A

  Toshiyuki Wakimoto, Shigeki Matsunaga, Akira Takai, Nobuhiro Fusetani

  Chemistry and Biology 9 (3) 309 - 319 1074-5521 2002 [Refereed][Not invited]
   

  Calyculin A isolated from the marine sponge Discodermia calyx is a potent inhibitor of protein phosphatases 1 and 2A. We attempted to elucidate its mode of binding to the enzymes by examining the activity of natural and chemically transformed derivatives. Ten natural derivatives including a new compound, hemicalyculin A, were provided. The structure of hemicalyculin A, which comprises the southern hemisphere of calyculin A, was firmly established by chemical methods. Six compounds were prepared by selective modifications of functional groups in calyculin A. The enzyme inhibitory activity of these compounds indicated that 17-phosphate, 13-hydroxyl, and the hydrophobic tetraene moieties were all necessary for binding to the enzymes. The derivatives lacking the peptide portion were less cytotoxic even when they possessed full enzyme inhibitory activity.
• The solution structures of calyculin A and dephosphonocalyculin A by NMR.

  KE Volter, GK Pierens, RJ Quinn, T Wakimoto, S Matsunaga, N Fusetani

  Bioorganic & Medicinal Chemistry Letters 9 (5) 717 - 722 0960-894X 1999/03 [Refereed][Not invited]
   

  The NMR solution structure of calyculin A (1) in chloroform exhibits intramolecular interactions, resembling the original crystal structure(1). In methanol, calyculin A has the hydrogen bonding moieties solvent exposed. Dephosphonocalyculin A in chloroform resembles calyculin A in chloroform and the crystal structure of calyculin A. Dephosphonocalyculin A in methanol resembles calyculin A in methanol. (C) 1999 Elsevier Science Ltd. All rights reserved.
• Octa- and nonaprenylhydroquinone sulfates, inhibitors of alpha 1,3-fucosyltransferase VII, from an Australian marine sponge Sarcotragus sp.

  T Wakimoto, A Maruyama, S Matsunaga, N Fusetani, K Shinoda, PT Murphy

  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 9 (5) 727 - 730 0960-894X 1999/03 [Refereed][Not invited]
   

  alpha 1,3-Fucosyltransferase (Fuc TVII) is a key enzyme in the biosynthesis of selectin ligands. We have isolated two inhibitors of Fuc TVII from a marine sponge Sarcotragus sp. They were characterized as octa- and nonaprenylhydroquinone sulfates on the basis of spectral data. These compounds inhibited Fuc-TVII with IC50 values of 3.9 and 2.4 mu g/mL, respectively. (C) 1999 Elsevier Science Ltd. All rights reserved.
• Erratum: Isolation of four new calyculins from the marine sponge Discodermia calyx (Journal of Organic Chemistry (1997) 62 (2640))

  S. Matsunaga, T. Wakimoto, N. Fusetani

  Journal of Organic Chemistry 62 9388  0022-3263 1997/12/01 [Refereed][Not invited]
  
• 31 Structure and Biological Activity of New Calyculins Isolated from the Marine Sponge Discodermia calyx

  Matsunaga Shigeki, Wakimoto Toshiyuki, Fusetani Nobuhiro

  Symposium on the Chemistry of Natural Products, symposium papers 天然有機化合物討論会 (39) 181 - 186 1997/07/20 

  The calyculins [calyculin A (1)], unique polyketides bearing a variety of functionalities isolated from the Japanese marine sponge Discodermia calyx, exhibit antitumor, smooth muscle contractile, and tumor promotion activities, which are most likely to be attributable to inhibition of protein phosphatases 1 and 2A. Eight known calyculins A(1)-H, which differ in the presence or absence of methyl group on C32 and in the geometry of C2-C3 and C6-C7 double bonds, display similar inhibitory activity against protein phosphatases 2A. Further examination of D. calyx extracts led to isolation of five more calyculin derivatives, calyculin J(2), calyculinamide A(3), calyculinamide F(4), des-N-methyl-calyculin A(5), and dephosphonocalyculin A(6). Structures of 2-6 were assigned on the basis of spectral data and chemical transformation. Calyculin J(2) had a bromine atom and an additional tetrahydrofuran ring. The structure including the absolute stereochemistry has been established by chemical transformation of calyculin A. Calyculinamide A(3) was found to have a terminal amide group instead of the nitrite in 1 by analysis of spectral data. This was further substantiated by hydrolysis of 1. Dephosphonocalyculin A (6) behaved a more polar compound than other calyculins in both normal phase and reversed phase chromatographies. The FAB mass spectrum exhibited an (M+H)^+ ion at m/z929, 80 units smaller than that of calyculin A, whereas the gross structure of 6, which was identical with that of 1 except for the phosphate ester on the C17 oxygen function, was easily assigned on the basis of the COSY, HMQC, and HMBC spectra. The structure including the absolute stereochemistry has been confirmed by chemical transformation of 6 and 1 to a common compound. Compounds 3, 4, 6 showed inhibitory activity against protein phosphatase 2A similar to that of calyculin A. However, IC_<50> values of 2 and 5 were 100 and 150 times higher than that of calyculin A.
• Isolation of dephosphonocalyculin A from the marine sponge, Discodermia calyx

  S Matsunaga, T Wakimoto, N Fusetani, M Suganuma

  Tetrahedron Letters 38 (21) 3763 - 3764 0040-4039 1997/05 [Refereed][Not invited]
   

  Fractionation of the EtOH extract of the marine sponge Discodermia calyx led to the isolation of dephosphonocalyculin A (2). The gross structure 2 was deduced on the basis of spectral data. The structure including the absolute stereochemistry has been confirmed by chemical transformation of 2 and calyculin A to a common compound. Dephosphonocalyculin A inhibits protein phosphatases 1 and 2A with IC50 values of 3.0 and 8.2 nM, respectively. (C) 1997 Elsevier Science Ltd.
• Isolation of four new calyculins from the marine sponge Discodermia calyx

  S. Matsunaga, T. Wakimoto, N. Fusetani

  Journal of Organic Chemistry 62 (8) 2640 - 2642 0022-3263 1997 [Refereed][Not invited]
  

MISC

• バクテリアが有する新たな非リボソームペプチド環化酵素

  松田研一, 脇本敏幸  バイオサイエンスとインダストリー  77-  (4)  306‐307  2019/07/10  [Refereed][Not invited]
  
• MyPR 未開拓天然物資源を求めて

  脇本敏幸  有機合成化学協会誌  77-  72  -74  2019  [Refereed][Invited]
  
• ペニシリン結合タンパク質によるペプチド環化

  松田研一, 倉永健史, 脇本敏幸  ファルマシア  55-  650  -654  2019  [Refereed][Not invited]
  
• クリルオイルに含まれる抗肥満成分の探索と作用機序解析

  海野 雄加, 山本 宙奈, 矢澤 一良, 脇本 敏幸, 斧 康雄  肥満研究  23-  (Suppl.)  177  -177  2017/09
• クリルオイル由来脂溶性成分による抗肥満効果の解明

  海野 雄加, 山本 宙奈, 高槻 修人, 矢澤 一良, 脇本 敏幸, 斧 康雄  日本薬学会年会要旨集  137年会-  (3)  194  -194  2017/03
• Genome Mining of Amino Group-Carrier Protein-Mediated Machinery: Insights into Biosynthesis of Unique Hydrazone Unit

  松田研一, 松田研一, 富田武郎, 志波優, 兼崎友, 吉川博文, 新家一男, 脇本敏幸, 葛山智久, 西山真  天然有機化合物討論会講演要旨集(Web)  59th-  2017
• Biosynthesis of Bioactive Compounds by Marine Sponge Symbiont Bacteria

  江上蓉子, 脇本敏幸, 阿部郁朗  バイオインダストリー  33-  (3)  11  -17  2016  [Refereed]
  
• 糸状菌メロテルペノイドの構造複雑化を担う分子基盤の解明

  松田侑大, 岩渕大輝, 脇本敏幸, 森貴裕, 淡川孝義, 阿部郁朗  天然有機化合物討論会講演要旨集(Web)  57th-  2015
• 複合培養を用いたStreptomyces属放線菌由来新規二次代謝産物の探索

  星野翔太郎, 張驪駻, 淡川孝義, 脇本敏幸, 尾仲宏康, 阿部郁朗  日本薬学会年会要旨集(CD-ROM)  135th-  2015
• 複合培養法によるStreptomyces属放線菌由来新規二次代謝産物の探索

  星野翔太郎, ZHANG Lihan, 淡川孝義, 脇本敏幸, 尾仲宏康, 阿部郁朗  日本放線菌学会大会講演要旨集  30th-  2015
• 複合培養法によりStreptomyces属放線菌から単離された新規macrolactam類の構造決定

  星野翔太郎, 張恵平, 林文晶, 岡田正弘, 脇本敏幸, 尾仲宏康, 阿部郁朗  日本生薬学会年会講演要旨集  62nd-  2015
• 複合培養法を利用したStreptomyces属放線菌からの新規薬用資源探索

  星野翔太郎, 岡田正弘, ZHANG Huiping, 林文晶, 脇本敏幸, 尾仲宏康, 阿部郁朗  食品薬学シンポジウム講演要旨集  6th-  2015
• 2S-Ep03 Chemical defense system of sponge-microbe association

  Wakimoto Toshiyuki  日本生物工学会大会講演要旨集  67-  266  -266  2015
• カイメンー共生バクテリア系の化学防御機構

  脇本敏幸, 阿部郁朗  バイオサイエンスとインダストリー  73-  49  -51  2015
• 海綿－共生微生物系の化学防御機構

  脇本敏幸, 江上蓉子, 阿部郁朗  化学と生物  53-  (8)  497  -499  2015
• 海綿動物の化学防御を担う共生菌

  江上蓉子, 脇本敏幸, 阿部郁朗  細胞工学  34-  412  -416  2015
• メディオマイシン生合成遺伝子の同定

  ZHANG L., 伊藤卓也, 脇本敏幸, 淡川孝義, 浅川義範, 阿部郁朗  日本放線菌学会大会講演要旨集  29th-  2014
• メディオマイシン生合成遺伝子の同定

  張驪駻, 伊藤卓也, 脇本敏幸, 淡川孝義, 浅川義範, 阿部郁朗  日本薬学会年会要旨集(CD-ROM)  134th-  2014
• ミコール酸含有微生物Tsukamurella pulmonisとの複合培養による,放線菌Streptomyces属由来新規生物活性物質の探索

  星野翔太郎, LIHAN Z., 淡川孝義, 脇本敏幸, 尾仲宏康, 阿部郁朗  日本放線菌学会大会講演要旨集  29th-  2014
• ミコール酸含有微生物との複合培養による,Streptomyces属放線菌由来新規生物活性物質の探索

  星野翔太郎, ZHANG Lihan, 淡川孝義, 脇本敏幸, 尾仲宏康, 阿部郁朗  天然薬物の開発と応用シンポジウム講演要旨集  20th-  2014
• ミコール酸含有微生物との複合培養を用いた,放線菌Streptomyces属由来新規生物活性物質の探索

  星野翔太郎, 張驪駻, 淡川孝義, 脇本敏幸, 尾仲宏康, 阿部郁朗  日本生薬学会年会講演要旨集  61st-  2014
• 異種発現系を用いた新規Teleocidin類の生産

  星野翔太郎, ZHANG Lihan, 淡川孝義, 伊藤卓也, 脇本敏幸, 阿部郁朗  日本農芸化学会大会講演要旨集(Web)  2014-  2014
• Teleocidin類の生合成機構の解明

  森貴裕, 淡川孝義, ZHANG Lihan, 星野翔太郎, 脇本敏幸, 森田洋行, 伊藤卓也, 石川淳, 阿部郁朗  天然有機化合物討論会講演要旨集(Web)  56th-  2014
• 糸状菌メロテルペノイドAustinol生合成に関わるスピロ環形成酵素の機能解析

  松田侑大, 淡川孝義, 脇本敏幸, 阿部郁朗, 松田侑大, 淡川孝義, 脇本敏幸, 阿部郁朗  日本生薬学会年会講演要旨集  60th-  2013
• 糸状菌メロテルペノイドの構造多様性創出機構の解明

  松田侑大, 淡川孝義, 脇本敏幸, 阿部郁朗, 松田侑大, 淡川孝義, 脇本敏幸, 阿部郁朗  天然有機化合物討論会講演要旨集(Web)  55th-  2013
• 異種発現系を利用した糸状菌二次代謝産物合成酵素遺伝子資源の有効活用

  淡川孝義, 松田侑大, 脇本敏幸, 阿部郁朗  食品薬学シンポジウム講演要旨集  5th-  2013
• 糸状菌メロテルペノイド生合成酵素の機能解析

  松田侑大, 淡川孝義, 脇本敏幸, 阿部郁朗  酵素工学研究会講演会講演要旨集  70th-  2013
• 糸状菌メロテロペノイドの環化様式制御による物質生産系の構築

  松田侑大, 淡川孝義, 伊藤崇敬, 脇本敏幸, 久城哲夫, 藤井勲, 海老塚豊, 阿部郁朗  生体触媒化学シンポジウム講演要旨集  16th-  2012
• Terretonin生合成経路における新規環化機構の解明

  淡川孝義, 松田侑大, 伊藤崇敬, 脇本敏幸, 海老塚豊, 阿部郁朗, 久城哲夫, 藤井勲  日本生薬学会年会講演要旨集  59th-  2012
• 糸状菌メロテロペノイドの環化様式制御による物質生産系の構築

  淡川孝義, 松田侑大, 伊藤崇敬, 脇本敏幸, 久城哲夫, 藤井勲, 海老塚豊, 阿部郁朗  酵素工学研究会講演会講演要旨集  68th-  2012
• 海洋天然物Calyculin A生合成遺伝子クラスターのクローニング

  江上蓉子, 脇本敏幸, 伊藤卓也, 浅川義範, 阿部郁朗  日本薬学会年会要旨集  132nd-  (2)  2012
• Calyculin A生合成遺伝子クラスターの探索

  脇本敏幸, 脇本敏幸, 江上蓉子, 江上蓉子, 淡川孝義, 淡川孝義, 伊藤卓也, 兼目裕充, 浅川義範, 阿部郁朗, 阿部郁朗  天然有機化合物討論会講演要旨集(Web)  54th-  2012
• Cytotoxic tetramic acid derivative produced by a plant type III polyketide synthase

  MORI Takahiro, WAKIMOTO Toshiyuki, MORITA Hiroyuki, ABE Ikuro  Bioscience & industry  69-  (4)  308  -310  2011/07/01  [Not refereed][Not invited]
  
• Total synthesis of UCS1025A

  Uchida Kenji, Ogawa Takahiro, Yasuda Yoshinori, Mimura Hiraku, Fujimoto Teppei, Fukuyama Tohru, Wakimoto Toshiyuki, Asakawa Tomohiro, Hamashima Yoshitaka, Kan Toshiyuki  Proceedings of the Symposium on Progress in Organic Reactions and Syntheses  37-  (0)  90  -90  2011  [Not refereed][Not invited]
   

  UCS1025A was isolated from the fungus Acremonium sp. KY4917 in 2000. Due to its potent telomerase inhibitory activity and antimicrobial activity, UCS1025A is expected to be a lead compound for the drug development. The structure of UCS1025A is composed of a pyrrolizidinone (azabicyclo[3.3.0]octanone) skeleton cross-linked by &gamma;-lactone and the hemiaminal at ring juncture. Herein, we will report the total synthesis of optically active UCS1025A which involves new strategies of the construction of pyrrolizidinone skeleton and the stereocontrolled hemiaminal moiety.
• フラン脂肪酸の抗炎症活性

  脇本 敏幸, 阿部 郁朗  脂質生化学研究  52-  103  -105  2010/05/25
• Total synthesis of SB-203207

  Hirooka Yasuo, Akao Yusuke, Asakawa Tomohiro, Kawamoto Yuichiro, Furuta Takumi, Wakimoto Toshiyuki, Fukuyama Tohru, Kan Toshiyuki  Proceedings of the Symposium on Progress in Organic Reactions and Syntheses  36-  (0)  82  -82  2010  [Not refereed][Not invited]
   

  Recently, researchers in SmithKline Beecham's group isolated SB-203207 (<B>1</B>) from Atreptomyces spesies, which shown to inhibit isoleucyl tRNA synthetase. The significant biologically activity and the complex structure of <B>1</B> has prompted us to investigate its total synthesis. In this symposium, we will report the asynmmetric total synthesis of SB-203207. Our synthesis features a desymmetric C-H insertion reaction for construction of a bicyclo [3,3,0]framework, stereoselective construction of sequential stereocenters, novel conversion from aldehyde to carboxylic acid and nitrile hydrolysis mediated vinylogous urea synthesis.
• ウイスキー中のアルドース還元酵素阻害活性成分 血中代謝物の活性

  根本 明日香, 諏訪 芳秀, 輿水 精一, 糠谷 東雄, 脇本 敏幸, 原口 博行, 矢部 千尋  糖尿病  49-  (Suppl.1)  S307  -S307  2006/04  [Refereed][Not invited]
  
• ビールの消化管運動促進作用 活性物質の構造決定

  山路 奈保子, 岩下 孝, 横尾 芳明, 根本 明日香, 小池 美奈子, 諏訪 芳秀, 桝屋 浩大, 脇本 敏幸, 辻 邦郎, 糠谷 東雄  アルコールと医学生物学  25-  135  -139  2005/09  [Refereed][Not invited]
   

  ビールから単離した2つの活性物質(Compound AとCompound B)の化学構造を解析した.質量分析とNMR解析では,2つの活性物質は同じ平面構造を有することが判明した.立体構造の解析で,Compound B(トランス体)は既知物質ファイトアレキシンのホルダチンA,Compound Aはそのシス体{新規化合物:アペリジン(仮称)}と構造決定された.2つの活性物質の化学合成を行ったところ,合成アペリジンおよび合成ホルダチンAの活性は,ムスカリンM3受容体に対する結合活性により確認された.以上より,ビールから単離した2つの活性物質(アペリジンとホルダチンA)は,ビールの消化管運動促進作用の活性本体として構造決定・同定された
• ウイスキー中のアルドース還元酵素阻害活性成分

  糠谷 東雄, 脇本 敏幸, 根本 明日香, 諏訪 芳秀, 輿水 精一, 原口 博行, 矢部 千尋  糖尿病  48-  (Suppl.2)  S90  -S90  2005/04  [Refereed][Not invited]
  

Books etc

• 天然物の化学 : 魅力と展望

  上村, 大輔 (カリクリンの生合成)
  東京化学同人 2016/06 (ISBN: 9784807915019) viii, 186, 4p

Research Projects

• 予知生合成科学・研究総括班

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2022/06 -2027/03 

  Author : 葛山 智久, 内山 真伸, 丸山 千登勢, 寺田 透, 渡辺 賢二, 勝山 陽平, 山崎 真巳, 脇本 敏幸, 大栗 博毅, 淡川 孝義, 荘司 長三
• 新規ペプチド修飾酵素の機能拡張による大環状複雑骨格の創出

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

  Date (from‐to) : 2022/06 -2027/03 

  Author : 脇本 敏幸, 松田 研一
• 希少微生物株のゲノムマイニングによる天然医薬品資源の探索

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2021/04 -2024/03 

  Author : 脇本 敏幸

   

  本研究では従来の天然物探索事例の少ない微生物株についてゲノム情報を取得し、ゲノム情報を起点とした新規天然物の探索を実施する。特に放線菌以外の菌株、中でも物質生産に秀でたグラム陰性菌を見出し、重点的に探索を進める。原位置培養方法を駆使して、未培養微生物株を収集するとともに、海綿動物や植物に共生する微生物株を重点的に探索標的とする。環境中より収集した菌株は16S rRNA配列によって系統解析し、放線菌以外で有望な微生物株を選別する。それらのゲノム解析を実施し、二次代謝物の生合成遺伝子を解析する。塩基配列と天然物の構造情報は現時点で完全には一致していないが、多くの代謝経路について経路を担う酵素や責任遺伝子が明らかになってきている。天然物の設計図である生合成遺伝子クラスターがゲノム上になければ、二次代謝産物が生産されることはないことを考えると、生合成遺伝子クラスターの有無は極めて重要な新規天然物探索の指標となる。本研究では新規微生物培養方法や次世代シークエンサーを駆使し、最先端の新規微生物二次代謝産物の探索を展開する。生合成遺伝子クラスターを起点とすることで様々なアドバンテージがある。第１に休眠遺伝子である場合はトランスポゾン変異などを導入して活性化を施すことができる。第２にひとたび新規天然物が得られれば、生合成遺伝子クラスターが紐づいてくるために、生合成経路の解析へ容易に展開できる。新規の二次代謝酵素が見出されれば生体触媒としての応用展開が期待できる。第３に生合成遺伝子クラスターには化合物の生合成経路のみならず、自己耐性機構が含まれている場合があるため、自己耐性機構から該当化合物の作用機序を明らかにすることができる。このように本研究では次世代型の新規天然物探索手法を最大限活用し、これまでにない包括的な探索研究を実施し、新規抗生物質や細胞毒性物質の発見を目指す。
• Redesigning Biosynthetic Machineries (International Activity Support Team)

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

  Date (from‐to) : 2016/06 -2022/03 

  Author : Abe Ikuro

   

  In this project, we have made a dramatic development from simply learning biosynthetic machineries to designing new blueprints for producing desired natural products. We established a new world-leading technology platform for creation of complex functional molecules by rational reconstruction of biosynthetic machineries as a new academic field. In order to strongly promote joint research with foreign countries, the International Activity Support Group invited outstanding foreign researchers and held lecture meetings on a regular basis. Furthermore, international symposiums were held with the aim of strengthening cooperation and exchange with the U.S., Germany, China, and other countries. We strategically promoted joint research with foreign countries, focusing on medium-term support for young researchers. Many of the results of joint research with foreign countries resulted in excellent co-authored papers.
• Biosynthetic gene cluster of bioactive natural products from marine invertebrates

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

  Date (from‐to) : 2018/04 -2021/03 

  Author : WAKIMOTO Toshiyuki

   

  Marine sponges are sessile animals harboring vast array of symbiont microorganisms. They inhabit in global oceans and the number of species are estimated to be 10,000. More than 20,000 bioactive natural products have been isolated from the marine sponges thus far. The search based on the randam screening is so common that cytotoxic compounds has been dominated and some of them exhibit significantly potent cytotoxicity with pM range of concentration. However, due to the scarcity and highly complex structure, most of them had not been developed to clinically useful anti-cancer agents. On the other hand, it has been suggested that many sponge-derived cytotoxins are produced by symbiont bacteria. In this study, we implanted the search for biosynthetic gene cluster of sponge-derived bioactive molecules and detailed analysis of biosynthetic pathway, and identification of producer symbiont.
• Production of rare bioactive natural products originating from uncultured bacteria

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

  Date (from‐to) : 2016/06 -2021/03 

  Author : WAKIMOTO Toshiyuki

   

  Marine sponges often contain potent cytotoxic compounds, which usually target molecules that are likely to be shared with the sponge itself. This fact in turn evokes the principle question of how marine sponges avoid self-toxicity. We previously identified an intriguing mechanism to prevent self-toxicity by the phosphorylation of the highly toxic calyculin A in the sponge holobiont, which is catalyzed by the phosphotransferase CalQ of a producer symbiont, “Candidatus Entotheonella” sp. However, the activating mechanism to dephosphorylate the stored phosphocalyculin A remains elusive. Here we show that the phosphatase specific to phosphocalyculin A is CalL, which is also encoded in the calyculin biosynthetic gene cluster. CalL represents a new clade and unprecedently coordinates the heteronuclear metals Cu and Zn. The CalL is localized in the periplasmic space of the sponge symbiont, where it is ready for the on-demand production of calyculin A in response to sponge tissue disruption.
• Redesigning Biosynthetic Machineries (Project Team)

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

  Date (from‐to) : 2016/06 -2021/03 

  Author : Abe Ikuro

   

  In this project, we have made a dramatic development from simply learning biosynthetic machineries to designing new blueprints for producing desired natural products. Based on the detailed analyses of the structural diversity of natural products from the viewpoint of genes, enzymes, and reactions, we established a new world-leading technology platform for creation of complex functional molecules by rational reconstruction of biosynthetic machineries as a new academic field. In order to facilitate the research plan, the project team formulated a research policy and provided a forum to actively promote exchanges, collaborations, and joint research among individual research promoters. Researchers from various fields collaborated and complemented each other in their areas of expertise to conduct joint research and create a new academic field.
• Biosynthetic study on bioactive compounds from marine sponges

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

  Date (from‐to) : 2017/07 -2019/03 

  Author : 脇本 敏幸, URIA AGUSTINUS

   

  海綿動物は多種多様な共生微生物を宿す付着性の多細胞動物である。世界中の海に生息し、１万種が現存するといわれる海綿動物からは、これまで２万以上の生理活性物質が単離、報告されてきた。ランダムスクリーニングに由来するため細胞毒性物質の数が多く、pM程度の濃度で強力な細胞毒性を示す化合物も少なくない。しかしながら、それらの多くが稀少かつ複雑な構造を有するために、抗がん剤として研究開発がなされた化合物は極めて少ない。一方で、それら海綿由来細胞毒性物質のほとんどは共生微生物によって生産されることが示唆されている。本研究では医薬品資源として有望な海綿由来稀少生理活性物質の生合成遺伝子および生産菌を同定し、可培養化や異種生産を目指した基礎的知見を得る。昨年度より海綿動物から微量成分として見出されたspongistatinの生合成遺伝子の特定を進めてきた。Spongistatinはtrans AT型PKSによる生合成経路が予想される化合物であり、その生合成遺伝子には特徴的な配列を有するketosynthase (KS)が含まれる。したがってKSの縮重プライマーを用いたtrans AT型PKSの探索を重点的に行った。その結果、海綿メタゲノムDNAよりtrans AT型PKSに属するKS配列を得た。さらに海綿メタゲノムライブラリーを構築し、得られたtrans AT型KS配列に基づくスクリーニングを行った。その結果、複数のヒットクローンを得ることに成功した。現在詳細なシークエンス解析を進めている。また、海綿共生微生物の可培養化を目指し、沖縄産海綿Theonella swinhoeiから様々な組成の培地を用いた培養を試みた。その結果、既知微生物と相同性が低い微生物を含む数百種類の微生物を単離した。その中にはPKSやNRPS遺伝子を有する微生物群も数多く含まれており今後さらに物質生産能の検証を進める。
• Bioactive secondary metabolites produced by sponge symbiont Entotheonella

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

  Date (from‐to) : 2015/04 -2018/03 

  Author : WAKIMOTO Toshiyuki, KURANAGA Takefumi, EGAMI Yoko

   

  Marine sponges are prolific sources of bioactive molecules as well as highly complex consortia, including significantly large populations of symbiotic bacteria. The symbiotic bacteria often cannot be cultured under normal laboratory conditions. To access the gene clusters responsible for the biosynthesis of sponge-derived bioactive natural products, homology-based screening of the sponge metagenome library has been implemented. We successfully obtained the biosynthetic gene clusters of cytotoxic compounds, calyculins isolated from the Japanese sponge Discodermia calyx by a metagenome mining approach. The single cell analysis revealed that all these gene clusters were encoded by the same filamentous bacterium, which was identified as Candidatus “Entotheonella sp.” based on the 16S rRNA sequence. In this study, we investigated new metabolites as well as other gene clusters responsible for the biosynthesis of bioactive peptides derived from Discodermia kiiensis and Theonella swinhoei.
• Collection and survey of biosynthetic genes of spore-derived biologically active substances and clarification of biosynthetic machinery

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

  Date (from‐to) : 2014/04 -2018/03 

  Author : Abe Ikuro

   

  By collecting sponges in the Pacific region and isolating and comparing biosynthesis genes, we elucidated its biosynthetic machinery and searched for origin of structural diversity of natural products. At the same time, we investigated the possibility of producing bacteria common to these sponges and investigated the universality of the distribution of symbiotic bacteria excelling in substance production. As a result, the importance of marine biological resources in the Pacific region was demonstrated, and great contribution was expected to future drug development.
• The mechanism regulating the function of a natural cytotoxin

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

  Date (from‐to) : 2015/04 -2017/03 

  Author : WAKIMOTO Toshiyuki, EGAMI Yoko

   

  Marine sponges are prolific sources of highly potent cytotoxic compounds, some of which are promising as anticancer drugs. However, the potent cytotoxicity would be harmful to the host sponge, because marine sponges are also animals. Therefore, it is possible that the self-resistance and activated defense systems underlie their biosynthetic mechanism. Wound-activated chemical defense systems are prevalent among terrestrial plants. The precursor molecule and the activating enzyme are compartmentalized in the tissue, which is readily disrupted by wounding to accomplish chemical defense. The similar activated defense strategy has also been found in a few marine sponges. In previous study, we obtained calyculin biosynthetic gene cluster from the metagenomic DNA of Japanese marine sponge Discodermia calyx and the deactivating enzyme was also clustered. The aim of this work is to identify the as-yet unknown enzyme involved in the activating process of the precursor molecule.
• Calyculin A生合成機構の解析

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2013/04 -2015/03 

  Author : 脇本 敏幸

   

  本研究では伊豆半島産チョコガタイシカイメンに含まれる細胞毒性物質カリクリンAの生合成経路の解明を目指した。カリクリンAはタンパク質脱リン酸化酵素1および2Aを特異的に阻害し、各種がん細胞に対してpMオーダーで増殖阻害活性を示す。我々はカリクリンAの生合成遺伝子クラスターの探索を進め、チョコガタイシカイメンよりメタゲノムライブラリーを作成、ポリケタイド合成酵素遺伝子に保存性の高い配列を足掛かりとしてスクリーニングを行った。その結果、全長150 kbpに及ぶ長大な生合成遺伝子クラスターの取得に至った。予想通り、本遺伝子クラスターはⅠ型ポリケタイド合成酵素と非リボソーム型ペプチド合成酵素をコードしており、それらのモジュールやドメインの構成はカリクリンAの構造と良い一致を示した。さらに、いくつかの推定修飾酵素について機能解析を試みた。遺伝子クラスター上流には３つのリン酸基転移酵素がコードされており、それらを大腸菌において異種発現し、酵素反応を試みた。その結果、３つのうち１つ（CalQ）がカリクリンAを基質として受け入れ、二リン酸化体を生成することを明らかにした。このことには海綿メタゲノムより同定した遺伝子クラスターがカリクリンA生合成遺伝子であることを支持している。カリクリンAの二リン酸体の生物活性を調べたところ、細胞毒性は1,000倍、PP2A阻害活性は20倍と著しく減弱しており、カリクリンAのプロドラッグ体であることが示唆された。したがって、カリクリン生合成における最終産物はカリクリンAではなく、本研究で見出されたホスホカリクリンAであることが明らかとなった。
• Identification and genome analysis of bacterial symbiont producing sponge-derived secondary metabolites

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

  Date (from‐to) : 2013/04 -2015/03 

  Author : WAKIMOTO Toshiyuki

   

  In this study, we tried to identify the bacterial symbiont producing sponge-derived secondary metabolites. Theonellidae family sponges are well known to contain diverse secondary metabolites. However, the real producer of them had been unknown, though it had been supposed to be symbiont bacteria. The identification of such bacteria could be a key issue to pave the way for the supply limitation of medicinally important sponge-derived compounds. Three Theonellidae sponge species were therefore subjected to genetic evaluation, cell separation and single cell analysis. As a result, the Entotheonella was identified to be the real producer of almost all secondary metabolites of three sponge spieces. Our findings provide not only the proof of a sponge-specific molecule synthesized by the unique and uncultured bacterium rather than sponge itself, but also useful insights into the supply of sponge-derived medicinally important compounds.
• Enzyme engineering for natural product biosynthesis

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

  Date (from‐to) : 2011/04 -2015/03 

  Author : ABE IKURO, WAKIMOTO Toshiyuki, AWAKAWA Takayoshi, MATSUDA Yudai, MORITA Hiroyuki, SUGIO Shigetoshi, NOGUCHI Hiroshi, KUSHIRO Testuo

   

  Several enzymes involved in the biosynthesis of natural products, such as plant polyketide synthase and CoA-ligase, exhibit remarkable catalytic versatility and substrate promiscuity. This makes the enzymes an excellent platform for enzyme engineering and further production of structurally divergent molecules. In this study, on the basis of the X-ray crystal structures of the enzymes, we succeeded in production of chemically and structurally divergent unnatural natural products, by structure-based enzyme engineering and precursor directed biosynthesis. In addition, we succeeded in elucidation of biosynthetic machinery of complex natural products including calyculin, teleocidin, and antimycin.
• Studies on biosynthetic assembly line of meroterpenoids

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

  Date (from‐to) : 2010/04 -2015/03 

  Author : ABE IKURO, MATSUDA Yudai, WAKIMOTO Toshiyuki, AWAKAWA Takayoshi, MORITA Hiroyuki

   

  Meroterpenoids are hybrid natural products of both terpenoid and polyketide origin. We identified several biosynthetic gene clusters of pharmaceutically important fungal meroterpenoids, which includes that is responsible for the production of pyripyropene A, terretonin, anditomin, and andrastin, through reconstituted biosynthesis in a heterologous fungal expression system. Interestingly, the biosynthetic assembly lines are rich sources for interesting enzymes and chemistry, such as previously unknown membrane-bound terpene cyclases and multifunctional oxygenases, for the generation of structurally divergent complex fungal meroterpenoids.
• Development of selective phosphatase inhibitor based on calyculin A

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

  Date (from‐to) : 2011 -2012 

  Author : TOSHIYUKI Wakimoto

   

  Many marine natural products exhibit highly potent cytotoxicity. They selectively inhibit the functions of either intracellular or membrane-bound target molecules, leading to cell death. These specific biological activities can provide valuable information on the biochemical research field. Therefore, we initiated this program aimed at the development of functionalized calyculin A, a protein phosphatase inhibitor, which was originally isolated from the marine sponge, Discodermia calyx. Based on the insights into the structural moieties essential for biological activity, the synthetic process toward library construction and fluorescent probe were developed.
• Research for useful marineorganisms in Asia and Pacific regions

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

  Date (from‐to) : 2009 -2011 

  Author : NAKAO Yoichi, HORI Kanji, SAKAI Ryuichi, FUSETANI Nobuhiro, MATSUOKA Shunji, FUKUZAWA Seketsu, WAKIMOTO Toshiyuki, FUJITA Masaki, TAKADA Kentarou

   

  In this project, a total 515 marine organisms were collected from 3 countries and preserved as the extract-, microbe-, and genome-libraries. Currently, evaluation for bioactivities of these extracts are being carried out. At the same time, thight collaborative relationship among the researchers of these countries and our group was founded, through this project.
• Studies on the biological activity of furan fatty acids

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

  Date (from‐to) : 2007 -2009 

  Author : WAKIMOTO Toshiyuki

   

  Furan fatty acid is known as naturally occurring in various organisms but rare fatty acid. The elucidation of biological activity of furan fatty acid has been limited to antioxidant activity in vitro due to paucity of this fatty acid. In this study, isolation and semi-synthesis of this fatty acid were established, which enable us to disclose more potent anti-inflammatory activity of furan fatty acid than that of EPA.