Published papers

The structure elucidation of large molecules remains a central challenge in natural products chemistry. This challenge has been addressed through spectroscopic methods as well as degradative and synthetic approaches, which have provided effective solutions. Recent advances in genome analysis have enabled an orthogonal approach to structure elucidation based on biosynthetic gene information. In this study, we report the structure elucidation of an unprecedentedly large cyanobactin, terukufazoline A (1), through integration of spectroscopic analysis, metagenome-guided biosynthetic information, chemical degradation, and total synthesis. Terukufazolines A (1) and B (2) were discovered from an undescribed marine cyanobacterium, and the intractable NMR and tandem MS data for 1 prompted us to incorporate biosynthetic gene information. Metagenomic analysis identified the cyanobactin biosynthetic gene cluster, whose core peptide sequence enabled the assignment of the amino acid sequence of 1. The absolute configuration was established by degradation-based analyses, and the proposed structures were verified by convergent total syntheses of 1 and 2.

Two linear lipopeptides mabuniamide B (1) and C (2) were isolated from an Okeania sp. marine cyanobacterium collected from the coast of Okinawa. Their structures were elucidated by spectroscopic analyses, and their absolute configurations of amino acid residues were determined using Marfey's analysis of the hydrolysates of 1 and 2. In addition, 1 and 2 inhibited uric acid production in cultured AML12 cells.

Two cyclic peptides, bisezakiamides A (1) and B (2), were isolated from a marine cyanobacterium from the coastal waters of Okinawa, Japan. Both compounds contain an α-hydroxy acid residue and a β-amino acid. The structures of 1 and 2 were elucidated by detailed spectroscopic analyses, including various two-dimensional NMR techniques and application of Marfey's analysis. Both compounds exhibited growth-inhibitory activity against HeLa S3 cells, with IC50 values of 8.7 and 5.8 μM for compounds 1 and 2, respectively.

Madangolide (1) is a 17-membered cyclic enamide isolated from the marine cyanobacterium Lyngbya bouillonii. Since its discovery in 1999, the relative and absolute configurations of 1 have remained unknown. Here, we report the isolation, structural determination, and biological activity of a new analog of madangolide (1), madangolide B (2), from a marine Moorena sp. cyanobacterium. The planar structure of 2 was established using conventional two-dimensional NMR spectroscopy. The absolute configuration was clarified by comparing the experimental and theoretical data, including the chemical shifts, homonuclear and heteronuclear coupling constants, and ECD spectra. Based on the similarity of the NMR data, we proposed an absolute configuration of madangolide (1) and verified it through degradation and derivatization reactions. This study demonstrates the effectiveness of modern computational chemistry in revealing the stereochemistry of complex natural products with conformational flexibility.

Ouhanamide (1), a new linear lipopeptide consisting of a long-chain fatty acid with a β-branched methyl group, was isolated from a marine Okeania sp. cyanobacterium. The overall structure was elucidated by a combination of various spectroscopic analyses, degradation reactions, and derivatizations. Ouhanamide (1) showed selective antiparasitic activity (IC50 = 1.2 ± 0.1 μM against Trypanosoma brucei rhodesiense and IC50 = 4.2 ± 0.9 μM against Plasmodium falciparum) without cytotoxicity at 10 μM against HeLa cells.

Shimojilide (1), a new cyclic lipopeptide, was isolated from a marine cyanobacterium Okeania sp. Its structure was elucidated by NMR and chemical derivatization, revealing a peptide–polyketide hybrid with a skipped diene and β-branched methyl group. Shimojilide (1) exhibited potent and selective antiproliferative activity with a unique JFCR39 fingerprint that poorly correlates with those of known anticancer agents, suggesting a distinct mechanism of action. Shimojilide (1) represents a promising lead for anticancer drug discovery.

Overweight and obesity are pathological conditions characterized by the accumulation of excessive fat. Adipokines, which are secreted by hypertrophied adipocytes, are known to increase the risk of various diseases. Therefore, identifying compounds that promote lipolysis in adipocytes is a promising way to prevent hypertrophy of these cells. Although numerous bioactive compounds have been isolated from marine cyanobacteria, reports on compounds exhibiting such activity remain limited. In this study, we report the discovery of zampamide (1), a linear lipopeptide that stimulates lipolysis in 3T3-L1 adipocytes. It was isolated from a marine cyanobacterium of the genus Caldora collected in Okinawa, Japan. The planar structure of 1 was elucidated primarily using two-dimensional NMR and mass spectrometry. The absolute configurations of the amino acid residues in 1 were determined after acid hydrolysis, followed by Marfey's analysis of the resulting hydrolysates. Additionally, the absolute configuration of the fatty acid moiety was determined by derivatization. Zampamide (1) increased the amount of free glycerol in a dose-dependent manner, demonstrating its lipolysis-promoting activity. These findings suggest that marine cyanobacteria are a valuable source of novel lipolysis-promoting agents for obese and overweight patients.

A novel 19-membered cyclic depsipeptide, hedopeptolide (1), was isolated from Okeania sp., a marine cyanobacterium collected in Okinawa, Japan. Its gross structure was determined by spectroscopic analyses, and the absolute configuration of the chiral centers was determined by single-crystal X-ray diffraction analysis following crystallization. Hedopeptolide (1) inhibited the lipopolysaccharide-induced nitric oxide production in RAW264.7 cells.

Sealgamide (1), a new antitrypanosomal lipopeptide, was isolated from a marine Okeania sp. cyanobacterium. Elucidation of its structure was challenging due to signal overlap caused by conspicuous rotamers but was ultimately achieved through partial hydrolysis and subsequent spectroscopic analyses. Sealgamide (1) exhibited moderate antitrypanosomal activity against Trypanosoma brucei rhodesiense (IC50 2.9 μM) while showing no antimalarial activity (IC50 > 25 μM) or cytotoxicity (IC50 > 30 μM). Furthermore, we discovered that an artificial C-terminal methyl ester analogue (2) exhibited notably enhanced antiparasitic activity.

Human African trypanosomiasis (HAT) is one of the most lethal of the neglected tropical diseases. While the discovery of a novel antitrypanosomal drug is highly desired, the creation of a superior lead compound is challenging. Herein we report ukabamide (1), which was isolated from a marine Moorena sp. cyanobacterium, as a selective and potent antitrypanosomal lipopeptide (IC50 = 34 ± 18 nM against Trypanosoma brucei rhodesiense). Its structure was elucidated by spectroscopic analyses and degradation reactions and confirmed by total synthesis. In addition, we prepared two modified analogs and revealed the importance of the fatty acid chain length for biological activity. These findings may provide design guidelines for an antitrypanosomal drug.

In this era of advanced technologies, is there still wisdom to be gleaned from nature? The answer is an unequivocal “Yes”. Discovering new natural products from nature, determining their structures, and revealing their biological activities ‐ this classical natural product chemistry is still a catalyst for innovation in a wide range of fields, including the expansion of chemical space, elucidation of life phenomena, and drug discovery. In this account, we describe a comprehensive bioorganic study of iezoside (1a), the novel marine natural product we recently discovered, including its discovery, synthesis, mode of action, and structure‐activity relationships.

Iezoside (1a) is a novel, potent sarco-/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor from marine cyanobacterium. This paper describes the synthesis of comprehensive iezoside (1a) analogs containing C-18/19 diastereomers, simplified analogs without the peptide unit, and aglycones. Evaluations of the antiproliferative activities against cancer cells and SERCA inhibitory activities of the synthesized analogs revealed how the absolute configurations at C-18/19, peptide, and the sugar unit contribute to each bioactivity.

The toxicity of polycavernoside E and its structurally related macrolides, akunolides A, B, and C, was evaluated in mice. These compounds were recently isolated from the marine cyanobacterium Okeania sp. collected in Okinawa, Japan. The minimal toxicity of polycavernoside E was estimated as 0.81 mg/kg by intraperitoneal injection, whereas those of akunolides A–C were >13 mg/kg. Owing to the considerable toxicity of polycavernoside E, the occurrence of this cyanobacterium should be considered in seaweed production environments.

Here, we report wajeepeptin (1), a new cyclic depsipeptide isolated from a marine Moorena sp. cyanobacterium. The structure was elucidated by a combination of spectroscopic analyses, X-ray diffraction analysis, and degradation reactions. Wajeepeptin (1) showed moderate cytotoxicity (IC50 = 3.7 μM against HeLa cells) and potent antitrypanosomal activity (IC50 = 0.73 ± 0.14 μM against Trypanosoma brucei rhodesiense).

Logging operations of Liriodendron tulipifera L., as timber trees, and fallen leaves in autumn from ornamental trees produce a large amount of leaf waste. In this study, the allelopathy of L. tulipifera fresh and fallen leaves was investigated for the development of potential applications of leaf waste. The extracts of fresh and fallen leaves of L. tulipifera showed growth inhibitory activity against weed species, Vulpia myuros (L.) C.C.Gmel., Echinochloa crus-galli (L.) P.Beauv., and Lolium multiflorum Lam., under laboratory conditions. The powder of L. tulipifera fresh and fallen leaves also inhibited the germination of E. crus-galli under greenhouse conditions. A potent allelochemical was isolated from fresh and fallen leaf extracts through a bioassay-guided separation process, and was identified as lipiferolide. Lipiferolide inhibited the growth of L. multiflorum and Lepidum sativum in a concentration-dependent manner. This investigation suggests that the leaf waste of L. tulipifera from logging operations and fallen leaves is potentially useful for the purpose of weed control, such as through the use of soil additive materials from leaves or the creation of foliar spray from leaf extracts. The development of weed control materials using L. tulipifera leaf waste may be a means with which to minimize waste, reducing environmental impacts and economic concerns.

Chamaecyparis obtusa (Siebold et Zucc.) Endl. is cultivated in the temperate region of East Asia for its high-quality and profitable timber. The timber-processing industry produces a large amount of waste, such as tree bark, branches, and leaves, and finding ways to minimize such waste is economically and environmentally efficient. In the present study, the allelopathic activity and allelochemicals of the C. obtusa leaves were investigated to develop potential application methods. C. obtusa leaves were phytotoxic and associated leaf extracts significantly suppressed the growth of the weeds; Echinochloa crus-galli, Phleum pratense and Lolium multiflorum under laboratory conditions. The leaf powder applied onto the soil surface also suppressed the germination of E. crus-galli and P. pratense under greenhouse conditions. Hinokiic acid and (+)-dihydrosesamin were isolated from the leaves and structurally identified. Both compounds suppressed the growth of cress and P. pratense in a concentration dependent manner. When the leaves of C. obtua were applied as a soil additive, hinokiic acid and (+)-dihydrosesamin in the leaves potentially cause the growth inhibitory activity by leaching into the soil. These experiments showed that the leaves and the leaf extracts of C. obtusa are phytotoxic. These experiments also demonstrated that the leaves and the leaf extracts of C. obtusa have growth inhibitory potential against several plant species, and the leaves produce allelochemicals. Therefore, the leaves and leaf extracts of C. obtusa may be useful for weed suppression purposes. The leaf biomass of C. obtusa is frequently large and the timber processing industry produces significant leaf waste. The development of weed control products using waste leaves may be a solution to minimize the timber processing waste to reduce environmental impact and provide economic value. However, leaf material should be evaluated for weed suppression and herbicidal activity as a soil additive and also possible as a foliar spray under field conditions.

The use of allelopathy developed as an alternative practice for weed management in agriculture. Oldenlandia corymbosa is an annual herb and notable for being applied in folk medicine. Its diverse range of pharmacological properties and several bioactive substances have been well reported. Nevertheless, the allelopathic potential of O. corymbosa has not yet been documented. Hence, we aim to explore the allelopathy of O. corymbosa for weed control purposes. The extracts of O. corymbosa were tested to determine their effects on the seedling growth of six tested species. Significant reductions in the growth of seedling were exhibited as the plant extract concentrations increased. The plant extracts of O. corymbosa were separated in several chromatography steps; two active substances isolated and characterized as hedyotiscone A and B. Hedyotiscone A and B significantly restricted the growth of cress seedling at concentrations higher than 0.3 mM. The I50 values of hedyotiscone A and B for the cress seedlings ranged from 0.023 to 0.131 mM and 0.167 to 0.810 mM, respectively. The inhibitory activity against cress seedlings indicates that hedyotiscone A and B contribute to the allelopathic effects and may be responsible for the allelopathic potential of O. corymbosa.

Amebiasis is an important cause of morbidity and mortality worldwide, and caused by infection with the protozoan parasite Entamoeba histolytica. Metronidazole is currently the first-line drug despite adverse effects and concerns on the emergence of drug resistance. Fumagillin, a fungal metabolite from Aspergillus fumigatus, and its structurally related natural and synthetic compounds have been previously explored as potential anti-angiogenesis inhibitors for cancers, anti-microbial, and anti-obese compounds. Although fumagillin was used for human amebiasis in clinical trials in 1950s, the mode of action of fumagillin remains elusive until now. In this report, we showed that fumagillin covalently binds to methionine aminopeptidase 2 (MetAP2) and non-covalently but abundantly binds to patatin family phospholipase A (PLA). Susceptibility against fumagillin of the amebic strains in which expression of E. histolytica MetAP2 (EhMetAP2) gene was silenced increased compared to control strain. Conversely, overexpression of EhMetAP2 mutants that harbors amino acid substitutions responsible for resistance to ovalicin, a fumagillin analog, in human MetAP2, also resulted in decrease in fumagillin susceptibility. In contrast, neither gene silencing nor overexpression of E. histolytica PLA (EhPLA) affected fumagillin susceptibility. These data suggest that EhPLA is not essential and not the target of fumagillin for its amebicidal activity. Taken together, our data have demonstrated that EhMetAP2 is the primary target for amebicidal activity of fumagillin, and EhMetAP2 represents a rational explorable target for the development of alternative therapeutic agents against amebiasis.

Polycavernoside E (1), a new polycavernoside analog, was isolated from a marine Okeania sp. cyanobacterium. The relative configuration was elucidated primarily by analyzing the two dimensional nuclear magnetism resonance (2D NMR) data. The absolute configuration was clarified by comparing the electronic circular dichroism (ECD) data of 1 with those of known analogs. Polycavernoside E (1) exhibited moderate antitrypanosomal activity against Trypanosoma brucei rhodesiense. Furthermore, the isolation of polycavernoside E (1) from marine cyanobacteria provides additional evidence that marine cyanobacteria, and not red algae, are responsible for the biosynthesis of polycavernosides.

Akunolides A (1), B (2), C (3), and D (4), new macrolide glycosides, were isolated from a marine Okeania sp. cyanobacterium. Their structures were elucidated by spectroscopic analyses and derivatization reactions. Akunolides A-D (1-4) are classified as 16-membered macrolide glycosides, which are relatively rare structures for marine cyanobacterium-derived natural products. Akunolides A-D (1-4) showed moderate antitrypanosomal activities against Trypanosoma brucei rhodesiense, with IC50 values ranging from 11 to 14 μM. Furthermore, akunolides A (1) and C (3) exhibited no cytotoxicity against normal human WI-38 cells even at a concentration of 150 μM.