Synthesis and structure-activity relationship study of antimicrotubule agents phenylahistin derivatives with a didehydropiperazine-2,5-dione structure.

Plinabulin (11, NPI-2358) is a potent microtubule-targeting agent derived from the natural diketopiperazine "phenylahistin" (1) with a colchicine-like tubulin depolymerization activity. Compound 11 was recently developed as VDA and is now under phase II clinical trials as an anticancer drug. To develop more potent antimicrotubule and cytotoxic derivatives based on the didehydro-DKP skeleton, we performed further modification on the tert-butyl or phenyl groups of 11, and evaluated their cytotoxic and tubulin-binding activities. In the SAR study, we developed more potent derivatives 33 with 2,5-difluorophenyl and 50 with a benzophenone in place of the phenyl group. The anti-HuVEC activity of 33 and 50 exhibited a lowest effective concentration of 2 and 1 nM for microtubule depolymerization, respectively. The values of 33 and 50 were 5 and 10 times more potent than that of CA-4, respectively. These derivatives could be a valuable second-generation derivative with both vascular disrupting and cytotoxic activities.

Streptobactin, a tricatechol-type siderophore from marine-derived Streptomyces sp. YM5-799.

A new catechol-type siderophore, streptobactin (1), was isolated from a culture broth of the marine-derived actinomycete Streptomyces sp. YM5-799. The structure of streptobactin was determined by NMR and MS analyses and ESIMS/MS experiments to be a cyclic trimer of benarthin. A dibenarthin (2), a tribenarthin (3), and benarthin (4) were also obtained. The production of 1 was regulated by an iron concentration in the culture. The iron-chelating activity of the compounds was evaluated by the chrome azurol sulfonate assay.

Efrapeptin J, a new down-regulator of the molecular chaperone GRP78 from a marine Tolypocladium sp.

A new down-regulator of the molecular chaperone GRP78, efrapeptin J, was isolated from a marine fungus, Tolypocladium sp. AMB18. The molecular formula of efrapeptin J was established as C(81)H(139)N(18)O(16)(+) by high-resolution FAB-MS. The structure was elucidated to be a linear pentadecapeptide containing a hexahydropyrrolo[1,2-a]pyrimidinium moiety by NMR and MS analyses. Efrapeptins F, G and J dose-dependently inhibited 2-deoxyglucose-induced luciferase expression in HT1080 human fibrosarcoma cells transfected with a luciferase reporter plasmid containing the GRP78 promoter. Efrapeptin J also inhibited the protein expression of GRP78 in HT1080 cells and MKN-74 human gastric cancer cells. Efrapeptin J induced cell death in HT1080 cells under endoplasmic reticulum stress.

Ascochytatin, a novel bioactive spirodioxynaphthalene metabolite produced by the marine-derived fungus, Ascochyta sp. NGB4.

Ascochytatin, a new spirodioxynaphthalene metabolite produced by a marine-derived fungus, was found from a screening program focused on the bacterial two-component regulatory system. The structure of ascochytatin was determined by spectroscopic methods, including NMR and MS. The relative stereochemistry was determined by an X-ray crystallographic analysis, and the absolute stereochemistry was determined by the modified Mosher's method.

New sulfoalkylresorcinol from marine-derived fungus, Zygosporium sp. KNC52.

A new sulfoalkylresorcinol (1) was isolated from the marine-derived fungus, Zygosporium sp. KNC52. The structure of 1 was elucidated by spectroscopic methods including MS and NMR, and the absolute stereochemistry was determined by the modified Mosher's method. Compound 1 inhibited FtsZ polymerization in vitro and exhibited weak antimicrobial activity against multi-drug-resistant bacteria.

Structural determination and proposed biosynthesis of alcanivorone, a novel alpha-pyrone produced by Alcanivorax jadensis.

A novel alpha-pyrone designated as alcanivorone was found in a culture broth of the marine bacterium, Alcanivorax jadensis, and its structure was determined by an analysis of 1D NMR, 2D NMR and MS data. Alcanivorone was produced by A. jadensis only when sodium pyruvate was added to the culture medium as a carbon source. Incorporation experiments using stable isotope-labeled pyruvate indicated that alcanivorone was biosynthesized from four molecules of pyruvate.

Three new polyketide-terpenoid hybrids from Penicillium sp.

Three novel hybrid polyketide-terpenoid metabolites were isolated from a Penicillium minioluteum strain. Their structures were determined by NMR spectroscopic analyses and X-ray crystallography. The proposed biosynthetic pathway including a unique retro-Claisen migration of methyl carbonate correlates the three compounds with berkeleydione and berkeleytrione.

Halobacillus faecis sp. nov., a spore-forming bacterium isolated from a mangrove area on Ishigaki Island, Japan.

A Gram-positive, spore-forming, rod-shaped halophilic bacterial strain, IGA7-4T, was isolated from a mangrove area on Ishigaki Island (Japan), and was characterized taxonomically using a polyphasic approach. Strain IGA7-4T was strictly aerobic and non-motile and formed central endospores. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain IGA7-4T is affiliated to the genus Halobacillus, and exhibits sequence similarities of 99.6-98.0% to the type strains of Halobacillus species. Levels of DNA-DNA relatedness between strain IGA7-4T and the type strains of Halobacillus species were 9.5-46.6%. The DNA G+C content of strain IGA7-4T was 46.5 mol%. The cell-wall peptidoglycan type (Orn-Asp), major cellular fatty acids (anteiso-C15:0, anteiso-C17:0, iso-C15:0 and iso-C16:0) and quinone type (MK-7) of the isolate support its affiliation to the genus Halobacillus. On the basis of phylogenetic analysis, phenotypic characteristics and chemotaxonomic data, the isolate represents a novel species of the genus Halobacillus, for which the name Halobacillus faecis sp. nov. is proposed. The type strain is IGA7-4T (=MBIC08268T=IAM 15427T=KCTC 13121T).

Urukthapelstatin A, a novel cytotoxic substance from marine-derived Mechercharimyces asporophorigenens YM11-542. I. Fermentation, isolation and biological activities.

Urukthapelstatin A, a novel cyclic peptide, was isolated from the cultured mycelia of marine-derived Thermoactinomycetaceae bacterium Mechercharimyces asporophorigenens YM11-542. The peptide was purified by solvent extraction, silica gel chromatography, ODS flash chromatography, and finally by preparative HPLC. Urukthapelstatin A dose-dependently inhibited the growth of human lung cancer A549 cells with an IC(50) value of 12 nM. Urukthapelstatin A also showed potent cytotoxic activity against a human cancer cell line panel.

Urukthapelstatin A, a novel cytotoxic substance from marine-derived Mechercharimyces asporophorigenens YM11-542. II. Physico-chemical properties and structural elucidation.

The new cyclic peptide antibiotic, urukthapelstatin A, has been isolated from a culture of Thermoactinomycetaceae bacterium Mechercharimyces asporophorigenens YM11-542. The structure of urukthapelstatin A was elucidated by NMR, MS, Marfey analysis, chiral HPLC and X-ray crystal analyses.

Tenacibactins A-D, hydroxamate siderophores from a marine-derived bacterium, Tenacibaculum sp. A4K-17.

Four new hydroxamate siderophores, tenacibactins A-D (1-4), were isolated from a culture broth of the marine-derived bacterium Tenacibaculum sp. A4K-17. The structures of these tenacibactins were determined by NMR analyses and ESIMS/MS experiments. The iron-binding (chelating) activity of 1-4 was evaluated by the chrome azurol sulfonate (CAS) assay.

New dihydrobenzofuran derivative, awajanoran, from marine-derived Acremonium sp. AWA16-1.

Awajanoran (1), a new dihydrobenzofuran derivative, was isolated from an agar-culture of Acremonium sp. AWA16-1, which had been isolated from sea mud collected at Awajishima Island in Japan. The structure of 1 was elucidated on the basis of a spectroscopic analysis. This compound inhibited the growth of A549 cells, the human lung adenocarcinoma cell line, with an IC50 value of 17 microg/ml, and also showed antimicrobial activity.

Awajanomycin, a Cytotoxic gamma-lactone-delta-lactam metabolite from marine-derived Acremonium sp. AWA16-1.

The new fungal metabolite awajanomycin (1), which has gamma-lactone-delta-lactam rings, was isolated from the marine-derived fungus Acremonium sp. AWA16-1, which had been collected from sea mud off Awajishima Island in Japan. The structure of 1 was elucidated by spectroscopic analysis and chemical methods. Awajanomycin (1) and its derivative (2) inhibited the growth of A549 cells with IC(50) values of 27.5 and 46.4 microg/mL, respectively.

Discovery of a marine bacterium producing 4-hydroxybenzoate and its alkyl esters, parabens.

Chemically synthesized 4-hydroxybenzoate (4HBA) is widely used in the chemical and electrical industries as a material for producing polymers such as those of the liquid crystal type. Its alkyl esters, called parabens, have been the most widely used preservatives by the food and cosmetic industries. We report here for the first time a microorganism, a marine bacterium, which biosynthesizes these petrochemical products. The marine bacterial strain, A4B-17, which was found to belong to the genus Microbulbifer on the basis of its rRNA and gyrB sequences, was isolated from an ascidian in the coastal waters of Palau. Strain A4B-17 was, surprisingly, found to produce 10 mg/liter of 4HBA, together with its butyl (24 mg/liter), heptyl (0.4 mg/liter), and nonyl (6 mg/liter) esters. We therefore characterized 23 other marine bacteria belonging to the genus Microbulbifer, which our institute had previously isolated from various marine environments, and found that these bacteria also produced 4HBA, although with low production levels (less than one-fifth of that produced by A4B-17). We also show that the alkyl esters of 4HBA produced by strain A4B-17 were effective in preventing the growth of yeasts, molds, and gram-positive bacteria.

Isolation of marine bacteria by in situ culture on media-supplemented polyurethane foam.

Polyurethane foam (PUF) supplemented with various agar media was used in situ to trap marine bacteria and it consequently provided a substrate on which they could be cultivated while exposed to natural seawater in the coral reef area. The bacterial population on the PUF blocks was analyzed by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments. Changing the composition of the cultivation medium in the PUF blocks and selecting different sampling sites resulted in different bacteria being detected on the PUF blocks. For example, iron-utilizing (IU) bacteria, siderophore-producing (SP) bacteria, and petroleum-degrading (PD) bacteria were isolated from PUF blocks and it was discovered that IU and SP contained iron and PD contained hydrocarbon. This method opens up the possibility for isolating novel and useful marine bacteria.

Catechol siderophore excretion by magnetotactic bacterium Magnetospirillum magneticum AMB-1.

Siderophore activity was detected in the culture supernatant of the magnetotactic bacterium Magnetospirillum magneticum AMB-1. Here we report the first structural elucidation of a siderophore produced by a magnetotactic bacterium. The structure of the purified compound was 3,4-dihydroxybenzoic acid as determined by nuclear magnetic resonance (NMR) and electro-spray ionization mass spectroscopy (ESI-MS).

Characterization of four Rhodococcus alcohol dehydrogenase genes responsible for the oxidation of aromatic alcohols.

Four genes were isolated and characterized for alcohol dehydrogenases (ADHs) catalyzing the oxidation of aromatic alcohols such as benzyl alcohol to their corresponding aldehydes, one from o-xylene-degrading Rhodococcus opacus TKN14 and the other three from n-alkane-degrading Rhodococcus erythropolis PR4. Various aromatic alcohols were bioconverted to their corresponding carboxylic acids using Escherichia coli cells expressing each of the four ADH genes together with an aromatic aldehyde dehydrogenase gene (phnN) from Sphingomonas sp. strain 14DN61. The ADH gene (designated adhA) from strain TKN14 had the ability to biotransform a wide variety of aromatic alcohols, i.e., 2-hydroxymethyl-6-methylnaphthalene, 2-hydroxymethylnaphthalene, xylene-alpha,alpha'-diol, 3-chlorobenzyl alcohol, and vanillyl alcohol, in addition to benzyl alcohol with or without a hydroxyl, methyl, or methoxy substitution. In contrast, the three ADH genes of strain PR4 (designated adhA, adhB, and adhC) exhibited lower ability to degrade these alcohols: these genes stimulated the conversion of the alcohol substrates by only threefold or less of the control value. One exception was the conversion of 3-methoxybenzyl alcohol, which was stimulated sevenfold by adhB. A phylogenetic analysis of the amino acid sequences of these four enzymes indicated that they differed from other Zn-dependent ADHs.

Mechercharmycins A and B, cytotoxic substances from marine-derived Thermoactinomyces sp. YM3-251.

A new cytotoxic substance named mechercharmycin A was isolated from marine-derived Thermoactinomyces sp. YM3-251. The structure of mechercharmycin A was determined by an X-ray crystallographic analysis to be cyclic peptide-like and bearing four oxazoles and a thiazole. Mechercharmycin B, a linear congener of mechercharmycin A, was also isolated from the same bacterium. Mechercharmycin A exhibited relatively strong antitumor activity, whereas mechercharmycin B exhibited almost no such activity.

Characterization of Sphingomonas aldehyde dehydrogenase catalyzing the conversion of various aromatic aldehydes to their carboxylic acids.

An aldehyde dehydrogenase gene, designated phnN, was isolated from a genome library of the 1,4-dimethylnaphthalene-utilizing soil bacterium, Sphingomonas sp. 14DN61. Escherichia coli expressing the phnN gene converted 1,4-dihydroxymethylnaphthalene to 1-hydroxymethyl-4-naphthoic acid. The putative amino acid sequence of the phnN gene product had 31-42% identity with those of NAD(+)-dependent short-chain aliphatic aldehyde dehydrogenases and a secondary alcohol dehydrogenase. The NAD(P)(+)-binding site and two consensus sequences involved in the active site for aldehyde dehydrogenase are conserved among these proteins. The PhnN enzyme purified from recombinant E. coli showed broad substrate specificity towards various aromatic aldehydes, i.e., 1- and 2-naphaldehydes, cinnamaldehyde, vanillin, syringaldehyde, benzaldehyde and benzaldehydes substituted with a hydroxyl, methyl, methoxy, chloro, fluoro, or nitro group were converted to their corresponding carboxylic acids. Interestingly, E. coli expressing phnN was able to biotransform a variety of not only aromatic aldehydes, but also aromatic alcohols to carboxylic acids.

Clonostachysins A and B, new anti-dinoflagellate cyclic peptides from a marine-derived fungus.

The two new anti-dinoflagellates, clonostachysins A and B, were obtained from a marine sponge derived fungus Clonostachys rogersoniana strain HJK9. Their chemical structures were determined by spectroscopic studies as highly N-methylated cyclic peptides of the nine amino acids. The absolute stereochemistry was elucidated by the advanced Marfey's method. Both clonostachysins A and B exhibited a selectively inhibitory effect on a dinoflagellate Prorocentrum micans at 30 microM, but had no effect on other microalgae and bacteria even at 100 microM.