Total synthesis of isoneoantimycin.

Antimycins are one of the well-known antifungal metabolites produced by Streptomyces bacteria. Neoantimycin and its analogues, the ring-expanded antimycins featuring a 15-membered tetraester ring, have been shown to be effective regulators of the oncogenic proteins GRP78/BiP and K-Ras. Isoneoantimycin was isolated from Streptomyces fradiae IFO12773 (ISP 5063) as a minor metabolite during the fermentation of neoantimycin and is the first reported antibiotic of the antimycin family without the macrolide core. In this study, we explored the total synthesis and stereochemical assignment of isoneoantimycin as an approach to perform structure-activity studies on neoantimycins. Taking the neoantimycin biosynthesis pathway into account, we presumed that the stereochemistry of isoneoantimycin is the same as that of neoantimycin. The synthesis of our target molecule with the (1S,2R,5S,6S,14R,15R,17S) configuration has been achieved by using chiral-pool building blocks. A comparison of the spectroscopic data between the synthetic and natural samples verified our presumption of the stereochemistry of natural isoneoantimycin.

Ligand-Dependant Selective Synthesis of Mono- and Dialkenylcarbazoles through Rhodium(III)-Catalyzed C-H Alkenylation.

The C-H alkenylation of N-acetylcarbazoles proceeds smoothly at the C1-position in the presence of a cationic Cp*Rh(III) catalyst to produce 1-alkenylcarbazoles. The use of a cationic CpE Rh(III) catalyst enables further alkenylation to give 1,8-dialkenylcarbazoles. The direct alkenylation procedure in combination with the ready removal of the acetyl directing group provides a straightforward synthetic pathway to 1- and/or 8-alkenyl-N-H-carbazole derivatives. One of 1-alkenyl-N-H-carbazoles obtained by the present C-H alkenylation/deacetylation exhibits solvatochromism.

Suppression of Linear Ubiquitination Ameliorates Cytoplasmic Aggregation of Truncated TDP-43.

TAR DNA-binding protein 43 (TDP-43) is a predominant component of inclusions in the brains and spines of patients with amyotrophic lateral sclerosis (ALS). The progressive accumulation of inclusions leads to proteinopathy in neurons. We have previously shown that Met1(M1)-linked linear ubiquitin, which is specifically generated by the linear ubiquitin chain assembly complex (LUBAC), is colocalized with TDP-43 inclusions in neurons from optineurin-associated familial and sporadic ALS patients, and affects NF-κB activation and apoptosis. To examine the effects of LUBAC-mediated linear ubiquitination on TDP-43 proteinopathies, we performed cell biological analyses using full-length and truncated forms of the ALS-associated Ala315→Thr (A315T) mutant of TDP-43 in Neuro2a cells. The truncated A315T mutants of TDP-43, which lack a nuclear localization signal, efficiently generated cytoplasmic aggregates that were colocalized with multiple ubiquitin chains such as M1-, Lys(K)48-, and K63-chains. Genetic ablation of HOIP or treatment with a LUBAC inhibitor, HOIPIN-8, suppressed the cytoplasmic aggregation of A315T mutants of TDP-43. Moreover, the enhanced TNF-α-mediated NF-κB activity by truncated TDP-43 mutants was eliminated in the presence of HOIPIN-8. These results suggest that multiple ubiquitinations of TDP-43 including M1-ubiquitin affect protein aggregation and inflammatory responses in vitro, and therefore, LUBAC inhibition ameliorates TDP-43 proteinopathy.

Rhodium(III)-Catalyzed Redox-Neutral Coupling of α-Trifluoromethylacrylic Acid with Benzamides through Directed C-H Bond Cleavage.

A rhodium(III)-catalyzed redox-neutral coupling of α-trifluoromethylacrylic acid with bezamides proceeds smoothly accompanied by amide-directed C-H bond cleavage to produce ß-[2-(aminocarbonyl)phenyl]-α-trifluoromethylpropanoic acid derivatives. One of the products can be transformed to a trifluoromethyl substituted heterocyclic compound. In addition, the redox-neutral coupling of α-trifluoromethylacrylic acid with related aromatic substrates possessing a nitrogen-containing directing group can also be conducted under similar conditions.

Total Syntheses and Configuration Assignments of JBIR-06 and Related Depsipeptides.

The first total syntheses of JBIR-06 and two analogous depsipeptides, 12-membered antimycin-class antibiotics, have been accomplished via Shiina macrolactonization. Comparison of the spectroscopic data of the synthesized compounds with those reported for natural products verified that the absolute configutation of the natural products was (2 S,4 S,6 S,7 R,14 S).

Evaluation of Inhibitory Activities of UK-2A, an Antimycin-Type Antibiotic, and Its Synthetic Analogues against the Production of Anti-inflammatory Cytokine IL-4.

The inhibitory activities of the antimycin-class antibiotics UK-2A, antimycin A, and splenocin B against the production of anti-inflammatory cytokine IL-4, which is related to IgE-mediated allergic responses in rat basophilic leukemia (RBL-2H3) cells, were evaluated. Although antimycin A and splenocin B showed cytotoxicity at concentrations at which IL-4 release from the cells was restricted, UK-2A was found to restrict IL-4 release without cytotoxicity. Three UK-2A analogues (4-6) were then synthesized and assessed. Compound 5 restricted IL-4 release dose-dependently without cytotoxicity, and its effect was more potent than that of UK-2A.

Iridium-Catalyzed Aerobic Coupling of Salicylaldehydes with Alkynes: A Remarkable Switch of Oxacyclic Product.

The iridium(III)/copper(II)-catalyzed dehydrogenative coupling of salicylaldehydes with internal alkynes proceeds efficiently under atmospheric oxygen through aldehyde C-H bond cleavage and decarbonylation. A variety of benzofuran derivatives can be synthesized by the environmentally benign procedure. DFT calculations suggest that this unique transformation involves the facile deinsertion of CO in the key metallacycle intermediate, which is in marked contrast to the corresponding rhodium(III) catalysis that leads to CO-retentive chromone derivatives.

Total syntheses and configuration assignments of JBIR-04 and unantimycin A.

First total syntheses of JBIR-04 and unantimycin A have been achieved. Comparison of our spectroscopic data with those reported for natural samples verified the structure of the natural products; (2S,4S,6S,7R,9S,28S) configuration was thus assigned via total synthesis.

Synthesis of Difluorinated Enynes through Sonogashira-Type Coupling.

The Sonogashira-type coupling of 2,2-difluoroethenyl tosylate with a variety of aliphatic and aromatic terminal alkynes proceeds smoothly even at room temperature to produce the corresponding difluorinated enyne derivatives. 2,2-Difluoroethenyl tosylate is a useful difluoroethenyl source because of its ready availability from 2,2,2-trifluoroethanol. Some of the obtained enynes exhibit strong fluorescence in the solid state. Further derivatization of a difluorinated enyne through Rh(III)-catalyzed oxidative coupling has also been examined.

Visualization analysis of the vacuole-targeting fungicidal activity of amphotericin B against the parent strain and an ergosterol-less mutant of Saccharomyces cerevisiae.

Here, we sought to investigate the vacuole-targeting fungicidal activity of amphotericin B (AmB) in the parent strain and AmB-resistant mutant of Saccharomyces cerevisiae and elucidate the mechanisms involved in this process. Our data demonstrated that the vacuole-targeting fungicidal activity of AmB was markedly enhanced by N-methyl-N″-dodecylguanidine (MC12), a synthetic analogue of the alkyl side chain in niphimycin, as represented by the synergy in their antifungal activities against parent cells of S. cerevisiae. Indifference was observed only with Δerg3 cells, indicating that the replacement of ergosterol with episterol facilitated their resistance to the combined lethal actions of AmB and MC12. Dansyl-labelled amphotericin B (AmB-Ds) was concentrated into normal rounded vacuoles when parent cells were treated with AmB-Ds alone, even at a non-lethal concentration. The additional supplementation of MC12 resulted in a marked loss of cell viability and vacuole disruption, as judged by the fluorescence from AmB-Ds scattered throughout the cytoplasm. In Δerg3 cells, AmB-Ds was scarcely detected in the cytoplasm, even with the addition of MC12, reflecting its failure to normally incorporate across the plasma membrane into the vacuole. Thus, this study supported the hypothesis that ergosterol is involved in the mobilization of AmB into the vacuolar membrane so that AmB-dependent vacuole disruption can be fully enhanced by cotreatment with MC12.

Antifungal thiopeptide cyclothiazomycin B1 exhibits growth inhibition accompanying morphological changes via binding to fungal cell wall chitin.

Cyclothiazomycin B1 (CTB1) is an antifungal cyclic thiopeptide isolated from the culture broth of Streptomyces sp. HA 125-40. CTB1 inhibited the growth of several filamentous fungi including plant pathogens along with swelling of hyphae and spores. The antifungal activity of CTB1 was weakened by hyperosmotic conditions, and hyphae treated with CTB1 burst under hypoosmotic conditions, indicating increased cell wall fragility. CTB1-sensitive fungal species contain high levels of cell wall chitin and/or chitosan. Unlike nikkomycin Z, a competitive inhibitor of chitin synthase (CHS), CTB1 did not inhibit CHS activity. Although CTB1 inhibited CHS biosynthesis, the same result was also obtained with a non-specific proteins inhibitor, cycloheximide, which did not reduce cell wall rigidity. These results indicate that the primary target of CTB1 is not CHS, and we concluded that CTB1 antifungal activity was independent of this sole inhibition. We found that CTB1 bound to chitin but did not bind to ß-glucan and chitosan. The results of the present study suggest that CTB1 induces cell wall fragility by binding to chitin, which forms the fungal cell wall. The antifungal activity of CTB1 could be explained by this chitin-binding ability.

Enhancement effect of N-methyl-N″-dodecylguanidine on the vacuole-targeting fungicidal activity of amphotericin B against the pathogenic fungus Candida albicans.

The alkylguanidium chain attached to the polyol lactone ring of niphimycin (NM) is considered a requisite for the fungicidal activity of NM characterized by vacuole membrane fragmentation and oxidative stress induction. The addition of N-methyl-N″-dodecylguanidine to the medium can enhance the vacuole-targeting fungicidal activity of amphotericin B (AmB), in which the lactone ring has no such alkylguanidium chain, on Saccharomyces cerevisiae cells. In this study, the enhancement effect of N-methyl-N″-dodecylguanidine on the vacuole-targeting fungicidal activity of AmB was examined against Candida albicans in RPMI 1640 medium at 37 °C. N-methyl-N″-dodecylguanidine was lethal to C. albicans cells and additionally enhanced the vacuole disruptive activity of AmB against this pathogenic fungus. N-methyl-N″-dodecylguanidine elevated the generation of cellular reactive oxygen species when added alone in a dose-dependent manner, but its enhancement effect on AmB lethality did not accompany amplification of oxidative stress induction. The fungal vacuoles were protected against the disruptive damage even if cells were treated with H(2)O(2) alone at a lethal concentration or treated with H(2)O(2) at a sublethal concentration in combination with AmB. N-methyl-N″-dodecylguanidine was ineffective in enhancing AmB lethality or AmB-induced vacuole disruption when cells had been pretreated with ergosterol. Ergosterol-dependent mechanism is thus considered to be a possible target of N-methyl-N″-dodecylguanidine in enhancing the vacuole-targeting fungicidal activity of AmB in C. albicans cells.

Evaluation of uridine 5'-eicosylphosphate as a stimulant of cyclic AMP-dependent cellular function.

Sporulation of the yeast Saccharomyces cerevisiae is negatively regulated by cyclic AMP (cAMP). This microbial cell differentiation process was applied for the screening of a substance that can elevate the intracellular cAMP level. Among nucleoside 5'-alkylphosphates, uridine 5'-eicosylphosphate (UMPC20) selectively and predominantly inhibited ascospore formation of the yeast cells. We suppose the inhibitory effect of UMPC20 could indeed reflect the elevation of the cellular cAMP level.

Targeted yeast vacuole disruption by polyene antibiotics with a macrocyclic lactone ring.

In this study, the polyene macrolide antibiotics amphotericin B (AmB), nystatin and filipin III were evaluated for their fungicidal activity and their ability to produce vacuole disruption as well as enhancement of these activities by allicin using Saccharomyces cerevisiae. Nystatin has a macrocyclic lactone ring analogous to AmB and their fungicidal activities were both increased by allicin, an allyl-sulfur compound, whereas filipin III, a pentaene macrolide, did not show an increase in fungicidal activity in the presence of allicin. Vacuole staining with the fluorescent probe FM4-64 showed that both AmB and nystatin induced vacuole membrane disintegration at their lethal concentrations; in addition, the vacuole disruptive effect was also enhanced by allicin. In contrast, filipin III did not affect vacuole morphology and addition of allicin had no effect despite filipin III localising to the cell cytoplasm. Isolated S. cerevisiae vacuoles were disrupted following treatment both with nystatin and AmB, though this activity was not potentiated in the presence of allicin. In contrast, filipin III had little effect on the vacuole architecture. This study reveals differential effects of polyene antibiotics on vacuoles in S. cerevisiae, which may be due to differences in the structure of the macrocyclic ring.

Selective inhibition of embryonic development in starfish by long-chain alkyl derivatives of UMP, TMP and AMP.

Nucleoside analogs have been evaluated as useful tools for the investigation of the mechanism of cell differentiation. We thus examined the effects of nucleoside 5'-alkylphosphates (1-10) on the morphogenetic development of starfish embryos. These nucleotide derivatives were all permissive for their development up to the blastula stage, but the derivatives with lauryl side chain selectively inhibited one of the following stages into bipinnaria larvae. Among them, uridine 5'-laurylphosphate (2) inhibited gastrulation of the blastula, as is the case with the antibiotic tunicamycin, suggesting its inhibitory activity on sulfated and non-sulfated glycoprotein syntheses. Unexpectedly, adenosine 5'-laurylphosphate (8) was evaluated as a novel class of inhibitor that can arrest the embryos exactly at the late gastrula stage, absolutely inhibiting cell differentiation involved in the development of gastrointestinal tract. This is the first report on the appearance of biological activity due to the structural modification of a naturally-occurring molecule, which is critical to the morphogenetic development of multicellular organisms.

Antimycin A-induced cell death depends on AIF translocation through NO production and PARP activation and is not involved in ROS generation, cytochrome c release and caspase-3 activation in HL-60 cells.

A respiratory inhibitor, antimycin A (AA), induced an apoptotic-like cell death characterized by nuclear and DNA fragmentation in human leukemia HL-60 cells. This cell death was significantly restricted by a nitric oxide synthase (NOS) inhibitor, N(G)-monomethyl-L-arginine (L-NMMA), and a poly(ADP-ribose) polymerase (PARP) inhibitor, 5-aminoisoquinoline (AIQ). Indeed, NO production and PARP overactivation were detected in the cells treated with AA. On the one hand, L-NMMA partly eliminated NO production and on the other, AIQ and L-NMMA also restricted PARP activation. Excessive signals related to PARP overactivation induce the translocation of an apoptosis-inducing factor (AIF) from the mitochondria to the nuclei, resulting in DNA fragmentation. In AA-treated cells, the nuclear translocation of AIF occurred. This translocation was restricted by pretreatment with AIQ and L-NMMA. Although pretreatment with ascorbic acid eliminated the reactive oxygen species (ROS) generation induced by the blockade of complex III by AA, the pretreatment did not protect the cells from AA-induced cell death. Furthermore, cytochrome c release or caspase-3 activation was not observed in the cells treated with AA. These results suggest that AA-induced cell death does not depend on respiratory inhibition and the succeeding cascades, but on NO production, PARP overactivation and AIF translocation.

Isolation of an Acremonium sp. capable of liquefying cross-linked poly(gamma-glutamic acid) hydrogels and the fungal enzyme involved in the disruption of gamma-ray irradiation-mediated cross-linking.

A microorganism capable of liquefying cross-linked poly(gamma-glutamic acid) (C-L PGA) was isolated and identified to be an Acremonium sp. The fungus produced an enzyme that can disrupt the cross-linkages of C-L PGA generated by gamma-ray irradiation. The enzyme can also liquefy C-L PGA prepared by chemical cross-linkage, suggesting the involvement of ester bonds in the gamma-ray irradiation-mediated cross-linking of PGA.

Synergistic fungicidal activities of amphotericin B and N-methyl-N"-dodecylguanidine: a constituent of polyol macrolide antibiotic niphimycin.

The synergy between the alkylguanidinium chain of niphimycin (NM), a polyol macrolide antibiotic, and polyene macrolide amphotericin B (AmB) without such an alkyl side chain was examined using N-methyl-N"-alkylguanidines as its synthetic analogs. Among the analogs, N-methyl-N"-dodecylguanidine (MC12) most strongly inhibited the growth of Saccharomyces cerevisiae cells and those of other fungal strains in synergy with AmB. MC12 itself was not lethal but the analog could be a cause of a rapid cell death progression of yeast cells in the presence of AmB at a nonlethal concentration. Their combined actions resulted in the generation of NM-like fungicidal activity that depended on plasma membrane disability and cellular reactive oxygen species production. We also found an aberrant vacuolar morphogenesis and an associated vacuolar membrane disability in cells treated simultaneously with MC12 and AmB, as in the case of NM-treated cells. These findings support the idea that the alkylguanidinium chain plays a major role in the fungicidal activity of NM in cooperation with the polyol lactone ring as its enhancer.

Synthesis of fluorescent molecular probes specific for the receptor of blepharismone, a mating-inducing pheromone of the ciliate Blepharisma japonicum.

Blepharismone (gamone 2) is a mating-inducing pheromone of the ciliate Blepharisma japonicum. N-Pyrenylbutyryl-blepharismone and N-biphenylacetyl-blepharismone, which are fluorescent derivatives of blepharismone, were synthesized as molecular probes for the gamone 2 receptor. Further, we proved that they have inhibitory activities against the blepharismone-induced monotypic pairing of B. japonicum.

Identification of phoslactomycin E as a metabolite inducing hyphal morphological abnormalities in Aspergillus fumigatus IFO 5840.

In our survey for antifungal compounds, a fermentation broth of Streptomyces sp. HA81-2 was found to inhibit the in vitro growth of Aspergillus fumigatus IFO 5840 accompanied by hyphal morphological abnormalities. One of the isolated antibiotics was identified as phoslactomycin E based on LC-MS and NMR spectral data. In a preliminary assay using the membrane fractions of A. fumigatus, phoslactomycin E was found to inhibit the activity of 1,3-beta glucan synthase.