The Isolation of Pyrroloformamide Congeners and Characterization of Their Biosynthetic Gene Cluster.

Dithiolopyrrolones are microbial natural products containing a disulfide or thiosulfonate bridge embedded in a unique bicyclic structure. By interfering with zinc ion homeostasis in living cells, they show strong antibacterial activity against a variety of bacterial pathogens, as well as potent cytotoxicity against human cancer cells. In the current study, two new dithiolopyrrolones, pyrroloformamide C (3) and pyrroloformamide D (4), were isolated from Streptomyces sp. CB02980, together with the known pyrroloformamides 1 and 2. The biosynthetic gene cluster for pyrroloformamides was identified from Streptomyces sp. CB02980, which shared high sequence similarity with those of dithiolopyrrolones, including holomycin and thiolutin. Gene replacement of pyfE, which encodes a nonribosomal peptide synthetase (NRPS), abolished the production of 1-4. Overexpression of pyfN, a type II thioesterase gene, increased the production of 1 and 2. Genome neighborhood network analysis of the characterized and orphan gene clusters of dithiolopyrrolones revealed a unified mechanism for their biosynthesis, involving an iterative-acting NRPS and a set of conserved tailoring enzymes for the bicyclic core formation.

A coumarin analogue NFA from endophytic Aspergillus fumigatus improves drought resistance in rice as an antioxidant.

BACKGROUND: Drought and its resulting oxidative damage are the major yield limiting factors for crops in arid and semi-arid regions. Recent studies have found that endophytic fungi coexisting in plants can alleviate biotic or abiotic damage to plant growth and development. In order to screen for the endophytes associated with drought stress, 12 strains of endophytic fungi with high antioxidant activity isolated from riparian plants Myricaria laxiflora were evaluated for their effects in rice by the crude extracts. RESULTS: Of the 12 endophytic fungi, Aspergillus fumigatus SG-17 functioned most effectively, with the crude extract exhibiting relatively higher antioxidant capacity both in vivo and in vitro. The subsequent MS and NMR analysis showed that the primary substance responsible for the antioxidant activity in the extract was (Z)-N-(4-hydroxystyryl) formamide (NFA), an analogue of coumarin. Enzyme activity assay in nerve cells SH-SY5Y showed that NFA could maintain the membrane integrity and regulate the antioxidase activity under oxidative stress. In rice suffering drought stress, NFA effectively alleviated the harm by regulating the contents of NADPH oxidases, antioxidants and heat shock proteins, all of which are closely related with the reactive oxygen species pathway. CONCLUSION: These findings indicated that some endophytes from plants often subjected to flooding and oxidative stress could enhance drought resistance by producing compounds such as NFA to regulate the oxidative pathway.

Poly(glyceryl monomethacrylate-co-ethylene glycol dimethacrylate) monolithic columns with incorporated bare and surface modified gluconamide fumed silica nanoparticles for hydrophilic interaction capillary electrochromatography.

This research article presents the preparation and characterization of monolithic capillary columns with incorporated bare fumed silica nanoparticles (FSNPs) and surface coated gluconamide FSNPs and their subsequent use in hydrophilic interaction capillary electrochromatography (HI-CEC) of small relatively polar solutes. The monolithic support was based on the in situ polymerization of glyceryl monomethacrylate (GMM) and ethylene glycol dimethacrylate (EDMA) yielding the poly(GMM-co-EDMA) monolith for the incorporation of bare and gluconamide-FNSPs. The poly(GMM-co-EDMA) monolith functioned as a true "support" for both types of polar FSNPs "stationary phases". In other words, monolithic capillary columns with "FSNPs stationary phases" were obtained in the sense that the contribution of the monolith proper to solute' retention was at its minimum. The gluconamide-FSNPs were obtained by reacting the FSNPs with the polar organosilane N-(3-triethoxysilylpropyl)gluconamide either by a pre- or on-column approach yielding p-gluconamide-FSNPs or o-gluconamide-FSNPs, respectively. While the p-gluconamide-FSNPs was coated by an oligosiloxane gluconamide layer as revealed by thermogravimetric analysis, the o-gluconamide-FSNPs are thought to be covered with a monomeric layer of gluconamide ligands as was manifested by the higher plate number obtained on the latter than on the former gluconamide-FSNPs incorporated monolithic columns. In the on-column modification process of FSNPs, the reaction was performed in a closed system whereby atmospheric water vapor are not available to cause the polymerization of the trifunctional organosilane N-(3-triethoxysilylpropyl)gluconamide. Also, the fact that the o-gluconamide-FSNPs incorporated monoliths were made from bare-FSNPs incorporated monoliths may indicate that the bare FSNPs were better dispersed into the monolithic matrix than the p-gluconamide-FSNPs, a condition that might have further contributed to the lower plate count obtained on p-gluconamide- than o-gluconamide-FSNPs incorporated monolithic columns. Overall, o-gluconamide-FSNPs stationary phases and to a lesser extent bare-FSNPs stationary phases proved useful in HI-CEC of small polar solutes, including DMF, formamide, thiourea, some phenols and nucleobases.

Synthesis and physicochemical characterization of the impurities of pemetrexed disodium, an anticancer drug.

A physicochemical characterization of the process-related impurities associated with the synthesis of pemetrexed disodium was performed. The possibility of pemetrexed impurities forming has been mentioned in literature, but no study on their structure has been published yet. This paper describes the development of the synthesis methods for these compounds and discusses their structure elucidation on the basis of two-dimensional NMR experiments and MS data. The identification of these impurities should be useful for the quality control during the production of the pemetrexed disodium salt.

Chemical analysis of bleach and hydroxide-based solutions after decontamination of the chemical warfare agent O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate (VX).

Detailed chemical analysis of solutions used to decontaminate chemical warfare agents can be used to support verification and forensic attribution. Decontamination solutions are amongst the most difficult matrices for chemical analysis because of their corrosive and potentially emulsion-based nature. Consequently, there are relatively few publications that report their detailed chemical analysis. This paper describes the application of modern analytical techniques to the analysis of decontamination solutions following decontamination of the chemical warfare agent O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate (VX). We confirm the formation of N,N-diisopropylformamide and N,N-diisopropylamine following decontamination of VX with hypochlorite-based solution, whereas they were not detected in extracts of hydroxide-based decontamination solutions by nuclear magnetic resonance (NMR) spectroscopy or gas chromatography-mass spectrometry. We report the electron ionisation and chemical ionisation mass spectroscopic details, retention indices, and NMR spectra of N,N-diisopropylformamide and N,N-diisopropylamine, as well as analytical methods suitable for their analysis and identification in solvent extracts and decontamination residues.

Cytotoxic psammaplysin analogues from a Suberea sp. marine sponge and the role of the spirooxepinisoxazoline in their activity.

Seventeen bromotyrosine-derived metabolites, including eight new compounds, were isolated from a Micronesian sponge of the genus Suberea. Four of the new compounds were psammaplysin derivatives (10-13), and the other four were ceratinamine derivatives (14-17). Of the compounds obtained, the psammaplysins exhibited cytotoxicity against human cancer cell lines (GI50 values down to 0.8 µM), while the ceratinamine and moloka'iamine analogues showed almost no activity. These results suggest that the spirooxepinisoxazoline ring system is a requirement for cytotoxicity and, therefore, may serve as an attractive molecular scaffold for the development of a potent anticancer agent.

Cytotoxic tetraprenylated alkaloids from the South China Sea gorgonian Euplexaura robusta.

Nine achiral tetraprenylated alkaloids, including three new compounds, named malonganenones I-K (1-3, resp.), together with six known analogs, 4-9, were isolated from the gorgonian Euplexaura robusta collected from Weizhou Island of Guangxi Province, China. The structures of compounds 1-3 were elucidated by extensive spectral analyses, especially of their 1D- and 2D-NMR data. Compounds 1, 4, 6, and 7 showed moderate cytotoxicities against K562 and HeLa tumor cell lines with IC(50) values ranging from 0.35 to 10.82 µM. Compound 6 also showed moderate inhibitory activity against c-Met kinase at a concentration of 10 µM.

Generation of continuous packed bed reactor with PVA-alginate blend immobilized Ochrobactrum sp. DGVK1 cells for effective removal of N,N-dimethylformamide from industrial effluents.

Effective removal of dimethylformamide (DMF), the organic solvent found in industrial effluents of textile and pharma industries, was demonstrated by using free and immobilized cells of Ochrobactrum sp. DGVK1, a soil isolate capable of utilizing DMF as a sole source of carbon, nitrogen. The free cells have efficiently removed DMF from culture media and effluents, only when DMF concentration was less than 1% (v/v). Entrapment of cells either in alginate or in polyvinyl alcohol (PVA) failed to increase tolerance limits. However, the cells of Ochrobactrum sp. DGVK1 entrapped in PVA-alginate mixed matrix tolerated higher concentration of DMF (2.5%, v/v) and effectively removed DMF from industrial effluents. As determined through batch fermentation, these immobilized cells have retained viability and degradability for more than 20 cycles. A continuous packed bed reactor, generated by using PVA-alginate beads, efficiently removed DMF from industrial effluents, even in the presence of certain organic solvents frequently found in effluents along with DMF.

Induced production of N-formyl alkaloids from Aspergillus fumigatus by co-culture with Streptomyces peucetius.

Co-culture of the fungus Aspergillus fumigatus with the bacteria Streptomyces peucetius led to the induction of production of formyl xanthocillin analogues. This mixed fermentation yielded two new metabolites, fumiformamide (1) and N,N'-((1Z,3Z)-1,4-bis(4-methoxyphenyl)buta-1,3-diene-2,3-diyl)diformamide (2), together with two known N-formyl derivatives and the xanthocillin analogue BU-4704. The structures were determined by spectroscopic methods and by comparison with literature. Cytotoxic activity of all the analogues was tested on the NCI-60 cell line screen, and compound 2 exhibited significant activity against several cell lines. The analogues did not show antimicrobial activity.

Diterpene formamides from the tropical marine sponge Cymbastela hooperi and their antimalarial activity in vitro.

Further investigations of the VLC (vacuum-liquid chromatography) fractions obtained from the dichloromethane solubles of the tropical marine sponge Cymbastela hooperi led to the isolation and characterization of five new diterpene formamides, 1-5. Compound 1 is one of the very few examples of a natural product that contains both formamide and isonitrile functionalities within the same molecule. In in vitro antiplasmodial bioassays, 1 was found to have moderate activity (IC(50) 0.5 microg/mL), 2 had weak activity (IC(50) 14.8 microg/mL), and 3-5 were inactive. The pattern of activity found for the metabolites investigated in the current study is consistent with previous findings for these classes of molecules.

Melanocins A, B and C, new melanin synthesis inhibitors produced by Eupenicillium shearii. I. Taxonomy, fermentation, isolation and biological properties.

New melanin synthesis inhibitors, melanocins A, B and C, were isolated from the fermentation broth and mycelium extract of Eupenicillium shearii F80695. Melanocin A, an isocyanide compound, inhibited mushroom tyrosinase and melanin biosynthesis of B16 melanoma cells with IC50 value of 9.0 nM and MIC value of 0.9 microM, respectively. Melanocin A also inhibited growth of Streptomyces bikiniensis. While, the structurally very related but non-isocyanide compounds melanocins B and C did not show inhibitory activity in these assays. Melanocins A, B and C showed potent antioxidant activity with scavenging activity of DPPH radical and superoxide anion radical.

Melanocins A, B and C, new melanin synthesis inhibitors produced by Eupenicillium shearii. II. Physico-chemical properties and structure elucidation.

New melanin synthesis inhibitors, melanocins A, B and C, were isolated from the fermentation broth and extract of mycelium of Eupenicillium shearii F80695. The structures of melanocins were established by spectroscopic methods. They are formamide compounds. In particular, melanocin A has an isocyanide group.

Isolation and characterization of carbinolamide and phenolic glucuronide conjugates of (+-)-N-methyl-N-(1-methyl-3,3-diphenylpropyl) formamide and N-formylmethamphetamine by FAB/MS, LC/MS/MS, and NMR.

The metabolic disposition of (+-)-N-methyl-N-(1-methyl-3,3- diphenyl-propyl)formamide, especially with regard to the formation of water soluble glucuronides, is described. The glucuronide conjugates, (+-)-N-hydroxymethyl-N-(1-methyl-3,3-diphenylpropyl)formamide glucuronide, (+-)-N-methyl-N-[1-methyl-3-(4'-hydroxyphenyl)-3-phenylpropyl]formamide glucuronide, and (+-)-N-methyl-N-[1-methyl-3-(4'-hydroxy-3'-methoxyphenyl)-3- phenylpropyl]formamide glucuronide were isolated from the bile of rats dosed with the parent compound. These conjugates were characterized spectroscopically by 1H-NMR, FAB/MS, and LC/MS/MS. Because it is becoming more common to isolate the intact glucuronide conjugates of xenobiotics, we investigated some common mass spectral fragmentation patterns of these conjugates, especially by LC/MS/MS. The fragmentation patterns for each of the conjugates were obtained under MS/MS conditions and compared. Specifically, the fragmentation patterns of phenolic glucuronide and an aliphatic O-glucuronide, in particular a carbinolamide glucuronide, were investigated. The data obtained from these studies was used to predict the nature of glucuronide conjugates obtained from rats dosed with the formamide analog, N-formylmethamphetamine. This is the first spectroscopic characterization of an intact carbinolamide glucuronide conjugate isolated from the bile of rats.

Purification and spectroscopic characteristics in N-methylformamide of the Azotobacter vinelandii Fe-Mo cofactor.

The iron-molybdenum cofactor from Azotobacter vinelandii can be removed from significant amounts of extraneous iron and other contaminants using anaerobic gel filtration. Electronic absorption spectra of the so-purified FeMoco along with analysis of the so-called 'easily complexed' iron are suggestive that FeMoco occupies at least two different states in N-methylformamide solution. Batch-related differences in spectral characteristics of independently isolated FeMoco samples are demonstrated. Non-cofactor iron, found in unpurified FeMoco, may affect the interpretation of ligand binding and other experiments probing FeMoco structure and reactivity. Oxidized FeMoco is shown to be clearly discernable from the semi-reduced species by means of electronic spectroscopy, and this method now forms a convenient analytical tool for study of the chemistry and electronic structure of FeMoco.

Deionization of formamide with Biorad AG501-X(D).

We have studied different conditions of the deionization of formamide with Biorad mixed bed resin AG501-X(D) and find that contrary to the popular usage, drying the resin before deionization produces the best results. In a typical deionization procedure, conductivity initially goes down, reaches a minimum plateau and finally goes up again. The initial rate of deionization, the minimum conductivity reached at the plateau and the rate of final rise in conductivity depend on whether the resin is dry or wet (i.e., straight from the bottle) and on the amount of resin used. In general, wet resin produced faster initial deionization, higher minimum conductivity and quicker final rise in conductivity. Surprisingly, a smaller amount (5%) of resin worked better than a larger amount (20%). With smaller amount of resin, although initial deionization was slower, the minimum conductivity achieved was lower and the final rise in conductivity was slower. This was partly due to the fact that the conductivity of formamide increases faster with increasing amount of water in it.

Studies on WF-5239, a new potent platelet aggregation inhibitor.

WF-5239 was produced by a fungal strain identified as Aspergillus fumigatus Fresenius. The substance was purified by solvent extraction followed by chromatography on silica gel and then crystallized (C9H9NO2, mp 142 approximately 145 degrees C). The chemical structure was determined from its physico-chemical properties as N-[2-cis(4-hydroxyphenyl)ethenyl]formamide. WF-5239 has inhibitory activity against rabbit platelet aggregation induced by arachidonic acid and collagen, with IC50 values of 1.25 and 5.0 micrograms/ml, respectively. Arachidonic acid induced mortality in mice was reduced by a single intraperitoneal dose of WF-5239 (30 mg/kg). These biological properties have been compared with those of aspirin.