Malbrancheamide B, a novel compound from the fungus Malbranchea aurantiaca

A new indole alkaloid, namely malbrancheamide B (2), was isolated from the culture medium and mycelia of the ascomycete Malbranchea aurantiaca along with malbrancheamide (1). Structural elucidation of 2 was carried out by a combination of mass spectrometry (MS) and (1)H and (13)C NMR spectroscopy analyses, as well as by comparison of the NMR data with those of 1. According to the conformational studies using molecular mechanics analyses, compound 2 exists in one preferred conformation, which was optimised by density functional theory (DFT) calculations. Compound 2 is the second chlorinated indole alkaloid possessing a bicyclo [2.2.2] ring with an unusual relative configuration at C12a in the bicyclo [2.2.2] diazaoctane ring system. So far, these structural features seem to be unique for the alkaloids biosynthesised by the fungus M. aurantiaca.     

Biomimetic total synthesis of malbrancheamide and malbrancheamide B

The biomimetic total syntheses of both malbrancheamide and malbrancheamide B are reported. The synthesis of the two monochloro species enabled the structure of malbrancheamide B to be unambiguously assigned. The syntheses each feature an intramolecular Diels-Alder reaction of a 5-hydroxypyrazin-2(1H)-one to construct the bicyclo[2.2.2]diazaoctane core, which has also been proposed as the biosynthetic route to these compounds.     

DKxanthene biosynthesis--understanding the basis for diversity-oriented synthesis in myxobacterial secondary metabolism

The DKxanthenes are a family of yellow pigments which play a critical role in myxobacterial development. Thirteen unique structures from Myxococcus xanthus DK1622 differ in the length of their characteristic polyene functionality, as well as the extent of methyl branching. We aimed to understand the mechanistic basis for this "molecular promiscuity" by analyzing the gene cluster in DK1622, and comparing it to the DKxanthene biosynthetic locus in a second myxobacterium, Stigmatella aurantiaca DW4/3-1, which produces a more limited range of compounds. While the core biosynthetic machinery is highly conserved, M. xanthus contains a putative asparagine hydroxylase function which is not present in S. aurantiaca. This observation accounts, in part, for the significantly larger metabolite family in M. xanthus. Detailed analysis of the encoded hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) assembly line provides direct evidence for the mechanism underlying the variable polyene length and the observed pattern of methyl functionalities.     

A cationic cyclisation route to prenylated indole alkaloids: synthesis of malbrancheamide B and brevianamide B, and progress towards stephacidin A

The synthesis of the prenylated indole alkaloids, malbrancheamide B and brevianamide B have been accomplished, starting with a prenylated proline derivative created using the Seebach ‘self-reproduction of chirality’ method, and using a cationic cascade sequence as the key step to form late-stage bridged diketopiperazine intermediates.     

Heterologous expression of a myxobacterial natural products assembly line in pseudomonads via red/ET recombineering

Natural products of microbial origin are widely used as pharmaceuticals and in agrochemistry. These compounds are often biosynthesized by multifunctional megasynthetases whose genetic engineering and heterologous expression offer considerable promise, especially if the natural hosts are genetically difficult to handle, slow growing, unculturable, or even unknown. We describe a straightforward strategy that combines the power of advanced DNA engineering (recombiogenic cloning) in Escherichia coli with the utility of pseudomonads as the heterologous host for the analysis and mutagenesis of known and unknown secondary metabolite pathways. The myxochromide S biosynthetic gene cluster from Stigmatella aurantiaca was rebuilt and engineered in E. coli to contain the elements required for expression in pseudomonads. The successful production in Pseudomonas putida, at unprecedented levels, demonstrates the feasibility of the new approach to the analysis and mutagenesis of these important pathways.     

Synthesis,molecular structure,and vibrational spectra of tetrakis(2-hydroxyethylammonium) chloride and its triethanolamine precursor and metabolite

A unique one-stage synthesis of tetrakis(2-hydroxyethylammonium) salts, precursors of new hypervalent silicon compounds, has been developed. The vibration spectra of tetrakis(2-hydroxyethylammonium) chloride and its triethanolamine precursor and metabolite have been studied by IR spectroscopy and quantum chemistry. Analysis of alterations in the modes with increasing number of HOCH₂CH₂ groups has been performed.     

Flavonoids from Daphne aurantiaca and their inhibitory activities against nitric oxide production

Chemical examination of the methanolic extract from the stem bark of Daphne aurantiaca led to the isolation of three new flavonoids (1-3), and 29 known flavonoids. All 32 compounds were isolated for the first time from Daphne aurantiaca. The isolates were tested for inhibitory activities against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages. Compounds 21 and 24 showed potent inhibitory activities against the production of NO with IC?? values of 0.006 and 0.076 μM, respectively.     

Biosynthesis of iso-fatty acids in myxobacteria

The fatty acid (FA) profiles of the myxobacteria Stigmatella aurantiaca and Myxococcus xanthus were investigated by acidic methanolysis of total cell extracts and GC or GC-MS analysis. The main components were 13-methyltetradecanoic acid (iso-15:0) and (Z)-hexadec-11-enoic acid (16:1, omega-5 cis). The biosynthesis of iso-FAs was investigated in several feeding experiments. Feeding of isovaleric acid (IVA) to a mutant impaired in the degradation of leucine to isovaleryl-CoA (IV-CoA)(bkd mutant) of M. xanthus only increased the amount of iso-odd FAs, whereas feeding of isobutyric acid (IBA) gave increased amounts only of iso-even FAs. In contrast, a bkd mutant of S. aurantiaca gave increased amounts of iso-odd and iso-even fatty acids in both experiments. We assumed that in S. aurantiacaalpha-oxidation takes place. [D(7)]-15-Methylhexadecanoic acid was synthesised and fed to S. aurantiaca as well as [D(10)]leucine and [D(8)]valine to elucidate this pathway in more detail. The iso-fatty acid was degraded by alpha- and beta-oxidation steps. [D(10)]Leucine was strongly incorporated into iso-odd and iso-even fatty acids, whereas the incorporation rates for [D(8)]valine into both types of fatty acids were low. Thus alpha-oxidation plays an important role in the biosynthesis of iso-fatty acids in S. aurantiaca. The incorporation rates observed after feeding of [D(10)]leucine and [D(8)]valine are the highest for iso-17:0 compared to the other acids. This indicates the central role of iso-17:0 in the biosynthesis of iso-FAs. The shorter homologues seem to be formed mainly by alpha-oxidation and beta-oxidation of this acid. After feeding of traces of unsaturated counterparts of this labelled FA occurred in the extracts indicating that desaturases are active in the biosynthesis of unsaturated fatty acids in S. aurantiaca.     

An Optimized Synthesis of 2-Methoxyestradiol,A Naturally Occurring Human Metabolite with Anticancer Activity

2-Methoxyestradiol, a naturally occurring human metabolite with demonstrated anticancer activity, has been synthesized in three steps and 76% yield from the bis(MOM) ether of 2-formylestradiol.     

Novel features in a combined polyketide synthase/non-ribosomal peptide synthetase: the myxalamid biosynthetic gene cluster of the myxobacterium Stigmatella aurantiaca Sga15

BACKGROUND: Myxobacteria have been well established as a potent source for natural products with biological activity. They produce a considerable variety of compounds which represent typical polyketide structures with incorporated amino acids (e.g. the epothilons, the myxothiazols and the myxalamids). Several of these secondary metabolites are effective inhibitors of the electron transport via the respiratory chain and have been widely used. Molecular cloning and characterization of the genes governing the biosynthesis of these structures is of considerable interest, because such information adds to the limited knowledge as to how polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) interact and how they might be manipulated in order to form novel antibiotics. RESULTS: A DNA region of approximately 50000 base pairs from Stigmatella aurantiaca Sga15 was sequenced and shown by gene disruption to be involved in myxalamid biosynthesis. Sequence analysis reveals that the myxalamids are formed by a combined PKS/NRPS system. The terminal NRPS MxaA extends the assembled polyketide chain of the myxalamids with alanine. MxaA contains an N-terminal domain with homology to NAD binding proteins, which is responsible during the biogenesis for a novel type of reductive chain release giving rise to the 2-amino-propanol moiety of the myxalamids. The last module of the PKS reveals an unprecedented genetic organization; it is encoded on two genes (mxaB1 and mxaB2), subdividing the domains of one module from each other. A sequence comparison of myxobacterial acyl-transferase domains with known systems from streptomycetes and bacilli reveals that consensus sequences proposed to be specific for methylmalonyl-CoA and malonyl-CoA are not always reliable. CONCLUSIONS: The complete biosynthetic gene cluster of the myxalamid-type electron transport inhibitor from S. aurantiaca Sga15 has been cloned and analyzed. It represents one of the few examples of combined PKS/NRPS systems, the analysis and manipulation of which has the potential to generate novel hybrid structures via combinatorial biosynthesis (e.g. via module-swapping techniques). Additionally, a new type of reductive release from PKS/NRPS systems is described.     

Chaetoglobosin L,a New Metabolite of Diplodia macrospora

From cultures of Diplodia macrospora a new metabolite of the cytochalasan family, Chaetoglobosin L (3) , has been isolated and its structure elucidated together with the known compounds diplosporin (1) and Chaetoglobosin K (2) .     

Utilizing DART mass spectrometry to pinpoint halogenated metabolites from a marine invertebrate-derived fungus

Prenylated indole alkaloids are a diverse group of fungal secondary metabolites and represent an important biosynthetic class. In this study we have identified new halogenated prenyl-indole alkaloids from an invertebrate-derived Malbranchea graminicola strain. Using direct analysis in real time (DART) mass spectrometry, these compounds were initially detected from hyphae of the fungus grown on agar plates, without the need for any organic extraction. Subsequently, the metabolites were isolated from liquid culture in artificial seawater. The structures of two novel chlorinated metabolites, named (-)-spiromalbramide and (+)-isomalbrancheamide B, provide additional insights into the assembly of the malbrancheamide compound family. Remarkably, two new brominated analogues, (+)-malbrancheamide C and (+)-isomalbrancheamide C, were produced by enriching the growth medium with bromine salts.     

(S)-citramalic acid,a useful chiral synthon for the synthesis of 15-deoxy-16(S)-hydroxy-16-methylprostaglandins

(S)-Citramalic acid, a readily available microbial metabolite has been efficiently transformed into (2S)-2-methyl-2-hydroxy-1-hexanol ($\text{7}$), an important chiral synthon for the synthesis of 15-deoxy-16(S)-hydroxy-16-methylprostaglandins.     

Biosynthesis of iso-fatty acids in myxobacteria: iso-even fatty acids are derived by alpha-oxidation from iso-odd fatty acids

The biosynthesis of iso-fatty acids was investigated in the myxobacteria Myxococcus xanthus and Stigmatella aurantiaca by feeding labeled precursors of these fatty acids and by conducting detailed analysis of the fatty acid profiles and fatty acid MS and NMR spectra. These experiments revealed that in S. aurantiaca all iso-even fatty acids are formed via alpha-oxidation of iso-odd fatty acids and not by using valine-derived isobutyryl-CoA.     

Gilmaniellin and Dechlorogilmaniellin,Two Novel Dimeric Oxaphenalenones

The structure and configuration of gilmaniellin, a metabolite of Gilmaniella humicola BARRON , has been shown to be 2 by X-ray analysis. By comparison of spectral data structure 1 has been assigned to dechlorogilmaniellin.     

High-throughput, accurate mass liquid chromatography/tandem mass spectrometry on a quadrupole time-of-flight system as a 'first-line' approach for metabolite identification studies

Throughput for drug metabolite identification studies has been increased significantly by the combined use of accurate mass liquid chromatography/tandem mass spectrometry (LC/MS/MS) data on a quadrupole time-of-flight (QTOF) system and targeted data analysis procedures. Employed in concert, these tools have led to the implementation of a semi-automated high-throughput metabolite identification strategy that has been incorporated successfully into lead optimization efforts in drug discovery. The availability of elemental composition data on precursor and all fragment ions in each spectrum has greatly enhanced confidence in ion structure assignments, while computer-based algorithms for defining sites of biotransformation based upon mass shifts of diagnostic fragment ions have facilitated identification of positions of metabolic transformation in drug candidates. Adoption of this technology as the 'first-line' approach for in vitro metabolite profiling has resulted in the analysis of as many as 21 new chemical entities on one day from diverse structural classes and therapeutic programs.     

Gadusol,a metabolite from fish eggs

Gadusol, a metabolite from the eggs of cod and other fishes, has been identified as 1,4,5-trihydroxy-2-methoxy-5-hydroxymethylcyclohex-1-en-3-one, related to the mycosporines and analogous compounds.     

Die Struktur von Cyclosporin C

The Structure of Cyclosporin C The structure of cyclosporin C ( 2 ), a minor antifungal metabolite from Trichoderma polysporum (Link ex Pers.) RIFAI has been elucidated. Hydrolytic cleavage and spectroscopic evidence show that cyclosporin C is a neutral oligopeptide of 11 amino acids linked together in a 33-membered ring. Cyclosporin C ( 2 ) differs from the main metabolite cyclosporin A ( 1 ) [2] [4] only by containing L-threonine in the place of L-α-aminobutyric acid as has been shown by the conversion of 2 into 1 . ¹³C-NMR. spectra and study of molecular models suggest that cyclosporin C ( 2 ) has the same molecular conformation as 1 , which is best described as a twisted β-pleated sheet held together in a conformationally stable form by intramolecular hydrogen bonding.     

A novel type of geosmin biosynthesis in myxobacteria

The biosynthesis of geosmin (1) and (1(10)E,5E)-germacradien-11-ol (2), two volatile terpenoid compounds emitted by the myxobacteria Myxococcus xanthus and Stigmatella aurantiaca, was investigated in feeding experiments with different labeled precursors. In these experiments, the volatiles released by the cell cultures grown on agar plates were collected with a closed-loop stripping apparatus (CLSA) and analyzed by GC-MS. [(2)H(10)]Leucine and [4,4,4,5,5,5-(2)H(6)]dimethylacrylate were fed to wild-type strains and bkd mutant strains, which are impaired in the degradation of leucine to isovaleryl-CoA. [(2)H(10)]Leucine was incorporated into 1 and 2 only by the wild-type strains via the biosynthetic pathway that involves leucine degradation and branching into the mevalonate pathway. Dimethylacrylyl-CoA (DMA-CoA) is an intermediate in the leucine degradation and in the recently discovered pathway from HMG-CoA to isovaleryl-CoA. The corresponding free acid, [4,4,4,5,5,5-(2)H(6)]dimethylacrylic acid, was incorporated into 1 and 2 only by the mutants impaired in leucine degradation. [4,4,6,6,6-(2)H(5)]Mevalonic acid lactone (12) was synthesized and fed to M. xanthus and S. aurantiaca wild-type strains and a double mutant strain of M. xanthus. This strain does not degrade leucine and is impaired in the reduction of 3-hydroxy-3-methylglutaryl-CoA to mevalonic acid. The mass spectral analysis of labeled 1 and 2 obtained in these feeding experiments led to a biosynthetic scheme to 1 with intermediate 2. This pathway differs from that observed in the liverwort Fossombronia pusilla and thus suggests microbial geosmin biosynthesis following a route different from that in liverworts. Our results are supported by a 1,2-hydride shift of the tertiary hydrogen atom at C-4a into the ring opposite to that in F. pusilla.     

Pseurotin, a new metabolite of Pseudeurotium ovalis Stolk having an unusual hetero-spirocyclic system.

The structure and absolute configuration of pseurotin ( 1 ), a new metabolite, isolated from culture filtrates of Pseudeurotium ovalis STOLK (Ascomycetes), has been shown to be 2-[1′(S), 2′ (S)-dihydroxhex-3′-ene-yl]-3-methyl-8(S)-methoxy-8-benzoyl-9(R)-hydroxy-(5S)-1-oxa-7-aza-spiro[4.4]non-2-ene-4, 6-dione ( 1 ), by spectral data and chemical transformations, and by X-ray analysis of its dibromo derivative 2 [1].