Frank Surup

Disciformycins A and B: 12-Membered Macrolide Glycoside Antibiotics from the Myxobacterium Pyxidicoccus fallax Active against Multiresistant Staphylococci**

Two macrolide glycosides with a unique scaffold were isolated from cultures of the myxobacterium Pyxidicoccus fallax. Their structures, including absolute configurations, were elucidated by a combination of NMR, MS, degradation, and molecular modeling techniques. Analysis of the proposed biosynthetic gene cluster led to insights into the biosynthesis of the polyketide and confirmed the structure assignment. The more active compound, disciformycin B, potently inhibits methicillin- and vancomycin-resistant Staphylococcus aureus.

Lenormandins A—G, new azaphilones from Hypoxylon lenormandii and Hypoxylon jaklitschii sp. nov., recognised by chemotaxonomic data

Seven new azaphilone pigments named lenormandins A–G were isolated from stromata (fruiting bodies) of the xylariaceous fungus, Hypoxylon lenormandii using preparative High Performance Liquid Chromatography (HPLC) and their structures were elucidated by NMR spectroscopy, high resolution mass spectrometry and other spectral methods. Their occurrence in herbarium specimens (including various type materials collected in the 19th and early 20th century) and several fresh collections was studied by analytical HPLC with diode array and mass spectrometric detection (HPLC-DAD/MS), revealing that this group of pigments is specific for Hypoxylon lenormandii from various geographic regions and otherwise only occurs in closely related taxa, i.e. H. sublenormandii and an undescribed fungus from Sri Lanka, for which we propose the name H. jaklitschii. The status of the new species was established by using molecular phylogenetic data based on ITS and partial beta-tubulin sequences, and by detailed morphological studies.

Rickenyls A–E, antioxidative terphenyls from the fungus Hypoxylon rickii (Xylariaceae, Ascomycota)

Our screening efforts for new natural products with interesting bioactivity have revealed the neotropical ascomycete Hypoxylon rickii as a prolific source. We isolated five secondary metabolites with a p-terphenyl backbone from the mycelial extract of a fermentation of this fungus in 70 l scale by using RP-HPLC, which were named rickenyls A-E (1-5). Their structures were elucidated by X-ray crystallography and NMR spectroscopy, complemented by HRESIMS. Two of the compounds contained a quinone core structure in ortho (2) and para-position (5), respectively. We obtained 2 spontaneously and by lead tetraacetate oxidation from 1. All compounds were screened for antimicrobial, antioxidative and cytotoxic activities. Rickenyl A (1) exhibited strong antioxidative effects and moderate cytotoxic activity against various cancer cell lines.

Deconins A-E: Cuparenic and Mevalonic or Propionic Acid Conjugates from the Basidiomycete Deconica sp. 471.

Bioassay-guided fractionation of antibacterial extracts from cultures of a basidiomycete from Northern Thailand, which represents a new species of the genus Deconica, yielded the terpenoid deconin A (1), whose structure was elucidated by spectral methods (NMR, HRMS) as a cuparenic/mevalonic acid conjugate. The absolute configuration of 1 was determined after saponification and comparison of specific rotations of the resulting cuparenic acid and mevalonolactone with authentic standards and literature data. Six minor congeners (2-7) were isolated and identified, and their antimicrobial and cytotoxic effects are reported. Compounds 1-4 are the first natural products featuring an unmodified mevalonic acid residue as a building block.

Gymnopalynes A and B, chloropropynyl-isocoumarin antibiotics from cultures of the basidiomycete Gymnopus sp.

A chlorinated isocoumarin with an acetylenyl side chain and its 3,4-dihydro derivative, named gymnopalynes A (1) and B (2), were isolated from cultures of a basidiomycete originating from the rain forest of Northern Thailand. The producing organism was identified as a species of Gymnopus (Marasmiaceae). Their structures were elucidated by spectroscopic methods including UV/vis and NMR spectroscopy as well as high-resolution mass spectrometry as 3-(3-chloroprop-1-yn-1-yl)-1H-isochromen-1-one (1) and 3-(3-chloroprop-1-yn-1-yl)-3,4-dihydro-1H-isochromen-1-one (2). The absolute stereochemistry of 2 was assigned as S by CD spectroscopy. Both compounds showed weak to moderate antimicrobial and pronounced cytotoxic activities.

Hypoxyvermelhotins A–C, new pigments from Hypoxylon lechatii sp. nov

A new species of Hypoxylon was discovered, based on material collected in French Guiana and recognised on the basis of new combination of morpholological characters in comparison with type and authentic material of macroscopically similar taxa. These findings were corroborated by the rather isolated positions of its ITS-nrDNA and beta-tubulin DNA sequences in molecular phylogenies. However, the most salient feature of this fungus only became evident by a comparison of its stromatal HPLC profile, revealing several secondary metabolites that were hitherto not observed in stromata of any other member of the Xylariaceae. Part of the stromata were subsequently extracted to isolate these apparently specific components, using preparative chromatography. Five metabolites were obtained in pure state, and their chemical structures were elucidated by means of high resolution mass spectrometry and nuclear magnetic resonance spectroscopy. They turned out to be tetramic acid derivatives of the so-called vermelhotin type. Aside from vermelhotin, previously isolated from cultures of endophytic fungi, we identified three novel congeners, for which the trivial names hypoxyvermelhotins A-C were proposed. Like vermelhotin, they constitute orange-red pigments and a preliminary biological characterisation revealed them to have rather strong cytotoxic and moderate to weak antimicrobial effects. These results further illustrate the high diversity of unique secondary metabolites in stromata of the hypoxyloid Xylariaceae, a family in which biological diversity seems to parallel the chemical diversity of their bioactive principles to a great extent.

Sporothriolide derivatives as chemotaxonomic markers for Hypoxylon monticulosum

During the course of a screening for novel anti-infective agents from cultures of tropical Xylariaceae originating from French Guiana and Thailand, pronounced antifungal activity was noted in extracts of cultures of Hypoxylon monticulosum. A bioassay-guided fractionation led to the known metabolite sporothriolide as active principle. In addition, three new derivatives of sporothriolide were isolated, for which we propose the trivial names sporothric acid, isosporothric acid and dihydroisosporothric acid. Their chemical structures were elucidated by high-resolution electrospray mass spectrometry in conjunction with two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy. From earlier studies on the biogenesis of the chemically similar canadensolides, we postulate that the new compounds were shunt products, rather than biogenetic precursors of sporothriolide. Interestingly, this compound class, as well as strong antifungal activities, was only observed in multiple cultures of H. monticulosum, but not in several hundreds of Hypoxylon cultures studied previously or concurrently. Therefore, sporothriolide production may constitute a species-specific feature with respect to Hypoxylon and the Xylariaceae, although the compound was previously reported from non-related fungal taxa.

Biosynthesis of Crocacin Involves an Unusual Hydrolytic Release Domain Showing Similarity to Condensation Domains

The crocacins are potent antifungal and cytotoxic natural compounds from myxobacteria of the genus Chondromyces. Although total synthesis approaches have been reported, the molecular and biochemical basis guiding the formation of the linear crocacin scaffold has remained unknown. Along with the identification and functional analysis of the crocacin biosynthetic gene cluster from Chondromyces crocatus Cm c5, we here present the identification and biochemical characterization of an unusual chain termination domain homologous to condensation domains responsible for hydrolytic release of the product from the assembly line. In particular, gene inactivation studies and in vitro experiments using the heterologously produced domain CroK-C2 confirm this surprising role giving rise to the linear carboxylic acid. Additionally, we determined the kinetic parameters of CroK-C2 by monitoring hydrolytic cleavage of the substrate mimic N-acetylcysteaminyl-crocacin B using an innovative high-performance liquid chromatography mass spectrometry-based assay.

Botryane, noreudesmane and abietane terpenoids from the ascomycete Hypoxylon rickii.

In the course of our screening for new bioactive natural products, a culture of Hypoxylon rickii, a xylariaceous ascomycete collected from the Caribbean island Martinique, was identified as extraordinary prolific producer of secondary metabolites. Ten metabolites of terpenoid origin were isolated from submerged cultures of this species by preparative HPLC. Their structures were elucidated using spectral techniques including 2D NMR and HRESIMS. Three of the compounds were elucidated as new botryanes (1-3) along with three known ones, i.e. (3aS)-3a,5,5,8-tetramethyl-3,3a,4,5-tetrahydro-1H-cyclopenta[de]isochromen-1-one (4), (3aS,8R)-3a,5,5,8-tetramethyl-3,3a,4,5,7,8-hexahydro-1H-cyclopenta[de]isochromen-1-one (5) and botryenanol (6). Further three new sesquiterpenoids featured a 14-noreudesmane-type skeleton and were named hypoxylan A-C (7-9); the diterpenoid rickitin A (10) contains an abietane-type backbone. Compounds 1, 2, 3, 7, and 10 showed cytotoxic effects against murine cells.

Concerted Action of P450 Plus Helper Protein To Form the Amino-hydroxy-piperidone Moiety of the Potent Protease Inhibitor Crocapeptin

The crocapeptins are described here as cyclic depsipeptides, isolated from cultures of the myxobacterium Chondromyces crocatus . Structure elucidation of the compounds revealed a cyanopeptolin-like skeleton, containing the characteristic amino-hydroxy-piperidone (Ahp)-heterocycle. Like the cyanopeptolins, the myxobacterial crocapeptins proved to be serine protease inhibitors. The nonribosomal origin of the peptide was confirmed by mutagenesis experiments, and the biosynthesis gene cluster was sequenced. It could be shown that the Ahp-heterocycle originates from a proline residue in the precursor molecule precrocapeptin, which is converted to crocapeptin by the tailoring enzymes CpnE and CpnF. Conversion of precrocapeptin isolated from a cpnF mutant into crocapeptin was achieved using recombinant CpnF, a cytochrome P450 enzyme responsible for hydroxylation of the proline residue in precrocapeptin. Addition of protein CpnE resulted in strongly increased conversion rates toward Ahp containing product. A mutant with 10-fold increased production of crocapeptin A was created through insertion of the Pnpt-promotor in front of the NRPS gene.