Karsten Krohn

Endophytic fungi: a source of novel biologically active secondary metabolites*

In the continual search by both pharmaceutical and agricultural industries for new products, natural selection has been found to be superior to combinatorial chemistry for discovering novel substances that have the potential to be developed into new industrial products. Since natural products are adapted to a specific function in nature, the search for novel secondary metabolites should concentrate on organisms that inhabit novel biotopes. Endophytic fungi inhabit such a biotope. In the course of the last 12 years, we have isolated ~ 6500 endophytic fungi from herbaceous plants and trees, screened them for biological activities, and have isolated and determined the structures of the biologically active compounds. Correlations were found between biological activity and biotope, e.g. a higher proportion of the fungal endophytes, in contrast to the soil isolates, inhibited at least one of the test organisms for antialgal and herbicidal activities. The substances isolated originated from different biosynthetic pathways: isoprenoid, polyketide, amino acid derivatives, and belonged to diverse structural groups: terpenoids, steroids, xanthones, chinones, phenols, isocumarines, benzopyranones, tetralones, cytochalasines, and enniatines. The potential role of the endophyte and its biologically active metabolites in its association with its host has been investigated. The fungal endophytes possess the exoenzymes necessary to colonize their hosts and they grow well in the apoplastic washing fluid of the host. When the roots of larch are colonized, the association with the host may be mutualistic, improving growth of the host and supplying the mycobiont with enough nourishment to extensively colonize the hosts roots. The concentrations of some plant defence metabolites are lower than in the control when the host is infected with a pathogen than with an endophyte. We hypothesize that the interaction fungal endophyte-plant host is characterized by a finely tuned equilibrium between fungal virulence and plant defence. If this balance is disturbed by either a decrease in plant defence or an increase in fungal virulence, disease develops. Not only must the endophyte synthesize metabolites to compete first with epiphytes and then with pathogens in order to colonize the host, but presumably also to regulate metabolism of the host in their delicately balanced association. The utilization of a biotope such as that of the fungal endophyte is one aspect of intelligent screening, another very important one is the taxonomy of the fungus in order to avoid redundant structural isolations. It is not a random walk through a random forest. Many groups of fungi in different biotopes are waiting to be exploited.

Biologically active secondary metabolites of endophytic Pezicula species

Endophytic Pezicula strains were isolated from living branches of ten deciduous and coniferous trees and tested for their fungicidal, algicidal and antibacterial activities. From the three biologically active strains that were selected for fermentation, five substances were isolated and their structures determined as ( R -mellein, (−)-mycorrhizin A, 2-methoxy-4-hydroxy-6-methoxymethyl-benzaldehyde, (+)-cryptosporiopsin and 4-epi-ethiosolide. They are strongly fungicidal and herbicidal, to a lesser extent algicidal and antibacterial. The synthesis of these metabolites in 85 isolates was dependent on conditions of culture and was also found to be taxonomically significant. The potential role of these substances is discussed with respect to mutualism and parasitism.

Dihydroisocoumarins from fungi : Isolation, structure elucidation, circular dichroism and biological activity

Five known and three new dihydroisocoumarins were isolated from different fungi. The new isocoumarins are 5-chloro-6-hydroxymellein, 5-chloro-4,6-dihydroxymellein and 5,6-dihydroxymellein. The absolute configuration of these secondary metabolites was confirmed by CD measurements and in two cases by X-ray structure analysis.

New Mono‐ and Dimeric Members of the Secalonic Acid Family: Blennolides A–G Isolated from the Fungus Blennoria sp.

Blennolides A-G (2-8), seven unusual chromanones, were isolated together with secalonic acid B (1) from Blennoria sp., an endophytic fungus from Carpobrotus edulis. This is the first reported isolation of the blennolides 2 and 3 (hemisecalonic acids B and E), the existence of which as the monomeric units of the dimeric secalonic acids had long been postulated. A compound of the proposed structure 4 (beta-diversonolic ester) will need to be revised, as its reported data do not fit those of the established structure of blennolide C (4). Other monomers, the blennolides D-F (5-7) seem to be derived from blennolides A (2) and B (3) by rearrangement of the hydroaromatic ring. The heterodimer 8, composed of the monomeric blennolide A (2) and the rearranged 11-dehydroxy derivative of blennolide E (6), extends the ergochrome family with an ergoxanthin type of skeleton. The structures of the new compounds were elucidated by detailed spectroscopic analysis and further confirmed by an X-ray diffraction study of a single crystal of 2. The absolute configurations were determined by TDDFT calculations of CD spectra, including the solid-state CD/TDDFT approach. Preliminary studies showed strong antifungal and antibacterial activities of these compounds against Microbotryum violaceum and Bacillus megaterium, respectively. They were also active against the alga Chlorella fusca and the bacterium Escherichia coli.

Absolute structural elucidation of natural products—A focus on quantum‐mechanical calculations of solid‐state CD spectra

In this review article we examine state-of-the-art techniques for the structural elucidation of organic compounds isolated from natural sources. In particular, we focus on the determination of absolute configuration (AC), perhaps the most challenging but inevitable step in the whole process, especially when newly isolated compounds are screened for biological activity. Among the many methods employed for AC assignment that we review, special attention is paid to electronic circular dichroism (CD) and to the modern tools available for quantum-mechanics CD predictions, including TDDFT. In this context, we stress that conformational flexibility often poses a limit to practical CD calculations of solution CD spectra. Many crystalline natural products suitable for X-ray analysis do not contain heavy atoms for a confidential AC assignment by resonant scattering. However, their CD spectra can be recorded in the solid state, for example with the KCl pellet technique, and analyzed possibly by nonempirical means to provide stereochemical information. In particular, solid-state CD spectra can be compared with those calculated with TDDFT or other high-level methods, using the X-ray geometry as input. The solid-state CD/TDDFT approach, described in detail, represents a quick and reliable tool for AC assignment of natural products.

Structure, bioactivities, biosynthetic relationships and chemical synthesis of the spirodioxynaphthalenes

Covering: 1989 to September 2010 The spirodioxynaphthalenes are a group of fungal secondary metabolites, consisting of a 1,8-dihydroxynaphthalene-derived spiroketal unit linked to a second oxidized naphthalene moiety, that show a great variety of biological activities. This review summarizes the research on the isolation, structure elucidation, biological activities, biosynthesis, and chemical synthesis of the spirodioxynaphthalenes published over the last 20 years. More than 100 fungal and plant metabolites are described, and over 100 references cited.

Structural and Stereochemical Studies of alpha-Methylene-gamma-lactone-Bearing Cembrane Diterpenoids from a South China Sea Soft Coral Lobophytum crassum

Four new alpha-methylene-gamma-lactone-bearing cembranoids, 20-acetylsinularolide B (6), presinularolide B (7), 3-dehydroxylpresinularolide B (8), and 3-dehydroxyl-20-acetylpresinularolide B (9), together with five known analogues, sinularolides B-E (1- 4) and 20-acetylsinularolide C (5), were isolated from a South China Sea soft coral Lobophytum crassum. Their structures and relative stereochemistry were established by a combination of detailed spectroscopic data analysis and chemical correlations. The structures of 1- 9 were further confirmed by an X-ray diffraction study on a single crystal of sinularolide B (1). The absolute configurations of sinularolide B (1) and presinularolide B (7) were determined by a novel solid-state CD/TDDFT approach and by a modified Moshers method, respectively. This study also revealed that the coupling constant between the lactonic methine protons ((3) J 1,2) varies considerably with different functional groups on the cembrane ring and that the determination of the stereochemistry of lactone ring fusion based on this coupling constant is risky. In a bioassay, sinularolides B and C (1 and 2) and new cembranoids 7 and 8 showed in vitro cytotoxicity against the tumor cell lines A-549 and P-388.

Bioactive nonanolide derivatives isolated from the endophytic fungus Cytospora sp.

Cytospolides F-Q (6-17) and decytospolides A and B (18 and 19), 14 unusual nonanolide derivatives, were isolated from Cytospora sp., an endophytic fungus from Ilex canariensis. The structures were elucidated by means of detailed spectroscopic analysis, chemical interconversion, and X-ray single crystal diffraction. The solution- and solid-state conformers were compared by the combination of experimental methods (X-ray, NMR) supported by DFT calculations of the conformers. Absolute configurations were assigned using the modified Moshers method and solution- and solid-state TDDFT ECD calculations. In an in vitro cytotoxicity assay toward the tumor cell lines of A549, HCT116, QGY, A375, and U973, the γ-lactone 17 demonstrated a potent growth inhibitory activity toward the cell line A-549, while nonanolide 16 with (2S) configuration showed the strongest activity against cell lines A-549, QGY, and U973. A cell cycle analysis indicated that compound 16 can significantly mediate G1 arrest in A549 tumor cells, confirming the important role of the C-2 methyl in the growth inhibition toward the tumor line. The discovery of an array of new nonanolides demonstrates the productivity of the fungus, and it is an example of chemical diversity, extending the nonanolide family by derivatives formed by ring cleavage, oxidation, esterification, and Michael addition.

Denbinobin inhibits nuclear factor-κB and induces apoptosis via reactive oxygen species generation in human leukemic cells

Denbinobin, a 1,4-phenanthrenequinone firstly isolated from the stems of Dendrobium moniliforme (Shi-Hu in Chinese medicine), has been reported to exhibit anti-tumoral and anti-inflammatory activities through mechanism(s) not yet fully understood. Because of the critical role of the transcription factor NF-kappaB and of ROS-induced activation of stress regulated kinases in tumorigenesis, we have investigated the effect of denbinobin on these pathways. We found that denbinobin is a potent inhibitor of TNFalpha and PMA-induced NF-kappaB activation, and that it can block the phosphorylation and degradation of IkappaBalpha by inhibiting TAK1 activity, an event lying upstream of IKK activation. Moreover, treatment with denbinobin not only elicited apoptotic signalling, including mitochondrial membrane dysfunction, activation of caspases and cleavage of poly(ADP-ribose) polymerase, but also induced intracellular reactive oxygen species (ROS) generation and sustained activation of the mitogen-activated kinases (MAPKs) ERK1+2, p38 and JNK 1+2. The apoptotic effects of denbinobin could be prevented by pre-treatment with the intracellular ROS scavenger N-acetyl-l-cysteine, but not by pharmacological inhibition of MAPKs, suggesting that intracellular ROS generation underlies denbinobin-induced apoptosis, and that this effect takes place in an MAPKs-independent pathway. To define the structural elements critical for these activities, a series of phenanthrenequinones with different substituents in the phenanthrene- and/or in the quinone ring were prepared and assayed for NF-kappaB inhibition and ROS production. In this way, the major structure-activity relationships and the structural elements critical for the activity of denbinobin could be established.

Xanthones and oxepino[2, 3-b]chromones from three endophytic fungi.

Three new metabolites, microsphaeropsones A-C (1-3) with a unique oxepino[2,3-b]chromen-6-one (ring-enlarged xanthone) skeleton, were isolated from the endophytic fungus Microsphaeropsis species, co-occurring with their putative biogenetic anthraquinoide precursors citreorosein (4) and emodin (5). From another Microsphaeropsis species, large amounts of fusidienol A (8 a), smaller amounts of emodin (5), the known aromatic xanthones 9 a and 9 b, the new 3,4-dihydrofusidienol A (8 b), and the new aromatic xanthone 9 c were isolated. The endophyte Seimatosporium species produced a new aromatic xanthone, seimatoxanthone A (10), and 3,4-dihydroglobosuxanthone A (12), closely related to alpha-diversolonic ester (13) from Microdiplodia sp.. The structures were determined mainly by extensive 1D and 2D NMR experiments and supported by X-ray single-crystal analysis of 1 and the oxidation product 7. The absolute configurations of the microsphaeropsones A-C (1-3) were established by comparison of the electronic and vibrational circular dichroism (ECD and VCD) spectra of 1 with time-dependent DFT (TDDFT) and DFT calculations by using either the solid-state structures or DFT-optimized geometries as inputs. Preliminary studies indicated that 1, 2, and enone 7 showed antibacterial, fungicidal, and algicidal properties.