Parallel Evolution of Asco- and Basidiomycete O-Prenyltransferases.

Prenyltransferases (PTs) are involved in the biosynthesis of a multitude of pharmaceutically and agriculturally important plant, bacterial, and fungal compounds. Although numerous prenylated compounds have been isolated from Basidiomycota (mushroom-forming fungi), knowledge of the PTs catalyzing the transfer reactions in this group of fungi is scarce. Here, we report the biochemical characterization of an O- and C-prenylating dimethylallyltryptophan synthase (DMATS)-like enzyme LpTyrPT from the scurfy deceiver Laccaria proxima. This PT transfers dimethylallyl moieties to l-tyrosine at the para-O position and to l-tryptophan at atom C-7 and represents the first basidiomycete l-tyrosine PT described so far. Phylogenetic analysis of PTs in fungi revealed that basidiomycete l-tyrosine PTs have evolved independently from their ascomycete counterparts and might represent the evolutionary origin of PTs acting on phenolic compounds in secondary metabolism.

19 F VT NMR: Novel Tm3+ and Ce3+ Complexes Provide New Insight into Temperature Measurement Using Molecular Sensors.

The front cover artwork is provided by Dr. Markus Plaumann's group at the Otto-von-Guericke University Magdeburg. The image shows the investigated 19 F labeled molecules, which can be used as temperature MR-sensors in different solvents. The direction of the chemical shift change is strongly solvent dependent. Read the full text of the Research Article at 10.1002/cphc.202300057.

Two fluorinated thulium complexes as molecular temperature sensors in MR applications.

19F-based magnetic resonance is a powerful tool to overcome several difficulties of standard 1H MR. We present the syntheses and characterization (including cell viability and stability tests) of two Tm3+ complexes. Both complexes allow the detection of temperature (ΔCT = -0.2319 ppm K-1 and -0.2122 ppm K-1) without a reference compound.

19 F VT NMR: Novel Tm3+ and Ce3+ Complexes Provide New Insight into Temperature Measurement Using Molecular Sensors.

In the past few decades, magnetic resonance spectroscopy (MRS) and MR imaging (MRI) have developed into a powerful non-invasive tool for medical diagnostic and therapy. Especially 19 F MR shows promising potential because of the properties of the fluorine atom and the negligible background signals in the MR spectra. The detection of temperature in a living organism is quite difficult, and usually external thermometers or fibers are used. Temperature determination via MRS needs temperature-sensitive contrast agents. This article reports first results of solvent and structural influences on the temperature sensitivity of 19 F NMR signals of chosen molecules. By using this chemical shift sensitivity, a local temperature can be determined with a high precision. Based on this preliminary study, we synthesized five metal complexes and compared the results of all variable temperature measurements. It is shown that the highest 19 F MR signal temperature dependence is detectable for a fluorine nucleus in a Tm3+ -complex.

Withanolides from Discopodium penninervium.

Discopodium penninervium is a medicinal plant endemic to Ethiopia. Its twigs extract led to the isolation of three new withanolides, discopodinolides A - C, and four known analogues. The structures of the compounds were elucidated using NMR, HRMS data analyses, and literature data. The relative configurations were defined by single-crystal X-ray crystallography and NOESY correlations. The antibacterial efficacy of the isolated compounds was evaluated against four commonly dispersed environmental strains of Escherichia coli, Bacillus subtilis, Bacillus pumilus, and Bacillus megaterium. Discopodinolides B and C exhibited moderate antibacterial activities against the pathogenic strains of E. coli, B. subtilis, and B. megaterium.

Alliacane-Type Secondary Metabolites from Submerged Cultures of the Basidiomycete Clitocybe nebularis.

Seven sesquiterpenoids, named nebucanes A-G (1-7), featuring a rare alliacane scaffold with unprecedented furan or pyrrole functions, were isolated from the fermentation broth of Clitocybe nebularis. Their structures were established on the basis of 1D/2D NMR spectroscopic analyses, HR-(+)-ESIMS spectra, and comparison of measured and calculated CD spectra for determination of the absolute configuration. Assessing the biological activities, nebucane D (4) exhibited antifungal effects against Rhodotorula glutinis, while nebucane G (7) displayed significant cytotoxicity against MCF-7 and A431 cell lines.

Formaldehyde as a Chemical Defence Agent of Fruiting Bodies of Mycena rosea and its Role in the Generation of the Alkaloid Mycenarubin†C.

Mycenarubin C, a previously unknown red pyrroloquinoline alkaloid, was isolated from fruiting bodies of the mushroom Mycena rosea and its structure was elucidated mainly by NMR spectroscopy and mass spectrometry. Unlike mycenarubin A, the major pyrroloquinoline alkaloid in fruiting bodies of M. rosea, mycenarubin C, contains an eight-membered ring with an additional C1 unit that is hitherto unprecedented for pyrroloquinoline alkaloids known in nature. Incubation of mycenarubin A with an excess of formaldehyde revealed that mycenarubin C was generated nearly quantitatively from mycenarubin A. An investigation into the formaldehyde content of fresh fruiting bodies of M. rosea showed the presence of considerable amounts of formaldehyde, with values of 5 µg per gram of fresh weight in fresh fruiting bodies. Although mycenarubin C did not show bioactivity against selected bacteria and fungi, formaldehyde inhibits the growth of the mycoparasite Spinellus fusiger at concentrations present in fruiting bodies of M. rosea. Therefore, formaldehyde might play an ecological role in the chemical defence of M. rosea against S. fusiger. In turn, S. fusiger produces gallic acid-presumably to detoxify formaldehyde by reaction of this aldehyde with amino acids and gallic acid to Mannich adducts.

Aminotenuazonic Acid: Isolation, Structure Elucidation, Total Synthesis and Herbicidal Activity of a New Tetramic Acid from Fruiting Bodies of Laccaria Species.

(5S,6S)-Aminotenuazonic acid, a new 3-acyltetramic acid, related to the well-known mycotoxin tenuazonic acid has been isolated from fruiting bodies of Laccaria bicolor. Its structure was mostly established by analysis of its 2D NMR and HR-(+)-ESI-MS spectra. A total synthesis starting from N-Boc-l-isoleucine gave (5S,6S)-aminotenuazonic acid in 8 % yield over nine steps (67 % de). The key steps of the total synthesis are a light-initiated Hofmann-Löffler-Freytag radical chain reaction and a Dieckmann cyclisation. The relative and absolute configurations of the natural product were determined by comparison of its NMR and CD spectra with those of the corresponding enantiopure synthetic compounds. Metabolic profiling of crude extracts of different mushrooms showed that aminotenuazonic acid is present in all four of the investigated Laccaria species. Aminotenuazonic acid shows phytotoxic activities against the root and shoot growth of Lepidium sativum, Pinus sylvestris and Arabidopsis thaliana comparable to those of tenuazonic acid.

E- and Z-Proxamidines, Unprecedented 1,3-Diazacyclooct-1-ene Alkaloids from Fruiting Bodies of Laccaria proxima.

Fruiting bodies of Laccaria proxima were screened for the presence of new secondary metabolites by means of HPLC-UV and LC-HR-(+)-ESIMS. Thus, two isomeric alkaloids with a highly unusual core structure, E-proxamidine and its Z-isomer, were isolated from Laccaria proxima. The proxamidines consist of an eight-membered heterocyclic ring system with a formamidine unit. The structures were established by 2D NMR spectroscopic methods, HR-(+)-ESIMS, and HR-(+)-ESIMS/MS. The proxamidines are probably biosynthetically derived from tryptophan, dimethylallyl pyrophosphate, and S-adenosylmethionine and the eight-membered ring of the proxamidines is likely to be generated by a rearrangement of the tryptophan sceleton. Metabolic profiling of fruiting bodies of some other Laccaria species revealed that the proxamidines appear in significant amounts only in L. proxima making the compounds suitable as chemotaxonomic markers. E-Proxamidine exhibits herbicidal activity against Lepidium sativum.

Nematicidal anthranilic acid derivatives from Laccaria species.

Three undescribed natural products, the anthranilic acid derivatives laccanthrilic acids A, B, and C, as well as the known (3S)-1,2,3,4-tetrahydro-3-ß-carboline-3-carboxylic acid were isolated from fruiting bodies of Laccaria laccata. The structures were established by 1D and 2D NMR spectroscopy, HR-(+)-ESIMS and chemical synthesis. The absolute configuration of laccanthrilic acids A and B was determined by GC-MS after hydrolytic cleavage and derivatisation of the resulting glutamic acid with methanol and Mosher's reagent and subsequent comparison with authentic synthetic samples of known absolute configuration. The absolute configuration of laccanthrilic acid C was determined by comparison of the CD spectra of laccanthrilic acids B and C with each other. Metabolic profiling of related species showed that the compounds are common in the genus Laccaria. Laccanthrilic acid B exhibited moderate nematicidal effects against Caenorhabditis elegans, which might explain to some degree the beneficial role of these fungi for the growth and survival of their host plants.

Mycenaflavin†A, B, C, and D: Pyrroloquinoline Alkaloids from the Fruiting Bodies of the Mushroom Mycena haematopus.

Four so far unknown pyrroloquinoline alkaloids, yellow mycenaflavins A, B, and C, and the purple mycenaflavin D, have been isolated from the fruiting bodies of Mycena haematopus. The structures of these new alkaloids were elucidated by NMR spectroscopy and HRMS (ESI+ ). The mycenaflavins are structurally related to mycenarubins and haematopodins, which have been previously identified in M. haematopus. However, compared with other known fungal pyrroloquinoline alkaloids, the mycenaflavins contain an additional double bond within the pyrroloquinoline moiety that accounts for the yellow colour of the monomeric mycenaflavins A, B, and C. The purple mycenaflavin D is the first known dimeric pyrroloquinoline alkaloid with a C-C bridge between the two pyrroloquinoline units. Although the minor pyrroloquinoline alkaloid constituent mycenaflavin A exhibits only moderate bioactivity against the soil bacterium Azoarcus tolulyticus, the major pyrroloquinoline alkaloid constituent haematopodin B is similarly active as the antibiotic gentamicin.

Anti-Staphylococcal Calopins from Fruiting Bodies of Caloboletus radicans.

Three new and seven known calopins were isolated from Caloboletus radicans. The structures of the new cyclocalopins, 8-deacetylcyclocalopin B (1), cyclocalopin A-15-ol (2), and 12,15-dimethoxycyclocalopin A (3), were mainly elucidated by NMR and MS data analysis. The stereochemistry of 1-3 was assigned based on NOE correlations and coupling constants and by comparison of their CD spectra with those of similar known calopins. While 1-10 were inactive against two cancer cell lines, they displayed anti-staphylococcal activity against methicillin-resistant Staphylococcus aureus strains (MRSA) with MIC values of 16-256 µg/mL. Moreover, some calopins were active against the fish pathogen Enterococcus faecalis F1B1.

Pelianthinarubins A and B, Red Pyrroloquinoline Alkaloids from the Fruiting Bodies of the Mushroom Mycena pelianthina.

Pelianthinarubin A (1) and pelianthinarubin B (2), two previously unknown pyrroloquinoline alkaloids, have been isolated from fruiting bodies of Mycena pelianthina. The structures of these alkaloids have been deduced from their HR-(+)-ESIMS and 2D NMR data. The absolute configurations of the pelianthinarubins A (1) and B (2) were assigned by analysis of the NOE correlations and coupling constants and by comparison of the CD spectra of 1 and 2 and of hercynine obtained by degradation of 1 with suitable compounds of known absolute configuration. The pelianthinarubins A (1) and B (2), which contain an S-hercynine moiety, differ considerably from the known pyrroloquinoline alkaloids from marine organisms and other Mycena species, such as the mycenarubins, the haematopodins, and the sanguinones.

Chemical ecology of fungi.

Fungi are widespread in nature and have conquered nearly every ecological niche. Fungi occur not only in terrestrial but also in freshwater and marine environments. Moreover, fungi are known as a rich source of secondary metabolites. Despite these facts, the ecological role of many of these metabolites is still unknown and the chemical ecology of fungi has not been investigated systematically so far. This review intends to present examples of the various chemical interactions of fungi with other fungi, plants, bacteria and animals and to give an overview of the current knowledge of fungal chemical ecology.

Evaluation of the anti-inflammatory and antioxidative potential of four fern species from China intended for use as food supplements.

Inflammation plays a major role in many diseases, for instance in arteriosclerosis, rheumatoid arthritis, autoimmune disorders and cancer. Since many plants contain compounds with anti-inflammatory activity, their consumption may be able to prevent the development of inflammatory-based diseases. Edible ferns are some of the most important wild vegetables in China and have traditionally been used both for dietary and therapeutic purposes. In this study we investigated the anti-inflammatory and antioxidant potential of fern extracts from Matteuccia struthiopteris, Osmundajaponica, Matteuccia orientalis and Pteridium aquilinum intended for use as nutraceuticals. Two modes of action were investigated: the inhibition of the pro-inflammatory gene expression of interleukin-1 beta (IL1-ß) and interleukin-6 (IL6), and the gene expression of iNOS by LPS-elicited macrophages. The results showed a decrease of IL1-ß gene expression for the five fern extracts. This effect was more pronounced for the extracts prepared from the roots of O. japonica (IC50 of 17.8 µg/mL) and the young fronds of M orientalis (50.0 µg/mL). Regarding the indirect measurement of NO, via iNOS gene expression, an interesting decrease of 50% was obtained with the extract of M. orientalis fronds at a low concentration (20 µg/mL) compared with P. aquilinum fronds (160 µg/mL) and leaves of O. japonica. The latter showed a higher decrease but at a high concentration of extract (160 µg/mL). The five fern extracts were also evaluated for their ability to scavenge 2,2-diphenyl-l-picrylhydrazyl (DPPH) radicals and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). All fern extracts exhibited antioxidant effects but the roots of O. japonica and the fronds of M orientalis were most efficient. The HPLC-MS analysis of the constituents of the fern extracts confirmed the presence of chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, kaempferol and apigenin, molecules known to exhibit antiinflammatory and/or antioxidant properties.

A free cyanohydrin as arms and armour of Marasmius oreades.

Cyanogenic plants and fungi are widespread in nature. Although the origin of hydrocyanic acid in plants has been studied in detail, little is known about its origin in fungi. Here, we report the identification of the cyanohydrin of glyoxylic acid as the precursor of hydrocyanic acid in the fungus Marasmius oreades and several other cyanogenic fungi. Moreover, a feeding experiment revealed glycine as biosynthetic precursor of the cyanohydrin of glyoxylic acid. Thus, the cyanogenesis of M. oreades and other fungi is fundamentally different from cyanogenesis in plants.

Loss of atomic nitrogen from even-electron ions? A study on benzodiazepines.

The fragment spectra of protonated nitro-substituted benzodiazepines show an unusual fragment [M + H - 14]+ , which is shown by accurate mass measurement to be due to the loss of a nitrogen atom. Our investigations show that this apparent loss of atomic nitrogen is rather an attachment of molecular oxygen to the [M + H - NO2 ]+• ion, which is the main fragment ion in these spectra. The oxygen attachment is exothermic, and rate constants have been derived. MSn spectra show that it is not easily reversible upon fragmentation of the adduct ion and that it is also observed with some secondary and tertiary fragments, which allows to limit the attachment site to the aromatic ring annulated to the diazepine moiety. Fragments of the oxygen adduct ion indicate that the O2 molecule dissociates in the adduct formation process, and the two oxygen atoms are bound to different sites of the ion. Comparison with radical cations generated by fragmentation of non-nitro-substituted benzodiazepines, none of which showed an oxygen attachment, and the fragmentation mechanisms involved in their formation indicates that the [M + H - NO2 ]+• ion is a distonic ion with the charge and radical site neighbored on the aromatic ring. From these results, we derive a proposal for the formation and structure of the [M + H - NO2 + O2 ]+• ion, which explains the experimental observations. Copyright © 2015 John Wiley & Sons, Ltd.

HR-MALDI-MS imaging assisted screening of ß-carboline alkaloids discovered from Mycena metata.

Fruiting bodies of Mycena metata were screened for the presence of new secondary metabolites by means of HPLC-UV, LC-HR-ESIMS, and high-resolution matrix-assisted laser desorption/ionization mass spectrometry imaging (HR-MALDI-MS imaging). Thus, a new ß-carboline alkaloid, 6-hydroxymetatacarboline D (1d), was isolated from fruiting bodies of M. metata. 6-Hydroxymetatacarboline D consists of a highly substituted ß-carboline skeleton, which is likely to be derived biosynthetically from l-tryptophan, 2-oxoglutaric acid, l-threonine, and l-proline. The structure of the alkaloid was established by 2D NMR spectroscopic methods and HR-ESIMS. Moreover, by extensive application of LC-HR-ESIMS, LC-HR-ESIMS/MS, and LC-HR-ESIMS(3) techniques we were able to elucidate the structures of a number of accompanying ß-carboline alkaloids, 1a-1c, 1e-1i, and 2a-2g, structurally closely related to 6-hydroxymetatacarboline D, which are present in M. metata in minor amounts. The absolute configuration of the stereogenic centers of the ß-carboline alkaloids was determined by GC-MS comparison with authentic synthetic samples after hydrolytic cleavage and derivatization of the resulting amino acids.

N-methylation of the amide bond by methyltransferase asm10 in ansamitocin biosynthesis.

Ansamitocins are potent antitumor agents produced by Actinosynnema pretiosum. As deduced from their structures, an N-methylation on the amide bond is required among the various modifications. The protein encoded by asm10 belongs to the SAM-dependent methyltransferase family. Through gene inactivation and complementation, asm10 was proved to be responsible for the N-methylation of ansamitocins. Asm10 is a 33.0 kDa monomer, as determined by gel filtration. By using N-desmethyl-ansamitocin P-3 as substrate, the optimal temperature and pH for Asm10 catalysis were determined to be 32 °C and 10.0, respectively. Asm10 also showed broad substrate flexibility toward other N-desmethyl-ansamycins and synthetic indolin-2-ones. Through site-directed mutagenesis, Asp154 and Leu155 of Asm10 were confirmed to be essential for its catalysis, possibly through the binding of SAM. The characterization of this unique N-methyltransferase has enriched the toolbox for engineering N-methylated derivatives from both natural and synthetic compounds; this will allow known potential drugs to be modified.