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Six new 16-residue peptaibols, acremopeptaibols A-F (1-6), along with five known compounds, were isolated from the cultures of the sponge-associated fungus Acremonium sp. IMB18-086 grown in the presence of the autoclaved bacterium Pseudomonas aeruginosa on solid rice medium. The peptaibol sequences were established based on comprehensive analysis of 1D and 2D NMR spectroscopic data in conjunction with HRESIMS/MS experiments. The configurations of the amino acid residues were determined by advanced Marfey's analysis. Compounds 1-6 feature the lack of the highly conserved Thr6 and Hyp10 residues in comparison with other members of the SF3 subfamily peptaibols. A plausible biosynthetic pathway of compounds 1-6 was proposed on the basis of genomic analysis. Compounds 1, 5, 7, and 10 exhibited significant antimicrobial activity against Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis, and Candida albicans. Compounds 7-10 showed potent cytotoxicities against the A549 and/or HepG2 cancer cell lines.

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Five new peptaibols, longibramides A-E (1-5) with 11 amino acid residues, were isolated from a fungus Trichoderma longibrachiatum Rifai DMG-3-1-1, which was isolated from a mushroom Clitocybe nebularis (Batsch) P. Kumm collected from coniferous forest in the subboreal area of northeast China. The structures of longibramides A-E were determined by their spectroscopic data (NMR and MS-MS spectra), their absolute configurations were determined by X-ray diffractions and Marfey's analyses. The X-ray diffractions of longibramides A, B, and the similar CD spectra of A-E showed that they all had α-helix conformations. Longibramides B and E showed moderate cytotoxicities against BV2 and MCF-7 cells and also showed some inhibitory effects against methicillin-resistant Staphylococcus aureus MRSA T144. L-trans-Hyp was not commonly found in natural peptaibols, which was the 6th or 10th amino acid residue in longibramides C-E. The X-ray diffractions of longibramides A and B afforded the accuracy conformations of their secondary structures, which maybe help to interpret the structure-activity relationships of the family of peptaibols in the future.

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A new peptaibol, RK-026A (1) was isolated from a fungus, Trichoderma sp. RK10-F026, along with atroviridin B (2), alamethicin II (3), and polysporin B (4) as a cytotoxic compound, which was selected by principal component analysis of the MS data from 5 different culture conditions. The structure of 1 was determined as a new atroviridin B derivative containing Glu at the 18th residue instead of Gln by NMR and HR-MS analyses including the investigation of detailed MS/MS fragmentations. 1 showed cytotoxicity toward K562 leukemia cells at an IC50 value of 4.1 µm.

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Fermentation of Acremonium tubakii W. Gams isolated from a soil sample collected from the University of Utah led to the isolation and characterization of six new linear pentadecapeptides, emerimicins V-X (1-6). Peptaibols containing 15-residues are quite rare, with only 22 reported. Genome mining and bioinformatic analysis were used to identify the emerimicin 60 kbp eme biosynthetic cluster harboring a single 16-module hybrid polyketide-nonribosomal peptide synthetase. A detailed bioinformatic investigation of the corresponding 15 adenylation domains, combined with 1D and 2D NMR experiments, LC-MS/MS data, and advanced Marfey's method, allowed for the elucidation and absolute configuration of all proteinogenic and nonproteinogenic amino acid residues in 1-6. As some peptaibols possess cytotoxic activity, a zebrafish embryotoxicity assay was used to evaluate the toxicity of the six emerimicins and showed that emerimicin V (1) and VI (2) exhibit the most potent activity. Additionally, out of the six emerimicins, 1 displayed modest activity against Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus faecium with MIC values of 64, 32, and 64 µg/mL, respectively.

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A peptide mixture named tolypin, originally isolated from species of the fungal genus Tolypocladium, was structurally characterised and sequences compared to those reported for efrapeptins isolated from strains of Tolypocladium inflatum. Chiral amino acid analysis, direct infusion, and online HPLC electrospray ionization tandem mass spectrometry provided composition, molecular weights of peptides, and series of diagnostic fragment ions. Sequences deduced from ESI-MS revealed that tolypins C-G are identical to efrapeptins C-G. The results were corroborated by ESI-MS and HPLC of an authentic efrapeptin sample from Eli Lilly Research Laboratories (USA). Comparison of the HPLC elution profiles of efrapeptin and tolypin indicated a pronounced microheterogeneity of the former. A high-resolution HPLC of authentic efrapeptin has not been published before. Close relationship and partial identity of sequences of tolypins and efrapeptins, which had previously been postulated, were definitely proven. The geographical origin of the two most important T. inflatum strains used for sequencing of efrapeptins/tolypins could unambiguously be clarified. A new minor compound, designated tolypin H1, was sequenced. High proportions of helicogenic Aib (α-aminoisobutyric acid) and l-isovaline, N-terminal acetyl-l-pipecolic acid and the unusual, amide-bound C-terminal residue, named (S)-2-amino-1-(1,5-diazabicyclo[4.3.0]non-5-ene-5-ylium)-4-methylpentane corresponding to 1-[(2S)-2-amino-4-methylpentyl]-2,3,4,6,7,8-hexahydropyrrolo[1,2-a]pyrimidin-1-ium, define these peptides as linear, cationic peptaibiotics.

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Fungi in the genus Trichoderma are notorious producers of secondary metabolites with diverse applications, such as antibacterial, antifungal, and plant growth-promoting properties. Peptaibols are linear peptides produced by such fungi, with more than 440 compounds described to date, including tricholongins, longibrachins, trichobrachins, and trichovirins. Peptaibols are synthesized by non-ribosomal peptide synthetases and they have several biological activities. Our research group isolated four peptaibols (6DP2, 6DP3, 6DP4, and 6DP5) with antifungal activity against the plant pathogen Colletotrichum gloeosporioides and the proteasome (a cancer chemotherapy target) from Trichoderma sp. P8BDA1F1, an endophytic fungus from Begonia venosa. The ethyl acetate extract of this endophyte showed activity of 6.01% and 75% against C. gloeosporioides and the proteasome, respectively. The isolated compounds were identified by MS/MS and compared to literature data, suggesting the presence of trilongins BI, BII, BIII, and BIV, which are peptaibols containing 20 amino acid residues. The minimum inhibitory concentration against C. gloeosporioides was 40 µM for trilongin BI, 320 µM for trilongin BII, 160 µM for trilongin BIII, and 310 µM for trilongin BIV. BI-BIV trilongins inhibited proteasome ChTL activity, with IC50 values of 6.5 ± 2.7; 4.7 ± 1.8; 6.3 ± 2.2; and 2.7 ± 0.5 µM, respectively. The compounds were tested ex vivo against the intracellular amastigotes of Leishmania (L.) infantum but showed no selectivity. It is the first report of trilongins BI-BIV with antifungal activity against C. gloeosporioides and the proteasome target.

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Leucinostatin Y, a new peptaibiotic, was isolated from the culture broth of the entomoparasitic fungus Purpureocillium lilacinum 40-H-28. The planar structure was elucidated by detailed analysis of its NMR and MS/MS data. The absolute configurations of the amino acids were partially determined by an advanced Marfey's method. The biological activities of leucinostatin Y were assessed using human pancreatic cancer cells, revealing the importance of the C-terminus of leucinostatins for preferential cytotoxicity to cancer cells under glucose-deprived conditions and inhibition of mitochondrial function.

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AIMS: The production of peptaibols, toxic secondary metabolites of Trichoderma, in the indoor environment is not well-documented. Here, we investigated the toxicity of peptaibols in the guttation droplets and biomass of Trichoderma strains isolated from problematic buildings. METHODS AND RESULTS: Seven indoor-isolated strains of T. atroviride, T. trixiae, T. paraviridescens and T. citrinoviride were cultivated on malt extract agar, gypsum boards and paperboards. Their biomass extracts and guttation droplets were highly cytotoxic in resting and motile boar sperm cell assays and in inhibition of somatic cell proliferation assays. The toxins were identified with HPLC/ESI-MS/MS as trichorzianines, trilongins, trichostrigocins and trichostrigocin-like peptaibols. They exhibited toxicity profiles similar to the reference peptaibols alamethicin, trilongins, and trichorzianine TA IIIc purified from T. atroviride H1/226. Particular Trichoderma strains emitted the same peptaibols in both their biomasses and exudate droplets. The trilongin-producing T. citrinoviride SJ40 strain grew at 37°C. CONCLUSIONS: To our knowledge, this is the first report of indoor-isolated Trichoderma strains producing toxic peptaibols in their guttation droplets. SIGNIFICANCE AND IMPACT OF THE STUDY: This report proves that indoor isolates of Trichoderma release peptaibols in their guttation droplets. The presence of toxins in these types of exudates may serve as a mechanism of aerosol formation for nonvolatile toxins in the indoor air.

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Peptaibols are an intriguing class of fungal metabolites due both to their wide range of reported bioactivities and to the structural variability that can be generated by the exchange of variable amino acid building blocks. In an effort to streamline the discovery of structurally diverse peptaibols, a mass spectrometry surface sampling technique was applied to screen the chemistry of fungal cultures in situ. Four previously undescribed peptaibols, all containing a rare threonine residue, were identified from a fungal culture (MSX53554), which was identified as Nectriopsis Maire (Bionectriaceae, Hypocreales, Ascomycota). These compounds not only increased the known threonine-containing peptaibols by nearly 20%, but also, the threonine residue was situated in a unique place compared to the other reported threonine-containing peptaibols. After the initial in situ detection and characterization, a large-scale solid fermentation culture was grown. The four peptaibols were isolated and characterized by mass spectrometry. In addition, one of the peptaibols was fully characterized by NMR and amino acid analysis using Marfey's reagent and exhibited moderate in vitro anticancer activity.

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Buildings that have been flooded often have high concentrations of Trichoderma spores in the air while drying. Inhaled spores and spore and mycelial fragments contain large amounts of fungal glucan and natural products that contribute to the symptoms associated with indoor mould exposures. In this study, we considered both small molecules and peptaibol profiles of T. atroviride, T. koningiopsis, T. citrinoviride, and T. harzianum strains obtained from damp buildings in eastern Canada. Twenty-residue peptaibols and sorbicillin-derived metabolites (1-6) including a new structure, (R)-vertinolide (1), were characterized from T. citrinoviride. Trichoderma koningiopsis produced several koninginins (7-10), trikoningin KA V, and the 11-residue lipopeptaibols trikoningin KB I and trikoningin KB II. Trichoderma atroviride biosynthesized a mixture of 19-residue trichorzianine-like peptaibols, whereas T. harzianum produced 18-residue trichokindin-like peptaibols and the 11-residue harzianin HB I that was subsequently identified from the studied T. citrinoviride strain. Two α-pyrones, 6-pentyl-pyran-2-one (11) and an oxidized analog (12), were produced by both T. atroviride and T. harzianum. Aside from exposure to low molecular weight natural products, inhalation of Trichoderma spores and mycelial fragments may result in exposure to membrane-disrupting peptaibols. This investigation contributes to a more comprehensive understanding of the biologically active natural products produced by fungi commonly found in damp buildings.

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Bergofungin is a peptide antibiotic that is produced by the ascomycetous fungus Emericellopsis donezkii HKI 0059 and belongs to peptaibol subfamily 2. The crystal structure of bergofungin A has been determined and refined to 0.84 Šresolution. This is the second crystal structure of a natural 15-residue peptaibol, after that of samarosporin I. The amino-terminal phenylalanine residue in samarosporin I is exchanged to a valine residue in bergofungin A. According to agar diffusion tests, this results in a nearly inactive antibiotic peptide compared with the moderately active samarosporin I. Crystals were obtained from methanol solutions of purified bergofungin mixed with water. Although there are differences in the intramolecular hydrogen-bonding scheme of samarosporin I, the overall folding is very similar for both peptaibols, namely 310-helical at the termini and α-helical in the middle of the molecules. Bergofungin A and samarosporin I molecules are arranged in a similar way in both lattices. However, the packing of bergofungin A exhibits a second solvent channel along the twofold axis. This latter channel occurs in the vicinity of the N-terminus, where the natural substitution resides.

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Filamentous fungi of the genus Stilbella are recognized as an abundant source of naturally occurring α-aminoisobutyric acid-containing peptides. The culture broth of Stilbella (Trichoderma) flavipes CBS 146.81 yielded a mixture of peptides named stilboflavins (SF), and these were isolated and separated by preparative TLC into groups named SF-A, SF-B, and SF-C. Although all three of these groups resolved as single spots on thin-layer chromatograms, HPLC analysis revealed that each of the groups represents very microheterogeneous mixtures of closely related peptides. Here, we report on the sequence analysis of SF-C peptides, formerly isolated by preparative TLC. HPLC coupled to QqTOF-ESI-HRMS provided the sequences of 10 16-residue peptides and five 19-residue peptides, all of which were N-terminally acetylated. In contrast to the previously described SF-A and SF-B peptaibols, SF-C peptaibols contain Ser-Alaol or Ser-Leuol, which are rarely found as C-termini, and repetitive Leu-Aib-Gly sequences, which have not been detected in peptaibols before. Taking the previously determined sequences of SF-A and SF-B into account, the entirety of peptides produced by S. flavipes (the 'peptaibiome') approaches or exceeds 100 non-ribosomally biosynthesized peptaibiotics. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

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The Chilean Sepedonium aff. chalcipori strain KSH 883, isolated from the endemic Boletus loyo Philippi, was studied in a polythetic approach based on chemical, molecular, and biological data. A taxonomic study of the strain using molecular data of the ITS, EF1-α, and RPB2 barcoding genes confirmed the position of the isolated strain within the S. chalcipori clade, but also suggested the separation of this clade into three different species. Two new linear 15-residue peptaibols, named chilenopeptins A (1) and B (2), together with the known peptaibols tylopeptins A (3) and B (4) were isolated from the semisolid culture of strain KSH 883. The structures of 1 and 2 were elucidated on the basis of HRESIMS(n) experiments in conjunction with comprehensive 1D and 2D NMR analysis. Thus, the sequence of chilenopeptin A (1) was identified as Ac-Aib(1)-Ser(2)-Trp(3)-Aib(4)-Pro(5)-Leu(6)-Aib(7)-Aib(8)-Gln(9)-Aib(10)-Aib(11)-Gln(12)-Aib(13)-Leu(14)-Pheol(15), while chilenopeptin B (2) differs from 1 by the replacement of Trp(3) by Phe(3). Additionally, the total synthesis of 1 and 2 was accomplished by a solid-phase approach, confirming the absolute configuration of all chiral amino acids as l. Both the chilenopeptins (1 and 2) and tylopeptins (3 and 4) were evaluated for their potential to inhibit the growth of phytopathogenic organisms.

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Six long-chain peptaibols, 1 - 6, were identified from agar cultures of a marine-derived Trichoderma longibrachiatum Rifai strain (MMS151) isolated from blue mussels. The structure elucidation was carried out using electrospray ionization ion trap mass spectrometry (ESI-IT-MS) and GC/EI-MS. The long-chain peptaibols exhibited the general building scheme Ac-Aib-Ala-Aib-Ala-Aib-XXX-Gln-Aib-Vxx-Aib-Gly-XXX-Aib-Pro-Vxx-Aib-XXX-Gln-Gln-Pheol and were similar or identical to recurrent 20-residue peptaibols produced by Trichoderma spp. Three new sequences were identified and were called longibrachins A-0, A-II-a, and A-IV-b. The isolated peptaibols were assayed for cytotoxic, antibacterial, and antifungal activities, and acute toxicity on Dipteran larvae.

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Four new 19-residue peptaibols, named tulasporins A-D (1-4), were isolated from the semi-solid cultures of Sepedonium tulasneanum. Their structures were elucidated on the basis of extensive ESI-HRMS(n) fragmentation studies as well as (1)H NMR spectroscopic analyses. Interestingly, the structures of tulasporins A-D (1-4) resemble those of chrysospermins isolated earlier from cultures of S. chrysospermur. Previously, it was hypothesized that the peptaibol production by Sepedonium species correlates with the morphology of the aleurioconidia, as exclusively round-shaped aleurioconidia forming species produced peptaibols. Since the investigated Sepedonium tulasneanum produces oval aleurioconidia, this study can be considered as the first report of peptaibols from a Sepedonium strain with oval-shaped aleurioconidia. Thus, it could be demonstrated that both round as well as oval aleurioconidia forming Sepedonium species are able to produce peptaibols. Tulasporins A-D (1-4), when tested against phytopathogenic fungi, exhibited good growth inhibitory activity against both Botrytis cinerea and Phytophthora infestans, while they were devoid of significant activity against Septoria tritici.

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In the course of searching for insecticides from soil microorganisms, we found that a fermentation broth of the fungus, Trichoderma brevicompactum FKI-6324, produced Trichopolyn VI, a new peptaibol, which possessed significant insecticidal potential. Spectroscopic analysis showed the compound to be a new trichopolyn I derivative. This paper describes the isolation, structure elucidation and biological activity of trichopolyn VI.

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Two new peptaibols, namely microbacterins A (1) and B (2), were isolated from the deep sea inhabited actinomycete Microbacterium sediminis spp. nov. YLB-01(T). The sequences of the amino acid residues were determined on the basis of intensive NMR and ESI-MS/MS spectroscopic analysis, in addition to the Marfey's method and CD and optical rotation data for the configurational assignment. Both 1 and 2 exhibited significant cytotoxic activities against a panel of human tumor cell lines.

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The Trichoderma citrinoviride VKPM F-1228 strain produces a complex of peptide-based antibiotics with antibacterial and antimycotic action. Synthesis of peptaibols is closely related to the conidiogenesis in the culture. The optimal procedure of the strain cultivation for production of peptaibols is stationary growing for 14 days at a temperature of 28 degrees C and pH 7.5 followed by formation of a dense mycelium film on the modified Saburo medium containing 30 gr/l of glucose and 12.5 gr/l of peptone. Eight individual peptaibols were extracted. The spectrum of their activity was estimated with the use of opportunistic bacteria and micromycetes as well as pathogenic clinical aspergilli. Compounds 9, 13, 14, 15 and 16 were shown active against opportunistic fungi and bacteria including methicillin resistant S. aureus, whereas compounds 9, 13 and 14 in addition showed antimycotic activity against clinical aspergilli.

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An extract of the filamentous fungus Bionectria sp. (MSX 47401) showed both promising cytotoxic activity (>90% inhibition of H460 cell growth at 20 µg/mL) and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). A bioactivity-directed fractionation study yielded one new peptaibol (1) and one new tetramic acid derivative (2), and the fungus biosynthesized diverse secondary metabolites with mannose-derived units. Five known compounds were also isolated: clonostachin (3), virgineone (4), virgineone aglycone (5), AGI-7 (6), and 5,6-dihydroxybisabolol (7). Compounds 5 and 7 have not been described previously from natural sources. Compound 1 represents the second member of the peptaibol structural class that contains an ester-linked sugar alcohol (mannitol) instead of an amide-linked amino alcohol, and peptaibols and tetramic acid derivatives have not been isolated previously from the same fungus. The structures of the new compounds were elucidated primarily by high-field NMR (950 and 700 MHz), HRESIMS/MS, and chemical degradations (Marfey's analysis). All compounds (except 6) were examined for antibacterial and antifungal activities. Compounds 2, 4, and 5 showed antimicrobial activity against S. aureus and several MRSA isolates.

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