The colorful fungi of the Chilean forests: Production, chemical characterization and possible applications of their pigments.

In Chile, as in the rest of the world, only a small fraction of the fungal diversity inhabiting the wide variety of its ecosystems is known. This diversity must hide an inestimable richness of species with interesting biotechnological potential, including fungal pigment producers. Recently, interest in filamentous fungi has increased significantly due to their importance as alternative sources of pigments and colorants that are environmentally and human health friendly. As a result, fungal pigments are gaining importance in various industrial applications, such as food, textiles, pharmaceuticals, cosmetics, etc. The increasing consumer demand for "green label" natural colorants requires the exploration of different ecosystems in search of new fungal species that are efficient producers of different pigment with a wide range of colors and ideally without the co-production of mycotoxins. However, advances are also needed in pigment production processes through fermentation, scale-up from laboratory to industrial scale, and final product formulation and marketing. In this respect, the journey is still full of challenges for scientists and entrepreneurs. This chapter describes studies on pigment-producing fungi collected in the forests of central-southern Chile. Aspects such as the exploration of potential candidates as sources of extracellular pigments, the optimization of pigment production by submerged fermentation, methods of pigment extraction and purification for subsequent chemical characterization, and formulation (by microencapsulation) for potential cosmetic applications are highlighted. This potential use is due to the outstanding bioactivity of most fungal pigments, making them interesting functional ingredients for many applications. Finally, the use of fungal pigments for textile and spalting applications is discussed.

Miniaturized Cultivation Profiling (MATRIX)-Facilitated Discovery of Noonazines A-C and Noonaphilone A from an Australian Marine-Derived Fungus, Aspergillus noonimiae CMB-M0339.

Subjecting the Australian marine-derived fungus Aspergillus noonimiae CMB-M0339 to cultivation profiling using an innovative miniaturized 24-well plate format (MATRIX) enabled access to new examples of the rare class of 2,6-diketopiperazines, noonazines A-C (1-3), along with the known analogue coelomycin (4), as well as a new azaphilone, noonaphilone A (5). Structures were assigned to 1-5 on the basis of a detailed spectroscopic analysis, and in the case of 1-2, an X-ray crystallographic analysis. Plausible biosynthetic pathways are proposed for 1-4, involving oxidative Schiff base coupling/dimerization of a putative Phe precursor. Of note, 2 incorporates a rare meta-Tyr motif, typically only reported in a limited array of Streptomyces metabolites. Similarly, a plausible biosynthetic pathway is proposed for 5, highlighting a single point for stereo-divergence that allows for the biosynthesis of alternate antipodes, for example, the 7R noonaphilone A (5) versus the 7S deflectin 1a (6).

New Azaphilones from the Marine-Derived Fungus Penicillium sclerotiorum E23Y-1A with Their Anti-Inflammatory and Antitumor Activities.

Nine new azaphilones, including penicilazaphilones I-N (1, 2 and 6-9), epi-geumsanol D (3) and penidioxolanes C (4) and D (5) were isolated from the culture of the marine-derived fungus Penicillium sclerotiorum E23Y-1A. The structures of the isolates were deduced from extensive spectroscopic data (1D and 2D NMR), high-resolution electrospray ionization mass spectrometry (HRESIMS), and electronic circular dichroism (ECD) calculations. All the azaphilones from P. sclerotiorum E23Y-1A were tested for their anti-inflammatory and antitumor activities. Penicilazaphilone N (9) showed moderate anti-inflammatory activity with an IC50 value of 22.63 ± 2.95 µM, whereas penidioxolane C (4) exhibited moderate inhibition against human myeloid leukemia cells (K562), human liver cancer cells (BEL-7402), human gastric cancer cells (SGC-7901), human non-small cell lung cancer cells (A549), and human hela cervical cancer cells, with IC50 values of 23.94 ± 0.11, 60.66 ± 0.13, 46.17 ± 0.17, 60.16 ± 0.26, and 59.30 ± 0.60 µM, respectively.

Azaphilones from the Fungus Penicillium multicolor LZUC-S2 and Their Antibacterial Activity.

Two new azaphilones, penimultiones A and B, together with seven known analogs were isolated from the culture of Penicillium multicolor LZUC-S2. Their structures were elucidated by detailed spectroscopic data analysis and chemical transformation. Penimultiones A and B belong to a rare class of azaphilones possessing a 1,3-dioxolane moiety. In addition, all compounds were evaluated for their antibacterial activity against five clinically bacterial strains in vitro, and three compounds showed potent antibacterial activity with minimum inhibitory concentration (MIC) values ranging from 12.5 to 50.0 µg/mL.

Scalable Total Synthesis of Acremolactone B.

Acremolactone B is a pyridine-containing azaphilone-type polyketide with herbicidal activity. We developed an aza-6π electrocyclization-aromatization strategy to construct the tetrasubstituted pyridine ring and achieved the first total synthesis of this molecule on a gram scale. A bicyclic intermediate was expeditiously prepared by using [2 + 2] photocycloaddition and chemoselective Baeyer-Villiger oxidation, which was further elaborated to a densely substituted aza-triene. An electrocyclization-aromatization cascade was exploited to forge the tetracyclic core of the natural product, and the side chain was introduced through diastereoselective acylation and reduction.

Azaphilone derivatives with anti-inflammatory activity from the mangrove endophytic fungus Penicillium sclerotiorum ZJHJJ-18.

Nine undescribed azaphilone derivatives, sclerazaphilones A-H (1-9), and three known analogues (10-12), were obtained and identified from the fermented rice cultures of a mangrove endophytic fungus Penicillium sclerotiorum ZJHJJ-18. 1D and 2D NMR, HRESIMS and spectral data indicated the chemical structures of 1-9, and their absolute configurations were assigned by experimental and computational analyses of electronic circular dichroism (ECD) spectra, and application of the chemical transformations. Compounds 1-4 were the first reported N-containing azaphilone derivatives with 5/6 dicyclic core. The bioassay results showed that compounds 3-5 exhibited effective inhibitory effects on the nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells with IC50 values in the range of 6.30-9.45 µM. Moreover, a molecular docking study was conducted to investigate the probable binding interaction of 3-5 with inducible nitric oxide synthase (iNOS).

Chemical characterization and microencapsulation of extracellular fungal pigments.

In this work, extracellular colored metabolites obtained from the filamentous fungi Talaromyces australis and Penicillium murcianum, isolated in the Andean-Patagonian native forests of Chile, were studied as prospect compounds to increase the sustainability of cosmetic products. The chemical and antioxidant properties of these natural pigments were characterized and strategies for their microencapsulation were also studied. UHPLC/MS-MS analyses indicated that the predominant metabolites detected in the cultures of P. murcianum were monascin (m/z = 411.15) and monashexenone (m/z = 319.10), while athrorosin H (m/z = 458.20) and damnacanthal (m/z = 281.05) were detected in cultures of T. australis. ORAC tests revealed that P. murcianum's metabolites had the greatest antioxidant properties with values higher than 2000 µmol of trolox equivalents/g. The fungal metabolites were successfully microencapsulated by ionic gelation into structures made of 1.3% sodium alginate, 0.2% chitosan, and 0.07% hyaluronic acid. The microencapsulation process generated structures of 543.57 ± 0.13 µm of mean diameter (d50) with an efficiency of 30% for P. murcianum, and 329.59 ± 0.15 µm of mean diameter (d50) and 40% efficiency, for T. australis. The chemical and biological characterization show the biotechnological potential of these fungal species to obtain pigments with antioxidant activity that could be useful in the cosmetic industry. The encapsulation process enables the production of easy-to-handle dry powder from the fungal metabolites, which could be potentially marketed as a functional cosmetic ingredient. KEY POINTS: • The predominant fungal pigments were of azaphilone and anthraquinoid classes. • The fungal pigments showed high antioxidant activity by ORAC assay. • Fungal pigment microcapsules obtained by ionic gelation were characterized.

Azaphilones as Activation-Free Primary-Amine-Specific Bioconjugation Reagents for Peptides, Proteins and Lipids.

Residue-selective bioconjugation methods for biomolecules are highly sought to expand the scope of their biological and medical applications. Inspired by the mechanism of the generation of natural vinylogous γ-pyridones (vPDNs), we have developed a novel unique azaphilone-based, activation-free primary-amine-selective bioconjugation method for biomolecules. Our strategy allows facile functionalization of primary amine groups in peptides and proteins, including the clinically used therapeutic antibody trastuzumab, by generating a highly stable vPDN linkage. Excellent chemoselectivity toward primary amines also enables the azaphilone derivatives to specifically modify the lipid components of Gram-positive bacteria while bypassing Gram-negative bacteria and mammalian cells. The new method shows significant advantages including chemoselectivity, efficiency, flexibility and biocompatibility, and therefore provides a valuable addition to the current toolbox for biomolecule conjugation.

Secondary metabolite biosynthetic diversity in the fungal family Hypoxylaceae and Xylaria hypoxylon.

To date little is known about the genetic background that drives the production and diversification of secondary metabolites in the Hypoxylaceae. With the recent availability of high-quality genome sequences for 13 representative species and one relative (Xylaria hypoxylon) we attempted to survey the diversity of biosynthetic pathways in these organisms to investigate their true potential as secondary metabolite producers. Manual search strategies based on the accumulated knowledge on biosynthesis in fungi enabled us to identify 783 biosynthetic pathways across 14 studied species, the majority of which were arranged in biosynthetic gene clusters (BGC). The similarity of BGCs was analysed with the BiG-SCAPE engine which organised the BGCs into 375 gene cluster families (GCF). Only ten GCFs were conserved across all of these fungi indicating that speciation is accompanied by changes in secondary metabolism. From the known compounds produced by the family members some can be directly correlated with identified BGCs which is highlighted herein by the azaphilone, dihydroxynaphthalene, tropolone, cytochalasan, terrequinone, terphenyl and brasilane pathways giving insights into the evolution and diversification of those compound classes. Vice versa, products of various BGCs can be predicted through homology analysis with known pathways from other fungi as shown for the identified ergot alkaloid, trigazaphilone, curvupallide, viridicatumtoxin and swainsonine BGCs. However, the majority of BGCs had no obvious links to known products from the Hypoxylaceae or other well-studied biosynthetic pathways from fungi. These findings highlight that the number of known compounds strongly underrepresents the biosynthetic potential in these fungi and that a tremendous number of unidentified secondary metabolites is still hidden. Moreover, with increasing numbers of genomes for further Hypoxylaceae species becoming available, the likelihood of revealing new biosynthetic pathways that encode new, potentially useful compounds will significantly improve. Reaching a better understanding of the biology of these producers, and further development of genetic methods for their manipulation, will be crucial to access their treasures.

Antimicrobial and antioxidant chlorinated azaphilones from mangrove Diaporthe perseae sp. isolated from the stem of Chinese mangrove Pongamia pinnata.

Many plants remain unexplored for their endophytic fungi that may possess potentially important phytochemicals. Consequently, we have focused to discover new natural products from endophytic fungus Diaporthe perseae sp. isolated from the stem of the Chinese mangrove Pongamia pinnata (L.) Pierre plant that led to the isolation of one new chlorinated isochromophilone G (1) along with six known azaphilones (2-7). The structures of the isolated compounds were elucidated by UV, NMR and Mass spectroscopic analysis. All the isolated compounds were screened for their antimicrobial and anti-oxidant activities.

Chaetoglobosins and azaphilones from Chaetomium globosum associated with Apostichopus japonicus.

Increasing attention has recently been focused on complex symbiotic associations, for instance coral and its symbionts. Sea cucumber, harboring diverse fungi, has also attracted more and more attention for their functional diversity. Here, secondary metabolites produced by Chaetomium globosum associated with sea cucumber, Apostichopus japonicus, were investigated using gene mining with third-generation sequencing technology (PacBio SMRT). Nine compounds, including one new compound cytoglobosin X (1), were isolated from cultures of Chaetomium globosum. Compound 1 was identified based on NMR data, HRESIMS, and ECD, and the absolute configurations were identified as 3S, 4R, 7S, 8R, 9R, 16S, 19S, 20S, and 23S. In an antimicrobial assay, compound 4 showed moderate activity against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus with MICs of 47.3 and 94.6 µM, respectively. Our results suggest that the microbiomes associated with sea cucumber could be an important resource for biodiversity and structural novelty, and the bioactive compounds may protect the host from pathogen microbial.

Azaphilones from the Red Sea Fungus Aspergillus falconensis.

The marine-derived fungus Aspergillus falconensis, isolated from sediment collected from the Canyon at Dahab, Red Sea, yielded two new chlorinated azaphilones, falconensins O and P (1 and 2) in addition to four known azaphilone derivatives (3-6) following fermentation of the fungus on solid rice medium containing 3.5% NaCl. Replacing NaCl with 3.5% NaBr induced accumulation of three additional new azaphilones, falconensins Q-S (7-9) including two brominated derivatives (7 and 8) together with three known analogues (10-12). The structures of the new compounds were elucidated by 1D and 2D NMR spectroscopy and HRESIMS data as well as by comparison with the literature. The absolute configuration of the azaphilone derivatives was established based on single-crystal X-ray diffraction analysis of 5, comparison of NMR data and optical rotations as well as on biogenetic considerations. Compounds 1, 3-9, and 11 showed NF-κB inhibitory activity against the triple negative breast cancer cell line MDA-MB-231 with IC50 values ranging from 11.9 to 72.0 µM.

Muyocopronones A and B: azaphilones from the endophytic fungus Muyocopron laterale.

Two new azaphilones, namely muyocopronones A (1) and B (2), were isolated from the cultures of an endophytic fungus Muyocopron laterale ECN279. Their structures were elucidated by extensive spectroscopic analysis. Their absolute configurations were determined using the modified Mosher's method and through comparisons of experimental and calculated electronic circular dichroism data. In addition, muyocopronone B (2) was found to exhibit a weak antibacterial activity against some Gram-positive bacteria.

Identification of anti-inflammatory polyketides from the coral-derived fungus Penicillium sclerotiorin: In vitro approaches and molecular-modeling.

Four new polyketides, including an unusual naphthoquinone derivative (1), two azaphilone analogous (2, 7) and an α-pyrone (12), were isolated from the gorgonian-derived fungus Penicillium sclerotiorum CHNSCLM-0013 together with nine known compounds. Their structures were identified based on the 1D, 2D NMR, HRESIMS, single crystal X-ray diffraction and the absolute configurations were determined by comparing the 1H NMR chemical shift and optical rotations with those reported in literature. In the bioassay, compounds 2, 6, 7, 10 and 12 exhibited significant inhibitory activities against the nitric oxide (NO) production in the LPS-induced macrophage RAW 264.7 with the IC50 values in the range of 2.5-18.0 µM. Compounds 2, 6 and 7 exhibited the possible mechanism of downregulating the expression of iNOS and COX-2 in mRNA level. The primary structure-activity relationship was also discussed based on the molecular-modeling. This study will make a contribution to the chemical diversities of polyketides especially the azaphilone derivatives and the discovery of potential anti-inflammatory agent from marine fungi.

Azaphilones from the Marine Sponge-Derived Fungus Penicillium sclerotiorum OUCMDZ-3839.

Four new azaphilones, sclerotiorins A-D (1-4), as well as the dimeric sclerotiorin E (5) of which we first determined its absolute configuration, and 12 known analogues (5-16) were isolated from the fermentation broth of Penicillium sclerotiorum OUCMDZ-3839 associated with a marine sponge Paratetilla sp.. The new structures, including absolute configurations, were elucidated by spectroscopic analyses, optical rotation, ECD spectra, X-ray single-crystal diffraction, and chemical transformations. Compounds 11 and 14 displayed significant inhibitory activity against α-glycosidase, with IC50 values of 17.3 and 166.1 µM, respectively. In addition, compounds 5, 7, 10, 12-14, and 16 showed moderate bioactivity against H1N1 virus.

Chlamyphilone, a Novel Pochonia chlamydosporia Metabolite with Insecticidal Activity.

Metabolites from a collection of selected fungal isolates have been screened for insecticidal activity against the aphid Acyrthosiphon pisum. Crude organic extracts of culture filtrates from six fungal isolates (Paecilomyces lilacinus, Pochonia chlamydosporia, Penicillium griseofulvum, Beauveria bassiana, Metarhizium anisopliae and Talaromyces pinophilus) caused mortality of aphids within 72 h after treatment. In this work, bioassay-guided fractionation has been used to characterize the main bioactive metabolites accumulated in fungal extracts. Leucinostatins A, B and D represent the bioactive compounds produced by P. lilacinus. From P. griseofulvum and B. bassiana extracts, griseofulvin and beauvericin have been isolated, respectively; 3-O-Methylfunicone and a mixture of destruxins have been found in the active fractions of T. pinophilum and M. anisopliae, respectively. A novel azaphilone compound, we named chlamyphilone, with significant insecticidal activity, has been isolated from the culture filtrate of P. chlamydosporia. Its structure has been determined using extensive spectroscopic methods and chemical derivatization.

Nitrogenated Azaphilone Derivatives through a Silver-Catalysed Reaction of Imines from ortho-Alkynylbenzaldehydes.

Nitrogenated azaphilones are interesting natural products with a wide range of applications. The structure of these compounds is characterized by the presence of an isoquinolinone framework. Here, we describe a new multicomponent silver-catalysed reaction that allows the transformation of simple imines derived from ortho-alkynylbenzaldehydes into complex nitrogenated azaphilone-type molecules in a straightforward way. This atom-economical process is high yielding, technically very simple and proceeds through a series of cascade processes that imply cycloisomerisation and formal cross-coupling reactions. This conceptually new process formally involves the synchronised catalytic generation and selective coupling of a nucleophile (isoquinolinone) and an electrophile (isoquinolinium). Some interesting colour properties of the synthesized azaphilone-type molecules are discussed.

New azaphilones from mangrove endophytic fungus Penicillium sclerotiorin SCNU-F0040.

Two new sclerotioramines (1 and 2) and a new natural product of sclerotioramine analog (3), together with seven known compounds have been isolated from the mangrove endophytic fungus Penicillium sclerotiorin SCNU-F0040. Their structures were identified based on the 1 D, 2 D NMR and HRESIM spectra. The absolute configurations of new compounds were deduced by specific rotation data and electronic circular dichroism spectra. All the isolated new compounds were tested on anti-diabetes activity by using a-glucosidase inhibition assay and anti-inflammatory activity by using cyclooxygenase inhibition assay, respectively. Compounds 1 and 2 have a-glucosidase inhibition activity with IC50 values of 102.3 and 217.5 µM. Compound 2 shows a moderate cyclooxygenase-2 inhibitory activity with an IC50 value of 47.8 µM.

Nitrogen Enriched Solid-State Cultivation for the Overproduction of Azaphilone Red Pigments by Penicillium sclerotiorum SNB-CN111.

Azaphilones are microbial specialized metabolites employed as yellow, orange, red or purple pigments. In particular, yellow azaphilones react spontaneously with functionalized nitrogen groups, leading to red azaphilones. In this study, a new two-step solid-state cultivation process to produce specific red azaphilones pigments was implemented, and their chemical diversity was explored based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and a molecular network. This two-step procedure first implies a cellophane membrane allowing accumulating yellow and orange azaphilones from a Penicillium sclerotiorum SNB-CN111 strain, and second involves the incorporation of the desired functionalized nitrogen by shifting the culture medium. The potential of this solid-state cultivation method was finally demonstrated by overproducing an azaphilone with a propargylamine side chain, representing 16% of the metabolic crude extract mass.

Novel chlorinated and nitrogenated azaphilones with cytotoxic activities from the marine algal-derived fungus Chaetomium globosum 2020HZ23.

Two novel chlorinated and nitrogenated azaphilones, namely N-butyl-2-aza-2-deoxychaetoviridin A (1) and N-hexyl-2-aza-2-deoxychaetoviridin A (2), along with a previously identified analogue, chaetoviridin A (3), were successfully obtained from Chaetomium globosum 2020HZ23, a marine algal-sourced endophytic fungus. The planar structures as well as the absolute configurations of these new metabolites were determined utilizing a synergistic approach that involved both spectroscopic techniques (1D/2D NMR and HRESIMS) and Density Functional Theory (DFT) calculations. Each compound was subject to in vitro cytotoxicity evaluation toward the A549 cancer cell line. Both compounds 1 and 2 demonstrated significant cytotoxicity, as evidenced by their respective IC50 values of 13.6 and 17.5 µM. Furthermore, 1 and 2 demonstrated potent cell migration inhibition, which elevated with increasing dose concentration. In contrast, compound 3 exhibited less cytotoxic activity relative to 1 and 2, suggesting that the cytotoxic potency escalates with N-substitution at the C-2 position and the introduction of a side chain. This finding could offer implications for future studies aimed at designing and refining lead compounds within this class.