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An integrated program of chemical profiling (GNPS) coupled with an expanded format 24-well-plate miniaturized cultivation profiling (MATRIX) utilizing traditional as well as grain/pulse and cereal media permitted rapid prioritization of Aspergillus terreusCMB-SWF012 as a source of unprecedented natural products. Scaled-up cultivation on rice and PDA yielded the rare tripeptides asterripeptides A–C (1–3), new indolo-sesquiterpene Michael adducts terreusides A and B (4and 5), and known precursors asterresin A (6) and (+)-giluterrin (7). Structures for 1–7were assigned by detailed spectroscopic and chemical analysis and biosynthetic considerations.

Chemical analysis of cultures of a Queensland mud dauber wasp nest-derived fungus, Talaromycessp. CMB-MW102, yielded the known dimeric oxaphenalenone duclauxin (1) along with a family of new 1-deoxy-d-glucosamine adducts, glyclauxins A–E (2–6). Despite 1D NMR spectra of 2–6being compromised by broadening of selected resonances, structures inclusive of absolute configuration were assigned on the basis of detailed spectroscopic analysis and biogenetic considerations, as well as biomimetic semisynthesis and chemical interconversion. For example, exposure of duclauxin (1) to synthetic 1-deoxy-d-glucosamine yielded glyclauxin B (3), while on handling and storage, glyclauxins C (4) and D (5) (bearing a 7-OMe moiety) proved chemically labile and underwent quantitative transformation to glyclauxins B (3) and A (2), respectively. These latter observations on chemical reactivity and stability informed a proposed biogenetic relationship linking all known members of the extended duclauxin family. Notwithstanding their potential status as artifacts, the detection of glyclauxins B (3) and A (2) in a fresh CMB-MW102 culture extract confirmed their natural product status.

Using a molecular networking guided strategy, chemical analysis of the Australian mullet fish gastrointestinal tract-derived fungus Amauroascussp. CMB-F713 yielded a family of polyketide pyrones, amaurones A–I (1–9), featuring an unprecedented carbon skeleton. Structures were assigned to 1–9by detailed spectroscopic analysis (including X-ray analysis of 1), biosynthetic considerations, and chemical interconversions. For example, the orthoacetate 5was unstable when stored dry at room temperature, transforming to the monoacetates 2and 3, while mild heating (40 °C) prompted quantitative conversion of 3to 2, via an intramolecular trans-acetylation. Likewise, during handling, the monoacetate 1was prone to intramolecular trans-acetylation, leading to an equilibrium mixture with the isomeric monoacetate amaurone J (10), confirmed when partial hydrolysis of the diacetate 2yielded the monoacetates 1and 10and the triol amaurone K (11).

A GNPS molecular networking approach mapped a library of 960 southern Australian marine sponges and prioritized Dysideasp. (CMB-01171) for chemical investigation. Although the published natural products literature on Australian Dysideasponges extends back over half a century and suffers from the perception of being near exhausted, fractionation of Dysideasp. (CMB-01171) led to the discovery of a family of 10 new biosynthetically and chemically related sesquiterpenes. Detailed spectroscopic analysis guided structure elucidation identified dysidealactams A–F (1–6), dysidealactones A and B (7and 8), and two solvolysis artifacts, 9and 10. The dysidealactams A–D (1–4) incorporate a rare glycinyl-lactam functionality, while dysidealactam E (5) is particularly noteworthy in incorporating an unprecedented glycinyl-imide moiety. In addition to expanding knowledge of Dysideanatural products, this study demonstrates the value of applying GNPS molecular networking to map chemical diversity and prioritize the selection of marine sponge extracts for more detailed chemical analysis.

Chemical analysis of the fungus Chrysosporiumsp. CMB-F294 isolated from the gastrointestinal tract of a market-purchased specimen of Mugilmullet yielded eight new alkaloids, belonging to a rare class of phenylpropanoid piperazines. Chrysosporazines F–M (1–8) occur as an equilibrium mixture of acetamide rotamers and feature unprecedented carbocyclic and heterocyclic scaffolds. Structures inclusive of absolute configuration were assigned by detailed spectroscopic analysis, supported by biosynthetic considerations. Structure–activity relationship studies determined that selected chrysosporazines were promising noncytotoxic inhibitors of the multidrug resistance efflux pump P-glycoprotein (P-gp), capable of reversing doxorubicin resistance in P-gp-overexpressing human colon carcinoma cells (SW620 Ad300). Chrysosporazine F (1) was particularly noteworthy, with a 2.5 μM cotreatment inducing a doxorubicin gain in sensitivity (GS 14) > 2-fold that of the positive control verapamil (GS 6.1).

Chemical profiling of soil-derived microbes collected under the auspices of the Australian citizen science initiative Soils for Science detected two fungi, Clonostachyssp. S4S-07771A07 and Coccidiodessp. S4S-14879B01, capable of producing pullenvalenes, a rare class of triterpene glycoside. Cultivation profiling followed by scaled up cultivation and fractionation of the former yielded the known pullenvalenes A–D (1–4) and the new analogues E–H (5–8), with structures secured by detailed spectroscopic analysis and biogenetic considerations. This study reveals that the pullenvalenes 1–8are produced by several genera of fungi (Clonostachys, Coccidiodesand Talaromyces) recovered from different geographic locations and substrates. We also draw attention to structural and biosynthetic similarities with the known Red Sea sponge metabolites neviotines A–D (9–12) and abudinols A–B (13–14), prompting speculation that the latter may be products of sponge-associated fungi.

The termite nest-derived fungus Trichoderma virensCMB-TN16 cultivated on rice-based media produced seven new first-in-class trimeric sesquiterpenes, trivirensols A–G (11–17). Structures inclusive of absolute configurations were assigned by detailed spectroscopic analysis and biosynthetic considerations. Although trivirensols exhibit no cytotoxicity to mammalian carcinoma cells, selected examples are bacteriostatic against vancomycin-resistant Enterococcus faecalis(VRE). Structure–activity relationship (SAR) investigations combined with in situchemical stability studies documented bacteriostatic activity for trivirensols A (11) and B (12) and the co-metabolite divirensols A (4), B (5), and G (10), all of which share a common terminal butenolide. Significantly, SAR studies also revealed bacteriostatic activity for trivirensols C (13) and G (17) and the co-metabolite divirensol C (6), all of which share a common hydrated butenolide terminal. Of note, when exposed to VRE cell cultures, the hydrated butenolides 6, 13, and 17undergo rapid in situdehydration to corresponding butenolides, suggesting hydrated butenolides are a pro-drug form of the butenolide VRE bacteriostatic pharmacophore.

A chemical investigation of the Australian termite nest-derived fungus Trichoderma virensCMB-TN16 yielded the known sesquiterpene gliocladic acid (1), together with two new acetylated analogues, 3-acetylgliocladic acid (2) and 14-acetylgliocladic acid (3), and seven new dimeric congeners, divirensols A–G (4–10). All metabolites were identified by detailed spectroscopic analysis, supported by biosynthetic considerations, and were assessed for antibacterial and cytotoxic properties. The divirensols are examples of an exceptionally rare class of dimeric sesquiterpene, likely linked via a highly convergent biosynthetic pathway. HPLC-DAD-MS analysis of the crude fungal extract detected ions attributed to putative monomeric biosynthetic precursors.

Chemical analysis of a fermentation of the Australian termite nest-derived fungus Trichoderma virensCMB-TN16 yielded five new acyclic nonapeptides, trichodermides A–E (1–5). Amino acid residues, configurations, and sequences were determined by a combination of spectroscopic (NMR and MS-MS) and chemical (C3Marfey’s) methods. The trichodermides adhere to the sequence homology pattern common to Trichoderma11 amino acid residue peptaibols; however, unlike other peptaibols the trichodermides do not exhibit antibacterial or antifungal activity and exhibit low to no cytotoxicity against mammalian cells. This variability in biological activity highlights the importance of knowing both planar structures and absolute configurations when interpreting structure–activity relationships.

Chemical profiling of extracts from a mud dauber wasp-associated fungus, Aspergillussp. (CMB-W031), revealed a remarkably diverse array of secondary metabolites, with many biosynthetic gene clusters being transcriptionally responsive to specific culture conditions. Chemical fractionation of a jasmine rice cultivation yielded many known fungal metabolites, including the highly cytotoxic (−)-stephacidin B and an unprecedented nonribosomal peptide synthase derived nitro depsi-tetrapeptide diketopiperazine, waspergillamide A (1). All structures were assigned by detailed spectroscopic analysis and, where appropriate, chemical degradation and Marfey’s analysis.

We validate the improved resolution and sensitivity of the C3Marfey’s method, including an ability to resolve all Ile isomers, against an array of amino acids commonly encountered in natural products and by comparison to an existing Marfey’s method. We also describe an innovative 2D C3Marfey’s method as an analytical approach for determining the regiochemistry of enantiomeric amino acid residues in natural products. The C3and 2D C3Marfey’s methods represent valuable tools for probing and defining the stereocomplexity of hydrolytically accessible amino acid residues in natural products.