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Chemical investigation of Australian pasture plant-derived

We report on the chemical analysis of a mud dauber wasp nest-associated fungus, Penicillium sp. CMB-MD22, leading to the discovery and structure elucidation of three known (1-3) and two new (4 and 5) anthrones, and a family of new and known bianthrones, neobulgarones 6-23. Detection and structure elucidation of 1-23 was supported by detailed spectroscopic analysis, as well as chemical (thermal) transformations, and global natural products social (GNPS) molecular networking. An empirical approach using HPLC retention times was effective at differentiating anti from syn bianthrone isomers, while a facile thermal equilibration was shown to favor anti over syn isomers. The neobulgarones 6-23 are natural products, and a crude extract rich in 6-23 exhibits selective antifungal activity against a co-isolated mud dauber wasp nest-associated fungus, suggestive of a possible ecological role as an antifungal chemical defense.

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

A GNPS molecular networking approach mapped a library of 960 southern Australian marine sponges and prioritized Dysidea sp. (CMB-01171) for chemical investigation. Although the published natural pr...

Chemical analysis of the fungus Chrysosporium sp. CMB-F294 isolated from the gastrointestinal tract of a market-purchased specimen of Mugil mullet 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 investigations into solid phase cultivations of an Australian sheep station pasture plant derived

The termite nest-derived fungus Trichoderma virens CMB-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 situ chemical 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 17 undergo rapid in situ dehydration to corresponding butenolides, suggesting hydrated butenolides are a pro-drug form of the butenolide VRE bacteriostatic pharmacophore.

Eleven new nitrogenous meroterpenoids, cinerols A-K (1-11), were isolated from the marine sponge Dysidea cinerea collected in the South China Sea, and their structures were determined by detailed spectroscopic analysis. Cinerols A (1) and B (2) feature a rare 5H-pyrrolo[1,2a]benzimidazole moiety, while cinerols C-G (3-7) are examples of rare meroterpene benzoxazoles. The cinerols are noncytotoxic to human melanoma A375 cells at the concentration of 32 μM; however, selected cinerols exhibit moderate inhibitory activity against one or more of protein-tyrosine phosphatase 1B, ATP-citrate lyase, and SH2 domain-containing phosphatase-1 with IC50 values of 2.8-27 μM.

Using a molecular networking guided strategy, chemical analysis of the Australian mullet fish gastrointestinal tract-derived fungus

A GNPS molecular networking approach mapped a library of 960 southern Australian marine sponges and prioritized

Chemical analysis of the fungus

Chemical analysis of a fermentation of the Australian termite nest-derived fungus Trichoderma virens CMB-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 (C3 Marfey’s) methods. The trichodermides adhere to the sequence homology pattern common to Trichoderma 11 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, Aspergillus sp. (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.

Six new (1-6) spongian-16-one analogues have been characterized from the Australian nudibranch species Goniobranchus collingwoodi, along with four known spongian-16-one derivatives. The structures and relative configuration were suggested by spectroscopic analyses informed by molecular modeling. Dissection of animal tissue revealed that the mantle and viscera differ in their terpene composition. Whole body extracts were not toxic to brine shrimp (Artemia sp.), but were unpalatable to palaemon shrimp (Palaemon serenus) at a concentration found within the nudibranch. Individual terpenes were not cytotoxic to human lung (NCIH-460), colorectal (SW620), and liver (HepG2) cancer cells.

The termite nest-derived fungus

Eleven new nitrogenous meroterpenoids, cinerols A-K (

A chemical investigation of the Australian termite nest-derived fungus Trichoderma virens CMB-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.

Cane toads are an invasive pest species in which all life stages employ cardiotoxic bufagenins as a chemical defense against predators. Curiously, the bufagenin profiles of eggs and tadpoles are more complex than those of parotoid secretion, the principle mechanism of toxin delivery in adult toads. In an effort to understand this complexity, we determined that selected strains of adult toad parotoid-gland-associated Gram-positive bacteria, Bacillus spp., were capable of biotransforming secreted bufagenins, marinobufagenin (1), telocinobufagenin (2), bufalin (3), and resibufagenin (4), to hydroxylated scaffolds commonly encountered in cane toad eggs and tadpoles. Scaled-up cultivation, preparative chromatography, and detailed spectroscopic analysis identified Bacillus sp. CMB-TD29 biotransformation products of 1, as 11α-hydroxymarinobufagenin (6), 12β-hydroxymarinobufagenin (7), and 17α-hydroxymarinobufagenin (8). Comparative bufagenin profiles across the cane toad life cycle suggest that bacterial biotransformation mediates the oxidative adaptation of adult toad bufagenins to hydroxylated bufagenins encountered in eggs and tadpoles. We speculate that knowledge of a relationship between the cane toad microbiome and bufagenin chemical defenses could inspire the development of a natural, nontoxic, environmentally sustainable bacterial biocontrol for this toxic invasive species.

Co-cultivation of Chaunopycnis sp. (CMB-MF028) and Trichoderma hamatum (CMB-MF030), fungal strains co-isolated from the inner tissue of an intertidal rock platform mollusc (Siphonaria sp), resulted in transcriptional activation of a rare class of 2-alkenyl-tetrahydropyran, chaunopyran A (7), and biotransformation and deactivation of the antifungal pyridoxatin (1), to methyl-pyridoxatin (8). This study illustrates the complexity of offensive and counter-offensive molecular defenses encountered during fungal co-cultivation, and the opportunities for activating new, otherwise transcriptionally silent secondary metabolites.

We validate the improved resolution and sensitivity of the C3 Marfey'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 C3 Marfey's method as an analytical approach for determining the regiochemistry of enantiomeric amino acid residues in natural products. The C3 and 2D C3 Marfey's methods represent valuable tools for probing and defining the stereocomplexity of hydrolytically accessible amino acid residues in natural products.

An Australian marine-derived isolate of Aspergillus versicolor (MST-MF495) yielded the known fungal metabolites sterigmatocystin, violaceol I, violaceol II, diorcinol, (-)-cyclopenol, and viridicatol, along with a new alkaloid, cottoquinazoline A (1), and two new cyclopentapeptides, cotteslosins A (2) and B (3). Structures for 1-3 and the known compounds were determined by spectroscopic analysis. The absolute configurations of 1-3 were addressed by chemical degradation and application of the C(3) Marfey's method. The use of "cellophane raft" high-nutrient media as a device for up-regulating secondary metabolite diversity in marine-derived fungi is discussed. The antibacterial properties displayed by A. versicolor (MST-MF495) were attributed to the phenols violaceol I, violaceol II, and diorcinol, while cotteslosins 2 and 3 were identified as weak cytotoxic agents.

An Australian estuarine isolate of an Acremonium sp. (MST-MF588a) yielded the two known compounds 19- O-acetylchaetoglobosin D ( 1) and 19- O-acetylchaetoglobosin B ( 2), as the sole cytotoxic principles, along with the known aromatic metabolite RKB 3564S ( 3), and a novel family of lipodepsipeptides, acremolides A-D ( 4- 7). Structures were assigned to 4- 7 on the basis of detailed spectroscopic analysis and chemical derivatization and by application of a new C 3 Marfey's method for amino acid analysis.

Chemical analysis of an Australian marine-derived Streptomyces sp. (CMB-M0150) yielded two new anthracycline antibiotics, aranciamycins I (1) and J (2), as well as the previously reported aranciamycin A (3) and aranciamycin (4). The aranciamycins 1-4, identified by detailed spectroscopic analysis, were noncytotoxic when tested against selected Gram-negative bacteria and fungi (IC50 >30 μM) and exhibited moderate and selective cytotoxicity against Gram-positive bacteria (IC50 >1.1 μM) and a panel of human cancer cell lines (IC50 > 7.5 μM). Significantly, 1-4 were cytotoxic (IC50 0.7-1.7 μM) against the Mycobacterium tuberculosis surrogate M. bovis bacille Calmette-Guerin.

Three new aromatic butenolides, gymnoascolides A-C (1-3), have been isolated from the Australian soil ascomycete Gymnoascus reessii and assigned structures on the basis of detailed spectroscopic analysis. The absolute configurations of gymnoascolides B (2) and C (3) at C-5 were solved using a combination of chemical derivatization and quantum chemical simulations.

An Australian isolate of Penicillium striatisporum collected near Shalvey, New South Wales, exhibited selective antifungal activity against Candida albicans versus Saccharomyces cerevisiae. Bioassay-directed fractionation yielded members of the rare class of fungal metabolites known as the calbistrins. These included a new example of this structure class, calbistrin E (1), as well as the known polyenes calbistrin C (2) and deformylcalbistrin A (3). Also recovered from P. striatisporum were new triene and butenolide acids, striatisporin A (4) and striatisporolide A (5), together with the known fungal metabolites versiol (6) and (+)-hexylitaconic acid (7). Structures for all metabolites were determined by detailed spectroscopic analysis.

A nematocidal agent present in a southern Australian marine sponge of the genus Echinodictyum has been isolated and identified by detailed spectroscopic analysis and total synthesis as the novel betaine (-)-echinobetaine A (6). Preliminary SAR investigations have been undertaken.

Investigations of a southern Australian marine sponge, Oceanapia sp., have yielded two new beta methyl branched bisthiocyanates, thiocyanatins D1 (3a) and D(2) (3b), along with two new thiocarbamate thiocyanates, thiocyanatins E1 (4a) and E2 (4b). The new thiocyanatins belong to a rare class of bioactive marine metabolite previously only represented by thiocyanatins A-C (1, 2a/b). Structures were assigned on the basis of detailed spectroscopic analysis, with comparisons to the known bisthiocyanate thiocyanatin A (1) and synthetic model compounds (5-7). The thiocyanatins exhibit potent nematocidal activity, and preliminary structure-activity relationship investigations have confirmed key characteristics of the thiocyanatin pharmacophore.

Bioassay-directed fractionation of two southern Australian sponges, Phoriospongia sp. and Callyspongia bilamellata, yielded two new nematocidal depsipeptides, identified as phoriospongins A (1) and B (2). The structures of the phoriospongins were determined by detailed spectroscopic analysis and comparison with the previously reported sponge depsipeptide cyclolithistide A (3), as well as ESIMS and HPLC analysis of acid hydrolysates. It is noteworthy that the unique and yet structurally related metabolites 1-3 are found in sponges spanning three taxonomic orders, Poescilosclerida, Haplosclerida, and Lithistida.

A Clathria sp. collected in the Great Australian Bight has yielded the novel metabolites clathrins A (6), B (7), and C (8). Structures were assigned to clathrins A-C on the basis of spectroscopic analysis. Clathrin A (6) represents a plausible biosynthetic intermediate that provides an inferred link between marine sesquiterpene/benzenoids and mixed terpene/shikimate biosynthesis.

The crude EtOH extract of an Echinodictyum sp. collected during trawling operations in the Great Australian Bight, Australia, displayed antibacterial and antiparasitic properties. Bioassay-directed fractionation yielded three novel sulfonic acids, the echinosulfonic acids A to C (1-3), and a new sulfone, echinosulfone A (4). Structures were assigned to these compounds on the basis of detailed spectroscopic analysis. It was determined that echinosulfonic acids A-C (1-3) and echinosulfone A (4) contributed to the antibacterial but not antiparasitic activity of the crude extract.

A Sigmosceptrella sp. from the Great Australian Bight, Australia, has yielded the new norditerpene cyclic peroxide, nuapapuin A (2a), and the norsesterterpene cyclic peroxide sigmosceptrellin D (3a), characterized as the corresponding methyl esters 2b and 3b. The crude methylated sponge extract also yielded the new norsesterterpene cyclic peroxide sigmosceptrellin E methyl ester (4). Relative stereochemistry about C2, C3, and C6 was assigned by established empirical rules and absolute stereochemistry by the advanced Mosher procedure. A plausible biosynthetic pathway has been proposed that rationalizes key transformations in the biosynthesis of all known norterpene cyclic peroxides and related norterpene ketones, dienes and sigmosceptrins.

Addition of NaCl to the solid-phase fermentation of an Australian isolate of Gymnoascus reessii resulted in enhanced production of chloropolyenylpyrroles, while the addition of NaBr suppressed chloropolyenylpyrrole production in favor of bromo and dechloro analogues. Access to a wider selection of polyenylpyrroles provided scope for SAR comparisons on this rare class of cytotoxic natural products, with the bromo- and dechloropolyenylpyrroles displaying significantly reduced cytotoxicity. These results suggest that the chloro substituent is a critical element in the pharmacophore for this rare class of natural product.

Investigation of two southern Australian marine sponges, Mycale spp., resulted in isolation of the known norsesterterpene mycaperoxide F methyl ester (5) together with a new norsesterterpene mycaperoxide G methyl ester (10) and a new norterpene ketone 11. All structures were secured by spectroscopic analysis and chemical derivatization. The absolute stereochemistry previously assigned to 5 by application of the Horeau procedure has been revised by application of the Mosher procedure.

Investigation of a southern Australian marine sponge, Mycale sp., resulted in isolation of the known norsesterterpenes 1-3 as well as two new isomeric norsesterterpenes, mycaperoxide C methyl ester (4) and mycaperoxide D methyl ester (5), and six new norterpenes (6-11).

Three new C25 furanoterpenes, hippospongins A−C (5−7); a new C24 furanoterpene, hippospongin D (8); and two new C22 furanoterpenes, hippospongins E and F (9 and 10), were isolated from a southern Australian collection of the marine sponge Hippospongia sp. The hippospongins possess structural features intermediate between C21 furanoterpenes and their putative biosynthetic precursors, C25 tetronic acids. All structures were secured by detailed spectroscopic analysis.

Chemical analysis of an Australian Streptomyces species yielded a range of known anthracyclines and biosynthetically related metabolites, including daunomycin (1), epsilon-rhodomycinone (2), 11-hydroxyauramycinone (3), 11-hydroxysulfurmycinone (4), aklavinone (5), bisanhydro-gamma-rhodomycinone (6), and the anthraquinone 7, as well as the hitherto unreported blanchaquinone (8). The structure assigned to 8 was secured by detailed spectroscopic analysis and correlation to known analogues, such as the anthraquinone 7. This account also represents the first natural occurrence of 3, 4, and 7 and the first spectroscopic characterization of 11-hydroxysulfurmycinone (4).

Two new antibacterial agents, rugulotrosin A (1) and B (2), were obtained from cultures of a Penicillium sp. isolated from soil samples acquired near Sussex Inlet, New South Wales, Australia. Rugulotrosin A (1) is a chiral symmetric dimer, and its relative stereostructure was determined by spectroscopic and X-ray crystallographic analysis. Rugulotrosin B (2) is a chiral asymmetric dimer isomeric with 1. Its structure was determined by spectroscopic analysis with comparison to the co-metabolite 1 and previously reported fungal metabolites. Both rugulotrosins A and B displayed significant antibacterial activity against Bacillus subtilis, while rugulotrosin A was also strongly active against Enterococcus faecalis and B. cereus.

Bioassay-directed fractionation of a Hymeniacidon sp. yielded as nematocidal agents the equilibrating E/Z bromoindole ethyl esters 1 and 2 and corresponding methyl esters 3 and 4. Also isolated for the first time as a natural product was an equilibrating mixture of seco-xanthine formamides, attributed the trivial name hymeniacidin (5). The structure for 5 was assigned on the basis of detailed spectroscopic analysis and total synthesis.

Chemical analysis of southern Australian marine sponges of the genera Higginsia and Spongosorites has yielded examples of the discorhabdin class of alkaloids. These include the known metabolites (+)-discorhabdin A (1), (+)-discorhabdin D (2), makaluvamine J (6), and damirone A (7), together with four new analogues, (+)-dihydrodiscorhabdin A (3), (+)-debromodiscorhabdin A (4), (+)-dihydrodiscorhabdin L (8), and (+)-discorhabdin X (5), with the latter compound being the first reported example of a thio heterocycle flanked by oxo-thio-acetal and azo-thio-acetal functionalities. Structures for the new compounds were assigned on the basis of detailed spectroscopic interpretation.

A Lyngbya sp. collected from intertidal rock platforms at Point Lonsdale, Victoria, Australia, has been found to contain the novel secondary [(1'Z)-3'-acetoxy-2'-bromo-1'-prop-1'-enyl]-2,5-dimethyldodecanoate [1]. The structure of 1 was determined by spectroscopic analysis and degradation.

Three new isomeric sesquiterpenes 6, 7, and 8 have been isolated from an Australian marine sponge, Arenochalina sp., and their structures determined by spectroscopic analysis and chemical correlation.

Two new isomeric norsesterterpene cyclic peroxides 3 and 5 have been isolated from an Australian marine sponge, Mycale (Carmia) cf. spongiosa, and their structures have been determined by spectroscopic analysis and chemical degradation.

Chemical analysis of an Australian marine-derived isolate of Penicillium bilaii, collected from the Huon estuary, Port Huon, Tasmania, yielded the known fungal aromatic polyketides citromycetin (1) and citromycin (2) together with two dihydro analogues, (-)-2,3-dihydrocitromycetin (3) and (-)-2,3-dihydrocitromycin (4). An Australian terrestrial isolate of Penicillium striatisporum collected near Shalvey, New South Wales, also yielded citromycetin (1), citromycin (2), and the new dihydro analogue (-)-2,3-dihydrocitromycetin (3), together with fulvic acid (5), anhydrofulvic acid (6), and a selection of new methoxylated analogues, 12-methoxycitromycetin (7), 12-methoxycitromycin (8), (-)-12-methoxy-2,3-dihydrocitromycetin (9), and 12-methoxyanhydrofulvic acid (10). P. bilaii also yielded the rare siderophore pistillarin (11), the known diketopiperazines cyclo-(L-Phe -L-Pro) (12), cyclo-(L-Pro-L-Tyr) (13), cyclo-(L-Pro-L-Val) (14), and cis-bis(methylthio)silvatin (15), and three new diketopiperazines, bilains A-C (16-18). The structures for the Penicillium metabolites 1- 18 were assigned by a combination of detailed spectroscopic analysis, including correlation with relevant literature data, chemical derivatization, degradation, and biosynthetic considerations. The citromycin polyketides 2 and 4 and the diketopiperazine 15 were weakly cytotoxic.

A Spongosorites sp. collected during trawling operations off the southern coast of Australia returned the new alkaloid dragmacidin E (3), the structure of which was secured by detailed spectroscopic analysis. Dragmacidin E (3), and its co-metabolite dragmacidin D (1) have been identified as potent inhibitors of serine-threonine protein phosphatases.

An Eryus sp. of marine sponge from the Great Australian Bight has yielded the first reported natural occurrence of a cyclonucleoside, N(3),5'-cycloxanthosine. The structure of N(3),5'-cycloxanthosine was confirmed by detailed spectroscopic analysis and total synthesis.