Computational Approaches for the Prediction of Environmental Transformation Products: Chlorination of Steroidal Enones.

There is growing interest in the fate and effects of transformation products generated from emerging pollutant classes, and new tools that help predict the products most likely to form will aid in risk assessment. Here, using a family of structurally related steroids (enones, dienones, and trienones), we evaluate the use of density functional theory to help predict products from reaction with chlorine, a common chemical disinfectant. For steroidal dienones (e.g., dienogest) and trienones (e.g., 17ß-trenbolone), computational data support that reactions proceed through spontaneous C4 chlorination to yield 4-chloro derivatives for trienones and, after further reaction, 9,10-epoxide structures for dienones. For testosterone, a simple steroidal enone, in silico predictions suggest that C4 chlorination is still most likely, but slow at environmentally relevant conditions. Predictions were then assessed through laboratory chlorination reactions (0.5-5 mg Cl2/L) with product characterization via HRMS and NMR, which confirmed near exclusive 4-chloro and 9,10-epoxide products for most trienones and all dienones, respectively. Also consistent with computational expectations, testosterone was effectively unreactive at these same chlorine levels, although products consistent with in silico predictions were observed at higher concentrations (in excess of 500 mg Cl2/L). Although slight deviations from in silico predictions were observed for steroids with electron-rich substituents (e.g., C17 allyl-substituted altrenogest), this work highlights the potential for computational approaches to improve our understanding of transformation products generated from emerging pollutant classes.

Broomeanamides: Cyclic Octapeptides from an Isolate of the Fungicolous Ascomycete Sphaerostilbella broomeana from India.

The genus Sphaerostilbella comprises fungi that colonize basidiomata of wood-inhabiting fungi, including important forest pathogens. Studies of fermentation cultures of an isolate (TFC201724) collected on the foothills of Himalayas, and closely related to S. broomeana isolates from Europe, led to the identification of a new cyclic octapeptide along with two closely related analogues (1-3) and four dioxopiperazines (4-7). The structure of the lead compound, broomeanamide A (1), was assigned mainly by analysis of 2D NMR and HRESIMS data. The structure consisted of one unit each of N-MeVal, Ala, N-MePhe, Pro, Val, and Ile and two N-MeLeu units. The amino acid sequence was determined on the basis of 2D NMR and HRESIMSMS data. NMR and HRMS data revealed that the other two new peptides have the same amino acid composition except that the Ile unit was replaced with Val in one instance (2) and the N-MeVal unit was replaced with Val in the other (3). The absolute configuration of 1 was assigned by analysis of the acid hydrolysate by application of Marfey's method using both C18 and C3 bonded-phase columns. Broomeanamide A (1) showed antifungal activity against Cryptococcus neoformans and Candida albicans, with MIC values of 8.0 and 64 µg/mL, respectively.

Anti-cryptococcal activity of preussolides A and B, phosphoethanolamine-substituted 24-membered macrolides, and leptosin C from coprophilous isolates of Preussia typharum.

Cryptococcus neoformans is a serious human pathogen with limited options for treatment. We have interrogated extracts from fungal fermentations to find Cryptococcus-inhibiting natural products using assays for growth inhibition and differential thermosensitivity. Extracts from fermentations of four fungal strains from wild and domestic animal dung from Arkansas and West Virginia, USA were identified as Preussia typharum. The extracts exhibited two antifungal regions. Purification of one region yielded new 24-carbon macrolides incorporating both a phosphoethanolamine unit and a bridging tetrahydrofuran ring. The structures of these metabolites were established mainly by analysis of high-resolution mass spectrometry and 2D NMR data. Relative configurations were assigned using NOESY data, and the structure assignments were supported by NMR comparison with similar compounds. These new metabolites are designated preussolides A and B. The second active region was caused by the cytotoxin, leptosin C. Genome sequencing of the four strains revealed biosynthetic gene clusters consistent with those known to encode phosphoethanolamine-bearing polyketide macrolides and the biosynthesis of dimeric epipolythiodioxopiperazines. All three compounds showed moderate to potent and selective antifungal activity toward the pathogenic yeast C. neoformans.

Campafungins: Inhibitors of Candida albicans and Cryptococcus neoformans Hyphal Growth.

Campafungin A is a polyketide that was recognized in the Candida albicans fitness test due to its antiproliferative and antihyphal activity. Its mode of action was hypothesized to involve inhibition of a cAMP-dependent PKA pathway. The originally proposed structure appeared to require a polyketide assembled in a somewhat unusual fashion. However, structural characterization data were never formally published. This background stimulated a reinvestigation in which campafungin A and three closely related minor constituents were purified from fermentations of a strain of the ascomycete fungus Plenodomus enteroleucus. Labeling studies, along with extensive NMR analysis, enabled assignment of a revised structure consistent with conventional polyketide synthetic machinery. The structure elucidation of campafungin A and new analogues encountered in this study, designated here as campafungins B, C, and D, is presented, along with a proposed biosynthetic route. The antimicrobial spectrum was expanded to methicillin-resistant Staphylococcus aureus, Candida tropicalis, Candida glabrata, Cryptococcus neoformans, Aspergillus fumigatus, and Schizosaccharomyces pombe, with MICs ranging as low as 4-8 µg mL-1 in C. neoformans. Mode-of-action studies employing libraries of C. neoformans mutants indicated that multiple pathways were affected, but mutants in PKA/cAMP pathways were unaffected, indicating that the mode of action was distinct from that observed in C. albicans.

Sphaerostilbellins, New Antimicrobial Aminolipopeptide Peptaibiotics from Sphaerostilbella toxica.

Sphaerostilbella toxica is a mycoparasitic fungus that can be found parasitizing wood-decay basidiomycetes in the southern USA. Organic solvent extracts of fermented strains of S. toxica exhibited potent antimicrobial activity, including potent growth inhibition of human pathogenic yeasts Candida albicans and Cryptococcus neoformans, the respiratory pathogenic fungus Aspergillus fumigatus, and the Gram-positive bacterium Staphylococcus aureus. Bioassay-guided separations led to the purification and structure elucidation of new peptaibiotics designated as sphaerostilbellins A and B. Their structures were established mainly by analysis of NMR and HRMS data, verification of amino acid composition by Marfey's method, and by comparison with published data of known compounds. They incorporate intriguing structural features, including an N-terminal 2-methyl-3-oxo-tetradecanoyl (MOTDA) residue and a C-terminal putrescine residue. The minimal inhibitory concentrations for sphaerostilbellins A and B were measured as 2 µM each for C. neoformans, 1 µM each for A. fumigatus, and 4 and 2 µM, respectively, for C. albicans. Murine macrophage cells were unaffected at these concentrations.

Photolysis of Trenbolone Acetate Metabolites in the Presence of Nucleophiles: Evidence for Metastable Photoaddition Products and Reversible Associations with Dissolved Organic Matter.

Photolysis of trenbolone acetate (TBA) metabolites in the presence of various nitrogen-, sulfur-, or oxygen-containing nucleophiles (e.g., azide, ammonia, or thiosulfate, respectively) results in rapid (half-lives ∼20-60 min), photochemically induced nucleophile incorporation across the parent steroid's trienone moiety. The formation of such nucleophile adducts limits formation of photohydrates, suggesting competition between the nucleophile and water for photochemical addition into the activated steroid structure. Analogous to previously reported photohydration outcomes, LC/MS analyses suggest that such photonucleophilic addition reactions are reversible, with more rapid elimination rates than thermal dehydration of photohydrates, and regenerate parent steroid structures. Beyond photonucleophilic addition pathways, we also found that hydroxylamine and presumed nucleophilic moieties in model dissolved organic matter (DOM; Fluka humic acid) can react via thermal substitution with TBA metabolite photohydrates, although this reaction with model DOM was only observed for photohydrates of trendione. Most nucleophile addition products [i.e., formed via (photo)reaction with thiosulfate, hydroxylamine, and ammonia] are notably more polar relative to the parent metabolite and photohydration products. Thus, if present, both nucleophilic adducts and bound residues in organic matter will facilitate transport and help mask detection of TBA metabolites in surface waters and treatment systems.

Arenicolins: C-Glycosylated Depsides from Penicillium arenicola.

During investigation of the secondary metabolism of four strains of Penicillium arenicola, two new depsides, arenicolins A (1) and B (2), were isolated and characterized. Their structures were established mainly by analysis of NMR and HRMS data and by comparison with known compounds. These depsides incorporate intriguing structural features, including dual alkyl side chains and a C-glycosyl unit, with 1 also containing an acylated 2-hydroxymethyl-4,5,6-trihydroxycyclohexenone moiety. Although the arenicolins were produced by all strains tested, arenicolin A (1) was obtained using only one of five medium compositions employed, while arenicolin B (2) was produced in all media tested. Neither compound showed antibacterial or antifungal activity, but 1 exhibited cytotoxicity toward mammalian cell lines, including colorectal carcinoma (HCT-116), neuroblastoma (IMR-32), and ductal carcinoma (BT-474), with IC50 values of 7.3, 6.0, and 9.7 µM, respectively.

Gakolanone: a new benzophenone derivative from Garcinia kola Heckel stem-bark.

Gakolanone (3',5'-digeranyl-2',4',6',3-tetrahydroxybenzophenone; 1), a novel benzophenone derivative was isolated from the hexane extract of Garcinia kola Heckel stem-bark along with three known 3-8'' linked biflavonoids: 3'',4',4''',5,5'',7,7''-heptahydroxy-3,8''-biflavanone (2); 3'',4',5,5'',5''',7,7''-heptahydroxy-4-methoxy-3,8''-biflavanone (3) and 4',4''',5,5'',7,7''-hexahydroxy-3,8''-biflavanone (4) from the ethanol extract. The compounds were characterized primarily using 1 D and 2 D nuclear magnetic resonance spectroscopy and mass spectrometry and by comparing with literature. The compounds were subjected to in-vitro alpha-amylase enzyme inhibitory assay using DNSA (3,5-dinitrosalicylic acid) reagent with acarbose used as the standard drug. All the compounds were found to show alpha-amylase inhibitory activities with IC50 of 21.4 ± 1.5, 9.9 ± 0.2, 15.3 ± 2.3, 12.9 ± 2.3 µg/mL respectively. All the compounds exhibited better alpha-amylase inhibitory activities than the standard drug, acarbose (IC50= 38.1 ± 8.3 µg/mL).

Intramolecular [2 + 2] Photocycloaddition of Altrenogest: Confirmation of Product Structure, Theoretical Mechanistic Insight, and Bioactivity Assessment.

While studying the environmental fate of potent endocrine-active steroid hormones, we observed the formation of an intramolecular [2 + 2] photocycloaddition product (2) with a novel hexacyclic ring system following the photolysis of altrenogest (1). The structure and absolute configuration were established by X-ray diffraction analysis. Theoretical computations identified a barrierless two-step cyclization mechanism for the formation of 2 upon photoexcitation. 2 exhibited progesterone, estrogen, androgen, and pregnane X receptor activity, albeit generally with reduced potency relative to 1.

Bioactive Rearrangement Products from Aqueous Photolysis of Pharmaceutical Steroids.

In an ongoing effort to study the environmental fate of endocrine-active steroid hormones, we report the formation of phenolic rearrangement products (3 and 4) with a novel 6,5,8,5-ring system following aqueous photolysis of dienogest (1) and methyldienolone (2). The structures were established by analysis of 2D NMR and HRMS data, and that of 3 was confirmed by X-ray diffraction analysis. These photoproducts exhibit progestogenic and androgenic activity, albeit with less potency than their parent compounds.

Wortmannin and Wortmannine Analogues from an Undescribed Niesslia sp.

In the course of our studies of coprophilous fungi as sources of antifungal agents, a strain of an undescribed species in the genus Niesslia (TTI-0426) was isolated from horse dung collected in Texas. An extract from fermentation cultures of this strain afforded two new antifungal wortmannin derivatives, wortmannins C and D (1 and 2), as well as four additional new related compounds, wortmannines B1-B4 (3-6), containing an unusual ring system. The structures of these metabolites were established mainly by analysis of HRESIMS and 2D NMR data. Relative configurations were assigned using NOESY data, and the structure assignments were supported by NMR comparison with similar compounds. Wortmannins C and D showed activity against Cryptococcus neoformans and Candida albicans in disk assays, but low MIC potency observed for 1 was suggested to be due in part to efflux processes on the basis of assay results for a Schizosaccharomyces pombe efflux mutant in comparison to wild-type.

Identification of cyclosporin C from Amphichorda felina using a Cryptococcus neoformans differential temperature sensitivity assay.

We used a temperature differential assay with the opportunistic fungal pathogen Cryptococcus neoformans as a simple screening platform to detect small molecules with antifungal activity in natural product extracts. By screening of a collection extracts from two different strains of the coprophilous fungus, Amphichorda felina, we detected strong, temperature-dependent antifungal activity using a two-plate agar zone of inhibition assay at 25 and 37 °C. Bioassay-guided fractionation of the crude extract followed by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) identified cyclosporin C (CsC) as the main component of the crude extract responsible for growth inhibition of C. neoformans at 37 °C. The presence of CsC was confirmed by comparison with a commercial standard. We sequenced the genome of A. felina to identify and annotate the CsC biosynthetic gene cluster. The only previously characterized gene cluster for the biosynthesis of similar compounds is that of the related immunosuppressant drug cyclosporine A (CsA). The CsA and CsC gene clusters share a high degree of synteny and sequence similarity. Amino acid changes in the adenylation domain of the CsC nonribosomal peptide synthase's sixth module may be responsible for the substitution of L-threonine compared to L-α-aminobutyric acid in the CsA peptide core. This screening strategy promises to yield additional antifungal natural products with a focused spectrum of antimicrobial activity.

Isolation, Synthesis, and Radical-Scavenging Activity of Rhodomelin A, a Ureidobromophenol from the Marine Red Alga Rhodomela confervoides.

A novel ureidobromophenol, rhodomelin A (1), was characterized from Rhodomela confervoides. Its structure was elucidated by spectroscopic analysis. Both enantiomers of 1 were synthesized using a convergent strategy starting from d/l-pyroglutamic acids, respectively, allowing assignment of the R-configuration for the naturally occurring isomer by chiral HPLC analysis. Rhodomelin A represents the first example of a naturally occurring ureidopyrrolidone alkaloid incorporating a γ-aminobutyric acid unit. The scavenging activity of 1 toward DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate)) radicals was assayed.

Environmental photochemistry of dienogest: phototransformation to estrogenic products and increased environmental persistence via reversible photohydration.

Potent trienone and dienone steroid hormones undergo a coupled photohydration (in light)-thermal dehydration (in dark) cycle that ultimately increases their environmental persistence. Here, we studied the photolysis of dienogest, a dienone progestin prescribed as a next-generation oral contraceptive, and used high resolution mass spectrometry and both 1D and 2D nuclear magnetic resonance spectroscopy to identify its phototransformation products. Dienogest undergoes rapid direct photolysis (t1/2 ∼ 1-10 min), forming complex photoproduct mixtures across the pH range examined (pH 2 to 7). Identified products include three photohydrates that account for ∼80% of the converted mass at pH 7 and revert back to parent dienogest in the absence of light. Notably, we also identified two estrogenic compounds produced via the A-ring aromatization of dienogest, evidence for a photochemically-induced increase in estrogenic activity in product mixtures. These results imply that dienogest will undergo complete and facile photolytic transformation in sunlit surface water, yet exhibit greater environmental persistence than might be anticipated by inspection of kinetic rates. Photoproduct mixtures also include transformation products with different nuclear receptor binding capabilities than the parent compound dienogest. These outcomes reveal a dynamic fate and biological risk profile for dienogest that must also take into account the composition and endocrine activity of its transformation products. Collectively, this study further illustrates the need for more holistic regulatory, risk assessment, and monitoring approaches for high potency synthetic pharmaceuticals and their bioactive transformation products.

Anti-Cryptococcus Phenalenones and Cyclic Tetrapeptides from Auxarthron pseudauxarthron.

Auxarthrones A-E (1-5), five new phenalenones, and two new naturally occurring cyclic tetrapeptides, auxarthrides A (7) and B (8), were obtained from three different solvent extracts of cultures of the coprophilous fungus Auxarthron pseudauxarthron. Auxarthrones C (3) and E (5) possess an unusual 7a,8-dihydrocyclopenta[a]phenalene-7,9-dione ring system that has not been previously observed in natural products. Formation of 1-5 was found to be dependent on the solvent used for culture extraction. The structures of these new compounds were elucidated primarily by analysis of NMR and MS data. Auxarthrone A (1) was obtained as a mixture of chromatographically inseparable racemic diastereomers (1a and 1b) that cocrystallized, enabling confirmation of their structures by X-ray crystallography. The absolute configurations of 7 and 8 were assigned by analysis of their acid hydrolysates using Marfey's method. Compound 1 displayed moderate antifungal activity against Cryptococcus neoformans and Candida albicans, but did not affect human cancer cell lines.

Benzophenone and Fimetarone Derivatives from the Coprophilous Fungus Delitschia confertaspora.

Studies of the genome-sequenced, flutimide-producing coprophilous fungus Delitschia confertaspora (ATCC 74209), originally obtained from a sample of rock hyrax (Procavia capensis) dung collected in Namibia, led to the discovery of three new highly aromatic natural products named delicoferones A-B (1-2) and fimetarone B (3). The new benzophenone derivatives 1 and 2 have a somewhat unusual skeleton that incorporates three aromatic rings linked via two ketone carbonyl groups, while 3 contains a spiro[chroman-3,7'-isochromene]-4,6'(8'H) skeleton reported only once previously. The structures of these compounds were assigned mainly by analysis of 2D NMR and HRESITOFMS data.

Biologically Active Secondary Metabolites from the Fungi.

Many Fungi have a well-developed secondary metabolism. The diversity of fungal species and the diversification of biosynthetic gene clusters underscores a nearly limitless potential for metabolic variation and an untapped resource for drug discovery and synthetic biology. Much of the ecological success of the filamentous fungi in colonizing the planet is owed to their ability to deploy their secondary metabolites in concert with their penetrative and absorptive mode of life. Fungal secondary metabolites exhibit biological activities that have been developed into life-saving medicines and agrochemicals. Toxic metabolites, known as mycotoxins, contaminate human and livestock food and indoor environments. Secondary metabolites are determinants of fungal diseases of humans, animals, and plants. Secondary metabolites exhibit a staggering variation in chemical structures and biological activities, yet their biosynthetic pathways share a number of key characteristics. The genes encoding cooperative steps of a biosynthetic pathway tend to be located contiguously on the chromosome in coregulated gene clusters. Advances in genome sequencing, computational tools, and analytical chemistry are enabling the rapid connection of gene clusters with their metabolic products. At least three fungal drug precursors, penicillin K and V, mycophenolic acid, and pleuromutilin, have been produced by synthetic reconstruction and expression of respective gene clusters in heterologous hosts. This review summarizes general aspects of fungal secondary metabolism and recent developments in our understanding of how and why fungi make secondary metabolites, how these molecules are produced, and how their biosynthetic genes are distributed across the Fungi. The breadth of fungal secondary metabolite diversity is highlighted by recent information on the biosynthesis of important fungus-derived metabolites that have contributed to human health and agriculture and that have negatively impacted crops, food distribution, and human environments.

Emestrins: Anti-Cryptococcus Epipolythiodioxopiperazines from Podospora australis.

Eleven emestrin-type epipolythiodioxopiperazines, including four new compounds, emestrins H-K (1-4), were isolated from the crude extracts of two strains of the coprophilous fungus Podospora australis. The structures of 1-4 were established primarily by analysis of NMR data, and the absolute configuration of C-6 in 1 was independently assigned using the modified Mosher method. Four of the known emestrins obtained (emestrins C-E and MPC1001C) were found to selectively inhibit the growth of Cryptococcus neoformans. These results also represent the first report of chemistry from any strain of P. australis.

Haenamindole and fumiquinazoline analogs from a fungicolous isolate of Penicillium lanosum.

Three amino acid-derived compounds, haenamindole (1) and 2'-epi-fumiquinazolines C (2) and D (3), were isolated from cultures of a fungicolous isolate of Penicillium lanosum (MYC-1813=NRRL 66231). Compound 1 was also encountered in cultures of P. corylophilum (MYC-418=NRRL 28126). Structure elucidation of these metabolites was based mainly on high resolution mass spectrometry and NMR data analysis. Haenamindole (1) was found to be a recently reported diketopiperazine-type metabolite that incorporates an unusual ß-Phe unit. Analysis of X-ray crystallographic data and the products of acid hydrolysis of 1 enabled a conclusive, slightly modified stereochemical assignment for haenamindole. Fumiquinazoline analog 2 is a new natural product, while related compound 3 has been previously reported only as a product of an in vitro enzymatic step and of a genetically engineered fungal culture. Compounds 1 and 3 showed antiinsectan activity against the fall armyworm Spodoptera frugiperda.

Effect of atmospheric carbon dioxide levels and nitrate fertilization on glucosinolate biosynthesis in mechanically damaged Arabidopsis plants.

BACKGROUND: Increased atmospheric carbon dioxide (CO2) levels predicted to occur before the end of the century will impact plant metabolism. In addition, nitrate availability will affect metabolism and levels of nitrogen-containing defense compounds, such as glucosinolates (GSLs). We compared Arabidopsis foliar metabolic profile in plants grown under two CO2 regimes (440 vs 880 ppm), nitrate fertilization (1 mM vs 10 mM) and in response to mechanical damage of rosette leaves. RESULTS: Constitutive foliar metabolites in nitrate-limited plants show distinct global patterns depending on atmospheric CO2 levels; in contrast, plants grown under higher nitrate fertilization under elevated atmospheric CO2 conditions have a unique metabolite signature. Nitrate fertilization dampens the jasmonate burst in response to wounding in plants grown at elevated CO2 levels. Leaf GSL profile mirrors the jasmonate burst; in particular, indole GSLs increase in response to damage in plants grown at ambient CO2 but only in nitrate-limited plants grown under elevated CO2 conditions. CONCLUSIONS: This may reflect a reduced capacity of C3 plants grown under enriched CO2 and nitrate levels to signal changes in oxidative stress and has implications for future agricultural management practices.