RESUMO
The endophytic fungus Pestalotiopsis virgatula, derived from the plant Terminalia chebula and previously found to produce a large excess of a single metabolite when grown in the minimal M1D medium, was induced to produce a variety of unusual metabolites by growing in potato dextrose broth medium. Analysis of the fermentation medium extract was performed using an HPLC-PDA-MS-SPE-NMR hyphenated system, which led to the identification of a total of eight metabolites (1-8), six of which are new. Most of the metabolites are structurally related and are derivatives of benzo[c]oxepin, rare among natural products. This includes dispiro derivatives 7 and 8 (pestalospiranes A and B), having a novel 1,9,11,18-tetraoxadispiro[6.2.6.2]octadecane skeleton. Relative and absolute configurations of the latter were determined by a combination of NOESY spectroscopy and electronic circular dichroism spectroscopy supported by time-dependent density-functional theory calculations (B3LYP/TZVP level). This work demonstrates that a largely complete structure elucidation of numerous metabolites present in a raw fermentation medium extract can be performed by the HPLC-SPE-NMR technique using only a small amount of the extract, even with unstable metabolites that are difficult to isolate by traditional methods.
Assuntos
Benzoxepinas/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Dicroísmo Circular/métodos , Endófitos/química , Espectroscopia de Ressonância Magnética/métodos , Compostos de Espiro/isolamento & purificação , Benzoxepinas/química , Estrutura Molecular , Compostos de Espiro/química , Terminalia/microbiologia , Fatores de TempoRESUMO
The extracts of five foliar fungal endophytes isolated from Pinus strobus (eastern white pine) that showed antifungal activity in disc diffusion assays were selected for further study. From these strains, the aliphatic polyketide compound 1 and three related sesquiterpenes 2-4 were isolated and characterized. Compound 2 is reported for the first time as a natural product and the E/Z conformational isomers 3 and 4 were hitherto unknown. Additionally, the three known macrolides; pyrenophorol (5), dihydropyrenophorin (6), and pyrenophorin (7) were isolated and identified. Their structures were elucidated by spectroscopic analyses including 2D NMR, HRMS and by comparison to literature data where available. The isolated compounds 1, 2, and 5 were antifungal against both the rust Microbotryum violaceum and Saccharomyces cerevisae.
Assuntos
Antifúngicos/química , Ascomicetos/química , Macrolídeos/química , Pinus/microbiologia , Policetídeos/química , Sesquiterpenos/química , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Sequência de Bases , Basidiomycota/efeitos dos fármacos , Endófitos/química , Compostos Heterocíclicos/química , Compostos Heterocíclicos/isolamento & purificação , Compostos Heterocíclicos/farmacologia , Cetonas/química , Cetonas/isolamento & purificação , Cetonas/farmacologia , Lactonas/química , Lactonas/isolamento & purificação , Macrolídeos/isolamento & purificação , Macrolídeos/farmacologia , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Folhas de Planta/química , Policetídeos/isolamento & purificação , Policetídeos/farmacologia , Saccharomyces/efeitos dos fármacos , Análise de Sequência de DNA , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/farmacologiaRESUMO
HPLC-SPE-NMR analysis of a crude extract of fermentation broth of cultured PESTALOTIOPSIS VIRGATULA isolate TC-320 from TERMINALIA CHEBULA Retz. (Combretaceae) disclosed the presence of a simple but unprecedented low-molecular-weight metabolite, 9-hydroxybenzo[ C]oxepin-3[1 H]-one, subsequently isolated by a targeted purification procedure.
Assuntos
Ascomicetos/química , Benzoxepinas/química , Cromatografia Líquida de Alta Pressão/métodos , Espectroscopia de Ressonância Magnética/métodosRESUMO
Direct hyphenation of analytical-scale high-performance liquid chromatography, photo-diode array detection, mass spectrometry, solid-phase extraction and nuclear magnetic resonance spectroscopy (HPLC-PDA-MS-SPE-NMR) has been used for accelerated dereplication of crude extract of Haplophyllum acutifolium (syn. Haplophyllum perforatum). This technique allowed fast on-line identification of six quinolinone alkaloids, named haplacutine A-F, as well as of acutine, haplamine, eudesmine, and 2-nonylquinolin-4(1H)-one. Acutine and haplacutine E, isolated by preparative-scale HPLC, showed moderate antiplasmodial activity with IC(50) values of 2.17+/-0.22 microM and 3.79+/-0.24 microM, respectively (chloroquine-sensitive Plasmodium falciparum 3D7 strain).
Assuntos
Alcaloides/isolamento & purificação , Quinolonas/isolamento & purificação , Rutaceae/química , Alcaloides/química , Animais , Cloroquina/farmacologia , Cromatografia Líquida de Alta Pressão , Resistência a Medicamentos/efeitos dos fármacos , Irã (Geográfico) , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Plasmodium falciparum/efeitos dos fármacos , Quinolonas/químicaRESUMO
The hyphenated technique HPLC-PDA-MS-SPE-NMR was used to assist targeted preparative-scale isolation of constituents of Lobelia siphilitica and Hippobroma longiflora (both Campanulaceae). This resulted in the isolation of two new alkaloids, (S)-2-[(2S,6R)-1-methyl-6-(2-oxo-2-phenylethyl)piperidin-2-yl]-1-phenylethyl acetate (3) and 6-[(E)-2-(3-methoxyphenyl)ethenyl]-2,3,4,5-tetrahydropyridine (4), the latter possessing a skeleton hitherto unseen among alkaloids of Lobelia and related genera. Lobeline (1), (1S,1'S)-2,2'-[(2R,6S)-1-methylpiperidine-2,6-diyl]bis(1-phenylethanol) (2), and lobetyolin (5) were also isolated. The structures of 1-5 were established using spectroscopic methods including homo- and heteronuclear two-dimensional NMR experiments and optical rotation data.