Production of Unusual Dispiro Metabolites inPestalotiopsis virgatulaEndophyte Cultures: HPLC-SPE-NMR, Electronic Circular Dichroism, and Time-Dependent Density-Functional Computation Study

Abstract

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.

Structural Diversity and Biological Activities of Novel Secondary Metabolites from Endophytes

“…They are new members of the rare benzannulated 6,6-spiroketal class of natural products and possess one or two γ -lactone units, representing the first occurrence of the γ -lactone units in the benzannulated 6,6-spiroketals. Jaroszewski and co-workers employed a hyphenated technique comprising HPLC-SPE-NMR to uncover some novel metabolites from Pestalotiopsis virgatula , an endophyte inhabiting the bark of Terminalia chebula [ 42 , 43 ]. Among them, pestalospiranes A and B ( 30 and 31 , Figure 5 ) have an unprecedented 1,9,11,18-tetraoxadispiro[6.2.6.2]octadecane skeleton in addition to the characteristic benzo[ c ]-oxepin motif [ 43 , 44 ].…”

“…Jaroszewski and co-workers employed a hyphenated technique comprising HPLC-SPE-NMR to uncover some novel metabolites from Pestalotiopsis virgatula , an endophyte inhabiting the bark of Terminalia chebula [ 42 , 43 ]. Among them, pestalospiranes A and B ( 30 and 31 , Figure 5 ) have an unprecedented 1,9,11,18-tetraoxadispiro[6.2.6.2]octadecane skeleton in addition to the characteristic benzo[ c ]-oxepin motif [ 43 , 44 ]. A bioinspired tandem dimerization-spiroketalization strategy to forge the unique dispiro skeleton of 31 has recently been described ( Scheme 2 ) [ 44 ].…”

NMR-based metabolomics: From sample preparation to applications in nutrition research

“…Despite the clear advantages of LC-NMR, it has not become routine in metabolomic analysis. More recent applications using liquid chromatography solid phase extraction hyphenated to NMR have demonstrated nicely the power of the technique in metabolite identification [19][20][21].…”

Bioactive potential of endophyticMyrotheciumsp. isolate M1-CA-102, associated withCalophyllum apetalum

“…Phongpaichit et al (2006) reported that some of the medicinal properties of host plants have also been exhibited by endophytic fungi. The fungal endophytes exist in the intercellular space of plant tissues, which are capable of producing a large number of natural products (Kesting et al, 2011).…”