qNMR for profiling the production of fungal secondary metabolites

Abstract

Analysis of complex mixtures is a common challenge in natural products research. Quantitative nuclear magnetic resonance spectroscopy offers analysis of complex mixtures at early stages and with benefits that are orthogonal to more common methods of quantitation, including ultraviolet absorption spectroscopy and mass spectrometry. Several experiments were conducted to construct a methodology for use in analysis of extracts of fungal cultures. A broadly applicable method was sought for analysis of both pure and complex samples through use of an externally calibrated method. This method has the benefit of not contaminating valuable samples with the calibrant, and it passed scrutiny for line fitting and reproducibility. The method was implemented to measure the yield of griseofulvin and dechlorogriseofulvin from three fungal isolates. An isolate of Xylaria cubensis (coded MSX48662) was found to biosynthesize griseofulvin in the greatest yield, 149 ± 8 mg per fermentation, and was selected for further supply experiments. Copyright © 2016 John Wiley & Sons, Ltd.

Keywords: 1H NMR; NMR; fungi; griseofulvin; qNMR; secondary metabolites.

Figure 1

Structures of compounds 1–3.

Figure 2

Solid substrate fermentations of MSX54665, G536, and MSX48662.

Figure 3

Selected sections of the ¹H NMR spectra of MSX54665, G536 and MSX48662 at 2.0 mg/mL in DMSO-d₆. Given are the sections useful for quantitation of the methoxy groups. The DMSO-d₅ peak is also included for comparison. Spectra were collected on a JEOL ECA-500 operating at 500 MHz with 2 dummy scans followed by 8 scans.

Figure 4

Selected sections of the ¹H NMR spectra of MSX54665, G536 and MSX48662 at 10, 5.0, and 2.0 mg/mL, respectively, in DMSO-d₆. Given are the sections useful for quantitation of the methoxy groups. The DMSO-d₅ peak is also included for comparison. Peak height of spectra were normalized to the DMSO-d₅ peak. Spectra were collected on a JEOL ECA-500, operating at 500 MHz with 2 dummy scans followed by 8 scans.