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LC-MS- and H NMR Spectroscopy-Guided Identification of Antifungal Diterpenoids from Sagittaria latifolia

Ravu, Ranga Rao, Jacob, Melissa R., Jeffries, Cynthia, Tu, Ying, Khan, Shabana I., Agarwal, Ameeta K., Guy, R. Kiplin, Walker, Larry A., Clark, Alice M., Li, Xing-Cong
Journal of natural products 2015 v.78 no.9 pp. 2255-2259
Cryptococcus gattii, Cryptococcus neoformans, Sagittaria latifolia, acid hydrolysis, animal pathogenic fungi, antifungal agents, chemical structure, chemotypes, diterpenoids, drugs, fractionation, inhibitory concentration 50, liquid chromatography, mass spectrometry, nuclear magnetic resonance spectroscopy, plant extracts, screening, spectral analysis
A dereplication strategy using a combination of liquid chromatography-mass spectrometry (LC-MS) and proton nuclear magnetic resonance spectroscopy (1H NMR) to facilitate compound identification towards antifungal natural product discovery is presented. This analytical approach takes advantage of the simplicity of semi-purified column fractions (CFs) of plant extracts, which were generated by an automated high throughput fractionation approach. These CFs contain only a few chromatographically tractable natural product compounds, which in most cases are the same chemotype of analogs, thus making it easy to interpret the LC-MS and 1H NMR data for structure identification. Antifungal screening showed that a CF derived from the plant extract of Sagittaria latifolia was active against the clinically important opportunistic fungal pathogen Cryptococcus neoformans with an IC50 value of 12.7 µg/mL, whereas the parent extract was inactive at 50 µg/mL, highlighting the importance of activity enrichment and the increased discovery potential by this fractionation approach. Dereplication analysis by LC-MS and 1H NMR indicated the presence of new compounds in this CF. In particular, 1H NMR played an important role in complementing LC-MS to rapidly characterize the structural type of such compounds. Subsequent fractionation of the plant extract resulted in the identification of two new isopimaradiene type of diterpenoids 1 and 2. The structures of 1 and 2 were determined by chemical methods and spectroscopic analysis as isopimara-7,15-dien-19-ol 19-O-a-L-arabinofuranoside and isopimara-7,15-dien-19-ol 19-O-a-L-(5'-acetoxy)arabinofuranoside, respectively. Compound 1 exhibited IC50s of 8.9 and 4.4 µM, respectively, against C. neoformans and Cryptococcus gattii. Its aglycone, isopimara-7,15-dien-19-ol (3), resulting from acid hydrolysis of 1 was also active against the two fungal pathogens with IC50s of 31.8 and 23.9 µM, respectively. This study demonstrates that utilization of the combined LC-MS and 1H NMR analytical tools is an improved chemical screening approach for hit prioritization in natural product drug discovery.