Isolation and on-line identification of marcfortines by HPLC-DAD-MS/MS

Marcfortines are a group of anti-parasitic compounds derived from fungi [1]. Salt of marcfortine A (1) and marcfortines B (2) and C (3) were isolated from the rice culture of the endophytic fungus Cladosporium sphaerospermum. According to the biological importance and chemical complexity of marcfortines, this study was conducted to monitor this class of compounds using HPLC-DAD-MS/MS techniques. Compounds 1–3 and MeOH extract were solubilized in 4:1 acetonitrile-H₂O solution and injected in HPLC equipped with two monolithic columns (C-18 Onyx™ 100×4.6mm) in line. A gradient method was adopted using H₂O-acetic acid 1% (phase A) and acetonitrile-acetic acid 1% (phase B), 15% of phase B in 5min., 15–50% of phase B in 5–20min. and more 5min. to return the initial conditions and stabilize the column, at 3 mL/min flow rate. The first elimination observed in MS/MS high resolution spectra of all compounds occurs at position 18 (CO group). The subsequent eliminations involve loss of NCH₅ for 1, and NH₃ for both 2 and 3. Four common ions m/z 188.1463 (C₁₃H₁₈N), m/z 176.1449 (C₁₂H₁₈N), m/z 174.1297 (C₁₂H₁₆N) and m/z 161.1213 (C₁₁H₁₅N) were observed for all the marcfortines. Therefore, these peaks make it possible to monitor this class of compounds. The presence of ion m/z 462.2728, and theirs fragments in MS/MS mode: m/z 434.2765 [M-CO]⁺; m/z 403.2367 [M-CO-NCH₅]⁺; m/z 188.1457; m/z 176.1450; m/z 174.1295; and m/z 161.1210, indicated the presence of marcfortine D (4). This is the first report of 4 from natural products sources, and also the first report of the isolation of the salt of marcfortine A (1) (identified by 2D NMR).

Acknowledgements: Fapesp, BIOprospecTA-Biota.

References: 1. Lee, B. H. et al. (2002) Curr. Top. Med. Chem. 2:779.