Isolation and Purification of Bioactive Molecules from P. luteoviolacea

Molecules 2–3 and 15 were isolated from extracts of P. luteoviolacea 2ta16. 10×100 mL cultures were grown as before and incubated for 24 h at 30 °C with shaking at 200 rpm. Cultures were pooled and extracted with an equal volume of ethyl acetate. The organic layer was dried with anhydrous MgSO₄, filtered and solvent was removed in vacuo. The residue was dissolved in 5 mL MeOH and filtered through a 0.2 µM vacuum filter. 2–3, and 15 were purified by semi-preparative scale HPLC using a reverse phase C₁₈ column (Phenomenex, 250 mm). Solvents used were water + 0.1% TFA (A) and MeCN + 0.1% TFA (B). An identical elution profile as described before for analytical scale experiments but at an increased flow rate of 2 mL/min was used for all semi-preparative purification experiments in this study. Guided by LC-MS/MS, fractions containing 2–3, and 15 were lyophilized. 500 µg of each product was dissolved in 50 µL CDCl₃. ¹H NMR, and ¹³C NMR spectra for 2 were obtained using a 600 MHz Varian NMR microprobe. Spectra of 3 and 15 were verified with literature values^{40 (link)}.