Pure filipin 3

To assay filipin in culture broths, 1 ml of culture was extracted with 1 ml of methanol, and further diluted with methanol to bring the absorbance at 338 nm in the range of 0.1 to 0.4 units. Control solutions of pure filipin III (Sigma) were used as control. To confirm the identity of filipin, an UV-visible absorption spectrum (absorption peaks at 356, 338, 320 and 311 nm) was routinely determined in a Hitachi U-2000 spectrophotometer. Quantitative determination of filipin was performed as previously described [18 (link)], using a Mediterranea Sea C18 column (4.6x150 mm, particle size, 3 mm) (Teknokroma).

Filipin and antimycin A production was assessed after growth at 30°C in YEME medium without sucrose. To assay filipin in culture broths, 1 volume of culture was extracted with 1 volume of methanol and was further diluted with methanol to bring the absorbance at 338 nm into the range of 0.1 to 0.4 units. Solutions of pure filipin III (Sigma) were used as controls. The identity of filipin was confirmed by analysis of its UV-visible absorption spectrum (absorption peaks at 356, 338, 320, and 311 nm). Quantitative determination of filipin was performed as previously described (26 (link)), using a Mediterranea Sea C₁₈ column (Teknokroma) (4.6 by 150 mm; particle size, 3 mm). For antimycin A production assessment, 1 volume of culture was extracted with 2 volumes of ethyl acetate and was dried by rotary evaporation. The pellet was then resuspended in methanol prior to HPLC analysis. The same chromatographic method was used for estimation of antimycin A production at 318 nm. Pure antimycin A (Sigma) was used as the standard.

To determine filipins production and metabolite purification, one volume of culture was extracted with two volumes of ethyl acetate, and the organic phase was sequentially treated with saturated NaCl solution and Na₂SO₄, vacuum dried, and resuspended in pure methanol. For routine determination of metabolite yield, cultures were extracted with one volume of methanol, and diluted when needed. Solutions of pure filipin III (Sigma) were used as control. Quantitative determination of filipins was assessed by reverse phase HPLC using a Waters 600 unit coupled to a diode array ultraviolet detector set at 338 nm equipped with a Mediterranean Sea C18 column (4.6 × 150 mm, particle size 3 µm) (Teknokroma). Elution was performed with a gradient (0.8 ml/min) of methanol-0.1% formic acid according to the following program (50:50 v/v 0–3 min, up to 90:10 v/v 3–12 min, 90:10 12–20 min, up to 100:0 v/v 20–21 min, 100:0 v/v 21–23 min, down to 0:100 v/v 23–24 min, 0:100 v/v 24–26 min, up to 50:50 v/v 26–27 min, 50:50 v/v 27–32 min). Retention time for Filipin III was 18.2 min. Thin layer chromatography was performed on silica 60 F254 plates (Merck), and elution was carried out with dichloromethane:methanol (4:1) (v/v). Plates were developed by spraying with phosphomolybdic acid:ethanol (1:9) (v/v) and heating.