Supernatant of N. spumigena (25 Litres) were applied to a reversed phase flash chromatography system
fitted with a C-18HS column (7.5 x 4.0 cm, i.d. 35-70 ƒÊm particle size, 60 . pore size). A step gradient of
10 to 100% aqueous methanol in 20% increments (500 mL per step) was used to elute different toxins
from the column and five fractions were collected. All fractions were dried under reduced pressure at
40‹ C and resuspended in 50 mL Milli-Q-Water and analyzed by HPLC. Column washing with 100%
methanol fraction showed interesting peak other than toxins. This fraction was passed through a C-18-HS
column (15 x 4.0 cm, i.d. 35-70 ƒÊm particle size, 60 A pore size). A step gradient of 10 to 100% aqueous
methanol in 10% increments (500 mL per step) was used to elute toxins from the column. The 90%
aqueous methanol fraction showed interesting peak on HPLC and was further purified by reversed phase
C18 HPLC column using the same solvent system with RI detector to yield 76 mg of compound 1 as
pale yellow liquid (Figure 1).