Biotransformation of danazol by Fusarium solani and Gibberella fujikuorii..., AZIZUDDIN, M. I. CHOUDHARY
Tokyo, Japan) kindly gifted a specific inhibitor of PEP, N -benzyloxycarbonyl-pro-prolinal.
Preparation of Fermentation Media: Two liters of medium for F. solani (ATCC 12823) was prepared
by mixing glucose (20 g), peptone (10 g), KH2 PO4 (10 g), yeast extract (10 g), glycerol (20 mL), and NaCl
(10 g). Medium of G. fujikuorii (ATCC 10704) was prepared by mixing glucose (160 g), NH4 NO3 (1 g),
KH2 PO4 (10 g), MgSO4 .7H2 O (2 g), and Giberella trace element solution (4 mL) into distilled water (2 L).
The fermentation medium thus obtained was distributed equally among 20 flasks of 250 mL capacity (100 mL
in each) and autoclaved.
Cultivation of the Microbes: Two-day-old spores of the both microbes were transferred into the broth
medium flasks (250 mL) of their respective media containing freshly prepared and autoclaved media (100 mL).
The seed flasks of the fungi were incubated on a shake table at 30 ◦ C for 2 days.
Inoculation of the Cultures: Broth cultures (100 mL) from 2-day-old seed flasks of the fungi were
equally distributed to 18 media flasks (250 mL) containing the respective media (100 mL). The incubation was
continued for a further 2 days for fungi.
Fermentation of Danazol (1): Danazol (1) (200 mg) was dissolved in DMSO (10 mL) and the
resulting solution was evenly distributed among 18 conical flasks containing shake cultures of microbes, and the
fermentation was continued for 12 days.
Extraction and Isolation: Each mycelium was filtered, washed with EtOAc (500 mL), and the broth
thus obtained was extracted with EtOAc (6 L). The ethyl extract was dried over anhydrous sodium sulfate
and concentrated in vacuo to afford a brown gum (approximately, 1 g for each fungi), which was adsorbed on
flash silica gel (3 g), subjected to column chromatography. Elution with EtOAc:pet. ether (5:5) afforded 2 and
EtOAc:pet. ether (6:4) afforded 3.
Prolyl Endopeptidase Inhibition Assay: PEP inhibition activity was assayed by a modified method
of Yoshimoto et al.;11 100 mM Tris (hydroxymethyl)-aminomethane HCl buffer containing 1 mM EDTA (pH
7.0, 247 μL), PEP (0.02 units/well) 15 μL, and a stock solution of the test compound in MeOH (8 μL, diluted
to the desired range of concentrations) were mixed in 96-well microplate and preincubated for 10 min at 30
◦ C. The reaction was initiated by adding 30 μL of 0.2 mM of N -benzyloxycarbonyl-Gly-Pro-pNA (in 40%
1,4-dioxane) as the substrate. The amount of released p-nitroaniline was measured spectrophotometrically, as
increase in absorption at 410 nm with a 96-well microplate reader (Molecular Devices, SpectraMax 340, USA).
The percentage inhibition was calculated by the following equation:
% Inhibition = 100 – [(O.D. of test compound / O.D. of control) × 100]
The potency of enzyme inhibitory activity was represented by the IC50 values, which were defined as the
concentration of the test compound that resulted in 50% inhibition of the enzyme with respect to the MeOH
control. Z -pro-prolinal was used as a positive control.
Results
17β-Hydroxy-2-(hydroxymethyl)-17α-pregn-4-en-20-yn-3-one (2): Colorless crystalline solid, mp 164-
165 ◦ C, [α]D25 –20◦ (c 0.1, CHCl3). UV (MeOH) λmax (log ε): 242 nm (4.16). IR (CHCI3)νmax : 3412 (OH),
3308 (OH), 1654 (C=O), 1620 cm−1 (C=C). FDMS m/z 342 [M+ , 100%]. EIMS m/z (rel. int. %): 342
947