Acetone) was added towards the cultures. The progress of conversion was
Acetone) was added towards the cultures. The progress of conversion was monitored by TLC. Right after biotransformations, the metabolites and remaining substrate have been extracted with methylene chloride. The organic options were dried with anhydrous magnesium sulphate, filtered, concentrated in vacuo and analysed by GC. Within the PPARβ/δ Agonist supplier analytical scale biotransformations using chosen strains, 0.two g of 1 dissolved in 2 ml of acetone was equally distributed among flasks with fungal cultures. The reactions have been carried out under the exact same conditions as in screening tests and continued until the substrate was metabolized. The progress of conversion was monitored by TLC. When the transformation completed, mycelia and broth had been extracted 3 instances with methylene chloride. The organic extracts had been combined, dried more than anhydrous magnesium sulphate and filtered, and the solvent was evaporated in vacuo. These crude extracts were analysed by TLC and GC and then chromatographed on a column of silica gel. Merchandise evaluation TLC of crude extracts was carried out with Merck Kieselgel 60 F254 plates, visualized by spraying them using a mixture of methanol in concentrated sulphuric acid (1:1 v: v) and heating to 120 till the colours created. Metabolites obtained within the analytical transformations had been separated by column chromatography on silica gel 60 (23000 mesh) eluting with all the exact same eluent as for TLC. GC analysis was performed making use of Hewlett Packard 5890A Series II GC instrument (FID, carrier gas H2 at flow price of two ml min-1) with DB-5MS column (crosslinked phenyl methyl siloxane, 30 m 9 0.32 mm 9 0.25 lm). The applied temperature system was 220 1 min-1, NMDA Receptor Antagonist Compound gradient four min-1 to 280 and then 30 to 300 3 min-1; injector and detector temperature had been 300 (for L. sulphureus temperature program was 215 1 min-1, gradient four min-1 to 280 after which 30 to 300 3 min-1). MS analyses have been performed on Varian CP-3800/Saturn 2000 apparatus using a Zebron ZB-5 MSI (30 m 9 0.25 mm 9 0.25 lm) column. The following temperature program was made use of: 220 1 min-1, gradient five min-1 to 300 five min-1. The NMR spectra had been recorded on a Bruker AvanceTM 600 MHz2021 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley Sons Ltd., Microbial Biotechnology, 14, 2187Microbial transformations of 7-oxo-DHEA (62 mg; 31 mol.), recognized 3b,17b-dihydroxy-androst-5en-7-one (two) (30 mg; 15 mol.), and a new product characterized as 3b,16b-dihydroxy-androst-5-en-7,17dione (six) (57 mg; 27 mol., Rt = 19.4 min). 3b,16b-Dihydroxy-androst-5-en-7,17-dione (six): white amorphous solid; 1H NMR (CD3OD, 600 MHz) d: 0.96 (3H, s, H-18), 1.27 (3H, s, H-19), three.14-3.18 (1H, m, H15a), 3.54.60 (1H, m, H-3a), three.94 (1H, t, J = eight.five Hz, H-16a), 5.72 (1H, d, J = 1.7 Hz, H-6). 13C NMR (CD3OD, 151 MHz) d: 14.9 (CH3, C-18), 17.7 (CH3, C19), 21.four (CH2, C-11), 31.9 (CH2, C-2), 32.1 (CH2, C12), 34.5 (CH2, C-15), 37.four (CH2, C-1), 39.9 (C, C-10), 41.1 (CH, C-14), 42.8 (CH2, C-4), 44.7 (CH, C-8), 48.2 (C, C-13), 51.6 (CH, C-9), 71.1 (CH, C-3), 75.4(CH, C16), 126.1 (CH, C-6), 169.six (C, C-5), 203.three (C, C-7), 220.7 (C, C-17). EI-MS m/z 318.5 [M]+(27), 290.4 (100), 192.five (48), 91.five (66), 77.4 (33). Biotransformation with Fusicoccum amygdali AM258 7-Oxo-DHEA (0.2 g) dissolved in two ml of acetone was evenly distributed among two flasks with 4 days old fungal cultures and incubated for additional 7 days. The regular process gave extracts, which have been purified on silica gel. Elution with acetone:et.
