Thin-layer And Gas Chromatography As Methods
THIN-LAYER AND GAS CHROMATOGRAPHY AS METHODS
TO DETERMINE REACTION PROGRESS OF MICROBIAL OXIDATIONS OF STEROID SUBSTRATES
E. Brzezowska
Agricultural University, Norwida 25, 50-375 Wrocław, Poland
The microbial transformation is often applied in organic synthesis because it is an inexpensive and convenient method, that could repleaced chemical synthesis. Enzymes can catalyse avariety of reactions on steroids (reduction, degradation, isomerisation, oxidation) [1].
Sclerophoma pythiophila is saprophytic fungus which commonly occurs on trees. It was used as a bioreagent to transformate steroid substrates for the first time. In previous studies we have found, that Sclerophoma pythiophila can easily transform prochiral ketones to optically active alcohols with high yield and high enantiomeric purity. Now this strain was used so as to investigate its ability to transform steroid compounds. The substrates were: testosterone, 19-nortestosterone and 1-dehydrotestosterone.
Fungus was incubated on 3 % glucose and 1 % peptone, shaken at 27° C in 300 ml Erlenmayer flasks with 50 ml medium. After 6 days of growth, 5mg of the substrate, dissolved in 1 ml of acetone were added to 50 ml of culture. The products were extracted with methylene chloride. Transformations were carried out from 2 to 14 days, depending on the reaction progress. Method of incubation was described previously [2]. The composition of crude mixtures was analysed by TLC and GC. TLC was carried out using Silicagel-60 plates with hexane-acetone as eluent. Steroids were detected on the plates with H2SO4-EtOH (1:1). GLC analysis was performed using a gas chrmatograph Hewlett Packard 5890 Series II, column Chrompack WCOT Fused Silica Ultra 1 (Crosslinked Methyl Silicone Gum) 25 m/0.32 mm/0.17 mm film thickness column (220°/2min, 5°/min-270°, 20°/min-300°/ 2min).
All examined substrates underwent transformation and we identified the reaction products on the basis of TLC and spectra analyses. TLC showed that all products obtained were less polar than the substrates themselves. The yield off all transformations was established on the basis of GLC.
The oxidation of secondary alcohols to ketones of steroid substrate is acommon transformation. The enzymatc conversion of C17 alcohol to ketone are often most catalyzed by 17b hydroxysteroid dehydrogenases. These enzymes are widely spread in living organisms. They were found in mammalian, microorganisms, bacteria, filamentous fungi and yests. They catalyze the interconversion of 17-hydroxy and 17-keto steroids [3].
To summarize Sclerophoma pythiophila is able to carried out an oxidation 17b alcohole to ketone of steroid substrate. Micobial transformations of steroid substrates are of practical use because they can be applied in pharmaceutical industry.
References
1. K. Faber Biotransformations in Organic Chemistry, Springer – Verlag, 1992, Germany.
2. E. Brzezowska, J. Dmochowska – Gładysz, T. Kołek and E. Nobilec J. Steroid Biochem. Molec. Biol. Vol. 46, no. 2, pp. 259-263, 1993.
3. B. A. Cooke, R. J. B. King Hormones and their Actions, Part I Elsevier Science Publishers BV 1988.
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