Microbial conversion of progesterone with Aspergillus sp.

Abstract

The conversion of progesterone (1) by Aspergillus sp. was studied. The major metabolites formed from progesterone were identified as 11α-hydroxyl progesterone (2), 15α-hydroxyl progesterone (3), 15β-hydroxypreg-1,4-dien-3,20-dione (4), 6β,11α-dihydroxypregesterone (5).Conversion products were characterized by spectroscopic methods including H NMR, C NMR and MS. Introduction Microbial conversion of steroids provide an important method of obtaining new steroid derivatives of potential pharmaceutical activity, which additionally fulfills green chemistry principles[1-2]. The major advantage of biological catalysts is their capability to catalyze novel reactions with regioand stereo-selectivity[3]. Since 11αhydroxylation of progesterone by Rhizopus arrhizus was patented in 1952[4]. A wide variety of microorganisms have been investigated and have been successfully applied to transform a wide range of steroids to produce functionalized compounds with therapeutic use and commercial value[5-7]. The Aspergillus genus is an important source of fungal pathogen which can cause severe seed yield loss in soybeans and other crops[8]. However, strains of Aspergillus genus had never used in the biotransformation of steroids. During our screening program for isolation of microorganism from Paris polyphylla Smith var. Yunnanenis capable of transforming progesterone, a strain of Aspergillus sp. was isolated from rhizome of Paris polyphylla Smith var. yunnanenis and applied for converting progesterone hydroxylation in different position. Materials and methods Chemicals Progesterone (1) was purchased from Yancheng Xinyi PharmC Qingdao). Thin layer chromatography (TLC) analysis was carried out on precoated plates with 0.20-0.25 mm thick silica gel GF254 (Qingdao) with detection by heating silica gel plates sprayed with 10% H2SO4 in EtOH. Isolation of transformed products The mycelium was filtered from the fermentation broth and extracted three times with ethanol, while the broth was extracted three times with EtOAc. That was evaporated under reduced pressure and brown crude (10.53 g) was obtained. Then the crude was subject to silica gel CC to afford Compound 2 (345 mg), 3 (123 mg), 4 (13 mg), 5 (18 mg) on elution with petroleum ether/EtOAc (9:1-1:9), Chemical structures of the products were determined by means of spectral data and mentioned in respective order. Results and discussion Fig. 1. The structures of Progesterone (1) and the biotransformed products 11α-hydroxyl progesterone (2), 15αhydroxyl progesterone (3), 15β-hydroxypreg-1,4-dien-3,20-dione (4), 6β,11α–dihydroxypregesterone (5). A.versicolor converted progesterone into the compound 2, 3, 4 and 5 (Fig. 1). All of them are more polar than the substrate itself. CNMR data of the substrate as well as the conversion products are listed in Table 1. The analytical data of compound 2, 3, 4 and 5 are mentioned in respective order. 11α-hydroxyprogesterone (2) Crystallized from methanol; m.p.: 155-157oC. H NMR (CDC l3, 400 MHz,δin ppm,multi.,J in Hz): 5.70(1H, m, H-4),0.65 (3H, s, H-18), 1.2 (3H, s, H-19), 4.99 (1H, m, H-11). ESI-MS: m/z 354.2 [M+Na]; m/z 369.2 [M+K]. Mass spectru m indicated a molecular of composition C21H30O3. It unequivocally confirms the structure o f 11α-hydroxyprogesterone[9,10]. 15α-hydroxyl progesterone (3) Crystallized from methanol; m.p.: 229-231C (methanol). H NMR (CDCl3, 400 MHz,δ in ppm,multi., J in Hz): 5.64 (1H, m, H-4), 4.10 (1H, m, H15β), 2.81 (1H, t, H-17α), 0.67 (3H, s, H-8), 1.17 (3H, s, H-19), 2.12 (3H, s, H-21).ESI-M S: m∕z: 353 [M+Na]. Mass spectrum indicated a molecular of composition C21H30O3. It is in accordance with the literature data for 15α-hydroxyl pregesterone[11]. 15β-hydroxypreg-1,4-dien-3,20-dione (4) Crystallized from methanol; m.p.: 229-234oC. H NMR (CDCl3, 400 MHz,δ in ppm, multi., J in Hz): 6.09 (1H, s, H-4), 7.06 (1H, d, H-1),