Microbiological hydroxylation of androst-1,4-dien-3,17-dione by Neurospora crassa

Nine hydroxy-derived androstadiene compounds were isolated from the fermentation broth of Neurospora crassa when incubated in the presence of androst-1,4-dien-3,17-dione (ADD; I) for 7 days. Hydroxylations at 6β, 7β, 11α, 14α- positions and 17-carbonyl reduction of the substrate were the characteristics observed in this biotransformation. Their structures were determined by spectroscopic methods as 17β-hydroxyandrost-1,4-dien-3-one (II), 14α-hydroxyandrost-1,4-dien-3,17-dione (III), 6β-hydroxyandrost-1,4-dien-3,17-dione (IV), 11α-hydroxyandrost-1,4-dien-3,17-dione (V), 6β,17β-dihydroxyandrost-1,4-dien-3-one (VI), 7β-hydroxyandrost-1,4-dien-3,17-dione (VII), 14α,17β-dihydroxyandrost-1,4-dien-3-one (VIII), 6β,14α-dihydroxyandrost-1,4-dien-3,17-dione (IX), and 11α,17β-dihydroxyandrost-1,4-dien-3-one (X). A new steroid substance, 6β,14α-dihydroxyandrost-1,4-dien-3,17-dione (IX), was also characterized during this study. The best fermentation condition was found to be 7-day incubation at 25°C and pH values of 5.0–6.0 in the presence of 0.05 g 100 mL^?1 of the substrate. At a concentration above 0.075 g 100 mL^?1, the biotransformation was completely inhibited.     

Influence of whole microalgal cell immobilization and organic solvent on the bioconversion of androst-4-en-3,17-dione to testosterone by Nostoc muscorum

The use of free, immobilized and reused immobilized cells of the microalga Nostoc muscorum was studied for bioconversion of androst-4-en-3,17-dione (AD) to testosterone in hexadecane. Among polymers such as agar, agarose, κ-carrageenan, polyacrylamide, polyvinyl alcohol, and sodium alginate that were examined for cell entrapment, sodium alginate with a concentration of 2% (w/v) proved to be the proper matrix for N. muscorum cells immobilization. The bioconversion characteristics of immobilized whole algal cells at ranges of temperatures, substrate concentrations, and shaking speeds were studied followed by a comparison with those of free cells. The conditions were 30 °C, 0.5 g/L, and 100 rpm, respectively. The immobilized N. muscorum showed higher yield (72 ± 2.3%) than the free form (24 ± 1.3%) at the mentioned conditions. The bioconversion yield did not decrease during reuse of immobilized cells and remained high even after 5 batches of bioreactions while Na-alginate 3% was used; however, reuse of alginate 2% beads did not give a satisfactory result.     

Studies on the microbial transformation of androst-1,4-dien-3,17-dione with Acremonium strictum

The strain of Acremonium strictum PTCC 5282 was applied to investigate the biotransformation of androst-1,4-dien-3,17-dione (I; ADD). Microbial products obtained were purified by preparative TLC and the pure metabolites were characterized on the basis of their spectroscopic features (¹³C NMR, ¹H NMR, FTIR, MS) and physical constants (melting points and optical rotations). The 15α-Hydroxyandrost-1,4-dien-3,17-dione (II), 17β-hydroxyandrost-1,4-dien-3-one (III), androst-4-en-3,17-dione (IV; AD), 15α-hydroxyandrost-4-en-3,17-dione (V), 15α,17β-dihydroxyandrost-1,4-dien-3-one (VI) and testosterone (VII) were produced during this fermentation. Formation of the 15α,17β-dihydroxy derivative of ADD is reported for the first time during steroid biotransformation. The bioconversion reactions observed were 1,2-hydrogenation, 15α-hydroxylation and 17-ketone reduction. From the time course profile of this biotransformation, ketone reduction and 1,2-hydrogenation were observed from the first day of fermentation while 15α-hydroxylation occurred from the third day. Optimum concentration of the substrate, which gave the maximum bioconversion efficiency, was 0.5 mg ml⁻¹ in one batch. The highest yield of the microbial products recorded in this work was achieved within the pH range 6.5–7.3 and at the temperature of 27 °C.     

Differential inhibition of androst-4-en-3,17-dione aromatization by carbon monoxide in the presence of estr-4-en-3,17-dione.

The aromatization of androst-4-en-3,17-dione or 17beta hydroxyandrost-4-en-3-one (testosterone) is not inhibited by carbon monoxide under normal incubation conditions, whereas the aromatization of corresponding 19-nor steroids (estr-4-en-3,17-dione and 17beta-hydroxyestr-4-en-3-one) is readily inhibited under the same conditions. A possible explanation was found when it was shown that androst-4-en-3,17-dione and testosterone could displace bound carbon monoxide from human placental microsomal cytochrome P-450. The 19-nor steroids did not displace carbon monoxide, even at very high concentrations. These C-18 compounds appeared to facilitate complex formation and reversed the effects of the C-19 steroids. A mutual antagonism was observed with regard to effects on the formation of the ce titrated. These observations suggested that the aromatization of androst-4-en-3,17-dione should be inhibited by carbon monoxide if sufficient concentrations of the 19-nor steroids were present in reaction flasks. This hypotheses was tested and positive results were obtained, providing strong evidence for the involvement of cytochrome P-450 in normal estrogen biosynthesis.     

Metabolism of androst-4-en-3,17-dione by the filamentous fungus Neurospora crassa

Microbial transformation of androst-4-en-3,17-dione (AD; I) using Neurospora crassa afforded six metabolites; 6beta,14alpha-dihydroxyandrost-4-en-3,17-dione (II), 6beta,9alpha-dihydroxyandrost-4-en-3,17-dione (III), 7alpha-hydroxyandrost-4-en-3,17-dione (IV), 9alpha-hydroxyandrost-4-en-3,17-dione (V), 14alpha-hydroxyandrost-4-en-3,17-dione (VI), and androst-4,6-dien-3,17-dione (VII). The steroid products were assigned by interpretation of their spectral data such as (1)H NMR, (13)C NMR, FTIR, and mass spectroscopy. The characteristic transformations observed were C-6beta, C-7alpha, C-9alpha, C-14alpha hydroxylations, and C6-C7 dehydrogenation. The best fermentation condition was found to be 6-day incubation at 25 degrees C and pH value of 5.0-6.5 according to TLC profiles. Time course study showed the accumulation of V and VI from the third day and IV from the fourth day of the fermentation. Optimum concentration of the substrate, which gave maximum bioconversion efficiency, was 3.5mM in one batch. Biotransformation was completely inhibited in a concentration above 7.0mM.     

Microbial transformation of androst-4-ene-3,17-dione by Beauveria bassiana

The microbial transformation of androst-4-ene-3,17-dione (I) by the fungus Beauveria bassiana CCTCC AF206001 has been investigated using pH 6.0 and 7.0 media. Two hydroxylated metabolites were obtained with the pH 6.0 medium. The major product was 11alpha-hydroxyandrost-4-ene-3,17-dione (II) whereas the minor product was 6beta,11alpha-dihydroxyandrost-4-ene-3,17-dione (III). On the other hand, four hydroxylated and/or reduced metabolites were obtained with the pH 7.0 medium. The major product was 11alpha,17beta-dihydroxyandrost-ene-3-one (V) and the minor products were 17beta-hydroxyandrost-ene-3-one (IV), 6beta,11alpha,17beta-trihydroxyandrost-ene-3-one (VI) and 3alpha,11alpha,17beta-trihydroxy-5alpha-androstane (VII). The products were purified by chromatographic methods, and were identified on the basis of spectroscopic methods. This fungus strain is clearly an efficient biocatalyst for 11alpha-hydroxylation and reduction of the 17-carbonyl group.     

Mycobacterium sp．高效转化植物甾醇为雄甾-1，4-二烯-3，17-二酮的诱变选育

采用紫外线、亚硝基胍复合诱变雄甾-4-烯-3,17-二酮（AD）和雄甾-1,4-二烯-3,17-二酮（ADD）的转化产生菌Mycobacterium sp．,结合平板筛选,获得一株遗传性状稳定单产ADD的突变菌株Mycobacterium sp．-11,其ADD质量浓度达到1246ms／L,比原始菌株（484mg／L）提高了150％,经初步优化后发酵液中ADD最高达到1430mg／L,发酵液中ADD质量占ADD、AD两产物质量总和的比例由70％提高到99．1％。     

Nucleosteroids: carbocyclic nucleoside analogs of androst-4-en-17 beta-ol

Steroidal nucleoside analogs were synthesized starting from testosterone. By reduction of the oxime of 17 beta-hydroxy-androst-4-en-3-one (testosterone), a mixture of the two amino epimers of C-3 were obtained. The 3 alpha-amino-androst-4-en-17 beta-ol was crystallized in 73% yield and coupled with 5-amino-4,6-dichloropyrimidine to give 3 alpha-(5'-amino-4'-chloro-pyrimidin-6'-yl)amino-androst-4-en-17 beta-ol. This compound was treated with triethyl orthoformate in acid media to give the corresponding purinyl steroid adduct 3 alpha-(6'-chloro-purin-9'-yl)-androst-4-en-17 beta-ol in 98% yield. This substance, in turn, was converted with good yield into the 6'-thio, 6'-methylamino, and 6'-diethyl aminopurinyl derivatives through nucleophilic reactions at C-6 of the purine nucleus.     

Gibberella intermedia C2转化4-雄甾烯-3、17-二酮的研究

甾体化合物具有独特的生理活性,已被广泛应用于抗炎、利尿、免疫、避孕及抗癌等领域。近些年,生物催化与转化在甾体药物中间体合成中发挥的作用日益强大。为了能够合成一些具有潜在价值的新型甾体化合物,以实验室菌种库中保藏的一株Gibberella intermedia C2为研究对象,选取了雄甾烷中一种有广泛用途的化合物4-雄甾烯-3、17-二酮(简称雄烯二酮,AD)为底物进行生物转化。转化液经提取分离,最终获得2个转化产物,经结构鉴定分别为15α-OH-AD和11α,15α-diOH-AD。转化机制研究发现,G.intermedia C2先将底物的15位羟基化生成15α-OHAD,再将其11位羟基化形成双羟基产物。赤霉菌能够特异性、有序地完成对AD的两步羟化反应。此外,通过工艺优化,确定了羟化4AD反应的最适工艺参数如下:发酵培养基的初始pH 6.5,装液量30ml/250ml,底物浓度6.0g/L,转化温度28℃,摇床转速220r/min,转化周期为84h。此时,底物AD的摩尔转化率达到81.5%。     

Proliferative effect of androst-4-ene-3,17-dione and its metabolites in the androgen-sensitive LNCaP cell line

As a therapeutic approach for the treatment of androgen-sensitive diseases, it would be tempting to lower the level of the potent androgens testosterone (T) and dihydrotestosterone (DHT) by using inhibitors of type 3 and type 5 17beta-hydroxysteroid dehydrogenases (17beta-HSDs). However, the efficiency of such a strategy will be optimal only if androst-4-ene-3,17-dione (Delta4-dione), the precursor of T, does not possess per se agonist activity on the androgen receptor (AR). To determine if the proliferative effect previously observed on AR(+) cells for Delta4-dione originates from its direct (per se) action on AR or from its transformation into a metabolite, we started a series of experimentations using the human prostate cancer LNCaP cell line, which expresses a highly sensitive AR. By real-time RT-PCR analysis, we detected type 1 5alpha-reductase (5alpha-R), a small amount of type 5 17beta-HSD, but not type 2 5alpha-R nor type 3 17beta-HSD. We then studied the transformation of labeled Delta4-dione in LNCaP cells after 1-7 days and the most important metabolite detected was 5alpha-androstane-3,17-dione (A-dione), which is the product of 5alpha-R activity. We measured only low levels of androsterone (ADT) and epi-ADT. This result was next confirmed by using an inhibitor of 5alpha-R that completely inhibited the transformation of Delta4-dione into A-dione, and consequently into ADT and epi-ADT. The proliferative effect of Delta4-dione (carefully purified) on LNCaP (AR(+)) cells was next determined in presence or absence of the 5alpha-R inhibitor. Although the cells proliferate in the presence of Delta4-dione only, no cell proliferation was observed with a combination of Delta4-dione and 5alpha-R inhibitor, suggesting that Delta4-dione is not androgenic per se. We next determined that A-dione and epi-ADT stimulated cell growth with the same pattern and potency as Delta4-dione, whereas ADT had a 3.5-fold lower proliferative activity. In conclusion, Delta4-dione is not in itself an agonist steroid on LNCaP (AR(+)) cells, and its proliferative activity appears to be mediated by its transformation into A-dione and/or into epi-ADT.     

Biotransformation of 4-androstene-3,17-dione by green cell suspension of Marchantia polymorpha: stereoselective reduction at carbon 17

The biotransformation of 4-androstene-3,17-dione by a green cell suspension of Marchantia polymorpha was studied. It was found that the cultured cells stereoselectively reduce the carbonyl group of 4-androstene-3,17-dione from the re-face at C-17 to form testosterone as the primary metabolite.     

1,4-Androstadiene-3,17-dione found in the urine of a boy

A steroid, that has not been detected so far in human urine or blood, was found in the urine of a boy with signs of epileptic convulsions. Only less than 0,5 mg of the substance could be isolated by extraction methods and purified by paper and thin layer chromatography. The unknown compound was investigated by circular dichroism, ultra-violet absorption, combined gas chromatographymass spectrometry, proton nuclear magnetic resonance and infra-red spectroscopy. The structure of the unknown was determined unequivocally as 1,4-androstadiene-3,17-dione.     

Highly efficient synthesis of boldenone from androst-4-ene-3,17-dione by Arthrobacter simplex and Pichia pastoris ordered biotransformation

Bioprocess and Biosystems Engineering - Boldenone (BD) is an important steroid hormone drug which is the derivative of testosterone. In this study, an ordered biotransformation method was proposed...