粉红单端孢Trichothecium roseum对雄甾-4-烯-3,17-二酮的生物转化

雄甾-4-烯-3,17-二酮（4AD）是甾体化合物合成过程中的关键中间体,其羟化产物通常具有良好的药理活性或作为工业生产甾体药物的重要中间体。利用粉红单端孢Trichothecium roseum对4AD进行生物转化,从其发酵提取物中共分离鉴定了3个4AD羟基化产物：6β-羟基-雄甾-4-烯-3,17-二酮（6β-OH-4AD,1）,14α-羟基-雄甾-4-烯-3,17-二酮（14α-OH-4AD,2）,6β,14α-双羟基-雄甾-4-烯-3,17-二酮（6β,14α-di-OH-4AD,3）,表明T. roseum对4AD的C-6β位和C-14α位具有较强的羟化能力,其中14α-OH-4AD(2)可作为合成强心甾类化合物毛地黄毒素的重要中间体,6β,14α-di-OH-4AD(3)可作为合成具有抗肿瘤活性的14α-羟基-雄甾-4-烯-3,6,17-三酮的重要中间体。提供了1株能够高效制备活性甾醇中间体14α-OH-4AD和6β,14α-di-OH-4AD的菌株,同时可为研究其他甾醇药物奠定基础。     

犁头霉11α-羟基化制备16β-甲基-11α，17α，21 三羟基孕甾-1，4 二烯 3，20 二酮

选育到一株对16β-甲基-17α,21-二羟基孕甾-1,4-二烯3,20-二酮(Ⅱa)11α-羟基化活性强的犁头霉A28菌株,并发现底物21乙酰化(Ⅱb)可明显提高11α-羟基化的能力。在适宜的转化条件下,Ⅱb投料浓度0.5%,产物16β-甲基-11α,17α,21-三羟基孕甾-1,4-二烯3,20-二酮(Ⅲ)收率为73%,结构经波谱分析确认。     

犁头霉11α-羟基化制备16β-甲基-11α,17α,21-三羟基孕甾-1,4-二烯-3,20-二酮

选育到一株对16β-甲基,17α,21-二羟基孕甾-1,4-二烯-3,20-二酮(Ⅱa)11α-羟基化活性强的犁头霉A28菌株,并发现底物21-乙酰化(Ⅱb)可明显提高11α-羟基化的能力.在适宜的转化条件下,Ⅱb投料浓度0.5%,产物16β-甲基-11α,11α,21-三羟基孕甾-1,4-二烯-3,20-二酮(Ⅲ)收率为73%,结构经波谱分析确认.     

黑曲霉催化雄甾-4-烯-3,17-二酮16β-羟基化

为进一步确定黑曲霉菌株TCCC41650的生物转化能力,以雄甾-4-烯-3,17-二酮(Androstenedione)为底物,利用黑曲霉菌株TCCC41650进行催化,产物经纯化、重结晶后,通过单晶衍射鉴定为16β-羟基雄甾-4-烯-3,17-二酮。转化条件为:培养液pH 6.0,乙醇添加量为2%,投料浓度为1‰时,72 h转化率为85.8%。目前甾体研究领域对于C16β-羟基化的微生物转化未见报道,研究结果为C16β-羟基甾体药物的研发奠定了基础。     

新月弯孢霉AS 3.4381对新型甾体底物C11β-羟基化

应用本实验室保藏的新月弯孢霉Curvularia lunataAS 3.4381对新型甾体化合物(Ⅰ)(16α,17β-二甲基-17-丙酰基雄甾-1,4-二烯-3-酮)作为底物进行生物转化C11β-羟基化反应的研究。实验研究结果表明,采用Ⅱ级发酵的工艺,收获新月弯孢霉菌丝体作为生物催化剂,在磷酸缓冲液介质体系中,对化合物Ⅰ的C11位实现β羟基化,生成皮质激素药物。测试数据TLC,MS,IR及1H NMR证明了该产物的化学结构,表明生物转化产物为C11β-羟基-16α,17β-二甲基-17-丙酰基雄甾-1,4-二烯-3-酮。     

分枝杆菌细胞裂解液催化甾体激素C_(1,2)位脱氢反应的研究

9β,11β-环氧-17α,21-二羟基-16β-甲基孕-1,4-二烯-3,20-二酮(Ⅳ)是生产9-氟甾体激素的关键前体,以9β,11β-环氧-17α,21-二羟基-16β-甲基孕-4-烯-3,20-二酮-21-醋酸酯(Ⅰ)为底物合成Ⅳ是工业化生产Ⅳ的重要方法。通过比较分枝杆菌全细胞转化法与细胞裂解液转化法,发现分枝杆菌全细胞只能将Ⅰ转化为9β,11β-环氧-17α,21-二羟基-16β-甲基孕-4-烯-3,20-二酮(Ⅱ),而细胞裂解液可以有效地将Ⅰ转化为Ⅳ,其反应机制为底物Ⅰ自发水解为中间体Ⅱ,Ⅱ在C_(1,2)位脱氢酶(KSTD)的催化作用下发生C_(1,2)位脱氢反应生成产物Ⅳ。为进一步提高产物Ⅳ的转化率,利用基因工程手段在分枝杆菌中分别过表达编码KSTD的关键基因:kst D、kst D3和kstD_M,提高脱氢反应效率,结果表明1 g/L底物Ⅰ在pH7.0的重组菌株MS136-kst D_M细胞裂解液中反应45h,Ⅳ的转化率为78.4%,比出发菌株提高了38.9%;并优化缓冲液pH,提高反应速率,结果表明1 g/L底物Ⅰ在pH7.5的重组菌株MS136-kstD_M细胞裂解液中反应45 h,Ⅳ的转化率为92.8%,比出发菌株提高了63.4%。     

微生物学方法制备16α-甲基-11α，17α，21-三羟基孕甾-1，4-二烯3，20-二酮

本文报道了简单节杆菌A69-2和球孢白僵菌AS69同时存在下对16α-甲基-17α-羟基孕甾-4．烯-3,20-二酮-21-醋酸酯(16MRSA)的协周转化作用。 这种协同转化怍用既能解除16α-甲基-11α,17a,21-三羟基孕甾-4-烯-3,20-二酮(16MllaHC)对球孢白僵菌AS69的11α羟化酶的抑制作用,又可降低高浓度的16M11aHC对节杆菌A69—2脱氢酶活性的影响,同时还能抑制节杆菌脱氢过程的副反应。在底物浓度为0.15％(W／V)时,l6α-甲基-11α,17a,21-三羟基孕甾-1,4-二烯-3,20-二酮(16MDHC)的收率约50%,故是制备1 6MDHC的一种理想的微生物学方法。     

A环饱和甾体的微生物转化作用III．倍他美松中间体16β-甲基-5a-△9(11)-孕甾烯-3β,17a, 21-三羟基一20一酮一21一醋酸酯的△1.4 作用

节杆菌9—2在一定浓度的氯化钻存在下转化16β一甲基一5a一△9(11)一孕甾烯-3β,17a,21-三羟基一20一酮21-醋酸酯(I)为16β-甲基-A1.4,9(11)-孕甾三烯-17a,21一二羟基-3,20-二酮(n)。转化的最适条件为氯化钴浓度0．08％;乙醇2一4％;pH 7—8;温度28—30℃。在此条件下,产物(II)的收率在60％以上。     

总枝状毛霉对去氢表雄酮的生物转化研究

从土样中筛选到1株能转化甾体的菌株,经形态观察,鉴定为总枝状毛霉(Mucor racemosus)。利用该菌株对去氢表雄酮进行生物转化研究,转化产物分离后,通过红外、质谱和二维核磁波谱分析,确定了化学结构。研究结果表明转化产物是7α-羟基去氢表雄酮和7β-羟基去氢表雄酮。     

A环饱和甾体的微生物转化作用IV．节杆菌9-2菌株的环氧化反应

经各项理化性质包括熔点、比旋值、紫外,红外吸收光谱、核磁共振谱、质谱及元素分析等项的鉴定,证明节杆菌9—2在低浓度(0．02％)的氯化钻存在下,氧化16β一甲基一3β、17a、21-三羟基一5a一△9(11)孕甾烯-20-酮-21-醋酸酯(I)的产物是16β一甲基9a,11a-环氧-17a,21-二羟基一△1,4,孕甾二烯一3,20一二酮(IV),收率48．8％。试验还发现氯化钻是调节产物16β-甲基-17a,21-二羟基△1,4,9(11)孕甾三烯-3,20-二酮(II)和(IV)累积量的有效因素;进一步讨论了该菌环氧化作用的机制。     

Biotransformation of 3b-Hydroxyandrost-5-en-17-one by Cell Suspension Cultures of Catharanthus roseus

Catharanthus roseus (L.) G. Don cell suspension cultures were used to transform 3b-hydroxyandrost-5-en-17-one, the products were isolated by chromatographic methods. Their structures were established by means of NMR and MS spectral analyses. Nine metabolites were respectively elucidated as: androst-4-ene-3,17-dione (Ⅰ), 6a-hydroxyandrost-4-ene-3,17-dione (Ⅱ), 6a,17b-dihydroxyandrost-4-en-3-one (Ⅲ), 6b-hydroxyandrost-4-ene-3,17-dione (Ⅳ), 17b-hydroxyandrost-4-en-3-one (Ⅴ), 15a,17b-dihydroxyandrost-4-en-3-one (Ⅵ), 15b,17b-dihydroxyandrost-4-en-3-one (Ⅶ), 14a-hydroxyandrost-4-ene-3,17-dione (Ⅷ), 17b-hydroxyandrost-4-ene-3,16-dione (Ⅸ). It is the first time to obtain the above compounds by biotransformation with Catharanthus roseus cell cultures.     

Steroid hydroxylation by Whetzelinia sclerotiorum, Phanerochaete chrysosporium and Mucor plumbeus

The fungi Whetzelinia sclerotiorum ATCC 18687, Phanerochaete chrysosporium ATCC 24725 and Mucor plumbeus ATCC 4740 were examined for their ability to perform steroid biotransformations under single phase, pulse feed conditions. The steroids 3beta-hydroxyandrost-5-en-17-one (dehydroepiandrosterone) (1), 17beta-hydroxyandrost-4-en-3-one (testosterone) (5), 3beta-hydroxypregn-5-en-20-one (pregnenolone) (3), pregn-4-ene-3,20-dione (progesterone) (9), 17alpha,21-dihydroxypregn-4-ene-3,11,20-trione (cortisone) (11), 17alpha,21-dihydroxypregna-1,4-diene-3,11,20-trione (prednisone) (14), and 3-hydroxyestra-1,3,5(10)-trien-17-one (estrone) (15) were fed to each fungus. The production of a number of novel metabolites is reported. Of the fungi investigated W. sclerotiorum performed the most interesting biotransformations and had a clear propensity for 2beta, 6beta/7beta and 15beta/16beta hydroxylations. P. chrysosporium was more prone functionalize steroids in the allylic position. Oxygen insertion at C-14 by M. plumbeus is reported for the first time. All three micro-organisms exhibited redox activity.     

Oxidation of 17alpha,20beta- and 17alpha,20alpha-dihydroxysipregn-4-en-ones--side products of biotransformation of progesterone by recombinant microorganisms expressing cytochrome P45017alpha

Progesterone biotransformation with recombinant yeast Yarrowia lipolytica E129A15 and Saccharomyces cerevisiae GRF18/YEp5117 alpha expressing bovine adrenocortical cytochrome P45017 alpha yielded 17 alpha-hydroxyprogesterone and two diols, 17 alpha, 20 beta- and 17 alpha, 20 alpha-dihydroxypregn-4-en-3-one. The oxidation of mixtures of the three steroids with chromic acid resulted in the cleavage of 17-20 bonds in the diols with the formation of androst-4-ene-3,17-dione. The biotransformation of pregn-4-ene-20 beta-ol-3-one by means of Y. lipolytica E129A15 was accompanied by the following reactions: the primary oxidation of these compounds to progesterone and the subsequent successive reactions of 17 alpha-hydroxylation and 20 alpha- and 20 beta-reduction. The results widen the possibilities for enzymatic and chemical modifications of steroids. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

Microbial transformation of hydrocortisone by Acremonium strictum PTCC 5282

The ability of a genus of cephalosporium-like fungus isolated from soil, Acremonium strictum PTCC 5282, for hydrocortisone biotransformation has been investigated. This potential had not been previously examined. The fermentation yielded 11beta,17beta-dihydroxyandrost-4-en-3-one, 11beta,17alpha,20beta,21-tetrahydroxypregn-4-en-3-one and 21-acetoxy-11beta,17alpha,20-trihydroxypregn-4-en-3-one. Each microbial metabolite was purified and characterized using spectroscopic methods.     

The facile bioconversion of testosterone by alginate-immobilised filamentous fungi

The potential for biotransformation of the substrate 17β-hydroxyandrost-4-en-3-one (testosterone) by six filamentous fungi, namely, Rhizopus oryzae ATCC 11145, Mucor plumbeus ATCC 4740, Cunninghamella echinulata var. elegans ATCC 8688a, Aspergillus niger ATCC 9142, Phanerochaete chrysosporium ATCC 24725 and Whetzelinia sclerotiorum ATCC 18687, was investigated. In this study both free cells and macerated mycelia immobilised in calcium alginate were utilised and the results (products, % yields, % transformation) were compared. In general the encapsulated cells of the microorganisms effectively generated products similar to those found using free cells. However, with immobilised macerated mycelia, isolation of the transformation products was expedited by the simple work up procedure, and their purification was facilitated by the absence of fungal secondary metabolites. Twenty seven analogues of testosterone were generated, wherein the androstane skeleton was functionalised at C-1β, -2β, -6β, -7α, -11α, -14, -15α, -15β and -16β by the moulds. Redox chemistry was also observed. Seven of the analogues, 6β,11α,17β-trihydroxyandrost-4-en-3-one, 6β,14α,17β-trihydroxyandrost-4-en-3-one, 2,6β-dihydroxyandrosta-1,4-diene-3,17-dione, 2β,16β-dihydroxyandrost-4-ene-3,17-dione, 2β,6β-dihydroxyandrost-4-ene-3,17-dione, 2β,15β,17β-trihydroxyandrost-4-en-3-one and 2β,3α,17β-trihydroxyandrost-4-ene, were novel compounds. Five others, namely, 7α,17β-dihydroxyandrost-4-en-3-one, 6β,14α-dihydroxyandrost-4-ene-3,17-dione, 15α,17β-dihydroxyandrost-4-en-3-one, 16β,17α-dihydroxyandrost-4-en-3-one and 2β,16β,17β-trihydroxyandrost-4-en-3-one, were fully characterised for the first time.     

Circulating neuroactive C21- and C19-steroids in young men before and after ejaculation

Twelve neuroactive and neuroprotective steroids, androgens and androgen precursors i.e. 3alpha,17beta-dihydroxy-5alpha-androstane, 3alpha-hydroxy-5alpha-androstan-17-one, 3alpha-hydroxy-5beta-androstan-17-one, androst-5-ene-3beta,17beta-diol, 3beta,17alpha-dihydroxy-pregn-5-en-20-one (17alpha-hydroxy-pregnenolone), 3beta-hydroxy-androst-5-en-17-one (dehydroepiandrosterone, DHEA), testosterone, androst-4-ene-3,17-dione (androstenedione), 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone), 3beta-hydroxy-pregn-5-en-20-one (pregnenolone), 7alpha-hydroxy-DHEA, and 7beta-hydroxy-DHEA were measured using the GC-MS system in young men before and after ejaculation provoked by masturbation. The circulating level of 17alpha-hydroxypregnenolone increased significantly, whereas the other circulating steroids were not changed at all. This fact speaks against the hypothesis that a drop in the level of neuroactive steroids, e.g. allopregnanolone may trigger the orgasm-related increase of oxytocin, reported by other authors.     

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.     

Reduction of the 20-Carbonyl Group of C-21 Steroids by Spores of Fusarium solani and Other Microorganisms. I. Side-Chain Degradation, Epoxide Cleavage, and Substrate Specificity

The spores of Fusarium solani reduced the C(2)-carbonyl group, 1-dehydrogenated ring "A" and cleaved the side chain of 16alpha, 17alpha-oxidopregn-4-ene-3, 20-dione (16alpha, 17alpha-oxidoprogesterone)(I) to give the following products: 20alpha-hydroxy-16alpha, 17alpha-oxidopregn-4-en-3-one(II); 20alpha-hydroxy-16alpha, 17alpha-oxidopregna-1, 4-dien-3-one(III); 16alpha-hydroxy-17a-oxa-androsta-1, 4-diene-3, 17-dione (16alpha-hydroxy-1-dehydrotestololactone)(IV); and 16alpha, 17beta-dihydroxy-androsta-1, 4-dien-3-one (16alpha-hydroxy-1-dehydrotestosterone)(V). When II was used as a substrate, it was metabolized into III, IV, and V at a slower rate than I. Furthermore, 16alpha-hydroxy-androst-4-ene-3, 17-dione (16alpha-hydroxyandrostenedione)(X) was transformed into IV and V. Pregn-4-ene-3, 20-dione (progesterone)(XII) was transformed into androsta-1, 4-diene-3, 17-dione (androstadienedione)(VIII) and 17a-oxa-androsta-1, 4-diene-3, 17-dione (1-dehydrotestololactone)(IX), while 17alpha-hydroxy-pregn-4-ene-3, 20-dione (17alpha-hydroxyprogesterone)(VI) was converted into its 1-dehydro analogue (VII) without accumulation of any 20-dihydro compounds. Substrate specificity in the 20-reductase system of F. solani, Cylindrocarpon radicicola, Septomyxa affinis, Bacillus lentus, and three strains of B. sphaericus are demonstrated. The 20-reductase is active only on steroids having the 16alpha, 17alpha-oxido, and Delta(4)-3-keto functions. Evidence of competition between side-chain degrading enzymes and the 20-reductase for the steroid molecule and evidence of side-chain degradation followed by epoxide cleavage (and not the reverse) are presented. A mechanism for the epoxide opening by nongerminating spores of F. solani is postulated.     

The degradation of beta-sitosterol by Pseudomonas sp. NCIB 10590 under aerobic conditions

The bacterial degradation of beta-sitosterol by Pseudomonas sp NCIB 10590 has been studied. Major biotransformation products included 24-ethylcholest-4-en-3-one, androsta-1,4-diene-3,17-dione, 3-oxochol-4-en-3-one-24-oic acid and 3-oxopregn-4-en-3-one-20-carboxylic acid. Minor products identified were 26-hydroxy-24-ethylcholest-4-en-3-one, androst-4-ene-3,17-dione, 3-oxo-24-ethylcholest-4-en-26-oic acid, 3-oxochola-1,4-dien-3-one-24-oic acid, 3-oxopregna-1,4-dien-3-one-20 carboxylic acid and 9 alpha-hydroxyandrosta-1,4-diene-3,17-dione. Studies with selected inhibitors have enabled the elucidation of a comprehensive pathway of beta-sitosterol degradation by bacteria.     

Transformation of androstane derivatives by Spirodela oligorrhiza

Spirodela oligorrhiza (duckweed) is capable of transforming some steroids of the androstane series. Hydrolysis of the acetates of testosterone and of 3β-hydroxyandrost-5-en-17-one by this species yielded the corresponding alcohols. Further transformation of testosterone and reduction of androst-4-ene-3,17-dione indicated the interconversions of the hydroxyl-ketone function on C-17 and reduction of the Δ⁴-double bond to the trans-A/B system. Only a trace amount of 3β-hydroxyandrost-5-en-17-one underwent further transformations.