Anna Panek

Microbial Baeyer-Villiger oxidation of steroidal ketones using Beauveria bassiana: Presence of an 11α-hydroxyl group essential to generation of D-homo lactones.

This paper demonstrates for the first time transformation of a series of 17-oxo steroidal substrates (epiandrosterone, dehydroepiandrosterone, androstenedione) by the most frequently used whole cell biocatalyst, Beauveria bassiana, to 11α-hydroxy-17a-oxa-d-homo-androst-17-one products, in the following sequence of reactions: 11α-hydroxylation and subsequent Baeyer-Villiger oxidation to a ring-D lactone. 11α-Hydroxyprogesterone, the product of the first stage of the progesterone metabolism, was further converted along two routes: hydroxylation to 6β,11α-dihydroxyprogesterone or 17β-acetyl chain degradation leading to 11α-hydroxytestosterone, the main metabolite of the substrate. Part of 11α-hydroxytestosterone underwent a rare reduction to 11α-hydroxy-5β-dihydrotestosterone. The experiments have demonstrated that the Baeyer-Villiger monooxygenase produced by the strain catalyzes solely oxidation of C-20 or C-17 ketones with 11α-hydroxyl group. 17-Oxo steroids, beside the 11α-hydroxylation and Baeyer-Villiger oxidation, also underwent reduction to 17β-alcohols; activity of 17β-hydroxysteroid dehydrogenase (17β-HSD) has significant impact on the amount of the formed ring-D δ-lactone.

Hydroxylation of DHEA and its analogues by Absidia coerulea AM93. Can an inducible microbial hydroxylase catalyze 7α- and 7β-hydroxylation of 5-ene and 5α-dihydro C19-steroids?

In this paper we focus on the course of 7-hydroxylation of DHEA, androstenediol, epiandrosterone, and 5α-androstan-3,17-dione by Absidia coerulea AM93. Apart from that, we present a tentative analysis of the hydroxylation of steroids in A. coerulea AM93. DHEA and androstenediol were transformed to the mixture of allyl 7-hydroxy derivatives, while EpiA and 5α-androstan-3,17-dione were converted mainly to 7α- and 7β-alcohols accompanied by 9α- and 11α-hydroxy derivatives. On the basis of (i) time course analysis of hydroxylation of the abovementioned substrates, (ii) biotransformation with resting cells at different pH, (iii) enzyme inhibition analysis together with (iv) geometrical relationship between the C-H bond of the substrate undergoing hydroxylation and the cofactor-bound activated oxygen atom, it is postulated that the same enzyme can catalyze the oxidation of C7-Hα as well as C7-Hβ bonds in 5-ene and 5α-dihydro C19-steroids. Correlations observed between the structure of the substrate and the regioselectivity of hydroxylation suggest that 7β-hydroxylation may occur in the normal binding enzyme-substrate complex, while 7α-hydroxylation-in the reverse inverted binding complex.

Biotransformations Utilizing β-Oxidation Cycle Reactions in the Synthesis of Natural Compounds and Medicines

β-Oxidation cycle reactions, which are key stages in the metabolism of fatty acids in eucaryotic cells and in processes with a significant role in the degradation of acids used by microbes as a carbon source, have also found application in biotransformations. One of the major advantages of biotransformations based on the β-oxidation cycle is the possibility to transform a substrate in a series of reactions catalyzed by a number of enzymes. It allows the use of sterols as a substrate base in the production of natural steroid compounds and their analogues. This route also leads to biologically active compounds of therapeutic significance. Transformations of natural substrates via β-oxidation are the core part of the synthetic routes of natural flavors used as food additives. Stereoselectivity of the enzymes catalyzing the stages of dehydrogenation and addition of a water molecule to the double bond also finds application in the synthesis of chiral biologically active compounds, including medicines. Recent advances in genetic, metabolic engineering, methods for the enhancement of bioprocess productivity and the selectivity of target reactions are also described.

Synthesis of dehydroepiandrosterone analogues modified with phosphatidic acid moiety.

Dehydroepiandrosterone (DHEA) and its metabolite 7α-OH DHEA have many diverse physiological, biological and biochemical effects encompassing various cell types, tissues and organs. In in vitro studies, DHEA analogues have myriad biological actions, but in vivo, especially in oral administration, DHEA produces far more limited clinical effects. One of the possible solutions of this problem is conversion of DHEA to active analogues and/or its transformation into prodrug form. In this article, the studies on the conversion of DHEA and 7α-OH DHEA into their phosphatides by the phosphodiester approach are described. In this esterification, N,N-dicyclohexylcarbodiimide (DCC) was the most efficient coupling agent as well as p-toluenesulphonyl chloride (TsCl).

Insight into the orientational versatility of steroid substrates—a docking and molecular dynamics study of a steroid receptor and steroid monooxygenase

Numerous steroids are essential plant, animal, and human hormones. The medical and industrial applications of these hormones require the identification of new synthetic routes, including biotransformations. The metabolic fate of a steroid can be complicated; it may be transformed into a variety of substituted derivatives. This may be because a steroid molecule can adopt several possible orientations in the binding pocket of a receptor or an enzyme. The present study, based on docking and molecular dynamics, shows that it is indeed possible for a steroid molecule to bind to a receptor binding site in two or more orientations (normal, head-to-tail reversed, upside down). Three steroids were considered: progesterone, dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. Two proteins were employed as hosts: the human mineralocorticoid receptor and a bacterial Baeyer–Villiger monooxygenase. When the steroids were in nonstandard orientations, the estimated binding strength was found to be only moderately diminished and the network of hydrogen bonds between the steroid and the host was preserved.

Biohydroxylation of 7‐oxo‐DHEA, a natural metabolite of DHEA, resulting in formation of new metabolites of potential pharmaceutical interest

Metabolism of steroids in healthy and unhealthy human organs is the subject of extensive clinical and biomedical studies. For this kind of investigations, it is essential that the reference samples of new derivatives of natural, physiologically active steroids (especially those difficult to achieve in the chemical synthesis) become available. This study demonstrated for the first time transformation of 7‐oxo‐DHEA—a natural metabolite of DHEA, using Syncephalastrum racemosum cells. The single‐pulse fermentation of substrate produced two new hydroxy metabolites: 1β,3β‐dihydroxy‐androst‐5‐en‐7,17‐dione and 3β,12β‐dihydroxy‐androst‐5‐en‐7,17‐dione, along with the earlier reported 3β,9α‐dihydroxy‐androst‐5‐en‐7,17‐dione and 3β,17β‐dihydroxy‐androst‐5‐en‐7‐one. Simultaneously, the same metabolites, together with small quantities of 7α‐ and 7β‐hydroxy‐DHEA, as well as the products of their reduction at the C‐17 were obtained after transformation of DHEA under pulse‐feeding of the substrate. The observed reactions suggested that this micro‐organism contains enzymes exhibiting similar activity to those present in human cells. Thus, the resulting compounds can be considered as potential components of the eukaryotic, including human, metabolome.

Hydroxylative activity of Aspergillus niger towards androst-4-ene and androst-5-ene steroids

&NA; Aspergillus niger, one of fungal species most frequently used for experimental and industrial‐scale biotransformations of various organic compounds, is generally known to transform steroids at 16&bgr; position. In this work, application of the strain A. niger KCH910 to bioconversion of dehydroepiandrosterone (DHEA), androstenediol and testosterone is described, with emphasis on the metabolic steps leading to the products. Evidence from this study indicated that incubated 5‐ene steroids underwent bioconversion within two metabolic pathways: oxidation by the action of 3&bgr;‐HSD (3&bgr;‐hydroxysteroid dehydrogenase) to 4‐ene steroids, and minor allylic hydroxylation to epimeric 7‐alcohols. Further transformation of the 3‐oxo‐4‐ene metabolites resulted in non‐selective 16‐hydroxylation. It is the first report on an A. niger strain able to introduce not only 16&bgr;‐ but also 16&agr;‐hydroxyl function into steroids. Graphical abstract Figure. No caption available. HighlightsTestosterone, DHEA, and androstenediol were transformed by A. niger KCH910.3‐Oxo‐4‐ene steroids were hydroxylated at 16&agr;‐ and 16&bgr;‐position.This is the first report on 16&agr;‐hydroxylation of steroids by A. niger strain.Hydroxylation of 5‐ene androstanes led to both 7&bgr;‐ and 7&agr;‐alcohols.3&bgr;‐hydroxy‐5‐ene steroids were converted by 3&bgr;‐HSD to 3‐oxo‐4‐ene steroids.

3β,11α-Dihy­droxy-17a-oxa-d-homoandrost-5-en-17-one

The title compound, C19H28O4, was prepared from DHEA (dehydroepiandrosterone) by its biotransformation using whole cells of the filamentous fungus Beauveria bassiana. The asymmetric unit contains two molecules. The lactone ring is trans-positioned to the neighboring six-membered ring. In the crystal structure, O—H⋯O hydrogen bonds form layers, which are linked to each other by O—H⋯O and C—H⋯O hydrogen bonds.

Metabolic fate of pregnene-based steroids in the lactonization pathway of multifunctional strain Penicillium lanosocoeruleum

BackgroundMetabolic activities of microorganisms to modify the chemical structures of organic compounds became an effective tool for the production of high-valued steroidal drugs or their precursors. Currently research efforts in production of steroids of pharmaceutical interest are focused on either optimization of existing processes or identification of novel potentially useful bioconversions. Previous studies demonstrated that P. lanosocoeruleum KCH 3012 metabolizes androstanes to the corresponding lactones with high yield. In order to explore more thoroughly the factors determining steroid metabolism by this organism, the current study was initiated to delineate the specificity of this fungus with respect to the cleavage of steroid side chain of progesterone and pregnenolone The effect of substituents at C-16 in 16-dehydropregnenolone, 16α,17α-epoxy-pregnenolone and 16α-methoxy-pregnenolone on the pattern of metabolic processing of these steroids was also investigated.Results and discussionAll of the analogues tested (except the last of the listed) in multi-step transformations underwent the Baeyer–Villiger oxidation to their δ-d-lactones. The activity of 3β-HSD was a factor affecting the composition of the product mixtures. 16α,17α-epoxy-pregnenolone underwent a rare epoxide opening with retention stereochemistry to give four 16α-hydroxy-lactones. Apart from oxidative transformations, a reductive pathway was revealed with the unique hydrogenation of 5-ene double bond leading to the formation of 3β,16α-dihydroxy-17a-oxa-d-homo-5α-androstan-17-one. 16α-Methoxy-pregnenolone was transformed to the 20(R)-alcohol with no further conversion.ConclusionsThis work clearly demonstrated that P. lanosocoeruleum KCH 3012 has great multi-functional catalytic properties towards the pregnane-type steroids. Studies have highlighted that a slight modification of the d-ring of substrates may control metabolic fate either into the lactonization or reductive and oxidative pathways. Possibility of epoxide opening by enzymes from this microorganism affords a unique opportunity for generation of novel bioactive steroids.