Alina Świzdor

Biotransformation XLV. Transformations of 4-ene-3-oxo steroids in Fusarium culmorum culture

The course of transformations of five 4-ene-3-oxo steroids with varying substituents at C-17 i.e.: 4-androsten-3-one, androstenedione, testosterone, progesterone and 17alpha-hydroxyprogesterone in Fusarium culmorum culture was investigated. All the substrates were hydroxylated either at 12beta and 15alpha, or at 15alpha or 6beta positions, depending on the structure of the substrate. The main product of 4-androsten-3-one transformation was 12beta,15alpha-diol. A similar 12beta,15alpha-diol was obtained from progesterone, but the main product of transformation of this substrate was 15alpha-hydroxyprogesterone. The products of hydroxylation at 6beta or 15alpha positions were isolated from 17alpha-hydroxyprogesterone. The androstenedione and testosterone transformation mixtures contained the same products (6beta-hydroxyandrostenedione, 6beta-hydroxytestosterone, 15alpha-hydroxyandrostenedione and 15alpha-hydroxytestosterone), but the quantities of 6beta- and 15alpha-alcohols varied, depending on the substrate used. During transformations of these two substrates, apart from hydroxylation, ketone-alcohol interconversion at C-17 occurred.

Baeyer-Villiger oxidation of DHEA, pregnenolone, and androstenedione by Penicillium lilacinum AM111

The Baeyer-Villiger monooxygenase (BVMO) produced by Penicillium lilacinum AM111, in contrast to other enzymes of this group known in the literature, is able to process 3beta-hydroxy-5-ene steroid substrates. Transformation of DHEA and pregnenolone yielded, as a sole or main product, 3beta-hydroxy-17a-oxa-d-homo-androst-5-en-17-one, a new metabolite of these substrates; pregnenolone was transformed also to testololactone. Testololactone was the only product of oxidation of androstenedione by P. lilacinum AM111. Investigations of the time evolution of reaction progress have indicated that the substrates stimulate activity of BVMO(s) of P. lilacinum AM111.

Studies on Baeyer-Villiger oxidation of steroids: DHEA and pregnenolone D-lactonization pathways in Penicillium camemberti AM83.

Penicillium camemberti AM83 strain is able to carry out effective Baeyer-Villiger type oxidation of DHEA, pregnenolone, androstenedione and progesterone to testololactone. Pregnenolone and DHEA underwent oxidation to testololactone via two routes: through 4-en-3-ketones (progesterone and/or androstenedione respectively) or through 3beta-hydroxy-17a-oxa-d-homo-androst-5-en-17-one. Analysis of transformation progress of studied substrates as function of time indicates that the 17beta-side chain cleavage and oxidation of 17-ketones to d-lactones are catalyzed by two different, substrate-induced, BVMOs. In the presence of a C-21 substrate (pregnenolone or progesterone) induction of the enzyme catalyzing cleavage at 17beta-acetyl chain was observed, whereas DHEA and androstenedione induced activity of the BVMO responsible for the ring-D oxidation; 5-en-3beta-alcohol was a more effective inducer that the respective 4-en-3-ketone.

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.

Asymmetric reduction of tetralones and their methoxy derivatives by Fusarium culmorum

Whole cells of the fungus Fusarium culmorum were used to carry out the bioreduction of 1- and 2-tetralones and their derivatives with a methoxy group in the aromatic ring. The process led to the corresponding (S)-alcohols. The microbial reduction system exhibited moderate activity and low enantioselectivity against 1-tetralones, but high activity and moderate selectivity against 2-tetralones. Reduction of 1-tetralones was accompanied by oxidation of the resulting alcohols (re-oxidation), but this was not observed in transformations of 2-tetralones. The effects of parameters such as concentration of the medium, time of substrate addition to the culture and time of incubation on the conversion and optical purity of the alcohols have been investigated. A young culture of the microorganism obtained after growth for 24 h exhibited higher (S)-reduction activity than that observed with 72-h-old cells. Doubling the concentration of ingredients in the cultivation medium limited both the (R)-reductive activity of the biocatalyst and the oxidation of the (S)-alcohols for the 1-tetralol series. Under modified reaction conditions, the yield and enantiomeric excess of (S)-alcohols were improved to 93–100% and 58–99%, respectively.

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).

The microbiological hydroxylation of patchoulol in Absidia coerulea and Mucor hiemalis

Patchoulol was subjected to transformation by Absidia coerulea AM93 and Mucor hiemalis AM450 strains. Both micro-organisms, which displayed differing vulnerability to the fungistatic action of the patchoulol, were capable of selective hydroxylations of the substrate. The major constituents of the mixtures after transformation were (8S)-8-hydroxypatchoulol and (9R)-9-hydroxypatchoulol; the transformation carried out by M. hiemalis resulted in the additional formation of (3R)-3-hydroxypatchoulol. The mixture of (8S)-8-hydroxypatchoulol and (9R)-9-hydroxypatchoulol can be used in the synthesis of patchoulenol, a compound with an odour very similar to that of a valuable fragrance, norpatchoulenol.

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.