Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx

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Bioorganic & Medicinal Chemistry

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Hydroxylation of DHEA and its analogues by Absidia coerulea AM93.Can an inducible microbial hydroxylase catalyze 7a- and7b-hydroxylation of 5-ene and 5a-dihydro C19-steroids?

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⇑ Corresponding author. Tel.: +48 713205252; fax: +48 713284124.E-mail address: anna.szpineter@up.wroc.pl (A. Panek).

Please cite this article in press as: Milecka-Tronina, N.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.11.05

Natalia Milecka-Tronina, Teresa Kołek, Alina Swizdor, Anna Panek ⇑Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland

a r t i c l e i n f o

Article history:Received 5 September 2013Revised 3 November 2013Accepted 28 November 2013Available online xxxx

Keywords:DHEA metabolismDehydroepiandrosterone 7-hydroxylaseEnzymatic steroid hydroxylationHydroxy-DHEAHydroxy-epiandrosteroneAbsidia coerulea

a b s t r a c t

In this paper we focus on the course of 7-hydroxylation of DHEA, androstenediol, epiandrosterone, and5a-androstan-3,17-dione by Absidia coerulea AM93. Apart from that, we present a tentative analysis ofthe hydroxylation of steroids in A. coerulea AM93. DHEA and androstenediol were transformed to themixture of allyl 7-hydroxy derivatives, while EpiA and 5a-androstan-3,17-dione were converted mainlyto 7a- and 7b-alcohols accompanied by 9a- and 11a-hydroxy derivatives. On the basis of (i) time courseanalysis of hydroxylation of the abovementioned substrates, (ii) biotransformation with resting cells atdifferent pH, (iii) enzyme inhibition analysis together with (iv) geometrical relationship between theC–H bond of the substrate undergoing hydroxylation and the cofactor-bound activated oxygen atom, itis postulated that the same enzyme can catalyze the oxidation of C7-Ha as well as C7-Hb bonds in 5-ene and 5a-dihydro C19-steroids. Correlations observed between the structure of the substrate and theregioselectivity of hydroxylation suggest that 7b-hydroxylation may occur in the normal bindingenzyme-substrate complex, while 7a-hydroxylation—in the reverse inverted binding complex.

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1. Introduction shown that the metabolism of DHEA can proceed through the fol-

The results of nearly 50 years of research have shown thatDHEA (dehydroepiandrosterone) is a multi-functional steroidimplicated in a broad range of biological processes (including dia-betes, obesity, atherosclerosis, anti-tumorigenesis, bone metabo-lism).1–3 Many studies have been undertaken as attempts toelucidate the mechanism(s) of DHEA action, pathways of itsmetabolism, and determination of the biological activity of itsmetabolites.4,5 The biological effects of DHEA and epiandroster-one (EpiA)–the 5a-dihydro derivative of DHEA–seem to be med-iated by its oxygenated metabolites.5–11 The 7-hydroxylatedderivatives of DHEA and EpiA were shown to mediate neuropro-tection, and 7b-hydroxy-EpiA was the most potent.9,10 Cyto-chrome P450 enzyme (CYP7B), highly expressed in rat, mousesheep, marmoset and human brain, catalyzes the 7a-hydroxyl-ation of DHEA and related steroids.12 The corresponding enzymeresponsible for formation of 7b-hydroxy-DHEA or -EpiA was notidentified in any of the investigated mammalian tissues. Somestudies indicated that P4507B1 could only generate the 7a isomerin human brain;13 other experiments have shown that yeast-ex-pressed human P4507B1 could produce both 7a- and 7b-hydro-xy-DHEA.14 Studies with the use of rat liver homogenate have

lowing sequence of reactions: 7a-hydroxylation, oxidation of thehydroxyl group at C-7, and finally, reduction of 7-oxo-DHEA to7b-hydroxy-DHEA.15 It can be postulated that an unknown epi-merase is involved in the inter-conversion process of the hydro-xyl group at C-7.16 Elucidation of the DHEA metabolism routesin mammalian brain is important for full explanation of its neuro-protective function.17

Literature reports provide a number of examples of microbialhydroxylations of DHEA and EpiA leading to 7-hydroxy deriva-tives.18 A significant part of reports on microbial transformationsof DHEA is concerned with regioselective oxidation leading to 7a-hydroxy-DHEA19,20 or, more often, to the mixture of 7a- and 7b-alco-hols.21–23 Among the 64 strains of filamentous fungi, which werefound able to catalyze oxidation of DHEA, more than half (38) carriedout the 7a- and 7b-hydroxylation of this steroid.22 Among the threestrains described in the literature capable of C-7 hydroxylation ofEpiA,23–25 Cunninghamella elegans and Rhizopus nigricans trans-formed the substrate to a mixture of 7a- and 7b-hydroxy-EpiA.24,25

Our previous studies demonstrated that 7a-hydroxy-EpiA was themain metabolite of transformation of EpiA by Mortierella isabellinaAM212; the strain catalyzed the 7a- and 7b-hydroxylation of DHEAand androstenediol.23

The current study presents results of transformation of DHEA,androstenediol, EpiA, and 5a-androstan-3,17-dione by the fungusAbsidia coerulea AM93. After the transformation of the substratesby this microorganism virtually no products other than the

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hydroxy derivatives were isolated, therefore the fungus was con-sidered an appropriate biocatalyst for the use in studies on thecourse of transformation of DHEA and EpiA to 7a- and 7b-hydroxyderivatives. Microbial transformations constitute a convenientmethod to determine the route in which the transformation ofthe substrates occurs, due to their simplicity of conducted experi-ments and easy monitoring of the course of reaction.

Although the efforts to isolate any of the fungal hydroxylaseshave failed and no X-ray structure of any of these enzymes hasbeen determined so far, the results obtained in the research onhydroxylation of steroids by whole-cell microorganisms providedample information on catalytic properties of hydroxylases. Themain aim of the presented study was detailed elucidation of thetransformation route of the substrates to the mixture of 7a-hydro-xy- and 7b-hydroxy derivatives. While there are literature reportson transformations of DHEA by cultures of filamentous fungi intomixtures of 7a- and 7b-hydroxy derivatives, only one of thesereports23 contains analysis of the transformation pathway ofDHEA. We expected that the study would reveal the reduction ofa keto group at C-7, including the conjugated keto group in7-oxo-DHEA. Such transformation is a postulated step in themetabolism of DHEA?7b-hydroxy-DHEA in mammalian tissues.

2. Materials and methods

2.1. Chemicals and reagents

DHEA (dehydroepiandrosterone, 3b-hydroxyandrost-5-en-17-one) (1), androstenediol (3b,17b-dihydroxyandrost-5-ene) (2),and epiandrosterone (3b-hydroxy-5a-androstan-17-one, EpiA) (3)standards were purchased from Sigma–Aldrich Chemical Co. 5a-Androstan-3,17-dione (4) was prepared by oxidation of EpiA (3)with chromium (VI) oxide. 7a-Hydroxy-DHEA (5), 7b-hydroxy-DHEA (6), 7-oxo-DHEA (7), 3b,7a,17b-trihydroxyandrost-5-ene(8), 3b,7b,17b-trihydroxyandrost-5-ene (9), 3b,17b-dihydroxyand-rost-5-en-7-one (10), 7a-hydroxy-EpiA (11), 9a-hydroxy-EpiA(13) and 11a-hydroxy-EpiA (14) were synthesized previously withpurity exceeding 99.1–99.5% as determined by elemental and GCanalysis.23

2.1.1. Preparation of 5a-androstan-3,17-dione (4)The mixture of 3 (2 g, 0.69 mmol) and 30 ml of acetone was

treated at room temperature with 1.5 ml of Jones reagent (CrO3,H2SO4) added by drops. After completion of the reaction, NaOHwas added, the liquid phase was separated, and then 40 ml ofwater was added. The resulting precipitate was filtered off to yield4 (1.84 g, 92%), which was found to be in excess of 99.2% purityfrom GC and elemental analysis; C19H28O2: calcd C, 79.10; H,9.76; found. C, 79.2; H, 9.91%; mp 126–130 �C (lit. 124–129 �C26);IR mmax(cm�1): 1740, 1714; 1H NMR (CDCl3) 0.87 (3H, s, 18-H),1.03 (3H, s, 19-H). For 13C NMR data please refer to Table 1.

2.2. Microorganism

The fungal strain Absidia coerulea AM93 used in this study wasobtained from the collection of the Institute of Biology and Botany,Medical University of Wrocław. The fungus was maintained onSabouraud 4% dextrose-agar slopes at 4 �C and freshly subculturedbefore use in the transformation experiments.

2.3. Conditions of cultivation and transformation

General experimental and fermentation details have beendescribed previously.27 Substrate was added to a 72-h-old cultureof the microorganism as an acetone solution for DHEA (1), EpiA (3)

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and 5a-androstan-3,17-dione (4) or methanolic solution for andro-stenediol (2), in concentration of 0.07 mmol/100 ml of medium.Each experiment was performed with three replications; the samesubstrate concentrations were used throughout, but in some casesa single experiment involved distribution of a dose of substrateamong several (6–10) flasks to help isolate the metabolites. The fi-nal concentrations of both solvents in the reaction medium were1 ml/100 ml.

2.4. Isolation and identification of metabolites

The products of biotransformation were extracted three timeswith chloroform. The organic extract was dried over anhydrousmagnesium sulfate, concentrated in vacuo and analyzed by TLCand GC. Transformation products were separated by column chro-matography on silica gel with hexane/acetone/ethyl acetate(1.4:1:0.5 v/v/v) for DHEA (1), hexane/acetone/chloroform(1:1:0.3 v/v/v) for androstenediol (2), hexane/acetone/methylenechloride (0.75:1:1.25 v/v/v) for EpiA (3), and hexane/acetone/chlo-roform (1.5:1:0.25 v/v/v) for 5a-androstan-3,17-dione (4) as elu-ents. TLC was carried out with Merck Kieselgel 60 F254 platesusing the same eluents. In order to develop the image, the plateswere sprayed with solution of methanol in concentrated sulfuricacid (1:1) and heated to 120 �C for 3 min. GC analysis was per-formed using Hewlett Packard 5890A Series II GC instrument(FID, carrier gas H2 at flow rate of 2 ml min�1) with ThermoTR-5MS column cross-linked 5% Phenyl Polisilphenylene-Siloxane,30 m � 0.25 mm � 0.5 lm (Polygen). The applied temperature pro-gram: 250 �C/1 min, gradient 5 �C/min to 300 �C/3 min; injectorand detector temperature was 300 �C. IR spectra were recorded inKBr discs on a Mattson IR 300 Spectrometer. The NMR spectra weremeasured in CDCl3, or, when the solubility of metabolites in chloro-form was low, in CD3OD. The spectra were recorded on a DRX300 MHz Bruker Avance spectrometer with TMS as internal stan-dard. Characteristic shift values in the 1H NMR and 13C NMR spectrain comparison to the starting compounds were used to determinestructures of metabolites, and in combination with DEPT analysisto identify the nature of the carbon atoms (Table 1). Partial signalassignment, where necessary, was achieved by performing 1H–1Hhomocorrelation experiments (COSY) and 1H–13C heterocorrelationexperiments (HSQC and HMBC). The stereochemistry of thehydroxyl group was deduced on the basis of NOESY experiments.Melting points (uncorrected) were determined on a Boetiusapparatus. Elemental analysis was performed on a Vario EL IIIanalyzer.

2.5. Time course experiments

Time course experiments were conducted in order to determinethe metabolic pathways. Conditions were identical to those in themain biotransformation experiments. One flask was harvestedafter 1, 2, 3, 4, 6, 8 h, and for substrate 2 after 24 h additionally.The 5 ml samples of reaction mixture were taken, extracted withchloroform and analyzed by GC and TLC as described in Section2.4. The presented values in all experiments are arithmetic meansof three measurements (Table 2); relative differences between theborder values have not exceeded 8%.

2.5.1. Transformation experiments with cycloheximideAlong incubation using DHEA (1) or EpiA (3) alone, several sets

of incubations were carried out in the presence of cycloheximide.Reactions were carried out in 100 ml Erlenmeyer flasks with30 ml of medium. Cycloheximide was added to a three-day-oldculture of the microorganism as a DMF (dimethylformamide)solution, in final concentration of 3.3 � 10�4 mmol/ml, simulta-neously with 0.02 mmol of 1 or 3 dissolved in 0.5 ml of acetone.

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Table 113C NMR data for starting materials 3, 4 and metabolites 12, 15–18

Carbon atom Compounds

3 12 4 15 16 17 18

1 36.94 36.79 38.47 38.06 38.11 31.57 39.902 31.41 31.38 38.10 37.99 37.91 37.77 38.243 71.10 70.92 211.59 211.33 210.85 211.15 211.854 37.88 37.60 44.56 44.00 43.98 44.58 45.035 44.96 42.05 46.58 39.03 43.81 38.45 47.276 28.44 38.78 28.59 36.81 38.76 27.24 29.077 30.90 74.78 30.51 66.20 74.24 21.58 30.258 35.05 42.92 34.93 39.02 42.66 37.32 34.069 54.53 52.52 53.86 45.38 51.82 75.57 59.94

10 35.87 35.12 35.79 35.81 35.28 40.35 37.3511 20.52 20.72 20.68 20.47 21.03 28.41 68.6212 31.69 31.52 31.46 31.10 31.44 27.13 42.9913 47.81 48.27 47.71 47.46 48.22 47.53 47.9214 51.43 51.03 51.21 45.68 50.87 44.19 50.2115 21.86 24.93 21.76 21.30 24.91 24.15 21.7616 35.75 36.00 35.79 35.72 35.94 35.78 35.7617 221.22 221.32 220.91 220.72 220.98 220.27 219.1518 13.81 14.04 13.78 13.47 14.03 12.74 14.5519 12.36 12.44 11.43 10.40 11.58 13.40 11.75

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Incubations of substrates were carried out for 8 h. In parallel, thetransformation of 1 or 3 with 0.5 ml of DMF/30 ml of mediumwas conducted. Reaction mixtures were extracted and analyzedby GC as described in Section 2.4.

2.5.2. Transformation of 7-hydroxy derivatives of DHEA (1) andEpiA (3)

Incubations of 7a-hydroxy-DHEA (5), 7b-hydroxy-DHEA (6),7a-hydroxy-EpiA (11), and 7b-hydroxy-EpiA (12) were carriedout in 100 ml Erlenmeyer flasks with 30 ml of medium. 0.02 mmolof the substrate in 0.5 ml of acetone was added to the grown cul-ture of the investigated microorganism. The transformation condi-tions were the same as in the standard experiment. After 8 hincubation of the substrate, the reaction mixture was extractedand analyzed using GC.

2.5.3. Transformation experiments by resting cellsThe fungal cells, cultivated as described in Section 2.3, were

harvested by filtration and washed with phosphate buffer solution.The transformation was carried out by resuspending the wet cells(3 g) in 100 ml of phosphate buffer in 300 ml Erlenmeyer flask.Substrates 1 and 3 were added to the cell suspensions as solutionsin acetone (concn 0.07 mmol/100 ml of medium). The experimentswere carried out in a 0.1 M phosphate buffer at pH 8.0, 7.4 and 6.8.After 4 and 6 h of incubation, 10 ml samples of the reaction mix-ture were taken, extracted with chloroform and analyzed by GCas described in Section 2.4.

2.5.4. Transformation of EpiA (3) in the presence ofcycloheximide by the fungal cultures inoculated 3 h previouslywith DHEA (1)

To the grown, 3–4 days old cultures of A. coerulea AM93 placed in100 ml Erlenmeyer flasks with 30 ml of medium, after 3 h incubationof 0.02 mmol of 1, 0.02 mmol of 3 was added simultaneously with3.3 � 10�4 mmol/ml of cycloheximide (as a DMF solution). After 1and 2 h of incubation of 3, 10 ml samples of reaction mixture weretaken, extracted with chloroform and analyzed by GC.

2.6. Crystal data collection

X-ray data of all substrates 1–4 were collected using KumaKM4CCD diffractometer (MoKa radiation; k = 0.71073 Å). Thestrategy for the data collection was evaluated by using the CrysAlis

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CCD software. Data reduction and analysis were carried out withthe CrysAlis RED program. The structure was solved by directmethods using SHELXS9728 program and refined using all F2 data,as implemented in the SHELXL97 program. The data were identicalwith those deposited in the Cambridge Crystallographic Data Cen-tre: for DHEA29 refcode: ZOYNAC01; for androstenediol30 refcode:KUMRUF; for EpiA31 refcode: ANDRON10; and for 5a-androstan-3,17-dione32 refcode: ANDION11.

3. Results

3.1. Products isolated in the course of transformations

3.1.1. Transformation of DHEA (1)After 8 h transformation of 120 mg of DHEA (1), the isolates

were (% mol): 7a-hydroxy-DHEA (5) (27.8 mg, 22%), 7b-hydroxy-DHEA (6) (62 mg, 49%) and 7-oxo-DHEA (7) (6.9 mg, 5.5%).

3.1.2. Transformation of androstenediol (2)After 48 h of transformation of 200 mg of androstenediol (2) the

following compounds were isolated (% mol): 3b,7a,17b-trihydr-oxyandrost-5-ene (8) (46.6 mg, 23%), 3b,7b,17b-trihydroxyandrost-5-ene (9) (95 mg, 45%) and 3b,17b-dihydroxyandrost-5-en-7-one(10) (6.3 mg, 3%).

3.1.3. Transformation of epiandrosterone (3)After 9 h transformation of 160 mg of epiandrosterone (3) the

following compounds were isolated (% mol): 7a-hydroxy-EpiA(11) (39.7 mg, 23.5%), 7b-hydroxy-EpiA (12) (30.3 mg, 18.0%), 9a-hydroxy-EpiA (13) (29.5 mg, 17.5%) and 11a-hydroxy-EpiA (14)(37.1 mg, 22%).

7b-Hydroxy-EpiA (12): mp 240–244 �C (Me2CO, cubes) (lit.237–238 �C25); C19H30O3: calcd C, 74.47; H, 9.87; found. C, 74.41;H, 9.88%. IR mmax(cm�1): 3420, 1739, 1H NMR (CDCl3) 0.85 (3H, s,19-H), 0.88 (3H, s, 18-H), 3.46 (1H, m, 7a-H), 3.58 (1H, m, 3a-H).

3.1.4. Transformation of 5a-androstan-3,17-dione (4)After 9 h transformation of 120 mg of 5a-androstan-3,

17-dione (4) the following compounds were isolated (% mol):7a-hydroxy-5a-androstan-3,17-dione (15) (25.3 mg, 20%),7b-hydroxy-5a-androstan-3,17-dione (16) (55.7 mg, 44%), 9a-hy-droxy-5a-androstan-3,17-dione (17) (22 mg, 17.4%) and 11a-hy-droxy-5a-androstan-3,17-dione (18) (11.4 mg, 9%).

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7a-Hydroxy-5a-androstan-3,17-dione (15): mp 160–163 �C(Me2CO, needles) (lit. 158–160 �C33); C19H28O3: calcd C, 75.10; H,9.32; found. C, 74.80; H, 9.21%. IR mmax(cm�1): 3393, 1740, 1716,1H NMR (CDCl3) 0.86 (3H, s, 18-H), 1.01 (3H, s, 19-H), 3.99 (1H,m, 7b-H).

7b-Hydroxy-5a-androstan-3,17-dione (16): mp 157–162 �C(Me2CO, needles); C19H28O3: calcd C, 75.10; H, 9.32; found. C,74.80; H, 9.21%. IR mmax(cm�1): 3340, 1738, 1714, 1H NMR (CDCl3)0.91 (3H, s, 18-H), 1.06 (3H, s, 19-H), 3.51 (1H, m, 7a-H).

9a-Hydroxy-5a-androstan-3,17-dione (17): mp 211–215 �C(Me2CO, needles) (lit. 212–214 �C33); C19H28O3: calcd C, 75.10; H,9.30; found. C, 74.80; H, 9.20%. IR mmax (cm�1): 3340, 1741, 1714,1H NMR (CDCl3), 0.88 (3H, s, 18-H), 1.14 (3H, s, 19-H).

11a-Hydroxy-5a-androstan-3,17-dione (18): mp 190–193 �C(Me2CO, needles) (lit. 191–193 �C34); C19H28O3: calcd C, 75.10; H,9.30; found. C, 74.80; H, 9.20%. IR mmax(cm�1): 3336, 1739, 1716,1H NMR (CDCl3) 0.88 (3H, s, 18-H), 1.13 (3H, s, 19-H), 4.00 (1H,m, 11b-H).

3.2. Structural identification of metabolites

Metabolism of DHEA (1), androstenediol (2) and EpiA (3) by A.coerulea AM93 yielded known metabolites identified as 7a-hydro-xy-DHEA (5), 7b-hydroxy-DHEA (6), 7-oxo-DHEA (7), 3b,7a,17b-trihydroxyandrost-5-ene (8), 3b,7b,17b-trihydroxyandrost-5-ene(9), 3b,17b-dihydroxyandrost-5-en-7-one (10), 7a-hydroxy-EpiA(11), 9a-hydroxy-EpiA (13) and 11a-hydroxy-EpiA (14) by com-parison of their spectral data with the literature values23 and onthe basis of identity of their Rt from GC and Rf from TLC with stan-dards available in our laboratory.

The metabolite 12 has a resonance at dH 3.58 ppm (m), con-firming that the 3b-OH group is maintained; a broad multipletat dH 3.46 ppm suggests hydroxylation at an equatorial positionof the steroid molecule. The shape and position of this signaland downfield shift of the C-18 and C-19 methyl signals by0.04 ppm and 0.03 ppm respectively, with respect to the sub-strate 3, correspond to the literature data of the steroidal 7b-alcohols.35 The stereochemistry of the newly introduced hydroxylgroup was inferred from NOESY correlation of H-7 (dH 3.46 ppm)

Figure 1. Crucial NOESY and HMBC correlations of 7b-hydroxy-EpiA (12), 7a-hydroxy-5a-androstan-3,17-dione (15), and 11a-hydroxy-5a-androstan-3,17-dione (18).

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and a-oriented H-9 (dH 0.74 ppm) and H-14 (dH 1.43 ppm)(Fig. 1). The appearance of a new methine carbon signal at dC

74.78 ppm, in combination with downfield shifts for C-6 (D10.34 ppm), C-8 (D 7.87 ppm) and c-carbon upfield shift for C-5(D 2.91 ppm), C-9 (D 2.01 ppm) and C-14 (D 0.4 ppm) furtherdetermine the product as 7b-hydroxy-EpiA (12). The spectraldata are in agreement with the literature.25 The 1H NMR spec-trum of 15 shows a new downfield signal for the oxygen-bearingmethine proton at dH 3.99 ppm. The signal shape and positionsuggest hydroxylation at an axial C-7 position of the steroid mol-ecule, in agreement with the literature data.33,36 The shift of C-18methyl signal (D 0.01 ppm) and C-19 methyl signal (D 0.02 ppm)in comparison to the spectrum of substrate 4 correspond to theshifts accompanying introduction of the 7a-hydroxyl group.35

This assignment of position of hydroxyl group was further sup-ported by the HMBC correlations of H-7 (dH 3.99 ppm) with C-5(dC 39.03 ppm) and C-14 (dC 45.68 ppm) (Fig. 1). The signal atdC 66.2 ppm and downfield shifts for C-6 (D 8.22 ppm), C-8 (D4.09 ppm), and c-gauche upfield shifts for C-5 (D 7.56 ppm), C-9 (D 8.48 ppm) and C-14 (D 5.53 ppm) constitute confirmationof the C-7-hydroxylation. The a-stereochemistry at C-7 OH groupwas supported on the basis of NOESY correlation between thegeminal H-7 (dH 3.99 ppm) and H-8 (dH 1.40 ppm) (Fig. 1). Finally,that metabolite was identified as 7a-hydroxy-5a-androstan-3,17-dione (15). The spectral data are in agreement with the lit-erature.33 The structure of 16 was determined as a result of com-parison of its spectroscopic data with that of the starting material4. The appearance of a new signal at dH 3.51 ppm suggests mono-hydroxylation, while the signal shape indicates an equatorialconfiguration of the introduced hydroxyl group. This observationis supported by the appearance of a new methine carbon signal atdC 74.24 ppm which, in combination with the downfield shift forthe C-6 (D 10.17 ppm) and C-8 (D 7.73 ppm) is an important con-firmation of 7-hydroxylation. The stereochemistry of the newlyintroduced OH group at C-7 was deduced as b (equatorial) onthe basis of NOESY correlation between H-7 (dH 3.51 ppm) andH-5 (dH 1.64 ppm), H-9 (dH 0.82 ppm), and H-14 (dH 1.41 ppm).Additionally, changes in signal shifts of the methyl groups at C-18 and C-19 correspond to those accompanying introduction ofthe 7b-hydroxyl groups into the substrate.35 In the 1H NMR spec-trum of 17, having visibly higher polarity on TLC than 4, theCHOH signal was absent and the 19-methyl group underwent asignificant downfield shift (D 11 ppm) suggesting the presenceof a non-protonated carbon, possibly due to functionalization atC-9. A new resonance at dC 75.57 ppm, the downfield shifts ofsignals C-10 (D 4.56 ppm) and C-11 (D 7.73 ppm) and c-gaucheupfield shifts of C-1 (D 6.9 ppm), C-5 (D 8.13 ppm), C-7(D 8.93 ppm), C-12 (D 4.33 ppm) and C-14 (D 7.02 ppm) signalsof this metabolite in comparison to the spectrum of substrate 4,confirmed hydroxylation at the 9a position. Additional supportwas the HMBC spectrum showing correlation of C-9 with H-12(dH 1.52 ppm). The spectral data of 17 are in agreement withthe literature.35,36 NMR spectra of 18 have new resonances atdH 4.00 ppm (m) and dC 68.62 ppm, indicating monohydroxyla-tion. In comparison to the substrate, the C-19 methyl signal ofthe product demonstrated significant downfield shift (D0.1 ppm) suggesting that hydroxylation had occurred at the11a-position. This was further supported by the 13C NMR of 18which showed downfield shifts for b-carbons C-9 (D 6.08 ppm)and C-12 (D 11.53 ppm) and the HMBC spectrum which showedlong-range couplings between H-11 (dH 4.00 ppm) and C-9(dC 59.94 ppm), C-12 (dC 42.99 ppm), and C-13 (dC 47.92 ppm).The stereochemistry of the newly introduced hydroxyl groupwas further supported by NOESY spectrum which showed corre-lation of 11b-H with the proton signals of C-18 (dC 14.55 ppm)and C-19 (dC 11.75 ppm) methyl groups (Fig. 1). The spectral data

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are in agreement with the literature for 11a-hydroxy-5a-andro-stan-3,17-dione.34

3.3. Determination of the metabolic pathway and the order ofhydroxylation reactions

In order to investigate pathways of hydroxylation of the sub-strates 1–4 by A. coerulea, composition of mixtures sampled after1, 2, 3, 4, 6, and 8 h incubation was studied. All the isolated mix-tures of metabolites obtained from the examined substrates con-tained both epimeric 7a- and 7b-alcohols; mixtures ofmetabolites obtained from EpiA (3) and 5a-androstan-3,17-dione(4) additionally contained the 9a- and 11a-hydroxy derivatives(Fig. 2, Table 2). An almost complete conversion of the hydroxy-ketones 1, 3 and the diketone 4 was observed after 8 h of incuba-tion, while the diol 2 was hydroxylated more than three timesslower (2, due to its low solubility in acetone, was introducedas a methanolic solution). Time course analysis of the transforma-tion of DHEA (1), EpiA (3) and 5a-androstan-3,17-dione (4)exhibits some similarities: during 2 h of incubation, 15% of thesubstrate was oxidized, and after subsequent 2 h the content ofthe hydroxy derivatives in the mixtures of metabolites was in-creased over three fold (Table 2). A rapid surge of the content ofhydroxy derivatives in the range 2–4 h of incubation indicatesthat the hydroxylating activity of the fungal culture increases inthe presence of the substrate; the extracts sampled after 1 h ofincubation contained small amounts of hydroxy derivatives onlyin case of the mixtures with DHEA (Table 2). The lack of the hy-droxy derivatives in extracts after 8 h of incubation of DHEA (1)or EpiA (3) in A. coerulea AM93 culture in the presence of cyclo-heximide—an inhibitor of de novo protein synthesis—indicatesthat hydroxylase(s) produced by the strain is(are) inducible en-zyme(s) (Table 3). The 7a-OH/7b-OH ratios of products in themixtures obtained from various substrates were different(Table 2): the mixtures obtained from 5-ene substrates (1, 2)

Figure 2. Products of transformation of C19-ste

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and 5a-androstan-3,17-dione (4) contained 2–2.5 times more ofthe product of 7b-hydroxylation than the 7a-alcohol; transforma-tion of EpiA (3) led to the prevalence of 7a-hydroxy-EpiA (11).However, the mixtures isolated after various times of incubationof the same substrate contained very similar 7a-OH/7b-OH ratios.In order to check whether the change of amount of 7a- and 7b-hydroxy derivatives obtained during the transformation of DHEA(1) and EpiA (3) is not due to 7a-OH ¡ 7b-OH interconversion,but is the result of structural differences of both substrates, 7a-,7b-hydroxy-DHEA (5, 6) and 7a-, 7b-hydroxy-EpiA (11, 12) werealso subjected to the transformation by the strain. The epimericC-7 alcohol was not identified in the extracts after 8 h incubationof 5, 6, 12 hydroxy derivatives, while the mixture after the trans-formation of 7a-hydroxy-EpiA (11) contained minor amounts of7b-hydroxy-EpiA (12) (3%) (Table 4).

The results of preliminary experiments showed that the con-centration of hydrogen ions influences the hydroxylating activityof A. coerulea AM93 culture (data not included). DHEA (1) and EpiA(3) were also subjected to transformation by the resting cultures inphosphate buffers at pH 8, 7.4 and 6.8 (Table 5). The composition ofthe mixtures isolated in this series of experiments shows that thehighest hydroxylating activity towards both substrates was exhib-ited by cultures at pH 8: after 4 h transformation of DHEA 46% ofthis substrate was oxidized at pH 8, 36% of the substrate was con-verted in cultures at pH 7.4, and 25% of DHEA was hydroxylated atpH 6.8. A similar reduction in the degree of conversion of the sub-strate while decreasing the pH of the culture was observed in thetransformation of EpiA. Analysis of the presented results indicatesthat the hydroxylating activity at pH 7.4 was almost 20% lowerthan at pH 8, while at pH 6.8 the hydroxylating activity was almost2 times lower. The 7a-OH/7b-OH ratios in the mixtures isolatedafter 4 h and 6 h incubation of DHEA at various pH values werevery similar; also in the mixtures of metabolites isolated in thisseries of experiments with EpiA (3) the 7a-OH/7b-OH ratiosshowed no significant differences (Table 5).

roids in the Absidia coerulea AM93 culture.

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Table 4Composition of crude mixtures obtained after 8 h transformations of hydroxyderivatives 5, 6, 11 and 12 by A. coerulea AM93

Substrate Compounds presents in the mixture (%) by GC

7a-Hydroxy-DHEA (5) 7a-Hydroxy-DHEA (5) 987b-Hydroxy-DHEA (6) —7-Oxo-DHEA (7) 2

7b-Hydroxy-DHEA (6) 7b-Hydroxy-DHEA (6) 1007a-Hydroxy-DHEA (5) —7-Oxo-DHEA (7) —

7a-Hydroxy-EpiA (11) 7a-Hydroxy-EpiA (11) 967b-Hydroxy-EpiA (12) 3.5

7b-Hydroxy-EpiA (12) 7b-Hydroxy-EpiA (12) 1007a-Hydroxy-EpiA (11) —

Table 3Composition of crude mixtures obtained after 8 h transformations of DHEA and EpiAby Absidia coerulea AM93 in the presence of a) 3.3 � 10�4 mmol/ml of cycloheximide,b) 0.5 ml DMF/30 ml of medium (control reaction)

Substrate DHEA EpiA

Compoundsa (%) DHEA 7a-OH 7b-OH EpiA 7a-OH 7b-OH

a) 100 — — 100 — —b) 11.5 24.5 61 18 20 15

a Determined by GC analysis.

Table 2Composition of crude mixtures obtained in transformations of 1–4 by Absidia coerulea AM93

Substrate Rt (min) Compounds present in the mixturea (%) Time of transformation (h) Average ratio of7a-OH:7b-OH

1 1b 2 2b 3 4 6 8

DHEA (1) 6.77 DHEA (1) 96.5 84.5 63.5 37 9.5 — 0.4:18.86 7a-Hydroxy-DHEA (5) 0.5 4.5 10 18 25 26.58.95 7b-Hydroxy-DHEA (6) 1.5 12 25 44.5 63 66.59.54 7-Oxo-DHEA (7) — Trace Trace Trace 1.5 6

Androstenediol (2) 6.93 Androstenediol (2) 100 98.5 95.5 89.5 79.5 70.4c 0.5:19.09 3b,7a,17b-Trihydroxyandrost-5-ene (8) — Trace 1.2 3 6 9.59.26 3b,7b,17b-Trihydroxyandrost-5-ene (9) — Trace 2.8 6 12.5 199.58 3b,17b-Dihydroxyandrost-5-en-7-one (10) — — Trace Trace Trace Trace

Epiandrosterone (3) 6.88 Epiandrosterone (3) 100 93 88.5 79 76.5 63 22.5 10 1.3:19.30 3b,7a-Dihydroxy-5a-androstan-17-one (11) — 2 3.5 5.5 5.5 10 21 24.59.36 3b,7b-Dihydroxy-5a-androstan-17-one (12) — 1.5 2.5 4.5 4.5 8 16.5 19.59.11 3b,9a-Dihydroxy-5a-androstan-17-one (13) — 1.5 2 4 5.5 8 17 209.15 3b,11a-Dihydroxy-5a-androstan-17-one (14) — 2 2.5 6.5 6.5 11 22.5 26

5a-Androstan-3,17-dione (4) 7.35 5a-Androstan-3,17-dione (4) 98.5 85.5 67.5 56.5 18.5 Trace 0.5:19.80 7a-Hydroxy-5a-androstan-3,17-dione (15) Trace 2.5 7 9.5 18.5 22.59.93 7b-Hydroxy-5a-androstan-3,17-dione (16) Trace 5.5 13.5 19 39 479.67 9a-Hydroxy-5a-androstan-3,17-dione (17) Trace 2.8 7.0 8.5 14.5 19.59.85 11a-Hydroxy-5a-androstan-3,17-dione (18) Trace 1.5 3.5 4.5 7.5 10

a Contents of compounds determined by GC analysis.b Mixtures of metabolites of 3 produced in the presence of cycloheximide by the cultures inoculated 3 h earlier with 1 (see Section 2.5.4.)c The mixture after 24 h contained: 12% of 2, 27% of 8, 56.5% of 9 and 4.5 of 10.

6 N. Milecka-Tronina et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

In order to test whether the cultures capable of 7a- and 7b-hydroxylation of DHEA (1) are also able to catalyze 9a- and 11a-hydroxylation of EpiA (3), we analyzed the composition of extractsafter 1 and 2 h of incubation of EpiA and cycloheximide (intro-duced simultaneously) in the cultures inoculated 3 h earlier withDHEA (Table 2). Extracts after 1 h of transformation contained7-hydroxy-EpiA, 9a- and 11a-hydroxy-EpiA. The 7a-OH/7b-OH/9a-OH/11a-OH ratios in the mixture after 2 h transformation ofEpiA (3) in the presence of cycloheximide (culture with hydroxyl-ating activity against DHEA (1)) and in the mixture after 3 h incu-bation of 3 in standard experiments, were very similar. The contentof the hydroxy derivatives in the mixtures after 2 h incubation ofEpiA was close to 20% (similar content of hydroxy derivativeswas observed in extracts after 3 h transformation of the substrateby cultures without previous inoculation with DHEA) (Table 2).

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4. Discussion

The strain of A. coerulea AM93 carried out effective hydroxyl-ation of the C19-steroids: DHEA (1), androstenediol (2), EpiA (3),and 5a-androstan-3,17-dione (4) (Fig. 2). The 5-ene substrates (1,2)—underwent regioselective C7-hydroxylation to the mixture ofepimeric allyl alcohols. During the transformation of 3 and 4, apartfrom the corresponding 7a- and 7b-alcohols, the 9a- and 11a-hy-droxy derivatives were also obtained (Table 2). Time course analy-sis of the transformation of substrates 1–4 and lack of thecorresponding hydroxy derivatives in the extracts after 8 h incuba-tion of DHEA (1) and EpiA (3) in the presence of cycloheximideindicate that hydroxylase(s) of this strain is(are) substrate-induced(Tables 2 and 3). Analogous dynamics of the increase in the amountof both C-7-epimeric hydroxy derivatives together with simulta-neous appearance of the 7a- and 7b-hydroxy derivatives in themetabolite mixtures of the tested substrates form the base forassumption that the 7a- and 7b-hydroxylations are catalyzed bythe same hydroxylase. Experiments in which the 7a- and 7b-hy-droxy derivatives of DHEA (5, 6) and EpiA (11, 12) were incubatedfor 8 h have shown that the 7a-OH ¡ 7b-OH interconversion doesnot affect the content of 7-hydroxy derivatives in the isolated mix-tures within the analyzed transformation period (Table 4).

The C7-Ha and C7-Hb bonds can be potentially located in anal-ogous positions with respect to the oxidating center of thehydroxylase in the enzyme-substrate complexes with participa-tion of the oxygen functions at C-3 or C-17. Participation of sub-stituents in the substrate molecule in the formation of theenzyme–substrate complex, proposed for the first time to explain6b- and 11a-hydroxylation of steroids by Aspergillus tamarii,37

was confirmed in a series of other enzymatic hydroxylation stud-ies of numerous steroids in cultures of other strains,18 and also bystudies using recombinant fission yeast with gene Cyp509C12isolated from Rhizopus oryzae.38 Yeast recombinant with geneCyp509C12 transformed progesterone to the mixture of 6b- and11a-hydroxy derivatives.

Figure 2 presents the diagrams of alternative pairs for com-plexes: (i) in the normal binding complex I the oxidation of theC7a-H occurs while the oxidation of the C7b-H takes place in thereverse inverted binding complex II, or (ii) in the normal bindingcomplex III the 7b-hydroxylation occurs, while the 7a-hydroxyl-

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ation takes place in the reverse inverted binding complex IV. Thedistances between the oxygen atom participating in the proposedenzyme-substrate complexes and the hydrogen atom in thehydroxylated position, calculated on the basis of X-ray structuresof substrates 1–4, have quite similar values: these distances arefalling into the 6.04–6.58 Å range for complexes I and II, and5.8–7.06 Å for III and IV. In the complexes I, II the differences ofthe distances are smaller (0.54 Å) than in the complexes III, IV(1.26 Å), however the analysis of the relationship between thestructure of the substrate and regioselectivity of the enzymatichydroxylation suggests that oxidation of the C7-Hb bond takesplace in the normal binding enzyme-substrate complex, whilethe oxidation of the C7-Ha bond occurs in the reverse invertedbinding complex (complexes III and IV).

Hydroxylation at C-7 of the 5-ene substrates, DHEA (1) andandrostenediol (2), is favored by the activating vicinity of p elec-trons; stereoelectronic interactions additionally prefer hydroxyl-ation of the allyl 7a-axial position. This is confirmed by theresults of several experiments on the enzymatic hydroxylation of3-oxo-4-ene steroids and steroidal 3b-alcohols with a double bondat C-5.18,20–23,39–42

Equatorial 7b-hydroxy derivatives predominated in the mix-tures of metabolites formed in transformation of DHEA (1) andandrostenediol (2) by A. coerulea AM93, which indicates thatthe equatorial 7b-hydrogen atom is located closer to the oxidat-ing center of the hydroxylase than the 7a-hydrogen atom. Theslightly higher content of the 7a-alcohol (8) formed from andro-stenediol (2) than the content of the corresponding 7a-alcohol(5) from DHEA may be associated with a spatial shift of the 7a-H of 2 by 0.39 Å to the enzyme oxidizing center (the distance dif-

Figure 3. Diagrams of enzyme–substrate complexes for 7a-, 7b-, 9a- and 11a-hydroxyhydrogen atoms calculated on the basis of X-ray structures of substrates.

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ference between C17�O M H7a in DHEA and androstenediol)(Fig. 3).

The main product of the transformation of EpiA (3) was 11a-hy-droxy-EpiA (14), which was accompanied by both epimeric C7-alcohols 11 and 12 as well as the 9a-hydroxy derivative 13. C11-Ha and C9-Ha atoms in the inverted binding enzyme-substratecomplex can be located in the similar positions with respect tothe oxidating center of the hydroxylase, in analogy to the hydrogenatoms at C-7 in the above-examined complexes, and it is probablethat the same enzyme catalyzes the hydroxylation at C-7 as well asat 9a- and 11a- positions (complex V Fig. 3). The hypothesis is sup-ported by the course of transformation of EpiA (3) in the presenceof cycloheximide, in a culture with hydroxylating activity againstDHEA (1) (the culture inoculated 3 h earlier with DHEA (1))(Table 2). The culture with the DHEA 7a- and 7b-hydroxylaseactivity catalyzed the 7a-, 7b-, 9a- and 11a-hydroxylation of EpiA.High content of 11a-hydroxy-EpiA (14) in the metabolite mixturesof 3 suggests that among the four oxidized hydrogen atoms, the11a-atom lies most close to the oxidative center of the enzyme.Such assumption allows explaining why the mixtures resultingfrom transformation of EpiA contain 7a-OH/7b-OH ratios substan-tially differing from the metabolite content for the remaining sub-strates (Table 2). The C3�O M H7b distance in EpiA is the longest(7.06 Å) from among those on Figure 3. Markedly higher contentof the 7b-hydroxy derivative of 5a-androstan-3,17-dione (16), incomparison to EpiA (Table 2) can be explained by the fact thatthe 7b-hydrogen atom of 4 is located 0.28 Å closer to the oxidatingcenter of the enzyme than the 7b-H in EpiA (complex III, Fig. 3);additionally, the C-3 carbonyl group exhibits stronger directing ef-fect than the 3b-hydroxyl group. The stronger directing effect of

lation with distances between the oxygen atom at C-3 or C-17 and hydroxylated

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the carbonyl group can be seen e.g. in the transformation of 5a-androstan-17-one and 5a-androstan-17b-ol by Calonectriadecora.43

The assumption that the same enzyme from A. coerulea cata-lyzes 7a- as well as 7b-hydroxylation of 5-ene (DHEA) and 5a-dihydro-substrate (EpiA) is also coherent with analysis of transfor-mation results of both compounds by the resting cultures of themicroorganism at various [H+] concentrations (Table 5). Withinthe chosen range of pH values, hydroxylating activity of the cul-tures changed in the 1–0.55 range. At the same time, the metabo-lite mixtures isolated after 4 h and 6 h incubation of the substrateat diverse pH values exhibited very similar 7a-OH/7b-OH ratios,not differing from those observed in transformations by the grow-ing cultures (Tables 2 and 5). Hydroxylation with two participatingselective enzymes would be quite unlikely to result in the lack ofdifference in the 7a-OH/7b-OH DHEA as well as 7a-OH/7b-OHEpiA ratios in transformations carried out by cultures characterizedby such diverse degree (0–0.45) of inhibition of the hydroxylatingactivity.

The total picture formed by experimental data presented aboveis best explained by assumption that the same enzyme from A.coerulea catalyzes 7a- as well as 7b-hydroxylation of 5-ene and5a-dihydro substrates. This hypothesis of an action of a singlehydroxylase cannot be, however, established for sure on the basisof the available data. The conclusive evidence would be either aset of crystal structures of steroid-enzyme complexes or a studyof action of a pure isolated enzyme. Neither of these evidenceswas presented so far for fungal hydroxylases.

5. Conclusions

The strain A. coerulea AM93, similarly to human P4507B1, cata-lyzes 7-hydroxylation of DHEA as well as EpiA.14,44 The extractsfrom cells infected with the recombinant mouse Cyp7b cDNAintroduced into a vaccinia virus vector converted DHEA to 7a-hy-droxy- (major product) and 7b-hydroxy-DHEA.45 Transformationscarried out by microorganisms can serve as models in studies onenzymatic hydroxylation, being suitable tools for elucidating met-abolic routes and hydroxylase capability.46,47 On the basis of timecourse analysis of the hydroxylation of DHEA and EpiA by A. coeru-lea AM93 together with geometrical relationships between the C-Hbond of the substrate undergoing hydroxylation and the cofactor-bound activated oxygen atom it is reasonable to assume that thesame enzyme catalyzes the oxidation of C7-Ha and C7-Hb bondsin DHEA and EpiA; such assumption needs further verification onthe basis of purification of a fungal hydroxylase and solution ofits crystal structure or experiments involving the purified singleenzyme.

Table 5Contribution of 7a- and 7b-hydroxy derivatives in the mixtures after 4 and 6 h ofincubation of DHEA and EpiA in resting cultures at different pH values

Substrate Time of transformation

4h 6h

pH Compoundsa (%)

DHEA DHEA 7a-OH 7b-OH DHEA 7a-OH 7b-OH8.0 53.5 13 32.5 4.5 27 66.57.4 64 9.5 24 24.5 21 536.8 74.5 7 17.5 49.5 14 34.5

EpiA EpiA 7a-OH 7b-OH EpiA 7a-OH 7b-OH8.0 58 13 10 11 22.5 17.57.4 66 11.5 8.5 30.5 18 146.8 78.5 6.5 5 55 12.5 9.5

a Determined by GC analysis.

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The presented results can be used in further research on identi-fication of the gene encoding the hydroxylase or isolation of thepure hydroxylase from this fungus. When the complexity of tasksof purification of fungal P450 systems and their heterologousexpression is fully taken into consideration, the results of the cur-rent study can be estimated as further steps on the way towardsunderstanding of the fungal 7-hydroxylase activity.

Acknowledgement

This research was supported financially by the European Unionwithin the European Regional Development Found (Grant No.POIG.01.03.01-00-158/09-02).

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