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

Article abstract of DOI:10.1111/cbdd.12813

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.

Full text of DOI:10.1111/cbdd.12813

Received Date : 03-Mar-2016
Revised Date : 24-May-2016
Accepted Date : 26-Jun-2016
Article type : Research Article
Biohydroxylation of 7-oxo-DHEA, a natural metabolite of DHEA, resulting in formation
of new metabolites of potential pharmaceutical interest
Running title: Biohydroxylation of 7-oxo-DHEA
*
Alina Świzdor, Anna Panek, and Natalia Milecka-Tronina
Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida
25, 50-375 Wrocław, Poland
*
Corresponding author: Alina Świzdor, Department of Chemistry, Wrocław University of
Environmental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland. Tel.: (+48
713205252, Fax: +48713284124, e-mail: alina.swizdor@up.wroc.pl
Key words: 7-oxo-DHEA; biotransformations; hydroxylations; Syncephalastrum racemosum;
DHEA
ABSTRACT
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 derivatives of new derivatives of natural, physiologically active steroids
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/cbdd.12813
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(especially those difficult to achieve in the chemical synthesis) become available. This paper
demonstrated for the first time transformation of 7-oxo-DHEA – a natural metabolite of
DHEA, by 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 microorganism 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.
Introduction
For a long time, DHEA (dehydroepiandrosterone) was perceived as an insignificant product
of metabolical degradation of steroids, but nowadays it is the object of intensive research.
Many of the biological functions of DHEA are associated with the activity of its metabolites,
which, like DHEA itself, initially were considered inactive. Among them, 7-oxygenated
derivatives are predominantly mentioned - 7α-hydroxy-DHEA and its 7β-hydroxy epimer
(1,2). Both compounds, as well as 7-oxo-DHEA – an intermediate in the DHEA→7-hydroxy-
DHEA conversion – are identified in many mammalian tissues. 7-Oxo-DHEA was assigned a
significant role in the induction and regulation of thermogenesis (3). Also, its beneficial effect
in the treatment of obesity is indicated (4). This metabolite is also regarded as a natural
antiglucocorticoid (5). In many countries, 7-oxo-DHEA is available commercially under the
trade name 7-Keto Fuel® or 7-Keto-DHEA, and is used as a food supplement for athletes to
increase the metabolic rate and improve the BMI index (6). There are no unequivocal results
indicating that 7-oxo-DHEA can improve mental capacity, although in some studies its
precognitive effect was observed, modulating the brain activity and delaying the progression
of neurodegenerative diseases (7).
In contrast to DHEA, 7-oxo-DHEA is not a precursor of biologically active androgens or
estrogens, which, if present in the body at a level significantly above physiological norm,
would increase the risk of hormone-dependent diseases. Thus, this compound is considered to
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be a safer steroid that could replace the DHEA therapy (8). As DHEA, it may also have
antiproliferative and cytotoxic activity for some tumor cell types (9).
For many years, our team has conducted research on microbial functionalization of
compounds of natural origin, including steroid systems (10-20). Among these
transformations, the hydroxylation reactions in an inactivated position of a transformed
compound are of particular importance. The use of biotransformation as a tool for
modification of the molecules makes it possible to obtain derivatives that are found in the
nature in minor amounts, as well as new analogues in amounts which not only allow for
determination of their structure, but also for biological assays. Biotransformations are often
the source of derivatives with enhanced biological activity, and hence with more effective
therapeutic effects relative to the parent substrates. Microorganisms express multienzyme
systems that ensure regio- and stereoselectivity of the reactions, and products obtained with
their participation are frequently not available at the conditions of classical chemical
transformations. Biotransformations using microorganisms can be also used as models useful
in determining the metabolic pathways of natural and xenobiotic compounds, including drugs,
in mammals (21,22).
As it is reported by extensive literature sources, DHEA is transformed in cultures of
microorganisms, as well as in mammalian tissues, usually to a mixture of both C-7-epimeric
hydroxy derivatives. These microbial transformations have been reviewed recently (23,24). In
some of these processes the 7-oxo-DHEA is also identified (16,20,25).
Monitoring of the metabolism of DHEA and its derivatives in mammals would be difficult
due to their very low (~ ng/ml) concentration (26), rapid conversion to hormones or inactive
conjugates, and a high content of other lipid compounds in the environment. So far, in body
fluids of mammals there were identified only a few metabolites of 7-hydroxy-DHEA
containing several hydroxyl groups in their structure (27). These compounds showed more
than thirty times higher anti-inflammatory activity and higher stability compared to the 7β-
hydroxy-DHEA in model studies of immune-dependent diseases such as multiple sclerosis,
lung injury, chronic inflammation of the prostate and colon (27). As demonstrated in the
study, the main metabolites of 7-oxo-DHEA were the 7-hydroxylated derivatives of DHEA,
but at the same time a number of other unidentified structures were also detected (6). The
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3
β,16α-dihydroxy-androst-5-en-7,17-dione was described as an excretory product in the urine
of a patient with adrenal carcinoma (28).
In the light of the reasoning above it is justified to conduct studies on the possibilities of
formation of new 7-oxo-DHEA derivatives with potential biological activity as a result of
microbial transformations. For this purpose, two studies were carried out. The first one
included experiments with 7-oxo-DHEA as the biotransformation substrate, and the second
one – with DHEA as the substrate. In the second study, influence of the substrate supply
mode to the microorganism culture was also investigated. The earlier literature reports on
transformations of natural compounds such as diosgenin (29) and ursolic acid (30,31)
encouraged us to the use of filamentous fungus Syncephalastrum racemosum. Another
indication for this microorganism was that it has minimal human pathogenicity (32), making
it a good candidate to be investigated in the bioconversion of products for potential
pharmacological use.
Methods and Materials
Chemicals
3
β-Hydroxy-androst-5-en-7,17-dione (1) was prepared from the DHEA 3-acetate by oxidation
with chromium(VI) oxide and subsequent base-catalyzed hydrolysis of 3β-acetoxy-androst-5-
en-7,17-dione (7-oxo-DHEA acetate). DHEA 3-acetate, DHEA (6) and androstenediol
(3β,17β-dihydroxyandrost-5-ene) were purchased from Sigma-Aldrich Chemical Co.
Chemical standards: 7α-hydroxy-DHEA (7), 7β-hydroxy-DHEA (8), 3β,7α,17β-trihydroxy-
androst-5-ene (9), 3β,7β,17β-trihydroxy-androst-5-ene (10) and 3β,17β-dihydroxy-androst-5-
en-7-one (5) were synthesized in our previous work (16).
Preparation of 3β-hydroxy-androst-5-en-7,17-dione (7-oxo-DHEA) (1)
To a solution of the DHEA 3-acetate (800 mg) in 32 ml of chloroform at 60 °C, a mixture of
acetic acid (8 mL), acetic anhydride (2 mL) and CrO (6 g) in chloroform (44 mL) was added
3
dropwise and then the mixture was stirred for further 2 h at 60 °C. After cooling to room
temperature, the solution was treated with 140 mL of 10% oxalic acid and extracted with
diethyl ether three times. The organic layers were combined, washed with water and saturated
NaHCO . After drying (MgSO ) and evaporation of solvent, crude 7-oxo-DHEA acetate was
3
4
saponificated with Na CO in methanol according to the procedure described earlier (33). The
2
3
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mixture of products was subjected to column chromatography in hexane:acetone (2:1 v/v) as
eluent. 7-Oxo-DHEA (307 mg, 42% yield) was found to be in excess of 99.1 % purity
following GC and elemental analysis, anal. C 75.49, H 8.72%, calcd for C H O C 75.46, H
1
9
26
3
8.67%.
Microorganism
The fungal strain, Syncephalastrum racemosum AM105, used for biotransformation was
obtained from the collection of the Department of Pharmaceutical Biology and Botany of the
Wrocław Medical University, Poland. It was maintained on Sabouraud 4% dextrose-agar
slopes and freshly subcultured before use in the transformation experiments.
Conditions of cultivation and transformation
Bioconversions of substrates were conducted according to the standard two-stage
fermentation procedure. Erlenmeyer flasks (300 mL), each containing 100 mL of a medium
-1
-1
consisting of glucose 30 g L and aminobak 10 g L , were inoculated with 5 mL of starter
cultures and incubated for 3 days at 25 °C in a rotary shaker. The same conditions were used
for the biotransformation stage. After growth period of microorganism, substrate as a 5%
solution in acetone was added to each of the cultures (single-feeding), to give a final substrate
-1
concentration of up to 0.50 g L . The experiment was performed with four replications.
Incubation was continued for up to 96 h after addition of the substrate. Alternatively, a 5%
solution of the substrate was pulse-fed to the growing fungus in portions of 10, 20, 30 and
40% at 48, 60, 72 and 84 h, respectively, after inoculation. The fermentation was allowed to
proceed for 3 days (after last feed).
Isolation and identification of products. At the end of incubation period, the cells were
collected by centrifugation and washed with 0.05 M sodium phosphate buffer (pH 7.0).
Mycelia and broth were extracted three times with chloroform. The organic extracts were
combined, dried over anhydrous magnesium sulfate, filtered, and the solvent was
subsequently evaporated in vacuo to give a brown gum. This crude extract was analyzed by
TLC and GC and then chromatographed on a column of silica with the same eluent as for
TLC.
TLC was carried out with Merck Kieselgel 60 F254 plates and visualized by spraying them
with a mixture of methanol in concentrated sulfuric acid (1:1 v:v) and heating to 120 °C until
the colors developed. GC analysis was performed using Hewlett Packard 5890A Series II GC
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-1
instrument (FID, carrier gas H at flow rate of 2 mL min ) with DB-5MS column, 30 m x
2
0
.320 mm x 0.25 μm. The applied temperature program was 220 °C/1 min, gradient 2 °C/min
to 270 °C and then 30 °C/min to 300 °C/5 min; injector and detector temperature was 300 °C.
IR spectra were determined using Mattson IR 300 Thermo Nicolet spectrophotometer using
KBr pellets. The NMR spectra were measure in CDCl . The spectra were recorded on a
3
TM
1
13
Bruker Avance 300 MHz spectrometer. Characteristic shift values in the H NMR and C
NMR spectra in comparison to the starting compounds were used to determine structures of
metabolites, in combination with DEPT analysis to identify the nature of the carbon atoms.
Elemental analysis was performed on Vario EL III analyzer.
Products isolated in the course of transformation of 7-oxo-DHEA (1)
After 96 h transformation of 7-oxo-DHEA (1) (0.2 g), yielded mycelial and broth extracts
were combined and purified on silica gel with the use of a mixture of acetone:ethyl
acetate:methylene chloride:2-propanol (0.3:1.5:1.5:0.2 v/v/v/v) as eluent. The following
compounds were isolated (% mol): 1β,3β-dihydroxy-androst-5-en-7,17-dione (2) (34 mg,
18%), 3β,12β-dihydroxy-androst-5-en-7,17-dione (3) (20 mg, 11%), 3β,9α,-dihydroxy-
androst-5-en-7,17-dione (3) (40 mg, 21%) and 3β,17β-dihydroxy-androst-5-en-7-one (5) (12
mg, 6%).
1
β,3β-Dihydroxy-androst-5-en-7,17-dione (2): amorphous solid; C H O : calcd. C,71.67;
19
26
4
-1 1
H, 8.23; found. C, 71.91; H, 8.39%; IR (KBr) νmax 3520, 1740, 1673, 1630, 1408 cm ; H
NMR (CDCl , 300 MHz) δ 0.90 (3H, s, H-18), 1.25 (3H, s, H-19), 3.61 (1H, dd, J = 11.4 Hz,
3
1
3
4
.4 Hz, H-1α), 3.70-3.76 (1H, m, H-3α), 5.85 (1H, br s, H-6); C NMR (CDCl , 75 MHz) δ
3
220.5 (C, C-17), 200.9 (C, C-7), 162.8 (CH, C-5), 128.1 (CH, C-6), 75.9 (CH, C-1), 66.3
(CH, C-3), 50.2 (CH, C-9), 47.4 (C, C-13), 45.5 (CH, C-8), 44.8 (CH, C-14), 44.5 (C, C-10),
4
2.2 (CH , C-2), 41.7 (CH , C-4); 35.5 (CH , C-16); 30.9 (CH , C-12), 24.2 (CH , C-15), 23.1
2
2
2
2
2
(
CH , C-11), 13.8 (CH , C-18), 11.7 (CH , C-19).
2 3 3
3
β,12β-Dihydroxy-androst-5-en-7,17-dione (3): amorphous solid; C H O : calcd. C,71.67;
19
26
4
-1 1
H, 8.23; found. C, 71.70; H, 8.43%; IR (KBr) νmax 3570, 1742, 1670, 1633, 1400 cm ; H
NMR (CDCl , 300 MHz) δ 0.98 (3H, s, H-18), 1.24 (3H, s, H-19), 3.66-3.71 (1H, m, H-3α),
3
1
3
3
.78 (1H, dd, J = 11.2 Hz, 4.7 Hz, H-12α), 5.75 (1H, br s, H-6); C NMR (CDCl , 75 MHz) δ
3
222.2 (C, C-17), 200.4 (C, C-7), 166.1 (CH, C-5), 125.9 (CH, C-6), 72.1 (CH, C-3), 70.3
(
CH, C-12), 51.4 (C, C-13), 48.7 (CH, C-9), 43.9 (CH, C-8), 43.4 (CH, C-14), 41.8 (CH , C-
2
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4), 38.5 (C, C-10), 36.3 (CH , C-1), 35.5 (CH , C-16); 31.1 (CH , C-2), 28.1 (CH , C-11),
2 2 2 2
2
4.0 (CH , C-15), 17.3 (CH , C-19), 8.1 (CH , C-18).
2
3
3
3
β,9α-Dihydroxy-androst-5-en-7,17-dione (4): amorphous solid; C H O : calcd. C, 71.67;
1
9
26
4
-1 1
H, 8.23; found. C, 71.72; H, 8.31%, IR (KBr) νmax 3452, 1740, 1670, 1629, 1406 cm ; H
NMR (CDCl , 300 MHz) δ 0.90 (3H, s, H-18), 1.38 (3H, s, H-19), 3.68-3.74 (1H, m, H-3α),
3
1
3
5
.82 (1H, br s, H-6); C NMR (CDCl , 75 MHz) δ 220.3 (C, C-17), 199.0 (C, C-7), 166.7
3
(CH, C-5), 125.8 (CH, C-6), 78.4 (C, C-9), 69.6 (CH, C-3), 49.3 (CH, C-8), 47.4 (CH, C-13),
4
4.2 (C, C-10), 41.8 (CH , C-4), 40.6 (CH, C-14), 35.6 (CH , C-16), 30.8 (CH , C-2), 28.7
2
2
2
(
CH , C-1); 26.6 (CH , C-11), 26.6 (CH , C-12), 23.7 (CH , C-15), 20.7 (CH , C-19), 12.9
2
2
2
2
3
(
CH , C-18).
3
3
β,17β-Dihydroxy-androst-5-en-7-one (5): white plates; C H O : calcd. C,74.96; H, 9.27;
19
28
3
-
1
1
found. C, 74.56; H, 9.36%; IR (KBr) νmax 3310, 1660, 1630, cm ; H NMR (CDCl , 300
3
MHz) δ 0.75 (3H, s, H-18), 1.20 (3H, s, H-19), 3.63-3.70 (1H, m, H-3α), 3.65 (1H, t, J=10.7
1
3
Hz, 17α-H),.5.67 (1H, br s, H-6); C NMR (CDCl , 75 MHz) δ 200.9 (C, C-7), 165.7 (C, C-
3
5), 126.0 (CH, C-6), 81.0 (CH, C-17), 70.5 (CH, C-3), 49.9 (CH, C-9), 45.3 (CH, C-8), 45.1
(
CH, C-14), 45.0 (CH, C-14), 43.5 (C, C-13), 41.8 (CH , C-4), 38.4 (C, C-10), 36.2 (CH , C-
2
2
1), 35.5 (CH , C-12); 31.2 (CH , C-2); 30.9 (CH , C-16), 25.7 (CH , C-15), 21.0 (CH , C-11),
2 2 2 2 2
1
7.4 (CH , C-19), 11.0 (CH , C-18).
3
3
Products isolated in the course of transformation of DHEA (6)
After 48 h transformation of DHEA (0.2 g), the resulting mycelial and broth extracts were
combined and purified on silica gel with the use of a mixture of acetone:ethyl
acetate:methylene chloride (0.5:1.5:1 v/v/v) as eluent. The following compounds were
isolated (% mol): 7α-hydroxy-DHEA (7) (22 mg, 10%), 7β-hydroxy-DHEA (8) (32 mg,
15%), 3β,7α,17β-trihydroxy-androst-5-ene (9) (12 mg, 5%), 3β,7β,17β-trihydroxy-androst-5-
ene (10) (14 mg, 6%). The NMR data of these compound were agreed with the literature (16)
and their R and R were identical with standards available in our laboratory.
t
f
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Time course experiments. The progress of the reaction was monitored by GC analysis. At
regular intervals, 5-mL samples of the conversion medium were taken, extracted and analyzed
by comparison of the GC and TLC data with those of authentic samples. Conditions of the
reaction were identical to those in the main biotransformation experiments.
Results and Discussion
7-Oxo-DHEA (1) was prepared from DHEA 3-acetate by oxidation with chromium(VI) oxide
and subsequent base-catalyzed hydrolysis of 3β-acetoxy-androst-5-en-7,17-dione (7-oxo-
DHEA acetate). The identity of 7-oxo-DHEA was confirmed by comparison of its
spectroscopic data with those of an authentic standard synthesized by us in our previous
studies (16).
With the synthesis of the substrate completed, our attention was turned toward its
biotransformation by S. racemosum. Three-days incubation of 1 with this microorganism
yielded several potential metabolites based on GC analysis comparison with the control
extracts, but only four metabolites were present in sufficient quantity for characterization
(Figure 1).
The structures of these compounds were determined as a result of comparison of their
1
spectroscopic data with that of the starting material (1). Thus, the H NMR spectrum of
metabolite 2 exhibited a new resonance at δ 3.60 ppm (dd, J = 4.4 Hz, J = 11,4 Hz). The
H
shape of this signal suggested that it belongs to an axial proton coupled with only two
protons, therefore it can be either the signal of 1α or 12α proton. The presence of multiplet at
δ 3.69-3.75 ppm (m) and olefinic resonance at δ 5.84 ppm confirmed that the molecule
H
H
1
3
retains the 3β-hydroxy-androstene skeleton. Correspondingly, the C NMR spectrum of 2
showed two oxygenated carbon signals at δ 75.9 ppm and δ 66.3 ppm. The first one showed
C
C
long-range (HMBC) correlations with H-19 (s, δ 1.24 ppm), and should be assigned to C-1.
H
Additionally, in the HMBC spectrum, the proton signal at δ 1.24 ppm correlated with the
H
carbons at δ 50.2 ppm (C-9) and δ 162.8 ppm (C-5). The NOE enhancement of H-1 (δ
C
C
H
3
.60 ppm) with H-3 (δ 3.72 ppm) was observed in the NOESY spectrum indicating that the
H
1-OH should be located in the β-configuration. Further evidence for 1β-hydroxylation was
provided by downfield shifts of C-2 (Δ 11.1 ppm) and C-10 (Δ 8.2 ppm) signals. This was
coupled with upfield shift of C-19 signal from δ 17.4 ppm to δ 11.7 ppm due to a γ-gauche
C
C
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effect. All these results fully confirmed the structure of metabolite 2 as 1β,3β-dihydroxy-
1
androst-5-en-7,17-dione. The H NMR spectrum of 3 revealed a new signal at δ 3.78 ppm
H
indicating hydroxylation. The characteristic multiplicity of this signal (dd) suggested the
hydroxyl group could be introduced at 12-position. It was confirmed by downfield shift for C-
1
1 (Δ 7.6 ppm) and C-13 (Δ 3.6 ppm). The β (equatorial) stereochemistry of the OH group at
3
C-12 was deduced from the γ-upfield shift of Me-18 from δ 13.7 ppm to δ 8.1 ppm and J
C
C
interaction between the proton signals of C-18 methyl group resonating at δ 0.98 ppm and C-
H
1
2 at δ 70.3 ppm. The rest of the proton and carbon signals was quite similar to the substrate
C
1
with upfield shift (Δ 0.08 ppm) for the 18-methyl protons’ signal. Thus, the structure of
metabolite 3 was deduced to be 3β,12β-dihydroxy-androst-5-en-7,17-dione. The new
1
3
chemical shift at δ 78.4 ppm in C NMR of 4 suggested the presence of a hydroxyl group,
C
1
whilst no proton resonance was observed in H NMR spectrum in the range of 3.5-4.5 ppm.
The data obtained confirmed that the hydroxyl group should be attached to a tertiary carbon.
The downfield shifts of signals: C-10 (Δ 5.8 ppm), C-11 (Δ 6.0 ppm), C-8 (Δ 3.6 ppm), and γ-
gauche upfield shifts of C-1 (Δ 7.6 ppm), C-12 (Δ 3.5 ppm), C-14 (Δ 3.7 ppm) signals of this
metabolite in comparison to the spectrum of substrate 1, indicated hydroxylation at the 9α
position. The proposed structure, 3β,9α-dihydroxy-androst-5-en-7,17-dione (4), was
supported by the downfield shift of the C-19 methyl signal from δ 1.23 ppm to δ 1.38 ppm,
H
H
with respect to 1, that was consistent with the reference shift value (34) The spectroscopic
data of 4 were in agreement with those in the literature (35). Structure of the product 5 was
1
determined on the basis of its H NMR shift values being identical to the literature data of the
standard (16).
Composition of crude mixture obtained after transformation of 7-oxo-DHEA is presented
in Table 1. As is apparent from these data, 7-oxo-DHEA underwent mainly hydroxylations;
no reduction of the conjugated 7-ketone was observed. All of the biochemical attack
processes observed in the transformation of this substrate occurred with equatorial
stereochemistry (1β-,12β-) with exception of axial functionalization of tertiary carbon at the
9
α-position. Aside from oxidation, a minor reduction pathway was observed with
transformation of the C-17 ketone to a C-17β-alcohol generating 3β,17β-dihydroxy-androst-5-
en-7-one (5). Two of the three hydroxy derivatives of 7-oxo-DHEA are new compounds (2
and 3). 1β-Hydroxylase activity, although rarely demonstrated by microorganisms, has been
observed in the transformation of the ursolic acid by S. racemosum (31). The metabolites
containing a hydroxyl group at 9α or 12β position were isolated after transformation by this
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microorganism of some less common steroids of the androstane series (35) and diosgenin
29). 3β,9α-Dihydroxy-androst-5-ene-7,17-dione (4) with a yield of 37% was previously
obtained as a sole transformation product of 7-oxo-DHEA 3β-acetate by Absidia regnieri
35).
(
(
In order to determine if the obtained metabolites of 7-oxo-DHEA could be final
metabolites of DHEA, we used DHEA (6) as a test substrate. It was found that cultures of S.
racemosum under the same conditions metabolize 6 to known compounds identified as 7α-
hydroxy-DHEA (7), 7β-hydroxy-DHEA (8), 3β,7α,17β-trihydroxy-androst-5-ene (9) and
3
β,7β,17β-trihydroxy-androst-5-ene (10) by comparison of their spectroscopic data with the
literature values (16) and on the basis of identity of their R from GC and R from TLC with
t
f
standards available in our laboratory. Based on the time course experiment analysis, it was
found that both allylic alcohols derived from DHEA appeared at the same time and their
contents in the reaction mixture rose rapidly during the first hours of the transformation
(
Table 2). After two days, also reduction products of the carbonyl group at C-17 of these
hydroxy derivatives were present in the extracts as well as small quantities of 7-oxo-DHEA
1).
(
Androstenediol (3β,17β-dihydroxy-androst-5-ene) was not found in any of the mixtures,
which may suggest that the identified triols (9 and 10) arise directly from the 7α-hydroxy- (7)
and 7β-hydroxy-DHEA (8). More than five times lower content of 7-oxo-DHEA (1) indicates
that further metabolism of the resulting allyl alcohols is mainly connected with the activity of
1
7β-HSD. The consequence of this is also a small content of hydroxy derivatives of 7-oxo-
DHEA (2-4) in the extracts which, after four-days transformation, reached a total max. value
of 12%.
The obtained results prompted us to carry out an experiment in which the method of
administration of DHEA was modified (single- vs. pulse-feeding method), having in mind the
fact that most microbial hydroxylases are enzymes induced by the presence of the substrates
specific to these enzymes (36-38). On the other hand, the feeding-pulse protocol could extend
the viability of the cells and their suitability for biotransformation processes (39). In the
modified regime of DHEA supply to the microbial cells, the same metabolites were obtained
as in the standard transformation, however, the quantitative composition of the mixtures of
metabolites in both experiments differed significantly (Table 2). Under the substrate pulse-
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feeding conditions, the content of the hydroxy derivatives containing 7-oxo group was more
than three times higher. Moreover, the profile of these metabolites was the same as that
obtained from 7-oxo-DHEA as the starting transformation substrate.
Conclusion
In summary, the undertaken biotransformations of 7-oxo-DHEA resulted in formation of two
new hydroxy derivatives: 1β,3β-dihydroxy-androst-5-ene-7,17-dione (2) and 3β,12β-
dihydroxy-androst-5-en-7,17-dione (3), as well as two known compounds: 3β,9α-dihydroxy-
androst-5-ene-7,17-dione (4) and 3β,17β-dihydroxy-androst-5-en-7-one (5). To the best of our
knowledge, this is the first report on biotransformation of 7-oxo-DHEA. The studies also
demonstrated that the hydroxy derivatives of 7-oxo-DHEA may be simultaneously the final
metabolites of DHEA transformation. Reactions observed during the transformation of DHEA
suggest that the studied microorganism contains enzymes exhibiting similar activity to those
present in animals and human eukaryotic cells. We are convinced that the achieved high
conversion rate of this substrate combined with minimal human pathogenicity of S.
racemosum, after process optimization, can lead to new approaches for the efficient
biosynthesis of steroids with potential biological activity.
Acknowledgments
This project was supported by Wrocław Centre of Biotechnology, The Leading National
Research Centre (KNOW) program for years 2014-2018.
Conflict of Interest
All authors declare no conflict of interest.
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Figure legends
Figure 1. Hydroxylation of 7-oxo-DHEA (1) by S. racemosum.
Table 1. Metabolites identified following transformation of 7-oxo-DHEA
a
Compounds
R (min)
t
%
7
1
3
3
-oxo-DHEA (1)
9.83
15.31
11.77
13.29
10.25
7
24
17
32
8
β,3β-dihydroxy-androst-5-en-7,17-dione (2)
β,12β-dihydroxy-androst-5-en-7,17-dione (3)
β,9α-dihydroxy-androst-5-en-7,17-dione (4)
β,17β-dihydroxy-androst-5-en-7-one (5)
3
a
contents of compounds in crude extracts determined by GC analysis.
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Table 2. Products accumulation during the conversion of DHEA by S. racemosum depending
on the time of transformation and the method of the substrate feeding.
a
Steroids, contents (%)
Time
b
Hydroxyderivatives
(h) DHEA (6)
7-oxo-DHEA (1)
2
3
4
5
7
8
9
10
c
6
2
8
6
84
4
9
-
-
-
-
-
-
-
-
-
-
-
c
c
c
1
4
9
24
-
38
26
21
3
36
-
-
16
13
-
4
6
8
34 11
29 10
-
1
4
2
6
-
-
2
2
7
d
9
6
-
5
8
7
10
15 27
a
c
b
determined by GC analysis in crude extracts. standards obtained in our previous work (16).
d
single-feeding of substrate. pulse-feeding of substrate
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