Biotransformation of testosterone by Ulocladium chartarum MRC 72584

Article abstract of DOI:10.3184/174751918X15341764332783

The incubation of testosterone 1 with Ulocladium chartarum MRC 72584 has been reported. U. chartarum MRC 72584 hydroxylated testosterone 1 at C-7β, C-6β, C-14α and C-12β, accompanied by a 5α-reduction and oxidations at C-6 and at C-17.

Full text of DOI:10.3184/174751918X15341764332783

4
44 RESEARCH PAPER
VOL. 42 AUGUST, 444–446
JOURNAL OF CHEMICAL RESEARCH 2018
Biotransformation of testosterone by Ulocladium chartarum MRC 72584
Kudret Yildirim*, Ali Kuru and Şengül Yılmaz
Chemistry Department, Sakarya University, 54187 Sakarya, Turkey
The incubation of testosterone 1 with Ulocladium chartarum MRC 72584 has been reported. U. chartarum MRC 72584 hydroxylated
testosterone 1 at C-7β, C-6β, C-14α and C-12β, accompanied by a 5α-reduction and oxidations at C-6 and at C-17.
Keywords: testosterone, Ulocladium chartarum, hydroxylation, 5α-reduction
Steroids are biologically and pharmaceutically important
substances that possess many physiological activities. Steroid-
based drugs are now extensively used for some health problems
due to their anti-inflammatory, anti-microbial, anti-tumour,
anti-estrogenic, anti-allergenic, anti-diabetic, anti-HIV and
at δ 208.16 ppm, suggesting that hydroxylation followed by
C
oxidation to a ketone had taken place at C-6. NMR data of 3
5
were in accordance with the literature.
The third metabolite was identified as 17β-hydroxy-5α-
1
13
androstane-3,6-dione 4. The H NMR and C NMR spectra of
4 revealed the same changes as for compound 3, except that the
1
anti-convulsant properties. The preparation of steroids by
conventional chemical synthetic routes has some disadvantages,
and microbial steroid biotransformations are widely used to
overcome these.
metabolite retained the C-17 resonance of 1 at δ 81.07 ppm.
C
5
NMR data of 4 were consistent with the literature.
1,2
The fourth metabolite was identified as 7β-hydroxyandrost-
4-ene-3,17-dione 5. The NMR spectrum of 5 had characteristic
resonances at δ 3.53 ppm (1H, m) and δ 74.21 ppm, indicating
Ulocladium species are widely distributed and are mostly
saprotrophs, including some plant pathogens and food spoilage
organisms. A few Ulocladium species have attracted attention
H
C
6
13
the presence of a 7β-hydroxyl group. The C NMR spectrum
of 5 showed a downfield shift for C-8 (Δ 7.08 ppm) while there
was a γ-gauche upfield shift for C-9 (Δ 2.98 ppm), indicating the
3
as potential sources of enzymes and as bio-control agents. In
this study, testosterone 1 was incubated with U. chartarum
MRC 72584 to investigate its metabolism.
13
presence of a 7β-hydroxyl group. The C NMR spectrum of 5
lacked the C-17 resonance of 1 at δ 81.25 and had a new carbon
C
Results and discussion
atom resonance at δ 220.70 ppm, suggesting that an oxidation
C
Incubation of testosterone 1 with U. chartarum MRC 72584 for
had taken place at C-17. NMR data of 5 were comparable to the
6
5
d afforded seven metabolites (Table 1). The first metabolite
literature.
was identified as androst-4-ene-3,17-dione 2 (Fig. 1). The
The fifth metabolite was identified as 7β,17β-
1
3
13
C NMR spectrum of 2 lacked the C-17 resonance of 1 at δC
dihydroxyandrost-4-ene-3-one 6. The C NMR spectrum
8
1.25 ppm and showed a new resonance at δ 220.50 ppm,
of 6 had two characteristic resonances at δ 74.92 and 81.09
C
C
ppm, suggesting the presence of 7β- and 17β-hydroxyl groups,
indicating an oxidation at C-17 (Table 2). The absence of the
13
respectively. The C NMR spectrum of 6 showed a downfield
shift for C-8 (Δ 7.48 ppm) and a γ-gauche upfield shift for C-9
(Δ 3.81 ppm), which were in agreement with the presence of
characteristic 17αH resonance (1H, t, J = 8.5 Hz) of 1 at δ
H
3
.65 ppm confirmed the oxidation of C-17. NMR data of 2 were
4
comparable to the literature.
1
a 7β-hydroxyl group. The H NMR spectrum of 6 possessed
Thesecondmetabolitewasidentifiedas5α-androstane-3,6,17-
1
a resonance at δ 3.63 (1H, t, J = 8.5 Hz), assigned to a
trione 3. Its H NMR spectrum, 3 lacked the 4-H resonance (1H,
H
1
7β-hydroxyl group. NMR data of 6 were consistent with the
s) of 1 at δ 5.74, indicating that the hydrogenation of the double
H
7
literature.
bond in ring A had taken place. The alkene resonances of 1 at
δC 123.87 and 171.52 ppm were replaced by signals for C-4 and
The sixth metabolite was identified as 14α,17β-
dihydroxyandrost-4-ene-3-one 7. The metabolite 7 had a
C-5 (δ 41.09 and 57.39 ppm), respectively. A comparison of the
C
13
C NMR spectrum of 3 with that in literature showed that the
reduction of the α,β-unsaturated system had taken place from
5
13
R1
the α-face. In the C NMR spectrum of 3, the C-17 resonance
of 1 at δ 81.25 ppm was replaced by a signal at δ 219.54 ppm,
R2
R4
C
C
indicating that an oxidation had taken place at C-17, and the
C-6 resonance of 1 at δ 32.63 ppm was replaced by resonance
C
H
R3
Table 1 Yields of metabolites
O
H
O
R1
Substrate
Testosterone 1 Androst-4-ene-3,17-dione 2
α-Androstane-3,6,17-trione 3
7β-Hydroxy-5α-androstane-3,6-dione 4
β-Hydroxyandrost-4-ene-3,17-dione 5
Metabolite
Yield (%)
O
3
4
9
3
1
2
5
R =R =H , R =H, R =βOH,αH
R =R =H , R =H, R =O
3 R =O
1
2
2
3
4
1
5
4 R =βOH,αH
1
1
2
2
3
4
1
R =βOH,αH, R =H , R =H, R =O
1
2
2
3
4
7
11
6 R =βOH,αH, R =H , R =H, R =βOH,αH
1 2 2 3 4
7
β,17β-Dihydroxyandrost-4-ene-3-one 6 11
4α,17β-Dihydroxyandrost-4-ene-3-one 7 12
2β,17β-Dihydroxyandrost-4-ene-3-one 8 9
7 R =R =H , R =OH, R =βOH,αH
1
2
2
3
4
1
8 R =H , R =βOH,αH, R =H, R =βOH,αH
1 2 2 3 4
1
Fig. 1 Chemical structures of testosterone 1 and its metabolites.
*
Correspondent. E-mail: kudrety@sakarya.edu.tr

JOURNAL OF CHEMICAL RESEARCH 2018 445
13
Table 2 C NMR data determined in CDCl for compounds 1–8
3
C atom
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
9
35.48
33.74
199.64
123.87
171.52
32.63
31.33
35.58
53.69
38.45
20.10
36.21
42.61
50.24
23.14
29.51
81.25
10.93
17.17
35.44
33.69
199.40
123.58
170.54
32.38
31.04
34.89
53.57
38.48
20.44
30.07
47.33
50.58
21.55
35.44
220.50
13.52
17.21
37.35
36.82
210.78
41.09
57.39
208.16
45.22
37.21
53.34
37.87
20.84
30.97
48.00
51.39
21.56
35.54
219.54
13.72
12.52
37.84
37.16
211.35
41.07
57.27
208.86
45.88
35.98
53.25
37.84
21.12
36.77
43.26
51.02
23.00
29.98
81.07
10.99
12.42
35.56
33.84
199.17
124.94
166.75
42.45
74.21
42.66
50.71
38.01
20.30
31.15
48.00
50.46
24.92
35.90
220.70
17.32
13.92
35.65
33.91
199.36
124.72
167.48
42.24
74.92
43.06
49.88
38.02
20.55
36.28
43.51
50.69
26.33
30.72
81.09
11.10
35.59
33.81
199.89
123.67
171.24
32.48
28.44
38.64
46.65
38.78
19.60
32.53
46.85
83.19
26.01
29.44
78.41
14.83
17.12
35.45
33.77
199.72
123.97
170.86
32.62
30.83
34.57
52.49
38.48
29.27
78.75
46.88
48.50
23.01
29.81
81.57
6.0
1
0
1
1
1
2
1
3
1
4
1
5
1
6
1
7
1
8
19
17.32
17.19
1
3
new carbon atom resonance at δ 83.19 ppm in its C NMR
Experimental
C
spectrum although it did not show any new signals in the range
–5 ppm in its H NMR spectrum, indicating the presence
of a tertiary hydroxyl group. The downfield shifts for C-8
Testosterone
1 was purchased from Sigma-Aldrich (Istanbul,
1
3
Turkey). Solvents were of analytical grade and were purchased from
Merck (Istanbul, Turkey). Potato dextrose agar (PDA) and agar for
PDA slopes and ingredients for liquid medium were also purchased
from Merck (Istanbul, Turkey). The steroids were separated by
column chromatography on silica gel 60 (Merck 107734), and
eluted with increasing concentrations of ethyl acetate in n-hexane.
Steroid mixtures from the column were then separated by column
chromatography on aluminium oxide 90 active neutral (activity
stage I, Merck 101077), using smaller columns for 2–3 h. Thin layer
chromatography (TLC) was carried out with 0.2 mm thick Merck
Kieselgel 60 F254 TLC plates using ethyl acetate/n-hexane (1:1) as
eluent. TLC plates were dipped into an anisaldehyde/H SO reagent
(
Δ 3.06 ppm) and C-15 (Δ 2.87 ppm) and γ-gauche upfield shifts
for C-7 (Δ 2.89 ppm) and C-16 (Δ 0.07 ppm) were in accordance
1
with the presence of a hydroxyl group at C-14. In its H NMR
spectrum, the metabolite 7 showed a typical downfield shift
(
Δ 0.63 ppm) for the 17α-H resonance and this suggested its
8
diaxial interaction with a 14α-hydroxyl group. This shift
confirmed the presence of a 14α-hydroxyl group. NMR data of
8
7
were in accordance with the literature.
The seventh metabolite was identified as 12β,17β-
dihydroxyandrost-4-ene-3-one 8. The C NMR spectra of
13
2
4
and heated to 120 °C for 3 min to visualise the spots. Infrared spectra
8
showed a typical resonance at δ 78.75 ppm, indicating the
C
were recorded using a Perkin Elmer Spectrum Two spectrometer.
9
presence a 12β-hydroxyl group. The downfield shifts for C-11
1
H
NMR spectra were recorded in deuteriochloroform with
(
Δ 9.17 ppm) and C-13 (Δ 4.27 ppm) and γ-gauche upfield shifts
tetramethylsilane as an internal standard reference at 300 MHz with a
Varian Mercury 300 spectrometer. C NMR spectra were recorded in
for C-9 (Δ 1.20 ppm) and C-14 (Δ 1.74 ppm) were in accordance
13
13
with the presence of a 12β-hydroxyl group. The C NMR
deuterochloroform at 75 MHz with a Varian Mercury 300 spectrometer
and are reported in Table 2. Chemical shifts are given in ppm (δ
scale) and coupling constants (J) are given in Hz. Melting points were
determined by an Electrothermal IA 9200 melting point apparatus and
are uncorrected.
U. chartarum MRC 72584 was obtained from TUBITAK,
Marmara Research Center, Food Science and Technology Research
Institute, Culture Collection Unit, Kocaeli, Turkey. Stock cultures
were maintained at 4 °C on PDA slopes. The biotransformation
experiment was performed in duplicate and run with a control flask
containing non-inoculated sterile medium and the substrate. After 5
d of incubation, the control was harvested and analysed by TLC. No
metabolites were detected in the control.
spectra of 8 showed a resonance at δ 81.57 ppm, suggesting
C
9
that the 17β-hydroxyl group of 1 was retained. NMR data and
10
the melting point of 8 were comparable to literature.
As can be seen from Table 1, testosterone 1 was hydroxylated
by U. chartarum MRC 72584 at C-6β, C-7β, C-12β and C-14α.
An oxidation at C-6 following the hydroxylation of 1 at C-6β, a
5
α-reduction before or after this hydroxylation and an oxidation
at C-17 were also observed.
Reports of 5α-reduction and hydroxylation of testosterone 1 at
2
,11,12
C-6β and C-14α by fungi are very common.
However, fungal
9
,10,13,14
hydroxylations of 1 at C-7β and C-12β are very rare.
In a previous work, Botryosphaerica obtusa hydroxylated
13
1
at C-7β, C-11α, C-12β and C-15α positions. In another
work, Botrytis cinerea hydroxylated 1 at C-7β and reduced its
9
Biotransformation of testosterone 1
The liquid medium for U. chartarum MRC 72584 was prepared by
14
C4–C5 double bond from the α-face. Rhizomucor tauricus
mixing glucose (20 g), peptone (5 g) and yeast extract (5 g) in 1 L of
10
hydroxylated 1 at C-6β and C-12β while a Penicillium isolate
15
distilled water. The medium was evenly distributed among 10 culture
hydroxylated 1 at C-1α, C-6β, C-12β and C-15β. We have now
shown that U. chartarum MRC 72584 hydroxylated 1 at C-6β,
C-7β, C-12β and C-14α and in addition to this, a 5α-reduction
and oxidations at C-6 and C-17 were also observed. Our work
on steroid biotransformations by U. chartarum MRC 72584 and
some other fungi is in progress.
flasks of 250 mL capacity (100 mL in each) and autoclaved for 20 min
at 121 °C. Spores freshly obtained from a PDA slope were transferred
aseptically into each flask containing sterile medium in a biological
safety cabinet. After cultivation at 28 °C for 3 d on a rotary shaker
(160 rpm), testosterone 1 (1 g) dissolved in DMF (10 mL) was evenly
distributed aseptically among the flasks. The biotransformation of

4
46 JOURNAL OF CHEMICAL RESEARCH 2018
−1
1
1
was carried out in 10 flasks for 5 d under the same conditions. The
(vmax cm ): 3450, 1660 and 1640; H NMR (300 MHz, CDCl ): δ 0.89
3
fungal mycelium was separated from the broth by filtration under
vacuum and the mycelium was rinsed with ethyl acetate (500 mL).
The broth was extracted three times each with ethyl acetate (1 L).
The organic extract was dried over anhydrous sodium sulfate and the
solvent evaporated in vacuo to give a brown gum (1343 mg), which was
then chromatographed on silica gel.
(3H, s, 18H), 1.18 (3H, s, 19H), 4.28 (1H, t, J = 7.8 Hz, 17αH), 5.70 (1H,
s, 4H).
Elution with 70% ethyl acetate in n-hexane afforded 12β,17β-
dihydroxyandrost-4-ene-3-one 8, which was crystallised from acetone
10
as needles; yield 95 mg (9%); m.p. 120–121 °C (lit. 121–125 °C);
−1
1
IR (vmax cm ): 3370, 1735 and 1655; H NMR (300 MHz, CDCl ): δ
3
Elution with 30% ethyl acetate in n-hexane afforded androst-4-ene-
0.80 (3H, s, 18H), 1.16 (3H, s, 19H), 3.48 (1H, dd, J = 5.0 and 11.0 Hz,
3
3
,17-dione 2, which was crystallised from acetone as prisms; yield
12αH), 3.85 (1H, t, J = 8.5 17αH), 5.71 (1H, s, 4H).
4
−1
0 mg (3%); m.p. 173–174 °C (lit. 170–172 °C); IR (v cm ): 1736
max
1
and 1440; H NMR (300 MHz, CDCl ): δ 0.91 (3H, s, 18H), 1.21 (3H, s,
Acknowledgement
3
19H), 5.75 (1H, s, 4H).
1
We are grateful to Raşit Fikret Yılmaz for running the H and
Elution with 40% ethyl acetate in n-hexane afforded the unreacted
13
C NMR spectra.
1
starting material (80 mg), which was identified by comparison of its H
and C NMR spectra with those of an authentic sample.
13
Received 8 July 2018; accepted 22 July 2018
Paper 1805512
https://doi.org/10.3184/174751918X15341764332783
Published online: 15 August 2018
Further elution with 40% ethyl acetate in n-hexane afforded
5
α-androstane-3,6,17-trione 3, which was crystallised from
16
ethyl acetate as plates; yield 44 mg (4%); m.p. 193–194 °C (lit.
−1
1
196–197 °C); IR (vmax cm ): 1740, 1720 and 1700; H NMR (300 MHz,
CDCl ): δ 0.88 (3H, s, 18H), 0.97 (3H, s, 19H).
3
References
Elution with 50% ethyl acetate in n-hexane afforded 17β-hydroxy-
1
S.P. Kolet, S. Niloferjahan, S. Haldar, R. Gonnade and H.V. Thulasiram,
Steroids, 2013, 78, 1152.
5
α-androstane-3,6-dione 4 which was crystallised from acetone as
16
needles; yield 95 mg (9%); m.p. 233–234 °C (lit. 230–231 °C); IR
2
M.V. Donova and O.V. Egorova, Appl. Microbiol. Biotechnol., 2012, 94,
1423.
−1
1
(
1
vmax cm ):3550 and 1710; H NMR (300 MHz, CDCl ): δ 0.80 (3H, s,
3
8H), 1.00 (3H, s, 19H), 3.67 (1H, d, J = 8.5 Hz, 17αH).
Further elution with 50% ethyl acetate in n-hexane afforded
3
4
5
6
7
8
9
B. Andersen and M. Hollensted, Int. J. Food. Microbiol., 2008, 126, 172.
A. Świzdor, A. Panek and N. Milecka-Tronina, Steroids, 2017, 126,101.
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7
β-hydroxyandrost-4-ene-3,17-dione 5, which was crystallised
6
from acetone as plates; yield 32 mg (3%); m.p. 222–223 °C (lit.
−1
1
218–220 °C); IR (vmax cm ):3465, 1670 and 1620; H NMR (300 MHz,
CDCl ): δ 0.94 (3H, s, 18H), 1.23 (3H, s, 19H), 3.53 (1H, m, 7αH), 5.76
3
(1H, s, 4H).
Elution with 60% ethyl acetate in n-hexane afforded 7β,17β-
dihydroxyandrost-4-ene-3-one 6, which was crystallised from acetone
10
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11 N. Nassiri-Koopaei and M.A. Faramarzi, Biocatal. Biotransform., 2015,
33, 1.
12 P. Fernandes, A. Cruz, B. Angelova, H.M. Pinheiro and J.M.S. Cabral,
Enzyme Microb. Tech., 2003, 32, 688.
7
as prisms; yield 117 mg (11%); m.p. 200–201 °C, (lit. 197.5–199.5 °C);
−
1
1
IR (vmax cm ): 3415, 1660 and 1610; H NMR (300 MHz, CDCl ): δ
3
0
.80 (3H, s, 18H), 1.21 (3H, s, 19H), 3.44 (1H, m, 7αH), 3.63 (1H, t, J =
.5 Hz, 17αH), 5.75 (1H, s, 4H).
1
3
K.E. Smith, S. Latif and D.N. Kirk, J. Steroid Biochem., 1990, 35, 115.
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5 Y.M. Shebany, Egypt. J. Chem., 2012, 47, 141.
8
1
4
Further elution with 60% ethyl acetate in n-hexane afforded 14α,17β-
dihydroxyandrost-4-ene-3-one 7, which was crystallised from acetone
as needles; yield 128 mg (12%); m.p. 180–181 °C (lit. 181–185 °C); IR
1
16 M. Numazawa, A. Mutsumi and M. Ogata, Chem. Pharm. Bull., 1988, 36,
8
3381.