Biotransformation of androst-4-ene-3,17-dione by some fungi

Article abstract of DOI:10.3184/174751917X15064232103083

The incubations of androst-4-ene-3,17-dione with Aspergillus candidus MRC 200634, Aspergillus tamarii MRC 72400, Aspergillus wentii MRC 200316 and Mucor hiemalis MRC 70325 for 5 days are reported. A. candidus MRC 200634 mainly hydroxylated androst-4-ene-3,17-dione at C-11α, C-15α and C-15β whilst A. wentii MRC 200316 hydroxylated it mainly at C-6β. A. tamarii MRC 72400 showed predominately a Baeyer–Villiger monooxygenase activity. M. hiemalis MRC 70325 hydroxylated the substrate at C-14α and reduced most of it at C-17.

Full text of DOI:10.3184/174751917X15064232103083

594 RESEARCH PAPER
VOL. 41 OCTOBER, 594–597
JOURNAL OF CHEMICAL RESEARCH 2017
Biotransformation of androst-4-ene-3,17-dione by some fungi
Kudret Yildirim*, Ali Kuru, Ece Keskin, Aylin Salihoglu and Neslihan Bukum
Chemistry Department, Sakarya University, 54187, Sakarya, Turkey
The incubations of androst-4-ene-3,17-dione with Aspergillus candidus MRC 200634, Aspergillus tamarii MRC 72400, Aspergillus wentii
MRC 200316 and Mucor hiemalis MRC 70325 for 5 days are reported. A. candidus MRC 200634 mainly hydroxylated androst-4-ene-
3,17-dione at C-11α, C-15α and C-15β whilst A. wentii MRC 200316 hydroxylated it mainly at C-6β. A. tamarii MRC 72400 showed
predominately a Baeyer–Villiger monooxygenase activity. M. hiemalis MRC 70325 hydroxylated the substrate at C-14α and reduced
most of it at C-17.
Keywords: androst-4-ene-3,17-dione, Aspergillus, Mucor, biotransformation
Microbial steroid biotransformations have been widely used to
prepare more valuable and functionalised compounds such as
steroid drugs and hormones due to the high regio- and stereo-
selectivities of these reactions. Efforts are still being made to
improve the efficiency of fungal steroid biotransformations and to
find new useful reactions and microorganisms.1
Incubation of androst-4-ene-3,17-dione
candidus MRC 200634 for 5 days afforded six metabolites
(Table 2) identified as 17β-hydroxyandrost-4-en-3-one 2,
1
with A.
R6
R3
O
O
Androst-4-ene-3,17-dione 1,
a 4-en-3-oxo-steroid, is an
important metabolite in androgen metabolism and is also used
as a starting material for the preparation of pharmaceutically
important androgens, anabolic drugs and other compounds, such
as spironolactone.2
H
R4
R5
O
R2
O
R1
A number of microorganisms have been used for the
biotransformation of androst-4-ene-3,17-dione 1.1,3 Most
metabolites obtained from the microbial biotransformations
of androst-4-ene-3,17-dione 1 are either biologically active
compounds or important intermediates in the synthesis of steroidal
drugs.4
1 R1 = R2 = R3 = R5 = H2, R4 = H, R6 = O
9
2 R1 = R2 = R3 = R5 = H2, R4 = H, R6 = βOH, αH
3 R1 = R2 = R3 = R5 = H2, R4 = OH, R6 = O
4 R1 = R2 = R5 = H2, R3 = βH, αOH, R4 = H, R6 = O
5 R1 = R2 = R3 = H2, R4 = H, R5 = βOH, αH, R6 = βOH, αH
6 R1 = R2 = R5 = H2, R3 = βH, αOH, R4 = H, R6 = βOH, αH
7 R1 = R2 = R3 = H2, R4 = H, R5 = βH, αOH, R6 = βOH, αH
8 R1 = R2 = R5 = H2, R3 = βOH, αH, R4 = H, R6 = O
10 R1 = βOH, αH, R2 = R3 = R5 = H2, R4 = H, R6 = O
11 R1 = R2 = R3 = R5 = H2, R4 = OH, R6 = βOH, αH
12 R1 = R3 = R5 = H2, R2 = βH, αOH, R4 = H, R6 = βOH, αH
13 R1 = R2 = R3 = R5 = H2, R4 = H, R6 = βCOCH3, αH
In the present work, androst-4-ene-3,17-dione 1 was incubated
with Aspergillus candidus MRC 200634, Aspergillus tamarii
MRC 72400, Aspergillus wentii MRC 200316 and Mucor hiemalis
MRC 70325 for 5 days in order to investigate its metabolism.
The structures of the metabolites (Fig. 1), which were known
1
were established by comparing their H NMR and 13C NMR
spectra (Table 1) with those of the starting material and the
literature values.5–11 All of the compounds retained the resonances
assigned to the 4-ene-3-keto moiety of 1.
Fig. 1 Chemical structures for compounds 1–13.
Table 1 13C NMR data determined in CDCl3 for compounds 1–12
Carbon atom
1
2
3
4
5
6
7
8
9
10
37.07
34.13
200.42
126.44
168.40
72.66
37.07
29.31
53.51
37.99
20.18
31.14
47.98
50.77
21.65
35.84
220.90
13.72
19.51
11
35.57
33.80
200.03
123.64
171.39
32.48
28.43
38.76
46.62
38.65
19.59
32.48
46.84
83.27
29.56
26.01
78.41
14.87
17.12
12
1
2
3
4
5
6
7
8
35.62
33.84
199.33
123.07
170.35
32.50
31.20
35.07
53.78
38.58
20.24
30.68
47.45
50.76
21.68
35.69
220.43
13.64
17.31
35.51
33.81
199.85
123.68
171.68
32.72
31.41
38.57
53.76
36.28
20.52
35.56
42.69
50.32
23.22
30.21
81.44
11.00
17.30
35.62
33.83
199.54
124.07
170.02
32.26
25.50
37.85
46.75
38.59
19.04
24.42
52.48
80.67
30.19
33.02
218.37
17.25
17.81
37.03
33.84
200.29
124.67
170.56
33.31
30.18
34.48
59.04
39.97
68.58
42.76
47.98
49.94
21.65
35.72
218.81
14.61
18.28
35.72
33.92
199.68
123.89
171.17
32.65
31.00
31.42
54.22
38.75
20.52
37.82
42.19
55.10
69.06
43.34
81.07
13.70
17.28
37.32
34.10
200.65
124.30
171.74
33.56
31.05
35.22
59.04
39.96
68.68
43.46
48.26
49.69
23.17
30.30
80.83
12.23
18.30
35.54
33.77
200.01
123.47
171.84
32.74
32.02
35.18
53.82
38.58
20.41
36.51
44.08
58.08
72.11
42.27
78.41
12.53
17.38
34.88
33.70
199.71
122.44
171.93
31.78
31.40
30.86
56.55
39.24
67.80
40.85
46.69
52.25
21.61
35.25
219.41
15.79
20.97
35.25
33.61
199.19
123.81
169.55
32.15
30.17
37.72
52.25
38.27
21.62
38.74
82.67
45.43
19.61
28.32
171.24
19.88
17.19
35.88
33.80
199.46
126.50
168.67
40.88
67.63
39.65
44.85
38.45
20.33
35.24
42.66
45.13
22.62
30.07
81.30
10.87
16.95
9
10
11
12
13
14
15
16
17
18
19
* Correspondent. E-mail: kudrety@sakarya.edu.tr

JOURNAL OF CHEMICAL RESEARCH 2017 595
ene-3,17-dione and 17a-oxa-D-homo-androst-4-ene-3,17-
Table 2 Metabolite yields following chromatography
8
dione 9. The structure of 2 was identified by comparison of its
NMR spectra with those of a previously isolated metabolite. The
metabolite 8 had characteristic new resonances at δ 4.46 ppm
(1H, m) and δ 67.80 ppm, and showed downfield shiHfts for C-9
(ΔδC 2.77 ppmC) and C-12 (Δδ 10.17 ppm) and a γ-carbon upfield
shift for C-8 (ΔδC 4.21 ppmC) in its 13C NMR spectrum, which
were consistent with the presence of an 11β-hydroxyl group.9
The metabolite 9 showed a downfield shift (ΔδC 35.22 ppm) for
the C-13 resonance, which was in accordance with the insertion
of an oxygen atom adjacent to this position on ring D. This was
coupled with downfield shifts (ΔδH 0.46 ppm and ΔδC 6.24 ppm)
for the 18-methyl resonance. The lack of C-17 resonance of 1 at
δC 220.43 ppm and the presence of a new resonance at δC171.24
ppm suggested the formation of a lactone for ring D.10
Fungus
A. candidus
MRC 200634
Metabolite
Yield (%)
7
5
8
10
3
13
17β-Hydroxyandrost-4-en-3-one 2
14α-Hydroxyandrost-4-ene-3,17-dione 3
11α-Hydroxyandrost-4-ene-3,17-dione 4
15β,17β-Dihydroxyandrost-4-en-3-one 5
11α,17β-Dihydroxyandrost-4-en-3-one 6
15α,17β-Dihydroxyandrost-4-en-3-one 7
A. tamarii
MRC 72400
5
4
60
17β-Hydroxyandrost-4-en-3-one 2
11β-Hydroxyandrost-4-ene-3,17-dione 8
17a-Oxa-D-homo-androst-4-ene-3,17-dione 9
A. wentii
MRC 200316
8
68
14α-Hydroxyandrost-4-ene-3,17-dione 3
6β-Hydroxyandrost-4-ene-3,17-dione 10
14α-Hydroxyandrost-4-ene-3,17-dione 3
14α,17β-Dihydroxyandrost-4-en-3-one 11
7α,17β-Dihydroxyandrost-4-en-3-one 12
M. hiemalis
MRC 70325
10
60
6
Incubation of androst-4-ene-3,17-dione 1 with A. wentii
MRC 200316 for 5 days afforded two metabolites (Table 2)
identified as 6β-hydroxyandrost-4-ene-3,17-dione 10 and
14α-hydroxyandrost-4-ene-3,17-dione 3. The metabolite 10
showed new resonances at δH 4.36 ppm (1H, t, J = 3 Hz) and
δC 72.66 ppm, and demonstrated a downfield shift for C-7 (ΔδC
5.87 ppm) and a γ-carbon upfield shift for C-8 (ΔδC 5.76 ppm) in
its 13C NMR spectrum, revealing the presence of a 6β-hydroxyl
group.7 The structure of 3 was identified by comparison of its
NMR spectra with those of a previously isolated metabolite.
Incubation of androst-4-ene-3,17-dione 1 with M. hiemalis
MRC 70325 for 5 days afforded three metabolites (Table 2)
identified by their NMR spectra as 14α-hydroxyandrost-4-ene-
3,17-dione 3, 14α,17β-dihydroxyandrost-4-en-3-one 11 and
7α,17β-dihydroxyandrost-4-en-3-one 12. The metabolite 11
14α-hydroxyandrost-4-ene-3,17-dione 3, 11α-hydroxyandrost-
4-ene-3,17-dione 4, 15β,17β-dihydroxyandrost-4-en-3-one
5, 11α,17β-dihydroxyandrost-4-en-3-one
6
and 15α,17β-
dihydroxyandrost-4-en-3-one 7. In the 13C NMR spectrum of 2,
the resonance attributed to the 17-ketone (δC 220.43 ppm) of 1 was
replaced by that for a CH(OH) (δC 81.44 ppm). The metabolite 3
showed a new carbon atom resonance at δC 80.67 ppm in its 13
C
NMR spectrum whilst it did not show any new resonance in the
range 3–5 ppm in its 1H NMR spectrum, indicating the presence
of a tertiary hydroxyl group. The downfield shifts for C-8 (ΔδC
2.78 ppm) and C-15 (ΔδC 8.51 ppm) and γ-gauche upfield shifts
for C-7 (ΔδC 5.70 ppm) and C-16 (ΔδC 2.67 ppm) compared to 1
were in accordance with the presence of a 14α-hydroxyl group.
The metabolite 4 had characteristic resonances at δH 4.08 ppm
(1H, dt, J = 5.0 and 10.0 Hz) and δC 68.58 ppm, which were
consistent with the presence of an 11α-hydroxyl group.5,6 The 13C
NMR spectrum of 4 showed downfield shifts for C-9 (ΔδC5.26
ppm) and C-12 (ΔδC 12.08 ppm) and a γ-carbon upfield shift
for C-8 (ΔδC 0.59 ppm), which were further consistent with the
presence of an 11α-hydroxyl group.6 The metabolite 5 had new
resonances at δH 4.20 ppm (1H, ddd, J = 7.8, 5.7 and 2.5 Hz) and
δC 69.06 ppm, which were in accordance with the presence of a
15β hydroxyl group.7 Its 13C NMR spectrum, on comparison to 1,
showed downfield shifts for C-14 (ΔδC 4.34 ppm) and C-16 (Δδ
7.65 ppm) whereas it showed a γ-gauche upfield shift for C-8 (ΔδCC
3.65 ppm). These changes were further in accordance with the
presence of a 15β hydroxyl group. The 13C NMR spectrum of 5
lacked the C-17 resonance of 1 at δC 220.43 ppm and had a new
carbon atom resonance at δC 81.07 ppm, suggesting the presence
of a 17β-hydroxyl group. The metabolite 6 had new resonances at
δH 3.99 ppm (1H, dt, J = 5.0 and 10.0 Hz) and δC 68.68 ppm, whilst
the 13C NMR spectrum of 6 showed downfield shifts for C-9 (ΔδC
5.26 ppm) and C-12 (ΔδC 12.78 ppm), indicating the presence of
an 11α-hydroxyl group. The lack of the C-17 resonance of 1 at δC
220.43 ppm and the presence of a new carbon atom resonance at
δC 80.83 ppm suggested the presence of a 17β-hydroxyl group.
The metabolite 7 showed characteristic resonances at δH 4.10 ppm
(1H, dt, J = 4.0 and 10.0 Hz) and δC 72.11 ppm, and demonstrated
downfield shifts for C-14 (ΔδC 7.32 ppm) and C-16 (ΔδC 6.58 ppm)
in its 13C NMR spectrum, indicating the presence a 15α-hydroxyl
group.8 The 13C NMR spectrum of 7 lacked the C-17 resonance
of 1 at δC 220.43 ppm and had a new carbon atom resonance at
δC 78.41 ppm, indicating the presence of a 17β-hydroxyl group.
Incubationofandrost-4-ene-3,17-dione 1with A. tamariiMRC
72400 for 5 days afforded three metabolites (Table 2) identified
as 17β-hydroxyandrost-4-en-3-one 2, 11β-hydroxyandrost-4-
showed a new carbon atom resonance at δC 83.27 ppm in its 13
C
NMR spectrum whereas it did not show any new signal in its 1H
NMR spectrum. The 13C NMR spectrum of 11 had downfield
shifts for C-8 (ΔδC 3.69 ppm) and C-15 (ΔδC 7.88 ppm) whereas it
had γ-gauche upfield shifts for C-7 (Δδ 2.77 ppm) and C-16 (ΔδC
9.68 ppm), which indicated the presencCe of a 14α-hydroxyl group.
The 13C NMR spectrum of 11 lacked the C-17 resonance of 1 at δ
220.43 ppm and had a new methine carbon atom resonance at δCC
78.41 ppm, consistent with the presence of a 17β-hydroxyl group.
The metabolite 12 had NMR resonances at δH 3.97 ppm (1H, bs)
and δC 67.63 ppm, and compared to 1 the 13C NMR spectrum of
12 showed downfield shifts for C-6 (ΔδC 8.38 ppm) and C-8 (ΔδC
4.58 ppm) and γ-carbon upfield shifts for C-9 (Δδ 8.93 ppm) and
C-14 (ΔδC 5.63 ppm), which were consistent withCthe presence of
a 7α-hydroxyl group. The lack of the C-17 resonance of 1 at δC
220.43 ppm and the presence of a new carbon atom resonance
at δC 81.30 ppm indicated the presence of a 17β-hydroxyl group.
As can be seen from Table 2, A. candidus MRC 200634
mainly hydroxylated androst-4-ene-3,17-dione 1 at C-11α, C-15α
and C-15β, accompanied by a minor hydroxylation at C-14α.
In a previous work, A. candidus MRC 200634 had been shown
to hydroxylate pregn-4-ene-3,20-dione 13 at C-11α and C-15β,
accompanied by a minor hydroxylation at C-14α.12 These results
suggested that A. candidus MRC 200634 might have metabolised
androst-4-ene-3,17-dione 1 in a different way since the substrate
lacked the C-17 side chain of pregn-4-ene-3,20-dione 13.
A. tamarii MRC 72400 showed a high Baeyer-Villiger mono-
oxygenase (BVMO) activity on androst-4-ene-3,17-dione 1,
accompanied by a minor hydroxylation at C-11β and a minor
reduction at C-17. In previous work, A. tamarii MRC 72400 had
also shown a high BVMO activity on 17β-hydroxyandrost-4-
en-3-one 2.13 Although A. tamarii is known for its high BVMO
activity on pregn-4-ene-3,20-dione 13, another A. tamarii strain
showed low BVMO activities on androst-4-ene-3,17-dione 1
and 17β-hydroxyandrost-4-en-3-one 2.14

596 JOURNAL OF CHEMICAL RESEARCH 2017
speed for each fungus, androst-4-ene-3,17-dione 1 (1 g) dissolved in
DMF (10 mL) was evenly distributed aseptically among the flasks.
The biotransformation of the substrate by each fungus was carried out
in 10 flasks for 5 days. The fungal mycelium was then 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 1 L of ethyl acetate. The organic extract was dried over anhydrous
sodium sulfate and the solvent evaporated in vacuo to give a brown gum
which was then chromatographed on silica gel. All biotransformation
experiments were performed in duplicate and run with a control flask
containing non-inoculated sterile medium and the substrate. After 5
days of incubation, the control was harvested and analysed by TLC. No
metabolites were detected in controls.
A. wentii MRC 200316 mainly hydroxylated androst-4-ene-
3,17-dione 1 at C-6β, accompanied by a minor hydroxylation
at C-14α. In previous work, 17β-hydroxyandrost-4-en-3-one 2
was hydroxylated by A. wentii MRC 200316 in the same way
in lower yields.15 In that work, however, the same fungus only
hydroxylated pregn-4-ene-3,20-dione 13 at C-11α in a high yield.
M. hiemalis MRC 70325 hydroxylated androst-4-ene-
3,17-dione 1 predominantly at C-14α and reduced most of
the substrate at C-17. This was accompanied by a minor
hydroxylation at C-7α. In previous work, 17β-hydroxyandrost-
4-en-3-one 2 was metabolised by M. hiemalis MRC 70325 in
a similar way in higher yields.16 In another study, a different
M. hiemalis strain hydroxylated pregn-4-ene-3,20-dione 13
predominantly at C-14α. This was accompanied by some minor
hydroxylations at various carbon atoms.17
Incubation of androst-4-ene-3,17-dione 1 (1 g) with A. candidus MRC
200634 for 5 days on a rotary shaker (150 rpm) at 28 °C afforded a brown
gum (2071 mg), which was then chromatographed on silica gel. Elution
with 30% ethyl acetate in n-hexane afforded the unreacted starting
In short, A. candidus MRC 200634, A. tamarii MRC
72400, A. wentii MRC 200316 and M. hiemalis MRC 70325
metabolised androst-4-ene-3,17-dione 1 in different ways. A.
tamarii MRC 72400 showed predominantly BVMO activity
on androst-4-ene-3,17-dione 1 whereas the rest of the fungi
hydroxylated it at different sites.
1
material (180 mg), which was identified by comparison of its H and
13C NMR spectra with those of an authentic sample.
Elution with 40% ethyl acetate in n-hexane afforded
17β-hydroxyandrost-4-en-3-one 2 (71 mg, 7%), which was crystallised
from acetone as prisms; m.p.158–159 °C (lit.20 154–155 °C); IR (vmax
/
cm–1): 3210, 1660 and 1620; δH 0.81 (3H, s, 18-H), 1.18 (3H, s, 19-H), 3.66
(1H, t, J = 8.5 Hz, 17α-H), 5.71 (1H, s, 4-H).
Experimental
General experimental details
Elution with 50% ethyl acetate in n-hexane afforded
14α-hydroxyandrost-4-ene-3,17-dione
3
(53 mg, 5%), which
Androst-4-ene-3,17-dione
1 was purchased from Sigma-
was crystallised from methanol as plates; m.p. 261–262 °C (lit.21
255–260 °C); IR (vmax/cm–1) 3460, 1735 and 1660; δH 1.03 (3H, s, 18-H),
1.21 (3H, s, 19-H), 5.73 (1H, s, 4-H).
Aldrich (Istanbul, Turkey). Solvents which were of analytical
grade, potato dextrose agar (PDA) and agar for PDA slopes
and ingredients for liquid medium were purchased from Merck
(Istanbul, Turkey). The steroids were separated by column
chromatography on silica gel 60 (Merck 107734), eluting
with increasing concentrations of ethyl acetate in n-hexane.
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
a p-anisaldehyde/H2SO4 reagent and heated to 120 °C for 3
minutes in order to visualise the spots. Infrared spectra were
recorded using a Perkin-Elmer Spectrum Two spectrometer.
1H and 13C NMR spectra were recorded in deuteriochloroform
with tetramethylsilane as an internal standard reference at
300 MHz and 75 MHz, respectively, with a Varian Mercury
300 spectrometer. Chemical shifts are given in ppm (δ scale),
coupling constants (J) are given in Hz. Melting points were
determined by an Electrothermal IA 9200 melting point
apparatus and are uncorrected.
Elution with 70% ethyl acetate in n-hexane afforded
11α-hydroxyandrost-4-ene-3,17-dione
4
(85 mg, 8%), which
was crystallised from acetone as needles; m.p. 242–243 °C (lit.22
240–241 °C); IR (vmax/cm–1)3430, 1735 and 1665; δH 0.94 (3H, s, 18-H),
1.33 (3H, s, 19-H), 4.08 (1H, dt, J = 5.0 and 10.0 Hz, 11β-H), 5.74 (1H, s,
4-H).
Further elution with 70% ethyl acetate in n-hexane afforded 15β,17β-
dihydroxyandrost-4-en-3-one 5 (106 mg, 10%), which was crystallised
from acetone as needles; m.p. 200–201 °C (lit.7 201–205 °C); IR (vmax
/
cm–1) 3435 and 1660; δH 1.05 (3H, s, 18-H), 1.23 (3H, s, 19-H), 3.54 (1H,
t, J = 8.5 Hz, 17α-H), 4.20 (1H, ddd, J = 7.8, 5.7 and 2.5 Hz, 15α-H), 5.74
(1H, s, 4-H).
Elution with pure ethyl acetate afforded 11α,17β-dihydroxyandrost-4-
en-3-one 6 (32 mg, 3%), which was crystallised from acetone as cubes;
m.p. 172–173 °C (lit.7 168–172°C); IR (vmax/cm–1) 3390, 1660 and 1610;
δH 0.80 (3H , s, 18-H) 1.33 (3H , s, 19-H), 3.68 (1H, t, J = 8.5 Hz, 17α-H),
3.99 (1H, dt, J = 5.0 and 10.0 Hz, 11β-H), 5.72 (1H, s, 4-H).
Further elution with pure ethyl acetate afforded 15α,17β-
dihydroxyandrost-4-en-3-one 7 (138 mg, 13%), which was crystallised
from acetone as needles; m.p. 95–96 °C (lit.8 93–94 °C); IR (vmax/cm–1)3395
and 1665; δH 0.80 (3H, s, 18-H), 1.20 (3H, s, 19-H), 3.88 (1H, t, J = 8.5 Hz,
17α-H), 4.10 (1H, dt, J= 4.0 and 10.0 Hz, 15β-H), 5.72 (1H, s, 4-H).
Incubation of androst-4-ene-3,17-dione 1 (1 g) with A. tamarii MRC
72400 for 5 days on a rotary shaker (180 rpm) at 24 °C afforded a
brown gum (2023 mg), which was then chromatographed on silica gel.
Elution with 30% ethyl acetate in n-hexane afforded the unchanged
starting material (105 mg), which was identified by comparison of its
1H and 13C NMR spectra with those of an authentic sample.
Elution with 40% ethyl acetate in n-hexane afforded
17β-hydroxyandrost-4-en-3-one 2 (51 mg, 5%), which was identified
by comparison of its 1H and 13C NMR spectra with those of a
previously isolated metabolite.
Elution with 50% ethyl acetate in n-hexane afforded
11β-hydroxyandrost-4-ene-3,17-dione 8 (42 mg, 4%), which was
crystallised from methanol as prisms; m.p. 194–195 °C, (lit.9
196–197 °C); IR (vmax/cm–1) 3420, 1740 and 1655; δH 1.18 (3H, s, 18-H),
1.47 (3H, s, 19-H), 4.46 (1H, m, 11α-H), 5.70 (1H, s, 4-H).
General fermentation details
A. candidus MRC 200634, A. tamarii MRC 72400, A. wentii MRC
200316 and M. hiemalis MRC 70325 were 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 liquid medium for A. candidus
MRC 200634 was prepared by mixing sucrose (30 g), NaNO3 (3 g),
K2HPO4 (1 g), KCl (0.5 g), MgSO4·7H2O (0.5 g) and FeSO4·7H2O
(0.01 g) in distilled water (1 L).18 The liquid medium for A. tamarii MRC
72400 was prepared by dissolving malt extract (30 g) in distilled water
(1 L).14 The liquid medium for A. wentii MRC 200316 was prepared by
mixing sucrose (15 g), glucose (15 g), polypeptone (5 g), MgSO4·7H2O
(0.5 g), KCl (0.5 g), K2HPO4 (1 g) and FeSO4·7H2O (0.01 g) in distilled
water (1 L) and the pH was adjusted to 7.2.19 The liquid medium for M.
hiemalis MRC 70325 was prepared by mixing glucose (20 g), peptone
(10 g) and yeast extract (10 g) in distilled water (1 L).17 Each medium was
evenly distributed among 10 culture flasks of 250 ml capacity (100 mL in
each) and autoclaved for 20 minutes at 121 °C. Spores freshly obtained
from PDA slopes were transferred aseptically into each flask containing
sterile medium in a biological safety cabinet. After a three-day
cultivation on a rotary shaker at an appropriate temperature and rotation
Elution with 60% ethyl acetate in n-hexane afforded 17a-oxa-
D-homo-androst-4-ene-3,17-dione 9 (634 mg, 60%), which was

JOURNAL OF CHEMICAL RESEARCH 2017 597
crystallised from methanol as needles; m.p. 205–206 °C (lit.23
209–210 °C); IR (vmax/cm–1) 1720 and 1665; δH 1.17 (3H, s, 19-H), 1.36
(3H, s, 18-H), 5.76 (1H, s, 4-H).
Received 9 August 2017; accepted 20 September 2017
Paper 1704941
https://doi.org/10.3184/174751917X15064232103083
Published online: 5 October 2017
Incubation of androst-4-ene-3,17-dione 1 (1 g) with A. wentii MRC
200316 for 5 days on a rotary shaker (150 rpm) at 27 °C afforded a
brown gum (2299 mg), which was then chromatographed on silica gel.
Elution with 50% ethyl acetate in n-hexane afforded
6β-hydroxyandrost-4-ene-3,17-dione 10 (718 mg, 68%), which
was crystallised from acetone as prisms; m.p. 195–196 °C; (lit.21
190–193 °C); IR (vmax/cm–1) 3450, 1730 and1665; δH 0.92 (3H, s, 18-H),
1.37 (3H, s, 19-H), 4.36 (1H, t, J = 3 Hz, 6α-H), 5.83 (1H, s, 4-H).
Further elution with 50% ethyl acetate in n-hexane afforded
14α-hydroxyandrost-4-ene-3,17-dione 3 (84 mg, 8%), which was
identified by comparison of its 1H and 13C NMR spectra with those of a
previously isolated metabolite.
Incubation of androst-4-ene-3,17-dione 1 (1 g) with M. hiemalis
MRC 70325 for 5 days on a rotary shaker (100 rpm) at 26 °C afforded a
brown gum (2378 mg), which was then chromatographed on silica gel.
Elution with 30% ethyl acetate in n-hexane afforded the unconverted
starting material (50 mg), which was identified by comparison of its 1H
and 13C NMR spectra with those of an authentic sample.
References
1
2
3
M.V. Donova and O.V. Egorova, Appl. Microbiol. Biotechnol., 2012, 94, 1423.
A. Malaviya and J. Gomes, Appl. Biochem. Biotechnol., 2009, 158, 374.
P. Fernandes, A. Cruz, B. Angelova, H.M. Pinheiro and J.M.S. Cabral,
Enzyme Microb. Tech., 2003, 32, 688.
4
5
M. Heidary and Z. Habibi, J. Mol. Catal. B: Enzym., 2016, 126, 32.
D.N. Kirk, H.C. Toms, C. Douglas, K.A. White, K.E. Smith, S. Latif and
R.W.P. Hubbard, J. Chem. Soc., Perkin Trans. 2, 1990, 1567.
J.W. Blunt and J.B. Stothers, Org. Magn. Reson., 1977, 9, 439.
P.C. Peart, W.F. Reynolds and P.B. Reese, J. Mol. Catal. B: Enzym., 2013,
95, 70.
6
7
8
9
P.C. Peart, K.P. McCook, F.A. Russell, W.F. Reynolds and P.B. Reese,
Steroids, 2011, 76, 1317.
M.T. Yazdi, H. Arabi, M.A. Faramarzi, Y. Gasemi, M. Amini, S. Shokravi
and F.A. Mohseni, Phytochemistry, 2006, 65, 2205.
10 J. R. Hanson and A.C. Hunter, Phytochemistry, 1998, 49, 2349.
11 T. Janeczko, J. Dmochowska-Gładysz, E. Kostrzewa-Susłow, A. Białońska
and Z. Ciunik, Steroids, 2009, 74, 657.
12 K. Yildirim and A. Kuru, J. Chem. Res., 2015, 39, 546.
13 K. Yildirim, A. Uzuner and E.Y. Gulcuoglu, Collect. Czech. Chem.
Commun., 2011, 76, 743.
14 D.R. Brannon, J. Martin, A.C. Oehlschlager, N.N. Durham and L.H. Zalkow,
J. Org. Chem., 1965, 30, 760.
15 K. Yildirim, I. Kupcu and F. Gulsan, Z. Naturforsch., C: J. Biosci., 2010,
65, 688.
16 K. Yildirim, H. Saran, O.F. Dolu and A. Kuru, J. Chem. Res., 2013, 37, 566.
17 T.N.H. Antoniou, B.P.G. Curran and K.E. Smith, Microbiol., 1994, 140, 1633.
18 M.L. Mascotti, A.A. Orden, F.B. Bisogno, G. Gonzalo and M. Kurina-Sanz,
J. Mol. Catal. B: Enzym., 2012, 82, 32.
19 M. Miyazawa, T. Takahashi, K. Sakata and I. Horibe, J. Chem. Technol.
Biotechnol., 2008, 83, 1006.
Elution with 50% ethyl acetate in n-hexane afforded
14α-hydroxyandrost-4-ene-3,17-dione 3 (106 mg, 10%), which was
identified by comparison of its 1H and 13C NMR spectra with those of a
previously isolated metabolite.
Elution with 60% ethyl acetate in n-hexane afforded 14α,17β-
dihydroxyandrost-4-en-3-one 11 (638 mg, 60%), which was
crystallised from ethyl acetate as prisms; m.p. 184–185 °C (lit.21
181–185 °C); IR (vmax/cm–1)3435, 1660 and 1615; δH 0.93 (3H, s, 18-H),
1.22 (3H, s, 19-H), 4.32 (1H, t, J = 8.5 Hz, 17α-H), 5.74 (1H, s, 4-H).
Elution with 70% ethyl acetate in n-hexane afforded 7α,17β-
dihydroxyandrost-4-en-3-one 12 (64 mg, 6%), which was crystallised
from dichloromethane as plates, m.p. 213-214 °C (lit.7 205–206 °C); IR
(vmax/cm–1)3360, 1660 and 1610; δH 0.79 (3H, s, 18-H), 1.20 (3H, s, 19-H),
3.70 (1H, t, J = 8.5 Hz, 17α-H), 3.97 (1H, bs, 7β-H), 5.79 (1H, s, 4-H).
20 M. Nagasawa, H. Hashiba, N. Watanabe, M. Bae, G. Tamura and K. Arima,
Agr. Biol. Chem., 1970, 34, 801.
21 J.R. Hanson, H. Nasir and A. Parvez, Phytochemistry, 1996, 42, 411.
22 M.I. Choudhary, S. Sultan, M.T.H. Khan, A. Yasin, F. Shaheen and A.U.
Rahman, Nat. Prod. Res., 2004, 18, 529.
Acknowledgements
This work was financially supported by the Sakarya University
Scientific Research Foundation (Project number: 20155001008),
Turkey. We are grateful to Rasit Fikret Yilmaz and Yavuz Derin
for running the 1H and 13C NMR spectra.
23 T. Kołek, A. Szpineter and A. Świzdor, Steroids, 2008, 73, 1441.