568 JOURNAL OF CHEMICAL RESEARCH 2013
and heated to 120 °C for 3 minutes in order to visualise the spots.
Infrared spectra were recorded using a Shimadzu IR Prestige-21 ins-
trument. Elemental analysis was performed using a Thermo Finnigan
Flash EA 1112 elemental analyser. 1H NMR spectra were recorded in
deuteriochloroform with tetramethylsilane as an internal standard
reference at 300 MHz with a Varian Mercury 300 spectrometer. 13C
NMR spectra were recorded in deuteriochloroform at 75 MHz 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.
M. hiemalis MRC 70325 was obtained from TUBITAK, Marmara
Research Center, Food Science and Technology Research Institute,
Culture Collection Unit, Kocaeli, Turkey. Stock cultures were main-
tained at 4 °C on PDA slopes. Biotransformation experiments were
run with control flasks containing non-inoculated sterile medium and
one of the substrates. After 5 days of incubation, all controls were
harvested and analysed by TLC. No metabolites were detected in the
controls.
The third metabolite had characteristic resonances at δH
3.80 ppm11 (1H, dt, J = 5 and 10 Hz) and δC 67.47 ppm12,
confirming the presence of an 11α-hydroxyl group. The 13C
NMR spectrum of the metabolite showed a downfield shift for
C-12 (∆ 9.87 ppm) whereas it showed a γ-gauche upfield shift
for C-13 (∆ 0.61 ppm), further confirming the presence of an
11α-hydroxyl group. The metabolite showed a new resonance
(1H, bs) at δH 3.57 ppm and a downfield shift (∆ 0.08 ppm) for
the 6-H resonance of pregnenolone 3, indicating the presence
of a 7α-hydroxyl group. The 13C NMR spectrum of the meta-
bolite showed downfield shifts for C-6 (∆ 1.62 ppm) and C-8
(∆ 4.75 ppm) whereas it showed γ-gauche upfield shift for C-9
(∆ 2.11 ppm) and C-14 (∆ 8.01 ppm). These shifts confirmed
that 7α-hydroxylation had taken place indicating that the
metabolite was 3β,7α,11α-trihydroxypregn-5-en-20-one 12.
Incubation of testosterone 1 with M. hiemalis MRC 70325
mainlyafforded14α,17β-dihydroxyandrost-4-en-3-one7(62%),
accompanied by androst-4-en-3,17-dione 4 (3%), 14α-hydrox-
yandrost- 4-en-3,17-dione 5 (9%) and 6β-hydroxyandrost-4-
en-3,17-dione 6 (2%) as minor metabolites. Incubation of
dehydroepiandrosterone 2 with M. hiemalis MRC 70325 mainly
afforded 3β,7α-dihydroxyandrost-5-en-17-one 9 (72%), accom-
panied by 3β,17β-dihydroxyandrost-5-ene 8 (6%) as a minor
metabolite. Incubation of pregnenolone 3 with M. hiemalis
MRC 70325 mainly afforded 3β,7α-dihydroxypregn-5-en-20-one
11 (64%), accompanied by 3β,14α-dihydroxypregn-5-en-7,20-
dione 10 (3%) and 3β,7α,11α-trihydroxypregn-5-en-20-one 12
(11%) as minor metabolites. 3β,14α-Dihydroxypregn-5-en-
7,20-dione 10 was identified as a new metabolite.
Incubation of testosterone 1 with M. hiemalis MRC 70325
mainly afforded 14α-hydroxylation. Whilst in previous work,8
incubation of progesterone with M. hiemalis also afforded
mainly 14α-hydroxylation.8 However, incubations of dehydro-
epiandrosterone 2 and pregnenolone 3 with M. hiemalis MRC
70325 mainly afforded 7α-hydroxylation. These results sug-
gested that M. hiemalis was able to hydroxylate testosterone 1
and progesterone at C-14α as a consequence of the presence of
a 4-en-3-one moiety. This could be one of the possible struc-
tural requirements for an 14α-hydroxylase to accept a steroid
molecule as a substrate. M. hiemalis MRC 70325 failed to
hydroxylate dehydroepiandrosterone 2 and pregnenolone 3
at C-14α due to the absence of a 4-en-3-one moiety in these
substrates. These results also suggested that the presence of the
C-17 side chain in pregnenolone 3 and progesterone had
no influence on the hydroxylation patterns at those positions
by M. hiemalis. Incubations of some steroids with Mucor
piriformis13,14 and Mucor racemosus15–17 gave some similar
results. These fungi mainly hydroxylated 4-en-3-one steroids
at C-14α whereas they mainly hydroxylated 5-en-3β-hydroxy
steroids at C-7α. The presence of the C-17 side chain also had
no influence on the hydroxylation patterns at C-7α and C-14α
by these fungi.
Biotransformation of testosterone 1: The liquid medium8 (PYG
broth) 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).
The 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 a PDA slope, were transferred
aseptically into each flask containing sterile medium in a biological
safety cabinet. After cultivation at 26 °C for 3 days on a rotary shaker
(100 rpm), testosterone 1 (1 g) dissolved in 10 mL of DMF was evenly
distributed aseptically among the flasks. The biotransformation of 1
was carried out in 10 flasks for 5 days under the same conditions. The
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 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 (1945 mg), which
was then chromatographed on silica gel. Elution with 30% ethyl
acetate in hexane afforded androst-4-en-3,17-dione 4 (30 mg, 3%),
which was crystallised from ethyl acetate as prisms, m.p. 172–173 °C
(lit.18 174–176 °C), vmax/cm−1 1732, 1720 and 1615; δH 0.90 (3H, s,
18-H), 1.20 (3H, s, 19-H), 5.74 (1H, s, 4-H).
Elution with 50% ethyl acetate in hexane afforded 14α-hydroxy-
androst-4-en-3,17-dione 5 (95 mg, 9%), which was crystallised from
methanol as plates, m.p. 260–261 °C (lit.10 255–260 °C), vmax/cm−1
3417, 1730, 1660 and 1620; δH 1.00 (3H, s, 18-H), 1.20 (3H, s, 19-H),
5.72 (1H, s, 4-H).
Elution with 60% ethyl acetate in hexane afforded 6β,17β-dihy-
droxyandrost-4-en-3-one 6 (21 mg, 2%), which was crystallised from
ethyl acetate as prisms, m.p. 210–212 °C (lit.10 215–220 °C), vmax/cm−1
3495, 1660 and 1620; δH 0.81 (3H, s, 18-H), 1.38 (3H, s, 19-H), 3.66
(1H, t, J = 8.5 Hz, 17α-H), 4.35 (1H, bs, 6α-H), 5.81 (1H, s, 4-H).
Further elution with 60% ethyl acetate in hexane afforded 14α,17β-
dihydroxyandrost-4-en-3-one 7 (654 mg, 62%), which was crystalli-
sed from ethyl acetate as prisms, m.p. 183–184 °C (lit.10 181–185 °C),
vmax/cm−1 3447, 1662 and 1640; δH 0.90 (3H, s, 18-H), 1.20 (3H, s,
19-H), 4.29 (1H, t, J = 8.2 Hz, 17α-H), 5.71 (1H, s, 4-H).
Biotransformation of dehydroepiandrosterone 2: Under the same
conditions, the incubation of dehydroepiandrosterone 2 (1 g) with a
M. hiemalis MRC 70325 culture for 5 days afforded a brown gum
(1776 mg), which was then chromatographed on silica gel. Elution
with 60% ethyl acetate in hexane afforded 3β,17β-dihydroxyandrost-
5-ene 8 (61 mg, 6%), which was crystallised from acetone as prisms,
m.p. 175–177 °C (lit.19 180–183 °C), vmax/cm−1 3470 and 1655; δH 0.76
(3H, s, 18-H), 1.02 (3H, s, 19-H), 3.54 (1H, m, 3α-H), 3.64 (1H, t,
J = 8.5 Hz, 17α-H), 5.35 (1H, d, J = 5 Hz, 6-H).
Elution with 90% ethyl acetate in hexane afforded 3β,7α-dihy-
droxyandrost-5-en-17-one 9 (760 mg, 72%), which was crystallised
from acetone as needles, m.p. 180–181 °C (lit.19 182–183, °C),
vmax/cm−1 3423, 1736, 1660 and 1606; δH 0.89 (3H, s, 18-H); 1.03
(3H, s, 19-H); 3.52 (1H, m, 3α-H); 3.93 (1H, bs, 7β-H); 5.58 (1H, d,
J = 5 Hz, 6-H).
In conclusion, we have shown that M. hiemalis MRC 70325
hydroxylated testosterone 1 mainly at C-14α whereas it mainly
hydroxylated dehydroepiandrosterone 2 and pregnenolone
3 at C-7α. Work on further steroid biotransformation by
M. hiemalis and other fungi is in progress.
Experimental
Testosterone 1, dehydroepiandrosterone 2 and pregnenolone 3 were
purchased from Sigma-Aldrich (Istanbul, Turkey). Solvents were of
analytical grade and were purchased from Merck (Istanbul, Turkey).
Potato dextrose agar 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) eluting with increasing concentrations of ethyl
acetate in hexane. TLC was carried out with 0.2 mm thick Merck
Kieselgel 60 F254 TLC plates using ethyl acetate/hexane (1:1) as
eluent. TLC plates were dipped into an anisaldehyde/H2SO4 reagent
Biotransformation of pregnenolone 3: Under the same conditions,
the incubation of pregnenolone 3 (1 g) with a M. hiemalis MRC 70325
culture for 5 days afforded a brown gum (1873 mg), which was then
chromatographed on silica gel. Elution with 90% ethyl acetate in
hexane afforded 3β,14α-dihydroxypregn-5-en-7,20-dione 10 (33 mg,