Baeyer-villiger oxidation of some steroids by Aspergillus tamarii MRC 72400

Article abstract of DOI:10.1135/cccc2011008

Biotransformations of epiandrosterone (1), dehydroepiandrosterone (2), testosterone (3), progesterone (4) and pregnenolone (5) by Aspergillus tamarii MRC 72400 for 5 days have been reported and the results of these incubations have been compared with previously published data obtained with Aspergillus tamarii QM 1223. A. tamarii MRC 72400 showed higher Bayer-Villiger monooxygenase activities than A. tamarii QM 1223 did. Apart from pregnenolone (5), A. tamarii MRC 72400 metabolized these steroids in different ways. Incubation of epiandrosterone (1) afforded 3β,11β-dihydroxy-5α-androstan-17- one (6) (3%) and 3β-hydroxy-17a-oxa-D-homo-5α-androstan-17-one (7) (9.5%). Incubation of dehydroepiandrosterone (2) afforded 3β-hydroxy-17a- oxa-D-homoandrost-5-en-17-one (8) (28%), testolactone (9) (6%), 3β,7β-dihydroxyandrost-5-en-17-one (10) (13%) and 3β,7α- dihydroxyandrost- 5-en-17-one (11) (24%). Incubation of testosterone (3) afforded testolactone (9) (58%). Incubation of progesterone (4) also afforded testolactone (9), however in higher yield (86%). Incubation of pregnenolone (5) afforded 3β-hydroxy-17a-oxa-D-homoandrost- 5-en-17-one (8) (25%) and testolactone (9) (27%).

Full text of DOI:10.1135/cccc2011008

Oxidation of Some Steroids by A. tamarii MRC 72400
743
BAEYER–VILLIGER OXIDATION OF SOME STEROIDS BY
Aspergillus tamarii MRC 72400
1,
2
3
Kudret YILDIRIM *, Ahmet UZUNER and Emine Yasemin GULCUOGLU
Chemistry Department, Sakarya University, 54187, Sakarya, Turkey;
1
2
e-mail: kudrety@sakarya.edu.tr, azuner@gmail.com, 3eyasemingulcuoglu@gmail.com
Received January 12, 2011
Accepted March 28, 2011
Published online May 25, 2011
Biotransformations of epiandrosterone (1), dehydroepiandrosterone (2), testosterone (3),
progesterone (4) and pregnenolone (5) by Aspergillus tamarii MRC 72400 for 5 days have
been reported and the results of these incubations have been compared with previously pub-
lished data obtained with Aspergillus tamarii QM 1223. A. tamarii MRC 72400 showed higher
Bayer–Villiger monooxygenase activities than A. tamarii QM 1223 did. Apart from
pregnenolone (5), A. tamarii MRC 72400 metabolized these steroids in different ways. Incu-
bation of epiandrosterone (1) afforded 3β,11β-dihydroxy-5α-androstan-17-one (6) (3%) and
3β-hydroxy-17a-oxa-D-homo-5α-androstan-17-one (7) (9.5%). Incubation of dehydroepi-
androsterone (2) afforded 3β-hydroxy-17a-oxa-D-homoandrost-5-en-17-one (8) (28%), testo-
lactone (9) (6%), 3β,7β-dihydroxyandrost-5-en-17-one (10) (13%) and 3β,7α-dihydroxy-
androst-5-en-17-one (11) (24%). Incubation of testosterone (3) afforded testolactone (9)
(58%). Incubation of progesterone (4) also afforded testolactone (9), however in higher yield
(86%). Incubation of pregnenolone (5) afforded 3β-hydroxy-17a-oxa-D-homoandrost-
5-en-17-one (8) (25%) and testolactone (9) (27%).
Keywords: Steroids; Biotransformation; Biocatalysis; Aspergillus tamarii; Baeyer–Villiger
oxidation.
A number of investigations involving microbial biotransformations of a
wide range of steroidal substrates have been carried out due to their poten-
tial for the preparation of more valuable and functionalized compounds
such as steroid drugs and hormones1–3. There are still enormous efforts to
increase the efficiency of microbial steroid biotransformations and to find
new useful microorganisms and reactions1. For example, some of these ste-
roid biotransformations have been carried out using Aspergillus wentii4,
Penicillium digitatum5 and Aspergillus terreus6. A. wentii showed some high
hydroxylase activities whereas P. digitatum and A. terreus showed some low
5α-reductase and Baeyer–Villiger monooxygenase (BVMO) activities, respec-
tively.
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 6, pp. 743–754
© 2011 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2011008

744
Yildirim, Uzuner, Gulcuoglu:
Baeyer–Villiger oxidations of some steroids afford steroidal lactones,
which are important compounds because of their anticarcinogenic7,8
,
antiandrogenic9,10 and antihypercholestrolemic activites11. Although chem-
ical syntheses of these lactones are possible, the microbial biotransform-
ations are much more convenient due to environmental concerns12. The
degradation of 17β-acetyl side chain of some C21 steroids and ring D oxida-
tion of some androgens are carried out by BVMOs 13. Many bacteria and
fungi have these enzymes and they catalyze the insertion of an oxygen
atom next to a keto function and convert different ketones to correspond-
ing esters and lactones14.
Aspergillus tamarii Kita is a dark greenish-brown fungus usually found on
substrates yielding isolates of Aspergillus flavus Link, Aspergillus parasiticus
Speare, and related fungi15. It produces some toxic metabolites such as
aflatoxins B1 and B2, and cyclopiazonic acid15,16
.
As far as biotransformations by A. tamarii are concerned, A. tamarii QM 1223
has been the only strain of this fungus used for steroid biotransform-
ation17–25. This strain mainly showed BVMO activities on different steroidal
substrates and has attracted considerable interest due to its ability to trans-
form progesterone (4) into testolactone (9) in high yield. In this study we
have investigated the biotransformation of epiandrosterone (1),
dehydroepiandrosterone (2), testosterone (3), progesterone (4) and pregne-
nolone (5) by A. tamarii MRC 72400 in order to extend previous results on
steroid biotransformation by A. tamarii. A. tamarii MRC 72400 showed
higher BVMO activities on these steroids than A. tamarii QM 1223 did.
RESULTS AND DISCUSSION
The analyzed metabolites resulting from incubations of steroids with A.
tamarii MRC 72400 are shown in Scheme 1. Incubation of epiandro-
sterone (1) with A. tamarii afforded two metabolites. The first metabolite
had a new resonance at δH 4.39 ppm (1 H, bs), which was in accordance
with the characteristic 11α-H resonance26. Its 13C NMR spectrum, on com-
parison to 1, showed downfield shifts for C-18 (∆ 2.11 ppm), C-19
(∆ 3.3 ppm), C-9 (∆ 4.18 ppm), and C-12 (∆ 9.09 ppm) whereas it showed
γ-gauche upfield shifts for C-8 (∆ 4.12 ppm) and C-13 (∆ 0.98 ppm), which
was consistent with 11β-hydroxylation27. The metabolite had a resonance
at δH 3.55 ppm (1 H, tt, J = 5.0 and 12.0 Hz) confirming that the 3β-
hydroxy group was maintained. All these results suggested that the metabo-
lite was 3β,11β-dihydroxy-5α-androstan-17-one (6).
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 6, pp. 743–754

Oxidation of Some Steroids by A. tamarii MRC 72400
745
SCHEME 1
Metabolism of steroidal substrates by Aspergillus tamarii MRC 72400
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746
Yildirim, Uzuner, Gulcuoglu:
The 1H NMR spectrum of the second metabolite showed an important
downfield shift (∆ 0.45 ppm) for the 18-methyl resonance at δH 0.84 ppm
of the starting material. The 13C NMR spectrum of the metabolite de-
monstrated important downfield shifts for C-13 (∆ 35.49 ppm) and C-18
(∆ 6.34 ppm) whereas it demonstrated an important upfield shift for C-14
(∆ 5.06 ppm), which was in accordance with heteroatom insertion into
ring D via Baeyer–Villiger oxidation. The metabolite had a resonance at δH
3.53 ppm (1 H, tt, J = 5.0 and 11.0 Hz), indicating that the 3β-hydroxy
group was retained. These results indicated that the metabolite was 3β-
hydroxy-17a-oxa-D-homo-5α-androstan-17-one (7).
During the time course experiment, the 18-methyl signal of epiandro-
sterone (1) shifted from 0.84 to 1.29 ppm and comparison of the methyl
group integrations in the 1H NMR spectrum indicated that 10% of
epiandrosterone (1) had been converted to 3β-hydroxy-17a-oxa-D-homo-
5α-androstan-17-one (7) by 24 h. At 48 h, the 18-methyl signal of epiandro-
sterone (1) shifted from 0.84 to 1.29 and 1.10 ppm, and comparison of the
methyl group integrations in the 1H NMR spectrum suggested that
lactonization had been completed and that 5% of remaining
epiandrosterone (1) had been converted to 3β,11β-dihydroxy-5α-andro-
stan-17-one (6). Further comparison of the methyl group integrations in
the 1H NMR spectra suggested that hydroxylation had been completed
by 72 h and no more reactions were observed after that time. Time course
experiment results indicated that epiandrosterone (1) was mainly converted
to the corresponding lactone 7 by a BVMO. In addition, an independ-
ent minor 11β-hydroxylation pathway took place (Scheme 1). A. tamarii
QM 1223 only showed a 11β hydroxylase activity on the same substrate
and the yield18 was higher than that from A. tamarii MRC 72400.
Incubation of dehydroepiandrosterone (2) with A. tamarii afforded four
1
metabolites. The H NMR spectrum of the first metabolite showed a signifi-
cant downfield shift (∆ 0.47 ppm) for the 18-methyl resonance at δH
0.87 ppm of the starting material. The 13C NMR spectrum of the metabolite
had significant downfield shifts for C-13 and C-18 (∆ 35.86 ppm and
∆ 6.24 ppm, respectively) whereas it had an important upfield shift for C-14
(∆ 5.1 ppm), which was consistent with steroidal D lactone formation. This
metabolite had resonances at δH 3.54 ppm (1 H, tt, J = 5.0 and 11.0 Hz) and
δH 5.35 ppm (1 H, d, J = 5.0 Hz), indicating that the 3β-hydroxy group and
the double bond at C5-C6 was maintained. All these results suggested that
the metabolite was 3β-hydroxy-17a-oxa-D-homoandrost-5-en-17-one (8).
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 6, pp. 743–754

Oxidation of Some Steroids by A. tamarii MRC 72400
747
The 1H NMR spectrum of the second metabolite also had a significant
downfield shift (∆ 0.50 ppm) for the 18-methyl resonance at δH 0.87 ppm
of the starting material. The 13C NMR spectrum of the metabolite showed
important downfield shifts for the C-13 and C-18 (∆ 35.01 ppm and
∆ 6.52 ppm, respectively) whereas it showed an important upfield shift for
C-14 (∆ 6.08 ppm), which was in accordance with insertion of an oxygen
atom adjacent to this position on the ring D. The metabolite lacked the res-
onances of 2 at δH 3.46 ppm (1 H, tt, J = 5.0 and 12.0 Hz) and δH 5.35 ppm
(1 H, d, J = 5.0 Hz) and had a new resonance at δH 5.78 ppm (1 H, bs),
suggesting that the conversion of a 5-ene-3β-hydroxy steroid into a 4-ene-
3-keto steroid had taken place. These results suggested that the metabolite
was testolactone (9).
1
The H NMR spectrum of the third metabolite had the 7α-H resonance
at δH 3.95 ppm (1 H, dt, J = 4.0 and 8.0 Hz) and showed an upfield shift
(∆ 0.03 ppm) for the 6-H resonance (1 H, d, J = 5.0 Hz) at δH 5.35 ppm of
the starting material, which was consistent with 7β-hydroxylation28. Its
13C NMR spectrum, in comparison to 2, showed downfield shifts for C-6
(∆ 4.93 ppm) and C-8 (∆ 8.69 ppm) whereas it showed a γ-gauche upfield
shift for C-9 (∆ 1.93 ppm), which are comparable with literature values29.
The metabolite had a resonance at δH 3.55 ppm (1 H, tt, J = 5.0 and
10.0 Hz), indicating that the 3β-hydroxy group was maintained. All these
results indicated that the metabolite was 3β,7β-dihydroxyandrost-5-en-
17-one (10).
1
The H NMR spectrum of the fourth metabolite had the 7β-H resonance
signal at δH 3.96 ppm (1 H, bs, Wh = 11.0 Hz) and showed a significant
downfield shift (∆ 0.27 ppm) for the 6-H resonance (1 H, d, J = 5.0 Hz) at
δH 5.35 ppm of the starting material, which was in accordance with 7α-
hydroxylation28. Its 13C NMR spectrum, in comparison to 2, showed down-
field shifts for C-6 (∆ 2.12 ppm) and C-8 (∆ 5.88 ppm) whereas it showed
a γ-gauche upfield shift for C-9 (∆ 8.37 ppm), which are comparable with
literature values29. The metabolite had a resonance at δH 3.56 ppm (1 H,
m, Wh = 22.0 Hz) suggesting that the 3β-hydroxy group retained. These
results suggested that the metabolite was 3β,7α-dihydroxyandrost-5-en-
17-one (11).
During the time course experiment, the 18-methyl signal of dehydroepi-
androsterone (2) shifted from 0.87 to 1.34 ppm and comparison of the
1
methyl group integrations in the H NMR spectrum indicated that 35% of
dehydroepiandrosterone (2) had been converted to 3β-hydroxy-17a-oxa-
D-homoandrost-5-en-17-one (8) after 24 h. By 48 h, the 18-methyl signals
of dehydroepiandrosterone (2) shifted from 0.87 to 1.34, 1.37, 0.89 and
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 6, pp. 743–754

748
Yildirim, Uzuner, Gulcuoglu:
0.90 ppm, and further comparison of the methyl group signals suggested
that 5% of 3β-hydroxy-17a-oxa-D-homoandrost-5-en-17-one (8) had been
oxidized to testolactone (9) and that most of the remaining dehydro-
epiandrosterone (2) had been hydroxylated at C-7β and C-7α. Further com-
1
parison of the methyl group integrations in the H NMR spectra suggested
that no more reactions took place after 72 h. According to time course ex-
periment results, A. tamarii MRC 72400 first showed a BVMO activity on
dehydroepiandrosterone (2) and this was followed by oxidation of some
of the initial product and independent minor hydroxylations at C-7
(Scheme 1). Both 7α- and 7β-hydroxylase activities of A. tamarii MRC 72400
were independent from its BVMO activity and its 7α-hydroxylase activity
was higher than its 7β-hydroxylase one. A. tamarii QM 1223 showed the
same activities23. However, its 7α-hydroxylase activity was fully concomi-
tant with its BVMO one and afforded a 7α-hydroxylated lactone. The yields
of 8, 9 and 10 from A. tamarii MRC 72400 were higher than those from
A. tamarii QM 1223. The yield of 9 was lower than that of 8 and it was
thought that this was due to possible inhibitory effects of allylic hydroxy-
lations at C-7 on the isomerase enzyme as in the literature23.
Incubation of testosterone (3) with A. tamarii afforded only testolactone
1
(9), which was identified by comparison of its H and 13C NMR spectra with
that of an authentic sample.
During the time course experiment, the 18-methyl signal of testosterone
(3) shifted from 0.77 to 0.93 ppm 26 and comparison of the methyl group
1
integrations in the H NMR spectrum indicated that 60% of testosterone (3)
was converted to androst-4-ene-3,17-dione (12) after 8 h of incubation. Af-
ter 24 h, the presence of the 18-methyl signal of testosterone (3) shifted
from 0.77 to 1.37 ppm and further comparison of the methyl group inte-
grations in the 1H NMR spectrum indicated that 100% of androst-4-ene-
3,17-dione (12) had been converted to testolactone (9). Further comparison
1
of the methyl group integrations in the H NMR spectra suggested that no
more reactions took place after 48 h. Time course experiment indicated that
testosterone (3) was first converted to androst-4-ene-3,17-dione (12) and
this compound was then converted to testolactone (9) by a BVMO activity
(Scheme 1) as it was in the incubation of this substrate with Penicillium
lilacinum30. A. tamarii QM 1223 showed the same activities accompanied by
an independent minor 11β-hydroxylation pathway and the yield17 of 9 was
lower than that from A. tamarii MRC 72400.
Incubation of progesterone (4) with A. tamarii also afforded testolactone
1
(9), which was identified by comparison of its H and 13C NMR spectra with
that of an authentic sample.
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Oxidation of Some Steroids by A. tamarii MRC 72400
749
During the time course experiment, the 18-methyl signal of progesterone
(4) shifted from 0.66 to 0.77 and 0.93 ppm 26, and comparison of the meth-
1
yl group integrations in H NMR spectrum indicated that 90% of progester-
one (4) had been converted to testosterone (3) and androst-4-ene-3,17-
dione (12) at 8 h. After 24 h, the presence of the 18-methyl signals at δH
0.66 ppm and δH 1.37 ppm, and further comparison of the methyl group
1
integrations in H NMR spectrum indicated that all of testosterone (3) had
been converted to androst-4-ene-3,17-dione (12), which was then fully
transformed to testolactone (9). By 48 h, further comparison of the methyl
group integrations in the 1H NMR spectrum suggested that lactonization
had been completed during the first 24 h. No further changes were ob-
1
served in the methyl group integrals of the H NMR spectra after 72 h. Ac-
cording to time course experiment results for progesterone (4), a BVMO
first converted progesterone (4) to testosterone acetate, which was not ob-
served during the time course experiment due to the presence of high levels
of an esterase enzyme activity31. This ester was then hydrolyzed to give tes-
tosterone (3). The oxidation of testosterone (3) at C-17 gave androst-
4-ene-3,17-dione (12). Finally, 12 was fully converted to testolactone (9) by
BVMO activity (Scheme 1). Apart from a minor 11β-hydroxylation path-
way, A. tamarii QM 1223 showed the same activities on the substrate and
the yield17 of 9 was lower than that from A. tamarii MRC 72400.
Incubation of pregnenolone (5) with A. tamarii afforded two metabolites.
The first metabolite was 3β-hydroxy-17a-oxa-D-homoandrost-5-en-17-one
1
(8), which was identified by comparison of its H and 13C NMR spectra with
that of an authentic sample. The second metabolite was testolactone (9),
which was also identified by comparison of its 1H and 13C NMR spectra
with that of an authentic sample.
During the time course experiment, the 18-methyl signal of pregneno-
lone (5) shifted from 0.60 to 1.34 ppm and comparison of the methyl
group integrations in the 1H NMR spectrum indicated that 50% of preg-
nenolone (5) had been converted to 3β-hydroxy-17a-oxa-D-homoandrost-
5-en-17-one (8) in 24 h. By 48 h, the presence of the double bond signal
at δH 5.78 (1 H, bs), three 18-methyl signals at δH 0.60, 1.34 and 1.37 ppm
1
and further comparison of the methyl group integrations in the H NMR
spectrum indicated that 55% of 3β-hydroxy-17a-oxa-D-homoandrost-5-en-
17-one (8) had been converted to testolactone (9). After 72 h, no further
1
changes were observed in the methyl group integrals of the H NMR spec-
tra. Time course experiment results for pregnenolone (5) suggested that
3β-hydroxy-17a-oxa-D-homoandrost-5-en-17-one (8) occured as the first
lactone and that some of it was then converted to testolactone (9)
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 6, pp. 743–754

750
Yildirim, Uzuner, Gulcuoglu:
(Scheme 1). A. tamarii QM 1223 showed the same activities with lower
yields23.
In conclusion, we have shown that apart from pregnenolone (5), A.
tamarii MRC 72400 metabolized these steroids in different ways when com-
pared with A. tamarii QM 122317,18,23. A. tamarii MRC 72400 did not show
any 11β-hydroxylase activity on testosterone (3) and progesterone (4), and
its 7α-hydroxylase activity on dehydroepiandrosterone (2) was independ-
ent from its BVMO activities. A. tamarii MRC 72400 showed low BVMO and
11β-hydroxylase activities on epiandrosterone (1) whereas A. tamarii QM
1223 only showed a high 11β-hydroxylase activity on the same substrate.
We have also shown that A. tamarii MRC 72400 showed higher BVMO ac-
tivities than A. tamarii QM 1223 did and that this strain may at least be
an effective substitute for A. tamarii QM 1223 in the production of testo-
lactone (9) from progesterone (4).
EXPERIMENTAL
Epiandrosterone, dehydroepiandrosterone, testosterone, progesterone and pregnenolone
were purchased from Fluka (Istanbul, Turkey). A. tamarii Kita MRC 72400 was obtained from
TUBITAK (Marmara Research Center, Food Science and Technology Research Institute, Cul-
ture Collection Unit, Kocaeli, Turkey). Stock cultures were maintained at 4 °C on PDA
slopes. Solvents were of analytical grade and were purchased from Merck (Istanbul, Turkey).
Potato dextrose agar and agar for PDA slopes and malt extract for liquid medium were also
purchased from Merck (Istanbul, Turkey).
The steroids were separated by column chromatography on silica gel 60 (Merck 107734)
with increasing concentrations of ethyl acetate in hexane as eluent. TLC was carried out
with 0.2 mm thick Merck Kieselgel 60 F254 TLC plates using ethyl acetate/hexane (1:1,
v/v) as eluent. In order to develop the chromatograms, TLC plates were dipped into an
anisaldehyde–H2SO4 reagent and heated to 120 °C for 3 min. Infrared spectra (wavenumbers
in cm–1) were recorded using a Shimadzu IR Prestige-21 apparatus. Optical rotation measure-
ments were carried out on a WXG-4 polarimeter. Optical rotation values are given in 10–1
deg cm2 g–1. Elemental analysis was performed using a Thermo Finnigan Flash EA 1112 ele-
mental analyser. High-resolution mass spectra (HRMS) were determined on a Bruker
Daltonics Apex III mass spectrometer operating in the electrospray mode. 1H NMR spectra
were recorded in deuteriochloroform with tetramethylsilane as an internal standard refer-
ence 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 deter-
mined by an Electrothermal IA 9200 melting point apparatus and are uncorrected.
Biotransformation experiments were run with control flasks containing non-inoculated
sterile medium and one of the substrates. After five days of incubation, all controls were also
harvested and analysed by TLC. No metabolites were detected in controls.
Time course experiments23 were conducted in order to determine the metabolic pathway.
Conditions were identical to those in main biotransformation experiments except that each
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 6, pp. 743–754

Oxidation of Some Steroids by A. tamarii MRC 72400
751
individual steroidal substrate (300 mg) dissolved in DMF (6 ml) was evenly distributed be-
tween 6 flasks (each containing 100 ml of medium). One flask was harvested after 8 h. Then
every 24 h one flask was harvested and extracted. TLC analysis was performed immediately
on the isolated mixture. Following 6 h under high vacuum, the product 1H NMR spectra
were determined in CDCl3 to confirm the steroidal nature of the extracts.
Biotransformation of Epiandrosterone (1) by A. tamarii MRC 72400
Spores freshly obtained from PDA slopes were transferred aseptically into 10 Erlenmeyer
flasks of 250 ml containing 100 ml of sterile 3% malt extract medium17 in a biological safety
cabinet. After cultivation at 24 °C for 3 days on a rotary shaker (180 rpm), epiandrosterone
(1; 500 mg, 1.72 mmol) dissolved in 10 ml of DMF was evenly and aseptically distributed
among the flasks. The biotransformation of the substrate was carried out in 10 flasks for five
days under the same conditions. The fungal mycellium was separated from the broth by
filtration under the vacuum and the mycellium was rinsed with ethyl acetate (500 ml). The
broth was then 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 (716 mg), which was then chromatographed on silica gel. Elution with 30% ethyl ace-
tate in hexane afforded the unreacted starting material (300 mg, 60%), which was identified
by comparison of its 1H and 13C NMR spectra with those of an authentic material.
Elution with 40% ethyl acetate in hexane afforded 3β,11β-dihydroxy-5α-androstan-17-one
(6; 16 mg, 3%), which was crystallized from diethyl ether as needles, m.p. 228–231 °C,
[α]2D0 +83, c 0.1, CHCl3 (lit.32 gives m.p. 235–238 °C, [α]2D0 +84.5, c 1, C2H5OH). HRMS for
C
19H30O3: calculated 306.2196, found 306.2206. IR: 3470 (O–H), 1741 (C=O). 1H NMR
(300 MHz, CDCl3): 1.06 s, 3 H (H-19); 1.10 s, 3 H (H-18); 3.55 tt, 1 H, J(3,2) = 5.0, J(3,4) =
12.0 (H-3); 4.39 bs, 1 H (H-11). 13C NMR (75 MHz, CDCl3): 219.94, 70.96, 68.06, 58.43,
52.90, 46.92, 45.72, 40.48, 37.57, 36.83, 35.74, 35.33, 31.24, 31.09, 30.81, 27.84, 21.65,
15.86, 15.51.
Elution with 50% ethyl acetate in hexane afforded 3β-hydroxy-17a-oxa-D-homo-
5α-androstan-17-one (7; 50 mg, 9.5%), which was crystallized from acetone as needles, m.p.
170–173 °C, [α]2D0 –43, c 0.1, CHCl3 (lit.6 gives m.p. 173–174 °C, [α]D20 –45, c 0.1, CHCl3).
HRMS for C19H30O3: calculated 306.2196, found 306.2182. IR: 3438 (O–H), 1724 (C=O).
1H NMR (300 MHz, CDCl3): 0.78 s, 3 H (H-19); 1.29 s, 3 H (H-18); 3.53 tt, 1 H, J(3,2) = 5.0,
J(3,4) = 11.0 (H-3). 13C NMR (75 MHz, CDCl3): 171.65, 83.39, 70.99, 53.01, 46.22, 44.13,
39.24, 37.84, 37.73, 36.70, 35.44, 31.24, 30.55, 28.59, 28.21, 21.97, 20.09, 19.73, 12.11.
Biotransformation of Dehydroepiandrosterone (2) by A. tamarii MRC 72400
Under similar conditions, the incubation of dehydroepiandrosterone (2; 500 mg, 1.734 mmol)
with A. tamarii afforded a brown gum (765 mg), which was then chromatographed on silica
gel. Elution with 30% ethyl acetate in hexane afforded the unchanged starting material (50 mg,
10%), which was identified by comparison of its 1H and 13C NMR spectra with those of an
authentic material.
Elution with 50% ethyl acetate in hexane afforded 3β-hydroxy-17a-oxa-D-homoandrost-
5-en-17-one (8; 148 mg, 28%), which was crystallized from ethyl acetate as needles, m.p.
234–235 °C, [α]2D0 –92, c 0.1, CHCl3 (lit.6 gives m.p. 234–235 °C, [α]D20 –95, c 0.1, CHCl3). For
C
19H28O3 (304.4) calculated: 74.96% C, 9.27% H; found: 75.11% C, 9.10% H. IR: 3448
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752
Yildirim, Uzuner, Gulcuoglu:
(O–H), 1720 (C=O), 1657 (C=C). 1H NMR (300 MHz, CDCl3): 0.98 s, 3 H (H-19); 1.34 s, 3 H
(H-18); 3.54 tt, 1 H, J(3,2) = 5.0, J(3,4) = 11.0 (H-3); 5.35 d, 1 H, J(6,7) = 5.0 (H-6). 13C NMR
(75 MHz, CDCl3): 171.97, 140.49, 120.49, 83.35, 71.40, 48.85, 46.57, 41.58, 38.77, 36.79,
36.48, 34.33, 31.17, 30.95, 28.67, 21.86, 19.97, 19.72, 19.19.
Elution with 60% ethyl acetate in hexane afforded testolactone (9; 32 mg, 6%), which
was crystallized from methanol as needles, m.p. 210–211 °C, [α]2D0 +44, c 0.1, CHCl3 (lit.6
gives m.p. 206–207 °C, [α]2D0 +48, c 0.1, CHCl3). For C19H26O3 (302.4) calculated: 75.46% C,
8.67% H; found: 75.32% C, 8.49% H. IR: 1718 (C=O), 1618 (C=C). 1H NMR (300 MHz,
CDCl3): 1.17 s, 3 H (H-19); 1.37 s, 3 H (H-18); 5.78 bs, 1 H (H-4). 13C NMR (75 MHz,
CDCl3): 199.46, 171.43, 169.70, 123.99, 82.80, 52.41, 45.59, 38.87, 38.41, 37.89, 35.39,
33.71, 32.29, 30.33, 28.45, 21.76, 20.00, 19.76, 17.32.
Elution with 80% ethyl acetate in hexane afforded 3β,7β-dihydroxyandrost-5-en-17-one
(10; 69 mg, 13%), which was crystallized from ethyl acetate–petroleum ether as needles,
m.p. 207–209 °C, [α]2D0 +65, c 0.1, CHCl3 (lit.4 gives m.p. 208–209 °C, [α]D20 +67, c 0.1, CHCl3).
For C19H28O3 (304.4) calculated: 74.96% C, 9.27% H; found: 74.85% C, 9.23% H. IR: 3239
(O–H), 1740 (C=O), 1665 (C=C). 1H NMR (300 MHz, CDCl3): 0.90 s, 3 H (H-18); 1.08 s, 3 H
(H-19); 3.55 tt, 1 H, J(3,2) = 5.0, J(3,4) = 10.0 (H-3); 3.95 dt, 1 H, J(7α,8) = 8.0, J(7α,6) =
J(7α,4β) = 4.0 (H-7); 5.32 bs, 1 H (H-6). 13C NMR (75 MHz, CDCl3): 221.40, 143.16, 125.76,
72.32, 70.74, 51.19, 48.20, 47.65, 41.60, 40.10, 36.85, 36.51, 35.89, 31.37, 31.13, 24.08,
20.26, 18.96, 13.44.
Elution with pure ethyl acetate in hexane afforded 3β,7α-dihydroxyandrost-5-en-17-one
(11; 127 mg, 24%), which was crystallized from ethyl acetate–petroleum ether as needles,
m.p. 173–175 °C, [α]2D0 –77, c 0.1, CHCl3 (lit.4 gives m.p. 174–175 °C, [α]D20 –73, c 0.1, CHCl3).
For C19H28O3 (304.4) calculated: 74.96% C, 9.27% H; found: 74.81% C, 9.20% H. IR: 3422
(O–H), 3325 (O–H), 1736 (C=O), 1672 (C=C). 1H NMR (300 MHz, CDCl3): 0.89 s, 3 H (H-18);
1.03 s, 3 H (H-19); 3.56 m, 1 H, Wh = 22 (H-3); 3.96 bs, 1 H, Wh = 11 (H-7); 5.62 d, 1 H
J(6,7β) = 5.0 (H-6). 13C NMR (75 MHz, CDCl3): 221.48, 145.75, 122.95, 70.69, 63.86, 46.97,
44.73, 42.31, 41.76, 37.29, 37.05, 36.75, 35.65, 31.00, 30.88, 21.71, 19.87, 18.08, 13.05.
Biotransformation of Testosterone (3) by A. tamarii MRC 72400
Under similar conditions, the incubation of testosterone (3; 500 mg, 1.734 mmol) with A.
tamarii afforded a brown gum (738 mg), which was then chromatographed on silica gel. Elu-
tion with 30% ethyl acetate in hexane afforded the unchanged starting material (125 mg,
25%), which was identified by comparison of its 1H and 13C NMR spectra with those of an
authentic material.
Elution with 60% ethyl acetate in hexane afforded testolactone (9; 304 mg, 58%), which
was identified by comparison of its 1H and 13C NMR spectra with those of an authentic ma-
terial.
Biotransformation of Progesterone (4) by A. tamarii MRC 72400
Under similar conditions, the incubation of progesterone (4; 500 mg, 1.59 mmol) with A.
tamarii afforded a brown gum (782 mg), which was then chromatographed on silica gel. Elu-
tion with 60% ethyl acetate in hexane afforded testolactone (9; 414 mg, 86%), which was
identified by comparison of its 1H and 13C NMR spectra with those of an authentic material.
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 6, pp. 743–754

Oxidation of Some Steroids by A. tamarii MRC 72400
753
Biotransformation of Pregnenolone (5) by A. tamarii MRC 72400
Under similar conditions, the incubation of pregnenolone (5; 500 mg, 1.58 mmol) with A.
tamarii afforded a brown gum (730 mg), which was then chromatographed on silica gel. Elu-
tion with 30% ethyl acetate in hexane afforded the unreacted starting material (205 mg,
41%), which was identified by comparison of its 1H and 13C NMR spectra with those of an
authentic material.
Elution with 50% ethyl acetate in hexane afforded 3β-hydroxy-17a-oxa-D-homoandrost-
5-en-17-one (8; 120 mg, 25%), which was identified by comparison of its 1H and 13C NMR
spectra with those of an authentic material.
Elution with 60% ethyl acetate in hexane afforded testolactone (9; 129 mg, 27%), which
was identified by comparison of its 1H and 13C NMR spectra with those of an authentic ma-
terial.
We are grateful to Fatih Sonmez and Hulya Demirhan for running the 1H and 13C NMR spectra.
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