Baeyer-villiger oxidation of some C19 steroids by penicillium lanosocoeruleum

Article abstract of DOI:10.3390/molecules181113812

The biotransformation of androsterone (1), epiandrosterone (2), androstanedione (3) and DHEA (dehydroepiandrosterone) (4) by Penicillium lanosocoeruleum-a fungal species not used in biotransformations so far-were described. All the substrates were converted in high yield (70%-99%) into D ring d-lactones. The oxidation of 1 produced 3a-hydroxy-17a-oxa-D-homo-5a-androstan- 17-one (5). The oxidation of 2 led to 3β-hydroxy-17a-oxa-D-homo-5a- androstan-17-one (6). The biotransformation of 3 resulted in the formation of 3a-hydroxy-17a-oxa-D-homo-5a-androstan-17-one (5) and 17a-oxa-Dhomo- 5a-androstan-3,17-dione (7). An analysis of the transformation progress of the studied substrates as a function of time indicates that the Baeyer-Villiger monooxygenase of this fungus does not accept the 3β-hydroxy-5-ene functionality of steroids. In this microorganism steroidal 3β-hydroxy- dehydrogenase (3β-HSD) was active, and as a result DHEA (4) was transformed exclusively to testololactone (8). Apart from the observed oxidative transformations, a reductive pathway was revealed with the C-3 ketone being reduced to a C-3a-alcohol. It is demonstrated for the first time that the reduction of the 3-keto group of the steroid nucleus can occur in the presence of a ring-D lactone functionality.

Full text of DOI:10.3390/molecules181113812

Molecules 2013, 18, 13812-13822; doi:10.3390/molecules181113812
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Baeyer-Villiger Oxidation of Some C19 Steroids by
Penicillium lanosocoeruleum
Alina Świzdor
Department of Chemistry, Wroclaw University of Environmental and Life Sciences, Norwida 25,
Wroclaw 50-375, Poland; E-Mail: alina.swizdor@up.wroc.pl; Tel.: +48-71-3205-252;
Fax: +48-71-3284-124
Received: 8 September 2013; in revised form: 28 October 2013 / Accepted: 30 October 2013 /
Published: 7 November 2013
Abstract: The biotransformation of androsterone (1), epiandrosterone (2), androstanedione
(3) and DHEA (dehydroepiandrosterone) (4) by Penicillium lanosocoeruleum—a fungal
species not used in biotransformations so far—were described. All the substrates were
converted in high yield (70%–99%) into D ring δ-lactones. The oxidation of 1 produced
3α-hydroxy-17a-oxa-D-homo-5α-androstan-17-one (5). The oxidation of 2 led to
3β-hydroxy-17a-oxa-D-homo-5α-androstan-17-one (6). The biotransformation of 3 resulted
in the formation of 3α-hydroxy-17a-oxa-D-homo-5α-androstan-17-one (5) and 17a-oxa-D-
homo-5α-androstan-3,17-dione (7). An analysis of the transformation progress of the
studied substrates as a function of time indicates that the Baeyer-Villiger monooxygenase
of this fungus does not accept the 3β-hydroxy-5-ene functionality of steroids. In this
microorganism steroidal 3β-hydroxy-dehydrogenase (3β-HSD) was active, and as a result
DHEA (4) was transformed exclusively to testololactone (8). Apart from the observed
oxidative transformations, a reductive pathway was revealed with the C-3 ketone being
reduced to a C-3α-alcohol. It is demonstrated for the first time that the reduction of the
3-keto group of the steroid nucleus can occur in the presence of a ring-D lactone functionality.
Keywords: steroidal lactones; biotransformation; Baeyer-Villiger oxidation;
DHEA;
androsterone;
epiandrosterone;
5α-androstan-3,17-dione;
3β-HSD;
Penicillium lanosocoeruleum

Molecules 2013, 18
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1. Introduction
Steroids represent an important class of natural products with varying pharmacological properties.
Minor modifications in their structure may affect their bioactivity, consequently, research on the
preparation of potentially useful steroid analogues is being continued for development of processes
exploitable by the pharmaceutical industry. Biotransformations are powerful tools for generation of
novel steroidal drugs, as well as for efficient production of steroidal key intermediates. In the majority
of cases, these modifications have been achieved by the use of whole-cells microorganisms. The use of
enzymes ensures the high regio- and stereoselectivity of the reaction to be performed, and
biotransformations are more environmentally friendly in comparison with chemical syntheses.
Microbial transformations of various steroids have been reviewed recently [1,2].
Currently research efforts in production of steroids of pharmaceutical interest are focused on
either optimization of existing processes or identification of novel potentially useful bioconversions.
It is not surprising that several microorganisms have been reported to exhibit Baeyer-Villiger
oxidation properties. Only in a few cases the isolated enzymes have been used [1,3,4]. Recently,
cyclopentadecanone monooxygenase (CPDMO) isolated from Pseudomonas sp., involved in the
catabolism of cyclopentadecanone in Nature, was also found to accept steroids as substrates [3]. It has
been screened for the Baeyer-Villiger oxidation of a large number of steroids but the products
were formed in low yields. The use of the chimeric enzyme PASTMO (a hybrid of phenylacetone
monooxygenase (PAMO) and bacterial steroid monooxygenase (STMO) from Rhodococcus
rhodochrous) in the biotransformation of progesterone afforded the final lactone (testololactone) with
a moderate (19%) conversion [4]. Among microorganisms, fungal species of the Penicillium [5–10],
Aspergillus [11–20] and Fusarium genera [21–23] are especially known to transform 4-en-3-oxo and
3β-hydroxy-5-ene C21 as well as C19 steroids. Enzymes performing oxidation belong to the family of
NADPH-dependent steroid Baeyer-Villiger monooxygenases (BVMOs) containing FAD as a cofactor.
The catalytic activity of this class of enzymes is manifested in the transformation of C-20 ketones into
the corresponding acetates, which then hydrolyze to the related C-17 alcohols, followed by oxidation
to ketones, and finally ring-D Baeyer-Villiger oxidation gives lactones.
Steroidal lactones are important compounds due to their biological activity such as anticancer and
antiandrogenic action. It was found that they could inhibit the 5α-reductase, and as a result of this,
block the conversion of testosterone to 5α-dihydrotestosterone [24] and therefore have therapeutic
potential for the treatment of androgen-dependent diseases. Steroidal testolactone as an inhibitor of
steroidal aromatase [25], is used in the clinical treatment of breast cancer [25] and a few forms of
precocious puberty [26]. Although chemical synthesis routes to steroidal ring-D lactones using
peracids [27,28] are possible, they often result in a larger variety of products (e.g., regioisomeric
lactones, epoxylactones or epoxyketones).
Microbial steroid BVMOs are steroid-induced enzymes, which utilize atmospheric oxygen as
oxidant. Their catalytic properties exhibit differences [1], even within the same species of
microorganism. For example, Penicillium citreo-viride oxidized androstenedione, progesterone
and DHEA (dehydroepiandrosterone) to testololactone, while pregnenolone was not transformed;
conversion of DHEA to testololactone occurred through the 3β-hydroxysteroid dehydrogenase/5-ene-4-
ene isomerase pathway resulting in the generation of a 4-en-3-oxo system in the steroid ring-A [7].

Molecules 2013, 18
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This same pathway was observed during the transformation of DHEA by P. griseopurpureum and
P. glabrum [8]. In contrast to P. citreo-viride, BVMO(s) from P. lilacinum accepted 3β-hydroxy-5-ene
substrates [6]. The enzyme was able to carry out the degradation of 17β-acetyl side chain and ring-D
lactonization of 3β-hydroxy-5-ene as well as 4-en-3-oxo substrates with preference for lactonization of
5-ene steroids. In this way DHEA was converted only to 3β-hydroxy-17a-oxa-D-homo-androst-5-en-
17-one (a D-lactone with conserved ring-A moiety), whereas pregnenolone was also transformed to
testololactone (a D-lactone with a 3-oxo-4-ene system). P. camemberti was able to carry out the
oxidation of pregnenolone and DHEA to testololactone, which could be formed via two routes:
through 4-en-3-ketone or 3β-hydroxylactone [9]. In most cases of transformations described in
literature, the steroids, besides the Baeyer-Villiger oxidation, also underwent hydroxylation, reduction
or dehydrogenation [8,12,15,16,19,23,29]. The mixtures of metabolites formed during the
transformations led to difficulties in product purification. The processes were usually slow (2–5 days)
and the productivity was low.
In the light of our interest [6,9,29] in the microbial Baeyer-Villiger oxidation of steroids, this
article reports the results of the transformation of a series of androstanes, namely DHEA,
epiandrosterone, androsterone and androstanedione, by Penicillium lanosocoeruleum KCH 3012. To
the best of the author’s knowledge, this is the first report of the use of P. lanosocoeruleum in
biotransformations.
2. Results and Discussion
The P. lanosocoeruleum KCH 3012 strain produces a Baeyer-Villiger monooxygenase which is
able to carry out the regioselective ring-D lactonization of 5α-dihydro-steroids (1–3) as well as
unsaturated steroid 4 (Scheme 1, Table 1). The corresponding lactones 5 and 6, were formed with high
yield (70%–96%) as the sole products from epimeric 3-hydroxy substrates—androsterone (1) and
epiandrosterone (2). Two lactones: 3α-hydroxy-17a-oxa-D-homo-5α-androstan-17-one (5) and
17a-oxa-D-homo-5α-androstan-3,17-dione (7) were isolated after transformation of a 3,17-diketo
compound—androstanedione (3). DHEA (4) was converted to testololactone (8).
Scheme 1. Structures of substrates and products of their transformations.
O
O
O
O
O
O
R
R
O
HO
H
H
4
8
1 R=α-OH
2 R=β-OH
3 R=O
5 R=α-OH
6 R=β-OH
7 R=O

Molecules 2013, 18
Table 1. The results of transformation of 1–4 and 7 by P. lanosocoeruleum.
13815
Time
(h)
Yield
Starting material
Androsterone (1)
Compounds
(%) a,b
-
48
Androsterone (1)
3α-Hydroxy-17a-oxa-D-homo-5α-androstan-17-one (5)
Epiandrosterone (2)
98 (96)
19 (17)
75 (70)
-
Epiandrosterone (2)
Androstanedione (3)
48
24
3β-Hydroxy-17a-oxa-D-homo-5α-androstan-17-one (6)
Androstanedione (3)
17a-Oxa-D-homo-5α-androstan-3,17-dione (7)
3α-Hydroxy-17a-oxa-D-homo-5α-androstan-17-one (5)
DHEA (4)
40 (37)
59 (55)
-
DHEA (4)
24
48
Testololactone (8)
96 (91)
24 (23)
71 (67)
17a-Oxa-D-homo-
17a-Oxa-D-homo-5α-androstan-3,17-dione (7)
5α-androstan-3,17-dione (7)
3α-Hydroxy-17a-oxa-D-homo-5α-androstan-17-one (5)
a Determined by GC analysis of the crude chloroform extracts. b Isolated yield in parentheses.
The structures of the obtained products were established by using spectroscopic techniques (IR,
13
1H-NMR and C-NMR). The assumed structures were confirmed by comparison of the characteristic
shift values of selected, diagnostic signals of products and starting compounds. Resonance signals in
1
both H-NMR and 13C-NMR spectra of all metabolites 5–8 suggested the formation of the ring-D
lactones. The absence of the carbonyl group signals at δC ca. 220 ppm, the presence of new signals at
δC ca. 171 ppm and downfield shifts of the C-13 resonance signals with respect to the substrates,
confirmed insertion of an oxygen atom into the ring-D. The oxygen insertion into ring-D of all
metabolites was supported by significant downfield shifts for both the 18-methyl protons’ signals in
13
1H-NMR and the C-18 signals in C-NMR. The bands at 1714–1720 cm−1 in the IR spectra of the
products confirmed the δ-lactone structures. The absence of the 3α-H resonance (m) from the 1H-NMR
spectrum of the metabolite 8 coupled with an increase in the C-19 methyl resonance signal
(∆ 0.14 ppm relative to substrate 4) and a shift of the signal of olefinic proton from δH 5.36 ppm to
δH 5.75 ppm indicated isomerization of the double bond with its formation between C-4 and C-5 and
oxidation of the C-3 alcohol to a ketone. Double bond migration into ring-A was fully supported in the
13C-NMR spectrum with downfield shifts observed for C-4 (∆ 81.8 ppm) and C-5 (∆ 28.1 ppm). Signal
C-5 was visible at δc 169.1 ppm and its position was consistent with the position of a β-carbon
conjugated with the carbonyl group. Oxidation of the 3β-OH group was fully supported by the loss of a
13
methine signal at δc 71.5 ppm in the starting material C-NMR spectrum, being replaced by a new
non-protonated resonance signal in the product spectrum at δc 199.4 ppm. All these observations for
metabolites 5–8 were in agreement with those reported in the literature [6,17,18,30].
3α-Hydroxy-17a-oxa-D-homo-5α-androstan-17-one (5) was previously obtained in 53% yield by
transformation of androsterone using Aspergillus tamarii KITA, but it was one of the six metabolites
produced from this substrate. During that transformation, substrate as well as 3α-hydroxy-17a-oxa-D-
homo-5α-androstan-17-one also underwent hydroxylation, giving a mixture of 1β- and 11β-hydroxy
products [16]. Epiandrosterone was converted to 3β-hydroxy-17a-oxa-D-homo-5α-androstan-17-one
(6) with 10% yield by Aspergillus terreus [18]. 17a-Oxa-D-homo-5α-androstan-3,17-dione (7) was
previously obtained with 29% yield as one of the three metabolites by transformation of

Molecules 2013, 18
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5α-pregna-3,20-dione using Aspergillus tamarii KITA [11]. Testololactone (8) was the only product of
the transformation of C19 or C21 3-oxo-4-ene steroids or DHEA and androstenediol by P. citreo-viride [7],
as well as progesterone by A. terreus [18]. This lactone was prepared with 32%–86% yield, depending
on the substrate and the used biocatalyst.
The composition of mixtures sampled after various transformation periods was studied in order to
investigate the metabolic pathways of 3 and 4 (Table 2). The reaction mixture after 8 h of incubation of
androstanedione (3) contained, apart from the substrate, 3% of androsterone (1) and 2% of 3-keto-D-
lactone 7. That result indicated that substrate 3 underwent transformation via two routes: through
stereoselective ketone reduction at C-3 and Baeyer-Villiger oxidation in ring-D. The rapid increase in
the contents of D-lactones 5 and 7 between 8 and 12 h of the incubation indicates that compound 1 as
well as compound 3 are the inducers of the BVMO responsible for the lactonization of C-17-ketones.
In the product mixture obtained after 12 h and 24 h of transformation of androstanedione (3) the
3-oxo-lactone 7 prevailed, but its content decreased with prolonged reaction time, and consequently
after 48 h of incubation, the main metabolite of 3 was 3α-hydroxy-17a-oxa-D-homo-5α-androstan-17-
one (5). Because the amount of accumulating androsterone (1) did not exceed 10%, it can be assumed
that lactone 5 was formed mainly from lactone 7 (Scheme 2). It was shown in a separate experiment
that the 3-keto-lactone 7 was converted by P. lanososoeruleum to the 3α-hydroxy-lactone 5. This is
apparently the first time that the reduction of the 3-keto group has been observed in the presence of the
lactone functionality in ring-D.
Table 2. Time course of the transformation of androstanedione (3) and DHEA (4).
Time of transformation (h)
Rt
Substrate
Compounds present in the mixture (%) a
(min)
4
8
12 24
48
-
Androstanedione (3)
3.75 Androstanedione (3)
3.15 Androsterone (1)
100 95 26
5
-
-
3
8
-
3α-Hydroxy-17a-oxa-D-homo-5α-
7.19
7.60
-
-
24 40
40 54
59
39
androstan-17-one (5)
17a-Oxa-D-homo-5α-androstan-3,17-
dione (7)
-
2
DHEA (4)
3.39 DHEA (4)
85
15
-
26
1
-
4.42 Androstenedione (9) b
8.42 Testololactone (8)
63 58
4
11 40 94
3β-hydroxy-17a-oxa-D-homo-androst-5-
en-17-one c
6.77
-
-
-
-
a Determined by GC analysis. b Identified in GC and TLC on the basis of standard. c The standard obtained in
our previous work [6].
The analysis of composition of mixtures after transformation of DHEA (4) indicated that the first
stage of the process was the oxidative conversion of 3β-hydroxy-5-ene to 3-oxo-4-ene functionality
(Scheme 3, Table 2). Androstenedione (9) was the only metabolite identified after 4 h incubation of 4;
the mixtures after 8 h and 12 h incubation contained a significant amount of 9. After 12 h of reaction,
the content of androstenedione in the reaction mixtures started to decrease. Simultaneously, the content
of testololactone (8) increased. The production of 8 reached a maximum at 24 h of the experiment.

Molecules 2013, 18
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3β-Hydroxy-17a-oxa-D-homo-androst-5-en-17-one was not identified in any of the reaction mixtures,
which suggests that the BVMO from P. lanosocoeruleum does not accept 3β-hydroxy-5-ene steroids
as substrates.
Scheme 2. D-Lactonization pathway of androstanedione (3) in P. lanosocoeruleum.
O
HO
H
1
O
O
O
O
O
HO
O
O
H
H
H
5
7
3
Scheme 3. D-Lactonization pathway of DHEA (4) in P. lanosocoeruleum.
O
O
O
O
HO
O
O
4
9
8
3. Experimental
3.1. General Conditions of Cultivation and Transformation
General experimental and fermentation details were described previously [6]. Each substrate
was added to a 72-h-old culture of the microorganism as a DMSO solution, in concentration of
0.35 mmol/100 mL of medium. Each experiment was performed with four replications.
3.2. Chemicals and Reagents
The substrates androsterone (3α-hydroxy-5α-androstan-17-one, 1), epiandrosterone (3β-hydroxy-
5α-androstan-17-one, 2) and dehydroepiandrosterone (3β-hydroxyandrost-5-en-17-one, DHEA, 4)
were purchased from Sigma-Aldrich (Poznań, Poland). Androstanedione (5α-androstan-3,17-dione, 3) was
obtained by oxidation of androsterone (1) with Jones’ reagent according to the known procedure [11]
and was of high purity (>99% following GC and elemental analysis, C19H28O2 calcd. C, 79.12; H 9.78;
1
found. C, 79.36; H. 9.84%. H-NMR: 0.89 (3H, s, 18-H), 1.03 (3H, s, 19-H). 13C NMR: 11.4
(C-19), 13.8 (C-18), 20.7 (C-11), 21.7 (C-15), 28.6 (C-6), 30.5 (C-7), 31.4 (C-12), 34.9 (C-8), 35.8
(C-16), 35.8 (C-10), 38.0 (C-2), 38.4 (C-1), 44.5 (C-4), 46.6 (C-5), 47.7 (C-13), 51.2 (C-14), 53.8

Molecules 2013, 18
13818
1
(C-9), 211.6 (C-3), 220.9 (C-17). H- and 13C-NMR data were found to be identical to that of
androstanedione reported in the literature [31,32]. Androstenedione (9) was purchased from Steraloids
Inc. (Newport, RI, USA). 3β-Hydroxy-17a-oxa-D-homo-androst-5-en-17-one was obtained previously
in our laboratory by transformation of DHEA by Penicillium lilacinum AM111 [6]. The last two of the
aforementioned compounds were used as analytical standards for the time course experiments. Solvents
were of analytical grade.
3.3. Microorganism
The fungal strain Penicillium lanosocoeruleum KCH 3012 used in this study was taken from the
collection of the Department of Chemistry, Wrocław University of Environmental and Life Sciences
(Wrocław, Poland). The fungus was maintained on Sabouraud 4% dextrose-agar slopes at 4 °C and
freshly subcultured before use in the transformation experiments.
3.4. Time Course Experiments
Time course experiments were conducted in order to determine the metabolic pathways. Conditions
were identical to those in the main biotransformation experiments. One flask was harvested after 4, 8,
12 h, and every consecutive flask—after another 24 h incubation of substrate. Reaction mixtures were
extracted and analyzed by GC and TLC as described above.
3.5. Isolation and Identification of the Products
The products of biotransformation were extracted three times with ethyl acetate. The organic
extracts were dried over anhydrous magnesium sulfate, concentrated in vacuo and analyzed by TLC
and GC. Transformation products were separated by column chromatography on silica gel with
hexane/acetone (3:1 v:v) for androsterone (1), hexane/acetone/2-propanol (1:1:0.15 v:v:v) for
epiandrosterone (2), hexane/acetone/ethyl acetate (1:0.6:0.3 v:v:v) for androstanedione (3) and
hexane/acetone (1:1 v:v) for DHEA (4) and as eluents. TLC was carried out with Kieselgel
60 F254 plates (Merck, Darmstadt, Germany) using the same eluents. In order to develop the image, the
plates were sprayed with solution of methanol in concentrated sulfuric acid (1:1) and heated to 120 °C
for 3 min. GC analysis was performed using Hewlett Packard 5890A Series II GC instrument (FID,
carrier gas H2 at flow rate of 2 mL min−1) with a HP-1 column cross-linked methyl siloxane,
30 m × 0.53 mm × 1.5 μm film thickness. The following program was used in the GC analysis:
220 °C/1 min, gradient 4 °C/min to 260° and 30 °C/min to 300°/2 min; injector and detector
temperatures were 300 °C. Elemental analysis was performed on vario EL III analyzer. IR spectra
were recorded in KBr disc on a Mattson IR 300 Spectrometer. The NMR spectra were measured in
CDCl3 and recorded on a DRX 300 MHz Bruker Avance spectrometer with TMS as internal standard.
1
13
Characteristic H- and C-NMR shift values 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.

Molecules 2013, 18
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3.6. Products Isolated in the Course of Transformations
3α-Hydroxy-17a-oxa-D-homo-5α-androstan-17-one (5). C19H30O3, Found: C, 74.51; H, 9.86. requires
1
C, 74.47; H, 9.87; IR νmax(cm−1): 3462, 1718. H-NMR δ (ppm): 0.73 (3H, s, 19-CH3), 1.28 (3H, s,
13
18-CH3), 4.05 (1H, t, J = 2.7 Hz, 3β-H), C-NMR δ (ppm): 172.0 (C-17), 83.4 (C-13), 66.2 (C-3),
53.0 (C-9), 46.3 (C-14), 39.3 (C-12), 38.4 (C-5), 37.9 (C-8), 36.0 (C-10), 35.5 (C-4), 31.9 (C-1), 30.5
(C-7), 28.9 (C-2), 28.6 (C-16), 28.1 (C-6), 21.5 (C-11), 20.1 (C-18), 19.6 (C-15), 11.0 (C-19). The
spectroscopic data corresponded to those described in literature [17].
3β-Hydroxy-17a-oxa-D-homo-5α-androstan-17-one (6). C19H30O3, Found: C, 74.52; H, 9.89. requires
1
C, 74.47; H, 9.87; IR νmax(cm−1): 3443, 1720. H-NMR δ (ppm): 0.76 (3H, s, 19-CH3), 1.29 (3H, s,
13
18-CH3), 3.56 (1H, m, 3α-H), C-NMR δ (ppm): 171.6 (C-17), 83.4 (C-13), 71.2 (C-3), 52.9 (C-9),
46.1 (C-14), 44.3 (C-5), 39.2 (C-12), 37.7 (C-8), 37.6 (C-4), 36.5 (C-1), 35.4 (C-10), 31.2 (C-2), 30.6
(C-7), 28.7 (C-16), 28.3 (C-6), 22.0 (C-11), 20.1 (C-18), 19.6 (C-15), 12.1 (C-19). The spectroscopic
data corresponded to those described in literature [18].
17a-Oxa-D-homo-5α-androstan-3,17-dione (7). C19H28O3, Found: C, 74.88; H, 9.29. requires C, 74.96;
1
H, 9.27; IR νmax(cm−1): 1721, 1714. H-NMR δ (ppm): 0.97 (3H, s, 19-CH3), 1.31 (3H, s, 18-CH3);
13C-NMR δ (ppm): 211.7 (C-3), 171.3 (C-17), 83.3 (C-13), 52.2 (C-9), 46.0 (C-5), 45.9 (C-14), 44.0
(C-4), 39.0 (C-12), 38.0 (C-1), 37.9 (C-2), 37.6 (C-8), 35.4 (C-10), 30.2 (C-7), 28.6 (C-16), 28.5 (C-6),
22.2 (C-11), 20.1 (C-18), 19.5 (C-15), 11.2 (C-19). The spectroscopic data corresponded to those
described in literature [30].
Testololactone (17a-oxa-D-homo-androst-4-en-3,17-dione) (8). C19H26O3, Found: C, 75.48; H, 8.68.
1
requires C, 75.46; H, 8.67; IR νmax(cm−1): 1717, 1664. H-NMR δ (ppm): 1.17 (3H, s, 19-CH3), 1.35
13
(3H, s, 18-CH3), 5.75 (1H, s, 4-H); C-NMR δ (ppm): 199.4(C-3), 171.2 (C-17), 169.1 (C-5), 124.0
(C-4); 82.7 (C-13), 52.4 (C-9), 45.9 (C-14), 39.2 (C-12), 38.4 (C-10), 38.0 (C-8), 35.3 (C-1),
33.9 (C-2), 32.4 (C-6), 30.4 (C-7), 28.7 (C-16), 30.0 (C-11), 20.0 (C-18), 19.9 (C-15), 17.45 (C-19).
The spectroscopic data were in agreement with those of authentic sample isolated by us in previous
studies [6].
4. Conclusions
The results obtained in this study indicate that the P. lanosocoeruleum KCH 3012 strain is a
promising fungus that may be used in commercial processes, and which offers a new route to
potentially active steroidal lactones. Among all the microorganisms that have been examined so far,
this strain demonstrates the highest activity of Baeyer-Villiger monooxygenase against 5α-saturated
steroids of the androstane series. Taking into account the catalytic abilities of this
strain explored so far and its high 3β-HSD activity, one should keep in mind that it can potentially
modulate the estrogen/androgen balance in Nature.
Acknowledgments
This research was supported financially by the European Union within the European Regional
Development Found (grant No. POIG.01.03.01-00-158/09).

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Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 5–8 are available from the authors.
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