Baeyer-Villiger oxidation of DHEA, pregnenolone, and androstenedione by Penicillium lilacinum AM111

Article abstract of DOI:10.1016/j.steroids.2008.07.008

The Baeyer-Villiger monooxygenase (BVMO) produced by Penicillium lilacinum AM111, in contrast to other enzymes of this group known in the literature, is able to process 3β-hydroxy-5-ene steroid substrates. Transformation of DHEA and pregnenolone yielded, as a sole or main product, 3β-hydroxy-17a-oxa-d-homo-androst-5-en-17-one, a new metabolite of these substrates; pregnenolone was transformed also to testololactone. Testololactone was the only product of oxidation of androstenedione by P. lilacinum AM111. Investigations of the time evolution of reaction progress have indicated that the substrates stimulate activity of BVMO(s) of P. lilacinum AM111.

Full text of DOI:10.1016/j.steroids.2008.07.008

steroids 7 3 ( 2 0 0 8 ) 1441–1445
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Baeyer-Villiger oxidation of DHEA, pregnenolone, and
androstenedione by Penicillium lilacinum AM111
∗
´
Teresa Kołek, Anna Szpineter, Alina Swizdor
Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
The Baeyer-Villiger monooxygenase (BVMO) produced by Penicillium lilacinum AM111, in
contrast to other enzymes of this group known in the literature, is able to process 3␤-
hydroxy-5-ene steroid substrates. Transformation of DHEA and pregnenolone yielded, as a
sole or main product, 3␤-hydroxy-17a-oxa-d-homo-androst-5-en-17-one, a new metabolite
of these substrates; pregnenolone was transformed also to testololactone. Testololactone
was the only product of oxidation of androstenedione by P. lilacinum AM111.
Investigations of the time evolution of reaction progress have indicated that the substrates
stimulate activity of BVMO(s) of P. lilacinum AM111.
Received 2 June 2008
Received in revised form
24 July 2008
Accepted 25 July 2008
Published on line 5 August 2008
Keywords:
© 2008 Elsevier Inc. All rights reserved.
Biotransformation
DHEA
Pregnenolone
Baeyer-Villiger oxidation
Steroidal lactones
Penicillium lilacinum
1.
Introduction
Literature reports indicate that the strains of genus Penicil-
lium are able to carry out transformations of steroid substrates
Recent years have brought, along with intensive research
on the role and biological activity of DHEA (3␤-hydroxy-
androst-5-en-17-one) and pregnenolone, many studies on
their metabolism and the role and activity of the metabolites,
including those which are not sex hormones [1–11]. DHEA
derivatives with oxygen function at C-7, mainly 7␣- and 7␤-
hydroxy-DHEA, were identified in many mammalian organs
and tissues (e.g. brain, liver, skin) [1–8,12,13]. 7␣-Hydroxy-
DHEA was often found as the main metabolite product of
microbiological transformations of DHEA [14–22].
by means of reduction, hydroxylation or Baeyer-Villiger oxi-
dation [23–32]. Penicillium decumbens strain has been used
for the reduction of double bonds, particularly the conver-
sion of 4-en-3-oxosteroids to 5␣-3-ones [24,25]. In view of the
fact that P. decumbens is one of only a few microorganisms
known to perform this conversion, it occupies a central role
in the metabolism of steroids. Some studies focused on 15␣-
hydroxylation of 3-ethyl-gone-4-en-3,17-dione catalyzed by
Penicillium raistrickii [26,27]. Various strains of genus Penicillium
carry out Baeyer-Villiger oxidation [26–32].
In an effort to obtain new metabolites of DHEA and preg-
nenolone, different from any known derivatives with oxygen
function at the C-7, screening tests were carried out and the
strain Penicillium lilacinum AM111 was selected for further stud-
ies.
Baeyer-Villiger monooxygenases (BVMOs) are flavoen-
zymes that catalyze the Baeyer-Villiger reaction by insertion
of an oxygen atom next to a keto function thus converting
ketones to corresponding esters or lactones. BVMOs are pro-
duced by numerous bacteria (e.g. of the genera Actinetobacter,
∗
Corresponding author. Tel.: +48 71 3205252; fax: +48 71 3284124.
´
E-mail address: alina.swizdor@up.wroc.pl (A. Swizdor).
0039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2008.07.008

1442
steroids 7 3 ( 2 0 0 8 ) 1441–1445
Arthobacter, Nocardia, Rhodococcus, Streptomyces) and fungi (e.g.
of the genera Aspergillus, Culvularia, Fusarium, Penicillium) [33].
The strain P. lilacinum was the source of the first isolated
BVMO responsible for oxidation of androstenedione to testolo-
lactone [28]. BVMOs carry out Baeyer-Villiger degradation of
17␤-acetyl side chain of C-21 substrates and ring D oxidation
of androstenedione [33]. Substrates of the reactions described
above were usually 4-en-3-oxo steroids.
DHEA and pregnenolone were subjected to transformation
by fungus Penicillium citreo-viride, which was shown to convert
androstenedione and progesterone to testololactone [31]. In
a culture of this strain, DHEA was oxidized to testololactone,
while pregnenolone was not transformed.
2.3.
Isolation and identification of the products
The products of transformation were extracted from the
mixtures three times with 20 ml of chloroform. Transforma-
tion products were separated by column chromatography
on silica gel with ethyl acetate/methylene chloride/acetone
(3:1:1) as eluent. TLC was carried out with Merck Kiesel-
gel 60 F254 plates using the same eluent. In order to
develop the image, the plates were sprayed with solu-
tion of methanol in concentrated sulphuric acid (1:1) and
heated to 120 ◦C for 3 min. GC analysis was performed using
Hewlett Packard 5890A Series II GC instrument (FID, car-
rier 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 ␮m film
thickness. Applied temperature program: 190 ◦C/1 min, gra-
dient 4 ◦C/min to 235 ◦C/5 min and 30 ◦C/min to 300 ◦C/3 min;
injector and detector temperature was 300 ◦C. Retention times
of the identified compounds are given in Table 1. Infrared
spectra were recorded in KBr discs 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. Optical rotation measure-
ments were carried out on Autopol IV automatic polarimeter
(Rudolph).
2.
Experimental
2.1.
Materials
2.1.1. Substrates
DHEA, pregnenolone, androstenedione, and progesterone
standard were purchased from Sigma–Aldrich Chemical Co.
The microorganism P. lilacinum AM111 used in this study was
obtained from the collection of the Institute of Biology and
Botany, Medical University of Wrocław. Originally, the AM111
strain was isolated from synthetic fibres.
2.4.
AM111
Biotransformations of DHEA (1) by P. lilacinum
After 36 h incubation of 100 mg DHEA in P. lilacinum AM111
culture, 92 mg of 3␤-hydroxy-17a-oxa-d-homo-androst-5-en-
17-one (4) have been isolated (Fig. 1).
2.2.
Conditions of cultivation and transformation
Fungi were incubated on 3% glucose and 1% aminobac, in
300 ml Erlenmeyer flasks with 100 ml of medium. After cul-
tivation at 25 ◦C for three days on a rotary shaker, 20 mg of the
substrate, dissolved in 1 ml of acetone, was added. Transfor-
mation of the substrate was carried out in 5 flasks under the
same conditions. The reaction progress was monitored by TLC
and GC. Each test was carried out in at least three independent,
parallel experiments.
2.4.1. 3ˇ-Hydroxy-17a-oxa-d-homo-androst-5-en-17-one
(4)
mp 227–230 ◦C; [˛]D20 = −93.9 (c 0.1 in CHCl3); IR ꢀmax(cm−1):
3446, 1716, 1653, 1215; 1H NMR: ı (ppm): 0.96 (s, 19-H3), 1.30 (s,
18-H3), 2.59 (m, 16␣-H), 2.68 (m, 16␤-H), 3.52 (m, Wh = 26.74 Hz,
3␣-H), 5.33 (t, 6-H), 13C NMR: ı (ppm): 171.5 (C-17), 140.6 (C-5),
120.8 (C-6), 71.5 (C-3), 83.2 (C-13), 49.0 (C-9), 46.7 (C-14), 41.9
(C-4), 38.9 (C-12), 36.9 (C-1), 36.6 (C-10), 34.4 (C-2), 31.5 (C-7),
Table 1 – Composition of crude mixture obtained in transformation by P. lilacinum AM111
Substrate
Rt (min)
Compounds present in mixture (%)a
Time of substrate incubation
6 (h)
12 (h)
24 (h)
30 (h)
DHEA (1)
4.38
8.04
DHEA (1)
93
7
66
34
14
86
–
100
3␤-Hydroxy-17a-oxa-d-homo-androst-5-en-17-one (4)
Androstenedione (3)
Pregnenolone (2)
5.44
9.40
Androstenedione (3)
Testololactone (5)
98
2
85
15
30
70
3
96
6.43
7.80
4.38
5.44
8.04
9.40
Pregnenolone (2)
85
15
–
–
–
75
9
5.5
9.5
–
58
–
7
12
15
3
51
–
2
7
23
12
Progesterone (6)b
DHEA (1)b
Androstenedione (3)b
3␤-Hydroxy-17a-oxa-d-homo-androst-5-en-17-one (4)
Testololactone (5)
–
–
a
Determined by GC analysis.
Identified in GC and TLC on the basis of standards.
b

steroids 7 3 ( 2 0 0 8 ) 1441–1445
1443
Fig. 1 – Baeyer-Villiger oxidation of DHEA (1), pregnenolone (2) and androstenedione (3); Pathways of pregnenolone
transformations: via DHEA or via progesterone to lactones 4 or 5.
31.1 (C-8), 28.8 (C-16), 21.9 (C-11), 20.1 (C-18), 19.9 (C-15), 19.3
(C-19).
3.
Results and discussion
The structures of the obtained products were determined by
means of the IR, 1H NMR and 13C NMR spectra. Assumed struc-
tures were confirmed by comparison of the chemical shifts
of selected, diagnostic signals in the NMR spectra of the sub-
strate and the products with the literature data. Resonance
signals in both 1H NMR and 13C NMR spectra of 4 were consis-
tent with the formation of the ring D lactone. Downfield shift
(35.7 ppm) of the C-13 resonance signal with respect to the sub-
strate is consistent with insertion of an oxygen atom adjacent
to this position on the ring D. This is coupled with the down-
field shift in the 18-methyl resonance signal of ıH 0.43 ppm
and ıC 6.6 ppm. Absence of signal of the 17-carbonyl group at
ıC = 221 ppm and presence of a signal at ıC = 171.5 ppm are con-
sistent with D lactone formation [34]. Proton NMR signals at
ı: 3.52(m) and 5.33(t), in analogy to the spectrum of DHEA (1),
indicate the presence of 3␤-OH group and double bond at C-5
in the product. The band at 1716 cm−1 in the IR spectrum of
the product 4 confirms the ␦-lactone structure, and the band
at 3446 cm−1 indicates that the hydroxyl group is present. The
spectral data of 5 were in agreement with those reported in
the literature [30,31,35].
2.5.
Biotransformations of pregnenolone (2) by P.
lilacinum AM111
After a three-day transformation of 100 mg of pregnenolone (2)
the following ingredients were isolated: 28 mg of an unreacted
substrate, 42 mg of 3␤-hydroxy-17a-oxa-d-homo-androst-5-
en-17-one (4) and 15 mg of testololactone (5) (Fig. 1). No
increase in the transformation rate was observed for incuba-
tion period longer than three days.
2.5.1. 2.5.1.Testololactone (5)
mp 209–210 ◦C; [˛]D20 = +46.2 (c 0.16 CHCl3), literature [27] mp
207–209 ◦C; [˛]D20 = +43; IR ꢀmax (cm−1): 1716, 1666, 1614, 1214;
1H NMR ı (ppm): 1.16 (s, 19-H3), 1.35 (s, 18-H3), 2.59 (m, 16␣-
H), 2.68 (m, 16␤-H), 5.75 (s, 4-H); 13C NMR: ı (ppm): 199.2 (C-3),
171.1 (C-17), 169.2 (C-5), 124.1 (C-4), 82.7 (C-13), 52.5 (C-9), 45.7
(C-14), 39.1 (C-12), 38.4 (C-10), 38.0 (C-8), 35.5 (C-1), 33.8 (C-2),
32.4 (C-6), 30.5 (C-7), 28.6 (C-16), 21.9 (C-11), 20.1 (C-18), 19.9
(C-15), 17.44 (C-19).
2.6.
Biotransformations of androstenedione (3) by P.
The strain P. lilacinum AM111 produces the Baeyer-Villiger
monooxygenase(s), which is able to carry out ring D oxidation
and degradation of 17␤-acetyl side chain of 3␤-hydroxy-
5-ene as well as 4-en-3-oxo steroid substrates. DHEA (1)
was oxidized to 3␤-hydroxy-17a-oxa-d-homo-androst-5-en-
lilacinum AM111
After two-day incubation of 100 mg of androstenedione (3) in
P. lilacinum AM111 culture, 90 mg of testololactone (5) were
isolated (Fig. 1).

1444
steroids 7 3 ( 2 0 0 8 ) 1441–1445
17-one (4) as the sole product, while androstenedione (3)
was converted, with high yield, into testololactone (5). Trans-
formation of pregnenolone (2), apart from 3␤-hydroxy-17a-
oxa-d-homo-androst-5-en-17-one (4), led to small amounts of
testololactone (Fig. 1). Analysis of the reaction mixture compo-
sition as function of time indicates that the steroid substrate
induces activity of the enzymes responsible for the ring D oxi-
dation as well as for the degradation of 17␤-acetyl side chain
(Table 1).
After 6 h transformation of DHEA the content of hydroxy-
lactone 4 in the reaction mixture was 7%, and after additional
6 h reached 34%. Androstenedione was converted respectively
into 2% and 15% of testololactone.
(5), is competitive with respect to the oxidation of 3␤-hydroxyl
group, the first stage of the pregnenolone (2) transformation
to progesterone (6).
The compound obtained by us, 3␤-hydroxy-17a-oxa-d-
homo-androst-5-en-17-one (4), is a new metabolite of DHEA
(1) and pregnenolone (2). The literature examples of micro-
biological oxidation of Baeyer-Villiger type of both substrates
led to testololactone (5) [29,31]. One of the strains of the genus
Penicillium – P. citreo-viride oxidized DHEA (1) to testololactone
(5), while it did not exhibit any enzymatic activity towards
pregnenolone (2) [31]. The strain P. lilacinum AM111 used by us
synthesizes BVMO(s) which, differently from other enzymes of
this group known in the literature, accepts both 3␤-hydroxy-
5-ene substrates: DHEA (1) and pregnenolone (2).
Among the products of the transformation of pregnenolone
(2), apart from 4 and 5, also progesterone (6), DHEA (1),
and androstenedione (3) were isolated. The time evolution of
the transformation of this substrate indicates that the com-
pound undergoes oxidation reactions of the Baeyer-Villiger
type along two pathways: through DHEA (1) to hydroxylactone
4 or, after conversion to progesterone (6), through androstene-
dione (3) to testololactone (5) (Table 1, Fig. 1). Oxidation of 2
and 6 likely proceeds according to the following sequence of
reactions: oxidative esterification of 17␤-acetyl chain to 17␤-
acetoxy-3␤-hydroxy-androst-5-ene or testosterone acetate,
ester bond hydrolysis leading to 17␤-alcohols, subsequently
oxidized to 17-oxo products (DHEA or androstenedione),
which undergo D-ring Baeyer-Villiger oxidation yielding D-
ring lactones 4 and 5. Among the products resulting from the
transformation of 2, only progesterone (6), androstenedione
(3), and DHEA (1) were identified in the reaction mixture. In
accord with the literature examples of progesterone to testolo-
lactone oxidation, testosterone acetate was not identified due
to its fast hydrolysis catalyzed by the active esterase present
in the culture [36]; testosterone was also usually not identified
[31].
The mixture of products after 6 h incubation of preg-
nenolone (2) contained, apart from the substrate, only
progesterone (6) (ca. 15%) (Table 1). Only after 12 h of the
elapsed reaction time, DHEA (1) and androstenedione (3) were
identified – products of the elimination of the 17␤-acetyl side
chain and subsequent oxidation of the 17␤-OH group. Higher
content of androstenedione (3) in the mixture, as compared
to DHEA (1), indicates that progesterone (6) is oxidized faster
than pregnenolone (2). 3␤-Hydroxy-17a-oxa-d-homo-androst-
5-en-17-one (4) is identified in the reaction mixture earlier
and in larger amount than testololactone (5), which suggests
that the BVMO responsible for the ring D oxidation prefers the
3␤-hydroxy-5-ene substrate. This assumption is supported by
the observed faster oxidation of DHEA (1) in comparison with
androstenedione (3) (Table 1).
Steroidal lactones possess useful biological properties,
such as anticancer, antiandrogenic, and antihypercholes-
terolemic activity [37–40]. The results obtained in this study
indicate that the strain of P. lilacinum AM111 is a promis-
ing fungus that may be used in commercial processes, and
which offers a potential new route to novel biologically active
steroids.
r e f e r e n c e s
[1] Mao Q, Lu S-F, Simon NG. Dehydroepiandrosterone and its
metabolites: differential effects on androgen receptor
tracking and transcriptional activity. J Steroid Biochem Mol
Biol 2006;99:50–8.
[2] Muller C, Pompon D, Urban P, Morfin R. Inter-conversion of
7␣- and 7␤-hydroxy- dehydroepiandrosterone by the human
11␤-hydroxysteroid dehydrogenase type 1. J Steroid Biochem
Mol Biol 2006;99:215–22.
[3] Marwah A, Gomez FE, Marwah P, Ntambi JM, Fox BG, Lardy
H. Redox reaction of dehydroepiandrosterone and its
metabolites in differentiating 3T3-L1 adipocytes: a liquid
chromatographic-mass spectrometric study. Arch Biochem
Biophys 2006;456:1–7.
[4] Tang W, Eggertsen, Chiang JYL, Norlin M. Estrogen mediated
regulation of CYP7B1: a possible role for controling DHEA
levels in human tissues. J Steroid Biochem Mol Biol
2006;100:42–51.
[5] Chalbot S, Morfin R. Neurosteroids: metabolism in human
intestine microsomes. Steroids 2005;70:319–26.
[6] Kim S-B, Chalbot S, Pompon D, Jo D-H, Morfin R. The human
cytochrome P4507B1: catalytic activity studies. J Steroid
Biochem Mol Biol 2004;92:383–9.
[7] Zenk JL, Frestedt JL, Kuskowski A. HUM5007, a novel
combination of thermogenic compounds, and
3-acetyl-7-oxo-dehydroepiandrosterone: each increases the
resting metabolic rate of overweight adults. J Nutr Biochem
2007;18:629–34.
[8] Jellinck PH, Croft G, McEvans BS, Gottfried-Blackmore A,
Jones G, Byford V, et al. Metabolism of
The composition of the mixture of pregnenolone (2)
transformation products indicates that from the moment of
identification of Baeyer-Villiger oxidation products in the mix-
ture, the amount of 4-ene-3-oxo products does not increase;
their total content, in the investigated incubation period, is
close to 15%, i.e. the amount of progesterone in the initial stage
of the transformation (Table 1). It is probable that the oxidation
of the 17␤-OH group, one of the stages in the cycle of reactions
leading from pregnenolone to 3␤-hydroxy-17a-oxa-d-homo-
androst-5-en-17-one (4) and progesterone to testololactone
dehydroepiandrosterone by rodent brain cell lines:
relationship between 7-hydroxylation and aromatization. J
Steroid Biochem Mol Biol 2005;93:81–6.
[9] Jellinck PH, Kaufmann M, Gottfried-Blackmore A, Croft G,
Byford V, McEvans BS, et al. Dehydroepiandrosterone (DHEA)
metabolism in brain: identification by liquid
chromatography/mass spectrometry of the delta-4-isomer
of DHEA and related steroid formed from androstenedione
by mouse BV2 microglia. J Steroid Biochem Mol Biol
2006;98:41–7.

steroids 7 3 ( 2 0 0 8 ) 1441–1445
1445
[10] Jellinck PH, Kaufmann M, Gottfried-Blackmore A, McEvans
BS, Jones G, Bulloch K. Selective conversion by microglia of
dehydroepiandrosterone to 5-androstenediol—a steroid
with inherent estrogenic properties. J Steroid Biochem Mol
Biol 2007;107:156–62.
[24] Holland HL, Nguyen DH, Pearson NM. Bioconversion of
corticosteroids by Penicillium decumbens ATCC 10436. Steroids
1995;60:646–9.
[25] Irrgang S, Schlosser D, Schmauder H-P. The steroid
15-hydroxylase of Penicillium raistrickii I 477 is inducible.
Biotechnol Lett 1992;14:33–8.
[11] Lardy H. A postulated role for
16␣-hydroxydehydroepiandrosterone in the prevention of
respiratory distress syndrome. Med Hypotheses
2006;66:107–9.
[26] Irrgang S, Schlosser D, Fritsche W. Involvement of
cytochrome P-450 in the hydroxylation of
13-ethyl-gone-4-ene-3,17-dione by Penicillium raistrickii. J
Steroid Biochem Mol Biol 1997;60:339–46.
[27] Fried J, Thoma RW, Klingsberg A. Oxidation of steroids by
microorganisms. III. Side chain degradation, ring D-cleavage
and dehydrogenation in ring A. J Am Chem Soc
1953;75:5764–5.
[12] Morfin M, Courchay G. Pregnenolone and
dehydroepiandrosterone as precursors of native
7-hydroxylated metabolites which increase the immune
response in mice. J Steroid Biochem Mol Biol 1994;50:91–
100.
[13] Lardy H, Marwah A, Marwah P. Transformation of DHEA and
its metabolites by rat liver. Lipids 2002;37:1187–91.
[14] Dodson RM, Nicholson RT, Muir RD. Microbial
transformations IV. The oxidation of
dehydroepiandrosterone at C-7. J Am Chem Soc
1959;81:6295–7.
[15] Defaye G, Luche MJ, Chambaz EM. Microbiological 7- and
15-hydroxylation of 19 steroids. J Steroid Biochem
1978;9:331–6.
[16] Crabb TA, Dawson PJ, Williams RO. Microbial
transformations. Part 3. The oxidation of androstene
derivatives with Cunninghamella elegans. J Chem Soc Perkin
Trans I 1980:2535–41.
[28] Prairie RL, Talalay P. Enzymatic formation of testololactone.
Biochemistry 1963;2:203–8.
[29] Viola F, Caputo O, Balliano G, Delprino L, Cattel L. Side chain
degradation and microbial reduction of different steroids by
Aspergillus aureogulgens. J Steroid Biochem 1983;19:1451–8.
[30] Bartman´ ska A, Dmochowska-Gładysz J, Huszcza E. Steroids’
transformations in Penicillium notatum culture. Steroids
2005;70:193–8.
[31] Liu H-M, Li H, Shan L, Wu J. Synthesis of steroidal lactones
by Penicillim citreo-viride. Steroids 2006;71:931–4.
[32] Sebek OK, Reineke LM, Peterson DH. Intermediates in the
metabolism of steroids by Penicillium lilacinum. J Bacteriol
1962;83:1327–31.
[17] Madyastha KM, Joseph T. Transformation of
dehydroepiandrosterone and pregnenolone by Mucor
piriformis. Appl Microbiol Biotechnol 1995;44:339–43.
[18] Cottilon A-C, Morfin R. Transformation of
3-hydroxy-steroids by Fusarium moniliforme 7␣-hydroxylase. J
Steroid Biochem Mol Biol 1999;68:229–37.
[19] Wilson MR, Gallimore WA, Reese PB. Steroid transformation
with Fusarium oxysporum var. cubense and Colletotrichum
musae. Steroids 1999;64:834–43.
[33] Holland HL. Organic synthesis with oxidative enzymes.
Weinheim: Verlag Chemie; 1992. p. 241–54.
[34] Hanson JR, Hunter AC. The hydroxylation of steroidal D
lactones by Cephalosporium aphidicola. Phytochemistry
1998;49:2349–53.
[35] Hunter AC, Carragher NE. Flexibility of the endogenous
progesterone lactonisation pathway in Aspergillus tamarii
KITA: transformation of a series of cortical steroid
analogues. J Steroid Biochem Mol Biol 2003;87:301–8.
[36] Fonken GS, Murray HC, Reineke LM. Pathway of
progesterone oxidation by Cladosporium resinae. J Am Chem
Soc 1960;82:5507–8.
[20] Kołek T. Biotransformation XLVII: transformations of 5-ene
steroids in Fusarium culmorum culture. J Steroid Biochem Mol
Biol 1999;71:83–90.
[21] Romano A, Romano D, Ragg E, Costantino F, Lenna R,
Gandolfi R, et al. Steroid hydroxylation with Botryodiplodia
malorum and Colletotrichum lini. Steroids 2006;71:429–
34.
[22] Hunter AC, Mills PW, Dedi C, Dodd HT. Predominant allylic
hydroxylation at carbons 6 and 7 of 4 and 5-ene
functionalized steroids by thermophilic fungus Rhizomucor
tauricus IMI23312. J Steroid Biochem Mol Biol
2008;108:155–63.
[37] Brodie AMH, Njar VCO. Aromatase inhibitors in advanced
breast cancer: mechanism of action and clinical
implications. J Steroid Biochem Mol Biol 1998;66:1–10.
[38] Braustein GD. Aromatase and gynecomastia. Endocr-Relat
Cancer 1999;6:315–24.
[39] Baran JC. The synthesis, stereochemistry, and biology of
16-hetero and 17-oxa-d-homo steroids. J Med Chem
1967;10:1039–47.
ˇ
[40] Djurendic´ EA, Sakacˇ MN, Zavisˇ MP, Gakovic´ AR, Canadi JJ,
[23] Holland HL, Dore S, Xu W, Brown FM. Formation of
5-steroids by biotransformation involving the 5 reductase
activity of Penicillium decumbens. Steroids 1994;59:642–7.
Andric´ SA, et al. Synthesis and biological evaluation of some
new A,B-ring modified steroidal D-lactones. Steroids
2008;73:681–8.