Microbial transformation of nandrolone decanoate by acremonium strictum

Article abstract of DOI:10.1002/ardp.200500235

Estr-4-en-3,17-dione II, 17β-hydroxyestr-4-en-3-one III, 15α-hydroxyestr-4-en-3,17-dione IV, and 15α,17β-dihydroxyestr- 4-en-3-one V were produced by microbial transformation of nandrolone decanoate I in the culture of Acremonium strictum PTCC 5282. Bioconversion characteristics observed were ester hydrolysis, oxidation, and hydroxylation. Each microbial product was purified chromatographically and characterized on the basis of spectral data obtained from ¹H-NMR, ¹³C-NMR, FT-IR, MS, and physical constants such as melting point and optical rotation.

Full text of DOI:10.1002/ardp.200500235

Arch. Pharm. Chem. Life Sci. 2006, 339, 473–476
M. Tabatabaei Yazdi et al.
473
Short Communication
Microbial Transformation of Nandrolone Decanoate by
Acremonium Strictum
Mojtaba Tabatabaei Yazdi1, Seyedeh Maryam Zanjanian1,*, Mohammad Ali Faramarzi1,
Mohsen Amini2, Amir Amani1, Khosrou Abdi2
1 Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences,
Tehran, Iran
2 Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran,
Iran
Estr-4-en-3,17-dione II, 17b-hydroxyestr-4-en-3-one III, 15a-hydroxyestr-4-en-3,17-dione IV, and
15a,17b-dihydroxyestr-4-en-3-one V were produced by microbial transformation of nandrolone
decanoate I in the culture of Acremonium strictum PTCC 5282. Bioconversion characteristics
observed were ester hydrolysis, oxidation, and hydroxylation. Each microbial product was puri-
fied chromatographically and characterized on the basis of spectral data obtained from 1H-NMR,
13C-NMR, FT-IR, MS, and physical constants such as melting point and optical rotation.
Keywords: Acremonium strictum / Microbial transformation / Nandrolone decanoate / Steroid /
Received: November 8, 2005; Accepted: December 9, 2005
DOI 10.1002/ardp.200500235
strictum was reported by Ambrus and coworkers [7].
Yoshihama and Nakakoshi reported steroid hydroxyla-
tions at various positions included 6b [6, 8, 9], 7a [6], 7b
[10], 11a [6, 8, 9], 14a [6, 11], 15b [9, 10], 17a [6, 9], and 17b
[10] with A. strictum. Our previous works on bioconver-
sion of two pregnane-based steroids (hydrocortisone and
progesterone) describe microbial modification of C-15
and side chain transformations by a strain of A. strictum
isolated from soil [12, 13]. In the present study, we exam-
ined this strain for biotransformation of nandrolone
decanoate to investigate the bioconversional effect of A.
strictum PTCC 5282 on an estrane-like steroid.
Introduction
Since the first comprehensive study by Mammoli and Ver-
celone, who described the involvement of microorgan-
isms in steroid bioconversions, a series of reports have
been published focusing on various aspects of microbial
steroid transformations, some of them describing the
production of pharmaceutical steroids. For example,
Murray and Peterson patented 11a-hydroxylation of pro-
gesterone by Rhizopus arrhizus. Today, it is assumed that
almost all steroids can be microbially hydroxylated [1].
Fungi are mainly involved in steroid bioconversion pro-
cesses [2–6].
Acremonium strictum, a cephalosporium-like fungus, is
able to hydroxylate different sites of the steroid back-
bone. Microbial addition of 1a-hydroxyl group using A.
Results and discussion
Biotransformation of 1 g nandrolone decanoate I by Acre-
monium strictum PTCC 5282 resulted in four steroid com-
pounds II-V. During a cultivation period of six days (150
rpm at 258C), the strain converted the substrate I into
204 mg of II, 166 mg of III, 49 mg of IV, and 56 mg of V,
respectively. Microbial products were isolated and puri-
fied using chloroform extraction followed by preparative
TLC with chloroform/acetone (8:2) and crystallization in
methanol. Chemical structures of the products (Fig. 1)
Correspondence: Mojtaba Tabatabaei Yazdi, Department of Pharma-
ceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medi-
cal Sciences, P. O. Box 14155-6451, Tehran 14174, Iran.
E-mail: mtabataba@sina.tums.ac.ir
Fax: + 98 21 664-61178
* This paper is dedicated to the memory of Dr. Seyedeh Maryam Zanja-
nian, deceased June 21, 2003.
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

474
M. Tabatabaei Yazdi et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 473–476
1
nyl group in 13C-NMR, absence of H-17 signal in H-NMR
around d 3.5–4.5, and the occurrence of the molecular
ion at m/z 272 indicated oxidation of C-17 in compound
III resulting in compound II. These two metabolites were
identified as 17b-hydroxyestr-4-en-3-one and estr-4-en-
3,17-dione, respectively, and have been previously
reported by Hanson and coworkers [14].
Deshielding effect of 15a-hydroxyl group in 5a-andros-
tanes on C-14, C-15, and C-16 were reported as d +7.2,
+50.2, and +12.4, respectively [15]. We have also shown
these effects as d +6.6, +49, and +12.9 in 5a-pregnanes
[13]. Our observation was very similar to those found for
IV (in comparison to II) suggesting the hydroxylation at
C-15. As it is shown in Table 1, they occur as d +6.6, +48.4,
and +10.9, respectively.
The 13C-NMR spectrum of compound V is similar to
that of 15b,17b-dihydroxyestr-4-en-3-one [10] indicating
probable a– and b–effects of the 15-hydroxyl group [15].
A c-deshielding effect (d +0.27) for 15a-OH in comparison
to d –0.06 for 15b-OH has been already reported in 15-
hydroxyandrostanes [16] suggesting the position of 15-
OH in compound V as a-orientation. Confirmation of this
observation resulted from the measurement of optical
rotation [17].
Figure 1. The structures of the substrate and biotransformed
metabolites. Nandrolone decanoate I, estr-4-en-3,17-dione II,
17b-hydroxyestr-4-en-3-one III, 15a-hydroxyestr-4-en-3,17-
dione IV, and 15a,17b-dihydroxyestr-4-en-3-one V.
Table 1. 13C-NMR signals of the substrate and the metabolites
(d in ppm downfield from TMS, in CDCl3).
These results proved the ability of A. strictum to med-
iate 15a-hydroxylation. However, no transformation of
the decanoyl side chain was detected except an ester
hydrolysis. However, 17b-hydroxyl oxidation by A. stric-
tum has not been observed before.
15a-Hydroxylation of estrane-based steroids has been
reported when using Aspergillus carneus [17], Penicillium
sp. [17], some strains of Fusarium sp. [3, 17], and Glomerella
cingulata [17]. It was also observed that several species of
Fusarium sp., Giberella sp., and Glomerella sp. have the abil-
ity to mediate the same transformation reaction of vari-
ous types of steroids [3, 5, 6, 17]. In addition to our pre-
vious work [12, 13], we describe A. strictum PTCC 5282 as a
suitable biocatalyst for transformation of some selected
sites in steroids D-ring.
Carbon atom
Compounds
III
I
II
IV
V
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
27.4
36.4
199.7
124.5
166.2
35.3
31.7
40.1
49.4
42.4
26.5
36.5
42.6
49.3
23.2
30.5
82.1
12.0
26.5
36.4
199.7
124.8
165.8
35.7
31.2
39.9
49.5
42.4
25.6
29.8
47.6
50.1
21.6
35.2
220.4
13.0
26.1
36.3
199.9
124.5
166.7
35.4
30.3
40.7
49.7
42.5
26.5
36.5
42.9
49.5
23.1
30.6
81.6
11.1
26.5
36.4
199.9
124.7
165.7
35.4
31.2
39.8
49.4
42.4
25.7
30.9
50.7
56.7
70.0
46.1
216.0
15.4
26.1
36.4
199.9
124.5
166.3
35.4
30.8
39.9
49.6
42.5
26.6
36.6
44.5
57.8
72.2
31.2
78.8
12.6
The authors acknowledge Dr. Gholamreza Zarrini regarding
fungal studies. They also thank Mr. Afshin Dalvandi for his
kind collaboration during spectroscopic work. The helpful
were determined by H-NMR, 13C-NMR, FT-IR, and MS. advice of Dr. Helmut Brandl (University of Zꢀrich, Zꢀrich, Swit-
Additionally, physical characteristics such as melting zerland) is also acknowledged.
point and optical rotation were determined. 13C-NMR
1
data of the substrate as well as the products are listed in
Table 1.
Experimental
Loss of side chain in compound III was approved
according to the molecular ion at m/z 274 and change in
the H-17 signal of the 1H-NMR spectrum (from d 4.5 in the
Chemicals
Nandrolone decanoate was kindly donated by Iran Hormone
substrate to d 3.7). Chemical shift of d 220.4 for 17-carbo-
pharmaceutical Co. (Tehran, Iran), which had been purchased
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Arch. Pharm. Chem. Life Sci. 2006, 339, 473–476
Biotransformation of Nandrolone by Acremonium Strictum
475
1
from Gedeonrichter (Budapest, Hungary). Sabouraud dextrose
agar and broth were purchased from Merck (Darmstadt, Ger-
many). All reagents and solvents were of analytical grade.
(33), 85 (65), 83 (100); H-NMR (CDCl3) d 0.92 (3H, s, H-18), 5.86
(1H, s, H-4); Rf in chloroform/acetone (8:2): 0.77.
17b-Hydroxyestr-4-en-3-one III
Instruments
Yield 16.6%; m.p. 112–1158C, [a]D + 558 (MeOH), lit [19] m. p. 111–
112 and 123–1248C (dimorphic crystals), [a]D + 55.28; IR mmax
3451, 2934, 1675, 1630 cm–1; MS (EI) m/z (%) 274 (70) (M+,
C18H26O2), 256 (18), 215 (25), 197 (22), 173 (24), 147 (36), 110 (100),
79 (36); 1H-NMR (CDCl3) d 0.82 (3H, s, H-18), 3.67 (1H, t, J = 8.8 Hz,
H-17), 5.83 (1H, s, H-4); Rf in chloroform/acetone (8:2): 0.54.
Melting point (m.p.) was determined on a Reichert-Jung hot
stage melting point apparatus (Leica AG, Reichert Division,
Vienna, Austria). Optical rotation was measured in 1-dm cells on
a Perkin-Elmer 142 automatic spectropolarimeter (Perkin Elmer,
Beaconsfield, UK). 1H- and13C-nuclear magnetic resonance (NMR)
spectra were recorded using FT-NMR Varian Unity plus spectro-
meter at 400 and 100 MHz (Varian Inc., Palo Alto, CA, USA),
respectively, in CDCl3 with tetramethylsilane (TMS) as internal
standard. Chemical shifts (d) were given in parts per million
(ppm) relative to TMS. Coupling constant (J) were given in Hertz
(Hz). Infrared (IR) spectra were recorded on a Magna-IR 550 Nico-
let FT-IR spectrometer (Nicolet, Madison, WI, USA). Mass spectra
(MS) were obtained with a Finnigan MAT TSQ-70 instrument (Fin-
nigan MAT, San Jose, CA, USA) by electron impact (EI) at 70 eV.
15a-Hydroxyestr-4-en-3,17-dione IV
Yield 4.9%; m.p. 195–1998C, [a]D + 1558 (MeOH), lit [17] m.p. 200–
2018C, [a]D + 1628; IR mmax 3450, 2920, 1652, 1625 cm–1; MS (EI) m/
z (%) 288 (100) (M+, C18H24O3), 272 (13), 260 (22), 215 (18), 160 (12),
147 (17), 110 (19), 90 (21); 1H-NMR (CDCl3) d 0.95 (3H, s, H-18), 3.02
(1H, dd, J = 8.2 Hz, J = 8 Hz, H-16), 4.43 (1H, dd, J = 8.2 Hz, J = 7.8
Hz, H-15), 5.83 (1H, s, H-4); Rf in chloroform/acetone (8:2): 0.39.
15a,17b -Dihydroxyestr-4-en-3-one V
Fungal strain
Acremonium strictum PTCC 5282 [12] was maintained at 48C on
Sabouraud 4% dextrose agar slant and freshly subcultured
before use in transformation experiments. The organism was
transferred to fresh medium every two months.
Yield 5.6%; m.p. 138–1418C, [a]D + 91.58, lit [17] m. p. 136–1398C,
[a]D + 958; IR mmax 3404, 2928, 1663, 1620 cm–1; MS (EI) m/z (%) 290
(30) (M+, C18H26O3), 272 (51), 218 (12), 163 (22), 149 (55), 110 (100),
97 (52), 81 (41); 1H-NMR (CDCl3) d 0.83 (3H, s, H-18), 3.92 (1H, dd, J
= 8 Hz, H-17), 4.12 (1H, m, H-15), 5.82 (1H, s, H-4); Rf in chloro-
form/acetone (8:2): 0.08.
Incubation conditions
Ten 500-mL Erlenmeyer flasks, each containing 100 mL of liquid
medium of Sabouraud 2% dextrose broth, were inoculated with
freshly obtained spores from agar slant cultures and incubated
for 12 h at 258C in a rotary shaker (150 rpm). Spores were col-
lected with sterile normal saline solution containing 0.1%
Tween 80. Nandrolone decanoate (1 g) was dissolved in 20 mL of
absolute ethanol and 2 mL of the ethanol solution was added to
each 500 mL Erlenmeyer flask; incubation continued for 6 days
under the same conditions. Sterile controls were processed simi-
larly.
References
[1] L. L. Smith in Biotransformations, (Eds.: H. J. Rehm, G.
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were crystallized from methanol.
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