Synthesis of steroidal lactone by penicillium citreo-viride

Article abstract of DOI:10.1016/j.steroids.2006.06.005

The biotransformations of a series of steroids by the fungus penicillium citreo-viride A.C.C.C. 0402 have been investigated, and the conversion to the same product testolactone (1) was observed from progesterone (2), dehydroepiandrosterone (3), 4-androstene-3, 17-dione (4), 5-androstene-3, 17-diol (5) with the exception of pregnenolone (6) and 3β-hydroxy-5, 16-pregnadien-20-one (7). The possible metabolic pathways of the biotransformations were also discussed in the paper and the fungus penicillium citreo-viride A.C.C.C. 0402 was isolated during screening stains from samples collected from Zhengzhou, Henan province of China.

Full text of DOI:10.1016/j.steroids.2006.06.005

steroids 7 1 ( 2 0 0 6 ) 931–934
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Synthesis of steroidal lactone by penicillium citreo-viride
Hong-Min Liua,∗, Heping Lia, Lihong Shana, Jian Wub
a
New Drug Research & Development Center, Zhengzhou University, Zhengzhou 450052, China
Bioengineering Department Zhengzhou University, Zhengzhou 450052, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
The biotransformations of a series of steroids by the fungus penicillium citreo-viride A.C.C.C.
0402 have been investigated, and the conversion to the same product testolactone (1) was
observed from progesterone (2), dehydroepiandrosterone (3), 4-androstene-3, 17-dione (4),
5-androstene-3, 17-diol (5) with the exception of pregnenolone (6) and 3␤-hydroxy-5, 16-
pregnadien-20-one (7). The possible metabolic pathways of the biotransformations were
also discussed in the paper and the fungus penicillium citreo-viride A.C.C.C. 0402 was isolated
during screening stains from samples collected from Zhengzhou, Henan province of China.
© 2006 Elsevier Inc. All rights reserved.
Received 29 April 2006
Received in revised form
20 June 2006
Accepted 21 June 2006
Published on line 12 September 2006
Keywords:
Biotransformation
Lactone
Penicillium citreo-viride
Incubation
Testolactone
1.
Introduction
Because of the important bioactivities of the testolac-
tones, scientists were prompted to synthesize these steroidal
Steroidal lactones possess useful biological activities such
as anticancer, antibacterial, anticareinogen and antiandro-
genic activites [1–4], and 16-oxa analogues of estrane series
express antihypercholestrolemic activities [5]. Plants of genus
Withania are known to exhibit a variety of pharmacological
activites mainly due to the presence of withanolides, which
are steroidal lactones. Testolactones are moderately specific
first generation inhibitors of human aromatase activity and
thereby may contribute to the prevention of hormone depen-
dent tumors such as breast cancer [6], prostatic hyperplasia
and prostate cancer [7]. Testolactones are used as therapeu-
tic agents in disorders caused by imbalance between estro-
gen and androgen action, e.g. gynecomastia [8] or precocious
puberty [9]. Aromatase inhibitors are also essential tools for
studying the role of estrogens in adults, or during develop-
ment [10].
lactones. Although chemical ways of synthesis of steroidal
lactones such as Baeyer-Villiger oxidation are possible, the
biotransformation is thought to be the more environmental
method, especially nowadays, green chemistry is advocated
so frequently. In fact a variety of fungi [11–17] have been iden-
tified which can stereospecifically degrade the 17␤-acetyl side
chain of progesterone 2 in high yield to give the ring D testolo-
lactone 1 and the metabolic pathway was also discussed [18].
Recently testosterone (8) was transformated into 1 by Peni-
cillium notatum has been reported [19]. With the aim of syn-
thesizing steroidal lactones and doing some further work, we
have endeavored to screen microorganisms capable of pro-
ducing steroidal lactones, and a common strain of penicillium
citreo-virid A.C.C.C. 0402 which could transform 2–5 into 1
was isolated during screening stains from samples collected
from Zhengzhou, Henan province, while 6 and 7 have not any
∗
Corresponding author. Tel.: +86 371 67767200; fax: +86 371 67767200.
E-mail addresses: liuhm@public2.zz.ha.cn, liuhm@zzu.edu.cn (H.-M. Liu).
0039-128X/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2006.06.005

932
steroids 7 1 ( 2 0 0 6 ) 931–934
changes. Penicillium genus have been reported to be used in the
biotransformations using steroids as the substrates, but the
attention was focused mainly on 15␣-hydroxylation catalyzed
by Penicillium raistrickii [20,21] and 5␣-reductase of Penicillium
decumbens [22,23] and Penicillium crustosum [24]. Here we first
reported on the microbial transformation using the strain of
penicillium citreo-viride.
Table 1 – The biotransformations of the substrates of 2–7
Substrates
Yield of 1 (%)
Yield of 4 (%)
2
3
4
5
6
7
60.9
65.4
73.6
32.5
–
8.4
<0.5
–
45.2
–
–
–
2.
Experimental
(–) Means that no product was detected on TLC.
2.1.
Instrumental methods
isolated from the mycelium. The mycelial broth was then
extracted for five times with ethyl acetate. The organic extract
altogether was concentrated into about 300 ml, which was
then washed with saturated aqueous NaHCO3, brine and
water, respectively, for three times, then dried with sodium
sulfate and the solvent was evaporated under reduced pres-
sure to give the mixed products. The mixture was sepa-
rated on silica gel column chromatography by using ether
acetate/chloroform (1:1) as eluant, and the solvent was col-
lected in aliquots (10 ml) and analysed by thin layer chro-
matography to identify the separated metabolite fractions.
The solvent systems used for running the TLC plates was also
ether acetate/chloroform (1:1), and the TLC was visualized by
spraying the plates with 50% sulfuric acid solution and heating
in an oven at 100 for 3 min until the colors developed.
Sterilization was carried out in an HVE-50 Hirayama auto-
clave. Aseptic operation was carried out in ClassIIA/B3 Bio-
logical safety cabinet from Forma Scientific. Incubation was
carried out on an HZQ-Q orbital shaker. Melting points (mp)
were determined on a XT5 melting point apparatus, which
was uncorrected. Infrared (IR) spectra was recorded using
KBr discs on a Bruker Vectror-22 spectrometer. Mass spec-
tra (MS) was obtained on an Esquire3000 mass spectrometer
by electrospray ionization (ESI). The 1H and 13C nuclear mag-
netic resonance (NMR) spectra were obtained using a Bruker
Avance DPX-400 spectrometer at 400 and 100 MHz, respec-
tively, with tetramethylsilane (TMS) as internal standard in
DMSO-d6. Chemical shifts (ı) were given in parts per mil-
lion (ppm) relative to TMS. Coupling constants (J) were given
in hertz (Hz). Chromatography was performed with ether
acetate/chloroform (1:1) and visualized by spraying the plates
with 50% sulfuric acid solution and heating in an oven at 100 ◦C
until the colors developed. Thin layer chromatography (TLC)
was performed on 0.25 mm thick layer of silica gel G (Qingdao
Marine Chemical Factory, China).
2.5.
Incubation of 2–7
The biotransformations of the substrates of 2–7 were, respec-
tively, shown in Table 1 according to Sections 2.3 and 2.4,
and the results were shown in Fig. 1. All of the substrates
of 2, 3, 4 and 5 could be converted into testolactone 1, mp
2.2.
Microorganism
201.2–202.1 ◦C; IR(KBr): 3420.1, 2954.6, 1719.8, 1667.2 cm−1 1H
;
NMR (CDCl3, 400 MHz) ı: 5.76 (s, H4), 1.18 (s, H18), 1.36 (s, H19)
ppm; 13C NMR (CDCl3, 400 MHz) ı: 38.9 (C1), 32.3(C2), 199.1(C3),
171.1(C4), 124.1(C5), 35.5 (C6), 33.8(C7), 37.9 (C8), 52.5 (C9), 38.4
(C10), 19.8 (C11), 30.4 (C12), 82.7 (C13), 45.7 (C14), 21.8 (C15),
28.5 (C16), 169.3 (C17); 17.4 (C18), 20.0 (C19); HRMS(ESI) m/z:
The strain of penicillium citreo-viride A.C.C.C. 0402 was isolated
from the samples collected from the suburb of Zhengzhou,
Henan province, PRC, and identified by Bioengineering Depart-
ment Zhengzhou University. Stock cultures were maintained
at 4 ◦C on agar slopes composed of peptone (1.2%), dextrose
(3.0%), yeast extract (0.1%), KH2PO4 (0.13%), agar (2.0%) (pH 4.5).
C19H
26O3[M + H+], calcd. 303.1960, found 303.1957, [M + Na+],
calcd. 325.1780, found 325.1771, [M + K+] calcd. 341.1579, found
343.1827.
2.3.
Conditions of cultivation and transformation
Compound 4, mp 172.6–173.5 ◦C; IR(KBr): 2952.1, 2919.2,
1735.3, 1664.3, 1616.3, 1378.2 cm-1; 1H NMR (CDCl3, 400 MHz)
ı: 5.75 (s, H4), 0.92 (s, H18), 1.22 (s, H19) ppm; 13C NMR (CDCl3,
400 MHz) ı: 35.5 (C1), 33.8 (C2), 199.1 (C3), 124.0 (C4), 170.2 (C5),
32.4 (C6), 31.2 (C7), 35.0 (C8), 53.7 (C9), 38.5 (C10), 20.2 (C11), 35.6
(C12), 47.4 (C13), 50.7 (C14), 21.6 (C15), 31.7 (C16), 220.2 (C17);
13.6 (C18), 17.6 (C19); HRMS(ESI) m/z: C19H26O2[M + H + ], calcd.
287.2006, found 287.2018.
Spores freshly obtained from peptone dextrose agar slopes
were transferred aseptically into 10, 500 ml Erlenmeyer flasks
containing 100 ml of sterile peptone dextrose broth in a bio-
logical safety cabined and were incubated for 48 h at 27.5 ◦C.
The cultures were shaken at 210 rpm on an orbital shaker.
Then 1.0 g substrate dissolved in 20 ml acetone was evenly dis-
tributed among the flasks under sterile conditions and incu-
bated for a further 5 days at the same conditions after which
the metabolites were extracted from the broth.
3.
Results and discussion
2.4.
Extraction and separation of metabolites
A series of different structural steroids were found to yield the
same product 1 in the biotransformation by penicillium citreo-
virid which has not been reported, so we have investigated the
scope of the use of penicillium citreo-virid for steroid transfor-
mation and discussed the metabolic pathways.
The mycelium was separated from the broth by filtration
under vacuum, and rinsed in ultrasonic with ethyl acetate
(100 ml × 5) to ensure that all of the available products were

steroids 7 1 ( 2 0 0 6 ) 931–934
933
Fig. 1 – The biotransformations of 2–5 by penicillium citreo-virid.
Fig. 2 – Metabolic pathways of the biotransformations of the substrates by penicillium citreo-virid.

934
steroids 7 1 ( 2 0 0 6 ) 931–934
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tions of the substrates in penicillium citreo-virid were shown
in Fig. 2. Conversion of 4 by penicillium citreo-virid led to a
single product 1 in a considerable yield, that was to say peni-
cillium citreo-virid could add an oxygen atom into D-ring of
the steroidal substrates; 3 and 5 could be transformed into
4 beside 1, that was to say penicillium citreo-virid could oxi-
date 3ß-hydroxy-5-ene derivatives to 4-en-3-one derivatives
of the steroidal substrates, respectively, and 2 could be also
changed into 4 and 1, that was to say penicillium citreo-virid
could degrade the 17␤-acetyl side chain of the steroidal sub-
strates, so we concluded that 4 is a necessary intermediate for
the biotransformations, in that way the biotransformation of
4 could carry out quickly and get the highest yield compare to
other substrates. The 3 was changed 4 first, then 4 was trans-
formed into 1, in fact very small quantity of 4 was detected by
TLC, so we could convince that the first step was taken place
fastly. The 5 and 2 also produced 4 first, respectively, through
different two steps, and their final product was the same prod-
uct 1. But we found that there was no any changes in the
transformation of 6 and 7, we concluded that the 3-hydroxy of
6 and 7 could not be oxidated into corresponding 3-carbonyl
derivatives because of the hindrance of the 17-acetyl of the
substrates which was different from the 3 and 5, while 3-
carbonyl derivative was thought to be the key intermediate,
so the biotransformation of 6 and 7 could not be finished. The
3-hydroxy of 3 and 5 could be easily oxidated into 3-carbonyl
corresponding derivatives and then the next step could carry
out quickly.
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In conclusion, this work investigated the biotransforma-
tions of a series of steroids by the fungus penicillium citreo-viride
A.C.C.C. 0402 and gave their metabolic pathways, respectively,
and we believed it would prompt further investigation about
the biotransformation and metabolic pathway of the steroidal
lactones.
Acknowledgement
We are grateful to NNSF of PRC for financially supporting of
this research.
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