Microbial transformation of 5α,6α-epoxy-3β-hydroxy-16-pregnen-20-one by Trichoderma viride

Article abstract of DOI:10.1016/j.steroids.2006.12.009

Fermentation of 5α,6α-epoxy-3β-hydroxy-16-pregnen-20-one (4) with Trichoderma viride under aerobic condition yielded 3β,5α,6β-trihydroxy-16-pregnen-20-one (5) and 3β,5α,6β,15β-tetrahydroxy-16-pregnen-20-one (6). Each microbial metabolite was characterized by spectroscopic methods. Compounds 6 and 3β,5α,15β-trihydroxy-16-pregnen-6,20-dione (7) are reported for the first time.

Full text of DOI:10.1016/j.steroids.2006.12.009

steroids 7 2 ( 2 0 0 7 ) 509–513
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Microbial transformation of 5␣,6␣-epoxy-3␤-hydroxy-
16-pregnen-20-one by Trichoderma viride
Hong-Min Liu∗, Wenzhong Ge, Heping Li, Jian Wu
Zhengzhou University, Department of Chemistry, No. 75 Da Xue North Road, ZhengZhou, Henan 450052, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Fermentation of 5␣,6␣-epoxy-3␤-hydroxy-16-pregnen-20-one (4) with Trichoderma viride
under aerobic condition yielded 3␤,5␣,6␤-trihydroxy-16-pregnen-20-one (5) and 3␤,5␣,6␤,
15␤-tetrahydroxy-16-pregnen-20-one (6). Each microbial metabolite was characterized by
spectroscopic methods. Compounds 6 and 3␤,5␣,15␤-trihydroxy-16-pregnen-6,20-dione (7)
are reported for the first time.
Received 7 April 2006
Received in revised form
11 December 2006
Accepted 15 December 2006
Published on line 21 January 2007
© 2007 Published by Elsevier Inc.
Keywords:
Microbial transformation
Hydroxylation
Polyhydroxy steroids
Trichoderma viride
1.
Introduction
producing extracellular enzymes, but there are few references
to steroid hydroxylations with Trichoderma viride.
In recent years, many interesting polyhydroxy steroids have
been isolated from marine sources (starfish) [1–3]. Some 15␤-
hydroxylated steroids have been identified in human urine
[4,5]. The biological activities of these compounds could well
be important and useful in physiology and medicine. Thus,
an increasing number of 15␤-hydroxy steroids have been pre-
pared by chemical methods [6–8].
Microbial transformation is able to produce different
hydroxylated steroids that would be difficult to synthesize
by classic chemical methods [9]. Microbial hydroxylation of
steroids has already replaced synthetic chemistry in large-
scale commercial preparation of pharmacologically active
steroids. Several isolates of Trichoderma viride play an impor-
tant role in the decomposition of organic materials [10,11] by
With the aim of synthesis of 15␤-hydroxylated steroids and
some marine steroids, we have endeavored to screen microor-
ganisms capable of producing 15-hydroxy steroids with
5␣,6␣-epoxy-3␤-hydroxy-16-pregnen-20-one as substrate. In
our studies on obtaining various physiologically important
steroid derivatives by microbial transformations, we isolated
a common strain of T. viride from soil using 5␣,6␣-epoxy-3␤-
hydroxy-16-pregnen-20-one as the substrate.
In this study, T. viride was applied to convert 5␣,6␣-expoxy-
3␤-hydroxy-16-pregnen-20-one (4) into polyhydroxy steroids.
This fermentation led to the formation of two metabolites.
The characterization of the metabolites was performed by var-
ious spectroscopic methods. Compound 6 and compound 7
are reported for the first time.
∗
Corresponding author. Tel.: +86 371 67767200; fax: +86 371 67767200.
E-mail address: liuhm@zzu.edu.cn (H.-M. Liu).
0039-128X/$ – see front matter © 2007 Published by Elsevier Inc.
doi:10.1016/j.steroids.2006.12.009

510
steroids 7 2 ( 2 0 0 7 ) 509–513
2.2.2. 3ˇ-Acetoxy-5,6-epoxy-16-pregnen-20-one (3)
To solution of compound (712 mg, 2 mmol) in
2.
Experimental
a
2
dichloromethane (10 mL), a mixture of formic acid (1.2 mL)
and aqueous hydrogen peroxide (30%, 1 mL) was added.
The mixture reaction stood for 45 min at 60 ◦C. After TLC
showed total conversion of the starting material, the solution
was cooled to room temperature. The aqueous layer was
extracted with dichloromethane. The combined organic
layer was washed successively with saturated aqueous
NaHCO3, brine and water, and finally dried with anhydrous
Na2SO4. The products (707 mg) were achieved by removal of
dichloromethane under reduced pressure as a mixture of ␣
2.1.
Instrumental methods
Melting points (mp) were determined on a XT5 melting point
apparatus and were uncorrected. Infrared (IR) spectra were
recorded using KBr discs on a Bruker Vectror-22 spectrome-
ter. Mass spectra (MS) were obtained on a Esquire3000 mass
spectrometer by electrospray ionization (ESI). Optical rota-
tions were measured on solutions of methanol in 1-dm cells
at 20 ◦C on a Perkin-Elmer 341 automatic spectropolarimeter.
The 1H and 13C nuclear magnetic resonance (NMR) spectra
were obtained using a Bruker Avance DPX-400 spectrometer at
400 and 100 MHz, respectively, with tetramethylsilane (TMS) as
internal standard in DMSO-d6. Chemical shifts (ı) were given
in parts per million (ppm) relative to TMS. Coupling constants
(J) were given in hertz (Hz). Thin layer chromatography (TLC)
was performed on 0.25 mm thick layer of silica gel G (Qing-
dao Marine Chemical Factory, China). Chromatography was
performed with petroleum ether (bp 60–90 ◦C)/acetone (7:3)
or chloroform/methanol (8:1) and visualized by spraying the
plates with 50% sulfuric acid solution and heating in an oven at
100 ◦C for 3 min until the colors developed. All measurements
were made on a Rigaku RAXIS-IV image plate area detector
and ␤ isomers [13]. Yield: 95%; ␣ isomer: mp 155–158 ◦C; [˛]D
20
−21.7 (c 0.12, MeOH); MS (ESI) m/z [M + Na]+ 395.3; IR (KBr)
ꢁmax: 2944, 2868, 1732, 1660, 1584, 1451, 1373, 1250, 1040 cm−1
;
1H NMR (DMSO-d6) see Table 1; 13C NMR (DMSO-d6) ı: 196.2
(C20), 169.8 (CH3COO), 154.2 (C17), 145.1 (C16), 70.8 (C3), 64.9
(C5), 62.4 (C6) ppm.
2.2.3. 5˛,6˛-Epoxy-3ˇ-hydroxy-16-pregnen-20-one (4)
To a solution of compound 3 (744 mg, 2 mmol) in acetone
(10 mL), aqueous sodium hydroxide (10%, 2 mL) was added and
the mixture was refluxed for 3 h. Then, acetone was evapo-
rated, the mixture was extracted with ethyl acetate, and the
combined organic extracts were washed with saturated brine,
water and finally dried with anhydrous Na2SO4. The ␤-epoxy
isomer was easily hydrolyzed in presence of sodium hydrox-
ide, and the product left in the water phase in the course
of extraction by acetate, so the solution was evaporated to
dryness only to give 568 mg of compound 4. Yield: 76%; mp
˚
using graphite monochromatic Mo K␣ radiation (ꢀ = 0.71073 A).
The data were collected at a temperature of 18 1 ◦C using
the ω-2Â scan technique and corrected for Lorenz-polarization
effects. A correction for secondary extinction was applied. The
two structures were solved by direct methods and expanded
using Fourier technique. The hydrogen atoms bonded to car-
bons in bpfp were geometrically placed and allowed to ride on
their parent carbon. The final cycle of full-matrix least-squares
refinement was based on observed reflections and variable
parameters. All calculations were performed using SHELXL-97
Program.35
140–143 ◦C; [˛]D −13.3 (c 0.15, MeOH); MS (ESI) m/z [M + Na]+
20
353; IR (KBr) ꢁmax: 2938, 2853, 1730, 1709, 1451, 1368, 1262, 1151,
1039 cm−1; 1H NMR (DMSO-d6) see Table 1; 13C NMR (DMSO-d6)
see Table 2.
2.2.4. 3ˇ,5˛,15ˇ-Trihydroxy-16-pregnen-6,20-dione (7)
NBS (445 mg, 2.5 mmol) was added to a solution of compound
6 (364 mg, 1 mmol) in a mixture of acetone (10 mL), water
(1 mL) and acetic acid (0.2 mL). The mixture was stirred for
45 min, poured into saturated aqueous NaCl and extracted
with ethyl acetate (3× 20 mL). The organic layer was washed
successively with saturated aqueous NaHCO3 (2× 10 mL) and
aqueous NaCl (2× 10 mL), dried with anhydrous Na2SO4, and
the solvent was evaporated under reduced pressure to afford
2.2.
Syntheses of compounds
2.2.1. 3ˇ-Acetoxy-5,16-pregnadien-20-one (2)
Compound 2 was synthesized by a literature procedure [12].
mp 172–174 ◦C; [˛]D −30 (c 0.11, MeOH); MS (ESI) m/z [M + Na]+
20
379; IR (KBr) ꢁmax: 2944, 2905, 2865, 1730, 1661, 1583, 1438,
1370, 1248, 1307 cm−1; 1H NMR (DMSO-d6) see Table 1; 13C NMR
(DMSO-d6) ı: 196.3 (C20), 169.8 (CH3COO), 154.4 (C17), 145.2
(C16), 140.1 (C5), 121.9 (C6), 73.3 (C3), 56.0 (C14), 50.0 (C9), 45.6
(C13) ppm.
305 mg of compound 7; compound 7 was crystallized with
methanol. Yield: 82%; mp 262–265 ◦C; [˛]D
−92.5 (c 0.16,
20
MeOH); MS (ESI) m/z [M + Na]+ 385.0; IR (KBr) ꢁmax: 3399, 2936,
1698, 1660, 1588, 1423, 1370, 1225, 1088, 1056, 1027 cm−1
;
Table 1 – The most significant 1H NMR chemical shifts of compounds (ı ppm, J Hz, in DMSO-d6)
Compound
H-3
H-6
H-15
H-16
H-18
H-19
H-21
2
4.45, m
4.72, m
4.72, m
3.55, m
3.77, m
3.81, m
3.71, m
5.36, d (J 4.4)
2.93, d (J 4.4)
3.08, d (J 1.6)
2.86, d (J 3.2)
3.33, br
6.88, br
6.86, br
6.86, br
6.86, br
6.87, d (J 1.0)
6.77, d (J 2.8)
6.78, d (J 3.2)
0.85, s
0.78, s
0.79, s
0.78, s
0.80, s
1.10, s
1.08, s
1.01, s
1.07, s
0.94, s
1.08, s
1.05, s
1.08, s
0.71, s
2.08, s
2.00, s
2.20, s
2.20, s
2.20, s
2.24, s
2.25, s
3(␣)
3(␤)
4
5
6
3.35, d (J 3.2)
4.46, dd (J 5.2, 2.8)
4.45, m
7

steroids 7 2 ( 2 0 0 7 ) 509–513
511
with freshly obtained spores from agar slope cultures and
incubated for 2 days at 27 ◦C in a rotary shaker (150 rpm). 5␣,6␣-
epoxy-3␤-hydroxy-16-pregnen-20-one (1 g) was dissolved in
20 mL acetone. Two milliliters of the acetone solution was
added to each 500-mL Erlenmeyer flask. Incubation was con-
tinued for 4 days at the same conditions.
Table 2 – 13C NMR signals of some compounds (ı ppm, in
DMSO-d6)
C
4
5
6
7
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
31.7
27.8
67.4
39.9
65.1
57.8
30.8
27.6
42.0
34.6
19.8
34.3
45.3
56.0
31.4
145.1
154.0
15.5
15.5
196.1
26.9
31.7
31.1
65.6
40.8
74.4
74.0
34.2
28.3
44.9
38.0
20.4
34.6
45.8
55.7
31.7
145.2
154.4
15.8
16.0
196.1
26.9
31.8
31.1
65.9
40.8
74.6
74.3
32.9
25.7
45.7
38.3
20.3
34.9
46.4
59.1
71.3
145.1
155.3
21.6
16.0
197.4
27.4
29.5
30.6
65.4
35.9
79.5
212.5
40.3
32.6
44.6
42.4
20.8
34.6
46.8
58.8
71.0
144.7
155.2
21.9
13.8
197.5
27.6
2.5.
Biotransformation, product isolation and analysis
All of the fermentation media were extracted exhaustively
with ethyl acetate and filtered to separate the broth from the
mycelium. The extract was evaporated under reduced pres-
sure. The transformation products were separated on silica
gel column chromatography by using CHCl3/CH3OH (8:1) as
eluant. Two metabolites (5) 291 mg, (6) 457 mg and the uncon-
verted substrate (4) 122 mg were isolated and characterized by
melting points and optical rotations and identified by spectral
data (IR, MS, 1H NMR, 13C NMR).
2.6.
3ˇ,5˛,6ˇ-Trihydroxy-16-pregnen-20-one (5)
This compound was crystallized with methanol to give col-
orless needles; mp 248–250 ◦C; [˛]D +13.8 (c 0.13, MeOH);
20
MS (ESI) m/z [M + Na]+ 371.1; IR (KBr) ꢁmax: 3433, 2933, 1647,
1582, 1373, 1275, 1158, 1078, 1032 cm−1; 1H NMR (DMSO-d6) see
Table 1; 13C NMR (DMSO-d6) see Table 2.
1H NMR (DMSO-d6) see Table 1; 13C NMR (DMSO-d6) see
Table 2.
2.3.
Maintenance and growth of microorganism
2.7.
(6)
3ˇ,5˛,6˛,15ˇ-Tetrahydroxy-16-pregnen-20-one
The steroid-converting fungus was isolated during screen-
ing among 96 stains from samples collected from various
areas of China. The strain was maintained on potato –
2% – dextrose agar slope, grown at 27 ◦C, stored at 4 ◦C
and freshly subcultured before using in the transformation
experiment. The selected strain was identified as a genus
T. viride.
This metabolite was crystallized from methanol to give color-
less needles; mp 313–315 ◦C; [˛]D −57 (c 0.10, MeOH); MS (ESI)
20
m/z [M + Na]+ 387.1; IR (KBr) ꢁmax: 3386, 2941, 1677, 1593, 1436,
1367, 1124, 1081, 1036 cm−1
;
1H NMR (DMSO-d6) see Table 1;
13C NMR (DMSO-d6) see Table 2.
2.4.
Incubation conditions
3.
Results and discussion
Ten 500-mL Erlenmeyer flasks, each containing 100 mL of
sterilized potato – 2% – dextrose broth, were inoculated
Biotransformation of 5␣,6␣-epoxy-3␤-hydroxy-16-pregnen-
20-one (4) was carried out in the T. viride culture. The isolated
Scheme 1 – Reagents and conditions: (a) (CH3CO)2O, NH4Cl, Py, 135 ◦C; (b) CrO3, HOAc, CH2ClCH2Cl, r.t.; (c) AcONa, 90 ◦C; (d)
HCOOH, CH2Cl2, H2O2, 60 ◦C; (e) NaOH, CH3OH, 60 ◦C; (f) NBS, CH3COCH3, r.t.

512
steroids 7 2 ( 2 0 0 7 ) 509–513
Fig. 1 – X-ray diffraction analysis diagram of 5.
Fig. 2 – X-ray diffraction analysis diagram of 7.
microorganism was able to transform this substrate into some
polyhydroxy steroids (5) and (6).
at C-15. The assignment was confirmed by 2D-NMR spectra.
The stereochemistry of 15-OH group should be ␤-configuration
from the NOESY spectrum and coupling constants in the 1H
NMR.
In order to confirm the configuration of C-15 hydroxyl
group, the compound 6 was oxidated to 6-ketone derivative
(7) by using NBS as a selective oxidant. The X-ray diffrac-
tion analysis of compound 7 (Fig. 2) showed that 15-hydroxyl
group had the ␤-configuration. Therefore, the stereochemistry
of compound 6 was fully confirmed.
The synthesis of the substrate (4) is shown in Scheme 1. The
most significant 1H NMR chemical shifts and 13C NMR signals
of compounds are presented in Tables 1 and 2, respectively.
The compound 3 was obtained in high yield and in short
time by using hydrogen peroxide as oxidant. It was interest-
ing to be find that the ␤-epoxy configuration of compound 3
was partly isomerized into ␣-epoxy (4) in the presence of the
base. That may be because that the ␤-epoxy ring opened in
the base then closed in more stable state as ␣-epoxy configu-
ration. This was confirmed by the 1H NMR of compound 4 at ı
2.86 (d, J = 3.2 Hz, H-6).
r e f e r e n c e s
The mass spectra of the metabolites (5) and (6) showed sig-
nals for the [M + Na]+ at m/z 371.1 and 387.1, which suggested
the incorporation of one and two oxygen atoms, respectively,
into the substrates. The IR spectra indicated the existence of
at least one hydroxyl group in the compounds 5 and 6.
In compound 5, three protons that resonated at ı 4.49, 4.20
and 3.71 in the 1H NMR were exchangeable with D2O, which
indicated the presence of three hydroxyls in the molecule. The
13C NMR spectra data (ıC-20 196.1, ıC-16 145.2, ıC-17 154.4) sug-
gested the presence of a carbonyl function and a double bond,
which were also supported by 1H NMR (ıH-16 6.87) and IR spec-
tra. This structure assignment was finally confirmed by X-ray
diffraction analysis (Fig. 1).
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