M. Koshimura et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 72–77
73
Table 1
ene-3,17-dione, pregnenolone, DHEA and estradiol were purchased
from Tokyo Chemical Industry Co., Ltd.
13C NMR data for transformation products determined in CDCl3 or CD3ODa.
Carbon atom
Compounds
2.2. Microorganism and medium
3
5
6a
38.6
7
8
9
10a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
37.2
31.6
71.6
42.2
37.4
33.3
37.2
31.6
71.6
42.2
35.8
31.0
71.2
41.9
38.2 127.1
31.3 116.0
70.6 156.3
42.4 117.5
Gelasinospora retispora GRK002 was provided by Professor
Emeritus Naoshi Nakamura, University of the Ryukyus. Yeast and
mold medium (YM medium) [0.3% yeast extract, 0.3% malt extract
and 0.5% peptone in distilled water] was used for the biotransfor-
mation experiment.
32.3
72.4
43.0
142.3
122.3
32.6
33.3
51.9
37.8
21.8
37.9
43.8
52.7
24.4
30.6
82.5
11.5
19.9
199.9
124.8
170.0
34.1
30.2
34.5
59.2
40.0
68.7
42.9
47.9
50.0
21.7
35.7
218.4
14.6
18.3
141.0
120.9
30.8
31.5
50.2
36.6
20.4
31.4
47.5
51.8
21.9
35.9
221.2
13.5
19.4
141.4
120.5
30.4
27.6
50.6
36.8
20.3
32.7
46.7
55.9
67.3
46.7
223.5
17.5
19.3
146.6
123.6
64.3
37.2
42.6
37.5
20.1
31.3
47.1
44.9
21.9
36.9
221.1
13.3
18.3
165.7 140.1
125.3 68.3
200.2 27.2
43.6 34.3
55.7 45.4
35.7 132.7
68.3 37.6
41.9 38.0
48.0 44.6
44.5 50.9
23.8 23.9
35.7 30.7
14.5 11.7
2.3. Growth and biotransformation conditions
1. An Erlenmeyer flask (1000 ml) containing 300 ml of YM
medium was inoculated with a suspension of G. retispora, and incu-
bated at 26 ◦C for 4 days under shaking (90 rpm). After full growth
of the microorganisms, 100 mg of substrate dissolved in 2 ml of
methanol was added to the grown cultures. The incubation was
then continued for 14 days at 26 ◦C. The culture was filtered and
the broth was extracted twice with ethyl acetate (EtOAc) and con-
centrated in vacuo, and the crude extract was fractionated by HPLC.
2. The fungi were incubated at 26 ◦C in 300 ml Erlenmeyer flasks
with 150 ml of YM medium. After 4 days incubation, 20 mg of DHEA
(3) and estradiol (4) was added. The metabolites were extracted
with EtOAc after 6 days.
17.1
–
White plates; m.p. 196–197 ◦C; IR (KBr): ꢀmax cm−1 3445, 1738,
1667; 1H NMR (CDCl3) ı 5.76 (1H, s, H-4), 4.08 (1H, ddd, J = 15.0,
10.5, 5.0 Hz, H-11), 1.35 (3H, s, H-19), 0.95 (3H, s, H-18); 13C NMR
(CDCl3) see Table 1; MS (EI) m/z 302 [M]+.
Medium without G. retispora was also prepared to act as a con-
trol. Metabolites were not detected in the control.
2.5. Biotransformation of pregnenolone (2)
2.3.1. Biotransformation of androst-4-ene-3,17-dione (1) at
different pH values
The EtOAc extract was separated by normal phase HPLC (n-
hexane–EtOAc, 3:2) to afford 3, 6, and 7.
For these experiments 200 ml Erlenmeyer flasks were used,
filled with 100 ml of YM medium (pH 5–9) and inoculated with G.
retispora at 26 ◦C. After 4 days incubation, substrate (1, 10 mg) was
added to the medium and further cultivated for 6 days. After incu-
bation, the mycelia were removed by filtration, and filtered culture
was extracted with EtOAc. The extracts were concentrated in vacuo
and analyzed on GC and 1H NMR.
White needles; m.p. 148–149 ◦C; IR (KBr): ꢀmax cm−1 3445,
1739, 1650; 1H NMR (CDCl3) ı 5.42 (1H, d, J = 5.0, H-6), 3.55 (1H,
m, H-3), 1.05 (3H, s, H-19), 0.90 (3H, s, H-18); 13C NMR (CDCl3) see
Table 1; MS (EI) m/z 288 [M]+.
2.3.2. Transformation of androst-4-ene-3,17-dione (1) at the
increased temperature
White plates; m.p. 174–176 ◦C; IR (KBr): ꢀmax cm−1 3448, 1653;
1H NMR (CD3OD) ı 5.25 (1H, d, J = 5.0, H-6), 3.47 (1H, t, J = 9.0, H-17),
3.30 (1H, m, H-3), 0.94 (3H, s, H-19), 0.65 (3H, s, H-18); 13C NMR
(CD3OD) see Table 1; MS (EI) m/z 290 [M]+.
Each 200 ml Erlenmeyer flasks containing 100 ml of YM medium
(pH 5–7) were inoculated with suspension of G. retispora and then
incubated for 4 days at 34 ◦C on a rotary shaker (90 rpm). Androst-
4-ene-3,17-dione (1, 10 mg) was added to medium. After 6 days
incubation, the culture and mycelia were separated by filtration,
and the filtered culture medium was extracted with EtOAc. The
EtOAc extracts were analyzed on GC and 1H NMR.
White plates; m.p. 172–173 ◦C; IR (KBr): ꢀmax cm−1 3446, 1736,
1653; 1H NMR (CDCl3) ı 5.46 (1H, d, J = 5.5, H-6), 4.62 (1H, t, J = 5.0,
H-15), 3.61 (1H, m, H-3), 1.25 (3H, s, H-18), 1.14 (3H, s, H-19); 13C
NMR (CDCl3) see Table 1; MS (EI) m/z 304 [M]+.
2.3.3. Transformation of androst-4-ene-3,17-dione (1) at the
higher substrate loadings
To produce 5, cultures were transferred into 1000 ml flask
containing 300 ml of YM medium at 26 ◦C for 4 days. Androst-4-
ene-3,17-dione (1, 500 mg or 1000 mg) was added as substrate, and
the mixture was incubated for 6 days. After the incubation period,
the grown culture was removed by filtration and the filtrate was
extracted with EtOAc. The crude oil was analyzed by GC and 1H
NMR.
2.6. Conversion of DHEA (3)
The transformation products were separated by HPLC (n-
hexane–EtOAc, 1:5) to give 8 and 9.
White plates; m.p. 180–181 ◦C; IR (KBr): ꢀmax cm−1 3390, 1734,
1643; 1H NMR (CDCl3) ı 5.65 (1H, dd, J = 5.4, 1.95, H-6), 3.96 (1H, br
s, H-7), 3.59 (1H, m, H-3), 1.02 (3H, s, H-19), 0.89 (3H, s, H-18); 13
NMR (CDCl3) see Table 1; MS (EI) m/z 304 [M]+.
C
2.4. Biotransformation of androst-4-ene-3,17-dione (1)
The crude extract obtained from the biotransformation of 1 was
separated by normal phase HPLC (n-hexane–EtOAc, 1:3) to give two
fractions. The first fraction was purified by HPLC (n-hexane–EtOAc,
1:15) to afford 5.
2.6.2. 3ˇ,11˛-Dihydroxyandrost-5-ene-7,17-dione (9)
White plates; m.p. 129–131 ◦C; IR (KBr): ꢀmax cm−1 3440, 1735,
1656; 1H NMR CDCl3) ı 5.80 (1H, s, H-6), 4.18 (1H, ddd, J = 15.0,