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H. Zhang et al. / Tetrahedron 69 (2013) 184e189
dione dissolved in 0.5% tween-80 (v/v 2%) was added into the cell
culture with final concentration being 1 g/L. The transformation
was carried out at different temperatures range from 20 ꢀC to 40 ꢀC
for 15 h. The products were extracted out and analyzed as described
above to measure the yield of testolactone.
Similar to the previous procedure, after androst-4-ene-3,17-
dione was added into the cell culture, the pH was adjusted to dif-
ferent pH from 3 to 9. The reaction mixture was incubated at 30 ꢀC
for 15 h, and the products were extracted out and analyzed as
described above to measure the yield of testolactone.
Similar to the previous procedure, androst-4-ene-3,17-dione
dissolved in different co-solvents (2%, v/v) was added into the re-
action mixture and the transformation was carried out at the
optimal temperature and pH for 12 h. The products were extracted
out and analyzed as described above to measure the yield of
testolactone.
1H), 0.82 (s, 3H); 13C NMR: CDCl3, 600 MHz,
d (ppm): 186.4 (C, C3),
169.2 (C, C5), 155.9 (CH, C1), 127.5 (CH, C2), 123.9 (CH, C4), 81.5 (CH,
C17), 52.6 (C, C9), 50.2 (C, C14), 43.6 (C, C13), 43.1 (C, C10), 36.4 (CH,
C8), 35.6 (CH2, C12), 33.2 (CH2, C6), 32.8 (CH2, C7), 30.4 (CH2, C16),
23.6 (CH3, C19), 22.5 (CH2, C15), 18.7 (CH2, C11), 11.2 (CH3, C18);
ESIMS: m/z calcd for C19H26O2: 286.42; found: 286.83.
4.3.3. Biotransformation of androst-1,4-diene-3,17-dione (ADD,
2). The transformation of androst-1,4-diene-3,17-dione followed
the same procedure as described in Section 4.3.2. Androst-1,4-
diene-3,17-dione (120 mg) was used and testolactone (7) was
obtained (102.2 mg, 82% yield). 1-Dehydrotestosterone (8) was
detected in the process at 3 h by HPLC (eluent: ethanol/water 4:6,
flow rate: 0.6 mL/min, detector: UV 254 nm).
4.3.4. Biotransformation of dehydroepiandrosterone (DHEA, 3). The
transformation of DHEA (3) followed the same procedure as de-
scribed in Section 4.3.2. DHEA (120 mg) was used and testolactone
(7) was obtained (97.4 mg, 78% yield). ADD (2) and 1-
dehydrotestosterone (8) were detected in the bioconversion of
DHEA (3) at 6 h by HPLC (eluent: ethanol/water 4:6, flow rate:
0.6 mL/min, detector: UV 254 nm and 215 nm).
All the following transformations of the steroids (1e6) were
carried out under the optimal conditions.
4.3.2. Biotransformation
of
androst-4-ene-3,17-dione
(4-AD,
1). Androst-4-ene-3,17-dione (120 mg) dissolved in DMSO (2.4 mL)
was added into the cell cultures (120 mL) prepared as described
above at 30 ꢀC. The resulting mixture was incubated at 30 ꢀC and
the reaction was followed by TLC and HPLC (eluent: ethanol/water
4:6, flow rate: 0.6 mL/min, detector: UV 254 nm). After 15 h, the
substrate was consumed and a single product was detected. The
mixture was extracted with ethyl acetate (120 mLꢁ3), and the
organic extract was dried over anhydrous sodium sulfate. The sol-
vent was removed and the residue was purified by silica gel chro-
matography with ethyl acetate/petroleum ether (7:3) as eluent to
give a white solid (104.3 mg, 83% yield), which was identified as
4.3.5. Biotransformation of progesterone (4). The transformation of
progesterone (4) followed the same procedure as described in
Section 4.3.2. Progesterone (100 mg) was used as substrate and
testolactone (7) was obtained (88.9 mg, 93% yield). ADD (2) and
1-dehydrotestosterone (8) were detected in the bioconversion of
progesterone at 4.5 h by HPLC (eluent: ethanol/water 5:5, flow rate:
0.6 mL/min, detector: UV 254 nm).
testolactone (7). 1H NMR: CDCl3, 600 MHz,
d
(ppm): 7.02 (d,
4.3.6. Biotransformation of testosterone (5). The transformation of
testosterone (5) followed the same procedure as described in Sec-
tion 4.3.2. Testosterone (180 mg) was used as substrate and testo-
lactone (7) was obtained (184.4 mg, 98% yield). ADD (2) and
1-dehydrotestosterone (8) were detected in transformation of
testosterone at 4.5 h by HPLC (eluent: ethanol/water 4:6, flow rate:
0.6 mL/min, detector: UV 254 nm).
J¼10.2 Hz, 1H), 6.26 (dd, J¼10.2, 1.8 Hz, 1H), 6.09 (s, 1H), 2.72e2.55
(m, 2H), 2.50e2.40 (m, 2H), 2.16e1.96 (m, 4H), 1.70e1.65 (m, 2H),
1.62e1.50 (m, 2H),1.48e1.42 (m,1H),1.39 (s, 3H),1.30e1.26 (m,1H),
1.22 (s, 3H), 1.16e1.09 (m, 1H); 13C NMR: CDCl3, 600 MHz,
d (ppm):
185.9 (C, C3), 170.8 (C, C17), 167.2 (C, C5), 154.3 (CH, C1), 128.1 (CH,
C2),124.2 (CH, C4), 82.4 (C, C13), 50.9 (C, C9), 45.6 (CH, C14), 43.0 (C,
C10), 38.9 (CH2, C12), 38.0 (CH2, C6), 32.3 (CH, C8), 32.1 (CH2, C1),
28.5 (CH2, C16), 23.4 (CH3, C19), 20.2 (CH2, C15), 20.1 (CH3, C18),
18.7 (CH2, C11); ESIMS: m/z calcd for C19H24O3: 300.39; found:
301.30. The X-ray crystal structure of the product was presented in
Fig. 5.
4.3.7. Biotransformation of pregnenolone (6). The transformation of
pregnenolone followed the same procedure as described in Section
4.3.2. Pregnenolone (160 mg) was used as substrate and testolactone
(7) was obtained (115.2 mg, 76% yield). 4-AD (1) was detected in the
transformation of pregnenolone at 4.5 h by HPLC (eluent: ethanol/
water 5:5, flow rate: 0.6 mL/min, detector: UV 254 nm and 205 nm).
During the transformation, two intermediates were detected at
3 h by TLC and HPLC. In a separate experiment, they were isolated
and identified as androst-1,4-diene-3,17-dione (ADD, 2) and 1-
dehydrotestosterone (8). 1H NMR for 8: CDCl3, 600 MHz,
d (ppm):
Acknowledgements
7.06 (d, J¼10.2 Hz, 1H), 6.24 (d, J¼11.4 Hz, 1H), 6.073 (s, 1H), 3.65 (t,
J¼8.4 Hz, 1H), 2.49e2.44 (m, 1H), 2.37 (dd, J¼13.2, 3.6 Hz, 1H),
2.08e2.05 (m, 1H), 1.97e1.94 (m, 1H), 1.89e1.85 (m, 1H), 1.78e1.75
(m, 1H), 1.71e1.63 (m, 3H), 1.62e1.60 (m, 1H), 1.47e1.45 (m, 1H),
1.37e1.30 (m, 1H), 1.24 (s, 3H), 1.12e1.00 (m, 3H), 0.98e0.94 (m,
This work was financially supported by National High-Tech
Research & Development Program of China (863 Program, No.
2011AA02A211), Tianjin Municipal Science & Technology Com-
mission (10ZCZDSY06700) and Chinese Academy of Sciences.
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Fig. 5. X-ray crystal structure of testolactone.