Biotransformations of steroids to testololactone by a multifunctional strain Penicillium simplicissimum WY134-2

Article abstract of DOI:10.1016/j.tet.2013.11.039

The biotransformations of a range of steroidal compounds, including 17α-hydroxy progesterone, progesterone, testosterone, androst-4-ene-3,17- dione (AD), pregnenolone, and dehydroepiandrosterone (DHEA), by Penicillium simplicissimum WY134-2 have been investigated. In all the cases, testolic acid and testololactone were detected, and the acid was converted to the lactone when pH was adjusted to 1, leading to isolation of testololactone in 25%-96% yields. Especially for progesterone and testosterone, the isolated yields were 93% and 96% with substrate concentration being 3 g/L, suggesting that P. simplicissimum WY134-2 may be used for the synthesis of testololactone. The results revealed the multi-functional catalytic activity of P. simplicissimum WY134-2 toward steroids for the first time. The possible reaction pathways of steroids promoted by this strain were discussed.

Full text of DOI:10.1016/j.tet.2013.11.039

Tetrahedron 70 (2014) 41e46
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Biotransformations of steroids to testololactone by a multifunctional
strain Penicillium simplicissimum WY134-2
,
,
a,
*
Beibei Yang a b, Yu Wang a, Xi Chen a, Jinhui Feng a, Qiaqing Wu a , Dunming Zhu
,
*
Yanhe Ma a
a National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial
Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
b University of Chinese Academy of Sciences, Beijing 100049, China
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 range of steroidal compounds, including 17a-hydroxy progesterone, pro-
Received 10 September 2013
Received in revised form 7 November 2013
Accepted 14 November 2013
Available online 20 November 2013
gesterone, testosterone, androst-4-ene-3,17-dione (AD), pregnenolone, and dehydroepiandrosterone
(DHEA), by Penicillium simplicissimum WY134-2 have been investigated. In all the cases, testolic acid and
testololactone were detected, and the acid was converted to the lactone when pH was adjusted to 1,
leading to isolation of testololactone in 25%e96% yields. Especially for progesterone and testosterone, the
isolated yields were 93% and 96% with substrate concentration being 3 g/L, suggesting that P. sim-
plicissimum WY134-2 may be used for the synthesis of testololactone. The results revealed the multi-
functional catalytic activity of P. simplicissimum WY134-2 toward steroids for the first time. The possi-
ble reaction pathways of steroids promoted by this strain were discussed.
Keywords:
Steroids
Biotransformation
Testolactone
Testolic acid
Ó 2013 Elsevier Ltd. All rights reserved.
Penicillium simplicissimum
Metabolic pathways
1. Introduction
biosorption of lead and copper ions,5 degradation of polyethylene,6
one-step conversion of wheat straw to sugars,7 and so on. Some
The derivatives of steroids formed through chemical synthesis
and microbial transformation are often potential active pharma-
ceutical ingredients (APIs) or key intermediates.1e3 Compared to
chemical modification, microbial transformation has high chemo-,
regio-, and stereoselectivity, and is more ecologically friendly. As
such, microbial transformation offers an effective and economical
way to synthesize steroidal derivatives. For example, some strains,
such as Curvularia lunata have been employed for the production of
cortisol from 11-deoxycortisol in industrial processes.4 However,
there is a considerable shortage of effective strains for production
of steroidal compounds of pharmaceutical importance. Therefore,
we initiated our effort to search for new microbial strains with
extraordinary ability of converting steroids. In this course, an iso-
lated strain Penicillium simplicissimum WY134-2, obtained from the
soil samples collected from Sichuan province of China, was found to
enzymes, e.g., lipase, xylanase, and vanillyl-alcohol oxidase, from P.
simplicissimum have been isolated, purified, and characterized.8e10
However, to the best of our knowledge, P. simplicissimum has not
been applied in the biotransformation of steroids. In addition,
steroidal lactones have conspicuous medicinal properties, such as
anticancer, antibacterial, anticarcinogen, and antiandrogen.11e14
Testololactone can suppress the synthesis of estrogen by inhibit-
ing the aromatase enzyme.15 Such function indicates that testolo-
lactone may be used to treat breast cancer,16 prostatic hyperplasia,
and prostate cancer.17 It can also be used as therapeutic agents for
disorders caused by the imbalance between estrogen and androgen
action, such as gynecomastia18 and precocious puberty.19 As such, P.
simplicissimum WY134-2 was examined toward the bioconversion
of 17a-hydroxy progesterone (1), progesterone (2), testosterone (3),
androstenedione (4), pregnenolone (5), and DHEA (6) in detail and
convert 17
a
-hydroxy progesterone to testololactone with high
the possible transformation pathways were conjected.
yield.
Up to now, there are numerous literature concerning the bio-
technological applications of P. simplicissimum, such as the
2. Results and discussion
In the more detailed study of transformation of 17a-hydroxy
progesterone (1) by P. simplicissimum WY134-2, testolic acid
was also formed. The acid was completely converted to
* Corresponding authors. Tel.: þ86 22 84861963; fax: þ86 22 84861996; e-mail
addresses: wu_qq@tib.cas.cn (Q. Wu), zhu_dm@tib.cas.cn (D. Zhu).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.11.039

42
B. Yang et al. / Tetrahedron 70 (2014) 41e46
testololactonedwhen the pH of fermentation culture was adjusted
to 1.0. In order to improve the efficacy of such conversion, the ef-
fects of initial pH and temperature on this bioconversion were in-
vestigated by using the cells growing at 30 ꢀC as catalyst and 17
a-
hydroxy progesterone (1) as substrate. The results are presented in
Figs.1 and 2. It is obvious that the optimal initial pH was 8.0 and the
yield of testololactone was highest when the biotransformation
temperature was 30 ꢀC.
Fig. 3. Effect of co-solvents on the biotransformation of 17a-hydroxy progesterone (1)
by P. simplicissimum WY134-2 (initial pH 8.0, 30 ꢀC for 48 h, final concentration of
solvents was 2% (v/v)).
in the biotransformations of steroids,20e22 the effect of different
concentrations of Tween 80 on the bioconversions of 17a-hydroxy
progesterone (1) by P. simplicissimum WY134-2 was studied (Fig. 4).
Obviously, the addition of Tween 80 promoted this conversion, and
the yield of testololactone was highest when substrate was dis-
solved in 1% Tween 80.
Fig. 1. pH dependence of the biotransformation of 17
a-hydroxy progesterone (1) by P.
simplicissimum WY134-2 (1% Tween 80 as emulsifier, 30 ꢀC for 48 h).
Fig. 2. Temperature dependence of the biotransformation of 17a-hydroxy pro-
gesterone (1) by P. simplicissimum WY134-2 (1% Tween 80 as emulsifier, initial pH 8.0,
reaction time 48 h).
Fig. 4. Effect of different concentrations of Tween 80 on the biotransformation of 17a-
hydroxy progesterone (1) by P. simplicissimum WY134-2 (initial pH 8.0, 30 ꢀC for 48 h).
The bioconversions of six steroids (1e6) were studied at pH 8.0
and 30 ꢀC by using cells growing at 30 ꢀC. Substrates diffused in 1%
Tween 80 were added after cells growing in fermentation medium
for 16e20 h (dry cell weight: 9.75 g/L). Different substrate con-
centrations ranging from 1 g/L to 3 g/L were initially screened by
analyzing the reaction mixtures with TLC. The biotransformations
were performed at the optimized substrate concentration and the
products were isolated. The results are summarized in Table 1. It
can be seen that testololactone was isolated in 93% and 96% yields
for substrates progesterone (2) and testosterone (3), respectively.
Owing to the low solubility of steroidal compounds in aqueous
medium, some co-solvents including DMF, DMSO, acetone, and
methylene dichloride were used to test the organic solvent toler-
ance of P. simplicissimum WY134-2. The effect of co-solvents on the
biotransformation of 17a-hydroxy progesterone (1) by P. sim-
plicissimum WY134-2 is shown in Fig. 3. It was clear that all the
tested solvents inhibited the activity of P. simplicissimum WY134-2.
Methylene dichloride could completely inhibit the conversion of
17a-hydroxy progesterone (1). Owing to the wide use of Tween 80

B. Yang et al. / Tetrahedron 70 (2014) 41e46
43
Scheme 1.23 Firstly, the side chain was oxidized by BaeyereVilliger
monooxygenase to yield testosterone acetate, which was hydro-
lyzed to testosterone (3). Then AD (4) was obtained by the de-
hydrogenation of testosterone (3). Finally, AD (4) was oxidized by
BaeyereVilliger monooxygenase to give testololactone (7), which
could be hydrolyzed to testolic acid (8). The hydrolysis of testolo-
lactone to testolic acid, which was confirmed by our experiment,
was also reported by Holmlund.24,25
According to the proposed reaction pathway, AD (4) was an
intermediate of the conversion from progesterone (2) to testolo-
lactone (7). However, the transformation efficiency of AD (4) to
testololactone (7) was far behind that of progesterone (2). There are
two possible reasons for such unusual phenomenon. First, the D-
ring lactonization may be inhibited by higher concentration of AD
(4), as evidenced by the lower conversion at higher AD (4) con-
centration. Second, progesterone (2) rather than AD (4) might be
a better inducer for the BaeyereVilliger monooxygenase produced
by P. simplicissimum WY134-2, leading to higher enzyme activity of
the strain. A similar observation was reported for the bioconversion
Table 1
Biotransformation of steroids 1e6 to testololactone (7)
Substrates
Concentration
(g/L)
Reaction
time (h)
Isolated
yield (%)
17 -Hydroxyprogesterone (1)
a
1
3
3
1
1
2
48
24
24
24
48
24
56
96
93
78
25
53
Progesterone (2)
Testosterone (3)
Androst-4-ene-3,17-dione (4)
Pregnenolone (5)
Dehydroepiandrosterone (6)
Such effective bioconversion of steroids indicated that P. sim-
plicissimum WY134-2 might be a potential strain for the industrial
production of testololactone.
During the conversion of progesterone (2) to testololactone (7),
two intermediates were detected at 2 h, which were identified as
testosterone (3) and androstenedione (4) by comparing HPLC
chromatographs with authentic samples (Fig. 5). The time course of
Fig. 5. HPLC analysis of the progesterone (2) biotransformation by P. simplicissimum WY134-2 (above, authentic samples; below, reaction mixture taken after incubation at 30 ꢀC for
10 h since the introduction of substrate; initial pH 8.0, 30 ꢀC, 1% Tween 80).
these compounds present in reaction mixture was investigated and
the results are shown in Fig. 6. Thus, the bioconversion course of
progesterone to testolic acid and testololactone was proposed as in
of progesterone (2) to
affinis, in which androst-1,4-diene-3,17-dione (ADD) was proposed
as an intermediate. Whereas ADD couldn’t be transformed to D1
D
1-testololactone catalyzed by Septomyxa
-
testololactone when it was the starting substrate for S. affinis under
identical conditions except when progesterone (2) was either
present at the initiation of bioconversion or the cells had been pre-
induced with progesterone (2).26 It was suggested that pro-
gesterone (2) rather than ADD might be a better inducer of the
enzyme responsible for the formation of
The yield of testololactone from 17 -hydroxy progesterone (1)
was lower than that from progesterone (2), which might indicate
that the existence of 17 -OH may increase the steric hindrance,
which hindered the BaeyereVilliger monooxygenation of side
chain. During the conversion of 17 -hydroxy progesterone to tes-
D
1-testololactone.
a
a
a
tololactone catalyzed by P. simplicissimum WY134-2, no in-
termediate was detected. In accordance with the biotransformation
of 17
Cylindrocarpon radicicola ATCC 11011,27 a possible transformation
mechanism of 17 -hydroxy progesterone (1) to testolic acid (8) by
a-hydroxy progesterone (1) to testololactone (7) catalyzed by
a
P. simplicissimum WY134-2 was proposed (Scheme 1). With the
oxidation by BaeyereVilliger monooxygenase and hydrolysis by
esterase, an unstable intermediate was obtained, which was
Fig. 6. Time course profile for the biotransformation of progesterone (2) by P. sim-
plicissimum WY134-2 (initial pH 8.0, 30 ꢀC, 1% Tween 80).

44
B. Yang et al. / Tetrahedron 70 (2014) 41e46
O
O
OH
O
H
H
H
H
H
H
H
H
H
H
H
H
H
O
O
O
O
O
O
O
2
3
O
OH
O
O
H
H
H
OH
pH=1~2
H
H
H
H
O
O
4
8
7
O
O
OH
HO
OH
OH
O
H
H
H
H
H
H
H
O
1
Scheme 1. D-Lactonization pathways of steroidal 4-en-3-ketones.
quickly transformed to AD (4). Then the transformation of AD (4) to
testolic acid (8) proceeded as described above.
followed by the hydrolysis of ester and dehydrogenation of C-17
hydroxy group yielded DHEA (6). BaeyereVilliger mono-
Among these steroidal substrates, the yield of testololactone
from pregnenolone (5) was lowest. Comparing the structure of
pregnenolone (5) and progesterone (2), it is possible that the oxi-
oxygenation of DHEA (6) gave
androst-5-en-17-one (9), which was oxidized by 3-
roid dehydrogenase (3 -HSD) to give testololactone (7). Finally,
3
b
-hydroxy-17a-oxa-
D-homo-
b-hydroxyste-
b
dation of 3
b
-OH and/or D5/4 isomerization in those two com-
testololactone was hydrolyzed to testolic acid (8).
pounds might be the rate-determining steps.
Some other microbial strains have been reported to transform
steroidal compounds to testololactone (7). For example, Aspergillus
tamari KITA transformed 17a-hydroxy progesterone (1) and deox-
There were different reported transformation pathways for the
conversion of pregnenolone (5) and DHEA (6) to testololactone
(7).28 In the bioconversion process of pregnenolone (5) catalyzed by
ycorticosterone to testololactone at substrate concentration of 1 g/L
P. simplicissimum WY134-2, 3
b
-Hydroxy-17a-oxa-
D
-homo-androst-
in 5 days. Some byproducts, including 17,20(R)-dihydroxypregna-
5-en-17-one (9) was detected as an intermediate, which also could
be converted to testololactone (7). Therefore, the bioconversion
course of pregnenolone (5) was proposed in Scheme 2. Firstly, the
side chain was cleaved through BaeyereVilliger monooxygenation,
4-en-3-one and
conversion of 17
D
1-testololactone, were observed in the bio-
a
-hydroxy progesterone to testololactone by this
fungi.29 Rhizopus stolonifer (ATCC 10404) converted testosterone to
give testololactone with substrate final concentration being 0.7 g/L
O
O
O
O
H
H
H
H
H
H
H
H
H
HO
HO
HO
6
5
OH
O
O
O
O
H
H
H
OH
H
H
H
H
H
H
pH=1~2
O
O
O
HO
8
7
9
Scheme 2. D-Lactonization pathways of steroidal 5-en-3-alcohols.

B. Yang et al. / Tetrahedron 70 (2014) 41e46
45
in 5 days. In this conversion, four byproducts were detected.30
There were some penicillium genus, which could give rise to tes-
tololactone. For instance, Penicillium camembertii AM83 could
convert steroids 2e6 to give testololactone (7). Among these ste-
roidal substrates, the highest yield of testololactone was 94% as
analyzed by GC. However, the final concentration of substrates was
only 0.2 g/L.28 Such low substrate concentration limited the ap-
plication of P. camembertii AM83 for the industrial production of
testololactone. Penicillium notatum could transform steroids 2, 5,
and 6 to yield testololactone (7) with substrate final concentration
at 4 ꢀC on Potato Dextrose Agar slants. Spores was washed with
3 ml of sterilized water and added to flat-bottomed flask (250 ml)
with 50 ml of the seed culture consisting of glucose (3%), corn steep
liquor (1.5%), NaNO3 (0.3%), MgSO4$7H2O (0.05%), KCl (0.05%),
FeSO4$4H2O (0.002%), K2HPO4$3H2O (0.2%), and KH2PO4 (0.1%) (pH
6.5). The seed culture was shaken at 200 rpm and 30 ꢀC for 14e16 h.
Then 1 ml of fungus suspension was transferred to the flat-
bottomed flask (100 ml) with 20 ml of transformation culture
medium, whose components were the same as seed culture. The
resulting mixture was cultured for 24 h and the substrate was
added for biotransformation.
being 0.33 g/L. Yet the presence of a substituent at 17a-position is
a barrier to lactonization by P. notatum.31 In our study, P. sim-
plicissimum WY134-2 could transform six steroidal compounds to
testololactone. Especially, the isolated yield reached 96% and 93%
for substrates progesterone and testosterone at 3 g/L in 24 h, re-
spectively. Testololactone was the sole isolated product for sub-
4.3. Biotransformation
4.3.1. Optimization of biotransformation conditions. The initial pH of
transformation culture was adjusted to different pH ranging from 4
to 9, and the cell suspension was transferred to transformation
culture medium, which was cultured for 24 h as described in Sec-
strates 1e4. Only trace of byproduct 3b-Hydroxy-17a-oxa-D-homo-
androst-5-en-17-one was detected when the substrates were 5
and 6.
tion 4.2. 17a-Hydroxy progesterone (1, final concentration of 1 g/L)
In our previous report, we found that Fusarium oxysporum
SC1301 could convert steroids 2e6 and ADD to give testolactone,
dissolved in 1% Tween-80 (v/v 4%) was then added to the trans-
formation culture and the bioconversion was implemented at
30 ꢀC, 200 rpm for 48 h. The pH of the reaction mixture was ad-
justed to 1.0 with concentrated hydrochloric acid and the reaction
mixture was vibrated at 40 ꢀC for 24 h. The mixture was extracted
with ethyl acetate (20 mlꢁ3). The yield of testololactone was
measured by HPLC analysis.
which is also called as
substrates being 1 g/L.32 Compared to F. oxysporum SC1301, P.
simplicissimum WY134-2 lacks the
1-dehydrogenase. F. oxysporum
SC1301 could not transform 17 -hydroxy progesterone (1) to tes-
tolactone, it might be due to the steric hindrance resulting from the
D
1-testololactone, with final concentration of
D
a
hydroxyl group at 17
a
-position.
Similar to the optimization of initial pH, after 17a-hydroxy
progesterone (1) was added to the transformation culture, the
bioconversion was carried out at different temperature from 20 to
40 ꢀC and the optimal pH obtained above. After incubation for 48 h,
the products were extracted and analyzed as described above to
measure the yield of testololactone.
3. Conclusions
P. simplicissimum WY134-2, isolated from soil samples, could
convert steroidal compounds 17 -hydroxy progesterone (1), pro-
a
gesterone (2), androst-4-ene-3,17-dione (3), testosterone (4),
pregnenolone (5), and dehydroepiandrosterone (6) to furnish the
same product, testololactone. Such biotransformations include
Similar to the previous optimization of initial pH, 17a-hydroxy
progesterone (1) dissolved in different co-solvents (2% v/v) was
added to the transformation culture with substrate final concen-
tration being 1 g/L. The transformation was implemented at opti-
mal initial pH and optimal temperature for 48 h. The products were
extracted and analyzed as described above to measure the yield of
testololactone.
oxidation of C-3 OH group and isomerization of
D
5/4, a selective
BaeyereVilliger oxidation of steroidal side chain and ring-D, de-
hydrogenation and hydrolysis of ester. It has been demonstrated for
the first time that P. simplicissimum has great multi-functional
catalytic properties toward the transformation of steroidal com-
pounds. Compared with other microorganisms reported previously,
such as P. camembertii AM83, P. notatum and A. tamari, P. sim-
plicissimum WY134-2 exhibited higher activity and specificity to
give product testololactone (7). The results revealed that P. sim-
plicissimum WY134-2 may be a useful strain for production of
testololactone (7).
Similar to the optimization of initial pH, 17a-hydroxy pro-
gesterone (1) dissolved in different concentrations of Tween 80 (4%
v/v) was added to the transformation culture with substrate final
concentration being 1 g/L. The transformation was implemented at
optimal initial pH and optimal temperature for 48 h. The products
were extracted and analyzed as described above to measure the
yield of testololactone.
All of the following bioconversions of steroidal compounds
(1e6) were carried out under the optimal conditions.
4. Experimental
4.1. General procedures
4.3.2. Biotransformation of 17a-hydroxy progesterone (1). 17a-Hy-
droxy progesterone (180 mg) dissolved in 1% Tween 80 (8 ml) was
added to the culture (180 ml) prepared as described above at 30 ꢀC.
The reaction was analyzed by TLC and HPLC. After incubation for
48 h, the substrate was consumed and a single product was
detected. Taking into consideration the unstability of this product
(testolic acid), the pH of reaction mixture was adjusted to 1.0, and
the mixture was shaken at 200 rpm and 40 ꢀC for 24 h to ensure the
conversion of testolic acid to testololactone. The reaction mixture
was extracted with ethyl acetate (180 mlꢁ3), and the organic ex-
tract was dried over sodium sulfate. The solvent was evaporated
under reduced pressure to give yellow crude product, which was
purified by silica gel chromatography with ethyl acetate/petroleum
ether (1:3) as eluent to give testololactone (7) as a white solid
(91.6 mg, 56% yield), which was identified as testololactone (2). 1H
Steroids 3, 4, and 5 were kindly donated by Tianjin Jinyao Group
Co., LTD. Other steroidal compounds and solvents were obtained
from commercial sources. Silica gel (Qingdao Haiyang ChemicalCo.,
200e300 mesh) was used for the column chromatography. The
thin-layer chromatography (TLC) was performed by developing in
solvent mixture of ethyl acetate and petroleum ether (1/3, v/v), and
visualizing by UV light (254 nm). The high performance liquid
chromatography (HPLC) analysis was performed on an Agilent 1200
system with an Eclipse XDB-C18 column (4.6ꢁ150 mm, eluent:
acetonitrile/water 6:4, flow rate: 0.5 ml/min, detector: UV254 nm).
4.2. Microorganism, maintenance, and cultivation
P. simplicissimum WY134-2 was isolated from soil samples col-
NMR: (600 MHz, CDCl3)
d: 1.09e1.16 (1H, m), 1.17 (3H, s, 18-H3),
lected from Sichuan province of China. The fungus was maintained
1.25e1.36 (2H, m), 1.38 (3H, s, 19-H3), 1.40e1.41 (1H, m), 1.53e1.59

46
B. Yang et al. / Tetrahedron 70 (2014) 41e46
(1H, m), 1.65e1.83 (4H, m), 2.00e2.08 (4H, m), 2.33e2.46 (4H, m),
2.56e2.62 (1H, m), 2.68e2.73 (1H, m), 5.76 (1H, s, 4-H); 13C NMR:
same procedure as described in Section 4.3.2. Progesterone (final
concentration being 3 g/L) was added to the transformation cul-
tures in flasks (20 ml each). Every 2 or 4 h since the introduction of
substrate, three flasks samples of reaction mixture were taken, pH
was adjusted to 1.0 and the flasks were shaken for 24 h at 200 rpm
and 40 ꢀC. The reaction mixtures were extracted with ethyl acetate.
The steroid contents in the extract were measured by HPLC
analysis.
CDCl3, 150 MHz,
d (ppm): 199.3, 171.3, 169.4, 124.3, 82.4, 52.7, 45.9,
39.2, 38.7, 38.2, 35.7, 34.0, 32.5, 30.6, 28.7, 22.0, 20.3, 20.1, 17.6.
HRMS (ESIþ): C19H26O3Na [MþNa]þ: calcd 325.1780, found
325.1766.
4.3.3. Biotransformation of progesterone (2). The bioconversion of
progesterone followed the same procedure as described in Section
4.3.2. Progesterone (420 mg) was added to the transformation
culture (140 ml). After incubation for 24 h, testololactone (7) was
obtained (387.7 mg, 96% yield).
During the transformation, two intermediates were detected at
2 h by TLC and HPLC, and identified as testosterone (3) and
androstenedione (4) by comparing HPLC chromatographs with
authentic samples.
Acknowledgements
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).
References and notes
4.3.4. Biotransformation of testosterone (3). The bioconversion of
testosterone followed the same procedure as described in Section
4.3.2. Testosterone (360 mg) was added to the fermentation culture
(120 ml). After incubation for 24 h, testololactone (7) was obtained
(351.3 mg, 93% yield).
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4.3.6. Biotransformation of pregnenolone (5). The bioconversion of
pregnenolone followed the same procedure as described in Section
4.3.2. Pregnenolone (260 mg) was added to the culture (260 ml).
After incubation for 48 h, testololactone (7) was obtained (62.8 mg,
25% yield).
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During the conversion, an intermediate, which was isolated and
identified as
(9) in a separate experiment, was detected at 3 h by HPLC. 1H
NMR: CDCl3, 600 MHz, (ppm): 0.96 (3H, s, 19-H3), 1.30 (3H, s, 18-
H3), 3.51 (1H, m, 3-H), 5.33 (1H, m, 6-H); 13C NMR: CDCl3, 150 MHz,
(ppm): 171.5, 140.7, 120.6, 83.2, 71.5, 49.0, 46.7, 41.9, 38.9, 36.9,
3b-Hydroxy-17a-oxa-D-homo-androst-5-en-17-one
ꢀ
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d
d
36.6,34.5, 31.5, 31.1, 28.8, 22.0, 20.1, 19.9, 19.3. HRMS (ESIþ):
C
19H28O3Na [MþNa]þ: calcd 327.1936, found 327.1943.
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4.3.7. Biotransformation of DHEA (6). The bioconversion of DHEA
followed the same procedure as described in Section 4.3.2. DHEA
(240 mg) was added to the transformation culture (120 ml). After
incubation for 24 h, testololactone (7) was obtained (133.4 mg, 53%
yield). During the transformation, an intermediate was detected at
2 h by HPLC. Its retention time analyzed by HPLC was the same as
the intermediate of pregnenolone (5). In a separate experiment,
yanova, B.; Angelova, B. World J. Microbiol. Biotechnol. 2010, 26, 1009e1014.
€
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this intermediate was isolated and identified as 3
oxa- -homo-androst-5-en-17-one (9).
b-Hydroxy-17a-
D
ꢀ
31. Bartmanska, A.; Dmochowska-G1adysz, J.; Huszcza, E. Steroids 2005, 70,
193e198.
4.3.8. Time course for the biotransformation of progesterone (2). The
time course for the bioconversion of progesterone followed the
32. Zhang, H.; Ren, J.; Wang, Y.; Sheng, C.; Wu, Q.; Diao, A.; Zhu, D. Tetrahedron
2013, 69, 184e189.