Increased yield of biotransformation of exemestane with β-cyclodextrin complexation technique

Article abstract of DOI:10.1016/j.steroids.2013.07.009

In this study, 6-methylenandrosta-4-ene-3,17-dione and Hydroxypropyl- β-cyclodextrin (HP-β-CD) were used to form a complex, which could be then biotransformed by Arthrobacter simplex ATCC6946 to obtain the antitumor drug exemestane. The complex was analyzed by UV, DSC and TG techniques, while the products were analyzed by HPLC, NMR and MS. These results confirmed that the β-cyclodextrin not only improved the water-solubility of 6-methylenandrosta-4-ene-3,17-dione, but also greatly enhanced the biocompatibility during the biotransformation process. This result may be applied to other precursors which have poor aqueous solubility in the biotransformation processes.

Full text of DOI:10.1016/j.steroids.2013.07.009

Steroids 78 (2013) 1148–1151
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Increased yield of biotransformation of exemestane with b-cyclodextrin
complexation technique
Guang Li a,b, Fushuang Li a, Le Deng c, Xiaolan Fang b, Hui Zou a, Kangpin Xu a, Tian Li b, GuiShan Tan a,
⇑
a College of Pharmaceutical, Central South University, Changsha 410081, China
b YueYang Vocational Technical College, Yueyang 414000, China
c College of Life Science, Hunan Normal University, Changsha, Hunan 410081, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, 6-methylenandrosta-4-ene-3,17-dione and Hydroxypropyl-b-cyclodextrin (HP-b-CD) were
used to form a complex, which could be then biotransformed by Arthrobacter simplex ATCC6946 to obtain
the antitumor drug exemestane. The complex was analyzed by UV, DSC and TG techniques, while the
products were analyzed by HPLC, NMR and MS. These results confirmed that the b-cyclodextrin not only
improved the water-solubility of 6-methylenandrosta-4-ene-3,17-dione, but also greatly enhanced the
biocompatibility during the biotransformation process. This result may be applied to other precursors
which have poor aqueous solubility in the biotransformation processes.
Received 28 February 2013
Received in revised form 20 July 2013
Accepted 26 July 2013
Available online 21 August 2013
Keywords:
b-Cyclodextrin complexation technique
Exemestane
Ó 2013 Elsevier Inc. All rights reserved.
Biotransformation
Arthrobacte simplex
Cyclodextrin
1. Introduction
substrate supply [4–5]. Meanwhile, immobilized cell systems have
taken a commonplace in steroid bioconversion investigations [6].
Biotransformation technology and application of steroid com-
pounds has developed more than 50 years ago [1]. Specific micro-
bial reactions for this biotransformation have been used in
numerous partial syntheses of steroids for various drug evalua-
tions. The potential advantages of a biotransformation process over
chemical methods include exquisite stereo- and region-selectivity,
and a potential reduction in the total number of chemical synthetic
steps. However, there are also shortcomings with biotransforma-
tion. The targets that are natural or non-natural starting substrates
are often sparingly soluble in water (usually between 10À5 and
10À6 mol/L) and limit their availability to the biocatalyst in its
aqueous environment. Starting substrate and intermediates can
inactivate or inhibit enzyme activity and is often deleterious to
the cells.
Many methods and techniques have been employed to over-
come this problem, such as micronized steroid substrate [2]; or
use of organic solvents, which could be a water-miscible or a
water-immiscible solvent, to dissolve a substrate with low aqueous
solubility (forming a second phase) [3]. The addition of certain sur-
factants can also improve the number of bioconversions limited by
Aqueous two-phase system is also promising [7]. Organic solvent
tolerant bacteria are microorganisms that thrive in organic sol-
vents of high concentrations, which is convenient to the industry
[8]. Beside, cyclodextrins (CDs) are increasingly being used for
enhancing the biocatalytic capacity of biocatalysts: microbial cells
or enzymes to organic substrates [9–10].
Exemestane (6-methylen-androsta-1,4-diene-3,17-dione) is a
lipophilic steroid drug. This irreversible aromatase inhibitor was
shown to be
a potent and selective-inhibitor of aromatase
in vitro and in vivo. The introduction of a 1,2-double bond in the
A ring of steroid molecule increase the aromatase inactivator affin-
ity for the aromatase enzyme, which leads to increased therapeutic
potency [11]. Its traditional preparing method is chemical synthe-
sis, the dehydrogenation of 6-methylen-androsta-4-ene-3,17-
dione (For simplicity, it is named substrate throughout the text)
may be carried out by treatment with a toxiferous dehydrogena-
tion agent, e.g. dichlorodicyanobenzoquinone (DDQ), selenium
dioxide or chloranil. The purification process have to use chro-
matograph gel column because of the numerous by-product, and
the production is lower than 50% [12].
In this study, an enhancement of exemestane production from
substrate complexed with HP-b-CD by Arthrobacter simplex cul-
tures was investigated (Fig. 1). This result maybe be applied to
other steroid precursors which have poor aqueous solubility in
the biotransformation processes.
⇑
Corresponding author. Tel.: +86 0731 82650395.
E-mail addresses: tgs395@yahoo.com.cn, tgs395@aliyun.com (G. Tan).
0039-128X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2013.07.009

G. Li et al. / Steroids 78 (2013) 1148–1151
1149
O
O
O
O
triethyl orthoformate,p-TSA
aluminium isopropoxide
N-methylaniline,H+
Arthrobacter
simplex
cyclohexanone
toluene
THF,ethanol
O
O
HO
O
1
2
3
4
Fig. 1. The partiall synthesis equation of exemestane.
was autoclaved at 121 °C for 20 min after pH adjusted to 7.0. The
culture was placed in an orbital shaker incubator set to 150 rpm
at 30 2 °C for about 20 h. These cultures was used for the second-
ary culture preparation in the biotransformation process.
2. Materials and methods
2.1. Regents
A. simplex ATCC6946 were purchased from ATCC, stored in 4 °C
in nutrient agar plate. Dehydroepiandrosterone were purchased
from Fangtong Pharmaceutical Co., Ltd., China. Hydroxypropyl-b-
cyclodextrin (HP-b-CD), methyl-b-cyclodextrin (ME-b-CD), sulfo-
butylether-b-Cyclodextrin (SBE-b-CD), were purchased from Qian-
hui Fine Chemical Co., Ltd., China, while the other reagents were
purchased from Sinopharm Chemical Reagent Co., Ltd.
2.5. Biotransformation process
S-complex was added to above-mentioned cultures as control,
and the same molar quantity of substrate, as fine powder, was
aseptically added to the cultures and the fermentation was contin-
ued at 33 2 °C. After 4 h intervals, aliquots of 1.0 ml were with-
drawn and extracted with two aliquots (1 + 1 ml) of ethyl
acetate. The organic phase separated after centrifugation
(5000 rpm, 5 min) was collected and dried over sodium sulfate.
The ethyl acetate was evaporated at 50 °C by rotary evaporation,
re-dissolved in 1 ml of ethyl acetate and used for qualitative anal-
ysis by TLC and/or HPLC. The end product was crystaled in etha-
nol–water resolution, then analyzed by nuclear magnetic
resonance spectrometry and mass spectrometry.
2.2. Preparation of substrate (6-methylen-androsta-4-ene-3,17-dione)
Substrate was prepared in our laboratory, and the purity is over
95%. Briefly, 6.0 g (0.02 mol) dehydroepiandrosterone (1) were
added under nitrogen to 60 ml toluene, and the reaction mixture
was stirred at 120 °C for about 1 h. After cooling at room temper-
ature, 12 ml cyclohexanone was introduced into the reaction flask
and the mixture were kept for another 20 min. Then 1.2 g alumi-
num isopropoxide was added, flux for 180 min, cooling to room
temperature, crystallize used acetone, dried, 3.39 g androsta-4-
ene-3,17-dione (2) was recovered as a yellowish solid with a chem-
ical purity >89%.
2.0 g androsta-4-ene-3,17-dione (0.01 mol), dry THF 13 ml, eth-
anol 2 ml, anhydrous triethyl orthoformate 2 ml, p-toluenesulfonic
acid monohydrate 26 mg, the reaction mixture was stirred at 40 °C
for about 1 h. Another p-toluenesulfonic acid monohydrate 50 mg
was added, stirred for about 2 h. After cooling to room tempera-
ture, 1 ml N-methylaniline and 1 ml 40% formaldehyde aqueous
solution were added into the reaction flask and the mixture was
stirred at 40 °C for about 4 h. After cooed to room temperature,
10 ml 37% HCl was added and the stirring was continued for fur-
ther 2 h. The solution was transferred into a separating funnel
and the mixture was extracted with EtOAc. The organic phases
were dried over Na2SO4. After purified by chromatography on silica
gel, 0.84 g 6-methylenandrosta-4-ene-3,17-dione (3) was recov-
ered as a white solid with a chemical purity >95% m.p. 159–163 °C.
2.6. Determination of the dissolubility of substrate and complex
We dissolved 30 mg of substrate in ethanol, diluted quantita-
tively and stepwise with ethanol to obtain a series of solution hav-
ing a known concentration of 0.05, 0.04, 0.02, 0.015, 0.01, 0.005 mg
per ml, respectively. The absorption value was determined at
259 nm using ethanol as blank solution. Lastly, we obtained regres-
sion equation using concentration as abscissa, absorption value as
ordinate, respectively
Excessive substrate-b-CD and substrate is dilute in distil water,
30 2 °C, 120 rpm shaking for 12 h, centrifugate, filtrate, dilute,
then determine absorption value at 259 nm using distil water as
blank solution, account according above regression equation using
to obtain the dissolubility of substrate and complex.
Thin layer chromatography (TLC): Samples was spotted onto sil-
ica gel thin-layer chromatography (TLC) plates and then run under
an elution mixture of dichloromethane:N-hexane:ethyl acetate
(1:2:1).Visualized under UV 254 nm.
HPLC analysis was performed on a C18 column (SinoChrom
ODS-AP,
5
l
m, 250 mm  4.6 mm i.d. Dalian Elite Analytical
2.3. Preparation of substrate–b-cyclodextrin (b-CD) complex (S-
complex)
Instruments Co., Ltd.). The mobile phase was acetonitrile–metha-
nol-0.05 M KH2PO4 (35:50:65, v/v), pH 4.5. The flow-rate was set
at 1.5 ml/min. The UV detector was set at 247 nm and the com-
puter equipped with N2010 software (ZheJiang University, china).
S-complex was prepared by the co-evaporation method. Briefly,
0.1 mol of b-CD (HP-b-CD, ME-b-CD, SBE-b-CD, respectively) was
dissolved in suitable quantity of water and 0.1 mol of substrate
methanol resolution were drop wised under stir and continuous
stirred for 4 h. Then, methanol was evaporated at 50 °C by a rotary
evaporator (Buchi, Switzerland). The S-complex was formed and
kept at 4 °C. The complex is analyzed by using ultraviolet absorp-
tion spectrum (UV), gravitational thermal analysis (GTA) and dif-
ferential scanning calorimetry (DSC).
3. Results
3.1. Dissolubility of substrate and complex
The maximum absorption value of substrate and complex was
found at 259 nm, using ethanol as blank solution, obtained regres-
sion equation using y = 42.4129xÀ0.08107, correlation coefficient
0.9987.
2.4. The microorganisms and culture preparation
Calculate dissolubility of substrate and complex in water using
above regression equation using, the dissolubility of substrate, ME-
b-CD-substrate complex, HP-b-CD-substrate complex, SBE-b-CD-
substrate complex is 0.074 mmol/l, 6.932 mmol/l, 8.53 mmol/l
The microorganisms A. simplex was inoculated in 200 ml flasks
containing 40 ml nutrient medium: glucose, 0.7%; peptone, 0.7%;
corn steep liquor, 1.0%; KH2PO4, 0.1%; tap water, then the medium

1150
G. Li et al. / Steroids 78 (2013) 1148–1151
2
DSC
TG
DSC
TG
100
80
60
40
20
0
8
6
1
100
0
80
4
-1
60
-2
2
40
-3
0
20
-4
-2
0
-5
0
100 200 300 400 500 600 700 800
Temperature( oC)
0
100 200 300 400 500 600 700 800
Temperature(oC)
(a)
(b)
4
2
DSC
TG
100
80
60
40
20
0
0
-2
-4
-6
0
100 200 300 400 500 600 700 800
Temperature(oC)
(c)
Fig. 2. TG-DSC trace of substrate (a), HP-b-CD (b), substrate-b-CD (c).
and 5.65 mmol/l, respectively, the dissolubility of complex is 94,
115, 76 times higher than the dissolubility of substrate, respec-
tively, the effectiveness of HP-b-CD is the best, so HP-b-CD is se-
lected at after experiment.
3.2. TG-DSC analysis of substrate and s-substrate
From Fig. 2A, it is clear that the TG curve of substrate occurs
phase-transition at 159 °C, there is a heat absorbing peak at this
temperature, and the peak is disappear in the Fig. 2C. We also
can learn from Fig. 2B, the HP-b-CD have loss 80.2% of its weight
during 280 °C–355 °C. However, in Fig. 2C, the S-complex only loss
55.3% of its weight in the same temperature scope. The DSC curve
of HP-b-CD have two heat absorption peak at 293 °C and 345 °C,
weightlessness just happen at this point. But the weightlessness
is fewer in the Fig. 2C. This indicates that the substrate molecule
have bond to the cavity of HP-b-CD. The substrate and HP-b-CD
have transform to a S-complex. As a result, the dissolubility of S-
complex is higher than substrate.
TLC: The Rf value of substrate and exemestane is 0.7 and 0.6,
respectively.
Fig. 3 presents the HPLC graph of biotransformation production,
the retention time of exemestane and the residual substrate appear
in 12 min and 16 min, respectively, the production is 84%.
Fig. 3. The HPLC trace of conversion production.
100
80
60
40
20
su b stra te
co m p le x
3.3. MS:[M]+296.18(100%)
0
NMR: 1H-NMR(500 MHz,CDCl3), d: 0.95(s, 3H), 1.18(s, 3H),
1.30(m, 1H), 1.40(m, 2H), 1.41(m, 2H), 1.60(m, 2H), 1.90(m, 2H),
2.0(m, 2H), 2.20(s, 1H), 4.93(s, 1H),5.00(s, 1H),6.10(d, 1H, 4-
H),6.18(dd, 1H, 2-H), 7.01(d,1H,1-H).
0
10
20
30
T /h
40
50
60
Fig. 4. The conversion efficiency of substrate (0.5 mg/ml) and HP-b-CD-substrate
complex (0.5 mg/ml as substrate).

G. Li et al. / Steroids 78 (2013) 1148–1151
1151
Table 1
The conversion production (%) with substrate of various concentration.
Adding mode
The concentration (mg/ml as substrate)
1.0
2.0
4.0
10.0
20.0
40.0
S-complex
Substrate
91.11 8.20
22.58 5.21
93.71 8.50
20.82 5.21
80.18 7.55
15.46 2.21
75.58 5.32
14.90 3.58
66.88 3.00
10.29 1.14
55.03 5.77
4.35 0.28
13C-NMR(500 MHz, CDCl3), d: 12.80, 18.68, 20.81, 12.80, 18.68,
21.02, 28.68, 30.13, 34.31, 34.60, 38.24, 42.63, 46.63, 48.90, 49.78,
111.45, 121.78, 126.83, 144.27, 153.01, 166.20, 185.34, 205.90.
is more utility than HP-b-CD. By increasing the water solubility
of substrates, the higher bioconversion efficiency can be obtained.
The Mechanism for the improvement of efficiency is complex.
Some researchers presume that all b-CD-induced effects exerted
upon the microbial cells are primarily a result of interactions with
the cell wall and membrane, and then the plasmalemma oxidase
transforms the steroid substrate [9]. According to our observation,
The improvement of efficiency also ascribe to the biotoxicity of
complex is lower than the substrate, because the fermentation
broth apparently attenuate at the later stage of fermentation when
using substrate and this is not happen when using complex. The
conversion production reduce after 48 h because the exemestane
is further degradated by A. simplex, so the process is no more than
48 h. The mechanism for the improvement of efficiency need be
study further See Table 1.
To our knowledge, this is the first report of biotransformation
for the production of exemestane with b-cyclodextrin complexa-
tion technique. The complex structure of the substrate molecule
is sensitive to chemicals. The method has provided adequate tools
for the preparation of exemestane. This is a significant improve-
ment over the known method which is based on the two-phase
system [14].
3.4. Bioconversion efficiency
Fig. 4 presents the maximum yield of exemestane when added
the medium contained 0.5 mg/ml substrate and S-substrate
0.5 mg/ml as substrate, respectively under optimized conditions.
Low solubility of the substrates in the media often leads to poor
mass transfer. Substrate has low solubility in water. After complex-
ation, the solubility of substrate was increased. The maximum
yield of exemestane produced from S-complex was 91.1% which
was about 3 times higher than free substrate.
Poor solubility and low transport rates of the substrates and
products to and from cells are the limiting steps in the biotransfor-
mation process. Complexation with cyclodextrin substantially en-
hanced the conversion byA. simplex in an aqueous fermentation
system. The form of complex increased the specific dehydrogenate
activity with no influence on cell growth [13]. Our results demon-
strate the enhancement of production by increasing the solubility
of the precursor with the maximum yield of 93.7%.
When the test was repeated with increasing concentration of
substrate and S-complex, the maximum biotransformation pro-
duction decreased gradually. Because the steroid substrate was
harmful to the cells, the toxicity increased when the concentration
was increasing, so it damaged the dehydrogenate activity of cells.
However, the increase of S-complex was not as harmful as the
increasing substrate, when the concentration was higher, the dif-
ference is more evident. This indicated that the addition of ME-b-
CD not only improved the dissolubility of substrate, but also in-
creased biocompatibility, resulting in the sustainable activity of
cell when the concentration increasing.
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The method of dehydrogenation of steroid using A. simplex has
been widely utilized, but the preparation of exemestane mostly
using chemical technology because the substrates is sparingly sol-
uble in water.
D1-Dehydrogenation using resting cells has been
shown to follow a substrate inhibition type kinetics, where oxygen
is a second substrate and the inhibition effect is exerted at the level
of electron transfer along the respiratory chain. So improving the
dissolubility of substrate is critical in biotransformation process.
In this study, the dissolubility of complex is over 100 times
higher than the dissolubility of substrate, thermo-gravimetric ana-
lytical and differential thermal analysis also demonstrate that the
substrate have formed complex with b-CD.
The results obtained from the current study, strengthen our
view that cyclodextrins can be used in the preparation of exemes-
tane because of their solubilizing action. The derivative of b-CD is
various, we only studied three types, maybe the other type b-CD