Synthesis of 3β-methyl ether of dehydroepiandrosterone by biotransformation of 3β-methyl ether of cholesterol with cells of mycobacteria Mycobacterium sp.

Article abstract of DOI:10.1007/s11172-019-2711-0

3p-Methyl ether of dehydroepiandrosterone was obtained by microbiological transformation of 3?-methyl ether of cholesterol with Mycobacterium sp. Androstane-3,17-dione, androst-4-ene-3,17-dione, and androsta-1,4-diene-3,17-dione were minor transformation products.

Full text of DOI:10.1007/s11172-019-2711-0

Russian Chemical Bulletin, International Edition, Vol. 68, No. 12, pp. 2355—2358, December, 2019
2355
Synthesis of 3β-methyl ether of dehydroepiandrosterone by biotransformation of
3β-methyl ether of cholesterol with cells of mycobacteria Mycobacterium sp.
V. A. Andryushina,а T. S. Stytsenko,а N. V. Karpova,а V. V. Yaderets,а
I. V. Zavarzin,b and D. V. Kurilovb
aFederal Research Center "Fundamentals of Biotechnology", Russian Academy of Sciences,
korp.1, 7 prosp. 60-letiya Oktyabrya, 117312 Moscow, Russian Federation.
Е-mail: andryushina@rambler.ru
bN. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences,
47 Leninsky prosp., 119991 Moscow, Russian Federation.
Е-mail: kur-dv@mail.ru
3-Methyl ether of dehydroepiandrosterone was obtained by microbiological transformation
of 3-methyl ether of cholesterol with Mycobacterium sp. Androstane-3,17-dione, androst-4-
ene-3,17-dione, and androsta-1,4-diene-3,17-dione were minor transformation products.
Key words: dehydroepiandrosterone, 3-methyl ether of cholesterol, 3-methyl ether of
dehydroepiandrosterone, androstane-3,17-dione, androst-4-ene-3,17-dione, androsta-1,4-
diene-3,17-dione, microbiological transformation, mycobacterium Mycobacterium sp., gas-liquid
chromato-mass spectrometry.
Dehydroepiandrosterone (3-hydroxyandrost-5-en-
17-one) 1 is an endogenous steroid hormone possessing
a wide range of physiological activity, for example, anti-
inflammatory, antiproliferative, antidepressant, and neu-
roprotective.1
used for the treatment and prevention of cardiovascular
diseases.6 There are known examples of its use in repro-
ductology.7 Compound 1 is also a valuable intermediate
in the synthesis of drospirenone and a number of promis-
ing anticancer agents.8
The synthesis of dehydroepiandrosterone 1 from cho-
lesterol by oxidation of dibromocholesterol acetate with
chromic acid in acetic acid was first described9 in the
1930s. It was widely used with small modifications even
on an industrial scale for the synthesis of androgen hor-
mones. However, this method is a multistage one, com-
mercially inefficient, and environmentally unfriendly,
since it produces a lot of waste.
It is known that compound 1 can be obtained by
chemical modifications of androstenedione10 or 16-de-
hydropregnenolone acetate (pregn-16-en-3-ol-20-one
acetate).11 For a long time, the main starting material for
the preparation of compound 1 was the 16-dehydropreg-
nenolone acetate,11 which is used for the industrial syn-
thesis of androgens and corticoids. A six-step synthesis of
steroid 1 from diosgenin with the intermediate generation
of 16-dehydropregnenolone acetate in a 44% total yield
is described in patent.12
Compound 1 also can be obtained by biotechnological
methods. Thus, steroid 1 was obtained in no more than
6% yield13 during transformation of cholesterol with rest-
ing Pseudomonas convexa cells. Also, dehydroepiandros-
terone 1 can be obtained microbiologically using recom-
binant strains of Mycobacterium smegmatis with blocked
enzyme 3-hydroxysteroid dehydrogenase/5-4-isomer-
Numerous literature data show that compound 1 has
a protective effect against the development of obesity,
cancer, diabetes, disorders of the adaptation process,
memory, immune and cardiovascular systems, depression,
Alzheimer's disease and other pathologies associated with
aging.1,2 In the human body, dehydroepiandrosterone 1
is the main intermediate in the synthesis of testosterone,
estradiol, and androstenedione.2 Recently, compound 1 was
found to be the only source of sex steroids in women in the
pre- and postmenopausal periods. It is also believed that
various hormone-deficient symptoms are associated with
a sharp age-related decrease in steroid 1 concentration.3,4
There are numerous data on the use of compound 1 in
the treatment of diseases associated with age-related
changes in hormonal levels.5 In addition, steroid 1 and its
metabolite (dehydroepiandrosterone sulfate) are widely
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2355—2358, December, 2019.
1066-5285/19/6812-2355 © 2019 Springer Science+Business Media, Inc.

2356 Russ. Chem. Bull., Int. Ed., Vol. 68, No. 12, December, 2019
Andryushina et al.
Scheme 1
ase14,15 or by microbiological transformation of sterols of
animal and plant origin, which include the stage of protec-
tion of the C(3) hydroxy group.13,16 Since sterols can be
considered as the most promising and affordable type of
steroid raw materials (especially for Russia, where phyto-
sterol can be obtained in unlimited amounts from forest
waste), the option of microbiological synthesis of com-
pound 1 from sterols becomes an actual issue.
Taking into account the above, it can be concluded
that the use of cholesterol derivatives is promising for the
preparation of compound 1. We suggested a biotechno-
logical method for its synthesis from 3-methyl ether of
cholesterol (2). The latter was transformed with the culture
Mycobacterium sp. to 3-methyl ether of dehydroepi-
androsterone (3) (Scheme 1). Hydrolysis of ether 3
upon heating with dilute hydrochloric acid gave the target
product 1.16
the availability of the hydrophobic steroid substrate, we used
Tween-80 and hydroxypropyl-β-cyclodextrin (HPCD)
additives.
It was found that the mycobacteria are able to almost
completely transform the starting compound 2 to form the
main product 3 and a number of minor compounds, namely,
androst-4-ene-3,17-dione (4), androsta-1,4-diene-3,17-
dione (5), and androstane-3,17-dione (6) (Scheme 2).
Preliminary analysis was performed by TLC. The
qualitative and quantitative composition of microbiological
transformation products was determined by GLC-MS.
The target product 3 was isolated preparatively and
characterized by physicochemical methods, which con-
firmed its chemical structure.
The data on the biotransformation of 3-methyl ether
of cholesterol (2) in the presence of Tween-80 are pre-
sented in Table 1.
Note that, within the framework of the approach in-
volving microbiological preparation of dehydroepiandros-
terone 1, it is necessary to solve a number of key problems
to successfully implement the biotransformation under
consideration, namely, to efficiently eliminate the side
chain while maintaining the 5-bond and the hydroxy
group at position C(3). In this case, one should take into
account the high hydrophobicity of the ethers used in the
transformation.17
In our work, we used bacterial cultures from the col-
lection of the laboratory of physiologically active com-
pounds of the Federal Research Center "Fundamentals of
Biotechnology" of the Russian Academy of Sciences,
Mycobacterium sp. R-77 and Mycobacterium sp. S-11094.
The starting compound 2 (considering its high hydro-
phobicity) was used in a finely dispersed state. To improve
As it is seen from Table 1, in the case of cell culture
Mycobacterium sp. R-77 the content of the main product 3
after completion of the transformation was higher by 14.5%.
Also, a lower content of the starting ether and byproducts
in the final reaction mixture (a total of 22.5%) was observed
as compared to the case when Mycobacterium sp. S-11094
was used (37%).
It is known that cyclodextrins are widely used in vari-
ous industries as efficient solubilizers or stimulators of
bioconversion processes. We used this technique in our
previous works.18,19 There is also literature data on the
effect of cyclodextrins on the structure of the cell wall of
microorganisms and, as a result, on the increase of the
efficiency of bioconversion processes.20 Therefore, it
seemed advisable to study the effect of HPCD on the
transformation rate of ether 2 and the accumulation of the
Scheme 2
i. Mycobacterium sp. S-11094 or R-77.

3β-Methyl ether of dehydroepiandrosterone
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 12, December, 2019
2357
Table 1. Microbiological transformation of 3-methyl ether of cholesterol (2) (2 g L–1 loading)
Culture
used
Time/h
Conversion
degree (%)
Relative content of steroids (%)
2
3
4
5
6
Mycobacterium sp. S-11094
Mycobacterium sp. R-77
92
92
2
2
92
95
2
2
2.7
1.1
63.0
77.5
33.6
1.2
0.7
20.2
—
Traces
Table 2. Effect of HPCD on the transformation of 3-methyl ether of cholesterol (2) with cells
Mycobacterium sp. R-77 (2 g L–1 loading)
Application form
Time/h
Conversion
degree (%)
Relative content of steroids (%)
2
3
4
5
6
Finely dispersed
suspension
In the presence
of HPCD
92
88
2
2
94
97
2
2
4.2
77.6
18.0
0.2
Traces
0.3
81.5
17.5
0.5
0.2
by TLC. A 30% ethanolic solution of NaOH was added to bring
the pH of the reaction medium to a neutral value, then water
(50 mL) was added and the resulting mixture was extracted with
benzene (50 mL). The benzene extract was concentrated under
reduced pressure, the residue was additionally washed with
acetone (20 mL) and dried. The dry residue (5.1 g) was dissolved
in ethyl acetate (100 mL) at 20 С with stirring. The undissolved
precipitate was filtered off, the ethyl acetate filtrate was concen-
trated under reduced pressure, and the residue obtained was dried
to obtain ether 2 (4.82 g, 90.4%), m.p. 86—87 С (cf. Ref. 21:
m.p. 81—82 С). 1H NMR (CDCl3),  (characteristic signals are
only given): 0.70 (s, 3 H, C(18)Н3); 0.97 (s, 3 H, C(19)Н3); 3.08
(m, 1 H, H(3)); 3.36 (s, 3 H, ОMe); 5.38 (br.s, 1 H, H(6)).
Microbiological transformation. The bacterial cultures Myco-
bacterium sp. R-77 and Mycobacterium sp. S-11094 were used in
the work. The indicated bacterial cultures were stored on agar
slants, the composition of which included the following compo-
nents (g L–1): glucose — 20.0, soy flour — 10.0, citric acid — 2.2,
urea — 0.5, NH4Cl — 1.0, KH2PO4 — 0.5, MgSO4 — 0.5, FeSO4 —
0.05, CaCO3 — 1.5 (medium pH 6.8—7.2).
main reaction products when using Mycobacterium sp.
R-77. The results are presented in Table 2.
As it follows from Table 2, the use of HPCD in the
transformation medium led to both a more complete
conversion of the starting ether and a reduction in the
transformation time. However, HPCD did not exert
a critical effect on the transformation of 3-methyl ether of
cholesterol by Mycobacterium sp. R-77 at a 2 g L–1 loading.
In conclusion, mycobacteria Mycobacterium sp. effi-
ciently transform 3-methyl ether of cholesterol into
3-methyl ether of dehydroepiandrosterone with minor
admixtures of androst-4-ene-3,17-dione, androsta-1,4-
diene-3,17-dione, and androstane-3,17-dione. It was
found that the use of HPCD in the transformation medium
leads to a more complete conversion of the starting ether
2 and a reduction in the transformation time.
Experimental
The biomass of mycobacteria aged 10—14 days obtained on
the agar slopes was transferred into 750-mL conical flasks with
100 mL of the medium of the same composition and grew on
a rocking shaker for 94—98 h at 28—30 С with stirring at a rate
of 220 rpm. Next, the seed material of mycobacteria in an amount
of 20 vol.% was transferred into flasks equipped with chippers
with a transformation medium of the following composition
(g L–1): glucose — 20.0, soy flour — 10.0, citric acid — 2.2, urea —
0.5, NH4Cl — 1.0, KH2PO4 — 0.5, MgSO4 — 0.5, FeSO4 — 0.05,
CaCO3 — 1.5 (medium pH 6.8—7.2). The concentration of
Transformation products were preliminary analyzed by TLC
on Sorbfil plates (Imid Ltd, Russia).
Analytical study was performed on an Agilent Technologies
instrument composed of a 7890 gas chromatograph (HP-5 col-
umn, 50 m × 320 μm × 1.05 μm) and a 5975 C mass selective
detector with a quadrupole mass analyzer.
Preparative isolation of the target product was carried out on
silica gel 0.060—0.200 mm, 40 Å (Acros, USA).
The melting point was measured in a capillary on an OptiMelt
MPA-100 instrument (USA).
the starting compound (substrate) 2 (loading) was 2 g L–1
.
1H NMR spectra were recorded on a Unity+400 spectrom-
eter (Varian) with an operating frequency of 400 MHz.
Preparation of 3-methyl ether of cholesterol (2). Methyl
orthoformate (2.5 mL) and 57% HClO4 (2 drops) were added to
a suspension of cholesterol (5 g, 12.93 mmol) in anhydrous
benzene (5 mL) with vigorous stirring. The reaction mixture upon
stirring at 20 С became entirely homogeneous and the reaction
was completed within 2 h. The reaction progress was monitored
Compound 2 was introduced into the reaction medium in the
form of a finely dispersed suspension with the addition of
Tween-80 (0.7 g L–1) or HPCD (KLEPTOSE, France) in a 1 : 1
molar ratio. The flasks with the reaction medium were placed on
a rocking shaker under conditions similar to these of growth.
The transformation products were analyzed by TLC (sorbent
silica gel) and GLC-MS. To carry out the TLC analysis, we col-
lected aliquots of the culture liquid (1 mL) at intervals determined

2358
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 12, December, 2019
Andryushina et al.
by the objectives of the experiment. Steroids were extracted with
a four-fold volume of ethyl acetate or chloroform. Steroid com-
pounds were separated by TLC, using a hexane—acetone solvent
system (2 : 1) as the eluent.
To evaluate the residual starting sterols and their micro-
biological transformation products, the chromatograms were
visualized with a 1% solution of vanillin in 10% HClO4, followed
by heating until colored spots appeared.
To isolate the products of transformation, the culture liquid
(60 mL, with a loading of the starting substrate of 2 g L–1) was
extracted twice with an equal volume of ethyl acetate. Then, the
ethyl acetate extract was concentrated under reduced pressure
on a rotary evaporator. The oily residue obtained after evapora-
tion of the solvent was dried in an oven at 60 С.
Hydrolysis of 3-methyl ether of dehydroepiandrosterone (3).
A solution of 8% hydrochloric acid (200 mL) was added to the
culture liquid containing compound 3 (100 mL). The mixture
was stirred for 1 h at 80 C. The completion of the reaction was
determined by TLC analysis. Dehydroepiandrosterone 1 was
isolated by extraction with chloroform. The chloroform extract
was treated with activated carbon, the carbon was filtered off,
the filtrate was evaporated in vacuo. Acetone was added to the
crystalline residue, compound 1 was collected by filtration, the
precipitate was dried at 60 C to obtain compound 1, m.p.
142—144 C (cf. Ref. 13: m.p. 140—141 C).
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Received April 26, 2019;
in revised form August 21, 2019;
accepted August 31, 2019