280
S. D. Shuvalova et al.
The transformation process was performed in a phos-
Culture growth and substrate transformation. The
culture of Curvularia lunata fungus from the collection of
the Bioengineering Center (Russian Academy of Sciences,
Moscow) was maintained in tubes with agar medium. Me-
dium composition (%): glucose, 0.4; yeast extract, 0.4; malt
extract, 1; agar, 2.5 (pH 6.2). The mycelium was grown in
media H and A; medium H (%%): saccharose, 2; microbial
biomass inoculate, 0.25; sodium nitrate, 0.2; dibasic ammo-
nium phosphate, 0.3; monobasic potassium phosphate, 0.1;
potassium chloride, 0.05; magnesium sulfate, 0.05 (pH 6);
medium A (%%): glucose, 2; soybean meal, 0.5; yeast ex-
tract, 0.5; monobasic potassium phosphate, 0.5; sodium chlo-
ride, 0.5 (pH 6.5).
14a-Hydroxyandrost-4-ene-3,17-dione (II). An aque-
ous suspension of fungal spores (2 ml) was introduced into
750-ml flasks containing 100 ml of medium A. The first in-
oculate was grown for 48 h. This seeding material (3 ml) was
introduced into flasks with medium H and the second inocu-
late was grown for 36 h. Mycelium was filtered from the me-
dium, washed with water, and transferred into 100 ml of
0.5% glucose solution (for other media, see the text above).
The main transformation product II was accumulated by
conducting the process at a substrate load of 4 g/liter. After a
72-h incubation, the cultural liquid with mycelium was ex-
tracted with ethyl acetate. After evaporation, the oily residue
was crystallized from ether to isolate compound II; yield
55%; m.p., 247 – 249°C (reported data: m.p., 252 – 254°C
[8]).
phate buffer (pH 6.2), physiological solution, and 0.5% glu-
cose solution. For an initial steroid concentration below
4 g/liter, these media proved to be equivalent. At a greater
steroidal load, the transformation time increases from 48 to
120 h (Table 1); conducting the process in glucose solution
hinders autolysis of the mycelium.
The initial substrate was introduced into the transforma-
tion medium by various methods: (i) DMF solution; (ii) solu-
tion in methanol containing 10% CaCl2; (iii) dry powder
ground in a mortar. It was found that DMF inhibits the
hydroxylation process; the two other methods were equiva-
lent (Table 2). Introduction of a surfactant (Sorbital S 20) at a
concentration of 10 – 30 mg% together with the substrate did
not increase the yield of hydroxylated product II (Table 2).
Thus, investigation of the process of AD hydroxylation
by the washed mycelium of C. lunata fungus showed that the
transformation is accompanied by the formation of side
products
(11b-hydroxyandrost-4-ene-3,17-dione
and
14a,17b-dihydroxyandrost-4-en-3-one). Optimum hydroxy-
lation conditions were established under which the target
product 14a-hydroxyandrost-4-ene-3,17-dione is obtained
with a yield of 55% (see the experimental part).
EXPERIMENTAL PART
The TLC analysis of compounds I – IV was performed
on Silufol UV-254 plates (Czech Republic) eluted in a meth-
ylene chloride – acetone (7 : 3) system; he spots were devel-
oped by 1% vanillin solution in 10% aqueous hydrochloric
acid. The IR and UV absorption spectra were recorded on a
Specord M-80 and Specord UV-VIS spectrophotometers, re-
spectively (Germany). The mass spectra were obtained with
a Finnigan SSQ-710 spectrometer (USA) with a system of
direct sample injection into the ion source operated at an
The side transformation products were isolated from
mother liquor solutions in ether combined after several runs.
The oily residue (8.2 g) obtained upon solvent evaporation
was chromatographed on a column filled with 100 g silica
gel 40/100 m to isolate compounds II – IV.
11b-Hydroxyandrost-4-ene-3,17-dione (III). Com-
pound III is eluted first with chloroform; yield 0.56 g (6.8%);
m.p., 185 – 187°C; IR spectrum (nmax, cm – 1): 3400 (OH),
1
electron beam energy of 70 eV. The H NMR spectra were
1
1650 (CO), 1740 (CO), 1610 (C=C); H NMR spectrum in
measured on a Unity Plus 400 spectrometer (Varian, USA)
with a working frequency of 400 MHz. Quantitative analysis
of the reaction mixtures was performed by HPLC in a Gilson
chromatograph (France) using C-18 columns.
CDCl3 (d, ppm): 1.139 (s, 3H, 18-CH3), 1.441 (s, 3H,
19-CH3), 4.437 (m, 11-H), 5.674 (d, 4-H); mass spectrum,
m/z: 302, 284, 269, 189, 163, 124, 123; reported data: m.p.,
183 – 187°C [6]; mass spectrum, m/z (Irel): 302 (86), 284
(19), 269 (23), 189 (42), 163 (100), 124 (76), 123 (82) [7].
TABLE 1. Effect of Androst-4-ene-3,17-dione Transformation
Conditions on Product Composition (HPLC Data)
Substrate
concentra-
tion, g/liter
Relative product yield, %
TABLE 2. Effect of the Method of Androst-4-ene-3,17-dione (I)
Introduction on the Yield of 14a-Hydroxylated Derivative II
Batch
No.
Process
time, h
II
III
IV
Surfactant (Sorbital
S 20) additive
Form of I
Yield of II, %
1
2
3
4
5
6
1.5
1.5
2.0
4.0
4.0
6.0
40
66
69.4
52.4
53.4
59
6.7
6.5
6.1
7
15.7
24.7
16.9
16.0
18.5
23.8
Ground I
Ground I
–
+
–
52
55
30
48
48
DMF solution
72
66.8
60
6.8
6.5
Methanol solution
with 10% CaCl2
120
–
58
Shuvalova
