Biotransformations of steroid compounds by Chaetomium sp. KCH 6651

Article abstract of DOI:10.1016/j.steroids.2009.02.006

Biotransformations of steroid compounds: androstenedione, testosterone, progesterone, pregnenolone and DHEA using Chaetomium sp. 1 KCH 6651 strain as a biocatalyst were investigated. The microorganism proved capable of selective hydroxylation of the steroid substrates. Androstenedione was converted to 14α-hydroxyandrost-4-en-3,17-dione (in over 75% yield) and 6β-hydroxyandrost-4-en-3,17-dione (in low yield), while testosterone underwent regioselective hydroxylation at 6β position. Progesterone was transformed to a single product-6β,14α-dihydroxypregnan-4-en-3,20-dione in high yield, whereas biotransformation of DHEA resulted in the formation of 7α-hydroxy derivative, which was subsequently converted to 7α-hydroxyandrost-4-en-3,17-dione.

Full text of DOI:10.1016/j.steroids.2009.02.006

Steroids 74 (2009) 657–661
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Biotransformations of steroid compounds by Chaetomium sp. KCH 6651
Tomasz Janeczkoba,∗, Jadwiga Dmochowska-Gładysza,1, Edyta Kostrzewa-Susłowa,1
,
b
Agata Biało n´ ska , Zbigniew Ciunik
a
Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland
b
Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 October 2008
Received in revised form 28 January 2009
Accepted 18 February 2009
Available online 4 March 2009
Biotransformations of steroid compounds: androstenedione, testosterone, progesterone, pregnenolone
and DHEA using Chaetomium sp. 1 KCH 6651 strain as a biocatalyst were investigated. The microorgan-
ism proved capable of selective hydroxylation of the steroid substrates. Androstenedione was converted
to 14␣-hydroxyandrost-4-en-3,17-dione (in over 75% yield) and 6␤-hydroxyandrost-4-en-3,17-dione (in
low yield), while testosterone underwent regioselective hydroxylation at 6␤ position. Progesterone was
transformed to a single product—6␤,14␣-dihydroxypregnan-4-en-3,20-dione in high yield, whereas bio-
transformation of DHEA resulted in the formation of 7␣-hydroxy derivative, which was subsequently
converted to 7␣-hydroxyandrost-4-en-3,17-dione.
Keywords:
Biotransformation
Hydroxylation
Androstendione
Progesterone
DHEA
©
2009 Elsevier Inc. All rights reserved.
Chaetomium sp
1. Introduction
Hydroxylation of tertiary carbon atoms is a convenient
way of obtaining anti-cancerogenic compounds of high activ-
ity, especially against breast and uterine cancer. Such properties
are observed, among others, in the case of derivatives of
androstenedione: 6␤,14␣-dihydroxyandrost-4-en-3,17-dione and
14␣-hydroxyandrost-4-en-3,6,17-trione [13,14].
Introduction of a hydroxyl group at 7␣ position of androstene-
dione leads to 7␣-hydroxyandrost-4-en-3,17-dione. This com-
pound has found application in industry for synthesis of diuretic
compounds [14,15].
Microbial transformations of steroids provide an important
method of obtaining new steroid derivatives of potential phar-
maceutical activity, which additionally fulfills green chemistry
principles [1,2]. Monooxygenases and reductases identified in
microorganisms and in mammals are very similar to each
other. Therefore, using microbial models it is possible to trace
and to predict metabolism of tested compounds in higher
organisms [3–6].
Over the last decade, there have been suggestions that some
steroids, such as pregnenolone and progesterone and their sulphate
derivatives—e.g. allopregnenolone, may act as allosteric modula-
tors of neuronal receptors [7,8]. Moreover, these compounds are
also capable of limiting the severity of spinal cord damages. Pro-
gesterone plays also an important role in repair of myelin sheath of
damaged nerves [9]. In many cases, hydroxy derivatives are char-
acterized with much higher biological activity than the less polar
substrate. For example, 7-hydroxy derivative of dehydroepiandros-
terone (DHEA), which is found in human body, is several times
more active than DHEA itself, as far as immunoprotective and
immunoregulatory properties are concerned [10–12].
2
. Experimental
2.1. Materials
The substrates: androst-4-en-3,17-dione (1), 17␤-hydroxy-
androst-4-en-3-one
(2),
␤-hydroxypregnan-5-en-20-one (4) and 3␤-hydroxyandrost-
-en-17-one (5) were purchased from Fluka. The fungal strain
pregnan-4-en-3,20-dione
(3),
3
5
Chaetomium sp. KCH 6651 was obtained from Department of
Phytopathology of Agriculture University of Kraków.
2.2. Screening procedure
Three hundred milliliters Erlenmeyer flasks, each containing
00 ml of the medium consisting of 3 g glucose and 1 g aminobak
dissolved in water, were inoculated with a suspension of microor-
∗ Corresponding author. Tel.: +48 71 3205252; fax: +48 71 3284124.
1
E-mail address: janeczko13@interia.pl (T. Janeczko).
1
Tel.: +48 71 3205252; fax: +48 71 3284124.
0
039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2009.02.006

6
58
T. Janeczko et al. / Steroids 74 (2009) 657–661
◦
ganisms and then incubated for 3–7 days at 25 C on a rotary shaker.
After full growth of the culture (about 12 g of cell dry weight/l)
Road, Cambridge CB2 1EZ, UK (fax: +44 1223 336033 or e-mail:
deposit@ccdc.cam.ac.uk).
2
0 mg of a substrate dissolved in 1 ml of acetone was added. After
2.5. Spectral data of isolation metabolites
1, 3, 6 and 9 days of incubation under the above conditions, portions
of 10 ml of the transformation mixture were taken out and extracted
2
.5.1. 14˛-Hydroxyandrost-4-en-3,17-dione (6)
with CHCl3 (3× 10 ml). The extracts were dried over MgSO , con-
4
1
H NMR (CDCl ) ı (ppm); 1.03 (s, 3H, 18-CH ) 1.20 (s, 3H, 19-
3
3
centrated in vacuo and analyzed on GC. All the experiments were
repeated three times.
−
1
CH ) 5.73 (s, 4-H). IR ꢄmax (cm ): 3492 (OH), 2930 (C–H), 1728 and
3
+
1658 (C O); GC–MS m/z (int., %) 302 [M ], 302 (89), 285 (100), 273
(
(
75), 251 (35), 227 (25), 213 (39), 183 (19), 183 (15), 149 (39), 123
44), 108 (54), 91 (72), 79 (39), 55 (31).
2.3. Preparative biotransformation
The same transformation were performed on the preparative
2
.5.2. 6ˇ,17ˇ-Dihydroxyandrost-4-en-3-one (7)
scale in 2000 ml flasks, each containing 500 ml of the cultivation
medium. The cultures were incubated under the same condi-
tions and then 200 mg of substrate dissolved in 2 ml of acetone
was added to the grown cultures. After 9 days of incubation the
1
H NMR (CDCl ) ı (ppm); 0.81 (s, 3H, 18-CH ), 1.38 (s, 3H, 19-
3
3
CH ), 3.65 (t, 1H, J = 8.4 Hz, H-17␣), 4.34 (br s, 1H, W = 15.1 Hz, H-
6
3
h
−
1
␣), 5.81 (s, 1H, H-4); IR ꢄmax (cm ): 3392 (OH), 2940 and 2881
(C–H), 1666 (C O).
mixtures were extracted with CHCl3 (3× 300 ml), dried (MgSO )
4
and concentrated in vacuo. The transformation products were
separated by column chromatography and analyzed (TLC, GC,
GC–MS).
2.5.3. 6ˇ-Hydroxyandrost-4-en-3,17-dione (8)
1
H NMR (CDCl ) ı (ppm); 0.93 (s, 3H, 18-CH ) 1.39 (s, 3H, 19-
3
3
CH ) 4.39 (br s, 1H, W = 16.7 Hz, H-6␣), 5.82 (s, 1H, H-4); IR ꢄmax
3
h
−
1
(
(
cm ): 3398 (OH), 2930 (C–H), 1731 and 1661 (C O); GC–MS m/z
int., %) 302 [M ], 302 (100), 286 (34), 273 (79), 246 (15), 232 (5),
2
.4. Analytical methods
+
2
14 (19), 189 (9), 176 (5), 149 (19), 137 (36), 124 (24), 91 (32), 79
The course of biotransformation was controlled by means of
(17), 67 (39), 55 (15).
TLC. Composition of product mixtures was established by GC.
Products were separated by column chromatography using sil-
ica gel (Kieselgel 60, 230–400 mesh, Merck) and hexane/acetone
mixture (2:1, v/v) as the eluent. Analytical TLC was carried out
on silica gel G (Merck) with different developing systems. Com-
pounds were detected by spraying the plates with H SO /CH OH
2.5.4. 6ˇ,14˛-Dihydroxypregnan-4-en-3,20-dione (9)
Crystal data: C21H30O , M = 346.45, colourless needle, crys-
w
4
tal dimensions 0.20 mm × 0.10 mm × 0.10 mm, monoclinic, space
◦
group P2 , a = 7.449(2), b = 10.116(2), c = 11.866(2) Å, ˇ = 94.35(3) ,
1
3
−3
,
2
4
3
V = 891.6(3) Å , Z = 2, Dc = 1.291 Mg m
T = 100(2) K, R = 0.046,
mixture (1:1, v/v). GC analysis was performed using a Hewlett-
Packard 5890A (Series II) GC instrument fitted with a flame
ionization detector (FID). The HP-5 (crosslinked phenyl methyl
siloxane) capillary column (30 m × 0.32 mm × 0.25 ␮m) was used to
determine the composition of product mixtures. The following tem-
wR = 0.074 (for 1181 reflections with I > 2ꢅ(I)) for 226 variables.
CCDC No. 697460.
2.5.5. 3ˇ,7˛-Dihydroxyandrost-5-en-17-one (10)
1
H NMR (CDCl3) ı (ppm); 0.86 (s, 3H, 18-CH3), 0.99 (s, 3H, 19-
◦
◦
◦
perature programme was used: 200 C (0 min)/10 C/min/270 C
CH3), 3.56 (m, 1H, Wh = 26.4 Hz, H-3␣), 3.96 (s br, 1H, Wh = 14.2 Hz,
◦
◦
−1
+
(
0 min)/30 C/min/300 C (5 min). The NMR spectra were recorded
H-7␤), 5.62 (dd, 1H, J = 5.2, 1.5 Hz, H-6); IR ꢄmax (cm ): 3393 (OH),
on DRX 500 MHz Bruker spectrometer and measured in CDCl3.
Optical rotations were measured on an Autopol IV automatic
polarimeter (Rudolph). MS analyses were performed on a Varian
Chrompack GC CP-3800 Saturn 2000GC/MS/MS with an ionizing
energy of 70 eV. IR spectra were recorded on Mattson IR 300 Thermo
Nicolet spectrophotometer. MS analyses were performed on a Var-
ian Chrompack GC CP-3800 Saturn 2000GC/MS/MS with an ionizing
energy of 70 eV.
2931 (C–H), 1731 (C O); GC–MS m/z (int., %) 304 [M ], 304 (9), 286
(100), 271 (31), 253 (15), 197 (3), 175 (5), 159 (9), 143 (6), 131 (11),
105 (7), 91 (12), 79 (7), 67 (9), 55 (5).
2.5.6. 7˛-Hydroxyandrost-4-en-3,17-dione (11)
1
H NMR (CDCl3) ı (ppm); 0.91 (s, 3H, 18-CH3) 1.21 (s, 3H, 19-
−
1
CH3) 4.09 (s, 1H, H-7␤), 5.82 (s, 1H, H-4); IR ꢄmax (cm ): 3521 and
3379 (OH), 2932 (C–H), 1731 and 1659 (C O), 1607 (C C).
Crystallographic measurement was performed at 100 K using
an Oxford Cryosystem device on
a
Kuma KM4CCD ꢀ-axis
3. Results and discussion
diffractometer with a graphite-monochromated Mo K␣ radia-
tion (ꢁ = 0.71073 Å). The data were corrected for Lorentz and
polarization effects. No absorption correction was applied. Data
reduction and analysis were carried out with the CrysAlis CCD
and CrysAlis Red programs [16]. Structure was solved by direct
methods (program SHELXS97) and refined by the full matrix
least-squares method on all F2 data using the SHELXL97 pro-
grams [17]. Non-hydrogen atoms were refined with anisotropic
displacement parameters; hydrogen atoms were placed in cal-
culated positions or found in ꢂꢃ maps. Before the last cycle
of refinement all H atoms were fixed and were allowed to
ride on their parent atoms. The Friedel pairs were merged
before the final refinement. The absolute structure was chosen
on the basis of known absolute configuration of the sub-
strate.
3.1. Identification of products
The products structures were determined by means of IR, 1H
NMR, 13C NMR and correlation spectroscopy (HQMC). All the prod-
ucts obtained proved to be secondary alcohols. The locations and
orientations of the introduced hydroxyl groups were determined
on the basis of the chemical shifts and the shapes of CHOH signals
1
13
in the H NMR spectra and also confirmed by C NMR and corre-
lation NMR spectroscopy (Table 1). For compound 9 the structure
was additionally confirmed by crystallographic analysis.
3.1.1. 14˛-Hydroxyandrost-4-en-3,17-dione (6)
The mass of the molecular ion (M 302) is larger by 16 than the
one for androstenedione (1), which indicates hydroxylation of the
+
Crystallographic data for crystal 9 in this paper, have been
deposited with the Cambridge Crystallographic Data Centre as
supplementary publication CCDC 697460. Copies of the data can
be obtained, free of charge, on application to CCDC, 12 Union
substrate. Whereas, lack of a signal in the range 3–5 ppm in the
H NMR spectrum indicates that the hydroxylation occurred at a
tertiary carbon atom. The singlet of 18-CH3 is significantly shifted
1
towards lower field by 0.13 ppm, and also the signals of 19-CH and
3

T. Janeczko et al. / Steroids 74 (2009) 657–661
659
Table 1
signal of a vinyl proton at C-6 is shifted by 0.26 ppm towards
lower field compared to the substrate, which indicates the pres-
ence of an adjacent polar substituent. Additionally, the chemical
shifts of the methyl groups – 18-CH3 and 19-CH3 – 0.99 and
1
3 C NMR chemical shifts of products in CDCl3.
Atom number
Products
6
7
8
10
35.9
11
36.2
0
.86 ppm, respectively, confirm that the product is 7␣-hydroxy-
1
2
3
4
5
6
7
8
9
37.9
33.6
200.6
123.5
171.7
33.1
32.5
37.9
46.6
38.7
19.0
35.4
52.7
78.1
24.3
29.6
220.3
17.2
35.4
33.8
37.4
34.3
DHEA (10). The spectral data observed are consistent with the lit-
erature data for this compound [21].
31.2
71.2
41.9
146.7
123.6
64.3
37.2
42.7
37.3
20.1
31.4
47.1
44.9
21.9
36.9
220.0
13.3
18.3
34.1
200.2
126.3
168.2
72.8
37.0
29.9
53.5
37.8
20.5
37.9
42.8
50.4
23.1
30.4
81.5
11.0
199.1
126.8
167.6
31.4
67.0
39.9
45.1
38.3
20.0
30.8
47.2
45.4
21.1
35.5
220.6
13.3
16.9
200.6
126.5
168.5
72.8
35.9
29.7
53.8
38.3
20.4
37.1
47.9
50.9
21.9
31.4
220.8
13.9
19.7
3
.1.5. 7˛-Hydroxyandrost-4-en-3,17-dione (11)
In the 1H NMR spectrum of 11 the signal of 4-H is shifted
towards lower field by 0.08 ppm, compared to androstenedione (1).
At ı = 4.09 ppm there is a signal visible of the shape typical for 7␣-
hydroxy derivatives of 4-en steroids [22]. The signals of angular
methyl groups are in consistency with the literature [18]. In the IR
1
0
1
1
1
2
−
1
spectrum the hydroxyl group is visible at 3321 cm , the carbonyl
1
3
−
1
−1
.
1
4
groups in the A-ring at 1659 cm and in the D-ring at 1731 cm
.2. Interpretation of using results
Previous research on optically pure bicyclic enones [23] showed
1
5
1
6
3
1
7
1
8
19
17.7
19.4
that the strain Chaetomium sp. KCH 6651 contains active monooxy-
genases. This was an inspiration for further study on this
microorganism as a biocatalyst in transformations of steroid com-
pounds.
The strain Chaetomium sp. KCH 6651 appeared to have an abil-
ity to hydroxylate tertiary carbon atoms in the tested steroid
substrates, which rarely occurs in microbial transformations. The
transformation of androstenedione (1) gave 14␣-hydroxyderivative
4
-H are slightly shifted, which suggest the presence of 14␣ hydroxyl
group [18]. Additionally, the structure of product 6 is confirmed by
the IR spectrum: there is a strong absorption band of a hydroxyl
group visible at 3492 cm , the carbonyl group in the A ring is
confirmed by the signal at 1658 cm and the carbonyl group in
the D-ring by the signal at 1728 cm . The spectral data obtained
for 6 are consistent with the literature data for 14␣-hydroxy-
androstenedione (6) [19].
−1
−1
−1
(6) in the yield over 75% and 6␤-hydroxyandrostenedione (8) in 14%
yield. Small amounts of testosterone – the product of androstene-
dione reduction – were observed in the reaction mixture after 24 h.
Testosterone under these conditions underwent 6␤-hydroxylation
and then again oxidation to give 6␤-hydroxyandrostenedione
3.1.2. 6ˇ,17ˇ-Dihydroxyandrost-4-en-3-one (7)
1
In the H NMR, signals of 4-H and 19-CH3 protons are shifted
towards lower field by 0.10 and 0.20 ppm, compared to the sub-
strate. These shifts and a wide singlet of an equatorial proton at
ı = 4.33 ppm, bound with secondary carbon atom, indicate that
hydroxyl group is located at C-6 and 6␤ oriented. A triplet at
(
Scheme 1).
Testosterone (2) in the culture of Chaetomium sp. KCH 6651
underwent hydroxylation at 6␤ position. In the course of the trans-
formation, gradual oxidation of the hydroxyl group at C-17 in the
resulting 6␤-hydroxytestosterone (7) was also observed, leading to
3
.65 ppm present in the spectrum, which is typical for 17␤ steroid
alcohols, described in the literature, unequivocally confirms that
␤-hydroxytestosterone (7) is the isolated metabolite. The NMR
spectral data are highly consistent with the literature [20].
6
(
␤-hydroxyandrostenedione (8). After a short time of the reaction
24 h) the presence of androstenedione (1) was also noticed, which
was next transformed to 14␣-hydroxyandrost-4-en-3,17-dione (6)
6
−1
In the IR spectrum, a signal at 3392 cm indicates the presence
(
Scheme 1, Table 2).
In the course of transformation of both androstenedione and
−
1
of a hydroxyl group, whereas a strong absorption band at 1666 cm
is typical for ␣,␤-unsaturated ketone in a six-member ring.
testosterone in the culture of the tested strain we have observed
two competitive processes: oxidation and reduction at C-17, as
well as hydroxylation of non-activated carbon atoms. In the case of
the transformation of androstenedione the hydroxylation is more
effective than the reduction of C-17 carbonyl group. This leads to
high yield of 14␣-hydroxyandrost-4-en-3,17-dione (6). Whereas
testosterone undergoes in similar degree oxidation at C-17 and
hydroxylation, which results in similar yields of the isolated
3
.1.3. 6ˇ-Hydroxyandrost-4-en-3,17-dione (8)
The molecular ion peak (M 302) observed in the GC-MS spec-
+
trum of this compound indicates that there is an androstenedione
oxidation product. In the 1H NMR of 8 the singlet of 19-CH3 is
shifted towards lower field by 0.18 ppm and the one of 4-H by
0.08 ppm compared to androstenedione, which indicate the pres-
ence of 6␤ hydroxyl group. The structure is confirmed by the IR
1
7
4␣-hydroxy derivative 6 and the sum of hydroxy derivatives
and 8.
Progesterone (3) in the culture of Chaetomium sp. KCH 6651
−
1
spectrum: a hydroxyl group is visible at 3398 cm , a strong band
−1
at 1661 cm confirms the presence of a carbonyl group in the A-
ring, whereas a signal at 1731 cm shows the carbonyl group in the
−1
underwent complete consumption after 3 days of the reac-
tion. Enzymatic system of the tested microorganism transformed
D-ring. The spectral data obtained for 6␤-hydroxyandrostenedione
(
8) are highly consistent with the literature [18].
Table 2
3
.1.4. 3ˇ,7˛-Dihydroxyandrost-5-en-17-one (10)
The molecular ion (M 304) in the GC-MS spectrum of 10 indi-
Composition of the reaction mixture after 4-day transformation of androstenedione
(1) and testosterone (2) in Chaetomium sp. KCH 6651 culture.
+
cates hydroxylation of substrate 5. In the 1H NMR an additional
signal at ı = 3.96 ppm (s br, 1H, W = 14.2 Hz, H-7␤) is observed,
in the region specific for secondary alcohols. The multiplet at
Products obtained
Yield of product
h
From substrate (1)
From substrate (2)
3
.56 ppm (m, 1H, W = 26.4 Hz, H-3␣), which has an identical
6␤-Hydroxytestosterone (7)
–
75%
14%
19%
39%
21%
h
14␣-Hydroxyandrostenedione (6)
shape and chemical shift as H-3␣ in the substrate spectrum,
confirms the retained A-ring structure of the substrate (5). The
6␤-Hydroxyandrostenedione (8)

6
60
T. Janeczko et al. / Steroids 74 (2009) 657–661
Scheme 1. Transformations of androstenedione (1) and testosterone (2) in Chaetomium sp. KCH 6651 culture.
Fig. 1. Crystal structure of 6␤,14␣-dihydroxyprogesterone (9).
it to
dihydroxyprogesterone (9) (Fig. 1, Scheme 2), and in a high yield
78%). Compound 9 may be used for instance as a substrate in syn-
a single product of a double hydroxylation—6␤,14␣-
(
thesis of 6␤,14␣-dihydroxyandrostendione, the compound of a high
androgenic activity, which is used as a breast cancer inhibitor.
The strain Chaetomium sp. KCH 6651, as we mentioned earlier,
possess a special ability for monohydroxylation of C-19 steroids in
Scheme 2. Transformation of progesterone (3) and pregnenolone (4) in Chaetomium
sp. KCH 6651 culture.
6␤ and 14␣ positions. Probably, in the transformation of proges-
terone take part at the same time monooxygenases responsible for
both 6␤-hydroxylation of testosterone and 14␣-hydroxylation of
androstenedione.
Pregnenolone (4) in the culture of the same strain underwent
complexed enzymatic processes, leading to several different prod-
ucts. Due to their low yields, they were not isolated (Scheme 2).
The result of the pregnenolone (4) transformation was surpris-
ing. We expected a course of the biotransformation similar to
either progesterone or DHEA (which is also a 5-en steroid com-
pound). After 1 day of DHEA (5) transformation by Chaetomium
sp. KCH 6651 (Scheme 3) the allyl 7␣-hydroxy derivative (10)
was obtained, which in the next 24 h was transformed to 7␣-
hydroxyandrostendione (11)—the compound used in industrial
synthesis of diuretics [15].
The results of our research prove strongly that steric factors asso-
ciated with substrates have influence on location and configuration
of the introduced hydroxyl group. If the carbonyl group is present in
a steroid substrate, as in the case of androstendione, the hydroxyl
group is introduced in 14-␣ position by the enzymatic system of
Chaetomium sp. KCH 6651. Whereas, if there is a hydroxyl group
Scheme 3. Transformation of DHEA (5) in the culture of Chaetomium sp. KCH 6651.

T. Janeczko et al. / Steroids 74 (2009) 657–661
661
in the substrate at C-17, then oxidation at C-6 occurs. In the case
of progesterone, which possesses an acyl group at C-17, double
hydroxylation at 6␤ and 14␣ takes place at the same time.
[8] Young J, Corpechot C, Haug M, Gobaille S, Baulieu EE, Robel P. Suppressive effects
of dehydroepiandrosterone and 3-beta-methyl-androst-5-en-17-one on attack
towards lactating female intruders by castrated male mice. II. Brain neuros-
teroids. Biochem Biophys Res Commun 1991;174:892–7.
The course of transformation of 5-en steroids by the tested strain
is much more complicated. DHEA (5) undergoes hydroxylation at
activated allyl position, however, after the next 24 h the product of
double bond isomerisation along with oxidation at C-3 is observed.
In the case of pregnenolone (4), several products are formed. This
is probably due to the two effects superimposed: the substrate may
be transformed in a similar way to both progesterone (3) and DHEA
[9] Fernandes M, Cruz A, Angelova B, Pinheiro HM, Cabral JMS. Microbial con-
version of steroid compounds: recent developments. Enzyme Microb Technol
2003;32:688–705.
[
10] Lafaye P, Chmielewski V, Nato F, Mazie J-C, Morfin R. The 7␣-hydroxysteroids
produced in human tonsils enhance the immune response to tetanus tox-
oid and Bordetella pertussis antigens. Biochim Biophys Acta 1999;1472:
222–31.
[
11] Morfin R. Involvement of steroids and cytochromes P(450) species in the
triggering of immune defenses. J Steroid Biochem Mol Biol 2002;80:273–
90.
12] Morfin R, Courchay G. Pregnenolone and dehydroepiandrosterone as precursors
of native 7-hydroxylated metabolites which increase the immune response in
mice. J Steroid Biochem Mol Biol 1994;50:91–100.
(
5).
Therefore, each of the tested substrates, which are very impor-
[
tant compounds in biochemistry of higher organisms, has its
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