The microbiological hydroxylation of 3α,5-cycloandrostanes by Cephalosporium aphidicola

Article abstract of DOI:10.1016/S0031-9422(99)00415-X

The microbiological hydroxylation of some 3α,5-cycloandrostanes by the fungus, Cephalosporium aphidicola has been shown to take place at C-2α and C-14α and a 6β-alcohol was oxidized to the 6-ketone.

Full text of DOI:10.1016/S0031-9422(99)00415-X

Phytochemistry 52 (1999) 1279±1282
The microbiological hydroxylation of 3a,5-cycloandrostanes by
Cephalosporium aphidicola
Caroline S. Bensasson, James R. Hanson*, Yvan Le Huerou
School of Chemistry, Physics and Environmental Science, University of Sussex, Brighton, Sussex, BN1 9QJ, UK
Received 18 May 1999; accepted 14 July 1999
Abstract
The microbiological hydroxylation of some 3a,5-cycloandrostanes by the fungus, Cephalosporium aphidicola has been shown
to take place at C-2a and C-14a and a 6b-alcohol was oxidized to the 6-ketone. # 1999 Elsevier Science Ltd. All rights
reserved.
Keywords: Microbiological hydroxylation; Cephalosporium aphidicola; Steroids; 3a,5-cycloandrostanes
1. Introduction
ated (Prochazka, Budesinsky & Prekajski, 1974) at C-
2a, C-11a and C-7b by R. nigricans whilst the 6b-
The microbiological hydroxylation of steroids pro-
vides a useful mild synthetic method for obtaining
access to rare steroids (Mahato & Majumdar, 1993).
The cyclopropane ring of the 3a,5-cyclosteroids is sen-
sitive to a variety of chemical reagents restricting
many chemical transformations. The ®ssion of a cyclo-
propane ring in the presence of an adjacent radical has
provided a probe for enzyme mechanism (Suckling,
1988; Nonhebel, 1993). Chemical studies have shown
that the cyclopropane ring of a 3a,5-cyclocholestane
undergoes ®ssion induced by an adjacent C-6 radical
(Cristol & Barbour, 1968). In continuation of our stu-
dies (Bensasson, Chevolot, Hanson & Quinton, 1999)
on the application of Cephalosporium aphidicola to the
microbiological hydroxylation of steroids, we have
examined the transformation of the 3a,5-cycloandros-
tanes 1, 3 and 6.
methoxy
analogue
was
hydroxylated
(Thoa,
Prochazka, Budesinsky & Kocovsky, 1978) at C-1b.
The fungus, Calonectria decora, has been shown
(Chambers et al., 1975) to hydroxylate 6b-hydroxy-
3a,5-cycloandrostan-17-one 1 at C-11a and the corre-
sponding diketone 3 at C-2a, C-11a, C-15a and C-19.
2. Results and discussion
Incubation of 6b-hydroxy-3a,5-cycloandrostan-17-
one 1 with C. aphidicola for 8 days gave six metab-
olites (see Table 1) which were separated by chroma-
tography. The ®rst metabolite to be isolated was 14a-
hydroxy-3a,5-cycloandrostane-6,17-dione 4. The 13C-
NMR spectrum (see Table 2) possessed a carbonyl sig-
nal at dC 209.9 in place the secondary alcohol at dC
73.3 and a tertiary alcohol at dC 81.0 in place of a
methine signal in the starting material. Hence the 6b-
alcohol had been oxidized to a ketone. The location of
the new tertiary alcohol at C-14a followed from the
down®eld shift of the signals assigned to C-8, C-13
and C-15 ꢀDd 7.6, 5.4 and 11.8 ppm, respectively)
together with g-gauche shieldings for the signals
assigned to C-7, C-12 and C-16 ꢀDd 3.0, 6.6 and 5.7
Prior studies (Holland, Chernishenko, Conn,
Munoz, Manoharan & Zawadski, 1990) have shown
that 17b-hydroxy-3a,5-cycloandrostane was hydroxyl-
ated at C-2a and C-7b by Rhizopus arrhizus. 6b-
Hydroxy-3a,5-cycloandrostan-17-one 1 was hydroxyl-
* Corresponding author.
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00415-X

1280
C.S. Bensasson et al. / Phytochemistry 52 (1999) 1279±1282
Table 1
Hydroxylation of 3a,5-cycloandrostanes by C. aphidicola
The third metabolite was 6b,14a-dihydroxy-3a,5-
cycloandrostan-17-one 2. The 13C-NMR spectrum
showed that one C±H resonance of the starting ma-
terial had been replaced by a tertiary alcohol ꢀdC 82.1).
This was located at C-14a from the pattern of down-
®eld shifts and g-gauche shieldings of the resonances
assigned to C-8, C-13 and C-15 on the one hand and
C-7, C-12 and C-16 on the other. The fourth and ®fth
metabolites were identi®ed as 3b,7a- and 3b,7b-dihy-
droxyandrost-5-en-17-one 10 and 11 (Dodson,
Nicholson & Muir, 1959; Crabb, Dawson & Williams,
1980; Bensasson, Hanson & Hunter, 1998) from their
1H- and 13C-NMR spectra. The ®nal metabolite was
the known 3b,5a,6b-trihydroxyandrostan-17-one 12
(Bensasson, Hanson & Hunter, 1998).
Substrate
Product % Yield
6b-Hydroxy-3a,5-cycloandrostan-17-one
14a-Hydroxy-3a,5-cycloandrostane-6,17-dione
3b,14a-Dihydroxyandrost-5-en-17-one
6b,14a-Dihydroxy-3a,5-cycloandrostan-17-one
3b,7a-Dihydroxyandrost-5-en-17-one
3b,7b-Dihydroxyandrost-5-en-17-one
3b,5a,6b-Trihydroxyandrostan-17-one
3a,5-Cycloandrostane-6,17-dione
1
4
2
6
9
2
10
15
13
9
10
11
12
3
14a-Hydroxy-3a,5-cycloandrostane-6,17-dione
2a-Hydroxy-3a,5-cycloandrostane-6,17-dione
3a,5-Cycloandrost-6-en-17-one
4
9
6
5
6
6b,7a-Dihydroxy-3a,5-cycloandrostan-17-one
3b,7a-Dihydroxyandrost-5-en-17-one
7
10
15
7
Incubation of 3a,5-cycloandrostane-6,17-dione
gave 14a-hydroxy-3a,5-cycloandrostane-6,17-dione
3
4
ppm, respectively). The second metabolite, 3b,14a-
dihydroxyandrost-5-en-17-one 9, lacked the 1H- and
13C-NMR signals associated with the cyclopropane
ring possessing instead signals characteristic of a sub-
and 2a-hydroxy-3a,5-cycloandrostane-6,17-dione 5.
The H-NMR spectrum of the latter possessed a new
1
CH(OH) signal at dH 4.63 as a doublet (J = 12.5 Hz)
of triplets (J = 4 Hz). The location of this hydroxyl
group at C-2 followed from the down®eld shifts of the
signals assigned to C-1 and C-3 ꢀDd 8.4 and 2.7 ppm,
respectively) and a g-gauche shielding for the signal
assigned to C-4 ꢀDd 2.3 ppm) when compared to the
starting material 3. The stereochemistry of the hy-
droxyl group followed from the multiplicity of the
CH(OH) resonance (one diaxial and two axial: equa-
torial couplings) and from the NOE enhancement (ca.
1%) of the signal on irradiation of the H-19 resonance
ꢀdH 1.04).
stituted
3b-hydroxyandrost-5-en-17-one.
These
1
included H-NMR signals at dH 3.54 (tt, J = 4.5 and
11 Hz) and 5.42 (d, J = 2.2 Hz) assigned to H-3 and
H-6 and 13C-NMR signals at dC 73.0, 142.1 and 122.2
assigned to C-3, C-5 and C-6. The location of the sec-
ond hydroxyl group at C-14a followed from the down-
®eld shifts of the resonances assigned to C-8, C-13 and
C-15 ꢀDd 5.7, 6.1 and 12.9 ppm, respectively) and the
g-gauche shieldings of the signals assigned to C-7, C-
12 and C-16 ꢀDd 4.7, 5.6 and 9.9 ppm, respectively)
when compared to 3b-hydroxyandrost-5-en-17-one 8.
Incubation of 3a,5-cycloandrost-6-en-17-one 6 with
C. aphidicola gave 6b,7a-dihydroxy-3a,5-cycloandro-
stan-17-one 7. In the 1H-NMR spectrum of this bio-
transformation product, the alkene C±H signals had
been replaced by two CH(OH) signals at dH 3.86 and
3.22. This metabolite was then identi®ed by compari-
son with an authentic sample of the diol (Cambie,
Thomas & Hanson, 1975). No hydroxylation products
were obtained from the incubation of 3a,5-cycloandro-
stan-17-one.
Table 2
13C-NMR Spectra of steroids 1±9
Carbon atom Steroid
1
2
3
4
5
6
7
8
9
1
33.6 31.2 33.4 33.8 41.8 31.4 33.1 37.2 37.2
25.4 25.4 25.8 26.2 71.9 25.0 24.9 31.5 31.6
24.7 24.7 35.5 36.1 38.2 25.7 24.5 71.4 71.5
11.6 12.2 11.8 12.5 9.5 14.7 9.4 42.2 42.2
38.9 39.3 46.3 46.8 46.4 42.6 35.5 141.3 141.6
73.3 73.9 208.5 209.9 207.4 132.9 77.1 120.8 120.7
35.9 33.7 43.4 38.9 43.9 124.8 70.0 31.5 31.2
29.9 32.7 34.3 37.5 34.4 35.9 33.9 31.5 34.2
47.8 41.3 46.2 40.0 48.5 46.3 39.7 50.3 44.0
42.9 43.6 46.7 46.7 45.8 36.7 42.9 36.7 36.8
21.9 21.0 22.1 21.2 22.5 21.5 21.8 20.4 20.4
31.7 25.4 31.4 25.1 31.7 31.8 31.4 30.8 25.1
47.9 53.3 47.8 53.3 48.2 48.4 47.7 47.5 54.5
51.4 82.0 51.9 81.0 52.2 50.1 45.8 51.8 82.2
21.7 33.7 21.6 33.5 22.0 21.8 21.2 21.8 33.6
35.8 30.5 35.7 30.2 36.1 35.9 35.8 35.8 30.5
2
Products involving the cleavage of the cyclopropane
ring were detected in the fermentations involving 6b-
hydroxy-3a,5-cycloandrostan-17-one 1 and in the fer-
mentation in which the 6b,7a-diol 7 was formed. The
possibility was explored that these were artefacts aris-
ing from an acid-catalysed reversal of the i-steroid
reaction in the acidic medium. 6b-Hydroxy-3a,5-
cycloandrostan-17-one 1 was shaken for 8 days with
sterile medium at the natural pH (4.5) and in media in
which the pH had been adjusted to pH 1.5 and 3.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
1
The crude extract was then examined by H-NMR.
In both of the latter, the cyclo-steroid was converted
to 3b-hydroxyandrost-5-en-17-one 8 whilst at the natu-
ral pH there was a 20% conversion. It therefore seems
probable that the metabolites containing the 3b-hydro-
221.3 219.3 220.0 218.7 221.2 220.7 221.2 221.3 218.7
13.9 18.5 13.7 18.2 14.2 13.9 13.6 13.5 17.6
20.2 20.6 19.7 20.0 20.0 17.8 20.2 19.4 19.4

C.S. Bensasson et al. / Phytochemistry 52 (1999) 1279±1282
1281
xyandrost-5-ene moiety arose via an acid-catalysed
rather than from a microbial cleavage of the cyclopro-
pane ring. The biotransformations with C. aphidicola
follow the pattern of other fungal hydroxylations of
3a,5-cyclo-steroids in which the cyclopropane ring is
not readily cleaved by the micro-organism even though
microbial reaction occurs at an adjacent centre. This
particular biotransformation provides access to C-2a
and C-14a. Chemical hydroxylation of C-14a normally
involves oxidation with chromium trioxide (Saint-
Andre et al., 1952) and would be precluded by the pre-
sence of the cyclopropane ring. The formation of the
6b,7a-diol from the 6,7-ene may involve an acid-cata-
lysed cleavage of the 6a,7a-epoxide.
mined at 75 MHz. IR Spectra were recorded as nujol
mulls. Chromatography was carried out on silica,
Merck 9385. Light petroleum refers to the fraction
b.p. 60±808. Extracts were dried over anhydrous
sodium sulfate. Cephalosporium aphidicola was cultured
as described previously (Hanson & Nasir, 1993).
3.1. Biotransformation of 6b-hydroxy-3a,5-
cycloandrostan-17-one 1
The substrate 1 (2g) in DMSO (25 cm3) and EtOH
(5 cm3) was evenly distributed between 50 ¯asks of a 3
day old culture of C. aphidicola. After a further 8
days, the mycelium was ®ltered and the broth was
extracted with EtOAc. The extract was dried and the
solvent was evap. to give a residue which was chro-
matographed on silica. Elution with 30% EtOAc: pet-
rol gave the starting material (181 mg). Elution with
35% EtOAc: petrol gave 14a-hydroxy-3a,5-cycloan-
3. Experimental
1H-NMR Spectra were recorded in deuteriochloro-
form at 300 MHz and 13C-NMR spectra were deter-

1282
C.S. Bensasson et al. / Phytochemistry 52 (1999) 1279±1282
drostane-6,17-dione 4 (48 mg) which crystallized from
EtOAc: petrol as needles, m.p. 265±2708 (found: C,
75.5; H, 8.8. C19H26O3 requires C 75.5; H, 8.7%), nmax
3440, 1739, 1696 cm 1; dH 0.78 (1H, t, J = 4.5 Hz, H-
3), 1.05 and 1.06 (each 3H, s H-18 and H-19). Elution
with 37% EtOAc: petrol gave 3b,14a-dihydroxyan-
drost-5-en-17-one 9 (114 mg) which crystallized from
EtOAc: petrol as needles, m.p. 205±2098 (Saint-Andre,
MacPhillamy, Nelson, Shabica & Scholz, 1952, 2158),
nmax 3386, 3200, 1729 cm 1; dH 1.03 (3H, s, H-18),
1.05 (3H, s, H-19), 3.54 (1H, tt, J = 4.5 and 11 Hz,
H-3), 5.42 (1H, d, J = 2.2 Hz, H-6). Elution with
40% EtOAc: petrol gave 6b,14a-dihydroxy-3a,5-
cycloandrostan-17-one 2 (194 mg) which crystallized
from EtOAc: petrol as needles, m.p. 118±1208 (found:
C, 74.3; H, 9.7. C19H28O3 requires C, 74.9; H, 9.3%),
nmax 3413, 1733 cm 1; dH 1.06 (3H, s, H-18), 1.11 (3H,
s, H-19), 3.39 (1H, t, J = 3 Hz, H-6). Elution with
45% EtOAc: petrol gave 3b,7b-dihydroxyandrost-5-en-
17-one 11 (269 mg) which crystallized from EtOAc:
petrol as needles, m.p. 2078 (Dodson et al., 1959, 215±
2168), identi®ed by comparison (IR and NMR) with
an authentic sample. Elution with 60% EtOAc: petrol
gave 3b,7a-dihydroxyandrost-5-en-17-one 10 (292 mg)
which crystallized from chloroform as needles, m.p.
1778 (Dodson et al., 1959, 1828), identi®ed by compari-
son (IR and NMR) with an authentic sample. Elution
with 20% MeOH: EtOAc gave 3b,5a,6b-trihydroxyan-
drostan-17-one 12 (180 mg) which crystallized from
MeOH: EtOAc as prisms, m.p. 298±3018 (Holland &
Diakow, 1979), identi®ed by comparison (IR and
NMR) with an authentic sample.
3.3. Biotransformation of 3a,5-cycloandrost-6-en-17-one 6
The substrate 6 (1.2 g) in DMSO (30 cm3) and
EtOH (10 cm3) was evenly distributed between 50
¯asks of C. aphidicola 3 days after inoculation. After a
further 8 days the mycelium was ®ltered and the broth
was extracted with EtOAc. The extract was dried and
the solvent evap. to give a residue which was chro-
matographed on silica. Elution with 10% EtOAc: pet-
rol gave the starting material 6 (360 mg). Elution with
80% EtOAc: petrol gave 6b,7a-dihydroxy-3a,5-
cycloandrostan-17-one 7 (332 mg) which crystallized
from EtOAc: petrol as needles, m.p. 1708 (Cambie et
al., 1975, 1728); nmax 3450, 1730 cm 1; dH 0.27 (1H, dd
J = 5 and 8 Hz, H-4), 0.52 (1H, t, J = 5 Hz, H-4),
0.93 (3H, s, H-18), 1.07 (3H, s, H-19), 3.22 (1H, d, J
= 3 Hz, H-6), 3.86 (1H, m, H-7). Elution with EtOAc
gave 3b,7a-dihydroxyandrost-5-en-17-one 10 (170 mg)
identical to the material described above.
References
Bensasson, C. S., Hanson, J. R.,
Phytochemistry, 49, 2355.
& Hunter, A. C. (1998).
Bensasson, C. S., Chevolot, Y., Hanson, J. R., & Quinton, J. (1999).
Phytochemistry, 50, 25.
Cambie, R. C., Thomas, P. W., & Hanson, J. R. (1975). Journal of
the Chemical Society Perkin Trans, 1, 323.
Chambers, V. E. M., Denny, W. A., Jones, Sir Ewart R. H.,
Meakins, G. D., Miners, J. O., Pinhey, J. T., & Wilkins, A. L.
(1975). Journal of the Chemical Society Perkin Trans, 1, 1359.
Crabb, T. A., Dawson, P. J., & Williams, R. O. (1980). Journal of
the Chemical Society Perkin Trans, 1, 2535.
3.2. Biotransformation of 3a,5-cycloandrostane-6,17-
dione 3
Cristol, S. J., & Barbour, R. V. (1968). Journal of the American
Chemical Society, 90, 2832.
The substrate 3 (1.5 g) in DMSO (25 cm3) and
EtOH (5 cm3) was evenly distributed between 50 ¯asks
of C. aphidicola 3 days after inoculation. After a
further 8 days the mycelium was ®ltered and the broth
was extracted with EtOAc. The extract was dried and
evap. and the residue chromatographed on silica.
Elution with 30% EtOAc: petrol gave the starting ma-
terial 3 (125 mg). Elution with 45% EtOAc: petrol
Dodson, R. M., Nicholson, R. T., & Muir, R. D. (1959). Journal of
the American Chemical Society, 81, 6295.
Hanson, J. R., & Nasir, H. (1993). Phytochemistry, 33, 831.
Holland, H. J., & Diakow, M. A. (1979). Canadian Journal of
Chemistry, 57, 436.
Holland, H. J., Chernishenko, M. J., Conn, M., Munoz, A.,
Manoharan, T. S., & Zawadski, M. A. (1990). Canadian Journal
of Chemistry, 68, 696.
Mahato, S. B., & Majumdar, I. (1993). Phytochemistry, 34, 883.
Nonhebel, D. C. (1993). Chemical Society Reviews, 22, 347.
Prochazka, Z., Budesinsky, M., & Prekajski, P. (1974). Collection of
Czechoslovak Chemical Communications, 39, 984.
Saint-Andre, A. F., MacPhillamy, H. B., Nelson, J. A., Shabica, A.
C., & Scholz, C. R. (1952). Journal of the American Chemical
Society, 74, 5506.
gave 14a-hydroxy-3a,5-cycloandrostane-6,17-dione
4
(136 mg) identical to the material described above.
Elution with 75% EtOAc: petrol gave 2a-hydroxy-
3a,5-cycloandrostane-6,17-dione 5 (32 mg) which crys-
tallized from EtOAc: petrol as needles, m.p. 241±2438
(found: C, 75.5; H, 8.8. C19H26O3 requires C, 75.5; H,
8.7%); nmax 3401, 1734, 1697 cm 1; dH 0.92 (3H, s, H-
18), 1.04 (3H, s, H-19), 4.63 (1H, dt, J = 12.5 and 4
Hz, H-2).
Suckling, C. J. (1988). Angewandte Chemie (International Edition in
English), 27, 537.
Thoa, H. K., Prochazka, Z., Budesinsky, M., & Kocovsky, P.
(1978). Collection of Czechoslovak Chemical Communications, 43,
2305.