The microbiological hydroxylation of 4β-hydroxy-4α-methyl-5α androstanes by Cephalosporium aphidicola

Article abstract of DOI:10.1016/S0031-9422(98)00456-7

Some 4β-hydroxy-4α-methyl-5α-androstanes were synthesized and their microbiological hydroxylation by Cephalosporium aphidicola was examined in order to explore the possibility of a biosynthetically patterned methylcarbinol: vicinal glycol biotransformation; however the substrates were hydroxylated mainly at C-7.

Full text of DOI:10.1016/S0031-9422(98)00456-7

PERGAMON
Phytochemistry 50 (1999) 25±30
The microbiological hydroxylation of 4b-hydroxy-4a-methyl-5a-
androstanes by Cephalosporium aphidicola
Caroline S. Bensasson, Yvan Chevolot, James R. Hanson *, Jacques Quinton
The School of Chemistry, Physics and Environmental Science, The University of Sussex, Brighton, Sussex BN1 9QJ, U.K.
Received 26 March 1998
Abstract
Some 4b-hydroxy-4a-methyl-5a-androstanes were synthesized and their microbiological hydroxylation by Cephalosporium
aphidicola was examined in order to explore the possibility of a biosynthetically patterned methylcarbinol: vicinal glycol
biotransformation; however the substrates were hydroxylated mainly at C-7. # 1998 Published by Elsevier Science Ltd. All
rights reserved.
Keywords: Microbiological hydroxylation; Cephalosporium aphidicola; Steroids; 4b-hydroxy-4a-methyl-5a-androstanes
1. Introduction
dition of methylmagnesium iodide to steroidal ketones
shows a marked preference for the formation of the
tertiary alcohol with an equatorial methyl group [3]
although this has not been examined previously
with steroidal 4-ketones as substrates. The steroidal
4-ketones were prepared via the 4-enes. 17b-
Hydroxyandrost-4-ene (5) [4], free from double bond
isomers, has become readily available from testos-
terone (4) by using a selective reduction with sodium
borohydride in a mixture of tri¯uoroacetic acid, acetic
acid, acetonitrile and dichloromethane [5].
The biosynthesis of the diterpenoid aphidicolin (1)
by the fungus, Cephalosporium aphidicola, involves the
ecient hydroxylation of the equatorial 17-methyl of
3a,16b,18-trihydroxyaphidicolane to a€ord the 16b,17-
glycol of aphidicolin [1]. The transformation of a
methylcarbinol to a vicinal glycol exempli®ed by this
step, is dicult to achieve regiospeci®cally by chemical
means. Consequently we have explored [2] the possi-
bility of using this organism for a biosynthetically-
directed biotransformation to achieve this reaction
using steroidal methylcarbinols as test substrates.
4b,17b-Dihydroxy-4a-methyl-5a-androstane (2) and
Hydroboration of 17b-acetoxyandrost-4-ene (6) [6]
and oxidation of the borane with alkaline hydrogen
peroxide gave
a mixture of 4a,17b-dihydroxy-5a-
4b,17b-dihydroxy-4a,17a-dimethyl-5a-androstane
(3)
androstane (7) and 4b,17b-dihydroxy-5b-androstane
(8). However the protecting acetoxyl group at C-17
had been hydrolysed. Oxidation of the borane with
chromium trioxide [7] to avoid the alkaline conditions
and form the 4-ketone directly was not successful.
Attempts to circumvent the hydrolysis of the 17b-
acetate by oxidizing the borane with sodium
perborate [8] were also unsuccessful. Hence the 17b-
hydroxyl group was protected as its sterically more
hindered 17b-pivaloate (9). The mixture of 17b-
pivaloyloxy-4a- and 4b-alcohols obtained from the
hydroboration, was oxidized with chromium trioxide
to a mixture of 17b-pivaloyloxy-5a- and 5b-androstan-
4-ones, (10) and (11), without further puri®cation. This
mixture was then epimerized at C-5 to a€ord the more
were potential substrates since not only is there a
formal similarity between these steroids binding in
both their normal and reverse modes when compared
to aphidicolin but also the methyl groups at C-4 are
equatorial substituents.
2. Results and discussion
The substrates (2) and (3) were prepared from tes-
tosterone (4) and 17a-methyltestosterone (13). The ad-
* Author to whom correspondence should be sent.
0031-9422/98/$ - see front matter # 1998 Published by Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(98)00456-7

26
C.S. Bensasson et al. / Phytochemistry 50 (1999) 25±30
Table 1. Incubation of steroids with C. aphidicola
Substrate
Product
% Yield
4b,17b-Dihydroxy-4a-methyl-5a-androstane (2)
starting material
20
37
10
7
4b-hydroxy-4a-methyl-5a-androstan-17-one (16)
4b,7a-dihydroxy-4a-methyl-5a-androstan-17-one (17)
4b,15a,17b-trihydroxy-4a-methyl-5a-androstane (19)
starting material
4b,17b-Dihydroxy-4a,17a-dimethyl-5a-androstane (3)
16
15
4b,7a,17b-trihydroxy-4a,17a-dimethyl-5a-androstane (18)

C.S. Bensasson et al. / Phytochemistry 50 (1999) 25±30
27
Table 2. 13C NMR spectra of 4b-hydroxy-4a-methylandrostanes
(determined in CDCl3 at 75 MHz)
stable 5a-androstan-4-one (12) by re¯uxing with
methanolic sodium hydroxide. The C-5 epimers may
be readily distinguished by the position of the H-19
Compound
1
resonance in the H NMR spectrum [9]. The pivaloyl-
Carbon
2
3
16
17
18
19
oxy group was hydrolysed at the same time. Treatment
of the ketone with methylmagnesium iodide in ether
gave 4b,17b-dihydroxy-4a-methyl-5a-androstane (2).
We were unable to detect any of the 4b-methyl isomer.
A similar sequence was applied to 17a-methyltestos-
terone (13) except that there was no need to protect
the tertiary 17b-hydroxyl group. Thus the ring A un-
saturated ketone was reduced to the 4-ene (14) with
sodium borohydride in a mixture of tri¯uoroacetic
acid, acetic acid, acetonitrile and dichloromethane and
thence via hydroboration, oxidation and isomerization,
converted to the 4-ketone (15). Reaction with methyl-
magnesium iodide gave 4b,17b-dihydroxy-4a,17a-
dimethyl-5a-androstane (3).
1
38.74
18.11
40.91
72.17
53.84
20.05
31.94
35.08
55.84
36.67
20.42
36.71
42.82
51.23
23.31
30.53
81.94
11.11
14.05
30.83
38.81
18.16
40.97
72.20
53.93
20.15
32.13
35.96
55.82
36.72
20.51
39.05
45.42
50.93
23.20
31.66
81.72
14.10
13.98
30.86
25.78
38.69
18.07
40.97
72.12
53.79
20.33
31.22
34.62
55.80
36.77
19.76
36.71
47.68
51.65
21.71
35.85
221.45
13.80
14.02
30.87
38.47
18.03
41.20
72.13
45.94
29.23
66.90
38.73
47.53
36.95
19.55
35.76
47.46
45.94
21.31
31.13
221.10
13.48
12.95
30.78
38.45
17.93
40.82
72.03
45.81
28.75
67.14
39.88
47.21
36.70
19.75
38.45
45.20
44.55
22.45
31.06
81.47
13.53
12.78
30.24
25.43
39.37
18.56
41.43
71.32
54.27
20.31
33.18
35.34
56.16
37.07
21.03
37.58
44.63
59.30
72.14
43.14
78.67
13.16
14.41
31.05
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
4-Me
17-Me
The stereochemistry at C-4 followed from an examin-
ation of the 1H NMR spectra of the 4b-hydroxy-4a-
methyl-5a-androstanes. Whilst there was no n.O.e.
enhancement of the C-4 methyl group signal on ir-
radiation of the C-10 methyl group signal, the trans-

28
C.S. Bensasson et al. / Phytochemistry 50 (1999) 25±30
annular 1:3-diaxial interaction with a 4b-hydroxyl group
produced a signi®cant down®eld shift (Dd 0.24 ppm) in
the position of the C-10 methyl group signal when com-
pared to 17b-hydroxy-5a-androstane [9].
the fermentation whilst those involved in aphidicolin
biosynthesis may only be present for a limited period,
for example during the idiophase, of the fermentation.
The substrates were incubated with C. aphidicola for
8 days and the metabolites were then isolated and sep-
arated by chromatography. The results are tabulated
(see Table 1). The metabolites were identi®ed from
3. Experimental
1H NMR spectra were recorded in deuteriochloro-
form at 300 or 500 MHz. 13C NMR spectra were
determined 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 sodium sulfate.
Jones reagent refers to a solution of chromium trioxide
(26.72 g) in conc. sulfuric acid (23 cm3) diluted to
100 cm3 with water. Cephalosporium aphidicola was
cultured as described previously [13].
1
their H and 13C NMR spectra (see Table 2).
The major metabolite from the incubation of
4b,17b-dihydroxy-4a-methyl-5a-androstane (2) was the
17-ketone (16). The H-17a signal at dH 3.61 had dis-
1
appeared from the H NMR spectrum whilst the 13C
NMR spectrum (see Table 2) showed a new carbonyl
resonance at dC 221.45 ppm. The second metabolite to
be isolated from the column, was assigned the struc-
ture of 4b,7a-dihydroxy-4a-methyl-5a-androstan-17-
one (17). The H-17a signal had disappeared and a car-
bonyl signal had appeared at dC 221.1 ppm. The 13C
NMR spectrum also contained a CH(OH) resonance
at dC 66.70 ppm in place of a methylene carbon.
Compared to the starting material, the signals assigned
to C-6 and C-8 had moved down®eld (Dd 8.81 and
3.65 ppm respectively) whilst there were g-gauche
up®eld shifts for the signals assigned to C-5, C-9 and
C-14 (Dd 7.90, 8.31 and 5.29 ppm respectively).
Consequently the new alcohol was located at C-7. The
1H NMR spectrum showed a CH(OH) signal at dH
4.09 (doublet, J 2.5 Hz) resembling that of a 7a-
alcohol [10]. The third metabolite to be isolated was
assigned the structure of 4b,15a,17b-trihydroxy-4a-
methyl-5a-androstane (19). Compared to the starting
material, the 13C NMR spectrum revealed down®eld
shifts for the signals assigned to C-14 and C-16 (Dd
8.07 and 12.61 ppm, respectively) whilst there was a
g-gauche up®eld shift for the signal assigned to C-17
3.1. 17b-Pivaloyloxyandrost-4-en-3-one
Testosterone (3) (5 g) in dry pyridine (50 cm3) was
treated with pivaloyl chloride (5 g) at room temp.
overnight. The mixture was poured into dil. HCl
(200 cm3) and the steroid was recovered in EtOAc.
The extract was washed with dil. HCl, aq. NaHCO3,
H2O and dried. The solvent was evap. to give 17b-
pivaloyloxy-androst-4-en-3-one (4.53 g) which crystal-
lized from petrol as prisms, m.p. 155±1578 (found: C,
77.6; H,10.1; C24H36O3 requires C, 77.4; H, 9.7%), IR,
1
1
n max 1722, 1679, 1617 cm ; H NMR dH 0.85 (3H, s,
H-18), 1.20 (9H, s OPiv), 1.25 (3H, s, H-19), 4.57 (1H,
t, J 8.5 Hz, H-17), 5.73 (1H, s, H-4).
3.2. 17b-Pivaloyloxyandrost-4-ene (9)
NaBH4 (1.3 g) was gradually added to a stirred mix-
ture of CF3CO2H (8 cm3), HOAc (8 cm3) and CH3CN
(8 cm3) which was cooled in an ice-bath. 17b-
Pivaloyloxyandrost-4-en-3-one (2.4 g) in CH2Cl2
(40 cm3) was added to the mixture which was then stir-
red for 6 h (TLC control). Sat. aq. NaHCO3 was
added to neutralize the mixture. The steroid was
extracted with CH2Cl2. The extract was thoroughly
washed with H2O and dried. The solvent was evap. to
give 17b-pivaloyloxyandrost-4-ene (9)-(1.84 g) which
crystallized from MeOH as needles, m.p. 114±1168
1
(Dd 3.27 ppm). The H NMR spectrum showed a new
signal at dH 4.27 ppm as a triplet (J, 9 Hz) of doublets
(J, 3 Hz) consistent with the presence of a 15a-alcohol.
Incubation of 4b,17b-dihydroxy-4a,17a-dimethyl-5a-
androstane (3) gave the corresponding 7a-alcohol (18).
The presence of the 7a-alcohol was established by the
down®eld shifts of the 13C NMR resonances of C-6
and C-8 (Dd 8.6 and 3.9 ppm, respectively) and the
g-gauche shieldings for C-5, C-9 and C-14 (Dd 8.12,
8.61 and 6.38 ppm, respectively) when compared to the
starting material. The H-7b proton resonance (dH
3.95 ppm) showed a comparable multiplicity to other
7a-alcohols [10].
In conclusion the transformations which we have
observed with these substrates have followed the
typical pattern of steroidal hydroxylations by this
organism [11, 12] rather than a biosynthetically pat-
terned pathway. It may be that the systems responsible
for steroidal transformations are more readily accessible
to the exogenous substrates and are present throughout
(found: C, 80.4; H, 11.1; C24H38O2 requires C, 80.4;
1
H, 10.7%), IR, nmax 1733 cm
;
1H NMR dH 0.82
(3H, s, H-18), 1.02 (3H, s, H-19), 1.19 (9H, s, OPiv),
4.57 (1H, t, J 8.5 Hz, H-17), 5.27 (1H, br.s. H-4).
3.3. Preparation of 17b-hydroxy-5a-androstan-4-one
(12)
17b-Pivaloyloxyandrost-4-ene
(3 g)
in
THF
(100 cm3) was treated with 1 M borane in THF

C.S. Bensasson et al. / Phytochemistry 50 (1999) 25±30
29
(20 cm3) at 08 for 4 h under N2. H2O (10 cm3) was
added dropwise at 08 followed by 10%, aq. NaOH
(30 cm3) and 30% aq. H2O2 (30 cm3). The mixture was
stirred for 1 h. Na2SO3 (6 g) was then added followed
by HOAc (3 cm3), H2O (150 cm3), dil. HCl (150 cm3)
and EtOAc (150 cm3). The mixture was stirred for a
further 15 min and then the organic layer was separ-
ated, washed with H2O and dried. The solvent was
evap. to give a residue which was taken up in Me2CO
(75 cm3) and treated with the Jones reagent (3 cm3)
dropwise. The mixture was stirred for 45 min and then
MeOH was added until the soln. remained a green
colour. After 30 min the solvents were evap. and the
residue was suspended in H2O. The steroids were
extracted with EtOAc. The extract was washed with aq
NaHCO3 and H2O. The solvent was evap to give a
gum (1.5 g) which was dissolved in MeOH (50 cm3).
This soln. was treated with NaOH (8 g) in H2O
(20 cm3) under N2 and heated under re¯ux for 1 h.
The soln was cooled and neutralized with dil HCl. The
MeOH was evap. and the steroids were recovered in
EtOAc. The extract was washed with aq NaHCO3,
H2O and dried. The solvent was evap to give 17b-
hydroxy-5a-androstan-4-one (1 g) which was recrystal-
lized from petrol as needles, m.p. 1218 (lit. [14], 1258),
give 17b-hydroxy-17a-methylandrost-4-ene (14) (2 g)
which crystallized from petrol as needles, m.p. 98±1008
(found: C, 82.8; H, 11.4; C20H32O requires C, 83.3; H,
11.2%), IR, nmax 3361 cm ; H NMR dH 0.88 (3H, s,
H-18), 1.03 (3H, s, H-19), 1.20 (3H, s, H-20), 5.27
(1H, br s, H-4).
1
1
3.6. 17b-Hydroxy-17a-methyl-5a-androstan-4-one (15)
17b-Hydroxy-17a-methylandrost-4-ene (3 g) in dry
THF (100 cm3) was treated with 1 M borane in THF
(20 cm3) at 08 for 6 h under N2. H2O (10 cm3) was
added dropwise at 08 followed by 10% aq. NaOH
(30 cm3) and 30% aq. H2O2 (30 cm3). The mixture was
stirred for 1 h. Na2SO3 (6 g) was then added followed
by HOAc (3 cm3), H2O (150 cm3), dil. HCl (150 cm3)
and EtOAc (150 cm3). The mixture was stirred for a
further 15 min and then the organic layer was separ-
ated, washed with H2O and dried. The solvent was
evap. to give a residue which was taken up in Me2CO
(75 cm3) and treated with the Jones reagent (3 cm3)
dropwise. The mixture was stirred for 45 min and then
MeOH was added until the soln. remained a green
colour. After 30 min the solvents were evap. and the
residue was suspended in H2O. The steroids were
extracted with EtOAc. The extract was washed with
aq. NaHCO3 and H2O. The solvent was evap. to give
a gum (1.5 g) which was dissolved in MeOH (50 cm3).
This soln. was treated with NaOH (8 g) in H2O
(20 cm3) under N2 and heated under re¯ux for 1 h.
The soln. was cooled and neutralized with dil HCl.
The MeOH was evap. and the steroids were recovered
in EtOAc. The extract was washed with aq NaHCO3,
H2O and dried. The solvent was evap. to give 17b-
hydroxy-17a-methyl-5a-androstan-4-one (1.36 g) which
was recrystallized from EtOAc: petrol as needles, m.p.
1
1
IR nmax 3526, 1712 cm ; H NMR dH 0.74 and 0.75
(each 3H, s, H-18 and H-19), 3.61 (1H, t, J 8.5 Hz,
H-17).
3.4. 4b,17b-Dihydroxy-4a-methyl-5a-androstane (2)
17b-Hydroxy-5a-androstan-4-one (1 g) in dry THF
(30 cm3) was treated with 3 M methylmagnesium
iodide in Et2O (8 cm3) under N2 at room temp. over-
night. 10% Aq. NH4Cl (100 cm3) was added and the
mixture was left to stand for 1 h. The steroid was
extracted with EtOAc and the extract was washed
with brine and dried. The solvent was evap. to give
4b,17b-dihydroxy-4a-methyl-5a-androstane (2) (0.7 g)
which was recrystallized from MeOH as needles, m.p.
186±1898 (found: C, 78.4; H, 11.4. C20H34O2 requires
149±1518 (found: C, 77.2; H, 10.5. C20H32O2 requires
1
C, 76.7; H, 10.5%) IR nmax 3344, 1711 cm
;
1H
NMR dH 0.76 (3H, s, H-19), 0.85 (3H, s, H-18), 1.22
(3H, s, H-20).
1
1
C, 78.4; H, 11.7%); IR, nmax 3383 cm . H NMR dH
0.73 (3H, s, H-18), 1.03 (3H, s, H-19), 1.15 (3H, s,
4-Me), 3.61 (1H, t, J 8.5 Hz, H-17).
3.7. 4b,17b-Dihydroxy-4a,17a-dimethyl-5a-androstane
(3)
3.5. 17b-Hydroxy-17a-methylandrost-4-ene (14)
17b-Hydroxy-17a-methyl-5a-androstan-4-one (0.8 g)
in dry THF (30 cm3) was treated with 3 M methyl-
magnesium iodide in Et2O (8 cm3) under N2 at room
temp. overnight. 10% Aq. NH4Cl (100 cm3) was added
and the mixture was left to stand for 1 h. The steroid
was extracted with EtOAc and the extract was washed
with brine and dried. The solvent was evap. to
give 4b,17b-dihydroxy-4a,17a-dimethyl-5a-androstane
(3) (0.3 g) which was recrystallized from MeOH as
needles, m.p. 155±1578 (found: C, 78.6; H, 11.5.
C21H36O2 requires C, 78.8; H, 11.3%); IR, nmax
NaBH4 (1.3 g) was gradually added to a stirred mix-
ture of CF3CO2H (8 cm3), HOAc (8 cm3) and CH3CN
(8 cm3) which was cooled in an ice-bath. 17b-Hydroxy-
17a-methylandrost-4-en-3-one (2.4 g) in CH2Cl2
(40 cm3) was added to the mixture which was then stir-
red for 4 h. (TLC control). Sat. aq. NaHCO3 was
added to neutralize the mixture. The steroid was
extracted with CH2Cl2. The extract was thoroughly
washed with H2O and dried. The solvent was evap. to

30
C.S. Bensasson et al. / Phytochemistry 50 (1999) 25±30
1
1
3400 cm ; H NMR dH 0.84 (3H, s, H-18), 1.03 (3H,
s, H-19), 1.15 (3H, s, 4-Me), 1.20 (3H, s, H-20).
tion was continued for a further 8 days. The mycelium
was ®ltered and the broth was extracted with EtOAc.
The extract was dried and the solvent evaporated to
give a residue which was chromatographed on silica.
Elution with 12% EtOAc:petrol gave the starting ma-
terial (120 mg). Elution with 25% EtOAc: petrol gave
4b,7a,17b-trihydroxy-4a,17a-dimethyl-5a-androstane
(18) (110 mg) which crystallized from CHCl3 as prisms,
3.8. Biotransformation of 4b,17b-dihydroxy-4a-methyl-
5a-androstane (2)
The substrate (2) (970 mg) 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
chromatographed on silica. Elution with 10%
EtOAc:petrol gave 4b-hydroxy-4a-methyl-5a-andro-
stan-17-one (16) (360 mg) which crystallized from
EtOAc:petrol as prisms, m.p. 196±1988 (found: C,
78.9; H, 10.2. C20H32O2 requires C, 78.9; H, 10.6%);
1H NMR dH 0.86 (3H, s, H-18), 1.05 (3H, s, H-19),
1.17 (3H, s, Me-4). Elution with 13% EtOAc:petrol
gave the starting material (190 mg). Elution with 45%
EtOAc:petrol gave 4b,7a-dihydroxy-4a-methyl-5a-
androstan-17-one (17) (93 mg) which crystallized from
EtOAc:petrol as prisms, m.p. 203±2058 (found: C,
74.9; H, 10.1. C20H32O3 requires C, 75.0; H, 10.1%);
1H NMR dH 0.87 (3H, s, H-18), 1.07 (3H, s, H19),
1.21 (3H, s, Me-4), 4.09 (1H, d, J 2.5 Hz, H-7).
Further elution gave 4b,15a,17b-trihydroxy-4a-methyl-
5a-androstane (19) (72 mg) which crystallized from
EtOAc:petrol as prisms, m.p. 263±2658 (found: C,
74.2; H, 10.9. C20H34O3 requires C, 74.5; H, 10.6%);
m.p. 168±1708 (found: C, 70.8; H, 10.5. C21H36O3.
1
H2O requires C, 71.1; H, 10.8%), IR, n max 3495 cm
;
dH 0.84 (3H, s, H-18), 1.04 (3H, s, H-19), 1.15 (3H, s,
Me-4), 1.22 (3H, s, H-20), 3.95 (1H, d, J 2 Hz, H-7b).
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