Steroid transformations with Fusarium oxysporum var. cubense and Colletotrichum musae

Article abstract of DOI:10.1016/S0039-128X(99)00067-7

The utility of two locally isolated fungi, pathogenic to banana, for steroid biotransformation has been studied. The deuteromycetes Fusarium oxysporum var. cubense (IMI 326069, UAMH 9013) and Colletotrichum musae (IMI 374528, UAMH 8929) had not been examined previously for this potential. In general, F. oxysporum var. cubense effected 7α hydroxylation on 3β-hydroxy- Δ⁵-steroids, 6β, 12β, and 15α hydroxylation on steroidal-4-ene-3-ones, side-chain degradation on 17α,21-dihydroxypregnene-3,20-diones, and 15α hydroxylation on estrone. Both strains were shown to perform redox reactions on alcohols and ketones.

Full text of DOI:10.1016/S0039-128X(99)00067-7

Steroids 64 (1999) 834–843
Steroid transformations with Fusarium oxysporum var. cubense and
Colletotrichum musae
Maureen R. Wilson1, Winklet A. Gallimore, Paul B. Reese*
Chemistry Department, University of the West Indies, Mona, Kingston 7, Jamaica, West Indies
Received 26 August 1998; received in revised form 24 March 1999; accepted 12 April 1999
Abstract
The utility of two locally isolated fungi, pathogenic to banana, for steroid biotransformation has been studied. The deuteromycetes
Fusarium oxysporum var. cubense (IMI 326069, UAMH 9013) and Colletotrichum musae (IMI 374528, UAMH 8929) had not been
examined previously for this potential. In general, F. oxysporum var. cubense effected 7␣ hydroxylation on 3␤-hydroxy-⌬5-steroids, 6␤,
12␤, and 15␣ hydroxylation on steroidal-4-ene-3-ones, side-chain degradation on 17␣,21-dihydroxypregnene-3,20-diones, and 15␣ hy-
droxylation on estrone. Both strains were shown to perform redox reactions on alcohols and ketones. © 1999 Elsevier Science Inc. All rights
reserved.
Keywords: Fusarium oxysporum var. cubense; Colletotrichum musae; Biotransformation; Hydroxylation; Steroid; Deuteromycete
1. Introduction
high humidities, yellow-orange masses (1 mm in diameter)
appear on the tissue surface [12,13]. There are no reported
secondary metabolites from the fungus and C. musae has
not been used previously in biotransformations. However,
other strains of Colletotrichum have participated in side-
chain cleavage, dehydrogenation, and 11 and 14 hydroxy-
lation of androstane and pregnane derivatives [14]. Hy-
droxylation of an A-nor steroid in the 12-position has also
been observed [15].
During the course of our investigations into the chemis-
try of fungi [16], we decided to study the potential of these
two locally isolated deuteromycetes for biotransformation
of a series of functionalized androstanes, pregnanes, and an
estrane.
Fusarium oxysporum var. cubense (E.F. Smith) Snyder
et Hansen is a specialized parasite of the higher plants
(Musaceae family) responsible for Panama disease of ba-
nanas. The spread of this disease is quite rapid, causing
great financial loss [1,2]. There are no literature reports on
the isolation of secondary metabolites from, or biotransfor-
mation of substrates by, this fungus. However, a series of
naphthoquinones [3], trichothecenes [4], and polyketides [5]
has been isolated from other F. oxysporum subspecies. In a
similar manner, related subspecies have also been exploited
in side-chain cleavage [6], hydrogenation [7], oxidation
with isomerization [8], and hydroxylation in the 6␤ [9], 12␤
[10], and 15␣ [11] positions of steroids.
Colletotrichum musae (Berkeley et Curtis) von Arx, also
known as Gloeosporium musarum, is a widely distributed
pathogen that causes anthracnose of banana (Musa spp.) and
abaca (Musa textilis). The microorganism effects crown and
finger stalk rot, indicated when blackening and softening of
the tissues occur and this is exacerbated during ripening. At
2. Experimental
1H and 13C NMR spectra were determined at 200 and 50
MHz, respectively, on a Bruker AC 200 instrument (Bruker,
Billerica, MA, USA). The multiplicities of the carbon atoms
were determined by the attached proton test and the distor-
tionless enhancement by polarization transfer experiments.
The solvents used were deuterated chloroform with tetra-
methylsilane as the internal standard. 13C NMR data can be
found in Table 1. Mass spectral data were obtained at an
* Corresponding author. Tel.: ϩ876-927-1910; fax: ϩ876-977-1835.
E-mail address: pbreese@uwinmona.edu.jm (P.B. Reese)
1 Present address: Sugar Industry Research Institute, Kendal Road,
Mandeville, Manchester, Jamaica.
0039-128X/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(99)00067-7

M.R. Wilson et al. / Steroids 64 (1999) 834–843
835
Table 1
13C NMR resonances of transformed steroids determined in CDCl3
2* 3* 4* 8* 9* 11*
6
12*
35.6
13
35.7
14*
35.3
16
34.6
17
19
20
22*
23
24
1
2
3
37.3 37.7 37.7 35.7 35.4 37.0
37.0
31.3
34.7 155.8 154.6 126.0 126.7
33.7 127.4 127.9 118.3 112.9
37.3
31.5
71.7
42.3
27.3
72.8
37.7
27.1
71.8
35.8
27.3
73.0
36.4
30.9 35.0 34.1
33.8
33.9
33.5
33.7
71.2 199.3 199.0 199.8 199.2 199.3 198.7 200.0 199.5 186.7 186.1 148.2 153.6
41.9 123.9 124.1 128.8 123.8 124.1 123.5 124.3 124.8 124.4 124.9 121.2 115.2
4
5
146.8 164.7 146.6 146.3 170.3 170.5 162.0 170.7 170.3 170.3 169.0 167.8 167.3 165.6 138.0 137.9 140.9
6
7
8
9
120.3 126.3 120.5 123.7
32.8
32.6
34.9
53.6
38.5
20.8
38.6
43.9
59.1
75.2
31.2
60.9
14.3
17.5
33.6
32.2
33.5
51.6
38.2
26.5
78.9
48.0
57.4
74.6
30.3
58.5
9.8
73.8
36.0
30.3
53.2
37.7
20.3
36.5
42.5
50.1
23.4
27.4
82.3
12.1
18.6
32.4
31.1
34.7
53.9
38.4
20.3
36.6
42.9
53.4
74.0
37.0
32.5
31.3
35.1
53.8
38.6
20.3
30.7
47.5
50.8
21.7
35.7
32.1
30.7
34.6
53.3
38.2
33.3
32.3
36.9
62.3
38.1
32.0
30.9
36.3
63.3
38.3
33.5
32.3
36.5
60.0
42.4
32.2
31.9
36.0
60.9
29.2
26.7
37.9
43.7
29.5 121.4
66.9 200.6
67.5
35.6
43.0
37.3
20.1
36.0
42.1
43.6
23.3
27.4
82.4
11.5
18.1
65.2
37.4
42.2
29.7
20.7
38.2
43.8
49.6
22.9
24.4
63.6
13.0
18.2
27.6
38.4
43.8
31.7
32.0
50.3
36.6
20.7
36.6
42.7
51.4
23.5
30.6
81.9
11.0
19.5
35.4
43.2
36.3
19.9
30.8
47.1
44.6
21.7
35.5
44.2
45.6
38.3
20.4
30.5
47.7
49.8
24.0
35.5
10
11
12
13
14
15
16
17
18
19
20
21
Ac
Ac
Ac
42.3 137.7 131.1
19.8 212.1 207.5 211.0 207.3
25.8
36.6
42.5
49.5
22.9
27.2
26.1
31.6
51.1
57.0
70.2
46.3
30.7
49.2
53.1
71.3
42.9
51.7
51.3
48.2
23.4
29.6
50.5
50.4
49.8
21.6
35.9
51.3
51.4
48.2
23.5
33.6
50.5
50.1
49.7
21.5
35.9
220.2 220.0
79.4 220.3 214.1
13.2
17.4
84.2 216.8
84.9 216.2
82.3 221.2
11.5
13.0
18.1
13.6
17.2
13.7
17.3
14.8
17.1
15.5
17.1
72.6
64.1
14.6
17.3
15.7
18.7
72.9
64.3
14.7
18.9
15.5
17.1
209.7 207.5 208.0
31.6
31.4
21.3
31.4
21.0
21.1
21.2
21.2
21.1
21.1
21.2
21.3
21.1
21.4
20.9
21.1
20.8
20.6
20.7
Ac 170.3 170.1 170.4
170.7 169.3 169.5 170.3
170.6 171.1 170.8
169.7
169.5
170.7
Ac 170.5
Ac
170.6
171.2
An asterisk (*) denotes that data on the acetate is presented.
ionizing voltage of 70 eV on a Kratos AEI MS-50 instru-
ment (Kratos Analytical Instruments, Chestnut Ridge, NY,
USA) for high-resolution electron impact ionization (EI)
and an MS-12 for chemical ionization (CI) spectrometry.
Infrared (IR) data were obtained by using a Perkin–Elmer
Fourier transform spectrophotometer (Norwalk, CT, USA)
and are for KBr disks. Melting points were obtained on a
Thomas–Hoover apparatus with open capillary tubes. Ul-
traviolet (UV) spectra were recorded on a Hewlett–Packard
HP 8452A diode array spectrophotometer (Cupertino, CA,
USA). Optical rotations were performed on a Perkin–Elmer
241 mc polarimeter. TLC was effected on Merck
(HF254ϩ366) glass or polyester-backed plates (0.25 mm
thick). Visualization of steroids both fed steroids and me-
tabolized products was done by spraying the plates with a
mixture of methanol-concentrated sulfuric acid (1:1) and
heating in an oven at 60°C for 2 min until the colors
developed. In some instances, visualization was effected
with the aid of a Spectroline UV lamp model ENF-24. Flash
chromatography was performed by using Merck Kieselgel
silica (40–63 ␮m diameter). Petrol refers to the petroleum
fraction boiling between 60°C and 80°C. Steroids were
obtained from Steraloids, Inc. (Wilton, NH, USA). Fermen-
tations were performed on a Gallenkamp Orbital Shaker. A
Janke and Kunkel Ultra-Turrax T25 homogenizer (IKA
Works, Inc., Wilmington, NC, USA) was used to break up
the fungal cells. pH readings were taken with a Schott-
Gerate pH meter (Schott, Mainz, Germany).
2.1. Fusarium oxysporum var. cubense
F. oxysporum var. cubense was isolated from the rhi-
zome of a banana plant showing the symptoms of Panama
disease and was identified by Dr Phyllis L. Coates Beck-
ford. Dr D. Brayford of the International Mycological In-
stitute, UK, confirmed the identification (IMI 326069). A
voucher specimen has been deposited with the University of
Alberta Microfungus Collection and Herbarium (UAMH
9013). The fungus was maintained on oatmeal agar slants
composed of rolled oats 10 g, MgSO4⅐7H2O 1 g, KH2PO4
1.5 g, NaNO3 1 g, agar 18 g/l taken to pH 5.6. Fourteen-
day-old mycelium was used to inoculate 500-ml conical
flasks, each containing 125 ml of medium consisting of 40 g
of glucose, 5 g of yeast extract, 5 g of NH4NO3, 2 g of
KH2PO4, 1 g/l MgSO4⅐7H2O, pH 6.9. In a typical fermen-
tation (1.9 g steroid fed) the fungus in 20 flasks was allowed
to grow on a rotary shaker at 90 rev./min (at 27°C) and 36 h
after inoculation, 190 mg of steroid in 19 ml of hot ethanol
was fed to 19 flasks. Feeding was repeated at 84 h. At 108
hours, 380 mg of steroid in 19 ml of hot ethanol was fed.
This procedure was repeated at 120, 132, and 144 h, and
harvest was performed at 180 h. At each feed, 1 ml of hot
ethanol was added to the growing fungus in the single

836
M.R. Wilson et al. / Steroids 64 (1999) 834–843
control flask. At harvest, the pH of the solution as well as
the mass of the dry mycelium were noted. The fermentation
beer was extracted with chloroform (ϫ3) and the total
extract was dried with anhydrous sodium sulfate and con-
centrated in vacuo. The mycelium was homogenized in the
solvent, and heated to boiling with constant stirring. The
mixture was filtered, dried, and concentrated. Mycelial and
broth extracts were combined before chromatography. The
mycelial residue was dried at 90°C in an oven for 12 h.
3␤,7␣-dihydroxypregn-5-en-20-one (6), 10 mg, which re-
sisted crystallization; IR (film) ␯max 3300, 1707, 1695, 1678
cmϪ1; 1H NMR (CDCl3) ␦ 0.65 (3H, s, H-18), 1.01 (3H, s,
H-19), 2.15 (3H, s, H-21), 2.58 (1H, t, J ϭ 8 Hz, H-17␣),
3.61 (1H, m, w/2 ϭ 18 Hz, 3-H␣), 3.85 (1H, t, J ϭ 2 Hz,
H-7␤), 5.61 (1H, dd, J ϭ 2, 6 Hz, H-6) [20].
2.4. Pregn-4-ene-3,20-dione (7)
Fermentation of 7 involved the use of 1.52 g of the
steroid. A dry mycelial mass of 64.47 g and organic extract
of 2.56 g were obtained. Column chromatography with 30%
ethyl acetate in petrol gave 255 mg of the fed compound.
Increasing the polarity of the eluting solvent to 50%, then
70% ethyl acetate in petrol gave 1.13 g of biotransformed
product that was acetylated. Chromatography on the acety-
lated mass (1.21 g), by using 30% ethyl acetate in petrol,
gave 15␣-acetoxypregn-4-ene-3,20-dione, 1.01 g, which
crystallized from acetone as cubes, m.p. 175–177°C, [␣]D
168° (c ϭ 1.04, CHCl3), lit [7]. m.p. 177–179°C, [␣]D 178°;
2.2. 3␤-Hydroxyandrost-5-en-17-one (1)
A fermentation by using 1.90 g of 1 yielded an organic
extract of 3.59 g and dry mycelial mass of 63.88 g. Column
chromatography with 30% ethyl acetate in petrol gave 150
mg of unconverted material. Elution with 50%, then 70%
ethyl acetate in petrol gave 1.30 g of biotransformed prod-
uct, which was acetylated by using 10 ml of pyridine and 10
ml of acetic anhydride. The mixture was stirred overnight at
60°C. Chromatography on the acetylated mass (1.39 g), by
using 20% ethyl acetate in petrol, gave 3␤,7␣,17␤-triace-
toxyandrost-5-ene, 30 mg, which crystallized from acetone
as cubes, m.p. 156–158°C, [␣]D Ϫ152° (c ϭ 0.60, CHCl3),
lit [17]. m.p. 163–164°C, [␣]D Ϫ220°; IR ␯max 2942, 1738,
IR ␯
2900, 1735, 1700, 1660, 1620 cmϪ1; UV (CHCl3)
max
1
␭
244 nm, log ⑀ ϭ 4.11; H NMR (CDCl3) ␦ 0.75 (3H,
max
s, H-18), 1.15 (3H, s, H-19), 1.51 (1H, d, J ϭ 8 Hz, H-14),
2.05 (3H, s, CH3CO2), 2.15 (3H, s, H-21), 4.97 (1H, dt, J ϭ
4, 10 Hz, H-15␤), 5.75 (1H, s, H-4).
1
1734, 1728, 1243 cmϪ1; H NMR (CDCl3) ␦ 0.82 (3H, s,
H-18), 1.05 (3H, s, H-19), 2.04 (9H, s, 3 CH3CO2), 4.65
(1H, t, J ϭ 8 Hz, H-17␣), 4.71 (1H, m, w/2 ϭ 8 Hz, H-3␣),
5.00 (1H, t, J ϭ 6 Hz, H-7␣), 5.60 (1H, d, J ϭ 6 Hz, H-6).
Further elution with 25% and 30% ethyl acetate in petrol
gave 3␤,7␣-diacetoxyandrost-5-en-17-one, 1.01 g, which
crystallized from acetone as cubes, m.p. 164–165°C, [␣]D
Ϫ172° (c ϭ 1.11, CHCl3), lit [18]. m.p. 168–170°C, [␣]D
Ϫ178°; MS (CI-NH3) m/z (%) 406 (1.7), 328 (25.1), 286
(28.9), 269 (100), 270 (64.3), 253 (6.5), 240 (4.5), 225 (6.8),
145 (4.8), 133 (9.2), 105 (4.5), 81 (7.0); IR ␯max 2900, 1739,
Further elution with the same system gave 12␤,15␣-
diacetoxypregn-4-ene-3,20-dione, 38 mg, which crystal-
lized from ethanol as cubes, 25 mg, m.p. 178–181°C, [␣]D
156° (c ϭ 0.58, EtOH), lit [21]. m.p. 181–184°C, [␣]D 134°
(CHCl3); IR (film) ␯
1735, 1729, 1711, 1669, 1615
240 nm, log ⑀ ϭ 4.18; H NMR
max
1
cmϪ1; UV (EtOH) ␭
max
(CDCl3) ␦ 0.98 (3H, s, H-18), 1.20 (3H, s, H-19), 2.05 (6H,
s, 2 CH3CO2), 2.10 (3H, s, H-21), 2.90 (1H, t, J ϭ 8 Hz,
H-16␣), 4.80 (1H, dd, J ϭ 6, 12 Hz, H-12␣), 4.97 (1H, dt,
J ϭ 4, 8 Hz, H-15␤), 5.75 (1H, s, H-4).
1
1730, 1723, 1250, 1235 cmϪ1; H NMR (CDCl3) ␦ 0.81
(3H, s, H-18), 1.00 (3H, s, H-19), 2.02 (6H, s, 2 CH3CO2),
4.61 (1H, m, w/2 ϭ 21 Hz, H-3␣), 5.12 (1H, t, J ϭ 4 Hz,
H-7␣), 5.56 (1H, d, J ϭ 6 Hz, H-6).
2.5. 17␤-Hydroxyandrost-4-en-3-one (10)
In the biotransformation of 10, 1.52 g of the steroid was
used. At harvest, 64.93 g of dried mycelium and 3.52 g of
organic extract were obtained. Column chromatography
with 25% ethyl acetate in petrol gave androst-4-ene-3,17-
dione (13), 40 mg, which crystallized from acetone as col-
orless cubes, m.p. 169–171°C, [␣]D 189° (c 1.17, CHCl3),
The fraction that eluted in 40% ethyl acetate in petrol
was composed of 3␤-acetoxyandrost-5-ene-7,17-dione, 30
mg, which crystallized from acetone as cubes, m.p. 175.5–
177°C, [␣]D Ϫ76.8° (c ϭ 1.17, CHCl3), lit [19]. m.p.
184°C, [␣]D Ϫ74°; IR ␯
3400, 1730, 1670, 1620 cmϪ1
;
max
UV (CHCl3) ␭max 240 nm, log ⑀ ϭ 4.12; 1H NMR (CDCl3)
␦ 0.91 (3H, s, H-18), 1.32 (3H, s, H-19), 2.05 (3H, s,
CH3CO2), 4.70 (1H, m, w/2 ϭ 18 Hz, H-3␣), 5.75 (1H, d,
J ϭ 2 Hz, H-6).
lit [22]. m.p. 169–170°C, [␣]D 205°; IR ␯
2940, 1740,
max
1670 cmϪ1; UV (CHCl3) ␭
240 nm, log ⑀ ϭ 4.20;
max
HRMS (EI) m/z (%) 286.1933 (100) [M]ϩ [C19H26O2], 271
(7), 244 (61), 201 (18), 148 (37), 124 (88), 91 (37), 79 (38);
1H NMR (CDCl3) ␦ 0.89 (3H, s, H-18), 1.21 (3H, s, H-19),
5.72 (1H, s, H-4).
2.3. 3␤-Hydroxypregn-5-en-20-one (5)
Increasing the polarity of the eluting solvent to 40% and
then to 50% gave 270 mg of fed material. Elution with 70%
ethyl acetate in petrol gave biotransformed product (1.11 g)
that was acetylated. Column chromatography on the mix-
ture (1.36 g), by using 20% ethyl acetate in petrol, gave
6␤,17␤-diacetoxyandrost-4-en-3-one, 25 mg, which failed
In this Experiment, 1.14 g of 5 was fed. On harvest, a dry
mycelial mass of 66.42 g and organic extract of 1.34 g were
obtained. Column chromatography with 30%, then 50%
ethyl acetate in petrol gave 850 mg of fed material. A
mixture of 60% ethyl acetate in petrol as the eluant gave

M.R. Wilson et al. / Steroids 64 (1999) 834–843
837
to crystallize; IR (film) ␯
1735, 1741, 1675, 1615, 1227
s, H-18), 1.48 (3H, s, H-19), 6.11 (1H, s, H-4), 6.24 (1H, d,
J ϭ 12 Hz, H-2), 7.65 (1H, d, J ϭ 12 Hz, H-1).
max
cmϪ1; UV (EtOH) ␭
239 nm, log ⑀ 4.16; 1H NMR
max
(CDCl3) ␦ 0.89 (3H, s, H-18), 1.29 (3H, s, H-19), 2.05 (6H,
s, 2 CH3CO2), 4.60 (1 H, t, J ϭ 8 Hz, H-17␣), 5.45 (1 H, t,
J ϭ 2 Hz, H-6␣), 5.95 (1H, s, H-4) [23].
Continued elution and increasing the concentration of ethyl
acetate in the eluant to 80% gave 260 mg of untransformed
steroid. Elution with pure ethyl acetate gave 17␣,20,21-trihy-
droxypregna-1,4-diene-3,11-dione [28] (19), 12 mg, which
crystallized from ethanol as plates, m.p. 210–212°C, [␣]D 38°
(c ϭ 0.4, CHCl3); IR ␷max 3365, 1740, 1720, 1660 cmϪ1; UV
(CHCl3) ␭max 246 nm (log ⑀ ϭ 4.01); HRMS m/z (%) 360.1937
[M-2]ϩ (30), 301 (34), 258 (29), 161 (21), 122 (100); 1H NMR
(CDCl3) ␦ 0.78 (3H, s, H-18), 0.92 (1H, t, J ϭ 8 Hz, H-8), 1.08
(1H, d, J ϭ 8 Hz, H-9), 1.43 (3H, s, H-19), 1.81 (1H, d, J ϭ
12 Hz, H-12␣), 2.49 (1H, d, J ϭ 12 Hz, H-12␤), 3.73 (3H, s,
H-20, H-21), 6.07 (1H, s, H-4), 6.21 (1H, dd, J ϭ 2, 10 Hz,
H-2), 7.70 (1H, d, J ϭ 10 Hz, H-1).
Further elution yielded 15␣,17␤-diacetoxyandrost-4-en-
3-one, 80 mg, which crystallized from acetone as cubes,
m.p. 149–151°C, [␣]D 116.5° (c ϭ 0.98, CHCl3), lit [24].
m.p. 149–153°C, [␣]D 106°, (MeOH); IR ␯max 2900, 1730,
1720, 1670, 1615 cmϪ1; UV (CHCl3) ␭
240 nm, log ⑀
max
1
4.20; H NMR (CDCl3) ␦ 0.88 (3H, s, H-18), 1.21 (3H, s,
H-19), 1.98 (3H, s, CH3CO2), 2.05 (3H, s, CH3CO2), 4.75
(1H, t, J ϭ 8 Hz, H-17␣), 4.95 (1H, m, w/2 ϭ 12 Hz,
H-15␣), 5.72 (1H, s, H-4); Increasing the polarity to 40%
ethyl acetate in petrol gave 15␣-acetoxyandrost-4-ene-3,17-
dione, 467 mg, which did not crystallize.
IR (film) ␯max 1735, 1669, 1610, 1239 cmϪ1; UV (EtOH)
2.8. 3-Hydroxyestra-1,3,5(10)-trien-17-one (21)
1
␭
240 nm, log ⑀ ϭ 4.11; H NMR (CDCl3) ␦ 1.05 (3H,
max
s, H-18), 1.25 (3H, s, H-19), 2.12 (3H, s, CH3CO2), 3.20
(1H, dd, J ϭ 8, 12 Hz, H-16␣), 5.25 (1H, m, /2 ϭ 20 Hz,
H-15␤), 5.75 (1H, s, H-4) [25].
In this fermentation, 1.14 g of 21 was fed. At harvest, a
dry mycelial mass 84.17 g and organic extract 2.54 g were
obtained. Column chromatography with 40% ethyl acetate
in petrol gave 690 mg of fed material and continued elution
provided 30 mg of biotransformed product. Column chro-
matography, eluting in 40% ethyl acetate in petrol, gave
w
2.6. 17␣,21-Dihydroxypregn-4-ene-3,11,20-trione (15)
3,15␣-dihydroxy-1,3,5(10)-estratrien-17-one (23), 3 mg; IR
A total mass of 760 mg of 15 was fed. At harvest, a dry
mycelial mass of 70.86 g and organic extract of 2.24 g were
obtained. Column chromatography with 40% ethyl acetate
in petrol gave androst-4-ene-3,11,17-trione (17), 80 mg,
crystallizing from acetone as cubes, m.p. 214–215°C, [␣]D
279° (c ϭ 0.80, CHCl3), lit [26]. m.p. 217–220°C, [␣]D
1
(film) ␯
2931, 1725, 1615 cmϪ1; H NMR (CDCl3) ␦
max
0.95 (3H, s, H-18), 2.85 (2H, m, w/2 ϭ 8 Hz, H-6), 3.05 (1H,
m, w/2 ϭ 6 Hz, H-15␤), 6.60 (1H, s, H-4), 6.65 (1H, d, J ϭ
8 Hz, H-2) 7.18 (1H, d, J ϭ 8 Hz, H-1).
The other steroid that eluted was acetylated and purified
with 20% ethyl acetate in petrol to give 3,17␤-diace-
toxyestra-1,3,5(10)-triene, 10 mg, as cubes from ethyl ace-
tate, m.p. 173–175°C, [␣]D 78° (c ϭ 0.13, CHCl3), lit [29].
m.p. 173–175°C; IR ␯max 2920, 1760, 1730, 1605 cmϪ1; 1H
NMR (CDCl3) ␦ 0.83 (3H, s, H-18), 2.05 (3H, s, CH3CO2),
2.35 (3H, s, CH3CO2), 2.91 (2H, d, J ϭ 6 Hz, H-6), 4.72
(1H, t, J ϭ 8 Hz, H-17␣), 6.80 (1H, s, H-4), 6.85 (1H, d, J ϭ
8 Hz, H-2), 7.28 (1H, d, J ϭ 8 Hz, H-1).
284° (c ϭ 0.51, CHCl3); IR ␯
1730, 1695, 1655, 1610
max
1
cmϪ1; UV (CHCl3) ␭
254 nm, log ⑀ ϭ 4.19; H NMR
max
(CDCl3) ␦ 0.90 (3H, s, H-18), 1.45 (3H, s, H-19), 2.35 (1H,
d, J ϭ 10 Hz, H-12␣), 2.48 (1H, d, J ϭ 10 Hz, H-12␤), 2.75
(1H, m, w/2 ϭ 8 Hz, H-9), 5.75 (1H, s, H-4).
Increasing the polarity of the solvent system to 60%, then
80% ethyl acetate in petrol gave 555 mg of the fed steroid.
Eluting with pure ethyl acetate gave 17␣,20,21-trihy-
droxypregn-4-ene-3,11-dione (16), 6 mg, which resisted
2.9. Colletotrichum musae
crystallization; IR (film) ␯
3350, 2900, 1738, 1708,
max
1667, 1607 cmϪ1; UV (EtOH) ␭
240 nm, log ⑀ ϭ 4.11;
max
C. musae was isolated from ripe bananas (Musa sp.)
showing symptoms of anthracnose infection. Dr Phyllis L.
Coates Beckford identified the fungus and this was con-
firmed by Dr P.F. Cannon, International Mycological Insti-
tute, UK (IMI 374528). A voucher specimen has been
deposited with the University of Alberta Microfungus Col-
lection and Herbarium (UAMH 8929) The fungus was
maintained on potato dextrose agar slants. Fourteen-day-old
mycelium was used to inoculate 500-ml flasks, each con-
taining 250 ml of a modification of Richard’s medium [30]:
50 g of sucrose, 10 g of KNO3, 5 g of KH2PO4, 0.25 g of
MgSO4⅐7H2O, 0.033 g of FeCl3⅐6H2O, and 10 g/l potato
starch. The pH was adjusted to 4.39 with aqueous NaOH. In
a typical fermentation the culture in 20 flasks was allowed
to grow at 27°C for 13 days with shaking at 200 rev./min.
1H NMR (CDCl3) ␦ 0.81 (3H, s, H-18), 1.46 (3H, s, H-19),
2.50 (1H, d, J ϭ 12 Hz, H-12␣), 2.68 (1H, d, J ϭ 12 Hz,
H-12␤), 3.78 (3H, s, H-21, H-20), 5.75 (1H, s, H-4).
2.7. 17␣,21-Dihydroxypregna-1,4-diene-3,11,20-trione
(18)
In the fermentation of 18, 760 mg of steroid was used.
An organic extract of 2.74 g and dry mycelial mass of 61.75
g were noted at harvest. Column chromatography with 40%
ethyl acetate in petrol gave androsta-1,4-diene-3,11,17-tri-
one [27] (20), 5 mg, which did not crystallize; IR (film) ␯
max
2985, 1743, 1717, 1711, 1604, 1591 cmϪ1; UV (EtOH)
1
␭
216 nm, log ⑀ ϭ 4.12; H NMR (CDCl3) ␦ 0.79 (3H,
max

838
M.R. Wilson et al. / Steroids 64 (1999) 834–843
Pulse feeding of 1 g of steroid was done during a 4-day
period to 19 flasks: 0.10 g on day 14, 0.20 g on day 15,
0.30 g on day 16, and 0.40 g on day 17. The steroid was
dissolved in 10 ml of ethanol for each feed. On day 21, the
contents of the flasks were filtered and the mycelium was
homogenized in ethyl acetate. The broth was extracted with
ethyl acetate (ϫ2). The broth and mycelial extracts were
combined, dried over sodium sulfate, and the solvents were
evaporated under reduced pressure. The mycelial residue
was dried at 90°C in an oven for 12 h.
2.10. 3␤-Hydroxyandrost-5-en-17-one (1)
The biotransformation of this steroid (1 g) yielded 27.63
g of dried mycelium and 4.1 g of organic extract. Trituration
with hexane separated the nonpolar fungal metabolites (3.25
g) from the steroidal portion (0.29 g). Column chromatog-
raphy by using 15% ethyl acetate in petrol yielded fungal
metabolites. Further elution with 22.5% ethyl acetate in
petrol gave the fed steroid, followed closely by 3␤,17␤-
dihydroxyandrost-5-ene (24), 145.5 mg, which crystallized
from methanol as cubes, m.p. 161–162°C, [␣]D Ϫ150° (c ϭ
4.0 ϫ 10Ϫ5, CHCl3), lit [31]. m.p. 177–179°C, [␣]D Ϫ55°
Scheme 1. Transformation of 3␤-hydroxyandrost-5-en-17-one by F. oxy-
sporum.
with 3␤-hydroxyandrost-5-en-17-one (1) as the substrate.
Hydroxylation was effected and in good yield. The untrans-
formed steroid was found associated with the mycelium at
harvest. Timed addition of the substrate was found to aug-
ment conversion of the xenobiotes. The other steroids were
subjected to fermentation under the same conditions. Prod-
ucts of biotransformation showed spectral properties con-
sistent with their proposed structure. 13C NMR shifts were
compared with those published by Blunt and Stothers [28].
(c ϭ 0.4, ipa); IR ␯
3450, 3200, 2920, 2850, 1440; UV
max
242 nm (log ⑀ ϭ 2.49); HRMS (EI) m/z (%)
(CHCl3) ␭
max
290.2248 (100) [M]ϩ [C19H30O2], 275 (18), 272 (62), 257
(41), 107 (80); 1H NMR (CDCl3) ␦ 0.78 (3H, s, H-18), 1.03
w
(3H, s, H-19), 3.52 (1H, m, /2 ϭ 18 Hz, H-3␤), 3.68 (1H,
t, J ϭ 9 Hz, H-17␤), 5.36 (1H, d, J ϭ 7.2 Hz, H-6).
2.11. 17␤-Hydroxyandrost-4-en-3-one (10)
3.2. 3␤-Hydroxy-⌬5-Steroids
Fermentation of 10 (1 g) yielded 10.48 g of the dried
mycelium and 1.52 g of organic extract. Column chroma-
tography of the acetylated extract, by using 20% ethyl
acetate in petrol, enabled the isolation of androst-4-ene-
3,17-dione (13), 29 mg, which was identified by comparison
with an authentic sample.
Results showed that these steroids were hydroxylated to
give mainly the 7␣ hydroxy compounds. Reduction of the
carbonyl group of dehydroisoandrosterone (1) occurred. It
would appear that the presence of a side chain at C-17 may
lower the hydroxylation rate, because 1 was metabolized in
very good yield, whereas conversion of pregnenolone (5)
was inferior. No side-chain cleavage was observed and the
reduction of the C-20 carbonyl of 5 was not seen.
2.12. 17␣,21-Dihydroxypregn-4-ene-3,11,20-trione (15)
Fermentation of 15 (1 g) yielded 12.50 g of dried my-
celium and 4.08 g of organic extract. Column chromatog-
raphy of this extract in 30% acetone in chloroform provided
the fed steroid (79 mg). Further elution enabled the isolation
of 17␣,20,21-trihydroxypregn-4-ene-3,11-dione (16) (10.4
mg), which was identified by comparison with an authentic
sample.
3.3. Bioconversion of 3␤-hydroxyandrost-5-en-17-one (1)
(Scheme 1)
The biotransformed compounds were characterized as
their acetates and their data were compared with that of the
3␤-acetate of 1.
3.3.1. 3␤,7␣,17␤-Triacetoxyandrost-5-ene
3. Results and discussion
NMR data showed that two new acetate groups were
present. The carbonyl at ␦ 220.6 (C-17) had disappeared
along with the methylene at ␦ 31.2 assigned to C-7. It was
concluded that the fungus had inserted a hydroxyl group at
C-7 and reduced the 17-ketone.
3.1. Fusarium oxysporum var. cubense
This fungus was found to biotransform a range of ste-
roids. The protocol for the bioconversion was determined

M.R. Wilson et al. / Steroids 64 (1999) 834–843
839
Scheme 2. Transformation of 3␤-hydroxypregn-5-en-20-one by F. oxy-
sporum.
3.3.2. 3␤,7␣-Diacetoxyandrost-5-en-17-one
Shifts in the 13C NMR showed that the C-17 carbonyl
was still present. The signal at ␦ 66.9 (CH) pointed to the
fact that a hydroxyl group had been inserted. The hydroxyl
group was determined to be in the 7␣ position [28].
Scheme 3. Transformation of pregn-4-ene-3,20-dione by F. oxysporum.
3.3.3. 3␤-Acetoxyandrost-5-ene-7,17-dione
3.6.1. 15␣-Acetoxypregn-4-ene-3,20-dione
The 13C NMR spectrum showed the presence of two
carbonyls and an acetate group. The disappearance of the
methylene assigned to C-7 was noted. The large shielding
effects experienced by the olefinic carbons suggested that
the double bond was in conjugation with a carbonyl. A
possible bioconversion sequence from 3␤-hydroxyandrost-
5-en-17-one (1) is that of allylic hydroxylation at C-7 to
form 3␤,7␣-dihydroxyandrost-5-en-17-one (2), which was
oxidized to 3␤-acetoxyandrost-5-ene-7,17-dione (3) or re-
duced to 3␤,7␣,17␤-trihydroxyandrost-5-ene (4). The inser-
tion of a hydroxyl group at the 7␣ position was in keeping
with the arrangement proposed by Bell et al. [32,33] for the
active sites available on the enzyme.
The spectra showed the appearance of a methine at ␦
75.2 and the disappearance of the methylene assigned to
1
C-15 in the 13C NMR spectrum. The H NMR confirmed
the 15␣ stereochemistry of the substituent [25].
3.6.2. 12␤,15␣-Diacetoxypregn-4-ene-3,20-dione
The spectra for this compound were similar to those of
the aforementioned steroid except for the presence of a
second acetate group. The signal for C-12 had shifted down-
field by 40.6 ppm. The second hydroxyl group was deter-
mined to have been inserted in the 12␤ position [28]. Pro-
gesterone (7) had thus been biotransformed to two
compounds 15␣-hydroxypregn-4-ene-3,20-dione (8) and
12␤,15␣-dihydroxypregn-4-ene-3,20-dione (9). It is pro-
posed that 8 is first formed by hydroxylation of 7 at position
15␣, which in turn undergoes further hydroxylation at C-12
to give 9.
3.4. Bioconversion of 3␤-hydroxypregn-5-en-20-one (5)
(Scheme 2)
3.4.1. 3␤,7␣-Dihydroxypregn-5-en-20-one (6)
Comparison of the NMR data with that of the fed steroid
showed that insertion of a hydroxyl group at C-7␣ had
occurred [28].
3.7. Bioconversion of 17␤-hydroxyandrost-4-en-3-one (10)
(Scheme 4)
3.7.1. Androst-4-ene-3,17-dione (13)
3.5. 3-Keto-⌬4-steroids
The 13C NMR data showed the presence of two car-
bonyls and the disappearance of the methine at ␦ 82.1,
which had been assigned to C-17. Oxidation of the C-17
hydroxyl group accounted for the new carbonyl at ␦
220.3. Representative absorbances in the IR spectrum for
the ␣,␤-unsaturated system and five-membered ring ke-
tone were seen.
The two examples used underwent hydroxylation at
C-15␣ in almost quantitative yield. Oxidation of the 17-
alcohol of 17␤-hydroxyandrost-4-en-3-one (testosterone)
(10) occurred. The hydroxylation of this steroid at the 6␤
position, an important mammalian reaction, was observed
[34].
The following biotransformed compounds of 10 were
purified and characterized as their acetates.
3.6. Bioconversion of pregn-4-ene-3,20-dione (7)
3.7.2. 15␣-Acetoxyandrost-4-ene-3,17-dione
(Scheme 3)
These spectra were quite similar to those of androst-4-
ene-3,17-dione (13). However, the methine signal at ␦ 71.3
in the 13C NMR indicated the presence of an acetoxy group
The biotransformed compounds were characterized as
their acetates and were compared with the fed compound.

840
M.R. Wilson et al. / Steroids 64 (1999) 834–843
Scheme 6. Transformation of 17␣,21-dihydroxypregna-1,4-diene-3,11,20-
trione by F. oxysporum.
downfield by 50.8 ppm. The bioconversion of 17␤-hy-
droxyandrost-4-en-3-one (10) therefore yielded 6␤,17␤-
dihydroxyandrost-4-en-3-one (11), 15␣,17␤-dihydroxyan-
drost-4-en-3-one (12), androst-4-ene-3,17-dione (13), and
15␣-hydroxyandrost-4-ene-3,17-dione (14).
Scheme 4. Transformation of 17␤-hydroxyandrost-4-en-3-one by F. oxy-
sporum.
3.8. 17␣,21-Dihydroxypregnenetriones
via substitution of a methylene. Hydroxylation had occurred
at position 15␣.
The two examples used underwent similar reactions. The
C-20 carbonyl was first reduced followed by oxidative deg-
radation to the androstanes.
3.7.3. 6␤,17␤-Diacetoxyandrost-4-en-3-one
Comparison of the spectra of this compound with those
for 10 indicated that one methylene group had been hy-
droxylated in the 6␤ position [28].
3.9. Bioconversion of 17␣,21-dihydroxypregn-4-ene-
3,11,20-trione (15) (Scheme 5)
3.7.4. 15␣,17␤-Diacetoxyandrost-4-en-3-one
The spectra showed that there had been insertion of a
hydroxyl group at C-15␣, the resonance of which moved
3.9.1. 17␣,20,21-Trihydroxypregn-4-ene-3,11-dione (16)
The NMR spectra of this polar compound showed that
the 20-carbonyl had been reduced. The molecular ion of this
steroid was absent, the largest fragment ion occurring two
atomic mass units less at m/z 360.1937, corresponding to a
formula of C21H28O5. This observation is not uncharacter-
istic of primary alcohols, which tend to lose two protons in
the ion chamber to form the corresponding aldehyde [35].
The base peak at m/z 122 represented diagnostic fragmen-
tation for polyfunctional steroids [36].
3.9.2. Androst-4-ene-3,11,17-trione (17)
13C NMR data showed a total of 19 carbons, indicating
loss of the acetyl group at C-17 with the formation of a
carbonyl. The steroid (15), when fed, was metabolized to
17␣,20,21-trihydroxypregn-4-ene-3,11-dione (16), which
probably underwent oxidative degradation to androst-4-ene-
3,11,17-trione (17).
3.10. Bioconversion of 17␣,21-dihydroxypregna-1,4-diene-
3,11,20-trione (18) (Scheme 6)
Scheme 5. Transformation of 17␣,21-dihydroxypregn-4-ene-3,11,20-trione
by F. oxysporum.

M.R. Wilson et al. / Steroids 64 (1999) 834–843
841
Scheme 8. Transformation of 3␤-hydroxyandrost-5-en-17-one by C. mu-
sae.
3.12.2. 3,17␤-Diacetoxyestra-1,3,5(10)-triene
1H NMR showed two acetate groups were present. The
absence of the 17-carbonyl and the presence of methine at ␦
82.3 were noted in the 13C NMR. Shift parameters indicated
that the compound was 3,17␤-diacetoxyestra-1,3,5(10)-
triene [28]. Hence, 3-hydroxyestra-1,3,5(10)-trien-17-one
(21) was biotransformed to 3,17␤-dihydroxyestra-1,3,
5(10)-triene (22) and 3,15␣-dihydroxyestra-1,3,5(10)-trien-
17-one (23) (Scheme 8). The hydroxylation proceeded very
poorly in this case. Casas–Campillo and Bautista [37] re-
ported that the degree of hydroxylation of estrones was
medium dependent and this could explain the poor biotrans-
formation.
Scheme 7. Transformation of 3-hydroxyestra-1,3,5(10)-trien-17-one by F.
oxysporum.
3.10.1. 17␣,20,21-Trihydroxypregna-1,4-diene-3,11-dione
(19)
Comparison of the NMR spectra of this compound with
that for the fed steroid showed that one of the carbonyls had
disappeared and a hydroxymethylene group was now
present.
3.13. Colletotrichum musae
C. musae had not been previously used to effect biotrans-
formations with any substrate. Richard’s medium [30] was
chosen over a modified Czapek–Dox broth because growth
of the fungus, although poor in both, was found to be better
in the former. An appropriate feeding protocol was deter-
mined by using 3␤-hydroxyandrost-5-en-17-one (1) as sub-
strate.
3.10.2. Androsta-1,4-diene-3,11,17-trione (20)
This was less polar than 18 and was produced in very
small quantities. The 13C NMR spectrum contained signals
for 19 carbons showing that cleavage of the side chain to
give a 17-carbonyl had occurred. It was proposed that in the
metabolism of 18 the trihydroxy compound, 17␣,20,21-
trihydroxypregna-1,4-diene-3,11-dione (19), first formed
underwent oxidative degradation to androsta-1,4-diene-
3,11,17-trione (20).
3.14. Bioconversion of 3␤-hydroxyandrost-5-en-17-one (1)
(Scheme 8)
3.11. Aromatic ring A steroid
3.14.1. 3␤,17␤-Dihydroxyandrost-5-ene (24)
In the proton NMR spectrum of the product, there were
two signals indicative of protons on carbons bearing hy-
droxyl groups. In the 13C NMR spectrum, the signal for the
carbonyl group had disappeared. A more polar metabolite
could be identified (m/z 302.1886, C19H26O3), but the quan-
tity isolated was inadequate for complete characterization.
In the example used, 15␣ hydroxylation was noted to
have taken place as well as the reduction of the carbonyl at
C-17.
3.12. Bioconversion of 3-hydroxyestra-1,3,5(10)-trien-17-
one (21) (Scheme 7)
3.15. Bioconversion of 17␤-hydroxyandrost-4-en-3-one
(10)
3.12.1. 3,15␣-Dihydroxyestra-1,3,5(10)-trien-17-one (23)
The signal that had been assigned to C-15 in the 13C
NMR spectrum had disappeared and was replaced by a
methine at ␦ 70.2. Comparison of NMR shift parameters led
to the conclusion that the hydroxyl group was at the 15␣
position [28].
3.15.1. Androst-4-ene-3,17-dione (13)
This compound was identified by comparison with an
authentic sample (mentioned above).
3.16. Bioconversion of pregn-4-ene-3,20-dione (7)
The following biotransformed compound was purified
and characterized as the acetate that was compared with the
3-acetate of 21.
Incubation of the fungus with this steroid yielded two
m
hydroxylated compounds [m/z 330.2204, C21H30O3,
/
z

842
M.R. Wilson et al. / Steroids 64 (1999) 834–843
330.2199, C21H30O3] neither of which was isolated in suf-
ficient quantity to enable its characterization.
References
[1] Kranz J, Schmutterer H, Koch W. Diseases, Pests and Weeds in
Tropical Crops. New York: John Wiley & Sons, 1978. pp. 202–4.
[2] Holliday P. Fungus Diseases of Tropical Crops. Cambridge, UK:
Cambridge University Press, 1980. pp. 171–3.
3.17. Bioconversion of 17␣,21-dihydroxypregn-4-ene-
3,11,20-trione (15)
[3] Tatum JH, Baker RA, Berry RE. Naphthoquinones produced by
Fusarium oxysporum isolated from citrus. Phytochemistry 1985;24:
457–9.
[4] Tseng TC, Lay LL. Mycotoxins produced by Fusarium spp. of Tai-
wan. Bot Bull Acad Sci 27:35–43. Chem Abs 1986;104:85017.
[5] Barton DHR, Cottier L, Freund K, Luini F, Magnus PD, Salaza I.
Total synthesis of bikaverin (6,11-dihydroxy-3,8-dimethoxy-1-meth-
yl-benzo[b]xanthene-7,10,12-trione). J Chem Soc [Perkin I] 1976;5:
499–503.
[6] Tom W, Abul–Hajj YJ, Koreeda MJ. Microdial oxidation of ecdys-
ones: convenient preparation of rubrosterone. J Chem Soc Chem
Commun 1975;24–5.
[7] Tamm Ch, Gubler A, Juhas G, Weiss–Berg E, Zurcher W. Uber die
substratspecifitat der Hydroxylierung von Androstan und Pregnand-
envaten durch Fusarium lini. Helv Chim Acta 1963;46:889–906.
[8] Vincke BJ, Devleeschouwer MJ, Dony J, Patriarche GJ. Efficiency of
bioconversion of steroids by Fusarium oxysporum into ⌬1,4-andros-
tadiene-3,17-dione using gas chromatography. Int J Pharm 1986;32:
61–9.
[9] Petzoldt K, Schering AG. 15␣-Hydroxy-4-androstene-3,17-dione.
German Patent DE 3,403,862. Chem Abs 1985;104:166905q.
[10] Schuepback M, Tamm Ch. Reactions with microorganisms. XII.
Conversion of toad poison (bufadienolide) by microorganisms. 2.
12␤-Hydroxyresibufogenin. Helv Chim Acta 1964;47:2217–26.
[11] Defaye G, Luche MJ, Chambaz EM. Microbial 7- and 15-hydroxy-
lations of C-19 steroids. J Steroid Biochem 1978;9:331–6.
[12] Holliday P. Fungus Diseases of Tropical Crops. Cambridge, UK:
Cambridge University Press, 1980. pp. 106–8.
[13] Kranz J, Schmutterer H, Koch W. Diseases, Pests and Weeds in
Tropical Crops. New York: John Wiley & Sons, 1978. pp. 196–8.
[14] Njar V, Shapiro S, Arunachalam T, Capsi E. Biotransformation of
progesterone to 14␣-hydroxypregna-1,4-diene-3,20-dione, a novel
fungal metabolite, by Colletotrichum antirrhini. J Steroid Biochem
1985;22:399–400.
3.17.1. 17␣,20,21-Trihydroxypregn-4-ene-3,11-dione (16)
This compound was identified by comparison with an
authentic sample (mentioned above).
Thus, F. oxysporum var. cubense biotransformed seven
steroids, i.e. 3␤-hydroxyandrost-5-en-17-one (dehydroisoan-
drosterone) (1), 3␤-hydroxypregn-5-en-20-one (pregnenolone)
(5), pregn-4-ene-3,20-dione (progesterone) (7), 17␤-hy-
droxyandrost-4-en-3-one (testosterone) (10), 17␣,21-dihy-
droxypregn-4-ene-3,11,20-trione (cortisone) (15), 17␣,21-di-
hydroxypregna-1,4-diene-3,11,20-trione (prednisone) (18),
and 3-hydroxyestra-1,3,5(10)-trien-17-one (estrone) (21).
From these experiments, it appears that the fungus is able to
effect 7␣ hydroxylation of 3␤-hydroxy-⌬5-steroids. The inser-
tion of a hydroxyl group at the C-15␣ position of 3-keto-⌬4-
steroids was performed in almost quantitative yield, and these
two reactions may have scope in synthesis. Reduction of the
C-20 carbonyl of the dihydroxypregnenetriones occurred, fol-
lowed by side-chain cleavage to form androstane derivatives.
However, there was no reduction of the conjugated carbonyl
group of pregnenolone (5) or progesterone (7). In general,
oxidation of alcohols and reduction of unconjugated ketones
also took place. Only four of the seven substrates were metab-
olized by C. musae, namely, 1, 7, 10, and 15. Products isolated
were those of oxidation or reduction, although ␣,␤-unsaturated
carbonyl functionalities in all fed compounds were left un-
touched. The formation of minute quantities of hydroxylated
steroids was noted. It is expected that the development of more
suitable fermentation conditions for Colletotrichum will pre-
cede improved yields of hydroxylated analogs.
[15] Levine SD, Principe PA. 12-Oxygenated-A-norandrost-3-enes. US
Patent 3,367,964. Chem Abs 1968;68:94651s.
[16] Hanson JR, Reese PB, Takahashi JA, Wilson MR. Biotransformation
of some stemodane diterpenoids by Cephalosporium aphidicola. Phy-
tochemistry 1994;36:1391–3.
[17] Morand P, Van Tongerloo A. 19-Hydroxysteroids. VI. Approaches to
the synthesis of 7,19-disubstituted androgens. Steroids 1973;21:65–
85.
[18] Dodson RM, Nicholson RT, Muir RD. Microbiological transforma-
tions. IV. The oxidation of dehydroisoandrosterone at C-7. J Am
Chem Soc 1959;81:6295–7.
[19] Billeter JR, Miescher K. Degradation products of the oxidation of
steroids. 4. Isolation of ⌬3,5-androstadiene-7,17-dione. Helv Chim
Acta 1948;31:629–32.
[20] Namboori K, Pereira L, Merchant JR. Fungal transformation of preg-
nenolone and progesterone with the marine fungus Cladosporium
herbarum. Indian J Biochem Biophys 1980;17:149–52.
[21] Dodson RM, Langbein G, Muir RD, Schubert A, Tamm Ch, Weiss–
Berg E. Identifizierung der als 6␤,15␣-dihydroxyprogesteron und
12␤,15␤-dihydroxyprogesteron beschriebenen Steroide mit 12␤,15␣-
dihydroxyprogesteron. Helv Chim Acta 1965;48:1933–40.
[22] Rao PN. Manganese dioxide oxidation: the optional introduction of a
⌬6-double bond with simultaneous cleavage of dihydroxyacetone or
17,20-glycol side chains in ⌬4-3- ketosteroids. J Org Chem 1961;26:
2149–52.
Acknowledgments
The authors thank Prof. John C. Vederas (University of
Alberta) for arranging mass spectral analyses. We are grate-
ful to Dr Phyllis L. Coates Beckford (Botany Department)
for helpful advice during all stages of the project and assis-
tance with isolation and maintenance of the strains. Fermen-
tations were carried out in the Biotechnology Center, Uni-
versity of West Indies.
This work was supported in part by funds secured under
a Canadian International Development Agency/Natural Sci-
ences and Engineering Research Council Research Fellow-
ship to P.B.R. Funding was also obtained from the Univer-
sity of West Indies Board for Graduate Studies and
Research & Publications Fund. M.R.W. and W.A.G. thank
the University of the West Indies for the granting of post-
graduate scholarships and tutorial assistantships.

M.R. Wilson et al. / Steroids 64 (1999) 834–843
843
[23] Holland HL, Chenchaiah PC, Thomas EM, Mader B, Dennis MJ.
Microbial hydroxylation of steroids. 9. Epoxidation of ⌬6-3-ketoster-
oids by Rhizopus arrhizus ATCC 11145, and the mechanism of the
6␤ hydroxylase enzyme. Can J Chem 1984;62:2740–7.
[24] Smith KE, Latif S, Kirk DN. Microbial transformations of steroids.
VI. Transformation of testosterone and androsterdione by Botryo-
sphaerica obtusa. J Steroid Biochem 1990;35:115–20.
[25] Smith KE, Latif S, White KA, Kirk DN. Microbial transformations of
steroids. I. Rare transformations of progesterone by Apiocrea chry-
sosperma. J Steroid Biochem 1988;31:83–9.
medium containing authentic amino acids. Nippon Shokubutsu Byori
Gakkaiho 34:235–41. Chem Abs 1968;70:55005.
[31] Ohnishi, S, Kosaki T, Osawa Y. Synthesis of 19-hydroxy-[7-
2H2]androstenedione for human metabolism studies. Steroids 1990;
55:5–9.
[32] Bell AM, Browne JW, Kasal A, Denny WA, Jones ERH, Meakins
GD. Microbial hydroxylation of steroids. VI. Hydroxylation of simple
mono- and dioxygenated 5␣-androstanes and of 3-oxoestranes with
the fungus Aspergillus ochraceus. J Chem Soc [Perkin I] 1972;23:
2930–6.
[26] Meister PD, Magerlein BJ, Murray HC, Peterson DH, Eppstein SH,
Marian–eigh H, Weintraub A, Reinecke LM. The 11-epimer of com-
pound F and other new oxygenated derivative of Reichstein’s com-
pound S: a new route to cortisone. J Am Chem Soc 1953;75:412–5.
[27] Hanson JR, Siverns M. 13C nuclear magnetic resonance spectra of
steroidal unsaturated ketones. J Chem Soc [Perkin I] 1975;19:
1956–8.
[33] Bell AM, Cherry PC, Clark IM, Denny WA, Jones ERH, Meakins
GD, Woodgate PD. Microbial hydroxylation of steroids. IV. Pattern
of dihydroxylation of monooxygenated 5␣-androstanes with cultures
of the fungus Calonectria decora. J Chem Soc [Perkin I] 1972;16:
2081–95.
[34] Davies HG, Green RH, Kelly DR, Roberts SM. Biotransformations in
Preparative Organic Chemistry. London: Academic Press, 1989. p.
177.
[28] Blunt JW, Stothers JB. 13C N.m.r spectra of steroids—a survey and
commentary. Org Magn Reson 1977;9:439–64.
[35] Silverstein R, Bassler G, Morrill T. Spectrometric Identification of
Organic Compounds. New York: John Wiley & Sons, 1991. p. 20.
[36] Bieman K. Mass Spectroscopy: Organic Chemical Applications. New
York: McGraw-Hill, 1962. p. 343.
[37] Casas-Campillo C, Bautista M. Microbiological aspects in the hy-
droxylation of estrogens by Fusarium monoliforme. Appl Microbiol
1965;13:977–84.
[29] Oppolzer W, Roberts DA. The enantioselective synthesis of (ϩ)-
estradiol from 1,3-dihydrobenzo[c]thiophene-2,2-dioxide by succes-
sive thermal sulphur dioxide extrusion and cycloaddition reactions:
preliminary communication. Helv Chim Acta 1980;63:1703–5.
[30] Egawa H, Masuko M, Ueyama A. Nitrogen utilization by plant
patogenic fungi. II. Unidentified ninhydrin-positive substance found
in the culture filtrate of plant-pathogenic fungi when cultured on a