BIOCATALYSIS AND BIOTRANSFORMATION
3
Table 1. 13C NMR data for testosterone
metabolites.
1
and its
200–201 ꢀC; IR ꢀmax 3445, 1680 cmÀ1; EA: calculated
for C19H28O4: C 71.22, H 8.81; found: C 71.10, H 8.73%;
1H NMR (CDCl3): dH 0.87 (3H, s, 18-H), 1.39 (3H, s, 19-
H), 3.50 (1H, dd, J = 5.0, 11.0Hz, 12a-H), 3.89 (1H, t, J =
8.5 Hz, 17a-H), 4.35 (1H, brs, 6a-H), 5.81 (1H, s, 4-H);
13C NMR (CDCl3): see Table 1.
C atom
1
2
3
4
5
6
7
1
2
35.48
33.74
36.97
34.09
36.37
34.19
36.93
34.24
35.68
33.89
37.04
34.19
35.02
34.13
3
199.64 200.33 200.41 214.07 199.61 200.47 200.26
4
5
123.87 126.34 126..32
171.52 168.06 168.32
34.61 123.76 126.38 126.53
39.91 171.07 168.14 167.67
6
7
8
9
10
11
12
13
14
15
16
17
18
19
32.63
31.33
35.58
53.69
38.45
20.10
36.21
42.61
50.24
23.14
29.51
72.58
37.17
29.34
53.57
38.00
20.19
31.19
47.57
50.81
21.64
35.71
72.96
37.09
29.75
53.64
38.01
20.56
38.01
42.88
50.44
23.25
30.42
81.64
11.07
19.51
29.96
69.63
37.81
59.24
36.50
20.26
42.25
43.29
51.29
23.04
29.44
81.12
10.96
22.98
32.72
32.14
35.29
53.81
38.63
20.50
36.56
44.31
58.43
72.46
42.57
78.70
12.56
17.48
72.90
38.04
29.17
53.81
38.15
20.27
36.98
42.43
46.94
34.91
69.85
80.62
11.88
19.52
72.81
37.43
28.84
52.46
37.95
29.33
78.99
46.98
48.51
23.07
30.01
81.87
6.02
Results
The first metabolite was identified as 6b-hydroxyan-
drost-4-en-3,17-dione 2. NMR spectra of 2 had new
resonances at dH 4.36 ppm (1H, brs) and dC 72.58 ppm
presence of a 6b-hydroxyl group (Hanson et al. 1996).
The 13C NMR spectrum of 2 lacked the C-17 resonance
of 1 at dC 81.25 ppm and had a new carbon atom res-
onance at dC 220.62 ppm, suggesting that an oxida-
tion had taken place at C-17.
81.25 220.62
10.93
17.17
13.71
19.46
19.44
ꢀC, lit (Hanson et al. 1996); mp 215–220 ꢀC; IR ꢀmax
3405, 1650, 1620 cmÀ1; EA: calculated for C19H28O3: C
The second metabolite was identified as 6b,17b-
1
1
dihydroxyandrost-4-en-3-one 3. The H NMR spectrum
74.96, H 9.27; found: C 74.82, H 9.13%; H NMR (CDCl3):
of 3 showed characteristic resonances at dH 3.65 ppm
(1H, t, J = 8.5 Hz) and dH 4.36 ppm (1H, brs), indicating
the presence of 17b- and 6b-hydroxyl groups, respect-
ively (Hanson et al. 1996). The 13C NMR spectrum of 3
had resonances at dC 72.96 and dC 81.64 ppm, which
were in agreement with the presence of 7b- and 17b-
hydroxyl groups, respectively.
dH 0.82 (3H, s, 18-H), 1.38 (3H, s, 19-H), 3.65 (1H, t, J =
8.5 Hz, 17a-H), 4.36 (1H, brs, 6a-H), 5.80 (1H, s, 4-H); 13C
NMR (CDCl3): see Table 1.
Elution with ethyl acetate in n-hexane (7:3) afforded
7b,17b-dihydroxyandrostan-3-one 4 (75 mg, 7%); crys-
ꢀ
tallized from ethyl acetate as needles; mp 202–203 C
ꢀ
lit (Farooq and Tahara, 2000); mp 196–197 C; IR ꢀmax
3325, 1710 cmÀ1; EA: calculated for C19H30O3: C 74.47,
The third metabolite was identified as 7b,17b-dihy-
droxyandrostan-3-one 4. The 1H NMR spectrum of 4
lacked the 4-H resonance of 1 at dH 5.74 ppm (1H, s),
suggesting the hydrogenation of the double bond in
ring A. The 13C NMR spectrum of 4 had two resonan-
ces at dC 69.93 ppm and dC 81.12 ppm, which were
typical for the presence of 7b- and 17b-hydroxyl
1
H 9.87; found: C 74.33, H 9.70%; H NMR (CDCl3): dH
0.74 (3H, s, 19-H), 0.83 (3H, s, 18-H), 3.65 (2H, m, 7a-H
and 17a-H); 13C NMR (CDCl3): see Table 1.
Elution with ethyl acetate in n-hexane (9:1) afforded
15a,17b-dihydroxyandrost-4-en-3-one 5 (53 mg,ꢀ 5%);
crystallized from acetone as needles; mp 96–97 C, lit
1
ꢀ
groups, respectively (Farooq and Tahara 2000). The H
(Peart et al. 2011); mp 93–94 C; IR ꢀmax 3400, 1660,
1650 cmÀ1; EA: calculated for C19H28O3: C 74.96, H
NMR spectrum of 4 showed two overlapping resonan-
ces at dH 3.65 ppm (2H, m), which were in agreement
with the presence of 7b- and 17b-hydroxyl groups.
The fourth metabolite was identified as 15a,17b-
dihydroxyandrost-4-en-3-one 5. The 13C NMR spectrum
of 5 showed two characteristic resonances at dH 3.89
ppm (1H, t, J = 8.5 Hz) and dH 4.11 ppm (1H, m), indi-
cating the presence of 17b- and 15a-hydroxyl groups,
respectively (Peart et al. 2011). The 13C NMR spectrum
of 5 showed two resonances at dC 72.46 and dC 78.70
ppm, further indicating the presence of 15a- and 17b-
hydroxyl groups, respectively.
The fifth metabolite was identified as 6b,16b,17b-
trihydroxyandrost-4-en-3-one 6. The 13C NMR spec-
trum of 6 had two resonances at dC 72.90 ppm and dC
80.62 ppm, which were characteristic (Hanson et al.
1996) for the presence of 6b- and 17b-hydroxyl
1
9.27; found: C 74.85, H 9.17%; H NMR (CDCl3): dH 0.80
(3H, s, 18-H), 1.20 (3H, s, 19-H), 3.89 (1H, t, J = 8.5 Hz,
17a-H), 4.11 (1H, m, 15b-H), 5.73 (1H, s, 4-H); 13C NMR
(CDCl3): see Table 1.
Elution with pure ethyl acetate afforded
6b,16b,17b-trihydroxyandrost-4-en-3-one
6 (34 mg,
3%); crystallized from ethyl acetate as cubes; mp
169–170 ꢀC; IR ꢀmax 3395, 1660 cmÀ1; EA: calculated
for C19H28O4: C 71.22, H 8.81; found: C 71.15, H 8.70%;
1H NMR (CDCl3): dH 0.88 (3H, s, 18-H), 1.39 (3H, s, 19-
H), 3.38 (1H, d, J = 7.3 Hz, 17a-H), 4.16 (1H, m, 16a-H),
4.34 (1H, brs, 6a-H), 5.81 (1H, s, 4-H); 13C NMR (CDCl3):
see Table 1.
Further elution with pure ethyl acetate afforded
6b,12b,17b-trihydroxyandrost-4-en-3-one
7 (23 mg,
2%); crystallized from ethyl acetate as needles; mp