276 J ournal of Natural Products, 2002, Vol. 65, No. 3
Hsu et al.
incubated with shaking on a rotatory shaker (HOTECH 706R),
operating at 250 rpm and 28 °C. After 72 h of incubation in
the above-described medium, 5 mL of stage I culture was used
as inoculum for stage II culture. Isosteviol 2 was added to each
flask 24 h later as 100 µL dimethylformamide (DMF) aliquots
each containing 10 mg of isosteviol in solution to give a final
concentration of 0.4 mg/mL of culture. Culture controls
consisted of fermentation blanks in which microorganisms
were grown under identical conditions but without substrate.
Substrate controls consisted of sterile medium containing the
same amount of substrate and incubated under the same
conditions. The fermentations were sampled for TLC analysis
at 24, 72, and 144 h. Samples of 4 mL of culture broth were
acidified with 6 N HCl and extracted with 1 mL of EtOAc-
butanol (9:1). The organic layer was spotted onto a TLC plate
with CHCl3-MeOH (10:1) as the solvent system. Eight me-
tabolites were reproducibly produced by Cunninghamella
bainieri, Actinoplanes sp., Mucor recurvatus, and Cunning-
hamella blakesleeana and displayed TLC Rf values at 0.42 (3),
0.40 (4), 0.37 (5), 0.27 (6), 0.41 (7), 0.36 (8), 0.48 (9), and 0.43
(10) as compared to 0.75 (2).
Micr obia l Meta bolism of Isosteviol (2) by Mu cor r e-
cu r va tu s (MR 36). The fermentation procedure was con-
ducted as described for C. bainieri except that the incubation
reaction was terminated 24 h after substrate addition. Biotrans-
formation of 2 (500 mg) produced 1.1 g of brown oil, which
was subjected to silica gel column chromatography using the
eluent CHCl3-CH3OH (10:1 and 9:1) to give 9 mg of 7 (4.0%)
and 5 mg of 8 (2.2%).
en t-12r,15r-Dih yd r oxy-16-k et ob eyer a n -19-oic
a cid
(7): brown oil; [R]25D -42.3° (c 1.04, MeOH); 1H NMR (C5D5N,
500 MHz) δ 0.98 (1H, td, J ) 13.0, 3.5 Hz, H-1), 1.07 (1H, dd,
J ) 13.5, 4.0 Hz, H-3), 1.12 (3H, s, CH3-20), 1.27-1.38 (2H,
m, H-5, H-7), 1.40 (3H, s, CH3-18), 1.45 (3H, s, CH3-17), 1.54
(1H, td, J ) 14.0, 3.5 Hz, H-11), 1.70 (1H, d, J ) 13.0 Hz,
H-1), 2.00-2.07 (3H, m, H-9, H-11, H-14), 2.20-2.23 (2H, m,
H-2, H-6), 2.41-2.48 (3H, m, H-3, H-6, H-14), 2.85 (1H, br d,
J ) 13.5 Hz, H-7), 4.03 (1H, s, H-12R), 4.62 (1H, s, H-15R);
13C NMR (C5D5N, 125 MHz), see Table 1; FABMS m/z 349 [M
- H]-; HRFABMS m/z 349.2024 (calcd for C20H29O5, 349.2015).
en t-7r,15r-Dih yd r oxy-16-k etobeyer a n -19-oic a cid (8):
oil; [R]25 -58.2° (c 0.67, MeOH); 1H NMR (C5D5N, 500 MHz)
D
Micr obia l Meta bolism of Isosteviol (2) by C. ba in ier i
(UI 3065). Cunninghamella bainieri (UI 3065) was grown in
twenty-five 125 mL stainless steel-capped DeLong flasks, each
containing 25 mL of medium. A total of 250 mg of isosteviol 2
(in 2.5 mL of dimethylformamide) was evenly distributed
among 25 flasks containing stage II cultures. The cultures
were incubated on a rotator shaker (250 rpm) at 28 °C for 6
days. The incubation mixture was pooled and acidified with 6
N HCl and then filtered to remove cells. The filtrate was
extracted three times with equal volumes of EtOAc-butanol
(9:1). The combined extracts were dried over anhydrous Na2-
SO4, and the solvent was evaporated to give 918 mg of a brown
residue. The crude residue was purified by chromatography
over a silica gel column using CHCl3-CH3OH (30:1) as the
eluent. After recrystallization with acetone, 67 mg of 3 (25.5%)
and 37 mg of 4 (14.1%) were obtained as white prisms and
white needles, respectively.
Micr obia l Tr a n sfor m a tion of Isosteviol (2) by Actin o-
pla n es sp . (ATCC 53771). Actinoplanes sp. bioconversion was
conducted as with C. bainieri preparative-scale reactions and
was terminated after 144 h following 250 mg of substrate
addition. Extraction as described above produced 560 mg of
brown oil. The crude residue was purified by chromatography
over a silica gel column using CHCl3-MeOH (10:1 and 9:1).
After recrystallization with acetone, 26 mg of 5 (5.8%) and 8
mg of 6 (1.7%) were obtained.
δ 1.12 (1H, m, H-1), 1.17 (3H, s, CH3-17), 1.19 (3H, s, CH3-
20), 1.20-1.37 (3H, m, H-3, H-11, H-12), 1.48 (3H, s, 18-CH3),
1.49 (1H, m, H-2), 1.69-1.75 (3H, m, H-1, H-11, H-12), 2.08-
2.15 (2H, m, H-9, H-14), 2.26 (1H, m, H-2), 2.46-2.56 (3H, m,
H-3, H-5, H-14), 2.63 (1H, m, H-6), 2.79 (1H, m, H-6), 4.53
(1H, br s, H-15R), 5.07 (1H, br s, H-7R); 13C NMR (C5D5N, 125
MHz), see Table 1; FABMS m/z 351 [M + H]+; HRFABMS m/z
351.2172 (calcd for C20H31O5, 351.2171).
Micr obia l Meta bolism of Isosteviol (2) by C. bla keslee-
a n a (ATCC 8688a ). Isosteviol (2, 250 mg), dissolved in 2.5
mL of DMF, was evenly distributed among 25 flasks containing
stage II cultures. Fermentation was stopped after 6 days. The
incubation mixtures were acidified with 6 N HCl and then
filtered to remove cells. The filtrate was extracted three times
with equal volumes of EtOAc-butanol (9:1). The combined
extracts were dried over anhydrous Na2SO4, and the solvent
was evaporated to give 573 mg of a brown residue. The crude
residue was chromatographed on silica gel. Elution with
CHCl3-CH3OH (40:1) yielded 12 mg of 3 (4.6%). Elution with
CHCl3-CH3OH (30:1) produced 22 mg of 9 (8.4%) and 74 mg
of 10 (28.2%).
en t-9r-Hyd r oxy-16-k etobeyer a n -19-oic a cid (9): white
needles (acetone); mp 246-248 °C; [R]25 -87.6° (c 0.5, CH3-
D
1
OH); H NMR (500 MHz, pyridine-d5) δ 1.09 (3H, s, CH3-17),
1.15 (1H, dd, J ) 13.0, 4.0 Hz, H-3), 1.19 (1H, dd, J ) 11.5,
2.5 Hz, H-14), 1.29 (3H, s, CH3-20), 1.39 (3H, s, CH3-18), 1.45-
1.52 (3H, m, H-1, H-7, H-12), 1.58 (1H, m, H-2), 1.75 (2H, dd,
J ) 9.5, 3.5 Hz, H-11), 1.98 (1H, m, H-12), 2.02 (1H, d, J )
18.5 Hz, H-15), 2.15-2.34 (6H, m, H-1, H-2, H-5, H-6, H-7),
2.43 (1H, d, J ) 13.0 Hz, H-3), 2.77 (1H, dd, J ) 11.5, 4.0 Hz,
H-14), 3.00 (1H, dd, J ) 18.5, 4.0 Hz, H-15); 13C NMR (C5D5N,
125 MHz), see Table 1; FABMS m/z 335 [M + H]+; HRFABMS
m/z 335.2206 (calcd for C20H31O4, 335.2222).
en t-11r,12r-Dih yd r oxy-16-k etobeyer a n -19-oic
a cid
(5): prisms (acetone); mp 221-223 °C; [R]25 +17.7° (c 0.5,
D
MeOH); 1H NMR (C5D5N, 500 MHz) δ 1.11 (1H, m, H-3), 1.24-
1.28 (3H, m, H-1, H-5, H-14), 1.33 (3H, s, CH3-20), 1.36 (3H,
s, CH3-17), 1.38 (3H, s, CH3-18), 1.49-1.56 (2H, m, H-2, H-7),
1.66 (1H, m, H-7), 1.82 (1H, d, J ) 10.5 Hz, H-9â), 1.84 (1H,
d, J ) 18.0 Hz, H-15), 1.85-1.88 (2H, m, H-6), 2.29 (1H, m,
H-2), 2.45-2.49 (2H, m, H-3, H-14), 2.91 (1H, dd, J ) 18.0,
3.5 Hz, H-15), 3.18 (1H, br d, J ) 13.5 Hz, H-1), 3.86 (1H, d,
J ) 3.5 Hz, H-12R), 4.01 (1H, dd, J ) 10.5, 3.5 Hz, H-11R);
13C NMR (C5D5N, 125 MHz), see Table 1; FABMS m/z 351
[M + H]+; HRFABMS m/z 351.2167 (calcd for C20H31O5,
351.2171).
An tih yp er ten sive Testin g. The antihypertensive activity
was determined as described previously.16-19
Ack n ow led gm en t. The authors are grateful to Dr. J ohn
P. N. Rosazza, Division of Medicinal and Natural Products
Chemistry, College of Pharmacy, University of Iowa, for kindly
providing the strains. Financial support by research grants
(NSC 88-2314-B-038-147 and NSC 88-2314-B-038-109) from
the National Science Council of the Republic of China is
gratefully acknowledged.
en t-11r,12r,17-Tr ih yd r oxy-16-k etobeyer a n -19-oic a cid
(6): amorphous powder (acetone); mp 134-136 °C; [R]25
D
+34.6° (c 0.5, MeOH); 1H NMR (C5D5N, 500 MHz) δ 1.09 (1H,
m, H-3), 1.26-1.32 (2H, m, H-1, H-5), 1.34 (3H, s, CH3-20),
1.37 (3H, s, CH3-18), 1.50 (1H, br d, J ) 14.5 Hz, H-2), 1.61
(1H, m, H-7), 1.72 (1H, d, J ) 13.0 Hz, H-7), 1.93 (1H, d, J )
18.5 Hz, H-15), 1.94 (1H, d, J ) 10.5 Hz, H-9â), 2.09-2.11
(3H, m, H-6, H-14), 2.29 (1H, d, J ) 13.5 Hz, H-2), 2.45 (1H,
d, J ) 12.0 Hz, H-3), 2.55 (1H, dd, J ) 11.5, 3.5 Hz, H-14),
2.97 (1H, dd, J ) 18.5, 3.5 Hz, H-15), 3.19 (1H, d, J ) 14.0
Hz, H-1), 4.06 (1H, dd, J ) 10.5, 3.5 Hz, H-11R), 4.11 (1H, d,
J ) 3.5 Hz, H-12R), 4.32, 4.43 (each 1H, d, J ) 10.5 Hz, CH2-
OH); 13C NMR (C5D5N, 125 MHz), see Table 1; FABMS m/z
367 [M + H]+; HRFABMS m/z 367.2110 (calcd for C20H31O6,
367.2120).
Refer en ces a n d Notes
(1) Avent, A. G.; Hanson, J . R.; de Oliveira, B. H. Phytochemistry 1990,
29, 2712-2713.
(2) Bracht, A. K.; Alvarez, M.; Bracht, A. Biochem. Pharmacol. 1985,
34, 873-882.
(3) Yamamoto, N. S.; Kelmer Bracht, A. M.; Ishii, E. L.; Kemmelmeier,
F. S.; Alvarez, M.; Bracht, A. Experimentia 1985, 41, 55-57.
(4) Ishii, E. L.; Schwab, A. J .; Bracht, A. Biochem. Pharmacol. 1987, 36,
1417-1433.
(5) Cheng, Y. W. Antihypertensive Effects of Isosteviol. M.S. Thesis,
Department of Pharmacology, College of Medicine, National Cheng
Kung University, Tainan, Taiwan, 1999.