506
A. Martinez et al. / Phytochemistry 117 (2015) 500–508
were shaken in CERTOMAT
R
B. Braun orbital shakers.
pooled, the cells were washed thoroughly with water, and the liq-
uid was saturated with NaCl and extracted continuously with
CH2Cl2 for four 6-h periods. Dry fungal cells were washed repeat-
edly with CH2Cl2. Both extracts were pooled, dried with dry
Na2SO4, and evaporated under reduced pressure. The resulting
mixture of compounds was chromatographed on a silica-gel col-
umn to provide 108 mg of starting material (3, 72%), and 24 mg
of methyl 3b,7b,30-trihydroxyoleana-9(11),12-dien-28-oate (5,
15%).
Commercially available reagents were used without further purifi-
cation. Merck silica-gel 60 (0.040–0.063 mm, Ref. 1.09385) was
used for flash chromatography. CH2Cl2 (Fisher, Ref. D/1852/17) or
n-hexane (Merck, Ref. 1.04374), with increasing amounts of
Me2CO (Fisher, Ref. A/0600/17), MeOH (Fisher, Ref. M/4000/17),
or AcOEt (Fisher, Ref. E/0900/17), were used as eluents (all the sol-
vents had an analytical reagent grade purity). Merck silica-gel 60
aluminium sheets (Ref. 1.16835) were used for TLC, and spots were
rendered visible by spraying with H2SO4–AcOH, followed by heat-
ing to 120 °C, and also visualized under UV at 254 nm.
4.6.1. Methyl 3b,7b,30-trihydroxyoleana-9(11),12-dien-28-oate (5)
25
Syrup; [
a]
+70 (c 1, CHCl3); IR (film) mmax 3457, 2947, 1723,
D
1240, 760 cmÀ1; for 1H NMR (400 MHz, CDCl3) see Table 1; for
13C NMR (100 MHz, CDCl3) see Table 2; HRESIMS m/z 501.3567
[M+1]+ (calcd. for C31H49O5, 501.3580).
4.2. Organism, media and culture conditions
R. miehei (CECT 2749) was obtained from the Spanish Type
Culture Collection (CECT), Departamento de Microbiología,
Universidad de Valencia, Spain. Medium PDA containing 4 g/l of
potato peptone, 20 g/l of glucose, 2 g/l of agar, at pH 5.6, was used
to store the microorganisms. In all microbial transformation exper-
iments, a medium of potato dextrose broth (Scharlau 02-483) was
used for microorganism proliferation. Erlenmeyer flasks (250 ml)
containing 90 ml of sterilized medium were inoculated with 1 ml
of microorganism suspended in saline suspension (9%).
Incubations were maintained at 28 °C with gyratory shaking
(150 rpm) for 6 days, after which the substrates (5–10%) in EtOH
were added.
4.7. Treatment of methyl oleanolate (3) with NBS
Methyl oleanolate (3, 1 g, 2.12 mmol) was dissolved in CCl4
(25 ml), and NBS (378 mg, 2.12 mmol) and a catalytic amount of
AIBN, were added. The reaction was kept for 1 h at reflux. Then,
the reaction mixture was treated with an aqueous solution of
NaHCO3, and the organic phase was extracted repeatedly with
CH2Cl2, dried with dry Na2SO4, and evaporated under reduced
pressure. The chromatographic purification gave 785 mg of methyl
3b-hidroxyoleana-9(11),12-dien-28-oate
(6,
79%)
(Garcia-
Granados et al., 2004).
4.3. Isolation of oleanolic (1) and maslinic (2) acids
4.8. Incubation of 6 with R. miehei
Oleanolic (3b-hydroxyolean-12-en-28-oic acid, 1) and maslinic
(2a,3b-dihydroxyolean-12-en-28-oic acid, 2) acids were isolated
Product 6 (500 mg, 1.07 mmol) was dissolved in EtOH (16 ml),
distributed among 8 Erlenmeyer flask culture (R. miehei) and incu-
bated for 13 days. Then, we proceeded as described above in the
incubation of substrate 3 with the same microorganism. After
chromatographic purification by flash column, 310 mg of starting
material (6, 62%), 59 mg of metabolite 5 (11%), and 44 mg of
from solid wastes resulting from olive-oil production, which were
extracted in a Soxhlet with hexane and EtOAc successively (Garcia-
Granados, 1998). Both acids were purified from these mixtures by
column chromatography over silica gel, eluting with
a
CHCl3/MeOH or CH2Cl2/acetone mixtures of increasing polarity
(Martinez et al., 2013).
methyl 3b,7b,15a,30-tetrahydroxyoleana-9(11),12-dien-28-oate
(7, 8%), were isolated.
4.4. Esterification of oleanolic acid (1)
4.8.1. Methyl 3b,7b,15
a,30-tetrahydroxyoleana-9(11),12-dien-28-
Oleanolic acid (1, 7.1 g, 15.3 mmol) was dissolved in THF
(25 ml) and then, 14.2 ml of a solution of NaOH 5 N were added.
The reaction was kept for 3 h at reflux. Then, 2.84 ml of CH3I were
added to the reaction mixture, keeping it under reflux 2 h.
Afterwards, the reaction mixture was washed with water, neutral-
ized with a HCl solution, and extracted repeatedly with CH2Cl2. The
organic phase was dried with dry Na2SO4, and evaporated under
reduced pressure. After chromatographic purification of the mix-
ture, 6.5 g of methyl 3b-hydroxyolean-12-en-28-oate (methyl
oleanolate, 3, 90%) were isolated (Garcia-Granados et al., 2000).
oate (7)
White solid; mp 134 °C; [a] mmax 3360,
25 +5 (c 1, CHCl3); IR (KBr)
D
1722, 1374, 1243, 756 cmÀ1; for 1H NMR (500 MHz, CDCl3) see
Table 1; for 13C NMR (125 MHz, CDCl3) see Table 2; HRESIMS m/z
517.3521 [M+1]+ (calcd. for C31H49O6, 517.3529).
4.9. Incubation of methyl maslinate (4) with R. miehei
Methyl maslinate (4, 600 mg, 1.27 mmol), was dissolved in
EtOH (40 ml), distributed among 20 Erlenmeyer flask culture (R.
miehei) and incubated for 13 days. Next, we proceeded as described
above in the incubation of substrate 3 with the same microorgan-
ism. The resulting mixture of compounds was chromatographed on
a silica-gel column to provide 378 mg of starting material (4, 63%),
4.5. Esterification of maslinic acid (2)
Maslinic acid (2, 12.3 g, 26 mmol) was dissolved in THF (25 ml)
and then, 24.6 ml of a solution of NaOH 5 N were added, keeping
the reaction for 3 h at reflux. Then, 4.92 ml of CH3I were added
to the reaction mixture, keeping it under reflux 2 h more. This mix-
ture was treated as described above in the esterification of oleano-
74 mg of methyl 2
a,3b,30-trihydroxyolean-12-en-28-oate (8, 12%),
41 mg of methyl 2
a,3b-dihydroxyoleana-9(11),12-dien-28-oate (9,
7%) (Garcia-Granados et al., 2004). The rest of the fractions (50 mg,
8%) were acetylated with Ac2O (2 ml) in pyridine (4 ml). After the
reaction was maintained for 24 h at room temperature, it was
diluted with cold H2O, extracted with CH2Cl2, washed with satu-
rated aqueous KHSO4, and dried over dry Na2SO4.
Chromatography on silica gel of the reaction mixture gave 28 mg
lic acid (1) to give 11.4 g of methyl 2a,3b-dihydroxyolean-12-en-
28-oate (methyl maslinate 4, 90%) (Garcia-Granados et al., 2000).
4.6. Incubation of methyl oleanolate (3) with R. miehei
Methyl oleanolate 3 (150 mg, 0.32 mmol) was dissolved in EtOH
(10 ml), distributed among 5 Erlenmeyer flask cultures (R. miehei),
and incubated for 13 days. Then, the cultures were filtered and
of methyl 2a,3b-diacetoxy-11-oxoolean-12-en-28-oate (10) and
16 mg of methyl
2a,3b-diacetoxy-12-oxo-13R-oleanan-28-oate
(11).