Biotransformation of oleanolic and maslinic acids by Rhizomucor miehei

Article abstract of DOI:10.1016/j.phytochem.2013.05.011

Microbial transformation of oleanolic acid by Rhizomucor miehei produced three metabolites. A known compound, a 30-hydroxyl derivative (queretaroic acid), and two 7β,30- and 1β,30-dihydroxylated metabolites, respectively. The action of the same fungus (R. miehei) on maslinic acid produced an olean-11-en-28,13β-olide derivative, a metabolite hydroxylated at C-30, an 11-oxo derivative, and two metabolites with an 11α,12β- epoxy group, hydroxylated or not at C-30. Their structures were elucidated by extensive analyses of their spectroscopic data, and also by chemical correlations.

Full text of DOI:10.1016/j.phytochem.2013.05.011

Phytochemistry xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Biotransformation of oleanolic and maslinic acids by Rhizomucor miehei
a,
a
a
a
Antonio Martinez a, , Francisco Rivas , Alberto Perojil , Andres Parra , Andres Garcia-Granados ,
⇑
⇑
Antonia Fernandez-Vivas b
a Departamento de Química, Orgánica Facultad de Ciencias, Universidad de Granada, E-18071 Granada, Spain
b Departamento de Microbiología, Facultad de Ciencias, Universidad de Granada, E-18071 Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Microbial transformation of oleanolic acid by Rhizomucor miehei produced three metabolites. A known
compound, a 30-hydroxyl derivative (queretaroic acid), and two 7b,30- and 1b,30-dihydroxylated metab-
olites, respectively. The action of the same fungus (R. miehei) on maslinic acid produced an olean-11-en-
28,13b-olide derivative, a metabolite hydroxylated at C-30, an 11-oxo derivative, and two metabolites
Received 12 March 2013
Received in revised form 6 May 2013
Available online xxxx
with an 11
analyses of their spectroscopic data, and also by chemical correlations.
Ó 2013 Elsevier Ltd. All rights reserved.
a,12a-epoxy group, hydroxylated or not at C-30. Their structures were elucidated by extensive
Keywords:
Biotransformation
Biohydroxylation
Triterpenoids
Oleanolic acid
Maslinic acid
Rhizomucor miehei
1. Introduction
Microbial transformations of triterpenoids have been developed
primarily in the last 10 or 15 years to produce new and useful com-
pounds (Muffler et al., 2011; Parra et al., 2009b). Biotransformation
methods can be used as an alternative to the traditional chemical
synthesis in search of new production routes for fine chemical,
pharmaceutical, and agrochemical compounds. Several biotrans-
formations of oleanolic acid (1), using different microorganisms,
have been published (Hikino et al., 1969, 1971, 1972a,b; Zhang
et al., 2005; Wang et al., 2006; Choudhary et al., 2008; Sun et al.,
2010; Capel et al., 2011; Liu et al., 2011; Zhu et al., 2011). Recently,
the first biotransformation of maslinic acid by Cunninghamella
blakesleana has been described (Feng et al., 2012). This paper con-
centrates on the production of new oleanolic and maslinic acid
derivatives by biotransformation processes, using Rhizomucor
miehei.
Triterpenoids are a large and structurally diverse group of natural
products (Hill and Connolly, 2012) that display nearly 200 distinct
skeletons. These compounds have been studied for their anti-
neoplastic, anti-inflammatory, anti-ulcerogenic, antimicrobial,
anti-plasmodial, antiviral (anti-HIV), hepato- and cardio-protective,
analgesic, anti-mycotic, immunomodulatory, and tonic effects
(Shanmugam et al., 2012; Cassels and Asencio, 2011; Rios, 2010;
Yadav et al., 2010; Kuo et al., 2009; Petronelli et al., 2009; Dzubak
et al., 2006; Akihisa and Yasukawa, 2006; Ovesna et al., 2004).
Oleanolic (3b-hydroxyolean-12-en-28-oic acid, 1) and maslinic
(2a,3b-dihydroxyolean-12-en-28-oic acid, 2) acids are natural
pentacyclic triterpenoid compounds (Pollier and Goossens, 2012;
García-Granados et al., 1998a,b). These acids are present in high
concentrations in olive-pomace oil, being the main components
of the protective wax-like coating of the olive skin. Our group
has reported a method to obtain large amounts of these com-
pounds from olive-pressing residues (García-Granados, 1998c).
Both triterpenic acids and some closely related compounds display
remarkable pharmacological activities such as antitumor, antibac-
terial, anti-HIV, anti-inflammatory, antioxidant, and hepatoprotec-
tive (Pollier and Goossens, 2012; Salvador et al., 2010; Wolska
et al., 2010; Wang et al., 2010; Reyes-Zurita et al., 2011; Huang
et al., 2011; Parra et al., 2009a, 2011).
2. Results and discussion
2.1. Biotransformation of oleanolic acid (1)
Biotransformation of oleanolic acid (1) with R. miehei for
13 days yielded a mixture of metabolites. Chromatography on a sil-
ica-gel column of this mixture resulted in recovery of 64% of the
substrate (1), and isolation of metabolites 3 (5%), 4 (6%), and 5
(5%) (Fig. 1).
Metabolite 3 had a molecular formula of C30H48O4, indicating
that the microorganism had inserted an oxygen atom onto the sub-
strate (1). The main difference of the 1H NMR spectra of compounds
⇑
Corresponding authors. Tel.: +34 958 240481; fax: +34 958 248437.
E-mail addresses: aramon@ugr.es (A. Martinez), frivas@ugr.es (F. Rivas).
0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.05.011
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2
A. Martinez et al. / Phytochemistry xxx (2013) xxx–xxx
Fig. 1. Metabolites isolated from the incubation of oleanolic acid (1) with Rhizomucor miehei.
1 and 3 was the presence in the latter of an AB system signal (dH
3.55 and 3.43), which was due to the coupling of two geminal pro-
tons to a hydroxyl group placed on a primary carbon. This signal
suggested the hydroxylation, by the fungus, at one of the seven
methyl groups of the substrate (1). The position of the microbial
hydroxylation was deduced by comparison of the NMR data (13C,
HSQC, and HMBC) of metabolite 3 and substrate 1. The analysis of
the HMBC of metabolite 3 ruled out the possibility that the new hy-
droxyl group was located at C-23, C-24, C-25, C-26, or C-27. There-
fore, this hydroxyl group may occur only at C-29 or C-30. The 13C
NMR chemical shifts of these carbon atoms, for the substrate 1,
were significantly different (dC 33.6 and 24.0, respectively), which
made it easier to locate the position of the new hydroxyl group. A
in vitro antimicrobial activity against six Gram-positive and twelve
Gram-negative bacterial species as well as three yeasts of Candida
species, being active against 95% of all of them (Kuete et al., 2007).
The molecular formula (C30H48O5) of the last metabolite iso-
lated (5) confirmed that the microorganism had inserted two oxy-
gen atoms in the molecule of the substrate (1). The AB system
signal (dH 3.55 and 3.42) that appeared in the 1H NMR spectrum
of metabolite 5 suggested the presence of a 30-hydroxyl group in
the molecule, being confirmed again by the correlations existing
in the HMBC spectrum of this metabolite (5), similar to those ob-
served in metabolite 4 (Fig. 2). In the 1H NMR spectrum of this
metabolite (5), a new signal also appeared at dH 3.34 (1H, dd,
J = 10.8, 4.9 Hz), due to a geminal proton to an equatorial hydroxyl
group. The correlations observed in the HMBC spectrum of this
metabolite (5) between this signal (dH 3.34) and C-2, C-9, C-10,
and C-25, suggested that the new hydroxyl group was placed at
C-1 (Fig. 2), being confirmed by the analysis of the 13C NMR spec-
trum of metabolite 5 compared with that of metabolite 3, where
hydroxyl group at C-29 would result in a
c-effect on C-30 (dC
19.8) (Shao et al., 1989), whereas a hydroxyl group at C-30 a
c-ef-
fect on C-29 (dC 28.8) (Yang et al., 1998). These data confirmed that
metabolite 3 had a structure of 3b,30-dihydroxyolean-12-en-28-oic
acid, also called queretaroic acid, a natural product isolated from
two Mexican cacti, Lemaireocereus queretaroensis and Lemaireocere-
us beneckei (Djerassi et al., 1956), and from Machaerocereus eruca
(Yang et al., 1998). Queretaroic acid (3) has also been obtained from
the hydroxylation of oleanolic acid (1) by Escherichia coli coexpress-
ing a cytochrome P450 gene from Nonomuraea recticatena and the
Pseudomonas redox partner camAB (Fujii et al., 2006). This
compound (3) showed anti-tumour properties inhibiting 32Pi-
incorporation into phospholipids of HeLa cells enhanced by TPA
(12-O-tetradecanoylphorbol-13-acetate) (Kinoshita et al., 1999).
Metabolite 4 had a molecular formula of C30H48O5, implying
that two oxygen atoms had been inserted onto the substrate (1)
by the microorganism. In the 1H NMR spectrum of metabolite 4 ap-
peared an AB system signal (dH 3.56 and 3.44) similar to that of
metabolite 3, suggesting the presence of a 30-hydroxyl group in
the molecule, being confirmed by the correlations of this signal
with those of C-19, C-20, C-21, and C-29 in the HMBC spectrum
of metabolite 4 (Fig. 2). In the 1H NMR spectrum of this metabolite
(4) a new signal also appeared at dH 3.86 (1H, dd, J = 11.2, 5.0 Hz),
due to a geminal proton to an equatorial hydroxyl group. This
second hydroxylation performed by the microorganism on a sec-
ondary carbon would be located on C-1, C-7, C-15, C-16, C-21, or
C-22. The correlations observed in the HMBC spectrum of this
metabolite (4) between this signal (dH 3.86) and C-6, C-8, C-14,
and C-26, suggested that the new hydroxyl group was placed at
C-7 (Fig. 2), being confirmed by the analysis of the 13C NMR spec-
trum of metabolite 4 compared with that of metabolite 3, where
effects
a (on C-1), b (on C-2 and C-10), and c (on C-3, C-5, and C-
25) were observed. These data confirmed the structure of
1b,3b,30-trihydroxyolean-12-en-28-oic acid for metabolite 5.
2.2. Biotransformation of maslinic acid (2)
Biotransformation of maslinic acid (2) by R. miehei for 13 days
produced a mixture of metabolites difficult to separate. Chroma-
tography on a silica-gel column of this mixture resulted in recovery
of 42% of the substrate (2), and isolation of metabolites 6 (0.5%)
and 7 (11%). Acetylation of the rest of the fractions yielded the
2a,3b-diacetoxy derivatives 8–12 (Fig. 3).
The molecular formula of metabolite 6 (C30H48O4) indicated
that this metabolite had two hydrogen atoms less than the sub-
strate (2). Its 1H NMR spectrum showed two double doublets (dH
6.04 and 5.42) corresponding to the signals of two olefinic protons,
which were correlated with the signal of H-9 (dH 1.94) in its COSY
spectrum. In the 13C NMR spectrum of this metabolite (6) appeared
two signals of methyne double bond carbon (dC 135.5 and 127.3)
that corresponded to a HC@CH system, and also a signal of a qua-
ternary oxygenated carbon at 89.9 ppm. These spectroscopic data
suggested that metabolite 6 had a structure of 28,13b-oleanolide
with a double bond between C-11 and C-12. In its HMBC spectrum
the correlations between H-11 and C-9, and between H-12 with C-
9 and C-13, confirmed this structure (Fig. 4). Therefore, metabolite
6 was 2a,3b-dihydroxyolean-11-en-28,13b-olide. A general mech-
effects
a
(on C-7), b (on C-6 and C-8), and
c
(on C-5 and C-26), were
anism to rationalize the formation of this metabolite (6) could be
the introduction of a hydroperoxyl group on the allylic position
at C-11, by the microorganism (Fig. 5). Then there would be the
loss of a hydrogen peroxide molecule, and the subsequent attack
observed. These data are consistent with a structure of 3b,7b,30-
trihydroxyolean-12-en-28-oic acid for metabolite 4. A 7b-hydroxy
derivative of oleanolic acid, called canthic acid, was tested for
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A. Martinez et al. / Phytochemistry xxx (2013) xxx–xxx
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Fig. 2. Key HMBC (C?H) correlations in metabolites 4 and 5.
of the carboxyl group of C-28 to the carbon atom of C-13 to form a
28,13b-olide group with a double bond at C-11. Moodley et al.
(2011) reported that an 11-en-28,13b-olide derivative of oleanolic
acid, with a structure similar to that of metabolite 6, presented
good antibacterial activity (MICs in the range 0.06–0.12 mg/mL)
for all the bacterial strains studied, and declared that the lactone
ring contributes significantly to the expression of antibacterial
activity in these types of triterpenes.
Metabolite 7 had a molecular formula of C30H48O5, with an ex-
tra oxygen atom with respect to that of the substrate (2). The AB
system signal (dH 3.55 and 3.43) that appeared in the 1H NMR
spectrum, and the correlations of this signal with those of C-19,
C-20, C-21, and C-29 observed in the HMBC spectrum of metabolite
7, confirmed the presence of a 30-hydroxyl group in the molecule.
The third derivative (10) had a molecular formula of C34H50O7.
Its 1H NMR spectrum showed two singlets (dH 5.63 and 2.37) cor-
responding to the signal of an olefinic proton at C-12, and to the
signal of H-9, respectively. In the HSQC spectrum, these signals
were correlated with those that appeared at dC 128.1 and 61.6,
respectively. In the 13C NMR spectrum of this metabolite (10) also
appeared a signal of a carbonyl group at dC 199.7, and the other sig-
nal of the double bond (dC 168.8). The correlations observed in the
HMBC spectrum of this compound (10) between the signal of H-9
(dH 2.37) and C-11, and the signal of H-12 (dH 5.63) and C-9, C-14,
and C-18 (Fig. 4), suggested the presence of an
a,b-unsaturated
system on the C ring of the molecule, with a carbonyl group at
C-11 and a double bond between C-12 and C-13. Therefore,
compound 10 had a structure of 2a,3b-diacetoxy-11-oxoolean-
Therefore, metabolite 7 had a structure of 2
an-12-en-28-oic acid.
a
,3b,30-trihydroxyole-
12-en-28-oic acid. A mechanism to explain the formation of this
compound (10) could be, as was the case for metabolite 6, the
introduction of a hydroperoxyl group on the allylic position at
C-11, by the microorganism. Then, there would be the loss of a
molecule of H2O to form a carbonyl group at this position
The rest of the fractions obtained from the chromatographic
separation of the mixture of metabolites from the biotransforma-
tion with R. miehei of substrate 2 were combined and acetylated,
yielding derivatives 8–12 (Fig. 3). The study of 1H NMR, 13C NMR,
HSQC, and HMBC spectra of the acetoxy derivatives 8 and 9 con-
(Fig. 5). This a,b-unsaturated carbonyl group system on the C ring
was previously achieved in the biotransformation of oleanolic acid
(1) with Cunninghamella blakesleeana (Hikino et al., 1969), and this
compound (10) was chemically synthesised through the modifica-
tion of oleanolic acid (Zhao et al., 2010).
firmed the structures of 2
a,3b-diacetoxyolean-11-en-28,13b-olide
(8), and 2 ,3b,30-triacetoxyolean-12-en-28-oic acid (9), respec-
a
tively, for these derivatives. To confirm the structures of metabolite
6 and its acetoxy derivative 8, both were chemically synthesised
from maslinic acid (2). Bromination of maslinic acid (2) with Br2/
CCl4 originated the monobrominated derivative 13 (Pereda-Miran-
da et al., 1992). This compound (13) had a molecular formula of
The fourth derivative isolated (11) had a molecular formula
C34H50O7, which indicated that the microorganism had inserted
an oxygen atom onto the substrate. In the IR spectrum, there were
no signals of hydroxyl groups. In the 1H NMR spectrum, no signals
of double bond protons appeared, but signals of two geminal pro-
tons to an oxygen atom (dH 3.02) were present, these being corre-
lated, in the HSQC spectrum, with the signals of two oxygenated
carbons (dC 57.1 and 52.4). Also, the presence of a signal of a totally
substituted oxygenated carbon at dC 87.4, in the 13C NMR spec-
trum, revealed the existence of a lactone group between C-28
and C-13. The correlations observed in the HMBC spectrum of this
compound (11) between the signals of H-11 and H-12 (dH 3.02)
with C-9, C10, C-13, and C-14 (Fig. 4), suggested the presence of
an epoxy group at C-11 and C-12. Therefore, compound 11 had a
C30H48BrO4. The position and stereochemistry of the bromine atom
was deduced from the NMR data for this compound (13): at dH 4.30
a signal due to the axial proton geminal to the bromine atom at
C-12, and at dC 56.2 a signal of a methynic carbon atom assigned
to C-12. Also, the presence of a signal of a totally substituted oxy-
genated carbon at dC 91.7, revealed the existence of a lactone group
between C-28 and C-13. These data, together with those gained
from the analysis of the HMBC spectrum, which showed a correla-
tion between the signal of H-12 (dH 4.30) and C-9, C-11, C-13, and
C-14 (Fig. 4), confirmed that compound 13 was 12
,3b-dihydroxyolean-28,13b-olide. The proposed mechanism to
explain the formation of this compound (13) could be the initial
formation of a bromonium ion at C-12/C-13 by the -face and
a-bromine-
2a
structure of 2a,3b-diacetoxy-11a,12a-epoxyolean-28,13b-olide.
Pereda-Miranda and Delgado (1990) described the chemical epox-
idation of an olean-11-en-28,13b-olide compound that originated
a
the subsequent attack of the carboxyl group of C-28 to the carbon
atom of C-13 to form the 28,13b-olide group (Fig. 6). Acetylation of
the bromine derivative (13) originated the diacetoxy derivative 14,
which was treated with DBU/xylene to give a compound with a
double bond at C-11, whose spectroscopic properties matched
with those of the diacetoxy derivative 8. The hydrolysis of com-
pound 8 with KOH in methanol/water gave metabolite 6 (Fig. 6).
an 11
of the diacetoxy derivative 8 with peracetic acid resulting an
11 ,12 -epoxy derivative, with identical spectroscopic properties
a,12a-epoxiderivative. Thus, we achieved the epoxidation
a
a
to those of compound 11 (Fig. 6).
The last derivative isolated (12) had a molecular formula of
C
36H52O9. Its 1H NMR spectrum showed signals similar to those de-
tected in compound 11, due to an 11a,12a-epoxy group, along
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A. Martinez et al. / Phytochemistry xxx (2013) xxx–xxx
Fig. 3. Metabolites isolated and acetylated derivatives achieved from the incubation of maslinic acid (2) with Rhizomucor miehei.
with a new AB system signal with doublets centred at dH 4.20 and
3.78, as identified in compound 9 for an acetoxy group at C-30.
These data indicated that the structure of compound 12 was
3. Concluding remarks
The biotransformation processes carried out reveal that
2a,3b,30-triacetoxy-11a,12a-epoxyolean-28,13b-olide.
R. miehei can perform remarkable functionalizations on these
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A. Martinez et al. / Phytochemistry xxx (2013) xxx–xxx
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Braun orbital shakers. 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 solvents had an analytical reagent grade purity). Merck
TLC silica-gel 60 (ref. 1.16835) was rendered visible by spraying
with H2SO4–HOAc–H2O, followed by heating to 120 °C.
4.2. Isolation of starting materials
Oleanolic acid (3b-hydroxyolean-12-en-28-oic acid, 1) and
maslinic acid (2a,3b-dihydroxyolean-12-en-28-oic acid, 2) were
isolated from solid wastes of olive-oil production, extracting in a
Soxhlet successively with hexane and EtOAc. The EtOAc extracts
were concentrated under reduced pressure, and the solid powder
obtained (10 g) was chromatographed on silica-gel by flash column
chromatography to give 2 g of 1, and 6 g of 2.
Fig. 4. Key HMBC (C?H) correlations in compounds 6, 10, 11, and 13.
4.2.1. 3b-Hydroxyolean-12-en-28-oic acid (oleanolic acid, 1)
White solid; mp 306–8 °C; [
a] +80 (c 1, CHCl3); IR (KBr) mmax
D
3438, 2930, 2869, 1690 cmÀ1 1H NMR (500 MHz, CD3OD) d 5.25
;
(1H, dd, J = 3.6, 3.6 Hz, H-12), 3.16 (1H, dd, J = 11.2, 4.8 Hz, H-3),
2.84 (1H, dd, J = 13.6, 3.7 Hz, H-18), 1.99 (1H, ddd, J = 13.4, 13.4,
3.6 Hz, H-16), 1.15 (3H, s, 3H-27), 0.97 (3H, s, 3H-23), 0.93 (6H,
s, 3H-25 and 3H-30), 0.90 (3H, s, 3H-29), 0.80 (3H, s, 3H-26),
0.77 (3H, s, 3H-24), 0.74 (1H, d, J = 11.5 Hz, H-5); for 13C NMR
(125 MHz, CD3OD), see Table 1; HRLSIMS m/z 479.3504 [M+Na]+
(calcd. for C30H48O3Na, 479.3501).
4.2.2. 2
White solid; mp 267-9 °C; [
(KBr) mmax 3386, 2936, 2867, 1690 cmÀ1
a
,3b-Dihydroxyolean-12-en-28-oic acid (maslinic acid, 2)
a]
+54 (c 1, CHCl3:MeOH, 2:1); IR
D
;
1H NMR (300 MHz, CD3-
OD) d 5.26 (1H, dd, J = 3.5, 3.5 Hz, H-12), 3.62 (1H, ddd, J = 11.3,
9.6, 4.5 Hz, H-2), 2.91 (1H, d, J = 9.6 Hz, H-3), 2.86 (1H, dd,
J = 14.1, 3.9 Hz, H-18), 2.02 (1H, ddd, J = 13.3, 13.3, 3.9 Hz, H-16),
1.70 (1H, dd, J = 14.0, 14.0 Hz, H-9), 1,17 (3H, s, 3H-27), 1.02 (3H,
s, 3H-23), 1.01 (3H, s, 3H-25), 0.95 (3H, s, 3H-30), 0.91 (3H, s,
3H-29), 0.82 (3H, s, 3H-26), 0.81 (3H, s, 3H-24); for 13C NMR
(75 MHz, CD3OD), see Table 1; HRLSIMS m/z 495.3458 [M+Na]+
(calcd. for C30H48O4Na, 495.3450).
Fig. 5. The general mechanism proposed for the formation of the metabolite 6 and
compound 10.
triterpenoid compounds. This microorganism has achieved
hydroxylation reactions at non-activated positions, oxidation reac-
tions of a hydroxyl group and the double bond, and formation of a
lactone group from the carboxyl group present in these triterpenic
acids. Some of these metabolites isolated present remarkable bio-
logical properties, such as queretaroic acid, or have very similar
structures of compounds that have noteworthy biological
activities.
4.3. Organism, media and culture conditions
R. miehei CECT 2749 was obtained from the Spanish Type Cul-
ture Collection (CECT), Departamento de Microbiología, Universi-
dad de Valencia, Spain. Medium YEPGA containing 1% of yeast
extract, 1% of peptone, 2% of glucose, and 2% of agar, at pH 5,
was used to store the microorganisms. In all microbial transforma-
tion experiments, 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 a dense microorganism suspension. Incubations were main-
tained at 28 °C with gyratory shaking (150 rpm) for 6 days after
which the substrates (5–10%) in EtOH were added.
4. Experimental
4.1. General experimental procedures
Measurements of NMR spectra were made in a VARIAN Inova
unity (300 MHz 1H NMR), and a VARIAN direct drive (500 and
600 MHz 1H NMR) spectrometers. Assignments of 13C NMR chem-
ical shifts (Table 1) were made with the aid of distortionless
enhancement by polarization transfer (DEPT) using a flip angle of
135°. Several programs were used for COSY, HSQC, and HMBC
experiments. IR spectra were recorded in a MATTSON SATELLITE
FT-IR spectrometer. High-resolution mass spectra were made in a
WATERS LCT Premier XE spectrometer or by LSIMS (FAB) ionization
mode in a MICROMASS AUTOSPEC-Q spectrometer (EBE geometry).
Melting points (mp) were determined using a Kofler (Reichert)
apparatus and are uncorrected. Optical rotations were measured
on a Perkin-Elmer 431 polarimeter at 25 °C. Incubations of sub-
strates with the microorganism were shaken in CERTOMAT R B.
4.4. Oleanolic acid incubation with R. miehei
Oleanolic acid 1 (500 mg) was dissolved in EtOH (24 ml), dis-
tributed among 12 Erlenmeyer flask cultures (R. miehei) and incu-
bated for 13 days. Then, the cultures were filtered and pooled, the
cells were washed thoroughly with water, and the liquid was sat-
urated with NaCl and extracted continuously with CH2Cl2 for four
6-h periods. Dry fungal cells were washed repeatedly with CH2Cl2.
Both extracts were pooled, dried with dry Na2SO4, and evaporated
under reduced pressure. The resulting mixture of compounds was
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A. Martinez et al. / Phytochemistry xxx (2013) xxx–xxx
Fig. 6. The mechanism proposed for the formation of the compound 13, and the chemical correlation of compounds 6, 8, and 11.
chromatographed on a silica-gel column to obtain 320 mg of start-
ing material (1, 64%), 25 mg of 3b,30-dihydroxyolean-12-en-28-oic
acid (queretaroic acid, 3, 5%), 32 mg of 3b,7b,30-trihydroxyolean-
12-en-28-oic acid (4, 6%), and 28 mg of 1b,3b,30-trihydroxyole-
an-12-en-28-oic acid (5, 5%).
CD3OD) d 5.27 (1H, m, J = 3.5, 3.5 Hz, H-12), 3.55 and 3.42 (2H,
AB system, J = 10.5 Hz, 2H-30), 3.34 (1H, dd, J = 10.8, 4.9 Hz, H-1),
3.18 (1H, dd, J = 11.5, 4.9 Hz, H-3), 2.79 (1H, dd, J = 14.0, 4.7 Hz,
H-18), 2.44 (1H, ddd, J = 19.3, 3.5, 3.5 Hz, H-11a), 2.09 (1H, ddd,
J = 19.3, 11.0, 3.5 Hz, H-11b), 1.18 (3H, s, 3H-27), 0.97 (3H, s, 3H-
25), 0.95 (3H, s, 3H-23), 0.89 (3H, s, 3H-29), 0.81 (3H, s, 3H-26),
0.76 (3H, s, 3H-24), 0.61 (1H, d, J = 10.8 Hz, H-5); for 13C NMR
(150 MHz, CD3OD), see Table 1; HRESIMS m/z 489.3578 [M+H]+
(calcd. for C30H49O5, 489.3580).
4.4.1. 3b,30-Dihydroxyolean-12-en-28-oic acid (queretaroic acid, 3)
White solid; mp 159 °C; [
a] +10 (c 1, CHCl3); IR (KBr) mmax
D
3386, 1689, 1460, 1034, 668 cmÀ1
;
1H NMR (600 MHz, CD3OD) d
5.27 (1H, dd, J = 3.5, 3.5 Hz, H-12), 3.55 and 3.43 (2H, AB system,
J = 10.9 Hz, 2H-30), 3.15 (1H, dd, J = 10.9, 4.9 Hz, H-3), 2.82 (1H,
dd, J = 13.8, 3.8 Hz, H-18), 1.18 (3H, s, 3H-27), 0.97 (3H, s, 3H-
23), 0.94 (3H, s, 3H-25), 0.89 (3H, s, 3H-29), 0.82 (3H, s, 3H-26),
0.78 (3H, s, 3H-24); 1H NMR (500 MHz, C5D5N) d 5.53 (1H, dd,
J = 3.5, 3.5 Hz, H-12), 4.03 and 3.95 (2H, AB system, J = 10.9 Hz,
2H-30), 3.48 (2H, m, H-3 and H-18), 1.36 (3H, s, 3H-27), 1.27
(3H, s, 3H-29), 1.25 (3H, s, 3H-23), 1.05 (6H, s, 3H-24 and 3H-
26), 0.91 (3H, s, 3H-25); for 13C NMR (150 MHz, CD3OD, or
125 MHz, C5D5N), see Table 1; HRESIMS m/z 473.3627 [M+H]+
(calcd. for C30H49O4, 473.3631).
4.5. Maslinic acid incubation with R. miehei
Maslinic acid (2, 2 g) was dissolved in EtOH (100 ml), distrib-
uted among 50 Erlenmeyer flask cultures (R. miehei) and incu-
bated for 13 days. Then, the cultures were filtered and pooled,
the cells were washed thoroughly with water, and the liquid
was saturated with NaCl and extracted continuously with CH2Cl2
for four 6-h periods. Dry fungal cells were washed repeatedly
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 column to
4.4.2. 3b,7b,30-Trihydroxyolean-12-en-28-oic acid (4)
obtain 845 mg of starting material (2, 42%), 10 mg of 2
dihydroxyolean-11-en-28,13b-olide (6, 0.5%), and 225 mg of
,3b,30-trihydroxyolean-12-en-28-oic acid (7, 11%). The rest of
a,3b-
White solid; mp 161 °C; [
a] +41 (c 1, CHCl3); IR (KBr) mmax
D
3356, 1694, 1461, 1036, 996, 756, 668 cmÀ1
;
1H NMR (500 MHz,
2a
CD3OD) d 5.34 (1H, dd, J = 3.6, 3.6 Hz, H-12), 3.86 (1H, dd,
J = 11.2, 5.0 Hz, H-7), 3.56 and 3.44 (2H, AB system, J = 10.8 Hz,
2H-30), 3.18 (1H, dd, J = 11.3, 5.5 Hz, H-3), 2.83 (1H, dd, J = 14.0,
4.7 Hz, H-18), 1.27 (3H, s, 3H-27), 1.01 (3H, s, 3H-23), 0.96 (3H,
s, 3H-25), 0.92 (3H, s, 3H-29), 0.85 (3H, s, 3H-26), 0.80 (3H, s,
3H-24); for 13C NMR (125 MHz, CD3OD), see Table 1; HRESIMS
m/z 489.3586 [M+H]+ (calcd. for C30H49O5, 489.3580).
the fractions (200 mg) were dissolved in pyridine (4 ml) and
Ac2O (2 ml). After the reaction was maintained for 24-h at room
temperature, it was diluted with cold H2O (50 ml), extracted
with CH2Cl2, washed with saturated aqueous KHSO4, and dried
over dry Na2SO4. Chromatography on silica-gel of the reaction
mixture gave 8 mg of 2
(8, 0.3%), 30 mg of 2 ,3b,30-triacetoxyolean-12-en-28-oic acid
(9, 1.2%), 26 mg of ,3b-diacetoxy-11-oxoolean-12-en-28-oic
acid (10, 1%), 6 mg of ,3b-diacetoxy-11 ,12 -epoxyolean-
28,13b-olide (11, 0.2%), and 23 mg of ,3b,30-triacetoxy-
11 ,12 -epoxyolean-28,13b-olide (12, 0.9%).
a,3b-diacetoxyolean-11-en-28,13b-olide
a
2
a
4.4.3. 1b,3b,30-Trihydroxyolean-12-en-28-oic acid (5)
2
a
a
a
White solid; mp 171 °C; [
a]
D + 43 (c 1, CHCl3); IR (KBr) mmax
2a
3375, 1693, 1460, 1040, 995, 756, 668 cmÀ1
;
1H NMR (600 MHz,
a
a
Please cite this article in press as: Martinez, A., et al. Biotransformation of oleanolic and maslinic acids by Rhizomucor miehei. Phytochemistry (2013),
http://dx.doi.org/10.1016/j.phytochem.2013.05.011

A. Martinez et al. / Phytochemistry xxx (2013) xxx–xxx
7
Table 1
13C NMR spectroscopic data for compounds 1–14.
Position
1
2
3a
3b
4
5
6
7
8
9
10
44.6
11
43.7
12
43.8
13
46.4
14
43.8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
39.9
27.9
79.7
39.9
56.8
19.5
33.8
40.6
49.1
38.2
24.5
123.7
145.2
42.9
28.9
24.1
47.6
42.8
47.3
31.6
34.9
34
48.1
69.5
84.4
40.5
56.7
19.6
33.8
40.6
49
39.8
39.3
39.7
27.9
79.5
39.6
54
30.4
73.9
46.2
49.9
38.2
24.6
124.1
144.5
44.3
32.3
24.8
47.5
42.8
42.7
36.2
29.9
33.2
28.7
16.4
15.9
10.7
26.4
181.7
28.1
66.3
80.5
38.2
76.8
39.8
54.5
19.2
34.1
41
46.2
68.8
83.9
39.4
55
17.8
31.2
41.8c
53.2
37.7
127.3
135.5
89.9
41.5c
25.5
21.4
44.2
50.7
37.4
31.6
34.5
27.3
28.4
16.2
19.3
19.2
18.4
180.2
33.4
23.7
48.1
69.5
84.4
40.6
56.7
19.6
33.9
40.5
49
39.2
24.3
123.7
145
42.9
28.8
24.6
47.5
42.1
42.5
36.1
29.8
33.3
29.3
17.5
17.1
17.7
26.5
181.8
28.1
66.3
43.8
69.9
80.6
39.6
54.6
17.7
31.1
41.8c
53.1
37.5
44
27.9
79.8
39.8
56.8
19.5
34
40.6
49.1
38.2
24.5
123.9
145
42.9
28.9
24.4
47.4
42.2
42.6
36.1
29.9
33.4
28.8
16.3
15.9
17.8
26.4
180.5
28.1
66.3
28.5
78.5
40.2
56.2
19.2
33.7
39.8
48.5
37.8
24.2
123.1
145.2
42.6
28.8
24.5
47.1
42
70.1
80.8
39.5
55
18.3
32.6
39.5
47.7
38.3
23.6
69.9
80.8
39.5
54.8
17.5
32.8
45.2
61.6
38.3
199.7
128.1
168.8
43.6
27.9
22.7
46.1
41.5
44.3
30.8
31.8
33.8
28.6
17.7
17.5
19.4
23.7
182.5
33
69.5
80.4
39.5
54.4
17.6
31.1
41.5
50.7
37.6
52.4
57.1
87.4
40.7
26.8
21.4
43.9
49.7
37.8
31.6
34.4
27.1
28.2
17.3
18.3
20.1
18.9
179.3
33.3
23.7
170.8
69.5
80.4
39.6
54.5
17.6
31.1
41.5
50.8
37.6
52.4
57
87.3
40.7
26.8
21.4
43.7
48.9
32.9
34.9
30.5
26.8
28.2
17.3
18.3
20.1
19
69.1
83.9
39.4
55.5
17.9
34.7
42.7
45.7
38.1
30.7
56.2
91.7
43.6
29.3
21.5
45.7
52.5
40.1
32
70
80.5
39.5
55.1
17.7
34.5
42.7
45.6
37.9
30.7
56.1
91.5
43.6
29.3
21.4
45.6
52.4
40
49.7
44.3
28.1
39.3
24.6
127.6
135.1
89.6
41.5c
25.5
21.4
44.1
50.6
37.5
31.6
34.5
27.3
28.2
17.1
19.1
19.1
18.4
179.9
33.4
23.7
170.9
123.5
145.3
42.9
28.8
24
125
123
143.6
42.7
28.9
24.3
47.4
41.9
42.5
36.1
29.8
33.3
28.7
16.1
11.9
18
143.2
41.7
27.7
23.2
46.4
40.6
41.5
34
47.6
42.7
47.2
31.6
34.9
33.9
29.3
17.1
17.5
17.7
26.5
181.8
33.6
24
42.4
36.3
30
32
34
29.4
32
34
33.4
28.8
16.9
15.9
17.9
26.6
180.7
29.2
66
27.7
28.6
16.7
18.3
19.3
21.3
178.9
33.4
23.7
27.7
28.4
17.5
18.2
19.2
21.3
178.8
33.4
23.7
170.9
28.8
16.3
15.9
17.7
26.4
180.8
33.6
24
28.6
17.8
16.6
17.2
26.1
183.2
27.9
67.8
171.4
26.5
181.7
28.2
66.3
178.7
28.4
66.9
171.2
23.5
171.1
MeCOO
MeCOO
MeCOO
MeCOO
MeCOO
MeCOO
170.6
171
170.7
21.4
21
170.3
170.6
170.9
170.6
21.2
21
170.6
21.2
21
21.2
21
21.2
21
21.3
21
21
20.9
a
b
c
13C NMR data for compound 3 measured in CD3OD.
13C NMR data for compound 3 measured in C5D5N.
Interchangeable within the same column.
4.5.1. 2
a
,3b-Dihydroxyolean-11-en-28,13b-olide (6)
CDCl3) d 6.00 (1H, dd, J = 10.3, 1.4 Hz, H-12), 5.43 (1H, dd,
J = 10.3, 3.2 Hz, H-11), 5.14 (1H, ddd, J = 11.2, 9.5, 4.5, H-2), 4.74
(1H, d, J = 9.5 Hz, H-3), 2.24 (1H, dd, J = 12.3, 4.5 Hz, H-1), 2.05
and 1.98 (3H each, s, AcO groups), 1.79 (1H, dd, J = 13.5, 13.5 Hz,
H-19), 1.05 (9H, s, 3H-25, 3H-26 and 3H-27), 0.97 (3H, s, 3H-29),
0.91 (3H, s, 3H-24), 0.89 (3H, s, 3H-23), 0.87 (3H, s, 3H-30); for
13C NMR (75 MHz, CDCl3), see Table 1; HRESIMS m/z 555.3683
[M+H]+ (calcd. for C34H51O6, 555.3686).
White solid; mp 187 °C; [
a]
D
+26 (c 1, CHCl3); IR (KBr) mmax
3407, 1757, 1463, 1388, 1218, 1050, 755 cmÀ1
;
1H NMR (300 Mz,
CD3OD) d 6.04 (1H, dd, J = 10.3, 1.4 Hz, H-12), 5.42 (1H, dd,
J = 10.3, 3.2 Hz, H-11), 3.74 (1H, ddd, J = 11.2, 9.5, 4.5, H-2), 2.99
(1H, d, J = 9.5 Hz, H-3), 2.18 (1H, dd, J = 12.3, 4.5 Hz, H-1), 1.94
(1H, bs, H-9), 1.79 (1H, dd, J = 13.5, 13.5 Hz, H-19), 1.04 (6H, s,
3H-26 and 3H-27), 1.01 (3H, s, 3H-23), 0.96 (6H, s, 3H-25 and
3H-29), 0.87 (3H, s, 3H-30), 0.81 (3H, s, 3H-24); for 13C NMR
(75 MHz, CD3OD), see Table 1; HRESIMS m/z 471.3454 [M+H]+
(calcd. for C30H47O4, 471.3474).
4.5.4. 2
White solid; mp 192 °C; [
1740, 1370, 1247, 1041, 756 cmÀ1
a
,3b,30-Triacetoxyolean-12-en-28-oic acid (9)
a
]
D
+28 (c 1, CHCl3); IR (KBr) mmax
;
1H NMR (300 MHz, CDCl3) d
4.5.2. 2
White solid; mp 183 °C; [
3355, 1685, 1455, 1388, 1047, 752 cmÀ1
a
,3b,30-Trihydroxyolean-12-en-28-oic acid (7)
5.26 (1H, J = 3.1, 3.1 Hz, H-12), 5.10 (1H, ddd, J = 11.6, 10.3, 4.2,
H-2), 4.74 (1H, d, J = 10.3 Hz, H-3), 4.06 and 3.99 (2H, AB system,
J = 11.0 Hz, 2H-30), 2.80 (1H, dd, J = 13.7, 4.2 Hz, H-18), 2.06, 2.05
and 1.97 (3H each, s, AcO groups), 1.13 (3H, s, 3H-27), 1.05 (3H,
s, 3H-25), 0.92 (3H, s, 3H-29), 0.89 (6H, s, 3H-23 and 3H-24),
0.73 (3H, s, 3H-26); for 13C NMR (75 MHz, CDCl3), see Table 1; HRE-
SIMS m/z 615.3893 [M+H]+ (calcd. for C36H55O8, 615.3897).
a]
+31 (c 1, MeOH); IR (KBr) mmax
D
;
1H NMR (300 MHz,
CDCl3) d 5.28 (1H, dd, J = 3.3, 3.3 Hz, H-12), 3.61 (1H, ddd,
J = 10.1, 9.6, 4.4 Hz, H-2), 3.55 and 3.43 (2H, AB system,
J = 10.9 Hz, 2H-30), 2.91 (1H, d, J = 9.6 Hz, H-3), 2.82 (1H, dd,
J = 13.6, 4.1 Hz, H-18), 2.05 (1H, ddd, J = 13.4, 13.4, 3.8 Hz, H-16),
1.18 (3H, s, 3H-27), 1.01 (3H, s, 3H-23), 1.00 (3H, s, 3H-25), 0.89
(3H, s, 3H-29), 0.80 (6H, s, 3H-24 and 3H-26); for 13C NMR
(75 MHz, CDCl3), see Table 1; HRESIMS m/z 489.3576 [M+H]+
(calcd. for C30H49O5, 489.3580).
4.5.5. 2
White solid; mp 131 °C; [
1740, 1658, 1370, 1246, 1042, 756 cmÀ1
a
,3b-Diacetoxy-11-oxoolean-12-en-28-oic acid (10)
D + 20 (c 1, CHCl3); IR (KBr) mmax
1H NMR (300 MHz,
a
]
;
CDCl3) d 5.63 (1H, s, H-12), 5.22 (1H, ddd, J = 10.3, 10.3, 4.6 Hz,
H-2), 4.71 (1H, d, J = 10.3 Hz, H-3), 3.20 (1H, dd, J = 12.9, 4.8 Hz,
H-1), 2.97 (1H, dd, J = 13.8, 4.0 Hz, H-18), 2.37 (1H, s, H-9), 2.04
and 1.95 (3H each, s, AcO groups), 1.35 (3H, s, 3H-27), 1.23 (3H,
4.5.3. 2
White solid; mp 222 °C; [
1765, 1743, 1368, 1253, 1030, 896, 755 cmÀ1
a
,3b-Diacetoxyolean-11-en-28,13b-olide (8)
D + 12 (c 1, CHCl3); IR (KBr) mmax
1H NMR (300 MHz,
a]
;
Please cite this article in press as: Martinez, A., et al. Biotransformation of oleanolic and maslinic acids by Rhizomucor miehei. Phytochemistry (2013),
http://dx.doi.org/10.1016/j.phytochem.2013.05.011

8
A. Martinez et al. / Phytochemistry xxx (2013) xxx–xxx
s, 3H-25), 0.94 (6H, s, 3H-29 and 3H-30), 0.91 (6H, s, 3H-24 and
3H-26), 0.89 (3H, s, 3H-23); for 13C NMR (75 MHz, CDCl3), see Ta-
ble 1; HRESIMS m/z 571.3634 [M+H]+ (calcd. for C34H51O7,
571.3635).
for 13C NMR (75 MHz, CDCl3), see Table 1; HRESIMS m/z
635.2941 [M+H]+ (calcd. for C34H52BrO6, 635.2947).
4.8. Dehydrohalogenation of 14 with DBU
4.5.6. 2
White solid; mp 152 °C; [
1776, 1742, 1368, 1253, 1037, 931, 756 cmÀ1
a
,3b-Diacetoxy-11
a
,12
a
a
-epoxyolean-28,13b-olide (11)
After 200 mg of compound 14 were dissolved in xylene (10 ml),
2.5 ml of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) were added.
The reaction was kept for 13 h at reflux. Next, ethyl acetate was
added and the reaction mixture was extracted with aqueous HCl
(5%). The organic phase was treated with an aqueous solution of
Na2CO3 (5%), dried with dry Na2SO4, and evaporated under reduced
]
D
+19 (c 1, CHCl3); IR (KBr) mmax
;
1H NMR (300 MHz,
CD3OD) d 5.15 (1H, ddd, J = 10.3, 10.3, 4.5 Hz, H-2), 4.77 (1H, d,
J = 10.3 Hz, H-3), 3.02 (2H, m, H-11 and H-12), 2.30 (2H, m, H-1
and H-18), 2.12 (1H, ddd, J = 13.1, 13.1, 5.6 Hz, H-16), 2.05 and
1.98 (3H each, s, AcO groups), 1.85 (1H, dd, J = 13.5, 13.5 Hz, H-
19), 1.15 (3H, s, 3H-25), 1.08 (3H, s, 3H-27), 1.05 (3H, s, 3H-26),
0.99 (3H, s, 3H-29), 0.91 (6H, s, 3H-24 and 3H-30), 0.89 (3H, s,
3H-23); for 13C NMR (75 MHz, CD3OD), see Table 1; HRESIMS m/z
571.3641 [M+H]+ (calcd. for C34H51O7, 571.3635).
pressure. Finally, after purification, 170 mg of 2a,3b-diacetoxyole-
an-11-en-28,13b-olide (8) were isolated.
4.9. Saponification of 8
First, 20 mg of compound 8 were dissolved in a mixture of
methanol/water/KOH (100:70:5), and stirred for 12-h at room tem-
perature. Next, the mixture was diluted with water and extracted
repeatedly with CH2Cl2. From the column chromatography of the
mixture, 15 mg of compound 6 were isolated.
4.5.7. 2
Sirup; [
1242, 1036, 932, 756 cmÀ1
a
,3b,30-Triacetoxy-11
a
,12
a-epoxyolean-28,13b-olide (12)
a]
+14 (c 1, CHCl3); IR (film) mmax 1775,1741, 1370,
D
;
1H NMR (300 MHz, CD3OD) d 5.15
(1H, ddd, J = 10.3, 10.3, 4.5 Hz, H-2), 4.77 (1H, d, J = 10.3 Hz, H-3),
4.20 and 3.78 (2H, AB system, J = 10.5 Hz, 2H-30), 3.03 (2H, m, H-
11 and H-12), 2.32 (2H, m, H-1 and H-18), 2.06 (6H, s, AcO groups),
1.98 (3H, s, AcO group), 1.82 (1H, dd, J = 13.7, 13.7 Hz, H-19), 1.16
(3H, s, 3H-25), 1.10 (3H, s, 3H-27), 1.06 (3H, s, 3H-26), 1.02 (3H, s,
3H-29), 0.92 (3H, s, 3H-24), 0.89 (3H, s, 3H-23); for 13C NMR
(75 MHz, CD3OD), see Table 1; HRESIMS m/z 629.3693 [M+H]+
(calcd. for C36H53O9, 629.3690).
4.10. Epoxidation of 8
After 70 mg of 8 were dissolved in CH2Cl2 (10 ml), 2.5 ml of per-
acetic acid with a few drops of ethanol were added until the tur-
bidity disappeared. The reaction was kept under stirring for 24-h
at room temperature. Afterwards, 20 ml of water were added to
the reaction mixture, which was extracted repeatedly with CH2Cl2,
and washed with a saturated solution of NaHCO3. The organic
phase was dried with dry Na2SO4, and evaporated under reduced
pressure. After chromatographic purification of the mixture
4.6. Bromination of maslinic acid (2)
After 500 mg of maslinic acid (2) were dissolved in 5 ml of CCl4,
15 ml of a solution of bromine dissolved in CCl4 (8.7 Â 10À2 M)
were added. The mixture was kept for 15 min at room tempera-
ture, and afterwards the solvent was evaporated and purified on
40 mg of 2a,3b-diacetoxy-11a,12a-epoxyolean-28,13b-olide (11)
were isolated.
column, yielding 12
(13, 325 mg), as a white solid; mp 266 °C; [
IR (KBr) mmax 3327, 1760, 1465, 1392, 1216, 1134, 1048, 927,
755 cmÀ1 1H NMR (300 MHz, CDCl3) d 4.30 (1H, dd, J = 3.9,
2.3 Hz, H-12), 3.73 (1H, ddd, J = 11.3, 9.5, 4.6 Hz, H-2), 3.04 (1H,
d, J = 9.5 Hz, H-3), 2.41 (1H, ddd, J = 15.0, 12.4, 3.9 Hz, H-11b),
a
-bromo-2
a
,3b-dihydroxyolean-28,13b-olide
Acknowledgements
a]
+49 (c 1, CHCl3);
D
This work was financially supported by grants from the Ministe-
rio de Ciencia e Innovación (CTQ2009-13898) and the Consejería de
Innovación, Ciencia y Empresa of the Junta de Andalucía (FQM-
0139). We thank David Nesbitt for reviewing the English in the
manuscript.
;
2.17 (1H, ddd, J = 13.0, 13.0, 5.4 Hz, H-16
a), 2.06 (1H, dd, J = 12.4,
4.6 Hz, H-1 ), 1.45 (3H, s, 3H-27), 1.22 (3H, s, 3H-26), 1.04 (3H,
a
s, 3H-24), 1.00 (3H, s, 3H-29), 0.96 (3H, s, 3H-25), 0.90 (3H, s,
3H-30), 0.82 (3H, s, 3H-23); for 13C NMR (75 MHz, CDCl3), see Ta-
ble 1; HRESIMS m/z 551.2743 [M+H]+ (calcd. for C30H48BrO4,
551.2736).
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http://dx.doi.org/10.1016/j.phytochem.2013.05.011