New approaches to the structural modification of olean-type pentacylic triterpenes via microbial oxidation and glycosylation

Article abstract of DOI:10.1016/j.tet.2011.04.055

Microbial transformation of 4 olean-type pentacyclic triterpenes (OPTs), 3-oxo oleanolic acid (1), 3-acetyl oleanolic acid (2), oleanolic acid (3), and esculentoside A (4) was studied. After the screening of 12 strains of microbes, preparative biotransformation by two strains of Streptomyces griseus ATCC 13273 and Aspergillus ochraceus CICC 40330 resulted in the isolation of 10 metabolites. The microbial catalyzed high efficient regio-selective methyl oxidation and glycosylation were discovered, which could be provided as an alternative method to expand the structural diversity of OPTs. All the structures of the metabolites were elucidated unambiguously by ESI-MS, ¹H NMR, ¹³C NMR, and 2D-NMR spectroscopy.

Full text of DOI:10.1016/j.tet.2011.04.055

Tetrahedron 67 (2011) 4206e4211
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
New approaches to the structural modification of olean-type pentacylic
triterpenes via microbial oxidation and glycosylation
,
,
b
b,
Yu-Yao Zhu a, Li-Wu Qian a c, Jian Zhang a , Ji-Hua Liu , Bo-Yang Yu
*
*
a Department of Complex Prescription of Traditional Chinese Medicine, China Pharmaceutical University, 639# Long Mian Avenue, Nanjing 211198, People’s Republic of China
b Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, Ministry of Education, 24# Tongjia Xiang Road, Nanjing, 211198, People’s Republic of China
c Department of Natural Products, Anhui Institute of Materia Medica, Hefei 230022, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Microbial transformation of 4 olean-type pentacyclic triterpenes (OPTs), 3-oxo oleanolic acid (1), 3-acetyl
oleanolic acid (2), oleanolic acid (3), and esculentoside A (4) was studied. After the screening of 12 strains
of microbes, preparative biotransformation by two strains of Streptomyces griseus ATCC 13273 and
Aspergillus ochraceus CICC 40330 resulted in the isolation of 10 metabolites. The microbial catalyzed high
efficient regio-selective methyl oxidation and glycosylation were discovered, which could be provided as
an alternative method to expand the structural diversity of OPTs. All the structures of the metabolites
were elucidated unambiguously by ESI-MS, 1H NMR, 13C NMR, and 2D-NMR spectroscopy.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 6 January 2011
Received in revised form 30 March 2011
Accepted 15 April 2011
Available online 21 April 2011
Keywords:
Biotransformation
Pentacyclic triterpene
Glycosylation
Oxidation
Streptomyces griseus ATCC 13273
Aspergillus ochraceus CICC 40330
1. Introduction
microbial transformation of OPTs, 3-oxo oleanolic acid (1) was used
as the substrate, by the screening of 12 strains of microbes, we
Olean-type pentacyclic triterpenes (OPTs) are of the bioactive
natural products, which have been shown to possess a wide range
of pharmacological applications, such as anti-tumor,1 anti-HIV,2
anti-microbial,3 anti-diabetic,4,5 and anti-inflammatory activities.6
The structural diversity of the natural originated OPTs mainly at-
tributed to the substitution of hydroxyl or carbonyl groups to the
methyl or methenyl carbons of the skeleton and the formation of
corresponding glycosides.7 The presence of such functional groups,
especially at C-3, C-28, and C-30, was also essential for the
biological activities of OPTs.8e10 Hereby, the researches on the
structural modification of OPTs were focused on how to introduce
the functional groups to the skeleton with the aim to enrich the
structural diversities and explore more effective and/or less toxicity
derivatives.11,12 In view of the complex skeleton and less active
position, microbial transformation was employed as an efficient
method to conduct the modification of OPTs nowadays.13 And in
our previous studies, some novel microbial transformation of
pentacyclic triterpenoids such as methyl migration, asymmetric
found that there were two more polar metabolites in the cultures of
Streptomyces griseus ATCC 13273 and one more polar metabolite in
the cultures of Aspergillus ochraceus CICC 40330 with high yield. To
further investigate the biotransformation capability of the two
microbes and explore the influence of the substitutions on C-3 and
C-20 to the reactions, another three substrates 3-acetyl oleanolic
acid (2), oleanolic acid (3), and esculentoside A (4, Fig. 1) were
screened, the other seven metabolites were detected. In this paper,
we described the isolation and structural elucidation of all the 10
metabolites prepared in our research. It is envisioned that microbial
transformation could play a more important role in the structural
modification to expand the structural diversities of OPTs.
2. Results and discussion
3-Oxo oleanolic acid (1) (200 mg) was added into a 2-day-old
cultivation of S. griseus ATCC 13273 and two more polar metabolites
(5 and 6) were detected by HPTLC. Compound 5 was the main
metabolite (84.3 mg) during the initial 48 h’ incubation, while to
the end of the 120 h incubation, compound 6 reached the highest
amount (125.1 mg). Furthermore, when the same amount of com-
pound 1 incubated with A. ochraceus CICC 40330, another more
polar metabolite 7 (146.6 mg) was isolated.
ketone a
-hydroxylation, and methyl hydroxylation14e17 have been
reported individually. As a continuation work to explore the
* Corresponding authors. Tel.: þ86 25 86185157; fax: þ86 25 86185158 (J.Z.); tel.:
þ86 25 83271321; fax: þ86 25 83313080 (B.-Y.Y.); e-mail addresses: wilson1978@
163.com (J. Zhang), boyangyu59@163.com (B.-Y. Yu).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.04.055

Y.-Y. Zhu et al. / Tetrahedron 67 (2011) 4206e4211
4207
(dd, 1H, J1¼14.0 Hz, J2¼4.0 Hz, H-18) ppm (Fig. 2), this further evi-
denced that the methyl group of C-29 was oxidized to a carboxyl
group in the biotransformation. Based on all the spectroscopic data
and comparison with that of serratagenic acid,18 compound 5 was
identified as 3-oxo-olean-12-en-28,29-dioic acid.
COOH
COOH
H
H
COOH
COOH
O
O
Fig. 2. Key HMBC and NOESY correlations of compound 5.
Compound 6 was isolated as colorless powder, the HR-ESI-MS of
compound 6 showed an [MꢀH]ꢀ ion at m/z 499.3060 (calcd for
C30H43O6, 499.3065), which indicated an increment of 16 amu as
compared to compound 5. The 13C NMR spectrum of 6 exhibited
Fig. 1. The structure of substrates 1e4.
Compound 5 was isolated as colorless powder, the HR-ESI-MS of
compound 5 showed an [MꢀH]ꢀ ion at m/z 483.3088 (calcd for
C30H43O5, 483.3115), indicating a 30 amu mass increase to that of
compound 1. The 13C NMR spectrum exhibited a new signal at
180.9 ppm and only six characteristic methyl signals appeared at
26.6, 26.0, 21.6, 20.0, 17.2, and 15.0 ppm (Table 1) with the absence
of one methyl signal in the substrate. This evidence suggested that
one methyl group might be oxidized to a carboxyl group. Further-
more, by comparing the 13C NMR spectrum of 1, the carbon signal at
a new signal at
showed two new proton signals at
(d, 1H, J¼11.0 Hz) ppm. The DEPT experiment showed the presence
of a new CH2 signal at 75.5 ppm and the disappearance of a CH3
d
64.1 (Table 1), while the 1H NMR spectrum of 6
d
4.29 (d, 1H, J¼11.0 Hz) and 3.83
d
d
d
signal, confirming that 6 was a monohydroxylated metabolite of 5.
In the HSQC experiment, the new proton signals showed correla-
tions with the new carbon signal at
d 64.1 ppm, and in the HMBC
d
23.5 ppm attribute to C-30 shifted up-field 3.5 ppm to
the metabolite, indicating that the oxidization was on C-29. In ad-
dition, the three-bond correlation between 1.59 (s, 3H, H-30) ppm
d 20 ppm in
experiment, the new CH2 signals show long-range (two- and three-
bond) 1He13C correlations with C-3 and C-4, confirming C-23 or
C-24 as the site of hydroxylation. Furthermore, the relative ste-
reochemistry of the hydroxyl group was established as axial based
on NOESY data. The NOESY spectrum of 6 showed NOE enhance-
d
and 180.9 ppm was also observed in the HMBC spectrum. The rel-
ative stereochemistry of the carboxyl group was established as
equatorial based on NOESY experiments. The NOESY spectrum of
ments between the new proton signals with
d
1.10 ppm (s, 3H, H-
) (Fig. 3), in-
5 showed NOE enhancements between d 1.59 (s, 3H, H-30) and 3.45
25) and 2.77 ppm (td, 1H, J1¼14.5 Hz, J2¼6.0 Hz, H-2
b
dicating that hydroxyl group was substituted at C-24. Based on all
the evidences, metabolite 6 was characterized as 24-hydroxy-3-
oxo-olean-12-en-28,29-dioic acid.
Table 1
13C NMR spectral data of 5, 6, 8, 9 (recorded in C5D5N, 125 MHz)
Carbon
5
6
8
9
1
2
3
4
5
6
7
8
39.1
34.4
216.1
47.4
55.3
19.8
32.6
39.7
47.2
37.0
23.9
123.0
144.3
42.3
28.3
23.9
46.7
41.0
41.2
42.6
30.0
32.4
26.6
21.6
15.0
17.2
26.0
179.9
180.9
20.0
39.0
34.5
213.4
53.8
56.7
18.9
32.2
38.6
46.4
36.1
23.0
121.7
143.3
41.0
27.2
22.7
45.5
40.0
39.9
41.4
28.1
31.2
19.8
64.1
14.4
16.2
24.9
178.7
179.8
18.9
39.4
28.1
78.1
39.8
55.8
18.8
33.3
39.0
48.1
37.4
23.9
123.3
144.3
42.2
28.3
23.9
46.7
41.1
41.1
42.6
30.0
32.4
28.8
16.5
15.6
17.5
26.1
179.9
181.0
20.0
38.7
28.4
80.2
43.2
56.4
19.1
33.6
39.8
48.2
37.2
23.9
123.0
144.4
42.1
28.3
24.1
46.6
41.1
41.1
42.6
29.3
32.4
23.5
64.6
16.0
17.3
26.0
179.9
180.5
20.0
COOH
COOH
H
H
COOH
COOH
H
9
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
O
HOH2C
O
HOH2C
Fig. 3. Key HMBC and NOESY correlations of compound 6.
Compound 7 was isolated as colorless powder, the ESI-MS of
compound 7 showed an [MþNa]þ ion at m/z 639.8 corresponding to
the molecular formula of C36H56O8Na, indicating a 162 amu mass
increase of 1. It was suggested that a hexose moiety was introduced.
Whereas, the 13C NMR spectrum showed the presence of six new
signals at
are characteristic of
glucose moiety in 7 resonated as a doublet at
constant of 8.5 Hz for H-10 indicated that the stereochemistry of the
glycosidic linkage at C-10 of
-glucose is . The anomeric carbon at
95.8 was typical for an ester glycosidic linked sugar at C-28 carboxy
group. The location of the glucose moiety was further confirmed by
d
95.8, 79.4, 78.9, 74.1, 71.2, and 62.2 ppm (Table 2), which
-glucose.19 The anomeric proton H-10 of the
6.32 ppm. A coupling
b-D
d
D
b
d

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Y.-Y. Zhu et al. / Tetrahedron 67 (2011) 4206e4211
Table 2
oxidation of the C-29 methyl group and the glycosylation of the C-
13C NMR spectral data of 7, 10e14 (recorded in C5D5N, 125 MHz)
28 carboxyl group (Scheme 1). While 3-acetyl oleanolic acid (2) was
subjected to the cultures, to our surprise, no metabolite was
detected. The subsequent biotransformation of oleanolic acid (3)
was conducted; 200 mg of compound 3 was added into the 2-day-
old fermentation broth of the two microorganisms, respectively. As
we expected, two more polar products, compounds 8 (98.6 mg) and
9 (118.7 mg) were isolated from the cultures of S. griseus ATCC
13273 and one more polar metabolite compound 10 (137.6 mg) was
isolated from the culture of A. ochraceus CICC 40330.
The molecular formula of 8, C30H46O5, was established by the
[MꢀH]ꢀ ion at m/z 485.5 in negative ion mode of ESI-MS, indicating
a 30 amu mass increase following the transformation. The 13C NMR
spectrum showed the presence of a new carbon signal at 181.0 ppm
(Table 1) and the disappearance of a methyl group carbon signal at
33.1 ppm of C-29. Based on the similar 1H and 13C NMR data of 5
and comparison of the spectroscopic data in the literature,18 8 was
identified as serratagenic acid.
Carbon
7
10
11
12a
13
14
1
2
3
4
5
6
7
8
39.2
34.7
216.2
47.4
55.4
19.8
32.5
39.8
47.2
36.9
23.8
122.7
144.2
42.2
28.2
23.6
46.2
41.8
47.0
30.8
34.0
32.5
26.6
21.6
15.0
17.3
26.0
176.4
33.1
23.4
39.0
28.1
78.2
39.4
55.9
18.9
33.1
40.0
48.2
37.4
23.5
122.9
144.2
42.2
28.3
23.7
47.1
41.8
46.3
30.8
34.1
32.6
28.7
16.5
15.7
17.6
26.1
176.4
33.2
23.9
44.0
70.8
82.7
42.6
47.5
18.0
33.0
39.6
48.3
36.8
23.6
123.3
144.2
42.2
28.1
23.7
45.9
43.9
43.2
44.1
30.8
34.3
65.1
14.7
17.0
17.2
26.0
179.5
28.2
176.9
51.4
43.2
70.3
73.6
40.8
47.5
17.4
32.0
38.9
47.9
36.1
22.9
122.3
143.1
40.5
27.2
22.7
45.0
41.8
41.3
42.0
29.9
33.1
69.0
13.1
16.6
16.3
25.5
178.7
28.0
176.4
51.4
44.8
71.6
73.1
42.4
48.2
18.2
33.0
39.8
48.5
37.2
23.9
123.4
144.6
42.2
28.3
23.9
46.7
42.8
26.3
51.3
38.0
34.1
67.8
14.5
17.4
17.2
26.2
179.9
71.1
175.8
51.5
44.9
71.6
73.0
42.4
48.1
18.3
32.9
40.0
48.6
37.2
23.9
123.4
143.8
42.2
28.2
23.5
46.5
43.2
42.4
43.9
30.5
34.0
67.7
14.5
17.5
17.3
26.1
176.1
28.2
177.0
51.6
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
OCH3
The molecular formula of 9 was established as C30H46O6 by
HR-ESI-MS in which a quasimolecular ion was detected at m/z
501.3183 [MꢀH]ꢀ (calcd for C30H45O6, 501.3221), indicating
a 16 amu mass increase to that of compound 8 following the trans-
formation. The DEPT spectrum showed the presence of a new
methylene carbon signal at
d 64.6 ppm (Table 1), with the disap-
pearance of one methyl group carbon signals attribute to C-24, this
indicated that the C-24 methyl group might be oxidized to the cor-
responding hydroxymethyl group, which was similar to that of
compound 6, the HMBC and NOESY experiments further confirmed
the structure of the compound 9 as 3b,24-dihydroxy-olean-12-en-
28,29-dioic acid.
Glc 1
95.8
74.1
79.4
71.2
78.9
62.2
95.8
74.2
79.3
71.2
79.0
62.3
95.8
74.1
79.3
71.0
78.9
61.9
2
3
4
5
6
The molecular formula of 10, C36H58O8Na, was established by
the [MþNa]þ ion at 641.2 in positive ion mode of ESI-MS, indicating
a 162 amu mass increase in 3. The 13C NMR spectrum showed the
presence of six new signals at
d 95.8, 79.3, 79.0, 74.2, 71.2, and
62.3 ppm (Table 2), indicating the presence of a glucose moiety.
Based on all the observations and comparison of the spectroscopic
data with those reported in the literature,20 10 was established as
Xyl 1
106.5
75.1
78.3
70.7
67.0
2
3
4
5
28-O-b-D-glucopyranosyl,3b-hydroxy-olean-12-en-28-oate.
To the substrate of compound 3, the same reactions were ob-
served (Scheme 2), this indicated that the substituent group on C-3
was very crucial to the biotransformation. Another substrate,
esculentoside A (4), which contained sugar moiety on C-3 hydroxyl
group, methyl carboxyl group of C-30, and free hydroxyl group on
C-23 was used to test whether these substitutions would affect the
biotransformation. Compound 4 (200 mg) was added to the two
cultures, respectively, two main less polar products, 11 (24.6 mg)
and 12 (56.0 mg), were isolated from the culture of S. griseus ATCC
a
Recorded in CD3OD.
the chemical shift of C-28, which shifted up-field from
d
182.8 to
176.4 by 6.4 ppm. By comparison of the spectroscopic data with
those reported in the literature,20 compound 7 was established as
28-O-b-D-glucopyranosyl 3-oxo-olean-12-en-28-oate.
In the biotransformation of compound 1 with the two micro-
organisms, two reactions were observed: the regio-selective
COOH
COOH
COOH
COOH
Streptomyces griseus
ATCC 13273
+
O
O
HOH2C
6
5
COOH
Aspergillus ochraceus
CICC 40330
O
O
Glc
3-oxo oleanolic acid (1)
O
O
7
Scheme 1. Microbial transformation of 1.

Y.-Y. Zhu et al. / Tetrahedron 67 (2011) 4206e4211
4209
COOH
COOH
COOH
Streptomyces griseus
ATCC 13273
COOH
+
HO
HOH2C
HO
9
8
COOH
Aspergillus ochraceus
CICC 40330
HO
O
Glc
oleanolic acid (3)
O
HO
10
Scheme 2. Microbial transformation of 3.
13273 and one same product 11 (112.4 mg) was detected as the only
metabolite in the cultures of A. ochraceus CICC 40330.
to 12. After comparison of the NMR spectroscopic data with those
reported in the literature,17 13 was characterized as 2
,3 ,23,
b
b
The molecular formula of compound 11, C36H56O11, was estab-
lished by the [MꢀH]ꢀ ion at m/z 663.2 in negative ion mode of ESI-
MS, indicating a 162 amu mass loss in the transformation. This
indicated that one hexose moiety might be hydrolyzed, this was
further confirmed by the comparison of the spectroscopic data with
those reported in the literature,21 and compound 11 was identified
as esculentoside B. The ESI-MS of compound 12 showed the
[MꢀH]ꢀ ion at m/z 531.5 in negative ion mode, indicating another
132 amu mass loss in the following transformation. This suggested
that another pentose moiety might be hydrolyzed, this was further
confirmed by the comparison of the spectroscopic data with those
reported in the literature21 and the TLC analysis of the acidic hy-
drolysis experiment, 12 was identified as phytolaccagenin.
While 200 mg of compound 12 was subsequently used as the
substrate for the further biotransformation, one more polar me-
tabolite 13 (128.8 mg) was isolated from the culture of S. griseus
ATCC 13273 and another more polar metabolite 14 (167.8 mg) was
isolated from the culture of A. ochraceus CICC 40330.
Compound 13 was isolated as colorless power, the molecular
formula was established as C31H48O8 by HR-ESI-MS in which a qua-
simolecular ion was detected at m/z 547.3252 [MꢀH]ꢀ (calcd for
C31H47O8, 547.3276), suggesting a 16 amu mass increase compared
29-tetrahydroxy-olean-12-ene-28,30-dioic acid 30-methyl ester.
The molecular formula of 14 was established as C37H58O12 by HRMS
inwhicha quasimolecularionwas detected at m/z 693.3822 [MꢀH]ꢀ
(calcd for C37H57O12, 693.3855), indicating a 162 amu mass increase
of 12. The 13C NMR spectrum of 14 showed the presence of six new
signals at
dicating the presence of a glucose moiety. The anomeric carbon at
95.79 was typical for an ester glycosidic linked sugar at C-28 car-
boxy group. Based on the 1H and 13C NMR data and similar evidence
d 95.8, 79.3, 78.9, 74.1, 71.0, and 61.9 ppm (Table 2), in-
d
of 7 and 10, compound 14 was established as 28-O-b-D-glucopyr-
anosyl phytolaccagenin.
To the biotransformation of 4 (Scheme 3), the two microor-
ganisms showed different capability to the hydrolysis of the sugar
moiety, the glycosidase in the S. griseus ATCC 13273 had no selec-
tivity to the glucoside and the xyloside, while the selectivity of the
glycosidase in A. ochraceus CICC 40330 was much higher, which
could provide an alternative way to identify the sugar chain com-
position of glycosides. Compound 12 is the aglycone of 4, the main
structural differences between 12 and 1, 2, 3 were the different
substituent of the oxygen-bearing group to the skeleton, and this
greatly influenced the biotransformation of S. griseus ATCC 13273,
the oxidation of the C-29 methyl group was blocked at the
H3COOC
H3COOC
COOH
COOH
HO
Aspergillus ochraceus
HO
O
CICC 40330
Streptomyces griseus
ATCC 13273
O
Xyl
CH2OH
Xyl
CH2OH
Glc
Esculentoside B (11)
Esculentoside A (4)
Streptomyces griseus
ATCC 13273
H3COOC
H3COOC
CH2OH
COOH
H3COOC
O
Glc
COOH
HO
HO
HO
HO
O
HO
Aspergillus ochraceus
CICC 40330
Streptomyces griseus
ATCC 13273
HO
CH2OH
CH2OH
CH2OH
Phytolaccagenin (12)
14
13
Scheme 3. Microbial transformation of 4.

4210
Y.-Y. Zhu et al. / Tetrahedron 67 (2011) 4206e4211
hydroxymethyl step, and no C-24 oxidized product was detected,
the further zymologic studies and the applications are in progress.
In conclusion, two main reactions, the regio-selective oxidation of
the methyl group on C-4 and C-20 and the formation of glycosyl ester
of C-28 carboxyl group were observed; both of these two reactions
could provide a versatile strategy to expand the structural diversities
of OPTs. It is particularly noteworthy that the microbial catalyzed ox-
ygenation of unactivated sp3 CeH bonds of the methyl group in one
step could be achieved by the unique microbe S. griseus ATCC 13273,
which is rich in P450 enzymes.22,23 Compared with the synthetic
chemical methods,24 such attempts always involve the heavy metals
and tedious steps, to develop the green catalytic methods for the ox-
idation of unactivated sp3 CeH bonds will have widespread applica-
tion in synthetic chemistry and the novel oxidation capability and the
enzyme system of S. griseus deserves further exploration. To the nat-
ural OPTs, another feature of the structural diversities relies on the
sugar moiety linked to hydroxyl or carboxyl groups; the sugars at-
tached to skeletons often dictate key pharmacological properties and/
or molecular mechanism of action, although there were a number of
reports on the microbial glycosylation research,25 the high efficient
and the regio-selective glucosylation of the C-28 carboxyl group of A.
ochraceus CICC 40330 will take great advantages over other methods.
The OPTs represent a promising and expanding platform for
biologically active natural compounds whose potential is currently
only partly exploited, our work underlines the value of microbial
transformations as a powerful biocatalytic means to expand the
structural diversities and prepare novel derivatives of OPTs. Re-
actions observed were highly regio-specific and functional group
specific, and they occurred under the mildest of reaction conditions
without the need for blocking groups typically required in synthetic
chemistry. The facile biocatalytic oxidation of the methyl groups by
whole cells illustrates the advantages and properties of microbial
transformations very well in a reaction that is difficult to achieve
using synthetic chemical methods.
The purity of all the substrates was above 98% by the HPLC-ELSD
analysis.
3.3. Microorganisms
S. griseus ATCC 13273, Gliocladium deliquescens NRRL 1086, Asper-
gillus sp., Mucor sp., Beauveria sp., Rhizopus sp., and Gibberella sp. et al.
were obtained from a courtesy of Prof. John P.N. Rosazza of University
of Iowa, USA. A. ochraceus CICC40330, Absidia coerulea, Saccharomyces
cerevisiae, Rhizopus chinensis, and Penicillium raistrickii were pur-
chased from China Center of Industrial Culture Collection.
3.4. Analytical-scale biotransformation
3.4.1. Analytical-scale biotransformation of S. griseus ATCC 1327328
.
Cultures were grown by a two-stage procedure in 25 ml of soybean
meal glucose medium held in 125 ml culture flasks. The soybean
meal glucose medium contained (in g/L) 20 glucose, 5 yeast extract,
5 soybean meal, 5 NaCl, and 5 K2HPO4 in distilled water and was
adjusted to pH 7.0 with 6 N HCl before being autoclaved at 121 ꢁC
for 15 min. Cultures were incubated with shaking at 180 rpm at
28 ꢁC. A 10% inoculum derived from 48-h-old stage I cultures was
used to initiate stage II cultures, which were incubated for 24 h
before receiving 10 mg of substrates in 1 mL of acetone or methanol
and incubations were conducted as before. Substrate controls
consisted of sterile medium and substrates incubated under the
same conditions but without microorganism. Substrate-containing
cultures were generally sampled by removing 3 mL of the entire
culture at 24, 48, 72, and 120 h after addition of substrate. Cultures
were extracted with equal volume of EtOAc. The organic phase was
concentrated and spotted on silica gel HPTLC plate, which was
developed by chloroform/methanol (10:1, v/v). The results were
visualized by spraying with H2SO4 (10% in ethanol) and heating at
120 ꢁC for 1e2 min.
3.4.2. Analytical-scale biotransformations of A. ochraceus CICC
4033016. Microbial cultures were grown according to the standard
two-stage fermentation protocol. Screening experiments were
done, using 125 mL DeLong culture flasks. The culture flasks held
one fifth of their volume of the following medium: potato 200 g,
glucose 20 g, KH2PO4 3 g, MgSO4 0.75 g. The pH value of the me-
dium was adjusted to 7.0 using 6 N HCl before being autoclaved for
20 min at 121 ꢁC and 15 psi. The flasks were incubated at 200 rpm
at 28 ꢁC. A 10% inoculum derived from 72-h-old stage I cultures was
used to initiate stage II cultures, which were incubated for 24 h
before receiving 10 mg of substrate in 1 mL ethanol, and the in-
cubations were conducted as before. Two kinds of controls were
run synchronously with the fermentation and worked-up in the
same way. One was the blank culture in order to define and exclude
the indigenous secondary metabolites generated by microorgan-
isms. The other was the blank substrate control, i.e., 1 was added to
the sterile broth without microorganisms, to test whether 1 would
be chemically decomposed or spontaneously transformed under
broth and fermentation conditions. Substrate-containing cultures
were generally sampled by removing 3 mL of the entire culture at
24, 72, and 144 h after addition of substrate. The mycelium was
filtrated and the broth was extracted with EtOAc (125 mLꢂ3). The
extracts were concentrated in vacuum and analyzed by HPTLC
developed with CHCl3/MeOH, 10:1 and 5:1 (v/v), respectively, and
visualized by spraying with 10% sulfanilic acid in ethanol.
3. Experimental
3.1. General procedures
NMR spectra were recorded on a Bruker AV-500 spectrometer in
C5D5N or CD3OD solution with TMS as the internal standard and
chemical shifts were expressed in d (parts per million). ESI-MS and
HR-ESI-TOF-MS experiments were performed on an Agilent 1100
Series MSD Trap mass spectrometer and an Agilent 6210 ESI-TOF
spectrometer, respectively. All the solvents used for extraction
and isolation were of analytical grade. HPTLC was performed on
precoated silica gel GF254 plates. Separation and purification were
carried out by column chromatography on silica gel (200e300
mesh). Silica gel was purchased from Qingdao Marine Chemical
Group Co., PR China.
3.2. Substrate
Compound 1 was prepared by the oxidation of 3 with Jones
reagent and purified by crystallization in methanol. The 1H NMR
and 13C NMR spectral data were identical to previous reports.26
Compound 2 was synthesized by stirring 3 with acetic anhy-
dride in dry pyridine with the presence of 4-dimethylaminopyr-
idine and purified by crystallization in methanol. The 1H NMR and
13C NMR spectral data were identical to previous reports.27
Compound 3 was purchased from Qingze Pharmacy Co. Ltd.,
Nanjing China.
3.5. Preparative scale biotransformation, isolation, and
identification of biotransformation product16
Compound 4 was isolated from the roots of Phytolacca esculenta.
Its structure was characterized by 1H NMR, 13C NMR, and MS
spectra.21
Using 24-h-old stage II cultures, substrate (200 mg) was dis-
tributed evenly among thirty 125 mL culture flasks. Substrate-

Y.-Y. Zhu et al. / Tetrahedron 67 (2011) 4206e4211
4211
containing cultures were incubated for 5 days and then extracted
with equal EtOAc for three times. The organic solvent layer was
evaporated to dryness. The extract was subjected to silica gel col-
umn chromatography eluted with solvent system of chloroform/
methanol to afford the products. The structure was identified based
on its MS and NMR.
1H, H-2), 4.14 (d, 1H, J¼9.5 Hz, H-3), 3.67 (s, 3H, eOCH3), 1.25 (s, 3H,
H-25),1.13 (s, 3H, H-24),1.13 (s, 3H, H-27), 0.96 (s, 3H, H-26), 0.78 (s,
3H, H-29); the 13C NMR spectral data are summarized in Table 2.
3.5.9. 2b,3b,23,29-Tetrahydroxy-olean-12-ene-28,30-dioic acid 30-
methyl ester (13). White powder; HR-ESI-TOF-MS m/z 547.3252
[MꢀH]ꢀ (calcd for C31H47O8, 547.3276); 1H NMR (C5D5N)
d 5.68 (t,
3.5.1. 3-Oxo-olean-12-en-28,29-dioic acid (5). Colorless powders
(methanol); HR-ESI-TOF-MS m/z 483.3088 [MꢀH]ꢀ (calcd for
3H, J¼3.5 Hz, H-12), 4.52 (d, 1H, J¼3 Hz, H-2), 4.25 (d, 1H, J¼4.5 Hz,
H-3), 4.15 (d, 1H, J¼10.5 Hz, H-23), 3.96 (dd, 2H, J1¼15.9 Hz,
J2¼10.5 Hz, H-29), 3.71 (d, 1H, J¼10.5 Hz, H-23), 3.67 (s, 3H,
eOCH3), 1.59 (s, 3H, H-25), 1.36 (s, 3H, H-24), 1.27 (s, 3H, H-27), 1.11
(s, 3H, H-26); the 13C NMR spectral data are summarized in Table 2.
C30H43O5, 483.3115); 1H NMR (C5D5N)
d
5.56 (t, 1H, J¼3.5 Hz, H-12),
3.45 (dd, 1H, J1¼14.0 Hz, J2¼4.0 Hz, H-18), 1.59 (s, 3H, H-30), 1.26 (s,
3H, H-27), 1.14 (s, 3H, H-23), 1.02 (s, 3H, H-25), 1.01 (s, 3H, H-24),
0.91 (s, 3H, H-26); the 13C NMR spectral data are summarized in
Table 1.
3.5.10. 28-O-b-D-Glucopyranosyl phytolaccagenin (14). Colorless
powders (methanol); HR-ESI-TOF-MS m/z 693.3822 [MꢀH]ꢀ (calcd
3.5.2. 3-Oxo-24-hydroxy-olean-12-en-28,29-dioic acid (6). Color-
less powders (methanol); HR-ESI-TOF-MS m/z 499.3060 [MꢀH]ꢀ
for C37H57O12, 693.3855); 1H NMR (C5D5N)
d
6.30 (d, 1H, J¼8.1 Hz,
H-10), 5.60 (t, 1H, J¼3.3 Hz, H-12), 4.52 (br s, 1H, H-2), 4.38e4.13 (m,
7H), 3.98 (d, 1H, J¼9.5 Hz, H-3), 3.69 (d, 1H, J¼10.5 Hz, H-23), 3.59
(s, 3H, eOCH3), 3.19 (dd, 1H, J1¼14.0 Hz, J2¼4.0 Hz, H-18), 1.61
(s, H-25), 1.35 (s, H-24), 1.22 (s, H-27), 1.19 (s, H-26) and 1.17
(s, H-29); the 13C NMR spectral data are summarized in Table 2.
(calcd for C30H43O6, 499.3065); 1H NMR (C5D5N)
d
5.54 (t, 1H,
J¼3.5 Hz, H-12), 4.30 (d, 1H, J¼11.0 Hz, H-24
a), 3.83 (d, 1H,
J¼11.0 Hz, H-24 ), 3.44 (dd, 1H, J1¼14.0 Hz, J2¼4.0 Hz, H-18), 1.59 (s,
b
3H, H-30), 1.47 (s, 3H, H-23), 1.25 (s, 3H, H-27), 1.10 (s, 3H, H-25),
1.03 (s, 3H, H-26); the 13C NMR spectral data are summarized in
Table 1.
Acknowledgements
3.5.3. 28-O-b-D-Glucopyranosyl 3-oxo-olean-12-en-28-oate (7). Colorless
This work was supported by the National Nature Science
Foundation of China (NSFC No. 20602040, 30672603). Thanks also
given to the financial support from the State Administration of
Foreign Expert Affairs of China (No. 111-2-07) and the ‘111 Project’
from the Ministry of Education of China.
powders (methanol); ESI-MS m/z 639.8 [MþNa]þ; 1H NMR (C5D5N)
d
6.32 (d, 1H, J¼8.5 Hz, H-10), 5.45 (t, 1H, J¼3.5 Hz, H-12), 4.48e4.02 (m,
6H), 3.21 (dd, 1H, J1¼14.0 Hz, J2¼4.0 Hz, H-18), 1.21 (s, 3H, H-27), 1.13 (s,
3H, H-23), 1.12 (s, 3H, H-25), 1.01 (s, 3H, H-24), 0.93 (s, 3H, H-30), 0.92 (s,
3H, H-29), 0.89 (s, 3H, H-26); the 13C NMR spectral data are summarized
in Table 2.
References and notes
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d
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for C30H45O6, 501.3221); 1H NMR (C5D5N)
d
5.55 (t, 1H, J¼3.5 Hz, H-
12), 4.47 (d, 1H, J¼11.0 Hz, H-24
a
), 3.65 (d, 1H, J¼11.0 Hz, H-24
b),
3.43 (dd, 1H, J1¼14.0 Hz, J2¼4.0 Hz, H-18), 1.59 (s, 3H, H-30), 1.53 (s,
3H, H-23), 1.27 (s, 3H, H-27), 0.99 (s, 3H, H-26), 0.87 (s, 3H, H-25);
the 13C NMR spectral data are summarized in Table 1.
3.5.6. 28-O-
b
-
D
-Glucopyranosyl
3b-hydroxy-olean-12-en-28-oate
(10). Colorless powders (methanol); ESI-MS m/z 641.2 [MþNa]þ;
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6.32 (d, 1H, J¼8.1 Hz, H-10), 5.45 (t, 1H, J¼3.4 Hz,
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3.21 (dd, 1H, J1¼14.1 Hz, J2¼4.4 Hz, H-18), 1.24 (s, 3H, H-27), 1.22 (s,
3H, H-23), 1.13 (s, 3H, H-25), 1.02 (s, 3H, H-24), 0.93 (s, 3H, H-30),
0.90 (s, 3H, H-29), 0.89 (s, 3H, H-26); the 13C NMR spectral data are
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3.5.7. Esculentoside
B (11). White powder; ESI-MS m/z 663.6
[MꢀH]ꢀ; 1H NMR (C5D5N)
5.59 (t, 1H, J¼3.5 Hz, H-12), 5.05 (d, 1H,
d
J¼9.4 Hz, H-10), 3.62 (s, 3H, eOCH3), 1.55 (s, 3H, H-25), 1.32 (s, 3H,
H-24),1.24 (s, 3H, H-27),1.18 (s, 3H, H-26),1.04 (s, 3H, H-29); the 13C
NMR spectral data are summarized in Table 2.
3.5.8. Phytolaccagenin (12). Colorless powders (methanol); ESI-MS
m/z 531.5 [MꢀH]ꢀ; 1H NMR (C5D5N)
d
5.32 (s, 1H, H-12), 4.29 (br s,
28. Betts, R. E.; Walters, D. E.; Rosazza, J. P. N. J. Med. Chem. 1974, 17, 599e602.