Biocatalysis of cycloastragenol by filamentous fungi to produce unexpected triterpenes

Article abstract of DOI:10.1002/adsc.201100511

The biocatalysis of cycloastragenol, a natural tetracyclic triterpenoid with anti-aging activity, by cultured whole cells of three strains of filamentous fungi, namely Cunninghamella elegans AS 3.1207, Syncephalastrum racemosum AS 3.264 and Doratomyces stemonitis AS 3.1411 produced 15 metabolites. Thirteen of them are new compounds. The structures of these metabolites were fully characterized on the basis of HR-ESI-MS analyses together with 1D and 2D NMR spectroscopy. The three fungal strains exhibited significant biocatalytic preferences: C. elegans enabled hydroxylation reactions, particularly on the 28- and 29-CH₃ groups; S. racemosum efficiently catalyzed a complicated rearrangement reaction to form the unusual ranunculane skeleton, which was further substituted with diverse side chains at C-19; D. stemonitis mainly led to carbonylation reactions, especially on 3-OH. It is particularly noteworthy that S. racemosum also catalyzed an unexpected ring expansion reaction to generate the rare 9(10)a-homo-19-nor-cycloartane skeleton. Biocatalysis was proved powerful in the structural diversification of cycloastragenol for future structure-activity relationship studies. Copyright

Full text of DOI:10.1002/adsc.201100511

FULL PAPERS
DOI: 10.1002/adsc.201100511
Biocatalysis of Cycloastragenol by Filamentous Fungi to Produce
Unexpected Triterpenes
Wen-zhi Yang,a Min Ye,a,* Fei-xia Huang,a Wen-ni He,a and De-an Guoa,*
a
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38
Xueyuan Road, Beijing 100191, Peopleꢀs Republic of China
Fax : (+86)-10-8280-2024; phone: (+86)-10-8280-1516; e-mail: yemin@bjmu.edu.cn or gda5958@163.com
Received: June 30, 2011; Published online: January 24, 2012
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adcs.201100511.
Abstract: The biocatalysis of cycloastragenol, a natu- form the unusual ranunculane skeleton, which was
ral tetracyclic triterpenoid with anti-aging activity, by further substituted with diverse side chains at C-19;
cultured whole cells of three strains of filamentous D. stemonitis mainly led to carbonylation reactions,
fungi, namely Cunninghamella elegans AS 3.1207, especially on 3-OH. It is particularly noteworthy that
Syncephalastrum racemosum AS 3.264 and Dorato- S. racemosum also catalyzed an unexpected ring ex-
myces stemonitis AS 3.1411 produced 15 metabolites. pansion reaction to generate the rare 9(10)a-homo-
Thirteen of them are new compounds. The structures 19-nor-cycloartane skeleton. Biocatalysis was proved
of these metabolites were fully characterized on the powerful in the structural diversification of cycloas-
basis of HR-ESI-MS analyses together with 1D and tragenol for future structure-activity relationship
2D NMR spectroscopy. The three fungal strains ex- studies.
hibited significant biocatalytic preferences: C. ele-
gans enabled hydroxylation reactions, particularly on
the 28- and 29-CH3 groups; S. racemosum efficiently Keywords: biocatalysis; cycloastragenol; filamentous
catalyzed a complicated rearrangement reaction to fungi; ring expansion; tetracyclic triterpenoids
Introduction
Cycloastragenol, or (20R,24S)-3b,6a,16b,25-tetrahy-
droxy-20,24-epoxycycloartane (CA, 1), is the genuine
Structural modification of bioactive natural products sapogenin of astragaloside IV, a major bioactive con-
is usually necessary to improve solubility, enhance ef- stituent of Astragalus plants.[4–6] Astragaloside IV ex-
ficacy, or reduce toxicity. Chemical approaches are hibits various pharmacological properties, such as
the routine solution but often encounter challenges anti-inflammatory,[7] anti-viral,[8] anti-aging,[9] anti-oxi-
when facing regio- or stereo-selectivity problems. dant,[10] and so on. Cycloastragenol could delay the
Moreover, multi-step reactions often result in low onset of cellular aging by increasing telomerase activi-
yield of the final products. Biocatalysis, by means of ty,[11] up-regulate the immune system by inducing IL-2
enzyme systems, shows high selectivity and efficiency, release,[12] and enhance the antiviral function of
and has been widely used in pharmaceutical synthe- human CD8+ T lymphocytes.[13] CA has been consid-
sis.[1] It is generally operated under mild conditions, ered as a promising new generation of anti-aging
and the most popularly used biological systems in- agent. However, few analogues of CA are known to
clude fungi, bacteria, and cultured plant suspension us, so far. Its structural modification is thus of great
cells. A number of filamentous fungi was reported to necessity for further evaluation of structure-activity
catalyze complex transformations that were difficult relationships. Recently, Kuban et al.[14] reported the
to realize by chemical approaches. For example, C. el- formation of a complicated rearrangement product of
egans AS 3.1207 was able to convert steroidal sapo- CA with a novel ranunculane framework by Cunning-
nins into pregnenolones,[2] and S. racemosum AS hamella blakesleeana NRRL 1369, which demonstrat-
3.264 could convert paeoniflorin into albiflorin.[3] Bio- ed the power of biocatalysis in structural diversifica-
catalysis is attracting more and more interests of re- tion of cycloastragenol to produce novel derivatives.
searchers.
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Wen-zhi Yang et al.
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Figure 1. Chemical structures of compounds 1–16 (* new compound).
This paper describes the biocatalysis of cycloastra- tract was separated by silica gel column chromatogra-
genol by three strains of filamentous fungi, namely phy, and then purified by semi-preparative HPLC-RI
Cunninghamella elegans AS 3.1207, Syncephalastrum to obtain compounds 2 (4 mg), 3 (5 mg), 4 (100 mg), 5
racemosum AS 3.264 and Doratomyces stemonitis AS (100 mg), 6 (20 mg), 11 (3 mg) and 12 (12 mg) from
3.1411, and structure elucidation of the 15 biotrans- the culture broth of C. elegans, compounds 3 (10 mg),
formed products. The biocatalytic features of the 9 (31 mg), 10 (3.5 mg), 12 (360 mg), 13 (10 mg), 14
three fungal strains were also discussed.
(7 mg), 15 (15 mg) and 16 (5.5 mg) from S. racemo-
sum, and compounds 7 (730 mg), 8 (4.5 mg) and 12
(5.5 mg) from D. stemonitus. The structures of com-
pounds 1–16 are shown in Figure 1. The NMR spec-
troscopic data for 16 are given in Table 1, the data for
Results and Discussion
Cycloastragenol (1) as the substrate for biocatalysis 1–15 are given in Table 2 (1H NMR for 1–8), Table 3
was obtained by Smith degradation of astragaloside (1H NMR for 9–15) and Table 3 (13C NMR for 1–15),
IV. A preliminary screening test revealed that CA respectively.
could be efficiently metabolized by three fungal
strains, Cunninghamella elegans AS 3.1207, Syncepha-
lastrum Racemosum AS 3.264, and Doratomyces ste- Ring Expansion Product 16
monitis AS 3.1411. Scaled-up biocatalytic fermenta-
tions of these three stains were then separately car- Compound 16 was deduced to possess the molecular
ried out. After 6 days of incubation with CA, the formula C30H48O5 based on its HR-ESI-MS data
fungal broth was extracted with ethyl acetate. The ex- (found: m/z=977.70692, calcd. for [2M+H]+:
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Adv. Synth. Catal. 2012, 354, 527 – 539

Biocatalysis of Cycloastragenol by Filamentous Fungi to Produce Unexpected Triterpenes
Table 1. 1D and 2D NMR[a] spectroscopic data for compound 16 (J in Hz).
No. 13C
1H
HMBC
1H-1H
NOESY
COSY
1
2
41.1 2.30, 2H, m
34.7 1.92, 1H, m (a); 2.18, 1H, m C-1, C-3, C-4, C-5, C-10
C-3, C-5, C-10, C-19
H-2
H-1, H-3
H-5, H-19, H-28
H-29; H-3
(b)
3
4
5
6
7
8
9
77.9 3.83, 1H, brd (12.0)
43.4
H-2
H-2 (b), H-5, H-28
59.8 2.67, 1H, d (5.6)
73.4 4.30, 1H, m
38.2 2.16, 2H, m
41.9 2.56, 1H, m
138.2
C-3, C-4, C-6, C-10, C-28, C-29
H-6
H-1, H-3, H-28
H-8, H-28, H-29
H-15 (a)
H-5, H-7
H-6, H-8
H-7
C-5, C-6, C-8, C-9
C-6, C-9
H-6, H-29
10 135.1
11 127.6 5.66, 1H, s
12 39.1 2.10, 1H, m (a); 2.25, 1H, m C-9, C-13, C-15
C-10, C-14
H-12
H-11
H-19, H-12 (a)
H-18, H-21; H-30
(b)
13 43.7
14 45.8
15 44.6 1.94, 1H, m (a); 2.23, 1H, m C-8, C-13, C-16, C-17, C-30
(b)
H-16
H-7, H-18; H-30
H-21, H-30
16 72.9 5.08, 1H, brs
C-13
H-17, H-18
H-18
17 57.2 2.53, 1H, d (8.0)
C-13, C-14, C-16, C-18, C-20, C-21,
C-22
18 19.0 1.30, 3H, s
C-12, C-14, C-17
H-8, H-12 (a), H-15 (a), H-
22
19 127.9 6.18, 1H, s
20 87.0
21 28.4 1.35, 3H, s
C-5, C-8, C-9, C-11
C-18, C-22
H-1, H-11
H-12 (a), H-17, H-22 (a), H-
24
22 34.9 1.68, 1H, m (a); 3.12, 1H, m C-20, C-21, C-24, C-25
H-23
H-18, H-26; H-18
(b)
23 26.4 2.08, 1H, m (a); 2.33, 1H, m C-22, C-25
(b)
H-22, H-24 H-24; H-26
H-23 H-21, H-23 (a), H-26, H-27
24 81.8 3.93, 1H, dd (5.2, 9.0)
25 71.3
C-25
26 27.1 1.34, 3H, s
C-24, C-25, C-27
H-22 (a), H-23 (b), H-24, H-
27
27 28.1 1.62, 3H, s
28 26.1 1.79, 3H, s
29 15.9 1.20, 3H, s
30 17.9 0.85, 3H, s
C-24, C-25, C-26
C-3, C-4, C-5, C-29
C-3, C-4, C-28
C-8, C-15
H-24, H-26
H-3, H-5, H-6, H-29
H-2 (a), H-28, H-6, H-8
H-12 (b), H-15 (b), H-17
[a] 1H and 13C NMR data were recorded in pyridine-d5 at 400 MHz and 100 MHz, respectively.
1
977.70782). The H NMR spectrum displayed two sin- of 16 from CA were signals corresponding to ring B
glet signals for two tri-substituted olefinic systems and C carbons (Table 1).
(dH =5.66, 6.19), four oxygenated tertiary protons
1
Further analysis of the H-1H COSY and HSQC
(dH =5.08, 4.30, 3.93, 3.84), and seven methyl groups spectra resolved three spin-coupling systems: dH =
in the up-field region. The absence of characteristic 5.66 (dC =127.6)!dH =2.10, 2.25 (dC =39.1) (I), dH =
AX system proton signals in the high field region for 3.84 (dC =77.9)!dH =1.92, 2.18 (dC =34.7)!dH =2.30
[15]
19-CH2 implied cleavage of the 9,19-cyclopropane (dC =41.1) (II), and dH =2.67 (dC =59.8)!dH =4.30
ring. In the 13C NMR spectrum, four unsaturated car- (dC =73.4)!dH =2.16 (dC =38.2)!dH =2.56 (dC =
bons (dC =127.6, 127.9, 135.1, 138.2), and six oxygen- 41.9) (III). Spin system I was ascribed to a partial sec-
bearing methines (dC =87.0, 81.8, 77.9, 73.4, 72.9, tion of ring C according to the HMBC correlations of
71.3) were detected. dC =71.3 and 87.0 corresponded dH =2.10 and 2.25 (12-CH2) with dC =44.6 (C-15)/43.7
to two quaternary carbons according to the DEPT (C-13), and dH =1.30 (18-CH3) with dC =39.1 (C-12);
spectrum. The major differences in 13C NMR spectra II was attributed to C-3, C-2 and C-1 of ring A due to
Adv. Synth. Catal. 2012, 354, 527 – 539
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FULL PAPERS
Table 2. 1H NMR[a] spectroscopic data for compounds 1–8 (J in Hz).
H
1
1
2*
3*
4*
5*
6*
7
8*
2.01, 1H, m; 1.90, 1H, m; 2.01, 1H, m; 2.12, 1H, m; 2.17, 2H, m
2.09, 1H, m 1.97, 1H, m 2.05, 1H, m 2.18, 1H, m
2.03, 1H, m; 1.35, 1H, m; 1.34, 1H, m;
2.16, 1H, m 2.08, 1H, m 2.04, 1H, m
2
3
5
6
7
8
1.30, 1H, m; 4.20, 1H, dt 1.37, 1H, m; 1.26, 1H, m; 1.30, 1H, m; 1.70, 2H, m 2.54, 1H, m; 2.53, 1H, m;
1.71, 1H, m (4.8, 9.2)
3.71, 1H, dd 3.63, 1H, d
1.74, 1H, m
3.70, 1H, m
1.71, 1H, m
4.21, 1H, dd
(5.0, 11.4)
2.04, 1H, d
(8.0)
1.66, 1H, m
3.89, 1H, m
2.78, 1H, m 2.74, 1H, m
4.31, 1H, d
(9.2)
2.24, 1H, d
(10.0)
(4.6, 11.4)
1.78, 1H, d
(9.6)
(9.2)
1.89, 1H, d
(10.0)
1.79, 1H, d
(10.0)
1.82, 1H, d
(8.0)
2.25, 1H, d
(10.0)
2.27, 1H, d
(10.0)
3.85, 1H, m 3.81, 1H, dt 2.83, 1H, m
(4.8, 10.0)
3.84, 1H, dt
(3.8, 10.0)
3.97, 1H, m
3.66, 1H, dt 3.73, 1H, m 3.72, 1H, brs
(4.8, 11.0)
1.71, 1H, m; 1.70, 1H, m; 1.81, 1H, m; 1.66, 1H, m; 1.63, 1H, m; 1.61, 1H, m; 1.66, 1H, m; 2.13, 2H, m
1.88, 1H, m 1.86, 1H, m 1.88, 1H, m
2.00, 1H, m 1.97, 1H, m 1.93, 1H, m
1.89, 1H, m
1.89, 1H, m
1.82, 1H, m
1.95, 1H, m
1.87, 1H, m 1.79, 1H, m
1.97, 1H, m 1.90, 1H, m 2.07, 1H, m
11 1.28, 1H, m; 1.27, 1H, m; 2.12, 1H, m; 1.21, 1H, m; 1.20, 1H, m; 1.22, 1H, m; 1.05, 1H, m; 1.11, 1H, m;
2.04, 1H, m 2.04, 1H, m 2.44, 1H, dd 2.09, 1H, m
(5.6, 14.8)
2.02, 1H, m
2.07, 1H, m 2.05, 1H, m 2.04, 1H, m
12 1.71, 2H, m 1.63, 2H, m 4.26, 1H, brs 1.71, 2H, m
1.71, 2H, m
1.29, 1H, m; 1.68, 2H, m 1.67, 1H, m;
1.71, 1H, m
1.84, 1H, m
15 1.84, 1H, m; 1.80, 1H, m; 1.94, 1H, m; 1.83, 1H, m; 1.76, 1H, m; 1.81, 1H, m; 1.80, 1H, m; 1.75, 1H, m;
2.18, 1H, m 2.14, 1H, m 2.24, 1H, m
16 5.08, 1H, m 5.04, 1H, brs 5.01, 1H, m
2.18, 1H, m
5.07, 1H, brs 5.02, 1H, m
2.13, 1H, m
2.13, 1H, m 2.15, 1H, m 3.26, 1H, m
5.06, 1H, brs 5.07, 1H, m 5.27, 1H, m
17 2.60, 1H, d
(7.8)
2.55, 1H, d
(8.0)
3.31, 1H, d
(8.0)
2.59, 1H, d
(8.0)
2.52, 1H, d
(8.0)
2.58, 1H, d
(8.0)
2.58, 1H, d
(8.0)
2.82, 1H, d
(7.6)
18 1.50, 3H, s
19 0.39, 1H, d
(4.1); 0.67,
1H, d (4.1)
21 1.37, 3H, s
1.45, 3H, s
0.46, 1H, d
(4.1); 0.68,
1H, d (4.1)
1.34, 3H, s
1.83, 3H, s
0.52, 1H, d
(4.1); 0.66,
1H, d (4.1)
1.87, 3H, s
1.48, 3H, s
0.47, 1H, d
(4.1); 0.63,
1H, d (4.1)
1.38, 3H, s
1.43, 3H, s
0.41, 1H, d
(4.1); 0.63,
1H, d (4.1)
1.34, 3H, s
1.47, 3H, s
0.42, 1H, d
(4.1); 0.65,
1H, d (4.1)
1.38, 3H, s
1.47, 3H, s
0.41, 1H, d
(4.1); 0.73,
1H, d (4.1)
1.37, 3H, s
1.59, 3H, s
0.44, 1H, d
(4.1); 0.78,
1H, d (4.1)
1.37, 3H, s
22 1.75, 1H, m; 1.70, 1H, m; 2.00, 1H, m; 1.76, 1H, m; 1.74, 1H, m; 1.75, 1H, m; 1.73, 1H, m; 1.76, 1H, m;
3.17, 1H, m 3.12, 1H, m 2.66, 1H, m 3.17, 1H, m 3.15, 1H, m 3.16, 1H, m 3.16, 1H, m 3.22, 1H, m
23 2.11, 1H, m; 2.07, 1H, m; 2,12, 1H, m; 2.14, 1H, m; 2.08, 1H, m; 2.12, 1H, m; 2.09, 1H, m; 2.11, 1H, m;
2.37, 1H, m 2.35, 1H, m 2.35, 1H, m
24 3.93, 1H, dd 3.91, 1H, dd 4.07, 1H, m
2.37, 1H, m
3.94, 1H, dd
(5.6, 9.0)
2.34, 1H, m
2.38, 1H, m 2.37, 1H, m 2.38, 1H, m
3.87, 1H, brs 3.94, 1H, dd 3.94, 1H, dd 3.94, 1H, dd
(5.6, 9.0)
(5.6, 8.8)
(5.6, 9.0)
(5.6, 9.0)
(5.2, 9.0)
26 1.35, 3H, s
27 1.63, 3H, s
28 1.95, 3H, s
1.34, 3H, s
1.61, 3H, s
1.97, 3H, s
1.42, 3H, s
1.60, 3H, s
1.99, 3H, s
1.35, 3H, s
1.64, 3H, s
4.36, 1H, d
(11.2); 4.45,
1H, d (11.2)
1.32, 3H, s
1.31, 3H, s
1.59, 3H, s
2.19, 3H, s
1.35, 3H, s
1.63, 3H, s
10.12, 1H, s 1.81, 3H, s
1.35, 3H, s
1.64, 3H, s
1.34, 3H, s
1.58, 3H, s
1.80, 3H, s
29 1.42, 3H, s
30 1.07, 3H, s
1.46, 3H, s
1.02, 3H, s
1.44, 3H, s
1.41, 3H, s
4.30, 1H, d
(10.4); 4.85,
1H, d (10.4)
1.01, 3H, s
1.67, 3H, s
1.02, 3H, s
1.51, 3H, s
1.01, 3H, s
1.52, 3H,s
1.05, 3H, s
4.10, 1H, d
(11.6); 4.18,
1H, d (11.6)
[a]
The data were recorded at 400 MHz in pyridine-d5; * new compound.
the correlations of 28-/29-CH3 with C-3; III belonged pling systems. Thus, ring B was deduced as a seven-
to ring B involving three methines and one methylene membered ring (dC =127.9, 135.1, 59.8, 73.4, 38.2,
(dC-5 =59.8, dC-6 =73.4, dC-7 =38.2, and dC-8 =41.9). The 41.9, and 138.2), and the dH =6.18 signal was assigned
vinyl quaternary carbon at dC =135.1 correlated with to H-19. It could be assumed that during the biocata-
H-1, H-2, H-5 and dH =6.18 was assigned to C-10. lytic process, the 9,19-propane ring of cycloastragenol
The other unsaturated quaternary carbon at dC = was cleaved and the B ring expanded to form an un-
138.2 correlated with H-8, dH =6.18, dH =2.10 and usual 9(10)a-homo-19-nor-cycloartane skeleton.
A
dH =2.25 was ascribed to C-9. The HMBC correla- similar 9,10-seco-cycloartane sapogenin (secomacoro-
tions of dH =6.18 with C-5, C-8, C-9 and dC =127.6 in- genin B) had been reported from Astragalus species
dicated that it connected the above three spin-cou- by Isaev and co-workers.[16]
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Biocatalysis of Cycloastragenol by Filamentous Fungi to Produce Unexpected Triterpenes
Table 3. 1H NMR[a] spectroscopic data for compounds 9–15 (J in Hz).
H
1
9*
10*
11*
12
13*
14*
15*
2.04, 1H, m; 2.11, 1.99, 1H, m;
1H, m 2.10, 1H, m
2.08, 1H, m; 2.17, 1.94, 1H, m; 1.96, 1H, m;
1H, m 2.97, 1H, m 2.94, 1H, brs
1.97, 1H, m;
2.94, 1H, d
(8.4)
1.97, 1H, m;
2.94, 1H, d
(8.4)
2
3
5
1.43, 1H, m; 2.47, 1.53, 1H, m;
1.26, 1H, m; 2.02, 1.94, 1H, m; 1.97, 1H, m;
1H, m 2.10, 1H, m 2.23, 1H, m
4.23, 1H, dd (4.8, 3.80, 1H, brs 3.84, 1H, brs
11.4)
1.97, 1H, m;
2.22, 1H, m
3.84, 1H, brs
1.97, 1H, m;
2.22, 1H, m
3.84, 1H, brs
1H, m
3.72, 1H, brs
1.74, 1H, m
3.70, 1H, brs
1.92, 1H, d (9.6) 1.74, 1H, m
2.02, 1H, d (10.0) 2.34, 1H, d
(6.2)
2.35, 1H, d
(6.4)
2.35, 1H, d
(7.4)
2.35, 1H, d
(7.4)
6
7
4.07, 1H, m
1.90, 1H, m; 2.06, 1.79, 1H, m;
1H, m
2.55, 1H, t (8.4)
3.98, 1H, m
3.85, 1H, m
1.66, 1H, m; 1.86, 1.75, 1H, m; 1.76, 1H, m;
1H, m
1.81, 1H, m
4.20, 1H, m 4.21, 1H, brs
4.20, 1H, m
1.74, 1H, m;
2.10, 1H, m
2.68, 1H, d
(11.0)
4.20, 1H, m
1.74, 1H, m;
2.10, 1H, m
2.68, 1H, d
(11.0)
1.98, 1H, m
2.42, 1H, m
2.11, 1H, m 2.10, 1H, m
2.67, 1H, m 2.68, 1H, d
(11.2)
8
11
5.48, 1H, d
(10.0)
1.17, 1H, m; 2.07, 3.38, 1H, brs 3.39, 1H, brs
1H, m
3.39, 1H, brs
3.39, 1H, brs
12 2.66, 1H, d
6.26, 1H, d
1.62, 1H, m; 2.36, 1.65, 1H, m; 1.78,1H, m;
1.81, 1H, m;
2.40, 1H, d
(13.8)
1.93, 1H, m;
2.16, 1H, m
5.08, 1H, dd
(6.4, 13.8)
2.58, 1H, d
(7.8)
1.81, 1H, m;
2.40, 1H, d
(13.8)
1.93, 1H, m;
2.16, 1H ,m
5.08, 1H, dd
(6.4, 13.8)
2.58, 1H, d
(7.8)
(16.2); 2.74, 1H, (10.0)
d (16.2)
15 1.93, 1H, m; 2.26, 1.88, 1H, m;
1H, m
16 5.10, 1H, brs
1H, m
2.67, 1H, m 2.38, 1H, d
(13.8)
1.66, 1H, m; 1.97, 1.94, 1H, m; 1.93, 1H, m;
2.24, 1H, m
5.14, 1H, brs
1H, m
4.54, 1H, brs
2.16, 1H, m 2.16, 1H, m
5.08, 1H, m 5.07, 1H, brs
17 2.72, 1H, d (7.6) 2.84, 1H, d (8.0) 2.69, 1H, d (8.0) 2.57, 1H ,d
(8.0)
2.58, 1H, d
(8.0)
18 1.33, 3H, s
19 1.12, 1H, d (4.1); 0.63, 1H, d
1.44, 3H, s
1.41, 3H, s
1.55, 3H, s
1.48, 3H, s
1.48, 3H, s
3.66, 2H, m
1.48, 3H, s
3.66, 2H, m
0.43, 1H, d (4.1); 3.94, 1H, m; 3.61, 2H, m
1.83, 1H, d (4.1) (3.8); 0.97, 1H, 0.64, 1H, d (4.1) 4.05, 1H, brs
d (3.8)
21 1.30, 3H, s
22 1.68, 1H, m; 3.07, 1.75, 1H, m;
1H, m 3.22, 1H, m
23 2.06, 1H, m; 2.33, 2.10, 1H, m;
1H, brs 2.38, 1H, m
1.49, 3H, s
1.54, 3H, s
2.05, 1H, m; 2.69, 1.59, 1H, m; 1.69, 1H, m;
1H, m 3.12. 1H, m 3.17, 1H, m
1.63, 1H, m; 1.70, 2.04, 1H, m; 2.07, 1H, m;
1.41, 3H, s
1.45, 3H, s
1.41, 3H, s
1.68, 1H, m;
3.16, 1H, m
2.08, 1H, m;
2.34, 1H, m
3.91, 1H, dd
(5.2, 9.0)
1.41, 3H, s
1.68, 1H, m;
3.16, 1H, m
2.08, 1H, m;
2.34, 1H, m
3.91, 1H, dd
(5.2, 9.0)
1H, m
2.29, 1H, m 2.33, 1H, m
3.89, 1H, dd 3.92, 1H, dd
24 3.91, 1H, dd (5.6, 3.96, 1H, brs
9.0)
(5.2, 9.0)
(5.2, 9.0)
26 1.34, 3H, s
27 1.62, 3H, s
28 1.97, 3H, s
1.35, 3H, s
1.64, 3H, s
2.04, 3H, s
1.61, 3H, s
1.69, 3H, s
4.37, 1H, d
(11.2); 4.46, 1H,
d (11.2)
1.34, 3H, s
1.67, 3H, s
1.86, 3H, s
1.35, 3H, s
1.63, 3H, s
1.86, 3H, s
1.34, 3H, s
1.63, 3H, s
2.04, 3H, s
1.34, 3H, s
1.63, 3H, s
2.04, 3H, s
29 1.40, 3H, s
30 1.26, 3H, s
1’
1.48, 3H, s
1.00, 3H, s
1.35, 3H, s
0.98, 3H, s
1.16, 3H, s
0.89, 3H, s
1.18, 3H, s
0.85, 3H, s
3.47, 1H, m;
3.59, 1H, m
1.66, 2H, m
1.50, 2H, m
0.94, 3H, t
(7.3)
1.17, 3H, s
0.85, 3H, s
4.17, 2H, m
1.17, 3H, s
0.85, 3H, s
4.17, 2H, m
2’
3’
4’
5.59, 1H, s
1.70, 3H, s
1.73, 3H, s
5.59, 1H, s
1.70, 3H, s
1.73, 3H, s
5’
[a]
The data were recorded at 400 MHz in pyridine-d5; * new compound.
The NOE correlations of H-3/28-CH3, 29-CH3/H-6, genol. The NOE correlation between H-24 and 21-
H-6/H-8, H-8/18-CH3 and 30-CH3/H-17 confirmed CH3 verified the S-configuration of C-24. Based on
that the trans-conjunctions of A/B, B/C and C/D rings the above evidences, the structure of compound 16
of compound 16 were the same as those of cycloastra- was established as (20R,24S)-3b,6a,16b,25-tetraACHTUNGTRENNUNGroxy-
Adv. Synth. Catal. 2012, 354, 527 – 539
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531

Wen-zhi Yang et al.
FULL PAPERS
Table 4. 13C NMR[a] spectroscopic data for compounds 1–15.
C
1
2*
3*
4*
5*
6*
7
8*
9*
10*
11*
12
13*
14*
15*
1
2
3
4
5
6
7
8
9
31.5
32.8
78.3
42.4
54.0
68.3
38.8
47.3
21.0
41.4 31.5 31.2 31.7 30.4
71.2 33.1 32.7 32.7 33.2
84.0 78.3 72.3 80.2 71.1
42.5 42.5 46.2 45.0 55.8
54.1 54.3 49.7 54.2 50.6
68.2 69.1 67.8 68.7 66.8
38.8 39.6 38.0 39.1 37.5
47.1 48.4 47.4 47.4 47.8
20.2 21.2 20.5 22.0 20.3
28.6 30.0 29.1 29.4 28.5
26.5 38.7 26.0 26.2 26.1
33.3 72.8 33.4 33.3 32.2
45.0 50.7 45.0 45.0 45.0
46.2 46.3 46.5 46.1 46.3
46.7 49.8 47.0 46.8 46.8
73.4 72.8 73.4 73.4 73.3
58.4 52.2 58.5 58.4 58.5
21.6 21.1 21.9 21.7 21.8
31.1 31.7 31.6 31.4 31.2
87.2 87.5 87.2 87.2 87.2
28.2 27.5 28.2 28.2 28.2
34.9 38.3 35.0 34.9 34.9
26.5 26.0 26.4 26.4 26.4
81.7 83.5 81.7 81.7 81.7
71.2 70.8 71.2 71.2 71.2
27.1 27.1 27.1 27.1 27.2
28.7 27.5 28.2 28.2 28.5
31.8
35.8
31.8
35.9
31.2
28.2
31.0
32.7
77.9
42.2
53.6
68.3
38.7
45.8
25.5
33.3
31.1
32.6
72.3
46.5
49.6
67.5
38.0
46.6
20.2
29.5
29.3
33.1
77.4
42.3
57.7
67.7
37.4
41.1
29.3
33.2
77.3
42.4
57.7
67.6
37.3
41.0
29.3
33.1
77.3
42.4
57.7
67.7
37.3
41.0
29.3
33.1
77.3
42.4
57.8
67.5
37.3
40.9
216.8 216.8 77.8
50.5
53.5
69.1
38.4
48.2
21.2
28.5
26.1
33.2
45.0
46.0
47.0
73.4
58.5
22.1
31.0
87.2
28.5
34.9
26.4
81.7
71.2
27.1
28.2
50.5
53.6
69.2
38.9
48.3
21.3
28.5
27.2
32.6
45.7
51.5
39.9
73.9
58.7
23.3
30.7
87.3
28.5
35.0
26.4
81.8
71.2
27.1
28.1
28.5
20.4
63.1
42.6
53.5
65.7
36.4
41.9
39.7
35.0
132.9 132.4
134.9 135.2
132.5 131.2
135.3 136.8
10 29.9
11 26.3
12 33.4
13 45.0
14 46.2
15 46.8
16 73.4
17 58.4
18 21.6
19 31.0
20 87.2
21 28.2
22 34.9
23 26.4
24 81.7
25 71.2
26 27.1
27 28.6
28 29.4
29 16.1
30 20.2
1’
210.2 131.7 26.3
39.5
33.3
44.9
45.5
45.1
73.4
58.2
20.2
68.6
87.3
28.6
34.7
26.4
35.8
33.9
44.7
45.4
45.1
73.3
58.2
20.2
77.3
87.3
28.6
34.9
26.4
81.6
71.3
27.1
28.1
27.3
15.2
19.4
70.2
32.2
19.8
14.0
35.9
34.0
44.7
45.4
45.1
73.3
58.2
20.2
76.4
87.2
28.7
34.8
26.4
81.6
71.3
27.1
28.1
27.3
15.1
19.4
66.9
34.8
33.8
44.5
45.2
45.0
73.2
58.1
20.0
70.1
87.1
28.6
34.8
26.4
81.7
71.3
27.1
28.1
27.2
15.1
19.4
170.9
53.3
45.6
45.9
45.2
72.9
56.8
20.8
27.7
86.7
28.5
34.9
26.4
81.8
71.2
27.1
28.2
29.0
15.5
19.7
135.2 33.5
47.1
48.2
45.0
73.8
54.1
21.4
28.6
87.5
28.7
35.0
26.4
81.8
71.2
27.1
28.1
29.6
15.8
22.0
44.6
45.7
43.3
74.1
61.8
22.6
32.0
84.8
30.4
31.8
33.2
110.7 81.6
72.8
25.3
25.6
67.8
12.4
20.1
71.3
27.1
28.1
27.3
15.2
19.5
29.9 29.7 67.8 24.7 204.1 28.6
17.2 16.3 12.4 65.1 8.6
20.2 22.0 20.4 20.2 19.8
20.4
20.4
2’
3’
4’
122.6 21.0
136.3
25.7
5’
18.0
[a]
The data were recorded at 100 MHz in pyridine-d5; * new compound.
20,24-epoxy-9(10)a-homo-19-nor-cycloartane (Figure 1). Hydroxylation Products 2–5
1
It was named neoastragenol. The key HMBC, H-1H
COSY and NOE correlations are illustrated in Four monohydroxylated metabolites (2, 3, 4, 5) were
Figure 2.
identified with the same molecular formula C30H50O6
based on their HR-ESI-MS data.
1
Figure 2. Key HMBC, H-1H COSY and NOE correlations of compound 16.
532
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Adv. Synth. Catal. 2012, 354, 527 – 539

Biocatalysis of Cycloastragenol by Filamentous Fungi to Produce Unexpected Triterpenes
Figure 3. Key HMBC and NOE correlations of compounds 2–6, 8–11, and 13–15.
1
The H NMR spectra of 2 and 3 both displayed one H-12/18-CH3 indicated the a-configuration of 12-OH
more down-field oxygenated tertiary proton than 1, (Figure 3). Thus, compound 3 was identified as
suggesting that hydroxylation occurred on a methyl- (20R,24S)-3b,6a,12a,16b,25-pentahydroxy-20,24-ep-
ene carbon. Further comparison of the 13C NMR data
of 2 with those of 1 revealed significant down-field
ACHTUNGTRENNUNG
1
shifts of C-1 (+9.9ppm) and C-3 (+5.7 ppm), indicat- and 5 both showed two additional down-field proton
ing the hydroxylation at C-2 (dC =71.2). This was con- signals which formed AB coupling systems. In the
firmed by the HMBC correlations of H-2/C-3, H-1/C- meanwhile, one methyl signal of cycloastragenol has
2 and H-3/C-2. The NOE correlation of H-2/29-CH3 disappeared. The new proton signals of compound 4
suggested the a-configuration of 2-OH (Figure 3). appeared at dH =4.36 and 4.45 (J=11.2 Hz). In the
Therefore, compound
2
was characterized as 13C NMR spectrum of compound 4, significant
(20R,24S)-2a,3b,6a,16b,25-pentahydroxy-20,24-ep
cycloartane.
oxy-
changes of C-3 (ꢀ6 ppm), C-4 (+3.8 ppm) and C-5
(ꢀ4.3 ppm) were observed when compared with cy-
The additional hydroxy group of compound 3 was cloastragenol. This evidence indicated the presence of
speculated to be at C-12 according to the significant 28- or 29-OH, which was also suggested by the
down-field shifts of C-11 (+12.4 ppm) and C-13 (+ HMBC correlations of 28-H2 with C-3, C-4 and C-5
5.6 ppm) when compared to cycloastragenol. This was (Figure 3). According to the NOE correlation of H-6/
confirmed by the HMBC correlations of H-11/C-12, 29-CH3, the additional hydroxy group was determined
H-17/C-12, and 18-CH3/C-12. The NOE correlation of to be at C-28. Therefore, compound 4 was identified
Adv. Synth. Catal. 2012, 354, 527 – 539
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Wen-zhi Yang et al.
FULL PAPERS
as
(20R,24S)-3b,6a,16b,25,28-pentahydroxy-20,24-
Compound 9 was characterized as an isomer of 6
epoxy-cycloartane. Likewise, the additional hydroxy according to its HR-ESI-MS data (m/z=1009.69791,
group of compound 5 was determined to be at C-29 calcd. for [2M+H]+: 1009.69745). According to the
according to the NOE correlation between H-3 and DEPT spectrum, one CH2 signal of cycloastragenol
28-CH3 (dH =2.19). Therefore, the structure of 5 was disappeared, and it could be converted to a carbonyl
established as (20R,24S)-3b,6a,16b,25,29-pentahy- group. The 13C NMR spectrum also showed significant
droxy-20,24-epoxy-cycloartane.
changes for C-9 (+18.7 ppm), C-10 (+5.1 ppm) and
C-12 (+19.9 ppm) when compared to cycloastragenol,
indicating that the carbonyl group (dC =210.2) was lo-
cated at C-11. The HMBC correlations of H-12/C-11
and H-19/C-11 further confirmed this deduction
Carbonylation Products 6–9
Four carbonylated metabolites (6, 7, 8, 9) were identi- (Figure 3). In accordance, the characteristic H-19 sig-
fied according to the characteristic down-field carbon- nals (dH =1.83 and 1.12, J=4.1 Hz) shifted down-field
yl signals (dC >200 ppm) in their 13C NMR spectra.
remarkably due to a de-shielding effect of the carbon-
The molecular formula of 6 was determined as yl group. Therefore, compound 9 was identified as
C30H48O6 based on its HR-ESI-MS data (m/z= (20R,24S)-3b,6a,16b,25-tetrahydroxy-20,24-epoxy-cy-
1009.69873, calcd. for [2M+H]+: 1009.69745). The cloartan-11-one.
strong IR absorption at 1717 cmꢀ1 indicated the pres-
1
ence of a carbonyl group. The H NMR spectrum ex-
hibited an aldehyde proton signal at dH =10.12 (1H, Dehydrogenation (10) and Cyclization (11) Products
s) together with six methyl signals, indicating that one
methyl group of cycloastragenol was converted to a The molecular formula (C30H48O5) of the dehydrogen-
formyl group. When compared to cycloastragenol, the ation product (10) was established by HR-ESI-MS
13C NMR spectrum of compound 6 displayed signifi- data (m/z=1466.05808, calcd. for [3M+H]+:
cant changes for C-3 (ꢀ7.2 ppm), C-4 (+13.4 ppm) 1466.05780). Two cis-olefinic proton signals (dH =5.48,
and C-5 (ꢀ3.4 ppm). Thus, it could be deduced that d, J=10.0 Hz; dH =6.26, d, J=10.0 Hz) in the
either 28- or 29-CH3 was converted to aldehyde. This 1H NMR spectrum indicated the presence of a double
deduction was supported by the HMBC correlations bond. When compared to cycloastragenol, the
of H-5 (dH =1.82) and 28-/29-CH3 (dH =1.67) with 13C NMR spectrum showed significant changes for C
dC =204.1, as well as dH =10.12 with C-28/29 (dC = ring carbons (C-9, C-13, C-14, and C-15). Thus, the
8.6) and C-4 (dC =55.8). The NOE correlation of H-6/ double bond should be located at C-11 and C-12. This
29-CH3 confirmed the presence of the 28-aldehyde deduction was further confirmed by the HMBC corre-
group (Figure 3). Therefore, the structure of com- lations of H-17/C-12, 18-CH3/C-12, H-12/C-9 and H-
pound 6 was established as (20R,24S)-3b,6a,16b,25- 11/C-13 (Figure 3). Therefore, compound 10 was char-
tetrahydroxy-20,24-epoxy-cycloartan-28-carbaldehyde.
acterized as (20R,24S)-3b,6a,16b,25-tetrahydroxy-
Compound 7 was characterized as the previously 20,24-epoxy-cycloartan-11(12)-ene.
reported cyclopycnanthogenin [(20R,24S)-6a,16b,25-
Compound 11 was identified as a 16,24-epoxy de-
trihydroxy-20,24-epoxy-cycloartan-3-one] by compar- rivative of cycloastragenol. The HR-ESI-MS revealed
ing its NMR data with the literature values.[17]
its molecular formula as C30H48O6 (m/z=505.35218,
1
The HR-ESI-MS of compound 8 provided evidence calcd. for [M+H]+: 505.35236). Its H NMR spectrum
for the molecular formula of C30H48O6 (m/z= displayed two new proton signals due to a CH2 group
1009.69461, calcd. for [2M+H]+: 1009.69745). In the (dH =4.37 and 4.46, J=11.2 Hz), suggesting that one
1H NMR spectrum, the signal for one methyl group of methyl group of cycloastragenol was hydroxylated.
cycloastragenol disappeared and two new signals cor- The carbon signals for ring A were almost identical to
responding to a CH2 group were observed (dH =4.10, those of compound 4, suggesting that the hydroxy
d, J=11.6 Hz; dH =4.16, d, J=11.6 Hz). It could thus group was introduced at C-28. This deduction was
be inferred that compound 8 was a derivative of 7, confirmed by the HMBC correlations of H-28/C-3
with a hydroxy group introduced at one methyl and H-28/C-4, as well as the NOE correlation of 29-
group. The 13C NMR spectral data were very similar CH3/H-6. When compared to 4, the signal for H-24
to those of 7 except for C-14 (+5.5 ppm) and C-15 had disappeared. In contrast, a new down-field qua-
(ꢀ7.1 ppm), indicating that the new hydroxy group ternary carbon signal at dC =110.7 corresponding to
was substituted at C-30. The HMBC correlations of an acetal carbon was observed in the 13C NMR spec-
H-30/C-8, H-15/C-30 and H-8/C-30 further confirmed trum. Considering that 11 had one more unsaturation
this deduction (Figure 3). Based on the above evi- degree than 4, it could be deduced that a new ring
dence, compound 8 was identified as (20R,24S)- linking C-16 and C-24 was formed in compound 11.
6a,16b,25,30-tetrahydroxy-20,24-epoxy-cycloartan-3-
one.
Although the HMBC correlation of H-16/C-24 was
not detected, the 13C NMR data of the 16,24;20,24-di-
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Adv. Synth. Catal. 2012, 354, 527 – 539

Biocatalysis of Cycloastragenol by Filamentous Fungi to Produce Unexpected Triterpenes
epoxy sub-structure complied very well with those of served. They were numbered as 5’, 4’, 3’, 2ꢀ and 1’, re-
the known cycloalpigenin [(20R,24R)-16b,24:20,24- spectively. This sub-structure was characterized as an
diepoxy-cycloartane-3b,7b,25-triol].[18] Therefore, we isopentenyl group according to the HMBC correla-
deduced the R-configuration of C-24. Based on the tions of H-4’/C-5’, H-4’/C-1’, H-5’/C-4’, H-1’/C-2ꢀ and
above evidence, compound 11 was identified as H-1’/C-3’. The isopentenyl group was connected to C-
(20R,24R)-3b,6a,25,28-tetrahydroxy-16b,24:20,24-
diepoxy-cycloartane.
19 through an ether bond, according to the HMBC
correlations of H-1’/C-19 and H-19/C-1’. The HMBC
correlation of H-19/C-12 and the NOE correlation of
H-19/18-CH3 confirmed the 19b-side chain was con-
nected to C-11 (Figure 3). Finally, compound 14 was
identified as (20R,24S)-3b,6a,16b,25-tetrahydroxy-19-
ACHTUNGTRENNUNG
Rearrangement Products 12–15
The characteristic 9,19-cyclopropane ring of cycloas- isopentenyloxy-ranunculan-9(10)-ene.
tragenol were cleaved in four biotransformed metabo-
The molecular formula of compound 15 was estab-
lites (12, 13, 14, 15) as the signals corresponding to lished as C32H52O7 on the basis of its HR-ESI-MS
19-CH2 disappeared. They were elucidated to possess data (m/z=549.37849, calcd. for [M+H]+: 549.37858).
the unusual ranunculane skeleton through a compli- The IR absorption at 1739 cmꢀ1 indicated the pres-
cated rearrangement.[14] To the best of our knowledge, ence of an ester carbonyl group. The 1H and
only two compounds with this novel skeleton had 13C NMR spectra showed signals attributed to the ra-
been reported so far.[14,19]
nunculane skeleton, together with signals for one car-
Compound 12 possessed the molecular formula of bonyl group (dC =170.9) and one methyl group (dH =
C30H50O6 according to its HR-ESI-MS data (m/z= 2.13). The latter two signals indicated the presence of
1013.73266, calcd. for [2M+H]+: 1013.72875). By an acetoxy group. The acetoxy group was connected
1
comparing its H and 13C NMR data with those in the to C-19 (dC =70.1) based on the HMBC correlation of
literature, it was identified as the known (20R,24S)- H-19/C-1’. Moreover, the 19b-acetoxy substituent at-
3b,6a,16b,19,25-pentahydroxy-ranunculan-9(10)-ene
tached to C-11 of the triterpene skeleton was con-
firmed by the HMBC correlation of H-19/C-12 and
(spectra measured in CD3OD).[14]
The molecular formula of compound 13 was estab- the NOE correlation of H-19/18-CH3. Based on the
lished as C34H58O6 based on its HR-ESI-MS data above evidence, compound 15 was characterized as
(m/z=1125.85414, calcd. for [2M+H]+: 1125.85395). (20R,24S)-3b,6a,16b,25-tetrahydroxy-19-acetoxy-ran-
1
Its H and 13C NMR data were very similar to those
of 12 except that C-19 (dC =76.4) was shifted down-
field by 8.7 ppm, and that it showed additional signals
ACHTUNGTRENuNUNG nculan-9(10)-ene.
corresponding to one methyl (dH =0.94, t, J=7.3 Hz; Biocatalytic Features of the Three Fungal Strains
dC =14.0) and three methylenes [(dH =1.50, m; dC =
19.8), (dH =1.66, m; dC =32.2), (dH =3.47, m, dH = The biocatalysis of three strains of filamentous fungi
3.59, m; dC =70.2)]. These carbons were numbered as (Cunninghamella elegans, Syncephalastrum racemo-
4’, 3’, 2’, 1’ from up-field to down-field, respectively. sum and Doratomyces stemonitis) on cycloastragenol
This sub-structure was characterized as an n-butyl showed significant preferences. The biotransformation
group linked to an oxygen atom, according to the reactions mainly involved hydroxylation (type I), rear-
HMBC correlations of H-4’/C-3’, H-4’/C-2’, H-3’/C-4’, rangement (type II), and carbonylation (type III). In
H-3’/C-1’, H-1’/C-3ꢀ and H-1’/C-2’. The HMBC corre- addition, dehydrogenation, cyclization, and a novel
lations of H-1’/C-19 and H-19/C-1ꢀ indicated that the ring expansion reaction were also observed
n-butyl group was attached to 19-OH of the ranuncu- (Scheme 1).
lane skeleton. The NOE correlation of H-19/18-CH3
C. elegans preferred to catalyze hydroxylation reac-
further confirmed the b-configuration of the side tions, particularly on 28- and 29-CH3 (4, 10.00%
chain at C-11 (Figure 3). Therefore, compound 13 was yield; 5, 10.00%). Hydroxylation on methylene was
identified as (20R,24S)-3b,6a,16b,25-tetrahydroxy-19- also observed as minor reactions (C-2 for 2, 0.40%
butoxy-ranunculan-9(10)-ene.
yield; C-12 for 3, 0.50%), which showed a-stereose-
The HR-ESI-MS data of 14 established its molecu- lectivity. The total percentage conversion of hydroxyl-
lar formula as C35H58O6 (m/z=575.43122, calcd. for ation reaction added up to more than 20%. In addi-
for [M+H]+: 575.43061). According to its 1H and tion, C. elegans could also catalyze carbonylation (6,
13C NMR spectra, compound 14 should also contain a 2.00%), rearrangement (12, 1.20%), and the rare
ranunculane skeleton. Aside from signals attributed 16,24-cyclization reaction (11, 0.30%).
to the triterpene skeleton, additional signals corre-
S. racemosum displayed a strong capacity to cata-
sponding to two methyls [dH =1.73 (dC =25.7), dH = lyze the complicated rearrangement reaction to pro-
1.70 (dC =18.0)], two vinyl carbons (dC =136.3, 122.6) duce the unusual ranunculane skeleton. Compound
and one methylene (dH =4.17; dC =66.9) were also ob- 12 was obtained in a yield of 17.14%. Moreover, n-
Adv. Synth. Catal. 2012, 354, 527 – 539
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
535

Wen-zhi Yang et al.
FULL PAPERS
Scheme 1. Biocatalytic reactions of Cunninghamella elegans AS 3.1207, Syncephalastrum racemosum AS 3.264 and Dorato-
myces stemonitis AS 3.1411 on cycloastragenol (CA, 1).
butylated (13, 0.48%), isopentenylated (14, 0.33%)
D. stemonitis tended to catalyze the carbonylation
and acetylated (15, 0.71%) derivatives of compound reaction on cycloastragenol. The carbonylation at 3-
12 were also produced. The total percentage conver- OH was the major reaction as compound 7 was ob-
sion of the arrangement reaction reached 18.7%. S. tained in a high yield of 44.24%. The combination of
racemosum could also catalyze hydroxylation (3, hydroxylation and carbonylation yielded compound 8
0.48%), carbonylation (9, 1.48%), and dehydrogena- (0.27% yield). The rearranged product 12 was also
tion (10, 0.17%) reactions on cycloastragenol. It is obtained in 0.33% yield.
particularly noteworthy that S. racemosum realized
Some of the above biocatalytic reactions, such as
the unexpected ring expansion reaction to produce rearrangement, cyclization, and ring expansion could
the unusual 9(10)a-homo-19-nor-cycloartane triter- be difficult for chemical approaches. In addition, each
pene skeleton (16, 0.26%).
of the three fungal strains exhibited its biocatalytic
preference, which could be used to synthesize differ-
536
asc.wiley-vch.de
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2012, 354, 527 – 539

Biocatalysis of Cycloastragenol by Filamentous Fungi to Produce Unexpected Triterpenes
ent types of cycloastragenol derivatives. It is also Preparation of Cycloastragenol
noteworthy that all three strains could convert cyclo-
Cycloastragenol (CA) was prepared from astragaloside IV
astragenol into compound 12 through a rearrange-
ment reaction. Kuban et al reported the same reac-
tion by a Cunninghamella species very recently.[14] It
appeared that different fungal strains contained simi-
lar enzymes that catalyzed this complicated rear-
rangement reaction.
by Smith degradation.[21,22] Typically, an amount of 1 g astra-
galoside IV was dissolved in 90 mL methanol, 60 mL water
containing 5 g NaIO4 was then added, and the solution was
stirred overnight at room temperature. The reaction mixture
was concentrated to about 50 mL under reduced pressure,
and then extracted by an equal volume of ethyl acetate for
three times. The separated ethyl acetate layer was washed
with an equal volume of ethyl acetate-saturated water twice,
and the organic layer was evaporated to dryness. The resi-
due was dissolved in 80 mL 75% methanol, and 1 g NaBH4
was added. The solution was left to stand for 24 h at room
temperature, 80 mL water were added, the pH adjusted to
2.0 with 1 mol/L H2SO4, then the mixture was allowed to
stand for another 24 h. The reaction mixture was extracted
with 150 mL ethyl acetate thrice, and the ethyl acetate layer
was washed with ethyl acetate-saturated water thrice. The
organic layer was then evaporated to dryness to yield the
crude product; yield: 750 mg. The crude product was sepa-
rated by silica gel column chromatography and eluted with
chloroform-methanol (50:1, 30:1, v/v). Following TLC detec-
tion, the 30:1 fraction afforded CA; yield: 384 mg. The
purity was above 98% as detected by HPLC-RI. The struc-
ture was identified by 1H and 13C NMR. Following the
above procedure, a total of 5 g CA was prepared for bio-
transformation experiments.
Conclusions
In summary, the biocatalysis of three strains of fila-
mentous fungi on cycloastragenol was carried out to
obtain 15 transformed products, including 13 new
compounds. Cunninghamella elegans, Syncephalas-
trum racemosum, and Doratomyces stemonitis pre-
ferred to catalyze hydroxylation, rearrangement, and
carbonylation reactions, respectively. In addition, an
unexpected ring expansion product was obtained
from C. elegans. These biocatalytic reactions could be
difficult for chemical approaches. The synthesis of an
array of novel cycloastragenol derivatives allows for
the future study on telomerase activation activity-
structure relationships for the discovery of anti-aging
drug candidates.
Screening of Fungal Strains
Experimental Section
A total of 13 different fungal strains, including Alternaria al-
ternata AS 3.4578, Doratomyces stemonitis AS 3.1411, Fusa-
rium avenaceum AS 3.4594, Mucor spinosus AS 3.3450, Rhi-
zopus chinensis CICC 3043, Syncephalastrum racemosum
AS 3.264, Cunninghamella elegans AS 3.1207, Mucor fragilis
AS 3.2215, Phyllosticta pirina AS 3.2886, Colletotrichum lini
AS 3.4486, Fusarium sambucinum AS 3.4602, Rhizoctonia
solani AS 3.2888, and Saccharomyces cerevisiae ACCC 2168
were screened in the preliminary test for their abilities to
metabolize cycloastragenol.
Apparatus and Reagents
Melting points were determined on an XT4A apparatus
(uncorrected). A Perkin-Elmer 243B polarimeter was used
to measure the optimal rotations at 208C. IR spectra were
obtained with a Thermo Nicolet Nexus 470 FT-IR spectrom-
1
eter. H, 13C and 2D NMR experiments were performed on
a Bruker Avance III (400 MHz) NMR spectrometer in pyri-
dine-d5. The HR-ESI-MS data were acquired on a Bruker
APEX II Fourier transform ion cyclotron resonance (FT-
ICR) mass spectrometer. Column chromatography was per-
formed on silica gel (200–300 mesh, Qingdao Marine Chem-
ical Corporation, Qingdao, China), ODS (Fuji Silysia Chem-
ical Ltd., Kasugai, Japan), and Sephadex LH-20 (Pharmacia
Biotech AB, Sweden).
Astragaloside IV (purity> 98%, Sanleng Biotechnological
Co. Ltd., Guilin, China), sodium periodate (analytical grade,
Sinopharm Chemical Reagent Co. Ltd., China), glucose,
methanol, acetonitrile (analytical grade, Beijing Chemical
Factory, China), ultra-pure water (Millipore, Bedford, MA,
USA), and potatoes freshly purchased from the local super-
market were used for the biotransformation experiments.
All the 13 fungal strains were purchased from China Gen-
eral Microbiological Culture Collection Center. Fermenta-
tions were carried out in potato media consisting of 20 g of
potato extract (prepared from 200 g of potato slices extract-
ed in boiling water for 30 min), 20 g of glucose, and
1000 mL of water. The media were sterilized at 1218C and
1.06 kg/cm2 for 30 min.[20] The strains were maintained on
potato dextrose agar slants at 48C.
Scaled-Up Biotransformation Experiments
The fungal strains (Cunninghamella elegans, Syncephalas-
trum racemosum and Doratomyces stemonitis) had been
sub-cultured for three times on potato dextrose agar slants
before use to obtain maximal enzyme activities. The pre-ac-
tivated fungi were incubated in a 250-mL flask (containing
100 mL potato culture medium) for two days to obtain a
stock inoculum. The scaled-up biotransformation fermenta-
tions were carried out in 1000-mL Erlenmeyer flasks con-
taining 400 mL potato culture medium. For each flask, 5 mL
of the inoculum were added. Two days later, CA in 95%
ethanol (12.5 mgmLꢀ1) was added to the cultures at a final
concentration of 62.5 mgmLꢀ1. The fungi were incubated at
258C on a rotary shaker (120 rpm) in the dark. After 6 days
of incubation, the cultures were pooled and filtered, and the
supernatant was extracted with an equal volume of ethyl
acetate thrice. The organic layer was concentrated to dry-
ness for metabolite isolation.
Adv. Synth. Catal. 2012, 354, 527 – 539
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
537

Wen-zhi Yang et al.
FULL PAPERS
AHCTUNGTRENNUNG
Isolation of Transformed Products
The isolation process mainly contained three steps, prelimi-
nary isolation on a silica gel column with chloroform-metha-
nol as the elution solvent, removing of pigments over Sepha-
dex LH-20 column, and purification by semi-preparative
HPLC. An Alltech 426 HPLC system with an RI 2000 de-
tector was used for semi-preparative HPLC. Samples were
separated on an Agilent Zorbax SB-C18 chromatographic
column (9.4ꢂ250 mm, 5 mm) eluted by mixtures of methanol
(HPLC grade, Mallinkrodt Baker, Phillipsburg, NJ) and
water.
Cunninghamella elegans: A total of 1.00 g of CA was
added to the cultures. After 6 days of incubation, 2.45 g of
ethyl acetate extract were obtained from the culture super-
natant. The extract was separated on a silica gel column and
eluted with chloroform-methanol (60:1 to 1:1, v/v) to give
four fractions. After pigment removal, the fractions were pu-
rified by semi-preparative HPLC-RI. Fr. 1 yielded 11 (3 mg,
0.30%) while Fr. 2 yielded 2 (4 mg, 0.40%), 3 (5 mg, 0.50%),
4 (100 mg, 10.00%), 5 (100 mg, 10.00%), 6 (20 mg, 2.00%),
and 12 (12 mg, 1.20%).
Syncephalastrum racemosum: A total of 2.10 g of CA was
added to the cultures. After 6 days of incubation, 4.51 g of
ethyl acetate extract were obtained from the culture super-
natant. The extract was separated on a silica gel column and
eluted with chloroform-methanol (60:1, 30:1, 20:1, 10:1, 5:1,
2:1, 0:1, v/v) to give 28 fractions. By preparative HPLC-RI
purification, 12 (360 mg, 17.14%) was obtained from Fr. 24–
26 while compounds 3 (10 mg, 0.48%), 9 (7 mg, 0.33%), and
16 (5.5 mg, 0.26%) from Fr. 20–23; 9 (24 mg, 1.14%), 10
(3.5 mg, 0.17%), 15 (15 mg, 0.71%), 14 (7 mg, 0.33%), and
13 (10 mg, 0.48%) were obatined from Fr. 16–19.
Doratomyces stemonitis: A total of 1.65 g of CA was
added to the cultures. After 6 days of incubation, 3.38 g of
ethyl acetate extract were obtained from the culture super-
natant. The extract was separated on a silica gel column and
eluted with chloroform-methanol (60:1, 30:1, 20:1, 10:1, 5:1,
2:1, 0:1, v/v) to give 29 fractions. The fractions were purified
with preparative HPLC-RI yielding 7 (730 mg, 44.24%)
from Fr. 11–16 while compounds 12 (5.5 mg, 0.33%) and 8
(4.5 mg, 0.27%) were obtained from Fr. 23–27.
ꢀ
ꢀ
2933, 2873 (nCꢀH), 1465, 1447 (das C H), 1379 (ds C H of
CH3), 1039 (nCꢀO) cmꢀ1
; HR-ESI-MS: m/z=1013.72944,
calcd. for [2M+H]+: 1013.72875, corresponding to
C30H50O6; 1H NMR (400 MHz, pyridine-d5) and 13C NMR
(100 MHz, pyridine-d5) data, see Table 2,Table 3, and
Table 4.
AHCTUNGTREG(NNUN 20R,24S)-3b,6a,16b,25,29-Pentahydroxy-20,24-epoxy-cy-
cloartane (5): white powder (MeOH); mp 243–2448C; [a]2D0:
+32.1 (c 0.17, MeOH); IR (KBr): nmax =3395 (nOꢀH), 2935,
ꢀ
ꢀ
2893 (nCꢀH), 1443 (das C H), 1379 (ds C H of CH3), 1031
(nCꢀO) cmꢀ1
;
HR-ESI-MS: m/z=1013.73324, calcd. for
[2M+H]+: 1013.72875, corresponding to C30H50O6;
1H NMR (400 MHz, pyridine-d5) and 13C NMR (100 MHz,
pyridine-d5) data, see Table 2, Table 3, and Table 4.
AHCTUNGERTG(NNUN 20R,24S)-3b,6a,16b,25-Tetrahydroxy-20,24-epoxy-cycloar-
tan-28-aldehyde (6): white powder (MeOH); mp 241–
2428C; [a]2D0: +30.9 (c 0.15, MeOH); IR (KBr): nmax =3399
ꢀ
(nOꢀH), 2969, 2938, 2871 (nCꢀH), 1717 (nC O), 1447 (das C H),
=
1377 (ds C H of CH3), 1080, 1035 (nCꢀO) cmꢀ1; HR-ESI-MS:
ꢀ
m/z=1009.69873, calcd. for [2M+H]+: 1009.69745, corre-
1
sponding to C30H48O6; H NMR (400 MHz, pyridine-d5) and
13C NMR (100 MHz, pyridined5) data, see Table 2, Table 3,
and Table 4.
AHCTUNGERTG(NNUN 20R,24S)-6a,16b,25-Trihydroxy-20,24-epoxy-cycloartan-3-
one (7): white powder (MeOH); mp 230–2318C; [a]2D0:
+30.9 (c 0.15, MeOH); IR (KBr): nmax =3384 (nOꢀH), 2987,
ꢀ
2935, 2871 (nCꢀH), 1707 (nC O), 1463, 1448 (das C H), 1380
=
(ds C H of CH3), 1114, 1039 (nCꢀO) cmꢀ1
;
1H NMR
ꢀ
(400 MHz, pyridine-d5) and 13C NMR (100 MHz, pyridine-
d5) data, see Table 2, Table 3, and Table 4.
AHCTUNGERTG(NNUN 20R,24S)-6a,16b,25,30-Tetrahydroxy-20,24-epoxy-cycloar-
tan-3-one (8): white powder (MeOH); mp 149–1508C; [a]2D0:
+71.2 (c 0.07, MeOH); IR (KBr): nmax =3421 (nOꢀH), 2966,
ꢀ
ꢀ
2932, 2883 (nCꢀH), 1708 (nC O), 1450 (das C H), 1380 (ds C
=
H
of CH3), 1073, 1022 (nCꢀO) cmꢀ1
;
HR-ESI-MS:
m/z=1009.69461, calcd. for [2M+H]+: 1009.69745, corre-
1
sponding to C30H48O6; H NMR (400 MHz, pyridine-d5) and
13C NMR (100 MHz, pyridine-d5) data, see Table 2, Table 3,
and Table 4.
AHCTUNGERTG(NNUN 20R,24S)-3b,6a,16b,25-Tetrahydroxy-20,24-epoxy-cycloar-
Structure Characterization
tan-11-one (9): white powder (MeOH); mp 250–2518C;
[a]2D0: +86.8 (c 0.08, MeOH); IR (KBr): nmax =3422 (nOꢀH),
ACHTUNGTRENNUNG(20R,24S)-2a,3b,6a,16b,25-Pentahydroxy-20,24-epoxy-cyclo-
ꢀ
ꢀ
2966, 2928 (nCꢀH), 1462 (das C H), 1377 (ds C H of CH3),
artane (2): white powder (MeOH); mp 228–2298C; [a]2D0:
+38.2 (c 0.15, MeOH); IR (KBr): nmax =3413 (nOꢀH), 2969,
1110, 1058, 1034 (nCꢀO) cmꢀ1
;
HR-ESI-MS: m/z=
1009.69791, calcd. for [2M+H]+: 1009.69745, corresponding
ꢀ
ꢀ
2935, 2878 (nCꢀH), 1450 (das C H), 1377 (ds C H of CH3),
to C30H48O6; H NMR (400 MHz, pyridine-d5) and 13C NMR
1
1057, 1033 (nCꢀO) cmꢀ1
; HR-ESI-MS: m/z=1013.72970,
calcd. for [2M+H]+: 1013.72875, corresponding to
C30H50O6; 1H NMR (400 MHz, pyridine-d5) and 13C NMR
(100 MHz, pyridine-d5) data, see Table 2, Table 3, and
Table 4.
(100 MHz, pyridine-d5) data, see Table 2, Table 3, and
Table 4.
AHCTUNGERTG(NNUN 20R,24S)-3b,6a,16b,25-Tetrahydroxy-20,24-epoxy-cycloar-
tan-11(12)-ene (10): white powder (MeOH); mp 229–2308C;
[a]2D0: +102.0 (c 0.05, MeOH); IR (KBr): nmax =3409 (nOꢀH),
ACHTUNGTRENNUNG(20R,24S)-3b,6a,12a,16b,25-Pentahydroxy-20,24-epoxy-cy-
cloartane (3): white powder (MeOH); mp 162–1638C; [a]2D0:
+35.8 (c 0.15, MeOH); IR (KBr): nmax =3422 (nOꢀH), 2967,
H
3045 (nCꢀ of cyclopropane ring), 2979, 2931, 2874 (nCꢀH),
ꢀ
ꢀ
1708, 1639 (nC C), 1460 (das C H), 1376 (ds C H of CH3),
=
1062, 1033 (nCꢀO) cmꢀ1
; HR-ESI-MS: m/z=1466.05808,
ꢀ
ꢀ
2932, 2877 (nCꢀH), 1459 (das C H), 1381 (ds C H of CH3),
1073, 1019 (nCꢀO) cmꢀ1; HR-ESI-MS: m/z=507.36772, calcd.
for [M+H]+: 507.36801, corresponding to C30H50O6;
1H NMR (400 MHz, pyridine-d5) and 13C NMR (100 MHz,
pyridine-d5) data, see Table 2, Table 3,and Table 4.
calcd. for [3M+H]+: 1466.05780, corresponding to
C30H48O5; 1H NMR (400 MHz, pyridine-d5) and 13C NMR
(100 MHz, pyridine-d5) data, see Table 2, Table 3, and
Table 4.
538
asc.wiley-vch.de
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2012, 354, 527 – 539

Biocatalysis of Cycloastragenol by Filamentous Fungi to Produce Unexpected Triterpenes
ACHTUNGTRENNUNG
Acknowledgements
ACHTUNGTRENNUNG
=
This work was supported by the 985 Project of Peking Uni-
versity Health Science Center (No. 985-2-119-121), Beijing
Natural Science Foundation (No. 7102103), and Beijing
NOVA Program (No. 2009B02).
ꢀ
ꢀ
3384 (nOꢀH), 2928 (nCꢀH), 1459 (das C H), 1377 (ds C H of
CH3), 1099, 1037 (nCꢀO) cmꢀ1; HR-ESI-MS: m/z=505.35218,
calcd. for [M+H]+: 505.35236, corresponding to C30H48O6;
1H NMR (400 MHz, pyridine-d5) and 13C NMR (100 MHz,
pyridine-d5) data, see Table 2, Table 3, and Table 4.
ACHTUNGTRENNUNG(20R,24S)-3b,6a,16b,19,25-Pentahydroxy-ranunculan-
References
9(10)-ene (12): white needle powder (MeOH); mp 309–
3108C; [a]2D0: +34.2 (c 0.10, MeOH); IR (KBr): nmax =3478,
[1] B. M. Nestl, B. A. Nebel, B. Hauer, Curr. Opin. Chem.
Biol. 2011, 15, 187–193.
[2] X. J. He, X. L. Wang, B. Liu, L. N. Su, G. H. Wang,
G. X. Qu, Z. H. Yao, R. H. Liu, X. S. Yao, J. Mol.
Catal. B: Enzym. 2005, 35, 33–40.
[3] X. X. Liu, X. C. Ma, C. H. Huo, S. H. Yu, Q. Wang,
Chin. J. Chin. Mater. Med. 2010, 35, 872–875.
[4] F. Fathiazad, M. K. Khosropanah, A. Movafeghi, Nat.
Prod. Res. 2010, 24, 1069–1078.
[5] E. Polat, E. Bedir, A. Perrone, S. Piacente, O. Alankus-
Caliskan, Phytochemistry 2010, 71, 658–662.
[6] Y. Kong, M. M. Yan, W. Liu, C. Y. Chen, B. S. Zhao,
Y. G. Zu, Y. J. Fu, M. Luo, M. Wink, J. Sep. Sci. 2010,
33, 2278–2286.
3387 (nOꢀH), 2972, 2945, 2888 (nCꢀH), 1635 (nC C), 1453, 1427
=
ꢀ
ꢀ
(das C H), 1379 (ds C H of CH3), 1092, 1044, 1027, 1005
(nCꢀO) cmꢀ1
;
HR-ESI-MS: m/z=1013.73266, calcd. for
[2M+H]+: 1013.72875, corresponding to C30H50O6;
1H NMR (400 MHz, pyridine-d5) and 13C NMR (100 MHz,
pyridine-d5) data, see Table 2, Table 3, and Table 4.
ACHTUNGTRENNUNG(20R,24S)-3b,6a,16b,25-Tetrahydroxy-19-butoxy-ranuncu-
lan-9(10)-ene (13): white powder (MeOH); mp 130–1318C;
[a]2D0: +34.7 (c 0.09, MeOH); IR (KBr): nmax =3451, 3421-
ꢀ
(nOꢀH), 2963, 2932, 2869 (nCꢀH), 1628 (nC C), 1454 (das C H),
=
1377 (ds C H of CH3), 1100, 1057 (nCꢀO) cmꢀ1; HR-ESI-MS:
ꢀ
m/z=1125.85414, calcd. for [2M+H]+: 1125.85395, corre-
1
sponding to C34H58O6; H NMR (400 MHz, pyridine-d5) and
13C NMR (100 MHz, pyridine-d5) data, see Table 2, Table 3,
[7] W. J. Zhang, P. Hufnagl, B. R. Binder, J. Wojta,
Thromb. Haemostasis 2003, 90, 904–914.
[8] Y. Y. Zhang, H. Y. Zhu, C. G. Huang, X. L. Cui, Y. J.
Gao, Y. Huang, W. F. Gong, Y. Zhao, S. S. Guo, J. Car-
diovasc. Pharmacol. 2006, 47, 190–195.
and Table 4.
ACHTUNGTRENNUNG(20R,24S)-3b,6a,16b,25-Tetrahydroxy-19-isopentenyloxy-
ranunculan-9(10)-ene (14): white powder (MeOH); mp 124–
1258C; [a]2D0: +9.6 (c 0.13, MeOH); IR (KBr): nmax =3420
ꢀ
(nOꢀH), 2969, 2934, 2871 (nCꢀH), 1630 (nC C), 1451 (das C H),
=
1379 (ds C H of CH3), 1062 (nCꢀO) cmꢀ1; HR-ESI-MS:
[9] X. Li, D. L. He, L. L. Zhang, X. F. Cheng, B. W. Sheng,
ꢀ
m/z=575.43122, calcd. for [M+H]+: 575.43061, correspond-
ing to C35H58O6; 1H NMR (400 MHz, pyridine-d5) and
13C NMR (100 MHz, pyridine-d5) data, see Table 2, Table 3,
and Table 4.
Y. Luo, Urol. Res. 2006, 34, 277–282.
[10] S. Y. Gui, W. Wei, H. Wang, L. Wu, W. Y. Sun, W. B.
Chen, C. Y. Wu, J. Ethnopharmacol. 2006, 103, 154–
159.
[11] H. F. Valenzuela, T. Fuller, J. Edwards, D. Finger, B.
Molgora, J. Immunol. 2009, 182, 9030.
ACHTUNGTRENNUNG(20R,24S)-3b,6a,16b,25-Tetrahydroxy-19-acetoxy-ranuncu-
lan-9(10)-ene (15): white powder (MeOH); mp 211–2128C;
˙
[a]2D0: +1.1 (c 0.37, MeOH); IR (KBr): nmax =3407 (nOꢀH),
[12] E. Yesilada, E. Bedir, I. Calis, Y. Takaishi, Y. Ohmoto,
J. Ethnopharmacol. 2005, 96, 71–77.
[13] S. R. Fauce, B. D. Jamieson, A. C. Chin, R. T. Mitsuya-
su, S. T. Parish, H. L. Ng, C. M. R. Kitchen, O. O. Yang,
C. B. Harley, R. B. Effros, J. Immunol. 2008, 181, 7400–
7406.
[14] M. Kuban, G. ꢃngen, E. Bedir, Org. Lett. 2010, 12,
4252–4255.
[15] I. Kitagawa, H. K. Wang, A. Takagi, M. Fuchida, I.
Miura, M. Yoshikawa, Chem. Pharm. Bull. 1983, 31,
689–697.
ꢀ
2966, 2933, 2873 (nCꢀH), 1739 (nC O), 1453 (das C H), 1382
=
ꢀ
(ds C H of CH3), 1234 (nCꢀ of the ester), 1088, 1034 (nCꢀ
O
O) cmꢀ1; HR-ESI-MS: m/z=549.37849, calcd. for [M+H]+:
1
549.37858, corresponding to C32H52O7; H NMR (400 MHz,
pyridine-d5) and 13C NMR (100 MHz, pyridine-d5) data, see
Table 2, Table 3, and Table 4.
Neoastragenol
[(20R,24S)-3b,6a,16b,25-tetrahydroxy-
20,24-epoxy-9(10)a-homo-19-nor-cycloartane] (16): white
powder (MeOH); mp 241–2428C; [a]2D0: +60.0 (c 0.11,
MeOH); IR (KBr): nmax =3422 (nOꢀH), 2968, 2933, 2902 (nCꢀ
[16] I. M. Isaev, D. A. Iskenderov, M. I. Isaev, Chem. Nat.
ꢀ
ꢀ
H), 1720 (nC O), 1626 (nC C), 1447 (das C H), 1375 (ds C H
=
=
of CH3), 1179, 1034 (nCꢀO) cmꢀ1
; HR-ESI-MS: m/z=
Compd. 2010, 46, 36–38.
[17] M. A. Agzamova, M. I. Isaev, Chem. Nat. Compd.
1998, 34, 474–476.
977.70692, calcd. for [2M+H]+: 977.70782, corresponding to
C30H48O5; 1H NMR (400 MHz, pyridine-d5) and 13C NMR
(100 MHz, pyridine-d5) data, see Table 1.
[18] M. A. Agzamova, M. I. Isaev, Chem. Nat. Compd.
1995, 31, 589–595.
[19] Z. Ali, S. I. Khan, D. Ferreira, I. A. Khan, Org. Lett.
2006, 8, 5529–5532.
[20] M. Ye, G. Q. Qu, H. Z. Guo, D. A. Guo, Appl. Environ.
Microbiol. 2004, 70, 3521–3527.
[21] Z. Z. Cao, J. H. Yu, Acta Chim. Sin. 1983, 41, 1137–
1145.
Supporting Information
The results of the preliminary screening test of 13 fungal
strains, and the HR-ESI-mass, 1H NMR, 13C NMR, 1H-1H
COSY, HSQC, HMBC, NOESY spectra of 15 transformed
products together with cycloastragenol are available as Sup-
porting Information.
[22] J. N. Fang, Acta Chim. Sin. 1988, 46, 1101–1104.
Adv. Synth. Catal. 2012, 354, 527 – 539
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
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