Novel biotransformation of pentacyclic triterpenoid acids by Nocardia sp. NRRL 5646

Article abstract of DOI:10.1016/j.tetlet.2005.01.155

Six pentacyclic triterpene acids, ursolic acid, oleanolic acid, betulinic acid, 23-hydroxybetulinic acid, glycyrrhetinic acid, and senegenin, were metabolized by the microbe Nocardia sp. NRRL 5646 to selectively furnish their corresponding 28-methyl esters. Notably, ursolic acid (1) was converted to oleanolic acid methyl ester (4) via two intermediates, oleanolic acid (2), and ursolic acid methyl ester (3), which are formed by participation of 'retro-biosynthetic' methyl migration from C-19 to C-20. Senegenin (11) was selectively converted to a nortriterpene methyl ester, senegenic acid methyl ester (12), via an unprecedented C-C bond cleavage. The stereochemical assignments of compounds 11 and 12 were made unambiguously for the first time using 2D NMR spectroscopy.

Full text of DOI:10.1016/j.tetlet.2005.01.155

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 2337–2340
Novel biotransformation of pentacyclic triterpenoid acids by
Nocardia sp. NRRL 5646
Jian Zhang,a Zhi-Hong Cheng,a Bo-Yang Yu,a,* Geoffrey A. Cordellb and Samuel X. Qiuc,*
aDepartment of Complex Prescription of TCM, China Pharmaceutical University, Nanjing 210038, PeopleÕs Republic of China
bDepartment of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at
Chicago, 833 South Wood Street, Chicago, IL 60612, USA
cDepartment of Chemistry, PO Box 1134, Washington University, One Brookings Drive, St. Louis, MO 63130, USA
Received 15 December 2004; revised 26 January 2005; accepted 27 January 2005
Abstract—Six pentacyclic triterpene acids, ursolic acid, oleanolic acid, betulinic acid, 23-hydroxybetulinic acid, glycyrrhetinic acid,
and senegenin, were metabolized by the microbe Nocardia sp. NRRL 5646 to selectively furnish their corresponding 28-methyl
esters. Notably, ursolic acid (1) was converted to oleanolic acid methyl ester (4) via two intermediates, oleanolic acid (2), and ursolic
acid methyl ester (3), which are formed by participation of Ôretro-biosyntheticÕ methyl migration from C-19 to C-20. Senegenin (11)
was selectively converted to a nortriterpene methyl ester, senegenic acid methyl ester (12), via an unprecedented C–C bond cleavage.
The stereochemical assignments of compounds 11 and 12 were made unambiguously for the first time using 2D NMRspectroscopy.
Ó 2005 Elsevier Ltd. All rights reserved.
Natural products (NPs) are an incredibly diverse group
of small (usually molecular weight less than 1500 Da)
organic compounds isolated from a variety of natural
sources, principally plants. The reason that NPs capture
the imagination of organic chemists and pharmaceutical
scientists is because of their well documented and wide
ranging biological activities and their skeletal diversity
and intriguing functional group characteristics, which
render them as indispensable leads for probing biologi-
cal system status and for drug discovery with new bioas-
say systems. Recently, we demonstrated that microbial
transformation could be a fruitful tool to enhance the
structural diversity of natural triterpene glycosides.1
As a continuation of these efforts to expand the struc-
tural diversity of natural products using microbial trans-
formation, a group of pentacyclic triterpene acids (TAs)
were chosen for biotransformation study. TAs are very
widely distributed in the plant kingdom and have been
shown to possess a wide range of biological activities.
For example, ursolic acid (1) and betulinic acid (5) pos-
sess anti-cancer and anti-HIV activities.2,3 Betulinic acid
is in preclinical development. It is envisioned that bio-
transformation of these TAs may provide analogs,
which could be utilized for screening for new activities
or for studying SAR. A panel of microbes was utilized
to screen for their ability to transform the TAs. Nocar-
dia sp. NRRL 5646 was found to convert these sub-
strates efficiently to less polar metabolites. This
organism is widely used for transformation because of
its broad spectrum of enzymatic capability, including
carboxylic acid and aldehyde reduction, phenol methyl-
ation, and the noteworthy skeleton rearrangement of
quinovic acid glycosides via methyl migration.1 In this
report, the biotransformation results of six triterpenoid
acids, ursolic acid (1), oleanolic acid (2), betulinic acid
(5), 23-hydroxybetulinic acid (7), glycyrrhetic acid (9),
and senegenin (11), by Nocardia sp. are described. Each
of the six triterpene acids was incubated on a prepara-
tive scale with the Nocardia sp. according to the stan-
dard, two-stage fermentation protocol.1,4 The work-up
and isolation of the metabolites were carried out as de-
scribed previously.1
Keywords: Biotransformation; Nocardia sp. NRRL 5646; Pentacyclic
triterpenoid acids; ÔRetro-synthetic methyl migration; Senegenin; C–C
bond cleavage; 2D NMRspectroscopy; Natural products.
A 200 mg sample of 1 (C30H48O3, Mr = 456) was used
for the scale-up incubation and three metabolites, coded
MT-1 (2), MT-2 (3), and MT-3 (4), were formed from
the biotransformation, as evident from the HPTLC
analysis. Silica gel chromatographic separation of the
*
Corresponding authors. Tel.: +1 636 346 4322; fax: +1 636 536 4294
(S.X.Q.); tel.: +86 25 8530 2355; fax: +86 25 8331 3080 (B.-Y.Y.);
e-mail: sqiu@wustl.edu
0040-4039/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.01.155

2338
J. Zhang et al. / Tetrahedron Letters 46 (2005) 2337–2340
transformation residue resulted in the isolation of com-
pounds 2 (21%), 3 (9%), and 4 (13% yield).
wise to the final product oleanolic acid methyl ester (4)
via two intermediates, namely, oleanolic acid (2), and
ursolic acid methyl ester (3), which are further converted
to oleanolic acid methyl ester (4) through extended
incubation.
MT-1 (2) was obtained as a white powder. The positive
ion ESI-MS showed a quasimolecular ion at m/z
[M+H]+ 457.2, and a molecular formula C30H48O3was
deduced from the HR-ESI-MS, indicating that 2 has
the same molecular formula as that of the parent com-
pound 1. The structure of 2 was assigned as oleanolic
acid by comparison of its 1H and 13C NMRspectra with
literature data,5 as well as co-TLC comparison with an
authentic sample.
The source of the methyl group in the enzyme system of
Nocardia sp. (inter- or intramolecular) has yet been de-
fined. In subsequent experiments, simple alcohols such
as ethanol, glycerol, isopropanol, and butanol showed
no effect on the biotransformation, implying that meth-
ylation may not be catalyzed by the general lipases
or esterases, which might be inhibited by exogenous
alcohols. Thus, it would be suggested that methyl
ester formation could most likely result from an
S-adenosylmethionine-dependent methyltransferase, a
member of a large family of enzymes with broad biologi-
cal activity including transmethylation.11
MT-2 (3) and MT-3 (4) were isolated as colorless pow-
ders, and their structures were readily assigned to ursolic
acid methyl ester and oleanolic acid methyl ester, respec-
1
tively, by comparison of their H and 13C NMRdata
with the literature.6–9 Thus methylation at the C-28 car-
boxylic acid of ursolic acid (1) occurred through cataly-
sis by the enzyme system of Nocardia sp. to form the
corresponding ester 3. It is also notable (Scheme 1) that
two, skeleton-rearranged metabolites, oleanolic acid (2)
and oleanolic acid methyl ester (4), were also obtained
from this biotransformation involving a methyl migra-
tion. Biogenetically, the ursane and oleanane skeletons
share a common intermediate, and the ursane nucleus
is biosynthesized from an oleanane precursor via a
methyl migration from C-20 to C-19.10 The current
observation confirms the unique ability of this Nocardia
sp. to catalyze a Ôretro-biosyntheticÕ conversion from the
ursane to the oleanane skeleton. This is the second
example of a Ôretro-biosyntheticÕ transformation with
this organism. A similar conversion was observed in
the biotransformation utilizing quinovic acid glycosides
as substrates.1
Betulinic acid (5) and 23-hydroxybetulinic acid (7) (Fig.
1) are members of the so-called Ôlupane-typeÕ pentacyclic
triterpenes, which were shown to exhibit potent apopto-
tic and anti-HIV activities.12,13 Incubation of 5 and 7
with Nocardia sp. afforded their corresponding methyl
esters, betulinic acid methyl ester (6) and 23-hydroxybet-
ulinic acid methyl ester (anemosapogenin methyl ester,
8), respectively. The structures were identified on the ba-
sis of their almost super-imposable NMRdata ( Table 1)
with literature values for betulinic acid methyl ester,9,14
and 23-hydroxybetulinic acid.15
Glycyrrhetinic acid (9), a potent and selective inhibitor
of 11b-hydroxysteroid dehydrogenases,16 is the aglycone
of the saponins from the well-known Traditional Chi-
nese Medicine licorice (Glycyrrhiza glabra). Similarly, 9
was converted to its methyl ester, glycyrrhetinic acid
methyl ester (methyl glycyrrhetinate, 10) by incubation
To investigate the mechanism of this biotransformation,
several experiments were performed. Both oleanolic acid
(2) and ursolic acid methyl ester (3) were found to be
converted to oleanolic acid methyl ester (4) when incu-
bated with Nocardia sp. under the same conditions.
On the other hand, no appreciable reaction could be de-
tected for substrate 4 after extended incubation for four
days. This implies that ursolic acid (1) is converted step-
29
O
30
29
RO
20
21
22
30
12
19
17
21
19
18
20
18
13
14
12
26
11
10
11
O
25
26
22
28
COOR
1
9
2
25
13
14
17
28
1
4
16
2
9
8
15
16
2
27
8
4
10
15
7
5
3
HO
27
6
7
3
5
HO
6
R
24
1
23
23
24
5
R1 = CH3,
R1 = CH3,
R1 = CH2OH, R2 = H
R2 = H
R2 = CH3
9
R = H
6
7
8
10 R = CH3
COOH
COOCH
3
R1 = CH2OH, R2 = CH3
HO
HO
30
29
20
3
1
20
27
CH Cl
21
22
19
21
2
18
19
17
12
18
x
12
22
11
25
26
11
13
14
17
x
25
26
COOR
13
14
1
9
COOH
1
4
HO
HO
9
28
HO
HO
16
28
16
8
2
10
15
8
2
10
15
4
7
3
5
7
COOH
3
COOCH
5
6
3
6
24
COOH
24
COOH
23
23
HO
HO
Senegenic acid 28-methyl ester (12), R = CH3
Senegenic acid (13), R = H
x
Senegenin (11)
4
2
Scheme 1. Biotransformation of ursolic acid (1), oleanolic acid (2) and
their corresponding methyl esters (3 and 4) by Nocardia sp.
Figure 1. Structures of pentacyclic triterpene acids biotransformed by
Nocardia sp.

J. Zhang et al. / Tetrahedron Letters 46 (2005) 2337–2340
Table 1. NMRspectral data of compounds 5–12 and senegenic acida
2339
C
5
6
7
8
9
10
11
12
44.4
Senegenic acid (15)
1
39.3
28.3
78.2
38.5
55.9
18.8
34.9
41.2
50.2
37.4
21.3
26.2
38.7
42.9
30.3
32.9
56.7
47.8
49.7
39.3
28.3
78.1
39.5
55.9
18.8
34.8
41.1
50.9
37.1
21.1
26.0
38.6
42.7
30.1
32.4
56.8
47.6
49.8
39.2
27.9
73.6
42.9
48.9
18.6
34.6
41.2
50.5
37.6
21.3
26.2
38.7
42.9
30.3
32.9
56.7
47.8
49.7
39.1
27.9
73.4
42.7
48.8
18.6
34.5
41.0
50.9
37.4
21.2
26.0
38.6
43.0
30.1
32.3
56.8
47.6
49.8
39.5
26.7
77.8
37.6
55.6
17.0
32.1
44.1
61.0
36.1
39.8
43.7
44.9
71.8
76.0
54.0
52.0
18.5
39.5
38.4
57.3
37.2
21.1
24.0
130.7
137.0
21.9
31.9
45.3
39.9
41.9
30.9
34.6
32.2
180.8
13.4
18.1
20.8
2
27.0
78.0
37.8
55.5
17.0
32.7
44.1
61.1
37.5
199.2
124.5
165.7
45.2
28.2
28.8
35.8
40.7
36.5
42.8
34.0
39.8
29.4
21.2
16.7
18.1
18.8
16.1
20.7
178.6
51.8
70.5
74.8
52.5
51.7
19.4
37.8
38.6
56.7
35.8
20.4
44.8
129.7
143.3
20.5
20.7
46.0
38.8
41.3
30.0
32.9
30.4
180.0
11.0
16.5
17.7
46.8
179.8
31.7
23.4
71.2
75.5
53.4
51.5
18.0
38.8
37.8
56.8
36.6
20.7
23.8
130.0
136.5
21.4
31.0
44.9
39.2
41.0
30.3
33.7
31.7
180.3
13.2
17.6
20.3
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
OCH3
199.1
124.4
165.4
45.1
28.0
28.0
35.6
40.4
36.0
42.9
34.1
39.9
29.0
20.8
16.5
17.8
18.7
16.0
20.7
180.9
151.4
31.3
37.6
28.7
16.4
16.4
19.2
15.2
178.9
109.9
19.8
150.9
31.0
37.5
28.7
16.4
16.3
18.8
14.9
176.5
110.1
19.4
51.3
151.4
31.8
37.6
68.2
12.9
16.5
19.5
14.9
178.9
109.9
19.5
150.9
31.0
37.4
67.9
12.9
16.2
18.6
14.9
176.5
110.1
19.4
51.3
177.6
32.6
24.3
51.3
180.2
32.7
25.1
a Recorded in pyridine-d5 at 500 MHz (1H) and 125 MHz (13C).
with Nocardia sp. The structure of 10 was confirmed by
comparison of its NMRdata with those reported in the
literature.17,18
the C-23 position (Scheme 2). In addition, the small cou-
pling constant (e/e coupling) of the H-3 doublet indi-
cated that the vicinal hydroxyl groups at C-2 and C-3
should be b-oriented. Therefore, the structure of 12
was elucidated as senegenic acid 28-methyl ester, sug-
gesting a highly selective methyl esterfication between
the carboxylic acid functionalities at C-23 and C-28.
Since there are no NMRdata reported for 12, a com-
plete, unambiguous assignment was done through 2D
NMRtechniques ( Table 1).
Senegenin (11) is an artificial aglycone from the vigorous
ethanolic hydrochloride hydrolysis of the saponins from
Polygala senega.19,20 When 11 was incubated with
Nocardia sp., a less polar metabolite was detected by
HPTLC from the incubation mixture, which was sub-
jected to silica gel chromatography to afford 12 as a col-
orless powder. The molecular formula of 12 was
established as C30H46O6 by HR-ESI-FTMS in which a
pseudo-molecular ion was detected at m/z: 503.3223
[M+H]+ {calcd for C30H47O6: 503.3372}, revealing that
chloride has been eliminated during the biotransforma-
tion. The 13C NMRspectra of 4 (Table 1) revealed its
similarity with senegenic acid (13) based on the almost
identical NMRdata with the literature values for 13,21
except that one additional oxygenated methyl signal
was observed at d 51.3 and the 13C signal of the
C-28 carboxylic acid of 12 was shifted 2.6 ppm upfield
compared with that of 11.21 This implied that 12 was a
methyl ester of senegenic acid (13).19 There are two pos-
sibilities for the methyl ester location due to the presence
of two carboxylic acid residues. Conclusive evidence
from the HMBC spectrum allows assignment of the
methyl ester to the C-28 carboxylic acid, as the 1H signal
at d 4.61 (d, J = 3.0 Hz), assignable to H-3a, was shown
to be long range correlated to the 13C signal at d 180.3.
This signal was then assigned to the carbonyl group at
In conclusion, this is the first report that Nocardia sp.
NRRL 5646 can catalyze specific methyl esterification
at the C-28 position of pentacyclic triterpene acids. In
addition, the ursane skeleton was isomerized to the ole-
anane skeleton, consistent with the previous transforma-
tion of quinovic acid glycosides, which also possess an
aglycone with the ursane skeleton.1 This suggests the
presence of a unique enzyme system in Nocardia sp.
28
COOCH
3
HO
3
HO H
23
COOH
Scheme 2. Key H–C long-range correlations of 12 from HMBC.

2340
J. Zhang et al. / Tetrahedron Letters 46 (2005) 2337–2340
2. Kashiwada, Y.; Wang, H. K.; Nagao, T.; Yeh, S.; Lee, K.
H. J. Nat. Prod. 1998, 61, 1090–1095.
3. Pisha, E.; Lee, C. H.; Lee, I.-S.; Chagwedera, T. E.;
Farnsworth, N. R.; Cordell, G. A.; Beecher, C. W. W.;
Fong, H. H. S.; Kinghorn, A. D.; Pezzuto, J. M. Nat.
Med. 1995, 1, 1046–1051.
4. Betts, R. E.; Walters, D. E.; Rosazza, J. P. N. J. Med.
Chem. 1974, 17, 599–602.
5. Pancharoen, O.; Tuntiwachwuttikul, P.; Taylor, W. C.;
Picker, K. Phytochemsitry 1994, 35, 987–992.
6. Lontsi, D.; Sondengam, B. L.; Bodo, B.; Martin, M. T.
Planta Med. 1998, 64, 189–191.
7. Tkachev, A. V.; Denisov, A. Y.; Gatilov, Y. V.; Bagr-
yanskaya, I. Y.; Shoetsov, S. A.; Rybalova, T. V.
Tetrahedron 1994, 50, 11459–11488.
8. Kajima, H.; Ogura, H. Phytochemistry 1986, 25, 729–
733.
9. Kajima, H.; Tominga, H.; Sato, S. Phytochemistry 1987,
26, 1107–1111.
10. Wendt, K. U.; Schulz, G. E.; Corey, E. J.; Liu, D. R.
Angew. Chem., Int. Ed. 2000, 39, 2812–2833.
11. Herdricks, C. L. Anal. Biochem. 2004, 326, 100–
105.
catalyzing this unusual Ôretro-biosyntheticÕ conversion.
Strikingly, the 12-chloromethylene group of senegenin
(11) was eliminated during the biotransformation with
Nocardia sp. Although there are many reports on en-
zyme-catalyzed C–C bond cleavage reaction by lyases
from a variety of substrates such as aromatic alde-
hydes,22 indoles,23 and lignins.24 To our knowledge, this
is the first report that a C–C bond cleavage was cata-
lyzed by microbe Nocardia sp. In addition, the results re-
ported here further exemplify the biocatalytic efficacy of
Nocardia sp. toward complex natural products, allowing
one-step transformations that are presently very diffi-
cult, if not impossible, to be performed using chemical
methods, such as the conversion of 1 to 2, and of 11
to 12, in which skeleton rearrangements occur by methyl
migration and the elimination of a chloromethylene
group, respectively. Finally, the versatile catalytic capa-
bilities of Nocardia sp. to generate structural diversity in
already complex natural products are of pharmaceutical
interest and await further exploration.
12. Hata, K.; Hori, K.; Takahashi, S. J. Nat. Prod. 2002, 65,
645–648.
Acknowledgements
13. Ji, Z. N.; Ye, W. C.; Liu, G. G.; Hsiao, W. L. Life Sci.
2002, 72, 1–9.
14. Wenkert, E.; Baddeley, G. V.; Burfitt, I. R.; Moreno, L.
N. Org. Magn. Reson. 1978, 11, 337–343.
15. Chen, W. K.; Wang, B. Y.; Lu, D. Y.; Lin, Q.; Lin, L. Y.
Huaxue Xuebao 1983, 41, 739–745.
16. Su, X. D.; Lawrence, H.; Ganeshapillai, D.; Cruttenden,
A.; Purohit, A.; Reed, M. J.; Vicker, N.; Potter, B. V. L.
Bioorg. Med. Chem. 2004, 12, 4439–4457.
17. Hiroka, M.; Morishima, N.; Kaji, E.; Mori, Y.; Zen, S.
Yakugaku Zasshi 1989, 109, 544–559.
This work was supported by a grant from the State Min-
istry of Education (No 02118), and a grant from Jiangsu
Province Government, China, awarded to Bo-yang Yu.
The samples of ursolic acid, betulinic acid, 23-hydroxy-
betulinic acid, glycyrrhetinic acid, and senegenin were
courtesy of Professor Fu, G. M., Wang, J. R., Ye, W. C.
and Zhu, D. N. of China Pharmaceutical University.
Supplementary data
18. Shu, Y. H.; Zhao, Y. Y.; Zhang, R. Y. Yaoxue Xuebao
1985, 20, 193–197.
Experimental details for the incubation and separation
of the metabolites from incubation mixture, and detailed
1H and 13C NMRassignments of compounds 1–13. The
supplementary data is available online with the paper in
ScienceDirect. Supplementary data associated with this
article can be found, in the online version, at
doi:10.1016/j.tetlet.2005.01.155.
19. Pelletier, S. W.; Adityachaudhury, N.; Tomaz, M.; Rey-
nolds, J. J.; Mechoulam, R. J. Org. Chem. 1965, 30, 4234–
4247.
20. Pelletier, S. W.; Nakamura, S.; Shimizu, Y. Chem.
Commun. 1966, 20, 727–728.
21. Miyase, T.; Saitoh, H.; Shiokawa, K.-I. Chem. Pharm.
Bull. 1995, 43, 466–472.
22. Demir, A. S.; Pohl, M.; Janzen, E.; Muller, M. J. Chem.
Soc., Perkin Trans. 1 2001(7), 633–635.
23. Phillips, R. S.; Sundaraju, B.; Faleev, N. G. JACS 2000,
122, 1008–1014.
References and notes
24. Shimada, M.; Habe, T.; Umezawa, T.; Higuchi, T.;
Okamoto, T. Biochem. Biophy. Res. Commun. 1984, 122,
1247–1252.
1. Chen, Z.-H.; Yu, B.-Y.; Cordell, G. A.; Qiu, S. X. Org.
Lett. 2004, 6, 3163–3165.