Glucosidation of betulinic acid by Cunninghamella species

Article abstract of DOI:10.1021/np980432b

Microbial transformation of the antimelanoma agent betulinic acid (1) was studied. Preparative scale biotransformation with resting-cell suspensions of Cunninghamella species NRRL 5695 resulted in the production of a fungal metabolite of 1, which has been characterized as 28-O-β-D- glucopyranosyl 3β-hydroxy-lup-20(29)-en-28-oate (2) based on spectral and enzymic data. The in vitro cytotoxicity assay of metabolite 2 revealed no activity against several human melanoma cell lines.

Full text of DOI:10.1021/np980432b

J . Nat. Prod. 1999, 62, 761-763
761
Glu cosid a tion of Betu lin ic Acid by Cu n n in gh a m ella Sp ecies
†
‡
,†
Parnali Chatterjee, J ohn M. Pezzuto, and Samir A. Kouzi*
Division of Basic Pharmaceutical Sciences, School of Pharmacy, Northeast Louisiana University, Monroe, Louisiana 71209,
and Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612
Received October 2, 1998
Microbial transformation of the antimelanoma agent betulinic acid (1) was studied. Preparative scale
biotransformation with resting-cell suspensions of Cunninghamella species NRRL 5695 resulted in the
production of a fungal metabolite of 1, which has been characterized as 28-O-â-D-glucopyranosyl 3â-
hydroxy-lup-20(29)-en-28-oate (2) based on spectral and enzymic data. The in vitro cytotoxicity assay of
metabolite 2 revealed no activity against several human melanoma cell lines.
The lupane-type pentacyclic triterpene betulinic acid (1),
3
â-hydroxy-lup-20(29)-en-28-oic acid, is widely distributed
in nature. Considerable amounts of betulinic acid (up to
.5%) are available in the outer bark of a variety of tree
2
1
species that are valuable for timber purposes. A closely
related compound, betulin (3), is a major constituent of
white-barked birch trees (Betula species) with yields up to
2
,3
2
2% (dry wt). Compound 3 can easily be converted to 1
4
in high yield. The triterpene 1 has been shown to exhibit
a variety of biological activites,5
-11
including inhibition of
human immunodeficiency virus (HIV) replication in H9
9
10
lymphocyte cells, blockage of HIV-1 entry into cells, and
cytotoxicity against a variety of cultured human tumor
cells.11 Synthetic derivatives of 1 have also been investi-
gated as specific inhibitors of HIV-1.1
2,13
Recently, betulinic
acid (1) was identified as a melanoma-specific cytotoxic
agent in both in vitro cell culture and in vivo studies.14 The
antitumor activity of 1 was mediated by the induction of
apoptosis, as demonstrated by a variety of cellular re-
sponses.14 Due to its favorable therapeutic index in these
studies, betulinic acid (1) is currently undergoing preclini-
cal development for treatment or prevention of malignant
melanoma.
resting-cell suspensions of Cunninghamella species af-
forded metabolite 2 in 0.77% yield. The parent compound
(1) was recovered unchanged from the incubations in 92%
yield. Preliminary screening studies revealed that 1 is
somehow being trapped inside the cells or at the cell wall,
which may explain the low yield of its bioconversion to 2.
In addition, our studies showed that metabolite 2 is not
excreted extracellularly by Cunninghamella species. These
observations were based on the fact that we were not able
to recover any detectable quantities of 1 and 2 after
extraction of the aqueous buffer. As a result, recovery of 1
and 2 was accomplished by homogenizing the cells with
an ultrasonic cell disruptor at 20 kHz, followed by extrac-
tion of the cell homogenate with ethyl acetate.
An important factor in the evaluation of the efficacy and
safety of a drug is the study of its mammalian metabolism.
Because there have been no reports on the mammalian
metabolism of betulinic acid (1), a prospective approach was
undertaken to study the metabolism of 1, utilizing micro-
organisms as in vitro model systems. Selected microorgan-
isms, particularly fungi, have been utilized successfully as
in vitro models to mimic and predict the metabolic fate of
drugs and other xenobiotics in mammalian systems.1
In this study we report the isolation and structure elucida-
tion of 28-O-â-D-glucopyranosyl 3â-hydroxy-lup-20(29)-en-
5-17
Metabolite 2 was isolated and purified by repeated
column chromatography. The HRFABMS of metabolite 2
2
8-oate (2), a conjugated fungal metabolite of betulinic acid
(1) from resting-cell suspensions of Cunninghamella species
showed a molecular ion at m/z 641.3992 (calcd for C36
58 8
H O
NRRL 5695. This is the first report describing the biotrans-
formation of the triterpene 1 and the isolation of the new
glucopyranosyl ester 2.
A total of 13 fungal cultures was screened for the ability
to catalyze the bioconversion of betulinic acid (1). Cun-
ninghamella species NRRL 5695 was the only culture
capable of reproducibly bioconverting 1 to a more polar
metabolite (2). Preparative biotransformation of 1 using
Na, 641.4029). The IR spectrum of 2 showed absorptions
-1
13
1
at 3400 (OH) and 1734 cm (ester). The C and H NMR
spectra of 2 showed new proton and carbon signals with
chemical shifts that are characteristic of â-D-glucose. The
anomeric proton H-1′ of the glucose moiety in 2 resonated
as a doublet at 6.45 ppm. A coupling constant of 8.42 Hz
for H-1′ indicated that the stereochemistry of the glucosidic
linkage at C-1′ of D-glucose is â. The â stereochemistry at
C-1′ was further confirmed by enzymic hydrolysis of the
glucosidic linkage in 2 using â-glucosidase enzyme. Com-
parisons of the 13C NMR spectra of 1 and 2 indicated that
2 is a glucopyranosyl ester of 1 at C-28, based on the upfield
*
To whom correspondence should be addressed. Tel.: (318) 342-1693.
Fax: (318) 342-3286. E-mail: pykouzi@alpha.nlu.edu.
†
School of Pharmacy, Northeast Louisiana University.
College of Pharmacy, University of Illinois at Chicago.
‡
1
0.1021/np980432b CCC: $18.00
© 1999 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/03/1999

7
62 J ournal of Natural Products, 1999, Vol. 62, No. 5
Notes
Ta ble 1. 13C NMR Data for Compounds 1 and 2 in C5D5Na
has a primary alcohol instead of the carboxylic acid at C-28,
showed a remarkably reduced antimelanoma activity com-
pared to 1. Further microbial transformation studies of 1
are in progress.
carbon
1
2
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
39.1 t
28.1 t
78.0 d
39.4 s
55.7 d
18.6 t
34.7 t
40.9 s
50.8 d
37.3 s
21.0 t
25.9 t
38.4 d
42.4 s
31.0 t
32.7 t
56.3 s
47.6 d
49.5 d
150.7 s
30.1 t
37.5 t
28.5 q
16.3 q
16.3 q
16.2 q
14.8 q
178.7 s
110.3 t
19.4 q
39.1 t
28.1 t
78.0 d
39.4 s
55.7 d
18.6 t
34.5 t
41.0 s
50.8 d
36.8 s
20.9 t
25.8 t
38.2 d
42.6 s
30.7 t
32.1 t
56.8 s
47.3 d
49.6 d
150.7 s
30.0 t
37.3 t
28.5 q
16.3 q
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
determined in open capillary tubes with a Thomas-Hoover
capillary melting point apparatus and are uncorrected. Optical
rotations were measured with a Perkin-Elmer 241 digital
polarimeter. IR spectra were recorded in KBr using a Nicolet
Impact 400D IR spectrophotometer. Microbial cells were
homogenized using a Tekmar Sonic Disruptor TMX 600 at 20
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
C-28
C-29
C-30
C-1′ (D-glucose)
C-2′
1
13
5 5
kHz. H and C NMR spectra were obtained in C D N on a
J EOL-Eclipse 400 FT-NMR spectrometer operating at 400
and 100 MHz, respectively. The chemical shifts are reported
in parts per million, and the coupling constants (J values) are
in Hertz. Standard pulse sequences were utilized for COSY,
HETCOR, DEPT, and APT experiments. HRMS and LRMS
were obtained using a VG 70-SEQ mass spectrometer (VG
Analytical, Manchester, England) at the Mass Spectrometry
Facility, Department of Medicinal Chemistry, University of
Washington, Seattle, WA 98195.
b
b
b
b
Ch r om a togr a p h ic Con d ition s. TLC analyses were car-
ried out on precoated Silica G-25 UV254 plates (E. Merck,
Darmstadt). The adsorbent used for column chromatography
was Si gel 70-230 mesh 60 Å (Aldrich Chemical Co., Milwau-
16.2 qb
16.2 qb
14.8 q
175.2 s
110.0 t
19.2 q
95.3 d
74.2 d
79.4 d
70.8 d
78.7 d
62.0 t
kee, WI). The solvent system used for TLC was CHCl
9:1v/v) solution, and the visualization of TLC plates was
SO spray reagent. The
3
-MeOH
(
performed using anisaldehyde-H
2
4
spots were visualized by spraying the plate and then heating
it at 110 °C for 3 min in an oven.
C-3′
C-4′
C-5′
C-6′
Micr oor ga n ism s a n d Med ia . The microbial cultures were
obtained from the culture collection of the School of Pharmacy,
Northeast Louisiana University, and were originally from the
American Type Culture Collection (ATCC), Manassas, VA, or
the USDA Northern Regional Research Laboratories (NRRL),
Peoria, IL. UI cultures were obtained from the Department of
Medicinal and Natural Products Chemistry, College of Phar-
macy, University of Iowa, Iowa City, IA. All preliminary
screening experiments were carried out in a medium consisting
of dextrose, 20 g; yeast extract, 5 g; peptone, 5 g; NaCl, 5 g;
a
δ in ppm; multiplicity determined by DEPT experiments;
1
1
1
13
assignments are based on H- H and H- C chemical shift
correlation experiments and comparisons to the assignments of
b
1
.
Interchangeable within column.
shift of C-28 from 178.7 to 175.2 ppm (Table 1). The DEPT
experiment of 2 showed the appearance of a new CH group
at 62.0 ppm and five new CH groups at 95.3, 79.4, 78.7,
4.2, and 70.8 ppm (Table 1). Based on all the evidence,
metabolite 2 was characterized as 28-O-â-D-glucopyranosyl
2
2 4 2
K HPO , 5 g; and distilled H O, 1000 mL. Stock cultures of
fungi and bacteria were stored on slants of Mycophil and
Eugon (Difco, Detroit, Michigan) agar, respectively, at 4 °C.
The 0.1 M phosphate buffer (pH 7.2) used for resting-cell
7
1
3
1
3
â-hydroxy-lup-20(29)-en-28-oate. C and H NMR chemi-
suspensions of Cunninghamella species consists of K
2 4
HPO ,
1
cal shift assignments of metabolite 2 were based on H-
1
0.6 g; KH PO , 4.08 g; and distilled H O, 1000 mL. The 0.07
2
4
2
1
1
13
H and H- C correlation experiments and comparisons
M phosphate buffer (pH 5) used for the enzymic hydrolysis of
to the assignments of 1. The proton1
8,19
and carbon
20
metabolite 2 consists of K HPO , 7.0 g; KH PO , 4.08 g; and
2
4
2
4
assignments of 1 have been reported in the literature. The
C NMR assignments of 1 are listed in Table 1 for
comparative purposes.
2
distilled H O, 1000 mL.
1
3
Rea gen ts. Dimethylformamide (DMF) and pyridine were
stored over 4 Å molecular sieves. â-Glucosidase (one enzyme
unit liberates 1.0 µmol of â-D-glucose from salicin per min at
pH 5.0 at 37° C) was purchased from Sigma Chemical Co., St.
Louis, Missouri.
F er m en ta tion P r oced u r es. Microbial metabolism studies
were carried out by incubating the cultures with shaking on
an Innova 5000 Gyrotory Tier Shaker (New Brunswick Scien-
tific Co., NJ ), operating at 150 rpm, at 25 °C. Preliminary
screening experiments were carried out in 125-mL stainless
steel-capped DeLong culture flasks containing 25 mL of
medium. The media were sterilized at 121 °C and 18 psi for
The in vitro cytotoxicity of betulinic acid (1) and its
metabolite 2 was evaluated against four human melanoma
cell lines: MEL-1 (lymph node), MEL-2 (pleural fluid),
MEL-3 (liver), and MEL-7 (metastatic melanoma from
-
1
growing area). The ED50 values of 2 (>20 µg mL against
all four cell lines) indicated that metabolite 2 is not active
against the tested melanoma cell lines when compared to
1
1
. The ED50 values of 1 against MEL-1, -2, -3, and -7 were
.4, 1.2, 1.8, and 4.4 µg mL , respectively. The results
-
1
1
5 min. Fermentations were carried out according to a
strongly suggest that the free carboxylic acid group in 1 is
essential for cytotoxic activity against melanoma. The
antimelanoma activity was lost when compound 1 was
converted to the glucopyranosyl ester 2. These findings are
standard two-stage protocol. In general, the substrate was
added to the incubation media 5 days after inoculation of stage
II cultures as a 5% solution in DMF at a concentration of 0.1
mg/mL of stage II medium. Cultures were sampled at 24-h
intervals by extracting 3 mL of the broth with 3 mL of EtOAc.
The extracts were concentrated and evaluated by TLC. Sub-
strate controls were prepared by adding the substrate to sterile
medium and then incubated without microorganisms. Culture
controls consisted of fermentation blanks in which the micro-
consistent with the reported in vitro antitumor activity of
derivatives of 1 at C-28.1
1,21
The in vitro antimelanoma
activity of 1 was considerably attenuated when the car-
boxylic acid at C-28 was converted to the methyl ester, the
aldehyde, or a methyl group. In addition, betulin (3), which

Notes
J ournal of Natural Products, 1999, Vol. 62, No. 5 763
organisms were grown under identical conditions without the
addition of the substrate. Substrate-autoclaved culture controls
consisted of microbial cultures that were grown to maturity
Winchester, VA) at 100 rpm and 37 °C for 3 days. Substrate
controls consisted of the phosphate buffer solution to which
metabolite 2 was added in DMF and incubated at 37 °C
without the enzyme. After incubation, the aqueous solution
was extracted with 3 × 5 mL of n-BuOH. The organic solvent
(5-7 days), autoclaved for 30 min, and then incubated after
the substrate was added. Culture controls of the resting-cell
incubations of Cunninghamella species were prepared by
suspending the mycelium in a sterile 0.1 M phosphate buffer
2 2 4
was washed with distilled H O, dried over anhydrous Na SO ,
and evaporated in vacuo. TLC analysis of the residue showed
a single spot identical to an authentic sample of betulinic acid
(1).
(pH 7.2) containing 2% dextrose, and then incubated without
the addition of the substrate.
P r ep a r a tive Biotr a n sfor m a tion of 1 to 2. Betulinic acid
1) was obtained from Aldrich Chemical Co. (Milwaukee, WI).
In Vitr o Cytotoxicity Assa y. Cytotoxicity of 1 and 2 was
determined against four cultured human melanoma cell
lines: MEL-1 (lymph node), MEL-2 (pleural fluid), MEL-3
(liver), and MEL-7 (metastatic melanoma from growing area)
as described previously.14
(
The physical and spectral data of 1 have been reported in the
1
8-20
literature.
Twenty-four 2-L flasks, each containing 400 mL
of Cunninghamella species NRRL 5695 stage II culture, were
incubated on the shaker for 5 days to get as much cell mass
as possible. The mycelium from each flask was filtered under
vacuum through a B u¨ chner funnel, washed several times with
Ack n ow led gm en t. This work is supported by a grant from
the National Cancer Institute (R15 CA74244). The purchase
of the 400 MHz NMR spectrometer was funded by the
Louisiana Education Quality Support Fund and Northeast
Louisiana University.
sterile distilled H
dextrose (2% w/v) in 200 mL of sterile 0.1 M phosphate buffer
pH 7.2). A total of 0.96 g of 1 was then dissolved in 0.96 mL
2
O, and suspended in a 1-L flask containing
(
of DMF and distributed equally among 24 flasks containing
resting-cell suspensions of Cunninghamella species. After 9
days of incubation on the shaker, the suspensions were filtered
Refer en ces a n d Notes
(
1) Maurya, S. K.; Devi, S.; Pandey, V. B. Fitoterapia 1989, 60, 468-
and the cells washed with distilled H
2
O. The cells were then
469.
frozen before homogenization using a Tekmar sonic disruptor
(2) O’Connell, M. M.; Bentley, M. D.; Campbell, C. S. Phytochemistry
1
988, 27, 2175-2176.
at 20 kHz. The cell homogenate was extracted with 3 × 2.0 L
(
(
3) Cole, B. J . W.; Bentley, M. D. Holzforschung 1991, 45, 265-268.
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2
of EtOAc. The organic layer was dried over anhydrous Na -
4
SO , filtered, and evaporated in vacuo to afford a yellowish
residue. The residue was chromatographed on a Si gel column
using EtOAc-hexane and EtOAc-MeOH as eluting systems,
and 10-mL fractions were collected. Repeated column chro-
matography with a gradient of EtOAc-hexane (20:80 to 100:
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167-1170.
(
6) Yasukawa, K.; Takido, M.; Matsumoto, T.; Takeuchi, M. Oncology
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991, 48, 72-76.
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0
), followed by EtOAc-MeOH (100:1 to 100:10) afforded
homogeneous fractions that showed a single spot on TLC (R
.32). The homogeneous fractions were combined and evapo-
rated in vacuo to give metabolite 2.
(
f
1
991, 1075, 206-212.
0
(
9) Fujioka, T.; Kashiwada, Y.; Kilkuskie, R. E.; Cosentino, L. M.; Ballas,
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1
994, 57, 243-247.
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(
10) Mayaux, J . F.; Bousseau, A.; Panwels, R.; De Clerq, E.; Pecq, J . B.
afforded white needles (0.01 g, 0.77% yield); mp 203-205 °C;
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2
5
[
1
R]
D
-6.0°(c 0.5 g/100 mL, C
5
H
5
N); IR (KBr) νmax 3483, 2961,
-
1 1
735 cm ; H NMR (C D N, 400 MHz) δ 6.45 (1H, d, J ) 8.42
5 5
(
12) Kashiwada, Y.; Hashimoto, F.; Cosentino, L. M.; Lee, K. H. J . Med.
Chem. 1996, 39, 1016-1017.
Hz, H-1′), 4.87 (1H, br s, H-29), 4.72 (1H, s, H-29), 4.45-4.36
(
3H, m, H-4′, H-6′), 4.30 (1H, t, J ) 8.42 Hz, H-5′), 4.19 (1H,
(13) Evers, M.; Poujade, C.; Solers, F. J . Med. Chem. 1996, 39, 1056-
t, J ) 8.79 Hz, H-2′), 4.06-4.04 (1H, m, H-3′), 3.45-3.41(2H,
m, H-3, H-18), 2.69-2.65 (3H, m, H-13, H-16), 2.20-2.15 (2H,
m, H-22), 2.09-2.06 (2H, m, H-15), 1.93-1.87 (4H, m, H-2,
H-12), 1.83-1.76 (1H, m, H-19), 1.72 (3H, s, Me-30), 1.65-
1068.
(14) Pisha, E.; Chai, H.; Lee, I.; Chagwedera, T. E.; Farnsworth, N. R.;
Cordell, G. A.; Beecher, C. W. W.; Fong, H. H. S.; Kinghorn, A. D.;
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K.; Pezzuto, J . M. Nature Med. 1995, 1, 1046-1051.
1
.59 (2H, m, H-1), 1.50-1.48 (2H, m, H-6), 1.35-1.32 (5H, m,
H-7, H-9, H-11), 1.19 (3H, s, Me-23), 1.12 (3H, s, Me-24, 25 or
6), 1.01 (3H, s, Me-27), 0.98 (3H, s, Me-24, 25 or 26), 0.78
(15) Smith, R. V.; Rosazza, J . P. J . Nat. Prod. 1983, 46, 79-91.
(
16) Davis, P. J . In Antibiotics and Microbial Transformations; Lamba,
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2
7
0.
1
3
(
(
3H, s, Me-24, 25 or 26), 0.82-0.76 (1H, m, H-5); C NMR
(
17) Clark, A. M.; Hufford, C. D. Med. Res. Rev. 1991, 11, 473-501.
+
C
5
D
5
N, 100 MHz), see Table 1; FABMS m/z 641 [M + Na ] ;
(18) Ahmad, V. U.; Rahman, A. U. Pentacyclic Triterpenoids; Elsevier:
New York, 1994; pp 1102-1104.
HRFABMS m/z 641. 3992 (calcd for C36
58 8
H O
Na, 641.4029).
(19) Otsuka, H.; Fujioka, S.; Komiya, T.; Goto, M.; Hiramatsu, Y.;
En zym ic Hyd r olysis of 2 by â-Glu cosid a se En zym e. A
total of 3 mg (0.0048 mmol) of metabolite 2 was dissolved in
Fujimura, H. Chem. Pharm. Bull. 1981, 29, 3099-3104.
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Bull. 1980, 28, 1006-1008.
3
0 µL of DMF and added to 5 mL of 0.07 M phosphate buffer
(
21) Kim, D. S. H. L.; Pezzuto, J . M.; Pisha, E. Bioorg. Med. Chem. Lett.
(pH 5). The enzyme â-glucosidase (3.4 mg, 30 units/mg) was
1
998, 8, 1707-1712.
then added to the solution, and the mixture was incubated in
a Dubnoff Metabolic Shaker Incubator (Precision Scientific,
NP980432B