Biotransformation of methyl cholate by Aspergillus niger

Article abstract of DOI:10.1016/j.steroids.2009.01.002

Biotransformation of methyl cholate using Aspergillus niger was investigated. This led to the isolation of two derivatives: methyl 3α,7α,12α,15β-tetrahydroxy-5β-cholan-24-oate identified as a new compound, and a known compound methyl 3α,12α-dihydroxy-7-ox

Full text of DOI:10.1016/j.steroids.2009.01.002

Steroids 74 (2009) 483–486
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Steroids
journal homepage: www.elsevier.com/locate/steroids
Biotransformation of methyl cholate by Aspergillus niger
Amal Al-Aboudia,∗, Mohammad Y. Mohammadb, Salim Haddada, Rawhi Al-Fara,c
,
M. Iqbal Choudharyb, Atta-ur-Rahmanb
a Chemistry Department, University of Jordan, 1194 Amman, Jordan
b H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
c Faculty of Science and IT, Al-Balqa’a Applied University, Salt, Jordan
a r t i c l e i n f o
a b s t r a c t
Article history:
Biotransformation of methyl cholate using Aspergillus niger was investigated. This led to the isolation of
two derivatives: methyl 3␣,7␣,12␣,15␤-tetrahydroxy-5␤-cholan-24-oate identified as a new compound,
and a known compound methyl 3␣,12␣-dihydroxy-7-oxo-5␤-cholan-24-oate. The structure elucidation
of the new compound was achieved using 1D and 2D NMR, MS and X-ray diffraction.
© 2009 Elsevier Inc. All rights reserved.
Received 5 September 2008
Received in revised form
23 December 2008
Accepted 6 January 2009
Available online 17 January 2009
Keywords:
Biotransformation
Methylcholate
Aspergillus niger
Methyl 3␣,7␣,12␣,15␤-tetrahydroxy-5␤-
cholan-24-oate
Methyl 3␣,12␣-dihydroxy-7-oxo-5␤-
cholan-24-oate
1. Introduction
used for epimerization of the hydroxy groups at C-3 [6] and C-7
[7]. Regio- and stereo-specific hydroxylation of methylene carbons
Cholic acid is one of the main components of bile acids in
humans. Besides their importance as end products of choles-
terol metabolism, bile acids are essential in the digestion and
re-sorption of fat, fatty acids and lipid-soluble vitamins. Cholic acid
and its derivatives have received a great interest in pharmaceuti-
cal industry [1], due to their numerous therapeutic applications
in cholestatic liver disease [2] and in dissolution of cholesterol
gallstones, thus avoiding the need for surgery [3]. In addition to
their applications in pharmaceutical industry, they have been used
as chiral templates in asymmetric synthesis [1] and have been
observed to act as organogelators, offering potential applications,
not only in medicine, but also in cosmetics, material sciences and
environmental clean up [1].
Microbial transformation of bile acids has been used in the
preparation of pharmaceutically significant compounds [4]. In par-
ticular, it has been successfully used to enhance the selectivity
and to reduce the number of preparation steps. Biotransforma-
tion is considered as the method of choice for selective oxidation
of the hydroxy groups at C-3, C-7 or C-12 [5]. The method is also
of bile acids, an inaccessible reaction by chemical means, can be
achieved in one step by using microbial transformation [4].
Although there are many studies describing biotransformation
of cholic acid and many of its derivatives using bacterial and fungi
species, most of the studies employed bacterial strains [4]. Only
few studies were reported on biotransformation of cholic acid by
Fungal species. Carlstrom et al. reported that biotransformation of
cholic acid using Fungal species produced traces of hydroxylated
metabolites [8], while other bile acid derivatives (i.e. lithocholic,
deoxycholic, chenodeoxycholic and ursodeoxycholic acid) gave
good yields of different hydroxylated metabolites [9,10].
In this study, the ability of the Aspergillus niger to transform
methylcholate has been investigated for the first time. Two metabo-
lites, a new and a known one, have been isolated and characterized
by different spectroscopic and X-ray diffraction techniques.
2. Experimental
2.1. General methods
Methylcholate was purchased from Merck. Silica gel
(70–230 mesh, Merck) was used for column chromatography
(CC). Pre-coated silica gel plates (Merck, F254; 20 × 20, 0.25 mm
∗
Corresponding author. Tel.: +962 6 5355000/22022; fax: +962 6 5355560.
E-mail address: alaboudi@ju.edu.jo (A. Al-Aboudi).
0039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2009.01.002

484
A. Al-Aboudi et al. / Steroids 74 (2009) 483–486
Table 1
1H NMR and 13 C NMR data for compounds 1–3.a.
Position
Compound 1
Compound 2
Compound 3
1H NMR
13 C NMR
1H NMR
13 C NMR
1H NMR
13 C NMR
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
0.99, 1.81
1.45, 1.59
3.41
1.66, 2.29
1.42
1.53, 1.95
3.94
1.80
2.25
–
1.58
3.81
–
2.00
1.12, 1.76
1.32, 1.90
1.86
0.65
0.86
1.43
35.35
30.38
71.92
39.59
41.71
34.68
68.49
39.59
26.42
34.78
28.19
73.10
46.46
41.51
23.25
27.52
47.05
12.51
22.48
35.29
17.34
30.95
31.12
174.86
51.53
0.99, 1.82
1.46, 1.60
3.40
1.65, 2.30
1.43
1.52, 2.02
4.12
1.85
35.18
29.80
71.51
39.14
41.78
34.40
67.15
34.57
26.25
35.24
27.90
73.26
47.12
46.16
69.05
40.46
46.46
14.31
21.81
34.74
16.36
30.84
30.43
175.00
50.70
1.30, 1.75
37.45
31.06b
70.86
39.48
46.61
45.19
213.71
45.59
35.88
34.56
29.40c
72.54
46.48
45.41
29.66c
29.74c
46.61
14.67
22.74
34.51
17.45
34.11
30.82b
174.72
51.64
2.35b
3.57
1.34, 2.48
1.60
2.85 dd (J = 5.5, 13.2), 2.02
–
2.72 ␺t J = 12
2.35
2.32
–
1.58
3.85
–
1.82
4.30 t, J = 5.8
1.35, 2.38
1.82
0.91
0.93
1.81
0.98 d, J = 6.4
1.80
2.32
–
1.60, 1.70c
3.95
1.90
1.76, 1.20c
1.20, 1.34c
1.60
0.88
1.22
1.58
0.94 d J = 6.3
1.19, 1.85
2.25b
–
3.65
0.95 d, J = 5.9
1.35, 1.79
2.21, 2.37
–
3.64
3.68
a
Assignments were based on 2D NMR spectra (COSY, HMBC and HMQC).
Exchangeable assignments.
Exchangeable assignments.
b
c
and 0.50 mm) were used for TLC. Spots were detected by spraying
with anisaldehyde/sulfuric acid, followed by heating at 140 ◦C. 1H,
13C and 2D NMR spectra (CDCl3 solutions) were recorded using
Bruker Avance-300 or Bruker Avance-500 spectrophotometers.
Chemical shifts (ı) are reported in ppm with TMS as an internal
standard, and coupling constants (J) in Hz. EIMS were recorded on
Jeol JMS-600H mass spectrometer. High resolution mass spectra
(HRMS) were measured in positive ion mode by electrospray
ionization (ESI) technique on a Bruker APEX-4 instrument. The
samples were dissolved in acetonitrile, and infused using a syringe
pump with a flow rate of 2 ␮L/min. External calibration was
conducted using Arginine cluster in a mass range m/z 175–871. All
reagents used were of analytical grades.
metabolite was detected by TLC after 32 h of incubation, and its
amount continued to increase with the progress of fermentation.
The second metabolite was detected after 104 h.
The experiment was repeated under the same conditions using
23 conical flasks and 1.0 g methylcholate, dissolved in 23 ml ace-
tone. After 5 days, the fermentation products were filtered and
extracted three times with ethyl acetate. The residual mycelium was
washed with methanol. The combined ethyl acetate and methanol
solutions were dried over Na2CO3 and evaporated under vacuum
to yield 3.4 g of a gummy mixture.
2.4. Isolation of metabolites
The mixture was chromatographed on a column containing
160 g silica gel, packed in ethyl acetate/benzene (75:25, v/v). The
polarity of the eluent was increased gradually to pure ethyl acetate
then to 10:90 (v/v) methanol/ethyl acetate. Fractions collected from
the column were further purified either by crystallization or by
thin layer chromatography. This led to the isolation of two com-
pounds; methyl 3␣,7␣,12␣,15␤-tetrahydroxy-5␤-cholan-24-oate
(2, 420 mg) and methyl 3␣,12␣-dihydroxy-7-oxo-5␤-cholan-24-
oate (3, 50 mg).
2.2. Organism
Cultures of A. niger were obtained from the American Type Cul-
ture Collection (ATCC 10549). All cultures were grown on Sabouraud
dextrose agar (SDA) and stored at 4 ◦C.
2.3. Incubation of methylcholate (1)
A. niger was grown in shake cultures at 30 ◦C in two conical flasks
(250 mL), each containing 100 mL of a sterile medium compris-
ing of (per dm3) glucose (10 g), peptone (5 g), KH2PO4 (5 g), yeast
extract (5 g), glycerol (5 mL), and NaCl (5 g). The medium solution
was adjusted to pH 5.5. The medium was sterilized by autoclaving
at 121 ◦C for 15 min. Incubations were initiated by suspending the
surface growth from slants in sterile medium. After 2 days of incu-
bation in the above-described medium, methylcholate (1) (0.08 g),
dissolved in 2 ml of acetone, was equally distributed in the con-
ical flasks containing the medium. Culture controls consisted of
fermentation blanks in which microorganisms were grown under
identical conditions but without the substrate. Substrate controls
consisted of sterile medium containing the same amount of sub-
strate and were incubated under the same conditions. The first
2.5. Spectral data of the isolated compounds
The 1H NMR and 13C NMR data of compounds 1-3 are summa-
rized in Table 1. 2D-experiments (COSY, HMBC and HMQC) were
used to assign the chemical shifts of compounds (Fig. 1).
2.6. Crystallographic data of compound 2
The structure of compound 2 was unambiguously determined
by single-crystal X-ray diffraction technique. Compound 2 was
recrystallized from acetonitrile. A colorless crystal with dimensions
0.5 mm × 0.4 mm × 0.1 mm was selected for the crystallographic
measurements. C25H42O6. C2H3N (F.W. 479.64), monoclinic with

A. Al-Aboudi et al. / Steroids 74 (2009) 483–486
485
Fig. 1. Biotransformation of methyl cholate 1 using A. niger.
Fig. 2. ORTEPII program plot of compound 2 unit with thermal ellipsoids at 30% probability.
´
´
´
˚
˚
˚
space group = P21, a = 9.756(2) A, b = 8.3630(17) A, c = 16.242(3) A,
´
ˇ = 97.16 (10)◦, V = 1314.8(5) A , Z = 2, Dcalc = 1.212 mg/m3,
3
˚
´
˚
F(0 0 0) = 524, Mo K␣ = 0.71073 A. Unit cell dimensions were
Table 2
determined by least squares fit of 556 reflections. Diffractometer
used Mercury CCD (2 × 2 bin mode) and the data acquired with
Crystal Clear software [11]. The intensity data within (ꢁ) range of
2.53–27.87◦ were collected at 293(2) K. A total of 5369 reflections
were collected, of which 4295 reflections were judged observed on
the basis of I > 2ꢂ(I). The structure was solved by the direct methods
with all non-hydrogen atoms were refined anisotropically by a
full-matrix least-square calculation on F2 using the SHELXTL V5.03
package [12]. Hydrogen atoms were constrained to side on their
parent atoms with 1.2 times Ueq of the parent atom. The final factors
were R1 = 0.0456 and wR2 = 0.1110. Fig. 2 is an ORTEPII program
plot of the molecular unit with thermal ellipsoids at 30% probability.
Crystallographic data for compound 2 is presented in Table 2.
Crystal data and structure refinement for ‘Methyl 3 ␣,7␣,12 ␣,15␤-tetrahydroxy-5␤-
cholan-24-oate.acetonitrile’.
Identification code
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
RF-BAU
C27 H45 N O6
479.64
293(2) K
0.71073 Å
Monoclinic
P21
´
˚
Unit cell dimensions
a = 9.756(2) A
˛ = 90◦
´
˚
b = 8.3630(17) A
ˇ = 97.16(3)◦
´
˚
c = 16.242(3) A
ꢀ = 90◦
´
˚
3
Volume
1314.8(5) A
Z
2
Density (calculated)
Absorption coefficient
F(0 0 0)
1.212 Mg/m3
0.084 mm−1
524
2.7. Supplementary material
Crystal size
Theta range for data collection
Index ranges
0.2 mm × 0.15 mm × 0.15 mm
2.53–27.87◦
CCDC contains supplementary crystallographic data. This
data can be obtained free of charge via http://www.ccdc.
cam.ac.uk/data request/cif, by e-mailing data request@ccdc.
cam.ac.uk, or by contacting the Cambridge Crystallographic Data
Center, 12 Union Road, Cambridge CB2 1EZ, UK. Fax: +44 1223
336033.
−12 ≤ h ≤ 12, −7 ≤ k ≤ 10, −21 ≤ l ≤ 21
5853
5369 [R(int) = 0.0322]
98.5%
Reflections collected
Independent reflections
Completeness to theta = 27.87◦
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2\S(I)]
R indices (all data)
Absolute structure parameter
Largest diff. peak and hole
Numerical
0.988 and 0.982
Full-matrix least-squares on F2
5369/1/307
3. Results and discussion
0.980
R1 = 0.0456, wR2 = 0.1110
R1 = 0.0587, wR2 = 0.1194
1.0(9)
0.201 and −0.245 eÅ−3
Screening scale experiments have shown that A. niger was
capableScreening scale experiments have shown that A. niger was
capable of converting methylcholate (1) into two metabolites. Large

486
A. Al-Aboudi et al. / Steroids 74 (2009) 483–486
assigned to H-8 as it was coupled with the two vicinal axial protons
at C-9 (ı 2.35) and C-14 (ı 1.90) with coupling constants very close
in value (Fig. 3). The downfield doublet of doublet was assigned
to the axial proton at C-6. The observed splitting is the result of
two coupling constants; a larger one with the geminal proton (ı
2.02) and a smaller coupling constant is with the equatorial pro-
ton at C-5 (ı 1.60) [15]. The data were in good agreement with the
reported values of 3␣,12␣-dihydroxy-7-oxo-5␤-cholan-24-oic [15].
Compound 3 is believed to be a direct product of 7␣ oxidation of
compound 1, indicating the presence of 7␣-hydroxysteroid dehy-
drogenase (7␣-HSDH) in A. niger, so this organism can be used to
convert methyl cholate to the corresponding 7-oxo derivative in a
high regio-specific manner. There are many publications describing
using either hydroxysteroid dehydrogenases [16] or whole cell bio-
transformations that regio-specifically oxidize cholic acid at each of
the three possible positions. Most of the microorganisms reported
are anaerobic bacteria [4,5]. Moreover, Bovara et al showed that
cholic acid has a much lower affinity for 12␣-HSDH than 7␣-HSDH
[17], which might be the reason for not obtaining the 12-oxo methyl
cholate in this study. It is worth noting that compound 3 did not
react with 7␣-HSDH. A possible explanation is the presence of
15␤-hydroxy that prevents the steroid from properly fitting in the
enzyme binding site.
Fig. 3. Key COSY correlations in compound 3.
scale fermentation was thus carried out to produce sufficient quan-
tities of the metabolites for structure elucidation (Fig. 1). Two sets
of controls were used to ensure the authenticity of metabolites.
Metabolites were isolated from the culture medium by successive
ethyl acetate extraction. The extract was evaporated under vacuum.
The residue was fractionated by silica gel column chromatography.
Methyl 3␣,7␣,12␣,15␤-tetrahydroxy-5␤-cholan-24-oate (2)
was isolated as a colorless solid from the silica gel column. EIMS
of compound 2 showed a weak molecular ion peak at m/z 438
(C25H42O6), 16 u higher than the substrate, while the base peak
was at 402, which may result from the loss of two water molecules.
High resolution mass spectrometry showed a molecular ion peak
at m/z 461.290753 corresponding to the formula C25H42O6 + Na
(cal. 461.287360). Comparing the NMR spectral data of compound
2 with that of the parent compound 1 suggested that an extra
hydroxy group was present either at C-15 or C-16 as the two
upfield CH2’s at ı 23.2 and 27.5 characteristic for C-15 and C-16 in
methylcholate were absent and all chemical shifts of ring D were
shifted downfield. The downfield shift of 7␤-H (ꢃı = 0.18 ppm)
indicated hydroxylation at C-15. Moreover, the downfield shift of
the 18-methyl signal (ꢃı = 0.26 ppm) suggested ␤-hydroxylation
at ring D due to the pseudo 1,3-diaxial relationship between the
methyl and the hydroxy group, whereas such a shift is not observed
in the case of ␣-hydroxylation [13]. In addition the 1H and 13C NMR
spectra of compound 2 are in good agreement with those reported
[13] for methyl 3␣,7␣,15␤-trihydroxy-5␤-cholan-24-oate.
Acknowledgements
The first author would like to thank the Deanship of Scientific
Research at the University of Jordan for financial support. Thanks
are also due to Al-Balqa’ Applied University, Salt, Jordan for the use
of the X-ray diffractometer.
References
[1] Virtanen E, Kolehmainen E. Use of bile acids in pharmacological and
supramolecular applications. Eur J Org Chem 2004;16:3385–99.
[2] Colombo C, Setchell KDR, Podda M, Curcio L, Ronchi M, Giunta A. The effects of
ursodeoxycholic acid therapy in liver disease associated with cystic fibrosis. J
Pediatr 1990;117:482–9.
[3] Crosignani A, Setchell KDR, Invernizzi P, Larghi A, Rodrigues CMP, Podda
M. Clinical pharmacokinetics of therapeutic bile acids. Clin Pharmacokinet
1996;30:333–58.
The 2D NMR techniques (COSY-45, HMQC and HMBC) sup-
ported hydroxylation at C-15. The COSY spectrum showed cross
peaks between H-14 (ı 1.82) and H-15 (ı 4.30) and between H-15
(ı 4.30) and H-16 (ı 2.38). The HMBC spectrum showed interac-
tions between H-15 (␦ 4.30) and C-14 (␦ 46.16); H-17 (␦ 1.82) and
C-16 (␦ 40.46); H3-21 (␦ 0.98) and C-17 (ı 46.46). The position
and stereochemistry of hydroxylation were finally confirmed with
single-crystal X-ray diffraction (Fig. 2). All the bond angles and bond
lengths were found to be in the normal range. The hydroxyl group at
C-15 position was found to be ␤-oriented with respect to C-3, C-7
and C-12 hydroxy substituents. It is worth mentioning that 15␤-
hydroxylation was not reported in the biotransformation of any of
the bile acids using A. niger, but it was reported from microbio-
logical transformation of lithocholic acid (3␣-hydroxy-5␤-cholanic
acid) by Cunninghamella sp. [14], and deoxycholic acid by Pencillium
sp. [8].
[4] Bortolini O, Medici A, Poli S. Biotransformations on steroid nucleus of bile acids.
Steroids 1997;62:564–77.
[5] Fantin G, Ferrarini S, Medici A, Pedrini P, Poli S. Regioselective microbial oxida-
tion of bile acids. Tetrahedron 1998;54:1937–42.
[6] Canzi E, Maconi E, Aragozzini F, Ferrari A. Cooperative 3-epimerization of
chenodeoxycholic acid by Clostridium innocuum and Eubacterium lentum. Curr
Microbiol 1989;18:335–8.
[7] Hirano S, Masuda N. Epimerization of the 7-hydroxy group of bile acids by the
combination of two kinds of microorganisms with 7␣- and 7␤-hydroxysteroid
dehydrogenase activity, respectively. J Lipid Res 1981;22:1060–8.
[8] Carlstrom K, Kirk D, Sjovall J. Microbial synthesis of 1␤- and 15␤-hydroxylated
bile acids. J Lipid Res 1981;22:1225–34.
[9] Hayakawa S. Microbial transformation of bile acids, a unified scheme for bile
acid degradation and hydroxylation of bile acids. Zeitschrift Allgemeine Mikro-
biologie 1982;22:309–26.
[10] Sawada H, Kulprecha S, Nilubol N. Microbial production of ursodeoxycholic
acid from lithocholic acid by Fusarium equiseti M41. Appl Environ Mirob
1982;44:1249–52.
[11] Rigaku. Crystal clear. Rigaku Corporation, Tokyo, Japan; 2000.
[12] Bruker, SHELXTL (including XPREP, XCIF, XL, XP, XS) Version 6.10. Bruker AXS
Inc., Madison, WI, USA; 2001.
[13] Iida T, Hikosaka M, Kakiyama G, Shiraishi K, Schteingart C, Hagey L, et
al. Potential bile acid metabolites. 25. Synthesis and chemical properties
of stereoisomeric 3␣,7␣,16- and 3␣,7␣,15-trihydroxy-5␤-cholan-24-oic acids.
Chem Pharm Bull 2002;50:1327–34.
Methyl 3␣,12␣-dihydroxy-7-oxo-5␤-cholan-24-oate (3) was
also isolated by column chromatography and purified by TLC. Oxi-
dation of one of the hydroxy groups to a keto group was inferred
from the presence of two CHOH moieties, instead of three, in the 1
H
[14] Bettarello L, Bortolini O, Fantin G, Guerrini A. Mixed oxo-hydroxy bile acids as
NMR spectrum of compound 3, resonating at 3.57 and 3.95. Also,
13C NMR spectrum revealed the presence of an extra carbonyl group
at ı 213.17. The position of the oxidation was assigned at C-7 on the
basis of the splitting pattern in the region 2.7–2.9 ppm. It is well
known that oxo-hydroxy bile acids exhibit clear and specific signals
in this region, due to the deshielding effect of the carbonyl group on
the near-by protons. Compound 3 showed a pseudo triplet at ı 2.72
and a doublet of doublet at ı 2.85. The upfield pseudo triplet was
actual or potential impurities in ursodeoxycholic acid preparation: a 1H and 13
NMR study. Il Farmaco 2000;55:51–5.
C
[15] Kulprecha S, Nihira T, Shimomura C, Yamada K, Nilubol N, Yoshida T, et
al. 15␤-Hydroxylation of lithocholic acid by Cunninghamella sp. Tetrahedron
1984;40:2843–6.
[16] Riva S, Bovara R, Pasta P, Carrea G. Preparative-scale regio- and stereospecific
oxidoreduction of cholic acid and dehydrocholic acid catalyzed by hydroxys-
teroid dehydrogenases. J Org Chem 1986;51:2902–6.
[17] Bovarar R, Carrea G, Riva S, Secundo F. A new enzymatic route to the sun thesis
of 12-ketoursodeoxycholic acid. Biotechnol Lett 1996;18:305–8.