steroids 7 3 ( 2 0 0 8 ) 1385–1390
1389
other hand, can be precipitated with (NH4)2SO4 (65–95% sat-
uration) and dissolved in 0.5 mL of TEA-buffer and stored at
4 ◦C for 3 days without significant decrease of activity.
Since a key step of the synthesis of ursodeoxycholic acid
from cholic acid is the regioselective oxidation of the 12␣-
OH function and the oxidation aptitude follows the order
C7 > C12 > C3, it is necessary to protect the C7– and C3–OH func-
tions to obtain this goal. The partially purified 12␣-HSDH, first,
and 7␣-HSDH, after, were efficiently used in a new integrated
chemo-enzymatic synthesis of ursodeoxycholic acid 7 starting
from sodium cholate 1 (Scheme 2).
The first step was the treatment of sodium cholate 1 with
the partially purified 12␣-HSDH in the presence of a catalytic
amount of NAD+. The reduction of sodium pyruvate by lac-
tate dehydrogenase from rabbit muscle maintained an high
NAD+ concentration that afforded, after 12 h, 3␣,7␣-dihydroxy-
12-keto-5-cholan-24-oic acid 4 with an excellent yield (98%).
The subsequent Wolf–Kishner modified reduction [25] of
the crude 3␣,7␣-dihydroxy-12-keto-5-cholan-24-oic acid 4
gave, after 12 h at 110-135 ◦C, very good yield (90%) of the crude
chenodeoxycholic acid 5. This was transformed in the corre-
sponding sodium chenodeoxycholate 5a and used in the next
step without further purification.
On the other hand, the chenodeoxycholate 5a was
very efficiently transformed into the 3␣-hydroxy-7-keto-5-
cholan-24-oic acid 6 (97%) by the partially purified 7␣-HDSH,
using the same cofactor recycling system as for the 12␣-OH
oxidation.
Finally, the 7-keto function of 6 was chemically reduced
[6] with sodium in 2-butanol to give ursodeoxycholic acid 7
(82%) together with chenodeoxycholic acid 4 (15%). The over-
all yield of this new integrated chemo-enzymatic synthesis of
ursodeoxycholic acid (70%) was considerable.
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[5] Hoffmann AF. The preparation of chenodeoxycholic acid
and its glycine and taurine conjugates. Acta Chem Scand
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[6] Liu Z. Advance in methods for preparation of
ursodeoxycholic acid. Yaoxue Tongbao 1988;23:583–6;
Liu Z. Advance in methods for preparation of
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[7] Samuelsson B. Preparation of ursodeoxycholic acid and
3␣,7. 12␣-trihydroxycholanic acid. Acta Chem Scand
1960;14:17–20.
[8] Iida T, Chang FC. Potential bile acid metabolites. 3. A new
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[10] Guillemette A. Ursodeoxycholic acid. Ger. Offen. 1980; DE
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[11] Sawada H, Kinoshita S, Yoshita T, Taguchi H. Microbial
production of chenodeoxycholic acid precursor,
12-ketochenodeoxycholic acid, from dehydrocholic acid. Eur
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[12] Sawada H, Kinoshita S, Taguchi H. Production of
chenodeoxycholic acid by bioconversion of dehydrocholic
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[13] Bortolini O, Cova U, Fantin G, Medici A. A new
non-enzymatic route to chenodeoxycholic acid. Chem Lett
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[14] Macdonald IA, Mahony DE, Jellet JF, Meier CE.
NAD-dependent 3␣- and 12␣-hydroxysteroid dehydrogenase
activities from Eubacterium lentum ATCC no. 25559. Biochim
Biophys Acta 1977;489:466–76.
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4.
Conclusions
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[17] Carrea G, Bovara R, Longhi R, Riva S. Preparation of
12-ketochenodeoxycholic acid from cholic acid using
coimmobilized 12␣-hydroxysteroid dehydrogenase and
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efficiency. Enzyme Microb Technol 1985;7:597–
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In conclusion, the numerous screenings of microbial oxida-
tions carried out by our research group on different bile acids
allowed us to identify bacteria able to oxidize regioselectively
the OH functions of cholic acid. In particular, A. calcoaceticus
lwoffi showed the presence of 7␣- and 12␣-HSDHs, that were
also very easily separated. This permits a simple and effi-
cient application of these partially purified enzymes to the
synthesis of ursodeoxycholic acid. The key steps of this new
chemo-enzymatic route are the quantitative oxidation of the
12-OH function of cholic acid and of the 7-OH function of
chenodeoxycholic acid carried out by 12␣-HSDH and 7␣-HSDH,
respectively.
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r e f e r e n c e s
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acids from raw materials (ox and pig bile).
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