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amounts of them were observed when S. platensis were
References and notes
grown in YMS medium. This suggests that oxidation
of hydroxy compound 3 and 4 was catalyzed by a soy-
bean-peptone-inducible enzyme. Oxidation of alcohols
into carboxylic acids takes place in two steps via the for-
mation of aldehydes, and are in general catalyzed by
alcohol deshydrogenase and aldehyde oxidoreductase.25
However some examples described the cytochrome
P450-dependent conversion of alcohols and aldehydes
into carboxylic acid.26 In mammalian metabolism, some
cytochromes P450 are involved or supposed to be in-
volved in the formation of fexofenadine and carebastine,
namely, CYP 3A4,27 4F12 and 2J2.28 As the induction
of cytochrome P450 by soybean peptones has been re-
ported in a Streptomyces strain,18 it could be suggested
that the inducible enzyme involved in the oxidation of
hydroxyterfenadine is a cytochrome P450.
1. Markham, A.; Wagstaff, A. Drugs 1998, 55, 269–274.
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Clin. Pharm. 1993, 45, 191–193.
4. Fexofenadine: Allegra, carebastine: Ebastel.
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S. A.; Li, H. Tetrahedron Lett. 1998, 39, 2701–2704.
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19. Liquid culture media containing (g/L) glucose 16, yeast
extract 4, malt extract 10 (YM medium) and glucose 16,
yeast extract 4, malt extract 10 and soybean peptones 5
(YMS medium).
20. The microorganisms were cultured in a 250mL flask
containing 100mL of medium at 30°C in rotatory shaker
(200rpm). After 60h substrate was added (20mg) in DMF
(0.2mL) and the incubations were maintained at 30°C
with orbital shaking and samples (1mL) were withdrawn
in 24h intervals. Biomass was sedimented (15min,
13,500rpm), supernanants were micro-filtered (0.45lm)
and the filtrates were analyzed by HPLC.
Yeasts expressing human P450 have been previously
used to prepare drug metabolites. For example, an effi-
cient method has been obtained for the preparation of
40-hydroxydiclofenac from oxidation of diclofenac with
yeast expressing cytochrome P450 2C9.29 Likewise, ter-
fenadine was incubated with yeast expressing cyto-
chrome P450 3A4. Interestingly microsomes expressing
CYP 3A4were able to oxidize terfenadine but no bio-
transformation was observed upon incubation with their
parent recombinant cells. This discrepancy is likely to be
related to the incapacity of substrate to penetrate in in-
tact yeast cells and thus points out a possible limitation
in the use of yeast expressing human P450 to prepare
large quantities of metabolites.
The best results were obtained with A. corymbifera cul-
tured in YMS, where the complete transformations of
terfenadine and ebastine were achieved. Since, culture
and incubation were performed without phosphate
added, no formation of phosphorylated compounds
was observed. The preparative experiments carried out
in 1L of YMS culture medium on ebastine30 (180mg)
and on terfenadine31 (200mg) led to 164mg of carebas-
tine (86% yield) and to 193mg of fexofenadine (93%
yield).
21. The reactions were stopped after 96h by addition of a
mixture of AcOEt/Et2O/MeOH (5/5/1) (20mL), stirred
and filtered on Celite. The organic phases were dried on
MgSO4, concentrated and the extracts were reconstituted
in acetonitrile for HPLC analysis.
3. Conclusion
22. 1H NMR (CD3OD 250MHz) 1.2 (6H, s), 1.6–1.8 (6H, m),
1.8–2.0 (6H, m), 2.6 (1H, s), 3.3 (2H, s), 3.6 (2H, s), 7.1
(2H, d, J = 7Hz), 7.2 (6H, m), 7.5 (6H, m).
It was shown that soybean-peptones induce an oxidizing
activity involved in the biotransformation of terfenadine
into fexofenadine in S. platensis. Study is in progress to
verify that the enzyme is a monooxygenase. Based on
the reaction conditions, a bioconversion process was
developed for the production of fexofenadine and carebas-
tine by oxidation of terfenadine and ebastine by A. cor-
ymbifera. Using this methodology, synthesis of some
hydroxylated intermediate analogues is in progress in or-
der to study mammalian and microbial cytochromes P450.
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Catalysed Reactions; Academic Press: San Diego, 2002.
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H.; Reith, M. K.; Cheng, L. K. Drug Metab. Dispos. 1995,
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T.; Miyazaki, H.; Kamataki, T.; Funae, Y. J. Pharmacol.
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Acknowledgements
29. Othman, S.; Mansuy-Mouries, V.; Bensoussan, C.; Batt-
We thank Mrs A.Triclin for her technical assistance and
Laboratoire Pharmafarm for providing ebastine.
´
ioni, P.; Mansuy, D. C. R. Acad. Sci. Paris, Serie IIC
2000, 3, 751–755.