Stereoselective bioreduction to a chiral building block on a kilogram scale

Article abstract of DOI:10.2533/000942903777678894

The highly stereoselective bioreduction of ethyl cyclohexanone-2-carboxylate (1) on a kilogram scale is described. Ethyl(1S,2S)-trans-2-hydroxycyclohexane carboxylate (2) was prepared in 56% yield with a diastereomeric ratio (dr) of 99:1 and an enantiomer

Full text of DOI:10.2533/000942903777678894

FH-HES
574
CHIMIA 2003, 57, No. 9
FH – HES Fachhochschulen – Hautes Ecoles Spécialisées
Chimia 57 (2003) 574–576
© Schweizerische Chemische Gesellschaft
ISSN 0009–4293
S
tereoselective Bioreduction to a Chiral
Building Block on a Kilogram Scal
e
Christelle Jablonski-Lorina*, Vito Melillob, and Ernst Hungerbühlera*
Abstract: The highly stereoselective bioreduction of ethyl cyclohexanone-2-carboxylate (1) on a kilogram
scale is described. Ethyl(1S,2S)-trans-2-hydroxycyclohexane carboxylate (2) was prepared in 56% yield with
a diastereomeric ratio (dr) of 99:1 and an enantiomeric ratio (er) of 99.5:0.5 after rectification. The cell growth
and the biotransformation were performed in a large wave bioreactor equipped with a 200 l cell bag at
Novartis Pharma AG, while the different purification steps were performed at the FHBB (Muttenz). The fourth
semester chemistry students at the FHBB were actively involved in the preparation and optimization of the
entire process.
Keywords: Biotransformation·High stereoselectivity · Scale-up process · S. pombe yeast · Wavebioreactor
Background
Among the different technologies so far 10 of the biotransformation described in
developed (resolution of racemic mixtures, the Scheme for the synthesis of kilogram
Interest in asymmetric synthesis has in- use of natural products as chiral source, use amounts of hydroxyester 2. Ethyl(1S,2S)-
creased very rapidly during the last few of chiral reagents or catalysts, etc.), bio- trans-2-hydroxycyclohexane carboxylate
years due to the pharmaceutical, vitamin transformations involving enzymes or (2) is a versatile chiral building block and
and agro industries’ need to produce enan- microorganisms have been shown to be an an important intermediate in pharmaceuti-
tiomerically pure compounds rather than interesting alternative method offering cal drug synthesis obtained by stereoselec-
racemates.
highly stereoselective reactions with the tive reduction of the keto function of ethyl
In 1990, nearly 90% of all chiral syn- diastereomeric ratio (dr) and enantiomeric cyclohexanone-2-carboxylate (1) with an
thetic drugs sold were racemic [1]. The ratio (er) often higher than 98:2, under mild African beer yeast (Schizosaccharomyces
awareness of the differences in pharma- and cheap reaction conditions, as well as pombe).
cological properties of a chiral compound with environmentally safe procedures
The project was carried out not only for
with its enantiomer and its racemate in- (reactions take place in water). However, educational purposes but also for commer-
creased and the necessity to consider two the use of biotransformations on a large cial purposes in collaboration with our
enantiomers as two different compounds scale for the production of chiral building spin-off partner SynphaBaseAG (www.syn-
when screened as drug candidates emerged. blocks remains limited due to the high dilu- phabase.com).
tion required and the laborious work-up
procedures.
The chemical procedure had previously
been established and optimized on a small
one-liter scale in the wave bioreactor for
sterile cell culture by a group of students
during a semester project (17 working
days). The results were later confirmed at
Project Description
A considerable internal know-how on the FHBB on a volume of eight liters with-
yeast biocatalyzed reactions has been de- in a 20-l bag on the same reactor.
*Correspondence: Prof. Dr. E. Hungerbühler
Tel.: +41 61 467 43 88
Fax: +41 61 467 44 57
veloped over the last ten years at the chem-
Using the facilities of the fermentation
E-Mail: e.hungerbuehler@fhbb.ch
c.jablonski@fhbb.ch
istry department of the University of Ap- group of Dr. K. Memmert at Novartis
plied Sciences, Basel (FHBB) in the group Pharma AG, the reaction was successfully
aFachhochschule beider Basel (FHBB)
Gründenstrasse 40
led by Prof. Dr. E. Hungerbühler [2–4].
performed on a 80-l scale. A very good
CH–4132 Muttenz
In connection with our ongoing project linearity could be observed on the three
of asymmetric reduction of β-keto-esters different scales (1 l, 8 l, and 80 l).
[5], we investigated a scale-up with a factor
bSynphaBase AG
Gründenstrasse 40
CH–4132 Muttenz

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CHIMIA 2003, 57, No. 9
OH
(S)
OH
(S)
O
COOEt
(S)
COOEt
(R)
S. pombe
+
COOEt
Peptone
Yeast extract
Sugar
2
3
dr (2/3) 90:10, er (2) 99.5:0.5, yield: 56%
dr (2/3) 99:1 after rectification
Water, 31.5 °C
1
Baker’syeast
dr (2/3) 2:98, er (3) 97:3, yield: 60%
Scheme. Stereoselective bioreduction of the ethyl cyclohexanone-2-carboxylate (1) with
S. pombe
The major difficulties of the scale-up
were related to:
– the absolute sterile conditions required
during the growth phase of the S. pombe
cells and
– a different geometry of the 200 l cell bag
compared to that of the 2 l and 20 l bags re-
sulting in a change of the wave style and the
set-up parameters. The oxygen ratio used
during the scale-up biotransformation was
40% (air enriched in oxygen) compared to
20% during the previous experiments.
Procedure
60 l of media made from sugar, peptone
and yeast extract and 20 l of a concentrate
of sugar and calcium carbonate were steril-
ized (121 °C for 20 min) in a 100 l fer-
menter (Fig. 1).
Fig. 1. Fermentor of 100 l used for the media preparation (Novartis AG)
A 2 l sterile preculture of S. pombe cells
was mixed with 5 l of media in the 200 l bag
in the wave bioreactor (Fig. 2) and gradual-
ly grew to 60 l of media.
Crucial parameters such as pH control,
glucose concentration, and O2/CO2 concen-
tration were checked every hour. The com-
plete phase of cell growth was performed
without contamination, the quality of the S.
pombe culture checked on the microscope,
the growth rate evaluated by counting cell
numbers on the microscope and by measure-
ment of the optical density.
After addition of the concentrate of sug-
ar and calcium carbonate, 960 ml of com-
pound 1 were added dropwise. The bio-
transformation was achieved over 44 h. The
reaction mixture was saturated with sodium
chloride, the work-up procedure (Fig. 3) af-
forded the separation of the product from
the biomass. The crude material was then
purified by solvent extractions. The separa-
tion of the diastereoisomers cis/trans was
achieved by rectification.
Fig. 2. Wavebioreactor and cell bag of 200 l (Novartis AG)

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Acknowledgements
We would like to thank Dr. K. Memmert , M.
Henke, and E. Weber for the use of the facilities
at Novartis and for their help. We would like to
thank H. Briellman for his technical assistance
during the work-up process of the scale-up reac-
tion in the Verfahrenstechnik Zentrum at the
FHBB. We also thank Prof. Dr. C. de Virgilio for
providing the strain of S. pombe and for his sug-
gestion regarding the selection of the yeast. And
finally thanks to G. Aspin, our Professor for
English at the FHBB, for his help with the
language.
[1] R.E. Gawley, J. Aubé, in ‘Principles of
Asymmetric Synthesis’, Tetrahedron Or-
ganic Chemistry Series, Vol. 14, Ed. Per-
gamon, 1996.
[2] M. Bertau, M. Bürli, E. Hungerbühler,
Chimica Oggi 1998, 58–61.
[3] M. Bertau, M. Bürli, E. Hungerbühler,
Bioworld 1999, 1, 7–10.
[4] M. Bertau, M. Bürli, E. Hungerbühler, P.
Wagner, Tetrahedron Asymmetr, 2001, 12,
2103–2107.
[5] The reduction of β-ketoesters with the
Noyori catalyst led also to the correspon-
ding hydroxyester with a high stereoselec-
tivity – e.g. the ethyl (1S,2S)-trans-2-hy-
droxycyclopentane carboxylate was syn-
thesized with a dr and an er higher than
99:1.
Fig 3. Reaction work-up (Chemistry Depart-
ment FHBB)
Results
The results obtained for the scale-up at
Novartis were similar to the results ob-
tained on small scale at the FHBB.
668 g of a mixture of trans/cis-2-hy-
droxycyclohexane ethylester (2, 3) was
synthesized with a ratio of 90:10 and puri-
fied over a limited period of time. The crude
product was prepared in 64% yield.
The scale-up reaction finally enabled us
to obtain after distillation the pure product
2 in 56% yield with a diastereomeric ratio
(trans/cis) of 99:1 and a enantiomeric ratio
of 99.5:0.5.
It is worth noting that, in contrast to the
classical process, the biotransformation
could be achieved without addition of
antifoaming agent, which highly simplified
the purification steps.
Perspectives
It was shown that the process developed
on a small scale could be transferred to a
large-scale production of an enantiomeri-
cally pure product without significant diffi-
culties.
Further large scale biotransformations
requiring the same know-how might be
investigated in the future.
For more information please contact
the authors (e.hungerbuehler@fhbb.ch or
c.jablonski@fhbb.ch) and www.fhbb.ch
Received: July 29, 2003