Lyophilised yeasts: Easy-to-handle biocatalysts for stereoselective reduction of ketones

Article abstract of DOI:10.1016/S0957-4166(99)00379-1

The use of lyophilised yeasts as biocatalysts for the reduction of carbonyl compounds has been studied. First, a comparison of the performances of fresh and lyophilised cells of seven yeasts was performed using ethyl acetoacetate and acetophenone as typical substrates. Lyophilised cells gave from low to high conversions but, with a few exceptions, always good enantioselectivity, and they were then employed for the reduction of structurally different carbonyl compounds. Highly enantioselective reduction of carbonyls was often achieved, even with aromatic ketones. Both enantiomers of the alcohols were, in most cases, obtained with high enantiomeric excess by simply choosing a suitable yeast.

Full text of DOI:10.1016/S0957-4166(99)00379-1

Pergamon
Tetrahedron: Asymmetry 10 (1999) 3515–3520
Lyophilised yeasts: easy-to-handle biocatalysts for stereoselective
reduction of ketones
a,∗
a
a
b
Francesco Molinari, Raffaella Gandolfi, Raffaella Villa and Ernesto G. Occhiato
a
Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sezione Microbiologia Industriale,
Università degli Studi di Milano, via Celoria 2, 20133 Milano, Italy
Dipartimento di Chimica Organica ‘U. Schiff’ and Centro sulla Chimica e la Struttura dei Composti Eterociclici e loro
Applicazioni, C.N.R., Università di Firenze, via G. Capponi 9, 50121 Firenze, Italy
b
Received 12 July 1999; accepted 26 August 1999
Abstract
The use of lyophilised yeasts as biocatalysts for the reduction of carbonyl compounds has been studied. First,
a comparison of the performances of fresh and lyophilised cells of seven yeasts was performed using ethyl
acetoacetate and acetophenone as typical substrates. Lyophilised cells gave from low to high conversions but, with
a few exceptions, always good enantioselectivity, and they were then employed for the reduction of structurally
different carbonyl compounds. Highly enantioselective reduction of carbonyls was often achieved, even with
aromatic ketones. Both enantiomers of the alcohols were, in most cases, obtained with high enantiomeric excess
by simply choosing a suitable yeast. © 1999 Published by Elsevier Science Ltd. All rights reserved.
1. Introduction
The asymmetric reduction of carbonyl compounds by microbial dehydrogenases is a well recognised
method for the preparation of chiral alcohols. Biocatalysts can catalyse stereoselective reductions with
1
high selectivity and, additionally, they work under mild and safe conditions. These methods offer great
potential for the production of a number of chiral fine chemicals which have a relevance in different
areas such as the pharmaceutical and agrochemical industries. The use of whole microbial cells is
particularly advantageous for carrying out the desired reduction since they do not require addition of co-
factors for their regeneration. Saccharomyces cerevisiae has been employed for a number of asymmetric
2
–4
5
reductions.
Other microbial species, such as bacteria (Bacillus stearothermophilus, Gluconobacter
6
7,8
9
10
oxydans, various Lactobacilli, Thermoanaerobium brockii, Thermoanaerobium ethanolicus ) and
fungi (yeasts
11,12
and Geotrichum candidum13,14), possess dehydrogenases which have been successfully
employed for the stereoselective reduction of carbonyls.
∗
Corresponding author: Tel: 0039-0223955844; fax: 0039-0270630829; e-mail: francesco.molinari@unimi.it
0957-4166/99/$ - see front matter © 1999 Published by Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00379-1

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F. Molinari et al. / Tetrahedron: Asymmetry 10 (1999) 3515–3520
However, there is still a need for new biocatalysts that are able to perform highly stereoselective
reactions, and the potential of microbial bio-reductions appears to be still far from being fully exploited.
The reasons why an extensive use of microbial cells as catalysts has not yet been adequately accepted
are mostly related to the demand of reliable preparation techniques capable of producing high yields of
the desired products and to the low familiarity with the techniques of applied microbiology. The use of
dry cells may partially circumvent these problems by making available biocatalysts easy to use without
any microbiological facilities. Besides the easily available S. cerevisiae, acetone powder of the fungus
G. candidum has proven to be effective in the asymmetric reduction of different ketones furnishing
the corresponding (S)-alcohols with excellent selectivity.15 Therefore, it is attractive to look for other
microbial species to be used as dry cells and which are also able to furnish the opposite enantiomer.
The aim of this work was the evaluation of lyophilised yeasts as easy-to-handle biocatalysts for
stereoselective reduction of various prochiral carbonyls (Scheme 1). The yeasts were selected on the
basis of previous studies, which have shown their ability to perform highly selective reductions.10,16,17
Scheme 1.
2. Results and discussion
Ethyl acetoacetate and acetophenone were used as typical substrates to compare the performances
of fresh and lyophilised cells. The reduction of these substrates have been thoroughly studied with S.
2,3,7,18–20
21–23
cerevisiae and other yeasts,
also on a preparative scale.
Table 1 reports the molar conversion
and enantiomeric excesses obtained after 24 h in biotransformations performed with the same cell
concentration.
The conversions generally decreased when lyophilised cells were used, but only in a few cases did this
occur in a dramatic manner. As expected, the reduction of 1 is accelerated by the β-oxo group and much
1
higher yields are always obtained. On the other hand, the enantiomeric excesses increased in some cases
with the use of lyophilised cells.

F. Molinari et al. / Tetrahedron: Asymmetry 10 (1999) 3515–3520
3517
Table 1
Reduction of acetophenone and ethyl acetoacetate with fresh and lyophilised cells of yeasts. Molar
conversion (%) and enantiomeric excess (%) after 24 h
Lyophilised cells were stored and used over six months without noticeable loss of activity. The
reduction of various prochiral carbonyls is reported in Table 2. Structurally different compounds were
chosen to check the substrate specificity of the yeasts.
The yeasts followed Prelog’s rule in the reduction of 2-octanone 7, the only exception being Pichia
etchellsii. It has already been observed that P. etchellsii is able to reduce C4–C8 methylketones to the
16
corresponding (R)-alcohols, while S. cerevisiae gives poor yields and low enantiomeric excess of the
(
S)-alcohols, following Prelog’s rule.24,25 The attack from the re-face became less predominant as the
difference between the small group (methyl) and the large one decreased. With Candida utilis and Pichia
fermentans, the opposite enantiomer was obtained with shorter chain ketones as the difference of size
between the two substituents decreased.
The reduction of β-keto esters 1 and 8 generally occurred with high yields and enantioselectivity, while
large decreases of the molar conversions with 9 are probably due to the poor stability of the substrate.
In this latter case, lower stereoselectivity was also observed, probably as a consequence of the smaller
differentiation between the large group and the small group.
The reduction of different aromatic ketones 10–15 was also performed. Simple aromatic ketones are
usually very poor substrates for Bakers’ yeast-mediated reductions, often furnishing very low yields of
18,20
the desired chiral alcohol.
Only P. etchellsii, P. glucozyma and P. minuta gave the reduction of 10
and 11 with notable molar conversions, although with different trends. Pichia etchellsii furnished lower
yields when increasing the size of the aliphatic substituent from methyl to propyl, while P. glucozyma and
P. minuta allowed for good conversions also in the reduction of 10 and 11 (95 and 50%, respectively).
The aromatic ketones 12 and 13 are both substrates of interest for producing chiral intermediates.
The alcohol obtained by reduction of 12 can be transformed into the corresponding enantiomerically
pure epoxide. It has been reported that Bakers’ yeast reduction of 12 furnished the (R)-enantiomer (ee
1
9
≥
80%). Compound 12 was not very stable and in some cases the substrate disappeared without alcohol
formation (P. etchellsii and P. glucozyma), while with Kluyveromyces marxianus and P. fermentans very
high enantioselectivity was achieved. Prelog’s rule was followed in all cases.

Table 2. Reduction of compounds 3–15 with lyophilised yeasts. Molar conversion (%) and enantiomeric excess (%) after 24 h

F. Molinari et al. / Tetrahedron: Asymmetry 10 (1999) 3515–3520
3519
Enantioselective reduction of 13 gave access to chiral cyclic boronates after introduction of the
boronic acid on the enantiomerically pure alcohol. The reduction of 13 occurred always with high
enantioselectivity yielding the corresponding enantiomerically pure (S)-alcohol. The biotransformations
26
showed the same stereobias observed with fresh cells of S. cerevisiae and acetone powder of G.
15
candidum. Reduction of nitro derivatives 14 and 15 followed behaviours similar to that observed with
17
fresh cells, furnishing high molar conversions in the ketone reduction.
3. Conclusion
In conclusion, this work shows that lyophilised yeasts can be employed as enantioselective biocatalysts
for carbonyl reduction. In a few cases, dry cells were more effective than fresh cells in terms of
activity, but this drawback is largely recompensed by the easiness of working with lyophilised cells since
enantiomeric excesses were quite similar. Although a wider range of substrates needs to be investigated
to fully evaluate the steric and electronic effects affecting yields and stereoselectivity of yeast-mediated
reduction, the use of lyophilised yeasts is an easy and efficient procedure available for organic chemists
without any skill in the techniques of applied microbiology. This method is simple and also suited for
small-scale screening aimed at the selection of the ‘right’ yeast for a given substrate just by evaluating a
restricted number of strains, often furnishing both enantiomers of the desired alcohol.
4. Experimental
4.1. Material
Substrates 1–13 were from Aldrich, while 14–15 were synthesized as described before.27
.2. Microorganisms, media and culture conditions
4
The microorganisms were from CBS (Centraal Bureau voor Schimmelcultures, Baarn, The Nether-
lands), DPVPG (Dipartimento di Biologia Vegetale Perugia, Italy) and MIM (Microbiologia Industriale
Milano, Italy). They were cultured in 3.0 L fermenters with 1.0 L of malt broth pH 6.0 for 48 h at 27°C
and agitation speed 100 rpm. The amounts of lyophilised cells obtained were:
Candida utilis CBS 621 8.0 g/L
Kluyveromyces marxianus CBS 397 3.4 g/L
Pichia etchellsii CBS 2011 5.8 g/L
Pichia fermentans IMAP 2770 5.1 g/L
Pichia glucozyma CBS 5766 5.1 g/L
Pichia minuta CBS 1708 3.0 g/L
Saccharomyces cerevisiae type II Sigma 5.9 g/L
Fresh cells from submerged cultures were centrifuged and washed with 0.1 M phosphate buffer, pH
7.0. Washed cells were either directly used or lyophilised.
4.3. Biotransformation conditions
Reductions were carried out in 10 mL screw-capped test tubes with a reaction volume of 5 mL with
−1
cells (50 g L , dry weight) resuspended in 0.1 M phosphate buffer with 5% glucose, pH 7.0. After

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F. Molinari et al. / Tetrahedron: Asymmetry 10 (1999) 3515–3520
45 min of incubation, neat substrate (20 mM) was added and the incubation continued for 24 h under
magnetic stirring.
4.4. Analytical methods
Alcohol and ketone concentrations were determined by gas-chromatographic analysis on a Carlo Erba
Fractovap GC equipped with a hydrogen flame ionization detector. The column (diameter 3 mm, length
000 mm) was packed with Carbowax 1540 (10% on Chromosorb 80–100 mesh). Samples (0.2 ml) were
2
taken at intervals and added to an equal volume of an internal standard (1-heptanol) solution in methanol.
The stereochemical outcome of the transformations was expressed as enantiomeric excess (ee) of the
major enantiomer. The alcohols obtained by reduction of compounds 1–13 were extracted with ethyl
ether, dried and transformed into the corresponding butyrate esters by reaction with butyryl chloride in
5
6
dry CH Cl with 2% pyridine. The nitro-alcohols were analyzed as described before. The enantiomeric
2
2
composition was determined by gas-chromatographic analysis using a chiral capillary column (diameter
.25 mm, length 25 m, thickness 0.25 µ, DMePeBeta-CDX-PS086, MEGA, Legnano, Italy). The
0
absolute configuration was determined by comparison with authentic samples from Aldrich or obtained
as reported.
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