Baker's yeast catalyzed asymmetric reduction in glycerol

Article abstract of DOI:10.1016/j.tetasy.2006.07.026

Free and immobilized baker's yeast were successfully employed in the asymmetric hydrogenations of prochiral β-keto esters and ketones in glycerol. The activities with immobilized cells were always higher then with free cells while the enantioselectivity was very high (99%) with both catalysts. Using glycerol, a non-toxic, biodegradable and recyclable liquid, as an environmentally friendly solvent allowed easy separation of the product by simple extraction with diethyl ether.

Full text of DOI:10.1016/j.tetasy.2006.07.026

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Tetrahedron: Asymmetry 17 (2006) 2043–2045
Baker’s yeast catalyzed asymmetric reduction in glycerol
Adi Wolfson,* Christina Dlugy, Dorith Tavor, Janine Blumenfeld and Yoram Shotland
Green Processes Centre, Chemical Engineering Department, Sami Shamoon College of Engineering,
Bialik/Basel Sts., Beer-Sheva 84100, Israel
Received 23 May 2006; accepted 17 July 2006
Abstract—Free and immobilized baker’s yeast were successfully employed in the asymmetric hydrogenations of prochiral b-keto esters
and ketones in glycerol. The activities with immobilized cells were always higher then with free cells while the enantioselectivity was very
high (99%) with both catalysts. Using glycerol, a non-toxic, biodegradable and recyclable liquid, as an environmentally friendly solvent
allowed easy separation of the product by simple extraction with diethyl ether.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
backs: low solubility of the organic substrate, undesired
side reactions such as hydrolysis, and difficult separation
of the product. Thus the enantioselective reduction of var-
ious prochiral ketones with baker’s yeast was also studied
in different organic solvents.7–9 Although employing organ-
ic reaction medium helps overcome most of the associated
drawbacks of water, the main disadvantages of using such
solvents are their toxicity, which damage the cells and has a
severe environmental impact. Moreover, glucose, which is
usually added to the reaction mixture as a hydrogen source
and as an electron donor in the regeneration of the co-
factor9 has negligible solubility in organic solvents.
Chiral hydroxyl esters and alcohols are useful intermedi-
ates and auxiliaries in the production of fine chemicals.1,2
One of the most attractive methods to synthesis pure alco-
hols enantiomer is via catalytic enantioselective reduction
of the corresponding prochiral ketones (Fig. 1). Though
chiral transition metal complexes were successfully
employed for this purpose,3,4 the use of biocatalytic reduc-
tion has some advantages since it proceeds at room temper-
ature and does not require high pressures of hydrogen.5
Several oxidoreductases are capable of efficiently reducing
ketones with a combination of co-factors such as NADPH
or NADH. However using whole cells, such as baker’s
yeast (Saccharomyces cerevisiae), for chiral reduction is
more economically attractive due to its availability, low
costs and ease of handling and disposal.
Immobilized baker’s yeast (IBY) was also prepared to ease
the cells separation and tested in the asymmetric reduction
of b-keto esters in water10 and organic solvents.8 The prep-
aration of yeast beads with alginate is the most frequently
used immobilization technique due to the easy preparation
method, low cost and relatively high affinity of alginate to
water, and its ability to form a gel under mild condi-
tions.11,12 Besides improved separation of IBY from the
reaction mixture, their preparation also keeps water
around the yeast cells that is essential for their performance
but may also stimulate unwanted side reactions.
O
OH
H
FBY/IBY
glycerol
R1
R2
R1
R2
Figure 1. Baker’s yeast catalyzed asymmetric reduction of prochiral
ketones.
Different organic solvents such as hexane, toluene, ethyl
acetate,7 light petroleum,13 and liquefied petroleum gas14
were tested with or without the addition of a small amount
of water with both FBY and IBY. More recently IBY was
also tested in ionic liquids15 and fluorous media,16
which offers recyclable and more environmentally friendly
organic media and allows easy separation of the product.
Water is the natural solvent of choice for biocatalysis.6
However, performing the reduction of prochiral ketones
with free baker’s yeast (FBY) in water has several draw-
*
Corresponding author. Tel.: +972 8 6475731; fax: +972 8 6475636;
e-mail: adiw@sce.ac.il
0957-4166/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2006.07.026

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2044
A. Wolfson et al. / Tetrahedron: Asymmetry 17 (2006) 2043–2045
Nevertheless, their production is also associated with
employing large amounts of hazardous and volatile organic
solvents.
reaction with FBY, while the enantioselectivity of the
product, (S)-methyl hydroxybutyrate, was very high
(>95%) with both catalysts. The higher performance with
IBY might be attributed to the existence of water in the
IBY beads that maintained their performance. For
comparison, the reactions in water with both FBY and
IBY under the same conditions were completed after
20 h. Indeed, the addition of 5 wt % of water to glycerol
increased the activity without changing the enantio-
selectivity.
Glycerol is a non-toxic, biodegradable and recyclable
liquid manufactured as a by-product of the transesterfica-
tion of a triglyceride in the production of natural fatty
acid derivatives. These derivatives are utilized in many
areas from pharmaceuticals and the food industry to alter-
native fuels, for example, biodiesel, and thus as the produc-
tion of glycerol raises, its price decreases. Glycerol has also
promising physical and chemical properties, which allows
its use as an alternative environmentally friendly solvent.
It has very high boiling point and negligible vapour pres-
sure; it is stable up to its boiling temperature and compat-
ible with most organic and inorganic compounds, as well as
not requiring special handling or storage. The relatively
high polarity of glycerol allows the dissolution of glucose
and sucrose, yet it also dissolves organic compounds that
are poorly miscible in water. Different hydrophobic sol-
vents such as ethers and hydrocarbons, which are immisci-
ble in glycerol allow removal of the products by simple
extraction. Herein, we report on the asymmetric reduction
of prochiral b-keto esters and ketones with both FBY and
IBY in glycerol as a green medium (Fig. 1).17
The effect of the addition of various hydrogen sources to
the reaction mixture with regards to the performance of
free and immobilized cells in glycerol was also tested. The
addition of glucose (entry 2) or sucrose (entry 3) as an
energy source equally increased the activity of the reaction
when compared to the reaction without any additive (entry
1), keeping the high enantioselectivity. Conversely, the
addition of ethanol as an energy source did not change
either the activity or the enantioselectivity. Finally, repre-
sentative prochiral carbonyl compounds were also reduced
in glycerol under optimal conditions, using IBY and
sucrose (Table 1, entries 6–8). The reaction with ethyl aceto-
acetate was more active than the reaction with methyl
acetoacetate (entry 6). As expected, the activities of baker’s
yeast toward the reduction of aliphatic prochiral ketones,
2-octanone and 2-butanone (entries 7 and 8, respectively),
were lower then those obtained with b-keto esters but the
enantioselectivity remained very high.
2. Results and discussion
We started the investigation with the asymmetric reduction
of methyl acetoacetate in glycerol with both FBY and IBY
(Table 1, entries 1–5).17 As illustrated in Table 1, IBY
always yielded higher activity than the corresponding
Recovery of the product from the glycerol can be carried
out by extraction with several glycerol immiscible solvents,
such as diethyl ether, dichloromethane and ethyl acetate.
Table 1. Enantioselective reduction of prochiral ketone with baker’s yeast in glycerola
Entry
1
Substrate
Energy source (g)
No
FBY conversion (%)
61
ee (%)
IBY conversion (%)
87
ee (%)
O
O
>99 (S)
>99 (S)
OMe
OMe
O
O
O
O
O
O
2
Glucose
Sucrose
Ethanol
Noc
71
>99 (S)
>99 (S)
95 (S)
97 (S)
>99 (S)
—
96
>99 (S)
>99 (S)
>99 (S)
97 (S)
O
O
O
O
3
75 (17)b
67
99 (95)b
90
OMe
OMe
OMe
OEt
4
5
73
94
6
Sucrose
Sucrose
Sucrose
74b
—
99b
16
>99 (S)
97 (S)
O
7
O
8b
—
—
22
>99 (S)
a 50 mL glycerol, 10 g FBY, 1 g substrate, 5 g additive 37 ꢁC, 96 h.
b 48 h.
c Addition of 5 wt % water.

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A. Wolfson et al. / Tetrahedron: Asymmetry 17 (2006) 2043–2045
Table 2. Comparison of the performance of IBY in the asymmetric reduction of ethyl acetoacetate in recyclable reaction media
2045
Medium
Ethyl acetoacetate (g)
Energy source (g)
IBY (g)
t (h)
Conversion (%)
Isolated yield (%)
ee (%)
Water
Glycerol
Ionic liquid15
Fluorous phase16
1
1
1.3
0.25
5
5
7.9
1
10
10
10
2.5
20
48
72
41
100
99
70
92
90
—
25
99
99
95
95
100
The product extractions were carried out in the presence of
the yeast cells since part of the product existed in the cells.
The highest extraction yields (>90%) were obtained with
ethyl acetate, which is relatively less toxic than other gly-
cerol immiscible solvents.17 The product extractions were
completed in the presence of the yeast cells, since part of
the product existed in the cells.
ral carbonyl compounds with baker’s yeast. Both activity
and enantioselectivity were high and competitive with the
reactions in water. IBY showed higher activity than FBY
with the same enantioselectivity. However, it should be
taken into account that although the immobilization proce-
dure is simple it requires some effort and time. High
products extraction yields were obtained.
The performance of IBY in the asymmetric reduction of
ethyl acetoacetate in various green solvents was then com-
pared (Table 2). The results in Table 2 illustrate that per-
forming the reaction in glycerol is advantageous over
ionic liquids or fluorous media as it is more active and
enantioselective and it affords higher product isolation
yield by simple extraction.
References
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organic solvent instead of water in yeast catalyzed enantio-
selective reductions is the ease of isolation of the prod-
ucts.13,18 The asymmetric reduction of ethyl acetoacetate
with FBY for example, was successfully performed in var-
ious organic solvents.7,18 Employing light petroleum as the
solvent resulted in 70% product isolation yield. The separa-
tion procedure involved yeast filtration and wash, solvent
evaporation under reduced pressure and bulb to bulb distil-
lation. Conversely, using glycerol as the reaction media
allowed simple extraction of the product with ethyl acetate
and after removal of the solvent at room temperature un-
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a yeast fermenting aqueous system usually results in mod-
erate enantioselectivities.19 This is probably due to the fact
that the ketones are unnatural substrates for alcohol dehy-
drogenase from baker’s yeast. Reduction of 2-hexanone in
an ionic liquid yielded higher activity and similar enantio-
selectivity when compared with an aqueous system
(% ee = 80).15 Performing the reduction of 2-octanone
and 2-butanone in glycerol resulted in much higher enantio-
selectivities (% ee >97). The separation of 2-butanol from
an aqueous system is problematic since it forms azeotropic
mixture with water. Employing glycerol as the solvent
allowed the simple distillation of the alcohol with high iso-
lation yield due to the low vapour pressure of glycerol.
14. Johns, M. K.; Smallridge, A. J.; Trewhella, M. A. Tetra-
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17. The representative procedure for methyl acetoacetate reduc-
tion with FBY and IBY in glycerol: 10 g of FBY (SIGMA,
type II) or 50 g of IBY (prepared from 10 g of FBY as
described in 10) was added to a mixture of 50 mL of glycerol
(99% purity, purchased from Frutarom Ltd) in a 250 mL
bottle and shaken for 30 min. Then 5 g of energy source was
added and the bottle was shaken for 10 min before 1 g of
methyl acetoacetate was added. The bottle was shaken at
300 rpm for 96 h at 37 ꢁC. At the end of the reaction, the
products were extracted with ethyl acetate (3 · 50 mL). The
conversion and the enantiomeric excess of the product were
determined by GC analysis with Astec, Chiraldex G-TAꢂ
(30 m · 0.25 mm, 0.25 lm thickness).
3. Conclusions
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In conclusion, glycerol, which is a renewable and biode-
gradable green solvent was employed for the first time as
a reaction medium in the asymmetric reduction of prochi-