An effective method to use ionic liquids as reaction media for asymmetric reduction by Geotrichum candidum

Article abstract of DOI:10.1016/j.tetlet.2006.04.140

An effective method was developed to use an enzyme in ionic liquids; the asymmetric reduction of ketones by Geotrichum candidum in ionic liquids proceeded smoothly with excellent enantioselectivity when the cell was immobilized on water-absorbing polymer containing water, while the reaction without the polymer did not proceed.

Full text of DOI:10.1016/j.tetlet.2006.04.140

Tetrahedron Letters 47 (2006) 4619–4622
An effective method to use ionic liquids as reaction media
for asymmetric reduction by Geotrichum candidum
a,
a
a
*
Tomoko Matsuda, Yusuke Yamagishi, Shinichi Koguchi,
b
a
Noritaka Iwai and Tomoya Kitazume
a
Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology,
259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
Frontier Collaborative Research Center, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
4
b
Received 30 March 2006; revised 24 April 2006; accepted 27 April 2006
Available online 22 May 2006
Abstract—An effective method was developed to use an enzyme in ionic liquids; the asymmetric reduction of ketones by Geotrichum
candidum in ionic liquids proceeded smoothly with excellent enantioselectivity when the cell was immobilized on water-absorbing
polymer containing water, while the reaction without the polymer did not proceed.
ꢀ
2006 Elsevier Ltd. All rights reserved.
Ionic liquids have been considered as environmentally
friendly solvents for organic synthesis due to their non-
volatility and recyclable feature, so the reaction using
chemical catalysts in ionic liquids has been fully investi-
An alcohol dehydrogenase preparation from G. candi-
dum IFO 5767 (dried cell) was used for the reduction
4
because the microorganism has suitable enzymes for
transformation in nonaqueous solvent; for example,
the alcohol dehydrogenase can catalyze the reduction
1
gated and has gained great success. The reaction using
5
biocatalysts in ionic liquids has also been developed
since biocatalysts are renewable natural catalysts with
in supercritical carbon dioxide as well as in organic sol-
6
vents using water-absorbing polymer. It is also known
2
+
high enantio-, regio- and chemo-selectivities. However,
that the enzyme is NADH dependent, and NAD can
the kinds of biocatalysts used in ionic liquids have been
very limited, and most of those reported so far are
hydrolytic enzymes such as lipases. There are only a
few reports for alcohol dehydrogenase catalyzed reac-
be recycled to NADH by using 2-propanol as shown
in Scheme 1.
4
Reduction of o-fluoroacetophenone was examined first
as a model reaction to test the viability of the reaction
in ionic liquids. The results are shown in Table 1. First,
3
tion using ionic liquids in spite of the fact that asym-
metric reduction of ketones and oxidation of alcohols
are very important reactions for organic synthesis.
Therefore, a method to use alcohol dehydrogenase in
ionic liquids is necessary to be developed. Here we
report an effective method to use alcohol dehydrogenase
in ionic liquids. The asymmetric reduction of ketones by
Geotrichum candidum in ionic liquids proceeded
smoothly with excellent enantioselectivity when the cell
was immobilized on water-absorbing polymer contain-
ing water, while the reaction without the polymer did
not proceed.
+
dried cell, NAD and 2-propanol were added to an ionic
liquid, [bmim]PF6 or [emim]BF , followed by the
4
O
OH
R
R
(S)
Geotrichum candidum
NAD+
NADH
IFO 5767
dried cell
O
OH
Keywords: Asymmetric reduction; Alcohol dehydrogenase; Ionic
liquid; Geotrichum candidum; Water-absorbing polymer.
Scheme 1. Asymmetric reduction of ketone and recycling of coenzyme
catalyzed by Geotrichum candidum dried cell containing alcohol
dehydrogenases.
*
Corresponding author. Tel./fax: +81 45 924 5757; e-mail:
tmatsuda@bio.titech.ac.jp
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.04.140

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T. Matsuda et al. / Tetrahedron Letters 47 (2006) 4619–4622
Table 1. Effect of reaction conditions on the reduction of o-fluoroacetophenone
a
a
Entry
Ionic liquid
[bmim]PF
[emim]BF
[bmim]PF
[emim]BF
[bmim]PF
[emim]BF
H
2
O
Water-absorbing polymer
Yield (%)
ee (%)
1
2
3
4
5
6
6
ꢀ
ꢀ
+
+
+
+
ꢀ
ꢀ
ꢀ
ꢀ
+
+
0
0
84
<1
92
96
—
—
>99
—
>99
>99
4
6
4
6
4
[
a
bmim]: 1-butyl-3-methylimidazolium, [emim]: 1-ethyl-3-methylimidazolium. Reaction conditions: 16 h at 30 ꢁC.
Determined by GC analyses. The absolute configuration was determined to be S for all samples examined by comparing the GC retention times
4
with those of the authentic samples prepared in the literature methods.
substrate, and the mixture was stirred for 16 h at 30 ꢁC.
100
75
Unfortunately, the reaction did not proceed at all (en-
tries 1 and 2). To improve the activity, water was added
to the reaction mixture, then the yield increased drasti-
cally to give (S)-1-(o-fluorophenyl)ethanol when
50
25
0
[
bmim]PF was used (entry 3), while the reaction in
6
[
emim]BF did not proceed (entry 4). The difference in
4
yield between the two reactions can be attributed to
the difference in the water miscibility; water is not misci-
ble with [bmim]PF so that water and [bmim]PF form
0
2
4
6
8
10
6
6
two separate layers (a two-layer reaction), while
emim]BF is completely miscible with water. The results
imply that the water layer around the cell is necessary
for the reaction to proceed. Therefore, to create the
Time (h)
[
4
Figure 1. Time course of the reduction of o-fluoroacetophenone in
by immobilized G. candidum cell on water-absorbing
polymer. Reaction conditions: 30 ꢁC; 2 -fluoroacetophenone:
0.041 mmol, ionic liquid: 2 mL, immobilized cell: 1.0 g (immobilized
cell was prepared from dried cell: 30 mg, NAD : 5 mg, H
2-propnol: 0.10 mL and water-absorbing polymer: 150 mg). Yield and
ee were determined by GC analyses. Ee of the products was >99% for
all samples examined. The absolute configuration was determined to be
S for all samples examined by comparing the GC retention times with
[
emim]BF
4
0
water layer in [emim]BF , water-absorbing polymer
4
+
was used, so that polymer layer holding water can be
formed strongly. To realize the above idea, water-
absorbing polymer was added to the mixture of water
and cell to immobilize the cell around the polymer phys-
ically. The resulting powder holding water and enzyme
was added to the ionic liquids and used for the reaction.
The yield for the reaction using both ionic liquids (en-
tries 5 and 6) was improved. For the case of water
2
O: 1.0 mL,
4
those of the authentic samples prepared in the literature methods.
ate yield (entries 3 and 6), and when there was a sulfur
functionality on the anion, the reaction hardly pro-
ceeded (entries 4, 7, 9 and 10). On the other hand, the
kind of imidazolium cation did not affect the reaction
outcome. The hydrophobic property of ionic liquids
did not relate to the yield, either, when the cell was
immobilized on water-absorbing polymer, although it
strongly affected the yield of the reaction without using
water-absorbing polymer (Table 1, entries 3 and 4).
Enantioselectivity of the reaction was independent on
the kind of ionic liquids, and whenever the reaction
proceed, it was excellent (ee >99%).
immiscible [bmim]PF , the yield improves because of
6
the increase in the surface area between the ionic liquid
7
and water layers (entry 5). For the case of [emim]BF ,
4
the stabilizing effect of the enzyme by keeping water
around it using the polymer improves the reaction yield.
The water-absorbing polymer, sodium polyacrylate,
may also play the pH-stabilizing role. After all, the use
of water-absorbing polymer holding water improved
the yield for the reduction in ionic liquids for both water
miscible and immiscible from 0% to up to 96%. Impor-
tantly, the enantioselectivity was always very high as far
as the reaction proceeded. The absolute configuration of
the product was same as that for the reaction in water
and supercritical solvent.
The substrate specificity was investigated. As listed in
Table 3, the reduction in an ionic liquid, [emim]BF4,
by immobilized G. candidum cell on water-absorbing
polymer proceeded for various ketones. Acetophenone
derivatives, benzyl acetone, 2-hexanone, b-keto ester
and fluorinated epoxy ketone were used as substrates,
and it was found that all of them were reduced with
moderate to excellent yield, and the enantioselectivity
was excellent for all of the substrates tested (ee >99%).
This wide substrate specificity together with the excellent
enantioselectivity with the green solvent, ionic liquid,
will be a very valuable synthetic tool.
Figure 1 shows time course of the reduction of o-fluoro-
acetophenone in [emim]BF by immobilized G. candi-
4
dum cell on water-absorbing polymer. The yield
increased with the reaction time, which proved that
the alcohol dehydrogenase surely catalyzed the reduc-
tion in the ionic liquid, eliminating major contribution
of the progress of the reaction during the work up.
Other ionic liquids were also tested for the reaction, and
the results are shown in Table 2. The yield was strongly
ꢀ
ꢀ
affected by the anion; PF6 and BF4 gave the highest
In conclusion, an effective method using water-absorb-
ing polymer to immobilize the cell was developed for
ꢀ
yield (entries 1, 2, 5 and 11), (CF SO ) N gave moder-
3
2 2

T. Matsuda et al. / Tetrahedron Letters 47 (2006) 4619–4622
4621
Table 2. Effect of ionic liquids on the reduction of o-fluoroacetophenone by immobilized G. candidum cell on water-absorbing polymer
a
b
b
Entry
Ionic liquid
[bmim]PF
[bmim]BF
[bmim](CF
[bmim]CF
[emim]BF
[emim](CF
[emim]CF
[emim]NO
[emim]CH
[emim]C
[omim]PF
Hydrophobic property
Yield (%)
ee (%)
1
2
3
4
5
6
7
8
9
6
s
m
s
m
m
s
m
m
m
m
n
92
82
49
1
96
61
0
0
0
<1
88
>99
>99
>99
—
>99
>99
—
—
—
—
>99
4
3
SO
SO
2 2
) N
3
3
4
3
SO
SO
2 2
) N
3
3
3
3
OSO
OSO
3
1
1
0
1
2
H
6
3
6
[
bmim]: 1-butyl-3-methylimidazolium, [emim]: 1-ethyl-3-methylimidazolium, [omim]: 1-octyl-3-methylimidazolium. Reaction conditions are descri-
bed in Figure 1. Reaction time: 16 h.
a
2d
s: Hydrophobic, n: half hydrophobic, m: hydrophilic. The hydrophobicity was determined by the methods in the literature.
Determined by GC analyses. The absolute configuration was determined to be S for all samples examined by comparing the GC retention times
b
4
with those of the authentic samples prepared in the literature methods.
Table 3. Substrate specificity of the reduction system in [emim]BF
4
by immobilized G. candidum cell on water-absorbing polymer
b
b
e
Substrate
Yield (%)
ee (%)
Config.
O
X = o-F
o-Me
96
50
27
81
>99
>99
>99
>99
S
S
S
S
p-F
H
X
O
4
7
9
8
>99
>99
S
S
Ph
O
O
O
O
8
2
7
>99
S
O
a
c,d
d
3, 23
>99, >99
nd
O
F3C
Ph
Reaction conditions are described in Figure 1. Reaction time: 16 h.
a
Racemic mixture.
b
Determined by GC analyses.
c
d
e
19
Determined by F NMR.
Product is a mixture of two diastereomers.
Determined by comparison of the GC retention times with those for the authentic samples prepared in the literature methods.
4
4
+
the asymmetric reduction using alcohol dehydrogenases
in ionic liquids. The asymmetric reduction of ketones by
G. candidum in ionic liquids proceeded smoothly with
excellent enantioselectivity when the cell was immobi-
lized on water-absorbing polymer, while the reaction
without the polymer did not proceed. Due to the wide
substrate specificity and excellent enantioselectivity, this
reduction system promoting green chemistry will be a
powerful tool for the asymmetric synthesis.
previously. The dried cell (30 mg) and NAD (5 mg)
were suspended in H O (1.0 mL) and 2-propanol
2
(0.10 mL). The water-absorbing polymer (BL-100,
150 mg) was added and stirred quickly. The resulting
immobilized cell (1.0 g) was added to an ionic liquid
(2.0 mL), and then followed by addition of the substrate
(0.041 mmol). The reaction mixture was stirred for 16 h
at 30 ꢁC, extracted with ether (5 mL · 4), and analyzed
by GC with a chiral column (Chirasil-DEX CB or
CP-Cyclodextrin-B-2,3,6-M-19) and/or F NMR using
undecane (0.028 mmol) or a,a,a-trifluorotoluene
(0.041 mmol) as an internal standard.
1
9
Experimental procedure: G. candidum IFO 5767 was
cultivated, and the dried cell was prepared as described

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T. Matsuda et al. / Tetrahedron Letters 47 (2006) 4619–4622
Acknowledgements
Y.; Abe, Y.; Matsumoto, K.; Monda, K.; Hayase, S.;
Kawatsura, M.; Itoh, T. Tetrahedron Lett. 2006, 48, 1801–
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. Howarth, J.; James, P.; Dai, J. F. Tetrahedron Lett. 2001,
2, 7517–7519; Eckstein, M.; Filho, M. V.; Liese, A.; Kragl,
1
The authors are grateful to Osaka Yuki Kagaku Kogyo
Co., Ltd, for providing BL-100 (water-absorbing
polymer).
3
4
U. Chem. Commun. 2005, 1084–1085.
4
5
. Nakamura, K.; Matsuda, T. J. Org. Chem. 1998, 63, 8957–
8964.
. (a) Matsuda, T.; Harada, T.; Nakamura, K. Chem.
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References and notes
1
2
. Welton, T. Chem. Rev. 1999, 99, 2071–2083; Jain, N.;
Kumar, A.; Chauhan, S.; Chauhan, S. M. S. Tetrahedron
2
005, 61, 1015–1060.
. (a) Rantwijk, F. v.; Lau, R. M.; Sheldon, R. A. Trends
Biotechnol. 2003, 21, 131–138; (b) Kielbasinski, P.;
Albrycht, M.; Luczak, J.; Mikolajczyk, M. Tetrahedron:
Asymmetry 2002, 13, 735–738; (c) Kitazume, T. In Electro-
chemical Aspects of Ionic Liquids; Ohno, H., Ed.; John
Wiley & Sons: New Jersey, 2005; Chapter 10; pp 135–142;
6. (a) Nakamura, K.; Matsuda, T.; Harada, T. Chirality 2002,
14, 703–708; (b) Nakamura, K.; Inoue, Y.; Matsuda, T.;
Misawa, I. J. Chem. Soc., Perkin Trans. 1 1999, 2397–
2402.
7. For the reaction in hexane, the surface area between the
water layer and hexane was increased by immobilization
with water-absorbing polymer as in Ref. 6.
(
d) Tsukada, Y.; Iwamoto, K.; Furutani, H.; Matsushita,