A novel method for enzymatic asymmetric reduction of ketones in a supercritical carbon dioxide/water biphasic system

Article abstract of DOI:10.1016/j.tetlet.2009.06.063

A novel method to enable asymmetric reduction of ketones by an alcohol dehydrogenase from Geotrichum candidum in a supercritical carbon dioxide and water biphasic system is described. The addition of sodium bicarbonate improved the reactivity up to a prac

Full text of DOI:10.1016/j.tetlet.2009.06.063

Tetrahedron Letters 50 (2009) 4934–4936
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A novel method for enzymatic asymmetric reduction of ketones
in a supercritical carbon dioxide/water biphasic system
Tadao Harada a, , Yuki Kubota a, , Takashi Kamitanaka a,b, , Kaoru Nakamura c, Tomoko Matsuda d,
*
a Department of Materials Chemistry, Ryukoku University, Otsu, Shiga 520-2194, Japan
b Industrial Research Center of Shiga Prefecture, Kamitoyama, Ritto, Shiga 520-3004, Japan
c Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
d Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel method to enable asymmetric reduction of ketones by an alcohol dehydrogenase from Geotri-
chum candidum in a supercritical carbon dioxide and water biphasic system is described. The addition
of sodium bicarbonate improved the reactivity up to a practical level while retaining excellent
enantioselectivity.
Received 14 May 2009
Revised 10 June 2009
Accepted 12 June 2009
Available online 16 June 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Biotransformations in biphasic systems have been investigated
widely to improve reaction efficiency and to simplify workup pro-
cedure. Recent developments include the application of organic
solvent/water, ionic liquid/water, ionic liquid/supercritical carbon
dioxide (scCO2), and scCO2/water systems.1 Using a biphasic sys-
tem, substrate/product inhibition has been prevented, and workup
procedure has been simplified. Among the solvents used in bipha-
sic systems, scCO2 has the benefits of being environmentally
benign, non-flammable, of low toxicity, high availability, and ambi-
ent critical temperature which make it suitable for
biotransformations.1
scCO2/water biphasic system. Contrary to our expectations, with-
out any additive, the reaction hardly proceeded. However, we
found that the addition of sodium bicarbonate improved the reac-
tivity and yield of the product up to a practical level.
First, we attempted to react acetophenone with the crude alco-
hol dehydrogenase of G. candidum NBRC 5767 (APG5) in the pres-
ence of 2-propanol as the hydrogen source in a biphasic system
(2 mL of 0.1 M MES buffer (pH 7.0) and 8 mL of scCO2) at 35 °C
as shown in Scheme 1.4 Disappointingly, the yield was poor (4%).
The following reasons can be considered for the low yield.
However, the types of enzymes used in scCO2 are very limited,
and only lipases have been used widely and successfully.1 The
applicability of other kinds of enzymes such as dehydrogenases
needs to be investigated to broaden the variety of organic transfor-
mations that can be achieved. Therefore, we have investigated the
use of a dehydrogenase for asymmetric reduction in scCO2 and re-
ported previously that asymmetric reduction of ketones was possi-
ble when the resting cells of Geotrichum candidum NBRC 5767 were
used.2 However, this method still has the drawback that the resting
cells of the microbe are difficult to be stored for a long time. The
resting cells need to be used immediately after cultivation. The
second weak point is that a large amount of biocatalyst is neces-
sary; the ratio of biocatalyst mass to substrate was >100. On the
other hand, the use of a crude enzyme preparation (cell dried using
acetone) resolves the above-mentioned drawbacks; the biocatalyst
can be stored for a long period in a freezer, and the ratio of sub-
strate to biocatalyst was reduced to 1.3 Therefore, we attempted
to use the crude enzyme preparation, an alcohol dehydrogenase,
of G. candidum NBRC 5767 (APG5) for asymmetric reduction in
(1) The high pressure inactivates the enzyme.
(2) Lowered pH of the aqueous layer due to high density of
scCO2 inactivates the enzyme.
To examine if the high pressure deactivates the enzyme, a bi-
phasic system composed of supercritical fluoroform (scCHF3) and
water under the same reaction conditions was used with the cells
and substrate. The reaction in scCHF3 readily proceeded, and the
corresponding (S)-alcohol was obtained in 57% yield (>99% ee).
Therefore, the cause of the low yield of the reaction in scCO2 bipha-
sic system was not the high pressure.
To examine if the lowered pH deactivates the enzyme, the effect
of pre-treatment with the supercritical fluid/aqueous buffer bipha-
sic solvent on the enzyme activity was examined. After the pre-
treatment, the activity of the enzyme was analyzed by the reaction
in aqueous buffer under atmospheric pressure. It was found that
the activity was lost completely by the pre-treatment with scCO2
in 8 MPa scCO2/aqueous buffer for 3 h (0% yield). However, pre-
treated biocatalyst with scCHF3/aqueous buffer did not lose any
activity (76% yield). This strongly suggests that the lowered pH
caused by scCO2 irreversibly inactivated the enzyme. Since large
changes of pH in the aqueous phase of the scCO2/aqueous buffer
biphasic system have been reported,5 the effect of pH on enzyme
* Corresponding author. Tel./fax: +81 45 924 5757.
E-mail address: tmatsuda@bio.titech.ac.jp (T. Matsuda).
Tel./fax: +81 775 43 7461.
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.06.063

T. Harada et al. / Tetrahedron Letters 50 (2009) 4934–4936
4935
O
OH
Ph
Supercritical fluid / aqueous buffer biphasic system
Geotricum candidum
>99% ee (S)
Ph
NAD+
NBRC 5767
NADH
alcohol dehydrogenase
O
OH
scCO2/aqueous buffer: Without additive: 4% yield
With NaHCO3: 25% yield
scCHF3/aqueous buffer: Without additive: 57% yield
Scheme 1.
Table 1
70
60
50
40
30
20
10
0
Asymmetric reduction of ketones by dehydrogenase in scCO2/aqueous buffer or
hexane/ aqueous buffer
8 MPa
10 MPa
Reaction conditions
Substrates
scCO2/aqueous buffera
Hexane/ aqueous bufferb
Yieldd (%)
ee (%)
Yieldd (%)
ee (%)
Acetophenone
22
60
36
82
50
82
37
57
>99
>99
>99
>99
>99
>99
>99
>99
21
49
42
>99
>99
>99
o-Fluoroacetophenone
m-Fluoroacetophenone
m-Fluoroacetophenonec
m-Chloroacetophenone
m-Chloroacetophenonec
tert-Butyl acetoacetate
tert-Butyl acetoacetatec
0
0.2 0.4 0.6 0.8 1.0 1.2
NaHCO3 (M)
44
15
>99
>99
Figure 1. Effect of NaHCO3 on the reduction of o-fluoroacetophenone in the scCO2/
aqueous buffer biphasic system. Reaction conditions: o-fluoroacetophe-
none = 0.082 mmol, NAD+ = 1.3
lmol, NaHCO3 = 0-200 mg; APG5 = 20 mg, MES
a
Reaction conditions: substrate = 0.082 mmol, NAD+ = 1.3
lmol, APG5 = 20 mg,
buffer (0.1 M pH 7.0) = 2 ml, 2-propanol = 2.6 mmol, 8 MPa or 10 MPa scCO2, 5 h,
NaHCO3 = 150 mg, 2-propanol = 2.6 mmol, MES buffer (0.1 M, pH 7.0) = 2 mL,
35 °C.
10 MPa scCO2, 5 h, 35 °C.
b
Reaction conditions: Substrate = 0.082 mmol, NAD+ = 1.3
lmol, APG5 = 20 mg,
activity was examined. Indeed, the enzyme was deactivated at a
low pH (below 5), and no reaction proceeded under these condi-
tions. Therefore, the inactivation of the biocatalyst was due to
the lowered pH of the buffered solution by scCO2.5 The buffering
action of MES buffer was not sufficient for the system.
2-propanol = 2.6 mmol, MES buffer (0.1 M, pH 7.0) = 2 mL, hexane = 8 ml, 5 h, 35 °C.
c
Reaction conditions: NAD+ = 6.5
lmol.
d
Side reactions such as aldol condensation were not observed.
To overcome the undesired effect of scCO2, we examined the
effect of addition of basic salts to the reaction mixture. Among sev-
eral bases tested,6 we have found that sodium bicarbonate was the
best additive. The addition of 12 mmol of sodium bicarbonate in
2.0 mL of MES buffer (0.1 M, pH 7.0) solution containing 20 mg of
enzyme, 0.082 mmol of acetophenone, 2.6 mmol of 2-propanol,
and 0.013 mmol of NAD+ gave 25% of the corresponding (S)-alco-
hols (>99% ee) by the biphasic reaction in 8 MPa scCO2 for 3 h.
The addition of sodium bicarbonate improved the reactivity from
4% to 25% yield.
Next, the effect of changing the amount of sodium bicarbonate
on the reduction of o-fluoroacetophenone was examined in the
scCO2/aqueous buffer biphasic system at 8 MPa and 10 MPa. As
shown in Figure 1, the amount of sodium bicarbonate significantly
affected the yields of the reaction. The suitable amounts of sodium
bicarbonate at 8 MPa or 10 MPa were 0.77 M or 0.89 M, respec-
tively. The reproducibility of these data was very high. The phe-
nomenon is explained as follows: the amount of sodium
bicarbonate necessary to make the reaction mixture pH neutral is
very narrow and different at different pressures.
in scCO2 system gave yields that were slightly higher than those
obtained in the hexane system when using o-fluoroacetophenone
and tert-butyl acetoacetate as a substrate, and results were similar
to those obtained in the hexane system when using other
substrates.
Finally, the present biphasic system was applied to the alginate-
immobilized dehydrogenase. The asymmetric reduction of o-fluo-
roacetophenone with the immobilized enzyme in scCO2/water bi-
phasic system proceeded, and a 75% yield of the (S)-alcohol was
obtained in >99% ee.8
In conclusion, the addition of sodium bicarbonate enabled the
enzymatic asymmetric reduction of various ketones under scCO2/
water biphasic conditions of up to a pressure of 14 MPa. Free and
immobilized enzyme can be used in the system. We believe that
the present method can be applicable to other biocatalytic reac-
tions under scCO2 and will open up a new field for the use of scCO2
for biocatalysis.
References and notes
Moreover, the use of sodium bicarbonate enabled the enzymatic
asymmetric reduction even in 14 MPa scCO2/aqueous buffer bipha-
sic system (29% yield), whereas the reaction without addition of
bicarbonate hardly proceeded (5% yield).
To examine the applicability of the present biphasic system to
other substrates, several ketones were used as substrates. These
ketones were reduced to the corresponding (S)-alcohol in high
yields with excellent ee (>99%) as shown in Table 1. The scCO2/
aqueous buffer biphasic system was also compared with hexane/
aqueous buffer7 biphasic system. As shown in Table 1, the reaction
1. Hobbs, H. R.; Thomas, N. R. Chem. Rev. 2007, 107, 2786.
2. (a) Matsuda, T.; Harada, T.; Nakamura, K. Chem. Commun. 2000, 1367; (b)
Matsuda, T.; Watanabe, K.; Kamitanaka, T.; Harada, T.; Nakamura, K. Chem.
Commun. 2003, 1198; (c) Matsuda, T.; Marukado, R.; Mukouyama, M.; Harada,
T.; Nakrmaura, K. Tetrahedron: Asymmetry 2008, 19, 2272.
3. Matsuda, T.; Nakamura, K. J. Org. Chem. 1998, 63, 8957.
4. The reduction was conducted as described in Ref. 2a except that the crude
enzyme was used in place of the resting cells of the microbe and water-
adsorbent polymer was not used.
5. (a) Holmes, J. D.; Steytler, D. C.; Rees, G. D.; Robinson, B. H. Langmuir 1998, 14,
6371; (b) Holmes, J. D.; Ziegler, K. J.; Audriani, M.; Lee, C. T., Jr.; Bhargava, P. A.;
Steytler, D. C.; Johnston, K. P. J. Phys. Chem. B 1999, 103, 5703.

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T. Harada et al. / Tetrahedron Letters 50 (2009) 4934–4936
6. The following basic salts were used: NaHCO3, CaCO3, CaOAc, KHCO3, and
Na2CO3.
8. Conditions: APG5 = 20 mg (immobilized with 0.36 g of calcium alginate);
NAD+ = 6.5
mol; 2-propanol = 2.6 mmol; o-fluoroacetophenone = 0.082 mmol;
l
7. Asymmetric reduction using resting cells of Geotrichum candidum in a hexane/
aqueous buffer biphasic system has been reported; Nakamura, K.; Inoue, Y.;
Matsuda, T.; Misawa, I. J. Chem. Soc., Perkin Trans. 1 1999, 2397.
NaHCO3 = 140 mg; MES buffer (0.1 M pH 7.0) = 1.64 mL; scCO2 = 10 MPa;
35 °C, 5 h. The reaction without NaHCO3 afforded only 3% yield of the
alcohol.