Light-mediated regulation of asymmetric reduction of ketones by a cyanobacterium

Article abstract of DOI:10.1016/S0957-4166(02)00683-3

The stereochemical course of the asymmetric reduction of ketones by a photosynthetic microbe is largely regulated by light. Thus, we find that the enantioselectivity of the reduction of α,α-difluoroacetophenone with Synechococcus elongatus PCC 7942 increa

Full text of DOI:10.1016/S0957-4166(02)00683-3

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 13 (2002) 2529–2533
Light-mediated regulation of asymmetric reduction of ketones by
a cyanobacterium
Kaoru Nakamura* and Rio Yamanaka
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
Received 12 September 2002; accepted 15 October 2002
Abstract—The stereochemical course of the asymmetric reduction of ketones by a photosynthetic microbe is largely regulated by
light. Thus, we find that the enantioselectivity of the reduction of a,a-difluoroacetophenone with Synechococcus elongatus PCC
7
942 increases as a result of illumination with fluorescent light. Furthermore, DCMU, an inhibitor of photosynthesis, affects the
stereoselectivity, and, under illumination decreases the enantioselectivity of the reduction. © 2002 Elsevier Science Ltd. All rights
reserved.
1
. Introduction
ing stereoselectivity of microbial reductions and several
methods can be applied for a practical synthesis, many
reactions are still unsuited to these types of stereochem-
ical control and new strategies to increase the stereose-
lectivities of whole cell-mediated reactions are therefore
desirable.
Optically active compounds are useful intermediates for
pharmaceuticals, agrochemicals and liquid crystals and
hence asymmetric synthesis is one of the most impor-
tant topics in current organic chemistry. Chiral com-
pounds containing fluorine atoms are of particular
interest in the fields of medical and biological chemistry
because of their significant biological response proper-
ties compared with the parent molecules. Asymmetric
reduction of carbonyl compounds with biocatalysts is
now recognized as a valuable approach for the synthe-
Herein, we report a novel method for stereochemical
control of the asymmetric reduction with a photosyn-
thetic microbe: control by light. Enantioselectivities in
asymmetric reduction of ketones are improved by illu-
mination with fluorescent light. This is the first report
on light-mediated regulation of an asymmetric
reduction.
1
2
sis of optically active compounds. To date, two meth-
ods have been used for biocatalytic reduction: one
involves the use of isolated enzymes and the other
involves the use of microbial whole cells. In general,
enantiomeric purities are high for isolated enzymatic
systems. On the other hand, whole-cell-reactions usu-
ally afford unsatisfactory stereoselectivities, because
several enzymes in the cell participate in the reaction
and the stereochemical preferences are not the same for
all of the different enzymes: one enzyme may afford
one stereoisomer while another enzyme may produce its
antipode. To date, several methods have been devel-
oped for improving the selectivity of whole-cell-cata-
lyzed reactions by modifying the reaction conditions:
2
. Results and discussion
2
.1. Reduction of a,a-difluoroacetophenone by a
cyanobacterium
When a,a-difluoroacetophenone 1 was added to a sus-
pension of Synechococcus elongatus PCC 7942 and
shaken (140 rpm) in the dark, under standard condi-
tions for microbial reduction, the corresponding R-
alcohol, (R)-2 was obtained with poor enantio-
selectivities (20–30% ee).
3
addition of inhibitors of a specific enzyme, reaction in
organic solvents, use of hydrophobic polymers such as
XAD, and the treatment of microbial cells with acet-
one. Although these methods are effective for increas-
4
5
6
These results suggest that reduction of 1 by S. elongatus
was conducted by plural enzymes: one transformed the
substrates into the corresponding R-alcohol, while the
other afforded the antipode on reduction. To increase
*
Corresponding author. Tel.: +81 774 38 3201; fax: +81 774 38 3201;
e-mail: nakamura@scl.kyoto-u.ac.jp
0
957-4166/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(02)00683-3

2
530
K. Nakamura, R. Yamanaka / Tetrahedron: Asymmetry 13 (2002) 2529–2533
the poor enantioselectivity, we tested several previously
reported methods for stereochemical control. However,
the results of our studies using such methods were
unsatisfactory.
2.2. Effect of addition of DCMU on reduction of a,a-
difluoroacetophenone by a cyanobacterium
DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is
10
known to inhibit photosynthesis. This inhibitor is
widely used because of its exceptionally potent and
specific inhibitory effect on the Hill reaction (light-
induced transport of electrons from water to acceptors).
Therefore the use of DCMU as an additive under
illumination with light can change reaction conditions
so that they are similar to darkness with respect to the
electron transport system. Thus, under these conditions,
although the microbe is exposed to light, reducing
power resulting from photosynthesis (i.e. NADPH) is
not generated, just as in the dark.
Since the microbe we used is a kind of cyanobacteria
that grows under illumination, we tested the reaction
under illumination with light. We supposed that the
relative enzymatic activities were largely influenced by
the amount of light in the environment. Phototrophs
such as cyanobacteria perform photosynthesis when
exposed to light, and all enzymatic activities change
depending on whether the environment is dark or
7
light. The relative activities of enzymes that conduct
the reduction of 1 may also vary and reactions in the
light may afford different stereoselectivities from those
in the dark. In fact, when the microbes were incubated
−1
Actually, as shown in Fig. 1, enantioselectivity of the
reduction of 1 in the light decreased almost to the level
of that in the dark conditions when 10 mM of DCMU
was added to the system. DCMU also decreases the
chemical yield of the reduction down to similar levels as
those observed for the reaction completed in the dark.
−
2
with 1 under illumination (13.4 mmol photons m
s
(
1,000 lux)), (R)-2 was obtained with 68–70% ee.
Although the enantioselectivity is still not satisfactory,
the selectivity is much higher than that observed for the8
reaction completed in the dark (20–30% ee) (Table 1).
Thus, we have found that light can regulate the selectiv-
ity of asymmetric reduction and induced an increase in
the activity of the enzyme that catalyzes the production
of (R)-2. Recently, we reported that chemical yields in
the reduction of 2%,3%,4%,5%,6%-pentafluoroacetophenone9
by S. elongatus were improved by light illumination.
However, in this case, the enantioselectivity was not
affected by light environment. On the contrary, when
a,a-difluoroacetophenone is used as a substrate, both
the chemical and enantiomeric purities of the corre-
sponding alcohol depend on light. Illumination
improved not only the chemical yield, but also the
enantiomeric purity. This result shows that illumination
induces or activates enzymes that catalyze the conver-
sion of the ketone into (R)-2.
The effect of DCMU on enantioselectivity was investi-
gated by changing its concentrations, with the results
shown in Fig. 2. Thus, increasing the DCMU concen-
tration led to decreases in the chemical and enan-
tiomeric purities. This result undoubtedly indicates that
inhibition of photosynthesis causes a decrease in enzy-
matic activities that selectively produce the R-alcohol.
This is a strong evidence for the association of photo-
synthesis with enzymatic activities in reduction of the
ketone by S. elongatus PCC 7942 (Fig. 3).
The mechanism of stereochemical control is proposed
as follows: Under illumination, chlorophyll in
cyanobacteria captures light energy and generates the
reducing agent NADPH. The R-enzymes (the enzymes
that selectively produce R-alcohol) use thus generated
11
To clarify the mechanism for the observed stereochemi-
cal control in the microbial reduction, the effect of a
photosynthetic inhibitor was investigated.
NADPH selectively while the S-enzymes (the enzymes
that produce S-alcohol) would not use the same coen-
zyme. Light also changes the physiology of cyanobacte-
Table 1. Reduction of difluoroacetophenone by S. elongatus PCC 7942
Light
Days
Yield (%)
Ee (%)
Config.
On
On
Off
Off
1
4
1
4
88
\99
61
68
70
27
31
R
R
R
R
96
Substrate; 10 mmol, biocatalyst as dry weight: 1 g/L, medium; 20 mL.

K. Nakamura, R. Yamanaka / Tetrahedron: Asymmetry 13 (2002) 2529–2533
2531
the reduction under dark and consequently, both R-
and S-alcohols are produced. Under illumination, only
the R-enzymes are activated and, as stated above, these
produce the R-alcohol selectively.
2
.3. Reduction of acetophenone derivatives by the
cyanobacterium
Acetophenone derivatives such as acetophenone, a-
monofluoroacetophenone, a,a-difluoroacetophenone,
and a,a,a-trifluoroacetophenone were reduced by S.
elongatus PCC 7942, and the effect of illumination was
observed (Table 2).
Although acetophenone was reduced to the S-alcohol
1
2
with an excellent ee (>99% ee S) in the light, the
chemical yield was poor and the reduction could not
proceed under darkness. When a-monofluoroacetophe-
none was used as the substrate, the reduction under
illumination produced the R-alcohol with higher enan-
tioselectivity than that in the dark, just as in the
reduction of 1. However, in the reduction of a,a,a-tri-
fluoroacetophenone, no effect of light on enantioselec-
tivity was observed. Although illumination affects
chemical yield (63% under illumination and 19% in the
dark), enantioselectivities were not influenced by illumi-
nation conditions.
Figure 1. The effects of DCMU on reduction of a,a-
difluoroacetophenone by S. elongatus PCC 7942.
The difference in sensitivity to brightness between tri-
fluoroacetophenone and mono- and difluoroacetophe-
none can be explained by the idea that the quantity of
enzymes that can reduce trifluoroacetophenone is less
than that for mono- and difluoroacetophenone. In the
case of using Geotrichum candidum as the biocatalyst,
a,a,a-trifluoroacetophenone was reduced only by S-
enzyme and gave S-alcohol with high enantioselectiv-
1
3
ity.
On the contrary, reductions of a-mono-
fluoroacetophenone and a,a-difluoroacetophenone were
conducted by both R- and S-enzymes, resulting in the
production of low enantioselectivities. The bulkiness
of the trifluoromethyl group in a,a,a-trifluoroacetophe-
none may decrease the number of enzymes that can
participate in the reaction.
13
3
. Conclusion
We have developed a novel method to improve enan-
tioselectivities in the reduction of ketones by a
cyanobacterium (S. elongatus PCC 7942). The enan-
tiomeric purities of the products increased when using
light, and we found that light is a new regulator of
enantioselectivities in asymmetric reduction of ketones.
Adding DCMU, an inhibitor of photosynthesis,
decreased the chemical and enantiomeric purities of
reduction products even under illumination.
Figure 2. The effects of concentration of DCMU on reduc-
tion of a,a-difluoroacetophenone by S. elongatus PCC 7942.
ria through the photosynthetic process. These physio-
logical changes induce expression and/or activation of
enzymes. Thus, enzymes that produce the R-alcohol
become active. We are not able to determine which
mechanism is plausible at the present time. Under
darkness, the reducing ability may stem from the oxida-
tion of alcohols such as saccharides; the S-enzymes
preferentially use the thus generated reduced form of
the coenzyme. The R-enzymes can also participate in
These results suggest that physiological changes
through photosynthesis have effects on the enzymatic
activities in the reduction of ketones by cyanobacteria.
Cyanobacteria are photosynthetic algae that perform
photosynthesis with the aid of light energy. Light con-

2
532
K. Nakamura, R. Yamanaka / Tetrahedron: Asymmetry 13 (2002) 2529–2533
Figure 3. Inhibition mechanism of DCMU in reduction of ketones by S. elongatus PCC 7942.
Table 2. Reduction of acetophenone derivatives by S. elongatus PCC 7942
R
Light
Yield (%)
Ee (%)
Config.
CH3
CH3
On
Off
On
Off
On
Off
On
Off
3
n.r.
35
8.5
77
21
62
19
\99
–
R
R
R
R
R
R
R
R
CH F
71
27
66
14
63
61
2
CH F
2
CHF2
CHF2
CF3
CF3
Substrate; 10 mmol, biocatalyst as dry weight: 1 g/L, medium; 20 mL.
trols the life of algae. Besides driving photosynthesis—
the process supporting algal autotrophic life, light con-
trols the signals for circadian phenomena and
photomorphogenesis. These physiological phenomena
are usually induced by changes in the activities of
enzymes. Therefore, light is the most important regu-
lator for selectivities in photosynthetic whole-cell-cata-
lyzed reactions.
4. Experimental
4.1. General
Synechococcus elongatus PCC 7942 was obtained from
the Institut Pasteur. Gas chromatographic analyses
were performed using chiral GC-column (CP-cyclodex-
trin-B-2,3,6-M-19 [CPCD]; 25 m; He 2 mL/min)
1
4

K. Nakamura, R. Yamanaka / Tetrahedron: Asymmetry 13 (2002) 2529–2533
2533
equipped on Shimadzu GC-14B with C-R6A. Yields were
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and 25°C, and the resulting mixture was extracted with
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min: a,a-difluoroacetophenone; CPCD 120°C, ketone 4.1
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3
4
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1
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4
.4. Method used to investigate the effect of addition
of DCMU on the reduction of a,a-difluoroacetophenone
by S. elongatus PCC 7942
a,a-Difluoroacetophenone (10 mmol) was added to a
suspended culture of S. elongatus PCC 7942 (1 g/L as dry
weight) in BG-11 medium (20 mL) with or without
DCMU (10 mM) in DMSO (100 mL). The mixture was
shaken at 140 rpm and 25°C for 24 h under illumination
7
8
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−
1
−2
(
fluorescent light; 13.4 photons mmol s m ) or darkness
and the resulting mixture was extracted with ether and
eluted by Extrelut. The chemical and enantiomeric puri-
ties were determined by GC analyses.
1
1
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4
.5. Method used to investigate the effect of DCMU
1
concentration on the reduction of a,a-difluoroacetophe-
none by S. elongatus PCC 7942
a,a-Difluoroacetophenone (10 mmol) was added to a
suspended culture of S. elongatus PCC 7942 (1 g/L as dry
weight) in BG-11 medium (20 mL) with DCMU (0, 0.1,
5
, 100 mM) in DMSO (100 mL). The mixture was shaken
at 140 rpm and 25°C for 30 h under illumination
−
1
−2
(
fluorescent light; 13.4 photons mmol s m ) and the
resulting mixture was extracted with ether and eluted by
Extrelut. The chemical and enantiomeric purities were
determined by GC analyses.
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