Light mediated cofactor recycling system in biocatalytic asymmetric
reduction of ketone
Kaoru Nakamura* and Rio Yamanaka
Institute for Chemical Research, Kyoto University, Uji Kyoto 611-0011, Japan.
E-mail: nakamura@scl.kyoto-u.ac.jp; Fax: +81 774 38 3201; Tel: +81 774 38 3201
Received (in Cambridge, UK) 23rd April 2002, Accepted 28th June 2002
First published as an Advance Article on the web 15th July 2002
Reduction of an artificial ketone by Synechococcus elonga-
tus PCC 7942 proceeds smoothly by the aid of light. The
efficiency of the reaction is very high since the coenzyme
NADPH is regenerated by using light energy.
Biocatalytic reduction is a useful tool for obtaining optically
active alcohols, and many scientists have studied reactions
using isolated enzymes, microbes and plant cell cultures as
Scheme 1 Reduction of 2A,3A,4A,5A,6A-pentafluoroacetophenone with S.
elongatus PCC 7942.
biocatalysts.1 Reduction of substrates usually requires a large
input of energy, and in microbial reductions, carbohydrates such
as sugars have been used to recycle the coenzyme. These
reaction proceeds in an excellent chemical yield ( > 90%) with
high enantioselectivity ( > 99% ee) (Scheme 1).
Since the perfluorinated phenyl group has a striking stacking
ability with electron-rich arenes,8 optically active alcohols
having pentafluorophenyl moiety are potentially useful chiral
building blocks for fine chemicals.9
carbohydrates are generated through photosynthesis with sun-
light energy. In other words, we have been indirectly using light
energy for asymmetric reduction.
Now we propose the direct use of light energy for such
reactions by using a biocatalyst that falls into a new category,
the phototroph, because it can directly use light energy.
For the majority of redox enzymes, nicotinamide adenine
dinucleotide [NAD(H)] and its respective phosphate
[NADP(H)] are required. These cofactors are prohibitively
expensive if used in stoichiometric amounts. Since it is only the
oxidation state of the cofactor that changes during the reaction,
it may be regenerated in situ by using a second redox-reaction
to allow it to re-enter the reaction cycle. Usually, formate,2
glucose,3 and simple alcohols such as ethanol4 and 2-propanol5
are used to generate the oxidized form of the coenzyme to the
reduced form. These reductants originally stem from bio-
products of CO2 by the aid of sunlight with phototroph.
Phototrophs such as algae and plants capture light energy to
generate NADPH from NADP+through photosynthetic elec-
tron-transfer reactions. Subsequently, CO2 is converted into
sugar, generally using NADPH6.
We propose that the reducing power of NADPH generated
through photosynthesis also can be used in the reduction of
exogenous substrates such as unnatural ketones to yield useful
optically active alcohols. Thus, cofactor-recycling is no prob-
lem when photosynthetic living cells are used as biocatalysts for
reduction. Accordingly, we can use solar energy directly for
bioconversion of artificial substrates.
We focus on cyanobacteria (microalgae) since they belong to
both phototroph and microbe categories, in other words, they
are plant-like photosynthetic bacteria. Therefore, the growth
rate of cyanobacteria is as high as that of typical microbes. The
problem of using cultured plant cells is that they usually grow
very slowly.
An advantage of the proposed biotransformation system is its
high substrate/biocatalyst (s/b) ratio ( = 2.0), which is compara-
ble to a low s/b ratio of the other biocatalysts (bakerAs yeast:
0.003–0.02 and plant cell cultures of Marchantia polymorpha:
0.001), therefore, a cyanobacterium-catalyzed reaction is very
effective. This supports the idea that the reaction proceeds by a
cofactor-recycling system that uses light energy.
To clarify our proposal, we investigated the effect of light and
the cofactor-dependency on the reduction of ketones. We
examined the effect of light on the reduction of a ketone by a
microbe (Fig. 1). This figure illustrates the time-course of the
yields of the alcohol. The line with the circles shows the yields
of the reduction under illumination, and the line with the
triangles shows the yields of the reduction under darkness. The
reaction rate under illumination is higher than that under
darkness. Actually, the initial rate under illumination is about
four times higher than that in the dark.
Furthermore, the line with the squares shows the yields in the
reduction under illumination after an initial two days under
darkness. Apparently, the reaction increased rapidly under
illumination. This graph supports the assumption of a direct
effect on the reaction using light.
Next, we investigated cofactor-dependency in reducing
ketones by using the cyanobacterium. A cell free extract
(acetone-dried powder) was prepared from the cyanobacterium,
Now we propose a new system for cofactor-recycling, in
which cyanobacteria converts light energy to reducing power.
The advantage of this reaction is that light energy, which is a
cheap resource, can be used since the microalgae possesses a
system that is required for production of reduced coenzymes,
NADPH.
We tried to reduce ketones by using a cyanobacterium under
illumination (see Experimental†).
We already reported that aryl methyl ketones are reduced to
the corresponding S-alcohols by Synechococcus elongatus PCC
7942, which is one of the cyanobacteria, that is a photosynthetic
prokaryote, with high enantioselectivity ( > 96% ee).7 When
2A,3A,4A,5A,6A-pentafluoroacetophenone is used as a substrate, the
Fig. 1 Effect of light on reduction of 2A,3A,4A,5A,6A-pentafluoroacetophenone
with Synechococcus elongatus PCC 7942.
1782
CHEM. COMMUN., 2002, 1782–1783
This journal is © The Royal Society of Chemistry 2002