Asymmetric reduction of ketones with a germinated plant

Article abstract of DOI:10.1016/j.tetasy.2007.12.015

A germinated radish sprout was used as a novel type of biocatalyst for the asymmetric reduction of ketones. The reactions proceeded with high enantioselectivities (>99% ee). The biocatalyst is easily obtainable from commercially available vegetable seeds and is very easy to use.

Full text of DOI:10.1016/j.tetasy.2007.12.015

Available online at www.sciencedirect.com
Tetrahedron: Asymmetry 19 (2008) 157–159
Asymmetric reduction of ketones with a germinated plant
Kiyoko Matsuo,a Sei-ichiro Kawabe,a,* Yosuke Tokuda,a Takashi Eguchi,a
Rio Yamanakab and Kaoru Nakamurac,*
aCollege of Life Science, Kurashiki University of Science and the Arts, 2640 Nishinoura, Tsurajima-cho, Kurashiki,
Okayama 712-8505, Japan
bFaculty of Pharmaceutical Science, Himeji Dokkyo University, 7-2-1 Kami-Ohno, Himeji, Hyogo 670-8524, Japan
cInstitute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
Received 31 October 2007; accepted 6 December 2007
Abstract—A germinated radish sprout was used as a novel type of biocatalyst for the asymmetric reduction of ketones. The reactions
proceeded with high enantioselectivities (>99% ee). The biocatalyst is easily obtainable from commercially available vegetable seeds
and is very easy to use.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
The merits of using vegetable seeds as pre-biocatalysts are
as follows. (1) Vegetables seeds are obtainable all over the
world. (2) Seeds are well preserved for a long time. (3)
Seeds have an ability to germinate at any time of the year
if suitable conditions for the germination are provided.
Until now, many kinds of biocatalysts have been applied
for useful biotransformations.1 For asymmetric reductions,
isolated enzymes, microbes such as yeasts and fungi, and
plant cell cultures have been used.2 Microbes and plant cell
cultures are more useful than isolated enzymes due to the
existence of many enzymes that catalyze various reactions.
Furthermore, in the asymmetric reduction of substrates,
whole living cell systems do not require cofactors and
cofactor-regeneration systems since they already have these
requirements. However, it is not easy to obtain these cell
cultures for organic chemists who are not familiar with cell
cultivation. On the other hand, another category of bio-
catalysts baker’s yeast2 and vegetables,3 has been applied
to organic synthesis because these biocatalysts are easily
obtainable from markets and easily manipulated by organ-
ic chemists. However, an essential problem, the reproduc-
ibility of experiments with raw baker’s yeast and
vegetables, has remained since the conditions of these bio-
catalysts are easily changeable depending on the places
they come from. Moreover, even from the same area, it is
difficult to obtain the biocatalyst with the same condition
constantly throughout a year. To solve these problems,
we have developed a novel biocatalyst that does not depend
on the place and/or time of isolation and still universally
available. This is a sprout prepared from a vegetable seed.
2. Results and discussion
We have investigated asymmetric reductions by germinated
plants obtained from commercially available seeds. If con-
ditions of germination and growth for plants can be opti-
mized, uniform and reproducible germinated plants
should be easily obtained. The radish sprout was selected
as the biocatalyst since radish seed is one of the most
obtainable seeds and the germination of radish is very easy.
Indeed, the biocatalysts, germinated radish (Raphanus sat-
ivus L.), reduced ketones, a,a,a-trifluoroacetophenone and
o-chloroacetophenone4,5 to the corresponding alcohols
with high enantioselectivities.
Radish seeds were obtained commercially and the surface
of the seed was sterilized by rinsing with water and 1% so-
dium hypochlorite and soaked in water for a while. Then
the seeds were placed on sterilized wet paper filters in the
dark for 1–2 days at 20 °C to be germinated. A grain of ger-
minated radish was cultivated in 30 mL of LS medium6
with 3% sucrose for 10 days7 to obtain the uniform radish
sprout (Biocatalyst) (Fig. 1). The radish sprout (7 1 g)
was transferred to the flask containing 30 mL of LS
*
Corresponding authors. E-mail: nakamura@scl.kyoto-u.ac.jp
0957-4166/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.12.015

158
K. Matsuo et al. / Tetrahedron: Asymmetry 19 (2008) 157–159
weight of radish (right vertical axis), chemical yields, and
ee (left vertical axis); cultivation = 10 days and reac-
tion = 1 day in the reduction of trifluoroacetophenone
(data are the average of more than 3 experiments.). Sucrose
was added at cultivation and also during reaction of the
substrate.
10 days
under illumination
in LS medium at 20 ºC
Surface sterilized
Radish seeds
in water
1-2 days on the wet paper
in the dark at 20 ºC
The fresh weight of radish increased monotonically against
the amount of sucrose (0.4 g without sucrose and 7 g with
4% sucrose added) and the yield of the reduction also
increased from 40% (without addition of sucrose) to
100% (2 g of sucrose was added). The selectivity of the
reduction changed from (R) to (S) by adding sucrose at cul-
tivation time. The (S)-enzyme which gives the (S)-alcohol
will be produced largely compared to the (R)-enzyme by
the effect of sucrose. Then, the addition of sucrose exclu-
sively enhances the reduction of the ketone to the (S)-alco-
hol and changes the stereoselectivity from (R) to (S). On
the contrary, the addition of sucrose at the reaction time
changed the stereoselectivity of the reduction slightly; the
alcohol with 46% ee (S) was obtained from the reaction
in the presence of sucrose and that of 40% ee (S) is pro-
duced from the reaction without adding sucrose (sucrose-
cultivated radish was used in both the reactions; yields were
99% in both the cases). Thus, the change of stereoselectivity
is thought to be the result of producing (S)-enzymes at cul-
tivation time.
Figure 1. Preparation of the biocatalyst.
medium and 30 lL of a,a,a-trifluoroacetophenone solution
(10% in DMSO) was added. The reaction was conducted
under illumination (4000 Lux) for 1–4 days at 20 °C
(Scheme 1).
OH
Ph
O
F3C
>99% ee
OH
F3C
O
Ph
Cl
Cl
>99% ee
Scheme 1. Reduction of ketones with germinated radish.
Then, the reaction mixture was extracted with ether and
chemical yield and ee of trifluorophenylethanol were deter-
mined by GC analyses using an internal standard (naph-
thalene).8,9 Thus, the corresponding (S)-alcohol was
obtained in 30–40% yield with 90–100% ee. With the same
method, o-chloroacetophenone was reduced and afforded
the corresponding (S)-alcohol in >99% ee (Scheme 1).
Interestingly, although (S)-alcohols were obtained in both
reactions, the absolute configurations are different accord-
ing to definition. The difference in stereoselectivities of the
reduction is due to the existence of plural enzymes that par-
ticipate in the reduction. The same phenomenon has been
observed in the reduction with Geotrichum candidum.9
Sucrose affects the growth of the biocatalyst and also accu-
mulates the reducing power. On the contrary, the addition
of sucrose at the reaction time did not affect the chemical
yield or the enantioselectivity of the reaction regardless of
the pre-treatment of the biocatalyst.
The other substrate, o-chloroacetophenone was also used
to check the stereochemical conversion by the addition of
sucrose. However, the reduction of the substrate without
adding sucrose at cultivation time did not proceed regard-
less of adding sucrose at the reaction time.
It is noteworthy that the addition of sucrose largely affected
the growth of the plant and bioconversion of the ketone.
Figure 2 shows the effect of sucrose on the fresh
The moderate enantioselectivity of the product was im-
proved up to 100% ee by using long reaction times (3 days)
although the chemical yield of the alcohol decreased
slightly. This phenomenon is explainable with the idea of
enantioselective decomposition of (R)-alcohol by radish.
Indeed, the reaction of the racemic alcohol with radish
afforded (S)-alcohol in 80% ee after 3 days.
Yield (%)
ee (R+)%
Fresh w eigh(g)
100
80
10
60
8
6
4
2
0
40
3. Conclusion
20
0
To conclude, we present herein a novel method for asym-
metric reduction. The proposed system should be univer-
sally applicable not only for asymmetric reduction, but
also for other biocatalytic reactions. Other plant seeds
may be applicable with the present method.
-20
-40
-60
-80
-100
0
1
2
3
4
References
sucrose (%)
Figure 2. Effect of sucrose on yield, ee and fresh weight of radish on the
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medium6 with 0–4% sucrose (30 mL) was placed in an 100-ml
Erlenmeyer flask, which was covered with silicone caps and
sterilized (120 °C, 20 min). A germinated radish was added to
this Erlenmeyer flask, and was shaken for 10 days at 20–25 °C
at 120 rpm, 4000 Lux.
8. Reaction: Sterilized LS medium (30 mL) with the same sucrose
concentration, as used for germination, was freshly prepared in
a 100-ml Erlenmeyer flask. 10% (w/v) trifluoroacetophenone in
DMSO (30 lL) and a radish obtained as above were added to
the flask, and shaken for 24 h at 20–25 °C at 120 rpm,
4000 Lux. After the reaction, the resulting mixture was
extracted with ether (5 mL Â 2) and eluted by Extrelut
(Merck). The chemical yield and ee of trifluorophenylethanol
were determined by GC analyses using an internal standard
(naphthalene).9 Gas chromatograph: column; CP-cyclodex-
trin-B2,3,6-M-19; 25 m, trifluoroacetophenone; temperature
120 °C; retention time ketone, 2.94 min; (S)-alcohol,
15.64 min; (R)-alcohol, 16.36 min, o-chloroacetophenone;
temperature 130 °C, ketone, 7.67 min; (S)-alcohol, 15.04 min;
(R)-alcohol, 17.77 min.
6. LS medium: Linsmaier, E. M.; Skoog, F. Physiol. Planta. 1965,
18, 100–127.
7. Preparation of radish sprout: Radish seeds were obtained
commercially from Takii Co. Ltd, Kyoto, Japan. Surface
sterilized seeds were produced by 5 min rinsing in water,
10 min exposure to 1% sodium hypochlorite, and exhaustive
rinsing with sterilized water. The seeds were stored on
sterilized wet paper filters for 1–2 days in the dark at 20–
25 °C to provide the germinated radish. A portion of LS liquid
9. Matsuda, T.; Harada, T.; Nakajima, N.; Itoh, T.; Nakamura,
K. J. Org. Chem. 2000, 65, 157–163.