Highly enantioselective bioreduction of prochiral ketones by stem and germinated plant of Brassica oleracea variety italica

Article abstract of DOI:10.3109/10242422.2011.635302

An eco-friendly and environmentally benign asymmetric reduction of a broad range of prochiral ketones employing Brassica oleracea variety italica (stems and germinated plant) as a novel biocatalyst was developed. It was found that B. oleracea variety italica could be used effectively for enantioselective bioreduction in aqueous medium with moderate to excellent chemical yield and enantiomeric excess (ee). This process is more efficient and generates less waste than conventional chemical reagents or microorganisms. Both R- and S-configurations were obtained by these asymmetric reactions. The best ee were achieved for pyridine derivatives (92-99%). The ee in germinated plant reactions were significantly higher than those of stem reactions. The low cost and the easy availability of these biocatalysts suggest their possible use for large scale preparations of important chiral alcohols.

Full text of DOI:10.3109/10242422.2011.635302

Biocatalysis and Biotransformation, November–December 2011; 29(6): 328–336
ORIGINAL ARTICLE
Highly enantioselective bioreduction of prochiral ketones by stem
and germinated plant of Brassica oleracea variety italica
MEHDI MOHAMMADI, MARYAMYOUSEFI & ZOHREH HABIBI
Department of Chemistry, Faculty of Science, Shahid Beheshti University, G.C.,Tehran, Iran
Abstract
An eco-friendly and environmentally benign asymmetric reduction of a broad range of prochiral ketones employing Brassica
oleracea variety italica (stems and germinated plant) as a novel biocatalyst was developed. It was found that B. oleracea
variety italica could be used effectively for enantioselective bioreduction in aqueous medium with moderate to excellent
chemical yield and enantiomeric excess (ee). This process is more efficient and generates less waste than conventional
chemical reagents or microorganisms. Both R- and S-configurations were obtained by these asymmetric reactions. The
best ee were achieved for pyridine derivatives (92–99%). The ee in germinated plant reactions were significantly higher
than those of stem reactions.The low cost and the easy availability of these biocatalysts suggest their possible use for large
scale preparations of important chiral alcohols.
Keywords: bioreduction, prochiral ketones, Brassica oleracea variety italica
Introduction
pharmaceuticals due to their versatile chemical prop-
Plants represent a unique class of potential biocata-
lysts for transformation of important non-natural
substrates. Over the past decade, several investiga-
tions have been carried out on the biotransformation
of foreign substrates (Baladassare et al. 2000).These
processes are more efficient and generate less waste
than conventional chemical reagents, enzymes, or
microorganisms (Kumaraswamy & Ramesh 2003;
Ward &Young 1990;Yousefi et al. 2010). Reactions
using chemical methods involve the use of expensive
chiral reagents and environmentally hazardous heavy
metals, while reactions using isolated enzymes
require costly cofactors (NADH, NADPH) which
require efficient regeneration (Noyori 1994). Even
with whole cells such as baker’s yeast, which is, by
far,the most widely used microorganism for biotrans-
formation (yielding the corresponding products with
fair to excellent enantioselectivity), recovery of the
desired product might not be straightforward.
erties (Chartrain et al. 2001; Daußmann et al. 2006;
Huisman et al. 2010; Strauss et al. 1999). Asymmet-
ric reduction of the corresponding prochiral ketones
is an effective route to produce chiral alcohols (Kroutil
et al. 2004), and biocatalysis, involving either isolated
oxido-reductases or living organisms, is one of the
most promising methods due to its high enantiose-
lectivity, mild reaction conditions and environmental
compatibility (Faber 1997;Moore et al.2007;Schmid
et al. 2001). Among different biocatalysts, plants are
excellent alternatives to the use of isolated enzymes,
since the oxido-reductase, cofactor (NADPH) and its
regeneration system are all located inside the plant,
and the addition of the expensive cofactor can be
avoided (Nakamura et al. 2003). Excellent results
have been obtained in the reduction of natural
ketones, since they are easily accepted by plant cells
(Nagaoka 2004). Over the last decade, various exam-
ples of the reduction of prochiral ketones to chiral
alcohols have been reported using growing (Villa
et al. 1998) or immobilized (Naoshima & Akakabe
1991) plant cell cultures.
Considerable attention has been paid to the syn-
thesis of enantiomerically pure compounds as chiral
synthons, which are increasingly in demand for devel-
opment of modern drugs and agrochemicals. Chiral
alcohols are building blocks for numerous chiral
Recently, the feasibility of direct use of different
parts of plants has been investigated by using both
Correspondence: Zohreh Habibi, Department of Chemistry, Faculty of Science, Shahid Beheshti University, G.C., Tehran, Iran. Tel: +98-21-29902710.
Fax: +98-21-22431663. E-mail: z_habibi@sbu.ac.ir
(Received 23 March 2011; revised 26 June 2011; accepted 22 October 2011)
ISSN 1024-2422 print/ISSN 1029-2446 online © 2011 Informa UK, Ltd.
DOI: 10.3109/10242422.2011.635302

Enantioselective bioreduction of ketones by Brassica oleracea 329
freshly cut carrot root (Baladassare et al. 2000;Yadav
increase the contact of the substrate with the bio-
catalyst, the external layer was removed, and the rest
was carefully cut into small thin pieces (approxi-
mately 1 cm long thread-like pieces). B. oleracea
seeds were obtained commercially from Falate Iran
Co. Tehran, Iran. The reaction products were ana-
lyzed by gas chromatography using a Thermoquest-
Finnigan (USA) instrument equipped with a
HP-CHIRAL-20B column and flame ionization
detector. Nitrogen was used as the carrier gas at a
constant flow rate of 1.5 mL/min. The injector and
detector (FID) temperature were kept at 250°C and
280°C, respectively. HPLC analyses of some alco-
hols were performed using a Shimadzu LC-10AD
instrument. The column was Chiralcel OD, 4.6ϫ
250 nm.The eluents were hexane/2-propanol (HPLC
grade, 85:15) at 0.5 mL per min flow rate and mon-
itored at 254 nm wavelength. Optical rotations were
measured with a Perkin–Elmer 241 polarimeter
using chloroform as solvent. Configuration and
enantiomeric excess (ee) of all the compounds was
determined by GC and HPLC analyses as well as
comparing the optical rotations with those reported
et al. 2002) and germinated radish sprout (Matsuo
et al. 2008) in the reduction of homochiral ketones.
In the context of the present study, Suraèz-Franco
and co-workers have investigated the ability of B.
oleracea variety italica (broccoli) to reduce aromatic
aldehydes (Suárez-Franco et al. 2010).
However, a problem with this approach is the
reproducibility of experiments, since the properties
of these biocatalysts may depend on their origins.
Moreover, even if sourced from the same area, it is
difficult to obtain biocatalysts with the same proper-
ties consistantly throughout a year.To address these
problems, we have used both freshly cut stems of
B. oleracea and a sprout of vegetable seeds for reduc-
tion of different prochiral ketones (Figure 1).
Materials and methods
General
All ketones were purchased from Merck (Germany).
B. oleracea were obtained from a local market. To
O
O
O
N
O
N
N
1
5
2
3
4
8
O
O
O
O
NC
F3C
6
7
O
O
O
O
CF3
O2N
Br
9
10
11
12
O
O
O
O
Cl
Br
F
C6H11
Br
F
Br
13
17
14
15
16
O
O
O
O
O
O
Cl
S
O
O
Ph
O
18
19
20
Figure 1. Chemical structures of the investigated ketones.

330 M. Mohammadi et al.
in the literature. Authentic standards of racemic
alcohols were analysed by GC and HPLC before
analysis of the reaction mixtures. The NMR spectra
were recorded using a Bruker AVANCE-300 spec-
trometer. Control experiments were done wherein
only the solvent was added. All other chemicals were
analytical grade from various commercial sources.
Experiments were performed in duplicate.
temperature. Then the broccoli stems were filtered
off and washed with water.The combined filtrate was
extracted as previously mentioned. The compound,
1-(pyridin-4-yl) ethanol, was obtained after column
chromatography with silica gel.
Recycling of the B. oleracea stems
After completion of the reduction, the reaction mix-
ture was filtered and the B. oleracea stems washed
successively with water and then kept in distilled
water at 0°C for 2 days. Afterwards, the water was
decanted and the B. oleracea stems wiped with soft
tissue paper to remove the remaining water. These
stems were used further for another reduction reac-
tion. It was observed that the activity of the stems
was lost completely and no conversion was achieved
for 1-(pyridin-4-yl) ethanone after 10 days incuba-
tion. So it was concluded that the plant cells lose their
activity completely after one round of reduction.
Reduction of ketones with B. oleracea stems
The B. oleracea stems were rinsed with 5% sodium
hypochlorite solution and sterile distilled H2O, and
cut into small pieces with a sterile knife. In separate
experiments ketones 1–20 (50 mg) were individually
added to a suspension of the freshly cut B. oleracea
stems (10 g) in H2O (100 mL). The mixtures were
incubated in a shaker (200 rpm) at room tempera-
ture for 2–4 days, and the reaction process was mon-
itored by TLC. Each individual suspension was
filtered, and the residue washed with water. The
aqueous solutions were then extracted with EtOAc
(3ϫ150 mL), and the organic phases evaporated
under reduced pressure. The residues were loaded
on a short silica gel column, using n-hexane as elu-
ant, to afford the reduced products.
GC and HPLC analysis
1-(Pyridin-4-yl)ethanol. GC analysis, Oven:T1ϭ90°C
for 10 min, T2ϭ125°C (DTϭ1°C/min) for 5 min,
T3ϭ200°C (DTϭ5°C/min) for 5 min, Retention
time: tRϭ14.2 min, tSϭ15.02 min. (Nakamura et al.
2002).
Reduction of ketones with B. oleracea germinated seeds
B. oleracea seeds were obtained commercially and
were sterilized in three steps: 1. Rinsing with water
and 1% sodium hypochlorite and soaking in water
for a while; 2. Sterilizing with ethanol (60 s); 3.
Rinsing with H2O2 (10% v/v). Then the seeds were
placed on sterilized wet paper filters in dark for 3
days at room temperature to be germinated. A grain
of germinated seed was cultivated in 10 mL of MS
(Murashige & Skoog 1962) medium with 3% sucrose
for 8 days to obtain the biocatalyst. The B. oleracea
sprout was transferred to a culture tube containing
10 mL of MS medium and 10% (w/v) of each ketone
in 20 μL of DMSO as cosolvent. The reaction was
continued under illumination (4000 Lux) for 24 h
at room temperature. The final suspensions were
then filtered off and the filtrates extracted with
EtOAc (3ϫ50 mL).
1-(Pyridin-3-yl)ethanol. GC analysis, Oven:T1ϭ90°C
for 10 min, T2ϭ125°C (DTϭ1°C/min) for 5 min,
T3ϭ200°C (DTϭ5°C/min) for 5 min, Retention time:
tRϭ9.8 min, tSϭ10.7 min. (Nakamura et al. 2002).
1-(pyridin-2-yl)ethanol. GC analysis, Oven:T1ϭ90°C
for 10 min, T2ϭ125°C (DTϭ1°C/min) for 5 min,
T3ϭ200°C (DTϭ5°C/min) for 5 min, Retention
time: tRϭ11.9 min, tSϭ13.1 min. (Yang et al. 2006).
1-Phenylethanol. GC analysis, Oven: T1ϭ100°C for
10 min, T2ϭ170°C (DTϭ3°C/min) for 5 min,
Retention time: tR ϭ13.46 min, tS ϭ14.31 min.
(Nakamura et al. 2002).
1,2,3,4-Tetrahydronaphthalen-1-ol. GC analysis,
Oven:T1ϭ120°C for 5 min,T2ϭ130°C (DTϭ1°C/
min) for 10 min, T3ϭ200°C (DTϭ5°C/min),
Retention time: tR ϭ19.9 min, tS ϭ20.8 min.
(Matharu et al. 2006).
Preparative scale reduction
B. oleracea stems (200 g), after rinsing with 5%
sodium hypochlorite solution and distilled water,
were cut into small pieces and placed in a conical
flask. To the B. oleracea stems in water, 0.5 g of
2-acetylpyridine () was added, and the mixtures
incubated in a shaker (200 rpm) for 3 days at room
1-p-Tolylethanol. Retention time: GC analysis, Oven:
T1ϭ100°C for 5 min, T2ϭ130°C (DTϭ1°C/min)

Enantioselective bioreduction of ketones by Brassica oleracea 331
rotation with the reported same molecule (Shimizu
et al. 2007).
for 5 min, T3ϭ180°C (DTϭ3°C/min), Retention
time: tR ϭ8.8 min, tS ϭ10.2 min. (Bandini et al.
2006).
2-Chloro-1-(4-bromophenyl)ethanol. HPLC analysis,
Retention time: tR ϭ34.8 min, tS ϭ36.1 min. (Ban-
dini et al. 2006).
4-(1-Hydroxyethyl)benzonitrile. GC analysis, Oven:
T1ϭ120°C for 5 min, T2ϭ130°C (DTϭ1°C/min)
for 10 min, T3ϭ200°C (DTϭ5°C/min), Retention
time: tR ϭ26.57 min, tS ϭ28 min. (Liu & Wolf
2007).
2-Bromo-1-(4-bromophenyl)ethanol. HPLC analysis,
Retention time: tR ϭ29.9 min, tS ϭ31.3 min. (Ban-
dini et al. 2006).
1-(4-(Trifluoromethyl)phenyl)ethanol. GC analysis,
Oven:T1ϭ100°C for 5 min,T2ϭ125°C (DTϭ1°C/
min) for 10 min, T3ϭ220°C (DTϭ5°C/min) for 5
min, Retention time: tR ϭ11.66 min, tS ϭ11.87 min.
(Savile & Kazlauskas 2005).
1-(Thiophen-2-yl)ethanol. GC analysis, Oven:
T1ϭ100°C for 5 min, T2ϭ170°C (DTϭ2.5°C/
min) for 5 min, Retention time: tR ϭ15.8 min,
tS ϭ16.6 min. (Matharu et al. 2006).
2,2,2-Trifluoro-1-phenylethanol. GC analysis, Oven:
T1ϭ100°C for 5 min, T2ϭ130°C (DTϭ1°C/min)
for 5 min, T3ϭ180°C (DTϭ3°C/min), Retention
time: tR ϭ8.9 min, tS ϭ9.2 min. (Matsuo et al.
2008).
1-(Furan-2-yl)ethanol.GCanalysis,Oven:T1ϭ100°C
for 5 min, T2ϭ170°C (DTϭ2.5°C/min) for 5 min,
Retention time: tR ϭ14.9 min, tS ϭ15.6 min.
(Matharu et al. 2006).
Benzyl 3-hydroxy butanoate. HPLC analysis, Reten-
tion time: tR ϭ15.1 min, tS ϭ17.2 min. (Nakamura
et al. 2002).
1-(4-Nitrophenyl)ethanol. GC analysis, Oven:
T1ϭ120°C for 5 min,T2ϭ130° C (DTϭ1°C/min)
for 10 min, T3ϭ200°C (DTϭ5°C/min), Retention
time:tR ϭ18.2 min, tS ϭ18.9 min. (Savile & Kazlaus-
kas 2005).
Methyl 4-chloro-3-hydroxy butanoate. GC analysis,
Oven:T1ϭ120°C for 5 min,T2ϭ130°C (DTϭ1°C/
min) for 10 min, T3ϭ200°C (DTϭ5°C/min),
Retention time: tR ϭ15.8 min, tS ϭ16.6 min. (Curt
et al. 1992).
1-Mesitylethanol. GC analysis, Oven:T1ϭ100°C for
5 min, T2ϭ130°C (DTϭ1 °C/min) for 5 min,
T3ϭ180°C (DTϭ3°C/min), Retention time:
tR ϭ11.9 min, tS ϭ12.4 min. (Savile & Kazlauskas
2005).
Result and discussion
In recent years various whole plant cells such as carrot,
apple, cucumber, onion, potato, sweet potato, radish,
cauliflower, pumpkin, artichoke, fennel and banana
have been used in plant-mediated reductions for syn-
thesis of chiral secondary alcohols (Bruni et al. 2006).
For finding reductases from new common vegetables,
broccoli (B. oleracea variety italica) was selected as a
novel biocatalyst for asymmetric reductions.Asymmet-
ric reduction of various prochiral ketones such as aro-
matic ketones and β-ketoesters to their corresponding
optically active secondary alcohols with moderate to
excellent yields and high ee was achieved.This method
is an eco-friendly environmental reduction system with
easy available and inexpensive biocatalyst.
1-(4-Bromophenyl)ethanol. GC analysis, Oven:
T1ϭ100°C for 5 min, T2ϭ130°C (DTϭ1°C/min)
for 5 min, T3ϭ180°C (DTϭ3°C/min) for 10 min,
Retention time: tR ϭ10.1 min, tS ϭ10.9 min. (Naka-
mura et al. 2002).
1-(4-Fluorophenyl)ethanol. GC analysis, Oven:
T1ϭ100°C for 5 min, T2ϭ130°C (DTϭ1°C/min)
for 5 min, T3ϭ180°C (DTϭ3°C/min), Retention
time: tRϭ9.2 min, tSϭ9.8 min. (Bandini et al.
2006).
1-(2-Bromo-4-cyclohexylphenyl)ethanol.
[α]2D5Ϫ25°
In an attempt to avoid potential irreproducibility
of reduction reactions due to variations in origin and
age of the material, we have also used the sprout
prepared from broccoli seed as a novel biocatalyst.
Since the seeds are obtainable from any location and
(CHCl3, c 0.6) for 90% ee, determined by HPLC
analysis, Retention time: tR ϭ22.1 min, tS ϭ19.8
min. Relative configuration of this compound was
determined by comparison of the sign of it's specific

332 M. Mohammadi et al.
Table I. 1H NMR data of the hydroxylated methine of the products
(300 MHz, CDCl3).
can be germinated at any time of the year under
suitable conditions, this method (Figure 2) should
be reproducible regardless of location.
Chemical
shift
A series of ketones were tested with the freshly cut
stems of B. oleracea variety italica in aqueous solution
at room temperature for 2–3 days.The crude reaction
mixtures were initially visualized byTLC, followed by
purification by thin layer chromatography. Structures
of all alcohols were confirmed by NMR spectroscopic
analysis.The 1H NMR spectra indicated the presence
of one signal characteristic of protons attached to
hydroxylated carbon.The oxygenated methine signals
were observed at δH 4–5.5 ppm.The downfield chem-
ical shift of this proton are reported in Table I. The
reaction mixtures were also analyzed by chiral GC or
HPLC for ee determination.
The results of asymmetric reduction by plant
stems and germinated seed are given inTables II and
III. In order to dissolve solid ketones a small quantity
of DMSO was used as a co-solvent. Both S-form and
R-form alcohols were obtained, but both biocatalysts
afforded the same enantiomer for each ketone with
variable yields (10–100%) and ee (69–99%). Results
showed that the ee increased for almost all of the
alcohols employing germinated plant compared with
B. oleracea stems. This increase is highly remarkable
for entries 8 (ϩ25), 13 (ϩ14), entry 15 (ϩ18) and
entries 4 and 16 (ϩ10).
Products
(ppm)
J (Hz)
1-(pyridin-4-yl)ethanol
1-(pyridin-3-yl)ethanol
1-(pyridin-2-yl)ethanol
1-phenylethanol
1,2,3,4-tetrahydronaphthalen-
1-ol
1-p-tolylethanol
4-(1-hydroxyethyl)
benzonitrile
1-(4-(trifluoromethyl)phenyl)
ethanol
2,2,2-trifluoro-1-
phenylethanol
1-(4-nitrophenyl)ethanol
1-mesitylethanol
4.88
4.92
4.84
4.88
4.71
q, 6.45
q, 6.42
q, 6.54
q, 6.16
t, 3.83
4.85
4.92
q, 6.40
q, 5.99
4.97
5.03
q, 6.25
q, 6.75
4.95
–
4.84
4.87
4.98
q, 6.49
–
q, 6.43
q, 6.33
q, 6.43
1-(4-bromophenyl)ethanol
1-(4-fluorophenyl)ethanol
1-(2-bromo-4-
cyclohexylphenyl)ethanol
2-chloro-1-(4-fluorophenyl)
ethanol
4.91
d, d (8.19, 3.42)
br
2-bromo-1-(4-bromophenyl)
ethanol
4.89–4.92
1-(thiophen-2-yl)ethanol
1-(furan-2-yl)ethanol
benzyl 3-hydroxy butanoate
methyl 4-chloro-3-hydroxy
butanoate
4.99
5.08
4.19–4.25
4.22–4.27
q, 6.11
q, 6.05
m
m
Asymmetric reduction of acetophenones afforded
theR-chiralityatthecarbonatombearingthehydroxyl
group which follows anti-Prelog's rule. This result is
in accordance with reported configuration obtained
from potato (Solanum tuberosum), sweet potato (Ipo-
moea batatas) and apple (Malus pumila) which all fol-
lowed anti-Prelog's rule (Yang et al. 2008). Compared
with the results reported for baker’s yeast and other
microorganisms, the results are attractive since in the
reduction system of plant cell cultures the hydrogen
attack takes place preferentially from the re-face of
the carbonyl group to give the S-enantiomer of cor-
responding alcohol (Hamada et al. 1988; Hirata et al.
1982). As can be seen in Tables II and III four deriv-
atives of acetophenone (entries 9, 14–16) gave the
S-configuration and follow Prelog's rule but all the
other derivatives (entries 6–8,10–13,20) have R-
configuration. The enantioselectivities of reduction
were increased for all acetophenone derivatives by
using germinated seed especially for p-trifluoromethyl
acetophenone (95% ee vs. 70% ee).
An examination of the experimental results
reveals that among the acetophenone derivatives,
phenyl trifluoromethyl ketone, p-trifluoromethyl
acetophenone and p-fluoroacetophenone with elec-
tron-withdrawing substituents were efficiently
reduced with significant enantioselectivities. It seems
that the carbonyl group is activated by these sub-
stituents. On the other hand steric hindrance caused
poor chemical yield such as with mesityl acetophe-
none (8 and 11% in germinated seed and stems
respectively).
It is noteworthy that for one of the acetophenone
derivatives, 2-bromo-1-(4-methoxyphenyl) etha-
none, (result not shown) no reduction of the carbo-
nyl group was observed (Figure 3), instead the bromo
group was replaced by a hydroxyl with high yield
(75%) while in analogous compounds, 2-bromo-1-
(4-bromophenyl) ethanone and 2-chloro-1-(4-
fluorophenyl) ethanone the carbonyl group was
Figure 2. Asymmetric reduction of prochiral ketones by ger-
minated seed.

Enantioselective bioreduction of ketones by Brassica oleracea 333
Table II. Reduction of ketones with stems of B. oleracea variety italic.
Time of
conversion
(day)
Entry
Compounds
Products
Config.a
Yield(%)b
ee(%)b
1
2
3
4
5
1-(pyridin-4-yl)ethanone
1-(pyridin-3-yl)ethanone
1-(pyridin-2-yl)ethanone
1-phenylethanol
3,4-dihydronaphthalen-
1(2H)-one
1-(pyridin-4-yl)ethanol
1-(pyridin-3-yl)ethanol
1-(pyridin-2-yl)ethanol
1-phenylethanol
R
R
R
R
S
100
97
98
75
76
94
92
92
79
92
3
3
3
4
2
1,2,3,4-tetrahydronaphthalen-1-ol
6
7
8
1-p-tolylethanone
4-acetylbenzonitrile
1-(4-(trifluoromethyl)
phenyl)ethanone
2,2,2-trifluoro-1-
phenylethanone
1-p-tolylethanol
4-(1-hydroxyethyl)benzonitrile
1-(4-(trifluoromethyl)phenyl)ethanol
R
R
R
58
97
96
82
91
70
3
2
2
9
2,2,2-trifluoro-1-phenylethanol
1-(4-nitrophenyl)ethanol
S
97
65
94
85
3
3
10
1-(4-nitrophenyl)
ethanone
R
11
12
1-mesitylethanone
1-(4-bromophenyl)
ethanone
1-mesitylethanol
1-(4-bromophenyl)ethanol
R
R
11
78
96
89
2
3
13
14
1-(4-fluorophenyl)
ethanone
1-(2-bromo-4-
cyclohexylphenyl)
ethanone
1-(4-fluorophenyl)ethanol
R
S
95
47
78
94
3
2
1-(2-bromo-4-cyclohexylphenyl)
ethanol
15
16
17
2-chloro-1-(4-
fluorophenyl)ethanone
2-bromo-1-(4-
bromophenyl)ethanone
1-(thiophen-2-yl)
ethanone
2-chloro-1-(4-fluorophenyl)ethanol
2-bromo-1-(4-bromophenyl)ethanol
1-(thiophen-2-yl)ethanol
S
S
63
71
14
69
78
91
2
2
3
R
18
19
20
1-(furan-2-yl)ethanone
benzyl 3-oxobutanoate
methyl 4-chloro-3-
oxobutanoate
1-(furan-2-yl)ethanol
benzyl 3-hydroxy butanoate
methyl 4-chloro-3-hydroxy butanoate
R
S
R
10
69
74
95
82
76
3
2
3
aThe configurations were assigned by comparison of the sign of the specific rotation of each alcohol with those of the reported molecules.
bConversion and ee were determined by GC and HPLC analysis of crude extract.
reduced and no substitution was observed. At pres-
ent, we have no satisfactory explanation for this phe-
nomenon.
For investigating the reduction of halogen-
containing aromatic ketones, 4¢-bromo and
4¢-fluoroacetophenones were chosen as model sub-
strates. As mentioned above, the configuration of
the resulting alcohols were similar to that from
acetophenone. In stems of broccoli, yields were
higher than that of acetophenone, while ee for 4¢-
fluoroacetophenone (78%) were similar to acetophe-
none (79%) but higher for 4¢-bromoacetophenone
(89%). The ee obtained using the germinated seed
(Table III) were notably higher for 4¢-bromo and
4¢-fluoroacetophenones (94%, 92% respectively)
than acetophenone (89%). This phenomenon was
OH
Br
O
O
Br
O
OH
O
O
Figure 3. Biotransformation of 2-bromo-1-(4-methoxyphenyl)ethanone by stem and germinated plant of B. oleracea.

334 M. Mohammadi et al.
methyl-4-chloro-3-oxobutanoate both with plant
stems and germinated seed (76%, 81% ee respec-
tively).Incomparisonbenzyl-3-oxobutanoateafforded
a Prelog-like selectivity (82%, 81% ee respectively).
The yields of reactions were higher with plant stems
(74%, 69%) compared with germinated plant (56%,
52%).Yang and co-workers reported an S-configura-
tion for the reduced product of ethyl 4-chloroacetoac-
etate using several plant tissues among which the best
results were obtained with carrot (45.5% yield, 91.0%
ee) (Yang et al. 2008) Blanchard and Van De Weghe
(2006) also investigated reduction of 4-chloro and
4-trifluoromethyl acetoacetate. The Prelog rule was
obeyed by 4-chloroacetoacetate, as seen with carrot
and baker's yeast as biocatalyst which resulted in the
S-form of the alcohol (90%, 55% ee respectively) but
4-trifluoromethyl acetoacetate showed an anti-Prelog
selectivity (R-configuration) with 78%, 62% ee respec-
tively.With these substrates, germinated seeds did not
improve the ee values significantly.
also reported for 4¢-chloroacetophenone reduction
by different plant tissues (Bruni et al. 2006) indicat-
ing that halogenated aromatic ketones are better
substrates in plant cells than simple aromatic ketones.
The halogen (here bromide and fluoride) also
enlarges the difference between the size of two sub-
stituents of the carbonyl, and therefore, improves the
enantioselectivity of this reaction. Also, the increase
in ee values observed by using germinated seeds was
greater for 4¢-fluoroacetophenone (ϩ14% increase)
than for 4¢-bromoacetophenone (ϩ5% increase).
Reduction of β-ketoesters to chiral alcohols had
been studied extensively using several plant cell
biocatalysts (carrot, apple, cucumber, onion, potato
and radish) and baker’s yeast (Yang & Yao 2004).
In this current study, methyl-4-chloro-3-oxobu-
tanoate and benzyl-3-oxobutanoate were chosen as
model substrates for examining β-ketoester asym-
metric reduction. Results are presented in Tables II
and III. An anti-Prelog selectivity was observed for
Table III. Reduction of ketones with germinated seed of B. oleracea variety italic.
ee%(compared
with brassica
stems)b
Time of
conversion
(day)
Entry
Compounds
Products
Config.a
Yield(%)b
1
2
1-(pyridin-4-yl)ethanone
1-(pyridin-3-yl)ethanone
1-(pyridin-4-yl)ethanol
1-(pyridin-3-yl)ethanol
R
R
78
94
1
1
99(ϩ5)
98(ϩ6)
3
4
5
1-(pyridin-2-yl)ethanone
1-phenylethanol
3,4- dihydronaphthalen-
1(2H)-one
1-(pyridin-2-yl)ethanol
1-phenylethanol
1,2,3,4-tetrahydronaphthalen-1-ol
R
R
S
88
71
75
1
1
1
97(ϩ5)
89(ϩ10)
91(Ϫ1)
6
7
8
1-p-tolylethanone
4-acetylbenzonitrile
1-(4-(trifluoromethyl)
phenyl)ethanone
2,2,2-trifluoro-1-
phenylethanone
1-(4-nitrophenyl)
ethanone
1-p-tolylethanol
R
R
R
57
81
78
1
1
1
90(ϩ8)
97(ϩ6)
95(ϩ25)
4-(1-hydroxyethyl)benzonitrile
1-(4-(trifluoromethyl)phenyl)
ethanol
9
2,2,2-trifluoro-1-phenylethanol
S
89
64
1
1
93(Ϫ1)
89(ϩ4)
10
1-(4-nitrophenyl)ethanol
R
11
12
1-mesitylethanone
1-(4-bromophenyl)
ethanone
1-mesitylethanol
1-(4-bromophenyl)ethanol
R
R
8
60
1
1
99(ϩ3)
94(ϩ5)
13
14
1-(4-fluorophenyl)
ethanone
1-(2-bromo-4-
cyclohexylphenyl)
ethanone
1-(4-fluorophenyl)ethanol
R
S
85
37
1
1
92(ϩ14)
90(Ϫ4)
1-(2-bromo-4-cyclohexylphenyl)
ethanol
15
16
17
2-chloro-1-(4-
fluorophenyl)ethanone
2-bromo-1-(4-
bromophenyl)ethanone
1-(thiophen-2-yl)
ethanone
2-chloro-1-(4-fluorophenyl)
ethanol
2-bromo-1-(4-bromophenyl)
ethanol
S
S
61
72
11
1
1
1
87(ϩ18)
88(ϩ10)
95(ϩ4)
1-(thiophen-2-yl)ethanol
R
18
19
20
1-(furan-2-yl)ethanone
benzyl 3-oxobutanoate
methyl 4-chloro-3-
oxobutanoate
1-(furan-2-yl)ethanol
benzyl 3-hydroxy butanoate
methyl 4-chloro-3-hydroxy
butanoate
R
S
R
12
52
56
1
1
1
93(Ϫ2)
81(Ϫ1)
81(Ϫ5)
aThe configurations were assigned by comparison of the sign of the specific rotation of each alcohol with those of the reported molecules.
bConversion and ee were determined by GC and HPLC analysis of crude extract.

Enantioselective bioreduction of ketones by Brassica oleracea 335
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R-alcohols. According to the results pyridine deriva-
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and germinated seed (97–99%). Chemical yields for
furan (10–12%) and thiofen (11–14%) were lower
than pyridine derivatives (88–100%) but ee were com-
parable with those of pyridine derivatives (91–95%).
To demonstrate the potential for large scale prep-
aration using this bioreduction protocol we con-
ducted a scaled-up reduction for the best substrate
with 100% chemical yield i.e. 2-acetylpyridine (0.5
g) using stems of B. oleracea (200 g). The ee and
isolated yield of the product were 90% and 76%
respectively.
Conclusion
We have reported a straightforward and mild eco-
friendly process for the synthesis of chiral alcohols
using B. oleracea variety italica as biocatalyst with
excellent yields and high optical purity. The key
features of the new process are the use of a low-
cost enzymatic system, the ease of work-up, high
enantioselectivity and mild conditions (water as
reaction media and room temperature). Aromatic,
halogen-containing aromatic ketones and β-ke-
toesters were efficiently reduced to their corre-
sponding alcohols with the best results being
obtained for pyridine derivatives, especially
3-acetylpyridine. Both R-and S-alcohols could be
obtained with these asymmetric reactions. Germi-
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Acknowledgment
The authors are thankful to Iran National Science
Foundation (INSF), Tehran, Iran.
Declaration of interests: This work was supported
by Iran National Science Foundation (INSF),Tehran,
Iran. The authors report no conflicts of interest. The
authors alone are responsible for the content and writ-
ing of the paper.
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