Enantioselective reduction of acetophenone analogues using carrot and celeriac enzymes system

Article abstract of DOI:10.1016/j.cclet.2009.11.019

The enantioselective reduction of acetophenone analogues catalyzed by carrot and celeriac was performed in moderate conversions and excellent enantiomeric excesses. The steric factors and electronic effects of the substituents at the aromatic ring were found to significantly affect the efficiency of the enantioselective reduction of acetophenone analogues, while they had a little effect on the enantioselectivity of acetophenone analogues reduction. It was also found that the conversions of acetophenone analogues reduction at 33 °C by means of both biocatalysts were three times as great as those at room temperature.

Full text of DOI:10.1016/j.cclet.2009.11.019

Available online at www.sciencedirect.com
Chinese Chemical Letters 21 (2010) 305–308
www.elsevier.com/locate/cclet
Enantioselective reduction of acetophenone analogues
using carrot and celeriac enzymes system
a
b
c
Xiang Liu a, , Zheng Guang Pan , Jian He Xu , He Xing Li
*
a School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, China
b State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
c Department of Chemistry, Shanghai Normal University, Shanghai 200234, China
Received 17 August 2009
Abstract
The enantioselective reduction of acetophenone analogues catalyzed by carrot and celeriac was performed in moderate
conversions and excellent enantiomeric excesses. The steric factors and electronic effects of the substituents at the aromatic ring
were found to significantly affect the efficiency of the enantioselective reduction of acetophenone analogues, while they had a little
effect on the enantioselectivity of acetophenone analogues reduction. It was also found that the conversions of acetophenone
analogues reduction at 33 8C by means of both biocatalysts were three times as great as those at room temperature.
# 2009 Xiang Liu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Enantioselective reduction; Acetophenone analogues; Plant cells; Biocatalysis
There is a considerable interest in efficient routes to obtain chiral alcohols since they are useful building blocks for
the synthesis of drugs, agrochemicals, flavors and pigments. These alcohols can be obtained from the enantioselective
reduction of prochiral ketones by biological methods [1–4]. Among these biological methods, microbes and plant cell
cultures are more useful than isolated enzymes due to the existence of many enzymes that catalyze various reactions
[5,6]. In addition, the use of microbes and plant cell cultures is particularly advantageous for carrying out the desired
reduction since they do not require the addition of cofactors for their regeneration. It has been reported that reduction
of a carbonyl group may be performed using various plant biocatalysts [7–10]. Acetophenone is a kind of very
interesting model xenobiotic substrate for bioreduction, because it may give rise to both enantiomers of 1-
phenylethanol. These compounds, as well as their simple analogues, have been effectively used as building blocks for
asymmetric synthesis of drugs [11–14].
Here we would like to report the results of the reduction of acetophenone analogues catalyzed by carrot (Daucus
carota L.) and celeriac (Apium graveolens L.) and evaluate the substituent effects on activity and enantioselectivity in the
enzymatic reduction of these ketones. Aromatic ketones 1a–1g were used as the substrates for the biotransformations.
The bioreduction of the carbonyl group by the carrot’s and celeriac’s enzymes system proceeded enantioselectivity,
following the Prelog’s rule [15], which resulted in formation of (S)-alcohols predominantly (Scheme 1).
* Corresponding author.
E-mail address: liuxiang@jiangnan.edu.cn (X. Liu).
1001-8417/$ – see front matter # 2009 Xiang Liu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2009.11.019

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X. Liu et al. / Chinese Chemical Letters 21 (2010) 305–308
Scheme 1. Enantioselective reduction of 1a–1g catalyzed by carrot or celeriac.
Table 1
Bioreduction results of 1a–1g using carrot or celeriac as biocatalysts.
Entry
Substrate
Product
Biocatalyst and conditiona
Conversion (%)b
e.e. (%)b
1
2
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
A
A
A
A
A
A
A
B
B
B
B
B
B
B
97
26
12
39
18
43
48
100
37
19
58
28
65
71
96
99
99
95
94
96
93
99
99
99
92
90
93
94
3
4
5
6
7
1g
1a
1b
1c
1d
1e
1f
2g
2a
2b
2c
2d
2e
2f
8
9
10
11
12
13
14
1g
2g
a
A: Reductions were carried out at 33 8C for 28 h in distilled water (pH 7.0) using carrot as biocatalyst. B: Reductions were carried out at 33 8C for
28 h in 0.1 mol/L phosphate buffer (pH 6.2) using celeriac as biocatalyst.
b
The conversion of 1a–1g and the ‘‘e.e.’’ (enantiomeric excess) of 2a–2g were determined by chiral GC analysis of crude extracts.
The bioreduction results of 1a–1g were presented in Table 1. The results obtained showed that 1a was reduced to the
corresponding chiral alcohol with excellent conversion and enantioselectivity using both biocatalysts (entries 1 and 8
in Table 1), while the presence of a substituent at the aromatic ring could reduce the conversion of 1b–1g (entries 2–7
and 9–14 in Table 1), especially the presence of o-substituent (entries 5 and 12 in Table 1). Therefore, the steric factors
of these substituents were unfavorable to the conversion of 1b–1g. Besides the steric factors, the electronic effects of
these substituents played an important impact on the activity of both biocatalysts. The electron-withdrawing character
of chloro and nitro group enhanced the activity of these biocatalysts and improved conversion of the substrates,
especially these strong electron-withdrawing such as m-chloro and p-nitro group (entries 6, 7, 13 and 14 in Table 1).
On the other hand, the electron-donating character of methyl and methoxy group had an opposite impact on this
activity and made against conversion of the substrates (entries 2, 3, 9 and 10 in Table 1). The results of electronic
effects were in accordance with the results reported by Zhu et al. [16] and Uwai et al. [17]. However, there were few
changes in the enantiomeric excesses of 2b–2g, no matter whether the aromatic ring of 2b–2g possessed an electron-
withdrawing or electron-donating substituent. This observation was in accordance with the results of the bioreduction
of methoxyacetophenone and bromoacetophenone by means of plant cells [18].
Our research also focused on the influence of reaction temperature on the activity of acetophenone analogues
bioreduction. It was observed that the conversions of 1a–1g at 33 8C by means of both biocatalysts were three times as
great as those of 1a–1g at room temperature (Table 2). The bioreduction of acetophenone analogues were also tested at
other temperatures (27 8C, 30 8C and 36 8C) and the results obtained were not as good as those at 33 8C. This
suggested that these ketoreducatases of whole cell systems existing in carrot and celeriac possessed higher activity at
33 8C, although a lot of bioreductions using whole plant cells (including carrot [19,20] and celeriac [18]) were carried
out at room temperature. The reaction temperature was another important factor which affected the activity of these
two biocatalysts.
The results obtained indicate that the reduction activity of acetophenone analogues by means of carrot’s and
celeriac’s enzymes system depends mainly on the steric factors and electronic effects of the substituents at the

X. Liu et al. / Chinese Chemical Letters 21 (2010) 305–308
307
Table 2
The conversion of acetophenone analogues reduction catalyzed by carrot or celeriac at different reaction temperatures.
Entry
Substrate
Product
Reaction temperature (8C)
Conversion (%)
Carrot
Celeriac
1
2
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2d
2g
2a
2b
2c
2d
2e
2d
2g
33
33
33
33
33
33
33
r.t.a
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
97
26
12
39
18
43
48
35
10
5
100
37
19
58
28
65
71
36
12
6
3
4
5
6
7
1g
1a
1b
1c
1d
1e
1f
8
9
10
11
12
13
14
14
6
20
9
15
16
21
22
1g
a
r.t.: room temperature. The other reduction conditions were the same as those in Table 1.
aromatic ring, and also depends on reaction temperature. The influence of these substituents is less important for
enantioselelctivity.
1. Experimental
Fresh carrot roots and celeriac stems were purchased from a local market. The external layer of these roots or stems
was removed and the rest was carefully cut into small thin pieces (approximately 5 mm  5 mm  2 mm). Aromatic
ketones 1a–1g were obtained from Fluka and the racemic alcohol standards were prepared by chemical reduction of
the corresponding ketones with sodium borohydride in methanol. Other chemical reagents were of analytic purity and
purchased from local suppliers.
The small thin pieces of healthy roots or stems (10.0 g) were placed in flasks with 50 mL of 0.1 mol/L phosphate
buffer at pH 6.2 (celeriac) or distilled water, pH 7.0 (carrot). To this suspension, 50 mg of 1a dissolved in 0.5 mL
ethanol was added and shaken at 33 8C for 1 h or 28 h in an orbital shaker (120 r/min). The suspension was filtered and
the filtrate was extracted with ethyl acetate (3 Â 20 mL). The organic phases were dried over anhydrous sodium sulfate
and evaporated in vacuum. The final products were purified by column chromatography. The enantiomeric
composition of product was established by GC equipped with CP-Chirasil-Dex CB chiral capillary column
(25 m  0.32 mm  0.25 mm, Varian). Configuration of product was determined by the sign of the specific rotation.
Acknowledgments
This research was financially supported by the National Natural Science Foundation of China (No. 20672037), and
the National Special Fund for State Key Laboratory of Bioreactor Engineering (No. 2060204).
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