Asymmetric reduction of α-hydroxy aromatic ketones to chiral aryl vicinal diols using carrot enzymes system

Article abstract of DOI:10.1016/j.cclet.2012.04.001

Asymmetric reduction of α-hydroxy aromatic ketones was carried out by using carrot enzymes system, yielding corresponding chiral vicinal diols with special functional groups. The optimum reaction conditions were obtained after investigation of various influencing factors. Chiral aryl vicinal diols were produced with good yields and excellent enantiomeric excesses under appropriate conditions. Meanwhile, the steric factors and electronic effects of the substituents on the aromatic ring were shown to have an interesting influence on both yield and enantioselectivity.

Full text of DOI:10.1016/j.cclet.2012.04.001

Available online at www.sciencedirect.com
Chinese Chemical Letters 23 (2012) 635–638
www.elsevier.com/locate/cclet
Asymmetric reduction of a-hydroxy aromatic ketones to
chiral aryl vicinal diols using carrot enzymes system
a
a
b
Xiang Liu a, , Yi Wang , Hai Yan Gao , Jian He Xu
*
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
Received 23 December 2011
Available online 15 May 2012
Abstract
Asymmetric reduction of a-hydroxy aromatic ketones was carried out by using carrot enzymes system, yielding corresponding
chiral vicinal diols with special functional groups. The optimum reaction conditions were obtained after investigation of various
influencing factors. Chiral aryl vicinal diols were produced with good yields and excellent enantiomeric excesses under appropriate
conditions. Meanwhile, the steric factors and electronic effects of the substituents on the aromatic ring were shown to have an
interesting influence on both yield and enantioselectivity.
# 2012 Xiang Liu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Asymmetric reduction; Chiral aryl vicinal diol; Aromatic ketone; Carrot cell
Due to its high chemo-, regio-, and stereo-selectivity and non-toxicity, biocatalysis has become an ideal tool for
asymmetric and green synthesis [1–4]. Moreover, utilization of whole cells instead of isolated enzymes as biocatalysts
is a relatively simple handling that does not require complex protein separation procedures or the addition and/or
recycling of exogenous cofactors. There are extensive works on the asymmetric reduction catalyzed by whole cells
with excellent enantioselectivity and yield [5–7]. However, the whole-cell catalytic systems reported previously are
mainly generated from microbial sources (fungi, bacteria or yeasts) [8–10]. By comparison, less attention has been
given to the use of plants as a potential enzyme source for catalzying selected organic reactions [11–13].
Chiral aryl vicinal diols with special functional groups are potentially valuable synthetic intermediates for the
preparation of pharmaceuticals, agrochemicals and pheromones [14–16]. The biocatalytic reduction of carbonyl to
enantiopure secondary alcohols is one of the most convenient methods for establishing chirality. Herein we explored
the asymmetric reduction of prochiral a-hydroxy aromatic ketones catalyzed by carrot (Daucus carota L.) cells,
affording corresponding chiral aryl vicinal diols which were predominated in (R)-alcohols, following the Prelog’s rule
(Scheme 1) [17].
* Corresponding author.
E-mail address: liuxiang@jiangnan.edu.cn (X. Liu).
1001-8417/$ – see front matter # 2012 Xiang Liu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2012.04.001

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X. Liu et al. / Chinese Chemical Letters 23 (2012) 635–638
Scheme 1.
1. Experimental
Fresh and healthy carrot (D. carota L.) roots were purchased from a local market. The external layer of these roots
was removed and the rest was carefully cut into small thin pieces (approximately 5 mm  5 mm  2 mm). a-Hydroxy
aromatic ketones 1a–1e were synthesized through the transformation of a-bromo aromatic ketones. The racemic diol
standards were prepared by chemical reduction of the 1a–1e with sodium borohydride in ethanol [18]. (S)-2a and (R)-
2a were purchased from Aldrich. All other chemicals and reagents were purchased in analytic grade from local
suppliers.
General procedures for the asymmetric reduction of a-hydroxy aromatic ketones catalyzed by carrot cells are as
follows: the small thin pieces of fresh carrot roots (10.0 g) were placed in flasks with 50 mL of 0.1 mol/L phosphate
buffer or distilled water. To this suspension, 30–70 mg of 1a was added and shaken at 28–36 8C for 20–36 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 the 2c was established by HPLC equipped with Chiralcel OD-H column
(4.6 mm  250 mm; Daicel Chemical Industry, Ltd., Japan), and the others were established by HPLC equipped with
Chiralcel OB-H column (4.6 mm  250 mm; Daicel Chemical Industry, Ltd., Japan).
2. Results and discussion
The effect of substrate concentration on the yield and enantiomeric excess (e.e.) of 2a was investigated. As shown in
Fig. 1a, the yield of 2a was heavily influenced by the substrate concentration. The probable reason for this was that the
intracellular activity of carrot cells gradually decreased until being inactivated because of the toxicity of substrate to
the cells. On the other hand, the e.e. of 2a gave no noticeable difference at varied concentrations ranging from 0.6 g/L
to 1.4 g/L, keeping above 96.7% in all the cases tested. Therefore, a substrate concentration at 1.0 g/L was appropriate
when considering both yield and e.e. in this work.
Fig. 1b demonstrated that the effect of temperature on the yield and e.e. of 2a. As the reaction temperature was
increased, both yield and e.e. rose firstly and then diminished gradually. The yield and e.e. reached their peak values at
32 8C and 30 8C, respectively. On the one hand, the reaction rate increased as the temperature climbed up, which
enhanced the yield in a certain period of reaction time. But on the other hand, a too high temperature would inactivate
enzymes, therefore reducing the reaction rate. As a result, the yield of 2a declined in a certain period of reaction time.
Compared to the yield, the e.e. of 2a was influenced to a smaller extent by the reaction temperature. However, the e.e.
of 2a decreased slowly with the increase of temperature because the 3D conformations binding substrate molecules
and enzymes were partially destroyed under a high-temperature environment, leading to the generation of both the (R)-
alcohol and the (S)-alcohol at the same time. As seen from Fig. 1b, the optimum temperature for the tested cases was
32 8C. At this temperature, relatively high enantioselectivity was maintained.
As can be seen in Fig. 1c, the pH of reaction medium had a noticeable effect on the yield and e.e. of 2a. The yield
rose markedly as the pH of reaction medium increased. The pH of reaction medium affects the reaction mainly because
of its influence on the enzyme activity. If the pH is too high or too low, it can alter or damage the state of ionization of
charged amino acids existing in an enzyme that may play a crucial role in substrate binding and/or the catalytic action
itself, thus affecting enantioselectivity and activity of the enzyme. So an appropriate pH is important for enzyme
controlled reactions. It is interesting to note that both yield and e.e. of 2a reached their peak values (90.5% yield and
99.4% e.e.) in distilled water, rather than in the phosphate buffer system. The most probable reason for this was that the
phosphate might affect the ionization of amino acids which then influenced the binding between substrate molecules

X. Liu et al. / Chinese Chemical Letters 23 (2012) 635–638
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(b)
(a)
Yield
Yield
ee
ee
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Reaction temperature /oC.
Substrate concentration / (g/L)
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ee
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e.e.
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6.4
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Reaction time / h
Fig. 1. The effect of reaction conditions on the yield and e.e. of 2a. Reduction conditions: (a) 32 8C, distilled water, 28 h; (b) 1.0 g/L 1a, distilled
water, 28 h; (c) 1.0 g/L 1a, 32 8C, 28 h; and (d) 1.0 g/L 1a, 32 8C, distilled water.
and enzymes [19]. The different buffer systems and pH (7.2–8.2) were also introduced and the yields were even lower
than those in the phosphate buffer system. Therefore, the distilled water of pH 7.0 was chosen as the reaction medium.
Fig. 1d showed that the yield and e.e. changed with the reaction time. The yield quickly went up until 28 h because
the initial rate of the reaction was very fast. The e.e. of 2a gradually went down after 24 h. The reason for this may be
that some adverse reactions happened under such conditions and the product inhibition happened because the reaction
time was too long. This phenomenon indicated that the reaction time must be moderate. So a reaction time of 28 h was
selected for the subsequent experiments.
The effect of the substituents located on the aromatic ring on the yield and e.e. of aryl vicinal diols was also studied.
The optimum reaction conditions were applied to the asymmetric reduction of a series of a-hydroxy aromatic ketones
1a–1e, and the results were shown in Table 1.
As can be seen in Table 1, a series of substrates were successfully reduced, giving the corresponding diols 2a–2e in
good yields and excellent e.e. As the best model substrate, 1a can be effectively reduced with higher yield than the rest,
which indicated that the steric hindrance of the substituents on the aromatic ring were unfavorable to the yields of 2b–
2e, the reason for this was that these substituents obstructed the binding between substrate molecules and enzymes.
Besides the steric factors, the electronic effects of these substituents played an important impact on the activity of
carrot cells because the electron-withdrawing or electron-donating character affected hydrogen bonding between the
Table 1
The variation trends of yields and e.e. of 2a–2e.
Entry
Substituent on the aromatic ring
Yield (%)
e.e. (%)
2a
2b
2c
2d
2e
H
90.5
46.2
48.8
62.9
62.3
99.4
100
m-Cl
p-Cl
99.0
95.8
94.5
p-CH3
p-OCH3
Reaction conditions: 1.0 g/L substrates, 32 8C, distilled water, 28 h.

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X. Liu et al. / Chinese Chemical Letters 23 (2012) 635–638
enzyme and the substrates [20]. The yields of 2d and 2e were more attractive than those of 2b and 2c, which suggested
that substrates with electron-donating substituents were more accepted by plant tissue than those with electron-
withdrawing groups if considering the yield. Also, the toxicity of the substrate containing chlorine was another factor
affecting the yields. On the other hand, the substrates (1b, 1c) with electron-withdrawing groups provided their
products with higher e.e. than those of 1d and 1e. It seems that the electron-withdrawing groups could contribute to
higher enantiopurity. Therefore, the electronic effects, caused by the substituents on the aromatic ring, made these
ketoreducatases existing in carrot cells more efficient.
In conclusion, bio-reduction catalyzed by the ketoreducatases existing in carrot cells provides an attractive
approach in reducing a series of a-hydroxy aromatic ketones with good yields and excellent e.e. The configurations of
the products were the same as what we could expect from Prelog’s rule. The steric factors and electronic effects of the
substituents on the aromatic ring had a clear effect on the yield and enantioselectivity of the reaction. Therefore, our
experiment offers a very clean, simple and convenient method for preparing (R)-aryl vicinal diols which are important
chiral building blocks, being involved in the production of a wide range of fine chemicals and pharmaceutical
molecules.
Acknowledgments
This research was financially supported by the National Natural Science Foundation of China (No. 21176203), the
National Special Fund for State Key Laboratory of Bioreactor Engineering (No. 2060204) and the Fundamental
Research Funds for the Central Universities (No. JUSRP211A12).
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