Biocatalytic reduction of prochiral aromatic ketones to optically pure alcohols by a coupled enzyme system for cofactor regeneration

Article abstract of DOI:10.1016/j.tetlet.2010.12.069

A simple, highly efficient, and economical biphasic cell-free system was developed for biocatalytic reduction of prochiral aromatic ketones to furnish enantiopure alcohols. This system is characterized by using endogenous enzymes of the cell-free extract to form enzyme-coupled NADPH recycling system. Besides, it offered much higher productivity than whole cells and greatly simplified the preparation process of biocatalysts in comparison with isolated enzymes. Various prochiral aromatic ketones, especially α-substituted acetophenone derivatives, were reduced to chiral alcohols with excellent enantiomeric excess (ee) and moderate to good yield by this cell-free system.

Full text of DOI:10.1016/j.tetlet.2010.12.069

Tetrahedron Letters 52 (2011) 999–1002
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Biocatalytic reduction of prochiral aromatic ketones to optically pure
alcohols by a coupled enzyme system for cofactor regeneration
Zhen Yan , Yao Nie a, , Yan Xu a, , Xiang Liu , Rong Xiao c
a,
⇑
b
a
School of Biotechnology and Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan University, Wuxi 214122, China
School of Chemical and Material Engineering, Jiangnan University, Wuxi 21412, China
Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ 08854, USA
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple, highly efficient, and economical biphasic cell-free system was developed for biocatalytic reduc-
tion of prochiral aromatic ketones to furnish enantiopure alcohols. This system is characterized by using
endogenous enzymes of the cell-free extract to form enzyme-coupled NADPH recycling system. Besides,
it offered much higher productivity than whole cells and greatly simplified the preparation process of
biocatalysts in comparison with isolated enzymes. Various prochiral aromatic ketones, especially
Received 13 September 2010
Revised 9 December 2010
Accepted 17 December 2010
Available online 22 December 2010
a
-substituted acetophenone derivatives, were reduced to chiral alcohols with excellent enantiomeric
Keywords:
excess (ee) and moderate to good yield by this cell-free system.
Asymmetric carbonyl reduction
Cofactor regeneration
Enzyme-coupled system
Cell-free extract
Ó 2010 Published by Elsevier Ltd.
Alcohol dehydrogenases (ADH), also referred to as carbonyl
reductases (CR), are a class of nicotinamide dependent oxidore-
when the enzyme exhibits both reductive and oxidative abilities
that are a harsh requirement for a large number of ADH/CR.7
Enzyme-coupled approach introduces another enzyme, which
1
ductases, which can catalyze the reduction of carbonyl compounds
to produce alcohols. Due to their strict recognition of substrate and
hence high enantiomeric excess products, ADH/CR are especially
suitable for asymmetric synthesis of optically pure alcohols, which
are very important chiral building blocks in chemical and pharma-
8
increases the cost of reaction. Cell-free extract, which contains
virtually the functional enzymes inside the cell, was rarely
employed as a biocatalyst to conduct asymmetric reduction in
9
recent years, and cell-free extract from recombinant Escherichia
2
ceutical industry. Therefore, biocatalytic carbonyl reduction has
coli, which is widely used as expression host has hardly been
applied to perform carbonyl reduction so far. Herein, we presented
a cell-free system by using cell-free extract as a biocatalyst to
conduct carbonyl reduction. It should be noted that this method
contained an efficient enzyme-coupled system for NADPH regener-
ation. Besides, this cheap biocatalyst offered much higher produc-
tivity than whole cells and greatly simplified the preparation
process of biocatalysts in comparison with isolated enzymes.
We have recently demonstrated that Candida parapsilosis CCTCC
M203011 could achieve enantioselective conversion of racemic 1-
phenyl-1,2-ethanediol to produce (S)-1-phenyl-1,2-ethanediol
been extensively studied in recent years.3
Today, whole-cell biocatalysts and isolated enzymes that exhi-
bit ADH/CR activity are predominantly applied in producing enan-
tiopure alcohols through ketone reduction.4 However, they have
their own drawbacks. For whole-cell catalysts, the cellular mem-
brane often retards the entry of substrate into the cellular systems
and prevents the product from being released from the cellular
system for an easy recovery. Consequently, productivities of
whole-cell biocatalysis are severely compromised.5 For isolated
enzymes, costly and time-consuming process of enzyme prepara-
tion is the main shortcoming. Also, coenzyme regeneration should
be taken into serious consideration since the reactions usually
require a stoichiometric amount of expensive coenzyme. As a con-
sequence, several coenzyme recycling systems have been devel-
oped, typically substrate-coupled approach and enzyme-coupled
1
0
((S)-1b). In this Letter, we constructed a recombinant E. coli
BL21(DE3)(pETSCR1) overexpressing NADPH-dependent carbonyl
reductase SCR1 (GenBank accession No. FJ939565) which is from
C. parapsilosis CCTCC M203011. Then the cell-free extract (total sol-
uble protein 5 g/L) from the recombinant E. coli was applied for
asymmetric reduction of prochiral aromatic ketones.
6
approach. Substrate-coupled method can only be accomplished
As expected, cell-free extract alone was inefficacious in asym-
metric reduction of
Table 1). Besides, the reactions achieved limited improvement
with high ee but low yield when glucose and a low amount of
a-hydroxyacetophenone (1a) to form (S)-1b
⇑
Corresponding author. Tel.: +86 510 85918197; fax: +86 510 85918201.
E-mail address: yxu@jiangnan.edu.cn (Y. Xu).
Contributed equally to this Letter.
(
0
040-4039/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2010.12.069

1
000
Z. Yan et al. / Tetrahedron Letters 52 (2011) 999–1002
Table 1
(where yield ranged from 36.4% to 84.1%). Therefore, by adding
Discovery of NADPH recycling system
several hydrogen donors simultaneously, it is possible that multi-
ple cofactor recycling systems run in parallel and hence greatly en-
hance the cofactor regeneration. It provided us a potential and
powerful alternative to cope with the problem that coenzyme
regeneration brings to us. Also, since the coenzyme recycling sys-
tem was formed by the endogenous enzymes of the cell, E. coli
BL21(DE3) harboring other ADH/CR also, theoretically, has the po-
tential to form such enzyme-coupled cell-free system. In fact, this
system has been successfully used by other ADH/CRs by our group
(data not shown).
Substrate 1aa
Glucose
NADPH
NADP+
eeb,c (%)
Yieldc (%)
+
+
+
+
+
+
À
+
À
À
+
À
À
+
À
À
À
+
À
À
>99
>99
>99
>99
>99
8.5
5.6
4.8
92.9
93.1
À
+
À
+
À
+
a
Reaction mixture in 2 mL comprised 1 mL potassium phosphate buffer (pH 6.5,
.1 M), 1 mL cell-free extract (total protein 5 mg), 40 mM 1a with or without 10 g/L
glucose, 0.1 mM NADP and 0.1 mM NADPH.
0
+
b
The expensive coenzymes, such as NADP(H), are the major
impediment of the large application of oxidoreductases, therefore,
Sole product (S)-1b.
Yield and ee were determined by chiral HPLC.
c
+
we investigated the requirement of NADP for this cell-free system
+
(
Table 2). Since the NADP and NADPH contained in the cell-free
+
NADP /NADPH was added separately. However, the complete con-
extract show no detectable effect on the reduction (Table 2), we
herein defined the total turnover number (TTN) as the molar
amount of synthesized product per molar amount of added cofac-
tor. The maximum TTN of 1916 was afforded with 47.9% yield at
version was unexpectedly achieved with excellent ee and high
yield in the presence of both glucose and NADP /NADPH. Since
the molar amount of NADPH was much less than that of substrate
and complete conversion was also achieved when NADPH was
replaced by NADP , it can be deduced that NADPH was regenerated
during the process. The requirement of glucose in the complete
conversion of 1a suggested that glucose acted as a hydrogen donor
to perform the NADPH regeneration. Besides, carbonyl reductase
SCR1 had no oxidative activity toward sugars or alcohols (data
not shown). Therefore, by utilizing glucose, cell-free extract itself
formed an enzyme-coupled NADPH recycling system. In this sys-
tem, the endogenous enzymes of cell-free extract corresponded
to the second enzyme in the classic enzyme-coupled approach.
Because NADP and NADPH had the same effect on the necessary
cofactor regeneration, while NADP is more stable and economical
than its reductive counterpart, NADP was employed as the initial
coenzyme in the following experiments. To further confirm the
existence of NADPH recycling system, we detected the optical den-
sity change at 340 nm in an activity assay system containing only
+
+
+
0.01 mM NADP . When 0.02 mM NADP was added, however, the
cell-free system exhibited high yield (91.3%) and TTN of 1826.
Thus, the endogenous enzymes of cell-free extract, which are usu-
ally overlooked by most researchers could form an efficient en-
zyme-coupled NADPH recycling system. Also, this system greatly
reduced the costs by avoiding the introduction of another enzyme.
Compared with whole-cell system, this cell-free system per-
formed much higher productivity. As shown in the Figure 2, using
the cell-free system, 40 mM 1a (due to poor solubility of 1a, no
higher concentration was tested for aqueous phase reactions.)
was completely converted into the corresponding product of
92.9% yield within 6 h, while whole-cell system required 12 h to
accomplish the complete conversion of 10 mM 1a. The sharp con-
trast indicated that the substrate/product transportation was
greatly retarded by the cellular membrane, but the cell-free system
facilitated the interaction between enzyme and substrate/product,
resulting in higher productivity.
+
+
+
+
+
cell-free extract, NADP and glucose. The increase of optical density
was observed, indicating that the NADPH was generated in the
cell-free extract (For NADPH regeneration assay details see the
Supplementary data).
Interestingly, there were more than one enzyme, which could
achieve NADPH regeneration in this cell-free system. As illustrated
in the Figure 1, glucose and trehalose showed the best results ((S)-
The majority of ketones are highly hydrophobic, and thus pos-
sess poor solubility in aqueous media, which directly leads to
low substrate concentrations. We hence developed a water–organ-
ic solvent system for asymmetric reduction. Several alkanes, esters,
and other reagents were examined as an organic phase and ethyl
laurate was finally adopted as an organic phase to conduct further
investigation. By using biphasic system, upto 80 mM 1a was con-
verted into (S)-1b with 90.1% yield and >99% ee within 12 h, afford-
ing a TTN of 3604, while only 10.1% yield was achieved for the
aqueous system (Table 3). In comparison with the reported litera-
1
b yield at 93.0% and 92.4%, respectively). Fructose, ethanol, malt-
ose, and lactose also facilitated the conversion to some extent
1
0,11
ture involving bioreduction of 1a to form (S)-1b,
this cell-free
system exhibited the highest productivity (7.2 mmol/L/h).
In order to examine the generality of the cell-free biphasic sys-
tem, various aromatic ketones were tested as substrates (Table 3).
The range of substrates included
a-substituted acetophenone
derivatives (1a–9a), acetophenone (10a) and ortho-, meta-, and
Table 2
+
Effect of NADP concentration on reduction of 1a by cell-free system
NADP+a (mM)
eeb (%)
Yield (%)
TTN
0
0
0
0
0
0
0
.2
.1
.05
.02
.01
.005
>99
>99
>99
>99
>99
>99
-
93.2
93.0
92.7
91.3
47.9
11.7
-
186
372
742
1826
1916
936
-
a
Substrate concentration was 40 mM.
Sole product (S)-1b.
Figure 1. Effect of co-substrate on reduction of 1a by cell-free system. Co-substrate
concentration was 10 g/L. ( ) yield, (s) ee.
b

Z. Yan et al. / Tetrahedron Letters 52 (2011) 999–1002
1001
a-carbon played an important role in the reaction, because the re-
sults were in conformity with the general rules that the strength of
hydrogen bond is in order of hydroxyl > bromine > hydrogen.
Although substrates varied, the corresponding alcohols were syn-
thesized with >99% ee in all cases, even on the poor substrates
(10a, 13a–15a). The high ee value indicated that the cell-free ex-
tract, though complex, produced no side reaction. Besides, the bi-
phasic cell-free system showed much higher yield in comparison
with aqueous whole-cell system.
In conclusion, we presented a biphasic cell-free system, which
could accomplish asymmetric conversion of various aromatic ke-
tones, involving enzyme-coupled cofactor regeneration. This sys-
tem exhibited much higher productivity than whole-cell system
by breaking the cell membrane barrier. Compared with the isolated
enzyme biocatalysts, this system greatly simplified the preparation
of biocatalyst, which is usually a costly and time-consuming pro-
cess. Most importantly, by utilizing the endogenous enzymes of
cell-free extract to form coenzyme recycling system, NADPH
regeneration could run efficiently without adding extra enzyme.
Finally, by introducing ethyl laurate as an organic phase to form
a biphasic system, various prochiral aromatic ketones, especially
Figure 2. Time course of reduction of 1a. (d) cell-free system with 10 mM 1a. (j)
cell-free system with 40 mM 1a. (N) whole-cell system with 10 mM 1a. (.) whole-
cell system with 40 mM 1a.
a-substituted acetophenone derivatives, were reduced to chiral
alcohols with excellent ee and moderate to good yield. Thus, an en-
zyme-coupled system for cofactor regeneration has been devel-
oped for asymmetric bioreduction. By utilizing the endogenous
enzymes that are usually overlooked by most researchers, this sys-
tem, which is not restricted in carbonyl reductase SCR1, but other
ADH/CR, provides us a promising alternative to develop biocata-
lytic system for fine biochemical synthesis in a simple, economical,
and efficient way.
para-substituted acetophenones (11a–15a). Among all the aro-
matic ketones, 1a was more suitable for this cell-free system med-
iated asymmetric reduction, giving corresponding chiral alcohol of
9
0.1% yield in high substrate concentration. Besides, a clear trend
was observed that only -substituted acetophenone derivatives
could be reduced to chiral alcohols with excellent yield, indicating
substituents at -carbon, that is, hydroxyl or bromine had a great
a
a
effect on the enzyme activity by forming hydrogen bond between
the active site residues of the enzyme and such electron-with-
Acknowledgments
drawing groups at the
a-position of substrates. This was coincided
Financial support from the National Natural Science Foundation
of China (NSFC) (No. 20776060 and 30800017), the National High
Technology Research and Development Program of China (863 Pro-
gram) (No. 2006AA020104 and 2007AA02Z226), the National Key
Basic Research and Development Program of China (973 Program)
with another enzyme identified from the C. parapsilosis CCTCC
1
2
M203011. In addition, for
a-substituted acetophenone deriva-
tives, -bromo-substitution showed lower yield than
a
a
-hydroxy-
substitution. We proposed that the hydrogen bond between amino
acid of the active site of the enzyme and the substituent at
Table 3
Reduction of various aromatic ketones by cell-free system
OH
O
E. coli/SCR1
cell-free extract
R2
R2
R1
R1
Aromatic ketonea
R
R
Biphasic cell-free systemb
Aqueous whole-cell system
1
2
eec (%)
Yieldc (%)
eec (%)
Yieldc (%)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
ad,e
a
a
a
a
a
a
a
a
0a
1a
2a
3a
4a
5a
H
OH
OH
OH
OH
OH
Br
Br
Br
Br
H
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(R)
–
90.1
90.5
86.6
87.5
91.1
82.7
80.4
84.6
83.0
5.0
–
–
5.2
15.5
11.3
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
>99(S)
–
10.1
7.8
13.2
20.1
22.5
5.5
7.7
9.7
6.1
–
m-Cl
p-Cl
p-CH
p-OCH
m-Cl
p-Cl
p-CH
3
p-OCH
H
o-Cl
m-Cl
p-Cl
p-CH
p-OCH
3
3
3
H
H
H
H
–
–
–
–
–
–
–
–
–
>99(R)
>99(R)
>99(R)
3
3
H
–
–
a
b
c
Substrate concentration: 1a 80 mM, 2a–9a 25 mM, 10a–15a 10 mM; NADP+ was 0.02 mM; glucose concentration was 10 g/L; reaction time was 6 h.
Ethyl laurate/water = 1:4 (v/v).
Determined by chiral HPLC (1a–10a, 14a, 15a) or GC (11a–13a).
Glucose concentration was 20 g/L.
Reaction time was 10 h.
d
e

1
002
Z. Yan et al. / Tetrahedron Letters 52 (2011) 999–1002
(
Nos. 2009CB724706 and 2011CB710802), the Program of Intro-
ducing Talents of Discipline to Universities (111 Project) (111-2-
6), and the Natural Science Foundation of Department of Science
4. (a) Goldberg, K.; Schroer, K.; Lutz, S.; Liese, A. Appl. Microbiol. Biotechnol. 2007,
6, 237–248; (b) Goldberg, K.; Schroer, K.; Lutz, S.; Liese, A. Appl. Microbiol.
7
Biotechnol. 2007, 76, 249–255.
0
5.
Chen, R. R. Z. Appl. Microbiol. Biotechnol. 2007, 74, 730–738.
and Technology of Jiangsu province, PR China (No. BK2008528) are
greatly appreciated.
6. Wichmann, R.; Vasic-Racki, D. In Advances in Biochemical Engineering
Biotechnology; Springer: Berlin, 2005; Vol. 92, pp 225–260.
7.
(a) Musa, M. M.; Ziegelmann-Fjeld, K. I.; Vieile, C.; Zeikus, J. G.; Phillips, R. S. J.
Org. Chem. 2007, 72, 30–34; (b) de Gonzalo, G.; Lavandera, I.; Faber, K.; Kroutil,
W. Org. Lett. 2007, 9, 2163–2166; (c) Bisogno, F. R.; Lavandera, I.; Kroutil, W.;
Gotor, V. J. Org. Chem. 2009, 74, 1730–1732.
Supplementary data
8
.
(a) Voss, C. V.; Gruber, C. C.; Faber, K.; Knaus, T.; Macheroux, P.; Kroutil, W. J.
Am. Chem. Soc. 2008, 130, 13969–13972; (b) Zhu, D. M.; Ankati, H.; Mukherjee,
C.; Yang, Y.; Biehl, E. R.; Hua, L. Org. Lett. 2007, 9, 2561–2563; (c) Iyer, R. B.;
Bachas, L. G. J. Mol. Catal. B: Enzym. 2004, 28, 1–5; (d) Hirano, J.; Miyamoto, K.;
Ohta, H. Tetrahedron Lett. 2008, 49, 1217–1219; (e) Mertens, R.; Greiner, L.; van
den Ban, E. C. D.; Haaker, H.; Liese, A. J. Mol. Catal. B: Enzym. 2003, 24–25, 39–52.
(a) Utaka, M.; Yano, T.; Ema, T.; Sakai, T. Chem. Lett. 1996, 1079–1080; (b)
Ishihara, K.; Higashi, Y.; Nakajima, N.; Utaka, M.; Nakamura, K. J. Ferment.
Bioeng. 1996, 81, 266–268; (c) Keinan, E.; Hafeli, E. K.; Seth, K. K.; Lamed, R. J.
Am. Chem. Soc. 1986, 108, 162–169; (d) Tsuchiya, S.; Miyamoto, K.; Ohta, H.
Biotechnol. Lett. 1992, 14, 1137–1142; (e) Ishihara, K.; Nakajima, N. Biosci.,
Biotechnol., Biochem. 2006, 70, 3077–3080; (f) Yang, W.; Xu, J. H.; Pan, J.; Xu, Y.;
Wang, Z. L. Biochem. Eng. J. 2008, 42, 1–5.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.12.069.
References and notes
9
.
1
2
.
.
Forrest, G. L.; Gonzalez, B. Chem. Biol. Interact. 2000, 129, 21–40.
(a) Schmid, A.; Dordick, J. S.; Hauer, B.; Kiener, A.; Wubbolts, M.; Witholt, B.
Nature 2001, 409, 258–268; (b) Schoemaker, H. E.; Mink, D.; Wubbolts, M. G.
Science 2003, 299, 1694–1697; (c) Patel, R. N. Coord. Chem. Rev. 2008, 252, 659–
7
01; (d) Wolberg, M.; Villela, M.; Bode, S.; Geilenkirchen, P.; Feldmann, R.;
Liese, A.; Hummel, W.; Muller, M. Bioprocess Biosyst. Eng. 2008, 31, 183–191; (e)
Ema, T.; Ide, S.; Okita, N.; Sakai, T. Adv. Synth. Catal. 2008, 350, 2039–2044; (f)
Kizaki, N.; Yasohara, Y.; Hasegawa, J.; Wada, M.; Kataoka, M.; Shimizu, S. Appl.
Microbiol. Biotechnol. 2001, 55, 590–595.
10. Nie, Y.; Xu, Y.; Mu, X. Q. Org. Process Res. Dev. 2004, 8, 246–251.
11. (a) Wei, Z. L.; Lin, G. Q.; Li, Z. Y. Bioorg. Med. Chem. 2000, 8, 1129–1137; (b) Wei,
Z.; Li, Z.; Lin, G. Tetrahedron 1998, 54, 13059–13072; (c) Tsujigami, T.; Sugai, T.;
Ohta, H. Tetrahedron: Asymmetry 2001, 12, 2543–2549; (d) Hasegawa, J.; Ogura,
M.; Tsuda, S.; Maemoto, S.; Kutsuki, H.; Ohashi, T. Agric. Biol. Chem. 1990, 54,
1819–1827.
3
.
(a) Moore, J. C.; Pollard, D. J.; Kosjek, B.; Devine, P. N. Acc. Chem. Res. 2007, 40,
1
412–1419; (b) Matsuda, T.; Yamanaka, R.; Nakamura, K. Tetrahedron:
Asymmetry 2009, 20, 513–557; (c) De Wildeman, S. M. A.; Sonke, T.;
Schoemaker, H. E.; May, O. Acc. Chem. Res. 2007, 40, 1260–1266.
12. Nie, Y.; Xu, Y.; Mu, X. Q.; Wang, H. Y.; Yang, M.; Xiao, R. Appl. Environ. Microbiol.
2007, 73, 3759–3764.