One-pot, single-step deracemization of 2-hydroxyacids by tandem biocatalytic oxidation and reduction

Article abstract of DOI:10.1039/c3cc46240d

A facile and efficient one-pot, single-step method for deracemizing a broad range of 2-hydroxyacids to (R)-2-hydroxyacids was established by combination of resting cells of an (S)-hydroxyacid dehydrogenase-producing microorganism and an (R)-ketoacid reductase-producing microorganism.

Full text of DOI:10.1039/c3cc46240d

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One-pot, single-step deracemization of 2-hydroxyacids
by tandem biocatalytic oxidation and reduction†
Cite this: DOI: 10.1039/c3cc46240d
ab
ab
ab
ab
ab
Ya-Ping Xue, Yu-Guo Zheng,* Ya-Qin Zhang, Jing-Lei Sun, Zhi-Qiang Liu
Received 15th August 2013,
ab
and Yin-Chu Shen
Accepted 23rd September 2013
DOI: 10.1039/c3cc46240d
www.rsc.org/chemcomm
A facile and efficient one-pot, single-step method for deracemizing a synthesis due to the diverging reaction conditions required for each
9,10
broad range of 2-hydroxyacids to (R)-2-hydroxyacids was established transformation. Moreover, it is very difficult to meet the require-
by combination of resting cells of an (S)-hydroxyacid dehydrogenase- ments of such double selectivities. To date, no general comparable
producing microorganism and an (R)-ketoacid reductase-producing process has been reported for the deracemization of a broad range
10
microorganism.
of racemic 2-HAs in a one-pot, single-step method. Tsuchiya et al.
reported the deracemization of mandelic acid by biocatalytic
The resolution of racemates is the most current approach to obtain oxidation–reduction. The asymmetric oxidation of (S)-mandelic acid
1,2
enantiomerically pure compounds. Enantiopure 2-hydroxyacids by Alcaligenes bronchisepticus required aerobic conditions, while
2-HAs) are particularly important building blocks for the prepara- Streptococcus faecalis employed for the reduction of the inter-
tion of pharmaceuticals and fine chemicals. Their classical kinetic mediate benzoylformate was inactivated under these conditions.
resolution by enzymatic or non-enzymatic catalysts is abundantly Recently, we established high-throughput screening methods to
documented. However, the yield of a kinetic resolution is limited to isolate a stereoselective 2-HA dehydrogenase-producing strain for
(
3
11
only 50%. As a consequence, alternative methods able to directly asymmetric oxidation of 2-HAs. Pseudomonas aeruginosa CCTCC M
convert a racemate mixture to a single enantiomer (deracemization) 2011394 harboring FMN-dependent (S)-2-HA dehydrogenase was iso-
4
are highly advantageous.
lated. Asymmetric oxidation of a wide range of racemic 2-HAs by
Tandem oxidation and reduction reactions in a one-pot, single- P. aeruginosa CCTCC M 2011394 produced the corresponding 2-keto-
12
step procedure operate the two processes concurrently in one pot, acids (2-KA) and (R)-2-HAs with excellent ee. Therefore, we envisaged
circumventing the often time-consuming and yield-reducing iso- that we might be able to couple our asymmetric oxidation of racemic
lation and purification of intermediates in the multiple synthesis 2-HAs with the opposite stereoselective reduction of 2-KAs for the
system. This approach is one of the most attractive deracemization efficient oxidoreductive deracemization to (R)-2-HAs (Scheme S1, ESI†).
methods which allow complete transformation of a racemate into a
In the initial experiments we attempted to screen microbes
2
single stereoisomeric product. One enantiomer is usually oxidized featuring a broad substrate spectrum with satisfactory 2-KA
to the ketone while the other enantiomer remains unchanged. The reductase (2-KAR) activity and (R)-stereoselectivity. A total of 124
ketone is then reduced to the opposite enantiomer subsequently. strains of microbes were isolated, of which 16 strains reduced
The net result is the conversion of the racemate to a single 2-KAs to the corresponding (R)-2-HAs. According to the overall
enantiomer in potentially 100% yield with 100% ee (enantiomeric performance including conversion, ee of the product, reaction
5
excess). This promising technology has been successfully applied to stability and substrate specificity, the best strain, designated
6
7
the deracemization of secondary alcohols and amines. The combi- ZJB5074, was chosen for further studies. According to the physio-
nation of an oxidation and a reduction reaction in a cascade allows logical and biochemical characterization as well as the compar-
8
performing deracemization in an economic and efficient fashion.
ison of the 18S rDNA gene sequence, strain ZJB5074 was identified
Unfortunately, it is still a challenge for chemists to run as Saccharomyces cerevisiae (Table S1 and Fig. S1, ESI†). The whole
oxidation and reduction reactions simultaneously in organic cells of S. cerevisiae ZJB5074 exhibit high 2-KAR activity at pH 7.5–
8
.0 and at 30–35 1C using benzoylformic acid as the substrate.
a
Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014,
China. E-mail: zhengyg@zjut.edu.cn; Fax: +86-571-88320630;
Tel: +86-571-88320630
Interestingly, the ee (%) values of the product were all kept above
99% and showed little variation in the temperature range from
25 to 50 1C and pH range from 6.0 to 9.0 (Fig. S2, ESI†), which is
b
Engineering Research Center of Bioconversion and Biopurification of Ministry of
Education, Zhejiang University of Technology, Hangzhou 310014, China
Electronic supplementary information (ESI) available: Scheme S1, Fig. S1–S3,
in contrast to the previous observation that enantioselectivity
decreased at higher reaction temperatures and pH during the
bioreduction by S. ellipsoideus GIM2.105. The bioreduction of
†
13
Table S1, and detailed experimental procedure. See DOI: 10.1039/c3cc46240d
This journal is c The Royal Society of Chemistry 2013
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-ketoacids mediated by resting cells of S. cerevisiae ZJB5074 Table 1 Substrate specificity of S. cerevisiae ZJB5074
ChemComm
a
2
proceeded smoothly because of the existence of a regeneration
system within the biocatalyst, which avoids the extra addition of
expensive cofactors. However, co-substrates are needed for cofactor
14
recycling. The addition of co-substrates such as glycerol, fructose,
lactose and glucose to the reaction media remarkably enhanced the Entry
b
c
Substrate
X
n
Relative activity
ee (%)
conversion efficiency (Fig. S2, ESI†). Glycerol was the most suitable
co-substrate for S. cerevisiae ZJB5074. When 10 g L of glycerol was
added to the reaction system, the yield of (R)-1a formed from its
racemate reached above 95% after 28 h of reduction, which was a
1
2
3
4
5
6
7
8
9
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
H
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
1.00
1.53
0.56
0.71
1.46
1.23
1.42
1.57
1.57
1.13
1.27
1.06
1.21
1.25
1.40
0.75
0.59
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.8
99.9
99.9
99.9
99.9
99.4
99.5
99.3
À1
2-Cl
3-Cl
4-Cl
2-F
140% increase compared to that obtained without addition of a
4-F
2,4-F
3,5-F
4-Br
3-OH
4-OH
4-CH
4-OCH
3-OCH
co-substrate. The positive effect on this resting-cell catalyzed reac-
tion was mainly achieved through the cellular metabolism of
15
glycerol to promote the regeneration of coenzymes. The cofactor 10
1
1
1
1
2
3
requirements in this reduction reaction were also investigated. The
activities of the permeabilized S. cerevisiae ZJB5074 with NADPH as
a cofactor were much higher than those with NADH or without 14
3
3
3
-4-OH
1
1
1
5
6
7
4-CF
H
H
3
addition of a cofactor, suggesting that an NADPH-dependent
reductase catalyzing asymmetric reduction was contained in the
whole cells of S. cerevisiae ZJB5074 (Fig. S3, ESI†). The operational
stability of S. cerevisiae ZJB5074 was evaluated by successive batch
bioreduction of 2a 20 times. Compared with the first batch, no
obvious decrease in ee and activity was observed, indicating that
S. cerevisiae ZJB5074 was a reusable biocatalyst with excellent
operational stability. To explore the catalytic capacity of S. cerevisiae
ZJB5074, the bioreduction of various 2-ketoacids was performed.
S. cerevisiae ZJB5074 exhibited a broad substrate spectrum and
efficiently catalyzed the bioreduction of all the 2-ketoacids tested
with excellent enantioselectivity (ee > 99%). The nature of the
substituent, position of the substituent, number of substituents
on the benzene ring, and the length of the carbon chain between
the hydroxy group and the benzene ring had some effect on the
a
Reaction conditions: 5 gdcw per L S. cerevisiae ZJB5074, 10 mM 2a–2q,
À1
1
0 g L glycerol, pH 7.5 (potassium phosphate, 100 mM), 30 1C, 180 rpm.
b
c
Determined by chiral RP-HPLC. The ee value of (R)-1.
activity but little effect on the enantioselectivity. Among all the Fig. 1 Deracemization of racemic 1b in one-pot biocatalysis by combination of
-HAs tested, substrates 2b (Table 1, entry 2) and 2e–2o (Table 1, resting cells of P. aeruginosa ZJB1125 and S. cerevisiae ZJB5074. (A) A one-pot,
2
two-step procedure; (B) a one-pot, single-step procedure.
entries 5–15) were efficiently reduced by S. cerevisiae ZJB5074 with
higher activity. S. cerevisiae ZJB5074 exhibited a relatively low activity
on substrates 2c and 2d (Table 1, entries 3 and 4) and substrates types of resting cells simultaneously (one-pot, single-step). The
with a longer carbon chain between the hydroxy group and the reaction yield of (R)-1b reached as high as 98.67% in a short period
benzene ring (Table 1, entries 16 and 17), the relative activities on 2p of reaction time (24 h) (Fig. 1B). Oxidation was the faster step of the
and 2q were only 75% and 59% of that on 2a, respectively.
overall reaction because 2b was detected. The results indicated that
The unique characteristics of S. cerevisiae ZJB5074 provide a solid the oxidation and reduction reactions can be run simultaneously in
basis for combining with P. aeruginosa CCTCC M 2011394 to one pot in parallel without compartmental separation. The two
deracemize 2-HAs via a one-pot process. The biocatalytic deracemi- microorganisms show high compatibility with each other and are
zation of 1b (20 mM) was performed by an oxidation–reduction one- suitable for single-step deracemization via an oxidation–reduction
pot, two-step strategy and a one-pot, single-step strategy. Fig. 1A concurrent reaction in the same reactor. Compared with the two-
shows a typical time course for production of (R)-1b from racemic 1b step reaction, the single-step reaction for deracemization has a
by P. aeruginosa CCTCC M 2011394 and S. cerevisiae ZJB5074. After simpler procedure, shorter reaction time and higher productivity.
6
h of oxidation, (S)-1b was almost completely converted to its Although oxidoreductase needs a cofactor to take part in the
corresponding ketoacid by P. aeruginosa CCTCC M 2011394 in the reaction, each of the resting-cell biocatalysts has its own in vivo
first step. The (R)-isomer of 1b was not oxidized during the entire cofactor regeneration system. (S)-2-HA dehydrogenase in P. aeruginosa
period examined. After removal of resting cells of P. aeruginosa ZJB1125 is a FMN-dependent flavoprotein that oxidizes (S)-2-HAs to
12
CCTCC M 2011394 by centrifugation, the second step reaction was 2-KAs, resulting in the reduction of FMN. Flavoproteins are known
started by adding resting cells of S. cerevisiae ZJB5074 and glycerol. to modulate the reoxidation of reduced flavin by molecular oxygen to
16
2
b, the oxidized by-product of (S)-1b, was gradually reduced to (R)-1b. a great extent. In the case of S. cerevisiae ZJB5074, NADPH was
After 28 h of reduction, the yield of (R)-1b formed from its racemate regenerated by addition of glycerol. So, there is no need to add the
was 92.28%. To develop a more simplified and practical process, expensive cofactor and/or the oxidoreductase for cofactor regenera-
deracemization of racemic 1b to (R)-1b was conducted using two tion in the tandem oxidation and reduction reactions.
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Table 2 Biocatalytic deracemization of various racemic 2-HAs in one-pot con- 1.5 with 6.0 M HCl and the desired product was extracted with 1.0 L of
a
current catalysis
ethylacetate. The extract obtained was concentrated under reduced
pressure and yielded 3.18 g of (R)-1b (99.5% ee, 90% isolated yield).
The results confirmed that deracemization of racemic 2-HAs in a one-
pot, single-step oxidation–reduction process by combination of resting
cells of P. aeruginosa CCTCC M 2011394 and S. cerevisiae ZJB5074 for
production of (R)-2-HAs was feasible in practical applications.
Deracemization of racemic 2-HAs to (R)-2-HAs was successfully
realized via concurrent asymmetric oxidation and stereoselective
reduction employing two resting-cell biocatalysts. One biocatalyst
was P. aeruginosa CCTCC M 2011394 which catalyzed the stereo-
selective oxidation of (S)-2-HAs to corresponding 2-ketoacids and
b
c
Entry Substrate
X
n
Time (h) (R)-1 (%) ee (%)
another was S. cerevisiae ZJB5074 which reduced the 2-ketoacids to
R)-2-HAs. This proposed one-pot, single-step deracemization
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
rac-1a
rac-1b
rac-1c
rac-1d
rac-1e
rac-1f
rac-1g
rac-1h
rac-1i
rac-1j
rac-1k
rac-1l
rac-1m
rac-1n
rac-1o
rac-1p
rac-1q
H
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
25
24
33
33
25
24
24
24
33
21
24
27
30
21
27
24
33
92.67
98.67
90.94
92.65
92.46
92.15
98.53
93.43
85.29
62.24
65.59
75.09
67.65
55.91
79.64
60.95
66.69
99.9
99.5
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.9
99.9
98.7
99.9
99.5
99.3
(
2-Cl
3-Cl
4-Cl
2-F
method leads to the development of a new tool for the preparation
of (R)-2-HAs.
Financial support from NSFC (No. 31170761), ‘‘973’’ Project
4-F
(
2011CB710806), ‘‘863’’ Project (2011AA02A210) and the Key Inno-
vation Research Group Programs of Zhejiang Province (No.
011R09043) is gratefully acknowledged. We would like to thank
2,4-F
3,5-F
4-Br
3-OH
4-OH
4-CH
4-OCH
3-OCH
2
0
1
2
3
4
5
6
7
Dr Mingjie Jin from Michigan State University for his assistance.
3
3
Notes and references
3
-4-OH
4-CF
H
H
3
1
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a
Reaction conditions: 10 gdcw per L P. aeruginosa CCTCC M 2011394,
À1
5
gdcw per L S. cerevisiae ZJB5074, 20 mM racemic 1a–1q, 10 g L glycerol,
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c
(
R)-1, determined by chiral RP-HPLC. The ee value of (R)-1.
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catalytic deracemization of a range of 2-HAs was performed in a one-
pot, single-step procedure. The results are given in Table 2. For
deracemization of 1a–1i (Table 2, entries 1–9), the enantiomeric
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high yields (85.29–98.67%) within 24–33 h. In the case of 1j–1q
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4
5
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(
2
prepared in a one-pot and single-step procedure with good yield and
excellent ee. Deracemization by stereoinversion in one-pot concurrent
catalysis thus overcomes the limitation of the maximum theoretical
yield of 50% encountered during kinetic resolution of racemic 2-HAs.
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of (R)-HAs for practical application, a 1.0 L scale one-pot concurrent
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7
8
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1
17
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2
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1
9
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This journal is c The Royal Society of Chemistry 2013
Chem. Commun.