Biocatalytic Preparation of Chiral Sulfoxides through Asymmetric Reductive Resolution by Methionine Sulfoxide Reductase A

Article abstract of DOI:10.1002/cctc.201800279

Here we report an environmentally friendly method for the preparation of chiral sulfoxides under high substrate concentration using recombinant methionine sulfoxide reductase A from Pseudomonas monteilii (pmMsrA) as a biocatalyst. Our results show that this enzyme can effectively accomplish the preparation of (R)-sulfoxides with approximately 50 % yield and 94–99 % enantiomeric excess through asymmetric reductive resolution of racemic sulfoxide. With the establishment of the enzyme regeneration system, the initial substrate concentration could be increased 40–100 times compared to our original report. The (R)-sulfoxides were obtained with high enantioselectivity under the substrate concentration up to 200 mm (approximately 32 g L^?1), representing a quite high substrate concentration in biocatalytic preparation of chiral sulfoxides. Moreover, this system showed fairly good activity and enantioselectivity towards a series of ortho- and para-substituted phenyl methyl sulfoxides under high substrate concentration.

Full text of DOI:10.1002/cctc.201800279

Accepted Article
Title: Biocatalytic preparation of chiral sulfoxides through asymmetric
reductive resolution by methionine sulfoxide reductase A
Authors: Liaotian Peng, Yuanmei Wen, Yu Chen, Zhimei Yuan, Yang
Zhou, Xiaoling Cheng, Yongzheng Chen, and Jiawei Yang
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To be cited as: ChemCatChem 10.1002/cctc.201800279
Link to VoR: http://dx.doi.org/10.1002/cctc.201800279
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10.1002/cctc.201800279
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Biocatalytic preparation of chiral sulfoxides through asymmetric
reductive resolution by methionine sulfoxide reductase A
Liaotian Peng,[a] Yuanmei Wen,[a] Yu Chen,[b] Zhimei Yuan,[a] Yang Zhou,[a] Xiaoling Cheng,[a]
Yongzheng Chen*[b] and Jiawei Yang*[a]
Abstract: Here we report an environmentally friendly method for the
and greener alternative in stereoselective synthesis due to
multiple advantages, such as high stereoselectivity and mild
reaction conditions.[10-12]
preparation of chiral sulfoxides under high substrate concentration
using recombinant methionine sulfoxide reductase
A
from
pseudomonas monteilii (pmMsrA) as biocatalyst. Our results show
that this enzyme can effectively accomplish the preparation of (R)-
sulfoxides with approximately 50% yields and 94-99% enantiometric
Most of reported biocatalytic approaches for preparation of chiral
sulfoxides focused on the asymmetric oxidation of sulfides by
13-15]
excess through asymmetric reductive resolution of racemic sulfoxide. whole-cell systems or isolated enzymes.[10,
For instance,
With the establishment of the enzyme regeneration system, the
initial substrate concentration could be increased 40-100 times
comparing to our original report. The (R)-sulfoxides was obtained
with high enantioselectivity under the substrate concentration up to
200 mM (approximately 32 g/L), representing a quite high substrate
concentration in biocatalytic preparation of chiral sulfoxides.
Moreover, this system showed fairly good activity and
enantioselectivity towards a series of ortho- and para-substituted
phenyl methyl sulfoxides under high substrate concentration.
Goundry and co-workers reported a BVMO-NADPH-KRED
enzyme system which achieved 94% of stereoselective
oxidation of sulfide after 24 h reaction at substrate concentration
of 40 g/L (approximately 130 mM). However, biocatalytic
approaches in asymmetric oxidative or reductive resolution of
rac-sulfoxides to prepare enantiopure sulfoxides were less
reported. Xu and colleagues reported a fed-batch reaction of
oxidative resolution process to synthesize (S)-phenyl methyl
sulfoxide by using resting cells of Rhodococcus sp. ECU0066 as
catalyst. The (S)-enantiomer in 37.8 mM (approximately 5.3 g/L)
concentration and 93.7% ee was obtained, suggesting that
asymmetric resolution could also be an effective approach to
prepare optically pure sulfoxides.[17] Recently, several studies on
the asymmetric reduction of rac-sulfoxides catalyzed by bacterial
cells under anaerobic condition has been reported. The ability of
the bacterial dimethyl sulfoxide reductase in catalyzing the
asymmetric reduction of sulfoxides was revealed.[18-21] In some
cases, high enantioselectivity was shown, while the substrate
Introduction
Chiral sulfoxides are valuable organosulfur compounds and
have been widely used as important intermediates in
asymmetric synthesis and as bioactive ingredients in
pharmaceutical industry.[1-6] In the asymmetric synthesis,
optically active sulfoxides are used as stereogenic centers and
may also induce chirality in successive reaction steps.[1-2] In the
medicinal and pharmaceutical industry, enantiopure sulfoxides
have significant commercial value in the synthesis of
pharmaceuticals such as the anti-carcinogenic compound
sulforaphane and the antiulcer medicine esomeprazole.[4-6] In
general, two strategies could be used for the synthesis of
enantiopure sulfoxides, including asymmetric oxidation of
prochiral sulfides and asymmetric resolution of racemic (rac)
sulfoxides.[7-8] Many methods for synthesis of chiral sulfoxides
have been established using chemical and biological catalysts.[8-
10] In particular, biocatalytic approach is becoming more useful
concentration (less than
1 g/L) was far from industrial
application.[21-23] Thus, biocatalytic reductive resolution of rac-
sulfoxides for chiral sulfoxides preparation has not yet been
established.
Methionine sulfoxide reductase (Msr) is another class of
enzymes that biologically involve in reducing the sulfoxide
group.[24-25] This enzyme family is important protein repair
enzyme that catalyze the reduction of methionine sulfoxide to
methionine and protect cells from oxidative stress.[26-27] Among
them, MsrA is specific to the (S)-enantiomer of methionine
sulfoxide,[28] which implied the potential application in (R)-
sulfoxides preparation through reductive resolution of rac-
29]
sulfoxides. In our previous reports,[22,
an MsrA gene from
Pseudomonas monteilii CCTCCM 2013683 (pmMsrA) has been
screened and recombinant expressed in E. coli. Several (R)-
sulfoxides with ee>93% were prepared under substrate
concentration of 2-5 mM using whole-cell system expressing
pmMsrA protein. However, the catalytic efficiency decreased
dramatically when the substrate concentration higher than 5 mM.
In this research, we investigated and optimized the catalytic
conditions of recombinant pmMsrA. Then, an enzyme
regeneration system was developed based on the general
catalytic mechanism of MsrA, resulting in a great improvement in
the catalytic efficiency and initial substrate concentration. Finally,
approximately 50% yield and 94-99% ee of (R)-1a-1g (Scheme
[a] L. Peng, Y. Wen, Z. Yuan, Y. Zhou, X. Cheng, Prof. Dr. J. Yang
Department of Biochemistry
Zunyi Medical University
Zunyi 563000, (P. R. China)
E-mail: yangjw@zmc.edu.cn
[b] Y. Chen, Prof. Dr. Y. Chen
Generic Drug Research Centre of Guizhou Province, Green
Pharmaceuticals Engineering Research Centre of Guizhou Province
School of Pharmacy
Zunyi Medical University
Zunyi, 563000, (P. R. China)
E-mail: yzchen@zmc.edu.cn
*Corresponding authors
Supporting information for this article is given via a link at the end of the
document.
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10.1002/cctc.201800279
ChemCatChem
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1) were obtained under the substrate concentration of 50-200
mM (10-32 g/L).
B
A
1
0.8
0.6
0.4
0.2
0
kDa
116.0
66.2
45
35
25
18.4
14.4
5
6
7
8
9
10
11
pH
C
D
1
1
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
Scheme 1. Enantioselective reduction of rac-sulfoxides 1a-1g to sulfides 2a-
2g by pmMsrA, yielding optically pure sulfoxides (R)-1a-1g.
10
20
30
40
50
0
1
2
4
8
12
16
Results and Discussion
T (℃)
Concentration (%)
Figure 1. Purification and characterization of pmMsrA. A, Gel-electrophoresis
of recombinant pmMsrA before and after purification, arrow shows the
pmMsrA protein band. B-C, Optimization of pH (B) and temperature (C) for
pmMsrA activity. D, Effect of methanol (●), ethanol (◆), isopropanol (※), 1,4-
dioxane (▲) and acetonitrile (■) on pmMsrA activity.
Purification and characterization of pmMsrA
The recombinant pmMsrA protein was induced by 0.2 mM of
IPTG for 14 h and purified through the His-tag (Figure 1A). The
pH and temperature optima were determined to establish
optimal conditions for pmMsrA, by using rac-1a (phenyl methyl
sulfoxide, Scheme 1) as substrate. Activities of pmMsrA from pH
5 to pH 11 in 50 mM of phosphate buffer were analyzed and the
results showed that the optimal pH for pmMsrA was 8.0 to 9.0
(Figure 1B). The enzyme had more than 60% of activity between
pH 7.0 and 10.0. Activities from 20 to 55℃ of reaction
temperature were then analyzed. The results indicated that
pmMsrA was most active under 30℃ and remained almost 80%
of activity between 20 and 40℃. The activity decreased
dramatically when the reaction temperature higher than 40℃
and less than 1% of activity remained under 55℃ (Figure 1C),
indicating that the catalysis of pmMsrA was temperature
sensitive. Taken together, pH 8.0 and 30℃ were considered as
the optimal reaction conditions. In addition, organic solvents are
frequently used to increase the solubility of substrates in the
water-phase biocatalytic reaction system, while it could inhibit
the enzyme activity. Thus, resistance of pmMsrA to 5 usually
used organic solvents, e.g. methanol, ethanol, isopropanol, 1,4-
dioxane and acetonitrile were studied (Figure 1D). The pmMsrA
enzyme exhibited a certain resistance to 4 of these reagents
(e.g. methanol, ethanol, isopropanol and 1,4-dioxane),
remaining more than 75% of activity under 8% of each solvent in
the reaction mixture.
Investigation of the enzyme regeneration for pmMsrA
biocatalytic reaction system
In our previous report, the whole-cell expressing recombinant
pmMsrA could convert the rac-1a to (R)-1a only when the initial
substrate concentration was no more than 5 mM.[22] Based on
the reported catalytic mechanism of MsrA, the reductant was
needed to break the intramolecular disulfide bond of MsrA after
catalysis, in order to regenerate the enzyme.[30-31] Thus, we
intended to establish a pmMsrA regeneration system (Scheme
2), aimed to prepare (R)-sulfoxide efficiently under high
substrate concentration. Since thioredoxin (Trx) was the general
reductant for MsrA regeneration in vivo,[31-32] we firstly tested the
effect of the pmMsrA-Trx system. The whole-cell co-expressing
pmTrx and pmMsrA proteins were applied to reduce the rac-1a
at cell density of 15 g/L. However, the conversion of (S)-1a was
still incomplete when the substrate concentration was higher
than 8 mM. After 10 h of reaction, the ee of (R)-1a was only 56%
under the substrate concentration of 10 mM (Figure 2). Based
on the previous reports, the additional Trx reductase and
cofactor NADPH were also needed to achieve the completely
MsrA regeneration.[33] Considering the complexity and high cost
of cofactor in this system, we turned to use dithiothreitol (DTT) to
break the disulfide bond of pmMsrA for its regeneration in vitro.
Thus, the pmMsrA-DTT system was then investigated. Ten μM
of pure pmMsrA enzyme was used to reduce rac-1a at 8 mM
and 10 mM of concentration, with 5 mM of DTT in the reaction
system. After only 2 h of reaction, the reduction of (S)-1a was
almost completely carried out under substrate concentration of
10 mM, while the (R)-1a was scarcely transformed. The ee of
(R)-1a reached approximately 90% after reaction, which is much
better pmMsrA-Trx system (Figure 2). Theoretically, after one
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10.1002/cctc.201800279
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round of reductive reaction by pmMsrA, one DTT molecule
should be used for the reduction of enzyme itself. We
speculated that DTT could be a key factor for increasing the
substrate concentration in our reaction system. Therefore, we
adjusted the concentration of DTT equal to the substrate
concentration, which result in an excessive DTT amount in the
reaction system. The reduction of (R)-1a at concentration of 50
mM was then performed to test our pmMsrA-DTT system under
high substrate concentration. The result manifested that the (S)-
1a was completely reduced with high enantioselectivity (E>200),
producing (R)-1a with ee>99% after 2 h of reaction. These
results suggest that the pmMsrA-DTT reaction system was
capable for efficient preparation of (R)-1a under high substrate
concentration.
system. The (S)-1a was almost completely converted while the
(R)-enantiomer was rarely reduced (approximately 4%, E=94),
obtaining 93% ee of (R)-1a after 2 h of reaction (Figure 3A).
More importantly, the crude enzyme-DTT system exhibited an
extremely high efficiency, in which (S)-1a was completely
reduced with high enantioselectivity (E>200) after only 30 min of
reaction. Approximately 25 mM of (R)-1a with ee>99% were
obtained (Figure 3B), strongly supporting the crude pmMsrA
enzyme-DTT system as an efficient asymmetric reductive
resolution strategy for (R)-sulfoxide preparation. Afterwards, we
further optimized the amount of DTT in the reaction mixture to
obtain a more economical system. Although the theoretical
amount of DTT should be equal to the substrate of (S)-sulfoxide,
it could be consumed by factors like other proteins in the
reaction mixture. Hence, the ratio of DTT/rac-1a from 0.5 to 1.0
in the reaction mixture were performed to investigate the
minimum concentration of DTT. The results indicated that when
the ratio of DTT/rac-1a was 0.5 (in which DTT amount was equal
to the (S)-1a), the reduce of (S)-1a was incomplete (ee of (R)-1a
was 80%, Figure 3C). However, when the ratio of DTT/rac-1a
reached 0.6, the (S)-1a was completely reduced and the ee of
(R)-1a reached 99% after reaction. Consequently, the suggested
amount of DTT in our crude pmMsrA enzyme-DTT system was
0.6-fold of rac-sulfoxide substrate.
CysA-SH
CysB-SH
CysA-SOH
CysB-SH
MsrA
MsrA
H2O
CysA-S
CysB-S
MsrA
A
100
80
60
40
20
0
25
20
15
10
5
Scheme 2. Design of pmMsrA regeneration system based on general catalytic
mechanism of MsrA.
100
80
60
40
20
0
0
15
30
60
90
120
Reaction time (min)
B
100
80
60
40
20
0
25
20
15
10
5
pmMsrA-Trx
pmMsrA-DTT
0
15
30
60
90
120
Reaction time (min)
Figure 2. The ee compare of (R)-1a after reaction by two pmMsrA
regeneration system under substrate concentration of 8, 10 and 50 mM.
C
100
80
60
40
20
0
Although the pure pmMsrA-DTT system has been worked for
asymmetric reduction of rac-sulfoxide, the enzyme purification
process was tedious and time consuming. Thus, we attempt to
apply the DTT in whole-cell and crude enzyme catalysis. Whole-
cell at density of 15 g/L and crude enzyme (containing 1.6 g/L of
total protein) extracted from the same amount of cells were used
as catalyst. The products were analyzed at different reaction
time (from 15 min to 2 h) under the substrate concentration of 50
mM. The results suggested that DDT also worked in whole-cell
0.5
0.6
0.7
0.8
0.9
1
Ratio: DTT/rac-1a
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10.1002/cctc.201800279
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Figure 3. Optimization of pmMsrA-DTT reaction system. A-B, Analysis of 1a
after conversion by whole cell-DTT (A) and crude enzyme-DTT (B) systems,
respectively. (▲) ee of (R)-1a; (■) Concentration of (R)-1a; (●) Concentration
of (S)-1a. C, ee compare of (R)-1a after reaction by crude enzyme-DTT
system with DTT/rac-1a from 0.5 to 1.0.
Asymmetric reductive resolution of sulfoxides with
substituents at aromatic ring
In our previous work, ortho- and para-chloro substitution on
aromatic ring of rac-1a did not affect the activity and
enantioselectivity of pmMsrA under low substrate concentration
(2 mM).[22] In this study, we further tested the catalytic properties
of several other substitution by electron-withdrawing or donating
groups at these two positions under high substrate concentration
(50-200 mM, Table 1). Rac-1b-1g were firstly added into the
crude pmMsrA enzyme-DTT system at concentration of 50 mM.
The data showed that (S)-1b-1g were almost completely
conversed under this substrate concentration and (R)-1b-1g with
94-99% ee were obtained by our system, exhibiting a wide
substrate scope. We then increased the initial concentration of
rac-1b to 200 mM (approximately 32 g/L) to test the activity of
our system. After 4 h of reaction, (R)-1b was also successfully
obtained with high enantioselectivity (99% ee, E>200), which is
quite similar with the preparation of (R)-1a (Table 1). These data
suggests that our system was capable to efficiently prepare (R)-
sulfoxides with electron-withdrawing or donating substituents at
the aromatic ring under high substrate concentration. We further
performed all the reactions at preparative scale and 43-46%
isolated yields of (R)-1a-1g were obtained (Table 1). The high
initial substrate concentration, catalytic rate and operability, as
well as wide substrate scope of our system implies a potential
future for industrial application.
Investigation of substrate tolerance of crude pmMsrA
enzyme-DTT system
In biocatalytic preparation of chiral sulfoxides, substrate
concentration is always the bottleneck for the industrial
application. Thus, the substrate tolerance of our crude pmMsrA
enzyme-DTT system was investigated using rac-1a from 50-200
mM (7-28 g/L) as substrate. We increased the protein
concentration to 2.5 g/L and the reaction time to 4 h, to ensure
the full conversion under high substrate concentration. The
results exhibited that the reductive resolution was successfully
carried out under every substrate concentration (Figure 4). The
(S)-1a can be completely reduced with high enantioselectivity
(E>200) at the substrate concentration up to 200 mM, and
approximately 98 mM of (R)-1a with ee>99% was detected after
reaction. Compared with our previous report using whole-cell
system,[22] this crude pmMsrA enzyme-DTT system greatly
improved the substrate concentration of rac-1a to 40 times. As
the best of our knowledge, 200 mM was the highest substrate
concentration in synthesis of chiral sulfoxides catalyzed by
single recombinant enzyme. Although the maximum yield of the
kinetic resolution was lower than the asymmetric oxidation of the
prochiral sulfide, the initial substrate concentration of our system
was much higher than most of the reported bio-oxidation
strategies (usually less than 30 mM[10, 13]), which ensured a
relatively high preparation efficiency. Moreover, compared with
the high cost of NAD(P)H in most of the oxidation systems, the
low-cost DTT in our system make it more economical for future
industrial applications. Additionally, compared with the current
biocatalytic approaches in chiral sulfoxide synthesis,[13, 16-17] our
system greatly shorten the reaction time to only 4 h, indicating
an quite high catalytic rate. Taken together, our study strongly
suggests that asymmetric reductive resolution of rac-sulfoxides
could be an effective strategy for green synthesis of chiral
sulfoxides.
Table 1. Biocatalytic preparation of (R)-1a-1g by our asymmetric reductive
resolution system (t=4 h).
Conc.
(mM)
Conversion
(%) [a]
ee
Yield
Entry
Substrate
E [c]
(%) [b]
(%) [d]
1
2
3
4
5
6
7
8
9
1a
1b
1c
1d
1e
1f
50
50
51
52
52
51
51
51
52
51
51
>99
>99
>99
>99
>99
94
>200
116
--
--
50
116
44
45
45
46
44
43
44
50
>200
>200
70
50
50
100
80
60
40
20
0
100
80
60
40
20
0
1g
1a
1b
50
98
91
200
200
>99
>99
>200
>200
[a] Conversion of 1a-1g to 2a-2g was determined by GC analysis after
reaction. [b] The ee value of 1f was determined by GC with CP-ChiraSil-
DEX CB column and others were determined by HPLC with OD-H chiral
column. [c] Calculation of E-value was as follows: E=ln[(1-c)(1-ee)]/ln[(1-
c)(1+ee)], where
c denoted the conversion. [d] Isolated yield was
50
80
100
120
160
200
determined as the ratio of the remaining amount of 1a-1g after purification
to its initial amount.
Substrate concentration (mM)
Figure 4. Analysis of 1a after reaction by crude pmMsrA enzyme-DTT system
under different substrate concentration. (▲) ee of (R)-1a; (■) Concentration of
(R)-1a; (●) Concentration of (S)-1a.
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10.1002/cctc.201800279
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containing heat-inactivated pmMsrA peptide was used as control. The
decrease of OD415 was used to describe the relative pmMsrA activity.
Conclusions
In summary, we purified the recombinant pmMsrA protein and
optimized its reaction condition as pH 8.0 at 30℃. Through
using the DTT as reductant, a pmMsrA enzyme regeneration
system with high catalytic efficiency and operability was
Conversion of rac-1a-1g
The substrates rac-1a-1g were commercially available with purity of
analytic grade. Whole-cell after IPTG induction, crude enzyme or pure
pmMsrA enzyme were added into the reaction buffer (50 mM phosphate,
pH 8.0) at concentrations as described in the main text. Substrates 1a-1g
were then added to the reaction mixture. Biocatalysis was performed at 5
mL of reaction volume in a 25 mL glass vials with screw caps in the
incubator at 30℃. After incubation with shaken at 300 rpm from 15 min to
4 h, the reaction mixture was extracted two times by using 10 mL of ethyl
acetate with vigorous shaking. The organic layers were separated by
centrifugation and then dried by anhydrous sodium sulfate. For the
successfully established. Enantiopure sulfoxides in
R
configuration was prepared with high enatioselectivity through
asymmetric reductive resolution by this pmMsrA-DTT system.
Our research represents a significant improvement in the aspect
of substrate concentration (up to 200 mM) and catalytic rate
(within
4 h) in green synthesis of chiral sulfoxides. The
establishment and application of pmMsrA-DTT system strongly
supports that asymmetric reductive resolution of rac-sulfoxides
could be an alternative strategy for green synthesis of optically
pure sulfoxides. In addition, since Msr protein family was wildly
existed in prokaryotes and eukaryotes, our report of pmMsrA in
chiral sulfoxide preparation will undoubtedly contribute to
discovering more Msr homologues as potential biocatalysts for
green synthesis of chiral sulfoxides.
separation and purification of sulfoxides after reaction,
4 parallel
reactions (4×5 mL, 200 mM of substrate) of 1a-1b and 10 parallel
reactions of 1c-1g (10×5 mL, 50 mM of substrate) were performed for 4
h. After extracting and collecting the reaction mixture, the remaining
sulfoxide 1a-1g was purified by silica gel column chromatography with
petroleum ether/ethyl acetate (3:1) as elution solvent.
Analytical methods
Quantification and conversion of 1a-1g to 2a-2g after reaction was
determined by GC with Angilent 7820 GC (Agilent Technologies, Santa
Clara, CA) using a HP-5 column (30 m×0.32 mm×0.25 μm) and an FID
detector. DMSO was added as an internal standard for GC analysis. The
temperature setting was programmed as follows: T0: 50℃; dT/dt: 30℃
/min, T1: 130℃, 0.5 min; dT/dt: 20℃/min, T2: 150℃, 1.5 min; dT/dt:
10℃/min, T3: 155℃, 1.5 min; dT/dt: 20℃/min, T4: 170℃, 3 min; split
ratio 1:5. The ee value of 1f was determined by GC using a CP-ChiraSil-
DEX CB column (25 m×0.25 mm×0.25 μm) and other sulfoxides were
determined using a ShimadzuTM Prominence HPLC on a DaicelTM OD-H
chiral column (250× 4.6 mm, 5 μm) with UV detection at 254 nm,
calculated by the equation: ee=[([R]-[S])/([R]+[S])]×100%. Calculation of
E-value for enantioselectivity was as follows: E=ln[(1-c)(1-ee)]/ln[(1-
c)(1+ee)], where c denoted the conversions. Isolated yield of chiral
sulfoxides 1a-1g was determined as the ratio of the remaining sulfoxides
after reaction and separation to its initial amount. Optical rotation data of
sulfoxides was determined by RUDOLPH research analytical Autopol I-
Experimental Section
Protein expression and purification
The expression plasmid of pmMsrA was constructed in our previous
work.[22] For the co-expression of pmMsrA and pmTrx, these two genes
were cloned into the co-expressing plasmid pRSFDute-1 (Novagen).
Overnight culture of E. coli BL21(DE3) cells containing expression
plasmid was diluted 1:100 in 1 Liter of LB medium (1% of peptone, 0.5%
of yeast extract, and 1% of NaCl) with 50 mg/L of kanamycin and grown
at 37°C until it reached an optical density at 600 nm (OD600) of 0.6. Cells
were induced with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG)
for 14 h at 20°C , and harvested by centrifugation at 5000 rpm for 15 min.
After frozen at -20℃ for more than 1 h, lysis buffer (50 mM phosphate, 1
mg/mL of lysozyme and 5 μg/mL Dnase) was added and cells were
incubated at room temperature for 1 h. The crude enzyme were obtained
by centrifuging for 0.5 h at 15000 rpm at 4°C . Purification of pmMsrA was
performed through the His6-tag in the recombinant protein. The soluble
crude enzyme was mixed with pre-equilibrated Ni-sepharose resin for 2 h.
The flow through was removed, and the column was washed with 8
column volumes of wishing buffer (50 mM phosphate, pH 7.4, 150 mM
NaCl and 10 mM imidazole). The protein was eluted with 2 column
volumes of elution buffer (50 mM phosphate, pH 7.4, 150 mM NaCl and
100 mM imidazole). Proteins were quantified by the method of Bradford
and analysed by SDS-PAGE for purity check.
AP. The absolute configuration of 1a-1c and 1e was assigned by
13, 33, 34]
comparison rotation data with the references,[9,
and other
compounds was assigned by the above analogies. 1H and 13C NMR
spectra were recorded on a Brucker-300 (300/75 MHz) spectrometer
using CDCl3 as a solvent and TMS as an internal standard.
Spectral and ee data of the biosynthesized compounds
Compound (R)-1a: [α]25D=+152.7 (c=1.0, CHCl3) for (R), 99% ee; lit:
[α]25D=+142.8 (c=0.53, acetone) for (R), 99% ee.[13] DaicelTM OD-H chiral
column (250×4.6 mm, 5 μm) at 25℃ with a flow rate of 1 mL/min UV and
the mobile phase used was 7% isopropanol: 93% n-hexane. Retention
time: 17.0 min for (R)-1a and 25.1 min for (S)-1a. 1H NMR (400
MHz,CDCl3): δ 2.65-2.66 (m, 3H), 7.41-7.48 (m, 3H), 7.56-7.59 (m, 2H);
13C NMR (100 MHz, CDCl3): δ 43.8, 123.4, 129.3, 131.0, 145.4.
Relative activity assay of pmMsrA
Relative activity of pmMsrA under different conditions were tested by a
colorimetric assay based on previous report[33] with modifications.
Specifically, 0.5 μM of pmMsrA,1 mM of DTT and 1mM of rac-1a were
put into the 1 mL of reaction system in 50 mM of phosphate buffer. After
20 min of reaction under different conditions (pH, temperature and
organic solvents), the reaction mixture was diluted with 50 mM of
phosphate buffer (pH 8.0) by 20 times. And then, 100 μL of diluted
samples were transfer into the 96-well plate, and another 100 μL of
reaction mixture (4 mM DTNB, 50 mM phosphate buffer, pH 8.0) were
added. After 10 min of incubation at 37℃, the plate were put into the
microplate reader to detect the absorption at 415 nm. A same system
Compound (R)-1b: [α]25D=+131.6 (c=1.0, CHCl3) for (R), 99% ee; lit:
[α]25D=−128.6 (c=1.5,acetone) for (S), 98.6% ee.[34] DaicelTM OD-H chiral
column (250×4.6 mm, 5 μm) at 25℃ with a flow rate of 1 mL/min and the
mobile phase used was 7% isopropanol: 93% n-hexane. Retention time:
1
16.6 min for (R)-1b and 18.2 min for (S)-1b. H NMR (400 MHz, CDCl3):
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10.1002/cctc.201800279
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δ 2.56 (s, 3H), 7.06 (t, J = 8.3 Hz, 2H), 7.50 (dd, J = 7.8 Hz, 5.3 Hz, 2H);
13C NMR (100 MHz, CDCl3): δ 43.6, 116.4, 125.6, 140.4, 163.9.
Keywords: biocatalysis• reductive resolution• chiral sulfoxides•
methionine sulfoxide reductase A (MsrA)• regeneration
Compound (R)-1c: [α]25D=+128.5 (c=1.0, CHCl3) for (R), 99% ee; lit:
[α]25D=+138.8 (c=0.66, acetone) for (R), 96%ee.[9] DaicelTM OD-H chiral
column (250×4.6 mm, 5 μm) at 25℃ with a flow rate of 1 mL/min and the
mobile phase used was 7% isopropanol: 93% n-hexane. Retention time:
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16.2 min for (R)-1c and 19.3 min for (S)-1c. H NMR (400 MHz, CDCl3):
δ 2.22 (s, 3H), 2.54 (s, 3H), 7.15 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.1 Hz,
2H); 13C NMR (100 MHz, CDCl3): δ 21.4, 43.8, 123.5, 130.0, 141.7,
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Compound (R)-1d: [α]25D=+152.7 (c=1.0, CHCl3) for (R), 99% ee;
DaicelTM OD-H chiral column (250×4.6 mm, 5 μm) at 25℃ with a flow
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follows: T0: 50℃; dT/dt: 30℃/min, T1: 130℃, 0.5 min; dT/dt: 20℃/min,
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170℃, 8 min; split ratio 1:5. Retention time: 6.595 min for (R)-1f and
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= 7.9 Hz, 2H), 7.32 (t, J = 7.6 Hz, 1H), 7.50 (d, J = 7.7 Hz, 1H), 10.09 (s,
1H); 13C NMR (100 MHz, CDCl3): δ 40.8, 117.1, 120.3, 124.3, 127.0,
132.6, 155.1
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We are grateful for financial supports from National Natural
Science Foundation of China (No. 31460230 and No. 21262051),
Guizhou Science and Technology Department (No. QKHLHZ-
2014-7586, No. QKHRCTD-2014-4002, QKHRC-2016-4029,
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of Pharmacy (GNYL-2017-006), the Fifth Batch of Talent Base in
Guizhou Province, and Program for Outstanding Youth of Zunyi
Medical University. We are also grateful for the English writing
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Entry for the Table of Contents (Please choose one layout)
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An efficient method for preparation of
chiral sulfoxides at high substrate
concentration (up to 200 mM) was
developed using pmMsrA as
Liaotian Peng, Yuanmei Wen, Yu Chen,
Zhimei Yuan, Yang Zhou, Xiaoling
Cheng, Yongzheng Chen* and Jiawei
Yang*
biocatalyst. After establishing enzyme
regeneration system (crude enzyme-
DTT), this enzyme effectively
Page No. – Page No.
accomplished the preparation of (R)-
1a-1g with 43-46% of yields and 94-
99% ee through asymmetric reductive
resolution of racemic sulfoxide.
Biocatalytic preparation of
chiral sulfoxides through
asymmetric reductive
resolution by methionine
sulfoxide reductase A
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