Identification of MsrA homologues for the preparation of (R)-sulfoxides at high substrate concentrations

Article abstract of DOI:10.1039/c9ob00384c

Here we report a methionine sulfoxide reductase A (MsrA) homologue with extremely high substrate tolerance and a wide substrate scope for the biocatalytic preparation of enantiopure sulfoxides. This MsrA homologue which was obtained from Pseudomonas alcaliphila (named paMsrA) showed good activity and enantioselectivity towards a series of aryl methyl/ethyl sulfoxides 1a-1k, with electron-withdrawing or electron-donating substituents at the aromatic ring. Chiral sulfoxides in the R configuration were prepared with approximately 50% of yield and up to 99% enantiomeric excess through the asymmetric reductive resolution of racemic sulfoxide catalyzed by the recombinant paMsrA protein. More importantly, kinetic resolution has been successfully accomplished with high enantioselectivity (E > 200) at initial substrate concentrations up to 320 mM (approximately 45 g L^-1), which represents a great improvement in the aspect of the substrate concentration for the biocatalytic preparation of chiral sulfoxides.

Full text of DOI:10.1039/c9ob00384c

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DOI: 10.1039/C9OB00384C
Organic & Biomolecular Chemistry
PAPER
Identification of MsrA Homologues for Preparation of (R)-
Sulfoxides at High Substrate Concentration
Jiawei Yang,a, b Yuanmei Wen, Liaotian Peng, Yu Chen, Xiaoling Cheng and Yongzheng Chen*
a
a
b, c
a
b, c
Received,
Accepted
Here we report a methionine sulfoxide reductase A (MsrA) homologue with extremely high substrate tolerance and wide
substrate scope in the biocatalytic preparation of enantiopure sulfoxides. This MsrA homologue which was from
pseudomonas alcaliphila (named paMsrA) showed good activity and enantioselectivity towards a series of aryl methyl/ ethyl
sulfoxides 1a-1k, with electron-withdrawing or donating substituents at the aromatic ring. Chiral sulfoxides in R configration
were prepared with approximately 50% of yield and up to 99% enantiomeric excess through asymmetric reductive resolution
of racemic sulfoxide catalyzed by the recombinant paMsrA. More importantly, kinetic resolution have been successfully
accomplished with high enantioselectivity (E>200) at an initial substrate concentration up to 320 mM (approximately 45
g/L), which represents a great improvement in aspect of substrate concentration in biocatalytic preparation of chiral
sulfoxides.
DOI: 10.1039/x0xx00000x
www.rsc.org/
On the other hand, kinetic resolution of rac-sulfoxide has become an
Introduction
effective approach for enantioselective synthesis of chiral sulfoxides,
8
a, 10
11
including biological
and chemical ways. Among them,
biocatalytic kinetic resolution approach containing two major
strategies: the oxidative resolution which asymmetrically oxidize the
Chiral sulfoxides are a group of valuable organosulfur chemicals
widely used as important intermediates in asymmetric synthesis and
medicinal chemistry. Many sulfoxides have high biological activity
8
a
rac-sulfoxides to sulfones,
and the reductive resolution which
1
asymmetrically reduce the rac-sulfoxides to the corresponding
across a broad range of indications, such as antiulcer agent
1
0
sulfides. For instance, Xu and colleagues developed a fed-batch
reaction of oxidative resolution process using the Rhodococcus sp.
ECU0066 cells, and the (S)-phenyl methyl sulfoxide in 37.8 mM
2
3
esomeprazole, potassium channel activator aprikalim, immuno-
4
suppressor oxisurane, etc. Because of the importance of chiral
sulfoxides in pharmaceutical industry, as well as their use as chiral
8
a
(approximately 5.3 g/L) concentration and 93.7% ee was obtained.
5
auxiliaries in asymmetric synthesis, strategies for their preparation
Abo and colleagues developed an electrochemical enzymatic
deoxygenation system for preparation of chiral sulfoxides by utilizing
6
in high optical purity were greatly concerned. As a green approach,
biocatalysis has been emerging as an important tool in industrial
synthesis of bulk chemicals, pharmaceuticals and agrochemical
1
0a, 10c
DMSO reductase.
Recently, we have established a pmMsrA-DTT
system which is able to prepare (R)-sulfoxides with approximately 45%
yield and 94–99% ee at substrate concentration up to 200 mM (32
7
intermediates. The strategies for biocatalytic preparation of
enantiopure sulfoxides can be summarised in two categories,
asymmetric oxidation of prochiral sulfides and asymmetric
1
0h
g/L). This result represents the highest substrate concentration in
synthesis of chiral sulfoxides catalyzed by single recombinant
enzyme, which is much higher than most of the reported bio-
6
c, 8
resolution of racemic (rac) sulfoxides.
At present, most reported
biocatalytic approaches focus on the asymmetric oxidation of
sulfides. A lot of enzymes including the BVMO, cytochrome P450
monooxygenase family, peroxidase and toluene dioxygenase have
been used to synthesize chiral sulfoxides through whole-cell system
9
oxidation strategies.
Methionine sulfoxide reductase A (MsrA) is a class of enzyme that
specifically reduces the (S)-enantiomer of methionine sulfoxide to
methionine, which is an important protein repairing enzyme that
6
c, 9
or isolated enzyme.
1
2
protects cell from oxidative stress. Thus, it is practicable to develop
a high efficient biocatalytic reductive resolution approach for
enantioselective synthesis of (R)-sulfoxides by MsrA enzyme.
Recently, Nosek and co-workers have designed a MsrA-oxaziridine
oxidant biphasic reaction system and accomplished up to 90% yield
a. Department of Biochemistry, Zunyi Medical University, Zunyi 563000, Guizhou
province, P.R. China.
1
3
b.
of (R)-sulfoxides. Although the theoretical maximum yield of the
kinetic resolution is only 50%, the development of enantioselective
reduction-oxidation system strongly supports that MsrA could also
be used as an excellent biocatalyst for preparing (R)-sulfoxides in
Key Laboratory of Biocatalysis & Chiral Drug Synthesis of Guizhou Province,
Generic Drug Research Center of Guizhou Province, Green Pharmaceuticals
Engineering Research Center of Guizhou Province.
School of Pharmacy, Zunyi Medical University, Zunyi, 563000, Guizhou province,
P.R. China.
c.
*Corresponding author.
high yields. Considering the MsrA protein family is widely existed in
Electronic Supplementary Information (ESI) available:See
DOI: 10.1039/x0xx00000x
12, 14
living species,
this study has tested the activities of several
This journal is © The Royal Society of Chemistry 20xx
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O rP gl ea na si ce &d Bo i on mo to al e dc juu l sa tr mC ha er mg i ins ts ry
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Journal Name
○
pmMsrA homologues aimed to obtain MsrA enzymes with better together, pH 8.0 and 30 C were considered as the optimal reaction
catalytic performance. Finally, an MsrA homologue from conditions.
View Article Online
DOI: 10.1039/C9OB00384C
Pseudomonas alcaliphila (paMsrA) with wider substrate scope and
extremely high substrate tolerance was successfully obtained.
Table 1. Comparing of pmMsrA with 4 homologues.
Name
Organism
Length Identity a)
Previous report
pmMsrA Pseudomonas monteilii 222 aa --
O
S
pcMsrA
pfMsrA
paMsrA P. alcaliphila
P. cremoricolorata
P. fluorescens
221 aa 90%
215 aa 81%
217 aa 72%
212 aa 60%
O
S
pmMsrA
S
+
R2
R1
R2
R1
R2
R1
(R)-1a-1g
rac-1a-1g
0-200 mM
2a-2g
vhMsrA
Vibrio hyugaensis
5
up to 99% ee, E>200
a)
Sequence identities with pmMsrA
This report
O
S
O
paMsrA
A
pcMsrA pfMsrA paMsrA vhMsrA Ladder
kDa
80
130
00
S
S
+
R2
1
R1
R2
R1
R2
R1
(R)-1a-1k
up to 99% ee, E>200
1
2
a-2k
rac-1a-1k
7
0
1
00-320 mM
55
4
0
1
a, 2a: R =Ph, R =CH
3
1g, 2g: R1=o-OHPh, R2=CH3
35
20
1
2
1
b, 2b: R =p-CH Ph, R =CH
1h, 2h: R =o-NHCHOPh, R =CH
3
1
3
2
3
1
2
1
1
1
1
c, 2c: R =p-FPh, R =CH
1i, 2i: R =m-NH Ph, R =CH
1 2 2 3
1
2
3
d, 2d: R =p-CHOPh, R =CH
1j, 2j: R1=2-Naphthyl, R2=CH3
1k, 2k: R =Ph, R =CH CH
1
2
3
B
C
e, 2e: R =p-BrPh, R =CH
1
1
0.8
0.6
0.4
0.2
0
1
2
3
1
2
2
3
f, 2f: R =o-NH Ph, R =CH
1
2
2
3
0.8
0
.6
Scheme 1. Enantioselective reduction of rac-sulfoxides 1a-1k to
sulfides 2a-2k by recombinant paMsrA protein, yielding optically
pure sulfoxides (R)-1a-1k.
0.4
0.2
0
4
5
6
7
8
9
10
11
20
25
30
35
40
45
50
55
60
pH
T (℃)
Results and discussion
Figure 1. Recombinant expression and characterization of 4 MsrA
homologues. A) Gel electrophoresis of 4 recombinant MsrA enzymes,
arrow indicates the MsrA protein band. Optimization of B) pH and C)
Screening and characterization of MsrA homologues
In our previous study, a methionine sulfoxide reductase A gene was temperature for pcMsrA (
cloned from Pseudomonas monteilii (pmMsrA) and the recombinant reaction system.
protein was used as biocatalyst for preparation of (R)-sulfoxides at
■
), pfMsrA (▲), paMsrA (●) and vhMsrA (◆)
1
0h
substrate concentration up to 200 mM. In this study, we tested the
activity of several pmMsrA homologues, in order to find MsrA Catalytic analysis of MsrA homologues on kinetic resolution of rac-
proteins with better catalytic properties for preparing chiral sulfoxides
sulfoxides. After sequence alignment using pmMsrA as template,
four pmMsrA homologues with sequence identities from 60%-90% To test whether these MsrA enzymes could accomplish the kinetic
were selected from GenBank database, and named as pcMsrA, resolution of rac-sulfoxides at high substrate concentration, the rac-
pfMsrA, paMsrA and vhMsrA. The detailed protein sequences 1a was used as substrate at the concentration of 100 mM (14 g/L).
information was summarized in Table 1. After 14 h of induction by The crude enzyme-DTT system which we established before was
1
0h
isopropyl-β-D-thiogalactopyranoside (IPTG), all four recombinant used as the reaction system. Crude MsrA enzymes (containing
proteins were successfully expressed (Figure 1A). The rac-1a (phenyl approximately 30-55% of MsrA recombinant proteins) were used as
methyl sulfoxide, Scheme 1) was used as substrate to test the activity biocatalysts at the concentration of 3 g/L of total protein. The results
of these enzymes, as well as pH and temperature optima to establish showed that three of the MsrA homologues (pcMsrA, paMsrA and
the reaction system. The results showed that all these four vhMsrA) exihibited similar activities on rac-1a compared with
recombinant MsrA proteins were active on rac-1a. Activities of pmMsrA (Table 2). After 4 h of reaction, (S)-1a was reduced with high
MsrAs from pH 5 to pH 11 in 50 mM phosphate buffer were then enantioselectivity (E>200) and approximately 50% of (R)-1a with
analyzed. All four MsrA enzymes showed similar pH profiles and the ee>90% were obtained by these MsrA homologues. To further
optimal pH was 8.0 (Figure 1B). These enzymes had more than 70% compare the activity of pmMsrA and these homologues, kinetic
○
of activity from pH 7.5 to pH 9.0. Activities from 20 to 60 C of resolution of rac-1f and rac-1g at the concentration of 100 mM was
1
0h
reaction temperature were also analyzed. These enzymes were most then performed. In our previous report, the pmMsrA accomlished
○
active at 30 C (Figure 1C). However, the activities of these four the kinetic resolution of rac-1f and rac-1g at the concentration of 50
enzymes decreased significantly when the reaction temperature mM. When concentration reached 100 mM, the reduction of these
○
10h
reached 40 C, which is quite similar with the pmMsrA. Taken two compounds was imcomplete and only less than 30% convension
was detected after reaction with pmMsrA (Table 2). However, two
2
| J. Name., 2018, 00, 1-3
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homologues (pcMsrA and paMsrA) exihibited fairly good activity not not working for pmMsrA. The results showed thaVtiewthAertipclaeMOnslirnAe
towards rac-1f, which were much better than pmMsrA (Table 2). In was active on some of these compounds atDsOuIb:s1t0r.a10te39c/oCn9cOeBn0t0ra3t8i4oCn
particular, (R)-1f with ee>99% (E>200) was obtained after the of 100 mM, eihibiting wider substrate scope compared with our
reaction by paMsrA. More importantly, paMsrA also accomplished previously reported pmMsrA. Among these substrates, the (S)-1h-1j
the kinetic reselution of rac-1g, in which (R)-1g with ee>99% (E>200) were reduced with good enantioselectivity and the (R)-1h-1j with 69-
was obtained after reaction by this enzyme (Table 2). Considering the 99% ee were obtained (Table 3). The high catalytic activity and
content of MsrA proteins in crude enzymes were different due to enatioselectivity of paMsrA on substrates rac-1a-1j indicates that the
their different expression level, we then purified these recombinant substitution on the benzene ring of 1a affect the enzyme activity
proteins and applied 5 μM of pure MsrA enzymes to further compare slightly, exhibiting an excellent compatibility on this series of
their activities. After 1 h of reaction with rac-1g, 6.8-18% of substrates. However, the substitution on the methyl group of 1a
conversions were detceted (Supporting Information). As expected, affected the activity of paMsrA seriously. Among the substrates rac-
the conversion of rac-1g by paMsrA was obviously higher than other 1k-1n (in which the methyl group was replaced by ethyl,
four MsrA homologuses. Taken together, these results suggested chloromethyl, ethenyl and isopropyl groups, respectively), only rac-
that the catalytic properties of paMsrA enzyme was much better 1k was active for the enzyme (Table 3). The (R)-1k with ee>99% was
than pmMsrA and other tested homologuses. Therefore, the paMsrA abtained with high enantioselectivity at the substrate concentration
was selected for our further research on kinetic resolution of rac- of 100 mM. The conversion of other three compounds were very low
sulfoxides.
even at the substrate concentration of 10 mM. These results suggest
that a methyl or ethyl group was essential for the activity of MsrA
Table 2. Kinetic resolution analysis of rac-1a, 1f, 1g by pmMsrA and enzymes. In addition, we tested the activity of an alkyl sulfoxide rac-
4
homologues. (t=4h, substrate concentration=100 mM)
1
o. The paMsrA enzyme exhibited high activities on both R and S
configurations of 1o, in which 91% and 77.8% of conversions were
obtained after reaction at the substrate concentrations of 10 mM
and 100 mM, respectively. This result demonstrates that the
structure of substrate could affect the enantiaoselectivity of MsrA
enzyme seriously, while further investigations were needed for
explanation of the machenism. Overall, the paMsrA enzyme showed
remarkably wide substrate scope for a series of aromatic sulfoxides
1a-1k at high substrate concentration. Finally, we performed the
reactions of 1a-1k on preparative scale, and 42-51% isolated yields
of (R)-1a-1k were obtained (Table 3).
Substrate
Enzyme
Conversion (%) a)
ee (%) b)
E c)
pmMsrA
pcMsrA
pfMsrA
paMsrA
vhMsrA
pmMsrA
pcMsrA
pfMsrA
paMsrA
vhMsrA
pmMsrA
pcMsrA
pfMsrA
paMsrA
vhMsrA
50.4
50.3
37.0
50.7
48.2
29.2
47.4
11.3
50.2
21.3
15.9
30.7
7.8
>99
>99
57
>200
>200
119
O
S
>99
90
n.d.d)
>200
>200
1
a
O
S
88
>200
>200
n.d.
>99
n.d.
n.d.
n.d.
n.d.
>99
n.d.
NH2
1
f
Table 3. Biocatalytic preparation of (R)-1a-1o by paMsrA
O
S
Concentration
(mM)
Conversion
%)
ee
(%)
Yield
(%)
Substrate
E
a)
b)
(
OH
O
S
50.1
13.1
>200
1
g
1
00
50.7
51.0
50.7
50.4
50.0
>99
>99
>99
>99
>99
>200
44
43
43
43
44
a)
Conversion of 1a, 1f, 1g to the corresponding products 2a, 2f, 2g
was determined by GC after reaction. The ee value of 1g was
determined by GC with CP-ChiraSil-DEX CB column and others were
determined by HPLC with AD-H or OD-H chiral column. Calculation
of E value was as follows: E=ln[(1-c)(1-ee)]/ln[(1-c)(1+ee)], where c
denotes the conversion. "n.d." means not determined due to low
conversion.
b)
1a
O
S
c)
100
>200
>200
>200
>200
1b
O
d)
S
1
00
F
1
c
O
S
Investigation of substrate scope of paMsrA
O
100
In our previous report,10h we found that the pmMarA was capable of
efficiently preparing a series of (R)-sulfoxides 1a-1g, with electron-
withdrawing or donating substituents at the aromatic ring. To test
the substrate scope of paMsrA, we also tested these sulfoxides as
substrates at a higher concentration (100 mM). Crude paMsrA
enzyme was used as biocalyst at the concentration of 3 g/L of total
protein (containing approximately 55 μM of paMsrA protein in the
reaction mixture). The results showed that all the (S)-enationmers
were almost completely converted after reaction, and (R)-1a-1g with
ee>99% were obtained by this enzyme (Table 3). Then, we test the
activity of paMsrA on some other compounds rac-1h-1o, which were
1
d
e
O
S
1
00
Br
1
O
S
100
50.2
>99
>200
45
NH2
1
f
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O
quite similar to rac-1a (Figure 2). The (R)-1b withVie9w5%ArticeleeOwnlianes
prepared at 300 mM (43 g/L) of substrate cDoOnIc: e10n.t1r0a3t9io/Cn9aOnBd00(R38)-41Cc
S
1
00
50.1
51.1
>99
95
>200
78
46
43
OH
with 99% ee was prepared at 280 mM (45 g/L) of substrate
concentration. To the best of our knowlege, 280-320 mM was the
highest susbstrate concentration in biocatalytic preperation of chiral
sulfoxides. The substrate tolerance paMsrA was 50 times higher than
1
g
O
S
NH
100
6
c
most of reported native enzymes. Even compared with our recently
O
reported pmMsrA, the substrate concentration of paMsrA was still
1
h
1
.6-2.0 times higher, representing a significant improvement in the
O
S
aspect of substrate tolerance. The extremely high substrate
tolerance and wide substrate scope of paMsrA implies an excellent
industrial prospect. Compared with asymmetric oxidation systems,
oxygen is not needed in the MsrA biocatalytic system, which would
greatly simplify the process for future industrial application at large
scale. Moreover, high cost cofactors like NAD(P)H were not needed
in the MsrA reaction system, which also makes it an economical
strategy.
1
00
50.4
>99
69
>200
44
NH2
1
i
O
S
1
1
00
00
44.4
50.9
28
51
44
1
j
O
S
>99
>200
100
1
k
O
80
S
O
S
Cl
60
n.d. c)
1
0
0
7.4
--
--
--
--
40
1
l
1a
O
S
20
1
11.6
n.d.
n.d.
0
100
150
200
250
300
320
350
1
m
Substrate concentration (mM)
1
00
80
O
S
1
1
0
0
33.7
--
--
O
S
6
0
1
n
O
1b
91.0
77.8
n.d.
n.d.
--
--
--
--
40
20
0
S
1
00
1
o
a)
The ee value of 1g-1h was determined by GC with CP-ChiraSil-DEX
CB column and others were determined by HPLC with AD-H or OD-H
chiral column. Isolated yield was determined as the ratio of the
100
150
200
250
300
320
b)
Substrate concentration (mM)
1
00
remaining amount of 1a-1k after purification to its initial amount.
The reaction time for 1c, 1d and 1o was 1 h, for 1i was 0.5 h, and for
8
6
4
2
0
0
0
0
0
c)
others was 4 h. "n.d." means not determined.
O
S
F
Investigation of substrate tolerance of paMsrA
1c
Substrate tolerance is a key issue in biocatalytic preparations of chiral
1
0h
sulfoxides for industrial application. In our previous work, the
pmMsrA accomplished the kinetic resolution of sulfoxides with high
enantioselectivity up to 200 mM of substrate concentration. Our
100
150
200
250
280
300
Substrate concentration (mM)
above results about paMsrA implied that this enzyme may have Figure 2. Conversion (●) and ee (▲) analysis of rac-1a-1c after
better performance at high substrate concentration. Thus, rac-1a reaction by paMsrA at different substrate concentrations. Crude
concentrations from 100-350 mM (14-49 g/L) was used to test the enzyme concentration: 3 g/L; DTT amount: 0.6-fold of substrate; t=4
substrate tolerance of paMsrA. The results demonstrated that the h.
reductive resolution of 1a was successfully accomplished at
substrate concentration up to 320 mM (45 g/L) (Figure 2). The (S)-1a
was completely reduced with high enantioselectivity (E>200) at this Conclusions
substrate concentration, and approximately 160 mM of (R)-1a with
ee> 99% was detected after reaction. We further tested the
substrate tolerance of rac-1b-1c for paMsrA, and the results were
In summary, four pmMsrA homologues were screened from
GenBank database and the corresponding recombinant
4
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ARTICLE
proteins were expressed. Three of them showed good activity (pH 8.0). Then, diluted samples (100 μL) were transferVrieewdAirntitcole Oan9lin6e-
on rac-sulfoxides. More importantly, an MsrA from P. alcaliphila well plate, and another 100 μL of reaction mDOixIt:u1r0e.10(439m/Cm9ODBT0N0B38, 45C0
species (paMsrA) exhibited remarkably excellent catalytic mM phosphate buffer, pH 8.0) were added. The plate was incubated
under 37 ◦C for 10 min, and a microplate reader was used to detect
the absorption at 415 nm (OD415). Proteins extracted from cells
containing plasmid pET-28a was used as the control. The decrease of
OD415 was used to describe the relative MsrA activities.
properties on a series of sulfoxides 1a-1k. The kinetic resolution
of rac-sulfoxides was successfully accomplished at up to 320
mM (45 g/L) of substrate concentration catalyzed by paMsrA
recombinant enzyme. Our study strongly supports that
asymmetric reductive resolution of rac-sulfoxides by MsrA
would become an effective strategy for green synthesis of
optically pure sulfoxides.
Biocatalytic kinetic resolution of rac-sulfoxides 1a-1o
The substrates rac-1a-1o were commercially available with analytical
grade purity. The process of kinetic resolution was based on our
1
0h
previously reported MsrA-DTT system.
The DTT was added into
Experimental
the reaction buffer (50 mM phosphate, pH 8.0) at the concentration
of 0.6-fold of substrate. The crude MsrA enzymes were used at the
concentration of 3 g/L of total protein, and the pure enzymes were
used at the concentration of 5 μM. For reaction of rac-1e and 1g, the
substrates were dissolve in 200 μL of methanol and then added into
the reaction mixture. For reaction of other substrates, the rac-
sulfoxides were directly added into the reaction mixture. Kinetic
resolution of rac-1a-1o was performed at the substrate
concentration of 10-320 mM as described in the section of "Results
and Discussions". Biocatalytic reactions were performed at 5 mL of
reaction volume in a 25 mL glass vial with a screw cap in the incubator
at 30 ◦C with shaking at 300 rpm. After incubation for 1-4 h as
described in the main text, the reaction mixture was extracted twice
by using 10 mL of ethyl acetate with vigorous shaking. The organic
layers were collected by centrifugation and then dried by anhydrous
sodium sulfate. For separation and purification of sulfoxides after
reaction, 5 parallel reactions (5×5 mL, 100 mM of substrates) were
performed. After extracting and collecting the reaction mixture, the
remaining sulfoxides 1a-1k were purified by silica gel column
chromatography with petroleum ether/ethyl acetate (3:1) as elution
solvent.
Screening, cloning and recombinant expression of pmMsrA
homologues
The protein BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi)
was applied to search MsrA homologues using protein sequence of
pmMsrA as template. Four MsrA protein sequences with sequence
identity from 60%-90% were selected randomly. After obtaining the
corresponding nucleotide sequences by tblastn program, the DNA
fragments of these MsrA genes were synthesized. The DNA fragment
was digested by BamH I and Hind III endonuclease and ligated into
the plasmid pET-28a. Afterwards, the recombinant plasmids were
transformed into E. coli DH5α cells. To induce the soluble expression
of recombinant enzymes, the plasmids were transformed into E. coli
BL21(DE3) cells. An overnight culture of BL21(DE3) cells containing
expression plasmid was diluted 1:100 in 1 L LB medium (1% peptone,
0
.5% yeast extract, and 1% 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 10 min. After freezing the cells at -20
◦C over 1 h, lysis buffer (50 mM phosphate, 1 mg/mL lysozyme, and
5
μg/mL DNase) was added and cells were incubated at room
Analytical methods
temperature for 1 h. The crude enzyme was obtained by centrifuging
for 15 min at 15000 rpm at 4 ◦C. Purification of MsrAs were
performed through the His -tag in the recombinant proteins. The
6
Quantification of 1a–1o and 2a–2o after reaction were determined
by GC with Agilent 7820 GC (Agilent Technologies, Santa Clara, CA)
using a HP-5 column (30 m×0.32 mm×0.25 μm) with an FID detector.
DMSO was added as an internal standard. The temperature setting
was programmed as follows: T0: 50 ◦C; dT/dt: 30 ◦C/min, T1: 130 ◦C,
soluble crude enzymes were mixed with pre-equilibrated Ni-
Sepharose resin for 2 h. The flowthrough 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
recombinant proteins were eluted by 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
subjected to SDS-PAGE gel electrophoresis. The intensities of protein
bands was quantified by the software BandScan 5.0 for estimating
the content of recombinant MsrA proteins in the crude enzyme.
0
1
.5 min; dT/dt: 20 ◦C/min, T2: 150 ◦C, 1.5 min; dT/dt: 10 ◦C/min, T3:
55 ◦C, 1.5 min; dT/dt: 20 ◦C/min, T4: 170 ◦C, 3 min; split ratio 1: 5.
The ee value of 1g-1h was determined by GC using a CP-ChiraSil-DEX
CB column (25 m×0.25 mm×0.25 mm), and other sulfoxides were
TM
determined using a Shimadzu Prominence HPLC on a Daicel AD-H
or 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-1k 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-AP. The absolute
configuration of 1a-1g, 1k was assigned by comparison of the
rotation data with previous reports and 1h-1j was assigned by the
Relative activity assay of pmMsrA
Relative activity of MsrA recombinant enzymes under different pH
and temperature conditions was determined by a colorimetric assay
based on literatures with modifications.10h, 15 Specifically, MsrA crude
enzyme (0.5 mg/mL), DTT (1 mM), and rac-1a (1 mM) were put into
1
2
mL reaction system containing 50 mM of phosphate buffer. After
0 min reaction under different pH and temperature conditions, the
1
13
above analogies. H and C NMR spectra were recorded on a
reaction mixture was diluted 20 times by 50 mM phosphate buffer
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
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O rP gl ea na si ce &d Bo i on mo to al e dc juu l sa tr mC ha er mg i ins ts ry
Page 6 of 9
ARTICLE
Journal Name
Brucker-300 (300/75 MHz) spectrometer using CDCl
TMS as an internal standard.
3
as a solvent and hexane. Retention time: 12.6 min for (R)-1f and 19.1 min for (S)-1f.
View Article Online
DOI: 10.1039/C9OB00384C
1
H NMR (400 MHz, CDCl ): δ2.89 (s, 3H), 4.71 (s, 2H), 6.67-6.74 (m,
3
1
3
2
1
H), 7.19-7.23 (m, 2H); C NMR (100 MHz, CDCl
17.7, 123.6, 126.3, 132.5, 147.4.
3
): δ 38.2, 117.6,
Spectral and ee data of 1a-1k after kinetic resolution and
purification
D
Compound (R)-1g: [α]25 =+128.9 (c=2.0, CHCl
3
) for (R), 99% ee; lit:
Compound (R)-1a: [α]25D=+138.2 (c=2.0, CHCl
α]25D=+152.7 (c=1.0, CHCl ) for (R), 99% ee;10h Daicel OD-H chiral
) for (R), 99% ee; lit:
3
α]25D=+133.6 (c=1.0, CHCl
) for (R), 94% ee.10h CP-ChiraSil-DEX CB
[
3
[
3
column (25 m×0.25 mm×0.25 μm) and programmed as follows: T0:
column (250×4.6 mm, 5 μm) at 25℃ with a flow rate of 1 mL/min,
and the mobile phase used was 7% isopropyl alcohol/93% n-
hexane. Retention time: 15.4 min for (R)-1a and 20.7 min for (S)-1a.
1H NMR (400 MHz, CDCl3): δ 2.62 (s, 3H), 7.35-7.44 (m, 3H), 7.53-
5
1
0℃; dT/dt: 30℃/min, T1: 130℃,0.5 min; dT/dt: 20℃/min, T2:
50℃, 5 min; dT/dt: 10℃/min,T3: 155℃, 2 min; dT/dt: 20℃/min,
T4: 170℃, 2 min; split ratio1: 5. Retention time: 6.397 min for (R)-
1
3
1
g and 6.604 min for (S)-1g. H NMR (400 MHz, CDCl
3
):δ 2.89 (s,
7
1
.55 (m, 2H); 13C NMR (100 MHz, CDCl3): δ 43.6, 123.3, 129.2,
H), 6.99 (t, J = 7.9 Hz, 2H), 7.32 (t, J = 7.6 Hz, 1H), 7.50 (d, J = 7.7
30.9, 145.3.
13
Hz, 1H), 10.09 (s, 1H); C NMR (100 MHz, CDCl
3
): δ 40.9, 117.5,
1
20.3, 124.5, 126.2, 132.8, 155.9.
Compound (R)-1b: [α]25D=+115.1 (c=2.0, CHCl
α]25D=+128.5 (c=1.0, CHCl ) for (R), 99% ee.10h Daicel OD-H chiral
) for (R), 99% ee; lit:
3
[
3
D
Compound (R)-1h: [α]25 =+117.17 (c=2.0, CHCl
3
) for (R), 95% ee;
column (250×4.6 mm, 5 μm) at 25℃ with a flow rate of 1 mL/min,
and the mobile phase used was 7% isopropyl alcohol/ 93% n-
hexane. Retention time: 13.9 min for (R)-1c and 16.0 min for (S)-1c.
CP-ChiraSil-DEX CB column (25 m×0.25 mm×0.25 μm) and
-1
programmed as follows: T0: 50℃; dT/dt: 30℃min , T1: 130℃, 0.5
-1
min; dT/dt: 20℃/min, T2: 150℃, 5 min; dT/dt: 10℃min ,T3: 155℃,
1H NMR (400 MHz, CDCl
H),7.21-7.25 (m, 2H),7.42-7.46 (m, 2H); 13C NMR (100 MHz, CDCl
): δ 2.30-2.32 (m, 3H), 2.58-2.61 (m,
3
2
min; dT/dt: 20℃/min, T4: 170℃, 8 min; split ratio1: 5. Retention
3
3
):
1
time: 14.727 min for (R)-1h and 14.367 min for (S)-1h. H NMR (400
δ 21.1, 43.6, 123.3, 129.8, 141.3, 141.9.
MHz, CDCl
(
(
3
):δ 2.13 (s, 3H), 2.83 (s, 3H), 7.06 (t, J=7.5 Hz, 1H), 7.24
d, J=6.7 Hz, 1H), 7.39 (d, J=7.9 Hz, 1H), 8.29 (d, J=8.3 Hz, 1H), 10.40
s, 1H); 13C NMR (100 MHz, CDCl
): δ 24.8, 40.9, 123.0, 123.8, 125.6,
Compound (R)-1c: [α]25D=+120.1 (c=2.0, CHCl
) for (R), 99% ee; lit:
3
3
[
α]25D=+131.6 (c=1.0, CHCl ) for (R), 99% ee.10h Daicel OD-H chiral
3
1
28.1, 132.5, 139.8, 169.1.
column (250×4.6 mm, 5 μm) at 25℃ with a flow rate of 1 mL/min,
and the mobile phase used was 7% isopropyl alcohol/ 93% n-
D
Compound (R)-1i: [α]25 =+140.55 (c=2.0, CHCl
3
) for (R), 99% ee;
hexane. Retention time: 14.9 min for (R)-1b and 16.1 min for (S)-1b.
1H NMR (400 MHz, CDCl
): δ 2.68 (s, 3H), 7.19 (t, J = 8.5 Hz, 2H),
.60-7.63 (m, 2H); 13C NMR (100 MHz, CDCl
): δ 44.1, 116.8 (d, J=
2.6 Hz, 2C), 125.9 (d, J= 8.9 Hz, 1C), 141.0 (d, J= 2.9 Hz, 2C), 164.3
Daicel 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 20% isopropyl
3
7
2
3
alcohol/ 80% n-hexane. Retention time: 20.7 min for (R)-1i and 26.2
1
min for (S)-1i. H NMR (400 MHz, CDCl
3
): δ 2.61 (s, 3H), 4.16 (s, 2H),
(d, J=251.5 Hz, 1C).
6
.66-6.68 (m, 1H), 6.67 (d, J =7.5 Hz, 1H), 6.94 (s, 1H), 7.15 (t, J =7.8
Hz, 1H); 13C NMR (100 MHz, CDCl
3
): δ 43.5, 108.8, 112.3, 117.2,
Compound (R)-1d: [α]25D=+135.01 (c=2.0, CHCl
α]25D=+152.7 (c=1.0, CHCl ) for (R), 99% ee.10h Daicel AD-H chiral
3
) for (R), 99% ee; lit:
1
29.9, 146.1, 148.0.
[
3
column (250×4.6 mm, 5 μm) at 25℃ with a flow rate of 1 mL/min,
and the mobile phase used was 10% isopropyl alcohol/ 90% n-
hexane. Retention time: 24.1 min for (R)-1d and 22.7 min for (S)-1d.
D
Compound (R)-1j: [α]25 =+172.9 (c=2.0, CHCl
3
) for (R), 99% ee;
Daicel 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% isopropyl
alcohol/93% n-hexane. Retention time: 16.1 min for (R)-1j and 22.2
1H NMR (400 MHz, CDCl
): δ 2.73 (s, 3H), 7.77 (d, J=8.2 Hz, 2H), 7.99
d, J=8.2 Hz, 2H), 10.02 (s, 1H); 13C NMR (100 MHz, CDCl
): δ 43.7,
3
(
3
1
min for (S)-1j. H NMR (400 MHz, CDCl3): δ 1.16 (t, J=7.4 Hz, 1H),
1
24.2, 130.4, 138.1, 152.3, 191.2.
2
(
1
.70-2.79 (m, 1H), 2.83-2.92 (m, 1H), 7.46-7.52 (m, 3H), 7.57-7.59
m, 2H); 13C NMR (100 MHz, CDCl3): δ 6.1, 50.3, 124.2, 129.2, 131.0,
43.1.
Compound (R)-1e: [α]25D=+97.3 (c=2.0, CHCl
α]25D=+102.3 (c=1.0, CHCl ) for (R), 99% ee.10h Daicel AD-H chiral
3
) for (R), 99% ee; lit:
[
3
column (250×4.6 mm, 5 μm) at 25℃ with a flow rate of 0.8 mL/min,
and the mobile phase used was 7% isopropyl alcohol/ 93% n-
hexane. Retention time: 32.6 min for (R)-1e and 31.4 min for (S)-1e.
D
Compound (R)-1k: [α]25 =+52.16 (c=2.0, CHCl
[
3
) for (R), 69% ee; lit:
α]25D=+55 (c=0.53, CHCl
3
) for (R), 58% ee.16 Daicel OD-H chiral
column (250×4.6 mm, 5 μm) at 25℃ with aflow rate of 1 mL/min,
and the mobile phase used was 20% isopropyl alcohol/80% n-
hexane. Retention time: 14.5 min for (R)-1k and 16.7 min for (S)-1k.
1H NMR (400 MHz, CDCl3): δ 2.75 (s, 3H), 7.53-7.57 (m, 3H), 7.84-
1H NMR (400 MHz, CDCl
d, J=8.5 Hz, 2H); 13C NMR (100 MHz, CDCl
32.5, 144.7.
): δ 2.65 (s, 3H), 7.46 (d, J=8.5 Hz, 2H), 7.59
): δ 43.9, 125.1, 125.3,
3
(
3
1
7
.94 (m, 2H), 7.93 (d, J= 8.6 Hz, 1H), 8.17 (s, 1H); 13C NMR (100 MHz,
CDCl3): δ 119.4, 124.0, 127.3, 127.8, 128.0, 128.4, 129.6, 132.8,
34.3, 142.6.
D
Compound (R)-1f: [α]25 =+139.8 (c=2.0, CHCl
3
) for (R), 99% ee; lit:
[
α]25D=+144.1 (c=1.0, CHCl ) for (R), 98% ee.10h Daicel OD-H chiral
column (250×4.6 mm, 5 μm) at 25℃ with a flow rate of 1 mL/min,
3
1
and the mobile phase used was 20% isopropyl alcohol/ 80% n-
6
| J. Name., 2018, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Page 7 of 9
O rP gl ea na si ce &d Bo i on mo to al e dc juu l sa tr mC ha er mg i ins ts ry
Journal Name
ARTICLE
Siedlecki, P. Tomlin, K. Vare, Org. Process Res. DVeievw.A2rt0ic1le7O,n2li1ne:
Conflicts of interest
There are no conflicts to declare.
1
07-113.
DOI: 10.1039/C9OB00384C
10 (a) M. Abo, M. Tachibana, A. Okubo, S. Yarnazaki, Bioorg
Med Chem 1995, 3, 109-112; (b)S. P. Hanlon, D. L. Graham,
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Acknowledgements
This work was supported by National Natural Science
Foundation of China (No. 31460230, No. 21562054 and No.
2
000, 3, 823-828; (d) D. R. Boyd, N. D. Sharma, A. W. T. King,
S. D. Shepherd, C. C. R. Allen, R. A. Holt, H. R. Luckarift, H.
Dalton, Org. Biomol. Chem. 2004, 2, 554-561; (e) M.
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1262051), Guizhou Science and Technology Department
QKHRC-2016-4029, QKHPTRC-2016-5801, QKHZC-2019-2579,
and QKHPTRC-2019-5101), Guizhou Education Department
QSHZZ-2012-169, GNYL-2017-006), Program for Outstanding
(
(
Youth of Zunyi Medical University (17zy-001), Zunyi Science and
Technology Bureau (No. ZSKH-2017-17 and ZSKH-2018-3)， the
Fifth Batch of Talent Base in Guizhou Province (2016). We are
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0, 3284-3290.
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1
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Organic & Biomolecular Chemistry
Page 8 of 9
View Article Online
An MsrA homologue with extremely high substrate tolerance and wide s
D
u
O
b
I:
s
1
t
0
r
.1
a
039/C9OB00384C
t
e
scope for the biocatalytic preparation of enantiopure sulfoxides

Page 9 of 9
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C9OB00384C
8
0x39mm (300 x 300 DPI)