Discovery and application of methionine sulfoxide reductase B for preparation of (S)-sulfoxides through kinetic resolution

Article abstract of DOI:10.1016/j.catcom.2019.105908

Here we report a methionine sulfoxide reductase B (MsrB) enzymatic system for the preparation of (S)-sulfoxides through kinetic resolution of racemic (rac) sulfoxides. Eight MsrB homologue recombinant proteins were expressed and their activities on asymmetric reduction of rac-sulfoxides were analyzed. Among these MsrB homologue proteins, one protein from Acidovorax species showed good activity and enantioselectivity towards several aryl-alkyl sulfoxides. The (S)-sulfoxides were prepared with 93–98% enantiomeric excess through kinetic resolution at initial substrate concentration up to 50 mM. The establishment of MsrB catalytic kinetic resolution system provides a new efficient green strategy for preparation of (S)-sulfoxides.

Full text of DOI:10.1016/j.catcom.2019.105908

Journal Pre-proof
Discovery and application of methionine sulfoxide reductase B
for preparation of (S)-sulfoxides through kinetic resolution
Yuanmei Wen, Liaotian Peng, Yang Zhou, Tao Peng, Yu Chen,
Xiaoling Cheng, Yongzheng Chen, Jiawei Yang
PII:
S1566-7367(19)30369-3
DOI:
https://doi.org/10.1016/j.catcom.2019.105908
CATCOM 105908
Reference:
To appear in:
Catalysis Communications
Received date:
Revised date:
Accepted date:
16 October 2019
12 December 2019
13 December 2019
Please cite this article as: Y. Wen, L. Peng, Y. Zhou, et al., Discovery and application
of methionine sulfoxide reductase B for preparation of (S)-sulfoxides through kinetic
resolution,
Catalysis
Communications
(2019),
https://doi.org/10.1016/
j.catcom.2019.105908
This is a PDF file of an article that has undergone enhancements after acceptance, such
as the addition of a cover page and metadata, and formatting for readability, but it is
not yet the definitive version of record. This version will undergo additional copyediting,
typesetting and review before it is published in its final form, but we are providing this
version to give early visibility of the article. Please note that, during the production
process, errors may be discovered which could affect the content, and all legal disclaimers
that apply to the journal pertain.
© 2019 Published by Elsevier.

Journal Pre-proof
Discoveryand application of methionine sulfoxide reductase B for
preparation of (S)-sulfoxides through kinetic resolution
Yuanmei Wena, Liaotian Penga, Yang Zhoua, Tao Penga, Yu Chen b, c, Xiaoling
Chenga, Yongzheng Chenb, c and Jiawei Yang*a, b
a
Department of Biochemistry, Zunyi Medical University, Zunyi 563000, Guizhou
province, P.R. China；
b
Generic Drug Research Centre of Guizhou Province; Green Pharmaceuticals
Engineering Research Centre of Guizhou Province；
c
School of Pharmacy, Zunyi Medical University, Zunyi, 563000, Guizhou province,
P.R. China；
*Corresponding author: Jiawei Yang
Phone: 86-851-28642710
E-mail: yangjw@zmu.edu.cn.
1

Journal Pre-proof
Abstract
Here we report a methionine sulfoxide reductase B (MsrB) enzymatic system for the
preparation of (S)-sulfoxides through kinetic resolution of racemic (rac) sulfoxides.
Eight MsrB homologue recombinant proteins were expressed and their activities on
asymmetric reduction of rac-sulfoxides were analysed. Among these MsrB
homologue proteins, one protein from Acidovorax species showed good activity and
enantioselectivity towards several aryl-alkyl sulfoxides. The (S)-sulfoxides were
prepared with 93-98% enantiomeric excess through kinetic resolution at initial
substrate concentration up to 50 mM. The establishment of MsrB catalytic kinetic
resolution system provides a new efficient green strategy for preparation of
(S)-sulfoxides.
Keywords
Methionine sulfoxide reductase B; (S)-sulfoxides; Kinetic resolution; Biocatalysis
2

Journal Pre-proof
1. Introduction
Chiral sulfoxides are valuable organosulfur compounds and being used in a wide
range of industrial fields [1, 2]. For instance, optically active sulfoxides are important
in asymmetric synthesis, either as chiral building blocks or as stereogenic centers [3,
4]. Moreover, enantiopure sulfoxides have been widely used as pharmaceuticals in
which usually only one of the enantiomers is responsible for the desired bioactivity [5,
6]. Thus, the enantioselective preparation of chiral sulfoxides is highly interested in
both asymmetric synthesis and pharmaceutical industry. Chiral sulfoxides could be
obtained through chemical or biological methods [7, 8]. In the past few years,
biocatalysis has been emerging as a valuable synthetic tool for the preparation of high
added-value compounds, as it has many advantages including that enzymes can afford
high efficiency and enantioselectivity under mild reaction conditions [9-11]. The
general strategies for preparation of enantiopure sulfoxides include asymmetric
oxidation of prochiral sulfides and kinetic resolution of racemic (rac) sulfoxides [7, 8].
Although the asymmetric oxidation is a more straightforward method [12-14], the
biocatalytic kinetic resolution is becoming an effective approach for enantioselective
preparation of chiral sulfoxides [15, 16]. In this approach, the unwanted sulfoxide
enantiomer is transformed while leaving the desired enantiomer. Although the major
drawback of kinetic resolution is the 50% of theoretical maximum yield, the
development of reductase-oxidant biphasic reaction systems provide an alternative
technique for overcoming this bottleneck [17].
In previous studies, several methionine sulfoxide reductase A enzymes have been
discovered and applied for the preparation of (R)-sulfoxides through reductive
3

Journal Pre-proof
resolution of rac-sulfoxides [15, 16, 18]. A series of aryl-alkyl sulfoxides in R
configuration were prepared with approximately 50% of yield and 99% enantiomeric
excess (ee) at the substrate concentration up to 320 mM (approximately 45 g/L) [16].
The extremely high substrate tolerance and enantioselectivity indicates that reductive
resolution would become an effective strategy for green synthesis of chiral
(R)-sulfoxides. On the other hand, attempting for preparation of (S)-sulfoxides by
reductive resolution has also been reported years ago [19-22]. For instance, there were
some cases using bacterial dimethylsulfoxide (DMSO) reductase for preparation of
the (S)-sulfoxides and showed good enatioselectivity and substrate scope [22].
Although the substrate concentrations in these examples was still far from industrial
scale, these reports provide an alternative reductive resolution strategy for preparation
of (S)-sulfoxides.
Methionine sulfoxide reductase (Msr) is a class of enzymes which reduce methionine
sulfoxide (Met-O) to methionine (Met) in cells [23, 24]. These enzymes play an
important role in protein repairing and protect cells from oxidative stress [25, 26].
There are three types of Msr proteins: MsrA which specifically reduces the
(S)-enantiomer of Met-O; MsrB which specifically reduces the (R)-Met-O in peptide
and fRMsr, of which there are two forms: one that reduces the free (S)-Met-(O) and
one that reduces the free (R)-Met-O, and is also called fRMsr [27, 28]. Considering
the high activity of MsrA shown in the preparation of (R)-sulfoxides through
reductive resolution, we hypothesized that MsrB or fRMsr could be similarly
exploited for the preparation of (S)-sulfoxides. Recently, Nosek and colleagues
investigated several MsrB homologues for preparation of (S)-sulfoxides and
concluded that the low activity of natural MsrBs prevented them from application for
4

Journal Pre-proof
kinetic resolution of sulfoxides [22]. However, the Msr enzymes belong to a
superfamily of proteins widely expressed in living species [29, 30]. Therefore, there
could be MsrB or fRMsr forms with higher specific activity. We thus tested the
activities of several MsrB and fRMsr homologues aimed to obtain Msr enzymes for
preparation of (S)-sulfoxides. Finally, an MsrB homologue with good activity and
enantioselectivity was successfully obtained.
2. Experimental
2.1 General
Commercial chemicals were purchased from Aladdin Bio-Chem Technology Co., Ltd.
(Shanghai, China), or Sangon Biotech Co., Ltd.(Shanghai, China). All sulfoxides were
purchased from TCI Co., Ltd. (Shanghai, China), and J&K Scientific Co., Ltd.
(Beijing, China).
2.2 Screening, cloning and expressing of recombinant MsrB and fRMsr homologues
The ORF of MsrB and fRMsr from Pseudomonas moneilii (pmMsrB and pmfRMsr)
were amplified by PCR using genomic DNA of Pseudomonas monteilii CCTCC
(China Center for Type Culture Collection) M2013683 we isolated before as template
[31]. The gene sequences have been submitted to the Genbank database (accession
number: MN312201 for pmMsrB and MN312202 for pmfRMsr). The protein BLAST
program (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was applied to search MsrB
homologues using protein sequence of pmMsrB as template. Seven MsrB protein
5

Journal Pre-proof
sequences with sequence identity from 55%-92% were selected randomly. After
obtaining the corresponding nucleotide sequences by tblastn program, the DNA
fragments of these MsrB genes were synthesized. Then, the DNA fragments were
inserted into the multiple cloning site (MCS) of the pET-28a. To induce the soluble
expression of recombinant proteins, the recombinant plasmids were transformed into
E. coli BL21 (DE3) cells. After overnight culturing, cells containing expression
plasmid was diluted 1:100 into 1 L of 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
absorbance at 600 nm (A600) of 0.6. Cells were then induced by 0.2 mM
isopropyl-β-D-thiogalactopyranoside (IPTG) for 14 h at 20 ◦C and harvested by
centrifugation at 2810 g 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, pH 7.0) was
added and cells were incubated at room temperature for 1 h. The soluble total proteins
were obtained by centrifuging for 15 min at 17000 g at 4 ◦C and defined as crude
enzyme. Proteins were quantified by the method of Bradford and subjected to
SDS-PAGE to check their expressions.
2.3 Relative activity assay
Relative activities of MsrB from Acidovorax sp. KKS102 (akMsrB) under different
pH and temperature conditions were determined by a colorimetric assay according to
our previous report [16]. Briefly, MsrB crude enzyme (0.5 mg/mL), DTT (1 mM), and
racemic methyl phenyl sulfoxide (rac-1a,1 mM) were assembled in 1 mL reaction
system. After 20 min of reaction, the mixture was diluted 20 times with 50 mM
phosphate buffer (pH 8.0). Then, 100 μL diluted samples were transferred into a
6

Journal Pre-proof
96-well plate, and another 100 μL of reaction mixture (4 mM DTNB, 50mM
phosphate buffer, pH 8.0) were added. The plate was incubated under 37 ◦C for 10
min, and a microplate reader was used to determine the absorbance at 415 nm (A415).
Proteins extracted from cells containing plasmid pET-28a was used as control. The
decrease of A415 was used to describe the relative activities of akMsrB.
2.4 Biocatalytic kinetic resolution of rac-sulfoxides
The process of kinetic resolution was based on our previously reported MsrA-DTT
system [15]. Whole-cell containing recombinant Msr protein was used as catalysts at
the cell density of 30 g/L (fresh weight). DTT was added into the reaction buffer (50
mM phosphate) at the concentration of 2-fold of substrates. Biocatalytic reactions
were performed at 5 mL of reaction volume with shaking at 300 rpm. After incubation,
the reaction mixture was extracted twice with 5 mL of ethyl acetate each time with
vigorous shaking. The organic layers were collected by centrifugation and then dried
by anhydrous sodium sulfate. For separation and purification of sulfoxides 1a-1c after
reaction, 20-30 parallel reactions (20, 30, 10 mM of rac-1a-1c, respectively) were
performed. After extracting and collecting the reaction mixture, the remaining
sulfoxides 1a-1c were purified by silica gel column chromatography with petroleum
ether/ethyl acetate (3:1) as elution solvent.
2.5 Analytical methods
Quantification of 1a–1g and 2a–2g after reaction were determined by GC with
Agilent 7820 GC (Agilent Technologies, Santa Clara, CA) using a HP-5 column (30
7

Journal Pre-proof
m×0.32 mm×0.25 μm) with an FID detector. DMSO was used as an internal standard.
The ee value of 1g was determined by GC using a CP-ChiraSil-DEX CB column (25
m×0.25 mm×0.25 mm). For other sulfoxides, ee was determined using a Shimadzu
Prominence HPLC on a DaicelTM AD-H or OD-H chiral column (250×4.6 mm, 5μm )
with UV detection at 254 nm. The percent ee was calculated by the
equation: %ee=[([R] -[S])/([R]+[S])]×100%. The quantification of two enantiomers of
residual 1b after different reaction time was determined by HPLC on OD-H chiral
column. 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. Optical rotation
data of sulfoxides was determined by RUDOLPH research analytical Autopol I-AP.
The absolute configuration of 1a-1g were assigned by comparison of the rotation data
1
13
and the retention time in HPLC with previous reports [12]. H and C NMR spectra
were recorded on a Brucker-300 (300/75 MHz) spectrometer using CDCl3 as a solvent
and TMS as an internal standard.
3. Results and Discussion
3.1 Identification of Msr proteins for asymmetric reduction of sulfoxide
In our previous reports, an MsrA protein from Pseudomonas monteilii CCTCC
M2013683 strain was successfully used to reduce the (S)-sulfoxides for the
preparation of (R)-sulfoxides [15, 32]. Considering there are other two types of Msr
enzymes (MsrB and fRMsr) active on the (R)-sulfoxides in vivo, these two genes
(named as pmMsrB and pmfRMsr) were cloned from this bacteria and recombinantly
expressed in E. coli. After induction by IPTG, both pmMsrB and pmfRMsr
8

Journal Pre-proof
recombinant proteins were successfully expressed in soluble form (Figure 1A). Then,
we used rac-1a as substrate to test their activities. The pmMsrB showed good activity
and moderate enationselectivity towards the substrate. After 16 h of reaction, 60% of
1a was transformed to the corresponding sulfide 2a, and the (S)-1a with 41% of ee
was obtained. In the meanwhile, the pmfRMsr was almost inactive on the substrate 1a
(Table 1). Thus, the MsrB protein was considered for our further researches on kinetic
resolution of rac-sulfoxides.
Figure 1 Recombinant expression of methionine sulfoxide reductase homologues.
The soluble total proteins were subjected to SDS-PAGE gel electrophoresis. Arrows
indicated the recombinant proteins. The names of proteins were corresponding to that
in Table 2.
Table 1 Asymmetric reduction analysis of rac-1a by methionine sulfoxide reductases.
(t=16 h, substrate concentration=2 mM)
9

Journal Pre-proof
a
b
Conversion(%)
ee (%)
E c
3
Enzyme
pmMsrB
60.5
4.6
41.4
n.d.d
91.6
57.2
8.0
pmfRMsr
ppMsrB
pkMsrB
pvMsrB
nrMsrB
paMsrB
akMsrB
ecMsrB
--
15
8
58.0
47.3
22.7
17.1
32.5
60.8
20.4
2
n.d.
--
5
27.6
96.0
n.d.
16
--
aConversion of 1a to the corresponding product 2a was determined by GC after reaction.
b The ee value was 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 denotes the
conversion.
d "n.d." means not determined due to low conversion.
3.2 Screening of MsrB homologues for preparation of (S)-sulfoxides
The MsrB belong to a superfamily of proteins widely expressed in living species [28].
Thus, we tested the activities of several pmMsrB homologues aimed to obtain MsrB
enzymes with better catalytic properties. After sequence alignment using pmMsrB as
template, seven pmMsrB homologues with sequence identities from 55%-92% were
selected from GenBank database, and named as ppMsrB, pkMsrB, pvMsrB, nrMsrB,
paMsrB, akMsrB and ecMsrB. The detailed protein sequences information was
10

Journal Pre-proof
summarized in Table S1. The sequence alignment of these proteins was carried out as
shown in Supplementary Material (Figure S1). After induction, all seven recombinant
proteins were successfully expressed (Figure 1B). The rac-1a was then used to test
their activities and whole-cell containing recombinant Msr proteins were used as
catalysts at the cell density of 30 g/L (fresh weight). The results showed that all these
MsrB proteins were active on this substrate (Table 1). Among all tested MsrB
enzymes, four proteins (pmMsrB, ppMsrB, pkMsrB and akMsrB) were found to have
good activity and enantioselectivity. After reaction by these four enzymes, 47.3%-60.8%
of 1a was transformed to the corresponding sulfide 2a, and the (S)-1a with
41.4%-96.0% of ee was obtained (Table 1). In addition, considering that the
expression of paMsrB and pvMsrB was obviously lower than other enzymes (Figure
1B), we increased their cell densities from 30 g/L to 50g/L to test their activities.
However, the conversions were only slightly improved (less than 10%), which
indicates that the difference in expression level among these enzymes is not the key
issue for their catalytic activities.
Therefore, we select these four MsrB proteins (ppMsrB, pkMsrB, paMsrB and
akMsrB) to investigate their substrate specificity by testing their activity on several
other aryl-alkyl sulfoxides 1b-1g. Although the substrate structure affected their
activity and enantiaoselectivity, all four enzymes did exhibited catalytic properties on
these sulfoxides (Table 2 and S2), suggesting a certain level of compatibility for
different sulfoxide substrates. Specifically, one MsrB protein from Acidovorax sp.
KKS102 (named akMsrB) showed high activity and enantiaoselectivity on the
substrates rac-1a-1c, in which the (R)-1a-1c were almost completely transformed and
(S)-1a-1c were obtained with ee>90% (Table 1 and 2).
11

Journal Pre-proof
Table 2 Kinetic resolution analysis of rac-1b-1c by 4 MsrB recombinant proteins.
(t=16 h, substrate concentration=2 mM)
ee(%)
38.8
80.3
52.3
96.4
E
3
6
2
9
Substrate
Enzyme
pmMsrB
ppMsrB
pkMsrB
akMsrB
Conversion (%)
57.2
63.8
69.7
69.2
1b
pmMsrB
ppMsrB
pkMsrB
akMsrB
26.5
47.4
42.6
67.2
6.2
1
6
6
8
50.9
44.0
93.1
1c
3.3 Catalytic property analysis of akMsrB
Considering that akMsrB showed the best catalytic properties among these
homologues, this enzyme was selected for further analysis. We firstly measured the
relative activities of akMsrB at temperatures ranging from 20 to 60°C. The maximum
activity was observed at 35°C. Moreover, this enzyme exhibited good thermo-stability
as more than 85% of activity retained at reaction temperature up to 50 °C. The effect
of pH on the activity of akMsrB was then tested with pH values ranging from 4.0 to
11.0. The results showed that this enzyme was sensitive to pH and the optimum pH
was 7.0 (Figure 2). Taken together, pH 7.0 and 35°C were considered as the optimal
reaction conditions for akMsrB.
12

Journal Pre-proof
In the above reactions catalyzed by akMsrB, we found that the conversions of
rac-1a-1c to 2a-2c were higher than 50%. These data mean that both the R and S
enantiomers could be reduced by the akMsrB whole-cell catalyst. To further
understand the details of reaction, we chose rac-1b as substrate to investigate the
time-dependent changes of the two enantiomers. The reactions were performed at a
higher substrate concentration of 20 mM. After 6 h of reaction, over 80% of (R)-1b
and 13.1% of (S)-1b were reduced to the sulfide (Figure 3). After 16 h of reaction, the
(R)-1b was almost completely reduced (approximately 99%), while 17.3% of (S)-1b
was also converted to the corresponding sulfide. These results indicate that the
akMsrB whole-cell catalyst could show activities both on (S) and (R)-sulfoxides,
while the reduction of (R)-sulfoxide was much faster than (S)-sulfoxide. It is known
that under stress conditions like stationary phase growth, E. coli cells upregulate
sulfoxide reducing enzymes (e.g. MsrA) [33], which may thus diminish the selectivity
factors. Therefore, we analyzed the catalytic activity of E. coli cells containing only
pET-28a plasmid. As shown in Figure 3, 13.2% and 16.3% of (S)-1b was converted to
the corresponding sulfide after 6 h and 16 h of reaction, respectively. These
conversions were very close to that by cells containing akMsrB enzymes. Thus, these
results suggest that the activity of akMsrB enzyme on (S)-sulfoxide probably very low
and the real enationselcetivity could be much higher. In addition, the (R)-1b was only
slightly converted (5.1% and 6.7% after 6 h and 16 h of reaction, respectively) by the
cells containing pET-28a plasmid, which also proved that the reduction of (R)-1b was
predominantly catalyzed by the recombinant enzyme other than the endogenous
(R)-sulfoxide reductase of the cells. We further purified this recombinant enzyme and
tried to test its enationselcetivity. Unfortunately, the activity of pure recombinant
akMsrB was extremely low and less than 10% of conversion was observed. Further
13

Journal Pre-proof
studies need to perform to investigate the mechanism and improve the stability of
akMsrB enzyme during purification and reaction.
Figure 2 Optimization of temperature and pH for akMsrB activity.
100
80
60
40
20
0
0
5
10
15
20
25
30
35
40
Reaction time (h)
Figure 3 Analysis of residual sulfoxides in R (●) and S (■) configurations and the ee
values (▲) of 1b after reaction by whole-cell containing akMsrB recombinant protein
(solid lines) or only pET-28a plasmid (dashed lines) after different reaction time.
3.4 Substrate tolerance analysis of akMsrB on kinetic resolution
The initial substrate concentration is an important issue in biocatalytic preparation of
chiral sulfoxides, we thus investigated the substrate tolerance of akMsrB whole-cell
14

Journal Pre-proof
catalyst. Concentrations of rac-1a-1c from 2 to 80 mM were used to test the enzyme
tolerance to the substrates. The results demonstrated that the kinetic resolution of
1a-1c was successfully accomplished with good enantioselectivities (E=13～53) at
substrate concentrations up to 20, 50 and 10 mM, respectively. Under these substrate
concentrations, the conversions of rac-1a-1c were 52.9%, 55.9% and 65.3%, and
products (S)-1a-1c with 92.7%, 93.3% and 94.7% of ee were obtained, respectively
(Figure 4). Compared to reported DMSO reductase system [19-22], our research
represents a big improvement in the aspect of initial substrate concentration. More
importantly, comparing to the membrane-associated multi-subunit DMSO reductase
[22], soluble recombinant MsrB was much easier to be prepared. The simpler
molecular structure of MsrB would exhibit great advantages in the subsequent process
like protein engineering for further improvement of the catalytic properties. Finally,
1
we performed these reactions of rac-1a-1c on preparative scale. The rotation data, H
and 13C NMR spectra of purified residual 1a-1c after reaction were determined and
presented in Supplementary Material.
15

Journal Pre-proof
100
80
60
40
20
0
2
4
8
10 15 20 25 30
100
80
60
40
20
0
2
4
8
10 30 40 50 60 80
100
80
60
40
20
0
2
4
8
10
15
20
Substrate concentration (mM)
Figure 4 Conversion (■) and ee (▲) analysis of rac-1a-1c after reaction by akMsrB at
different substrate concentrations. t=16 h.
4. Conclusion
In summary, this study reports the first successful application of methionine sulfoxide
reductase for the preparation of (S)-sulfoxides through kinetic resolution. The protein
from Acidovorax sp. KKS102 (named akMsrB) showed good activity on several
rac-sulfoxides. The kinetic resolution of rac-sulfoxides was successfully
accomplished at substrate concentrations up to 50 mM (7.8 g/L) when catalyzed by
akMsrB whole-cell catalyst. Our study supports that asymmetric reductive resolution
16

Journal Pre-proof
of rac-sulfoxides catalysed by MsrB could become an effective strategy for green
synthesis of optically pure sulfoxides. Further engineering of these MsrB is ongoing
to improve their catalytic performances, including substrate tolerance, substrate
specificity and stability.
Acknowledgements
This work was supported by National Natural Science Foundation of China
(31460230, 31960202 and 21562054), Guizhou Science and Technology Department
(QKHZC-2019-2579 and QKHPTRC-2019-5101), Department of Education of
Guizhou Province (JYSCXJH-2018-037 and 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 grateful for the English writing
assistance from Dr. Ming Zhuo at University of Texas Medical Branch, USA.
References:
[1] I. Fernandez, N. Khiar, Recent developments in the synthesis and utilization of
chiral sulfoxides, Chemical reviews, 103 (2003) 3651-3706.
[2] R. Bentley, Role of sulfur chirality in the chemical processes of biology, Chemical
Society Reviews, 34 (2005) 609-624.
[3] B.M. Trost, M. Rao, Development of chiral sulfoxide ligands for asymmetric
catalysis, Angewandte Chemie International Edition, 54 (2015) 5026-5043.
[4] S. Otocka, M. Kwiatkowska, L. Madaliꢀska, P. Kiełbasiꢀski, Chiral organosulfur
ligands/catalysts with a stereogenic sulfur atom: applications in asymmetric
synthesis, Chemical reviews, 117 (2017) 4147-4181.
17

Journal Pre-proof
[5] W.R. Goundry, B. Adams, H. Benson, J. Demeritt, S. McKown, K. Mulholland, A.
Robertson, P. Siedlecki, P. Tomlin, K. Vare, Development and scale-up of a
biocatalytic process to form a chiral sulfoxide, Organic Process Research
&Development, 21 (2017) 107-113.
[6] K. Okamoto, T. Nishino, Mechanism of inhibition of xanthine oxidase with a new
tight binding inhibitor, Journal of Biological Chemistry, 270 (1995) 7816-7821.
[7] J. Han, V.A. Soloshonok, K.D. Klika, J. Drabowicz, A. Wzorek, Chiral sulfoxides:
advances in asymmetric synthesis and problems with the accurate determination
of the stereochemical outcome, Chemical Society Reviews, 47 (2018)
1307-1350.
[8] T. Matsui, Y. Dekishima, M. Ueda, Biotechnological production of chiral organic
sulfoxides: Current state and perspectives, Applied microbiology and
biotechnology, 98 (2014) 7699-7706.
[9] R.A. Sheldon, P.C. Pereira, Biocatalysis engineering: the big picture, Chemical
Society Reviews, 46 (2017) 2678-2691.
[10] R.A. Sheldon, J.M. Woodley, Role of biocatalysis in sustainable chemistry,
Chemical reviews, 118 (2017) 801-838.
[11] R.N. Patel, Biocatalysis for synthesis of pharmaceuticals, Bioorganic &
medicinal chemistry, 26 (2018) 1252-1274.
[12] D. Linde, M. Cañellas, C. Coscolín, I. Davó-Siguero, A. Romero, F. Lucas, F.J.
Ruiz-Dueñas, V. Guallar, A.T. Martínez, Asymmetric sulfoxidation by
engineering the heme pocket of a dye-decolorizing peroxidase, Catalysis Science
& Technology, 6 (2016) 6277-6285.
[13] I. Bassanini, E.E. Ferrandi, M. Vanoni, G. Ottolina, S. Riva, M. Crotti, E. Brenna,
D. Monti, Peroxygenase-Catalyzed Enantioselective Sulfoxidations, European
18

Journal Pre-proof
Journal of Organic Chemistry, 2017 (2017) 7186-7189.
[14] Y. Zhang, F. Liu, N. Xu, Y.-Q. Wu, Y.-C. Zheng, Q. Zhao, G. Lin, H.-L. Yu, J.-H.
Xu, Discovery of two native Baeyer-Villiger monooxygenases for asymmetric
synthesis of bulky chiral sulfoxides, Appl. Environ. Microbiol., 84 (2018)
e00638-00618.
[15] L. Peng, Y. Wen, Y. Chen, Z. Yuan, Y. Zhou, X. Cheng, Y. Chen, J. Yang,
Biocatalytic Preparation of Chiral Sulfoxides through Asymmetric Reductive
Resolution by Methionine Sulfoxide Reductase A, ChemCatChem, 10 (2018)
3284-3290.
[16] J. Yang, Y. Wen, L. Peng, Y. Chen, X. Cheng, Y. Chen, Identification of MsrA
homologues for the preparation of (R)-sulfoxides at high substrate concentrations,
Organic & biomolecular chemistry, 17 (2019) 3381-3388.
[17] V. Nosek, J. Míšek, Chemoenzymatic deracemization of chiral sulfoxides,
Angewandte Chemie International Edition, 57 (2018) 9849-9852.
[18] C. Achilli, A. Ciana, G. Minetti, Kinetic resolution of phenyl methyl sulfoxides
by mammalian methionine sulfoxide reductase A, Tetrahedon Letters, 58 (2017)
4781-4782.
[19] S.P. Hanlon, D.L. Graham, P.J. Hogan, R.A. Holt, C.D. Reeve, A.L. Shaw, A.G.
McEwan, Asymmetric reduction of racemic sulfoxides by dimethyl sulfoxide
reductases from Rhodobacter capsulatus, Escherichia coli and Proteus species,
Microbiology, 144 (1998) 2247-2253.
[20] H.R. Luckarift, H. Dalton, N.D. Sharma, D.R. Boyd, R.A. Holt, Isolation and
characterisation of bacterial strains containing enantioselective DMSO reductase
activity: application to the kinetic resolution of racemic sulfoxides, Applied
microbiology and biotechnology, 65 (2004) 678-685.
19

Journal Pre-proof
[21] M. Abo, M. Tachibana, A. Okubo, S. Yamazaki, Enantioselective deoxygenation
of alkyl aryl sulfoxides by DMSO reductase from Rhodobacter sphaeroides fs
denitrificans, Bioorganic & medicinal chemistry, 3 (1995) 109-112.
[22] V. Nosek, J. Míšek,Enzymatic kinetic resolution of chiral sulfoxides-an
enantiocomplementary approach, Chemical Communications, 55 (2019)
10480-10483.
[23] N. Brot, H. Weissbach, Peptide methionine sulfoxide reductase: biochemistry and
physiological role, Peptide Science, 55 (2000) 288-296.
[24] S. Boschi-Muller, G. Branlant, Methionine sulfoxide reductase: chemistry,
substrate binding, recycling process and oxidase activity, Bioorganic chemistry,
57 (2014) 222-230.
[25] H. Weissbach, L. Resnick, N. Brot, Methionine sulfoxide reductases: history and
cellular role in protecting against oxidative damage, Biochimica et Biophysica
Acta (BBA)-Proteins and Proteomics, 1703 (2005) 203-212.
[26] C. Yin, L. Zheng, J. Zhu, L. Chen, A. Ma, Enhancing stress tolerance by
overexpression of a methionine sulfoxide reductase A (MsrA) gene in Pleurotus
ostreatus, Applied microbiology and biotechnology, 99 (2015) 3115-3126.
[27] C. Achilli, A. Ciana, G. Minetti, The discovery of methionine sulfoxide reductase
enzymes: An historical account and future perspectives, Biofactors, 41 (2015)
135-152.
[28] F. Etienne, D. Spector, N. Brot, H. Weissbacha, A methionine sulfoxide reductase
in Escherichia coli that reduces the R enantiomer of methionine sulfoxide,
Biochemical and Biophysical Research Communications, 300 (2003) 378-382.
[29] N. Rouhier, C.V. Dos Santos, L. Tarrago, P. Rey, Plant methionine sulfoxide
reductase A and B multigenic families, Photosynthesis research, 89 (2006)
20

Journal Pre-proof
247-262.
[30] J.A. Maupin-Furlow, Methionine Sulfoxide Reductases of Archaea, Antioxidants,
7 (2018) 124.
[31] J.W. Yang, D.J. Zheng, B.D. Cui, M. Yang, Y.Z. Chen, RNA-seq transcriptome
analysis of a Pseudomonas strain with diversified catalytic properties growth
under different culture medium, MicrobiologyOpen, 5 (2016) 626-636.
[32] J. Yang, Z. Yuan, Y. Zhou, J. Zhao, M. Yang, X. Cheng, G. Ou, Y. Chen,
Asymmetric reductive resolution of racemic sulfoxides by recombinant
methionine sulfoxide reductase from a pseudomonas monteilii strain, Journal of
Molecular Catalysis B: Enzymatic, 133 (2016) S588-S592.
[33] J. Moskovitz, M.A. Rahman, J. Strassman, S.O. Yancey, S.R. Kushner, N. Brot,
H. Weissbach, Escherichia coli Peptide Methionine Sulfoxide Reductase Gene:
Regulation of Expression and Role in Protecting Against Oxidative Damage,
Journal of Bacteriology, 177 (1995), 502-507.
21

Journal Pre-proof
Authors Contributions
Yuanmei Wen: Methodology, Investigation, Formal analysis, Conceptualization,
Writing-Original Draft;
Liaotian Peng: Methodology, Investigation;
Yang Zhou: Methodology, Investigation;
Tao Peng: Software, Validation
Yu Chen: Investigation;
Xiaoling Cheng: Data Curation, Investigation;
Yongzheng Chen: Conceptualization, Funding acquisition
Jiawei Yang: Supervision, Conceptualization, Writing-Review&Editing, Project
administration, Funding acquisition.
22

Journal Pre-proof
The authors declared that they have no conflicts of interest to this work.
23

Journal Pre-proof
Highlights
MsrB was firstly applied for (S)-sulfoxides preparation through kinetic resolution
MsrB from Acidovorax species showed good activity and enantioselectivity towards
several rac-sulfoxides
(S)-sulfoxides were prepared with 93-98% of ee at substrate concentration up to 50
mM
24