Chemoenzymatic Deracemization of Chiral Sulfoxides

Article abstract of DOI:10.1002/anie.201805858

The highly enantioselective enzyme methionine sulfoxide reductase A was combined with an oxaziridine-type oxidant in a biphasic setup for the deracemization of chiral sulfoxides. Remarkably, high ee values were observed with a wide range of substrates, thus providing a practical route for the synthesis of enantiomerically pure sulfoxides.

Full text of DOI:10.1002/anie.201805858

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Accepted Article
Title: Chemoenzymatic Deracemization of Chiral Sulfoxides
Authors: Jiri Misek and Vladimir Nosek
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201805858
Angew. Chem. 10.1002/ange.201805858
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Angewandte Chemie International Edition
10.1002/anie.201805858
COMMUNICATION
Chemoenzymatic Deracemization of Chiral Sulfoxides
[
a]
[a]
Vladimír Nosek, and Jiří Míšek*
Dedication
Abstract: A combination of the highly enantioselective enzyme
methionine sulfoxide reductase A and an oxaziridine-type oxidant in
a biphasic setup was utilized for deracemization of chiral sulfoxides.
Remarkably, high enantiomeric excess was achieved with a wide
range of substrates and thus provides a practical route for the
synthesis of enantiopure sulfoxides.
substrate methionine sulfoxide. This finding suggested that MsrA
might actually serve as an excellent biocatalyst for the
preparation of chiral sulfoxides by kinetic resolution. Indeed,
incubation of 0.1 mol% of the isolated enzyme and dithiothreitol
(DTT) as a reductant with the racemic sulfoxide 1a provided a
practically enantiopure product (ee >99%) at 50% conversion.
The conversion reached 50% within an hour and remained
constant for several hours, which indicated an extraordinarily
high level of enantioselectivity (see SI). Remarkably, the enzyme
tolerated a wide range of substrates (Table 1). All substrates
Chiral sulfoxides are important biologically active compounds
and several of them constitute the active component of
[
1,2]
marketed pharmaceuticals, such as Omeprazol.
Furthermore,
1
with the tested substituents at R reacted readily with the
they also became popular as chiral ligands, catalysts, and
enzyme, including o-, m-, p- substituted phenyls, as well as
[
3–8]
building blocks in asymmetric synthesis.
Therefore, the
various alkyl substituents. Methyl and ethyl substituents were
enantioselective preparation of chiral sulfoxide is of high interest
to both academia and industry. Asymmetric catalysis as the
most attractive way for the preparation of chiral compounds can
be in general applied in three modes: (1) synthesis of a chiral
compound from an achiral precursor; (2) kinetic resolution of a
chiral compound (and its dynamic version); and (3)
deracemization of a chiral compound. The first approach has
been studied extensively for the enantioselective synthesis of
chiral sulfoxides. Ever since the pivotal work using a chiral
2
tolerated at the R site. Sterically more demanding substituents
2
than the ethyl group at the R site caused a dramatic decrease
of the reaction rate.
Table 1. Substrate scope of kinetic resolution with MsrA.
[
9,10]
titanium catalyst for asymmetric oxidation of sulfides,
a
[
11–19]
[20,21]
plethora of other metal-based,
organocatalytic,
and
[
22–28]
enzymatic approaches have emerged. On the other hand,
kinetic resolution of chiral sulfoxides has been explored to much
O-
O-
MsrA (0.1 mol%)
+
S+
S+
S
[
29–32]
R1
R2
R1
R2
R1
R2
lesser extent.
chiral sulfoxides is concerned, to the best of our knowledge, this
approach has never been reported. Conceptually,
And as far as one-pot deracemization of
50 mM PBS, pH 7.5
DTT (4 eq.)
rac-1
(R)-1
2
3
7 °C, 1-5 h
deracemization is a challenging process as it requires a co-play
of at least two reaction pathways and the product reenters the
[
a]
[
33–35]
Entry
Product
Conversion [%]
50
ee [%]
>99
catalytic cycle.
chemical or enzymatic deracemizations of chiral molecules have
Therefore, only a handful of efficient
O-
S+
[
36–38]
been described to date.
It should also be pointed out that
1
despite the wealth of methods for asymmetric synthesis of chiral
sulfoxides, general protocols providing sulfoxides with high
enantiomeric purity (ee ≥99%) remain still scarce.
1
a
O-
S+
Herein, we report on the development of a highly efficient
deracemization method for the preparation of chiral sulfoxides.
This chemoenzymatic process utilizes the enantioselective
enzyme methionine sulfoxide reductase A and an oxaziridine-
based lipophilic oxidant in a biphasic system.
2
50
>99
1b
O-
S+
3
4
50
50
>99
>99
We have previously reported on a new fluorescent probe for the
[
39]
methionine sulfoxide reductase A (MsrA).
During further
1c
experimentation with the enzyme, rather low substrate
a
O-
S+
specificity was observed. On the other hand, enantioselectivity
was maintained at a very high level, similar to that of the natural
1
d
O-
S+
[a]
V. Nosek, Dr. J. Míšek
[
b]
Department of Organic Chemistry
Faculty of Science, Charles University in Prague
Hlavova 2030/8, 12843 Prague 2 (Czech Republic)
E-mail: misek@natur.cuni.cz
5
<2
n.d.
1e
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Angewandte Chemie International Edition
10.1002/anie.201805858
COMMUNICATION
O-
S+
O-
MsrA (0.1 mol%)
whole E. coli cells)
O-
(
S+
S+
S
6
50
50
50
50
>99
>99
>99
>99
+
aqueous buffer/decane
20:1)
37 °C, 5 h
(
1
f
Br
Br
Br
Br
O-
S+
rac-1f
(R)-1f
2f
7
8
9
conversion 50%
ee >99%
(without decane conversion <5%)
1
g
NC
MeO
HO
O-
S+
Scheme 1. Whole E. coli cell format of kinetic resolution with MsrA without
external reductant.
1
h
O-
S+
Development of the latter biphasic system prompted us to look
for a lipophilic oxidant capable of oxidizing the sulfide back to
the racemic sulfoxide and thus return it to the catalytic cycle.
1
i
O- 1j
S+
This approach would allow an
a
priori quantitative
First, t-butyl
1
0
50
50
>99
>99
deracemization of racemic sulfoxides.
hydroperoxide 3 and cumyl hydroperoxide 4 were tested, since
O- 1k
S+
[
c]
1
1
they possess large lipophilic groups and are known to oxidize
[
46]
sulfides to sulfoxides.
However, solubility of these
O-
S+
hydroperoxides in aqueous media was still relatively high,
leading to a lethal effect on the cells. Therefore, the tertiary
1
2
50
>99
[
47]
hydroperoxide 5 with a long alkyl chain was synthesized. The
addition of the C8 alkyl chain eliminated the toxicity to the cells
but the rate of sulfide oxidation was very low (conversion <5% in
1
l
[
a] reactions were performed at 10 µmol scale (6.5 mM substrate). [b] n.d. =
not determined. [c] 0.15 mol% of MsrA.
2
4 h). Finally, the saccharin-based oxaziridine 6, prepared in
[
48]
two simple steps from cheap starting materials (see SI), was
identified as the optimal oxidant. It proved to be compatible with
the cell culture and to exhibit sufficient reactivity. Under
optimized conditions, 0.65 mmol of racemic sulfoxide 1f (142
mg) was incubated with E. coli cells overexpressing MsrA in M9
minimal buffer (100 mL, OD600=5, 0.1 mol% MsrA), 1 equiv of
oxidant 6 and 5% (v/v) of decane. The yield of 82% after 24 h
was indicated by analytical HPLC and the sulfoxide 1f was
isolated in 79% (112 mg) yield with an ee of >99%. By using
these optimized conditions, a wide range of chiral sulfoxides was
deracemized at 0.65 mmol scale (Table 2). Excellent
enantioselectivity (ee ≥99 %) was attained in all cases and the
isolated yields ranged from moderate to high (52-90%) (Table 2,
entry 1-11). Apart form the recently reported elegant
deracemization of a specific allylic sulfoxide using Viedma
The enzyme MsrA employed in this study utilizes three
catalytically active cysteine groups for the reduction of
[
40,41]
sulfoxides.
Therefore, disulfide reducing agents, such as
DTT or TCEP (tris(2-carboxyethyl)phosphine), are preferred as
terminal reductants. However, while the need for such
stoichiometric reductants is unlikely to be a serious issue in the
laboratory setup, it may become a limiting factor for the practical
preparation of larger quantities of enantioenriched sulfoxides.
We hypothesized that E. coli cells, used for the production of the
enzyme, could provide sufficient reducing power through the
NADPH/TrxR/Trx system to drive the reaction without any
[
42–44]
external reductant.
The whole-cell format of the reaction
would also eliminate the need for the enzyme isolation. However, ripening, this is the first general method for efficient
[
49]
the initial attempts with the whole-cell catalysis failed, as the
observed conversion of the substrate 1f after 24 hours was less
then 5%. Detailed investigation of the system revealed that the
resulting sulfides are toxic for the cells. We hypothesized that
the relatively high concentration of the lipophilic product (formed
from 0.65 mM starting sulfoxide) could interfere with the cell
membrane integrity rather than affecting any specific cellular
pathways. To address this issue, we took advantage of the fact
that E. coli cells can be cultured in the presence of decane as a
deracemization of chiral sulfoxides.
O
O
OOH
OOH
Ph
OOH
S
N
Ph
O
C H
9
19
H25C12
3
4
5
6
[
45]
co-solvent.
We assumed that the lipophilic sulfide product
Figure 1. Lipophilic oxidants tested for deracemization.
would reside in the organic phase, which would prevent its direct
contact with the cells, so that its toxic effect should be eliminated.
Indeed, addition of 5% of decane (v/v) to the whole-cell culture
experiment provided practically enantiopure sulfoxide 1f (ee
To further demonstrate the utility of our method, nonsteroidal
anti-inflamatory drug sulindac 1m was deracemized (Table 2,
entry 12). Due to the low solubility of sulindac in aqueous media
and organic solvents, 1 equivalent of β-cyclodextrin was added
to the reaction mixture to enhance its solubility. Under these
>
99%) at 50% conversion, which matches the in vitro
experiments (Scheme 1). It is noteworthy that neither hexane
nor heptane was compatible with the cell culture.
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COMMUNICATION
O-
S+
conditions, racemic sulindac was deracemized providing the (R)-
enantiomer in 61% (140 mg) isolated yield and an ee of 97%.
7
5%
[
c],[d]
1
2
97
Table 2. Deracemization of chiral sulfoxides.
(
61%, 140 mg)
MsrA (0.1 mol%)
whole E. coli cells)
HOOC
(R)-Sulindac (1m)
F
(
O-
O-
(reduction pathway)
S+
S+
R1
R2
R1
R2
oxaziridine 6 (1 eq.)
[a] reactions were performed at 0.65 mmol scale (6.5 mM substrate). [b] 0.2
mol% of MsrA. [c] 0.3 mol% of MsrA. [d] β-Cyclodextrin (1 equivalent) was
added to the reaction mixture.
(oxidation pathway)
rac-1
(R)-1
aqueous buffer/decane
20:1)
7 °C, 24 h
(
3
In conclusion, the first biocatalytic deracemization of chiral
Entry
1
Product
HPLC yield
ee [%]
[
a]
sulfoxides has been developed (with ≤90% isolated yield and
(
isolated yield)
≥
97% ee). The enzyme MsrA showed an unprecedented level of
O-
S+
6
8%
enantioselectivity while keeping a remarkably wide substrate
scope. The combination of MsrA and oxaziridine oxidant 6 in a
biphasic setup enabled deracemization of various aryl-alkyl and
alkyl-alkyl sulfoxides.
99
(
(
62%, 62 mg)
1
a
O-
S+
75%
2
>99
70%, 70 mg)
Experimental Section
1
b
O-
S+
General procedure for deracemization of sulfoxides. A 2 mL
preculture of E. coli BL21(DE3) with msrA gene in pGEX vector
was used to inoculate 200 mL LB medium with ampicillin
(100 mg/L). The bacterial culture was incubated at 37 °C, 200
rpm until OD600 of 0.8 was reached. Then, the production of the
enzyme was induced with 0.5 mM IPTG and the culture was
incubated at 37 °C, 200 rpm for additional 2 h until OD600 of 2.5
was reached. Then, cells were harvested by centrifugation (3500
8
0%
3
4
(
(
72%, 72 mg)
9
9
1
c
O-
S+
5
5%
99
54%, 59 mg)
1
d
×
g, 30 min, 4 °C). The cell pellet was washed with M9 minimal
medium (48 mM Na HPO , 22 mM KH PO , 8.6 mM NaCl, 18.7
mM NH Cl, 2 mM MgSO , 0.1 mM CaCl , 0.4% glycerol,
O-
S+
2
4
2
4
8
2%
5
6
7
8
>99
99
4
4
2
(
79%, 112 mg)
pH 6.95) and centrifuged (3500 × g, 30 min, 4 °C). Racemic
sulfoxide (0.65 mmol) in M9 minimal medium (80 mL) and cells
resuspended in M9 minimal medium (20 mL) were added to a 1L
Erlenmeyer flask followed by a solution of oxaziridine 6 (230 mg,
1
f
Br
O-
S+
7
4%
(
61%, 65 mg)
0
.65 mmol) in decane (5 mL). The reaction mixture was
1
g
NC
MeO
HO
incubated at 37 °C, 200 rpm for 24 h. The conversion of the
reaction was determined by analytical HPLC. Then, 10%
solution of Na S O (40 ml) was added and the reaction mixture
2 2 3
was centrifuged (3500 × g, 30 min, 4 °C). The supernatant was
extracted with EtOAc (4 × 200 ml), the organic phase was
2 4
washed with H O (50 mL) and brine (50 ml), dried over MgSO ,
O-
S+
9
1%
99
(
(
90%, 99 mg)
1
h
O-
S+
6
2%
and concentrated by rotary evaporation under reduced pressure.
The crude product was purified by column chromatography on
99
52%, 52 mg)
93%
1
i
silica gel (typically cyclohexane/acetone
=
1/1). The
O- 1j
enantiomeric excess of the purified product was determined by
chiral HPLC. The absolute configuration of the sulfoxides was
determined by comparison of the HPLC retention times and the
optical rotation with the reported data.
[
b]
9
1
>99
99
S+
(75%, 75 mg)
O- 1k
72%
[
c]
0
1
S+
(59%, 64 mg)
O-
S+
8
9%
Acknowledgements
1
99
(
88%, 97 mg)
1
l
We acknowledge support of Czech Science Foundation (GACR
1
7-25897Y). Also, we would like to thank Prof. Kočovský for the
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Angewandte Chemie International Edition
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COMMUNICATION
help with the preparation of the manuscript and Dr. Veselý for an
[26]
N. M. Kamerbeek, A. J. J. Olsthoorn, M. W. Fraaije, D. B. Janssen,
Appl. Environ. Microbiol. 2003, 69, 419–426.
access to chiral HPLC.
[
[
[
[
27]
28]
29]
30]
S. Kato, H.-J. Yang, T. Ueno, S. Ozaki, Phillips George N., S.
Fukuzumi, Y. Watanabe, J. Am. Chem. Soc. 2002, 124, 8506–8507.
A. Riebel, G. de Gonzalo, M. W. Fraaije, J. Mol. Catal. B Enzym. 2013,
88, 20–25.
Keywords: biocatalysis • deracemization • oxidoreductases •
chiral sulfoxides • biphasis reactions
N. Komatsu, M. Hashizume, T. Sugita, S. Uemura, J. Org. Chem.
1
993, 58, 7624–7626.
J. Sun, C. Zhu, Z. Dai, M. Yang, Y. Pan, H. Hu, J. Org. Chem. 2004,
9, 8500–8503.
[
[
[
[
[
[
1]
2]
3]
4]
5]
6]
R. Bentley, Chem. Soc. Rev. 2005, 34, 609–624.
J. Legros, J. R. Dehli, C. Bolm, Adv. Synth. Catal. 2005, 347, 19–31.
I. Fernández, N. Khiar, Chem. Rev. 2003, 103, 3651–3706.
M. C. Carreno, Chem. Rev. 1995, 95, 1717–1760.
6
[
[
31]
32]
J. Wang, M. Frings, C. Bolm, Chem. – Eur. J. 2014, 20, 966–969.
H. R. Luckarift, H. Dalton, N. D. Sharma, D. R. Boyd, R. A. Holt, Appl.
Microbiol. Biotechnol. 2004, 65, 678–685.
B. M. Trost, M. Rao, Angew. Chem. Int. Ed. 2015, 54, 5026–5043.
Mikołajczyk,M., Drabowicz,J., Kiełbasinśki,P., Chiral Sulfur Reagents:
Applications in Asymmetric and Stereoselective Synthesis, CRC Press,
[
[
[
[
33]
34]
35]
36]
N. J. Turner, Curr. Opin. Chem. Biol. 2010, 14, 115–121.
C. Moberg, Acc. Chem. Res. 2016, 49, 2736–2745.
1
997.
W. Kroutil, K. Faber, Tetrahedron Asymmetry 1998, 9, 2901–2913.
C. V. Voss, C. C. Gruber, W. Kroutil, Angew. Chem. Int. Ed. 2008, 47,
[
[
7]
8]
M. Mellah, A. Voituriez, E. Schulz, Chem. Rev. 2007, 107, 5133–5209.
G. Sipos, E. E. Drinkel, R. Dorta, Chem. Soc. Rev. 2015, 44, 3834–
7
41–745.
C. J. Dunsmore, R. Carr, T. Fleming, N. J. Turner, J. Am. Chem. Soc.
006, 128, 2224–2225.
A. D. Lackner, A. V. Samant, F. D. Toste, J. Am. Chem. Soc. 2013,
35, 14090–14093.
3
860.
[
[
[
[
[
[
[
[
[
[
37]
38]
39]
40]
41]
42]
43]
44]
45]
46]
[
9]
P. Pitchen, E. Dunach, M. N. Deshmukh, H. B. Kagan, J. Am. Chem.
Soc. 1984, 106, 8188–8193.
2
[
[
10]
11]
F. D. Furia, G. Modena, R. Seraglia, Synthesis 1984, 1984, 325–326.
N. Komatsu, M. Hashizume, T. Sugita, S. Uemura, J. Org. Chem.
1
N. Makukhin, V. Tretyachenko, J. Moskovitz, J. Míšek, Angew. Chem.
Int. Ed. 2016, 55, 12727–12730.
1
993, 58, 4529–4533.
[
[
[
12]
13]
14]
K. Nakajima, M. Kojima, J. Fujita, Chem. Lett. 1986, 15, 1483–1486.
B. Saito, T. Katsuki, Tetrahedron Lett. 2001, 42, 3873–3876.
M. Palucki, P. Hanson, E. N. Jacobsen, Tetrahedron Lett. 1992, 33,
F. Tête-Favier, D. Cobessi, S. Boschi-Muller, S. Azza, G. Branlant, A.
Aubry, Structure 2000, 8, 1167–1178.
W. T. Lowther, N. Brot, H. Weissbach, J. F. Honek, B. W. Matthews,
Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 6463–6468.
C. H. Williams, FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 1995, 9,
7
111–7114.
K. Noda, N. Hosoya, R. Irie, Y. Yamashita, T. Katsuki, Tetrahedron
994, 50, 9609–9618.
C. Bolm, F. Bienewald, Angew. Chem. Int. Ed. Engl. 1996, 34, 2640–
642.
[
[
15]
16]
1
1
267–1276.
J. A. Chemler, Z. L. Fowler, K. P. McHugh, M. A. G. Koffas, Metab.
Eng. 2010, 12, 96–104.
2
[
[
[
17]
18]
19]
J. Legros, C. Bolm, Angew. Chem. Int. Ed Engl. 2004, 43, 4225–4228.
H. Egami, T. Katsuki, J. Am. Chem. Soc. 2007, 129, 8940–8941.
J. Fujisaki, K. Matsumoto, K. Matsumoto, T. Katsuki, J. Am. Chem.
Soc. 2011, 133, 56–61.
S. Lee, S. M. Kim, R. T. Lee, Antioxid. Redox Signal. 2013, 18, 1165–
1
207.
R. Aono, H. Kobayashi, K. J. N, K. Horikoshi, Biosci. Biotechnol.
Biochem. 1994, 58, 2009–2014.
[
[
[
[
[
[
20]
21]
22]
23]
24]
25]
S. Liao, I. Čorić, Q. Wang, B. List, J. Am. Chem. Soc. 2012, 134,
F. Fringuelli, R. Pellegrino, F. Pizzo, Synth. Commun. 1993, 23, 3157–
1
0765–10768.
3
163.
V. Mojr, V. Herzig, M. Buděšínský, R. Cibulka, T. Kraus, Chem.
Commun. 2010, 46, 7599–7601.
[
[
47]
48]
R. Kundu, Z. T. Ball, Org. Lett. 2010, 12, 2460–2463.
F. A. Davis, J. C. Towson, D. B. Vashi, R. ThimmaReddy, J. P.
McCauley, M. E. Harakal, D. J. Gosciniak, J. Org. Chem. 1990, 55,
T. Matsui, Y. Dekishima, M. Ueda, Appl. Microbiol. Biotechnol. 2014,
9
8, 7699–7706.
Z.-G. Zhang, R. Lonsdale, J. Sanchis, M. T. Reetz, J. Am. Chem. Soc.
014, 136, 17262–17272.
S. Ozaki, P. R. Ortiz de Montellano, J. Am. Chem. Soc. 1995, 117,
056–7064.
M. Anderson, S. Allenmark, Biocatal. Biotransformation 2000, 18, 79–
6.
1
254–1261.
[
49]
A. H. J. Engwerda, N. Koning, P. Tinnemans, H. Meekes, F. M.
2
Bickelhaupt, F. P. J. T. Rutjes, E. Vlieg, Cryst. Growth Des. 2017, 17,
4
454–4457.
7
8
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10.1002/anie.201805858
COMMUNICATION
Entry for the Table of Contents (Please choose one layout)
Layout 1:
COMMUNICATION
The
first
efficient
Vladimír Nosek, Jiří Míšek*
deracemization of chiral
sulfoxides
developed
has
been
Page No. – Page No.
(with
≤90%
Chemoenzymatic
Deracemization of Chiral
Sulfoxides
isolated yield and ≥97%
ee). The enzyme MsrA
showed an unprecedented
level of enantioselectivity
while keeping a remarkably
wide substrate scope. The
combination of MsrA and
oxaziridine-type oxidant in
a biphasic setup enabled
deracemization of various
aryl-alkyl and alkyl-alkyl
sulfoxides.
O-
S+
O-
S+
Enzymatic reduction
Chemical oxidation
R1
R2
R1
R2
rac
(R)
1
R =aryl, alkyl
2
yield up to 90%
ee ≥97%
R =methyl, ethyl
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