Regioselective Ring-Opening of Styrene Oxide Derivatives Using Halohydrin Dehalogenase for Synthesis of 4-Aryloxazolidinones

Article abstract of DOI:10.1002/adsc.201900786

A biocatalytic approach towards a range of 4-aryloxazolidinones is developed using a halohydrin dehalogenase from Ilumatobacter coccineus (HheG) as biocatalyst. The method is based on the HheG-catalyzed α-position regioselective ring-opening of styrene oxide derivatives with cyanate as a nucleophile, producing the corresponding 4-aryloxazolidinones in moderate to good yields. Synthesis of enantiopure 4-aryloxazolidinones is also achievable using chiral epoxide materials. (Figure presented.).

Full text of DOI:10.1002/adsc.201900786

Title: Regioselective Ring-Opening of Styrene Oxide Derivatives Using
Halohydrin Dehalogenase for Synthesis of 4-Aryloxazolidinones
Authors: Nanwei Wan, Jiawei Tian, Xiaoying Zhou, Huihui Wang,
Baodong Cui, Wenyong Han, and Yongzheng Chen
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To be cited as: Adv. Synth. Catal. 10.1002/adsc.201900786
Link to VoR: http://dx.doi.org/10.1002/adsc.201900786

10.1002/adsc.201900786
Advanced Synthesis & Catalysis
COMMUNICATION
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Regioselective Ring-Opening of Styrene Oxide Derivatives
Using Halohydrin Dehalogenase for Synthesis of 4-
Aryloxazolidinones
Nanwei Wan,* Jiawei Tian, Xiaoying Zhou, Huihui Wang, Baodong Cui, Wenyong
Han, and Yongzheng Chen*
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, China. * E-mail: nanweiwan@zmu.edu.cn; yzchen@zmu.edu.cn
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.((Please
delete if not appropriate))
enzymatic catalysis for the synthesis of
halohydrin oxazolidinones. One enzymatic route was reported by
Abstract. A biocatalytic approach towards a range of 4-
aryloxazolidinones is developed using
a
dehalogenase from Ilumatobacter coccineus (HheG) as
biocatalyst. The method is based on the HheG-catalyzed α-
position regioselective ring-opening of styrene oxide
derivatives with cyanate as a nucleophile, producing the
corresponding 4-aryloxazolidinones in moderate to good
yields. Synthesis of enantiopure 4-aryloxazolidinones is
also achievable using chiral epoxide materials.
Janssen and coworkers, which starts from epoxides
using a halohydrin dehalogenase (HHDH) from
Agrobacterium radiobacter (HheC) (Scheme 1a).[8] In
this method, cyanate (OCN-) is accepted by HheC as
a nucleophile to ring-opening of epoxides at β-
position, and the produced β-substituted alcohols are
further converted to 5-substituted oxazolidinones
undergoing spontaneous cyclization. Recently, Fasan
and coworkers have successfully synthesized 4-
substituted oxazolidinones via the P450-catalyzed
cyclization of carbonazidates (Scheme 1b).[9] This is
the first and unique report that synthesis of 4-
substituted oxazolidinones via enzymatic catalysis.
However, this method suffers from the low catalytic
efficiency (turnover numbers <100) and the substrate
carbonazidates are not easy to get. Compared with the
P450-method, the HHDH-method exhibits relative
high substrate tolerance and good enzyme stability. In
addition, the epoxides are cheap and widely available
materials. Because most of the characterized HHDHs
show the high β-position regioselectivity in the ring-
opening process of epoxides, synthesis of 4-
substituted oxazolidinones using HHDH has not been
reported until now.
Keywords: Oxazolidinones; Halohydrin dehalogenase;
Epoxides; Regioselectivity; Ring-opening
Heterocyclic compounds are not only key central
skeletons of a wide range of biologically active
compounds, but also serve as useful synthons in
synthetic organic chemistry. Oxazolidinones are a
class of oxygen and nitrogen containing heterocycles,
which have received significant attention in
medicinal chemistry due to their outstanding
biological activity.[1] Oxazolidinones have been used
as a new class of fungicides, antibacterials and
antimicrobial agents.[2] In addition, oxazolidinones
are valuable synthetic intermediates for a variety of
organic transformations, for example, as precursors
of β-amino acids.[3] 4-substituted oxazolidinones such
as 4-ary substituted oxazolidinones[4] and 4-
hydroxymethyl substituted oxazolidinones,[5] are one
important type of oxazolidinone derivatives.
Therefore many synthetic strategies have been
designed for the formation of 4-substituted
oxazolidinones.[5-6] However, these chemical methods
usually suffer from the need for harsh reaction
temperature, long reaction time, or the use of
expensive catalysts.
Biocatalysis is an important green synthetic
technology which have been widely used in the
organic synthesis due to its mild reaction
conditions.[7] Several efforts have been made on
Scheme 1. Biocatalytic routes for the synthesis of
oxazolidinones.
1
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10.1002/adsc.201900786
Advanced Synthesis & Catalysis
Recently, we have found that the HHDH from
Subsequently, the reaction conditions for the
production of 2a were investigated using the
recombinant E. coli (HheG) whole cells as
biocatalysts. We first evaluated the reaction pH of PB
buffer. As showed in Table 1 (entries 1-5), the
highest yield was found at pH 7.5 (entry 3). Though
the maximum ee of 2a was found at pH 6.0, the
corresponding yield was very low. Investigation of
reaction temperature was carried out next at the range
Ilumatobacter coccineus (HheG) showed high α-
position regioselectivity in the azide-mediated ring-
opening of styrene oxide derivatives, producing 2-
azido-2-aryl-1-ols in good yields.[10] Herein, we
further explored the HheG-catalyzed ring-opening
reaction of styrene oxide derivatives using cyanates
as a nucleophile and developed an alternative
biocatalytic route to 4-aryloxazolidinones in
moderate to good yields (Scheme 1c).
o
from 25 to 40 C in pH 7.5 PB buffer (entries 6-8).
o
We began our investigation with an analytical
reaction using styrene oxide 1a as a model substrate
(Scheme 2). The reaction was carried out in 5 mL PB
buffer (Na2HPO4-KH2PO4, 50 mM, pH 7.0)
containing 10 mM 1a, 15 mM NaOCN and 3 mL cell
free extract of Escherichia coli (HheG). After
We found that the first choice of 30 C was the
optimum temperature for both the yield and ee of 2a
(entry 3 vs entries 6-8), which was in according with
our previous study of HheG in the azide-mediated
ring-opening reaction.[10] It was clear that increasing
o
the temperature to 40 C caused significant decrease
o
reaction at 30 C for 6 h, the reaction mixture was
in the yield (entry 8). The equivalence ratio of
NaOCN to 1a was examined in the range from 0.5 to
6 (entries 9-12). The yield increased with the increase
of equivalence ratio and reached up to 64% at the
ratio 3:1. When we further increased the ratio to 6:1,
dramatic increase in the yield was not obtained (entry
12 vs entry 11). Reducing the equivalence ratio to
0.5:1 slightly improved the ee to 80%, but caused
20% decrease in the yield (entry 9 vs entry 3).
Although we expected to synthesize enantiopure 2a
via kinetic resolution of 1a, the enantioselectivity (E
value) in the cyanate-mediated ring-opening process
was really low to meet this goal. Hence, we chose the
ratio 3:1 for this reaction with the consideration of the
high yield. At last, a survey of cell density indicated
the highest yield was obtained using 15 g cdw/L cells
(entry 14). Higher cell density did not improve the
yield (entry 15).
extracted for chiral HPLC analysis. Compared to the
synthesized (R, S)-2a and (R, S)-3a, the enzymatic
reaction produced 2a in 32% yield and trace of 3a,
which indicated HheG remained high α-position
regioselectivity in the cyanate-mediated ring-opening
of 1a. Interestingly, HheG also exhibited moderate
enantioselectivity in this reaction, giving 78% ee of
(S)-2a. We also performed a control reaction using
the E. coli cells in the absence of HheG as
biocatalysts and found no production of 2a or 3a,
which indicated spontaneous ring-opening of 1a with
cyanate did not occur in this reaction.
Scheme 2. HheG-catalyzed ring-opening of 1a with
cyanate.
Table 1. Optimization of the reaction conditions for the production of 2a.
Cell Conc.
(g cdw/L)
Yield 2a
ee (S)-1a
(%)c)
<5
ee (R)-2a
(%)c)
85
Entrya)
pH
T (oC)
NaOCN:1a
E valued)
(%)b)
7
1
2
3
4
5
6
7
8
9
10
11
12
13
14
6.0
7.0
7.5
8.0
8.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
30
30
30
30
30
25
35
40
30
30
30
35
30
30
1.5:1
1.5:1
1.5:1
1.5:1
1.5:1
1.5:1
1.5:1
1.5:1
0.5:1
2:1
10
10
10
10
10
10
10
10
10
10
10
10
5
15
13
9
10
9
9
7
7
6
11
8
7
16
37
29
21
34
17
14
18
49
64
68
56
78
19
50
43
39
48
24
12
15
75
88
92
79
77
72
71
73
62
70
69
80
57
47
31
53
3:1
6:1
3:1
3:1
5
8
4
86
24
2
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10.1002/adsc.201900786
Advanced Synthesis & Catalysis
15
7.5
30
3:1
20
70
83
20
3
a) The reactions were performed in 5 mL PB buffer containing 10 mM 1a and analyzed after 6 h. b) The yields were
c)
determined by chiral HPLC using the calibration curve of racemic 2a. The ee values were determined by chiral GC or
HPLC.
d)
The E values were calculated from ee 1a and ee 2a using an online tool (http://biocatalysis.uni-
graz.at/enantio/cgi-bin/enantio.pl).
Because the substrate concentration was an
important indicator to evaluate a biocatalytic reaction,
the reaction courses for the substrate concentrations
from 5 to 50 mM were investigated under the
optimized reaction conditions (Table 1, entry 14). As
depicted in Figure 1, the reactions almost finished at
6 h. The yields did not increase obviously when the
reaction time was prolonged to 12 h or 24 h. Stable
yields (>75%) could be obtained at the substrate
concentrations of 5-30 mM. Obvious decrease in the
yield was observed when a higher substrate
concentration of 50 mM was conducted for this
reaction. With the comprehensive consideration of
the yield and substrate concentration, the reaction
was established to perform with epoxide
concentration at 30 mM.
Following identification of this optimized
catalytic system, we then started to investigate the
scope of this process (Table 2). In general, all the
tested styrene oxide derivatives 1a-1k could be
converted to the corresponding 4-aryloxazolidinones
2a-2k in moderate to good yields (entries 1-11). As
expected, the HheG exhibited good α-position
regioselectivity in these ring-opening reactions,
which was demonstrated by the ratio of 4-
aryloxazolidinone 2 to 5-aryloxazolidinone 3. It could
be found that both para- and meta-substitutions on 1a
were well tolerated (entries 2-11 vs entry 1). The
para-substituted epoxides gave relative higher yield
and regioselectivity than that of the meta-substituted
epoxides for the production of 4-aryloxazolidinones
(entries 6-8 vs entries 2-4). The epoxides with the
strong electron-withdrawing group or the bulk alkyl
group on the aromatic ring resulted in moderate
yields, while still retained good α-position ring-
opening regioselectivity (entries 10-11). It is
important to note that the 4-fluorophenyl substituted
oxazolidinone 2f, an important synthon for the
synthesis of tyrosine kinase inhibitors, was usually
prepared from the corresponding β-aminoalcohols
and diethyl carbonate under high temperature (100-
130 oC).[11] Using the enzymatic reaction, 2f could be
synthesized from the readily available epoxide 1f in
good yield under mild reaction condition (entry 6).
100
6 h
90
80
70
60
50
40
30
20
10
0
12 h
24 h
5
10
15
20
30
50
Concentration 1a (mM)
Figure 1. Reaction courses for the substrate concentrations
from 5 to 50 mM. Analytic yields were determined by
chiral HPLC with three parallel samples.
Table 2. Substrate scope for the synthesis of 4-aryloxazolidinones 2 catalyzed by HheG.
Entrya)
Substrate Product
R
H
m-F
m-Cl
m-Br
m-CH3
p-F
p-Cl
p-Br
p-CH3
p-CN
p-(CH3)3
Ratio 2:3b)
91:9
85:15
84:16
80:20
92:8
87:13
87:13
90:10
92:8
Yield 2 (%)c)
ee 2 (%)d)
31(S)
9(-)
1
2
3
4
5
6
7
8
9
77
66
65
46
49
67
70
58
47
32
29
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
7(-)
8(-)
<5(-)
30(-)
<5(-)
20(-)
15(-)
52(-)
6(-)
1g
1h
1i
2g
2h
2i
10
11
91:9
91:9
1j
2j
1k
2k
3
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10.1002/adsc.201900786
Advanced Synthesis & Catalysis
12e)
13e)
(R)-1a
(S)-1a
(S)-2a
(R)-2a
H
H
ND
ND
84
62
>99(S)
>99(R)
a) Reaction conditions: PB buffer (50 mM, pH 7.5) 30 mL, cell density 15 g cdw/L, epoxides conc. 1a-1k 30 mM, NaOCN
conc. 90 mM, reaction temperature 30 ˚C, reaction time 12 h. b) Determined by 1H NMR. c) Isolated yield. d) Configuration
were defined using commercial enantiopure (R)-2a and (S)-2a. The ee values were determined by chiral HPLC. e) Epoxides
conc. 15 mM, NaOCN conc. 45 mM. ND = not detected.
silica gel (petroleum ether/ethyl acetate = 1:1) to afford the
desired product 4-aryloxazolidinone 2.
When the chiral epoxides (R)-1a was used in the
reaction, the optically active (S)-2a was obtained in
84% yield and >99% ee (entry 12). Replacement of
(R)-1a with the opposite enantiomer (S)-1a also gave
enantiopure (R)-2a in good yield (entry 13). What
Acknowledgements
should be emphasized was that (S)-2a was a key
intermediate
for
the
synthesis
of
antihypercholesterolemic drug ezetimibe[4b] and K-
opioid receptor agonist CJ-15161 (Scheme 3).[12]
These results indicated enantiocomplementary
synthesis of chiral 4-aryloxazolidinones from chiral
epoxides was practicable for this enzymatic method.
We are grateful for financial support from the National Natural
Science Foundation of China (No. 21662050 and 31800663),
Guizhou Science and Technology Department (QKHRC-2016-
4029, QKHPTRC-2016-5801, and QKHZC-2019-2579), Program
for Outstanding Youth of Zunyi Medical University (17zy-001),
National First-Rate Construction Discipline of Guizhou Province
(Pharmacy) (YLXKJS-YX-04) and the Fifth Batch of Talent Base
in Guizhou Province (2016).
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In conclusion, we conducted a detailed study of the
HheG catalyzed ring-opening of styrene oxide
derivatives with cyanate. Our study has achieved for
the first time biocatalytic synthesis of 4-
aryloxazolidinones from cheap and readily available
epoxides. This synthetic route represents a great
improvement compared to the method via P450-
catalyzed cyclization of carbonazidates. In addition,
synthesis of enantiopure 4-aryloxazolidinones was
also achievable from chiral epoxide materials.
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Experimental Section
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aryloxazolidinones 2a-2k
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cells (HheG) was carried out using the previously reported
protocol.[10] To a 30 mL (25 mL for 1j, 20 mL for 1k)
suspension of 15 g cdw/L E. coli cells (HheG) in 50 mM
PB buffer (pH 7.5) was added solid NaOCN to a final
concentration of 90 mM. Then the epoxide 1 was added to
a final concentration of 30 mM using 300 μL of dimethyl
sulfoxide as co-solvent. The reaction mixture was stirred at
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o
250 rpm and 30 C. After reaction for 12 h, the mixture
was extracted using ethyl acetate (2 × 30 mL). The organic
phase was separated, dried over anhydrous Na2SO4,
filtered, and concentrated by rotary evaporation. The
residue was purified by flash column chromatography on
4
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10.1002/adsc.201900786
Advanced Synthesis & Catalysis
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5
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10.1002/adsc.201900786
Advanced Synthesis & Catalysis
COMMUNICATION
Regioselective Ring-Opening of Styrene Oxide
Derivatives Using Halohydrin Dehalogenase for
Synthesis of 4-Aryloxazolidinones
Adv. Synth. Catal. Year, Volume, Page – Page
Nanwei Wan,* Jiawei Tian, Xiaoying Zhou, Huihui
Wang, Baodong Cui, Wenyong Han, and
Yongzheng Chen*
6
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