Reprogramming Epoxide Hydrolase to Improve Enantioconvergence in Hydrolysis of Styrene Oxide Scaffolds

Article abstract of DOI:10.1002/adsc.202000898

Enantioconvergent hydrolysis by epoxide hydrolase is a promising method for the synthesis of important vicinal diols. However, the poor regioselectivity of the naturally occurring enzymes results in low enantioconvergence in the enzymatic hydrolysis of styrene oxides. Herein, modulated residue No. 263 was redesigned based on structural information and a smart variant library was constructed by site-directed modification using an “optimized amino acid alphabet” to improve the regioselectivity of epoxide hydrolase from Vigna radiata (VrEH2). The regioselectivity coefficient (r) of variant M263Q for the R-isomer of meta-substituted styrene oxides was improved 40–63-fold, and variant M263V also exhibited higher regioselectivity towards the R-isomer of para-substituted styrene oxides compared with the wild type, which resulted in improved enantioconvergence in hydrolysis of styrene oxide scaffolds. Structural insight showed the crucial role of residue No. 263 in modulating the substrate binding conformation by altering the binding surroundings. Furthermore, increased differences in the attacking distance between nucleophilic residue Asp101 and the two carbon atoms of the epoxide ring provided evidence for improved regioselectivity. Several high-value vicinal diols were readily synthesized (>88% yield, 90%–98% ee) by enantioconvergent hydrolysis using the reprogrammed variants. These findings provide a successful strategy for enhancing the enantioconvergence of native epoxide hydrolases through key single-site mutation and more powerful enzyme tools for the enantioconvergent hydrolysis of styrene oxide scaffolds into single (R)-enantiomers of chiral vicinal diols. (Figure presented.).

Full text of DOI:10.1002/adsc.202000898

Title: Reprogramming epoxide hydrolase to improve
enantioconvergence in hydrolysis of styrene oxide scaffolds
Authors: fulong li, Yanyan Qiu, Yucong Zheng, Feifei Chen, Xudong
Kong, Jian-He Xu, and Hui-Lei Yu
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To be cited as: Adv. Synth. Catal. 10.1002/adsc.202000898
Link to VoR: https://doi.org/10.1002/adsc.202000898

10.1002/adsc.202000898
Advanced Synthesis & Catalysis
Very Important Publication
FULL PAPER
DOI: 10.1002/adsc.202((will be filled in by the editorial staff))
Reprogramming Epoxide Hydrolase to Improve
Enantioconvergence in Hydrolysis of Styrene Oxide Scaffolds
Fu-Long Li, Yan-Yan Qiu, Yu-Cong Zheng, Fei-Fei Chen, Xu-Dong Kong, Jian-He
Xu* and Hui-Lei Yu*
State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Centre for Biomanufacturing and
Frontiers Science Center for Materiobiology and Dynamic Chemistry, East China University of Science and Technology,
Shanghai 200237, China. Fax: (+86)-21-6425-0840, Email: jianhexu@ecust.edu.cn; huileiyu@ecust.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))
Abstract. Enantioconvergent hydrolysis by epoxide hydrolase is a promising method for the synthesis of important
vicinal diols. However, the poor regioselectivity of the naturally occurring enzymes results in low enantioconvergence in
the enzymatic hydrolysis of styrene oxides. Herein, modulated residue No. 263 was redesigned based on structural
information and a smart variant library was constructed by site-directed modification using an “optimized amino acid
alphabet” to improve the regioselectivity of epoxide hydrolase from Vigna radiata (VrEH2). The regioselectivity
coefficient (r) of variant M263Q for the R-isomer of meta-substituted styrene oxides was improved 40–63-fold, and
variant M263V also exhibited higher regioselectivity towards the R-isomer of para-substituted styrene oxides compared
with the wild type, which resulted in improved enantioconvergence in hydrolysis of styrene oxide scaffolds. Structural
insight showed the crucial role of residue No. 263 in modulating the substrate binding conformation by altering the
binding surroundings. Furthermore, increased differences in the attacking distance between nucleophilic residue Asp101
and the two carbon atoms of the epoxide ring provided evidence for improved regioselectivity. Several high-value vicinal
diols were readily synthesized (>88% yield, 90%–98% ee) by enantioconvergent hydrolysis using the reprogrammed
variants. These findings provide a successful strategy for enhancing the enantioconvergence of native epoxide hydrolases
through key single-site mutation and more powerful enzyme tools for the enantioconvergent hydrolysis of styrene oxide
scaffolds into single (R)-enantiomers of chiral vicinal diols.
Keywords: epoxide hydrolase; enantioconvergent hydrolysis; regioselectivity; structural re-designing; vicinal diols
regiopreference, forming an enantioenriched
product with a 100% theoretical yield.[5] As
Introduction
regioselectivity is
enantioconvergence, regioselectivity coefficients
(%), and , are used to quantify the degree of
a
key determinant of
Chiral vicinal diols and epoxides are
ubiquitous synthons for the preparation of
versatile -adrenergic receptor agonist (-



regioselectivity, indicating the attack percentages
at carbon atoms Cand Cof an epoxide
enantiomer (either R- or S-configuration),
respectively.[6]
blocker) drugs and amino alcohols.[1] Much effort
has been devoted to exploring efficient
approaches for the synthesis of chiral vicinal
diols and epoxides.[2] Biocatalytic strategies can
provide environmentally friendly options, as
exemplified by the asymmetric reduction of
hydroxyketone using oxidoreductase. However,
oxidoreductases require expensive coenzymes
and lack the desired activity.[3] As an alternative
route, the hydrolysis of racemic epoxides using
cofactor-independent epoxide hydrolases (EHs)
shows great promise for the synthesis of chiral
vicinal diols. This can be conducted using either
Owing to the uniqueness and importance of
enantioconvergent hydrolysis in chiral synthesis, a
few enantioconvergent EHs from plants and microbes
have been discovered and characterized in recent
decades.[6,7] These enzymes can completely hydrolyse
the two enantiomers of a racemic epoxide into
enantioenriched chiral vicinal diols. However,
naturally occurring EHs generally suffer from
insufficient regioselectivity towards both enantiomers
of epoxide substrates, resulting in incomplete
enantioconvergence. Protein engineers aim to evolve
the performance of enzymes in terms of regio- or
enantioselectivity.[8] For example, the regioselectivity
factors (R, S) of engineered Kau 2 EH were
kinetic
resolution
or
enantioconvergent
hydrolysis.[4] Enantioconvergent hydrolysis can
simultaneously catalyse pairs of epoxide
enantiomers
with
complementary
1
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10.1002/adsc.202000898
Advanced Synthesis & Catalysis
improved by 17% and 87% toward (S)- and (R)-
contrast, mutating to Phe or Trp with bulky
enantiomers
of
para-chlorostyrene
oxide,
sidechains
caused
a
severe
decrease
in
respectively.[9] Furthermore, a three-site mutant of
PvEH1 also exhibited higher regioselectivity (R =
86%,S = 99%) than the wild-type toward styrene
oxide.[7f]
regioselectivity. Herein, to rationally and efficiently
construct variants for improved enantioconvergence
towards other styrene oxide scaffolds, an “optimized
amino acid alphabet” consisting of six pre-selected
residues (Ala-Val-Ser-Cys-Asn-Gln) was designed in
accordance with CAST (Combinatorial Active-site
In our previous study, a novel epoxide hydrolase
(VrEH2) discovered from Vigna radiata was shown to
enantioconvergently hydrolyse racemic para-
nitrostyrene oxide (rac-pNSO).[7a,7b] However,
unsatisfactory regioselectivity and strict substrate
specificity severely limited its application scope.
Subsequently, a semi-rational design strategy was
adopted that revealed a crucial residue (Met263) for
precise modulation of the regioselectivity.[10] This
resulted in near-perfect enantioconvergence toward
rac-pNSO after single-site mutation (M263N).
However, the enantioconvergence of VrEH2 must be
improved for the enzymatic hydrolysis of other
styrene oxides with different substitution groups.
Recently, a variant of PvEH1 was obtained by
reshaping its two substrate tunnels, exhibiting
enhanced enantioconvergence towards meta-
chlorostyrene oxide (>99% conversion, 95.6% ee).[11]
However, examples of perfect enantioconvergence
(>99% conversion, >99% ee) towards other
substituted styrene oxide have yet to be reported,
which severely limits the catalytic application of EHs
in the synthesis of important chiral intermediates.
Saturation Test) method and the previous studies.[12]
A
smart mutant library on the key residue (Met263) was
then constructed by site-directed mutagenesis using
the simplified amino acid alphabet.
Figure 1. Structural insight of the key residue (Met-263)
locating in the active centre, the optimized amino acid
alphabet for constructing a smart library was indicated.
The catalytic residue Asp101 oxyanion hole residues
(Tyr150 and Tyr232) were shown in grey.
Herein, to explore the enantioconvergent
potential of VrEH2 in the biohydrolysis of various
epoxide scaffolds, a smart mutant library was
constructed by redesigning the enzyme based on the
previously identified key site. The resultant variants
showed enhanced enantioconvergence towards a
series of styrene oxides. More importantly, the
regioselectivity modulation mechanism for differently
substituted styrene oxides was elucidated based on
structural information from variants and docking
results. This will aid the design of additional and
improved enantioconvergent EHs for the green and
efficient synthesis of high-value vicinal diols.
To verify the enantioconvergence of the
redesigned variants in the hydrolysis of epoxide
scaffolds, substrates 1a–17a bearing different
substituents were designed (Scheme S1, Figure 2)
and the enantiomeric excess (ee) of the corresponding
vicinal diols was determined (Figure S1). For styrene
oxide (1a), para-substituted 2a–6a, and ortho-
substituted 12a, the wild type (WT) showed partial
enantioconvergence, with eep values of 60%–93%.
Meanwhile, poor eep values of 8%–21% were
obtained for meta-substituted substrates 8a–11a,
except for the fluorinated compound 7a, clearly
indicating that the position and properties of the
substituent group on the benzene ring affected
enantioconvergence using the EHs. The eep values for
para- and meta-disubstituted substrates 13a and 14a
were clearly lower than those of para-
monosubstituted substrates 2a and 3a, indicating that
Results and Discussion
Constructing a smart mutant library by
reprogramming regioselectivity modulating
residue Met263
the
additional
meta-substituent
significantly
influenced product selectivity. The eep values of para-
and ortho-disubstituted epoxides 15a and 16a were
similar to those of para-monosubstituted substrates
2a and 3a, suggesting that an additional ortho-
substituent did not affect the regioselectivity.
The crucial regioselectivity modulating residue,
Met263 of VrEH2, was identified previously by
structure-guided semi-rational design (Figure 1).
Mutations on this site significantly modulated the
enantioconvergence of VrEH2 toward rac-pNSO. The
polar side chain of Asn263 from the M263Q mutation
formed a new hydrogen bond with the para-nitro
group of pNSO, enabling a clear shift in substrate
binding pose that led to improved regioselectivity. In
Among the redesigned variants, M263Q, M263V,
and M263N showed significantly improved
enantioconvergence towards a series of styrene
oxides. For para- and nonsubstituted substrates 1a–
2
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10.1002/adsc.202000898
Advanced Synthesis & Catalysis
Figure 2. The ee values of product (1b-17b) by enantioconvergent hydrolysis of substrate 1a-17a using WT and variants
M263Q, M263V or M263N.
Table 1 The regioselectivities of WT and variant M263Q towards two enantiomers of meta-substituted styrene oxides 8a-
11a.
Substrate
Enzyme
WT
α (%)a
75 ± 0.0
7 ± 0.0
β (%)a
25 ± 0.0
93 ± 0.0
r b
0.3
13
Fold (r)
--
(R)-8a
M236Q
43
WT
85 ± 0.1
98 ± 0.0
15 ± 0.1
2 ± 0.0
5.7
49
--
(S)-8a
M236Q
8.5
WT
73 ± 0.4
5 ± 0.1
27 ± 0.4
95 ± 0.1
0.3
19
--
(R)-9a
(S)-9a
M236Q
63
WT
90 ± 0.7
95 ± 0.2
10 ±0.7
5 ± 0.2
9
--
M236Q
19
2.1
WT
81 ± 0.3
8 ± 0.2
19 ± 0.3
92 ± 0.2
0.2
11
--
(R)-10a
(S)-10a
(R)-11a
(S)-11a
M236Q
55
WT
64 ± 0.3
90 ± 0.2
36 ± 0.3
10 ± 0.2
1.8
9
-
M236Q
5
WT
62 ± 0.1
4 ± 0.5
38 ± 0.1
96 ± 0.5
0.6
24
--
M263Q
40
WT
77 ± 0.3
94 ± 0.0
23 ± 0.3
6 ± 0.0
3.3
15
--
M263Q
4.5
a α, β (%) are regioselectivity coefficients indicating the attacking percentage of the Cα and Cβ of (S)-enantiomers or (R) -
enantiomers, respectively.
b r (regioselectivity ratio) is calculated as α/β (S) or β/α (R), which represents the specificity of the regioselectivity
attacking.
were more than 92% (Figure 2). Furthermore, the
5a and bulky substrate 17a, variant M263V
enantioconvergence of M263Q towards 7a and 12a–
showed better enantioconvergence, with a higher eep
16a was improved by an eep value of 80%–95%.
value (90%) than that of WT (eep, 57%–79%). In
These results indicated that redesigning the residue
particular, M263N was found to exhibit near-perfect
(M263) engineered enantioconvergence of VrEH2
enantioconvergence toward rac-6a, with the ee value
towards a series of substituted styrene oxides, which
of 6b improved to 99% (eep of WT = 93%). The
enantioconvergence of M263Q towards meta-
substituted styrene oxides 8a–10a increased
significantly expanded the substrate scope of VrEH2
in an enantioconvergent manner.
significantly, and the optical purities of 8b and 11b
3
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10.1002/adsc.202000898
Advanced Synthesis & Catalysis
Determination of regioselectivities toward para-
and meta-substituted styrene oxides to
characterize the improved enantioconvergence
were also simulated for para- or meta-substituted
styrene oxide substrates (R)-3a and (R)-8a (Figure 3).
For WT, the epoxide ring of (R)-3a and (R)-8a was
positioned between the catalytic residue (Asp101) and
oxygen hole residues (Tyr150, Tyr232). Furthermore,
the geometric distances between Asp101 and the C
(dCα) or C (dCβ) atoms of the epoxide ring were both
less than 3.5 Å, with distance differences (d) of only
0.2 Å. This indicated the two positions could be
equally attacked by catalytic residue, which was
consistent with the regioselectivity coefficients
detected experimentally. For M263V and M263Q, the
distance difference between Asp101 and the two
carbons of the epoxide ring was more than 0.8 Å, and
the geometric distance (dCβ < dCα) indicated that C
was more readily attacked compared with WT.
To characterize the improved
enantioconvergence of M263V and M263Q, the
regioselectivity ratios (r) towards para- and meta-
substituted styrene oxides were measured using
optically pure (S)- or (R)-epoxides as substrates,
respectively. The regioselectivity ratio (r) indicated
the difference in the enzyme attacking frequencies at
Cand Cof the epoxide ring, and was calculated
experimentally by measuring the molar ratio of diol
isomers obtained using an optically pure substrate.29
As shown in Table S1, both WT and M263V showed
high rS values of more than 49.0 for the S-enantiomer
of para-substituted substrates 1a, 3a, and 4a, while
the rR values of the wild-type for 1a, 3a, and 4a were
only 5.6, 4.3, and 3.5, respectively. Therefore, the
insufficient regioselectivity of WT toward R-
enantiomers 1a, 3a, and 4a was the main origin of
low enantioconvergence. Comparatively, the rR value
of variant M263V toward (R)-1a reached 32.3,
representing a 5.8-fold increase compared with the
WT, and the original regioselectivity toward (S)-1a
(rS = 49.0) was retained, causing an obvious
improvement of enantioconvergence. Furthermore,
the regioselectivity enhancement of variant M263V
toward (R)-enantiomers of substrates 3a and 4a also
led to improved enantioconvergence.
For meta-substituted styrene oxides 8a–11a, the
WT did not show a complementary regiopreference
toward either enantiomer, and the r values toward
both enantiomers were insufficient. Particularly for 8a,
9a, 10a, and 11a, the rR values were less than 1.0
owing to the poor optical purity of product (8%–21%
eep). Compared with the WT, the regioselectivity of
variant M263Q toward both enantiomers of substrates
8a, 9a, 10a, and 11a was improved (Table 1). The rR
values showed a 40–53-fold improvement towards the
R-enantiomer and afforded the complementary
regiopreference towards the S-enantiomer, greatly
enhancing enantioconvergence in the hydrolysis of
meta-substituted styrene oxides.
Figure 3. The preferred substrate binding conformations of
(R)-3a and (R)-8a with WT and variants. The distances
between the two carbon atoms (Cα and Cβ) of epoxide ring
and the nucleophile residue Asp101 were labelled.
Through structural observation, the introduction
of Val263 with a smaller side chain provided a larger
hydrophobic pocket. The change in binding
surroundings was more beneficial to the hydrophobic
para-substituted styrene oxide binding in a more
favourable conformation from analysis of the binding
energy (4.93 kcal/mol). Furthermore, the precisely
modulated conformation with the increased distance
difference consolidated the attacking preference at the
C atom of the epoxide ring, leading to improved
regioselectivity. For meta-substituted styrene oxide,
the C atom of the epoxide ring was readily attacked
in the WT binding conformation, which was
undesirable regarding distance difference. In contrast,
substituting Met263 into residues Gln263 with a polar
side chain significantly changed the original binding
environment, resulting in a substrate binding pose in
Structural evidence for origin of improved
regioselectivity
To further explore the origin of improved
regioselectivity toward para- or meta-substituted
styrene oxides, the crystal structures of variants
M263Q (PDB ID: 7CG6) and M263V (PDB ID:
7CG2) were both determined at a resolution of 2.0 Å
(Table S4). For variants M263V and M263Q, the
introduction of Val263 provided a smaller side chain
on the modulated site (Figure S3A), while the
Gln263 amides group showed an inverted orientation
and shifted towards catalytic residue Asp101 (Figure
S3B). These changes caused by the mutation at
Met263 would greatly affect substrate binding.
Binding conformations with the WT and variants
4
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10.1002/adsc.202000898
Advanced Synthesis & Catalysis
the opposite orientation compared with that of the WT (9 mL, pH 7.0, 100 mM) and 10% cosolvent dimethyl
(Figure S4). The geometric distance between Asp101 sulphoxide at 30 °C with stirring (400 r/min). To minimize
and the C (dCα) or C (dCβ) atoms of the epoxide the spontaneous hydrolysis, substrate was added by 5
ring indicated that the attacking preference reversed batches. All the reaction processes were monitored by
on C of epoxide ring, which greatly improved its detecting the substrate with TLC, and the final vicinal diol
regioselectivity towards C. These findings verified products were extracted with ethyl acetate and purified by
the importance of residue 263 in modulating the flash chromatography.
epoxide binding pose by altering the binding
surroundings, and a single mutation at this site could
Conclusion
achieve the improved enantioconvergence of VrEH2
in the hydrolysis of various styrene oxides with
different substituted groups or bulky substrates.
Redesigning a key residue of VrEH2 resulted in a
smart mutant library that produced a marked
improvement in the enantioconvergent hydrolysis of
structurally diverse styrene oxides. More efficient
catalytic tools for chiral vicinal diols synthesis were
also obtained. Structural analysis showed the crucial
role of residue 263 in modulating the epoxide binding
pose by precisely altering the substrate binding
environment. Furthermore, the increased difference in
attacking distances provided evidence to explain the
improved regioselectivity. More significantly, these
findings can be introduced into other homologous
enzyme families to further enhance their applicability
to the enantioconvergent hydrolysis of various
epoxides.
Preparation of chiral vicinal diols via
enantioconvergent hydrolysis of styrene oxides
Subsequently, to explore the potential of the
redesigned enzymes in the biopharmaceuticals field,
several vicinal diol products derived from substrates
1a, 3a, 6a, 8a, 13a, 14a, and 17a, which are important
blocks for the preparation of -blocker drugs[13], such
as denopamine, FAAH inhibitors, solabegron,
dichlorolsoproterenol, and pronethalol, were prepared
in an enantioconvergent manner. Then a 10-mL
preparative scale reaction was performed, to minimize
the effect of epoxides spontaneous hydrolysis, a fed-
batch mode (5×20 mM) was applied in the system. The
results of these reactions are shown in Table 2. The
enantioenriched vicinal diols can be prepared with
high isolated yields (>88%) and high ee values (90%-
98%), and the final optical purity of some products
Experimental Section
can be further improved by recrystallized method[7a]
.
Biological and chemical reagents
Compared to current pathways using metal catalysts
or (salen) CoIII complexes,[14] the enantioconvergent
manner provides a promising and green pathway for
the synthesis of pharmaceutically relevant chiral diols.
Racemic substrates 1a-5a and enantiopure (R)-1a and
(S)-1a were purchased from commercial companies, while
6a-17a were prepared according to chemical methods.8 The
enantiopure 3a, 4a, 8a, 9a, 11a, 12a were obtained by
preparative HPLC (Shanghai Chiralway Biotech Co., Ltd,
China). All designed oligonucleotide primers were
prepared by SangonTech (Shanghai, China). The E. coli
BL21 (DE3) were used for expression of protein. All used
restriction enzymes and biological reagents of molecular
experiment were purchased from New England Biolabs
(Beijing, China).
Table 2. Enzymatic synthesis of (R)-vicinal diols by
enantioconvergent hydrolysis of styrene oxides.a
STY (gL-
1d-1)
Isolated
Time
(h)
eep
Substrate Enzyme
yield
(%)
rac-1a
rac-3a
M263V
M263V
M263N
M263Q
M263Q
M263Q
M263V
1.5
3.0
5.0
4.5
7.5
2.5
12.0
90
94
90
98
90
90
92
90
199
126
Site-directed Mutagenesis
91
92
90
89
88
89
Mutants
were
constructed
by
site-directed
mutagenesis via PCR using PrimeSTAR. The site-directed
primers (Table S1) were designed by software NewDNA.
Nearly 100 ng recombinant plasmid pET28-VrEH2 was
used as template and the PCR was performed according to
the general procedures. After digestion by endonuclease
DpnⅠ,the PCR product was transformed into E. coli BL21
(DE3). All the resulting mutants were verified by sequence
analysis.
rac-6a
72.0
74.5
58.3
148.7
33.5
rac- 8a
rac-13a
rac-14a
rac-17a
Expression and purification of variants
The recombinant cells were grown in Luria-Bertani
medium containing 50 μg/mL kanamycin for nearly 2.5-3 h
o
in a shaker under 37 C and 180 rpm. The inducer IPTG
aThe reactions were performed, including (5×20 mM)
substrates and catalyst (20 g cdw/L), in aqueous phase
system (10 mL) consisting of potassium phosphate buffer
was added with a final concentration of 0.2 mM when the
OD600 value of the growing cell reached 0.6-0.8. The cells
5
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10.1002/adsc.202000898
Advanced Synthesis & Catalysis
were induced for expression of the aimed protein for 24 h 2.36 (br, 2H). 13C NMR (150 MHz, DMSO-d6) δ 143.0,
under 16 oC and 180 rpm. The lysis buffer (20 mM Tri-HCl 131.7, 128.6, 128.2, 73.5, 67.65.
buffer, 300 mM NaCl and 20 mM imidazole, 5 mM β-
mercaptoethanol, pH 8.0) was prepared for cells re-
suspending. After sonication disruption, the cell lysate was
centrifuged for 50 min (10,956 g) under 4 °C to obtain
(R)-1-(4-Cyanophenyl)-1,2-ethanediol (6b): white solid;
150 mg, yield 92%; 98% ee. [α]3D0 : -24.0 (c 1.0, EtOH). lit.
[17]. [α]D25 : -20.5 (c 0.8, EtOH).1H NMR (400 MHz, CDCl3)
δ 7.69-7.62 (m, 2H), 7.51 (d, J = 8.1 Hz, 2H), 4.89 (dd, J =
supernatant. The cell free extract was purified by HisTrap
8.0, 3.4 Hz, 1H), 3.81 (dd, J = 11.2, 3.5 Hz, 1H), 3.62 (dd,
Ni+ affinity column (GE-healthcare) as described
previously.[10]
J = 11.2, 8.0 Hz, 1H), 2.34 (br, 2H). 13C NMR (150 MHz,
DMSO-d6) δ 140.9, 136.2, 128.9, 126.7, 74.2, 68.0, 21.2.
Regioselectivity coefficient assay
(R)-1-(3-Chlorophenyl)-1,2-ethanediol (8b): colorless oil;
155 mg, yield 90%; 90% ee. [α]2D5 : -26.0 (c 1.0, EtOH). lit.
Using optically pure (S)- or (R)-epoxides as substrates,
the configuration of products was determined by the
attacking preference of the enzyme on the two carbon
atoms of epoxide ring (regioselectivity).[15] Thus the
regioselectivity coefficients can be deduced by calculating
molar ratio of the two enantiomers of product in hydrolysis
of enantiopure epoxides. Enantiopure substrate (10 mM, 50
μL) was added to potassium phosphate buffer (100 mM,
pH 7.0, 450 µL) containing purified enzyme (0.1 mg/mL)
and the reaction mixture was agitated at 30°C and 1000
rpm. Then the reaction mixture was extracted by an equal
volume of ethyl acetate when the reaction was converted
completely. The molar ratio of the two enantiomers of
product was analyzed by HPLC (Shimadzu LC-10AT) with
a chiral OD-H column (Daicel, 4.6 Φ × 250 mm).
25
[16]. [α]D : -22.5 (c 1.1, EtOH). H NMR (400 MHz, CDCl3)
δ 7.32 (s, 1H), 7.27-7.23 (m, 2H), 7.16 (dd, J = 6.3, 2.3 Hz,
1H), 4.72 (dd, J = 8.3, 3.2 Hz, 1H), 3.68 (dd, J = 11.5, 3.3
Hz, 1H), 3.63 (s, 2H), 3.55 (dd, J = 11.6, 8.4 Hz, 1H). 13C
NMR (100 MHz, CDCl3) δ 151.5 (dd, J = 49.0, 12.7 Hz,
1C), 149.0 (dd, J = 49.0, 12.7 Hz, 1C), 137.6 (dd, J = 5.0,
3.9 Hz, 1C), 122.0 (dd, J = 6.4, 3.7 Hz, 1C), 117.3 (d, J =
17.3 Hz, 1C), 115.1 (d, J = 17.9 Hz, 1C), 73.5, 67.8.
1
(R)-1-(3,4-Fluorophenyl)-1,2-ethanediol (13b): colorless
oil; 155 mg, yield 89%; 92% ee. [α]2D5 : -38.0 (c 1.0, EtOH).
1H NMR (400 MHz, CDCl3) δ 7.26 – 7.05 (m, 3H), 4.79
(dd, J = 8.1, 3.4 Hz, 1H), 3.75 (dd, J = 11.3, 3.5 Hz, 1H),
3.60 (dd, J = 11.3, 8.1 Hz, 1H), 2.22 (br, 2H). 13C NMR
(100 MHz, CDCl3) δ 151.4 (dd, J = 49.1, 12.7 Hz, 1C),
148.9 (dd, J = 48.8, 12.7 Hz, 1C), 139.8 – 135.0 (m, 1C),
Preparing chiral vicinal diols by enzymatic 122.0 (dd, J = 6.4, 3.7 Hz, 1C), 117.3 (d, J = 17.3 Hz, 1C),
enantioconvergent hydrolysis of styrene oxides
115.12 (d, J = 17.9 Hz, 1C), 73.5, 67.8.
(R)-1-(3,4-Dichlorophenyl)-1,2-ethanediol (14b): white
solid; 182 mg, yield 88%; 90% ee. [α]3D0 : -40.0 (c 1.0,
CHCl3). 1H NMR (400 MHz, CDCl3) δ 7.49 (d, J = 1.9 Hz,
1H), 7.46-7.39 (m, 1H), 7.20 (dd, J = 8.3, 1.9 Hz, 1H), 4.80
(dd, J = 8.0, 3.4 Hz, 1H), 3.77 (dd, J = 11.2, 3.5 Hz, 1H),
3.61 (dd, J = 11.2, 8.0 Hz, 1H), 2.14 (br, 2H). 13C NMR
(150 MHz, DMSO-d6) δ 145.3, 131.0, 130.4, 129.6, 128.8,
127.2, 72.9, 67.3.
A
representative enantioconvergent hydrolysis
reaction was carried out in a 10-mL reaction system. The
recombinant E.coli cells were re-suspended in buffer (pH
7.0, 100 mM potassium phosphate , containing 10% (v/v)
dimethyl sulfoxide) at a final concentration of 15 g cdw/L
(cdw = dry cell weight), the substrate (0.02 mol) was fed
by 5 batches and the total substrate loading reached 100
mM. The reaction was performed in a stirring reactor under
30°C and 400 r/min and the reaction process were (R)-1-(2-Naphthalenyl)-1,2-ethanediol (17b): white solid;
monitored by TLC. When the substrate was totally 167.5 mg, yield 89%; 90% ee. [α]3D0 : -32.0 (c 1.0, EtOH).
converted, the product was extracted using ethyl acetate (5 lit.[17]. [α]D23 : -28.6 (c 0.79, EtOH).1H NMR (400 MHz,
× 10 mL). The combined organic phase was washed with DMSO-d6) δ 7.93-7.82 (m, 4H), 7.49 (tdd, J = 9.0, 5.8, 3.4
saturated NaCl solution (2 × 50 mL), then dried over Hz, 3H), 5.38 (d, J = 4.2 Hz, 1H), 4.77 (t, J = 5.8 Hz, 1H),
anhydrous Na2SO4 for 12 h, then concentrated under 4.71 (dd, J = 10.3, 5.8 Hz, 1H), 3.53 (t, J = 5.9 Hz, 2H).
reduced pressure. The resulting residual was purified by 13C NMR (150 MHz, DMSO-d6) δ 141.6, 133.3, 132.8,
flash chromatography on silica gel eluted with petroleum 128.2, 127.9, 127.7, 126.4, 125.9, 125.5, 125.2, 74.4, 67.8.
ether/ethyl acetate. H and 13C NMR data and optical
rotation of the vicinal diols were listed as following:
1
Crystallization, structure determination and molecular
docking
(R)-1-Phenyl-1,2-ethanediol (1b): white solid; 124 mg,
After Ni+ column purification, the collected protein
was further purified using a gel filtration column (Superdex
75 Hiload 16/60) to obtain protein as monomer in solution.
Protein samples (VrEH2M263Q 16 mg/mL, VrEH2M263V 18
mg/mL) were added in 96 well sitting-drop plate and mixed
with equal volume (2 µL) of crystallization solution. The
crystal growing condition (0.15 M Tris-HCl, pH 8.5, 35%
PEG3350, 18 °C) of VrEH2M263Q and VrEH2M263V were
same as wild type, protein crystals could be observed in
diamond shaped after three days. Crystals were flash-
cooled with liquid nitrogen in crystallization solution
containing 10% (v/v) glycerol as cryoprotectant and used
yield 90%; 94% ee. [α]3D0 : -40.0 (c 1.0, EtOH). lit.[16]. [α]D25 :
-37.8 (c 1.0, EtOH). 1H NMR (400 MHz, CDCl3) δ 7.37 (d,
J = 4.4 Hz, 4H), 7.34-7.29 (m, 1H), 4.83 (dd, J = 8.2, 3.5
Hz, 1H), 3.77 (dd, J = 11.3, 3.5 Hz, 1H), 3.67 (dd, J = 11.3,
8.2 Hz, 1H), 2.21 (br, 2H). 13C NMR (150 MHz, CDCl3) δ
140.4, 128.5, 127.9, 126.1, 74.7, 68.0.
(R)-1-(4-Chlorophenyl)-1,2-ethanediol (3b): white solid;
157 mg, yield 91%; 90% ee. [α]3D0 : -64.0 (c 1.0, CHCl3). lit.
[16]. [α]D25 : -55.9 (c 1.0, CHCl3). 1H NMR (400 MHz, CDCl3)
δ 7.37-7.28 (m, 4H), 4.80 (dd, J = 8.1, 3.3 Hz, 1H), 3.75
(dd, J = 11.3, 3.3 Hz, 1H), 3.61 (dd, J = 11.2, 8.2 Hz, 1H),
6
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10.1002/adsc.202000898
Advanced Synthesis & Catalysis
for X-ray diffraction. The diffraction data of crystals were
collected at beamline BL17U1 of the Shanghai
Synchrotron Radiation Facility (SSRF),[18] and the software
HKL2000 was used for data indexing, integrating and
scaling.[19] The initial structure model of mutants was
obtained by molecular replacement using the VrEH2M263N
(PDB ID: 5Y6Y) as search template. Then the structure
Enzym. 2010, 65, 41-48. e) H. H. Ye, D. Hu, X. L. Shi, M. C. Wu,
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and refinement result are listed in Table S4. The molecular
docking was carried out using the software
AutodockingTools 1.5.6,[21] the substrates were docked into
the active site of WT and variants, respectively. The
coordinate of central grid was determined based on the
locations of the catalytic residues. The programs Autogrid 4
and Autodock 4 were performed by default.
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[9] M. Kotik, W. Zhao, G. Iacazio, A. Archelas, J. Mol. Catal. B-
Enzym. 2013, 91, 44-51.
[10] F. L. Li, X. D. Kong, Q. Chen, Y. C. Zheng, Q. Xu, F. F.
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This work was financially supported by The National
Key Research and Development Program of China
(2019YFA09005000), The
National
Natural
Science Foundation of China (21922804 & 21672063 &
21536004 & 21871085), and the Fundamental Research
Funds for the Central Universities (22221818014); We are
very grateful for the access to beamline BL17U1 at
Shanghai Synchrotron Radiation Facility and thank the
beamline staff for technical help. We also thank Simon
Partridge, PhD, for editing the English text of a draft of this
manuscript.
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FULL PAPER
Reprogramming Epoxide Hydrolase to Improve
Enantioconvergence in Hydrolysis of Styrene
Oxide Scaffolds
Adv. Synth. Catal. 2020 Volume, Page – Page
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