Biocatalytic asymmetric ring-opening of dihydroisoxazoles: a cyanide-free route to complementary enantiomers of β-hydroxy nitriles from olefins

Article abstract of DOI:10.1039/d0gc01445a

By combination of the cyanide-free synthesis of chiral nitriles and the Kemp elimination reaction catalyzed by aldoxime dehydratases, we herein report a new application of aldoxime dehydratase in the asymmetric ring-opening of 5-sub-4,5-dihydroisoxazoles

Full text of DOI:10.1039/d0gc01445a

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Paul T. Anastas et al.
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DOI: 10.1039/D0GC01445A
ARTICLE
Biocatalytic Asymmetric Ring-opening of Dihydroisoxazoles: A
Cyanide-free Route to Complementary Enantiomers of β-Hydroxy
nitriles from Olefins†
Received 00th January 20xx,
Accepted 00th January 20xx
Daijun Zheng,a and Yasuhisa Asano *
DOI: 10.1039/x0xx00000x
By combination of the cyanide-free synthesis of chiral nitriles and the Kemp elimination reaction catalyzed by aldoxime
dehydratases, we herein report a new application of aldoxime dehydratase in the asymmetric ring-opening of 5-sub-4,5-
dihydroisoxazoles to synthesize chiral β-hydroxy nitriles with broad substrate scope, excellent enantioselectivity (up to
99% ee), and good turnover number (up to 11 s–1). Upon simple isolation and treatment with an alkaline reagent, the
remaining chiral 5-sub-4,5-dihydroisoxazoles can be easily transformed into their corresponding β-hydroxy nitriles. Using
site-directed mutagenesis, a ferrous Heme-containing active site was confirmed and two possible deprotonation pathways
were proposed. To the best of our knowledge, this is the first enzymatic reaction used to construct a chiral hydroxyl group
and nitrile group in one-step starting from a simple alkene, which provides a novel and useful strategy for the synthesis of
complementary enantiomers of β-hydroxy nitriles.
Introduction
Chiral β-hydroxy nitriles are versatile building blocks that can
be easily transformed into chiral β-hydroxy acids, γ-hydroxy
amines, lactones etc,1-4 which are important motifs of
pharmaceutical and biologically active molecules (Scheme 1),
and can be used as potential monomers for the preparation of
biodegradable plastics.5
Due to the versatility of this family of compounds, the
methods used for their efficient synthesis have been
extensively explored over the last few decades. The most
common strategy used in organic synthesis involves
asymmetric transfer hydrogenation (ATH) to reduce keto-
nitrile precursors into their corresponding hydroxyl nitrile
compounds using transition-metal complexes such as
ruthenium (II)-complex6-10 and iridium-complexes11-13
. In
addition, alternative methods using the β-borylation of
organoborane compounds with acceptor of α, β-unsaturated
nitriles catalysed by Copper-complex following with oxidation
to synthesize corresponding alcohols have been reported.14-16
What’s more, the asymmetric cross aldol reaction of aldehydes
Scheme 1. Biologically-active molecules containing the motifs derived from β-hydroxy
nitriles.
with different nucleophilic addition donors and catalysts, such
as acetonitrile/Cu alkoxide-chiral phosphine complex,17
TMSCH2CN/copper fluoride,18 organolithium reagents,19
cyanomethylzinc bromide/DPMPM20 have been well
developed. Moreover, the region-selective ring-opening of
epoxide with cyanide was also a classic synthetic method
accessing to β-hydroxy nitriles.21 In general, all these synthetic
routes inevitably use costly chiral transition-metal complexes
as catalysts to construct the chiral centre. Nowadays,
benefitting from the great development of biotechnology and
green chemistry, a variety of synthetic strategies to β-hydroxy
nitriles have been developed using purified enzymes or whole
cell catalysts. Among them, lipases,2, 22, 23 reductases,1, 24-26
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resulting phenoxyl anion.41 It was also mentioned that the
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metabolism of leflunomide, an DOiIm: 1m0.1u0n39o/mD0oGdCu0la14t4o5rAy
therapeutic drug, could be transferred into teriflunomide via
isoxazole ring-opening catalysed by human cytochrome P450.
This provides the idea of synthesizing a chiral β-hydroxy nitrile
product using
a
prochiral substrate containing the
dihydroisoxazole motif. To verify this hypothesis, the substrate
5-phenyl-4,5-dihydroisoxazole was designed and tested in the
reaction with aldoxime dehydratase Oxd-B obtained from
Bacillus sp.OxB-1.42-44
Results and discussion
In the initial screening process, the reaction was carried out in
Scheme 2. (a) Enantioselective dehydration catalysed by aldoxime dehydratase.
(b) The Kemp elimination reaction catalysed by aldoxime dehydratase. (c)
Asymmetric ring-opening of dihydroisoxazole catalysed by aldoxime dehydratase.
the absence of
a reductant, which showed excellent
29
dehydrogenases,27 and nitrilases28,
have been mostly
enantioselectivity on the cleavage of the N-O bond as
expected, but the activity was barely satisfactory. In view of
the important role of
employed toward the synthesis of chiral β-hydroxy nitrile via
the kinetic resolution of racemic hydroxyl nitriles or the
asymmetric reduction of β-oxo-nitriles. In addition, few
publications have reported the use of monooxygenase for the
asymmetric hydroxylation of 3-phenylpropanenitrile30 or
a reductant in the aldoxime
dehydration45 and Kemp elimination reactions,33 which can
significantly enhance the enzyme activity, different reductants
were tested for the asymmetric ring-opening of 5-phenyl-4,5-
dihydroisoxazole (Fig. S1). The enhanced effect on the
enzymatic activity upon the addition of a reductant was
observed, which showed the activity can be improved 1.9–6.0
multi-enzymatic
biosynthesis
system
of
alcohol
dehydrogenase/halohydrin dehalogenase treating on α-halo
ketones and cyanide31 to form this family of chiral compounds.
In part, these biocatalytic methods overcome the use of costly
transition-metal catalysts, but are confined to a limited scope
of precursors, including keto-nitriles and racemic hydroxyl
nitriles, which are prepared using tedious synthetic processes
or inevitably use poisonous cyanide as a starting material. By
combination of cyanide-free synthesis of chiral nitriles
(Scheme 2a)32 and the Kemp elimination reaction (Scheme
2b)33 catalysed by aldoxime dehydratases (EC 4.99.1.5, EC
4.99.1.6, EC 4.99.1.7), we herein report a novel cyanide-free
enzymatic approach to construct a chiral hydroxyl group and
nitrile group in one-step toward the efficient synthesis of the
complementary enantiomers of β-hydroxy nitriles via the
asymmetric ring-opening of 5-sub-4,5-dihydroisozazoles
(Scheme 2c).
fold using
a variety of reductants. In addition, the
enantioselectivity of the enzyme was not affected by the
additives with the ee of the product being maintained at ≥99%
in each case. The reductant effect implies a ferrous-Heme
active state was involved in the reaction, which is consistent
with the sensitivity of Oxd B to oxygen.42
Aldoxime dehydratase is a class of Heme-containing enzyme
involved in the cyanogenesis pathway observed in nature,
which catalyses the dehydration of aldoximes to their
corresponding nitrile derivatives.34 Using the dehydration
function of the enzyme, we established a cyanide-free
platform for the synthesis of nitriles utilizing aldoxime
precursors.35-38 Recently, we discovered the enzyme catalyses
the Kemp elimination reaction via a -Fe-N- complex catalytic
pathway,33 which is different from the classic acid-base
mechanism.39, 40 In this enzymatic elimination reaction, the N-
O bond is initially opened with the action of the –Fe-N-
complex (Heme Fe-proximal histidine) followed by
deprotonation by a distal histidine residue to form the nitrile
triple-bond. Upon investigation of the catalytic mechanism, it
has been reported that the Kemp elimination reaction can also
occur based on the redox catalysis of Heme-containing P450-
BM3 via heterolysis to liberate an intermediate containing a
nitrogen radical moiety (Fe(III)-N·) and phenoxyl anion. The
product is formed by bond rotation and proton-transfer to the
Figure 1. (a) The pH effect on the activity of Oxd B in the catalytic asymmetric ring-
opening of 5-phenyl-4,5-dihydroisoxazole. The reactions were performed on a 500 µL
scale using 100 mM buffer (pH 3.0–10.0),
1 U Oxd-B, 20 mM 5-phenyl-4,5-
dihydroisoxazole, 0.05 mM FMN, and 1 mM Na2S2O5 at 30 °C for 30 min. The buffer
solutions used were Gly-HCl (pH 3.0), Citrate-buffer (pH 3.0, 4.0, 5.0, and 6.0), KPB
(pH 6.0, 7.0, and 8.0), Tris-HCl (pH 7.0, 8.0, and 9.0), and Gly-NaOH (pH 8.0, 9.0, and
10.0). (b) The control experiments were carried out without Oxd B at pH 9.0 and pH
10.0. The yield and ee were determined using chiral HPLC (n = 2).
2 | J. Name., 2012, 00, 1-3
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The classic Kemp elimination reaction can take place under
alkaline conditions via an acid-base mechanism.40 Accordingly,
this reaction was investigated in a range of different pH buffers
(Fig. 1a). Although the yield varied significantly at different pH
in a various buffer types, the enzyme could work at pH 5.0–
10.0 with the desired product formed in 2–36% yield, and the
optical purity of the product was maintained at ≥99% at pH
5.0–8.0. From the control results, spontaneous reactions were
detected in pH 9.0–10.0 (Fig. 1b), which caused a sharp
decrease in the product ee. Enlightened by these results, an
useful method to prepare racemic β-hydroxy nitriles is
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DOI: 10.1039/D0GC01445A
described from alkenes via
a 5-sub-4,5-dihydroisoxazole
intermediate46, 47 upon treatment with a methanolic NaOH
solution at room temperature (Scheme 3, and detailed data
are shown in the SI, Section 5). The result is consistent with the
previous reports that β-hydroxy nitriles were formed by
treating dihydroisoxazoles with bases (such as trimethylamine,
1,8-diazabicyclo[5.4.0]undec-7-ene,49 NaOMe,50 ring-
48
Figure 2. Time course of the asymmetric ring-opening of 5-phenyl-4,5-dihydroisoxazole
using Oxd B. The reactions were performed on a 500 µL scale using 100 mM KPB buffer
(pH 6.0), 1 U Oxd B, 20 mM 5-phenyl-4,5-dihydroisoxazole, 0.05 mM FMN, and 1 mM
Na2S2O5 at 30 °C. The yield and ee were determined using chiral HPLC (n = 2).
opening of 3-bromo-2-isoxazolines upon treatment with an
alkanethiolate.51
To further improve the product yield, the reaction was
monitored for a prolonged time of 3 h (Fig. 2). During the
enantioselective transformation of the substrate, the ee of the
substrate 5-phenyl-4,5-dihydroisoxazole (1a) increased to 99%
over 2 h and the product (2a) ee was maintained at 99%,
indicating the excellent catalytic enantioselectivity of Oxd B on
this substrate. Additionally, it is often beneficial to agitate a
reaction to improve the mass transfer process and as a result,
enhance the reaction rate. However, a ~5-fold decrease in the
yield of the β-hydroxy nitrile product was observed upon
shaking the reaction when compared to the static-state
reaction over 1 h. As reported in the dehydration of aldoximes,
the activity of Oxd B can be increased by 5-fold under
anaerobic conditions.42 Therefore, the oxygen sensitivity of
Oxd B seems to be the reason of the decrease in the enzymatic
activity in a shaking mode. By shaking, the enzyme is much
more susceptible to exposure to oxygen in the air, which leads
to the oxidation of ferrous-Heme to ferric-Heme, the resting-
state of Heme in the ring-opening reaction of
dihydroisoxazole. 52, 53
With the optimized reaction conditions, a series of substrates
were investigated using Oxd B (Table 1). Oxd B exhibited good
selectivity toward most aromatic substrates with desired
product S/R-2 formed in 48–99% ee. Among them, substrates
bearing an electron-donating group on the aryl ring showed
relatively higher ee values than those bearing an electron-
withdrawing group. In addition, the enzyme preferred
substrates bearing a substituent at the para-position (Table 1,
1d, 1g, and 1h), rather than the ortho- (Table 1, 1b and 1e) or
meta-position (Table 1, 1c and 1f). The selectivity toward
heterocyclic aromatic substrates (Table 1, 1j) was also very
excellent, but a slight decrease in activity was observed.
However, increasing the size of the aromatic ring to a
naphthalene group leads to the reaction conversion becoming
negligible (Table 1, 1i), which may be caused by the lower
solubility of the substrate in water. For aliphatic substrates
(Table 1, 1k and 1l), Oxd B exhibited a deterioration in the
reaction selectivity.
To establish an efficient catalytic system for aliphatic
substrates, another two aldoxime dehydratases obtained from
Pseudomonas chlororaphis B23 (Oxd A)54, 55 and Rhodococcus
sp. N-771 (Oxd RE),45, 56 were selected and investigated using a
range of different substrates. They were found to display
higher activity toward aliphatic substrates when compared
with aromatic substrates in the aldoxime dehydration reaction.
Scheme 3. Synthesis of dihydroisoxazoles and (rac)-β-hydroxy nitriles from alkenes.
This journal is © The Royal Society of Chemistry 20xx
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(a) For Oxd A, the reactions were performed on a 500 µL scale using 100 mM KPB
Table 1. Investigation of the substrate scope of Oxd A, B, and RE.a
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buffer (pH 7.0), 10 U Oxd A, 20 mM substrate, and 4 mM Na2S2O4 at 30 °C for 3 h
DOI: 10.1039/D0GC01445A
(n = 2); for Oxd B, the reactions were performed on a 500 µL scale using 100 mM
KPB buffer (pH 6.0), 20 U Oxd-B, 20 mM substrate, 0.05 mM FMN, and 1 mM
Na2S2O5 at 30 °C for 2 h (n = 2); and for Oxd RE, the reactions were performed on
a 500 µL scale using 100 mM KPB buffer (pH 7.0), 1 U Oxd RE, 6 mM substrate, 1
mM Na2S at 30 °C for 2 h (n = 2). (b) The conversion was calculated using conv =
ees/(ees+eep).57 (c) The ee was determined using chiral HPLC and chiral GC. (d)
The activity for substrate 1a was taken to be 100%. The relative activity for Oxd B
was determined on a 500 µL scale using 100 mM KPB buffer (pH 6.0), 1 U Oxd B,
20 mM substrate, 0.05 mM FMN, and 1 mM Na2S2O5 at 30 °C for 2 h (n = 2).
ee of
S/R-1
[%]
ee of
Relative
activity
[%] d
Conv.
[%] b
Prior to investigating the substrate scope, optimization of the
reductant was also carried out. Oxd A showed the best activity
with sodium dithionite (Na2S2O4), which was ~79-fold higher
than that observed in the control experiment performed in the
absence of a reductant (Fig. S7). Oxd RE preferred sodium
sulfide (Na2S), which exhibited a ~8.2-fold higher activity than
the control (Fig S3). The optimal reductant concentration for
Oxd A and Oxd RE were 4 mM (Fig. S8) and 1 mM (Fig. S4),
respectively. Upon monitoring the asymmetric ring-opening
reaction of 5-phenyl-4,5-dihydroisoxazole with time, Oxd A
could catalyse the reaction in 83% yield over 3 h, but gave a
nearly racemic product (Fig. S9). The same result was also
obtained using Oxd RE, the product was formed in 28% yield
and 53% ee at pH 6.0, and 48% yield and 55% ee at pH 7.0 over
Substrate (±)-1
Enzyme
S/R-2
[%] c
N
O
Oxd A
Oxd B
Oxd RE
Oxd A
Oxd B
Oxd RE
Oxd A
Oxd B
Oxd RE
Oxd A
Oxd B
Oxd RE
Oxd A
Oxd B
Oxd RE
Oxd A
Oxd B
Oxd RE
Oxd A
Oxd B
Oxd RE
Oxd A
Oxd B
Oxd RE
Oxd A
Oxd B
Oxd RE
Oxd A
Oxd B
Oxd RE
Oxd A
Oxd B
Oxd RE
Oxd A
Oxd B
Oxd RE
93
51
64
70
31
21
70
47
20
95
32
23
67
47
59
52
53
38
94
40
39
74
47
23
0.5
8
24 (S)
99 (S)
68 (S)
25 (R)
43 (S)
10 (R)
77 (S)
76 (S)
17 (S)
21 (S)
45 (S)
12 (S)
25 (R)
47 (S)
18 (R)
19 (S)
64 (S)
38 (S)
99 (S)
63 (S)
18 (S)
77 (S)
87 (S)
23 (S)
0 (---)
6 (---)
1 (---)
9 (S)
2 (R)
96 (R)
38 (R)
11 (S)
95 (R)
36 (S)
33 (R)
86 (R)
66 (R)
1 (R)
100
100
100
75
8
1a
N
O
1b
33
75
42
32
102
8
N
O
1c
N
O
2
h, respectively (Fig. S5). Our substrate specificity
1d
97 (R)
41 (R)
12 (S)
53 (R)
13 (S)
18 (R)
56 (R)
62 (R)
6 (R)
investigations show that Oxd exhibited very poor
A
36
74
55
93
55
38
59
101
28
61
79
25
36
0
enantioselectivity toward the substrates studied, though the
conversion is high (Table 1). Oxd RE showed relatively better
results toward aliphatic substrates when compared to
aromatic substrates. In addition, it also exhibited slightly
better enantioselectivity than Oxd B in the asymmetric ring-
opening of aliphatic substrates (Table 1; 1k and 1l).
Interestingly, Oxd A and Oxd RE exhibited different selectivity
toward ortho-substituted aromatic substrates when compared
with Oxd B (Table 1; 1b and 1e). What causes this difference in
selectivity compared with other substrates and Oxd B is still
not clear.
The kinetic parameters for both Oxd B and Oxd RE were
determined under the optimized reaction conditions (Table S2).
The turnover number of Oxd B reached 11 s–1 with a specific
activity of 18 U/mg, which is about one third of the activity
observed for (Z)-phenylacetaldoxime dehydration (53 U/mg)
using the assay method containing Na2S2O5 (SI, Section 3).
However, the Oxd RE activity was lower than Oxd B at 4 U/mg
with a turnover number of 3 s–1, ~15% of the activity observed
for the (Z)-phenylacetaldoxime dehydration reaction (30
N
Cl
O
1e
1f
1g
1h
1i
N
O
Cl
N
O
95 (R)
28 (R)
27 (R)
99 (R)
77 (R)
13 (---)
71 (---)
6 (---)
3 (R)
Cl
N
O
MeO
N
O
7
9
15
83
79
123
89
12
48
95
80
58
77
49
79
83
67
31
88
59
38
N
O
U/mg). In addition, the catalytic efficiency of Oxd B (Kcat/Km
=
1j
96 (S)
75 (S)
84 (R)
99 (R)
39 (R)
87 (R)
94 (R)
49 (R)
99 (R)
20 (R)
17 (S)
48 (S)
87 (S)
11 (S)
66 (S)
82 (S)
3.7 × 104 M–1s–1) is ~9-fold higher than Oxd RE (Kcat/Km = 4.3 ×
103 M–1s–1). An obvious substrate inhibition effect was
observed using 1 U of the enzyme upon increasing the
substrate concentration to 30 mM for Oxd B and 6 mM for Oxd
RE, but no any effect on the optical purity of the product (Fig.
S2 and S6). Therefore, a scale-up reaction was performed
based on a substrate concentration of 20 mM and 1 U Oxd B.
Under the optimal conditions, the reaction concentration
could reach 150 mM with the product formed with 50%
conversion and 99% ee (Table S1).
S
N
O
1k
1l
N
O
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DOI: 10.1039/D0GC01445A
a.
Figure 3. (a) Oxd B homology model and superposition to Oxd RE structure docking
with substrate 1a; (b) Comparison of the Oxd B mutants’ activity for substrate 1a. The
reactions were performed on a 500 µL scale using 100 mM KPB buffer (pH 6.0), 2 µg
of Oxd-B mutants, 20 mM 5-phenyl-4,5-dihydroisoxazole, 0.05 mM FMN, and 1 mM
Na2S2O5 at 30 °C for 10 min. The yield and ee were determined using chiral HPLC (n =
2).
To elucidate the reaction mechanism, a homology model of
Oxd B was established based on the crystal structure of Oxd-
RE.58 In addition, the corresponding residues located in the
catalytic pocket of the Heme were selected for an alanine-
scanning study (Fig. 3a). In this scanning study, L128A, T202A,
Q204A, H282A, F289A, and H306A mutants were constructed
via site-mutagenesis (SI, Section 7), which were purified as the
wild type of Oxd B to obtain the pure enzyme (SI, Section 2.2)
and used for activity comparison. To begin with, the activity of
the mutants toward (Z)-phenylacetaldoxime was assayed. As
reported in previous publication, no activity was detected for
the T202A, H282A, and H306A mutants, which are
corresponding to the S219, H299, and H320 residues in Oxd RE,
the crucial residues in the dehydration of butyraldehyde
oxime.33 These results reveal that the homology model of Oxd
B is workable to some extent, although the protein sequence
homology between Oxd B and Oxd RE is only 31%. After
verifying the reliability of the homology model, the mutants
were used in the 5-phenyl-4,5-dihydroisoxazole ring-opening
reaction. As expected, the H282A mutant showed no activity
toward the substrate because the proximal axial residue
ligating to iron is thought to affect the redox potential of the
Heme, thus leading to a great difference in the catalytic
Scheme 4. Proposed reaction mechanism for Oxd B-wild (a) and oxd B-H306A (b).
activity for bond cleavage.59-61 Surprisingly, the activity of the
mutants was determined to be 20% for T202A and 5% for
H306A when compared with wild-Oxd B (Fig. 3b), which is
similar to that reported in the Kemp elimination reaction.33 To
confirm the transformation in mutant of H306A, the amount of
Oxd B-H306A was increased 10-fold , and the product could be
obtained in 3.8% yield and 99% ee over 10 min, 21% yield and
95% ee in 1 h, respectively. This implied a discriminating
catalytic mechanism between the ring-opening of
dihydroisoxazole and aldoxime dehydration reactions. It seems
that the ring-strain in dihydroisoxazole could facilitate the
cleavage of the N-O bond via redox of ferrous-Heme without
the assistance of the other residues (T202 and H306 in the (Z)-
phenylacetaldoxime dehydration reaction), even though its
catalytic efficiency was slower than that observed under the
assistance of the other residues. In particular, weak activity
was detected for H306A, which is thought to play an important
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enantioselectivity (up to 99% ee). The reaction can be carried
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–1
DOI: 10.1039/D0GC01445A
out with a good turnover number (kcat up to 11 s ) and high
catalytic efficiency (kcat/km up to 3.7 × 104 M–1s–1). Using site-
directed mutagenesis analysis, the reaction was found to
mostly occur in the Heme-containing catalytic pocket involving
residues H282 and H306. The proximal histidine ligated to the
Heme plays a very important role in improving the redox
potential of ferrous-Heme, which ensures its reductive ability
to open the N-O bond. Without the assistance of any other
residues, the N-O bond can be also slowly opened via its redox
reaction with ferrous-Heme. Two kinds of deprotonation
pathways (histidine deprotonation and oxygen anion
deprotonation) seem to exist in the catalytic process, which
both contribute to the formation of the nitrile triple-bond.
Scheme 5. Preparative scale reaction using substrate 1a with Oxd B. The reaction
was performed on a 50 mL scale using 100 mM KPB buffer (pH 6.0), 500 U Oxd B,
100 mM 5-phenyl-4,5-dihydroisoxazole, 0.25 mM FMN, and 5 mM Na2S2O5 at 30°C
for 2 h. The isolated yield was calculated after purification by silica gel column
chromatography and the ee was determined by chiral HPLC.
Conflicts of interest
The authors declare no conflict of interest.
role in the next deprotonation step to form the nitrile triple-
bond (Scheme 4a). This suggests that in addition to the
efficient deprotonation step by histidine (H306), it may have
another route for deprotonation, which we refer to as the
oxygen anion deprotonation pathway.41 After ring-opening
upon attack of the ferrous-Heme, the resulting oxygen anion
will approach the hydrogen atom of H-C=N- (via single bond
rotation) followed by deprotonation and electron transfer back
to ferric-Heme to produce the active state of ferrous-Heme,
which undergoes the next catalytic cycle (Scheme 4b). We also
noticed that the product ee did not change significantly in the
presence of any mutants. This reveals the cooperative control
of the enantioselectivity with the surrounding residues of the
Heme, not only from certain residues in the catalytic pocket.
To verify the practicability of this novel method in organic
Acknowledgements
We are grateful for the support from the JST ERATO Asano
Active Enzyme Molecule Project, Japan (Grant Number
JPMJRER1102) and Grant-in-Aid for Scientific Research (S) from
the Japan Society for Promotion of Sciences (Grant
No.17H06169) awarded to Y. Asano.
References
1. H. Ankati, D. Zhu, Y. Yang, E. R. Biehl and L. Hua, J. Org. Chem.,
2009, 74, 1658-1662.
2. O. Pàmies and J.-E. Bäckvall, Adv. Synth. Catal., 2002, 344, 947-
952.
synthesis,
a preparative scale reaction of 5-phenyl-4,5-
3. S. D. Sharma, R. D. Anand and G. Kaur, Synth. Commun., 2004,
34, 1855-1862.
dihydroisoxazole (1a) was performed at 30 °C using 5 mmol of
substrate, 0.25 mM FMN, 500 U Oxd B (see the activity assay
method in SI, Section 3), and 5 mM Na2S2O5 in 50 mL KPB (pH
6.0, 100 mM) stirred slowly for 2 h. After purification by silica
4. J. M. Concellón and C. Concellón, J. Org. Chem., 2006, 71, 4428-
4432.
5. E. Bugnicourt, P. Cinelli, A. Lazzeri and V. Alvarez, eXPRESS
Polym. Lett., 2014, 8, 791-808.
6. T. Touge, T. Hakamata, H. Nara, T. Kobayashi, N. Sayo, T. Saito,
Y. Kayaki and T. Ikariya, J. Am. Chem. Soc, 2011, 133, 14960-
14963.
7. A. Kišić, M. Stephan and B. Mohar, Adv. Synth. Catal, 2014, 356,
3193-3198.
gel
phenylpropanenitrile (
ee, and (S)-5-phenyl-4,5-dihydroisoxazole (
in 48% yield and 99% ee. The obtained (S)-5-phenyl-4,5-
dihydroisoxazole ( 1a) was transformed into (S)-3-hydroxy-3-
phenylpropanenitrile ( 2a) in 95% yield and 94% ee upon
column
chromatography,
2a) was isolated in 42% yield and 99%
1a) was isolated
(R)-3-hydroxy-3-
R-
S-
S-
8. Y. Li, Z. Li, F. Li, Q. Wang and F. Tao, Org Biomol Chem, 2005, 3,
2513-2518.
S-
treatment with 2% NaOH methanol solution (10 mL) for 30 min 9. P. N. Liu, P. M. Gu, F. Wang and Y. Q. Tu, Org. Lett., 2004, 6,
(Scheme 5).
169-172.
10. W. K. Masahito, Murata.; Takao, Ikariya., J. Org. Chem., 2002,
67, 1712-1715.
11. H. Vazquez-Villa, S. Reber, M. A. Ariger and E. M. Carreira,
Angew. Chem. Int. Ed., 2011, 50, 8979-8981.
12. C. Chen, L. Kong, T. Cheng, R. Jin and G. Liu, Chem. Commun.,
2014, 50, 10891-10893.
13. O. Soltani, M. A. Ariger, H. Vázquez-Villa and E. M. Carreira, Org.
Lett., 2010, 12, 2893-2895.
14. J.-E. Lee and J. Yun, Angew. Chem. Int. Ed., 2008, 47, 145-147.
15. L. Zhu, T. Kitanosono, P. Xu and S. Kobayashi, Chem. Commun.,
2015, 51, 11685-11688.
Conclusion
In summary, by combination of cyanide-free synthesis of chiral
nitriles and the Kemp elimination reaction, we have achieved
the first enzymatic reaction to synthesize chiral β-hydroxy
nitriles from alkenes via the key step of asymmetric ring-
opening of dihydroisoxazoles catalysed by aldoxime
dehydratase with broad substrate scope and excellent
6 | J. Name., 2012, 00, 1-3
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Please do not adjust margins
Journal Name
ARTICLE
16. S. Mun, J.-E. Lee and J. Yun, Org. Lett., 2006, 8, 4887-4889.
17. Y. Suto, R. Tsuji, M. Kanai and M. Shibasaki, Org. Lett., 2005, 7,
3757-3760.
18. Y. Suto, N. Kumagai, S. Matsunaga, M. Kanai and M. Shibasaki,
Org. Lett., 2003, 5, 3147-3150.
50. A. Yashiro, Y. Nishida, K. Kobayashi and M. Ohno,VSieywnAlerttictl,e2O0n0lin0e,
2000, 361-362.
DOI: 10.1039/D0GC01445A
51. M. H. Seo, Y. Y. Lee and Y. M. Goo, Synth. Commun., 1994, 24,
1433-1439.
52. R.-Z. Liao and W. Thiel, J. Phys. Chem B, 2012, 116, 9396-9408.
19. J. Granander, J. Eriksson and G. Hilmersson, Tetrahedron: 53. E. Pinakoulaki, C. Koutsoupakis, H. Sawai, A. Pavlou, Y. Kato, Y.
Asymmetry, 2006, 17, 2021-2027.
Asano and S. Aono, J. Phys. Chem B, 2011, 115, 13012-13018.
20. K. Soai, Y. Hirose and S. Sakata, Tetrahedron: Asymmetry, 1992, 54. K. Oinuma, Y. Hashimoto, K. Konishi, M. Goda, T. Noguchi, H.
3, 677-680.
Higashibata and M. Kobayashi, J. Biol. Chem., 2003, 278, 29600-
29608.
55. J. Nomura, H. Hashimoto, T. Ohta, Y. Hashimoto, K. Wada, Y.
Naruta, K.-I. Oinuma and M. Kobayashi, Proc. Natl. Acad. Sci.,
2013, 110, 2810-2815.
21. D. Mitchell and T. M. Koenig, Synth. Commun., 1995, 25, 1231-
1238.
22. B. Xia, J. Xu, Z. Xiang, Y. Cen, Y. Hu, X. Lin and Q. Wu, ACS Catal.,
2017, 7, 4542-4549.
23. E. Garcıa-Urdiales, F. Rebolledo and V. Gotor, Tetrahedron: 56. Y. Kato, R. Ooi and Y. Asano, Arch. Microbiol., 1998, 170, 85-90.
́
Asymmetry, 2001, 12, 3047-3052.
24. M. L. Contente, I. Serra, F. Molinari, R. Gandolfi, A. Pinto and D.
Romano, Tetrahedron, 2016, 72, 3974-3979.
25. D. Zhu, H. Ankati, C. Mukherjee, Y. Yang, E. R. Biehl and L. Hua,
Org. Lett., 2007, 9, 2561-2563.
26. L.-J. Wang, C.-X. Li, Y. Ni, J. Zhang, X. Liu and J.-H. Xu, Bioresour.
Technol., 2011, 102, 7023-7028.
57. C. S. Chen, Y. Fujimoto, G. Girdaukas and C. J. Sih, J. Am. Chem.
Soc., 1982, 104, 7294-7299.
58. H. Sawai, H. Sugimoto, Y. Kato, Y. Asano, Y. Shiro and S. Aono, J.
Biol. Chem., 2009, 284, 32089-32096.
59. Z. J. Wang, H. Renata, N. E. Peck, C. C. Farwell, P. S. Coelho and
F. H. Arnold, Angew. Chem. Int. Ed., 2014, 53, 6810-6813.
60. T. K. Hyster and F. H. Arnold, Isr. J. Chem., 2015, 55, 14-20.
27. C. Rodriguez, W. Borzecka, J. H. Sattler, W. Kroutil, I. Lavandera 61. K. Kobayashi, M. Kubo, S. Yoshioka, T. Kitagawa, Y. Kato, Y.
and V. Gotor, Org. Biomol. Chem., 2014, 12, 673-681.
28. S. Kamila, D. Zhu, E. R. Biehl and L. Hua, Org. Lett., 2006, 8,
4429-4431.
Asano and S. Aono, ChemBioChem, 2006, 7, 2004-2009.
29. C. Mukherjee, D. Zhu, E. R. Biehl and L. Hua, Eur. J. Org. Chem.,
2006, 5238-5242.
30. Y. Chen, F. Lie and Z. Li, Adv. Synth. Catal., 2009, 351, 2107-
2112.
31. S.-Y. Chen, C.-X. Yang, J.-P. Wu, G. Xu and L.-R. Yang, Adv. Synth.
Catal, 2013, 355, 3179-3190.
32. R. Metzner, S. Okazaki, Y. Asano and H. Gröger, ChemCatChem,
2014, 6, 3105-3109.
33. Y. Miao, R. Metzner and Y. Asano, ChemBioChem, 2017, 18,
451-454.
34. M. Dadashipour and Y. Asano, ACS Catal., 2011, 1, 1121-1149.
35. T. Betke, P. Rommelmann, K. Oike, Y. Asano and H. Gröger,
Angew. Chem. Int. Ed., 2017, 56, 12361-12366.
36. T. Betke, J. Higuchi, P. Rommelmann, K. Oike, T. Nomura, Y.
Kato, Y. Asano and H. Gröger, ChemBioChem, 2018, 19, 768-
779.
37. C. Plass, A. Hinzmann, M. Terhorst, W. Brauer, K. Oike, H.
Yavuzer, Y. Asano, A. J. Vorholt, T. Betke and H. Gröger, ACS
Catal., 2019, 9, 5198-5203.
38. A. Hinzmann, S. Glinski, M. Worm and H. Gröger, J. Org. Chem.,
2019, 84, 4867-4872.
39. A. N. Alexandrova, D. Röthlisberger, D. Baker and W. L.
Jorgensen, J. Am. Chem. Soc., 2008, 130, 15907-15915.
40. M. L. Casey, D. S. Kemp, K. G. Paul and D. D. Cox, J. Org. Chem.,
1973, 38, 2294-2301.
41. A. Li, B. Wang, A. Ilie, K. D. Dubey, G. Bange, I. V. Korendovych,
S. Shaik and M. T. Reetz, Nat. Commun., 2017, 8, 14876.
42. Y. Kato, K. Nakamura, H. Sakiyama, S. G. Mayhew and Y. Asano,
Biochemistry, 2000, 39, 800-809.
43. Y. Kato and Y. Asano, Protein Expression Purif., 2003, 28, 131-
139.
44. Y. Asano and Y. Kato, FEMS Microbiol. Lett., 1998, 158, 185-190.
45. Y. Kato, S. Yoshida, S.-X. Xie and Y. Asano, J. Biosci. Bioeng.,
2004, 97, 250-259.
46. F. J. F. Castro, M. M. Vila, P. R. Jenkins, M. L. Sharma, G. Tustin,
J. Fawcett and D. R. Russell, Synlett, 1999, 798-800.
47. A. Pohjakallio, P. M. Pihko and U. M. Laitinen, Chem. - Eur. J.,
2010, 16, 11325-11339.
48. R. Huisgen and M. Christl, Chemische Berichte, 1973, 106, 3291-
3311.
49. A. Pohjakallio, P. M. Pihko and J. Liu, J. Org. Chem., 2010, 75,
6712-6715.
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