Halohydrin dehalogenase-catalysed synthesis of fluorinated aromatic chiral building blocks

Article abstract of DOI:10.1016/j.catcom.2021.106285

Kinetic resolution of a series of fluorinated styrene oxide derivatives was studied using halohydrin dehalogenase. A mutant HheC-W249P catalysed nucleophilic ring-opening with azide and cyanide ions with excellent enantioselectivity (E-values up to >200), which gives access to various enantiopure β-substituted alcohols and epoxides. It was found that the enzyme tolerates substrates in concentrations over 50 mM. However, different side reactions were observed at elevated concentrations and with prolonged reaction time. The biocatalytic azidolysis and cyanolysis of racemic 4-trifluoromethylstyrene oxide were performed on preparative scale, affording (R)-2-azido-1-(4-trifluoromethyl-phenyl)-ethanol in 38% yield and 97% ee, and (S)-3-hydroxy-3-(4-trifluoromethyl-phenyl)-propionitrile in 30% yield and 98% ee.

Full text of DOI:10.1016/j.catcom.2021.106285

Catalysis Communications 152 (2021) 106285
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Catalysis Communications
journal homepage: www.elsevier.com/locate/catcom
Short communication
Halohydrin dehalogenase-catalysed synthesis of fluorinated aromatic chiral
building blocks
a
d
a
c
b
ˇ ´b
´
Irena Dokli , Nevena Milcic , Petra Marin , Marina Svetec Miklenic , Martina Sudar ,
b
a,*
ˇ
´
´
Lixia Tang , Zvjezdana Findrik Blazevic , Maja Majeric Elenkov
a
ˇ
´
ˇ
Division of Organic Chemistry and Biochemistry Ruđer Boskovic Institute, Bijenicka c. 54, 10000 Zagreb, Croatia
b Faculty of Chemical Engineering and Technology, University of Zagreb, Savska c. 16, 10000 Zagreb, Croatia
c Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10000 Zagreb, Croatia
d University of Electronic Science and Technology, No.4, Section 2, North Jianshe Road, Chengdu, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Kinetic resolution of a series of fluorinated styrene oxide derivatives was studied using halohydrin dehalogenase.
A mutant HheC-W249P catalysed nucleophilic ring-opening with azide and cyanide ions with excellent enan-
tioselectivity (E-values up to >200), which gives access to various enantiopure β-substituted alcohols and ep-
oxides. It was found that the enzyme tolerates substrates in concentrations over 50 mM. However, different side
reactions were observed at elevated concentrations and with prolonged reaction time. The biocatalytic azidolysis
and cyanolysis of racemic 4-trifluoromethylstyrene oxide were performed on preparative scale, affording (R)-2-
azido-1-(4-trifluoromethyl-phenyl)-ethanol in 38% yield and 97% ee, and (S)-3-hydroxy-3-(4-trifluoromethyl-
phenyl)-propionitrile in 30% yield and 98% ee.
Biocatalysis
Epoxides
Halohydrin dehalogenase
Kinetic resolution
β-Substituted alcohols
1. Introduction
has been reported in combination with azide [10–14], nitrite [15], cy-
anide [16] and cyanate ions [17,18] as nucleophiles. Since the fluo-
Enantiomerically pure epoxides and their ring-opening products are
frequently used building blocks for the synthesis of pharmaceuticals.
Different resolution methods have been described including application
of organometallic catalysts [1] and biocatalysts [2]. Among them, bio-
catalytic transformations employing halohydrin dehalogenase (HHDH)
are very promising due to its capability to catalyse ring-opening re-
actions with several nucleophiles [3,4]. The usefulness of HHDHs in
organic synthesis has been demonstrated by various preparative scale
experiments [5,6] and an industrial application [7]. HHDHs are
cofactor-independent enzymes and can be expressed in recombinant
form in E. coli up to 60% of the total cellular protein content, allowing
the isolation of up to 160 mg of pure enzyme from 1 L culture [8]. Easy
preparation, stability and high selectivity of already discovered enzymes
make HHDHs attractive catalysts for laboratory scale synthesis. How-
ever, to establish the role as an industrial biocatalyst, protein engi-
neering is necessary in order to increase catalytic performances, expand
their substrate range and provide access to both enantiomers of desired
products [9].
roaryl moiety is an attractive pharmacophore [19,20], we were
interested in exploring the possibility of employing HHDH in the reso-
lution of fluorine-substituted styrene oxides with the aim of synthesising
the corresponding β-azido alcohol and β-hydroxy nitriles in enantiopure
form. The growing interest in fluorinated organics makes the develop-
ment of synthetic procedures leading to such compounds highly desir-
able. This task is not simple due to the difficulties of the incorporation of
the fluorine atom into organic molecules. One of the approaches to the
synthesis of fluorinated organics implies development of fluorination
methods using various fluorinating agents [21]. The main drawback of
this approach is often a high price and/or toxicity of the fluorinating
agent. Another approach consists of synthetic modifications of simple
commercially available fluorine containing compounds and a design of
new intermediates [22]. Such building blocks must possess both C-F
moiety and a functional group, which can be used for the incorporation
of C-F fragment into a target molecule. In addition, such compounds can
be modified in enantioselective fashion leading to optically active in-
termediates, for example alcohols, amines, amino alcohols and epoxides.
In this study, we report enantioselective ring-opening reaction of
Catalytic activity of HHDHs toward several styrene oxide derivatives
* Corresponding author.
E-mail address: majeric@irb.hr (M.M. Elenkov).
https://doi.org/10.1016/j.catcom.2021.106285
Received 26 October 2020; Received in revised form 17 January 2021; Accepted 18 January 2021
Available online 22 January 2021
1566-7367/© 2021 The Author(s).
Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

I. Dokli et al.
Catalysis Communications 152 (2021) 106285
epoxides 1a-1e (Scheme 1) in the presence of azide and cyanide ions
CHCl3). The NMR data were identical with synthesized racemic refer-
ence compounds.
catalysed by variant HheC-W249P [23].
2. Experimental
2.3. Preparative-scale cyanolysis
2.1. Kinetic resolution experiments – general procedure
To a solution of epoxide 1b (100 mg, 0.532 mmol, 20 mM final
concentration) in the mixture of DMSO (1.3 mL) and Tris-SO4 buffer
(14.7 mL, 0.5 M, pH 7.0), NaCN was added (26 mg, 0.532 mmol) fol-
lowed by addition of HheC-W249P (ca 40 mg in 10.6 mL buffer). The
reaction was carried out at room temperature on a magnetic stirrer at
1000 rpm. After 15 h of incubation the reaction mixture was extracted
with ethyl acetate (3 × 25 mL). The combined organic extracts were
dried with Na2SO4 and solvent removed by rotary evaporation. Column
chromatography of the residue (hexane/ethyl acetate 7:3) yielded pure
(S)-4b and (S)-1b.
To 2.0 mL of Tris-SO4 buffer (50 mM, pH 7.5) at room temperature,
125
μ
L of a stock solution of substrate in DMSO was added (5
μ
mol, final
L,
μL of cell-free
concentration 2 mM) followed by the addition of NaN3 or NaCN (125
7.5 mol). Reactions were initiated by the addition of 250
μ
μ
extract of enzyme in TEMG buffer (10 mM Tris-SO4, 1 mM EDTA, 1 mM
β-mercaptoethanol, 10% glycerol, pH 7.5). Reaction progress was fol-
lowed by periodically taking samples (0.5 mL) from the reaction
mixture. Samples were extracted with MTBE (1.0 mL) containing
mesitylene as internal standard, dried over anhydrous Na2SO4, and
analyzed by GC on HP-5 column to determine the conversion and
regioisomeric ratio. In parallel, control reactions were performed by
following the substrate consumption and product formation in the
absence of enzyme. The enantiomeric excess (ee) of the remaining ep-
oxides and products was determined by chiral GC and HPLC, respec-
tively, under conditions described in Supplementary data.
(S)-3-Hydroxy-3-(4-trifluoromethyl-phenyl)-propionitrile ((S)-4b)
23
was obtained in 30% yield (35 mg), 98% ee, [
α
]
D -45.3 (c = 0.54 in
CHCl3). The remaining (S)-2-(4-(trifluoromethoxy)phenyl)-oxirane ((S)-
23
1b) was obtained in 33% yield (33 mg), 61% ee, [
α]
D + 11.2 (c = 1.11
in CHCl3). The NMR data were identical with synthesized racemic
reference compounds.
3. Results and discussion
2.2. Preparative-scale azidolysis
3.1. Kinetic resolution of 1b catalysed by HheC and HheC-W249P mutant
To a solution of epoxide 1b (250 mg, 1.33 mmol, 50 mM final con-
centration) in the mixture of DMSO (1.3 mL) and Tris-SO4 buffer (20 mL,
0.5 M, pH 7.0), NaN3 was added (86 mg, 1.33 mmol) followed by
addition of HheC-W249P (ca 20 mg in 5.3 mL buffer). The reaction was
carried out at room temperature on a magnetic stirrer at 1000 rpm. After
7 h of incubation the reaction mixture was extracted with ethyl acetate
(3 × 25 mL). The combined organic extracts were dried with Na2SO4 and
solvent was removed by rotary evaporation. Column chromatography
(hexane/ethyl acetate 9:1) yielded pure (R)-2b and (S)-1b.
Among the wild-type HHDHs, the most commonly applied in bio-
catalysis is the enzyme from Agrobacterium radiobacter AD1 (HheC), due
to its remarkable stereoselectivity and broad nucleophile scope. HheC
was shown to catalyse azidolysis of styrene oxide, p-Cl- and p-NO2-sty-
rene oxide with excellent enantioselectivity (E > 200) [10]. Since in this
study we chose to focus on styrene oxide derivatives with fluorine
bearing groups, HheC was considered as a catalyst of choice.
Initially, the wild-type (WT) enzyme and the mutant W249P were
tested in the ring-opening reaction of 1b in the presence of azide and
cyanide ions (Table 1). Both reactions catalysed by HheC proceeded
with very high enantioselectivity, however azidolysis reaction was faster
compared to cyanolysis, as observed in previous studies [4,24] (Table 1,
(R)-2-Azido-1-(4-trifluoromethyl-phenyl)-ethanol ((R)-2b) was ob-
23
tained in 35% yield (117 mg), 97% ee, [
α
]
D -70.3 (c = 1.18 in CHCl3).
The remaining (S)-2-(4-(trifluoromethoxy)phe2n3yl)oxirane ((S)-1b) was
obtained in 35% yield (88 mg), 87% ee, [
α
]
+ 15.7 (c = 1.46 in
D
O
O
O
O
O
F
F3C
F3CO
F2HCO
F3CS
1a
1b
1c
1d
1e
Scheme 1. Racemic epoxides 1a-1e used as substrates.
Table 1
Ring-opening of rac-1b in the presence of sodium azide or sodium cyanide catalysed by HHDH.a
Entry
HHDH
NaNu
t (h)
Conversion (%)b
ees (%)c
eep (%)d
E-value
1
2
3
4
HheC
NaN3
NaCN
NaN3
NaCN
2
3
2
3
45 (39)
34 (22)
53 (50)
43 (31)
63 (S)
27 (S)
>99 (S)
45 (S)
>99 (R)
98 (S)
>200
>100
>200
>200
HheC
HheC-W249P
HheC-W249P
>99 (R)
>99 (S)
a
Conditions: 1b (2 mM), NaN3 or NaCN (3 mM), 250 μL cell-free extract HHDH, Tris-SO4 buffer (4 mL, 0.5 M, pH 7.0), 5% DMSO, total volume 5 mL.
b
c
d
Apparent conversion determined by GC (value in parentheses is conversion catalysed by enzyme = intrinsic).
Determined by GC.
Determined by HPLC.
2

I. Dokli et al.
Catalysis Communications 152 (2021) 106285
54–68% yield after purification [26], except commercially available 1a.
HheC-W249P-catalysed transformations of epoxides 1a–1e (2 mM) with
1.5 equivalent of NaN3 were carried out in Tris-SO4 buffer containing
5% DMSO for the solubility improvement. Reactions were performed
with cell-free extract at room temperature and monitored by GC anal-
ysis. Conversions determined by GC were always found higher
compared to calculated conversions (given in parentheses; calculated
according to the formula c = ees / (ees + eep)). The difference observed in
the enzyme-catalysed conversion of substrate (intrinsic) and conversion
measured by GC (apparent) is mainly the result of hydrolytic instability
of the epoxide. Spontaneous hydrolysis of substrates becomes significant
when the rate of biocatalytic reaction is low. Spontaneous azidolysis
leading to rac-2 was found to be negligible under the conditions used,
reflecting in very high product ee (97% ee for 2a and > 99% ee for 2b-2e,
60
50
40
30
20
10
0
HheC-W249P
HheC-WT
Table 2). To minor extent, spontaneous chemical reaction occurs at C
α
leading to rac-3, which was observed in control reaction performed
without enzyme. However, enzyme-catalysed azidolysis is β-regiose-
lective toward tested substrates (96–100% of β-attack). All kinetic res-
olutions occurred with high enantioselectivity toward the (R)-
enantiomer, yielding almost enantiopure products while leaving the (S)-
enantiomer of the epoxide behind. E-value was calculated from the
enantiomeric purity of substrate and product, as well as the percentage
of enzymatic conversion (intrinsic conversion) as already mentioned,
while absolute configurations were determined by chiral GC analysis
using the chemically prepared reference compounds of known config-
uration; for more information see Supplementary data.
0
20
40
60
80
100
120
t (min)
Fig. 1. Progress curves of biocatalytic azidolysis of 1b catalysed by WT and
mutant HheC.
Entries 1 and 2).
Residue Trp249 in HheC is known for its importance regarding the
catalytic activity and enantioselectivity. This residue is positioned in the
loop that forms the halide-binding site; therefore, its replacement
destabilises the site, leading to faster halide release and a higher reaction
rate [23,25]. The HheC-W249P mutant showed significantly higher
activity in the kinetic resolution of 1b (Table 1, Entries 3 and 4). While
both enzymes catalyse the formation of azido alcohol (R)-2b with >99%
ee, activity of W249P is considerably higher. 40% Conversion of rac-1b
was achieved already after 15 min, while 50% conversion after 1 h,
resulting in complete kinetic resolution of substrate (Fig. 1). Thus, (R)-
enantiomer was transformed to azido alcohol ((R)-2b, >99% ee), while
(S)-enantiomer remained as unreacted epoxide ((S)-1b, >99% ee). The
stereoselectivity of the cyanolysis reaction is unchanged compared to
azidolysis; however, inversion of the absolute configuration in the cor-
responding β-hydroxy nitrile 4b appears due to a different substituent
priority according to the Cahn–Ingold-Prelog convention. Due to supe-
rior catalytic properties, W249P was selected for further biocatalytic
studies.
This method is not applicable to ortho-substituted styrene oxides due
to very low enzyme activity, while meta-substituted derivatives could be
converted to products with high enantioselectivity (e.g. E = 128 in the
case of m-F and E > 200 with m-CF3-styrene oxide; unpublished data).
3.3. Kinetic resolution of epoxides 1a-1e in the presence of NaCN
catalysed by HheC-W249P
To further explore the potential of HheC-W249P for producing
β-hydroxy nitriles, we investigated the reaction of epoxides 1a-1e with
cyanide ion as a nucleophile. As illustrated in Table 3, HheC-W249P
showed activity with entire series of epoxides generating β-hydroxy
nitriles in completely regioselective fashion and with high enantiose-
lectivity (E > 100). However, the reaction rate strongly depends on the
nucleophile, and lower rate was observed with cyanide compared to
azide ion. Control experiments, without enzyme, revealed negligible
spontaneous cyanolysis reaction leading to rac-4, but again significant
spontaneous substrate hydrolysis.
3.2. Kinetic resolution of epoxides 1a-1e in the presence of NaN3
catalysed by HheC-W249P
Substrates used in this study were prepared from the corresponding
substituted benzaldehydes by using trimethylsulfoxonium iodide in
Table 2
Conversion of epoxides 1a-1e with sodium azide catalysed by HheC-W249P.a
Entry
Epoxide
R
Conversion (%)b
ee1 (%)c
ee2 (%)d
β-regioselectivitye
E-value
1
2
3
4
5
1a
1b
1c
1d
1e
F
55 (37)
53 (50)
60 (50)
52 (50)
53 (49)
56 (S)
97 (R)
82 (100)
97 (99)
93 (97)
81 (96)
97 (100)
>100
>200
>200
>200
>200
CF3
>99 (S)
>99 (S)
>99 (S)
96 (S)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
OCF3
OCHF2
SCF3
a
Conditions:1a-1e (2 mM), NaN3 (3 mM), 250 μL HheC-W249P, Tris-SO4 buffer (4 mL, 0.5 M, pH 7.0), 5% DMSO, total volume 5 mL, reaction time 2 h.
b
c
Apparent conversion determined by GC (value in parentheses is conversion catalysed by enzyme = intrinsic).
Determined by GC.
d
e
Determined by HPLC.
Apparent regioselectivity determined by GC (intrinsic value is in parentheses).
3

I. Dokli et al.
Catalysis Communications 152 (2021) 106285
Table 3
Conversion of epoxides 1a-1e with sodium cyanide catalysed by HheC-W249P.a
Entry
Epoxide
R
Conversion (%)b
ee1 (%)c
ee4 (%)d
β-regioselectivitye
E-value
1
2
3
4
5
1a
1b
1c
1d
1e
F
54 (47)
37 (27)
43 (33)
43 (36)
22 (19)
86 (S)
37 (S)
48 (S)
56 (S)
23 (S)
97 (S)
100
100
100
100
100
>100
>200
>100
>200
>100
CF3
>99 (S)
97 (S)
OCF3
OCHF2
SCF3
>99 (S)
98 (S)
a
b
c
Conditions: 1a-1e (2 mM), NaN3 (3 mM), 250 μL HheC-W249P, Tris-SO4 buffer (4 mL, 0.5 M, pH 7.0), 5% DMSO, total volume 5 mL, reaction time 2 h.
Apparent conversion determined by GC (value in parentheses is conversion catalysed by enzyme = intrinsic).
Determined by GC.
d
e
Determined by HPLC.
Apparent regioselectivity determined by GC.
3.4. Preparative scale reaction
sensitive to spontaneous hydrolysis; moreover, the spontaneous reaction
between the epoxide and nucleophile occurred. In order to minimize the
rate of chemical side-reactions and perform preparative scale trans-
formations, higher concentrations of enzyme and lower concentrations
of substrates were required. Azidolysis of epoxide 1b at 50 mM substrate
concentration gave the corresponding azido alcohol (R)-2b in 38% yield
and 97% ee and remaining epoxide (S)-1b in 30% yield and 87% ee,
respectively. Due to lower enzymatic activity in cyanolysis reaction,
very high catalyst loadings were required to obtain (S)-4b in 30% yield
and 98% ee. Nevertheless, preparative scale transformations require
further improvement in order to minimize the effect of chemical side-
reactions and increase productivity. This can be facilitated by a
detailed kinetic analysis of all reactions involved in the system. Thus,
further optimization will be done by employing enzyme reaction
engineering.
Tris-SO4 buffer containing 5% DMSO allows substrates to be dis-
solved in 2 mM concentration, which was considered too low for pre-
parative transformation. Therefore, by increasing substrate
concentration and decreasing the biocatalyst loading, we tried to
enhance the reaction and perform it on a preparative scale. It was found
that enzyme tolerates substrate 1b in concentration over 50 mM.
However, at elevated concentrations and with the prolonged reaction
time, optical purity and product yields are affected by chemical side
reactions (see Table S2 in the Supplementary data). Particularly,
chemical azidolysis that takes place at Cβ reduces the product ee, while
reaction at C
α increases the amount of α-regioisomer (3b). Besides,
substrate spontaneously hydrolyses giving the corresponding diol,
which becomes significant under these conditions over a period of
several hours.
Thus, preparative-scale experiments using 250 mg of rac-1b were
performed with 50 mM substrate concentration (9.4 g/L) and 1 equiv-
alent of NaN3, by using cell-free extract containing approximately 20 mg
(8 wt%) of HheC-W249P. To avoid the reduction of ee, the reaction was
conducted for 7 h and terminated at ca 45% conversion, allowing
isolation of (R)-2b in 38% yield and 97% ee and (S)-1b in 35% yield and
87% ee. Enzymatic reaction was very fast in the beginning but slowed
down significantly once conversion of 40% was reached (see Fig. S1 in
the Supplementary data). In order to achieve complete conversion of
(R)-1b and to obtain (S)-1b with >99% ee, the reaction time should be
prolonged.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
This work was financially supported by the Croatian Science Foun-
dation (HrZZ, IP-2018-01-4493).
Similarly, a reaction with NaCN was performed. However, a greater
amount of enzyme (40 wt%), lower substrate concentration (20 mM)
and a longer reaction time (15 h) were required to achieve comparable
conversion and to obtain (S)-4b in 30% yield and 98% ee. With a pro-
longed reaction time, a slight reduction of product ee was observed due
to chemical reaction of epoxide with NaCN, while product resulting
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.catcom.2021.106285.
from the Cα-attack was not detected. Practical limitations to scale-up
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