Control of enantioselectivity in the enzymatic reduction of halogenated acetophenone analogs by substituent positions and sizes

Article abstract of DOI:10.1016/j.tetlet.2020.151820

We utilized acetophenone reductase from Geotrichum candidum NBRC 4597 (GcAPRD), wild type and Trp288Ala mutant, to reduce halogenated acetophenone analogs to their corresponding (S)- and (R)-alcohols beneficial as pharmaceutical intermediates. Reduction by wild type resulted in excellent (S)-enantioselectivity for all of the substrates tested. Meanwhile, reduction by Trp288Ala resulted in high (R)-enantioselectivity for the reduction of 4′ substituted acetophenone and 2′-trifluoromethylacetophenone. In addition to that, we were able to control the enantioselectivity of Trp288Ala by the positions and sizes of the halogen substituents.

Full text of DOI:10.1016/j.tetlet.2020.151820

Tetrahedron Letters xxx (xxxx) xxx
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Control of enantioselectivity in the enzymatic reduction of halogenated
acetophenone analogs by substituent positions and sizes
Afifa Ayu Koesoema a, Daron M. Standley b, Shusuke Ohshima a, Mayumi Tamura a, Tomoko Matsuda a,
⇑
a Department of Life Science and Technology, School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta-cho Midori-ku, Yokohama 226-8501, Japan
b Department of Genome Informatics, Genome Information Research Center, Research Institute of Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We utilized acetophenone reductase from Geotrichum candidum NBRC 4597 (GcAPRD), wild type and
Trp288Ala mutant, to reduce halogenated acetophenone analogs to their corresponding (S)- and
(R)-alcohols beneficial as pharmaceutical intermediates. Reduction by wild type resulted in excellent
(S)-enantioselectivity for all of the substrates tested. Meanwhile, reduction by Trp288Ala resulted in
high (R)-enantioselectivity for the reduction of 40 substituted acetophenone and 20-trifluoromethy-
lacetophenone. In addition to that, we were able to control the enantioselectivity of Trp288Ala by
the positions and sizes of the halogen substituents.
Received 31 January 2020
Revised 5 March 2020
Accepted 9 March 2020
Available online xxxx
Keywords:
Enantioselectivity control
Alcohol dehydrogenase
Asymmetric reduction
Halogenated acetophenone analogs
Ó 2020 Elsevier Ltd. All rights reserved.
Introduction
needed as pharmaceutical intermediates. Thus, rational design
[16–20] was employed on ADH to produce both (S)- and (R)-1-phe-
Enantiomerically pure 1-phenylethanol analogs with halogen or
halogenated group substituents (2b-12b) are versatile pharmaceu-
tical intermediates [1]. For example, (S)-1-(30,40-dichlorophenyl)
ethanol ((S)-8b) is an intermediate of sertraline, an obsessive-com-
pulsive disorder medication [2,3]. Both (S)- and (R)-fluoro substi-
tuted 1-phenylethanol are potential intermediate of various
bioactive compounds [4]. (S)-1-(20-Fluorophenyl)ethanol ((S)-2b)
is an intermediate of JN-403, a nicotinic acetylcholine receptor
nylethanol analogs. However, to the best of our knowledge, sys-
tematic and precise studies investigating the correlation between
the halogen substituent positions and sizes with the enantioselec-
tivity of an ADH have not been conducted.
Our group utilized the acetone powder (dried cell) of Geotri-
chum candidum NBRC 4597 (APG4), containing several (S)-ADHs
and (R)-ADHs to reduce halogenated acetophenone analogs enan-
tioselectively [12]. We have purified and overexpressed one of
the (S)-ADH in APG4, Geotrichum candidum acetophenone reduc-
tase (GcAPRD) [21,22] and characterized it as a robust enzyme with
excellent (S)-enantioselectivity in reducing unsubstituted ace-
tophenone, 4-phenyl-2-butanone, various aliphatic ketones such
as 2-butanone and 3-hexanone [23], as well as various cyclic
ketones such as 2-tetralone and its analogs (5-MeO, 6-OH, 6-
MeO, 6-Cl, and 7-MeO) [24]. Crystal structure of GcAPRD has been
determined [23,25], and several rational mutational studies have
been done to modify the Zn2+ coordination dynamics (Val153)
[26], and to alter the substrate specificity as well as enantioselec-
tivity (Trp288) [27]. Changing Trp288, located in the small binding
pocket of GcAPRD, into smaller amino acid residue had enlarged
the pocket.
a7 agonist with therapeutic potential in a variety of neurological
disorders [5]. (S)-1-(30-Fluorophenyl)ethanol ((S)-3b) is an inter-
mediate of EGFR and HER2 kinase inhibitors, a potential agent
for cancer treatment [6]. (R)-1-(40-Fluorophenyl)ethanol ((S)-4b)
is an intermediate of anti-malaria drug [7], and gamma-secretase
modulator useful for Alzheimer’s disease treatment [8]. Trifluo-
romethyl substituted 1-phenylethanol such as (R)-1-(20-trifluo-
romethylphenyl)ethanol ((R)-12b), being bulkier and more
challenging to be synthesized, is needed as an intermediate of
lysophosphatidic receptors’ agonist for the treatment of pulmonary
fibrosis [4].
Alcohol dehydrogenases (ADHs) [4,9,10] have been employed to
reduce halogenated acetophenone analogs to produce the corre-
sponding (S)-alcohols [11–13]. There are only a few (R)-selective
ADHs existing in nature [14–16], although the (R)-alcohols are also
In this study, we investigated the reduction of halogenated ace-
tophenone analogs by GcAPRD wild type and Trp288Ala in detail
(Fig 1). The wild type reduced all of the halogenated acetophenone
analogs tested to their corresponding (S)-alcohols (ee >99%). Mean-
while, we could control Trp288Ala enantioselectivity based on the
⇑
Corresponding author.
E-mail address: tmatsuda@bio.titech.ac.jp (T. Matsuda).
https://doi.org/10.1016/j.tetlet.2020.151820
0040-4039/Ó 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: A. A. Koesoema, D. M. Standley, S. Ohshima et al., Control of enantioselectivity in the enzymatic reduction of halogenated ace-
tophenone analogs by substituent positions and sizes, Tetrahedron Letters, https://doi.org/10.1016/j.tetlet.2020.151820

2
A.A. Koesoema et al. / Tetrahedron Letters xxx (xxxx) xxx
Fig. 1. Asymmetric reduction of halogenated acetophenone analogs by GcAPRD wild type and Trp288Ala. a(S) of low ee for 1a and 3a. Fluoride, chloride, bromide, and
trifluoromethyl substituents are indicated with yellow, blue, green, and purple background, respectively. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
positions and sizes of halogen substituent in the phenyl ring. Enan-
tioselectivity shifted from (S) to (R), and we obtained the highest
(R)-enantioselectivity (ee up to 99%) for the reduction of 40 substi-
tuted acetophenone and 20-trifluoromethylacetophenone. GcAPRD
wild type and Trp288Ala are useful catalysts to produce beneficial
halogenated 1-phenylethanol analogs in (S)- and (R)-configuration.
negligible. Although substitutions mainly in the 20 and 30 position
(2a-3a, 5a-7a, and 9a-10a) displayed some substrate inhibition,
the reduction still proceeded with satisfactory yield. No effect on
positions and sizes of substituent on yield and enantioselectivity
was also observed in the reduction by ADHs from Saccharomyces
cerevisiae [29] and Haloferax volcanii [30].
For Trp288Ala-catalyzed reduction of 1a-12a, we employed glu-
cose/glucose dehydrogenase (GDH) as the cofactor regeneration
system because the reactivity of Trp288Ala towards 2-propanol
oxidation was poor. Trp288Ala-catalyzed reduction proceeded
with a satisfactory yield (Fig 2b), and the yields did not relate to
the kcat values and may be determined by the stability of
Trp288Ala in the presence of various substrates. The equilibrium
between oxidation–reduction is not related since the GDH shifted
the equilibrium position from glucose towards gluconolactone. It
was reported in the reduction by Lactobacillus brevis ADH that
enzyme stability was one of the factors affecting yield in the asym-
metric reduction of ketones [31,32].
The enantioselectivity of Trp288Ala-catalyzed reduction of sub-
stituted acetophenone analogs is mostly opposite from the wild
type. Only a single mutation changed the enantioselectivity signif-
icantly. For Trp288Ala-catalyzed reduction, the presence of halo-
gen substituents inverted the enantioselectivity from (S) for an
unsubstituted substrate (1a) to (R) for substituted substrates (2a-
12a, except for 3a) (Fig. 2d). Substrates with substitutions in the
20 position (2a, 5a, and 9a) were reduced with a moderate (R)-
enantioselectivity. The enantioselectivity got higher in (R)-configu-
ration when the substituent size was getting larger (ee (R):
F < Cl < Br). Thus, we performed the reduction of 12a with larger
trifluoromethyl group in the 20 position by Trp288Ala with the
hope of obtaining even higher (R)-enantioselectivity. To our
delight, the reduction proceeded with satisfactory yield and 99%
ee (R), completely opposite from the enantioselectivity achieved
by using the wild type. To the best of our knowledge, we were
the first to report the enantioselective reduction of 12a to the cor-
responding (R)-12b by using ADHs. As mentioned earlier, (R)-12b
is an intermediate of pulmonary fibrosis drug [4].
Results and discussions
At first, kinetic studies (Michaelis-Menten constant (Km) and
kcat determination) of GcAPRD wild type and Trp288Ala towards
halogenated acetophenone analogs were conducted (Table S2) to
assess the possibility of their preparative scale asymmetric
reduction. For the wild type, the presence of substitution in
the 20 position (2a, 5a, and 9a) decreased the Km compared with
1a. Kcat (sꢀ1) values for the wild type were comparable with 1a,
or higher for some substrates (8a, 10a, and 11a). We observed
substrate inhibitions for halogenated acetophenone analogs with
substitution in the 20 and 30 position, but not 40 position, except
7a.
Meanwhile, for Trp288Ala, the presence of substitution in the 20
position (2a, 5a, and 9a) also decreased the Km compared with 1a.
Favorably, kcat (sꢀ1) values for halogenated substrates increased
up to 15 times (9a) of that of 1a. Substitution in the 20 position
(2a, 5a, and 9a) had much higher kcat than 1a although it was
the most sterically hindered. Halogen substituents in the 20 posi-
tion could induce polarization of the carbonyl group by the pres-
ence of concomitant orbital overlap between the carbonyl oxygen
and halogen group, as observed in xylose reductase from Candida
tenuis [28]. Substrate inhibition was only observed for Br substitu-
tion in the 20 (9a) and 30 (10a) positions. The kinetic parameters of
the wild type and Trp288Ala were comparable to other ADHs for
halogenated acetophenone analogs reduction [17,20]. With satis-
factory kinetic parameters, we conducted the preparative scale
asymmetric reduction. The results are shown in Fig. 2. For the wild
type-catalyzed reduction of 1a-12a, we employed 2-propanol as
the cofactor regeneration system. The wild type-catalyzed reduc-
tion proceeded with satisfactory yield (Fig 2a) and excellent enan-
tioselectivity in (S)-configuration (Fig 2c) independent of the
positions and sizes of the halogen substituents. The reduction of
sterically bulky 20-trifluoromethylacetophenone (12a) also suc-
ceeded in giving the corresponding (S)-alcohol in 77% yield and
>99% ee.
Substrates with substitutions in the 30 position (3a, 6a, and 10a)
were reduced with (S)- or (R)-enantioselectivity depending on the
size of the substituents. Substrate with F-substitution (3a) was
reduced with (S)-enantioselectivity, Cl-substitution (6a) was
reduced with moderate (R)-enantioselectivity, and Br-substitution
(10a) was reduced with high (R)-enantioselectivity (ee: F (S), Cl
(R) < Br (R)). Substrates with substitutions in the 40 position (4a,
7a, 8a, and 11a) were reduced with the highest (R)-enantioselec-
tivity (92–96% ee (R)). It was clear that we were able to control
the reduction enantioselectivity of Trp288Ala systematically based
on the halogen substituent positions and sizes.
The yield may be affected more by the thermodynamic equilib-
rium position of the 2-propanol cofactor regeneration system
rather than the kinetic parameters. The substrate concentration
used in the reaction (~10 mM) was much higher than Km, and
the reaction time was long enough to make the effect from kcat
Please cite this article as: A. A. Koesoema, D. M. Standley, S. Ohshima et al., Control of enantioselectivity in the enzymatic reduction of halogenated ace-
tophenone analogs by substituent positions and sizes, Tetrahedron Letters, https://doi.org/10.1016/j.tetlet.2020.151820

A.A. Koesoema et al. / Tetrahedron Letters xxx (xxxx) xxx
3
Fig. 2. Asymmetric reduction of halogenated acetophenone analogs (1a-12a) by GcAPRD wild type, and Trp288Ala. Isolated yield of reduction by a wild type, and b
Trp288Ala. ee (%) of reduction by c wild type, and d Trp288Ala. Preparative scale reductions were done under the conditions described in supporting information. aReaction
yield and ee for reduction of 1a by wild type and Trp288Ala was published previously [27]. Positive and negative ee values indicates Prelog (S) and anti-Prelog (R)
enantiopreference, respectively. Fluoride, chloride, bromide, and trifluoromethyl substituents are indicated with yellow, blue, green, and purple background, respectively.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Reduction by other Trp288 mutants can give further insight into
the binding configurations of halogenated acetophenone analogs.
We employed three Trp288 mutants reported in our previous
works [24,27]–Trp288Phe, Trp288Met, and Trp288Val–to reduce
a representative substrate (12a). The ee values for Trp288Phe,
Trp288Met, and Trp288Val were >99% (S), 50% (S), and 81% (R),
respectively. As the size of the engineered pocket was increased,
the enantioselectivity shifted from S to R. These results correlated
with our previous observation that the size of the engineered
pocket affects the reduction enantioselectivity [24,27].
We then tried to determine the cause of the enantioselectivity
inversion of halogenated acetophenone analogs reduction by
Trp288Ala. We hypothesized the presence of interaction between
40-halogen substituent or 20-trifluoromethyl substituents, for
example 40-Cl, with the engineered pocket of Trp288Ala, resulting
in its very high (R)-enantioselectivity (Fig. S1a). Previously, by
docking simulation, we found an interaction between the butyl
group and the loop containing Gly94 in the engineered pocket of
Trp288Ala [27], explaining the >99% ee (S) for 1-phenylpentan-1-
one or butyl phenyl ketone reduction (Fig. S1b). To assess the pres-
ence of interaction between halogen substituent in the 40 position
and engineered pocket, we conducted the reduction of 1-(40-chlor-
ophenyl)-1-pentanone or butyl 40-chlorophenyl ketone (13a)
(Fig. S1c). The decrease of ee from >99% (S) in the reduction of
unsubstituted butyl phenyl ketone to 74% (S) in the reduction of
13a indicated the presence of some binding affinity between 40
Cl substituent and the engineered pocket.
To examine our hypothesis, we performed docking simulations
of the three representative substrates with high (R)-enantioselec-
tivity 4a, 7a, and 12a using the wild type crystal structure
[23,25] and the Trp288Ala model structure [24] (Fig 3) with the
addition of side chain flexibility as mentioned in supporting infor-
mation. Because the Trp288Ala mutant did not readily crystalize,
we modelled the Trp288Ala mutation on the wild type crystal
structure, as described in supporting information. The binding
energy values and details for the wild type and Trp288Ala can be
seen in Table S3 and Table S4, respectively. Consistent with the
experimental result, productive pro-(S) binding poses were found
for the docking of 4a (Fig. 3a), 7a (Fig. 3c), and 12a (Fig. 3e) in
the active site of the wild type. In these binding poses, the halogen
substituents of 4a, 7a, and 12a may interact through van der Waals
interactions with Leu264, while their methyl group interacted with
either Leu122 or Trp288 in accordance with the previously
reported interaction of methyl group in aliphatic ketones with
Trp288 [23].
We found a productive pro-R binding pose for the docking of
4a (Fig. 3b), 7a (Fig. 3d), and 12a (Fig. 3f) in the active site of the
Trp288Ala. The fluorine substituent of 4a and chlorine
substituent of 7a interacted through a C-Xꢁ ꢁ ꢁhydrogen bond
donor (C-Xꢁ ꢁ ꢁHBD interaction) with the NH2 group in the side
chain of Asn119 in the engineered pocket, while the methyl
substituent interacted with Leu264 in the large pocket. The
distances between substrates’ halogen substituents and Cd of
Asn119 (3.3
Å and 3.5 Å for 4a and 7a, respectively), as
Please cite this article as: A. A. Koesoema, D. M. Standley, S. Ohshima et al., Control of enantioselectivity in the enzymatic reduction of halogenated ace-
tophenone analogs by substituent positions and sizes, Tetrahedron Letters, https://doi.org/10.1016/j.tetlet.2020.151820

4
A.A. Koesoema et al. / Tetrahedron Letters xxx (xxxx) xxx
Fig. 3. Binding poses of a 4a in the active site of GcAPRD wild type crystal structure, b 4a in the active site of GcAPRD Trp288Ala model structure, c 7a in the active site of
GcAPRD wild type crystal structure, d 7a in the active site of GcAPRD Trp288Ala model structure, e 12a in the active site of GcAPRD wild type crystal structure, and f 12a in the
active site of GcAPRD Trp288Ala model structure. The CPK coloring was used except for the backbone of the receptor. NADH are shown in purple stick. Catalytic zinc is shown
in grey sphere, and the C4 of NADH with hydride to be transferred to the ketone is highlighted with white circle. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
well as C-Xꢁ ꢁ ꢁHBD angle (133° and 110° for 4a and 7a, respec-
Conclusions
tively), were among the average of other reported crystal struc-
tures in PDB containing halogenated ligands with C-Xꢁ ꢁ ꢁHBD
By the utilization of GcAPRD wild type and Trp288Ala, we could
interaction [33]. For 12a, the pro-R binding pose was stabilized
through a van der Waals interaction of the trifluoromethyl sub-
stituent with Asn119 and Leu122 in the engineered pocket of
Trp288Ala.
enantioselectively reduce the halogenated acetophenone analogs
to their corresponding (S)- and (R)-alcohols, pharmaceutically
important intermediates. Importantly, sterically bulky (S) and
(R)-12b were synthesized in >99% ee (S) and 99% (R) by the wild
type and Trp288Ala, respectively. We were able to clearly and sys-
tematically control the reduction enantioselectivity based on the
positions and sizes of halogen substituents in the phenyl ring for
the reduction by Trp288Ala.
In addition, we performed docking simulations of 20 (2a and
5a) and 30 (3a and 6a) substituted acetophenones with low
(R)-enantioselectivity (except 3a) in the model structure of
Trp288Ala, with the addition of side chain flexibility to Leu122
or Phe287, following the same protocol used for 40-substituted
acetophenones (Fig. S2 and Table S5, detailed discussions can
be found in supporting information). The crucial C-Xꢁ ꢁ ꢁHBD
interaction with Asn119, found in 40 substituted acetophenone
(Fig. 3b and d), could not be formed in the docking poses of 20
and 30 substituted acetophenone, which may result in their
low (R)-enantioselectivities. Altogether the docking simulations
have supported the importance of interactions with the halogen
substituents or trifluoromethyl group with the amino acids in
the engineered pocket for the high (R)-enantioselectivity
observed in the reduction of 4a, 7a, and 12a.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgments
The authors acknowledged Professor Miki Senda and Professor
Toshiya Senda from High Energy Accelerator Research Organiza-
Please cite this article as: A. A. Koesoema, D. M. Standley, S. Ohshima et al., Control of enantioselectivity in the enzymatic reduction of halogenated ace-
tophenone analogs by substituent positions and sizes, Tetrahedron Letters, https://doi.org/10.1016/j.tetlet.2020.151820

A.A. Koesoema et al. / Tetrahedron Letters xxx (xxxx) xxx
5
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Please cite this article as: A. A. Koesoema, D. M. Standley, S. Ohshima et al., Control of enantioselectivity in the enzymatic reduction of halogenated ace-
tophenone analogs by substituent positions and sizes, Tetrahedron Letters, https://doi.org/10.1016/j.tetlet.2020.151820