Molecular Catalysis 541 (2023) 113099
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Molecular Catalysis
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Review
Biocatalytic asymmetric reduction of prochiral bulky-bulky ketones
Auwal Eshi Sardauna a, 1, Muhammad Abdulrasheed a, 1, Alexis Nzila b, c, Musa M. Musa a, d, *
a
Department of Chemistry, King Fahd University of Petroleum and Minerals, Dhahran 31261, Saudi Arabia
Department of Bioengineering, King Fahd University of Petroleum and Minerals, Dhahran 31261, Saudi Arabia
c
Interdisciplinary Research Center for Membranes and Water Security, King Fahd University of Petroleum and Minerals, Dhahran 31261, Saudi Arabia
d
Interdisciplinary Research Center for Refining and Advanced Chemicals, King Fahd University of Petroleum and Minerals, Dhahran 31261, Saudi Arabia
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Alcohol dehydrogenases
Asymmetric reduction
Biocatalysis
Enantiopure alcohols
Bulky-bulky ketones
Biocatalytic asymmetric reduction of prochiral bulky-bulky ketones is an attractive approach to produce their
corresponding valuable enantiopure alcohols. These ketones contain alkyl or aryl groups that exhibit subtle
differences in size, and thus their asymmetric reduction is challenging. Bulky-bulky ketones include acyclic
ketones that comprise an aryl ring in each side of the ketone or aryl and another relatively bulky group such as
cyclohexyl or long chains; they also include prochiral cyclic ketones that comprise two moieties that are similar
in their sizes such as tetralones and tetrahydrofuran-3-one. This review summarizes recent examples of bio­
catalytic asymmetric reduction of bulky-bulky ketones, a transformation that is not easily accomplished not only
by enzymes, but also by organo- and organometallic catalysis. Moreover, it has identified gaps that limits the
efficiency of the biocatalytic asymmetric reduction of bulky-bulky ketones, and has proposed various strategies
to improve this efficiency.
1. Introduction
Catalytic asymmetric reduction of ketones is a straightforward
approach to obtaining optically active secondary alcohols [1–4].
Asymmetric reduction of bulky-bulky ketones, which comprise two
groups that exhibit similar sizes, is among the challenging tasks because
of the fact that the catalyst has to discern between two groups that
slightly vary in their sizes. Although examples of asymmetric transfer
hydrogenation of these substrates using transition metals have been
reported [5,6], biocatalytic asymmetric reduction using ketoreductases
(KREDs)/alcohol dehydrogenases (ADHs) remains an attractive
approach [7–9], apparently because biocatalytic reactions are con­
ducted under mild reaction conditions, which minimizes the formation
of by-products [10–12]. Moreover, transition metal-based catalysts are
expensive and tedious to prepare. Although enzymes seem to have po­
tentials as green catalysts, however, the environmental aspects of the
use of enzymes in chemical transformations have to be assessed based on
the reaction burden on the environment (i.e., the E factor) [13–15].
Prochiral bulky-bulky ketones include substrates that contain two
bulky substituents such as two aryl groups or aryl and a large alkyl group
and the prochiral carbonyl is not within a cycle, referred to as acyclic
bulky-bulky ketones throughout this article. They also include small
cyclic ketones, which contain two substituents that are almost spatially
symmetrical such as tetralones and tetrahydrofuran-3-one. Optically
active alcohols produced via asymmetric reduction of these bulky-bulky
ketones are important building blocks in pharmaceutically relevant
compounds (Fig. 1).
Alcohol dehydrogenases (EC 1.1.1.X, ADHs with X = 1, 2, or 252 will
be discussed in this review) are an important class of enzymes that
catalyze the interconversion of ketones and their corresponding opti­
cally active secondary alcohols [27–31]. They require either
nicotinamide-adenine dinucleotide (NAD+) or its phosphate (NADP+) as
a cofactor. ADHs in general have a high substrate specificity (i.e., limited
substrate scope). The progression in the biotechnology field using
site-directed mutagenesis and directed evolution along with molecular
dynamics simulations provided engineered mutants of ADHs that helped
in widening their substrate specificity to accept substrates that are not
accepted by wild-type ADHs. This review summarizes recent examples
of biocatalytic asymmetric reduction of prochiral bulky-bulky ketones
using whole cells or purified enzymes.
* Corresponding author.
E-mail address: musam@kfupm.edu.sa (M.M. Musa).
1
These Authors contributed equally
https://doi.org/10.1016/j.mcat.2023.113099
Received 30 December 2022; Received in revised form 26 February 2023; Accepted 18 March 2023
Available online 24 March 2023
2468-8231/© 2023 Elsevier B.V. All rights reserved.
A.E. Sardauna et al.
Molecular Catalysis 541 (2023) 113099
2. Discussion
2.1. Stereopreference of alcohol dehydrogenases
There are four possibilities to deliver the hydride of NAD(P)H to a
prochiral ketone in an ADH-catalyzed asymmetric reduction. Prelog
used a diamond lattice model to predict stereospecificity of ADHs [32].
According to Prelog’s rule, the majority of the known ADHs exhibit
Prelog stereopreference in which the cofactor delivers its hydride from
the re face of prochiral ketones, producing their corresponding
(S)-configured alcohols (Fig. 2). It should be noticed that, in some in­
cidents, the small substituent of the ketone exhibits higher
Cahn-Ingold-Prelog priority than that of the large substituent, which
leads to the formation of (R)-alcohol as the Prelog product. Keinan and
coworkers screened asymmetric reduction of various aliphatic acyclic
ketones using Thermoanaerobium brockii secondary ADH (TbSADH) and
they proposed a model for the active site. Their model suggests that
TbSADH exhibits two hydrophobic binding pockets that vary in their
sizes and in their affinities towards the alkyl groups with the smaller
pocket exhibiting higher binding affinity than that of the larger pocket
[33]. Heiss and Phillips observed the same trend in the asymmetric
reduction of ethynyl ketones using Thermoanaerobacter pseudoethanoli­
cus secondary ADH (TeSADH) [34].
The stereochemical outcome of asymmetric reduction of bulky-small
prochiral ketones can be generally predicted by the above-mentioned
model; however, that of the bulky-bulky prochiral ketones is chal­
lenging to predict. Moreover, because most ADHs exhibit two pockets
that vary in their sizes, reports of asymmetric reduction of bulky-bulky
Fig. 2. Stereochemistry in ADH-catalyzed asymmetric reduction of prochiral
ketones. RL is more sterically hindered than RS. ADR: adenine diribose. The
indicated (S)- and (R)-alcohols are when RL exhibits higher Cahn-Ingold-Prelog
priority than RS.
ketones by wild-type ADHs are seldom.
2.2. Biocatalytic asymmetric reduction of acyclic bulky-bulky ketones
2.2.1. Asymmetric reduction of acyclic bulky-bulky ketones using whole
cells
Spassov and coworkers tested ten strains of fungi (including yeast)
and bacteria, in the asymmetric reduction of substituted benzophenones
[35]. These strains are Corynespora cassiicola DSM 62,475, Debaryomyces
marama DSM 70,250, Absidia blakesleeana AlCC 70148A, Syncephalas­
trum racemosum ATCC 18,192, Rhizopus fusiformis CBS 26,630, Rhizopus
arrhizus ATCC 11,145, Enterobacter liquefaciens DSM 30,063, Corynes­
pora melonis CBS 12,925, Rhizopus tritici CBS 12,808 and Debaryomyces
phaffii IF0 1362. They showed that strains Debaryomyces marama DSM
Fig. 1. Structures of pharmaceutically important compounds containing optically active alcohols that are produced via asymmetric reduction of bulky-bulky ketones.
Bepotastine [16], Cloperastine [17,18], Montelukast [19], Carbinoxamine [20,21], Amprenavir [22], Fosamprenavir [22], MK-0449 [23], Cladosporol [24],
Chrysanthone [25], 3,6,9-Trihydroxy-3,4-dihydroanthracen-1(2H)-one [26].
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Molecular Catalysis 541 (2023) 113099
70,250, Corynespora cassiicola DSM 62,475, Rhizopus arrhizus ATCC 11,
145, Absidia blakesleana ATCC 70148A and Syncephalastrum racemosum
ATCC 18,192 reduced the para-substituted diaryl ketones. For instance,
4-chlorobenzophenone was reduced using Debaryomyces marama to the
corresponding (S)-alcohol using DMF as a cosolvent with an excellent
conversion and enantioselectivity (Scheme 1).
Chartrain and coworkers screened 129 microorganisms for their
abilities to produce optically active cyclohexyl(phenyl)methanol from
its prochiral ketone [36]. Only a single yeast strain, Candida magnolia
MY 1785, led to the formation of (R)-cyclohexyl(phenyl)methanol in
low yield and good enantioselectivity (Scheme 2). Two decades later,
Sahin and coworkers screened ten bacterial strains for their capabilities
to asymmetrically reduce cyclohexyl(phenyl)methanone [37]. They
identified Lactobacillus paracasei BD101 to be the most potent strain,
which produced the (S)-alcohol in excellent yield and enantioselectivity
(Scheme 2).
Chartrain and coworkers screened 310 microorganisms in the
asymmetric reduction of 3-cyclopentoxy-4-methoxybenzophenone,
eight of which successfully catalyzed the desired reaction to produce
either (R)- or (S)-corresponding alcohols [38]. Six of the strains, Asper­
gillus nidulans MF 121, Aspergillus nidulans MF 145, Bullera tsugae MF
1669, Hansenula holstii MY 1538, Mucor sulfa MF 37, and Rhodotorula
pilimanae ATCC 32,762, showed good to excellent enantioselectivities
(86 to >99% ee). The same research group reported the production of
(S)-diaryl alcohol by Rhodotorula pilimanae in preparative scale with an
excellent enantioselectivity, yet low yield (Scheme 3). Although all the
six strains showed good enantioselectivity, they all reduced the ketone
with very low yields (3–30%).
Cui, Qian and their coworkers reported an asymmetric reduction of
fluorenones using baker’s yeast displaying excellent enantioselectivity
(Scheme 4) [39]. To improve the solubility of these substrates, they
employed 10% (v/v) of water-miscible cosolvents. Except for acetoni­
trile, other cosolvents such as ethanol, THF, 1,4-dioxane, 1,2-dime­
thyoxyethane,
DMF,
and
DMSO
resulted
in
improved
enantioselectivities. While the reduction of 2-chlorofluorenone, 2-bro­
mofluorenone and 2-iodofluorenone gave the corresponding fluorenols
with excellent enantioselectivities, that of 2-fluorofluorenone resulted in
lower enantioselectivity (65% ee). This observation was attributed to the
comparable sizes of fluorine and hydrogen atoms, which restricts the
distinction of the two aromatic rings. Lastly, 2-methylfluorenone also
afforded a low fluorenol optical yield (52% ee) despite its similarity to
the chloro-substituted ketone. This suggests that electronic effect also
has an influence on enantioselectivity.
Homann, Zaks and their coworkers reported a fast method for
screening and selecting cells capable of enantioselectively reducing ke­
tone substrates [40]. They used multi-well plates to select 60 out of 300
microbes that are capable of asymmetric reduction of selected ketones.
4-(4-Trifluoromethoxy-benzoyl)-piperidine-1-carboxylic acid tert-butyl
ester and 4-(4-cyclopropylmethoxy-benzoyl)-piperidine-1-carboxylic
acid tert-butyl ester were reduced to their corresponding (S)- and
(R)-alcohols, respectively, by several bacterial strains with good to
excellent enantioselectivities (Scheme 5). They used these chiral alco­
hols as intermediates in the synthesis of an antiviral antagonist and an
anti-muscarinic receptor antagonist.
Interestingly, Sahin and coworkers used whole cells of Weissella
paramesenteroides N7 to reduce 1,2-diphenylethanone to (S)-1,2-diphe­
nylethanol with an excellent yield and enantioselectivity (Scheme 6)
Scheme 2. Asymmetric reduction of cyclohexyl(phenyl)methanone using
whole cells of Candida magnolia and Lactobacillus paracasei BD101.
Scheme 3. Asymmetric reduction of 3-cyclopentoxy-4-methoxybenzophenone
using whole cells of Rhodotorula pilimanae.
Scheme 4. Asymmetric reduction of fluorenones using baker’s yeast.
[41].
The use of whole cells in asymmetric reduction of ketones is an
attractive approach, however, the fact that more than one enzyme could
be involved in a certain transformation complicates the elucidation of
reaction mechanism.
2.2.2. Asymmetric reduction of acyclic bulky-bulky ketones using purified
ADHs
This part of the review discusses examples of asymmetric reduction
of acyclic bulky-bulky ketones using purified enzymes. Zhu and co­
workers reported the asymmetric reduction of diaryl ketones by
carbonyl reductase from the red yeast Sporobolomyces salmonicolor
AKU4429 (SsCR), an NADP+-dependent ADH [42]. They used the
wild-type enzyme to reduce 4-chlorobenzophenone and 4-methylbenzo­
phenone to produce their corresponding (R)-alcohols. They observed a
stereo-preference switch when mutant Q245P SsCR was used in the
asymmetric reduction of these substrates. They carried out other re­
actions with multiple substrates and ascertained that the Q245P mutant
switches the stereo-preference of the reduction of the diaryl ketones
especially those having para-substituents on one ring (Scheme 7). They
also showed that both conversion and stereoselectivity were dependent
on the cosolvent used.
Alcohol dehydrogenase from Thermoanaerobacter pseudoethanolicus
(TeSADH) or that from Thermoanaerobium brockii (TbSADH), NADP+dependent ADHs, are robust catalysts that has been shown to be
Scheme 1. Asymmetric reduction of 4-chlorobenzophenone using whole cells
of Debaryomyces marama.
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Molecular Catalysis 541 (2023) 113099
Scheme 5. Asymmetric reduction of substituted benzoyl piperidine esters using whole cells of various microbes.
Scheme 6. Asymmetric reduction of 1,2-diphenylethanone using Weissella paramesenteroides N7.
Scheme 7. Switching the stereopreference of Sporobolomyces salmonicolor AKU4429 (SsCR) in asymmetric reduction of aryl ketones.
identical [43]. Their wild types do not accept bulky-bulky diaryl ke­
tones. Expansion of the size of the large pocket of TeSADH through a
single point mutation in W110A allowed for the accommodation of
substrates containing an aryl group in one side such as 4-pheny-2-buta­
none and 1-pbenyl-2-propanone [44]. Mutation of I86, which lines the
smaller pocket of TeSADH, with A allowed for the accommodation of
acetophenone [45]. Subsequently, expanding the sizes of both pockets of
the active site of TeSADH in W110A/I86A mutant enabled this enzyme
to reduce 1,2-diphenylethanone with high ee and yield to produce its
(R)-alcohol (Scheme 8), which is an analog of anticancer agent com­
bretastatin [46].
Sun and coworkers used A85G/I86A TbSADH as a template and
conducted a combinatorial active-site double-code saturation muta­
genesis of residues G101, W110, L294, and C295 lining the substrate
binding pockets [47]. They identified A85Q/I86A/Q101A as an efficient
catalyst for the asymmetric reduction of diaryl ketones (Scheme 9).
Docking studies showed that W110 and Y267 are important residues for
the π–π interactions with the phenyl substituent of the substrate.
Guided by structural analysis and molecular dynamic simulations for
TbSADH, Sun, Nie and their coworkers used site-directed saturation
mutagenesis at A85 and I86, which line the binding pocket of the
enzyme, in order to expand substrate specificity to accommodate diaryl
ketones [48]. They used the asymmetric reduction of 4-chlorophenyl
pyridin-2-yl ketone (CPMK) as a model reaction. Mutants A85G, I86A,
I86S, I86E, I86T, I86L, and I86P were identified to be active towards
CPMK, favoring the (S)-alcohol except for I86P, which resulted in the
(R)-alcohol. Unlike single point mutations, which led to low conversions
and medium enantioselectivities in asymmetric reduction of CPMK,
combinational active site saturation led to improved conversions and
enantioselectivities with the double mutants A85G/I86L and A85V/I86S
giving the best results with kcat/Km of 703.52 and 219.17 s− 1 mM− 1,
respectively. These two mutants were then used in asymmetric
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Molecular Catalysis 541 (2023) 113099
reduction of a series of diaryl ketones and their performance was
compared with that of the WT-enzyme, which in general gives poor
conversions with these substrates (Scheme 10). The produced enantio­
pure alcohols can be used in the synthesis of antiallergic drugs such as
bepotasine and (S)-carbinoxamine.
Sun and coworkers used a proline induced loop-engineering test
(PiLoT) in switching the stereoselectivity and expanding substrate
specificity of TbSADH towards CPMK, which is not a substrate for wildtype TbSADH [49]. The crystal structure indicated that P84, which is
located at the interface of a β–strand and loop region, is a key residue in
the binding pockets. They carried out in silico saturation mutagenesis at
P84 plus deletion of P84 (ΔP84) and showed that variants P84G, P84S,
P84V, P84Y and ΔP84 were the feasible mutants with P84S favoring the
(S)-product and ΔP84 favoring the (R)-product. They used epistasis to
enhance protein activity and selectivity, which resulted in mutants
P84S/I86L and P84S/I86A having the highest conversion and enantio­
selectivity towards the formation of (S)-alcohol, while mutant
ΔP84/A85G TbSADH had the highest enantioselectivity towards
(R)-alcohol (Scheme 11). This important finding indicates that
non-bonding interactions control the stereopreference of ADHs. It also
shows the importance of mutations of P84 residue, which unlike other
amino acids has only one rotatable angle, of TbSADH in generating
flexible mutants in terms of substrate specificity and stereopreference.
They finally performed preparatory scale asymmetric reduction re­
actions of CPMK, which yielded the (S)-configured alcohol in 73% yield
and >99% ee. This study might serve as a guide to control substrate
Scheme 8. Asymmetric reduction of 1,2-diphenylethanone using W110A/
I86A TeSADH.
Scheme 9. Asymmetric reduction of ketones bearing bulky substituents on
both sides using A85G/I86A/Q101A TbSADH.
Scheme 10. Asymmetric reduction of diaryl ketones using TbSADH variants.
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Molecular Catalysis 541 (2023) 113099
Scheme 11. Switching the stereopreference of TbSADH in asymmetric reduction of CPMK.
specify and stereopreference for other ADHs.
Alcohol dehydrogenase from Kluyveromyces polyspora (KpADH), an
NADP+-dependent ADH has been identified as a potential robust diaryl
ketone reductase [50]. Asymmetric reduction of CPMK produced
(R)-(4-chlorophenyl)-(pyridin-2-yl)methanol (i.e., Prelog mode) in low
activity and moderate enantioselectivity (82% ee). Subsequent high
throughput screening resulted in identification of S237A KpADH, which
improved the enantioselectivity for this reaction to 96% ee [50]. Zhou, Ni
and their coworkers used iterative combinatorial mutagenesis strategy on
residues inside the substrate-binding pocket of KpADH to tune its stereo­
preference in asymmetric reduction of diaryl ketones [51]. They showed
that asymmetric reduction of CPMK using Q136N/F161V/S196G/
E214G/S237C KpADH produced the corresponding anti-Prelog
(S)-(4-chlorophenyl)-(pyridin-2-yl)methanol in 98% ee (Scheme 12). They
also showed that asymmetric reduction of CPMK using E214V/T215S
KpADH variant produced the corresponding (R)-alcohol in excellent
enantiopurity.
Ni, Xu and their coworkers carried out site-directed mutagenesis on
KpADH to identify residues essential in tuning its enantioselectivity and
substrate specificity [52]. The crystal structure of KpADH revealed that
substrate binding pockets consists F161, C165, S196, F197, F86, Y127,
M131, P133, Q136, E214, S237 and Q238 residues. Individual site
directed mutagenesis of these sites by alanine indicates that S237A has
the highest activity towards CPMK. Saturation mutagenesis at S237 led
to the mutants S237A, S237G, S237D, S237W and S237E that are
capable of reduction of CPMK to its corresponding (R)-alcohol with
activities higher than that of the wild-type enzyme. Kinetic and docking
analysis showed that S237G had the highest catalytic activity and the
lowest binding energy. They then tested the asymmetric reduction of
several diaryl ketones by using various S237 mutants (Scheme 13).
Ni and coworkers also reported a structural guided mutagenesis to
identify the molecular switch of key residues of KpADH responsible for
the switch of its stereo-preference in the asymmetric reduction of CPMK
[53]. Docking analysis revealed that residues E214 and S234 are vital in
manipulating this stereo-preference. They explored the synergetic effect
of these residues towards CPMK via combinatorial mutagenesis. Their
efforts resulted in E214Y/S237A KpADH, which is capable of reducing
CPMK to the corresponding (R)-alcohol (i.e., Prelog product) in 99% ee;
and E214G/S237C KpADH capable of reducing CPMK to the corre­
sponding anti-Prelog alcohol in 79% ee (Scheme 14). These findings
clearly reveals the minimal changes that are required to tune the ster­
eopreference of ADHs in asymmetric reduction of diaryl ketones. Kinetic
parameters showed that the designed mutants have higher catalytic
activities than that of the wild-type enzyme with E214V/S237A having
the best catalytic activity followed by E214Y/S237A.
Ni, Xu and their coworkers reported asymmetric reduction of various
substituted diaryl ketones (4-chlorophenyl-2-pyridinylmethanone, 3-chlor­
ophenyl-2-pyridinylmethanone, 2-chlorophenyl-2-pyridinylmethanone, 4bromophenyl-2-pyridinylmethanone,
4-trifluoromethylphenyl-2-pyr­
idinylmethanone and 4-methylphenyl-2-pyridinylmethanone) using
KpADH and concluded that the nature of the substituent at the para position
of these substrates is essential for the enantioselectivity of their KpADHcatalyzed asymmetric reduction [54]. They used a combination of single
point, site saturation, and combinatorial mutagenesis to tune stereo­
preference and stereoselectivity of KpADH in the reduction of para
substituted diaryl ketones. Among the tested mutants, they found that
T215S and E214Y exhibit the highest (S)-selectivity in the asymmetric
reduction of 4-trifluoromethylphenyl-2-pyridinylmethanone while T215R
and E214G exhibit the highest (R)-selectivity. Moreover, T215S and E214V
gave the highest (S)-selectivity, while the opposite stereopreference was
obtained using T215N and E214G with respect to 4-methylphenyl-2-pyridi­
nylmethanone. Combinatorial mutagenesis of both the (S)-selective and
(R)-selective mutants showed that E214I/T215S/S237A and
E214Y/T215S/S237A have the highest (S)-selectivity while
F161V/S196G/E214G showed the highest (R)-selectivity with respect to
4-trifluoromethylphenyl-2-pyridinylmethanone and 4-methylphenyl-2-­
pyridinylmethanone (Scheme 15).
Shao and coworkers used shrinking mutagenesis approach in ADH
from Lactobacillus kefir DSM 20,587 (LkADH), a well-known NADP+dependent anti-Prelog ADH [55], to generate mutants with extended
binding pockets that could reduce 4-chlorodiphenylketones (CPPK) and
other diaryl ketone analogs to their corresponding alcohols [56]. They
used iterative shrinking mutagenesis on five residues around the cata­
lytic triad (S143-Y156-K160), which were identified based on structural
information of the binding pockets. They concluded that
Y190P/I144V/L199V/E145C/M206F exhibits the highest conversion,
enantioselectivity and catalytic activity in asymmetric reduction of
CPPK (Scheme 16). They performed a scaled up reduction of CPPK and
CPMK using resting cells containing this mutant, which resulted in
excellent ee’s and high tolerance to these substrates. The produced
(R)-(4-chlorophenyl)(phenyl)methanol is a key building block for
levorotatory cloperastine, which exhibits better efficiency in cough
control when compared with racemic cloperastine [57].
Subsequently, Shao and coworkers further engineered the five-point
mutated LkADH to generate Y190P/I144V/L199V/E145C/M206F/
D150I LkADH, which exhibits improved hydrophobicity [58]. This
mutant showed a three-fold improvement in catalytic efficiency in the
asymmetric reduction of CPPK when compared with Y190P/I144V/­
L199V/E145C/M206F LkADH. This mutant can be used in the synthesis
of (S)-4-chlorophenylpyridylmethanol and (R)-4-chlorobenzhydrol,
Scheme 12. Asymmetric reduction of CPMK using Kluyveromyces polyspora ADH (KpADH) variants.
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Molecular Catalysis 541 (2023) 113099
Scheme 13. Asymmetric reduction of diaryl ketones using KpADH variants.
Scheme 14. Switching the stereopreference of KpADH in asymmetric reduction of CPMK.
which are intermediates in the manufacturing of pharmaceutical drugs
bepotastine and cloperastine, respectively. They achieved these con­
versions with high ee and high catalytic activity. Other diaryl ketones
were reduced using this mutant in poor to medium enantioselectivities.
Examples of biocatalytic asymmetric reduction of diaryl prochiral
ketones listed in this section indicate that it is possible to tune substrate
specificity of ADHs using modern tools of protein engineering to
accommodate such substrates, and thus producing additional important
optically active alcohols, which can be used as building blocks in
pharmaceutical products. Efforts should also be directed towards more
sterically hindered and hydrophobic ketones that mimic the very
interesting example of asymmetric reduction of (E)-methyl 2-(3-(3-(2(7-chloroquinolin-2-yl)vinyl)phenyl)-3-oxopropyl)benzoate to the cor­
responding (S)-alcohol, which is a precursor for motelukast that is used
to prevent symptoms of asthma (Fig. 1), using the ketoreductase CDX026 (Scheme 17) [59,60].
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Molecular Catalysis 541 (2023) 113099
Scheme 15. Switching stereopreference of KpADH in the asymmetric reduction of para-substituted diaryl ketones.
enantioselectivities (Scheme 20). In most of the cases, the tetralols were
produced in high enantio- and diastereoselectivities, yet yields that are
higher than 50%, which was explained by an in situ racemization of the
unreacted enantiomer of the tetralone substrate upon depletion of the
more reactive enantiomer via kinetic resolution (i.e., dynamic kinetic
resolution), which is commonly encountered in compounds containing
an activated C–H bond [64]. The production of more than one stereo­
isomers in few cases suggests the possibility of the involvement of more
than one enzyme in these strains.
Pava and coworkers synthesized feroxidin derivative, a natural
product from Aloe ferox, by reducing 5,6,7,8-tetrahydro-8-methyl-1,3-
Scheme 16. Asymmetric reduction of CPPK and CPMK using Y190P/I144V/
L199V/E145C/M206F LkADH.
2.3. Biocatalytic asymmetric reduction of cyclic ketones
2.3.1. Biocatalytic asymmetric reduction of tetralones using whole cells
Sicsic and coworkers reported asymmetric reduction of 1-tetralone
and its 4-substituted analogs as well as 2-tetralone using whole cells of
Sporobolomyces pararoseus and Rhodotorula rubra [61]. They reported
that reduction of 1-tetralone, 2-tetralone, and 4-methyl-1-tetralone
using R. rubra resulted in formation of their corresponding (S)-tetra­
lols with excellent conversions and high enantioselectivities, whereas
reduction of these using S. pararoseus resulted in marginal to no enan­
tioselectivity towards the formation of (R)-alcohols in low conversions.
Furthermore, reduction of the 4-substituted-1-tetralones using R. rubra
resulted in both cis- and trans-(S)-alcohols, while S. pararoseus afforded
only cis-products with (R)-configuration (Scheme 18), which led the
authors to propose the possibility of involvement of two reductases in
the latter that exhibit opposite stereopreferences and are sensitive to the
substituents at position four of 1-tetraloles.
Buisson, Azerad and their coworkers reported the use of various
strains of fungi (including yeast) to synthesize enantiomerically pure
substituted 1-tetralol-2-carboxyesters from their corresponding 1-tetra­
lones [62]. Asymmetric reductions of these substrates using these
strains resulted in enantiocomplementary production of their corre­
sponding alcohols, with high optical purities and good yields (Scheme
19). Subsequently, asymmetric reduction of 1- and 3-carbomethoxy-2-­
tetralones was reported using various microorganisms [63]. Notably,
five strains reduced 1-carbomethoxy-2-tetralone giving rise to cis-­
products only with (S)-configured alcohols, while Saccharomyces mon­
tanus and Aspergillus ochraceus afforded significant yields of trans
products (12 and 35%, respectively) with the opposite configuration of
alcohol (Scheme 20). On the other hand, reduction of 3-carbomethox­
y-2-tetralone produced three stereoisomers with good to high
Scheme 18. Asymmetric reduction of substituted 1-tetralones using Spor­
obolomyces pararoseus and Rhodotorula rubra.
Scheme 19. Enantiocomplementary biocatalytic asymmetric reduction of 2carboxyethyl-1-tetralone by microorganisms.
Scheme 17. Asymmetric reduction of (E)-methyl 2-(3-(3-(2-(7-chloroquinolin-2-yl)vinyl)phenyl)-3-oxopropyl)benzoate using CDX-026.
8
A.E. Sardauna et al.
Molecular Catalysis 541 (2023) 113099
Scheme 20. Asymmetric reduction of 1- and 3-carbomethoxy-2-tetralones using various microorganisms.
dimethoxynaphthalen-6-one using microorganisms [65], among which
Aspergillus niger and Beauveria bassiana showed the best enantiose­
lectivities giving rise to tetralols with high optical purities (Scheme 21).
Kroutil and coworkers reported the asymmetric reduction of 2-tetra­
lone using Rhodococcus ruber DSM 44,541 to (S)-2-tetralol in moderate
enantioselectivity and poor conversion [66]. Subsequently, they re­
ported the production of (R)-2-tetralol from 2-tetralone using Paracoccus
pantotrophus DSM 11,072 and Comamonas sp. DSM 15,091 in excellent
enantioselectivities (Scheme 22) [67]. However, like most of the other
strains, these cells showed no activity towards 1-tetralone, which is
more sterically hindered than 2-tetralone.
Janeczko and coworkers studied the effect of reaction time on the
stereopreference and enantioselectivity of selected whole cells towards
asymmetric reduction of 1- and 2-tetralones [68]. They observed a
time-dependent improvement in the ee of (S)-1-tetralol and (S)-2-tetralol
for these reactions. Interestingly, reduction of 2-tetralone using Absidia
cylindrospora KCh 336 afforded (S)-2-tetralol after three days and the
(R)-2-tetralol after nine days. They attributed this phenomenon to a
possible stereoinversion driven by various reductases that could be
present in this strain.
Zaks and his co-workers used whole cells of Pichia methanolica
58,403 to obtain (S)-1-tetralol from 1-tetralone with low yield and
excellent optical purity [40]. Recently, Kalay and Sashin described
optimized reaction conditions that led to the first gram-scale preparation
of 1-tetralol using whole cells of Lactobacillus paracasei BD101, which
enabled the production of 7.04 g of (R)-1-tetralol with excellent both
yield and optical purity (Scheme 23) [69].
Rao and his co-workers reported the asymmetric reduction of
substituted indanones and 2-tetralones using Daucus carota and baker’s
yeast, with better yields and enantioselectivities reported for the former
(Scheme 24) [70].
The scarcity of examples on biocatalytic asymmetric reduction of 1tetralones demands for isolation and characterization of ADHs from
strains that are capable of performing this function (e.g., Lactobacillus
paracasei BD101), which will guide in engineering other ADHs that can
accommodate such sterically demanding substrates.
2.3.2. Biocatalytic asymmetric reduction of tetralones using purified
enzymes
In an effort to synthesize 1,8-dihydroxynaphthalene-melanin, Müller
and coworkers used 2-tetralones as model substrates with tetrahydrox­
ynaphthalene reductase (T4HNR) from Magnaporthe grisea in order to
demystify the selectivity mechanism of asymmetric dearomatization of
1,3,6,8-tetrahydroxynaphthalene [71]. The unsubstituted 2-tetralone
was reduced to (S)-2-tetralol with a poor enantioselectivity, whereas
the substituted tetralones were all reduced with high enantioselectivities
except 6-methoxy-2-tetralone and 5-hydroxy-2-tetralone (Scheme 25).
They concluded that the interaction between methoxy group of the
substrates and a hydrophobic region of the enzyme consisting of L232,
I157, and I274 as well as the hydrogen bond between the hydroxyl
group of the substrates and carboxyl group of I274 controls the stereo­
preference of the enzyme in asymmetric reduction of substituted 2-tetra­
lones. Subsequently, they conducted asymmetric reduction of other
naphtholic substrates such as 1,4-naphthoquinones, flaviolin, lawsone
and 2,3-epoxy-1,4-naphthoquinones, which are key building blocks of
important natural products (Scheme 25) [72,73].
Musa and coworkers reported the asymmetric reduction of 2-tetra­
lone[44] and its substituted analogs using various mutants of TeSDAH
Scheme 21. Asymmetric synthesis of dimethyl analog of feroxidin using whole cells of Beauveria bassiana.
9
A.E. Sardauna et al.
Molecular Catalysis 541 (2023) 113099
Scheme 22. Asymmetric reduction of 2-tetralone using lyophilized Comamonas sp., Paracoccus pantotrophus, or Rhodococcus ruber.
residues. The variants mostly showed enhanced catalytic activities
especially V187S/I291F, which showed the best activity and stereo­
selectivity towards most of the tested substrates. Consequently, they
used resting E. coli whole cells expressing V187S/I291F to produce
(S)-7-fluoro-1-tetralol from its corresponding tetralone with excellent
conversion and enantioselectivity (Scheme 28).
Unlike biocatalytic asymmetric reduction of 2-tetralones, in which
few examples have been reported, it is obvious that biocatalytic asym­
metric reduction of 1-tetralones is challenging. Thus, more efforts using
progresses in the biotechnology field should be devoted to tackle this
challenge.
Scheme 23. Gram-scale synthesis of (R)-1-tetralol using Lactobacillus para­
casei BD101.
2.3.3. Biocatalytic asymmetric reduction of cyclic ketones containing
heteroatoms using whole cells
An interesting class of bulky-bulky ketones is cyclic ketones that
contain a heteroatom such as tetrahydrothiophene-3-one and
tetrahydrofuran-3-one; these ketones are spatially symmetrical and thus
not easy to be asymmetrically reduced. Konuki and coworkers reported
an approach for production of (R)-tetrahydrothiophene-3-ol from
tetrahydrothiophene-3-one via bioreduction using whole cells followed
by crystallization [77]. More specifically, they used Penicillium, Asper­
gillus,
or
Streptomyces
to
asymmetrically
reduce
tetrahydrothiophene-3-one to its corresponding (R)-alcohol in medium
to high enantioselectivities (70–92%). However, crystallization of the
produced alcohols using acetone/hexane led to significant improvement
in enantiopurity (Scheme 29).
Scheme 24. Asymmetric reduction of 1-oximino indanone using Daucus carota.
[74]. Four variants (W110G, W110A, W110V, and W110A/I86A) of
TeSADH were tested and found to be effective in the asymmetric
reduction of 2-tetralones (Scheme 26). The identity and the position of
the substituents were found to be crucial in the stereopreference of these
reactions. For example, the asymmetric reduction of 5-methoxy-,
8-methoxy-, and 7-hydroxy-2-tetralones using the tested mutants
resulted in the formation of their corresponding anti-Prelog tetralols.
Hydrophobic residues L107, Y267, and L294 were shown by docking in
the active site of W110G TeSADH to be crucial in controlling the ster­
eopreference of TeSADH in the asymmetric reduction of substituted
tetralones. Such small alteration in the structure of the active site shows
the peculiarity of enzymatic asymmetric reduction.
Matsuda and coworkers reported the asymmetric reduction of
unsubstituted 1-tetralone as well as 2-tetralone and its substituted an­
alogs using acetophenone reductase from Geotrichum candidum
(GcAPRD), an NAD+-dependent ADH.[75] The corresponding (S)-tet­
ralols were produced in good yields and excellent enantioselectivities.
Interestingly, mutant W288A resulted in a switch in the enantioprefer­
ence of GcAPRD in the asymmetric reduction of 2-tetralone, 5-methox­
y-2-tetralone, and 6-hydroxy-2-tetralone and the corresponding
anti-Prelog (R)-2-tetralols were produced (Scheme 27), which was
attributed to the presence of a hydrophobic/hydrophilic interaction of
the substituents with the enzyme binding pockets. Other mutants that
have similar selectivity to that of W288A are W288C, W288T, W288V,
W288S, and W288G of which W288S produced (R)-6-hydroxy-2-tetralol
with the highest ever-reported enantioselectivity (82% ee).
Zhang, Qin and their coworkers conducted site-directed mutagenesis
on an NAD+-dependent carbonyl reductase from Bacillus aryabhattai
(BaSDR1) to synthesize optically active halogenated 1-tetralols from
their corresponding 1-tetralone analogs [76]. More specifically, they
identified Q139, V187, Q237, F250 and I291 as target residues for en­
gineering. Mutation at these residues not only leads to enlarged catalytic
pocket to give enough steric flexibility to the tetralones, but also adjusts
hydrophobicity to increase the proximity between the substrates and the
2.3.4. Biocatalytic reduction of cyclic ketones containing heteroatoms using
purified enzymes
Liang and coworkers reported a biocatalytic asymmetric reduction of
tetrahydrothiophene-3-one to its corresponding (R)-alcohol with high
enantioselectivity [78]. They selected Lactobacillus kefir KRED after
screening various KREDs to accomplish this reduction. In efforts to
improve the enantioselectivity of L. kefir KRED-catalyzed asymmetric
reduction of tetrahydrothiophene-3-one, directed evolution and
throughput screening resulted in variant CDX-033, which enabled the
production of (R)-tetrahydrothiophene-3-ol in >99% ee and in kilogram
scale using an enzyme-coupled cofactor regeneration approach (Scheme
30).
Reetz and coworkers used triple-code-saturation mutagenesis
approach on A85, I86, W110, L294, and C295 sites of TbSADH, which
were identified as contact points based on docking of
tertrahydrothiofuran-3-one in the active site of TbSADH [79]. They
identified I86V/W110L/L294Q and I86N/C295N as the best variants for
asymmetric reduction of tertrahydrothiofuran-3-one to produce the
corresponding (S)-alcohol in 95% ee and (R)-alcohol in 99% ee,
respectively (Scheme 31). Docking studies revealed that sites 294 and
295 are crucial for switching the stereopreference of TbSADH. They
further performed asymmetric reduction of other difficult-to-reduce
cyclic
ketones
containing
heteroatoms
such
as
tertrahydrothiofuran-3-one using various mutants of TbSADH with high
enantioselectivities. Subsequently, Sun and coworkers used molecular
dynamics simulations along with molecular mechanics analysis to reveal
the importance of hydrogen bonds formed between the oxygen atom of
tetrahydrofuran-3-one and Q or N at 294 or 295 positions, respectively,
in controlling the stereopreference of TbSADH towards the asymmetric
reduction of tetrahydrofuran-3-one [80]. (S)-3-Hydroxytetrahydrofuran
10
A.E. Sardauna et al.
Molecular Catalysis 541 (2023) 113099
Scheme 25. Asymmetric reduction of 2-tetralones and 1,4-naphthoquinones using tetrahydroxynaphthalene reductase from Magnaporthe grisea.
Scheme 26. Asymmetric reduction of 2-tetralones using various mutants of TeSADH. X = W110G, W110A, W110V, or W110A/I86A.
Scheme 27. Asymmetric reduction of substituted 2-tetralones using W288A GcAPRD.
11
A.E. Sardauna et al.
Molecular Catalysis 541 (2023) 113099
Scheme 31. Stereopreference control of TbSADH-catalyzed asymmetric
reduction of tetrahydrofuran-3-one.
Scheme 28. Asymmetric reduction of 7-fluoro-1-tetralone using recombinant
E. coli cells expressing V187S/I291F BaSDR1.
Scheme 32. Enantiocomplementary asymmetric reduction of 4-(bromo­
methylene)cyclohexanone using TbSADH mutants.
Scheme 29. Asymmetric reduction of tetrahydrothiophene-3-one using Peni­
cillium, Aspergillus, or Streptomyces, followed by crystallization.
is possible to tune substrate specificity and stereopreference of ADHs
using protein-engineering tools to accommodate these substrates and
produce both enantiomers of their corresponding enantiopure alcohols
with high enantioselectivities. Protein engineering using directed evo­
lution or site-directed mutagenesis combined with molecular dynamics
simulations should provide more mutants of ADHs that are capable of
asymmetric reduction of bulky-bulky ketones with high efficiencies and
enantioselectivities to enable better industrial utilization of these
enzymes.
Declaration of Competing Interest
The authors declare no conflict of interest.
Scheme 30. Asymmetric reduction of tetrahydrothiophene-3-one using a
Lactobacillus kefir KRED variant.
Data availability
No data was used for the research described in the article.
serves as a precursor for amprenavir and fosamprenavir (Fig. 1), which
are protease inhibiters used in HIV/AIDS treatment.
Acknowledgments
2.3.5. Biocatalytic asymmetric reduction of other cyclic bulky-bulky
ketones
Reetz and coworkers reported the asymmetric reduction of 4-alkyli­
dene cyclohexanones to their corresponding axially chiral enantiopure
alcohols using TbSADH variants in high stereoselectivities [81]. They
showed that W110X TbSADH (X = A, E, M, T) reduced 4-alkylidene
cyclohexanones to the corresponding (R)-alcohols, whereas I86X
TbSADH (X = A, G, E, M, T) produced the corresponding (S)-alcohols
(Scheme 32). These mutants were identified using site saturation
mutagenesis for sites that are known to line the large and small pockets
of the active site of TbSADH (i.e., W110 and I86, respectively). Subse­
quently, molecular dynamics simulations showed that the stereo­
preferences of these TeSADH mutants towards 4-(bromomethylene)
cyclohexanone are not only controlled by the enlarged sizes of the two
pockets that resulted from W110T or I86A, but also by C–H interactions
of indole of the W110 and that of the cyclohexane ring of the substrate
[82]. This allows for a hydride transfer to the re face of the substrate
when using I86A TeSADH. It is worth mentioning that these substrates
cannot be asymmetrically reduced using transition metal catalysis.
The authors gratefully acknowledges funding by Deanship of Scien­
tific Research (DSR) at King Fahd University of Petroleum and Minerals
(KFUPM), project number DF191007.
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