Deracemization and Stereoinversion of Alcohols Using Two Mutants of Secondary Alcohol Dehydrogenase from Thermoanaerobacter pseudoethanolicus

Article abstract of DOI:10.1002/ejoc.202000728

We developed a one-pot sequential two-step deracemization approach to chiral alcohols using two mutants of Thermoanaerobacter pseudoethanolicus secondary alcohol dehydrogenase (TeSADH). This approach relies on consecutive non-stereospecific oxidation of alcohols and stereoselective reduction of their prochiral ketones using two mutants of TeSADH with poor and good stereoselectivities, respectively. More specifically, W110G TeSADH enables a non-stereospecific oxidation of alcohol racemates to their corresponding prochiral ketones, followed by W110V TeSADH-catalyzed stereoselective reduction of the resultant ketone intermediates to enantiopure (S)-configured alcohols in up to > 99 percent enantiomeric excess. A heat treatment after the oxidation step was required to avoid the interference of the marginally stereoselective W110G TeSADH in the reduction step; this heat treatment was eliminated by using sol-gel encapsulated W110G TeSADH in the oxidation step. Moreover, this bi-enzymatic approach was implemented in the stereoinversion of (R)-configured alcohols, and (S)-configured alcohols with up to > 99 percent enantiomeric excess were obtained by this Mitsunobu-like stereoinversion reaction.

Full text of DOI:10.1002/ejoc.202000728

European Journal of Organic Chemistry
Accepted Article
Accepted Article
Title: Deracemization and Stereoinversion of Alcohols Using
Two Mutants of Secondary Alcohol Dehydrogenase from
Thermoanaerobacter pseudoethanolicus
Authors: Sodiq A. Nafiu, Masateru Takahashi, Etsuko Takahashi,
Samir M. Hamdan, and Musa M. Musa
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202000728
Link to VoR: https://doi.org/10.1002/ejoc.202000728
0
1/2020

European Journal of Organic Chemistry
10.1002/ejoc.202000728
FULL PAPER
Deracemization and Stereoinversion of Alcohols Using Two
Mutants of Secondary Alcohol Dehydrogenase from
Thermoanaerobacter pseudoethanolicus
Sodiq A. Nafiu, [a] Masateru Takahashi, [b] Etsuko Takahashi, [b] Samir M. Hamdan, [b] and Musa M.
Musa*[a]
[
a]
S. A. Nafiu, Prof. M. M. Musa
Chemistry Department
King Fahd University of Petroleum and Minerals
Dhahran, 31261, KSA
E-mail: musam@kfupm.edu.sa
http://faculty.kfupm.edu.sa/CHEM/musam/index.html
Dr. M. Takahashi, E. Takahashi, Prof. S. M. Hamdan
Division of Biological and Environmental Sciences and Engineering
King Abdullah University of Science and Technology
Thuwal, 23955-6900, KSA
[
b]
Supporting information for this article is given via a link at the end of the document.
Abstract: We developed
deracemization approach for alcohols using two mutants of
Thermoanaerobacter pseudoethanolicus secondary alcohol
a
one-pot sequential two-step
ketones are less naturally abundant than alcohols; hence,
quantitative production of enantiopure alcohols using
deracemization approaches is more attractive than asymmetric
reduction of their ketones.
dehydrogenase (TeSADH). This approach relies on consecutive non-
stereospecific oxidation of alcohols and stereoselective reduction of
their prochiral ketones using two mutants of TeSADH with poor and
good stereoselectivities, respectively. More specifically, W110G
TeSADH enables a non-stereospecific oxidation of alcohol racemates
to their corresponding prochiral ketones, followed by W110V
TeSADH-catalyzed stereoselective reduction of the resultant ketone
intermediates to enantiopure (S)-configured alcohols in up to >99%
enantiomeric excess. A heat treatment after the oxidation step was
required to avoid the interference of the marginally stereoselective
W110G TeSADH in the reduction step; this heat treatment was
eliminated by using sol-gel encapsulated W110G TeSADH in the
oxidation step. Moreover, this bi-enzymatic approach was
implemented in the stereoinversion of (R)-configured alcohols, and
Deracemization strategies of alcohols using chemical catalysis[
and chemoenzymatic approaches[ 8 ] have been reported.
However, biocatalysis-based deracemization reactions are
preferable due to the following reasons: firstly, they possess high
stereo-, regio- and chemoselectivities, secondly, they can
overcome challenges such as profitability and sustainability in fine
chemical industries,[9,10] thirdly, they are environmentally benign
catalysts, and fourthly, they work effectively under mild conditions,
which eliminates side products that could result from undesirable
isomerization or epimerization. Therefore, several methods of
enzymatic deracemization of alcohols have been reported.
However, these methods use whole cells,[11] which complicates
the elucidation of deracemization mechanism, thus restricts
further developments of these approaches. Alternatively, they use
complicated systems that comprise multiple enzymes,[12] which
raises problems associated with compatibility among involved
enzymes and with the reaction conditions.
7]
(S)-configured alcohols with up to >99% enantiomeric excess were
obtained by this Mitsunobu-like stereoinversion reaction.
Biocatalytic asymmetric redox reactions mediated by alcohol
dehydrogenases (ADHs) is an important class of reactions.
Secondary ADH from Thermoanaerobacter pseudoethanolicus
Introduction
Optically active alcohols are important building blocks for
chemicals that play key roles in pharmaceutical, agrochemical,
and food industries.[1,2] Kinetic resolution (KR) is the commonly
used industrial method for the synthesis of enantiomerically pure
alcohols.[ 3 , 4 ] However, a maximum yield of 50% with high
enantioselectivity is a challenging drawback associated with this
approach. An optimum alternative that overcomes this hitch is to
obtain enantiopure alcohols via deracemization, which ensures
up to >99% yields of enantiopure alcohols from their racemates.
Among the deracemization strategies discussed in the literature
are cyclic deracemization, dynamic kinetic resolution,
(
TeSADH, EC 1.1.1.2), a nicotinamide-adenine dinucleotide
+
phosphate (NADP )-dependent ADH, is a robust ADH, and thus
an attractive choice as a biocatalyst.[13,14] Our group previously
developed a one-pot two-step deracemization approach for
[15]
secondary alcohols by employing a single mutant of TeSADH.
This approach relies on non-stereospecific oxidation of alcohol
racemates to their corresponding ketones, which in turn followed
by an enantioselective reduction employing the same enzyme
(
Scheme 1). The desired tuned enantioselectivities of the redox
reactions was accomplished by controlling the concentrations of
acetone and 2-propanol co-substrates in the oxidation and
reduction reactions, respectively. Enantiopure alcohols were
obtained in 20% to 87% ee when W110A TeSADH was used and
[
5]
stereoinversion and enantioconvergence. Although enantiopure
alcohols can be produced in high yields by asymmetric reduction
of their prochiral ketones,[6] this approach is not optimal since
47% to >99% when W110G TeSADH was used.
1
This article is protected by copyright. All rights reserved.

European Journal of Organic Chemistry
10.1002/ejoc.202000728
FULL PAPER
both mutants are from the same enzyme and thus compatibility is
not an issue.
Under the aforementioned deracemization conditions and using
two mutants of TeSADH, (rac)-4-(4ʹ-hydroxyphenyl)-2-butanol
[
(rac)-1a] was deracemized to obtain (S)-1a in >99% ee. (S)-4-(4ʹ-
Methoxyphenyl)-2-butanol [(S)-1b] was obtained from its
racemate in 98% ee (Table 1), compared to 72% ee and 87% ee
when the single enzymatic approach was employed using W110G
or W110A mutants of TeSADH, respectively. (S)-4-Phenyl-2-
butanol [(S)-1d] and (S)-1-phenyl-2-butanol [(S)-1e] were also
obtained in >99% ee using W110G and W110V TeSADHs. In all
cases, the percent of remaining ketone intermediate after the
reduction step was less than 0.5%. The currently reported bi-
enzymatic deracemization approach gave better results in terms
of ee than the previously reported single enzymatic approach
developed in our laboratory. 15]
Scheme 1. Scheme 1. One-pot two-step deracemization of alcohols using a
single TeSADH mutant. X = G or A. R = Phenyl-ring-containing substituents.
[
In an effort to improve the enantiopurities of the alcohols obtained
by this method, we explored the use of two mutants of TeSADH
that vary in their enantioselectivities, one for the non-
stereospecific oxidation step and the other for the stereoselective
reduction. Herein, we report the implementation of this approach
using W110G and W110V TeSADH mutants in the oxidation and
reduction steps, respectively, to accomplish deracemization of
racemic alcohols and stereoinversion of (R)-alcohols.
Table 1. Bi-enzymatic deracemization of racemic phenyl-ring containing
secondary alcohols.[
a]
Results and Discussion
The medium enantioselectivities encountered with few substrates
in the single enzymatic deracemization, shown in Scheme 1,
could be explained by the incomplete depletion of (R)-alcohol in
the oxidation reaction or the selectivity mistakes that could be
encountered in the second step by virtue of using a marginally
selective enzyme, or both. To improve the enantioselectivity of
this sequential deracemization approach, we conducted the
oxidation step using W110G TeSADH, which is known for its
marginal enantioselectivity with phenyl-ring-containing secondary
alcohols.[ This approach should ensure the maximum depletion
of both enantiomers of alcohol substrates. We selected 1-phenyl-
R1
R2
Entry
Substrat
e
Ketone
[%]
ee
[%][b]
1
2
3
4
5
6
7
(rac)-1a
(rac)-1a
(rac)-1b
(rac)-1b
(rac)-1c
(rac)-1d
(rac)-1e
p-HO-C
p-HO-C
6
H
H
4
(CH
(CH
2
2
)
2
2
CH
CH
CH
CH
CH
CH
CH
3
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
>99
>99[c]
98
6
4
)
3
3
3
3
3
2
p-MeO-C
p-MeO-C
6
H
H
4
(CH
(CH
2
2
)
2
6
)
2
98[c]
95
16]
4
C
C
C
6
H
6
H
6
H
5
5
5
CH
(CH
CH
2
2-propanol [(rac)-1c] because it was deracemized with low
2
)
2
>99
>99
efficiency using W110G or W110A TeSADHs in the previously
[
]
reported single enzymatic deracemization approach. 15
2
CH
3
Quantitative conversion of (rac)-1c to the corresponding ketone
was achieved; this step was carried out using acetone (3%, v/v),
which was used as a co-substrate and a co-solvent at the same
time. Upon full conversion of (rac)-1c to its ketone using W110G
TeSADH, a heat treatment to deactivate this mutant was applied
before proceeding with the enantioselective reduction step; this
step was necessary to prevent the intervention of W110G
TeSADH in the reduction step. W110V TeSADH was used for the
enantioselective reduction step because it was reported to have
high enantioselectivity in the reduction of phenyl-ring-containing
[
[
a] Unless stated, oxidation reactions were performed by using racemic alcohol
(rac)-1a–e (0.03 mmol)], W110G TeSADH (0.2 mg), NADP+ (1.0 mg), and
acetone (3%, v/v); reduction reactions were performed by using W110V
TeSADH (0.2 mg) and 2-propanol (5%, v/v). [b] The % ee of the acetate ester
derivative of the product was determined by using GC with a chiral stationary
phase. [c] Oxidation reactions were performed using sol-gel encapsulated
W110G TeSADH.
In order to overcome the heat treatment to inactivate the
enzyme used in the oxidation reaction, we used sol-gel
encapsulated W110G TeSADH to perform the oxidation step.[
Encapsulation of enzymes in porous sol-gel offers unique
properties such as large surface area and porosity.[ Affecting
the oxidation of (rac)-1a and (rac)-1b by sol-gel-encapsulated
W110G TeSADH enabled the complete oxidation to their
corresponding ketones. The reduction of the intermediate ketones
was then followed by using free W110V TeSADH after removal of
the sol-gel W110G TeSADH, thus avoiding the heat treatment
after the oxidation step. It was observed that deracemization of
17]
[
]
ketones. 16 The second step was affected by using 2-propanol
5%, v/v). Using these sequential redox reactions, (rac)-1c was
(
18]
deracemized to obtain (S)-1c in 95% ee, compared to 47% ee
when W110G TeSADH was used as a sole catalyst. This
significant improvement in the deracemization efficiency
encouraged us to implement this deracemization, using W110G
and W110V TeSADH, on other phenyl-ring-containing alcohols.
The utilization of two mutants of TeSADH is advantageous since
2
This article is protected by copyright. All rights reserved.

European Journal of Organic Chemistry
10.1002/ejoc.202000728
FULL PAPER
these two substrates using the sol-gel encapsulated enzyme Conclusion
(
entries 2 and 4 of Table 1) gave the same results in terms of ee
and percentage recovery when compared to the results obtained
by the free enzymatic deracemization approach. This sol-gel
encapsulated enzymatic deracemization approach could enable
deracemization of heat labile racemic alcohols without
decomposition. Moreover, it allows for enzyme recycling.
The ability of W110G TeSADH to deplete both enantiomers of
alcohols in the deracemization reactions described above
indicates that this approach can be used in stereoinversion of (R)-
alcohols, the slightly undesired enantiomer for W110G TeSADH,
In conclusion, the use of a marginally stereoselective W110G
mutant of TeSADH for the oxidation of alcohol racemates and the
highly stereoselective W110V TeSADH for the reduction of the
corresponding ketones enabled a one pot deracemization of
secondary alcohols in two steps. This approach was used in
deracemization of the phenyl-ring-containing racemic alcohols in
high efficiencies (up to >99% ee). It was also employed in the
stereoinversion of (R)-configured alcohols to give up to >99% ee
of (S)-configured alcohols. This approach is an attractive one for
industrial production of enantiopure alcohols from their racemates
(
i.e., Mitsunobu-like reaction). Thus, we extended this approach
to conduct stereoinversion reactions of (R)-1a, (R)-1c, and (R)-1d. because it uses an environmentally benign robust catalyst.
W110G TeSADH-catalyzed oxidation of (R)-alcohols, followed by
W110V TeSADH-catalyzed reduction of resultant prochiral
ketones offered (S)-alcohols in high ee’s (up to >99%), as shown
in Table 2. Whereas, (S)-1c and (S)-1d were previously obtained
Experimental Section
in 33% ee and 80% ee, respectively, using a single enzymatic
stereoinversion approach for their (R)-alcohols catalyzed by
W110G TeSADH.[19] Stereoinversion of undesired enantiomers
that result from transformations such as KR is economically and
environmentally pivotal in industrial sector. The currently reported
environmentally benign stereoinversion reaction of alcohols offers
an alternative to the well-known Mitsunobu stereoinversion
reaction, which, although known for its poor atom efficiency that
is evident by the production of stoichiometric amounts of
hydrazine and phosphine oxide by-products, still widely used.[20]
General
+
Sodium borohydride, NADP , (rac)-1c, (R)-1c, (S)-1c, (R)-1d, (S)-1d, 2a,
2b, 2d, and 2e were purchased from commercial sources and used without
further treatment. Whereas, (rac)-1a, (rac)-1b, (rac)-1d and (rac)-1e were
prepared from their corresponding ketones 2a, 2b, 2d, and 2e, respectively,
using sodium borohydride.[21] However, (R)-1a was prepared by Candida
antarctica lipase
[
B
(CaLB)-catalyzed KR of (rac)-1a. 16] All Gas
Chromatography (GC) analyses were conducted using a capillary gas
chromatography (GC) loaded with HP chiral-20B column (30 m, 0.32 mm
[
i.d.], 0.25 μm film thickness) using Helium as the carrier gas with a flame
ionization detector. NMR spectra were recorded on a JOEL JNM-LA500
FT NMR at 500 MHz ( H) and at 125 MHz (13C) at room temperature, using
1
3
deuterated chloroform (CDCl ) peak as an internal standard.
Table 2. Bi-enzymatic stereoinversion of (R)-configured phenyl-ring containing
[
a]
secondary alcohols.
Gene Expression and purification of W110G and W110V TeSADH
mutants
W110G and W110V TeSADHs were expressed and purified as reported
[
,22]
previously. 15
Deracemization of secondary alcohols
Alcohol racemates [(rac)-1a-e, 0.03 mmol] were added into a mixture
+
containing W110G TeSADH (0.2 mg) and NADP (2.0 mg, 2.7 µmol) in
Tris-HCl buffer solution (970 µL, 50 mM, pH 8.0) and acetone (30 µL, 0.41
mmol) in a 2.0-mL Eppendorf tube. The mixture was shaken at 50 °C at
Entry
Substrate
(rac)-1a
(rac)-1c
(rac)-1d
R
Ketone [%]
<0.5
ee [%]
>99
88
180 rpm for about 24 h until both enantiomers were oxidized; reactions
1
2
3
6 4 2 2
p-HO-C H CH CH
were monitored by GC. The solution obtained after the oxidation reaction
was subjected to heat treatment at 80 °C for 45 min to denature any
remaining W110G TeSADH. A fresh W110V TeSADH (0.2 mg) followed
by 2-propanol (50 µL, 0.65 mmol) were added to the same reaction vessel,
and further subjected to shaking for about 24 h at 180 rpm at 50 °C. The
percent conversion and ee were then evaluated by using a GC loaded with
a chiral stationary phase.
C
C
6
H
H
5
CH
CH
2
<0.5
6
5
2
CH
2
<0.5
99
[
a] Unless stated, oxidation reactions were performed using (R)-alcohol (0.03
+
mmol), W110G TeSADH (0.4 mg), and NADP (1.0 mg) in 970 µL Tris-HCl
buffer solution (50 mM, pH 8.0) containing acetone (3%, v/v), at 50 °C with
shaking at 180 rpm for 48 h; reduction reactions were performed using 2-
+
Preparation of sol-gel encapsulated W110G TeSADH
propanol (5%, v/v), W110V TeSADH (0.2 mg) and NADP (1.0 mg) at 50 °C with
shaking at 180 rpm for 24 h. [b] The % ee of the stereoinversion products was
determined using GC with a chiral stationary phase.
Sol-gel encapsulated W110G TeSADH was prepared with little
modification from the previously reported method.[17a] The Sol was
prepared by mixing 2.10 g of tetramethyl orthosilicate (TMOS), 0.47 g of
distilled water and three drops of 0.05 M HCl. The mixture was mixed
thoroughly until a homogeneous phase was achieved. The gels were then
processed by adding the previously prepared sol (1.5 mL) to a solution
The high efficiencies of the currently reported approach in
deracemization of racemic alcohols and in stereoinversion of (R)-
alcohols, when compared to those obtained using a single mutant
of TeSADH, justifies the use of two mutants especially that the
two mutants are for the same enzymes. Future efforts should be
devoted towards the design of new ADH mutants that exhibit
expanded substrate scopes and tuned stereoselectivities to
enable further improvement of the currently reported approach.
+
containing W110G TeSADH and NADP in a 15 mL Eppendorf tube. The
W110G TeSADH and NADP+ were prepared by adding Tris-HCl buffer
solution (50 mM, pH 8.0) to attain final concentration of 0.43 and 3.0
mg.mL-1 for the enzyme and coenzyme, respectively. The sol-gel was
3
This article is protected by copyright. All rights reserved.

European Journal of Organic Chemistry
10.1002/ejoc.202000728
FULL PAPER
subsequently protected with Parafilm and allowed to stand undisturbed for
Keywords: Alcohol dehydrogenases • deracemization • redox
reaction • stereoinversion • stereoselectivity
48h to allow complete formation of a sol-gel encapsulated W110G
TeSADH.
[
[
[
[
[
1]
2]
3]
4]
5]
W. Kroutil, H. Mang, K. Edegger, K. Faber, Curr. Opin. Chem. Biol. 2004,
, 120–126.
K. Goldberg, K. Schroar, S. Lutz, A. Liese, Appl. Microbiol. Biotechnol.
007, 76, 237–248.
Deracemization using sol-gel W110G TeSADH
Alcohol racemates [(rac)-1a or (rac)-b] (0.03 mmol) were introduced into
8
2
+
a mixture of sol-gel encapsulated W110G TeSADH and NADP (2.0 mg,
A. Liese, K. Seelbach, C. Wandrey, Industrial biotransformation, Wiley-
VCH, Weinheim, 2nd edn, 2006.
2.7 µmol) in Tris-HCl buffer solution (970 µL, 50 mM, pH 8.0) and acetone
(30 µL, 0.41 mmol) in a 2.0-mL Eppendorf tube. The mixture was shaken
U. Bornscheuer, R. Kazlauskas, Hydrolases in Organic Synthesis, Wiley-
VCH, Weinheim, 2nd edn, 2006.
at 50 °C at 180 rpm for about 24 h until both enantiomers were oxidized;
reactions were monitored by GC. The encapsulated W110G TeSADH was
removed from the reaction mixture before W110V TeSADH (0.2 mg of 4.6
mg/mL) and 2-propanol (50 µL, 0.65 mmol) were added to the same
reaction vessel, and further subjected to shaking for about 24 h at 180 rpm
Readers are referred to the following reviews on deracemization: a) M.
M. Musa, F. Hollmann, F. G. Mutti, Catal. Sci. Technol. 2019, 9, 5487–
5503; b) A. Diaz-Rodriguez, I. Lavandera, V. Gotor, Curr. Green Chem.
2015, 2, 192–211; c) V. Verho, J. E. Bäckvall, J. Am. Chem. Soc. 2015,
137, 3996–4009; d) G. A. Applegate, D. B. Berkowitz Adv. Synth. Catal.
2015, 357, 1619–1632; e) M. Rachwalski, N. Vermue and F. P. J. T.
2 4
at 50°C. The resulted organic layers were subjected to drying with Na SO
and further concentrated. The percent recovery and the ee of (S)-
configured alcohols were subsequently evaluated using a GC equipped
with a chiral column.
Rutjes, Chem. Soc. Rev. 2013, 42, 9268–9282; f) J. H. Lee, K. Han, M.
J. Kim and J. Park, Eur. J. Org. Chem. 2010, 999–1015; g) J. Steinreiber,
K. Faber, H. Griengle, Chem. Eur. J. 2008, 14, 8060–8072.
Stereoinversion of (R)-configured secondary alcohols
[6]
a) B. T. Cho, Chem. Soc. Rev. 2009, 39, 443–452; b) F. Hollmann, I. W.
C. E. Arends, D. Holtmann, Green Chem. 2011, 13, 2285–2313; c) Q.-A.
Chem, Z.-S. Ye, Y. Duan, Y.-G. Zhou, Chem. Soc. Rev. 2013, 42, 497–
511; d) F. Hollmann, D. J. Opperman, C. E. Paul Angew. Chem Int. Ed.
in press, doi.org/10.1002/anie.202001876.
(
R)-Configured alcohols [(R)-1a, (R)-1c and (R)-1d, 0.03 mmol] were
+
added into a mixture containing W110G TeSADH (0.4 mg) and NADP (2.0
mg, 2.7 µmol) in Tris-HCl buffer solution (970 µL, 50 mM, pH 8.0) and
acetone (30 µL, 0.41 mmol) in a 2.0-mL Eppendorf tube. The mixture was
shaken at 50 °C at 180 rpm for about 36 h until oxidation reaction was
complete; reactions were monitored by GC. The solution obtained after the
oxidation reaction was subjected to heat treatment at 80 °C for 45 min to
denature any remaining W110G TeSADH. A fresh W110V TeSADH (0.2
mg) followed by 2-propanol (50 µL, 0.65 mmol) were added to the same
reaction vessel, and further subjected to shaking for about 24 h at 180 rpm
at 50 °C. The percent conversion and ee were then evaluated by using a
GC loaded with a chiral stationary phase.
[7]
[8]
a) G. R. A. Adair, J. M. Williams, Chem. Commun. 2007, 2608–2609; b)
G. R. A. Adair, J. M. Williams, Chem. Commun. 2005, 5578–5579; c) Y.
Shimada, Y. Miyake, H. Matsuzawa, Y. Nishibayashi, Chem. Asian J.
2007, 2, 393–396.
a) K. Kedziora, A. Diaz-Rodriuez, I. Lavandera, V. Gotor-Fernández, V.
Gotor, Green Chem. 2014, 16, 2448–2453; b) D. Méndez-Sánchez, J.
Mangas-Sánchez, I. Lavandera, V. Gotor, V. Gotor-Fernández,
ChemCatChem 2015, 7, 4016–4020; c) E. Liardo, N. Rios-Lombardia, F.
Moris, J. González-Sabin, F. Rebolledo, Eur. J. Org. Chem. 2018, 3031–
3035.
[
[
[
9]
S. M. A. De Wildeman, T. Sonke, H. E. Schoemaker, O. May, Acc. Chem.
Res. 2007, 40, 1260–1266.
Determination of enantiomeric excess
10] J. C. Moore, D. J. Pollard, B. Kosjek, P. N. Devine, Acc. Chem. Res.
007, 40, 1412–1419.
The produced alcohols were converted to their corresponding acetate
esters by treatment with two drops of acetic anhydride and three drops of
pyridine prior to their analysis by the chiral GC. The following method was
used in the GC analysis: Initial oven temperature was 100 °C for 10 min to
2
11] a) C. E. Paul, I. Lavandera, V. Gotor-Fernández, W. Kroutil, V. Gotor,
ChemCatChem 2013, 5, 3875–3881; b) B. Li, Y. Nie, X. Q. Mu, Y. Xu, J.
Mol. Catal. B: Enzym. 2016, 129, 21–28; c) S. Venkataraman, A. Chadha,
J. Ind. Microbiol. Biotechnol. 2015, 42, 173–180; d) T. Sivakumari, A.
Chadha, RSC Adv., 2015, 5, 91594–91600; e) T. Saravanan, R.
Selvakumar, M. Doble, A. Chadha, Tetrahedron Asymmetry 2012, 23,
1
80 °C for 20 min at 5 °C/min; injector 220 °C, detector 230 °C; and the
Helium at 15 mL/min. The volume injected was 1.0 µL with split ratio of
0:1.
1
1
360–1368; f) T. Cazetta, P. J. S. Moran, A. R. Rodrigues, J. Mol. Cat.
Determination of absolute configuration of alcohols
B: Enzym. 2014, 109, 178–183; g) M.C. Fragnelli, P. Hoyos, D. Romano,
R. Gandolfi, A. R. Alcantara, F. Molinari, Tetrahedron 2012, 68, 523–528;
h) Y.-P. Xue, Y.-G. Zheng, Y.-Q. Zhang, J.-L.-Sun, Z.-Q. Liu, Y.-C. Shen,
Chem. Commun. 2013, 49, 10706–10708; i) C. V. Voss, C. C. Gruber,
W. Kroutil, Angew. Chem. Int. Ed. 2008, 47, 741–745; Angew. Chem.
2008, 120, 753–757.
The absolute configurations of the produced alcohols were elucidated by
comparing the chiral GC retention time of their acetate derivatives with
either their commercially available (S)- or (R)-acetate enantiomer or the
acetate derivatives of alcohols prepared by W110A TeSADH-catalyzed
asymmetric reduction of their ketones, which are reported to produce (S)-
alcohols,[23] and (R)-configured alcohols synthesized by CaLB-catalyzed
KR of racemic alcohols. 16]
[12] C. V. Voss, C. C. Gruber, K. Faber, T. Knaus, P. Macheroux, J. Am.
Chem. Soc. 2008, 130, 13969–13972.
[
[13] F. O. Bryant, J. Wiegel, L. G. Ljungdahl, Appl. Environ. Microbiol. 1988,
54, 460–465.
[
[
14] M. M. Musa, K. I. Ziegelmann-Fjeld, C. Vieille, R. S. Phillips, Org. Biomol.
Chem. 2008, 6, 887–892.
Acknowledgements
15] I. Karume, M. M. Musa, S. M. Hamdan, M. Takahashi, ChemCatChem
2016, 8, 1459–1463.
The authors acknowledge the support provided by the Deanship
of Scientific Research (DSR) at King Fahd University of Petroleum
and Minerals (KFUPM) for funding this work through project
number DF191007. The authors also thank Prof. Claire Vieille,
from the Department of Microbiology and Molecular Genetics as
well as Biochemistry and Molecular Biology at Michigan State
University, for providing the plasmids of TeSADH.
[16] J. M. Patel, M. M. Musa, D. A. Rodriguez, D. A. Sutton, V. V. Popik, R.
S. Phillips, Org. Biomol. Chem. 2014, 12, 5905–5910.
[
17] a) M. M. Musa, K. I. Ziegelmann-Fjeld, C. Vieille, J. G. Zeikus, R. S.
Phillips, Angew. Chem. 2007, 119, 3151–3154; Angew. Chem. Int. Ed.
2
007, 46, 3091–3094; b) L. M. Ellerby, C. R. Nishida, F. Nishida, S. A.
Yamanaka, B. Dunn, J. S. Valentine, J. I. Zink, Science 1992, 255, 1113–
115.
1
4
This article is protected by copyright. All rights reserved.

European Journal of Organic Chemistry
10.1002/ejoc.202000728
FULL PAPER
[
[
[
18] D. Koszelewski, N. Müller, J. H. Schrittwieser, K. Faber, W. Kroutil, J.
Mol. Catal. B-Enzym. 2010, 63, 39–44.
19] M. M. Musa, I. Karume, M. Takahashi, S. M. Hamdan, N. Ullah,
ChemistrySelect 2018, 3, 10205–10208.
20] a) J. S. Carey, D. Laffan, C. Thomson, M. T. Willams, Org. Biomol. Chem.
2006, 4, 2337–2347; b) K. C. K. Swamy, N. N. B. Kumar, E. Balaraman,
K. V. P. P. Kumar, Chem. Rev. 2009, 109, 2551–2651.
21] L. A. Gemal, L. J. Luche, J. Am. Chem. Soc. 1981, 103, 5454–5459.
22] O. Bsharat, M. M. Musa, C. Vieille, S. A. Oladepo, M. Takahashi, S. M.
Hamdan, ChemCatChem 2017, 9, 1487–1493.
[
[
[
23] M. M. Musa, K. I. Ziegelmann-Fjeld, C. Vieille, J. G. Zeikus, R. S. Phillips,
J. Org. Chem. 2007, 7, 451–460.
5
This article is protected by copyright. All rights reserved.

European Journal of Organic Chemistry
10.1002/ejoc.202000728
FULL PAPER
Entry for the Table of Contents
A one-pot two-step deracemization approach for alcohols that uses two mutants of Thermoanaerobacter pseudoethanolicus
secondary alcohol dehydrogenase that exhibit different extents of stereoselectivity is reported. This approach is also used in
stereoinversion of (R)-alcohols (i.e., Mitsunobu-like reaction).
Institute and/or researcher Twitter usernames: KFUPM
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
.
.
W. Kroutil, H. Mang, K.
K. Goldberg, K. Schroar, S. Lutz, A. Liese, Appl
A. Liese, K. Seelbach, C. Wandrey Industrial iotransformation, Wiley-VCH, Weinheim
U. Bornscheuer, R. Ka zlaus as, Hydrolases in Organic Synthesis, Wiley-VCH, Weinheim
E
degger, K. Faber, Curr. Opin. Chem.
B
iol. 2004, 8, 120–126.
.
Microbiol. Biotechnol. 2007,
7
6, 237–248.
,
b
,
,
2nd edn, 2006.
2nd edn, 2006.
k
.
Readers are referred to the follow ing reviews on deracemization: a) M. M. Musa, F. Hollmann, F. G. Mutti, Catal. Sci. Technol. 2019, 9,
a) B. T. Cho Chem. oc. Rev. 2009, 39, 443–452; b) F. Hollmann, I. W. C. E. Arends, D. Holtmann, Green Chem. 2011, 13, 2285–2313; c) Q.-A. Chem
a) G. R. A. Adair, J. M. Williams, Chem. Commun. 2007, 608–2609; b) G. R. A. Adair, J. M. illiams, Chem. Comm un. 2005, 5578–5579 c) Y. Shimada, Y. Miyake, H. Matsuzawa, Y. Nishibayashi, Chem.
a) K. Kedziora, A. Diaz- Rodriue z, I. Lavandera, V. Gotor-Fernández, Gotor, Green Chem. 2014, 16, 2448–2453; b) D. Méndez-Sánche z, J. Mangas-Sánchez, I. Lavandera, V. Gotor, V. otor-Fernández, Chem CatChem 2015, 7, 4016–4020
S. M. A. De Wildeman, T. Son e, H. E. Schoemaker, O. May Acc. Chem. Res. 2007, 0, 1260–1266.
J. C. Moore, D. J. Pollard, B. Kosjek P. N. Devine, Acc. Chem. Res. 2007, 40, 1412–1419.
a) C. E, Paul, I. Lavandera, V. Gotor-Fernández, W. Kroutil, V. Gotor, ChemCatChem, 013, 5, 3875–3881; b) B. Li
a) C. V. Voss, C. C. Gruber, W. Kroutil, Ange w. Chem. Int. Ed. 008, 47 741–745; b) C. V. Voss, C. C. Gruber, K. Faber, T. Knaus, P. Macheroux, J. Am. Chem. Soc. 2008, 130
F. O. Bryant, J. Wiegel, L. G. Ljungdahl, ppl. nviron. icrobiol. 1988 54, 460–465.
M. M. Musa, K. I. Ziegelmann-Fjeld, C. Vieille, R. S. Phillips Org. iomol. Chem. 2008, 6,
I. Karume, M. M. Musa, S. M. Hamdan, M. Ta ahashi, Chem CatChem 2016, 8, 1459–1463.
J. M. Patel, M. M. usa, D. A. Rodrigue z, D. A. Sutton, V. Popi R. S. Phillips, rg. Biomol. Chem. 2014, 12,
a) M. M. Musa, K. I. Ziegelmann-Fjeld, C. Vieille, J. G. Zei us, R. S. Phillips Angew. Chem. Int. Ed. 2007, 46, 091–3094; b) L. M. Ellerby
K. Domini M. Nicole, J. H. Schrittwieser, F. Kurt, K. Wolfgang J. Mol. Catal. B-Enzym. 2010, 63, 39–44.
M. M. Musa, I. Karume, M. Takahashi, S. M. Hamdan, N. Ullah, ChemistrySelect 2018, 3, 10205–10208.
a) J. S. Carey D. Laffan, C. Thomson and M. Willams, Org Biomol. Chem. 2006, 2337–2347; b) K. C. K. Swam
L. A. Gemal, L. J. Luche, J. Am. Chem. oc. 1981, 03, 5454–5459
ladepo, M. Ta ahashi, S. M. Hamdan, Chem CatChem 2017, 9, 1487–1493.
5
487–5503; b) A.
D
iaz-Rodrigue z, I. Lavandera, V. Gotor, Curr. Green Chem. 2015, 2, 192–211; c) V. Verho,
uan, Y.-G. Zhou, Chem. Soc. Rev. 2013, 42, 97–511 d) F. Hollmann, D. J. Opperman, C. E. Paul Ange w. Chem Int. Ed. in press, doi.org/10.1002/anie.202001876.
sian J. 007, 2, 393–396.
J. E. Bäc kvall, J. Am. Chem. Soc. 2015, 137, 3996–4009; d) G. A. Applegate, D. B. Ber kowitz Adv. Synth. Catal. 2015, 357, 1619–1632; e) M. Rachwalski, N. Vermue and F. P. J. T. Rutjes, Chem. Soc. Rev. 2013, 42, 9268–9282; f) J. H. Lee, K. Han, M. J. K im and J. Park, Eur. J. Org. Chem. 2010, 999–1015; g) J. Steinreiber, K. Faber, H. Griengle, Chem. Eur. J. 2008, 14, 8060–8072.
.
,
S
,
Z.-S. Ye, Y.
D
4
;
.
2
W
;
A
2
.
V
.
G
; c) E. Liardo, N. Rios-Lombardia, F. Moris, J. Gonzále z-Sabin, F. Rebolledo, Eur. J. Org. Chem. 2018, 3031–3035.
.
k
,
4
0.
,
1.
2
,
Y. Nie, X. Q. Mu, Y.
X
u, J. Mol. Catal. B: Enzym. 2016, 129
,
21–28
;
c) S. Ven
kataraman, A. Chadha, J. Ind. Microbiol. Biotechnol. 2015, 42, 173–180
;
d) T. Siva
kumari, A. Chadha,
R
SC Adv., 2015, 5, 91594–91600
;
e) T. Saravanan, R. Selva
kumar, M. Doble, A. Chadha, Tetrahedron Asymmetry 2012, 23,
1
360–1368; f) T. Cazetta, P. J. S. Moran, A. R. Rodrigues, J. Mol. Cat. B: Enzym. 2014, 109, 178–183; g) M.C. Fragnelli, P. Hoyos, D. Romano, R. Gandolfi, A. R. A lcantara, F. Molina ri, Tetrahedron 2012, 68, 523–528; h) Y.-P. Xue, Y.-G. Zheng, Y.-Q. Zhang, J.-L.-Sun, Z.-Q. Liu, Y.-C. Shen, Chem. Commun. 2013, 49, 10706–10708;
2.
2
,
,
13969–13972.
3.
A
E
M
,
4.
,
B
887–892.
5.
k
6.
M
V
.
k,
O
5905–5910.
7.
k
,
3
, C. R. Nishida, F. Nishida, S. A. Yamanaka, B. Dunn, J. S. Valentine, J. I. Zink, Science 1992, 255, 1113–1115.
8.
k,
,
9.
0.
,
T
.
.
4
,
y, N. N. B. Kumar, E. Balaraman and K. V. P. P. Kumar, Chem. Rev. 2009, 109, 2551–2651.
1.
S
1
.
2. O. Bsharat, M. M. Musa, C. Vieille, S. A.
O
k
3.
M. M. Musa, K. I. Ziegelmann-Fjeld, C. Vieille, J. G. Zeikus, R. S. Phillips, J. Org. Chem. 2007, 7, 451–460.
6
This article is protected by copyright. All rights reserved.