One-pot two-step chemoenzymatic deracemization of allylic alcohols using laccases and alcohol dehydrogenases

Article abstract of DOI:10.1016/j.mcat.2020.111087

A series of enantioenriched (hetero)aromatic secondary allylic alcohols has been synthesized through deracemization of the corresponding racemic mixtures combining a non-selective chemoenzymatic oxidation (laccase from Trametes versicolor and oxy-radical TEMPO) and a stereoselective biocatalyzed reduction (lyophilized cells of E. coli overexpressing an alcohol dehydrogenase, ADH). Both steps were performed in aqueous medium under very mild reaction conditions. After optimization, a sequential one-pot two-step protocol was set up, obtaining the corresponding chiral alcohols in moderate to high conversions (48–95%) and enantiomeric excess (65->99% ee). Depending on the ADH stereopreference, both antipodes from these valuable chiral synthons could be prepared, even at preparative scale (119?178 mg), in a straightforward manner.

Full text of DOI:10.1016/j.mcat.2020.111087

MolecularCatalysis493(2020)111087
Contents lists available at ScienceDirect
Molecular Catalysis
journal homepage: www.elsevier.com/locate/mcat
One-pot two-step chemoenzymatic deracemization of allylic alcohols using
laccases and alcohol dehydrogenases
Jesús Albarrán-Velo, Vicente Gotor-Fernández*, Iván Lavandera*
Organic and Inorganic Chemistry Department, Universidad de Oviedo, Avenida Julián Clavería 8, 33006, Oviedo, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords:
Alcohol dehydrogenases
Bioreduction
Deracemization
Laccases
One-pot synthesis
A series of enantioenriched (hetero)aromatic secondary allylic alcohols has been synthesized through derace-
mization of the corresponding racemic mixtures combining a non-selective chemoenzymatic oxidation (laccase
from Trametes versicolor and oxy-radical TEMPO) and a stereoselective biocatalyzed reduction (lyophilized cells
of E. coli overexpressing an alcohol dehydrogenase, ADH). Both steps were performed in aqueous medium under
very mild reaction conditions. After optimization, a sequential one-pot two-step protocol was set up, obtaining
the corresponding chiral alcohols in moderate to high conversions (48–95%) and enantiomeric excess (65-
> 99% ee). Depending on the ADH stereopreference, both antipodes from these valuable chiral synthons could
be prepared, even at preparative scale (119−178 mg), in a straightforward manner.
1. Introduction
Alternatively, biocatalytic tools have become very relevant along
the last decades as enzymes can promote extremely selective transfor-
Chiral allylic alcohols are versatile building blocks for organic
synthesis as they contain two moieties, an alcohol and a carbon-carbon
double bond, which can be subsequently modified in order to give ac-
cess to molecules with up to three contiguous chiral centers. In this
context, many different reactions have been designed where the hy-
droxyl group directs the transformation of the alkene moiety, such as
epoxidation [1], hydrogenation [2], dihydroxylation [3], or hydro-
formylation [4], among others. Also in the last years, different ap-
proaches have been developed to substitute the alcohol group by dif-
ferent nucleophiles [5], affording a broad spectrum of highly valuable
derivatives.
As a consequence, several chemical methodologies have been de-
scribed to synthesize these compounds in a chiral fashion [6]. Recent
examples involve the (stereoselective) formation of C-C bonds, such as
the alkenylation of aldehydes [7], the coupling of alcohols and alkynes
under redox neutral conditions [8], the aldol addition on α,β-un-
saturated ketones [9], the alkyl addition on α,β-unsaturated aldehydes
[10], and the Stille coupling reaction [11]. Other methods rely on the
selective creation of the C-O bond, such as the addition of N-hydro-
xyphthalimide to allenes [12], or the C-H bond, through the metal-
catalyzed reduction of enones [13–15] or vinyl epoxides [16]. Other
procedures involve the metal- or organocatalyzed isomerization of
oxiranes [17] and the kinetic resolution of racemic allylic alcohols
[18,19].
mations under very mild reaction conditions. For instance, en-
antioenriched allylic alcohols have been obtained through lipase-
mediated kinetic [20,21] and dynamic kinetic resolutions [22–25] of
the corresponding racemates. Also, oxidoreductases have been suc-
cessfully applied to obtain these chiral compounds through the oxida-
tive kinetic resolution catalyzed by alcohol dehydrogenases (ADHs)
[26] or alcohol oxidases [27] starting from the racemic alcohols, or the
bioreduction of the corresponding α,β-unsaturated ketones [28,29].
Due to the relevance of multi-step catalyzed syntheses, the design of
chemoenzymatic [30,31] or multienzymatic [32,33] protocols where
allylic alcohols are obtained or involved as intermediates has also been
envisaged.
An elegant manner to get access to enantioenriched sec-alcohols is
performing deracemization protocols starting from the racemic forms
[34–37]. From an atom economic point of view, this transformation is
ideal as both substrate and product are the same compound. Although
this approach is very simple, final compounds are frequently not ob-
tained in high enantiomeric excess (ee) and also by-products can be
formed, due to the presence of many different enzymes, since tradi-
tionally, whole-cell biocatalysts are employed. For instance, Chadha
and co-workers reported the deracemization of racemic allylic alcohols
through an oxidation-reduction strategy using a yeast [38,39]. Next,
Kroutil and co-workers demonstrated that deracemization of secondary
alcohols could be achieved via stereoinversion in a cascade fashion
⁎ Corresponding authors.
E-mail addresses: vicgotfer@uniovi.es (V. Gotor-Fernández), lavanderaivan@uniovi.es (I. Lavandera).
https://doi.org/10.1016/j.mcat.2020.111087
Received 8 May 2020; Received in revised form 13 June 2020; Accepted 16 June 2020
Availableonline07July2020
2468-8231/©2020ElsevierB.V.Allrightsreserved.

J. Albarrán-Velo, et al.
MolecularCatalysis493(2020)111087
Scheme 1. a) General representation of a linear deracemization concept. b) Linear deracemization of sec-allylic alcohols combining the laccase/TEMPO system with
an ADH. A, B: substrate (and product) enantiomers; I: intermediate.
[40]. For this, two enantiocomplementary ADHs are necessary with
different nicotinamide cofactor preference (NADH or NADPH), pro-
viding the final alcohols with excellent ee values. Whereas this method
is very smart, it can suffer from relatively limited substrate scope and
undesired crossed regeneration cofactor systems, lowering both con-
versions and selectivities. A subsequent approach is the use of linear
deracemizations [37], where the racemic sec-alcohol is transformed
into the corresponding ketone intermediate via non-selective oxidation,
followed by a stereoselective reduction using a chiral (bio)catalyst
(Scheme 1a). This system presents the advantage that just one selective
reaction is necessary to get access to the enantioenriched product,
broadening the possible scope of the reaction. On the contrary, it is not
possible to perform both steps in a cascade manner as the final product
would also be oxidized, therefore, one-pot sequential methodologies
must be strictly followed.
In the search for novel mild oxidative methods, we and others found
out that the combination of a laccase [41–44] with substoichiometric
amounts of the oxy-radical TEMPO is very suitable to achieve the oxi-
dation of secondary benzylic [45–49], allylic [50–52] and propargylic
alcohols [53,54] in aqueous medium employing oxygen as final elec-
tron acceptor. This transformation has been coupled to other (chemo)
enzymatic reactions to perform the amination of racemic alcohols
[48,49,52] and the isomerization of allylic alcohols into saturated
carbonyl compounds [50,51]. It has also been envisaged as the first step
in linear deracemizations of profenols [55], benzylic [47], and pro-
pargylic [53] alcohols when it was combined sequentially with alcohol
dehydrogenases [56]. Herein, the application of the laccase from Tra-
metes versicolor (LTv) and TEMPO with stereocomplementary ADHs is
disclosed, to accomplish the synthesis of valuable enantioenriched
allylic alcohols starting from the racemic compounds in a one-pot se-
quential mode (Scheme 1b).
Thin layer chromatographies (TLCs) were conducted with silica gel
precoated plates and visualized with UV and potassium permanganate
stain. Column chromatographies were performed using silica gel
(230–400 mesh).
The oxidation step mediated by the laccase/TEMPO catalytic system
was optimized in an open-to-air test tube using magnetic stirring, while
the bioreduction step was studied in 1.5 mL Eppendorf vial. Sequential
reactions were performed in sealed glass tubes of different sizes
(18 × 72 × 10), (18 × 109 × 10) or (28 × 137 × 20) mm] depending
on the reaction scale.
NMR spectra were recorded on a 300 MHz spectrometer. All che-
mical shifts (δ) are given in parts per million (ppm) and referenced to
the residual solvent signal as internal standard. IR spectra were re-
corded on a spectrophotometer on NaCl pellets. Gas chromatography
(GC) analyses were performed on standard gas chromatograph appa-
ratus equipped with a FID detector. High performance liquid chroma-
tography (HPLC) analyses were carried out in a chromatograph with UV
detector at a 210 nm wavelength. High resolution mass spectra (HRMS)
were obtained in a spectrometer using the ESI-TOF positive mode.
Measurement of the optical rotation was carried out at 590 nm in a
standard polarimeter.
2.2. Typical procedure for the synthesis of (E)-4-(het)arylbut-3-en-2-ones
Ketones 2b-p were synthesized following the procedure already
described by List and co-workers for ketones 2a, 2l and 2o [60]. To a
solution of the corresponding aldehyde (2.5 mmol) in acetone (6.3 mL),
morpholinium trifluoroacetate (0.5 mmol) was added, and the reaction
was stirred at 75 °C in a sealed tube overnight. After this time the re-
action mixture was cooled at room temperature, and a NaHCO3 satu-
rated aqueous solution (5 mL) and EtOAc (10 mL) were added. After
phase separation, the aqueous phase was extracted three times with
EtOAc (10 mL), and the combined organic phases dried over anhydrous
Na2SO4, filtered and the solvent evaporated under reduced pressure.
The reaction crude was purified by column chromatography on silica
gel (25% EtOAc/hexane), yielding the corresponding ketone 2b-p
(59–96% yield): 2b (92%, 406 mg), 2c (71%, 320 mg), 2d (61%,
251 mg), 2e (59%, 333 mg), 2f (78%, 344 mg), 2g (63%, 259 mg), 2h
(85%, 340 mg), 2i (77%, 339 mg), 2j (68%, 280 mg), 2k (72%,
266 mg), 2l (81%, 299 mg), 2m (75%, 255 mg), 2n (94%, 320 mg), 2o
(82%, 313 mg) and 2p (96%, 366 mg). The NMR data of these com-
pounds were in agreement with those reported in the literature [52,60].
2. Experimental
2.1. Material and methods
Laccase from Trametes versicolor (LTv, 0.66 U/mg) was purchased
from Sigma-Aldrich. Made in house ADHs overexpressed in E. coli and
later lyophilized [Ralstonia species (RasADH), Sphingobium yanoikuyae
(SyADH), Thermoanaerobacter species (ADH-T), Lactobacillus brevis
(LBADH), Thermoanaerobacter ethanolicus (TeSADH) and Rhodococcus
ruber (ADH-A)] were obtained as previously reported in the literature
[57–59], while evo-1.1.200 ADH was acquired from Evoxx technologies
GmbH. Glucose dehydrogenase (GDH-105) was purchased from Co-
dexis Inc. D-Glucose, NADPH, NADH, ketone 2a and all other reagents
for chemical transformations and product isolation/purification were
obtained from commercial sources and used as received. Citrate buffer
(pH 5, 50 mM) was saturated with molecular oxygen by bubbling it for
15 min prior to be used in the chemoenzymatic oxidation experiments.
2.3. Typical procedure for the synthesis of (E)-4-(het)arylbut-3-en-2-ols
Alcohols 1a-p were synthesized following the procedure described
by our research group [52]. A solution of NaBH4 (170.2 mg, 4.5 mmol)
in water (1 mL) was added dropwise to a stirring solution of the cor-
responding (3E)-4-(het)arylbut-3-en-2-one 2a-p (3 mmol) in MeOH
2

J. Albarrán-Velo, et al.
MolecularCatalysis493(2020)111087
(10 mL) at 0 °C. The reaction mixture was firstly stirred for 1 h in an ice
bath and then 2 h at room temperature. Afterwards, hot water (5 mL)
was added and the product was extracted with CH2Cl2 (3 × 15 mL), and
the combined organic phases were washed with brine (5 mL). The re-
sulting organic layer was dried over anhydrous Na2SO4, filtered and the
solvent evaporated under reduced pressure. The reaction crude was
purified by column chromatography on silica gel (50% Et2O/hexane,
83% EtOAc/hexane or 25% EtOAc/hexane), yielding the corresponding
alcohol 1a-p (46–99% yield): 1a (99%, 445 mg), 1b (88%, 469 mg), 1c
(64%, 351 mg), 1d (46%, 230 mg), 1e (57%, 387 mg), 1f (78%,
417 mg), 1g (71%, 354 mg), 1h (85%, 414 mg), 1i (81%, 435 mg), 1j
(76%, 380 mg), 1k (87%, 389 mg), 1l (91%, 409 mg), 1m (89%,
369 mg), 1n (87%, 361 mg), 1o (91%, 423 mg) and 1p (71%, 328 mg).
The NMR data of these compounds were in agreement with those re-
ported in the literature [52].
(2 min, 13,000 rpm). This extraction and centrifugation protocol was
repeated twice, and the organic layers were combined and dried over
Na2SO4.
Conversion values into the corresponding enantioenriched alcohols
1a-p were determined by taking an aliquot of the resulting suspensions
and injecting them for GC analysis (see the Supplementary data).
Derivatization of alcohols 1a-p as acetates with acetic anhydride was
necessary for the measurement of their enantiomeric excess values: An
aliquot of the corresponding alcohol 1a-p obtained in the bio-
transformation crude and dissolved in EtOAc, was placed in a 1.5 mL
Eppendorf vial. Then, DMAP (10 mg) and acetic anhydride (5 drops)
were added, and the mixture was shaken at 30 °C and 900 rpm for 2 h.
After this time, an aqueous solution of NaOH 10 M (200 μL) was added,
and the organic layer was separated by centrifugation (2 min,
13,000 rpm), dried over Na2SO4, filtered and transferred to a GC glass
vial for analysis.
2.4. Typical procedure for the oxidation of (E)-4-(het)arylbut-3-en-2-ols
using the laccase from Trametes versicolor-TEMPO catalytic system
2.6. Typical procedure for the deracemization of (E)-4-(het)arylbut-3-en-2-
ols at analytical scale
In an open-to-air test tube, TEMPO (1.2–4.1 mg, 10−33 mol%) was
added to a solution of the racemic alcohol 1a-p (0.08 mmol, 100 mM) in
a (biphasic mixture of) oxygen-saturated citrate buffer (pH 5, 50 mM),
studying the possibility of using MTBE (tert-butyl methyl ether) as co-
solvent (up to 50% v/v, for a total solution volume of 800 μL). The
reaction mixture was magnetically stirred for a few minutes to dissolve
all the reagents, and then the laccase from Trametes versicolor (7 mg, 4.6
U) was added. The mixture was stirred for additional 16 h at 30 °C
controlling the agitation speed at 150 rpm. After this time, the product
was extracted with EtOAc (2 × 2 mL), the organic phases were com-
bined, dried over Na2SO4 and filtered, and an aliquot was taken for the
determination of the conversion value by GC (see Tables S1 and S2 in
the Supplementary data).
In an open-to-air glass tube, TEMPO (4.1 mg, 33 mol%) was added
to a solution of the corresponding racemic alcohol 1a-p (0.08 mmol,
100 mM) in a biphasic mixture of an oxygen-saturated citrate buffer
50 mM pH 5 and MTBE (50% v/v, for a total volume of 800 μL). The
reaction mixture was magnetically stirred for a few minutes to dissolve
all the reagents, and then the laccase from Trametes versicolor (7 mg, 4.6
U) was added. The reaction was stirred for 16 h at 30 °C, observing the
complete evaporation of MTBE along this time. This fact led to a vo-
lume reduction from the initial 800 μL to 400 μL, and in consequence,
the substrate concentration increased from the initial 100 mM to ap-
proximately 200 mM.
To the resulting reaction crude containing the ketone intermediate,
different protocols have been applied depending on the alcohol dehy-
drogenase that is used. In the case of LBADH, 2-PrOH (0.3 mL), Tris·HCl
buffer 50 mM pH 8 (0.9 mL), MgCl2 (0.2 mL of a 10 mM solution in
Tris·HCl buffer 50 mM pH 8), and NADPH (0.2 mL of a 10 mM solution
in Tris·HCl buffer 50 mM pH 8) were added, leading to concentrations
of approximately 40 mM for the substrate, 1 mM for MgCl2, 1 mM for
NADPH and 15% v/v for 2-PrOH. At the same time, the addition of this
concentrated buffer to the reaction medium, caused an increase in the
pH from an initial value of 5 to approximately 7.5, therefore, further pH
adjustment was not required for the bioreduction reaction.
For ADH-T, 2-PrOH (0.3 mL), Tris·HCl buffer 50 mM pH 8 (1.1 mL),
and NADPH (0.2 mL of a 10 mM solution in Tris·HCl buffer 50 mM pH
8) were added, leading to concentrations of approximately 40 mM for
the substrate, 1 mM for NADPH and 15% v/v for 2-PrOH. At the same
time, the addition of this concentrated buffer to the reaction medium,
caused an increase in the pH from an initial value of 5 to approximately
7.5, therefore, further pH adjustment was not required for the bior-
eduction reaction.
In the case of ADH-A, 2-PrOH (0.3 mL), Tris·HCl buffer 50 mM pH 8
(1.1 mL), and NADH (0.2 mL of a 10 mM solution in Tris·HCl buffer
50 mM pH 8) were added, leading to concentrations of approximately
40 mM for the substrate, 1 mM for NADH and 15% v/v for 2-PrOH. At
the same time, the addition of this concentrated buffer to the reaction
medium, caused an increase in the pH from an initial value of 5 to
approximately 7.5, therefore, further pH adjustment was not required
for the bioreduction reaction.
2.5. Typical procedure for the bioreduction of (E)-4-(het)arylbut-3-en-2-
ones using overexpressed alcohol dehydrogenases
For E. coli/RasADH: In
a 1.5 mL Eppendorf vial, ketone 2a
(0.015 mmol, 25 mM) was dissolved in DMSO (10% v/v, 60 μL). Then,
Tris·HCl buffer 50 mM pH 7.5 (540 μL) containing glucose (50 mM),
GDH (10 U) and NADPH (1 mM) was added. Finally, overexpressed E.
coli/RasADH lyophilized cells (10 mg) were added. The reaction was
shaken at 30 °C and 250 rpm for 24 h, and extracted with EtOAc
(0.5 mL), separating the organic layer by centrifugation (2 min,
13,000 rpm). This extraction and centrifugation protocol was repeated
twice, and the organic layers were combined and dried over Na2SO4.
For the other ADHs: In a 1.5 mL Eppendorf vial, ketone 2a
(0.015 mmol, 25 mM) was dissolved in 2-PrOH (10% v/v, 60 μL). Then,
Tris·HCl buffer 50 mM pH 7.5 (540 μL) containing MgCl2 (1 mM, for E.
coli/LBADH and evo-1.1.200) and NADPH (1 mM for E. coli/LBADH, E.
coli/ADH-T, E. coli/SyADH, and E. coli/TeSADH) or NADH (1 mM for E.
coli/ADH-A and evo-1.1.200) was added. Finally, E. coli lyophilized
cells or evo-1.1.200 (10 mg) were added. The reaction was shaken at
30 °C and 250 rpm for 24 h, and extracted with EtOAc (0.5 mL), se-
parating the organic layer by centrifugation (2 min, 13,000 rpm). This
extraction and centrifugation protocol was repeated twice, and the
organic layers were combined and dried over Na2SO4.
General procedure under the optimized conditions: In a 1.5 mL
Eppendorf vial, the corresponding ketone 2a-p (0.024 mmol, 40 mM)
was dissolved in 2-PrOH (15% v/v, 90 μL). Then, Tris·HCl buffer 50 mM
pH 7.5 (390 μL for E. coli/LBADH; 450 μL for E. coli/ADH-T or E. coli/
ADH-A), MgCl2 (60 μL, 10 mM for E. coli/LBADH), and NADPH (60 μL,
10 mM, for E. coli/LBADH or E. coli/ADH-T) or NADH (60 μL, 10 mM,
for E. coli/ADH-A) were added. Finally, lyophilized cells containing the
overexpressed alcohol dehydrogenase (10 mg) were added. The reac-
tion was shaken at 30 °C and 250 rpm for 24 h, and extracted with
EtOAc (0.5 mL); the organic layer was separated by centrifugation
Finally, the corresponding E. coli cells overexpressing the alcohol
dehydrogenase (33.3 mg) were added. The sealed tube was closed and
the reaction was shaken at 30 °C and 250 rpm for 24 h. After this time,
the reaction was stopped by addition of distilled water or NaOH 10 M
aqueous solution (for alcohols 1k-l, 1.5 mL). The mixture was extracted
with EtOAc (5 mL) and the organic layer was separated by centrifuga-
tion (3 min, 4900 rpm). This extraction and centrifugation protocol was
performed twice and, finally, the organic layers were combined and
3

J. Albarrán-Velo, et al.
MolecularCatalysis493(2020)111087
dried over Na2SO4. Conversion and ee values were measured as pre-
viously described. The products were purified through column chro-
matography using the following eluents: 50% Et2O/hexane (1a-j), 83%
EtOAc/hexane (1k and 1l) or 25% EtOAc/hexane (1m-p).
reduction from the initial 8 mL to 4 mL, and as a consequence, the
substrate concentration increased from the initial 100 mM to approxi-
mately 200 mM.
To the resulting reaction mixture containing the ketone inter-
mediate 2a, 2-PrOH (3 mL), Tris·HCl buffer 50 mM pH 8 (11 mL), and
NADPH (2 mL of a 10 mM solution in Tris·HCl buffer 50 mM pH 8) were
added, leading to a concentration of approximately 40 mM for ketone
2a, 1 mM for MgCl2, 1 mM for NADPH and 15% v/v for 2-PrOH. At the
same time, the addition of this concentrated buffer to the reaction
medium, caused an increase in the pH from an initial value of 5 to
approximately 7.5, therefore, further pH adjustment was not required
for the bioreduction reaction.
Finally, overexpressed E. coli/ADH-T cells (333 mg) were added.
The glass tube was closed and the reaction shaken at 30 °C and 250 rpm
for 24 h. After this time, the reaction was stopped by addition of dis-
tilled water (20 mL). The mixture was extracted with EtOAc (50 mL)
and the organic layer was separated by centrifugation (3 min,
4900 rpm). This extraction and centrifugation protocol was performed
three times and, finally, the organic layers were combined and dried
over Na2SO4. Analysis through GC showed 76% of conversion and >
99% of enantiomeric excess (as acetate derivative). The product was
purified through column chromatography on silica gel (50% Et2O/
hexane), affording the alcohol (S)-1a in 74% yield.
2.7. Typical procedure for the deracemization of (E)-4-(het)arylbut-3-en-2-
ols at semi-preparative scale
In an open-to-air glass tube, TEMPO (8.2 mg, 33 mol%) was added
to a solution of the appropriate racemic alcohol 1d, 1f, 1g or 1j
(0.16 mmol, 100 mM) in a biphasic mixture of an oxygen-saturated ci-
trate buffer (pH 5, 50 mM) and MTBE (50% v/v, for a total volume of
1.6 mL). The reaction mixture was stirred for a few minutes to dissolve
all the reagents, and then the laccase from T. versicolor (14 mg, 9.2 U)
was added. The reaction mixture was stirred for 16 h at 30 °C, with the
complete evaporation of MTBE being observed during this time. This
led to a volume reduction from the initial 1.6 mL to 800 μL, and as a
consequence, the substrate concentration increased from the initial
100 mM to approximately 200 mM.
To the resulting reaction crude containing the ketone intermediate
(2d, 2f, 2g or 2j), different protocols were applied depending on the
alcohol dehydrogenase that was used. In the case of LBADH, 2-PrOH
(0.6 mL), Tris·HCl buffer 50 mM pH 8 (1.8 mL), MgCl2 (0.4 mL of a
10 mM solution in Tris·HCl buffer 50 mM pH 8), and NADPH (0.4 mL of
a 10 mM solution in Tris·HCl buffer 50 mM pH 8) were added, leading
to concentrations of approximately 40 mM for the substrate, 1 mM for
MgCl2, 1 mM for NADPH and 15% v/v for 2-PrOH. At the same time, the
addition of this concentrated buffer to the reaction medium, caused an
increase in the pH from an initial value of 5 to approximately 7.5,
therefore, further pH adjustment was not required for the bioreduction
reaction.
In the case of ADH-A, 2-PrOH (0.6 mL), Tris·HCl buffer 50 mM pH 8
(2.2 mL), and NADH (0.4 mL of a 10 mM solution in Tris·HCl buffer
50 mM pH 8) were added, leading to concentrations of approximately
40 mM for the substrate, 1 mM for NADH and 15% v/v for 2-PrOH. At
the same time, the addition of this concentrated buffer to the reaction
medium, caused an increase in the pH from an initial value of 5 to
approximately 7.5, therefore, further pH adjustment was not required
for the bioreduction reaction.
Finally, the corresponding E. coli cells overexpressing the alcohol
dehydrogenase (66.6 mg) were added. The sealed tube was closed and
the reaction was shaken at 30 °C and 250 rpm for 24 h. After this time,
the reaction was stopped by addition of distilled water (3 mL). The
mixture was extracted with EtOAc (10 mL) and the organic layer was
separated by centrifugation (3 min, 4900 rpm). This extraction and
centrifugation protocol was performed three times and, finally, the
organic layers were combined and dried over Na2SO4. Conversion va-
lues into the corresponding enantioenriched alcohols 1d, 1f, 1g and 1j
were determined by GC analyses. Derivatization of alcohols 1d, 1f, 1g
and 1j as acetates with acetic anhydride was necessary for the mea-
surement of their enantiomeric excess values. The products were pur-
ified through column chromatography using 50% Et2O/hexane as
eluent, yielding alcohols (R)-1d, (S)-1f, (R)-1g, and (R)-1jin 57-78%.
2.8.2. (R)-1f
In an open-to-air glass tube, TEMPO (52 mg, 33 mol%) was added to
a solution of the racemic alcohol 1f (1 mmol, 178 mg, 100 mM) in a
biphasic mixture of an oxygen-saturated citrate buffer (pH 5, 50 mM)
and MTBE (50% v/v, for a total volume of 10 mL). The reaction mixture
was magnetically stirred for a few minutes to dissolve all the reagents,
and then the laccase from T. versicolor (88 mg, 58 U) was added. The
reaction mixture was stirred for 16 h at 30 °C, being observed the
complete evaporation of MTBE during this time. This led to a volume
reduction from the initial 10 mL to 5 mL, and as a consequence, the
substrate concentration increased from the initial 100 mM to approxi-
mately 200 mM.
To the resulting crude reaction mixture containing the ketone in-
termediate 2f, 2-PrOH (3.75 mL), Tris·HCl buffer 50 mM pH
8
(21.25 mL), MgCl2 (5.1 mg, 0.025 mmol), and NADPH (21 mg,
0.025 mmol) were added, leading to concentrations of approximately
40 mM for ketone 2f, 1 mM for MgCl2, 1 mM for NADPH and 15% v/v
for 2-PrOH. At the same time, the addition of this concentrated buffer to
the reaction medium, caused an increase in the pH from an initial value
of 5 to approximately 7.5, therefore, further pH adjustment was not
required for the bioreduction reaction.
Finally, overexpressed E. coli/LBADH cells (415 mg) were added.
The glass tube was closed and the reaction shaken at 30 °C and 250 rpm
for 24 h. After this time, the reaction was stopped by addition of dis-
tilled water (20 mL). The mixture was extracted with EtOAc (50 mL)
and the organic layer was separated by centrifugation (3 min,
4900 rpm). This extraction and centrifugation protocol was performed
three times and, finally, the organic layers were combined and dried
over Na2SO4. Analysis through GC showed 78% of conversion and 95%
of enantiomeric excess (as acetate derivative). The product was purified
through column chromatography on silica gel (50% Et2O/hexane), af-
fording the alcohol (R)-1f in 76% yield.
2.8. Typical procedure for the deracemization of (E)-4-(het)arylbut-3-en-2-
ols at preparative scale
2.8.1. (S)-1a
2.9. Chemical synthesis of (R)-1-[(2R,3R)-3-(3-methoxyphenyl)oxiran-2-
In an open-to-air glass tube, TEMPO (41 mg, 33 mol%) was added to
a solution of the racemic alcohol 1a (0.8 mmol, 119 mg, 100 mM) in a
biphasic mixture of an oxygen-saturated citrate buffer (pH 5, 50 mM)
and MTBE (50% v/v, for a total volume of 8 mL). The reaction mixture
was magnetically stirred for a few minutes to dissolve all the reagents,
and then the laccase from T. versicolor (70 mg, 46 U) was added. The
reaction mixture was stirred for 16 h at 30 °C, being observed the
complete evaporation of MTBE during this time. This led to a volume
yl]ethan-1-ol (3)
L-(+)-Diethyl tartrate (DET, 175 μL, 0.848 mmol) was added to a
solution of Ti(OiPr)4 (241 μL, 0.848 mmol) in dry CH2Cl2 (5 mL) at
–20 °C. The solution was stirred during 30 min at –20 °C and then, a tert-
butyl hydroperoxide (tBuOOH) solution in decane (5.5 M, 250 μL,
1.38 mmol) was added dropwise. After additional 10 min of stirring at
–20 °C, a solution of (R)-1f (95% ee, 120 mg, 0.673 mmol) in dry CH2Cl2
4

J. Albarrán-Velo, et al.
MolecularCatalysis493(2020)111087
(2 mL) was added dropwise and stirred at –20 °C during 3 h. Afterwards,
the resulting mixture was quenched with water (10 mL) and left
warming up to room temperature during 30 min. The reaction was fil-
tered through a Celite pad and extracted with CH2Cl2 (3 × 15 mL). The
organic phases were combined and washed with brine (2 × 10 mL),
dried over anhydrous Na2SO4, filtered, and the solvent evaporated
under reduced pressure. The reaction crude was analyzed by 1H NMR
finding a 89% conversion and 83% diastereomeric excess. Then, the
reaction crude was purified by preparative thin layer chromatography
(75% Et2O/hexane), yielding the corresponding epoxide 3 (80% yield,
95% ee, 83% de).
reaction conditions considering, if possible, lower amounts of TEMPO
(10 or 20 mol%) and MTBE (0 or 20% v/v). Gladly, it was discovered
that complete conversions into (E)-4-phenylbut-3-en-2-one (2a) were
attained after 16 h, even just employing 10 mol% of TEMPO in the
absence of MTBE (see Table S1 in the Supplementary data), appearing
critical the use of a slow speed magnetic agitation (150 rpm) since it
avoided the split of the reactant and catalysts around the vial walls.
Next, the oxidation of allylic alcohols 1b-p (100 mM) was under-
taken searching for the lowest possible amounts of TEMPO and MTBE
(Table S2 in the Supplementary data for a complete optimization of the
reaction conditions). Although a clear trend cannot be observed, it was
possible to obtain quantitative conversions (> 97%) for all substrates
(Table 1), being feasible to diminish the TEMPO loading for aromatic
substrates 1h (entry 8, Table 1) and 1j (entry 10, Table 1), and for
heteroaromatic derivative 1m (entry 13, Table 1). The results achieved
for allylic alcohols 1b, 1g, 1o and 1p were especially remarkable (en-
tries 2, 7, 15, and 16, Table 1), as the quantity of TEMPO could be
lowered to 10 mol% and the organic cosolvent could be avoided.
(R)-1-[(2R,3R)-3-(3-Methoxyphenyl)oxiran-2-yl]ethan-1-ol (3) [61]
Yellow wax. Rf (75% Et2O/hexane): 0.62. IR (NaCl): 3470, 2979,
2965, 2915, 2873, 2847, 1737, 1604, 1586, 1492, 1459, 1441, 1257,
1238, 1154, 1139, 1036, 1025, 853, 801, 782, 732, 695, 591,
504 cm−1 1H NMR (300.13 MHz, CDCl3, major diastereoisomer): δ
.
1.31 (d, 3JHH =6.5 Hz, 3 H), 2.21 (br s, 1 H), 3.06 (dd, 3JHH = 2.6, 4JHH
3
=0.8 Hz, 1 H), 3.80 (s, 3 H), 3.93 (dd, JHH =2.1 Hz, 1 H), 4.10 (qd,
3JHH = 6.4, 3JHH =2.9 Hz, 1 H), 6.79–6.91 (m, 3 H), 7.23–7.28 (m, 1 H)
ppm. 13C NMR (75.5 MHz, CDCl3, major diastereoisomer): δ 160.0 (C),
138.8 (C), 129.7 (CH), 118.2 (CH), 114.1 (CH), 110.9 (CH), 65.7 (CH),
64.9 (CH), 55.4 (CH3), 54.7 (CH), 18.8 (CH3) ppm. HRMS (ESI+, m/z):
calcd for (C11H15O3)+ (M+H)+ 195.1016, found: 195.1014.
3.3. Bioreduction of (E)-4-(het)arylbut-3-en-2-ones
Once optimized the first step of the deracemization protocol, next
we focused on the bioreduction of the α,β-unsaturated ketones, starting
with (E)-4-phenylbut-3-en-2-one (2a) as the model substrate. A series of
alcohol dehydrogenases overexpressed in Escherichia coli were tried
such as the one from Rhodococcus ruber (ADH-A) [62], Thermo-
anaerobacter ethanolicus (TeSADH) [63], Thermoanaerobacter species
(ADH-T) [64], Ralstonia species (RasADH) [57,65], and Sphingobium
yanoikuyae (SyADH) [58,65,66], displaying Prelog selectivity, and the
ones from Lactobacillus brevis (LBADH) [67,68] and the commercially
available evo-1.1.200 ADH [69], acting with opposite selectivity
(Tables 2 and S3).
After enzymatic screening for the production of the alcohol (S)-1a,
ADH-A and ADH-T demonstrated complete control of the stereoselec-
tion, observing low to moderate conversions and selectivities with
TeSADH, RasADH, and SyADH. On the other hand, anti-Prelog evo-
1.1.200 displayed moderate selectivity, while LBADH led to the alcohol
(R)-1a in enantiomerically pure form. Then, key parameters for the
bioreduction as the amount of 2-propanol (5–30% v/v), the substrate
concentration (10−40 mM), and the enzyme loading (10−15 mg per
0.015 mmol of ketone) were studied, collecting the most relevant re-
sults in Table 2.
3. Results and discussion
3.1. Chemical synthesis of (E)-α,β-unsaturated ketones and racemic (E)-
allylic alcohols
Prior to the development of the enzymatic study, which includes the
laccase-mediated oxidation of the (het)aryl allylic alcohols 1a-p and the
bioreduction of the corresponding ketones 2a-p, the syntheses of both
families of organic compounds were performed following methods al-
ready reported in the literature (Scheme 2). Firstly, (E)-unsaturated
ketones 2a-p were synthesized in moderate to excellent yield after
column chromatography (59–96%) following the procedure already
described by List and co-workers for ketones 2a, 2l and 2o [60], con-
sisting in the aldol condensation between acetone and (hetero)aromatic
aldehydes catalyzed by the morpholinium trifluoroacetate salt. The
resulting ketones were then chemically reduced using sodium borohy-
dride, obtaining the racemic (E)-alcohols 1a-p after 3 h of reaction and
column chromatography purification on silica gel (46–99%) [52].
Subsequently, GC analytical methods to separate the ketone and alcohol
enantiomers were developed to measure in a later stage the enzymatic
reaction conversion and ee values (see the Supplementary data for de-
tails).
The conversion into optically active (E)-4-phenylbut-3-en-2-ol in-
creased until 86% when enhancing the hydrogen donor concentration
up to 20% v/v (entries 15–17), while the selectivity of the enzymes
slightly decreased. Therefore, as a compromise, 15% v/v of 2-propanol
and 40 mM of ketone concentration were selected as the best conditions
to achieve the bioreduction, as excellent selectivities and conversions
close to 80% were reached (entries 10, 12, and 14).
3.2. Laccase-mediated oxidation of (E)-4-(het)arylbut-3-en-2-ols 1a-p
With the best results in hand, and considering this substrate con-
centration as optimum to couple it with the chemoenzymatic aerobic
oxidation, the reductions of α,β-unsaturated ketones 2b-p catalyzed by
ADH-A, ADH-T and LBADH were carried out in order to produce opti-
cally active alcohols 1b-p. Gratifyingly, (S)- and (R)-alcohols were ob-
tained with more than 80% ee for fourteen out of the sixteen alcohols
(Table 3). In comparison with substrate 2a (entry 1), similar results
were found with the three enzymes for the majority of ketones, getting
conversions between 60–80% and selectivities > 90%. However, two
Recently, our research group described the oxidation of this family
of allylic alcohols using the catalytic system composed by the laccase
from Trametes versicolor and the oxy-radical TEMPO [52]. To achieve a
complete conversion in the oxidation of (E)-4-phenylbut-3-en-2-ol (1a,
100 mM), the transformation was performed at 30 °C using 4.6 units of
LTv and 33 mol% of TEMPO in a citrate buffer 50 mM pH 5.0 using
MTBE (50% v/v) as cosolvent to facilitate the solubility of the starting
material. However, in this contribution we have further optimized the
Scheme 2. Chemical synthesis of (E)-α,β-unsaturated ketones
2a-p and racemic (E)-allylic alcohols 1a-p.
5

J. Albarrán-Velo, et al.
MolecularCatalysis493(2020)111087
Table 1
Best conditions for the aerobic oxidation of racemic (het)aryl allylic alcohols 1a-p using the laccase/TEMPO system in the presence of TEMPO (10-33 mol%) and
MTBE (0-50% v/v) after 16 h at 30 °C and 150 rpm.
Entry
Alcohol [(Het)Ar]
TEMPO (mol%)
MTBE (% v/v)
c (%)a
1
2
3
4
5
6
7
8
1a (C6H5)
10
10
33
33
33
33
10
20
33
10
33
33
20
33
10
10
0
0
> 99
> 99
> 99b
> 99b
> 99b
98b
1b (4-OMe-C6H4)
1c (4-Cl-C6H4)
1d (4-F-C6H4)
1e (4-Br-C6H4)
1f (3-OMe-C6H4)
1g (3-F-C6H4)
1h (3-Me-C6H4)
1i (2-OMe-C6H4)
1j (2-F-C6H4)
1k (2-Pyridyl)
1l (3-Pyridyl)
1m (2-Furyl)
50
50
50
50
0
50
50
50
50
50
50
50
0
99
98
9
> 99b
99
10
11
12
13
14
15
16
99b
> 99b
99
1n (3-Furyl)
1o (2-Thienyl)
1p (3-Thienyl)
> 99b
> 99
> 99
0
a
Conversion values measured by GC analysis.
These results were already reported in the literature [52]. Lower conversions were obtained reducing the percentages of TEMPO and/or MTBE (see the Sup-
b
plementary data).
different facts can be highlighted. First, amongst the two Prelog en-
zymes (ADH-A and ADH-T), ADH-A seems to be a more robust bioca-
talyst leading to higher conversion values for selected substrates (4-Br,
3-Me and 2-OMe, entries 5, 8 and 9), which were slowly converted by
ADH-T (22–37% conversion). Second, it became clear that apart from
possible steric effects, these reductions depended on the electronic
character of the (het)aryl substituent. Hence, electron rich groups such
as methoxy at positions 2 (entry 9) and 4 (entry 2), or heteroaromatic
rings such as furan (entries 13 and 14) and thiophene (entries 15 and
16), limited the extent of the reaction, while electron-withdrawing
moieties such as fluorine (entries 7 and 10) and pyridine (entries 11 and
12), favored the enzymatic transformation.
Due to the fact that the enzymatic bioreduction of ketones derived
from furan (2m and 2n) and thiophene (2o and 2p) afforded lower
conversions, we decided to study in more detail these biotransforma-
tions varying the substrate and hydrogen donor concentrations (Fig. 1).
Compared to the initial results shown in Table 3 (entries 13–16) and in
Fig. 1 (conditions A), the decrease of 2-propanol concentration up to
10% v/v (conditions B), even at lower substrate concentration (25 mM,
conditions C), did not usually lead to better results. However, the use of
Table 2
Bioreduction of ketone 2a using overexpressed ADHs after 24 h at 30 °C and 250 rpm.
Entry
[Alcohol] (mM)
ADH (mg)a
2-PrOH (% v/v)
c (%)b
ee (%)b
1
2
3
4
5
6
7
8
25
25
25
25
25
10
25
10
25
40
25
40
25
40
25
25
25
ADH-A (10)
ADH-T (10)
LBADH (10)
ADH-A (10)
ADH-T (10)
ADH-T (10)
LBADH (10)
LBADH (10)
LBADH (15)
ADH-A (10)
ADH-T (10)
ADH-T (10)
LBADH (10)
LBADH (10)
ADH-A (10)
ADH-T (10)
LBADH (10)
5
5
5
64
65
54
75
78
85
74
79
74
79
83
77
83
75
85
86
86
99 (S)
> 99 (S)
> 99 (R)
> 99 (S)
> 99 (S)
> 99 (S)
97 (R)
> 99 (R)
> 99 (R)
> 99 (S)
94 (S)
> 99 (S)
> 99 (R)
> 99 (R)
92 (S)
94 (S)
> 99 (R)
10
10
10
10
10
10
15
15
15
15
15
20
20
20
9
10
11
12
13
14
15
16
17
a
The amount of ADH in mgs (per 0.015 mmol of ketone) is indicated in parentheses.
Conversion and enantiomeric excess values were measured by GC analysis. Major product enantiomer appears in parentheses.
b
6

J. Albarrán-Velo, et al.
MolecularCatalysis493(2020)111087
Table 3
Bioreduction of ketones 2a-p (40 mM) after 24 h at 30 °C and 250 rpm.
Entry
Ketone
LBADH
LBADH
ADH-T
ADH-T
ADH-A
ADH-A
c (%)a
ee (%)b
c (%)a
ee (%)b
c (%)a
ee (%)b
1
2
3
4
5
6
7
8
2a (C6H5)
75
51
77
70
78
78
79
72
56
80
94
89
67
64
66
66
> 99 (R)
70 (R)
92 (R)
94 (R)
94 (R)
93 (R)
98 (R)
95 (R)
88 (R)
99 (R)
99 (R)
99 (R)
86 (R)
90 (R)
76 (R)
88 (R)
77
50
77
75
29
51
83
37
22
83
88
86
69
66
67
66
> 99 (S)
60 (S)
94 (S)
96 (S)
94 (S)
93 (S)
99 (S)
95 (S)
90 (S)
99 (S)
> 99 (S)
99 (S)
82 (S)
90 (S)
78 (S)
86 (S)
79
49
77
70
78
75
80
69
60
81
93
89
64
61
64
63
> 99 (S)
60 (S)
99 (S)
96 (S)
94 (S)
94 (S)
99 (S)
96 (S)
90 (S)
99 (S)
> 99 (S)
99 (S)
82 (S)
90 (S)
78 (S)
93 (S)
2b (4-OMe-C6H4)
2c (4-Cl-C6H4)
2d (4-F-C6H4)
2e (4-Br-C6H4)
2f (3-OMe-C6H4)
2g (3-F-C6H4)
2h (3-Me-C6H4)
2i (2-OMe-C6H4)
2j (2-F-C6H4)
2k (2-Pyridyl)
2l (3-Pyridyl)
2m (2-Furyl)
9
10
11
12
13
14
15
16
2n (3-Furyl)
2o (2-Thienyl)
2p (3-Thienyl)
a
Conversion values measured by GC analysis.
Enantiomeric excess values were measured by GC analyses after derivatization of the reaction crudes with acetic anhydride. Major product enantiomer appears
b
in parentheses.
lower amounts of the ketone (conditions D), generally afforded higher
conversions, although at the expenses of slightly inferior ee values. Si-
milar trends were observed for all ADHs independently of their ste-
reopreference.
alcohols 1a-p, and the discovery of active and selective ADHs that could
be applied to the bioreduction of the corresponding α,β-unsaturated
ketones 2a-p, motivated us to initiate the search for the optimal con-
ditions towards the deracemization of racemic allylic alcohols 1a-p.
Taking into account the different reaction conditions required for both
steps, including pH (acidic for the laccase/TEMPO system and neutral
for the ADH), the agitation mode (magnetic stirring, 150 rpm, for the
oxidation in contrast to the orbital shaking, 250 rpm, preferred by the
ADHs), and also the substrate concentration (100 mM in the first stage
3.4. Deracemization of (E)-4-(het)arylbut-3-en-2-ols 1a-p
The successful development of aerobic oxidation reactions mediated
by the laccase from Trametes versicolor in combination with TEMPO for
Fig. 1. Effect of the substrate and 2-PrOH concentration in the
bioreductions of α,β-unsaturated ketones 2 m-p using: a) E.
coli/ADH-T (for 2 m-o) and E. coli/ADH-A (for 2p) for the
production of (S)-alcohols; and b) E. coli/LBADH for the pro-
duction of (R)-alcohols. Conditions A: [ketone] =40 mM and
[2-PrOH] = 15% v/v (see Table 3); conditions B: [ketone]
=40 mM and [2-PrOH] = 10% v/v (see Table S4); conditions
C: [ketone] =25 mM and [2-PrOH] = 10% v/v (see Table S4);
conditions D: [ketone] =25 mM and [2-PrOH] = 15% v/v
(see Table S4). For each pair, filled bar represents conversion
and dashed bar represents ee value.
7

J. Albarrán-Velo, et al.
MolecularCatalysis493(2020)111087
Table 4
Deracemization of alcohols 1a-p combining the laccase/TEMPO system and the corresponding ADH under the optimized conditions.
Entry
Alcohol 1a-p
R
ADH
c (%)a
ee (%)b
1
1a
1a
1a
1b
1b
1c
1c
1d
1d
1e
1e
1f
H
H
H
E. coli/LBADH
E. coli/LBADH
E. coli/ADH-T
E. coli/LBADH
E. coli/ADH-T
E. coli/LBADH
E. coli/ADH-A
E. coli/LBADH
E. coli/ADH-T
E. coli/LBADH
E. coli/ADH-A
E. coli/LBADH
E. coli/ADH-A
E. coli/LBADH
E. coli/ADH-T
E. coli/LBADH
E. coli/ADH-A
E. coli/LBADH
E. coli/ADH-A
E. coli/LBADH
E. coli/ADH-T
E. coli/LBADH
E. coli/ADH-A
E. coli/LBADH
E. coli/ADH-A
E. coli/LBADH
E. coli/ADH-T
E. coli/LBADH
E. coli/ADH-T
E. coli/LBADH
E. coli/ADH-T
E. coli/LBADH
E. coli/ADH-A
74 (52)
73 (55)
76 (51)
49 (22)
48 (20)
74 (62)
69 (54)
60 (44)
52 (40)
69 (56)
55 (39)
66 (52)
70 (57)
84 (62)
87 (68)
68 (52)
72 (58)
64 (55)
60 (49)
81 (65)
82 (66)
95 (87)
95 (83)
91 (76)
86 (70)
65 (48)
64 (48)
59 (42)
62 (44)
58 (47)
62 (49)
65 (51)
69 (52)
> 99 (R)
> 99 (R)
> 99 (S)
72 (R)
65 (S)
93 (R)
98 (S)
93 (R)
98 (S)
92 (R)
93 (S)
93 (R)
98 (S)
> 99 (R)
> 99 (S)
96 (R)
96 (S)
94 (R)
94 (S)
99 (R)
99 (S)
96 (R)
96 (S)
94 (R)
95 (S)
87 (R)
86 (S)
93 (R)
92 (S)
75 (R)
74 (S)
89 (R)
92 (S)
2c
3
4
5
6
7
8
9
4-OMe
4-OMe
4-Cl
4-Cl
4-F
4-F
4-Br
4-Br
3-OMe
3-OMe
3-F
3-F
3-Me
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
1f
1g
1g
1h
1h
1i
1i
1j
1j
3-Me
2-OMe
2-OMe
2-F
2-F
1k
1k
1l
2-Pyridyl
2-Pyridyl
3-Pyridyl
3-Pyridyl
2-Furyl
2-Furyl
3-Furyl
3-Furyl
2-Thienyl
2-Thienyl
3-Thienyl
3-Thienyl
1l
1m
1m
1n
1n
1o
1o
1p
1p
a
Conversion values measured by GC analysis. Isolated yields after column chromatography appear in parentheses.
b
Enantiomeric excess values were measured by GC analyses after derivatization of the reaction crudes with acetic anhydride. Major product enantiomer appears
in parentheses.
c
No cosolvent was used and TEMPO loading was diminished to 10 mol% in the first step.
and 40 mM in the second one), a one-pot two-step deracemization
protocol was followed. Hence, the aerobic oxidation of alcohols 1a-p
was stopped by a proper dilution of the reaction with Tris·HCl buffer
50 mM pH 8, leading to a pH adjustment of 7.5 and a substrate con-
centration of 40 mM to carry out the bioreduction step (Table 4).
In order to demonstrate a general methodology, the oxidation step
was achieved in the presence of TEMPO (33 mol%) and MTBE (50% v/
v), conditions that allowed the quantitative oxidation of alcohols 1a-p
(Tables S1 and S2 in the Supplementary data) [70], necessary to avoid
ee erosion in the whole process. After 16 h, the organic cosolvent was
evaporated, so it did not interfere in the second step. Then, over-
expressed LBADH was added to produce the alcohols (R)-1a-p (22–87%
yield, 72- > 99%), and ADH-T (20–68% yield, 65- > 99%) or ADH-A
(39–83% yield, 86- > 99%) to obtain (S)-1a-p after column chroma-
tography.
deracemization experiments (Scheme 3a) were performed with selected
alcohols 1d,f,g,j (0.16 mmol) and also at preparative scale with 1a and
1f (0.8–1.0 mmol) for the production of derivatives (S)-1a and (R)-1f,
and their optical rotation values were compared to those previously
described in the literature (see the Supplementary data), confirming in
this manner the proposed absolute configurations, which were in ac-
cordance with the stereoselectivity generally displayed by these ADHs
[62,64,67,68].
As a further extension of this synthetic approach, (R)-1f obtained at
preparative scale, was stereoselectively epoxidized employing modified
conditions of Sharpless methodology, providing epoxide 3 in high yield
(80%, Scheme 3b) and selectivity (95% ee, 83% de). This highly re-
active derivative is a synthon for biologically active natural compounds
Bryostatin 1, used as antiviral and anti-Alzheimer agent, and ampho-
tericin B, a potent antifungal agent [61].
Some of these allylic alcohols have been described as valuable
precursors of biologically active compounds. Thus, (S)-1c can provide
(R)-baclofen [71], which has been employed for the treatment of
spasticity, (S)-1e is a key building-block of α-amino amide dipeptidyl
peptidase IV inhibitors [72,73], used for the treatment of type 2 dia-
betes, and (R)-1b has been utilized as precursor of a derivative of cis-
calamenene terpenoid [74].
4. Conclusions
The search for asymmetric synthetic methods that can provide
highly valuable molecules under simple and mild conditions has largely
increased in the past years. Deracemization reactions are elegant stra-
tegies to produce chiral enantiopure compounds starting from the
corresponding racemates. Different biocatalytic approaches have been
To demonstrate the applicability of this method, semi-preparative
8

J. Albarrán-Velo, et al.
MolecularCatalysis493(2020)111087
Scheme 3. a) Deracemization examples at (semi)preparative scale. b) Epoxidation of enantioenriched alcohol (R)-1f.
designed using whole cell biocatalysts or by combining two ADHs with
opposite stereopreference and different cofactor recognition. However,
an interesting method is the linear deracemization via combination of a
non-selective oxidation with a stereoselective reduction step. Herein,
we have optimized the oxidation of a series of (het)aryl allylic sec-
ondary alcohols with the laccase from Trametes versicolor and the ra-
dical TEMPO, and the reduction of the corresponding α,β-unsaturated
ketone intermediates employing overexpressed alcohol dehy-
drogenases. It was observed that in the oxidation step it was possible to
diminish the quantity of TEMPO and MTBE for some of the substrates
by carefully controlling the agitation speed. Applying both steps in a
sequential one-pot mode, 16 enantioenriched alcohols (up to > 99% ee)
could be obtained with moderate to high isolated yields (up to 87%).
Finally, the application of this methodology at preparative scale was
also demonstrated, synthesizing a valuable epoxy alcohol that can be
utilized as intermediate of biologically active compounds.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.mcat.2020.111087.
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CRediT authorship contribution statement
Jesús Albarrán-Velo: Validation, Investigation, Writing - original
draft. Vicente Gotor-Fernández: Conceptualization, Writing - review
& editing, Supervision. Iván Lavandera: Conceptualization, Writing -
review & editing, Supervision.
Declaration of Competing Interest
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Acknowledgments
Financial support from the Spanish Ministry of Economy and
Competitiveness (MEC, Project CTQ2016-75752-R) and the Asturian
regional government (FC-GRUPIN-IDI/2018/000181) are gratefully
acknowledged. J.A.-V. also thanks the Asturian regional government for
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