Chemoenzymatic Deracemization of Secondary Alcohols by using a TEMPO-Iodine-Alcohol Dehydrogenase System

Article abstract of DOI:10.1002/cctc.201500816

A deracemization system for secondary alcohols was established after the analysis of individual steps and their compatibility in one pot. The chemical oxidation and bioreduction occurred in a sequential manner to yield 1-arylethanols and lineal aliphatic alcohols with excellent conversions and enantiomeric excess values. The oxidation step was performed by using 2,2,6,6-tetramethylpiperidin-1-oxyl and iodine. This chemical process was extremely favored by sonication, which allowed quantitative formation of the corresponding ketone intermediates after just 1 h. Simple destruction of iodine in the same pot allowed sequential bioreduction of the ketones by using either Prelog or antiPrelog enzymes, which led to the preparation of the enantiopure alcohols in excellent yields. Just a sec: The one-pot deracemization of secondary alcohols involving oxidation with 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) and iodine followed by alcohol dehydrogenase (ADH)-catalyzed bioreduction is described. 1-Arylethanols and lineal aliphatic alcohols are obtained with excellent conversions and enantiomeric excess values. LBADH=alcohol dehydrogenase from Lactobacillus brevis.

Full text of DOI:10.1002/cctc.201500816

DOI: 10.1002/cctc.201500816
Communications
Chemoenzymatic Deracemization of Secondary Alcohols
by using a TEMPO–Iodine–Alcohol Dehydrogenase System
Daniel MØndez-Sµnchez,[a] Juan Mangas-Sµnchez,[a, b] Ivµn Lavandera,[a] Vicente Gotor,[a] and
Vicente Gotor-Fernµndez*[a]
A deracemization system for secondary alcohols was estab-
lished after the analysis of individual steps and their compati-
bility in one pot. The chemical oxidation and bioreduction oc-
curred in a sequential manner to yield 1-arylethanols and lineal
aliphatic alcohols with excellent conversions and enantiomeric
excess values. The oxidation step was performed by using
2,2,6,6-tetramethylpiperidin-1-oxyl and iodine. This chemical
process was extremely favored by sonication, which allowed
quantitative formation of the corresponding ketone intermedi-
ates after just 1 h. Simple destruction of iodine in the same
pot allowed sequential bioreduction of the ketones by using
either Prelog or antiPrelog enzymes, which led to the prepara-
tion of the enantiopure alcohols in excellent yields.
substrate enantiomer into a nonchiral intermediate, which sub-
sequently reacts to give the final product with opposite config-
uration ideally to obtain a single enantiomer.[4] A few (chemo)-
enzymatic deracemization protocols have been described for
the asymmetric synthesis of broad families of organic com-
pounds, such as amino acid and amino derivatives.[4,5] Never-
theless, the deracemization of secondary alcohols has attracted
much attention on the basis of simple oxidation–reduction se-
quences and is usually accomplished by multienzymatic com-
binations.[4,6] With that purpose, the action and high selectivity
of oxidoreductases such as alcohol dehydrogenases (ADHs)
has been fully exploited.[7] Redox enzymes are useful biocata-
lysts for both the oxidation of alcohols and the selective reduc-
Enzymes have stormed over the
last decades as useful tools in
the search of sustainable meth-
odologies towards chiral com-
pounds in theoretical 100%
yield, breaking the inherent limi-
tations associated with classical
kinetic resolutions.[1] The possi-
bility of developing dynamic ki-
Scheme 1. Different pathways to deracemize secondary alcohols by combining a) two stereoselective steps,
b) one selective step for oxidation and one nonselective step for reduction, and c) one nonselective step for oxi-
dation and one selective step for reduction. The approach used in this article belongs to pathway c.
netic resolutions, deracemization
from racemates,[2] or stereoselec-
tive desymmetrizations starting
from prochiral or meso com-
pounds[3] allows a reduction in cost and the elimination of
waste, both of which are particularly attractive for the imple-
mentation of biocatalytic strategies in the chemical industry. In
this context, the most common deracemization strategies in-
volve stereoinversion or cyclic modes.
tion of ketones by using whole cells and semipurified or puri-
fied enzymes.[8] In addition, it is worth mentioning that these
fully enzymatic deracemizations occur under mild reaction con-
ditions that permit the presence of additional functional
groups in the reactive substrate such as esters[9] and carboxylic
acids.[10]
Deracemization of
a
racemate by stereoinversion
(Scheme 1a) consists in the selective transformation of one
The main difficulty of deracemization through stereoinver-
sion resides in the perfect combination of enantioselective oxi-
dation and stereoselective reduction steps without detriment
to the conversion or the enantiomeric excess of the target op-
tically active alcohol. In this sense, cyclic deracemization sys-
tems of secondary alcohols (Scheme 1b,c) provide less exigent
reaction conditions, as they make use of a nonselective pro-
cess. One approach can be the selective (enzymatic) oxidation
of one enantiomer into the ketone, followed by nonselective
chemical reduction in a stepwise fashion (Scheme 1b).[11] On
the other hand, it is also possible to achieve deracemization by
combining a nonselective oxidation step of the alcohol with
stereoselective (enzymatic) reduction of the prochiral ketone
[a] D. MØndez-Sµnchez, Dr. J. Mangas-Sµnchez, Dr. I. Lavandera, Prof. V. Gotor,
Dr. V. Gotor-Fernµndez
Organic and Inorganic Chemistry Department
Biotechnology Institute of Asturias (IUBA)
University of Oviedo
Avenida Juliµn Clavería s/n 33006 Oviedo (Spain)
E-mail: vicgotfer@uniovi.es
[b] Dr. J. Mangas-Sµnchez
Department of Biotechnology
University of Lund
PO Box 124, SE-221 00 Lund (Sweden)
Supporting Information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201500816.
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Communications
formed (Scheme 1c). This synthetic strategy has been satisfac-
torily reported by using a metal complex as the oxidant[12] and
by applying other redox biocatalysts such as laccases in combi-
nation with 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO).[13]
Clearly, in this case the oxidation reaction must be quantitative
to obtain a proper deracemization protocol.
Table 1. Bioreduction of acetophenone (2a, 40 mm).[a]
Entry
Oxidant (equiv.)
c [%][b]
ee [%][b]
Herein, we report a versatile and commonly applicable dera-
cemization strategy for secondary alcohols involving nonselec-
tive chemical oxidation by employing the iodine/TEMPO
system and a stereoselective bioreduction step. After optimiza-
tion of the individual processes, both were combined in a one-
pot and stepwise manner to yield the (R)- or (S)-alcohols de-
pending on the stereopreference of the ADH used in the re-
duction of the ketone intermediate.
1
2
3
4
5
6
7
–
>99
85
91
73
2
>99
96
>99
>99
n.d.
99
TEMPO (0.1)
PIDA (2)
TEMPO/PIDA (0.1/2)
TEMPO/NCS (0.1/1.5)
TEMPO/PIDA (0.1/2)[c]
TEMPO/I2 (0.1/2)[c]
85
>99
>99
[a] Bioreduction was performed by using ADH-A and 2-PrOH (6.4%v/v) in
the presence of the different oxidizing agents at 308C for 24 h at
250 rpm. [b] Conversion and enantiomeric excess values were measured
by GC analyses (see the Supporting Information for details). n.d.=not de-
termined. [c] Na2S2O3 saturated aqueous solution (200 mL) was supple-
mented before enzyme addition.
The oxidation of alcohols for the formation of carbonyl com-
pounds is a key reaction in organic synthesis.[14] Therefore,
a number of strategies have appeared in the literature involv-
ing metal or organic oxidants such as chromium(VI) salts, man-
ganese dioxide, activated dimethyl sulfoxides, and hypervalent
iodine reagents, among others. In the search for a simple and
compatible approach with the bioreduction process, the use of
a catalytic amount of TEMPO in combination with several oxi-
dizing agents was tested to oxidize the model substrate 1-phe-
nylethanol (1a). A premise for this study was the development
of the oxidative process in a buffer system, which is the
medium required for the ADH-catalyzed bioreduction
(Scheme 2). Tris-HCl buffer pH 7.5 and a catalytic amount of
ed as the oxidant, as it can be easily quenched afterwards by
sodium thiosulfate, which converts the excess amount of
iodine into harmless iodide anions (Table 1, entries 6 and 7).
With this treatment, the activity of ADH-A remained unaltered
in the stereoselective reduction of acetophenone and a total
conversion was obtained in the bioreduction step (Table 1,
entry 7). In summary, iodine was selected as a mild oxidant for
two main and important reasons. On the one hand, it allows
the simple reoxidation of TEMPO (0.1 equiv.) in aqueous
medium under mild reaction conditions. On the other hand,
the destruction of the excess amount of iodine into iodide
ions can be accomplished in one pot without detriment to the
enzymatic activity, which is in contrast to the inhibition or
competitive oxidative reverse reaction observed with other oxi-
dants tested in this study.
Scheme 2. Oxidation of (Æ)-1-phenylethanol (1a) with a catalytic amount of
TEMPO and different oxidizing agents in Tris-HCl buffer pH 7.5.
Once a convenient oxidation protocol was found, an exhaus-
tive optimization study was performed by searching for suita-
ble conditions for the complete oxidation of 1-phenylethanol
(Table 2). Different parameters that could affect the reactivity
of the catalytic TEMPO/iodine system were analyzed, such as
the amount of TEMPO, number of equivalents of iodine, sub-
strate concentration, pH, and temperature.
TEMPO were initially selected for screening. A series of oxi-
dants including sodium hypochlorite (NaOCl),[15] phenyliodi-
ne(III) diacetate (PIDA),[16] N-chlorosuccinimide (NCS),[17] and
iodine[18] were tested and used in slight excess amounts at dif-
ferent temperatures (30–508C), and significant conversions
into acetophenone (2a) were found.
Once the chemical oxidation of a series of secondary alco-
hols was explored, we focused on the bioreduction step of 2a
by using a Prelog ADH, such as the one from Rhodococcus
ruber (ADH-A),[19] and 2-PrOH (6.4%v/v) as the hydrogen donor
in a “coupled-substrate” approach.[20] To identify compatible
conditions with the oxidizing systems, the bioreduction of 2a
was studied in the presence of the previously tested chemical
oxidants by using favorable conditions for the overexpressed
ADH-A mediated reductions (Table 1).
The first attempts for the oxidation of 1a were performed
by using a 50 mm Tris-HCl buffer pH 7.5, a catalytic amount of
TEMPO (0.1–0.2 equiv.), and an excess amount of iodine (1.5–
2 equiv.), but low conversions were obtained (Table 2, en-
tries 1–3). The presence of a hydrophobic cosolvent such as
hexane (10%v/v; Table 2, entry 4) did not lead to any improve-
ment. A significant increase in the conversion values was
achieved at pH 9 (Table 2, entry 5), which allowed the 50%
conversion to be surpassed if 0.2 equivalents of TEMPO were
employed (Table 2, entry 6). This is due to the fact that TEMPO
works better under basic conditions. Unfortunately, the activity
of the system dramatically decreased at higher substrate con-
centrations (80-160 mm; Table 2, entries 7 and 8). Similar re-
sults were attained by using lower concentrations of the alco-
hol or higher temperatures (Table 2, entries 9–11). A similar
The enantiomeric excess (ee) of (S)-1-phenylethanol was
slightly affected by the presence of TEMPO or PIDA, which re-
sulted in a notable decrease in the conversion (Table 1, en-
tries 2–4) that dramatically diminished upon using NCS
(Table 1, entry 5). In the search for compatible redox conditions
for the overall deracemization process, iodine was then select-
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phatic (Table 3, entries 8 and 9) alcohols were finally selected
to have a broader overview of the potential of this oxidizing
system.
Table 2. TEMPO/iodine-catalyzed oxidation of (Æ)-1-phenylethanol (1a)
in aqueous medium.
Focusing on meta-substituted substrates, complete conver-
sions were found with a moderately activating (Table 3,
entry 2) or deactivating group (Table 3, entry 4). On the contra-
ry, a strong electron-withdrawing group such as the nitro func-
tionality led to poor 39% conversion upon using this oxidizing
system (Table 3, entry 3). Regardless of the position of the
chloro substituent, the catalytic system was active, and in all
cases quantitative conversions were found (Table 3, entries 4–
6). Nevertheless, the oxidation of substrate 1g with a chlorine
atom at the b position did not proceed at any extension
(Table 3, entry 7). This result can be explained, as these activat-
ed alcohols can be oxidized under strong conditions.[21] Finally,
aliphatic alcohols reacted effectively to yield 1-cyclohexyletha-
none (Table 3, entry 8) and 2-octanone (Table 3, entry 9) in
quantitative conversions.
Entry
TEMPO
[equiv.]
I2
1a
[mm]
pH
T
[8C]
t
[h]
c
[equiv.]
[%][a]
1
2
3
4[b]
5
6
7
8
9
10
11
12
13
14[c]
0.1
0.2
0.1
0.1
0.1
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
1.5
1.5
2
40
40
40
40
40
40
80
160
20
40
20
20
20
20
7.5
30
30
30
30
30
30
30
30
30
60
60
30
60
30
16
16
16
16
16
16
16
16
16
16
16
16
16
1
16
23
18
14
48
54
30
22
49
52
57
52
60
98
7.5
7.5
7.5
9
9
9
9
9
9
9
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
10
10
10
Once the TEMPO/iodine system was found to be efficient for
the complete chemical oxidation of secondary alcohols
(Table 3), E. coli/ADH-A, without the need of an external cofac-
tor,[19] was used in a stepwise fashion to reduce the corre-
sponding ketones on the basis of the excellent activity of this
enzyme under the conditions for the oxidative process shown
in Table 1. Gratifyingly, the corresponding (S)-alcohols were
produced in enantiopure form and with conversions over 95%
starting from the racemic forms (Table 4).
[a] Conversion values were measured by GC analyses (see the Supporting
Information for details). [b] Hexane (10%v/v) was used as the cosolvent.
[c] Ultrasound was used over a period of 1 h.
trend was observed by using Tris-HCl buffer pH 10 (Table 2, en-
tries 12 and 13). Remarkably, the oxidation of 1a was almost
complete after sonication of the reaction mixture for 1 h
(Table 2, entry 14).
Table 4. Deracemization of alcohols 1 (20 mm) with TEMPO (0.2 equiv.),
iodine (2 equiv.), and Escherichia coli/ADH-A with 2-PrOH.
Once the optimal conditions were found for the oxidation of
1a, extension of this methodology to a series of aromatic and
aliphatic racemic alcohols was considered (Table 3). Different
Table 3. TEMPO/iodine-catalyzed oxidation of racemic alcohols 1 (20 mm)
by using ultrasound in aqueous medium for 1 h.
Entry
R
c [%][a]
ee [%][b]
1
2
3
4
5
6
7
C6H5 (1a)
96
96
99
99
99
98
96
>99
>99
>99
>99
>99
>99
>99
3-MeOC6H4 (1b)
3-ClC6H4 (1d)
4-ClC6H4 (1e)
2-ClC6H4 (1 f)
cyclohexyl (1h)
(CH2)5Me (1i)
Entry
R1
R2
c [%][a]
1
2
3
4
5
6
7
8
9
C6H5 (1a)
Me
Me
Me
Me
Me
Me
CH2Cl
Me
Me
>99
>99
39
>99
>99
>99
<1
3-MeOC6H4 (1b)
3-O2NC6H4 (1c)
3-ClC6H4 (1d)
4-ClC6H4 (1e)
2-ClC6H4 (1 f)
2,4-Cl2C6H3 (1g)
cyclohexyl (1h)
(CH2)5Me (1i)
[a] Conversion values of the bioreduction reaction were measured by GC
analyses (see the Experimental Section for details). [b] Enantiomeric
excess values of the resulting deracemized alcohols were measured by
GC analysis on a chiral stationary phase (see the Supporting Information
for details).
>99
>99
[a] Conversion values were measured by GC analyses (see the Supporting
Information for details).
Trying to obtain their alcohol antipodes, antiPrelog ADHs
were tested in the bioreduction step of the sequential dera-
cemization (Table 5). For this study, two different semipurified
enzymes were considered: the NADPH-dependent ADH from
Lactobacillus brevis (LBADH)[22] and the NADH-dependent and
commercially available evo-1.1.200.[23] Satisfactorily, all the sub-
strates were recognized by both enzymes, so after the chemi-
cal oxidation and quenching, the stereoselective bioreduction
substitution patterns were chosen in the meta position of the
aromatic ring (methoxy, chloro, and nitro; Table 3, entries 2–4),
and we also studied the effect of the same substituent at dif-
ferent positions on the phenyl moiety (chloro group; Table 3,
entries 4–6). In addition, b-chlorinated (Table 3, entry 7) and ali-
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brevis (LBADH, 300 UmLÀ1) was purchased from Codexis. Evo-
1.1.200 (1716 UmgÀ1) was acquired from Evocatal. Escherichia coli
strains to overexpress ADH-A from Rhodococcus ruber were kindly
provided by Prof. Wolfgang Kroutil (University of Graz). GC analyses
were performed for conversion and enantiomeric excess measure-
ments (see the Supporting Information).
Table 5. Deracemization of alcohols 1 (20 mm) with TEMPO (0.2 equiv.),
iodine (2 equiv.), and antiPrelog ADHs with 2-PrOH.
Yield [%][a]
ee [%][b]
Chemical oxidations
Entry
R
ADH
1
2
3
4
5
6
7
8
C6H5 (1a)
C6H5 (1a)
LB
91
91
99
99
92
95
99
99
99
99
98
98
95
94
99
99
Iodine (30 mg, 1.5 equiv.) and TEMPO (2.5–5 mg, 0.1–0.2 equiv.)
evo-1.1-200
LB
evo-1.1-200
LB
evo-1.1-200
LB
evo-1.1-200
LB
evo-1.1-200
LB
evo-1.1-200
LB
were successively added over
a solution of alcohol 1a–
3-MeOC6H4 (1b)
3-MeOC6H4 (1b)
3-ClC6H4 (1d)
3-ClC6H4 (1d)
4-ClC6H4 (1e)
4-ClC6H4 (1e)
2-ClC6H4 (1 f)
2-ClC6H4 (1 f)
cyclohexyl (1h)
cyclohexyl (1h)
(CH2)5Me (1i)
(CH2)5Me (1i)
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
i (0.08 mmol, 20 mm) in 50 mm Tris-HCl buffer at pH 10 (4 mL) in
a 15 mL falcon tube. The mixture was sonicated for 1 h and con-
versions were measured by GC analysis.
General procedure for the bioreduction of ketones 2a–
i with ADH-A overexpressed in E. coli
9
10
11
12
13
14
E. coli/ADH-A cells (15 mg) and 2-PrOH (32 mL) were successively
added to an Eppendorf tube containing 2a–i (0.08 mmol) in
50 mm Tris-HCl buffer pH 10 (468 mL). The mixture was shaken at
308C and 250 rpm for 24 h. Then, the mixture was extracted with
EtOAc (2”500 mL), centrifuged (13000 rpm, 90 s), and dried
(Na2SO4). The substrate conversion and the enantiomeric excess of
alcohol 1a–i were measured by GC analysis.
evo-1.1-200
[a] Conversion values of the alcohols were measured by GC analysis (see
the Experimental Section for details). [b] Enantiomeric excess values of
the resulting deracemized alcohols were measured by GC analysis with
a chiral stationary phase (see the Supporting Information for details).
General procedure for the bioreduction of ketones 2a–
i with LBADH
of the corresponding ketones led to the enantiopure (R)-alco-
hols with conversions over 90%.
LBADH (10 mL, 3 U), 1 mm NADPH (60 mL of a 10 mm stock solu-
tion), 1 mm MgCl2 (60 mL of a 10 mm stock solution), and 2-PrOH
(32 mL) were successively added to an Eppendorf tube containing
2a–i (0.08 mmol) in 50 mm Tris-HCl buffer pH 10 (468 mL). The mix-
ture was shaken at 308C and 250 rpm for 24 h. Then, the mixture
was extracted with EtOAc (2”500 mL), centrifuged (13000 rpm,
90 s), and dried (Na2SO4). The substrate conversion and the enan-
tiomeric excess of alcohol 1a–i were measured by GC analysis.
In conclusion, chemoenzymatic deracemization strategies
offer significant advantages in chemical synthesis for the prep-
aration of enantiopure compounds with high yields. Dynamic
kinetic resolution has been fully exploited over the last two de-
cades, whereas other deracemization strategies involving the
use of redox processes in combination with chemical and en-
zymatic catalysts have been less explored. Herein, we have de-
veloped a deracemization protocol for secondary alcohols on
the basis of the chemical oxidation of the racemates and the
sequential bioreduction of the intermediate ketones formed in
the same vessel. A simple and cheap system such as TEMPO
together with iodine allowed the effective oxidation of the al-
cohols, which required ultrasound conditions for the fast quan-
titative formation of the ketones after just 1 h. This system was
fully compatible with the alcohol dehydrogenase-catalyzed
bioreduction of the resulting ketones, after quenching of the
excess amount of iodine with an aqueous Na2S2O3 saturated
solution. Gratifyingly, different Prelog and antiPrelog alcohol
dehydrogenases efficiently mediated the preparation of enan-
tiopure (S)- and (R)-alcohols with conversions over 90%, start-
ing from inexpensive racemates.
General procedure for the bioreduction of ketones 2a–
i with evo-1.1.200
Evo-1.1.200 (50 mL, 3 U), 1 mm NADH (60 mL of a 10 mm stock solu-
tion), 1 mm MgCl2 (60 mL of a 10 mm stock solution), and 2-PrOH
(25 mL) were successively added to an Eppendorf tube containing
2a–i (0.08 mmol) in 50 mm Tris-HCl buffer pH 10 (468 mL). The mix-
ture was shaken at 308C and 250 rpm for 24 h. Then, the mixture
was extracted with EtOAc (2”500 mL), centrifuged (13000 rpm,
90 s), and dried (Na2SO4). The substrate conversion and the enan-
tiomeric excess of alcohol 1a–i were measured by GC analysis.
Deracemization experiments by using ADH-A overexpressed
in E. coli
Iodine (30 mg, 1.5 equiv.) and TEMPO (2.5 mg, 0.1 equiv.) were suc-
cessively added over a solution of alcohol 1a–i (0.8 mmol, 20 mm)
in 50 mm Tris-HCl buffer pH 10 (4 mL). The mixture was sonicated
for 1 h at room temperature until complete conversion to ketone
2a–i. After this time, the reaction was stopped by adding a saturat-
ed Na2S2O3 aqueous solution (300 mL). An aliquot (500 mL) was
then withdrawn, and E. coli/ADH-A cells (15 mg) and 2-propanol
(32 mL) were successively added. The mixture was shaken at 308C
Experimental Section
General Methods
Chemical reagents were purchased from different commercial sour-
ces (Sigma–Aldrich, Acros, and Fluka) and were used without fur-
ther purification. The alcohol dehydrogenase from Lactobacillus
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Communications
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and 250 rpm for additional 24 h. Then, the mixture was extracted
with EtOAc (2”500 mL), centrifuged (13000 rpm, 90 s), and dried
(Na2SO4). The substrate conversion and enantiomeric excess of (S)-
alcohols 1a–i were measured by GC analysis.
Deracemization experiments by using LBADH
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Iodine (30 mg, 1.5 equiv.) and TEMPO (2.5 mg, 0.1 equiv.) were suc-
cessively added over a solution of alcohol 1a–i (0.8 mmol, 20 mm)
in 50 mm Tris-HCl buffer pH 10 (4 mL). The mixture was sonicated
for 1 h at room temperature until complete conversion to ketone
2a–i. After this time, the reaction was stopped by adding a saturat-
ed Na2S2O3 aqueous solution (300 mL). An aliquot (500 mL) was
then withdrawn, and LBADH stock (10 mL, 3 U) in 50 mm Tris-HCl
buffer pH 10, 1 mm MgCl2 (60 mL of a 10 mm stock solution), 2-
PrOH (32 mL), and 1 mm NADPH (60 mL of a 10 mm stock solution)
were successively added. The mixture was shaken at 308C and
250 rpm for additional 24 h. Then, the mixture was extracted with
EtOAc (2”500 mL), centrifuged (13000 rpm, 90 s), and dried
(Na2SO4). The substrate conversion and enantiomeric excess of (R)-
alcohols 1a–i were measured by GC analysis.
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Deracemization experiments by using ADH evo-1.1.200
Iodine (30 mg, 1.5 equiv.) and TEMPO (2.5 mg, 0.1 equiv.) were suc-
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in 50 mm Tris-HCl buffer pH 10 (4 mL). The mixture was sonicated
for 1 h at room temperature until complete conversion to ketone
2a–i. After this time, the reaction was stopped by adding a saturat-
ed Na2S2O3 aqueous solution (300 mL). An aliquot (500 mL) was
then withdrawn, and evo-1.1.200 (3 U), 10 mm NADH (50 mL),
10 mm MgCl2 (50 mL), and 2-PrOH (25 mL) were successively added.
The mixture was shaken at 308C and 250 rpm for additional 24 h.
Then, the mixture was extracted with EtOAc (2”500 mL), centri-
fuged (13000 rpm, 90 s), and dried (Na2SO4). The substrate conver-
sion and enantiomeric excess of (R)-alcohols 1a–i were measured
by GC analysis.
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Acknowledgements
Financial support from the Spanish Ministerio de Economía y
Competitividad (CTQ-2013-44153-P), the Principado de Asturias
(SV-PA-13-ECOEMP-42), and the University of Oviedo (UNOV-13-
EMERG-01) is gratefully acknowledged.
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Keywords: alcohols
oxidoreductases · TEMPO
·
biocatalysis
·
deracemization
·
[1] V. Kçhler, N. J. Turner, Chem. Commun. 2015, 51, 450–464.
Received: July 23, 2015
[2] a) N. J. Turner, Curr. Opin. Chem. Biol. 2010, 14, 115–121; b) A. Díaz-Ro-
dríguez, I. Lavandera, V. Gotor, Curr. Green Chem. 2015, 2, 192–211.
Published online on October 16, 2015
ChemCatChem 2015, 7, 4016 – 4020
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