Deracemization of sec-alcohols through sequential application of C. Albicans and Ketoreductases

Article abstract of DOI:10.21577/0103-5053.20180205

A biocatalytic cascade process was developed using immobilized cells of the wild type yeast Candida albicans CCT 0776 in calcium alginate beads and a commercially available ketoreductase. The aim was to promote deracemization by stereoinversion of (±)-1-arylethanols in high substrate concentration (above 100 mmol L^-1) to prepare the (R)-enantiomers of the alcohols (90-99percent), with a high enantiomeric excess (83-99percent) after 2 to 19 h. The (R)-1-(3-methoxyphenyl)ethanol, with 70percent yield and 91percent ee, obtained after 5 h was used to prepare (S)-1-(3-methoxyphenyl)-ethylamine with 60percent yield and 91percent ee after two steps, a key intermediate in the synthesis of (S)-rivastigmine.

Full text of DOI:10.21577/0103-5053.20180205

http://dx.doi.org/10.21577/0103-5053.20180205
J. Braz. Chem. Soc., Vol. 30, No. 4, 772-779, 2019
Printed in Brazil - ©2019 Sociedade Brasileira de Química
Article
Deracemization of sec-Alcohols through Sequential Application of
C. albicans and Ketoreductases
a
a
,a
Fábio D. Nasário, Paulo J. S. Moran and José Augusto R. Rodrigues *
a
Departamento de Química Orgânica, Instituto de Química, Universidade Estadual de Campinas,
3084-971 Campinas-SP, Brazil
1
A biocatalytic cascade process was developed using immobilized cells of the wild type yeast
Candida albicans CCT 0776 in calcium alginate beads and a commercially available ketoreductase.
The aim was to promote deracemization by stereoinversion of (± ±-1-arylethanols in high substrate
-1
concentration (above 100 mmol L ± to prepare the (R±-enantiomers of the alcohols (90-99%±, with
a high enantiomeric excess (83-99%± after 2 to 19 h. The (R±-1-(3-methoxyphenyl±ethanol, with
70% yield and 91% ee, obtained after 5 h was used to prepare (S±-1-(3-methoxyphenyl±-ethylamine
with 60% yield and 91% ee after two steps, a key intermediate in the synthesis of (S±-rivastigmine.
Keywords: oxidative kinetic resolution, deracemization, stereoinversion, ketoreductase,
alcohol dehydrogenase
Introduction
Biocatalytic processes using whole cells of
microorganisms or isolated enzymes are recognized as
useful tools in the preparation of important enantiopure
compounds such as chiral alcohols or amines. These
compounds are used as building blocks to synthesize natural
1
-5
products, pharmaceuticals, and agricultural chemicals
6
7
8
such as (S±-rivastigmine, taxol, (S±-fluoxetine, beta-
lactams antibiotics, lipid A and L-carnitine and
S±-beta-blockers as propranolol, metoprolol and
S±-carvedilol (Figure 1±.
9
,10
11
12
1
3
(
(
The use of enzymes was restricted to selective
modification of racemic mixtures in kinetic resolution
(
KR± procedures, resulting in a maximum 50% yield
Figure 1. Examples of drugs derived from chiral secondary alcohol
precursors.
1
4-20
of the enantiopure product
compounds.
or reduction of prochiral
2
1-23
In recent years, multi-enzymatic
methodologies have emerged as useful tools in the
preparation of chiral compounds with a theoretical 100%
yield. This is because enzymes largely operate under similar
conditions, such as neutral pH and moderate temperature,
aldehydes±, as well as the stereoselective oxidation of
1,26,27
alcohols.
The use ofADHs to promote deracemization
of racemic secondary alcohols, in substrate concentration
-1
range of 25-80 mmol L , has been studied using a single
2,24,25
28-32
33-35
and typically exhibit high substrate specificity.
microorganism, a consortium of two microorganisms,
whole cells of a natural or recombinant biocatalyst and an
Oxidoreductases, such as alcohol dehydrogenases
1
,36
6,37
(
ADHs, also known as ketoreductases (KREDs±±, depend
isolated ADH,
chemo-enzymatic
and enzymatic
+
38,39
on their (costly± nicotinamide cofactor (NAD(P± ± to
catalyze enantioselective reduction of ketones (and
processes
with a view to obtaining enantiopure
compounds exhibiting a 100% maximum yield, since
racemic substrates are more readily available starting
24
*
e-mail: jaugusto@iqm.unicamp.br
materials than prochiral substrates.

Vol. 30, No. 4, 2019
Nasário et al.
773
-
1
Biocatalytic deracemization of secondary alcohols,
which means conversion of a racemate into an enantiopure
compound mediated by enzymes, generally occurs
via two distinct mechanisms: stereoinversion or cyclic
deracemization. Deracemization via stereoinversion
involves the stereoselective oxidation of an enantiomer
into an intermediate ketone, which is subsequently
reduced to the alcohol, with the opposite configuration to
the enantiomer oxidized, obtaining a single enantiomer at
thickness±, using helium as the carrier gas (0.9 mL min ± and
a split ratio of 1:50. The injector and detector temperatures
were maintained at 230 and 280 °C, respectively. Column
temperature was kept at 60 °C for 1 min, increased to
-1
230 °C at a rate of 30 °C min , raised again to 280 °C at a
rate of 25 °C min and then maintained constant for 2 min.
One microliter of a 0.3 mg mL solution of the compound
or extracted reaction aliquots in ethyl acetate was injected.
Chiral GC flame ionization detection (FID± analysis
was conducted in an Agilent 6850 Series GC system
equipped with a Hydrodex -β-3P chiral capillary column
(25 m × 0.25 mm × 0.25 m±. The carrier gas was hydrogen
-1
-1
40
®
the end of the process. Cyclic deracemization systems,
on the other hand, use a low(non±-selective irreversible
reaction, such as partially selective reduction of a prochiral
ketone, combined with stereospecific oxidation of an
enantiomer. Thus, the enantiomer that is not oxidized
®
-1
(1 mL min ± and injector and detector temperatures were set
at 200 and 220 °C, respectively. Chiral high-performance
liquid chromatography (HPLC± analysis was carried out
4
1-43
accumulates after several cycles.
In this respect, the
®
yeast Candida sp. is widely reported as an important
in an Agilent Technologies 1200 series system, using a
source of oxidoreductases and mediates the synthesis of
15 cm × 2.1 mm × 5 μm Supelco Astec cellulose DMP
column obtained from Sigma-Aldrich , with a UV-Vis
44
®
a broad range of chiral compounds. In a previous study,
we reported deracemization of (± ±-1-phenylethanol via
stereoinversion, initially using a sequential process with
Candida albicans and Lactobacillus brevis to prepare
the (R±-enantiomer. Inverting the sequence of these
detector. Thin layer chromatography (TLC± analysis was
performed using aluminum plates coated with F254 nm
silica gel. The C. albicans CCT 0776 yeast strain was
purchased from the André Tosello Research Foundation
(Campinas, São Paulo State, Brazil± and stored at ultra-low
temperature (–80 °C± in 20% glycerol.
14
microorganisms produces an enantioenriched antipode.
Here, we report efficient deracemization via
stereoinversion of (± ±-1-arylethanols, in high substrate
Growth conditions of C. albicans, cell immobilization,
synthesis, characterization of the racemic alcohols (± ±-1a-i
and isolated products (R±-1a-i obtained via biocatalysis
-1
concentration (above 100 mmol L ±, using a cascade process
with Candida albicans CCT 0776 and different commercial
ketoreductases to produce enantioenriched (R±-alcohols.
Additionally, we demonstrate a practical application of this
deracemization procedure and the value of these compounds
in synthesizing the key intermediate drug (S±-rivastigmine,
14
were reported in a previous study.
Screening of ketoreductases: reduction of acetophenone
(2a)
45
used to treat Parkinson’s and Alzheimer’s disease.
The ketoreductases obtained from Codexis, as a
lyophilized pounder with 40-70% of the enzyme, were
screened in accordance with the producer’s protocol.
Approximately 10 mg of KREDs 1-5 (Table 1, entries
Experimental
General methods
1
-5± were weighed out into separate labelled vials, which
T h e k e t o n e s 2 a - i a n d A D H f r o m
Parvibaculum lavamentivorans (ADH-PL± were purchased
were added with 1 mL of a fresh KRED Recycle Mix N
-
1
-1
solution (containing 50 mmol L of 2a, 250 mmol L
®
®
-1
from Sigma-Aldrich and the KREDs from Codexis . All
the other commercial reagents and solvents were purchased
of potassium phosphate, 2 mmol L of magnesium
-1
+
-1
+
sulfate, 1.1 mmol L of NAD , 1.1 mmol L of NADP ,
®
-1
-1
from Sigma-Aldrich at the highest purity available and
80 mmol L of D-glucose and 10 U mL of GDH (glucose
dehydrogenase±, pH 7.0±. The reaction was mixed at 30 °C
and 180 rpm for 19 h. An aliquot of 250 μL of the reaction
mixture was extracted with 500 μL of ethyl acetate and
dried with sodium sulfate. Conversion and enantiomeric
excess were measured by GC-mass spectrometry (MS±
and GC-FID. For ADH-PL (Table 1, entry 25±, 15 μL
of enzyme suspension was used, while for KREDs 6-24
(Table 1, entries 6-24±, 10 mg of each enzyme was weighed
out into separate labelled vials, which were added with
used as received. Nuclear magnetic resonance (NMR±
spectra were recorded in an Avance spectrometer at 400
1
13
(
H NMR± and 100 MHz ( C NMR±. Chemical shifts were
reported in parts per million (ppm± and coupling constants
J± in hertz (Hz±. Optical rotation was measured using a
(
CarlWeiss POLAMATA polarimeter. Gas chromatography
®
(
GC± analyses were performed in an Agilent 6890 Series
GC system equipped with a J&W Scientific DB1 silica
capillary column (30 m × 0.25 mm i.d. × 0.25 m film

7
74
Deracemization of sec-Alcohols through Sequential Application of C. albicans and Ketoreductases
J. Braz. Chem. Soc.
-
1
0
.9 mL of KRED Recycle Mix P (containing 125 mmol L
The substrate concentration study with (± ±-1a
(35-200 mmol L ± was performed with 11 g of C. albicans
-1
-1
of potassium phosphate, 1.25 mmol L magnesium sulfate,
-1
+
-1
1
2
mmol L of NADP , pH 7.0± and 0.1 mL of 250 mmol L
immobilized in calcium alginate beads suspended in
approximately 10 mL of distilled water for 1 h. Next, 1 mL
of the reaction was collected, filtered through a 0.2 μm
regenerated cellulose membrane filter, and transferred
to a 2 mL vial containing 4 mg of the ketoreductase
KRED-P1-A04, and 25 mg of dry Recycle Mix P. The
amount of co-substrates were proportional to initial amount
of 1a, 4 equiv. of acetone in the first step, and 37 equiv. of
2-propanol in the second step.
a solution in 2-propanol. The reaction was mixed at 30 °C
and 180 rpm for 24 h. A 250 μL aliquot of the reaction
mixture was extracted with 500 μL of ethyl acetate and
dried with sodium sulfate. Conversion and enantiomeric
excess (ee± were measured by GC-MS and GC-FID.
General procedure for deracemization by stereoinversion
using immobilized C.albicans cells in calcium alginate beads
and a ketoreductase
Synthesis of (S)-1-(3-methoxyphenyl)ethylamine ((S)-3h)
It was added 0.5 mmol of substrate dissolved in
0
.150 mL of acetone as co-solvent to a 125 mL Erlenmeyer
Triphenylphosphine (1.2 equiv.± and phthalimide
(1 equiv.± were added to a solution of (R±-1h (0.23 mmol±
in 3 mL of dry tetrahydrofuran (THF±, under nitrogen
atmosphere and magnetic stirring. The solution was
cooled to 0 °C and diisopropyl azodicarboxylate (DIAD,
1.2 equiv.± was added. The mixture was heated to 25 °C
and stirred for approximately 3 h. The solvent was
removed by vacuum and the product was purified by flash
chromatography using a hexane/ethyl acetate gradient
as the mobile phase and a Biotage Isolera Spektra One
system. The protected amine was dissolved in THF (3 mL±
and ethanol (1 mL±, and hydrazine monohydrate (50 μL±
was added. The reaction was stirred at 66 °C for 2 h.
The white suspension formed was filtered and washed
with THF, and the organic solvent was evaporated in
vacuum, producing a light-yellow oil, (S±-3h, with 60%
flask containing 11 g of C. albicans immobilized in
calcium alginate beads suspended in approximately
1
3
0 mL of distilled water. The flask was incubated at
0 °C and 180 rpm in an orbital shaker, until complete
oxidative kinetic resolution (OKR± was achieved, which
was monitored by GC-MS (conversion± and GC-FID or
HPLC (ee±. Next, 1 mL of the reaction was collected,
filtered through a 0.2 μm regenerated cellulose membrane
filter, and transferred to a 2 mL vial containing 50 mg of
dry Recycle Mix N or 25 mg of dry Recycle Mix P, added
with 100 μL of 2-propanol and 4 mg of an anti-Prelog
KRED or 15 μL of ADH-PL. A procedure using 2 mg of
KRED instead of 4 mg exhibited no loss of efficiency. The
reaction was mixed at 30 °C and 180 rpm for 24 h.A 250 μL
aliquot of the reaction mixture was extracted with 500 μL of
ethyl acetate and dried with sodium sulfate. Conversion and
ee were determined by GC-MS and GC-FID or HPLC. The
process was scaled up using the total reaction volume from
the OKR step (10 mL±; 100 μL of ADH-PL was added with
+
yield (0.14 mmol±. MS (m/z, %±: 151 (M , 9±, 136 (100±,
1
109 (16±, 94 (10±, 77 (8±, 44 (12±; H NMR (400 MHz,
CDCl ± d 1.39 (d, 3H, J 6.8 Hz±, 1.86 (br s, 2H±, 3.82
3
(s, 3H±, 4.10 (q, 1H, J 6.8 Hz±, 6.78 (m, 1H±, 6.93 (m,
1
3
1
mL of 2-propanol and 250 mg of dry Recycle Mix P. The
2H±, 7.26 (m, 1H±; C NMR (100 MHz, CDCl ± d 25.5,
3
reaction was mixed at 30 °C and 180 rpm for 5 h. The product
was extracted with 10 mL ethyl acetate (3 times±, dried
with sodium sulfate and the solvent removed in vacuum,
producing a colorless liquid, (R±-1h, 70% yield (0.35 mmol±.
51.3, 55.2, 111.4, 112.2, 118.1, 129.5, 149.4, 159.9;
2
0
47
20
[α] –26.0 (c 1.0; CHCl ± 91% ee (S±, Lit. [α] –26.8
D
3
D
47
(c 1.15; CHCl ± ≥ 96% ee for the (S±-enantiomer. In order
3
to determine the enantiomeric excess, the enantioenriched
amine (S±-3h was acetylated and its GC chromatogram
compared to that obtained for the acetylated racemic
compound. Thus, chemical acetylation was carried out
using approximately 1 mg of the amine (S±-3h dissolved
in ethyl acetate (1 mL±, added with K CO (10 mg± and
+
MS (m/z, %±: 152 (M , 47±, 137 (44±, 135 (20±, 109 (100±,
1
9
4 (30±, 77 (33±, 43 (17±; H NMR (400 MHz, CDCl ±
3
d 1.48 (d, J 6.4 Hz, 3H±, 1.99 (s, 1H±, 3.81 (s, 3H±, 4.86 (q,
J 6.4 Hz, 1H±, 6.81 (m, 1H±, 6.94 (m, 2H±, 7.25 (t, J 8.0 Hz,
1
3
1
1
(
H±; C NMR (100 MHz, CDCl ± d 25.15, 55.23, 70.34,
3
2
3
20
10.92, 112.90, 117.70, 129.55, 147.62, 159.79; [α] +28
c 1.0; CHCl ± 91% ee (R±. Lit. [α] +29 (c 1.0; CHCl ±,
acetic anhydride (250 μL±. The mixture was shaken at
D
46
20
D
-1
30 °C and 900 rpm for 1 h and an aqueous 10 mol L
3
3
5
4% ee for the (R±-enantiomer. The ee was determined by
NaOH solution (200 μL± was added. The organic layer
was separated, dried with anhydrous sodium sulfate and
transferred to a GC glass vial for analysis. Enantiomeric
GC-FID, with an oven temperature of 100 °C for 3 min, then
increased to 180 °C at 1.5 °C min and maintained constant
-1
48
for 10 min; retention time: t = 22.95 min [(R±-enantiomer];
tS = 23.60 min [(S±-enantiomer].
excess was determined by GC-FID at temperatures of
120-160 °C (0.4 °C min ± and 160-180 °C (5 °C min ± and
R
-1
-1

Vol. 30, No. 4, 2019
Nasário et al.
775
kept constant for 5 min; t = 91.86 min [(S±-enantiomer];
tR 93.08 min [(R±-enantiomer].
The reactions were monitored for 4-24 h; based on Codexis’
screening protocol, however, in some reactions > 90%
conversion was detected after 1 h or, in certain cases, 15 min.
Of the seven KREDs that displayed anti-Prelog activity,
NADH-dependent KRED-NADH-110 converted 96%
of acetophenone (2a± to (R±-1a with 87% ee (Table 1,
entry 5±. It was selected to couple with resting cells of
S
Results and Discussion
Twenty-five commercial KREDs were screened for
acetophenone (2a± reduction to determine enantioselectivity
and conversion to 1-phenylethanol (1a±. As shown in
Table 1, all KREDs exhibited high conversion (90-98%±
in reducing 2a after 4-24 h. Twelve KREDs yielded the
enantiomerically enriched alcohol 1a with > 80% ee, while
five KREDs showed Prelog selectivity and produced (S±-1a
with 96-99% ee (Table 1, entries 2-4, 22 and 24±, and seven
KREDs displayed anti-Prelog selectivity producing (R±-1a
with 87-99% ee (Table 1, entries 5, 6, 13, 20, 21 and 25±.
1
4,29,44
C. albicans, a yeast capable of oxidizing alcohols,
and developing a deracemization process of (± ±-1a
by stereoinversion. Initially, we attempted to combine
C. albicans and the KRED in a one-pot process; however,
this was ineffective, likely due to competition between
enzymes with opposite stereoselectivity for the substrate.
The yeast was immobilized in calcium alginate beads to
ensure easier removal of the biocatalyst from the media
Table 1. Screening of commercial ketoreductases for reduction of acetophenone (2a±
1
a
entry
KRED
Cofactor
Recycling system
time / h
d
e
Conversion / %
ee / %
a
1
KRED-101
KRED-119
NADPH
NADPH
NADPH
NADH
GDH/glucose
GDH/glucose
GDH/glucose
GDH/glucose
GDH/glucose
2-propanol
2-propanol
2-propanol
2-propanol
2-propanol
2-propanol
2-propanol
2-propanol
2-propanol
2-propanol
2-propanol
2-propanol
2-propanol
2-propanol
2-propanol
2-propanol
2-propanol
2-propanol
2-propanol
2-propanol
19
19
19
19
19
4
93
93
98
96
96
96
98
96
98
98
98
97
96
98
98
96
98
97
97
96
96
95
94
96
90
4 (S±
98 (S±
> 99 (S±
> 99 (S±
87 (R±
> 99 (R±
3 (S±
a
2
a
3
KRED-130
a
4
KRED-NADH-101
KRED-NADH-110
KRED-P1-A04
KRED-P1-B02
KRED-P1-B05
KRED-P1-B10
KRED-P1-B12
KRED-P1-C01
KRED-P1-H08
KRED-P1-H10
KRED-P2-B02
KRED-P2-C02
KRED-P2-B02
KRED-P2-DO3
KRED-P2-D11
KRED-P2-D12
KRED-P2-G03
KRED-P2-H07
KRED-P3-B03
KRED-P3-G09
KRED-P3-H12
ADH-PL
a
5
NADH
b
6
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
NADPH
b
7
4
b
8
4
28 (S±
4 (S±
b
9
4
b
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
4
1 (R±
b
b
b
b
b
b
b
b
b
b
b
b
b
b
c
4
4 (S±
4
9 (S±
4
93 (R±
5 (S±
4
4
7 (S±
4
90 (R±
4 (S±
4
4
53 (S±
8 (R±
4
4
89 (R±
> 99 (R±
> 99 (S±
46 (S±
96 (S±
> 99 (R±
4
4
4
4
2
24
a
-1
b
c
1
0 mg of KRED, 1 mL of KRED Recycle Mix N with 50 mmol L of 2a; 10 mg of KRED or 15 μL of enzyme suspension, 0.9 mL of KRED Recycle
-
1
d
e
Mix P, 0.1 mL of 2-propanol, 25 mmol L of 2a; conversion measured by gas chromatography-mass spectrometry (GC-MS±; enantiomeric excess (ee±
determined by gas chromatography-flame ionization detector (GC-FID±. KRED: ketoreductase; NADH: nicotinamide adenine dinucleotide; ADH-PL:
alcohol dehydrogenases from Parvibaculum lavamentivorans; NADPH: nicotinamide adenine dinucleotide phosphate; GDH: glucose dehydrogenase.

7
76
Deracemization of sec-Alcohols through Sequential Application of C. albicans and Ketoreductases
J. Braz. Chem. Soc.
after oxidation (first step±. Besides there is the advantage
to reuse the beads during four oxidative kinetic resolution
A sequential process was proposed for deracemization
via stereoinversion: efficient OKR of (± ±-1a (0.5 mmol± was
observed after 40 min with C. albicans cells immobilized in
calcium alginate beads: 51% acetophenone (2a± and 49%
(R±-1-phenylethanol ((R±-1a±, with 94% ee. Subsequently,
(
OKR± cycles of (± ±-1-phenylethanol (1a± without activity
loss in each cycle.
Oxidative kinetic resolution (OKR± of
+
(
± ±-1-arylethanols was performed using immobilized
cells in Ca-alginate beads, with reaction times between
0 min and 16 h. For each substrate (1a-i± the OKR
the beads were removed and KRED-NADH-110, NAD(P± ,
D-glucose, and GDH were dispersed in 1 mL of the reaction
mixture.After 15 min of reduction, 90% of (R±-1a with 91%
ee was obtained, resulting in successful deracemization in
less than 1 h (Figure 2±. The anti-Prelog ketoreductases
KRED-P1-H10, KRED-P2-H07, KRED-P1-A04,
KRED-P2-C11 and ADH-PL, which are dependent of
NADPH (nicotinamide adenine dinucleotide phosphate±
cofactor, were also coupled with the immobilized yeast
cells. In this process, 2-propanol was used to regenerate
the NADPH cofactor, enabling efficient deracemization of
(± ±-1-arylethanols and yielding (R±-enantiomers (Table 3±.
The deracemization process was efficient and produced
(R±-1-arylethanols ((R±-1a-i± with excellent conversion
(up to 99%± and high ee (up to > 99%± in less than 20 h
(oxidative and reductive steps±. However, for 1f and 1g,
successful OKR with C. albicans cells immobilized in
Ca-alginate beads (Table 2, entries 6 and 7± was observed
in the first step, but no or low selective reduction of the
respective ketones (2f and 2g± was detected (second
step± with the KREDs studied after 24 h (Table 3,
entries 15 and 16±. The entire deracemization process of
4
time was optimized in order to obtain the best result
possible. The (R±-1a-i were obtained with conversion
of 49-79% and 75-99% ee, as well as the corresponding
ketone with conversion of 21-51% (Table 2±. For (± ±-1a
(
Table 2, entry 1±, OKR was reached after 40 min and
C. albicans immobilized in Ca-alginate beads oxidized
the (S±-enantiomer to ketone acetophenone (2a± with
5
1% conversion, obtaining 49% of (R±-1a with 94%
ee. OKR of (± ±-1-(4-chlorophenyl±ethanol ((± ±-1b±
and (± ±-1-(4-bromophenyl±ethanol (± ±-1d yielded the
(
7
R±-alcohols after a reaction of 4 and 6 h, with 79 and
5% ee, respectively, whereas the reaction was finalized
in 1 h for (± ±-1-(4-fluorophenyl±ethanol ((± ±-1c±, which
also exhibited a halogen in position 4 of the aromatic ring,
and (R±-1-(4-fluorophenyl±ethanol ((R±-1c± was obtained
with 57% conversion and 90% ee (entry 2-4, Table 2±.
The presence of a hydroxyl group in position 3 or 4 of
the aromatic ring (entry 7 and 9, Table 2± promoted a
significant increase in ORK time, from 40 min to 16 h.
Table 2. Oxidative kinetic resolution (OKR± of 0.50 mmol of 1-arylethanol with C. albicans cells immobilized in calcium alginate beads
Alcohol
a
entry
Substrate
R
time / h
b
c
Conversion / %
ee / %
c
1
2
3
4
5
6
7
8
(± ±-1a
(± ±-1b
(± ±-1c
(± ±-1d
(± ±-1e
(± ±-1f
(± ±-1g
(± ±-1h
(± ±-1i
H
0.67
4
49
73
57
79
55
49
49
57
53
94 (R±
c
4-Cl
79 (R±
c
4-F
1
90 (R±
c
4-Br
6
75 (R±
c
4-Me
4-OMe
4-OH
3-OMe
3-OH
1
93 (R±
c
c
2
> 99 (R±
> 99 (R±
16
2
c
93 (R±
d
9
16
96 (R±
a
0
.5 mmol of 1a-i, 0.150 mL of acetone, 11 g of C. albicans immobilized in calcium alginate beads, 10 mL of H O. The reactions were mixed at 30 °C and
2
b
c
1
80 rpm; conversion monitored by gas chromatography-mass spectrometry (GC-MS±; enantiomeric excess (ee± determined by gas chromatography-flame
d
ionization detector (GC-FID±; ee was determined by HPLC.

Vol. 30, No. 4, 2019
Nasário et al.
777
Figure 2. Deracemization by stereoinversion of (± ±-1-arylethanols using immobilized C. albicans cells in calcium alginate beads and an anti-Prelog KRED.
Table 3. Deracemization of (± ±-1-arylethanols by stereoinversion using immobilized C. albicans cells in calcium alginate beads and a commercial KRED
1
a-i
a
entry
Substrate
R
H
Reduction catalyst
time / h
e
Conversion / %
ee / %
b
f
1
1a
KRED-P1-A04
KRED-P1-H10
ADH-PL
2
2
90
98
97 (R±
92 (R±
95 (R±
82 (R±
83 (R±
84 (R±
94 (R±
95 (R±
95 (R±
89 (R±
84 (R±
79 (R±
96 (R±
95 (R±
b
f
f
f
f
f
f
f
f
f
f
f
f
f
2
d
3
9
92
b
4
1b
1c
1d
1e
4-Cl
4-F
KRED-P2-C11
KRED-P2-H07
ADH-PL
5
> 99
> 99
> 99
90
b
5
5
c
6
12
3
b
7
KRED-P1-A04
KRED-P2-C11
ADH-PL
b
8
3
92
c
9
7
91
b
1
1
1
1
1
1
1
1
1
1
2
0
1
2
3
4
5
6
7
8
9
0
1
4-Br
4-CH3
KRED-P1-A04
KRED-P2-C11
ADH-PL
19
7
> 99
> 99
> 99
96
b
c
14
2
b
b
b
c
KRED-P2-C11
KRED-P2-H07
KRED-P1-A04
ADH-PL
2
97
f
1f
1g
1h
4-OCH3
4-OH
24
40
19
3
50
> 99 (R±
f
52
87 (R±
b
b
d
b
b
f
3-OMe
KRED-P1-A04
KRED-P2-H07
ADH-PL
95
93 (R±
f
99
93 (R±
f
10
17
96
91 (R±
g
1i
3-OH
KRED-P1-A04
KRED-P2-H07
94
96 (R±
g
2
17
94
96 (R±
a
st
nd
1
step: 0.5 mmol of 1a-i, 0.150 mL of acetone, 11 g of immobilized C. albicans, 10 mL of H O; 2 step: 1 mL of the reaction, 25 mg of dry Recycle Mix
2
b
c
d
P, 0.100 mL of 2-propanol; 4 mg of KRED or 15 μL of ADH-PL; 10 mL of oxidative kinetic resolution (OKR± (first step±, 0.100 mL of ADH-PL, 1 mL
of 2-propanol and 250 mg of dry Recycle Mix P; conversion monitored by gas chromatography-mass spectrometry (GC-MS±; enantiomeric excess (ee±
determined by gas chromatography-flame ionization detector (GC-FID±; ee determined by HPLC. KRED: ketoreductase;ADH-PL: alcohol dehydrogenases
e
f
g
from Parvibaculum lavamentivorans.
(
4
1
± ±-1-phenylethanol ((± ±-1a± lasted about 2 h, including
0 min of OKR using immobilized C. albicans cells and
h of reduction with KRED-P1-A04 or KRED-P1-H10
was maintained (entries 3 and 19, Table 3±, leading to
(R±-1a and (R±-1h with excellent conversion (above 90%±,
91-95% ee and 70% yield of (R±-1h starting from 0.5 mmol
of racemate (± ±-1h.
(
(
entries 1 and 2, Table 3±, yielding (R±-1-phenylethanol
(R±-1a± with 90-98% conversion and 92-97% ee.
Substrate concentration was evaluated from
-
1
Reduction of acetophenone (2a± withADH-PL was slower,
lasting 8 h (entry 3, Table 3±. In this case, the complete
procedure took 9 h and provided 92% (R±-1a with 95% ee.
The process was scaled up (10 times± with (± ±-1a and
35-200 mmol L of (± ±-1a, using C. albicans cells
immobilized in Ca-alginate beads and 4 mg of
KRED-P1-A04 after 2 h (Table 4±.The process demonstrated
-
1
to be efficient for concentration until 110 mmol L ,
(
± ±-1h using the ADH-PL. Reproducibility of the results
furnishing (R±-1a (91-94%, 90-94% ee, entries 1-4,

7
78
Deracemization of sec-Alcohols through Sequential Application of C. albicans and Ketoreductases
J. Braz. Chem. Soc.
Table 4. Study of the substrate concentration in deracemization of (± ±-1a,
using C. albicans cells immobilized in Ca-alginate beads and 4 mg of
KRED-P1-A04 after 2 h
Methodologies with a chemoenzymatic or biocatalytic
step have been reported in the preparation of (S±-3h due to
6,48,50,51
the importance of this compound in organic synthesis;
however, none applied deracemization via stereoinversion
step with substrate concentration above 100 mmol L .
Substrate concentration
1a
a
entry
-1
-
1
b
c
(
(± ±-1a± / (mmol L ±
Conversion / %
ee / %
1
2
3
4
5
6
35
55
94
92
92
91
92
75
93 (R±
94 (R±
93 (R±
90 (R±
79 (R±
40 (R±
Conclusions
82
110
150
This study demonstrated the significant oxidative
potential of C. albicans CCT 0776 as an enantioselective
catalyst for the oxidation of secondary alcohols, especially
200
a
st
-1
1
step: (± ±-1a 35-200 mmol L , 4 equiv. of acetone, 11 g of immobilized
(
± ±-1-arylethanols, and its application in oxidation-
nd
C. albicans, 10 mL of H O, 1 h; 2 step: 1 mL of the reaction, 25 mg
2
reduction sequences for deracemization via stereoinversion
of those compounds coupled with commercially available
ketoreductases, yielding (R±-1-arylethanols, (R±-1a-e and
of dry Recycle Mix P, 37 equiv. of 2-propanol, 4 mg of KRED-P1-A04,
b
1
h; conversion monitored by gas chromatography-mass spectrometry
c
(GC-MS±; enantiomeric excess (ee± determined by gas chromatography-
flame ionization detector (GC-FID±.
(R±-1h-i, with 90-99% conversion and 79-99% ee, after
2-19 h. As an example of the applicability of this procedure,
Table 4±. Similar processes were proposed to deracemize
secondary alcohols such as 2-octanol, sulcatol, 2-decanol
we obtained (R±-1h from (± ±-1-(3-methoxyphenyl±ethanol
((± ±-1h±with70%yieldand91%ee,whichwasusedtoprepare
the (S±-rivastigmine precursor (S±-1-(3-methoxyphenyl±-
ethylamine((S±-3h±, with60%yieldand91%ee.Theseresults
represent a useful asymmetric green methodology applicable
in organic synthesis, since the immobilization of biocatalyst
provided the reuse of the catalyst besides the possibility to
-
1
and 1-phenylethanol, with 30-80 mmol L substrate
1
,36
concentration. Compared to the procedure developed
-1
here, we could successfully deracemize 110 mmol L of
± ±-1a and other (± ±-1-arylethanols into the (R±-enantiomer
(
with high conversion and ee, using commercially available
ketoreductases and immobilized yeast.
-1
increase the substrate concentration above 100 mmol L .
As an example of the applicability of this procedure, it
was obtained (R±-1h from (± ±-1-(3-methoxyphenyl±ethanol
Supplementary Information
(
(± ±-1h± with 70% yield and 91% ee, which was used to
prepare (S±-1-(3-methoxyphenyl±ethylamine ((S±-3h±, a key
intermediate of (S±-rivastigmine synthesis that is used to
Supplementary data are available free of charge at
http://jbcs.sbq.org.br as PDF file.
49
treat Parkinson’s and Alzheimer’s diseases. Synthesis of
S±-3h consisted of two steps: a Mitsunobu reaction, with
(
Acknowledgments
substitution of the hydroxyl group for phthalimide and
configuration inversion, followed by phthalimide hydrolysis
with hydrazine, yielding 60% (S±-3h with 91% ee. The
preparation of (S±-3h from (± ±-1h is shown in Figure 3.
The authors are grateful to FAPESP (grant 2016/12074-7,
SãoPauloStateFoundation±andCNPq(grant301212/2010-4±
for their generous financial support.
Figure 3. Synthesis of (S±-1-(3-methoxyphenyl±ethylamine ((S±-3h± from (± ±-1-(3-methoxyphenyl±ethanol ((± ±-1h±.

Vol. 30, No. 4, 2019
Nasário et al.
779
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