Continuous flow whole cell bioreduction of fluorinated acetophenone

Article abstract of DOI:10.1016/j.tet.2013.12.036

Several microorganism strains were used to reduce 2,2,2- trifluoroacetophenone (1) and 4′-Br-2,2,2-trifluoroacetophenone (3). Immobilized cells of Geotrichum candidum in calcium alginate led to conversion and enantiomeric excess higher than 99%. By using immobilized G. candidum cells under continuous flow conditions, the same conversion and enantiomeric excess were achieved in 90 min of residence time.

Full text of DOI:10.1016/j.tet.2013.12.036

Tetrahedron 70 (2014) 3239e3242
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Continuous flow whole cell bioreduction of fluorinated
acetophenone
Raquel de Oliveira Lopes a, Joyce Benzaquem Ribeiro a, Amanda Silva de Miranda a,
a
a
b
^
Gabriela Veloso Vieira da Silva , Leandro S.M. Miranda , Ivana Correa Ramos Leal ,
,
Rodrigo Octavio Mendonc¸ a Alves de Souza a
*
a Biocatalysis and Organic Synthesis Group, Chemistry Institute, Universidade Federal do Rio de Janeiro, CEP 22941 909 Rio de Janeiro, Brazil
b
ꢀ
Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, CEP 22941 909 Rio de Janeiro, Brazil
a r t i c l e i n f o
a b s t r a c t
Several microorganism strains were used to reduce 2,2,2-trifluoroacetophenone (1) and 40-Br-2,2,2-
trifluoroacetophenone (3). Immobilized cells of Geotrichum candidum in calcium alginate led to con-
version and enantiomeric excess higher than 99%. By using immobilized G. candidum cells under con-
tinuous flow conditions, the same conversion and enantiomeric excess were achieved in 90 min of
residence time.
Article history:
Received 21 October 2013
Received in revised form 5 December 2013
Accepted 11 December 2013
Available online 28 December 2013
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Continuous flow
Bioreduction
Chiral trifluoromethyl alcohols
1. Introduction
glucose, formic acid or dihydrogen, are necessary to perform the
reduction reaction. The reduction of the substrate accompanies the
Chiral trifluoromethyl alcohols play an important role in phar-
maceutical, veterinary, agrochemical, and material sciences based
on the influence of fluorine’s unique properties. Of particular rel-
evance is the emergence of drug candidates featuring fluorine
atoms, which often present a favorable therapeutic profile.1 Chiral
trifluoromethyl alcohols can be produced by asymmetric hydro-
genation of trifluoromethyl ketones catalyzed by organo-
boranes,2e5 (S)-binap,6 chiral organomagnesium amides,7 chiral
rhodium-(I)-complexes8 or chiral ruthenium-complexes.9,10 How-
ever, some chemical catalysts led to insufficient levels of enantio-
selectivity and low catalytic efficiencies.
oxidation of the coenzyme from NADH to NADþ. Then, the co-
enzyme has to be reduced to NADH, which can be driven by dif-
ferent hydrogen sources. After that, the next cycle of the ketone can
occur (Scheme 1).18e20
On the other hand, chiral trifluoromethyl alcohols can also be
produced by biocatalytic processes.11e17 Biocatalysis often offers
advantages over chemical synthesis, mainly due to higher enan-
tioselectivity, milder and safer reaction conditions and lower en-
vironmental impact. Biocatalysts for reduction can be isolated
enzymes and whole cells microorganisms, animals or plants. To
exhibit catalytic activities, the enzymes require a coenzyme, such as
NADH or NADPH from which a hydride is transferred to the sub-
strate carbonyl carbon. Hydrogen sources, as ethanol, 2-propanol,
Scheme 1. The cycle of asymmetric reduction of ketone to alcohol by alcohol de-
hydrogenase, the coenzyme (NADH) and cofactor regeneration (2-propanol).
In comparison to isolated enzymes, the whole cell methodology
has distinct characteristics. Enzymes used as whole cells are stable
once they are used in their natural environment. Furthermore, the
cells have internal coenzyme and cofactor regeneration, so that the
addition of cheap glucose is sufficient to drive the reaction.21e25
* Corresponding author. E-mail addresses: souzarod21@yahoo.com.br, sou-
zarod21@gmail.com (R.O. Mendonc¸ a Alves de Souza).
0040-4020/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.12.036

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R. de Oliveira Lopes et al. / Tetrahedron 70 (2014) 3239e3242
In this work, we report the use of eight different microorgan-
these microorganisms, we decided to increase the substrate con-
centration to 14.4 mM. According to the results showed in Table 2,
considering acetophenone 1, G. candidum preserved the conversion
and enantioselectivity achieved using 7.2 mM of substrate, and A.
niger achieved 96% conversion. Different results were obtained to
acetophenone 3, for which increasing the concentration to 14.4 mM
leads to significantly decrease on conversions.
isms (six yeasts strains and two filamentous fungi strains) for the
asymmetric reduction of 2,2,2-trifluoroacetophenone (1) and 40-Br-
2,2,2-trifluoroacetophenone (3) (Scheme 2). Different concentra-
tions of substrate were studied using free cells and immobilized
cells (only yeasts strains) under batch and continuous flow
conditions.
Scheme 2. Reduction of 2,2,2-trifluoroacetophenone (1) and 40-Br-2,2,2-trifluoroacetophenone (3) using whole cells microorganisms.
2. Results and discussion
In order to improve the method, yeasts were immobilized in
calcium alginate spheres and tested for the reduction of aceto-
phenone 1 and 3.21e25 The immobilization can influence enantio-
meric excess and conversion level,26 however according to the
results showed in Table 3, immobilized Candida sp., G. candidum, R.
minuta, and R. rubra were able to completely reduce 2,2,2-
trifluoroacetophenone (1). On the other hand, reduction with
immobilized Hansenula sp. and K. marxianus presented lower
conversions in comparison to free whole cells.
In the first step of this work, we tested eight different micro-
organisms (six yeasts strains and two filamentous fungi strains) for
the asymmetric reduction of 2,2,2-trifluoroacetophenone (1) and
40-Br-2,2,2-trifluoroacetophenone (3) using free cells and substrate
concentrations of 7.2 and 14.4 mM (final concentration in 5% glu-
cose solution). According to the results showed in Table 1, all mi-
croorganisms were able to reduce completely 2,2,2-
trifluoroacetophenone (1), and some microorganisms, such as
Geotrichum candidum and Aspergillus niger provided the S-enan-
tiomer (2b) in enantiomeric excess higher than 90%. Kluyveromyces
marxianus and Rhodotorula minuta were able to produce the S-en-
antiomer (2b) in 50% and 45% ee, respectively, while lower ee were
obtained with Hansenula sp. (24%) and Candida sp. (29%). Mucor
ramannianus and Rhodotorula rubra furnished the R-enantiomer
(2a) in 13% and 45% ee, respectively. Interestingly, for some mi-
croorganisms the switch from hydrogen to bromine in the 40 po-
sition leads to decreased conversions compared to the 2,2,2-
trifluoroacetophenone (1). G. candidum, R. minuta, and R. rubra
were able to reduce completely 40-Br-2,2,2-trifluoroacetophenone
(3). G. candidum provided the S-enantiomer in 96% ee, while R.
minuta and R. rubra provided the S-enantiomer and R-enantiomer
in 50% and 35% ee, respectively. All reactions were carried out for
24 h at 30 ꢀC under orbital shaking speed of 150 rpm in the orbital
shaker.
Considering the 40-Br-2,2,2-trifluoroacetophenone (3), immo-
bilized Candida sp., G. candidum, R. minuta, and R. rubra were able to
reduce it preserving the same conversion achieved by using free
whole cells. Immobilized Hansenula sp. and K. marxianus increased
conversions compared with whole cells. All immobilized microor-
ganisms preserved the enantiomeric excess achieved by using of
free whole cells.
Based onTable 3, high ee were achieved for the reduction of 2,2,2-
trifluoroacetophenone (1) and 40-Br-2,2,2-trifluoroacetophenone (3)
by using immobilized cells of G. candidum. Then, we decided to in-
crease the substrate concentration to 14.4 mM for this microorgan-
ism. The immobilized cells of G. candidum were able to reduce the
acetophenone 1 with high conversions and enantiomeric excess
while for the reduction of acetophenone 3, the conversion decreased
to 56%.
Based on the results obtained on the bioreduction of aceto-
phenones under bath conditions by using immobilized G. candidum
Table 1
Bioreduction of 2,2,2-trifluoroacetophenone (1) and 40-Br-2,2,2-trifluoroacetophenone (3) by using free cells (with substrates in a 7.2 mM final concentration)
Microorganism
2,2,2-Trifluoroacetophenone (1)
40-Br-2,2,2-trifluoroacetophenone (3)
Conversion (%)
ee (%)
Space-time
Conversion (%)
ee (%)
Space-time
yield [g/(L d)]
yield [g/(L d)]
A. niger
>99
>99
>99
>99
>99
>99
>99
>99
90 (S)
29 (S)
91 (S)
24 (S)
50 (S)
13 (R)
45 (S)
28 (R)
127
127
127
127
127
127
127
127
79
62
>99
39
68
91
94 (S)
1 (S)
96 (S)
74 (S)
18 (S)
1 (R)
145
114
183
72
125
167
183
183
Candida sp.
G. candidum
Hansenula sp.
K. marxianus
M. ramannianus
R. minuta
>99
>99
50 (S)
35 (R)
R. rubra
Reaction condition: Substrates were previously dissolved in 1 mL of ethanol and added to a mixture of microorganisms and 5% glucose solution to give 50 mL of a solution with
final substrate concentration of 7.2 mM. Reactions were carried out for 24 h at 30 ꢀC under a shaking speed of 150 rpm in the orbital shaker. Products were analyzed by (chiral)
gas chromatography (GC).
Based on the table above, higher ee were achieved for the re-
duction of 2,2,2-trifluoroacetophenone (1) and 40-Br-2,2,2-
trifluoroacetophenone (3) by using G. candidum and A. niger.
Then, in order to enhance the productivity of the reduction with
and based on previous results obtained by our group on the bio-
reduction of ketones in continuous flow with immobilized cells, we
decided to move forward towards a continuous flow methodology
for such transformation. For such, cells of G. candidum immobilized

R. de Oliveira Lopes et al. / Tetrahedron 70 (2014) 3239e3242
3241
Table 2
Bioreduction of 2,2,2-trifluoroacetophenone (1) and 40-Br-2,2,2-trifluoroacetophenone (3) by using free cells (with substrates in a 14.4 mM final concentration)
Microorganism
2,2,2-Trifluoroacetophenone (1)
40-Br-2,2,2-trifluoroacetophenone (3)
Conversion (%)
ee (%)
Space-time
Conversion (%)
ee (%)
Space-time
yield [g/(L d)]
yield [g/(L d)]
A. niger
G. candidum
96
>99
91 (S)
95 (S)
243
253
8
56
96 (S)
91 (S)
29
206
Reaction condition: Substrates were previously dissolved in 1 mL of ethanol and added to a mixture of microorganisms and 5% glucose solution to give 50 mL of a solution with
final substrate concentration of 14.4 mM. Reactions were carried out for 24 h at 30 ꢀC under a shaking speed of 150 rpm in the orbital shaker. Products were analyzed by
(chiral) gas chromatography (GC).
Table 3
Bioreduction of 2,2,2-trifluoroacetophenone (1) and 40-Br-2,2,2-trifluoroacetophenone (3) by immobilized cells (with substrates in a 7.2 mM final concentration)
Microorganism
2,2,2-Trifluoroacetophenone (1)
40-Br-2,2,2-trifluoroacetophenone (3)
Conversion (%)
ee (%)
Space-time
yield [g/(L d)]
Conversion (%)
ee (%)
Space-time yield
[g/(L d)]
Candida sp.
G. candidum
Hansenula sp.
K. marxianus
R. minuta
>99
>99
68
65
>99
>99
8 (S)
127
127
86
82
127
127
62
>99
76
97
97
20 (S)
93 (S)
12 (S)
4 (S)
61 (S)
42 (R)
114
183
140
178
178
180
96 (S)
43 (S)
75 (S)
20 (S)
43 (R)
R. rubra
98
Reaction condition: Substrates were previously dissolved in 1 mL of ethanol and added to a mixture of microorganisms and 5% glucose solution to give 50 mL of a solution with
final substrate concentration of 7.2 mM. Reactions were carried out for 24 h at 30 ꢀC under a shaking speed of 150 rpm in the orbital shaker. Products were analyzed by (chiral)
gas chromatography (GC).
in calcium alginate were packed in a glass column (Omnifit column;
volume: 12.3 mL) for the bioreduction of acetophenone 3 under
continuous flow conditions. We evaluated the substrate concen-
tration (7.2 and 14.4 mM) and different residence time on the
bioreduction of 40-Br-2,2,2-trifluoroacetophenone (3). We decided
for acetophenone 3 instead of acetophenone 1, due to its potential
as a building block for the interesting intermediates for the phar-
maceutical industry and cascade cross-coupling reactions.
According to the results showed on Table 4, using lower concen-
tration of substrate (7.2 mM) at 60 min of residence time and flow
rate of 45 mL/min, the S-enantiomer was achieved in 97% conver-
sion and enantiomeric excess higher than 99%. Using higher con-
centration of substrate (14.4 mM) at 90 min of residence time and
flow rate of 30 mL/min, conversion and enantiomeric excess were
up to 99%.
conversion and enantiomeric excesses could be observed with the
decrease in reaction time for 90 min.
4. Experimental
4.1. Materials
2,2,2-Trifluoroacetophenone, 40-Br-2,2,2-trifluoroacetophenone,
and (R)-2,2,2-trifluoro-1-phenylethanol were purchased from Sigma
Aldrich and racemates were obtained via NaBH4 reduction. The
products were analyzed by 1H and 13C NMR.
4.1.1. rac-2,2,2-Trifluoro-1-phenylethanol (2). 1H NMR (300 MHz,
CDCl3, TMS)
d
(ppm): 2.83 (s, 1H, OH); 4.98e5.05 (q, 1H, J¼6.7 Hz,
H1); 7.40e7.50 (m, 5H, H20, H30, H40, H50, H60).
13C NMR (75 MHz, CDCl3, TMS)
d (ppm): 72.4, 72.8, 73.2, 73.7 (q,
Table 4
J¼31.9 Hz, COH); 122.6 and 126.4 (d, J¼279.7 Hz, CF3); 127.7 (C20
Bioreduction of 40-Br-2,2,2-trifluoroacetophenone (3) in continuous flow by using
immobilized cells of G. candidum
and C60); 128.9 (C40); 129.8 (C30 and C50); 134.3 (s, C10).
Final concentration Residence Flow rate Conversion ee (%) Space-time
4.1.2. rac-1-(4-Bromophenyl)-2,2,2-trifluoroethanol (4). 1H NMR
of substrate (mM) time (min)
(
m
L/min) (%)
yield [g/(L d)]
(300 MHz, CDCl3, TMS)
d (ppm): 2.78 (s, 1H, OH); 5.00 (q, 1H,
7.2
30
60
30
90
90
45
90
30
88
97
47
>99 (S) 7695
J¼6 Hz, H1); 7.37 (d, 2H, J¼9 Hz, H30 and H50); 7.56 (m, 2H, H20 and
>99 (S) 4241
>99 (S) 8219
>99 (S) 5870
H60).
14.4
13C NMR (75 MHz, CDCl3, TMS)
d
(ppm): 72.5 (q, J¼31.5 Hz,
>99
COH); 118.6, 122.3, 126.0 and 129.8 (q, J¼280.5 Hz, CF3); 128.0 (C40);
129.3 (C20 and C60); 132.1 (C30 and C50); 133 (d, C10).
Mp¼52.5e53.6 ꢀC (StuartÔ melting point apparatus SMP3)
(mp¼55e56 ꢀC).2
Reaction condition: Substrates were previously dissolved in 1 mL of ethanol and
added to a mixture of microorganisms and 5% glucose solution to give a solution
with final substrate concentration of 7.2 mM or 14.4. Immobilized cells of G. can-
didum were used to fill the column (Omnifit column; volume: 12.3 mL), which was
heated at 30 ꢀC. Products were analyzed by (chiral) gas chromatography (GC).
3. Conclusion
4.2. Microorganisms, media, growth conditions, and
biotransformation
In conclusion we presented a highly efficient whole cell bio-
catalyzed reduction of trifluoroacetophenone 1 and 3. In the re-
action systems studied high conversion and good to excellent
enantiomeric excess (>99%) for the reduction of 1 were observed
for the S-enantiomer after 24 h using G. candidum, A. niger cells. In
the case of 3, only G. candidum presented high conversion and
excellent enantiomeric excess. Upon immobilization G. candidum
cells were essayed under continuous flow conditions and the same
K. marxianus, Hansenula sp., G. candidum, Candida sp., R. rubra, R.
minuta, and filamentous fungi, A. niger, Trichoderma harzianum, and
M. ramannianus, belong to the collection of the ‘Departamento de
Engenharia Bioquímica, Escola de Química, UFRJ’. Cells were
allowed to grow for 48 h at 30 ꢀC under a shaking speed of 150 rpm
in the orbital shaker in a medium containing 1% glucose, 0.5% yeast
extract, 0.5% peptone, 0.1% (NH4)2SO4, and 0.1% MgSO4$7H2O. After

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R. de Oliveira Lopes et al. / Tetrahedron 70 (2014) 3239e3242
that period, they were harvested by centrifugation, re-suspended in
distilled water and used for the reaction. After centrifugation, the
cells (12 g/L, dried weight) were added to the 250 mL Erlenmeyer
containing: 5% glucose in a final volume of 50 mL distilled water.
After 30 min of addition of the microorganisms, the substrate
(previously dissolved in 1 mL of ethanol) was added to the 50 mL of
the mixture to give a final solution with substrate concentration of
40-Br-2,2,2-trifluoroacetophenone (3): after 5 min at 100 ꢀC the
temperature was increase in 40 ꢀC/min to 140 ꢀC and kept 140 ꢀC
for 24 min. The elution order for 40-Br-2,2,2-trifluoroacetophenone
(3) was: 2,2,2-trifluoroacetophenone (3) (tR¼6.5 min), (S)-enan-
tiomer (4b) (tR¼25.9 min) and (R)-enantiomer (4a) (tR¼27.0 min).
4.6. Polarimeter
7.2 mM or 14.4 mM. The reaction was carried out for 24 h at 30 ꢀ
C
(R)-1-(4-Bromophenyl)-2,2,2-trifluoroethanol (4a): [
a
]
23 ꢂ37.4ꢀ
under a shaking speed of 150 rpm in the orbital shaker. After 24 h,
the mixture was centrifuged to separate the cells and the liquid
phase was extracted with ethyl acetate. The organic phase was
dried (anhydrous Na2SO4), filtered, and concentrated under vac-
uum. Products were analyzed by (chiral) gas chromatography (GC).
D
20
(c 1.06 Ethanol) (literature [a]
ꢂ27.5ꢀ).2 Optical rotations were
D
measured from CHCl3 solutions using a JASCO DIP-370 polarimeter
at the sodium D line (589 nm) operating at room temperature and
compared to literature.
Acknowledgements
4.3. Immobilized cells
Financial support from FAPERJ, FINEP, CAPES, and CNPq is
acknowledged.
After the growing process described above, the cells (12 g/L, dry
weight) were centrifuged and the precipitate was re-suspended in
3 mL of distilled water to obtain a cell-suspension. Then, a 1.5% w/v
sodium alginate aqueous solution in distilled water (final volume of
20 mL) was added to the cell-suspension, and the mixture (cell-
suspension and sodium alginate aqueous solution) was dropped
into a CaCl2 aqueous solution (0.1 M), forming calcium alginate
spheres. Spheres were filtered and washed with distilled water.
After that, the spheres were added to the 250 mL Erlenmeyer
containing 50 mL of a 5% glucose solution with substrate concen-
tration of 7.2 or 14.4 mM (substrates were previously dissolved in
1 mL of ethanol before they were added to the 5% glucose solution).
The reaction was carried out for 24 h at 30 ꢀC under a shaking speed
of 150 rpm in the orbital shaker. After 24 h, the mixture was filtered
to separate the cells and the liquid phase was extracted with ethyl
acetate. The organic phase was dried (anhydrous Na2SO4), filtered,
and concentrated under vacuum. Products were analyzed by (chi-
ral) gas chromatography (GC).
Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2013.12.036.
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^
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Products were analyzed by (chiral) gas chromatography (GC) on
column CYCLODEXB (29 mꢁ0.25 mmꢁ0.25
mm).
2,2,2-Trifluoroacetophenone (1): after 5 min at 100 ꢀC the
temperature was increase in 5 ꢀC/min to 120 ꢀC and kept at 120 ꢀC
for 9 min. The elution order for 2,2,2-trifluoroacetophenone (1)
was: 2,2,2-trifluoroacetophenone (1) (tR¼2.6 min), (S)-enantiomer
(2b) (tR¼15.0 min) and (R)-enantiomer (2a) (tR¼15.5 min).