Microbial and homogenous asymmetric catalysis in the reduction of 1-[3,5-bis(trifluoromethyl)phenyl]ethanone

Article abstract of DOI:10.1016/j.tetasy.2006.06.033

Two complementary approaches for the enantioselective reduction of 1-[3,5-bis(trifluoromethyl)phenyl]ethanone 1 are described and compared: microbial versus asymmetric reduction of ketones through asymmetric hydrogen transfer. Among the various microorgan

Full text of DOI:10.1016/j.tetasy.2006.06.033

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Tetrahedron: Asymmetry 17 (2006) 2000–2005
Microbial and homogenous asymmetric catalysis in the reduction of
1-[3,5-bis(trifluoromethyl)phenyl]ethanone
*
´ ´
Mirjana Gelo-Pujic, Frederic Le Guyader and Thierry Schlama
`
Rhodia-CRTL, BP 62, 85 rue des freres Perret, F-69192 Saint Fons cedex, France
Received 7 June 2006; accepted 20 June 2006
Available online 18 July 2006
Abstract—Two complementary approaches for the enantioselective reduction of 1-[3,5-bis(trifluoromethyl)phenyl]ethanone 1 are
described and compared: microbial versus asymmetric reduction of ketones through asymmetric hydrogen transfer. Among the various
microorganisms screened, Lactobacillus kefir and Aspergillus niger reduced ketone 1 to the corresponding (R)-alcohol (R)-2. The (S)-alco-
hol (S)-2 was obtained by reduction of 1 using homogenous asymmetric catalysis. The configuration of the alcohol in both the bioca-
talysis and hydrogen transfer approaches was controlled by the choice of the enzyme and by the configuration of ligands,
respectively. Both enantiomers were obtained in high yield and ee.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Substrates commonly reduced by oxidoreductases, either
isolated enzymes or whole microorganisms, are b-keto-
esters1 and b-diketones,2 then ketothioacetals3 and related
compounds, aliphatic ketones4 and aldehydes,5 cyclic6 and
polycyclic ketones7 and carbon–carbon double bonds.8
Stereoselective reductions of the carbonyl compounds by
bakers’ yeast (Saccharomyces cerevisiae) are one of the
most explored biotransformations.9–11
The synthesis of chiral secondary alcohols via the catalytic
reduction of the corresponding prochiral ketones is one of
the key transformations in the asymmetric organic synthe-
sis. Only a few major methods have appeared over the past
two decades; among them are the enantioselective reduc-
tions with molecular hydrogen, enantioselective reduction
with modified hydrides, enantioselective hydrogen transfer
and biocatalysis. Biocatalysis and enantioselective hydro-
gen transfer are two complementary methodologies fre-
quently used in our laboratory. Biocatalysis is today
commonly used in organic synthesis. Its benefits are in an
efficient and selective catalysis, including chemoselectivity,
regioselectivity, diastereoselectivity and enantioselectivity.
Moreover, the biocatalysts accept a broad range of ‘unnat-
ural’ substrates; they act under mild reaction conditions
and are biodegradable and thus environmentally friendly.
Enzymes that catalyze oxidation–reduction are cofactor
dependent alcohol dehydrogenases.
Enzymatic and microbial reductions of the alkyl aryl
ketones proceed in the majority of cases according to
Prelog’s rule12 generating alcohols in the (S)-configuration.
The enzyme transfers the pro-(R) hydrogen of the cofactor
to the re-face of a ketone. The majority of enzymes such as
HLADH and microorganisms such as bakers’ yeast, Geo-
trichum, Curvularia or Thermoanaerobium follow this rule,
while only a few microorganisms (Lactobacillus, Mucor
and Pseudomonas) have been described to possess enzymes
of the opposite specificity, that is, anti-Prelog’s specificity.
Acetophenones and their derivatives are generally poor
substrates for alcohol dehydrogenases. Although they are
reduced with high enantioselectivities, the yields are gener-
ally low. However, electron withdrawing groups adjacent
to a ketone moiety increase their reactivity.13–17 The exam-
ples of microbial reductions of aromatic trifluoromethyl
ketones,18 a-ketoacids and a-ketoesters,19 b-ketoesters20
and imidazoyl21 ketones have been described.
Abbreviations: ADH, alcohol dehydrogenase; ATCC, American Type
Culture Collection; BY, bakers’ yeast; DMAP, dimethyl-amino-pyridine;
DSMZ, Deutsche Sammlung fur Microorganismen und Zellen; HLADH,
horse liver alcohol dehydrogenase; NAD(P)+, nicotinamide adenine
dinucleotide (phosphate).
*
Corresponding author. Tel.: +33 4 72 89 69 88; fax: +33 4 72 89 68 94;
e-mail: Mirjana.Gelo-Pujic@eu.rhodia.com
0957-4166/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2006.06.033

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M. Gelo-Pujic et al. / Tetrahedron: Asymmetry 17 (2006) 2000–2005
2001
On the other hand, asymmetric hydrogen transfer from a
hydrogen donor is an oxido-redox process catalyzed by
an organometallic complex. One of the most efficient asym-
metric catalysts developed by Noyori22 contains a mono-
tosylated chiral diamine as the ligand associated to a
ruthenium complex. This system was shown to be very effi-
cient, enantioselective and independent of the substrate.
The mechanism of hydrogen transfer has been described
by Noyori.23 The monotosylated chiral diamine and the
ruthenium complex form an active catalyst with 18 elec-
trons, which loses two electrons in the presence of a base.
The 16 electron complex is then hydrogenated by the do-
nor, generally 2-propanol. This active catalyst is finally
able to reduce the aromatic ketone via a six-centre mecha-
nism.24 Both, 18- and 16-electron complexes have been iso-
lated and their structures have been determined by X-
ray.23a The results confirmed the hypothesis of the reduc-
tion by hydrogen transferred from ruthenium and not the
metal alcoholate as in the case of Meerwein–Ponndorf–
Verley reduction. The amino group is necessary for efficient
catalysis by complexing the carbonyl group of the sub-
strate. The catalytic system described has been efficiently
employed in reduction of various aromatic ketones.22
ADH) in the reduction of 1 was performed under different
pH conditions, cofactor concentration, cofactor regenera-
tion system and the addition of an organic cosolvent.
The substrate concentration was kept constant at 5 mg/
ml (20 mM). Under all conditions assayed only HLADH
and L. kefir ADH showed activity and transformed 1 in
a very moderate yield, but with 100% ee into the alcohol
2 (Fig. 2). Both enzymes reduced 1 but with the opposite
enantioselectivity. While HLADH gave the (S)-enantio-
mer, L. kefir ADH gave an anti-Prelog’s configuration,
that is, the R-alcohol. Product configurations and the
enantioselectivity of the enzymatic reductions were deter-
mined by a chiral GC comparison of the reduction prod-
ucts with the authentic samples.
OH
CF3
O
H
CF3
D
A
L
L
2
(S)-
CF3
H
.
k
e
f
i
r
CF3
NAD(P)+
OH
OH
NAD(P)H
1
CF3
We have applied two of the above described methodologies
in the reduction of 1-[3,5-bis(trifluoromethyl)phenyl]etha-
none 1. Commercially available oxido-reductases, as well
as microbial strains expressing these activities were screened.
On the other hand, numerous chiral ligands associated to the
ruthenium-p-cymene complex were screened under the con-
ditions of homogenous asymmetric catalysis. Our objective
was to evaluate the diversity of our strain collection and of
the (per)fluorosulfonyl-diamine ligands in the reduction
and secondly, to obtain the alcohol of (R)-configuration.
O
2
(R)-
CF3
Figure 2.
The results are summarized in Table 1. A little activity was
obtained without the recycling of the cofactor. When a sec-
ondary alcohol such as 2-propanol or cyclopentanol was
used, the reduction was more efficient. The secondary alco-
hol has a dual role: first it serves to recycle the cofactor and
secondly it enables better solubility of the substrate in the
aqueous medium. However, by increasing the concentra-
tion of 2-propanol added, the yield of reduction decreased
slightly. As the product of 2-propanol oxidation is acetone,
it could possibly act as a competitive substrate of the dehy-
drogenase. To check this possibility, the cofactor concen-
tration was increased for 35% (from 2 to 2.7 mM) and we
observed a 50% increase in the yield of reduction. Very
likely, both phenomena are taking place, that is, a substrate
competition and a cofactor depletion, but it was not inves-
tigated further.
1-[3,5-Bis(trifluoromethyl)phenyl]ethanone 1 is a key pre-
cursor of (R)-1-[3,5-bis-(trifluoromethyl)phenyl]ethanol
(R)-2, a sub-structure of the tachykinin NK1 receptor
antagonist L-754030, a potent anti-depressant as shown
in Figure 1. Its structure is closely related to the structure
of MK869, shown recently to be efficient in patients with
major depressive disorder.25
CF3
CF3
F3C
O
O
OH
O
N
F3C
Table 1. Reduction of 1 (20 mM) with HLADH and L. kefir ADH
H
N
ADH
NAD(P)H 2-Propanol Time GC
ee (%)
O
F
(mM)
(ll)
(h)
yield (%)
CF3
CF3
HN N
Figure 1.
L-754030
1
2
(R)-
L. kefir
0.2
2
2
2.7
1
1
2.5
100
25
100
100
100
48
48
44
66
72
72
15
20
24
49
0
>99 (R)
>99 (S)
>99 (S)
>99 (S)
—
HLADH
HLADH
HLADH
HLADHa
HLADHb
2. Results and discussion
2.1. Bio-reductions
0
—
Conditions: 0.1 M phosphate buffer pH 7.8, 4.4–5 U/ml of ADH at 30 ꢁC
and 1300 rpm.
a Reaction in n-hexane.
b Reaction in acetonitrile.
A screening of commercially available alcohol dehydrogen-
ases (HLADH, YADH, BYADH and Lactobacillus kefir

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2002
M. Gelo-Pujic et al. / Tetrahedron: Asymmetry 17 (2006) 2000–2005
Although the alcohol obtained under all conditions is
enantiomerically pure, the yield is rather low. According
to the literature data, the rate of enzymatic reduction can
be enhanced in organic–aqueous biphasic media.26 Reac-
tions performed with 1 as a substrate and in the phosphate
buffer–hexane medium did not give any product. In the
presence of a water miscible solvent, such as acetonitrile,
no product was obtained either. On the other hand, the
use of organic solvents instead of buffer media can control
the stereochemistry of enzymatic reduction.13,27 Unfortu-
nately, neither HLADH nor L. kefir ADH showed any
activity in pure n-hexane or acetonitrile under assayed
conditions.
Yield of (R)-alcohol (%)
ee of (R)-alcohol (%)
100
80
60
40
20
0
1
3
6
8
20
24
t (h)
These screening results revealed that L. kefir ADH is capa-
ble of selectively reducing 1 into the desired (R)-alcohol.
However, the purified enzyme was not very stable in solu-
tion and we observed a loss of activity when storing the en-
zyme even at À80 ꢁC. L. kefir is a lactic bacterium
frequently used in food biotechnology. All further experi-
ments were performed with the biocatalyst obtained by
growing L. kefir DSM 20587. The advantage of using the
whole cells instead of the isolated enzyme is in its ability
to perform the reactions without the external addition of
cofactors. This makes the whole procedure much easier.
Initial experiments were performed with 5 mM 1 and the
enantioselectivity of the microbial enzymatic system was
confirmed when compared to isolated enzyme. 5 mM
(1.2 g/l) 1 was completely transformed into the (R)-alcohol
within 16 h with excellent enantioselectivity (ee >99%). The
concentration of the biocatalyst used in these experiments
was 20–24 g/l of dry cell mass. Higher concentrations of ke-
tone seemed to inhibit the enzyme. For example, 100 mM 1
gave 13% of transformation in 16 h while 200 mM 1 gave
only 2% under the same conditions (Table 2).
Figure 3. L. kefir catalyzed reduction of 1.
2.2. Homogenous asymmetric catalysis
According to the synthesis patented by Merck,28 the (R)-
alcohol is available through the asymmetric reduction of
1 by using (S,R)-1-amino-2-indanol as the chiral ligand
associated to [Ru(p-cymene)Cl2]2. The described catalytic
system allowed us to obtain the (R)-alcohol in an excellent
yield and high enantiomeric excess.
We have prepared and screened numerous chiral ligands in
the reduction of 1 (Fig. 4). The ligands used in this study
were prepared by mixing a diamine with one equivalent
of the corresponding chlorosulfonyl compound in
dichloromethane.
OH
Ligand + [Ru(p-cymene)Cl2]2
CF3
Table 2. Reduction of 1 with L. kefir cells
1
1 (mM)
1 (g/l)
GC yield (%) (16 h)
GC yield (%) (96 h)
HCO2H/NEt3, 20 °C
1/ Ru / L = 100/1/1
[1] = 2 M
CF3
)-2
5
10
20
50
100
200
1.2
2.4
4.8
12
24
48
100
77
31
13
13
2
—
100
69
51
51
31
(
S
Figure 4. Reduction of 1 through homogeneous catalysis.
First, the perfluorosulfonylated ligands were screened in a
formic acid/triethylamine system. This was chosen based
on the previously studied reduction of acetophenone.29
Note that the absolute configurations of the ligands are
(S,S) and as a consequence the product obtained is the
(S)-alcohol. As the desired product is the (R)-alcohol, it
could be easily obtained with the corresponding (R,R)-
1,2-diamine as the ligand. Indeed, in the case of acetophe-
none, the asymmetric reduction using (S,R)-1-amino-2-ind-
anol afforded (R)-phenylethanol30 and, with (R,S)-1-
amino-2-indanol, (S)-phenylethanol30 was obtained. Table
3 summarizes the results of the reduction of BTA with the
ligands prepared in this study.
The kinetics of reductions catalyzed by L. kefir are pre-
sented in Figure 3. The reaction reaches a plateau at ca.
85–90% of conversion. The addition of the biocatalyst does
not influence the conversion; nor does the addition of
NADPH or 2-propanol for recycling the cofactor. We sus-
pect the alcohol formed to inhibit the enzyme. However
this is the subject of our ongoing study and will be reported
elsewhere.
Twenty other strains were screened for the alcohol dehy-
drogenase activity, but only Aspergillus niger cells showed
activity with 1 as the substrate. The activity was 10-fold
lower than the activity of L. kefir and the enantiomer ob-
tained has the (R)-configuration according to the anti-Pre-
log’s rule (results not shown).
The results indicate that none of the cyclohexane diamine-
based ligands 3–5 (entries 1–3) were efficient when com-
pared to the amino alcohol used by Merck that gave 90%

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M. Gelo-Pujic et al. / Tetrahedron: Asymmetry 17 (2006) 2000–2005
Table 3. Reduction of 1 with perfluorosulfonylated ligands
2003
Entry
Ligand
Time (h)
GC yield (%)
ee (%) (config)
H
NHSO2CF3
1
24
100
22 (S)
57 (S)
3
NH2
H
H
CF3
CF3
NHSO2
NH2
2
24
96
H
H
4
NHSO2Tol
3
4
24
6
94
67
71 (S)
5
NH2
H
H
Ph NHSO2CF3
31 (nd)
6
Ph NH2
H
CF3
CF3
H
Ph NHSO2
5
6
82
58 (S)
Ph NH2
H
7
H
Ph NHSO2Tol
6
7
6
6
99
71 (S)
73 (S)
8
Ph NH2
H
H
Ph
Ph
NHSO2
NH2
OMe
100
9
H
ee. However, it is important to note that the ligands
carrying an aromatic group on the sulfonyl function gave
better enantioselectivities.
the system of 2-propanol/KOH and compared with previ-
ously obtained results (Table 4).
From the results shown in Table 4, it is clear that the cat-
alytic system is more active with 2-propanol as a hydrogen
donor. On the other hand, the enantioselectivity was also
enhanced under these conditions. The results demonstrate
the possibility of preparing the (S)-alcohol with an im-
proved ee of 82% by performing the reaction at a lower
temperature, near 0 ꢁC.
The second series of reductions were performed with
ligands based on diphenylethylene diamine motif 6–9
(entries 4–7). The same effect was found as previously
described for cyclohexenediamine-based ligands. In order
to improve the enantioselectivity, the sulfonyl group needs
to be substituted by an aromatic ring. In addition, when
the aromatic ring was substituted by electron donating
groups, such as MeO, better enantioselectivities were ob-
tained. As all these assays were carried out in the system
of formic acid/triethylamine, the best results obtained (en-
try 7) still gave moderate ee of 73%. This ligand was tried in
3. Conclusion
This study has shown that both enantiomers of a secondary
alcohol could be obtained in good yield and high enantio-
meric excess by biocatalysis as an alternative method to the
existing chemical methods. Two complementary ap-
proaches were developed: microbial reduction of prochiral
ketones and asymmetric reduction through asymmetric
hydrogen transfer. Both methods were applied in the
reduction of 1.
Table 4. Reduction of 1 with diphenylethylene diamine-based ligands
under different reaction conditions
Entry
Reaction
medium
T
Time
(h)
GC
yield (%)
ee (%)
(config)
(ꢁC)
7
8
9
HCOOH/NEt3
HCOOH/NEt3
2-Propanol/KOH
2-Propanol/KOH
20
20
20
1
24
1
1
100
8
100
98
73 (S)
73 (S)
75 (S)
82 (S)
Microbial reduction gives a quantitative transformation
and the selectivities are generally very high. However, the
enzymes are cofactor dependent and the cofactor needs
10
4