Microbial reduction of ethyl 2-oxo-4-phenylbutyrate. Searching for R-enantioselectivity. New access to the enalapril like ACE inhibitors

Article abstract of DOI:10.1016/j.tetasy.2006.04.008

Herein, different microorganisms were tested in the enantioselective reduction of ethyl 2-oxo-4-phenylbutyrate in aqueous medium for the preparation of ethyl (R)-2-hydroxy-4-phenylbutyrate, a key intermediate in the production of angiotensin converting enzyme (ACE) inhibitors. The use of Pichia angusta led to the (R)-enantiomer in 81% ee.

Full text of DOI:10.1016/j.tetasy.2006.04.008

Tetrahedron: Asymmetry 17 (2006) 1186–1188
Microbial reduction of ethyl 2-oxo-4-phenylbutyrate.
Searching for R-enantioselectivity. New access to the
enalapril like ACE inhibitors
b
c
Paulo S. Bergo de Lacerda,a,b, Joyce Benzaquem Ribeiro, Selma G. F. Leite,
*
Maria Antonieta Ferrara,a,b Ricardo B. Coelho,b Elba P. S. Bon,b
Edson Luiz da Silva Limab and O. A. C. Antunesb
aFarManguinhos–Fundac¸a˜o Oswaldo Cruz, Rio de Janeiro, RJ 20000, Brazil
bInstituto de Quı´mica, UFRJ, Cidade Universita´ria CT Bl A, Rio de Janeiro, RJ 21945-970, Brazil
cEscola de Quı´mica, UFRJ, Cidade Universita´ria CT Bl E, Rio de Janeiro, RJ 21945-970, Brazil
Received 22 February 2006; accepted 7 April 2006
Abstract—Herein, different microorganisms were tested in the enantioselective reduction of ethyl 2-oxo-4-phenylbutyrate in aqueous
medium for the preparation of ethyl (R)-2-hydroxy-4-phenylbutyrate, a key intermediate in the production of angiotensin converting
enzyme (ACE) inhibitors. The use of Pichia angusta led to the (R)-enantiomer in 81% ee.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
marxianus (96%, 92% and 91%, respectively) high enantio-
selectivity was only observed for the (S)-enantiomer
(95% ee S. cerevisiae and 92% Dekera sp.) and was poor
for the (R)-enantiomer (32% ee K. marxianus). We envis-
aged that the production of ACE inhibitors of lisinopril/
enalapril type (Fig. 1) would involve the (R)-enantiomer
(via, e.g., the corresponding triflate), and as a result
decided to investigate the use of other microorganisms.
In our previously published results,1 we described the use
of Saccharomyces cerevisiae; Dekera sp.; Hansenula sp.
and Kluyveromyces marxianus in the reduction of ethyl 2-
oxo-4-phenylbutyrate, based on our expertise with these
microorganisms.2 However, although very high conver-
sions were obtained with S. cerevisiae, Dekera sp. and K.
NH2
Lisinopril
N
N
CO2H
H
O
R=
Enalapril
EtO2C
R
EtO2C
O
EtO2C
OTf
CH3
N
N
H
COOH
O
Figure 1. Process for the production of ACE inhibitors of lisinopril/enalapril type.
*
Corresponding author. Tel.: +55 21 39772461; fax: +55 21 25602518; e-mail: pbergo@far.fiocruz.br
0957-4166/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2006.04.008

P. S. B. de Lacerda et al. / Tetrahedron: Asymmetry 17 (2006) 1186–1188
1187
Several collections have become available in Brazil in
recent years and as Candida guilliermondii, Aureobasidium
pullulans,3 Pichia pastoris, Pichia anomala4 and Pichia
angusta5 have not been so used in microbial reductions,
these were chosen as potential tools for the desired trans-
formation. It is worth noting, however, that this empirical
search is based upon our previous experience that the
microorganism contains both pro-S and pro-R enzymes
and under particular circumstances, a particular activity
is preponderant.6 In addition, there are many literature
examples of the use of several Pichia sp.7 mediated enantio-
selective reductions.
of this process are the presence of ether in the medium
(ethyl ether–water 20:1), the large amount of cells required
for the process (1 g cell/12 mg substrate) and the toxicity of
the phenacyl chloride.
Since it was observed herein that by using P. angusta, 100%
conversion and 81% ee of ethyl (R)-2-hydroxy-4-phen-
ylbutyrate could be obtained and based on the hypothesis
that two different enzymes should be present, we have
decided to investigate the conversion x ee, that is, the kinet-
ics of this transformation. Other experiments, including
immobilization and other growing effects, are also cur-
rently under investigation.
2. Results and discussion
3. Conclusion
The results obtained: yield (Y) enantiomeric excess (ee) and
the relative main configuration (R or S), obtained in the
reduction of the ketoester by means of each strain, are
summarized in Table 1. Candida guilliermondii and Aureo-
basidium pullulans showed an (S)-preference with high
ee. For comparison purposes, other microorganisms includ-
ing K. marxianus were used. K. marxianus, P. pastoris and
P. anomala showed R preference with low ee. However,
using Pichia angusta 100% conversion and 81% ee of ethyl
(R)-2-hydroxy-4-phenylbutyrate were obtained (Scheme 1).
In conclusion, we have reported for the first time the prep-
aration of ethyl (R)-2-hydroxy-4-phenylbutyrate via micro-
bial reduction of ethyl 2-oxo-4-phenylbutyrate in aqueous
medium using an attractive process in terms of potential
industrial application and green chemistry.
4. Experimental
All microorganisms used were collected from different
fruits (Hansenula sp., Dekera sp., and K. marxianus), and
belong to the collection of the ‘Departamento de Engenha-
Table 1. Microbial reduction of 2-oxo-4-phenylbutyrate
Microorganism
Conversion (%)
% ee (configuration)
´
´
ria Bioquımica, Escola de Quımica, UFRJ’, purchased (S.
cerevisiae), belong to the collection of the ‘Departamento
S. cerevisiae
Dekera sp.
C. guilliermondii
A. pullulans
K. marxianus
P. pastoris
99a
89
100 (S)
100 (S)
100 (S)
60 (S)
35 (R)
35 (R)
35 (R)
81 (R)
´
´
de Bioquımica, Instituto de Quımica, UFRJ (P. pastoris)’,
or belong to the collection of ‘Instituto Nacional de Cont-
role de Qualidade em Sau´de (INCQS), FIOCRUZ’, and
are freely available upon request.
20a
92
82
95
94
100
P. anomala
P. angusta
Cells were allowed to grow for 48 h, under 150 rpm at
30 ꢁC in a medium containing 1% glucose, 0.5% of yeast ex-
tract, 0.5% peptone, 0.1% (NH4)2SO4 and 0.1% MgSO4.
After that period, they were harvested by centrifugation,
re-suspended in water and used in the reaction. The cells
(3.8 g/l, dried weight), which were centrifuged, were added
to the reaction medium which contained 5% glucose, and
0.1% MgCl2 in a final volume of 100 ml. After 30 min of
the addition of the microorganisms, ethyl 2-oxo-4-phen-
ylbutyrate (0.14% in volume) in 1% aqueous ethanol was
added to the medium. The reaction was carried out for
24 h at 30 ꢁC and 150 rpm. After 24 h, the medium was
centrifuged to separate the cells and the liquid phase then
extracted with ethyl acetate. The organic phase was dried
over anhydrous Na2SO4, filtered and concentrated under
vacuum. The ee and the absolute configuration of the
a 80% Decomposition occurred.
Chadha et al.8 reported that the enantioselective reduction
of 1 to (R)-2 could not be achieved by using S. cerevisiae,
but was made possible by using cell free aqueous extracts
of the callus of Daucus carota (wild carrot) with excellent
chemical yields (90%) and ees (99%). However, in this
process, a large amount of cells was needed, cell: sub-
strate = 100:1, while a reaction time of 10 days was
required.
S. cerevisiae preincubated in the presence of phenacyl chlo-
ride was used in the production of (R)-2 to give excellent
conversion (96%) and good ee (81%).9 The disadvantages
Candida guilliermondii,
Aureobasidium pullulans,
S. Cerevisae or
Kluyveromyces marxianus,
Pichia pastoris,
Pichia anomala or
Pichia angusta
Dekera sp.
(S)-preference
(R)-preference
EtO2C
OH
EtO2C
O
EtO2C
OH
Scheme 1.

1188
P. S. B. de Lacerda et al. / Tetrahedron: Asymmetry 17 (2006) 1186–1188
6. Procopiou, P. A.; Morton, G. E.; Todd, M.; Webb, G.
Tetrahedron: Asymmetry 2001, 12, 2005.
reaction were determined by chiral high-resolution chro-
matography, performed on a commercial available Cyclo-
dex B capillary column (30 m · 0.25 mm, i.d.), initial
temperature: 393 K (30 min), rate 2ꢁ/min, final tempera-
ture: 473 K. Ethyl (S)-2-hydroxy-4-phenylbutyrate and
rac-ethyl 2-oxo-4-phenylbutyrate were used as chromato-
graphic standards. The elution order was R (50.3 min)
followed by S (50.9 min). The reaction products were iso-
7. (a) Sugai, T.; Sakuma, D.; Kobayashi, N.; Ohta, H. Tetrahe-
dron 1991, 47, 7237; (b) Koul, S.; Crout, D. H. G.; Errington,
W.; Tax, J. J. Chem. Soc., Perkin Trans. 1995, 2969; (c) Sugai,
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R.; Carter, A. S.; Murrell, J. C.; Dalton, H.; Hallinan, K. O.;
Crout, D. H. T.; Holt, R. A.; Crosby, J. Biocatal. Biotrans.
1995, 12, 159; (e) Sugai, T.; Katoh, O.; Ohta, H. Tetrahedron
1995, 51, 11987; (f) Ikeda, H.; Sato, E.; Sugai, T.; Ohta, H.
Tetrahedron 1996, 52, 8113; (g) Molinari, F.; Bertolini, C.;
Aragozzini, F. Ann. Microbiol. Enzim. 1997, 47, 131; (h)
Fuganti, C.; Grasselli, P.; Mendozza, M.; Servi, S.; Zucchi, G.
Tetrahedron 1997, 53, 2617; (i) Ohtsuka, Y.; Katoh, O.; Sugai,
T.; Ohta, H. Bull. Chem. Soc. Jpn. 1998, 70, 483; (j) Molinari,
F.; Occhiato, E. G.; Aragozzini, F.; Guarna, A. Tetrahedron:
Asymmetry 1998, 9, 1389; (k) Molinari, F.; Bertolini, C.;
Aragozzini, F. Biocatal. Biotrans. 1998, 16, 87; (l) Goswami,
A.; Mirfakhrae, K. D.; Patel, R. N. Tetrahedron: Asymmetry
1997, 10, 4239; (m) Martinez, F.; Del Campo, C.; Sinisterra, J.
V.; Llamma, E. F. Tetrahedron: Asymmetry 2000, 11, 4651; (n)
Nanduri, V. B.; Hanson, R. L.; Goswami, A.; Wasylyk, J. M.;
LaPorte, T. L.; Katipally, K.; Chung, H. J.; Patel, R. N.
Enzyme Microbiol. Technol. 2001, 28, 632; (o) Goswami, A.;
Mirfakhrae, K. D.; Tobleben, M. J.; Swaminathan, S.; Patel,
R. N. J. Ind. Microbiol. Biotechnol. 2001, 26, 259; (p) Forzato,
C.; Gandolfi, R.; Molinari, F.; Nitti, P.; Pitacco, G.; Valentin,
E. Tetrahedron: Asymmetry 2001, 12, 1039; (q) Tsujigami, T.;
Sugai, T.; Ohta, H. Tetrahedron: Asymmetry 2001, 12, 2543;
(r) Lagos, F. M.; Del campo, C.; Llama, E. F.; Sinisterra,
J. V. Enzyme Microbiol. Technol. 2002, 30, 895; (s) Tripathi,
M. K.; Jinwal, U. K.; Roy, U.; Patra, A.; Roy, P. K.; Batra, S.;
1
lated and characterized by H nuclear magnetic resonance
(NMR) and infrared spectroscopy (IR). 1H NMR
(200 MHz/CDCl3): d 7.20–7.30 (m, 5H, Ar–H); 4.16–4.27
(m, 3H, CH(OH)CO and CH2(OCO)CH3); 2.73–2.82 (m,
2H, PhCH2CH2); 2.3 (s, OH); 1.93–2.09 (m, 2H, CH2CH2-
CHOH); 1.30 (t, 3H, COCH2CH3) and IR (film) m cmÀ1
:
3467 (m O–H), 1732 (m C@O) 1214 and 1100 (m C–O),
701 (m C@C).
Acknowledgements
Financial supports from FIOCRUZ, CNPq, FAPERJ and
CAPES, and analytical support from FIOCRUZ and
DQO-UFRJ are gratefully acknowledged.
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