Preparation the key intermediate of angiotensin-converting enzyme (ACE) inhibitors: High enantioselective production of ethyl (R)-2-hydroxy-4- phenylbutyrate with Candida boidinii CIOC21

Article abstract of DOI:10.1002/adsc.200700420

Forty microorganisms belonging to different taxonomicalgroups were used to catalyze the enantioselective reduction of ethyl 2-oxophenylbutyrate to afford the corresponding ethyl2-hy droxy-4-phenylbutyrate. Several microorganisms led to over 99% ee of ethyl(S)-2-hydroxy-4-phenylbutyrate. Especially, we firstly found that the Candida boidinii CIOC21 could be effectively used for the enantioselective preparation the ethyl (R)-2-hydroxy-4-phenylbutyrate in pure aqueous medium with 99% ee, a key intermediate in the production of angiotensin-converting enzyme (ACE) inhibitors.

Full text of DOI:10.1002/adsc.200700420

FULL PAPERS
DOI: 10.1002/adsc.200700420
Preparation the Key Intermediate of Angiotensin-Converting
Enzyme (ACE) Inhibitors: High Enantioselective Production of
Ethyl (R)-2-Hydroxy-4-Phenylbutyrate with Candida boidinii
CIOC21
Yongzheng Chen,a,b Hui Lin,a,b Xiaoying Xu,a Shiwen Xia,a,* and Lixin Wanga,*
a
Key Laboratory for Asymmetric Synthesis and Chirotechnology of Sichuan Province and Union Laboratory of
Asymmetric Synthesis, Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041, Peopleꢀs
Republic of China
Fax : (+86)-28-8521-1877; e-mail: swxia2008@hotmail.com
Graduate School of Chinese Academy of Sciences, Beijing 100039, Peopleꢀs Republic of China
b
Received: August 26, 2007; Revised: December 17, 2007; Published online: February 11, 2008
Abstract: Forty microorganisms belonging to differ- lective preparation the ethyl (R)-2-hydroxy-4-phenyl-
ent taxonomicalgroups were used to cataylze the
butyrate in pure aqueous medium with 99% ee, a
enantioselective reduction of ethyl 2-oxophenylbuty- key intermediate in the production of angiotensin-
rate to afford the corresponding ethyl2-hydroxy-4-
phenylbutyrate. Several microorganisms led to over
converting enzyme (ACE) inhibitors.
99% ee of ethyl( S)-2-hydroxy-4-phenylbutyrate. Es- Keywords: ACE inhibitors; bioreduction; Candida
pecially, we firstly found that the Candida boidinii boidinii; ethyl( R)-2-hydroxy-4-phenylbutyrate; Sac-
CIOC21 could be effectively used for the enantiose- charomyces cerevisiae
Introduction
last years.[2] Moreover, ethyl( R)-2-hydroxy-4-phenyl-
butyrate has been found to act as a versatile inter-
The growing interest in asymmetric synthesis has pro- mediate for the synthesis of angiotensin-converting
moted a great development in biotransformations in enzyme (ACE) inhibitors (Scheme 1), such as benaze-
organic synthesis and they have been used for the pril, enalapril, lisinopril, ramipril and quinapril, etc.,
[1]
synthesis of chiralcompounds. The optically pure a- which are widely used to treat hypertension and con-
hydroxy acid ester structuralunit is widespread in
naturalproducts and has been frequentyl used as a
gestive heart failure.[3].
A number of routes to ethyl2-hydroxy-4-phenybl u-
convenient building block in organic synthesis in the tyrate has been developed, including reduction of 2-
Scheme 1. Process for the production of ACE inhibitors.
426
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Adv. Synth. Catal. 2008, 350, 426 – 430

Preparation the Key Intermediate of Angiotensin-Converting Enzyme (ACE) Inhibitors
FULL PAPERS
oxo-4-phenylbutanoic acid and its derivates,[4] asym- ee was obtained by the reduction of 1 in aqueous
metric hydrogenation,[5] multistep asymmetric synthe- medium using Candida boidinii CIOC21.
[7]
sis,[6] chemicaland enzyme resoultion.
Especially
The effects of the cosolvents were subsequently in-
ethyl( S)-2-hydroxy-4-phenylbutyrate could be easily vestigated. Usually, a small amount of an organic sol-
prepared by the reduction of many microorganisms vent is often used to ensure sufficient solubility of
with excellent yield and enantiomeric excesses.[8] keto ester substrates.[11] From the results shown in
However, only few microorganisms were described as Table 2, addition of 10% ethanol or DMP increased
efficient biocatalysts in the reduction of ethyl 2-oxo- the activity but sharply decreased the enantioselectivi-
phenylbutyrate to ethyl (R)-2-hydroxy-4-phenylbuty- ty (entry 2 and entry 7). By contrast with ethanol,
rate,[7] Lacerda and co-workers reported the reduction DMP, MTBE and diisopropylether (entries 2, 3, 6, 7),
of ethyl2-oxo-4-phenybl utyrate with K. marxianus, P. correspondingly better reactivity and enantioselectivi-
anomala, P. angusta to give the ethyl( R)-2-hydroxy-4- ty were obtained in DMSO and DMF (entries 4, 5).
phenylbutyrate in low to moderate enantioselectivity However, (R)-2 with 92% yield and 99% ee was ob-
(35% ee, 35% ee, and 81% ee, respectively).[9] Besides, tained in aqueous medium using Candida boidinii
Chadha et al. reported that the enantioselective re- CIOC21. In conclusion, in terms of the reactivity and
duction of ethyl2-oxophenyblutyrate to ethyl( R)-2- enantioselectivity, the aqueous medium was the opti-
hydroxy-4-phenylbutyrate by using Daucus carota malmedium. Most importantyl, there have been sig-
(wild carota) with excellent chemical yield (90%) and nificant developments in the direct synthesis of ethyl
ee (99%).[10] However, in this process, a large amount (R)-2-hydroxy-4-phenylbutyrate using microbial cells
of cells was needed, while this procedure was time- under solvent-free conditions. Hence, it will contrib-
consuming (10 days). Herein, we first report the enan- ute to the development of green and sustainable syn-
tioselective preparation of ethyl (R)-2-hydroxy-4-phe- thetic processes.[12]
nylbutyrate through bioreduction of ethyl 2-oxophe-
nylbutyrate in pure aqueous medium with high activi-
ty and enantioselectivity (yield 92%, 99% ee) by Can- Conclusions
dida boidinii CIOC21 in a comparatively short time
(12 h).
In conclusion, a practical biocatalytic synthesis of op-
tically active ethyl (R)-2-hydroxy-4-phenylbutyrate
has been developed. The synthesis is based on a re-
duction of the corresponding ethyl2-oxo-4-phenybl u-
tyrate. The reaction proceeds smoothly in a pure
aqueous solvent media and gives the desired product
Results and Discussion
During the reduction of ethyl2-oxo-4-phenybl utyrate,
Candida boidinii CIOC21 was found to be a promis- (R)-2 with high conversion (92%) and an excellent
ing redox biocatalyst with great synthetic potential enantioselectivity (up to 99% ee). It is a simple and
due to its excellent chemical yield and enatiomeric environmentally-friendly method to prepare the opti-
excess. The results are summarized in Table 1. Some cally active ethyl (R)-2-hydroxy-4-phenylbutyrate, a
microorganisms (entries 22–29) showed an (S)-prefer- key intermediate in the production of angiotensin-
ence with moderate to high yields and excellent enan- converting enzyme (ACE) inhibitors. This example is
tioselectivity (93–99%). However, other microorgan- one more proof that the biocatalytic approach is a
isms (entries 30–38) showed (R)-preference. Especial- valuable method for the synthesis of optically active
ly, using Candida boidinii CIOC21, 92% yield and a-hydroxy acid esters. At the same time, (R)-2 and
99% ee of ethyl( R)-2-hydroxy-4-phenylbutyrate were (S)-2 with high yields and optical purities were ob-
obtained.
At the same time, (R)-2 and (S)-2 with high yields Future work in this area should be devoted to investi-
and opticalpurities were obtained respectiveyl, using gating the properties and characteristics of the reduc-
tained using different microbialstrains (Scheme 2).
Candida boidinii CIOC21 (92% yield, 99% ee) and tase in Candida boidinii CIOC21. A better under-
Saccharomyces cerevisiae CIOC SY2 (94% yield, 99% standing of the underlying biochemical mechanism
ee) (Scheme 2).
would also be of interest.
The best strain selected for further preparative syn-
thesis of the (R)-2 was Candida boidinii CIOC21
(92% yield, 99% ee). The effects of reaction time, re-
action temperature, pH value, and the substrate con-
centration were also investigated to optimize the reac-
tion conditions. Under the optimalconditions (12 h,
pH 7.0, 308C, substrate concentration 20 mM, wet
cells:substrate=20:1), (R)-2 with 92% yield and 99%
Experimental Section
General Remarks
1H and 13C NMR spectra were recorded on a Bruker-300
(300/75 MHz) spectrometer using CDCl3 as a solvent and
TMS as internalstandard. TLC was performed on galss-
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Yongzheng Chen et al.
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Table 1. Microbialreduction of 2-oxo-4-phenybl utyrate.
Entry
Microorganism
Time [h]
Yield [%][a]
ee [%][a]
[Absolute configuration]
1
2
3
4
5
6
7
8
Geotrichum candidum CIOC1035
Candida boidinii CIOC616
12
12
24
12
12
12
24
24
12
24
24
12
12
12
12
12
12
12
12
12
24
24
12
24
12
12
12
12
12
24
12
24
12
12
24
24
24
12
12
12
35
59
26
86
70
62
66
36
61
83
80
67
82
63
59
86
63
56
82
46
68
90
41
26
90
57
50
49
94
95
22
88
44
13
91
37
89
92
0
4 (S)
10 (S)
10 (S)
14 (S)
15 (S)
17 (S)
17 (S)
20 (S)
27 (S)
28 (S)
32 (S)
34 (S)
36 (S)
38 (S)
38 (S)
41 (S)
46 (S)
48 (S)
61 (S)
77 (S)
87 (S)
88 (S)
95 (S)
98 (S)
99 (S)
99 (S)
99 (S)
99 (S)
99 (S)
10 (R)
13 (R)
16 (R)
16 (R)
39 (R)
48 (R)
55 (R)
56 (R)
99 (R)
-
Variovoras paradoxus CIOC1842
Geotrichum candidum CIOC1175
Saccharomyces cerevisiae CIOC346
Trichosporon cutaneum CIOC25
Achrombaccter xylosoxidans subsp. denitrificans CIOC768
Alcaligenes faecalis CIOC924
Candida boidinii CIOC1644
Alcaligenes faecalis CIOC1799
Stenotrophomonas maltophilia CIOC1788
Candida boidinii CIOC26
Candida magnoliae CIOC1919
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Geotrichum candidum CIOC1062
Candida boidinii CIOC1647
Pichia farinosa CIOC1463
Candida boidinii CIOC1646
Geotrichum candidum CIOC616
Saccharomyces cerevisiae CIOC396
Geotrichum candidum CIOC108
Alcaligenes faecalis CIOC767
Saccharomyces cerevisiae CIOC1090
Saccharomyces cerevisiae CIOC S1
Microbacterium terregens CIOC1901
Yarrowia lipolytica CIOC1502
Geotrichum candidum CIOC CIOC11
Saccharomyces cerevisiae CIOC1396
Saccharomyces cerevisiae CIOC399
Saccharomyces cerevisiae CIOC SY2
Alcaligenes faecalis CIOC1786
Alcaligenes faecalis CIOC1837
Achrombaccter xylosoxidans subsp. denitrificans CIOC2679
Candida boidinii CIOC22
Trichosporon cutaneum CIOC570
Arthrobacter sp. CIOC1008
Alcaligenes faecalis CIOC2006
Achromobater xylosoxidans subsp. xylosoxidans CIOC2007
Candida boidinii CIOC21
Trichosporon cutaneum CIOC1795
Yarrowia lipolytica CIOC1506
0
-
[a]
Determined by GC analysis.
Scheme 2. Stereoselective reduction of keto ester 1.
backed silica plates. Column chromatography was per-
formed by using silica gel (200–300 mesh) with ethyl ace-
tate/petroleum ether as eluent. The enantiomeric excess was
determined by GC analysis on a Chirasil-Dex CB column.
All other reagents were purchased from commercial sources
and were used without further purification. Compound 1
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Preparation the Key Intermediate of Angiotensin-Converting Enzyme (ACE) Inhibitors
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Table 2. Reduction by Candida boidinii CIOC21: effect of
cosolvents on the synthesis of the ethyl (R)-2-hydroxy-4-
phenylbutyrate.
1.94–2.00 (1H, m, ArCH2CHH), 2.10–2.15 (1H, m,
ArCH2CHH), 2.74–2.81(2H, m, OCH2CH3), 2.81(1H, s,
OH), 4.17–4.25 (3H, m, ArCH2, CHOH), 7.17–7.32(5H, m,
Ar-H); 13C NMR (CDCl3, 75 Hz): d=175, 141, 129, 128, 125,
69, 61, 35, 30, 14; MS: m/z=208 (M+).
Entry Solvent [10% v/v]
Yield
[%][a]
ee [%][a]
Configuration
1
2
3
None
Ethanol98
Methyl tert-butylether
(MTBE)
DMF
92
29
99 (R)
R)
21 (R)
56 (
Determination of ee of the Product Formed
The absolute configurations were assigned by chiral phase
GC analysis using authentic (R)-2 as a standard under con-
ditions that gave almost near baseline resolution of the en-
antiomers (Scheme 3). The absolute configurations and opti-
calpurities of ethyl2-hydroxyphenyblutyrate produced by
microbialcellreductions were determined by GC (25 m”
0.25 mm, CP Chirasil-Dex CB column, Varian, USA. Tc =
1608C).
4
5
6
7
86
90
91
99
99 (R)
88 (R)
31 ( R)
69 (R)
DMSO
Diisopropylether
Dimethylphthaal te
(DMP)
[a]
Determined by GC analysis.
and ethyl( R)-2-hydroxy-4-phenylbutyrate were purchased
from Aldrich Chemical Co, and the rac-2-hydroxy-4-phenyl-
butyrate was prepared by reduction with NaBH4 in a metha-
nolmedium.
Cultivation of Microorganisms
Microorganisms screened were preserved in our laboratory.
Yeasts were grown in a medium containing 0.2% (w/v) glu-
cose, 2% (w/v) peptone, and 1% (w/v) yeast extract solution
(pH 6.8–7.0); bacteria were grown in a medium containing
0.1% (w/v) sodium chloride, 0.5% (w/v) beef extract, 2%
(w/v) peptone solution (pH 6.8). Strains were maintained on
nutrient agar slants at 48C. Erlenmeyer flasks (250-mL) con-
taining 100 mL of the appropriate sterilized cultivation
medium were inoculated with the tested microorganism and
incubated in an orbitalshaker (180 rpm) at 27 8C. After 48 h
of growth (yeast, bacteria), the cells were harvested by cen-
trifugation and washed twice with cool physiological saline
(0.85%).
Scheme 3. Chiral-phase GC analysis of a-keto ester reduc-
tion products. Samples were analyzed on a 25 m Chirasil-
Dex CB column. (a) Racemic 2 prepared by NaBH4 reduc-
tion of 1. (b) (R)-2 prepared by Candida boidinii CIOC21
reduction of 1 in aqueous medium. (c) (R)-2 prepared by
Candida boidinii CIOC21 reduction of
1 in aqueous
medium with 10% (v/v) DMSO as a cosolvent. (d) (S)-2 pre-
pared by Yarrowia lipolytica CIOC1502 reduction of 1.
Typical Procedure for the Microbial Screening
To a suspension of microorganisms cells (yeasts, bacteria) in
10 mL of 50 mM potassium phosphate buffer (pH 7.0), was
added keto ester 1 (0.1 mmol) and glucose 0.5 g. The reac-
tion mixtures were incubated in an orbitalshaker (160 rpm)
at 308C for 12 h. After centrifugation at 8000”g for 8 min,
the supernatant was saturated with NaCland then extracted
three times with ethylacetate. Chemicalyiedl and ee of the
product were determined by GC analysis.
Acknowledgements
We are grateful for financial support from the National Natu-
ral Science Foundation of China (No. 20672110).
Isolation and Characterization of the Products
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fied by flash chromatography (15% EtOAc/petroleum
ether), this gave isolated yields of 35 mg (0.168 mmol). This
concentrated sample was used for GC analysis to determine
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