A new chemo-enzymatic route to chiral 2-hydroxy-4-phenylbutyrates by combining lactonase-mediated resolution with hydrogenation over Pd/C

Article abstract of DOI:10.1039/b925402a

A new chemo-enzymatic route to both isomers of 2-hydroxy-4-phenylbutyric acid is reported. The key step is the lactonase-catalyzed hydrolysis of cis- and trans-2-hydroxy-4-phenyl-4-butyrolactones followed by hydrogenation over Pd/C to afford optically pure 2-hydroxy-4-phenylbutyric acid.

Full text of DOI:10.1039/b925402a

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A new chemo-enzymatic route to chiral 2-hydroxy-4-phenylbutyrates by
combining lactonase-mediated resolution with hydrogenation over Pd/Cw
Bing Chen,a Hai-Feng Yin,b Zhen-Sheng Wang,b Jia-Ying Liua and Jian-He Xu*a
Received (in Cambridge, UK) 2nd December 2009, Accepted 3rd February 2010
First published as an Advance Article on the web 25th February 2010
DOI: 10.1039/b925402a
A
new chemo-enzymatic route to both isomers of
same time and that the enantioselectivity of the enzyme was
only interfered with by the configuration of the alpha site
while the chirality at the gamma site of the lactone ring has
little, if any, influence on the optical purity of the products.
These results encouraged us to try the enzymatic hydrolysis of
a 3:1 mixture of cis- and trans-2-hydroxy-4-phenyl-4-butyrol-
actones. We found that both optically pure (2S)-cis- and
(2S)-trans-lactones could be obtained within almost the same
time (Fig. 1). The configuration of the gamma site did not affect
either the enantioselectivity nor the activity of the enzyme.
Based on these results, we designed a new chemo-enzymatic
route to optically pure 2-hydroxy-4-phenylbutyrate. The key
step is the enzymatic hydrolysis of a racemic mixture of
cis-/trans-2-hydroxy-4-phenyl-4-butyrolactone followed by direct
Pd-catalyzed hydrogenation of the products. In order to solve the
problems encountered with traditional methods, we set the
following goals: to obtain both isomers in high chemical and
optical purity; to identify the most efficient biocatalyst and to
adapt the method for potential industrial production.
2-hydroxy-4-phenylbutyric acid is reported. The key step is the
lactonase-catalyzed hydrolysis of cis- and trans-2-hydroxy-4-
phenyl-4-butyrolactones followed by hydrogenation over Pd/C
to afford optically pure 2-hydroxy-4-phenylbutyric acid.
2-Hydroxy-4-phenylbutyrate is an important precursor to
angiotensin-converting enzyme (ACE) inhibitors1 which have
the (S)-homophenylalanine moiety as the central pharmaco-
phore unit. These ACE inhibitors from the ‘pril-family’, such
as Enalapril, Lisinopril, Cilapril and Benazepril, greatly
expand the range of anti-hypertensive theurapeutics.2
Developing a cost effective process for the production of
these key building blocks is therefore very important.3 So far,
many routes to optically pure 2-hydroxy-4-arylbutyrates
have been developed, including: (a) classical resolution;4 (b)
asymmetric synthesis;5 and (c) asymmetric catalysis.3,6–9
Routes a and b have gradually been replaced by route c for
the consideration of atom-economy, operational-simplicity,
eco-friendliness and cost-efficiency. Asymmetric catalysis
includes, but is not limited to, three traditional methods: (1)
asymmetric hydrogenation using substrates such as ethyl
2,4-dioxo-4-phenylbutyrate3 and (E)-ethyl 2-oxo-4-arylbut-3-
enoates;6 (2) enzymatic hydrolysis of racemic substrates
including cyanohydrin7 and esters of 2-hydroxy-4-arylbutyrate;8
and (3) enzymatic reduction of 2-oxo-4-phenylbutyrate and
its derivatives.9 Asymmetric hydrogenation usually affords
products in moderate optical purity though the catalysts or
chiral ligands are not always readily available. The low
efficiency of the biocatalyst from time to time limits its
application in industrial production. For example, 2-oxo-
4-phenylbutyrate and its derivatives were reduced usually at
a substrate-to-catalyst ratio of around 1/25 to 1/100.9 All of
these results prompted us to develop a new process for
producing 2-hydroxy-4-phenylbutyrate and its esters based
on a highly efficient enzyme, Fusarium lactonase.
The synthesis started from Claisen condensation of aceto-
phenone 1 and diethyl oxalate (Scheme 1). (Z)-2-hydroxy-4-
oxo-4-phenylbut-2-enoic acid 2 was obtained in about 90%
Previously, we reported the Fusarium lactonase-catalyzed
bioresolution of cis- or trans-4-substituted 2-hydroxy-4-
butyrolactones.10 We also noted that the enzymatic hydrolysis
of cis- and trans-lactones were completed within almost the
Fig. 1 HPLC analysis of the same sample on Chiracel AD-H and
OJ-H columns before and after Fusarium lactonase-catalyzed hydrolysis
of
a mixture of cis-/trans-2-hydroxy-4-phenyl-4-butyrolactones.
Before enzymatic hydrolysis, cis-lactones are not separable on a
Chiracel OJ-H column (tR = 27.9 min) while trans-lactones show
two peaks—tR 19.5 min for (2R,4S)-isomer and tR 22.8 min for
(2S,4R)-isomer. After enzymatic hydrolysis, the (2R,4S)-lactone was
hydrolyzed and (2S,4R)-lactone remained intact. Cis-lactones are
separated on Chiracel AD-H column—tR 16.6 min for (2R,4R)-
isomer, tR 18.5 min for (2S,4S)-isomer and 14.8 min for racemic
trans-lactone. After enzymatic hydrolysis, (2R,4R)-lactone was
hydrolyzed and (2S,4S)-lactone remained.
a Laboratory of Biocatalysis and Bioprocessing, State Key Laboratory
of Bioreactor Engineering, East China University of Science and
Technology, 130 Meilong Road, Shanghai 200237, P. R. China.
E-mail: jianhexu@ecust.edu.cn; Fax: +86-21-64250840
b PepTech (Shanghai) Pharmaceutical Co., Ltd, Building #4,
Lane 388 Yindu Road, Shanghai 200231, P. R. China
w Electronic supplementary information (ESI) available: Materials
and methods; spectra for relative compounds. See DOI: 10.1039/
b925402a
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Scheme 1 Synthesis of 2-hydroxy-4-phenyl-4-butyrolactone.
Scheme 2 Synthesis of 2-hydroxy-4-phenylbutyrates through tandem
yield. Subsequent saponification and extraction with dichloro-
methane removed residual acetophenone from the product.
Acidification, NaBH4 reduction and cyclization afforded a
racemic mixture of cis-/trans-2-hydroxy-4-phenyl-4-butyrolactone
3 in a ratio of 3 to 1, which was pure enough for use in the
enzymatic resolution.
Fusarium lactonase-catalyzed hydrolysis of racemic cis-/trans-4-phenyl
2-hydroxyl-4-butyrolactones and hydrogenation over Pd/C.
catalyzed by these four reFPL variants.12 It was found that
reFPL3 was the most efficient lactonase (Table 1).
The residual (2S)-cis-/trans-lactone was then isolated (yield
33%) by simple extraction with ethyl acetate and submitted
directly to hydrogenation over 5% Pd/C (1 atm, room
temperature) in acetic acid (Scheme 2). The benzylic hydroxyl
group was reduced, and the stereo-configuration of the
a-hydroxyl group was left intact. After an overnight reaction,
both cis- and trans-substrates afforded (S)-2-hydroxy-4-
phenylbutyric acid 4. The crystalline acid was further
purified (yield 88% from the lactone) by crystallization from
dichloroethane.6 It was quantitatively converted to its ethyl
ester 5 using Me3SiCl/EtOH. Optical rotation measurement
([a]2D0 + 8.51, EtOH, c = 1.0; lit9, +7.51) confirmed the
configuration assignment of the 2-hydroxyl group to be
S. Chiral HPLC analysis (AD-H column) showed a purity of
>99% ee.
Enzymatic hydrolysis of lactone 3 was then carried out. The
biocatalyst (reFPL) used here was whole cells of recombinant
E. coli BL21 (DE3) expressing the Fusarium proliferatum
lactonase gene (GenBank accession number EU595535)
(Table 1).10 In a typical experiment, the substrate of 20 g LÀ1
(with 20% MeCN as a cosolvent) was catalyzed by 2.3 g
biocatalyst (reFPL4, dry cell weight) per liter. The pH was
controlled at 6.4–6.7 by NH4OH, while the reaction progress
was monitored by chiral HPLC. We found that reFPL-
catalyzed hydrolysis of trans-isomer was slightly faster than
that of cis-isomers. After 1 h, both (2S)-isomers of trans-/
cis- lactones were in >99% ee. This biotransformation was
conducted at a substrate-to-catalyst ratio of 8.7/1 and was
completed within 60 min, which was 102–103 more efficient
than other enzymatic or chemo-enzymatic routes.9
A modified procedure was applied to the preparation of
(R)-2-hydroxy-4-phenylbutyric acid. In a typical experiment,
the substrate at 20 g LÀ1 (20% MeCN) was catalyzed by 2.3 g
biocatalyst (reFPL4, dry cell weight) per liter. The pH was
controlled at 6.4–6.7 by NH4OH. The reaction process was
monitored by chiral HPLC and was terminated at around
30% conversion, in usually less than 30 min. The residual
undesired butyrolactones were extracted completely with ethyl
acetate, while the acids in the aqueous layer were collected,
acidified and cyclized. The lactones were then extracted
with ethyl acetate (isolated yield 37%) and submitted to
We also screened a set of biocatalysts for the purpose
of making the biotransformation process more efficient.
Commercially available lipases were tested at a 1 to 1, substrate-
to-catalyst ratio. However, after 24 h, only poor ee values
(o16%) were achieved with o21% conversion. We also
modified11 the reFPL with plasmids (pET28a/pET24a) and signal
peptide (with/without signal peptide) to generate four reFPL
variants: reFPLs 1–4. We monitored with chiral HPLC the
conversions and ee values of the residual substrate during the
hydrolysis process of cis-4-phenyl-2-hydroxyl-4-butyrolactone
Table 1 Screening of biocatalysts for asymmetric hydrolysis of 4-phenyl-2-hydroxy-4-butyrolactone (10 mM)a
Enzyme
Amount of enzymes
Time/h
Conv. (%)d
ee (%)
E
reFPL1b
10 IU
10 IU
10 IU
0.5
0.5
0.5
0.5
24
24
24
24
24
50.6
48.3
51.0
50.5
21.3
18.4
o10
o10
o10
o10
96.9e
95.3e
96.2e
92.8e
16.0f
348h
123h
366h
99.0h
reFPL2b
reFPL3b
reFPL4b
10 IU
Lipase OFc
Lipase AKc
Lipase L-1754c
Lipase AYSc
Lipase PPLc
Lipase Dc
18 mg (7.5 Â 102 IU)
18 mg (53.8 IU)
18 mg (77.9 IU)
18 mg (60.5 IU)
18 mg (8.5 IU)
18 mg (2.5 Â 102 IU)
—
g
g
15.3f
—
—
—
—
o1.0f
o1.0f
o1.0f
o1.0f
g
g
g
g
24
—
a
b
Reaction condition: 10 ml KPB buffer (50 mM, pH 6.4), 200 ml MeCN, 30 1C, 180 rpm; Whole cells of E. coli were used as a biocatalyst and
their amount was controlled at 10 IU towards racemic pantolactone; The amount of lipase was controlled at 18 mg while the activity was tested
c
using p-nitrophenyl butyrate as substrate; Conversion was calculated from formula c = ees/(ees + eep);13 e eep, analyzed by chiral HPLC AD-H
d
column; ees, analyzed by chiral HPLC AD-H column; Not calculated; E = ln[1 À c(1 + eep)]/ln[1 À c(1 À eep)].13
f
g
h
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Chem. Commun., 2010, 46, 2754–2756 | 2755

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hydrogenation over Pd/C, giving (R)-2-hydroxy-4-phenyl-
butyric acid. Chiral HPLC analysis of its ethyl ester showed
a 98% ee.
7 I. Osprian, M. H. Fechter and H. Griengl, J. Mol. Catal. B:
Enzym., 2003, 24–25, 89.
8 P. Kalaritis, R. W. Regenye, J. J. Oartridge and D. L. Coffen,
J. Org. Chem., 1990, 55, 812; A. Liese, U. Kragl, H. Kierkels and
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Sugai and H. Ohta, Agric. Biol. Chem., 1991, 55, 293; A. Chadha
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2000, 2, 1037.
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Catal., 2008, 350, 426; A. Chadha and B. Baskar, Tetrahedron:
Asymmetry, 2002, 13, 1461; P. S. B. Lacerda, J. B. Ribeiro,
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This process has two advantages. First, both (R)- and
(S)- acids were prepared in excellent optical purity using one
single biocatalyst. Secondly, the simplicity of this procedure
makes it very attractive for potential industrial applications.
The starting materials used are readily available and only one
purification step, the recrystallization of the acid, is needed in
the whole process.9
In conclusion, a new route to optically pure (R)- and (S)-2-
hydroxyl-4-phenylbutyric acids has been developed and has
potential for industrial applications.
The authors thank Dr James Ma and Dr Hongyan Liu for
informative discussion and proof-reading. This project is
supported by the National Natural Science Foundation of
China (grant nos. 20773038 & 20902023), the Ministry of
10 B. Chen, H.-F. Yin, Z.-S. Wang, J.-H. Xu, L.-Q. Fan and J. Zhao,
Adv. Synth. Catal., 2009, 351, 2959; X. Zhang, J.-H. Xu, Y. Xu and
J. Pan, Appl. Microbiol. Biotechnol., 2007, 75, 1087; B. Chen,
L.-Q. Fan, J.-H. Xu, J. Zhao and X. Zhang, Appl. Biochem.
Biotechnol., 2009, DOI: 10.1007/s12010-009-8819-1.
Science and Technology (grant nos. 2009CB724706
&
2009ZX09501-016), the China National Special Fund for State
Key Laboratory of Bioreactor Engineering (grant no.
2060204) and the Shanghai Leading Academic Discipline
Project (No. B505).
11 For reFPL1, the lactonase gene with the signal peptide sequence
was inserted into plasmid pET28a(+) which was digested with
NdeI and BamHI; for reFPL2, the lactonase gene with the signal
peptide sequence was inserted into plasmid pET24a(+) which was
digested with NdeI and BamHI; for reFPL3, the lactonase gene
without signal peptide sequence was inserted into plasmid
pET24a(+) which was digested with NdeI and BamHI; for
reFPL4, the lactonase gene without signal peptide sequence was
inserted into plasmid pET28a(+) which was digested with NdeI
and BamHI. The lactonase gene without signal peptide has now
been deposited in GenBank with an accession number of
EU596535, while the lactonase gene with signal peptidesequence
was shown in the Supplementary Information. All of the recombi-
nant lactonases were expressed in E. coli BL21(DE3).
Notes and references
1 R. A. Sheldon, Chimica Oggi, 1991, 35.
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hedron: Asymmetry, 2003, 14, 2659; B. Larissegger-Schnell,
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3 P. Herold, A. F. Indolese, M. Studer, H. P. Jalett, U. Siegrist and
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5 W.-Q. Lin, Z. He, Y. Jing, X. Cui, H. Liu and A.-Q. Mi,
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12 Please see Supplementary Informationw.
13 C.-S. Chen, Y. Fujimoto, G. Girdaukas and C. J. Sih, J. Am.
Chem. Soc., 1982, 104, 7294.
6 Q. Meng, L. Zhu and Z. Zhang, J. Org. Chem., 2008, 73, 7209.
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This journal is The Royal Society of Chemistry 2010
2756 | Chem. Commun., 2010, 46, 2754–2756