Chemo-enzymatic synthesis of (R)-and (S)-2-hydroxy-4-phenylbutanoic acid via enantio-complementary deracemization of (±)-2-hydroxy-4-phenyl-3- butenoic acid using a racemase-lipase two-enzyme system

Article abstract of DOI:10.1055/s-2005-871577

Deracemization of (±)-2-hydroxy-4-phenylbut-3-enoic acid was accomplished by lipase-catalyzed kinetic resolution coupled to mandelate racemase-mediated racemization of the non-reacting substrate enantiomer. Stepwise cyclic repetition of this sequence led

Full text of DOI:10.1055/s-2005-871577

1936
LETTER
Chemo-Enzymatic Synthesis of (R)- and (S)-2-Hydroxy-4-phenylbutanoic
Acid via Enantio-Complementary Deracemization of ( )-2-Hydroxy-4-phenyl-
3-butenoic Acid Using a Racemase–Lipase Two-Enzyme System
C
hemo-Enzyma
t
a
r
of
(
R
)-
b
and
(
S
)-2-H
y
a
droxy-4-ph
r
enylbutanoic
Acid Larissegger-Schnell, Wolfgang Kroutil, Kurt Faber*
Department of Chemistry, Organic & Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria
Fax +43(316)3809840; E-mail: Kurt.Faber@uni-graz.at
Received 4 May 2005
Although both of these methods have the clear merit of a
Abstract: Deracemization of ( )-2-hydroxy-4-phenylbut-3-enoic
acid was accomplished by lipase-catalyzed kinetic resolution
coupled to mandelate racemase-mediated racemization of the non-
reacting substrate enantiomer. Stepwise cyclic repetition of this
100% theoretical yield of a single stereoisomeric product,
its absolute configuration is determined by the enantio-
preference of the biocatalyst employed. Since mirror-
sequence led to a single enantiomeric product, the stereochemical image enzymes sensu stricto do not exist,8 production of
outcome of which could be controlled by switching between lipase-
both enantiomers through biocatalytic deracemization by
catalyzed acyl-transfer/ester hydrolysis reactions. Both enantio-
simple choice of the ‘matching enantiomer’ of the chiral
meric products were easily transformed into (R)- and (S)-2-
(bio)catalyst is virtually impossible.
hydroxy-4-phenylbutanoic acid, important building blocks for
In order to circumvent this limitation, we envisaged to ap-
ply lipase-catalyzed ester hydrolysis and ester formation
to our recently developed deracemization protocol9 based
on the enzymatic racemization of the non-reacting sub-
strate enantiomer using a racemase.10 Taking into consid-
eration that ester hydrolysis and esterification represent
reactions in opposite directions, products of opposite
configuration are usually obtained.
ACE-inhibitors.
Key words: deracemization, lipase, mandelate racemase, biocatal-
ysis, enzyme catalysis
(R)-2-Hydroxy-4-phenylbutanoic acid (3) is an important
building block for the production of a large variety of an-
giotensin converting enzyme (ACE) inhibitors having in
common the (S)-homophenylalanine moiety as the central
pharmacophore unit.1 These agents, often denoted as the
‘-pril family’, such as enalapril, lisinopril, cilapril or
benazepril, efficiently expand the range of antihyper-
tensiva, like b-blockers, A2-antagonists or Ca-channel
blockers. Due to the fact that many of these drugs have
lost patent protection (or soon will do so), the production
costs of the required building blocks has become a major
issue.
The enantio-complementary deracemization protocol was
realized as follows (Scheme 1): since 2-hydroxy-4-phe-
nylbutanoate (3) is not a substrate for mandelate race-
mase,11 the corresponding butenoic acid derivative (1),
which allows resonance stabilization of the a-carbanion
(occurring during mandelate racemase catalysis) via con-
jugation with the aryl-moiety,12 was chosen.13 Mandelate
racemase racemizes (R)-1 at a rate of 53% to that of its
natural substrate – mandelate – which corresponds to a
turnover frequency of approximately 500 sec–1.11
For the synthesis of 3 or close derivatives thereof in non-
racemic form, numerous strategies have been devised,
which can be categorized into (i) classic2 or (ii) kinetic
resolution of a racemate 3or (iii) asymmetric transforma-
tion of prochiral precursor.4 The majority of these routes
have one or more weak points,4m i.e. high cost of reagents
(e.g., chiral transition metal complexes), insufficient cata-
lyst selectivity or -activity, sensitivity of catalysts (e.g.,
asymmetric hydrogenation), or stability of starting mate-
rials (e.g., keto acids). The most dramatic limitation com-
mon for all strategies relying on kinetic resolution is the
maximum theoretical yield of 50% for a single enantio-
mer. In order to overcome this fundamental drawback,
two approaches – summarized under the term ‘deracem-
ization’5 – were recently proposed: (i) dynamic kinetic
resolution6 and (ii) microbial stereo-inversion.7
(S)-Series: in a first step, kinetic resolution of rac-1 in the
acyl-transfer mode was accomplished using Pseudomo-
nas sp. lipase in diisopropyl ether at the expense of vinyl
acetate as acyl donor9 to furnish (S)-2 and non-reacted
(R)-1 in excellent ee (>99%) at 50% conversion. The latter
was re-racemized without separation from formed prod-
uct using mandelate racemase14 in aqueous buffer. Step-
wise repetition of this two-enzyme procedure for three
cycles furnished (S)-2 in >99% ee and 68% overall yield
from the racemate. Alkaline hydrolysis of the O-acyl moi-
ety and catalytic reduction of the C=C bond gave (S)-3 as
the sole product in >99% ee.15
(R)-Series: access to (R)-3 started by enzymatic hydroly-
sis of rac-2 (obtained by acylation of rac-1) in aqueous
acetone for sufficient solubilization of the polar substrate.
Since Pseudomonas sp. lipase displayed low enantiose-
lectivity, Candida antarctica lipase B (Novozyme 435)
was chosen for perfect enantioselectivity, which produced
a mixture of (S)-1 and unreacted (R)-2. Re-racemization
SYNLETT 2005, No. 12, pp 1936–1938
0
1
.0
8
.2
0
0
5
Advanced online publication: 07.07.2005
DOI: 10.1055/s-2005-871577; Art ID: G12405ST
© Georg Thieme Verlag Stuttgart · New York

LETTER
Chemo-Enzymatic Synthesis of (R)- and (S)-2-Hydroxy-4-phenylbutanoic Acid
1937
OH
OH
OAc
CO2
OH
c, d
a
+
Ph
CO2H
Ph
CO2H
Ph
Ph
CO2H
52%
H
rac 1
(R)-1
(S)-2
(S)-3
ee >99%
68% from rac-1
b
sole product
ee >99%
e
3 cycles
OAc
CO2H
OH
OAc
OH
f
c, d
+
Ph
Ph
CO2H
Ph
CO2H
Ph
CO2H
52%
rac-2
(S)-1
(R)-2
(R)-3
ee >99%
53% from rac-2
b, e
sole product
2 cycles
ee >99%
Scheme 1 Reagents and conditions: (a) Pseudomonas sp. lipase (Amano PS-C-II), vinyl acetate, i-Pr2O, 25 °C; (b) mandelate racemase [EC
5.1.2.2], buffer pH 7.5, 30 °C; (c) MeOH, K2CO3, 0 °C; (d) MeOH, H2, Pd/C; (e) Ac2O, pyridine; (f) Candida antarctica lipase B (Novozyme
435), buffer pH 7.5, acetone, 30 °C.
of the former by mandelate racemase followed by re-
esterification and lipase-hydrolysis through two cycles
gave (R)-2 in >99% ee and 53% overall yield from rac-2.
Hydrolysis of the O-acetyl group and hydrogenation
furnished (R)-2 in >99% ee as the sole product.16
References
(1) (a) Sheldon, R. A. Chirotechnology; Marcel Dekker: New
York, 1993, 365. (b) Sheldon, R. A. Chim. Oggi 1991, 9, 35.
(2) Attwood, M. R.; Hassall, C. H.; Kröhn, A.; Lawton, G.;
Redshaw, S. J. Chem. Soc., Perkin Trans. 1 1986, 6, 1011.
(3) (a) Liese, A.; Kragl, U.; Kierkels, H.; Schulze, B. Enzyme
Microb. Technol. 2002, 30, 673. (b) Kalaritis, P.; Regenye,
R. W.; Partridge, J. J.; Coffen, D. L. J. Org. Chem. 1990, 55,
812. (c) Chadha, A.; Manohar, M. Tetrahedron: Asymmetry
1995, 6, 651. (d) Osprian, I.; Fechter, M. H.; Griengl, H. J.
Mol. Catal. B: Enzym. 2003, 24-25, 89. (e) Huang, S. H.;
Tsai, S. W. J. Mol. Catal. B: Enzym. 2004, 28, 65.
(f) Sugai, T.; Ohta, H. Agric. Biol. Chem. 1991, 55, 293.
(4) (a) Spindler, F.; Pittelkow, U.; Blaser, H. U. Chirality 1991,
3, 370. (b) Chang, C.-Y.; Yang, T.-K. Tetrahedron:
Asymmetry 2003, 14, 2239. (c) Chadha, A.; Manohar, M.;
Soundararajan, T.; Lokeswari, T. S. Tetrahedron:
The main limitation of this process lies in an indispens-
able aqueous-organic solvent switch, which necessitates a
consecutive sequence of steps rather than a dynamic pro-
cess. The latter is caused by the inactivity of mandelate
racemase in organic media at low water activity.17 The en-
zyme is remarkably stable and can be used repeatedly, in
particular in immobilized form, which facilitates its re-
covery.18 Although isolated overall yields were still below
the theoretical 100%, this threshold should be approach-
able using improved recovery procedures for the polar
hydroxycarboxylic acids (e.g., by ion exchange chroma-
tography) based on the fact that this process is virtually
free of side reactions. The possibility to determine the
stereochemical configuration of the sole product by a sim-
ple switch between the acyl-transfer- and hydrolysis-
mode demonstrates the flexibility of this process.
Asymmetry 1996, 7, 1571. (d) Fadnavis, N. W.; Radhika, K.
R. Tetrahedron: Asymmetry 2004, 15, 3443. (e) Schmidt,
E.; Blaser, H. U.; Fauquex, P. F.; Sedelmeier, G.; Spindler,
F. In Microbial Reagents in Organic Synthesis, Vol. 381;
Servi, S., Ed.; NATO ASI Series C, Kluwer Acad. Publ.:
Dortrecht, 1992, 377. (f) Blaser, H. U.; Jalett, H. P. Stud.
Surf. Sci. Catal. 1993, 78, 139. (g) Hummel, W.; Schütte,
H.; Schmidt, E.; Wandrey, C.; Kula, M. R. Appl. Microbiol.
Biotechnol. 1987, 26, 409. (h) Dao, D. H.; Kawai, Y.; Hida,
K.; Hornes, S.; Nakamura, K.; Ohno, A.; Okamura, M.;
Akasaka, T. Bull. Chem. Soc. Jpn. 1998, 71, 425.
(i) Kaluzna, I.; Andrew, A. A.; Bonilla, M.; Martzen, M. R.;
Stewart, J. D. J. Mol. Catal. B: Enzym. 2002, 17, 101.
(j) Fechter, M. H.; Griengl, H. In Enzyme Catalysis in
Organic Synthesis, 2nd ed., Vol. 2; Drauz, K.; Waldmann,
H., Eds.; Wiley-VCH: Weinheim, 2002, 947. (k) North, M.
Tetrahedron: Asymmetry 2003, 14, 147. (l) Wang, Y.-F.;
Chen, S.-T.; Liu, K. K.-C.; Wong, C.-H. Tetrahedron Lett.
1989, 30, 1917. (m) Herold, P.; Indolese, A. F.; Studer, M.;
Jalett, H. P.; Siegrist, U.; Blaser, H. U. Tetrahedron 2000,
56, 6497.
In summary, both enantiomers of 2-hydroxy-4-phenyl-
butanoic acid were obtained as single stereoisomers in
>99% ee via stepwise deracemization of the correspond-
ing ( )-2-hydroxy-4-phenylbut-3-enoic acid through a
lipase-racemase protocol. Whereas the S-enantiomer was
formed through lipase-catalyzed acyl-transfer, the R-
counterpart was produced via ester hydrolysis.
Acknowledgement
This study was performed in cooperation with BASF-AG
(Ludwigshafen) and B. Hauer and R. Stürmer are thanked for their
valuable contributions.
(5) For the concept of deracemization see: (a) Faber, K.
Chem.–Eur. J. 2001, 7, 5004. (b) Strauss, U. T.; Felfer, U.;
Faber, K. Tetrahedron: Asymmetry 1999, 10, 107.
(6) Huerta, F. F.; Laxmi, Y. R. S.; Bäckvall, J.-E. Org. Lett.
2000, 2, 1037.
Synlett 2005, No. 12, 1936–1938 © Thieme Stuttgart · New York

1938
B. Larissegger-Schnell et al.
LETTER
(7) Chadha, A.; Baskar, B. Tetrahedron: Asymmetry 2002, 13,
1461.
chromatography to yield (S)-1 (56 mg; 63%); mp 132–
133 °C; lit. mp 104 °C; [a]D20 +96.5 (c 0.27, MeOH, >99%
ee); lit. [a]D25 +85.2 (c 0.55, MeOH, 94% ee).
(8) Although several processes producing enantiomeric
products were reported depending on the choice of
biocatalyst, the search for e.g. ‘anti-Kazlauskas’ carboxyl
ester hydrolyses and ‘anti-Prelog’ alcohol dehydrogenases
still represents a major challenge in biocatalysis.
(9) Strauss, U. T.; Faber, K. Tetrahedron: Asymmetry 1999, 10,
4079.
(10) For a review on enzyme-catalyzed racemization see:
Schnell, B.; Faber, K.; Kroutil, W. Adv. Synth. Catal. 2003,
345, 653.
(S)-2-Hydroxy-4-phenylbutanoic Acid [(S)-3].
(S)-1 (50 mg, 0.28 mmol) was hydrogenated employing a
rubber balloon using a catalytic amount of Pd on C (10%, 5
mg) in MeOH for 10 min. Then the catalyst was filtered off
and the solvent was evaporated to yield (S)-3 (42 mg, 83%);
mp 115–117 °C; lit. mp 114 °C; [a]D20 +8.1 (c 1.0, EtOH,
>99% ee); lit. [a]D25 +7.5 (c 0.5, EtOH, 84% ee); chiral
HPLC analysis using the method described above showed a
single peak at t = 26.8 min.
(11) Felfer, U.; Goriup, M.; Koegl, M. F.; Wagner, U.;
Larissegger-Schnell, B.; Faber, K.; Kroutil, W. Adv. Synth.
Catal. 2005, 347, 951.
(12) Kenyon, G. L.; Hegeman, G. D. Biochemistry 1970, 9, 4029.
(13) rac-2-Hydroxy-4-phenyl-3-butenoic acid (rac-1) was
prepared according to: Nerdel, F.; Rachel, H. Chem. Ber.
1956, 89, 671; mp 137–139 °C, lit. mp 137 °C.
(14) For a large-scale preparation of mandelate racemase see:
Stecher, H.; Felfer, U.; Faber, K. J. Biotechnol. 1997, 56, 33.
(15) (S)-2-Acetoxy-4-phenyl-3-butenoic Acid [(S)-2] via
Deracemization of [rac-1].
(16) (R)-2-Acetoxy-4-phenyl-3-butenoic Acid [(R)-2] via
Deracemization of rac-1.
Acylation step (c): a solution of rac-1 (0.5g, 2.8 mmol) and
acetic anhydride (5 mL) in pyridine (0.2 mL) was kept at 0–
5 °C. After 6 h the solution was poured into ice-water (100
mL), which was acidified with HCl (3 M) to pH 1–2 and
extracted three times with EtOAc. The combined organic
layers were washed with H2O and brine, dried (Na2SO4) and
evaporated to yield rac-2 (0.45 g, 72%); mp 74–77 °C.
Kinetic resolution step (f): to a solution of rac-2 (0.45 g, 2.0
mmol) in acetone (4.5 mL) and phosphate buffer (45 mL, 50
mmol, pH 7.5), lipase from Candida antarctica B
(Novozyme 435, 1.8 g) were added and the mixture was
shaken for 24 h at 30 °C and 130 rpm. The reaction mixture
was filtered and the recovered lipase was dried for reuse. The
filtrate was evaporated from acetone, the residue was
acidified with HCl (3 M) to pH 1–2, extracted three times
with EtOAc, dried (Na2SO4) und evaporated. HPLC analysis
as described above showed a conversion of 50%; (S)-1: t =
36.2 min, (R)-2: t = 31.9 min. For the racemization step (b)
see above. After repeating (e) and (f) for two times and (b)
once, the residue was purified by flash chromatography to
yield (R)-2 as the sole product (0.33g, 53%); [a]D20 –114.3 (c
0.49, EtOH, >99% ee). (R)-2-Hydroxy-4-phenyl-3-butenoic
acid [(R)-1] was prepared as described for (S)-1. Yield 61%;
[a]D20 –71.9 (c 0.29, MeOH, ee >99%; lit. [a]D25 –90.6 (c 1.9,
MeOH). (R)-2-Hydroxy-4-phenylbutanoic acid [(R)-3] was
prepared as described for (S)-3. Yield 85%; [a]D20 –8.5 (c
1.0, EtOH, ee >99%); lit. [a]D25 –9.0 (c 1.0, EtOH). Chiral
HPLC analysis as described above showed a single peak at
t = 24.8 min. For spectroscopic and physical data of (R)-1–
3 and (S)-1–3 see: Chadha, A.; Manohar, M. Tetrahedron:
Asymmetry 1995, 6, 651.
Kinetic resolution step (a): to a solution of rac-1 (0.25 g, 1.4
mmol) in diisopropyl ether (25 mL), vinyl acetate (2.5 mL)
and lipase PS-C ‘Amano’ II (0.25 g) were added and the
mixture was shaken for 48 h at 25 °C and 150 rpm. The
enzyme was filtered and dried for reuse; the filtrate was
evaporated to dryness. HPLC analysis showed a conversion
of 50% [Chiralpak AD column, Daicel, heptane–2-PrOH–
CF3COOH, 90:10:0.1; 0.4 mL/min, 18 °C, (S)-2: t = 29.3
min, (R)–1: t = 42.6 min].
Racemization step (b): to a solution of (S)-2 and (R)-1
obtained from step (a) in Hepes buffer (10 mL, 50 mmol, pH
7.5, 10 mM MgCl2), mandelate racemase [EC 5.1.2.2] (1.5
g, prepared as described in ref. 14) rehydrated in 15 mL
Hepes buffer was added. The mixture was shaken for 24 h at
30 °C and 150 rpm. After centrifugation the solution was
acidified to pH 1–2 and extracted with EtOAc, dried
(Na2SO4) and evaporated. HPLC analyses showed complete
racemization of (R)-1; (S)-1: t = 36.2 min. After repeating
step (a) for three times and step (b) for two times, the residue
was purified by flash chromatography to yield (S)-2 as the
sole product (0.21g, 68% overall yield from rac-1); mp 80–
82 °C; mp lit. 82 °C; [a]D20 +100.3 (c 0.47, EtOH, >99% ee);
lit. [a]D25 +108.0 (c 0.36, EtOH).
(17) Although mandelate racemase is not deactivated in various
organic solvents, it is catalytically inactive at low water
activity; see: Pogorevc, M.; Stecher, H.; Faber, K.
Biotechnol. Lett. 2002, 24, 857.
(18) Strauss, U. T.; Kandelbauer, A.; Faber, K. Biotechnol. Lett.
2000, 22, 515.
(S)-2-Hydroxy-4-phenyl-3-butenoic Acid [(S)-1].
A mixture of (S)-2 (110 mg, 0.5 mmol), MeOH (4 mL) and
K2CO3 (0.5 g) was stirred at 0 °C. After 3–4 h the mixture
was acidified with HCl (3 M) to pH 1–2 and then extracted
three times with EtOAc. The organic layer was dried
(Na2SO4), evaporated and the residue was purified by flash
Synlett 2005, No. 12, 1936–1938 © Thieme Stuttgart · New York