Enantio-complementary deracemization of (±)-2-hydroxy-4- phenylbutanoic acid and (±)-3-phenyllactic acid using lipase-catalyzed kinetic resolution combined with biocatalytic racemization

Article abstract of DOI:10.1016/j.tet.2006.01.007

Deracemization of (±)-3-phenyllactic acid (1) and (±)-2-hydroxy-4-phenylbutanoic acid (2) was accomplished by lipase-catalysed kinetic resolution coupled to biocatalytic racemization of the non-reacting substrate enantiomers using Lactobacillus paracasei DSM 20008. Cyclic repetition of this sequence led to a single enantiomeric product from the racemate. Access to both enantiomers was achieved by switching between lipase-catalysed acyl-transfer and ester hydrolysis reactions. Both products constitute important building blocks for virus protease- and ACE-inhibitors, respectively.

Full text of DOI:10.1016/j.tet.2006.01.007

Tetrahedron 62 (2006) 2912–2916
Enantio-complementary deracemization of
(G)-2-hydroxy-4-phenylbutanoic acid and (G)-3-phenyllactic
acid using lipase-catalyzed kinetic resolution combined
with biocatalytic racemization
Barbara Larissegger-Schnell,a Silvia M. Glueck,b Wolfgang Kroutila and Kurt Fabera,
*
aDepartment of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28,
A-8010 Graz, Austria
bResearch Centre Applied Biocatalysis, Graz, Austria
Received 25 November 2005; revised 20 December 2005; accepted 4 January 2006
Available online 20 January 2006
Abstract—Deracemization of (G)-3-phenyllactic acid (1) and (G)-2-hydroxy-4-phenylbutanoic acid (2) was accomplished by lipase-
catalysed kinetic resolution coupled to biocatalytic racemization of the non-reacting substrate enantiomers using Lactobacillus paracasei
DSM 20008. Cyclic repetition of this sequence led to a single enantiomeric product from the racemate. Access to both enantiomers was
achieved by switching between lipase-catalysed acyl-transfer and ester hydrolysis reactions. Both products constitute important building
blocks for virus protease- and ACE-inhibitors, respectively.
q 2006 Elsevier Ltd. All rights reserved.
1. Introduction
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. For the synthesis
of 2 in nonracemic form, numerous strategies based on
(i) racemate resolution via crystallization9 or (ii) kinetic
resolution of a racemate10 or (iii) the asymmetric
transformation of a prochiral precursor11 have been devised.
3-Phenyllactate (1) and derivatives thereof are frequently
used in nonracemic form as components of pharmaceuticals
and natural antibiotic agents.1 They represent an integral
part of bioactive peptides, such as Aeruginosins2 and
Microcin,3 which were shown to be potent protease
inhibitors. The p-fluoro-analog of 1 is a key building
block for the synthesis of AG7088 (Ruprintrivir), a potent
rhinovirus inhibitor currently being tested in clinical trials to
treat the common cold.4 Several approaches to obtain 1
in nonracemic form have been reported.5 The majority of
them is based on racemate resolution6 or the asymmetric
The majority of these routes have one or more weak
points:4,5,11m high cost of reagents, such as chiral transition
metal complexes, insufficient catalyst selectivity or activity,
sensitivity of catalysts in asymmetric hydrogenation, or low
stability of starting materials, such as a-keto acids.
transformation of
precursor.4a,7
a suitable non-chiral synthetic
The most dramatic limitation common for all strategies
relying on kinetic resolution is the maximum theoretical
yield of 50% for a single enantiomer. In order to
overcome this fundamental drawback, two approaches—
summarized under the term ‘deracemization’12—were
recently proposed: (i) dynamic kinetic resolution13 and
(ii) microbial stereo-inversion.14
(R)-2-Hydroxy-4-phenylbutanoic acid (2) is an important
building block for the production of a large variety of
angiotensin converting enzyme (ACE) inhibitors having in
common the (S)-homophenylalanine moiety as the central
pharmacophore unit.8 These agents from the the ‘pril-
family’, such as Enalapril, Lisinopril, Cilapril or Benazepril,
efficiently expand the range of antihypertensives, like
Although both of these methods have the clear merit of a
100% theoretical yield of a single stereoisomeric product,
its absolute configuration is determined by the enantiopre-
ference of the biocatalyst employed. Since mirror-image
enzymes sensu stricto do not exist,15 production of both
Keywords: Enantio-complementary; Deracemization; Lipase; Biocatalytic
racemization; Biocatalysis.
*
Corresponding author. Tel.: C43 316 380 5332; fax: C43 316 380 9840;
e-mail: kurt.faber@uni-graz.at
0040–4020/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.01.007

B. Larissegger-Schnell et al. / Tetrahedron 62 (2006) 2912–2916
2913
enantiomers through biocatalytic deracemization by simple
choice of the ‘matching enantiomer’ of the chiral
(bio)catalyst is virtually impossible.
furnish a mixture of (S)-3 and unreacted (R)-1 in O99% ee
at 50% conversion. Biocatalytic racemization of (R)-1 was
accomplished without separation from the O-acetylated
product 3 using whole cells of Lactobacillus paracasei
DSM 20008 in aqueous buffer. After the mixture of rac-1
and (S)-3 thus obtained was subjected to two subsequent
cycles of lipase-catalysed kinetic resolution/biocatalytic
racemization as described above, (S)-3 was obtained in 56%
overall yield from the racemate as the sole product. Finally,
hydrolysis of the O-acetyl group gave (S)-3-phenyllactic
acid (1) without loss of optical purity in 69% yield and
O99% ee.
In order to circumvent this limitation, we envisaged to apply
lipase-catalyzed ester hydrolysis and ester formation via
acyl transfer to our recently developed deracemization
protocol16 based on the enzymatic racemization of the non-
reacting substrate enantiomer using a racemase.17 Taking
into consideration that ester hydrolysis and esterification
represent reactions in opposite directions, products of
opposite configuration are to be expected. The proof
of principle for this concept was recently verified by us
using a lipase–mandelate racemase two-enzyme process.18
However, the restricted substrate tolerance of mandelate
racemase (EC 5.1.2.2), which is unable to isomerize
3-phenyllactate (1) or 2-hydroxy-4-phenylbutanoate (2)
imposed severe limitations19 and forced us to employ a
suitable b,g-unsaturated synthetic precursor for 2-hydroxy-
4-phenylbutanoate (i.e., 2-hydroxy-4-phenylbut-3-enoate)
instead, which required additional synthetic steps and
thus unfavorably enhanced the overall complexity of the
process. Since this substrate-analog-trick could not be
applied to 3-phenyllactate (1), the latter compound could
not be deracemized by using the above mentioned
mandelate racemase–lipase protocol. In search for a suitable
biocatalyst for the racemization of a wide variety of
saturated aliphatic 2-hydroxycarboxylic acids, we recently
identified whole resting cells of Lactobacillus sp. as a
promising alternative.20
When this deracemization protocol was applied to
2-hydroxy-4-phenylbutanoic acid (rac-2), (S)-2 was
obtained in O99% ee in similar yield.
2.2. (R)-Series (Scheme 1)
It was expected, that lipase-catalysed hydrolysis of rac-O-
acetate esters 3 and 4 would give access to the
corresponding enantiomeric products, that is, (S)-1,2 and
nonreacted a-acetoxycarboxylic acids (R)-3,4. However,
initial attempts were hampered by solubility problems of
the rather polar substrates and insufficient enantioselec-
tivities of various lipases. For instance, Candida rugosa
lipase (Amano AY), various Pseudomonas sp. lipases
(Amano PS-C-I, PS-D), and Candida antarctica lipase A
displayed low enantioselectivity. After some experimen-
tation, we identified two lipases, that is, porcine pancreas
lipase (EC 3.1.1.3) for rac-3 and Candida antarctica
lipase B for rac-4, which showed excellent enantioselec-
tivity. In addition, solubility problems were overcome by
using acetone as co-solvent. Without separation of
materials, the cyclic sequence of acetylation/hydrolysis/
racemization was repeated two times to give (R)-3 and
(R)-4 in 98% and O99% ee, respectively, as the sole
products. Finally, deacylation of the latter compounds
furnished a-hydroxycarboxylic acids (R)-1 and (R)-2 in
74–75% yield. Biocatalysts could be recovered by
filtration and reused for the whole number of cycles
until completion of the process.
In order to demonstrate the general applicability of this
method, we envisaged to develop an enantio-complemen-
tary deracemization protocol as follows (Scheme 1).
The main limitation of this process lies in the neccessity to
switch between aqueous-organic solvent systems, which
furnishes a consecutive sequence of steps rather than a
dynamic process. Attempts to conduct the biocatalytic
racemization in situ in an organic solvent were unsuccess-
ful so far. 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 from the aqueous medium
(e.g., by ion exchange chromatography or continuous
extraction). Detailed analysis showed that this process is
virtually free of yield-limiting side reactions and that
microbial degradation of rac-1,2 during the racemization
step by was negligible.21 The possibility to determine the
stereochemical configuration of the sole product by a
simple switch between lipase-catalyzed acyl-transfer- and
hydrolysis-mode demonstrates the flexibility of this
process.
Scheme 1. Reagents and conditions: (a) Pseudomonas sp. lipase (Amano
PS-C-II), vinyl acetate, i-Pr2O, 25 8C; (b) Lactobacillus paracasei DSM
20008, buffer pH 6.5, 42 8C; (c) Ac2O, pyridine; (d) porcine pancreas lipase
(EC 3.1.1.3, for 3), Candida antarctica lipase B/Novozyme 435 (for 4),
buffer pH 7.5, acetone, 30 8C.
2. Results and discussion
2.1. (S)-Series (Scheme 1)
Kinetic resolution of (G)-1 via Pseudomonas sp. lipase
catalysed acyl-transfer using vinyl acetate as acyl donor
proceeded with excellent enantioselectivities (EO200) to

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3. Conclusion
ethyl acetate, the organic layer was dried (Na2SO4),
evaporated and the residue was purified by flash chromato-
graphy using dichloromethane/methanol (gradient from
0–10% MeOH) as eluent to yield (S)-1 (0.11 g, 69%); mp
122–123 8C; lit.22 mp: 120–121 8C; [a]2D0 K27.55 (c 1.0;
acetone, O99% ee); lit.23 [a]2D5 K27.80 (c 1.13; acetone);
HPLC analysis using the method described above showed
a single peak at TRet 24.53 min.
In summary, both enantiomers of 3-phenyllactic acid (1)
and 2-hydroxy-4-phenylbutanoic acid (2) were obtained as
single stereoisomers in O98% ee from the corresponding
racemates via a stepwise deracemization protocol consisting
of a lipase-kinetic resolution followed by biocatalytic
racemization. Whereas (S)-enantiomers were accessed
through lipase-catalysed acyl-transfer, the (R)-counterparts
were obtained via ester hydrolysis.
4.1.3. (S)-2-Acetoxy-4-phenylbutanoic acid (S)-4. In the
same manner as described above, rac-2 (0.5 g, 2.77 mmol)
was converted to (S)-4 (0.38 g) in a total yield of 62%;
mp: 28–30 8C; [a]2D0 K6.61 (c 0.5; acetone, O99% ee);
lit.24 [a]2D5 K11.0 (c 0.92; EtOH); HPLC analysis showed
a single peak at TRet 19.12 min using a Chiralpak AD
column (Daicel, heptane/2-propanol/CF3COOH 90:10:0.1;
0.4 mL/min, 18 8C).
4. Experimental
4.1. General
Determination of conversion and enantiomeric excess were
determined via HPLC on a chiral stationary phase. HPLC
analyses were carried out on a Jasco HPLC-system (pumps
PU-980, multi-wave-length-detector MD-910, autosampler
AS-950, degasser CMA/260) using a Chiralpak AD column
(column A, Daicel, 0.46 cm!25 cm). Compounds were
purified by flash chromatography on silica gel Merck 60
(230–400 mesh). Melting points were obtained on a
Gallenkamp melting point apparatus MFB-595 in open
capillary tubes, optical rotation values were measured on a
Perkin-Elmer polarimeter 341 at 589 nm (Na-line) in a 1 dm
4.1.4. (S)-2-Hydroxy-4-phenylbutanoic acid (S)-2. In the
same manner as described above, (S)-4 (0.22 g, 1 mmol)
was converted to (S)-2 (0.13 g; 73%); mp 115–117 8C; lit.23
mp: 114 8C; [a]D20 C8.1 (c 1.0; EtOH, O99% ee); lit.23 [a]2D5
C7.5 (c 0.5; EtOH, 84% ee). HPLC analysis using the
method described above showed a single peak at TRet
27.01 min.
cuvette and are given in units of 10K1 deg cm2 gK1
.
4.1.5. (R)-O-Acetyl-3-phenyllactic acid (R)-3. Acylation
step (c): a solution of rac-1 (0.5 g, 3 mmol) in acetic
anhydride (4 mL) and pyridine (0.25 mL) was kept at
0–5 8C. After 6 h the solution was poured into ice-water
(50 mL), which was acidified with HCl (3 M) to pH 1–2 and
extracted three times with ethyl acetate. The combined
organic layers were washed with H2O and brine, dried
(Na2SO4) and evaporated to yield rac-3 (0.50 g, 80%).
4.1.1. (S)-O-Acetyl-3-phenyllactic acid (S)-3. Kinetic
resolution step (a): to a solution of rac-1 (0.5 g, 3 mmol)
in diisopropyl ether (50 mL), vinyl acetate (5 mL) and
Pseudomonas sp. lipase (Amano PS-C-II, 0.5 g) were added
and the mixture was shaken for 48 h at 25 8C and 150 rpm.
The enzyme was filtered, washed and dried for reuse, the
filtrate was evaporated to dryness.
Kinetic resolution step (d): to a solution of rac-3 (0.5 g,
2.6 mmol) in acetone (21 mL) and phosphate buffer (35 mL,
pH 7.5; 50 mmol), lipase from porcine pancreas (EC 3.1.1.3,
5 g, Sigma Type II, crude) was added and the mixture was
shaken for 20 h at 30 8C and 150 rpm. After centrifugation,
acetone was evaporated from the filtrate. The residue was
acidified with HCl (3 M) to pH 1–2, extracted three times
with ethyl acetate, dried (Na2SO4) and evaporated.
Racemization step (b): (S)-3 and (R)-1 obtained from step
(a) were dissolved in bis-Tris-buffer (5 mL, 50 mmol,
pH 6.5, 10K2 M MgCl2) and the pH was adjusted to 6.5.
Then, lyophilized whole cells of Lactobacillus paracasei
DSM 2000820b [2 g, rehydrated in bis-Tris-buffer (12 mL,
50 mmol, pH 6.5, 10K2 M MgCl2) for 30 min] were added
and the mixture was shaken for 48–72 h at 42 8C and
120 rpm until racemization of (R)-1 was complete. After
centrifugation, the solution was acidified to pH 1–2 with
HCl (3 M) and rac-1 and (S)-3 were extracted three times
with ethyl acetate, dried with Na2SO4 and evaporated.
For the racemization step (b) see above.
After performing steps (c) and (d) twice and step (b) once,
the residue obtained by extraction was purified by flash
chromatography (to remove minor impurities emerging
from the cells) using dichloromethane/methanol (gradient
from 0–10% MeOH) to yield (R)-3 as the sole product
(0.25 g, 40%), oil; [a]2D0 C8.86 (c 1.39; acetone, 98% ee).
HPLC analysis using the method described above showed a
single peak at TRet 17.80 min.
After cyclic repetition of step (a) twice and step (b) once, the
residue obtained by extraction was purified by flash
chromatography (to remove minor impurities emerging
from the cells) using dichloromethane/methanol (gradient
from 0–10% MeOH) to yield (S)-3 as the sole product
(0.35 g, 56%, oil); HPLC analysis showed a single peak at
TRet 14.8 min using a Chiralpak AD column (Daicel,
heptane/2-propanol/CF3COOH 90:10:0.1; 0.5 mL/min,
18 8C); [a]2D0 K10.25 (c 1.79; acetone, O99% ee).
4.1.6. (R)-3-Phenyllactic acid (R)-1. In the same manner as
described for (S)-1, (R)-3 (0.15 g, 0.7 mmol) was converted
to (R)-1 (0.09 g, 75%); mp 124–125 8C; lit.22 mp: 120–
121 8C; [a]2D0 C26.95 (c 1.0; acetone, 98% ee); lit.22 [a]2D5
29.8 (c 1.13; acetone). HPLC analysis using the method
described above showed a single peak at TRet 20.38 min.
4.1.2. (S)-3-Phenyllactic acid (S)-1. Hydrolysis step: a
mixture of (S)-3 (0.2 g, 1 mmol), MeOH (8 mL) and K2CO3
(1 g) was stirred at 0 8C for 5 h. After acidification with HCl
(3 M) to pH 1–2, the product was extracted three times with

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2915
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4.1.7. (R)-2-Acetoxy-4-phenylbutanoic acid (R)-4. In the
same manner as described above, rac-2 (0.5 g, 2.8 mmol)
was converted to rac-4 (0.84 g, 87%).
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shaken for 24 h at 30 8C and 150 rpm. The lipase was
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was evaporated from acetone, the residue was acidified with
HCl (3 M) to pH 1–2, extracted three times with ethyl
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Acknowledgements
This study was performed within the Research Centre
Applied Biocatalysis. Financial support by BASF AG
(Ludwigshafen), TIG, FFG, Province of Styria and City of
Graz is gratefully acknowledged. B. Hauer and R. Stu¨rmer
(BASF-AG) are thanked for their valuable contributions.
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