An efficient preparative scale resolution of 3-phenylbutyric acid by lipase from Burkholderia cepacia (Chirazyme L1)

Article abstract of DOI:10.1016/S0957-4166(98)00055-X

Lipase from Burkholderia cepacia (Chirazyme L1) catalysed the highly enantioselective hydrolysis of racemic methyl 3-phenylbutyrate to afford (R)- (-)-methyl 3-phenylbutyrate of >98% ee (E>50). The resolution was performed at 150 g scale yielding 68.7 g of (R)-(-)-methyl 3-phenylbutyrate (>98% ee, 92% yield on enantiomer) and (S)-(+)-3-phenylbutyric acid of 89% ee.

Full text of DOI:10.1016/S0957-4166(98)00055-X

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 9 (1998) 1191–1195
An efficient preparative scale resolution of 3-phenylbutyric acid
by lipase from Burkholderia cepacia (Chirazyme L1)
Govindarajulu Varadharaj,a Keith Hazell b and Christopher D. Reeve b,∗
aFaculty of Applied Sciences, University of the West of England, Bristol, BS16 1QY, UK
bZeneca LifeScience Molecules, PO Box 2, Belasis Avenue, Billingham, Cleveland, TS23 1YN, UK
Received 26 January 1998; accepted 5 February 1998
Abstract
Lipase from Burkholderia cepacia (Chirazyme L1) catalysed the highly enantioselective hydrolysis of racemic
methyl 3-phenylbutyrate to afford (R)-(−)-methyl 3-phenylbutyrate of >98% ee (E>50). The resolution was
performed at 150 g scale yielding 68.7 g of (R)-(−)-methyl 3-phenylbutyrate (>98% ee, 92% yield on enantiomer)
and (S)-(+)-3-phenylbutyric acid of 89% ee. © 1998 Elsevier Science Ltd. All rights reserved.
1. Introduction
β-Aryl carboxylic acids are valuable synthetic intermediates for the preparation of a range of com-
pounds of biological interest. 3-Phenylbutyric acid was used in the synthesis of ar-juvabione, the aryl ana-
logue of juvabione, a sesquiterpene possessing juvenile hormone activity.1 (−)-Malyngolide, a δ-lactone
antibiotic of algal origin, was prepared in enantiomerically pure form from (R)-(−)-3-phenylbutyric
acid.2 All four stereoisomers of β-methylphenylalanine have been prepared in high enantiomeric purity
from (R)- and (S)-3-phenylbutyric acid.3 β-Aryl carboxylic acids have also been used for the preparation
of chiral 3-alkylindanones and their corresponding dihydrocoumarins.4
The preparation of enantiomerically enriched 3-phenylbutyric acid has been achieved using a number
of approaches. The asymmetric hydrogenation of β-methylcinnamic acid using BINAP–ruthenium(II)
complexes afforded (S)-(+)-3-phenylbutyric acid in 85% ee.5 Alternatively, the asymmetric conjugate ad-
dition of organometallic reagents to chiral α,β-unsaturated amides,4,6 esters7 and vinyl sulfoximines8 has
yielded both enantiomers of 3-phenylbutyric acid in high enantiomeric excess. (S)-(+)-3-Phenylbutyric
acid has also been obtained by enantioselective hydrolysis of racemic 3-phenylbutyronitrile by Mycobac-
terium strain A2777.9
∗
Corresponding author.
0957-4166/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00055-X

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G. Varadharaj / Tetrahedron: Asymmetry 9 (1998) 1191–1195
Here we describe the development of an efficient preparative scale resolution of 3-phenylbutyric acid
1 by lipase catalysed enantioselective hydrolysis of the corresponding methyl ester 2 (Scheme 1).
Scheme 1. Reagents: (i) methanol, cat. H2SO4, reflux, (ii) Burkholderia cepacia lipase (Chirazyme L1), 5 mM sodium phosphate
buffer, pH 7.6, 25°C
2. Results and discussion
To identify enzymes catalysing the hydrolysis of (rac)-2 a broad screen of over 60 commercially
available lipases, esterases and proteases was performed using a simple protocol. Twenty of the lipases
and esterases catalysed the hydrolysis of the substrate to varying extents, however none of the proteases
were found to hydrolyse the substrate. Enzymes showing an appreciable degree of hydrolysis were
investigated further in an autotitrator. Reactions were stopped at close to 50% conversion at which point
unhydrolysed 2 was isolated. The enantioselectivity of each enzyme was calculated from the extent of
conversion obtained by titration and the enantiomeric purity of unreacted 2 determined by chiral phase
HPLC (Table 1).
Lipases from Burkholderia cepacia, Pseudomonas sp., Pseudomonas fluorescens and Chromobac-
terium viscosum all exhibited high enough enantioselectivity to be of practical use for the resolution of
(rac)-2. However from a consideration of reaction rates and enzyme cost the lipase from Burkholderia
cepacia was selected as the most appropriate for preparative scale use. Further developments focused
on establishing the effect of enzyme concentration, substrate concentration and temperature on the
performance of the resolution reaction.
In view of the fact that enzyme cost can be an important factor in the overall economics of a resolution
process the effect of reducing the concentration of enzyme was studied. A series of resolution reactions
were performed at a fixed (rac)-2 concentration of 6.6 g L−1 and enzyme concentrations in the range 1.33
Table 1
Enantioselectivity of lipase and esterase catalysed hydrolysis of (Rac)-2

G. Varadharaj / Tetrahedron: Asymmetry 9 (1998) 1191–1195
1193
g L−1 to 0.166 g L−1. A resolution time of approximately 22 h was observed at an enzyme concentration
of 0.166 g L−1, giving a substrate to catalyst ratio of 40:1 and an 8-fold reduction in enzyme usage. The
efficiency of the resolution was further improved by increasing the concentration of (rac)-2 from 6.6 to
50 g L−1, a 7.5-fold increase in volume productivity. At a 40:1 substrate to catalyst ratio the resolution
time was 21 h and the high degree of enantioselectivity was preserved.
In order to counteract the inevitable increase in reaction time resulting from a decrease in enzyme
concentration the effect of temperature on the rate and enantioselectivity of the reaction was studied
(Table 2).
Table 2
Effect of temperature on the rate and enantioselectivity of Burkholderia cepacia lipase catalysed
resolution of (Rac)-2
A 5°C rise in reaction temperature from 25 to 30°C produced an increase in initial rate of hydrolysis
approximately in line with that expected from Q10≈2. Although the initial rate of hydrolysis was seen to
increase this was not reflected in a reduction in the overall resolution time. Indeed, a trend towards longer
total resolution times at increasing temperature was apparent suggesting that the enzyme is not stable to
elevated temperatures. The data obtained at 35°C further supports this view. Moreover, the increase in
temperature led to a progressive decrease in the enantioselectivity of the resolution reflected in a decline
in enantiomeric purity of unreacted 2 at equivalent extents of conversion. This can be rationalised on the
basis of increased conformational flexibility of the enzyme at elevated temperatures allowing binding and
hydrolysis of (R)-2 by the enzyme.
The defined reaction conditions were applied on a preparative scale (150 g (rac)-2) allowing the
isolation of 68.7 g of (R)-2 (92% isolated yield on enantiomer) of >98% ee (Figs 1 and 2) and >98%
chemical purity. The hydrolysis product, (S)-1 was not quantitatively recovered from the resolution
reaction however, isolation of a small sample followed by conversion to its methyl ester (S)-2, indicated
it to be of 89% ee.
Fig. 1. Extent of hydrolysis of (Rac)-2 by Burkholderia cepacia lipase as a function of time

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G. Varadharaj / Tetrahedron: Asymmetry 9 (1998) 1191–1195
Fig. 2. Chiral phase HPLC analysis of (Rac)-2 and (R)-2 obtained by lipase catalysed resolution
In conclusion, we have developed a simple and efficient resolution of 3-phenylbutyric acid using a
commercially available lipase. The process has been operated on a preparative scale allowing access to
both enantiomers in high enantiomeric purity. The high degree of enantioselectivity observed (E>50) in-
dicates that the enantiomeric purity of the (S)-enantiomer could be significantly improved by terminating
the reaction at approximately 45% conversion.
3. Experimental
1H NMR spectra were recorded on a Jeol FX90Q spectrometer in CDCl3. Optical rotations were
measured on a POLAAR 20 automatic polarimeter (Optical Activity Limited). GC was performed on
a Perkin–Elmer 8500GS system equipped with DB5 column (30 m×0.328 mm I.D. capillary column,
J&W Scientific) using a helium carrier gas at 8 psi (split volume 53 ml min−1) and a temperature
gradient of 100°C rising to 200°C at a rate of 20°C min−1. Retention times of 1 and 2 were 8.04 and
7.32 minutes respectively. Chiral phase HPLC was performed on a Hewlett Packard HP1050 system
equipped with a Chiralcel OJ column (25 cm×4.6 mm I.D. analytical column and 5 cm×4.6 mm I.D.
guard column, Daicel Industries Limited) eluted with hexane:2-propanol (90:10) at a flow rate of 1 ml
min−1. Compounds were detected by UV absorbance at 260 nm. Retention times of (R)- and (S)-2 were
9.55 and 11.54 minutes respectively. TLC was performed on precoated silica gel plates (Merck) eluted
with hexane:ethyl acetate (90:10). Rf values of 1 and 2 were 0.19 and 0.47 respectively.
3.1. (rac)-Methyl 3-phenylbutyrate (2)
(rac)-3-Phenylbutyric acid (180 g, 1.097 mol) was heated at reflux in methanol (500 ml) containing
catalytic H2SO4. On completion of the reaction the solution was cooled and the methanol evaporated.
The residue was taken up in heptane (250 ml) and washed successively with 10% sodium bicarbonate
and water. The organic phase was dried over anhydrous sodium sulphate and evaporated to afford (rac)-2
as a pale yellow oil (182.4 g, 93%) of >98% chemical purity (GC).
3.2. Enzyme screening
Screening reactions were performed in 5 ml of 50 mM sodium phosphate buffer, pH 7.6 containing
(rac)-2 (50mg) and enzyme (10 mg). Reactions were incubated at ambient temperature with stirring for
18 h. Hydrolysis of the substrate was detected by TLC.

G. Varadharaj / Tetrahedron: Asymmetry 9 (1998) 1191–1195
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3.3. Titration studies
Reaction profiles for the hydrolysis of (rac)-2 were obtained using a DL-21 autotitrator (Mettler
Toledo) using a pH stat function. Reactions were performed in 30 ml of 5 mM sodium phosphate buffer,
pH 7.6 containing (rac)-2 (0.2 g) at 25°C. Hydrolysis was initiated by the addition of enzyme (40 mg).
The pH was maintained at 7.6 by automatic titration of 0.1 M NaOH solution. Reactions were stopped at
approximately 50% hydrolysis and unreacted 2 extracted with ethyl acetate for analysis of enantiomeric
purity.
3.4. Preparative scale resolution of (rac)-2 by Burkholderia cepacia lipase
Compound (rac)-2 (150 g, 0.843 mol) and 5 mM sodium phosphate buffer, pH 7.6 (2.8 L) were placed
in a temperature controlled reactor at 25°C equipped with pH control. The reaction was initiated by
the addition of 3.75 g of enzyme dissolved in 200 ml of the above buffer. On completion unreacted 2
was isolated by extraction with 2×2 L of heptane. The pooled organic extract was dried over anhydrous
sodium sulphate and evaporated to afford a slightly turbid pale yellow oil. The oil was taken up in 100
ml of heptane and passed through a bed of basic alumina to remove proteinaceous material followed by
filtration through a 0.2 µ PTFE membrane. Removal of the solvent afforded (R)-2 (68.7 g, 92% isolated
23
yield on enantiomer) as a clear pale yellow oil in >98% ee, >98% chemical purity (GC) and [α]D −41
(c=0.988, toluene), lit11 [α]D −44 (c=1, benzene).
20
Acidification of a sample of the aqueous phase with H2SO4 followed by extraction with ethyl acetate
allowed the isolation of a sample of (S)-1. The sample was methylated following the general procedure
23
described above to afford (S)-2 as a pale yellow oil in 89% ee, >98% chemical purity (GC) and [α]D
+38.5 (c=1, toluene).
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