Enzymatic resolution of naproxen

Article abstract of DOI:10.1016/S0957-4166(03)00492-0

Trichosporon sp. (TSL), a newly found strain isolated from a locally fermented cottage cheese has been found to be highly stereoselective in the resolution of (S)-(+)-naproxen (ee >99%, E～500) from the corresponding racemic methyl ester. The process of resolution using whole cells has been scaled up to multi-kg level. Optimization of experimental conditions including downstream processing at 80-100 g/L substrate concentration with >90% recovery has been achieved. Changes in the physical parameters such as the particle size of the substrate play an important role in the resolution kinetics. A new strain of Trichosporon sp. having high cell density in cultivation (>60 g dry cell mass L^-1 in 14-16 h) is found to be sufficiently stable for two years in dry powder form at 5-8°C. The viability of the resolution process has been further improved by the development of a simple racemization process for the enriched (R)-(-)-ester.

Full text of DOI:10.1016/S0957-4166(03)00492-0

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 14 (2003) 2459–2465
Enzymatic resolution of naproxen
Surrinder Koul, Rajinder Parshad, Subhash C. Taneja* and Ghulam N. Qazi
Biotechnology Division, Regional Research Laboratory, Canal Road, Jammu-Tawi 180001, India
Received 29 May 2003; accepted 13 June 2003
Abstract—Trichosporon sp. (TSL), a newly found strain isolated from a locally fermented cottage cheese has been found to be
highly stereoselective in the resolution of (S)-(+)-naproxen (ee >99%, Eꢀ500) from the corresponding racemic methyl ester. The
process of resolution using whole cells has been scaled up to multi-kg level. Optimization of experimental conditions including
downstream processing at 80–100 g/L substrate concentration with >90% recovery has been achieved. Changes in the physical
parameters such as the particle size of the substrate play an important role in the resolution kinetics. A new strain of Trichosporon
sp. having high cell density in cultivation (>60 g dry cell mass L−1 in 14–16 h) is found to be sufficiently stable for two years in
dry powder form at 5–8°C. The viability of the resolution process has been further improved by the development of a simple
racemization process for the enriched (R)-(−)-ester.
© 2003 Elsevier Ltd. All rights reserved.
cades, in a patent, disclosed the preparation of (S)-(+)-
naproxen through enantioselective hydrolysis of alkyl
esters, using a biocatalyst from Bacillus thai and other
microorganisms.14 The resolution process was per-
formed at low substrate concentration and required
longer reaction time (5–6 days). Qu-Meng et al.15 also
reported bio-catalytic resolution of racemic esters at
mmol scale using crude isolated lipase from Candida
cylindraceae (CCL) and Mucor rhizopus. CCL was
shown to hydrolyse (S)-(+)-ester to (S)-(+)-naproxen
(acid) while other enzymes hydrolysed the (R)-(−)-ester
to the corresponding acid. Bianchi et al.16 in their
patent reported a yield of 19% for the (S)-(+)-naproxen
after 50 days of continuous reaction by using immobi-
lized CCL. Water soluble esters were prepared by Mat-
son et al.17 for kinetic resolution studies, by means of a
two stage extractive membrane, to obtain (S)-(+)-
naproxen. In the last few years, an increasing number
of enzymatic kinetic resolution reactions have been
reported for the preparation of (S)-(+)-naproxen
involving trans-esterification as well as hydrolytic
methods18–21 using nitrilases,22,23 isomerases24 and
lipases and esterases.25–41 Polyclonal antibodies have
also been utilized as stereoselective catalysts for the
hydrolysis of esters.42–45 Resolution has also been
reported with animal liver enzymes.46
1. Introduction
Aspirin, a first generation anti-inflammatory drug, dis-
covered 150 years ago, is still in use because of its
multiple biological activities. The introduction of
indomethacin in the 1960s as a second-generation anti-
inflammatory agent was followed by 2-arylacetic and
propanoic acids by Chen et al.1 and Adams et al.2
(known as 2-APA’s or profens) which had compara-
tively lower side effects such as gastrointestinal irrita-
tion, hepato-toxicity, papillary necrosis besides higher
therapeutic index. Parallel to the study on 2-aryl-
propanoic acids,3 another potent NSAID namely (S)-
(+)-6-methoxy-a-methyl-2-naphthaleneacetic
acid
(naproxen) possessing eleven and fifty five times higher
activity compared to phenylbutazone and aspirin
respectively was reported by Harrison et al.4 In terms of
biological activity, the (S)-enantiomer is thirty times
more active than the (R)-enantiomer.5 Naproxen, there-
fore is one of the early chiral molecules developed and
used as a single enantiomer.6 Besides several asymmet-
ric syntheses,7–11 the two most commonly used resolu-
tion methodologies being adopted for the preparation
of (S)-(+)-naproxen from its racemate are (a) resolution
via diasteroisomeric salt formation12,13 and (b) enzy-
matic kinetic resolution. The use of enzymes for their
kinetic resolution began in the 1970s when Gist Bro-
Such a large number of patents and publications
appearing in literature in the last decade and a half in
the area of enzymatic resolution of (S)-(+)-naproxen
underscores the importance of enzymatic processes,
* Corresponding author. Tel.: +91-(191)-2580329; fax: +91-(191)-
2548607/2543829; e-mail: sc taneja@yahoo.co.in
–
0957-4166/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0957-4166(03)00492-0

2460
S. Koul et al. / Tetrahedron: Asymmetry 14 (2003) 2459–2465
which are catalytic, simple, and environmentally
friendly.
showed triglyceride (triacetin) hydrolysing activity of 45
U/g dry cell mass and 75 U/g solid for lyophilized cell
free extract. After detailed experimentation an optimum
enzyme to substrate ratio of 85 U of enzyme per gram
of the substrate was established. Since we did not have
a prior knowledge of the effectiveness of TSL with
respect to various racemic alkyl esters of naproxen, for
that reason several esters of ( )-6-methoxy-a-methyl-2-
naphthaleneacetic acid were prepared to investigate its
behaviour.
2. Results and discussion
As a part of our study into the kinetic resolution of
drugs, their intermediates and chiral auxiliaries47–49
using indigenous microbial enzymes and also commer-
cial ones, we herein report a highly stereoselective
process for the preparation of (S)-(+)-naproxen using
whole cells of Trichosporon sp.(TSL), a newly found
strain isolated from a locally fermented cottage cheese.
The culture has been deposited in Deutsche Sammlung
von Mickroorganismen und Zellekulturen GmbH
Braunschweig, Germany (DSMZ11829) and its
hydrolytic activity has also been patented.50,51 The
effectiveness of the new yeast strain lies in its very high
selectivity for the (S)-enantiomer in hydrolyzing the
alkyl esters, particularly the methyl ester of ( )-6-
methoxy-a-methyl-2-naphthaleneacetic acid (Scheme 1).
Another advantage is its cultivation to high cell density
(>60 g dry cell mass L−1 in 14–16 h fermentation time).
Table 1 summarizes the results of the kinetic resolution
of various racemic alkyl esters using whole cell prepara-
tion of TSL. For the methyl ester the enzyme displayed
a very high selectivity (ee >99%, E ꢀ500) and a fairly
good rate of hydrolysis to produce (S)-(+)-naproxen.
However, the rate of hydrolysis as well as the selectivity
were influenced by the size of alkyl group of the ester
moiety. The lowest selectivity (ee ꢀ20%) was observed
when n-heptyl ester was used as a substrate. The
chloroethyl ester group (COOCH2CH2Cl) on the other
hand proved to be the least accepted substrate even
though a selectivity of 80% was recorded.
The isolated organism was identified as a strain of
Trichosporon sp. belonging to an imperfect yeast of the
family Cryptococaceae and sub-family Trichosporoidea.
The resting cells, as well as the ester hydrolase isolated
from it, were found to have stable biochemical charac-
teristics at temperatures in the range of 25–35°C and
pH 6–9. The isolated enzyme in its lyophilized state in
crude or cell free form retained its activity when stored
at 4°C for 2 years. Under optimized fermentation con-
ditions >60 g dry cell mass/L broth was obtained. It
2.1. Optimisation of reaction conditions
Since TSL showed both a high selectivity as well as a
high conversion rate for the methyl ester of racemic
naproxen, it was selected as the substrate for further
optimization studies such as the influence of particle
size of the substrate, substrate concentration, reaction
temperature and pH as well as effect of co-solvents to
improve overall time–space yields.
Scheme 1.
Table 1. Hydrolysis of racemic esters of ( )-6-methoxy-a-methyl-2-naphthaleneacetic acid using TSL (whole cell pellet)a
Entry (R)
Reaction time (h)
Conversion (%)
Product ee (%)
Enantioselectivity factor
CH3
C2H5
i-C3H7
CH2CH2Cl
CH2CH2OCH3
n-C4H9
t-C4H9
n-C7H15
CH2C6H5
48
48
96
96
96
96
96
96
96
45
44
45
11
28
20
34
15
25
>99
97
96
80
73
70
80
20
94
500
164
118
10
8
7
13
2
44
a Reaction conditions: pH 8.0 (0.1 M phosphate buffer) temp. 28°C, particle size 300–150 mm (50–100 mesh), conc. of substrate: 40 g/L, enzyme
85 U/g of substrate, conversions and enantiomeric purities were determined by chiral HPLC analysis. Racemic acid and most of the racemic
esters were resolved on a single chromatogram using a chiral HPLC Lichro Cart 250-4 (S,S)-Whelk-01 (5 mm) column.

S. Koul et al. / Tetrahedron: Asymmetry 14 (2003) 2459–2465
2461
2.2. Influence of the particle size of the substrate on
the reaction kinetics
The particle size of the substrate proved to be very
important in the overall kinetics of the reaction. The
racemic methyl ester, being highly insoluble in an
aqueous phase, was used in the form of a fine powder.
It was observed that commercial samples of racemic
methyl ester had wide variations in their particle size
from 650 to 150 mm (20–100 mesh). As a result the rate
of hydrolysis also varied from batch to batch. There-
fore the effect of the substrate particle size reduction
from 300 mm (50 mesh) to 150 mm (100 mesh) and
further to 75 mm (200 mesh) was studied. As depicted in
Figure 1, a direct relationship between the particle size
of the substrate and the rate of the hydrolysis was
observed which may be attributed to improved amalga-
mation and interactions between substrate and the
enzyme.
Figure 2. Effect of substrate concentration on the rate of
product formation.
Table 2. Effect of temperature variation on enantioselec-
2.3. Influence of substrate concentration on reaction
kinetics
tivity and hydrolytic activity of TSLa
S. No.
Temp. (°C)
Conversion (%) Product ee (%)
Keeping all other reaction parameters constant, the
substrate concentration was varied from 40 g/L to 140
g/L. The average rate of product formation recorded
was generally above or at 0.8 g/L/h for the first 24 h
irrespective of the substrate concentration up to ꢀ100
g/L (Fig. 2). However, the influence of a higher sub-
strate concentration beyond 100 g/L became apparent
when a significant drop in productivity was observed.
This can be attributed to a combination of various
factors such as higher viscosity and to a lesser extent
product inhibition.
1
2
3
4
5
6
7
25
28
30
32
35
42
45
35
45
45
45
45
47
43
99
99
99
99
97
80
60
a Conc. of the substrate 50 g/L, particle size 200 mesh, pH 8.0, time
30 h enzyme 85 U/g of the substrate.
Marginally better reactor rheology was created when
cell free extract in place of whole cell culture, was used.
However the overall reaction rate, did not show any
significant improvement (data not shown). Periodic
addition of substrate and removal of the product
through on line membrane filtration only marginally
improved the reaction rate. It is apparent from the
above studies that 80–100 g/L is the optimal concentra-
tion for TSL catalysed resolution.
2.4. Influence of temperature
Though live cells of TSL tolerated a wider range of
cultivation temperature (25–35°C) and were able to
withstand exposure to 45°C for 3 h, the optimum
activity of the enzyme in terms of selectivity and con-
version remained in a narrow range of 28–32°C (Table
2).
2.5. Influence of pH
Several buffers in the concentration range of 0.1–1.5 M
were used as media but showed no significant improve-
ment in the kinetics of the reaction. However at pH 7–8
an optimal reaction rate and selectivity was recorded.
As can be seen from the Table 3, pH values below 7 are
unfavorable in terms of both rate of hydrolysis and
selectivity. An increase beyond pH 8 marginally
improved the rate of the reaction but at the cost of
selectivity. The marginal loss of ee at pH 9.0 can be
attributed to competitive non-enzymatic hydrolysis in
basic conditions as observed in a separate set of reac-
tions carried out in the absence of enzyme where 3%
hydrolysis with 0% ee was observed, thus signifying a
retention of high selectivity by TSL even at higher pH.
Figure 1. Influence of particle sizes on the rate of reaction at
pH 8.0, temp 30°C, substrate concentration 50 g/L, enzyme
85 U/g of substrate.

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S. Koul et al. / Tetrahedron: Asymmetry 14 (2003) 2459–2465
Table 3. Influence of pH on the hydrolytic activity and
3. Experimental
enantioselectivitya
3.1. General
S. No.
pH
Conversion (%)
Product ee (%)
All the reagents used in the study were of analytical
grade. The authenticity of racemic esters prepared dur-
ing the study was confirmed by spectroscopic analysis
including NMR, LC/MS and of the resolved enan-
tiomers by optical rotation and chiral chromatographic
methods. Melting points recorded by capillary method
are uncorrected. The optical rotations were measured
on a Perkin–Elmer 241 polarimeter with chloroform as
the solvent. HPLC analyses were carried out on a
Lichro Cart 250-4 (S,S)-Whelk-01 (5 mm) column with
1
2
3
4
5
6
7
8
5.2–6.2
6.0
6.7
7.0–7.2
7.8
8.0
16
27
34
43
45
44
45
48
90
90
98
>99
>99
99
8.5
9.0
96
94
a Conc. of the substrate 50 g/L, particle size 200 mesh, time 30 h,
enzyme 85 U/g of the substrate.
a
mobile phase of hexane:isopropanol:acetic acid
(90:10:0.5%) and a flow rate of 1.2 mL/min using a
diode array detector (254 nm). Reactions were routinely
monitored on 0.25 mm silica gel plates (E. Merck)
using UV light for detection of the spots. The commer-
cial lipases like Porcine pancreatic lipase (PPL, enzyme
activity 0.23 U/mg solid with triacetin), Candida rugosa
lipase (CRL enzyme activity 0.190 U/mg solid with
triacetin) and Candida cylindraceae lipase (CCL,
enzyme activity 0.33 U/mg solid with triacetin) were
purchased from M/s Sigma Chemical, USA. Pseu-
domonas sp. lipase (PSL, enzyme activity 0.25 U/mg
solid with triacetin) was obtained as free gift form M/s.
Amano, Japan. The procured lipases were used as such
without purification. Metrohm pH Stat-718 was used
for monitoring the pH. Standard sieves of 50, 100 and
200 mesh were employed for obtaining the required
particle size of the substrate. For cell disintegration, a
Kinnomed Konsolt-AB (Sweden) disintegrator was
used.
2.6. Influence of co-solvents
The use of co-solvents was also studied with methyl
ester as the substrate. However, both non-polar and
polar solvents (up to 10% v/v) showed significant
decreases in reaction rates as well as in enantioselectiv-
ity (data not included).
2.7. Comparative hydrolytic activity of TSL vis-a`-vis
selected commercially available enzymes
The data of the hydrolytic activity and selectivity of
four commercially available enzymes namely PPL,
CCL, CRL, and PSL vis-a`-vis TSL is presented in
Table 4. While the PPL and PSL lipases failed to
hydrolyse the methyl ester of ( )-6-methoxy-a-methyl-
2-naphthaleneacetic acid under the experimental condi-
tions, CRL and CCL displayed comparable
enantioselectivity with respect to TSL. The latter how-
ever, showed distinct superiority with a far higher rate
of hydrolysis.
3.2. Cultivation of Trichosporon sp. (TSL)
A culture medium, comprising of 1.5% glucose, 0.05%
potassium dihydrogen phosphate (KH2PO4), 1.0% corn
steep liquor and 0.3% of urea (pH 6.8 before steriliza-
tion and pH 6.5 after sterilization) was prepared and
dispensed in shake flasks (200 mL each) and in a 10 L
stainless steel fermenter (SS 304S, working volume 7.5
L) and autoclaved. The pre-culture of the strain Tri-
chosporon sp. (DSMZ 11829) was prepared in the shake
flask by inoculating a loopful of the culture prepared
on solid agar medium and incubating the flask at 30°C
on a rotary shaker. The 24 h old pre-culture thus
produced was inoculated (5% v/v) into the culture
medium and fermentation carried out at 500 rpm, 0.5
volume/volume/min aeration rate and kept at a con-
stant temperature of 30°C for 14–18 h. The culture was
thereafter centrifuged to collect the yeast cells. The cell
pellet was washed twice with distilled water and the wet
cell mass of TSL thus obtained was lyophilized at
−40°C to obtain a dry powder that was found stable for
2 years when stored at 5–8°C.
2.8. Racemisation
Experiments were also carried out to racemise the
enriched methyl ester of (R)-(−)-6-methoxy-a-methyl-2-
naphthaleneacetic acid for its reutilization. Both
sodium metal and sodium methoxide were found effec-
tive in completely racemising the ester (98% yield), the
details are given in Section 3.
Table 4. Hydrolytic behaviour of TSL vis-a`-vis selected
commercial enzymes with ( )-6-methoxy-a-methyl-2-naph-
thaleneacetic acid methyl estera
Enzyme
Reaction
time (h)
Conversion
(%)
ee (%)
Configuration
PSL
PPL
CCL
CRL
TSL
60
60
60
60
24
–
–
14.3
7
45
–
–
–
–
S
S
S
99
99
>99
3.3. Preparation of cell free extract and crude enzyme
a Substrate conc. 40 g/L, pH 8.0 (0.1 M phosphate buffer), temp.
32°C, particle size ꢀ200 mesh, enzyme 85 U/g of substrate, for
commercial enzymes ratio of enzyme (dry powder):substrate 1:3.
The wet cell mass prepared as described above was
either sonicated (at 40% w/v solid suspension) for 5 min
or milled with glass beads of size ƒ 0.5 mm for 10 min

S. Koul et al. / Tetrahedron: Asymmetry 14 (2003) 2459–2465
2463
at 0–4°C. The sonicated or glass milled mixture was
centrifuged at 10,000g for 10 min at 0–4°C. For the
larger quantities (>500 g cell pellet) cell free extracts
were prepared in a continuous glass mill using a cell
disintegrator52 (volume 150 mL, glass beads, size ƒ 0.1
mm). A yeast cell suspension of 40% (w/v) was pre-
pared in a phosphate buffer 0.1 M at pH 7.0 keeping a
flow rate of 450 mL/h at 0–4°C. The glass-milled mix-
ture was centrifuged at 10,000g for 10 min at 4°C. The
cell free extract thus obtained was lyophilized at −40°C
with the resulting solid showing a hydrolysing activity
of 0.075 and 0.145 U/mg, respectively, using triacetin
and p-nitrophenyl acetate as assay substrates (one unit
of enzyme corresponds to liberation of 1 mmol of
organic acid from triacetin or hydrolysis of 1 mmol of
p-nitrophenyl acetate in 1 min). Protein estimation was
made according to Lowry’s method using BSA as the
reference protein.53
HPLC. The reaction was terminated after 48 h (conver-
sion ꢀ41%) by adding dilute HCl. The acidified con-
tents (pH 3) were extracted with ethyl acetate (5×600
mL). The combined organic layer was then extracted
with 5% sodium hydroxide solution (4×125 mL). The
aqueous alkaline portion was washed with ethyl ace-
tate:n-hexane (1:9, 3×100 mL) to remove the traces of
the enriched (R)-ester and thereafter acidified with
dilute hydrochloric acid. The resulting white precipitate
of the (S)-(+)-acid was filtered, washed with distilled
water and dried at 50°C to give (S)-(+)-naproxen as a
white powder (53.8 g) [h]D=+65.6 (c 1, CHCl3) ee
ꢀ99% (chiral HPLC Lichro Cart 250-4 (S,S)-Whelk-01
(5 mm) column). The organic layer and ethyl acetate:n-
hexane washings were pooled, washed with distilled
water (3×50 mL), dried over sodium sulfate and con-
centrated to furnish enriched (R)-ester (86.5 g).
3.5.2. Method B (suitable for multi-kg scale). The methyl
ester of ( )-6-methoxy-a-methylnaphthaleneacetic acid
(5.0 kg, 200 mesh), was added to an aqueous suspen-
sion, comprising of lyophilized cells of TSL (9.0 kg,
ꢀ85 U/g of substrate) in distilled water (44 L) and the
contents stirred at 600 rpm at a temperature 30 1°C
while maintaining the pH at 8.0 (substrate concentra-
tion approx. 80 g/L) with a pH stat using a 2 M NaOH
solution. Aliquots were regularly drawn to monitor the
progress of the reaction on a chiral HPLC. The reac-
tion was terminated after 48 h (43% conversion) with
the pH adjusted to 8.5, the contents centrifuged and the
cell mass washed thoroughly with 5% NaOH solution
(6 L) and re-centrifuged. This process was repeated
twice and the combined aqueous solution passed
through a filter membrane followed by extraction with
n-hexane:ethyl acetate (9:1, 2×3.5 L). The resolved (S)-
acid from the aqueous solution was precipitated by
adding 15% sulfuric acid (6 L), filtered, washed with
distilled water and dried in an air oven at 50°C to
furnish the (S)-naproxen (1674.5 g) mp 152–52.5°C
[h]D=+66.6 (c 1, CHCl3,), ee >99% (HPLC). The
organic solvent washings containing small amounts of
the (R)-ester were concentrated (40.5 g) and pooled
with the cell mass left after centrifugation. The cell
mass holding the enriched (R)-ester, with some small
amounts of the (S)-acid left unextracted after the
removal of bulk acid, was then partially dried at 50°C
(air oven). The dried mass (approx. 12 kg) was then
placed in an extractor (Dean Stork type) containing
toluene (40 L). Boiling and distillation of toluene (5 L)
removed traces of water from the cell mass and the
contents were refluxed at 110°C to dissolve the ester
and the acid adsorbed on it. The organic layer was
drained out and the solid mass extracted twice with
boiling toluene (2×20 L). After cooling, the combined
toluene extracts were extracted with 5% sodium
hydroxide solution (6 L) and the alkaline aqueous
portion washed with ethyl acetate:n-hexane (1:9, 1.5 L)
and then acidified to pH 4.0 with dilute hydrochloric
acid. The precipitate was filtered, washed with distilled
water and dried to give a white solid of (S)-(+)-
naproxen (164 g). The combined toluene layer [esti-
mated to contain 2.71 kg of enriched (R)-ester] was
washed with distilled water (2×2 L), dried over anhy-
3.4. Preparation of alkyl esters
3.4.1. Method A. In a typical procedure, a solution of
( )-6-methoxy-a-methyl-2-naphthaleneacetic acid (150
g, 650 mmol) in methanol (520 mL) was placed in a
three-necked round bottom flask fitted with a reflux
condenser. Concentrated sulfuric acid (10 mL) was
added with constant stirring while maintaining the tem-
perature at 60–70°C until the addition was complete (70
min). The contents were then heated on a water bath
for 20 min, cooled and solid filtered. The filtrate was
concentrated to one fourth of its original volume and
cooled again. The two crops of the solid were pooled
together, washed thoroughly with water and dried to
give the racemic methyl ester (146 g, yield 92%) mp
75°C.
3.4.2. Method B. In an alternative process the racemic
ester was prepared by treating the dichloromethane
solution of the acid chloride of ( )-6-methoxy-a-
methyl-2-naphthalene acetic acid (160 mmol) with dry
n-butanol (200 mmol). The resulting ester after normal
work up was purified by column chromatography on
silica gel using EtOAc:CH2Cl2 (9:1) as the eluant to
furnish the corresponding butyl ester (40.7 g, yield 93%)
mp 52–53°C.
3.5. Process for the kinetic resolution of the methyl
ester of ( )-6-methoxy-a-methyl-2-naphthalene acetic
acid
Two different methodologies depending upon the scale
of the reaction were adopted for the downstream pro-
cess [separation of (S)-(+)-naproxen from enriched (R)-
(−)-ester after the completion of the reaction].
3.5.1. Method A (suitable up to 500 g scale). Tri-
chosporon sp. (TSL, 280 g lyophilised powder, ꢀ85 U/g
of substrate) was added to a suspension of racemic
methyl ester (150 g, 200 mesh, substrate conc. 90 g/L)
in a sodium phosphate buffer (0.1 M, pH 8.0, 1650
mL). The contents were stirred at 30°C and the pH
maintained by a pH stat using 1 M NaOH solution.
Periodically aliquots were drawn and analysed on chiral

2464
S. Koul et al. / Tetrahedron: Asymmetry 14 (2003) 2459–2465
drous calcium chloride and the solution used directly
for racemisation reaction.
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3.6. Racemisation of (R)-enriched ester
A toluene solution comprising of enriched (R)-(−)-
methyl ester of 6-methoxy-a-methyl-2-naphthalene ace-
tic acid (approx. 2.7 kg, ꢀ11 mol) was initially
concentrated (5 L toluene distilled over) to remove the
last traces of water. To the remaining solution was
added either freshly cut sodium metal (40 g) or freshly
prepared sodium methoxide (prepared from 40 g
sodium metal and dry methanol). The contents were
then refluxed and reaction monitored by chiral HPLC.
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cooled, neutralized with concentrated sulfuric acid (50
mL) and washed with water until pH 7 was attained.
The organic layer was dried over anhydrous sodium
sulfate and concentrated at reduced pressure to furnish
the racemised ester (2.6 kg, 98% yield). The racemic
ester thus obtained was decolourised by activated car-
bon prior to micronisation and reuse for kinetic
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naproxen. The yeast organism, or the enzyme derived
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strate ratio of 85 U of enzyme per gram of the substrate
was optimal with the rate of hydrolysis not much
influenced up to a substrate concentration of 100 g/L.
Temperature in the range of 28–32°C and pH between
7 and 8 were found to be optimum for the best time
space yield and enantioselectivity. The use of a buffer
or an aqueous solution had no direct effect on the rate
of the hydrolysis or enantioselectivity. The use of co-
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