Biocatalyzed irreversible esterification in the preparation of S-naproxen

Article abstract of DOI:10.1016/j.molcatb.2010.01.014

Dimethyl carbonate was used as alcohol donor in the esterification of naproxen in the presence of immobilised lipase B from Candida antarctica (Novozym 435). The conjugation of hydrolysis of dimethyl carbonate and esterification of acid, created irreversible operative conditions so permitting the recovery of S-naproxen in high ee (>98%). The adopted procedure has a general use.

Full text of DOI:10.1016/j.molcatb.2010.01.014

Journal of Molecular Catalysis B: Enzymatic 65 (2010) 49–51
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Biocatalyzed irreversible esterification in the preparation of S-naproxen
R. Morrone∗, N. D’Antona, D. Lambusta, G. Nicolosi
CNR – Istituto di Chimica Biomolecolare, Via Paolo Gaifami 18, 95126 Catania, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 25 January 2010
Dimethyl carbonate was used as alcohol donor in the esterification of naproxen in the presence of
immobilised lipase B from Candida antarctica (Novozym 435). The conjugation of hydrolysis of dimethyl
carbonate and esterification of acid, created irreversible operative conditions so permitting the recovery
of S-naproxen in high ee (>98%). The adopted procedure has a general use.
Keywords:
Naproxen
NSAID
© 2010 Elsevier B.V. All rights reserved.
Profen
Lipase
Dimethyl carbonate
Irreversible esterification
1. Introduction
a low boiling point solvent or a volatile nucleophile (methanol) are
used. The removal of water by the use of salt hydrates [17,18] has
Profen drugs are 2-phenylpropionic acid derivatives and con-
stitute a structural class of NSAIDs widely used for the treatment
of inflammatory diseases, such as rheumatoid arthritis and
osteoarthritis. The anti-inflammatory property of profens resides
primarily in the S enantiomer [1–3] and, in order to obtain
the eutomer, numerous strategies have been developed, such as
asymmetric synthesis [4,5], biocatalytic kinetic resolution [6,7],
preferential crystallization [8] and chiral chromatography separa-
tions of racemates [9].
no real application since, due to the heterogenic operative condi-
tions, the damage to the enzyme occurs during the shaking of the
reaction mixture and moreover, the separation of water saturated
salts is practically impossible. In the past we have envisaged, in
an intrinsically chemical action, the possible procedure to over-
come the problem, and we have indicated a valid alcohol generator
system based on the reaction of water with alkyl orthoformates,
although two molecules of unreacted alcohol are wasted for each
ester molecule produced [13,19].
Among these procedures, the enantioselective catalysis by
lipases has furnished good results [10–12]. In this way, the direct
esterification in organic solvent of the specific profen of inter-
est, in presence of a lipase with R stereopreference, is a valuable
method because preliminary derivatizations are not required and
the eutomer (S-enantioform) is recovered as unreacted substrate.
However, this method needs to reach conversion values beyond
50% and therefore, the use of irreversible conditions is mandatory
[13–15]. Otherwise, due to the water produced in the esterification
reaction, at high conversion grade the favoured hydrolysis of the
enantioform preferentially recognised by the lipase occurs, with
consequent final damage of the enantiomeric excess of both ester
(R-form) and unreacted acid (S-form).
In this work, we report an improvement of the biocatalyzed irre-
versible esterification reaction using dimethyl carbonate as alcohol
donor. This new procedure has successfully been applied to obtain
S-naproxen.
2. Materials and methods
2.1. Materials
The chemicals and solvents were obtained from commercial
sources and used without further purification. The (S)-(+)-naproxen
was obtained from Sigma–Aldrich and racemic naproxen was
obtained by a reported procedure [20]. Novozym 435® (lipase from
Candida antarctica, CAL B), lipases from Candida rugosa and Pseudo-
mas cepacia were purchased from Sigma; Lipozyme (lipase from
Mucor meihei) was obtained from Fluka; Rhizomucor miehei lipase
from Genzyme. The conversion values and enantiomeric excesses
of the biocatalyzed reactions were determined by a Dionex HPLC
apparatus equipped with LiChroCART® 250-4 (S,S)Whelk-O1 col-
umn and UV detector at 254 nm. The analyses were performed
using a mixture of hexane, 2-propanol and acetic acid in the ratio
Different approaches have been suggested to remove the water
in a lipase catalyzed esterification, in order to shift the equilibrium
towards the products, but they are not practicable for preparative
purposes. The distillation of water [16] results impracticable when
∗
Corresponding author. Fax: +39 095 7338310.
E-mail address: raffaele.morrone@icb.cnr.it (R. Morrone).
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.01.014

50
R. Morrone et al. / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 49–51
Fig. 1. Biocatalyzed esterification of ( )-naproxen.
Table 2
of 80:20:0.5 as eluent. The enantiomeric ratio (E) was calculated
from the extent of esterification and the enantiomeric excess of
the unreacted acid using the equation reported in literature [21].
Esterification of ( )-naproxen in different solvents.
Solvent
Time (d)
Conv. %
eeAc
eeEst
E
Tetrahydrofuran
Diethyl ether
Di-isopropyl ether
tert-Butyl methyl ether
Toluene
1,2-Dichloropropane
1,4-Dioxan
Dichloromethane
tert-Amyl alcohol
Acetonitrile
2
2
1
1
1
1
4
4
7
3
2
0
24
26
42
34
21
12
28
15
29
21
–
10
10
25
15
15
8
23
11
29
20
–
32
28
34
29
56
59
59
62
71
73
–
2
2
2
2
4
4
5
5
8
8
2.2. General procedure for esterification of ( )-naproxen by lipase
Rac-naproxen (10 mg, 0.043 mmol) was dissolved in 1 ml of
solvent of choice and 40 mg of enzyme, 5.2 ␮l (0.129 mmol) of
methanol were added to the solution and kept in a shaker at 45 ◦C
and 300 rpm. At the opportune conversion value, the reaction was
stopped by filtering off the lipase. The progress of the reaction was
monitored by chiral HPLC on aliquots of 10 ␮l of the reaction mix-
ture taken periodically.
Chloroform
Reaction conditions: 10 mg (0.043 mmol)
( )-naproxen, 5.2 ␮l (0.129 mmol)
methanol, 40 mg Novozym 435, 1 ml solvent, 45 ◦C, 300 rpm.
2.3. (S)-(+)-Naproxen
sider it for an enantiomeric preparative resolution, the reaction was
repeated in different solvents as reported in Table 2.
To a solution of rac-naproxen (500 mg; 2.17 mmol) and dimethyl
carbonate (585.9 mg; 6.51 mmol) in acetonitrile (50 ml) was added
Novozym (2 g) and the resulting mixture was stirred at 45 ◦C and
300 rpm for 9 days. The reaction was stopped by filtering off the
enzyme and the filtrate was partitioned with saturated bicarbon-
ate solution. The aqueous phase was acidified to pH 2.0 with 2N
H2SO4 and extracted with tert-butyl methyl ether to give 130 mg
of unreacted (S)-(+)-naproxen (yield 52%; ee > 98%).
All the considered solvents, except tetrahydrofuran, allowed
to perform the esterification. Reactions carried out in acetonitrile
and chloroform gave the best results in terms of enantioselectiv-
ity (E = 8). Although in chloroform the reaction rate is higher than
in acetonitrile, this latter was chosen as solvent for further experi-
ments in view of the lesser toxicity.
3. Results and discussion
3.3. Reversible esterification
3.1. Screening of lipases
A resolution of ( )-naproxen by esterification catalyzed by
Novozym 435 was attempted in acetonitrile in presence of
methanol as nucleophile. In the adopted condition the equilib-
rium was reached in 7 days with a conversion value of substrate
approaching 55%. Any lengthening of the reaction time led to the
slow decline in the ee values, as indicated in Fig. 2.
In order to identify a suitable lipase possessing R stereopref-
erence, ( )-naproxen was subjected to direct esterification in
presence of immobilised [Candida antarctica (Novozym 435), Mucor
meihei (Lipozyme)] and not immobilised (Candida rugosa, Pseudo-
mas cepacia, Rhizomucor meihei) lipases (Fig. 1) in di-isopropyl ether
as solvent and methanol as nucleophile.
3.4. Irreversible esterification procedure
All the investigated lipases catalyzed the esterification of
naproxen but only Novozym 435 showed the desired R stereopref-
erence, as reported in Table 1. All the next reactions were carried
out in presence of Novozym 435 as catalyst.
The irreversible devised procedure contemplates the use of
a dialkylcarbonate as “chemical utilizer” of the water produced
during the biocatalyzed esterification reaction. In particular, the
3.2. Selection of solvent
With the intention of improving the enantioselectivity of
Novozym 435 in the esterification of naproxen, in order to con-
Table 1
Esterification of ( )-naproxen with different lipases.
Lipase from
Time (d)
1
Conv. %
26
Est. ee
28
Ac. ee
10
Stereopref.
R
Candida antarctica
(Novozym 435)
Candida rugosa
Pseudomonas cepacia
Mucor meihei
5
1
2
21
12
26
75
15
3
20
22
3
S
S
S
(Lipozyme)
Rhizomucor meihei
2
14
12
2
S
Fig. 2. Trend of biocatalyzed ( )-naproxen esterification under reversible condi-
tions. (ꢀ) Enantiomeric excess of unreacted S-naproxen; (᭹) enantiomeric excess of
R-naproxen methyl ester; (ꢁ) conversion value.
Reaction conditions: 10 mg (0.043 mmol)
( )-naproxen, 5.2 ␮l (0.129 mmol)
methanol, 40 mg lipase, 1 ml di-isopropyl ether, 45 ◦C, 300 rpm.

R. Morrone et al. / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 49–51
51
stopped and S-naproxen was present in the mixture of reaction
with a ee >98% (yield 52%) while the naproxen (R)-methyl ester was
recovered with low enantiomeric purity (ee 34%). In the adopted
reaction conditions, neither naproxen ester formation nor hydrol-
ysis of dimethyl carbonate was observed in absence of Novozym
435.
4. Conclusion
Fig. 3. Representation of biocatalyzed irreversible esterification process.
We have applied a new method to achieve the esterification of a
chiral acid in irreversible manner by the use of dimethyl carbonate
as alcohol donor. The procedure preserves the enantiorecognition
of the substrate during the entire esterification reaction, thus per-
mitting the recovery of unreacted substrate in enantiopure grade.
The procedure applied to the resolution of naproxen furnished S-
naproxen with ee > 98% and yield 52%. The adopted method has
general use, and work is in progress in our laboratory using this
approach in the esterification/resolution of valuable organic acids.
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Fig. 4. Trend of irreversible biocatalyzed esterification of ( )-naproxen. (ꢀ) Enan-
tiomeric excess of unreacted S-naproxen; (᭹) enantiomeric excess of R-naproxen
methyl ester; (ꢁ) conversion value.
coupling of two lipase catalyzed reactions working in synergy takes
place, as reported in Fig. 3.
In an initial step, lipase catalyses the reversible esterification
of the acid, generating water. At the same time, the produced
water hydrolyses the dialkylcarbonate in presence of the lipase
and as a consequence CO2 and two molecules of methanol are
generated allowing the progression of the esterification process in
irreversible manner. At the final stage of the reaction, the pres-
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(transalcoholysis) of the produced ester but without any effect on
the conversion and ee values. With this intention dimethyl carbon-
ate [22] was added to a solution of naproxen in acetonitrile and
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The progress of esterification was monitored by chiral HPLC (Fig. 4).
After 9 days, when the conversion value was 74%, the reaction was
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