JOURNALOF FERMENTATION ANDBIOENGINEERING

Vol. 81, No. 3, 269-271. 1996

Enzymatic Resolution of Racemic Ibuprofen by Lipase-Catalyzed

Esterification Reaction: Effects of Water Content and
Solid Supports
MIN GON KIM AND SUN BOK LEE*
Department of Chemical Engineering, Pohang University of Science and Technology, San 31, Hyoja Dong,
Pohang 790-784, Korea

Received 13 October 1995IAccepted 20 December 1995

For the production of optically pure $(+)-ibuprofen, enzymatic esterification of racemic ibuprofen was
investigated using Candida cylindracea lipase. It was found that the reaction rates and yields could be enhanced
by adding solid supports to the reaction medium. The effects of the amount of water added and the type and
amount of support used were also examined.

[Key words: S-ibuprofen, lipase, esterification, organic solvent, solid support]

There is an increasing trend towards the use of optical-
ly pure enantiomers for drugs and agrochemicals because
they are more effective and have fewer side effects com-
pared with their racemic mixtures. Desirable reasons for
producing optically active pure materials include: (i)
biological activity is often associated with only one enan-
tiomer, (ii) the unwanted enantiomer is considered as an
impurity; (iii) where the switch from racemate to enan-
tiomer is feasible, there is the opportunity effectively to
double the capacity of an industrial process (1). In the
case of ibuprofen [2-(4-isobutylphenyl) propionic acid],
only the S-(+)-enantiomer is known to be biologically
active.
In a previous paper, we examined the effects of organ-
ic additives and temperature on the enzymatic hydrolytic
resolution of racemic ibuprofen esters using Candida
cylindracea lipase, and found that the enantioselectivity
of the enzyme could be remarkably enhanced by adding
a small amount of a polar solvent such as N,N-dimethyl-
formamide, formaldehyde or formamide (2). In the
present study, we investigated the lipase-catalyzed synthe-
sis of the S-(+)-ibuprofen methyl ester from racemic
ibuprofen and methanol (R,S-ibuprofen+methanol+
S-ibuprofen methylester +R-ibuprofen) as an alterna-
tive process to the hydrolytic resolution of ibuprofen
esters. In this case, the reaction should be carried out in a
microaqueous organic medium because the esterification
reaction is the reverse of the hydrolysis reaction.
Although enzymatic enantioselective esterifications
(or transesterifications) of racemic 2-arylpropionic acids
have previously been attempted (3-6), the main draw-
back of the esterification process is that the enzyme reac-
tion rate in organic media is much lower than that in the
hydrolytic resolution process. In this work, we show that
the esterification reaction rate as well as the final conver-
sion can be appreciably enhanced by simply adding a
solid support material such as silica gel or Eupergit C to
the reaction medium.
The enzyme used was C. cylindracea lipase (Type VII,
L-1754) prepared by Sigma (St. Louis, USA). HPLC-
grade alcohols and optically pure S-ibuprofen were
purchased from Aldrich (Milwaukee, USA). Racemic
* Corresponding author.
269
ibuprofen was a gift from Kuk Jeon Medicines (Seoul,
Korea). Silica gel (S-0507, 230-400 mesh), celite (D-
3877) and Amberlite IRC-50 (16-50 mesh) were pur-
chased from Sigma, and Eupergit C from Rohm Pharma
(Weiterstadt, Germany). All the other chemicals and or-
ganic solvents used were of reagent grade.
For a typical esterification reaction, the reaction mix-
ture consisted of an organic solvent (lOml), water (0.3
ml), racemic ibuprofen (1 mmol), methanol (2 mmol),
and C. cylindracea lipase (0.3-0.5 g). The reactions were
carried out at 30C and reciprocally shaken at 150
rpm in a water bath. The optical purity of ibuprofen
was determined after extracting ibuprofen from the reac-
tion mixtures with carbon tetrachloride and phosphate
buffer (pH 7.2) as described previously (2).
The concentrations of ibuprofen and ibuprofen ester
were analyzed by HPLC using p-Bondapak CIB column
(Waters, USA). The eluent solution (pH 3.0) consisted
of 25% (v/v) distilled water, 75% (v/v) acetonitrile, and
20 ml/l phosphoric acid. The absorbance of samples was
detected at 246 nm. The optical purity of ibuprofen was
determined by HPLC with a ResolvosilR BSA-7PX
column (M.-Nagel, Germany). The samples were eluted
with 50mM phosphate buffer solution (pH 7.8) con-
taining 4.5% n-propanol and 4mM octanoic acid. The
enantiomeric ratio Q was calculated from the extent of
esterification (c) and the enantiomeric excess of the
product (ee) using the equation of Chen et al. (7):
E= In [l -c(l +ee)]
where ee=w
In [l -c(l -ee)]
[fl and [R] in the above equation represent the concen-
tration of the S and R isomer of ibuprofen, respectively.
Initial reaction rates were determined by polynomial
regression of the time-course profiles.
Time-course profiles of the esterification of racemic
ibuprofen obtained in the absence or presence of addi-
tives are shown in Fig. 1. When the enzyme was used as
a dry powder without addition of water or other addi-
tives (closed rectangles in Fig. l), the reaction rate was
very low (especially in the initial phase) and the equilibri-
um yield was only about half of the theoretically expect-
ed value (50%). When a small amount of water was ad-
ded to the reaction medium, however, both the reaction
270 KIM AND LEE J. FERMENT. BIOENG.,

60 TABLE 1. Effect of organic solvents on esterification of racemic

ibuprofen with methanol by C. cylindracea lipase
Final yield and
Initial enantiomeric ration
Organic solvent Log P
(Pm$T$re$me) Fin;l#ld E
n
Ethyl ether 0.85 0.4 <O.l n.d.
I-Heptanone 1.8 0.2 1.6 n.d.
Benzene 2.0 3.6 33.2 325
Chloroform 2.0 0.1 1.3 n.d.
Anisole 2.1 3.0 26.3 n.d.
Toluene 2.5 4.0 28.5 293
Butyl ether 2.9 6.0 29.3 21
Carbon tetrachloride 3.0 23 44.1 492
50 700 150 200 o-Xylene 3.1 7.8 45.3 510
Time (h) Cyclohexane 3.2 46 44.0 413
n-Hexane 3.5 30 42.2 433
FIG. 1. Effects of solid supports and salts on the esterification of n-Heptane 4.0 34 46.5 551
racemic ibuprofen with methanol catalyzed by C. cylindracealipase in n-Octane 4.5 34 45.4 514
cyclohexane. Symbols: (B) nothing added, (0) 0.3 ml water added;
and 0.3 ml water added in the presence of (0) silica gel (0.6 g), ( 0 ) B The final yield and enantiomeric ratio (k) were determined at a
Eupergit C (0.5 g), (A) celite (0.3 g), (0) Amberlite IRC-50 (0.6 g), (x ) reaction time of 200 h (n.d.: not determined).
sodium sulfate (0.6 g), and (+) magnesium sulfate (1 .Og). Reaction Reaction mixture: 1 mmol ibuprofen, 2 mmol methanol, 0.5 g
mixture: 1 mmol ibuprofen, 2mmol methanol, 0.3 g enzyme, and enzyme, 0.5 g silica gel, 0.3 ml water, and 10 ml solvent.
10 ml cyclohexane.
effects of the additives on the reaction were examined
rate and final yield were enhanced up to the initial water at the same initial water level (3% v/v). By adding
content of 3% (v/v). The time course of the reaction appropriate solid supports to the medium, both the reac-
obtained at an optimal water content (3% v/v) is shown tion rate and the final (equilibrium) conversion were
in Fig. 1 (closed circles) as an example. enhanced as compared to those obtained without additives.
It is well known that while water is essential for the The results shown in Fig. 1 indicate that silica gel and
catalytic activity of enzymes in organic media, excess Eupergit C can effectively enhance the catalytic efficiency
water molecules reverse the direction of the esterification of the enzyme. On the other hand, the salts were found
reaction. As a consequence, there is an optimal water to have little or even a negative effect when water was
level that maximizes the enzyme activity in organic media; present in the reaction mixture. Silica gel, Eupergit C,
control of the water content is thus important for and celite were observed to be uniformly dispersed in the
optimizing esterification reactions in organic solvents. reaction medium, whereas Amberlite IRC-50, sodium sul-
Recently, Halling et al. reported that the catalytic fate, and magnesium sulfate were aggregated by absorb-
activity of Candida rugosa lipase and chymotrypsin ing water in the reaction medium. The higher efficiencies
could be enhanced by the addition of solid salt hydrates of silica gel and Eupergit C appear to be in part attribut-
which buffer water activity in organic reaction media (8, able to effective dispersion of the powdered enzyme in
9). In this work, we examined the effects of various sup- the organic medium.
port materials, as well as salts, on the enzymatic esterifi- Since addition of silica gel to the reaction mixtures
cation of ibuprofen. It should be noted that the solid gave the best results among the solid supports tested, the
support was used as an additive; the enzyme was not esterification of racemic ibuprofen was carried out in

g1.
immobilized onto the support prior to use. various organic solvents in the presence of silica gel. The
The time courses of the reaction in the presence of results are shown in Table 1, from which it is seen that
solid supports or salts are compared in Fig. 1. The the initial reaction rates are roughly correlated with the

60 60
l
l l
50 l
50
l
l
l
3 II a
@ l
0 l 0
l eeee l e
.s?40 .g 40
>1
z z
ii h
30 30

1j , , , , , (a)
20
UN
20
W

0.0 1.0 2.0 3.0 4.0 5.0 6 0.0 1.0 2.0 3.0 4.0 5.0 6.0 0 3 6 9 12 0 3 6 9 72
Water added (% v/v) Water added (% v/v) Silica gel (% w/v) Silica gel (% w/v)

FIG. 2. Effect of water content on the initial reaction rate (a) and the final yield (b). Reaction mixture: 1 mmol ibuprofen, 2 mmol methanol,
0.5 g enzyme, 0.5 g silica gel, and 10 ml cyclohexane. Effect of amount of silica gel on the initial reaction rate (c) and the final yield (d). Reaction
mixture: 1 mm01 ibuprofen, 2 mmol methanol, 0.5 g enzyme, 10 ml cyclohexane, and 0.3 ml water. The final yield corresponds to the conversion
determined at a reaction time of 200 h.
VOL. 81, 1996
log P values (hydrophobicity of the solvent) estimated
by the Rekker method (10). The highest reaction rate
was attained when cyclohexane was employed as the reac-
tion medium. The stereoselectivity of C. cylindracea
lipase appears to be better in organic media than an aque-
ous solution. The enantiomeric ratio (E) was greater
than 400 in most of the hydrophobic solvents (Table 1).
The effects of varying the water content and the
amount of silica gel were also examined using cyclo-
hexane as the reaction medium. With a fixed amount of
silica gel, the initial reaction rate increased as more
water was added to the medium (Fig. 2a). However, the
final yield gradually decreased as the water content was
increased up to 3.5% (v/v), after which further increases
in the initial amount of water did not affect the equilib-
rium of the reaction (Fig. 2b). In view of the results
shown in Fig. 2a, increasing the amount of silica gel was
expected to reduce the water content, and thereby the
rate of the enzyme reaction, because of the water-sorp-
tion property of silica gel. However, when the amount
of silica gel in the reaction mixtures was varied with a
fixed water content, the reaction rate increased as more
silica gel was added up to a certain level (Fig. 2~). On
the other hand, the final yield changed as expected: the
yield gradually increased with larger amounts of silica
gel, presumably due to a reduction in water activity
(Fig. 2d).
In summary, we have shown that the esterification rate
of racemic ibuprofen could be enhanced markedly by
selection of a suitable organic reaction medium and by
the addition of water and a solid support. As an exam-
ple, when cyclohexane was used as the reaction medium,
the esterification rate obtained in the presence of 3%
(v/v) water and 5% (w/v) silica gel was about 16-fold
higher than that without adding water and a solid sup-
port to the reaction medium (Fig. 2).
NOTES 271
This work was supported by the Korean Ministry of Science and
Technology and Hyundai Pharmaceutical Co.
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