Direct enantioselective HPLC monitoring of lipase-catalyzed kinetic resolution of flurbiprofen

Article abstract of DOI:10.1002/chir.20798

The solvent versatility of Chiralpak IB, a 3,5-dimethylphenylcarbamate derivative of cellulose-based chiral stationary phase, is demonstrated in the direct enantioselective HPLC monitoring of lipase-catalyzed kinetic resolution of flurbiprofen in nonstandard HPLC organic solvents. Nonstandard HPLC organic solvents were used as the reaction media for the lipase-catalysis and in mean time as diluent to dissolve the difficult to dissolve enzyme substrate (the acid) and as eluent for the simultaneous enantioselective HPLC baseline separation of both substrate and product in one run without any further derivatization.

Full text of DOI:10.1002/chir.20798

CHIRALITY 22:597–603 (2010)
Direct Enantioselective HPLC Monitoring of Lipase-Catalyzed
Kinetic Resolution of Flurbiprofen
*
ASHRAF GHANEM
Australian Centre for Research On Separation Science (ACROSS), School of Chemistry, University of Tasmania,
Hobart, Tasmania, Australia
ABSTRACT
The solvent versatility of Chiralpak IB, a 3,5-dimethylphenylcarbamate
derivative of cellulose-based chiral stationary phase, is demonstrated in the direct enan-
tioselective HPLC monitoring of lipase-catalyzed kinetic resolution of flurbiprofen in
nonstandard HPLC organic solvents. Nonstandard HPLC organic solvents were used as
the reaction media for the lipase-catalysis and in mean time as diluent to dissolve the
‘‘difficult to dissolve’’ enzyme substrate (the acid) and as eluent for the simultaneous
enantioselective HPLC baseline separation of both substrate and product in one run
C
VV
without any further derivatization. Chirality 22:597–603, 2010.
2009 Wiley-Liss, Inc.
KEY WORDS: chiral separation; chiralpak IB; direct monitoring; enantioselective HPLC;
flurbiprofen; HPLC; kinetic resolution; lipase
INTRODUCTION
organic solvents are seldom explored in the lipase-cata-
lyzed kinetic resolution of racemates due to the difficulty
associated with the enantioselective analysis. Neverthe-
less, if analyzed, they are not directly monitored as both
substrate and product and not analyzed in one run.17,18
The latter render the calculated enantiomeric excesses of
both substrate and product inaccurate. This is due to the
different response factor of the detector towards the sub-
strate (acid) and product (esters) in case of lipase-cata-
lyzed esterification or vice versa in case of enantioselective
hydrolysis.
Lipase-catalyzed kinetic resolution of flurbiprofen in
nonstandard HPLC solvents such as dichloromethane
(DCM), ethyl acetate (EtOAc), methyl tert-butyl ether
(MtBE) and tetrahydrofuran (THF) might be the method
of choice to overcome solubility problem of the sub-
strate.19 However, such nonstandard solvents might cause
irreversible damage of the CSP by dissolving or swelling
of the chiral selector.20–22 Thus, reactions in nonstandard
HPLC organic solvents are usually monitored off-line.20
Yet, a new generation of chiral packing materials for re-
solution of enantiomers by chromatography has been
introduced by immobilizing the polysaccharide derivatives
onto the chromatographic matrices.23–28 Thus, series of
immobilized polysaccharide-derived CSPs namely Chiral-
pak IA, Chiralpak IB and most recently Chiralpak IC
became commercially available.29,30 Such immobilization
renders the polysaccharide derivatives insoluble in any
Flurbiprofen (R, S)-[2-(3-fluro-4-phenyl) phenyl] propi-
onic acid is one of the most prevalent nonsteroidal anti-
inflammatory drugs (NSAIDs).1,2 The S-enantiomer
exhibits most of its anti-inflammatory activity, while the
R-enantiomer enhances its gastrointestinal toxicity.3 Fur-
thermore, R-flurbiprofen blocks nociception in rats, indi-
cating a different mechanism of analgesia and suggesting
the use of this enantiomer, and possibly other R-aryl propi-
onates, as analgesics.4 Flurbiprofen is reported to be less
prone to undergo chiral inversion in vivo.5,6 Consequently,
the resolution of flurbiprofen is particularly important.
To date, kinetic resolution is still one of the major strat-
egies for resolution of racemic mixtures7 using either
chemical8 or enzymatic9 procedures. Indeed, enzymes are
remarkable catalysts, capable of accepting a wide range of
substrates, at the same time exhibiting highly chemo-,
regio-, and enantioselectivity. Moreover, the price and
availability of most classes of enzymes have significantly
decreased over the last few years making them more eco-
nomically attractive catalysts for the production of biologi-
cally active compounds.10 Of particular interest, lipases
were most commonly used as biocatalysts for the enzy-
matic resolution of racemates. Most lipases accept a broad
range of non-natural substrates and are thus very versatile
for applications in organic synthesis. They do not require
cofactors and are commercially available in free and immo-
bilized forms. In many cases, they exhibit good to excel-
lent stereoselectivity.11
It is widely suggested that the lipase-catalyzed esterifica-
tion be conducted in hydrophobic solvents.12,13 However,
the solubility of flurbiprofen is poor in the commonly used
hydrophobic solvents, such as isooctane and n-hexane.14
Thus, the reported lipase-catalyzed resolution of flurbipro-
fen has been performed in hydrophilic standard organic
Dedicated to Professor Schurig on the occasion of his 70’s birthday.
*Correspondence to: Ashraf Ghanem, Pfizer Research Fellow/Lecturer
Australian Centre for Research On Separation Science (ACROSS), School
of Chemistry, University of Tasmania, Private Bag 75, Hobart, Tasmania
7001, Australia. E-mail: aghanem@utas.edu.au
Received for publication 28 May 2009; Accepted 11 August 2009
DOI: 10.1002/chir.20798
Published online 6 November 2009 in Wiley InterScience
solvents, particularly, acetonitrile.14–16 Nonstandard HPLC (www.interscience.wiley.com).
C
VV
2009 Wiley-Liss, Inc.

598
GHANEM
Fig. 1. Lipase-catalyzed enantioselective esterification of racemic flurbiprofen (1) using n-butanol in nonstandard HPLC organic solvents.
mobile phase solvent21,22 and combines the benefits of the Scientific (Fair Lawn, NJ). n-butanol used in the synthesis
coated polysaccharide-type CSPs namely the broad appli- of ester was Analar grade and purchased from BDH chem-
cation scope and the high preparative potential- with icals (Poole, England). Lipases from Asperigillus niger
enhanced robustness, practically unlimited solvent com- (lipase AS), Burkholderia cepacia (formerly Pseudomonas
patibility and extended range of applications.21–24 Subse- cepacia) free (lipase PS) and immobilized on ceramic par-
quently, broadening the choice of solvents able to be used ticles (lipase PS-C) together with Candida rugosa (lipase
as mobile phase eluent or diluents.19,20
AYS) were gifts from Amano (Nagoya, Japan). Immobi-
Here, we report on the first direct enantioselective lized Candida antarctica lipase B (Novozym435), its free
HPLC monitoring of lipase-catalyzed enantioselective form (Novozym525), and immobilized Rhizomucor miehei
access to enantiomerically pure/enriched flurbiprofen lipase (lipozyme RM IM) were from Novo Nordisk (Bags-
using nonstandard HPLC organic solvents as diluent to vaerd, Denmark). Lipase type VII from Candida rugosa
dissolve the enzyme substrate and eluent to analyze both (CRL) was from Sigma (Steinhiem, Germany). Lipase
substrate and product in one run without further derivati- from Candida cylinderacea (CCL), lipase immobilized in
zation.
sol–gel-Ak on sintered glass from Mucor miehei (MML)
and flurbiprofen were purchased from Fluka (Buchs,
Switzerland).
EXPERIMENTAL
Instrumentation
Chromatographic Conditions
The mobile phase was consisted of n-hexane/methyl tert-
butyl ether/2- propanol/trifluroacetic acid (85:15:0.4:0.1
v/v/v/v) and the flow rate was 1.3 ml/min. The column was
operated at room temperature (248C). UV detection was set
at 254 nm.
The mobile phase for HPLC was filtered through a Milli-
pore membrane filter (0.45 ml) from Nihon Millipore
(Yonezawa, Japan) and degassed via Waters in-line degas-
ser AF. The HPLC system consisted of a Waters binary
pump, Model 1525, (Milford, MA), equipped with a dual k
absorbance detector model 2487 and an autosampler
model 717plus. The UV-detector was set at 300 nm. Chiral-
cel OD (4.6 mm 3 250 mm ID coated on 5 ml silica-gel)
and Chiralpak IB (4.6 mm 3 250 mm ID immobilized on 5
ml silica-gel) column was purchased from Chiral Technolo-
gies Europe (Illkirch, France). The enantiomeric excess
ee of the substrate (ees) and the product (eep) as well as
the conversion (Conv.) and enantioselectivity (E) were cal-
culated as previously described.31–33
Chemical Synthesis of Racemic Butyl Ester of
Flurbiprofen (2)
Few drops of conc. sulfuric acid were added to 0.04 M
racemic flurbiprofen (1) in benzene followed by the addi-
tion of 360 ll of n-butanol (0.04 mol, 3 M equiv.). The mix-
ture was stirred under reflux over night, solvent was
evaporated under vacuum and the residue was neutralized
with 10% sodium hydrogen carbonate. The ester was
extracted twice using 30 ml diethyl ether. The ether was
dried over sodium sulfate anhydrous, filtered, and evapo-
Materials
All solvents used as mobile phase in HPLC including 2- rated under vacuum to afford 0.46 g pale yellowish oil
propanol were HPLC-grade and purchased from Fisher (76% chemical yield).
Fig. 2. Baseline separation of racemic flurbiprofen (1) and its corresponding n-butyl ester 2 on Chiralpak IB. Chromatographic parameters including
the separation factor (a) and the resolution (Rs) are shown.
Chirality DOI 10.1002/chir

KINETIC RESOLUTION OF FLURBIPROFEN
599
TABLE 1. Enzymatic parameters including enantiomeric
excesses of both substrate 1 and product 2 ees and eep,
respectively, conversion and enantioselectivity (E) of
different lipases screened for the enantioselective
esterification of flurbiprofen (1) using
at zero time (control). Lipase was (50 mg) added and 50 ll
samples of the nonstandard HPLC organic solvent aliquot
were withdrawn at several time intervals. The samples
were filtered and used directly without dilution for direct
enantioselective HPLC analysis.
1-butanol in acetonitrile
Lipase
Time (h)
ees (%)
eep (%)
Conv. %
E
RESULTS AND DISCUSSION
Novozym435
Novozym525
Lipozyme
Lipase AS
Lipase PS
Lipase AYS
CRL
Lipase PS-C
CCL
MML
168
168
168
168
168
168
168
168
168
168
29.2
13.3
4.7
5.1
3.4
4.7
6.0
6.0
4.0
4.0
72.3
60.4
17.6
33.3
33.3
9.5
28.8
18.0
21.0
13.2
9.2
33.0
39.7
12.2
21.8
10.7
8.2
4.6
1.4
2.1
2.0
1.2
1.2
2.6
1.3
2.0
Organic solvents, as a reaction media, have an important
role in determining the enantioselectivity in a lipase-cata-
lyzed biotransformation reactions.34,35 Moreover, versatil-
ity of the organic solvents in the mobile phase plays a cru-
cial role in the achievement of the enantioselective HPLC
baseline separation of the enzyme substrate and product
in one run without further derivatization. These simultane-
ous baseline separations in one run is mandatory for the
precise determination of the enantiomeric excesses of sub-
strates and products. The latter is an important prerequi-
site for the detailed investigation of the enzyme-catalyzed
enantioselective resolution of racemates.19,20
For the direct monitoring of the lipase-catalyzed enantio-
selective esterification of flurbiprofen 1 (cf. Fig. 1) in non-
standard solvents such as DCM, ethyl acetate (EtOAc),
methyl tert-butyl ether (MtBE) and THF, the acid itself
(1) and its corresponding ester (2) should be simultane-
ously baseline separated in their racemic synthesized form
in one run without further derivatization using a chiral sta-
tionary phase that is compatible with these nonstandard
organic solvents. Indeed, these solvents can not be used
as mobile phase eluents in conventionally coated HPLC
columns (e.g., Chiralcel OD).20–22 Thus, the enantioselec-
tive HPLC separation of both synthesized racemic acid (1)
and its corresponding butyl ester (2) was investigated
using Chiralpak IB (the immobilized version of 3,5-di-
methylphenylcarbamate derivative of cellulose-based chi-
ral stationary phase, namely Chiralcel OD). Several investi-
gations were performed using standard organic solvents
consisting of n-hexane/2-propanol with different volume
ratios. However, these combinations were not able to con-
9.0
42.8
14.2
33.3
1H NMR (CDCl3) d 6.8–7.7 (m, 8H, aromatic), 3.9 (t,
2H), 3.6 (q, 1H), 1.5 (m, 2H), 1.4 (d, 3H), 1.2 (m, 2H), 0.8
(t, 3H).
13C NMR (CDCl3) d 130.7, 128.9, 128.4, 127.6, 126.9,
123.5, 115.4, 115.0, 64.8, 45.1, 30.6, 19.0, 18.3, 13.6, 12.2.
Lipase-Catalyzed Esterification of Flurbiprofen (1)
Ten milligrams (0.04 mmol) racemic flurbiprofen 1,
10.8 ll n-butanol (0.12 mmol, 3 equiv.), molecular sieves,
and 2.0 ml either standard (acetonitrile) or nonstandard
HPLC organic solvent (such as toluene, MtBE, EtOAc, THF
or DCM) were added and stirred in a 4 ml reaction vial. The
reaction mixture was thermostated at 408C. Sample (50 ll)
was withdrawn and injected at zero time (control).
Lipase (50 mg) was added and 50 ll sample was with-
drawn and directly injected to HPLC without dilution or
workup at several time intervals.
Lipase-Catalyzed Hydrolysis of Flurbiprofen
Butyl Ester (2)
Flurbiprofen butyl ester (12.2 mg; 0.04 mmol), 1.0 of ml duct the simultaneously baseline separation of both race-
solvent, and 1.0 ml of 0.1 M phosphate buffer (pH 7) were mic acid (1) and its corresponding butyl ester (2) in one
added to 4 ml reaction vial. The reaction mixture was ther- run. Thus, more investigations have been performed using
mostated at 408C and 50 ll sample of the nonstandard the aforementioned nonstandard solvents solely or in com-
HPLC organic solvent aliquot was withdrawn and injected bination with n-hexane in mobile phase composition using
TABLE 2. Enzymatic parameters including enantiomeric excesses of substrate 1 and product 2 ees and eep, respectively,
conversion and enantioselectivity (E) of some selected lipases screened for the enantioselective esterification of flurbiprofen
(1) using 1-butanol in several nonstandard organic solvents. Nr (no reaction)
Lipase
Solvent
Toluene
MtBE
Ethyl acetate
THF
DCM
Toluene
MtBE
Ethyl acetate
THF
DCM
Log P
Time (h)
ees (%)
eep (%)
Conv. %
E
Novozym435
2.50
1.30
0.68
0.49
1.30
2.50
1.30
0.68
0.49
1.30
6
96
120
Nr
72
87.7
84.8
14.5
67.6
71.8
63.8
56.4
54.1
18.5
14.5
16.1
5.2
15.6
96.1
75.7
8.8
91.3
81.8
98.2
69.9
14.6
54.0
43.5
11.2
25.8
39.0
263.5
6.1
Novozym525
16
120
120
Nr
120
12.6
46.2
21.4
3.0
Chirality DOI 10.1002/chir

600
GHANEM
Fig. 3. HPLC chromatograms (from T 5 0 to T 5 5d) showing the UV traces of both substrate 1 and product 2 during the direct HPLC monitoring
of the reaction progress of Novozyme525-catalyzed enantioselective esterification of 1 using 1-butanol in MtBE as nonstandard HPLC solvent.
different ratios for the simultaneous separation of 1 and 2
Upon the fulfillment of the enantioselective baseline sep-
in one run on Chiralpak IB. A simultaneous baseline sepa- aration of the enzyme substrate (1) and the expected
ration of both racemic flurbiprofen (1) and its correspond- product (2), yet, the enzymatic reaction can be easily and
ing butyl ester (2) was achieved in one run using n-hex- directly HPLC monitored in either standard or nonstan-
ane associated with MtBE as a nonstandard HPLC organic dard HPLC organic solvents. Thus, the screening of differ-
solvent, 2-propanol and trifluoroacetic acid in mobile phase ent lipases for the enantioselective esterification of flurbi-
composition (n-Hex/MtBE/2-PrOH/trifluoroacetic acid profen (Fig. 1) was first performed in acetonitrile (log P
(85:15:0.4:0.1 v/v/v/v) (Fig. 2).
20.34) as standard organic solvent. Of all 10 tested
Chirality DOI 10.1002/chir

KINETIC RESOLUTION OF FLURBIPROFEN
601
Fig. 4. Lipase-catalyzed enantioselective hydrolysis of butyl ester (2) in a biphasic system consisting of nonstandard solvent and phosphate
buffer pH 7.
lipases, only lipase from Candida antarctica lipase B CAL-
In terms of enantioselectivity (E), enantiomeric excess
B (Novozym435 and Novozym525) showed better activity of the resulting product (eep) (R-ester) at maximum con-
and enantioselectivity (also called enantiomeric ratio) E. version (43.5%), lipase from Candida antarctica B (CAL-B)
The rest did not show any promising results even after known as Novozym525 showed the best performance in
168 hrs. Results are shown in Table 1. Due to the R-stereo- MtBE having log P 1.30 (ees 5 75.7 %, eep 5 98.2 %, E 5
preference of Candida antarctica lipase B,15,18 the enantio- 263.5 after 120 hrs) (cf. Fig. 3). While in toluene (log P
selective esterification of racemic 1 [(RS)-1], (R)-1 was 2.50) Novozym525 showed very good enantioselectivity
the faster reacting enantiomer which was selectively and enantiomeric excess of unreacted substrate (ees) (S-
esterified to afford its corresponding butyl ester (R)-2 leav- acid) at maximum conversion (54.0%), in shorter time (ees
ing the second enantiomer of the substrate (S)-1 in enan- 5 96.1 %, eep 5 81.8 %, E 5 39.0 after 16 hrs). Log P in Ta-
tiomerically enriched form. This was achieved when using ble 2 is defined as the ratio of concentration of a substance
different lipases in presence of n-butanol in acetonitrile as in two immiscible phases at equilibrium namely octanol
standard organic solvent (cf. Table 1).
Based on the aforementioned results, two lipases were
selected for further investigation in nonstandard organic
solvents. Thus, lipase from Candida Antarctica CAL-B
(Novozym435) and its free form (Novozym525) were used
for the enantioselective esterification of racemic flurbipro-
fen 1 in nonstandard HPLC solvents such as toluene,
methyl tert-butyl ether (MtBE), ethyl acetate (EtOAc),
THF and DCM, results are summarized in Table 2. The
reactions were directly monitored by enantioselective
HPLC using Chiralpak IB without further workup or re-
moval of the nonstandard solvent. In comparison with the
lipase-catalyzed kinetic resolution of (1) in acetonitrile as
standard organic solvent, excellent enantioselectivities E,
enantiomeric excesses of both substrate (1) and product
(2) and faster reaction rates were observed when using
nonstandard HPLC organic solvents.
TABLE 3. Enzymatic parameters including enantiomeric
excesses of both substrate 2 and product 1 ees and eep,
respectively, conversion and enantioselectivity (E) of
different lipases screened for the enantioselective hydrolysis of
racemic flurbiprofen butyl ester (2) in a biphasic system con-
sisting of MtBE as organic solvent and phosphate buffer pH 7
Lipase
Time (h)
ees (%)
eep (%)
Conv. %
E
Novozym435
Novozym525
Lipozyme
Lipase AS
Lipase PS
Lipase AYS
CRL
Lipase PS-C
CCL
MML
72
96
24.9
25.6
9.0
12.8
12.3
13.1
11.1
11.7
11.7
9.3
22.5
19.9
14.2
50.0
77.7
41.1
93.7
62.5
13.2
17.0
52.4
56.3
0.3
0.2
0.1
0.2
0.1
0.1
0.4
0.3
1.9
1.8
1.4
3.4
9.0
2.7
34.8
4.8
1.4
1.5
168
168
168
168
168
168
168
168
Fig. 5. Selected HPLC chromatograms (from t 5 0 to t 5 3 d) showing
UV traces of both substrate 2 and product 1 during the direct HPLC mon-
itoring of the reaction progress of Novozym435-catalyzed enantioselective
hydrolysis of flurbiprofen butyl ester (2) in a biphasic system consisting
of MtBE and phosphate buffer pH 7.
Chirality DOI 10.1002/chir

602
GHANEM
TABLE 4. Enzymatic parameters including enantiomeric
capabilities to allow the exploration of the enantioselectiv-
ity of lipases toward ‘‘difficult to dissolve’’ substrates in
harsh organic solvents.
excesses of both substrate 2 and product 1 ees and eep,
respectively, conversion and enantioselectivity (E) of
selected lipases screened for the enantioselective hydrolysis
of racemic flurbiprofen butyl ester (2) in a biphasic system
consisting of different organic solvents and
ACKNOWLEDGMENTS
phosphate buffer pH 7
The author thanks Prof. Nabil Aboul-Enein, Prof. Aida
Al-Azouny, and Mr. M. Farrag, Medicinal and Pharmaceu-
tical Chemistry Department, Pharmaceutical and Drug
Industries Research Division, National Research Centre
(NRC), Dokki-Cairo, Egypt for their scientific supports
Time ees
eep
(%)
Conv.
%
Lipase
Solvent
(h)
(%)
E
Novozym435 Toluene
144
144
144
144
144
144
17.8
4.7
12.6
9.0
6.4
2.3
8.4
0.6
1.2
20.6
Ethyl acetate
DCM
8.9 89.9
9.0 33.5
6.6 39.1
8.1 88.8
8.2 13.3
2.1 and discussions.
Novozym525 Toluene
2.4
18.4
1.4
Ethyl acetate
DCM
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These biotransformation reactions were easily monitored
by direct enantioselective HPLC equipped with immobi-
lized chiral stationary phase, namely, Chiralpak IB and a
UV-detector. This direct enantioselective HPLC monitor-
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