Resolution of (R,S)-flurbiprofen catalysed by dry mycelia in organic solvent

Article abstract of DOI:10.1016/j.tet.2007.08.051

Mycelia of Aspergillus oryzae display high enantioselectivity towards (R)-flurbiprofen and can be efficiently used in pure organic solvent for the resolution of (R,S)-flurbiprofen through esterification. The use of the lyophilized mycelia facilitates the separation process so that in one step the two enantiomers of flurbiprofen, which are both valuable for pharmaceutical applications, can be easily separated. The biotransformation can be carried out in different apolar solvents using different primary alcohols as nucleophiles under very mild conditions.

Full text of DOI:10.1016/j.tet.2007.08.051

Tetrahedron 63 (2007) 11005–11010
Resolution of (R,S)-flurbiprofen catalysed
by dry mycelia in organic solvent
Patrizia Spizzo,a Alessandra Basso,a Cynthia Ebert,a Lucia Gardossi,a,
Valerio Ferrario,a,b Diego Romanob and Francesco Molinarib
*
aLaboratory of Applied and Computational Biocatalysis, Dipartimento di Scienze Farmaceutiche, Universitaꢀ degli Studi di Trieste,
Piazzale Europa 1, 34127 Trieste, Italy
bDipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sezione Microbiologia Industriale, Universitaꢀ degli Studi di
Milano, via Celoria 2, 20133 Milano, Italy
Received 8 May 2007; revised 27 July 2007; accepted 16 August 2007
Available online 22 August 2007
Abstract—Mycelia of Aspergillus oryzae display high enantioselectivity towards (R)-flurbiprofen and can be efficiently used in pure organic
solvent for the resolution of (R,S)-flurbiprofen through esterification. The use of the lyophilized mycelia facilitates the separation process so
that in one step the two enantiomers of flurbiprofen, which are both valuable for pharmaceutical applications, can be easily separated. The
biotransformation can be carried out in different apolar solvents using different primary alcohols as nucleophiles under very mild conditions.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
and applied to the asymmetric hydrolysis of racemic flurbi-
profen esters.5,6
Flurbiprofen is a cyclooxygenase inhibiting non-steroidal
anti-inflammatory drug (NSAID). Cyclooxygenase inhibit-
ing activity resides primarily in the (S)-enantiomer, whereas
the (R)-enantiomer has scarce anti-cyclooxygenase activity,
but it has been found to inhibit tumour growth in various
animal models.1
Immobilized lipase B from Candida antarctica (Novozym
435Ò) has been reported to exhibit a relatively high enantio-
preference towards the (R)-flurbiprofen and it has been ap-
plied to the flurbiprofen resolution via either esterification
or transesterification carried out in organic media.7–9
Industrially, (R)-flurbiprofen is obtained via the following
steps: (i) synthesis of a suitable activated derivative of flur-
biprofen, (ii) preparation of the corresponding amide of
(R,R)-thiomicamine to obtain a diastereomeric mixture,
(iii) second order asymmetric resolution and (iv) hydrolysis
of the optically pure amide. The process leads to a 73%
yield, although the preliminary production of (R,R)-thiomic-
amine has to be considered.2
In the past few years, the synthesis of ester bound has been
reported by different microorganism used as dry myce-
lia.10,11 Dry mycelia can show high enantioselectivity, stabil-
ity to temperature and organic solvent. The cell structure
may act as natural matrix that is able to protect the enzymes
from the possible negative action of external agents.
In the present study we describe the use of dry mycelia in
pure organic solvent in the resolution of (R,S)-flurbiprofen.
Dry mycelia of moulds are a precious source of enantio-
selective enzymes with interesting economic and techno-
logical benefits, such as improved stability while avoiding
costly and time consuming purifications.10,12,13
Several studies concerning the biocatalyzed resolution of ra-
cemic flurbiprofen have been also reported in the recent
years. The hydrolytic route has been pursued by starting
from a suitable flurbiprofen ester and, in some cases, with
the aid of chiral cyclodextrins that form selective inclusion
complexes.3,4 Novel lipases and esterases have been isolated
2. Results and discussion
A series of lyophilized mycelia of moulds have been
screened in the enantioselective esterification of 10 mM
(R,S)-flurbiprofen 1 with equimolar ethanol 2 in n-heptane
at 50 ꢀC (see Scheme 1 and Table 1).
Keywords: Mycelia; Flurbiprofen; Organic solvent; Enzymatic esterifica-
tion.
* Corresponding author. Tel.: +39 0405583103; fax: +39 04052572; e-mail:
gardossi@units.it
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.08.051

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P. Spizzo et al. / Tetrahedron 63 (2007) 11005–11010
O
O
O
O
OH
OH
Dry mycelia
OH
+
+
CH3
CH3
F
)-Flurbiprofen
CH3
F
( )-Flurbiprofen
F
)-Flurbiprofen ethyl ester
(
R,
S
(
R
Ethanol
S
1
2
3
1-S
Scheme 1. Enantioselective esterification of (R,S)-flurbiprofen 1 with ethanol 2 catalysed by dry mycelia in toluene.
Table 1. Screening of enantioselective esterification of (R,S)-flurbiprofen 1
with ethanol 2 catalysed by dry mycelia in n-heptanea
Table 3. Screening of different solvents in the enantioselective esterification
of (R,S)-flurbiprofen 1 with ethanol 2 catalysed by dry mycelium of Asper-
gillus oryzae MIM at 30 ꢀCa
Biocatalyst
Time Molar
(h) conversion (%) ethyl ester 3 (%)
ee (R)-flurbiprofen Eb
Solvent
Log P Time Molar ee (R)-flurbiprofen
(h) conversion (%) ethyl ester 3 (%)
Aspergillus oryzae
MIM
Aspergillus oryzae
CBS 102.07
Rhizopus oryzae
CBS 112.07
Rhizopus oryzae
CBS 260.28
Rhizopus oryzae
CBS 328.47
Rhizopus oryzae
CBS 391.34
24 40
85
82
20
42
46
25
21
17
Benzene
Toluene
Isooctane
n-Heptane
2.0
2.5
4.5
4.0
168
168
48
48
168
32
36
26
28
15
90
92
90
92
62
24 40
168 50
168 49
168 48
168 71
1.8
Pentadecane 7.5
3.6
4.0
a
Reaction conditions: 10 mM (R,S)-flurbiprofen 1, 10 mM ethanol 2,
30 g lꢁ1 of dry mycelia, 30 ꢀC.
c
A second set of experiments was performed in an attempt to
obtain the two enantiomers with higher volumetric yields
since they both are of pharmacological relevance. Toluene
was selected due to the higher solubility of (R,S)-flurbipro-
fen 1 in such solvent, as compared to n-heptane whereas ben-
zene was discarded because of its toxicity. This allowed us to
perform further experiments at 50 mM concentration of
(R,S)-flurbiprofen 1. A molar ratio of acid/alcohol of
1:0.55 was used to prevent the esterification proceeding
much further than 50% conversion.
a
Reaction conditions: 10 mM (R,S)-flurbiprofen 1, 10 mM ethanol 2,
30 g lꢁ1 of dry mycelia, 50 ꢀC, n-heptane.
b
c
E¼ln[1ꢁc(1+eep)]/ln[1ꢁc(1ꢁeep)], c¼molar conversion, eep¼enantio-
meric excess of product.14
Not determined, the above mentioned equation give reliable results except
for low and high extents of conversion.14
The two strains of Aspergillus oryzae gave the best results in
terms of rate and enantioselectivity. A. oryzae MIM was
tested in various organic solvents differing in their polarity.
Reactions were performed at 50 ꢀC (Table 2).
1-Octanol 4 (Scheme 2) was selected in order to increase the
hydrophobicity of the produced ester, thus favouring the
synthetic route of the reaction as compared to the hydrolytic
one.16,17
Solvents leading to significant molar conversions were
used to perform the esterification at 30 ꢀC (Table 3) since
it is known that enantioselectivity is inversely related to
temperature.15
Reactions were performed at 30 and 50 ꢀC (Figs. 3 and 4)
and data show how at 30 ꢀC reaction kinetics are unac-
ceptably low. This causes a low ee of the unreacted acid,
although no significant effect of the temperature on the
enantioselectivity of the biocatalyst was observed. As a mat-
ter of fact, at 40% conversion the ee of the (S)-flurbiprofen
1-S was about 60% in both cases.
The most promising results in terms of enantioselectivity
and also in terms of molar conversion were obtained in
n-heptane, benzene and toluene solvents with values of
log P comprised between 2 and 4 (Figs. 1 and 2).
Table 2. Screening of different solvents in the enantioselective esterification
of (R,S)-flurbiprofen 1 with ethanol 2 catalysed by dry mycelium of Asper-
gillus oryzae MIM at 50 ꢀCa
100
80
60
40
20
0
Solvent
Log P Time Molar ee (R)-flurbiprofen
(h) conversion (%) ethyl ester 3 (%)
b
Acetonitrile
Ethanol
Tetrahydrofuran
Diisopropyl ether
Benzene
ꢁ0.3 168
ꢁ0.2 168
0.5 168
1.9 168
<5
<5
<5
9
32
28
40
27
20
—
—
—
—
88
84
85
62
65
b
b
b
2.0
2.5
4.0
4.5
7.5
96
96
24
48
48
Toluene
n-Heptane
Isooctane
Pentadecane
0
1
00
20
0
3
00
400
500
Time (h)
a
Reaction conditions: 10 mM (R,S)-flurbiprofen 1, 10 mM ethanol 2,
30 g lꢁ1 of dry mycelia, 50 ꢀC.
Not determined.
Figure 1. Molar conversion of the esterification of (R,S)-flurbiprofen 1 with
ethanol 2 catalysed by dry mycelium (30 g lꢁ1) of Aspergillus oryzae MIM
at 30 ꢀC in n-heptane (:) and in toluene (6).
b

P. Spizzo et al. / Tetrahedron 63 (2007) 11005–11010
11007
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
0
100
20
0
3
00
400
500
0
100
200
300
400
Time (h)
500
600
700
800
900
Time (h)
Figure 2. Enantioselectivity (ee of the product (R)-flurbiprofen ethyl ester
3) in the esterification of (R,S)-flurbiprofen 1 with ethanol 2 catalysed by
dry mycelium (30 g lꢁ1) of Aspergillus oryzae MIM at 30 ꢀC in n-heptane
(:) and in toluene (6).
Figure 3. Molar conversion of the esterification of (R,S)-flurbiprofen 1 with
1-octanol 4 catalysed by dry mycelium (10 g lꢁ1) of Aspergillus oryzae
MIM in toluene at 30 ꢀC (>) and 50 ꢀC (A).
At 50 ꢀC, the esterification proceeds up to the maximum
conversion (about 55%) with 84% ee of the unreacted
(S)-flurbiprofen 1-S. It is noteworthy that molar conversion
was complete, although during the direct esterification water
is produced. Moreover Figure 5 shows the production and ee
of the (R)-flurbiprofen octyl ester 5 performed at 50 ꢀC.
100
90
80
70
60
50
40
30
20
10
0
Recovery of the unreacted (S)-flurbiprofen 1-S and its sepa-
ration from the ester were easily accomplished by removing
the biomass by centrifugation and extraction of the acid from
the organic solvent.
0
1
00
20
0
3
00
40
0
500
600
7
00
80
0
900
Time (h)
Results obtained with the dry mycelia were finally compared
with the data obtained using two commercial preparations of
lipase. Figures 6 and 7 report conversions and enantiomeric
excess obtained by using the lipase B from C. antarctica
(CalB) in its native form (Chirazyme L-2) and as immobi-
lized enzyme (Novozym 435Ò), at 50 ꢀC.
Figure 4. Enantioselectivity (ee of the residual (S)-flurbiprofen 1-S) in the
esterification of (R,S)-flurbiprofen 1 with 1-octanol 4 catalysed by dry
mycelium (10 g lꢁ1) of Aspergillus oryzae MIM in toluene at 30 ꢀC (>)
and 50 ꢀC (A).
Maximum conversion was achieved in all cases, but enan-
tiomeric excess of the (S)-flurbiprofen 1-S was lower
(about 50%) than what observed with A. oryzae MIM
(>80%). In the case of immobilized CalB, the reaction
was reversible, with enantiomeric excess (ee) of the sub-
strate reaching a maximum after 2 days, and progressively
decreasing as the reaction progressed further. A possible ef-
fect of the polymeric support on the equilibrium and, there-
fore, on the overall enantioselectivity of the transformation
can be ascribed to substrate adsorption as previously
pointed out in the case of 4-methyloctanoic acid esterifica-
tion with ethanol catalysed by Novozym 435Ò.18 As a mat-
ter of fact, we have verified that, after the first day of
reaction, about 5% of racemic (R,S)-flurbiprofen 1 is ad-
sorbed by the support in the experimental conditions used
for the enzymatic resolution.
100
90
80
70
60
50
40
30
20
10
0
60
50
40
30
20
10
0
0
100
200
300
Time (h)
400
500
600
700
Figure 5. Conversion (6) and ee (:) of the (R)-flurbiprofen octyl ester
5 in the esterification of (R,S)-flurbiprofen 1 with 1-octanol 4 catalysed
by dry mycelium (10 g lꢁ1) of Aspergillus oryzae MIM in toluene at
50 ꢀC.
O
O
O
OH
OH
O
Dry mycelia
OH
+
+
CH3
CH3
CH3
F
F
F
(R,S)-Flurbiprofen
1-Octanol
4
(R)-Flurbiprofen octyl ester
(S)-Flurbiprofen
1
5
1-S
Scheme 2. Enantioselective esterification of (R,S)-flurbiprofen 1 with 1-octanol 4 catalysed by dry mycelia in toluene.

11008
60
P. Spizzo et al. / Tetrahedron 63 (2007) 11005–11010
0.2 g lꢁ1 pH 5.8) added with Tween 80 (0.5%). Suspensions
of spores (1.6ꢂ104) were used as inoculum. The microor-
ganisms were also cultured in 10 l stirred tank reactor con-
taining 2 l of medium at 28 ꢀC, 200 rpm and aeration
1 vvm. Cells grown for 48 h in submerged cultures were har-
vested by filtration at 4 ꢀC, washed with phosphate buffer
(Kpi 0.1 M, pH 7.0) and lyophilized.
50
40
30
20
10
0
4.2. Enzymes and chemicals
Lipase B from C. antarctica (Chirazyme L-2) was obtained
from Roche, while Novozym 435Ò was kindly donated by
Novozymes.
0
25
50
75
1
00
125
1
50
175
200
Time (h)
Figure 6. Molar conversion of the esterification of (R,S)-flurbiprofen 1 with
1-octanol 4 catalysed by immobilized CalB (Novozym 435Ò) (-) and
native CalB (Chirazyme L-2) (,) in toluene at 50 ꢀC.
All chemicals were purchased from Sigma–Aldrich and
Fluka and were used without any further purification.
Log P of solvents was from the literature.19
100
90
80
70
60
50
40
30
20
10
0
4.2.1. Resolution via esterification with ethanol 2. The es-
terifications of (R,S)-flurbiprofen 1 with absolute ethanol 2
were carried out by suspending the dried cells (30 mg mlꢁ1
)
in 2.5 ml of different organic solvents (acetonitrile, ethanol,
tetrahydrofuran, diisopropyl ether, benzene, toluene, iso-
octane, n-heptane and pentadecane). Equimolar concentra-
tions (10 mM) of acid (6.1 mg, 0.025 mmol) and alcohol
(1.2 mg, 0.025 mmol) were added to the suspension and
the reaction mixture was maintained in a thermostatted
bath at 30 or 50 ꢀC under constant magnetic stirring. With-
drawals of 100 ml were taken from the reaction mixture
and analysed by gas chromatography. To each sample
100 ml of internal standard ((R,S)-flurbiprofen methyl ester,
2 mg mlꢁ1) was added and, after centrifugation, samples
were injected in the gas chromatography.
0
50
100
Time (h)
150
200
Figure 7. Enantioselectivity (ee of the residual (S)-flurbiprofen 1-S) of the
esterification of (R,S)-flurbiprofen 1 with 1-octanol 4 catalysed by immobi-
lized CalB (Novozym 435Ò) (-) and native CalB (Chirazyme L-2) (,) in
toluene at 50 ꢀC.
The work-up of the microbial-catalysed esterification with
ethanol is reported as an example: mycelium was removed
by centrifugation and the organic solution was extracted
twice with 4 ml of an aqueous NaOH solution (1%). The
organic extracts were evaporated to give the crude ester,
which was purified by flash chromatography (hexane/ethyl
acetate, 7:3). The aqueous extracts were brought to pH 1.0
with an aqueous HCl solution (5%) and re-extracted with
AcOEt and the organic extracts were dried over Na2SO4.
The solvent was evaporated and the unreacted (S)-flurbipro-
fen 1-S was purified by crystallisation.
3. Conclusions
Mycelia of A. oryzae are a valuable source of carb-
oxylesterases that display high enantioselectivity towards
(R)-flurbiprofen. The use of the lyophilized mycelium al-
lows to separate in one step the two enantiomers, which
are valuable for pharmaceutical application; recovery and
separation of the products are easy and fast. The biotrans-
formation can be carried out in different solvents (i.e.,
n-heptane or toluene) and with different primary alcohols
(i.e., ethanol or 1-octanol) under very mild conditions.
4.2.2. Resolution via esterification with 1-octanol 4. Res-
olution of (R,S)-flurbiprofen 1 was carried out in anhydrous
toluene (6 ml) at 30 and 50 ꢀC by direct esterification of
(R,S)-flurbiprofen 1 (73.3 mg, 50 mM, 0.3 mmol) with
1-octanol 4 (26 ml, 27.5 mM, 0.165 mmol) catalysed by
whole cells (10 mg mlꢁ1, 0.043 U/ml, p-nitrophenyl palmi-
tate units) of the dry mycelia of A. oryzae MIM under con-
stant stirring (orbital shaker, 150 rpm).
4. Materials
4.1. Microorganisms
A. oryzae MIM (Microbiologia Industriale Milano), A. ory-
zae CBS 102.07 (Centraalbureau voor Schimmelcultures,
Baarn, Holland), Rhizopus oryzae CBS 112.07, R. oryzae
CBS 260.28, R. oryzae CBS 328.47, R. oryzae CBS
391.34 were used throughout this study and routinely main-
tained on malt extract (8 g lꢁ1, agar 15 g lꢁ1, pH 5.5). The
microorganisms were cultured in 500 ml Erlenmeyer flasks
containing 100 ml of medium and incubated for 48 h at
28 ꢀC on a reciprocal shaker (100 rpm). The liquid media
contained a basal medium (BM: Difco yeast extract
1 g lꢁ1, (NH4)2SO4 5 g lꢁ1, K2HPO4 1 g lꢁ1, MgSO4$7H2O
Comparison between dry mycelia of A. oryzae MIM and
native lipase B from C. antarctica (Chirazyme L-2) and
immobilized lipase B (Novozym 435Ò) was carried out
at 50 ꢀC under the same experimental conditions using
Chirazyme L-2 (1 mg mlꢁ1, 0.02 U/ml, p-nitrophenyl pal-
mitate units) and Novozym 435Ò (35 mg mlꢁ1, 0.07 U/
ml, p-nitrophenyl palmitate units), respectively, in anhy-
drous toluene.

P. Spizzo et al. / Tetrahedron 63 (2007) 11005–11010
11009
4.3. Analytical methods
distilled water in order to solubilize the salts and finally dried
with Na2SO4. After solvent evaporation a white solid was
obtained (27 mg, 82%).
4.3.1. Resolution via esterification with ethanol 2. Molar
conversions were determined by GLC carried out on a Carlo
Erba Fractovap 2150 gas chromatograph equipped with
hydrogen flame ionisation detector; the column temperature
was kept at 190 ꢀC. The column (4ꢂ1500 mm) was packed
with OV-17. Retention time: (R,S)-flurbiprofen methyl ester
12.57 min, (R,S)-flurbiprofen ethyl ester 3 15.32 min.
The (S)-flurbiprofen 1-S and the (R)-flurbiprofen octyl ester
1
5 were characterized by H NMR, 13C NMR, 19F NMR,
FTIR, mass spectrometry and polarimetry. 1H and 13C
NMR spectra were registered with a Varian Gemini 200
spectrometer (200 MHz), the 19F NMR spectra were re-
corded with a Bruker AC 200F (188 MHz). Chemical shifts
were expressed in parts per million with respect to the tetra-
The absolute configuration of the ethyl ester was deter-
mined by comparison with the optical rotation of standards
of the optically pure enantiomers obtained by esterification
of optically pure (S)-flurbiprofen 1-S with ethanol 2 using
conventional esterification procedures.20 The enantiomeric
composition was determined by gas chromatography car-
ried out on a Dani 6500 gas chromatograph equipped with
hydrogen flame ionisation detector using a chiral capillary
column at 160 ꢀC (diameter 0.25 mm, length 25 m, thick-
ness 0.25 mm, DMePeBeta-CDX-PS086, MEGA, Legnano,
Italia). Retention time: (R)-flurbiprofen ethyl ester 3
50.2 min, (S)-flurbiprofen ethyl ester 51.4 min. The stereo-
chemical outcome of the transformations was expressed as
enantiomeric excess (ee) of the major enantiomer.
methylsilane signal for 1H and 13C NMR and Ar–CF3 for 19
F
NMR. Signal multiplicity is expressed as follow: s (singlet),
d (doublet), dd (double doublet), t (triplet), br t (broad trip-
let), q (quartet), m (multiplet).
Infrared spectra were recorded on a JASCO FTIR-200.
Mass spectra, carried out with electron impact method, were
registered at 70 eV using a ION TRAP GCQ FINNIGAN
mass spectrometer. Optical rotations were measured on
a JASCO DIP-1000.
1
4.4.1. (S)-Flurbiprofen 1-S. H NMR (CDCl3): d 1.59 (d,
3H, J¼7.3 Hz, CH3), 3.85 (q, 1H, J¼7.1 Hz, CH), 7.14–
7.24 (m, 2H, H-Ph), 7.38–7.60 (m, 6H, H-Ph). 13C NMR
(CDCl3): d 18.2, 45.0, 115.3, 115.8, 123.8, 123.9, 127.9,
128.2, 128.5, 128.6, 129.1, 129.2, 131.0, 131.1, 135.6,
141.0, 141.1, 157.4, 162.4, 179.6. 19F NMR (CDCl3):
d ꢁ117.67 (dd, J1¼8.4 Hz, J2¼11.3 Hz). FTIR (Nujol):
cmꢁ1 3000 (br), 1698, 1580, 1514, 1216, 765, 724, 698. EI
(CHCl3): m/z 244. [a]2D0 ꢁ1.46 (c 2.5, CH2Cl2).
4.3.2. Resolution via esterification with 1-octanol 4. Molar
conversion (referred to (R,S)-flurbiprofen 1) and enantio-
meric excess (ee) of (S)-flurbiprofen 1-S were evaluated
by RP-HPLC (HPLC Gilson 321 system equipped with
UV–vis Agilent DA and Gilson Dual length). Sample of
50 ml was diluted to 1 ml using the same mobile phase
(acetonitrile or hexane) of the HPLC analysis and immedi-
ately analysed. Molar conversions were calculated by ana-
lysing the samples at 260 nm, using a Phenomenex Gemini
5m C18 110A column (250ꢂ4.6 mm) with a gradient start-
ing from 50% of acetonitrile for 23 min, then for 15 min
with 100%. Trifluoroacetic acid (0.1% v/v) was added to
the mobile phases and a flow of 1 ml minꢁ1 was used. Re-
tention times: (R,S)-flurbiprofen 1 10.5 min, (R,S)-flurbiro-
fen octyl ester 5 33.6 min. Enantiomeric excess was
calculated by analysing the samples at 260 nm
using a Daicel ChiralpakÒ AD column (250ꢂ4.6 mm),
with an isocratic elution with a mobile phase hexane/
isopropanol¼80:20 and with a 1 ml minꢁ1 flow. Retention
time: (R)-flurbiprofen 4.8 min, (S)-flurbiprofen 1-S
6.3 min. The resolution factor (a) of the two enantiomers
is 0.61.
1
4.4.2. (R)-Flurbiprofen octyl ester 5. H NMR (CDCl3):
d 0.88 (br t, 3H, J¼6.6 Hz, CH3), 1.30 (s, 10H, CH2), 1.55
(d, 3H, J¼7.3 Hz, CH3), 1.62 (m, 2H, CH2), 3.77 (q, 1H,
J¼7.2 Hz, CH), 4.20 (t, 2H, J¼6.8 Hz, CH2), 7.11–7.21
(m, 2H, H-Ph), 7.36–7.65 (m, 6H, H-Ph). 13C NMR
(CDCl3): d 14.7, 18.9, 23.3, 26.5, 29.2, 29.8, 30.4, 32.4,
45.8, 65.7, 115.6, 116.0, 124.0, 124.1, 128.2, 128.5, 128.8,
128.9, 129.4, 129.5, 131.2, 131.3, 136.1, 142.5, 142.6,
157.7, 162.7, 174.6. 19F NMR (CDCl3): d ꢁ118.06 (dd,
J1¼7.9 Hz, J2¼11.2 Hz). FTIR (Nujol): cmꢁ1 1738, 1583,
1514, 1216, 1177, 766, 724, 697. EI (CHCl3): m/z 356.
[a]2D0 ꢁ1.96 (c 2.5, CH2Cl2). ee at maximum conversion
(55% conversion of (R,S)-flurbiprofen 1): 69%.
Acknowledgements
4.4. Extraction of (R,S)-flurbiprofen 1 and of
(R)-flurbiprofen octyl ester 5
Thanks are due to M.I.U.R. (Rome, Italy) and COST D-25
for financial support to L.G. and F.M. We thank Paolo
Martinuzzi for 19F NMR spectra and Gabriella Di Luca for
polarimetric measurements.
Dry mycelia of A. oryzae MIM were removed by filtration
and rinsed with toluene to recover eventually adsorbed
acid and esters. After partial removing of toluene the organic
pools were extracted with a saturated solution of NaHCO3.
References and notes
The organic phase, containing the (R)-flurbiprofen octyl es-
ter 5, was dried with Na2SO4, filtrated and the solvent was
evaporated. A yellow oil was obtained (53 mg, 90%). The
aqueous phase, containing the (S)-flurbiprofen 1-S, was
acidified to pH 2.0 with 6 N HCl. The extraction was carried
out with diethyl ether. Organic phase was then washed with
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