Enantioselective hydrolysis of (RS)-2-fluoroarylacetonitriles using nitrilase from Arabidopsis thaliana

Article abstract of DOI:10.1016/S0957-4166(01)00034-9

The enzymatic resolution of 2-fluoroarylacetonitriles (RS)-3 using nitrilase from the plant Arabidopsis thaliana is described. Racemic 2-fluoronitriles 3 are easily accessible from O-silylated aromatic cyanohydrins 2 by reaction with DAST. The nitriles (RS)-3 were hydrolysed with the nitrilase as a catalyst, not to the expected 2-fluoroarylacetic acids but to the corresponding (R)-2-fluoroarylacetamides (R)-5 as the main products. After optimization of reaction conditions (pH 9, 7°C), the enantiomeric excesses of (R)-5a,c and f (R=H, 3-Me, 3-OMe) could be improved to >99% by one recrystallization. The acid catalysed hydrolysis of (R)-5a,5c and 5f afforded the corresponding (R)-2-fluoroarylacetic acids (R)-4a,4c and 4f without racemization.

Full text of DOI:10.1016/S0957-4166(01)00034-9

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 279–285
Enantioselective hydrolysis of (RS)-2-fluoroarylacetonitriles using
nitrilase from Arabidopsis thaliana1
Franz Effenberger* and Steffen Oßwald†
Institut fu¨r Organische Chemie der Universita¨t Stuttgart, Pfaffenwaldring 55, D-70569 Stuttgart, Germany
Received 14 December 2000; accepted 17 January 2001
Abstract—The enzymatic resolution of 2-fluoroarylacetonitriles (RS)-3 using nitrilase from the plant Arabidopsis thaliana is
described. Racemic 2-fluoronitriles 3 are easily accessible from O-silylated aromatic cyanohydrins 2 by reaction with DAST. The
nitriles (RS)-3 were hydrolysed with the nitrilase as a catalyst, not to the expected 2-fluoroarylacetic acids but to the
corresponding (R)-2-fluoroarylacetamides (R)-5 as the main products. After optimization of reaction conditions (pH 9, 7°C), the
enantiomeric excesses of (R)-5a,c and f (R=H, 3-Me, 3-OMe) could be improved to >99% by one recrystallization. The acid
catalysed hydrolysis of (R)-5a,5c and 5f afforded the corresponding (R)-2-fluoroarylacetic acids (R)-4a,4c and 4f without
racemization. © 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
approach to the enantiopure a-fluorocarboxylic acids.
Nitrilases can be classified, depending on their substrate
specificity, into ‘aromatic nitrilases’, ‘arylacetonitrilases’
and ‘aliphatic nitrilases’.11 The nitrilases from Alcalige-
nes faecalis ATCC 8750,12 Alcaligenes faecalis JM313
and Pseudomonas fluorescens DSM 715514 were unam-
biguously identified as arylacetonitrilases.14 Nitrilases
derived from plants have not yet been investigated
systematically with respect to their substrate
specificities.
Since CꢀH and CꢀF bonds are comparable in their
steric demands but markedly different in their elec-
tronic properties, organofluorine compounds play an
important role in a variety of applications including
medicinal diagnostics, nuclear magnetic resonance
tomography, positron emission tomography (PET) and
so on.2–4 Organofluorine compounds have attracted
particular attention in biologically active compounds.5
Thus optically active a-fluorocarboxylic acids and their
derivatives are highly potent as analogues of phero-
mones,6 antirheumatics7 and as analgesics.8
To date, the nitrilase from Arabidopsis thaliana is the
only plant nitrilase known to be cloned, sequenced and
functionally expressed in E. coli,15 hence, this enzyme is
available in amounts sufficient for preparative applica-
tions. In a comprehensive investigation of the substrate
range of A. thaliana nitrilase16 a-fluoroarylacetonitriles
were found to be possible substrates for this enzyme.
Stereoselective syntheses have been developed especially
for a-fluorocarboxylic acids because of their biological
activity.5 The kinetic resolution of ethyl a-
fluorohexanoate9a and alkyl 2-fluorodecanoates9b is
reported for the application of enzymes in the prepara-
tion of optically active a-fluorocarboxylic acids. For the
preparation of optically active a-fluorophenylacetic
acids, however, neither an efficient enzymatic nor
efficient chemical procedure is so far known which
affords enantiopure fluoro acids. Because racemic a-
fluoronitriles are readily accessible by reaction of
unprotected or O-silylated cyanohydrins with diethyl-
aminosulfur trifluoride (DAST),10 the kinetic resolu-
tion of a-fluoronitriles appears to offer an easy
Herein, we report the kinetic resolution of a-fluoroaryl-
acetonitriles using nitrilase from A. thaliana and the
study of the reaction under a number of reaction
conditions.
2. Results and discussion
2.1. Preparation of 2-fluoroarylnitriles 3
The racemic 2-fluoronitriles
3
were prepared
analogously to the methodology developed by Le
Tourneau et al.10 In a one-pot reaction the starting
aldehydes 1 were reacted with trimethylsilylcyanide to
* Corresponding author. E-mail: franz.effenberger@po.uni-stutt-
gart.de
Part of dissertation, Universita¨t Stuttgart, 2000.
†
0957-4166/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00034-9

280
F. Effenberger, S. Oßwald / Tetrahedron: Asymmetry 12 (2001) 279–285
give the corresponding silylated cyanohydrins 2, which
subsequently gave the 2-fluoronitriles 3 with diethyl-
aminosulfur trifluoride (DAST) (Scheme 1).10a 2-
Fluoro-2-(methylphenyl)ethanenitriles 3b,c,d, which
have not been described so far, could be isolated by this
route in 66–79% yield. For the synthesis of 2-fluoro-2-
(3-methoxyphenyl)ethanenitrile 3f the method was
modified to improve the yield. The silylated cyanohy-
drin 2f17 was first distilled and subsequently fluorinated
with DAST at −78°C, giving 3f in 70% yield.
can be separated by chromatography on silica gel with
different eluents. The enantiomers of both the remain-
ing nitrile and acid (as methyl ester) could be separated
directly by gas chromatography. The enantiomeric
excess of the amide 5a was determined by gas chro-
matography after hydrolysis and subsequent esterifica-
tion of the intermediate acid with diazomethane.
Conversion and amide/acid ratio were determined by
HPLC. The configuration of the products was assigned
by comparison of specific rotation values of acid 4a and
nitrile 3a with literature data.10b,19
The 2-fluorocarboxylic acids 4, required as reference
compounds, were prepared by acid hydrolysis of the
nitriles 3. In the hydrolysis of 3a with concentrated
HCl, halogen exchange was observed and 2-chloro-2-
phenylacetic acid18 formed, as well as the desired 2-
fluoro-2-phenylacetic acid 4a. By using 49% sulfuric
acid instead of HCl, the fluoroacetic acids 4 were
obtained as the sole products in 26–64% yield.
At 41% conversion, nitrile 3a, acid 4a and amide 5a
were obtained in a ratio of 59:6:35. The amide 5a is
(R)-configured, with an e.e. of 76% (Table 1), whereas
the acid 4a (with an e.e. of 13%) and the main enan-
tiomer of the remaining nitrile 3a (with an e.e. of 36%)
were found to have (S)-configuration.
At higher conversion rates, the e.e.s of the (S)-nitrile
and the (S)-acid did not increase as expected. After 24
h and 84% conversion, nitrile 3a, acid 4a and amide 5a
were obtained in a ratio of 16:22:62 with e.e.s of 47, 42
and 59%, respectively. This result is caused by racem-
ization of the nitrile 3a under the reaction conditions,
as was demonstrated by using isolated (S)-3a.
2.2. Enzymatic hydrolysis of 2-fluoroarylnitriles 3 using
the nitrilase from Arabidopsis thaliana
Preliminary investigations of the nitrilase catalysed
hydrolysis of 2-fluorobenzyl cyanide 3a, as depicted in
Scheme 2, surprisingly resulted in the formation of
2-fluoro-2-phenylacetamide 5a as the main product
with acid 4a obtained as a side product. Normally the
nitrilase of A. thaliana reacts as nitrilase to give only
the carboxylic acid and not as hydratase/amidase yield-
ing the amide in the first step and the acid in the second
step.16 The acid 4a was extracted with sodium bicar-
bonate solution, while the mixture of nitrile and amide
Additional substrates (RS)-3b–3f have been hydrolysed
and investigated using the nitrilase of A. thaliana with
respect to the influence of aromatic ring substituents, R,
on the rates and enantioselectivities of the conversions
(Scheme 2, Table 1).
Scheme 1.
Table 1. Enantioselective hydrolysis of 2-fluoronitriles (RS)-3 to 2-fluoroacetamides (R)-5 and 2-fluoroacetic acids (S)-4
catalysed by the nitrilase from Arabidopsis thaliana
Substrate
Rel. activity (%)a
Conversion (%)b
Ratio 5:4
2-Fluoro acids
% E.e.
2-Fluoroamides
(R)-5 % E.e.
(S)-4
3a
3b
3c
3d
3e
3f
26
B0.1
35
4.3
2.8
23
41
–
33
21
34
21
85:15
–
80:20
70:30
85:15
82:18
4a
4b
4c
4d
4e
4f
13
–
14
B5
B5
11
5a
5b
5c
5d
5e
5f
76
–
76
22
B5
80
a Activity referred to butyronitrile.
b Conversion is defined as: conversion=[amide]+[acid]/[nitrile]0.

F. Effenberger, S. Oßwald / Tetrahedron: Asymmetry 12 (2001) 279–285
281
Table 2. Optimization of the reaction conditions using 2-
fluoro-2-phenylethanenitrile 3a as substrate
Table 1 shows that the substituent R sterically influ-
ences the enzyme activity. While 2-fluoro-2-(2-
methylphenyl)benzyl cyanide 3b was not accepted by the
nitrilase from A. thaliana, nitriles 3d and 3e, carrying
para-substitutedphenylrings, werehydrolysed6–10times
slowerthan3a. The2-fluorobenzylcyanides3cand3fwith
meta-substituted phenyl rings, however, were converted
with rates similar to compound 3a. In all cases, the ratio
(R)-amide 5/(S)-acid 4 is comparable, with the amide as
the main product.
pH Temp. (°C)
Conversion
(%)
Ratio 5a:4a
(R)-5a % e.e.
8.0
8.5
9.0
9.5
9.0
9.0
20
20
20
20
30
7
18
24
21
11
46
39
87:13
85:15
82:18
82:18
71:29
84:16
75
81
82
71
40
94
With respect to the amide (R)-5, the stereoselectivity is
satisfactory only for the nitriles 3a, 3c and 3f, yielding
the amides (R)-5a, 5c and 5f with an e.e. of approximately
80%. The enantiomeric excesses determined for the acids
(S)-4 are low in all cases (Table 1). In order to improve
the enantiomeric excess of the (R)-amides, the reaction
conditions have been varied, using 2-fluorobenzyl cyanide
3a as substrate. The results are summarized in Table 2.
The optimized reaction conditions were also applied to
substrates 3c and 3f (Scheme 3, Table 3). The nitrilase
catalysed hydrolysis gave the (R)-amides 5c and 5f with
e.e.s of 88 and 91%, respectively, and about 50% conver-
sion. E.e.s were improved by recrystallization from
petroleum ether/chloroform to afford 5c with an e.e. of
97% and 5f with e.e. of >99%. The total yield of both
amides after enzymatic hydrolysis and recrystallization
was in the range of 60–70%, referred to the conversion
of nitriles (R)-3c and (R)-3f.
As can be seen from Table 2, the variation of the pH value
from 8.0 to 9.5 at 20°C has almost no influence on the
enantiomeric excess. The best result at this temperature,
an e.e. of 82%, was obtained at pH 9.0, the pH optimum
of the nitrilase.16,20 The temperature, however, had a very
significant influence on the observed e.e. An increase in
e.e. from 40 to 82% was seen on lowering the reaction
temperature from 30 to 20°C. Lowering the temperature
further to 7°C led to a further increase in the e.e., which
increased to 94% at 39% conversion.
The acid catalysed hydrolysis of (R)-5a, 5c and 5f with
10% sulfuric acid proceeded without racemization, giving
the (R)-2-fluoroacetic acids (R)-4a, 4c and 4f in 80–90%
yield with enantiomeric excesses of 98 to >99% (Table 3).
Because both the sign of optical rotation values and the
retention order of 4c and 4f correspond with those of acid
(R)-4a, the (R)-configuration was also assigned to 4c and
4f.
On a preparative scale under optimized reaction condi-
tions (pH 9.0 at 7°C), (R)-2-fluoro-2-phenylacetamide
(R)-5a was isolated at 53% conversion with an e.e. of 92%.
After recrystallization from petroleum ether/chloroform
enantiomerically pure (R)-5a was obtained in 72% yield
(referred to conversion) (see Scheme 3, Table 3).
In summary, enantiomerically pure (R)-2-fluoroaryl-
acetamides 5 are accessible for the first time by the kinetic
resolution of racemic 2-fluoroarylnitriles (RS)-3 cat-
Scheme 2.
Scheme 3.

282
F. Effenberger, S. Oßwald / Tetrahedron: Asymmetry 12 (2001) 279–285
Table 3. Enzymatic hydrolysis of 2-fluoronitriles (RS)-3a,3c,3f to (R)-2-fluoroacetamides (R)-5a,5c,5f and their hydrolysis to
2-fluoroacetic acids (R)-4a,4c,4f
Substrates (RS)-3
Conv. (%)a
2-Fluoroacetamides (R)-5
2-Fluoroacetic acids (R)-4
% E.e.
Yield (%)b
% E.e.c
Yield (%)
% E.e.
3a
3c
3f
53
56
48
5a
5c
5f
92
88
91
72
61
67
\99
97
\99
4a
4c
4f
91
90
88
\99
98
\99
a Conversion for the enzymatic reaction of (RS)-3.
b Total yield of enzymatic reaction and recrystallization referred to reacted nitrile (R)-3.
c E.e. values after recrystallization.
alysed by a nitrilase from A. thaliana. (R)-2-Fluoroary-
lacetamides can easily be hydrolysed in 10% sulfuric
acid to give the corresponding (R)-2-fluoroarylacetic
acids 4 without any racemization. The enzymatic
hydrolysis of arylacetonitriles to the corresponding
amides is superior to chemical hydrolysis because no
decomposition products are found. The isolated nitrile
starting material can be easily racemized by heating at
50°C for 1 h in the buffer applied for the enzymatic
conversion and thus can be returned to the resolution.
Because racemic 2-fluoroamides are known to be fungi-
toxic,21 the optically pure forms are also expected to
have biological activity.
The reaction mixture was allowed to warm to room
temperature, stirred for a further 20 min, poured on ice
(100 mL) and vigorously stirred for 5 min. The organic
phase was separated and the aqueous phase was
extracted with diethyl ether (2×100 mL). The combined
organic phases were washed with a 5% aqueous
NaHCO3 solution and water to neutralize, dried
(Na2SO4) and concentrated. The crude products were
chromatographed on silica gel with petroleum ether/
ethyl acetate (90:10 for 3b, 3d, 93:7 for 3c).
3.2.1. 2-Fluoro-2-(2-methylphenyl)ethanenitrile 3b. Yield:
1
79% (light yellow oil). H NMR (250 MHz): l 2.46 (s,
3H), 6.19 (d, J=46.4 Hz, 1H), 7.25–7.58 (m, 4H). Anal.
calcd for C9H8FN (149.2): C, 72.47; H, 5.41; N, 9.39.
Found: C, 72.42; H, 5.47; N, 9.34%.
3. Experimental
3.1. Materials and methods
3.2.2. 2-Fluoro-2-(3-methylphenyl)ethanenitrile 3c. Yield:
1
72% (light yellow oil). H NMR (250 MHz): l 2.41 (s,
Melting points were determined on a Bu¨chi SMP-20
and are uncorrected. Unless otherwise stated, H NMR
3H), 6.00 (d, J=47.1 Hz, 1H), 7.29–7.37 (m, 4H). Anal.
calcd for C9H8FN (149.2): C, 72.47; H, 5.41; N, 9.39.
Found: C, 72.68; H, 5.40; N, 9.24%.
1
spectra were recorded on a Bruker AC 250 F (250
MHz) and ARX 500 (500 MHz) in CDCl3 with TMS as
internal standard. Chromatography was performed
using silica gel S (Riedel-de Haen), grain size 0.032–
0.063 mm. HPLC was performed on a Pharmacia LKB
with VWM 2141 detector using an RP-C18 Vertex
column (25 cm×4 mm) with Nucleosil 100, 5 mm
(Knaur). Optical rotations were measured in a ther-
mostated polarimeter with l=10 cm. Enantiomeric
excesses: GC separations were conducted with a Chi-
raldex G-TA (ICT) column (30 m×0.32 mm), carrier
gas hydrogen. All solvents were dried and distilled.
3.2.3. 2-Fluoro-2-(4-methylphenyl)ethanenitrile 3d. Yield:
1
66% (light yellow oil). H NMR (250 MHz): l 2.40 (s,
3H), 6.00 (d, J=47.3 Hz, 1H), 7.25–7.46 (m, 4H). Anal.
calcd for C9H8FN (149.2): C, 72.47; H, 5.41; N, 9.39.
Found: C, 72.76; H, 5.60; N, 9.22%.
3.3. 2-Fluoro-2-(3-methoxyphenyl)ethanenitrile 3f
To
a
solution of 2f17 (4.71 g, 20.0 mmol) in
dichloromethane (50 mL) at −78°C under an inert gas
atmosphere DAST (3.55 g, 22.0 mmol) was slowly
added dropwise, and the reaction mixture was stirred
for 1 h at −78°C. The mixture was poured on ice (100
mL) and was vigorously stirred for 5 min. The organic
phase was separated and the aqueous phase was
extracted with diethyl ether (2×100 mL). The combined
organic phases were washed with a 5% aqueous
NaHCO3 solution to neutralize, and water. The organic
extract was then dried (Na2SO4) and concentrated. The
residue was chromatographed on silica gel with
petroleum ether/ethyl acetate (85:15) to give 3f as a
3.2. Preparation of 2-fluoroethanenitriles 3b,c and d;
general procedure
According to a known procedure,10a to the respective
methylbenzaldehyde 1b–d (2.40 g, 20 mmol) and ZnI2
(10 mg, 0.031 mmol) at 0°C under an inert gas atmo-
sphere, trimethylsilyl cyanide (1.98 g, 20 mmol) was
slowly added dropwise with stirring. After warming to
room temperature, the reaction mixture was stirred for
a further 20 min. The reaction mixture was mixed with
anhydrous dichloromethane (25 mL), cooled to 0°C,
and a solution of DAST (3.55 g, 22 mmol) in
dichloromethane (10 mL) was slowly added dropwise.
1
light yellow oil (2.32 g, 70%). H NMR (250 MHz): l
3.84 (s, 3H), 6.02 (d, J=46.9 Hz, 1H), 7.06–7.42 (m,
4H).

F. Effenberger, S. Oßwald / Tetrahedron: Asymmetry 12 (2001) 279–285
Table 4. Physical data and elemental analysis of 2-fluoroacetic acids 4
283
Compd
Mp (°C)
1H NMR (250 MHz, CDCl3) l
Elemental analysis (calcd/found)
Mol. formula
C
H
N
(mol. weight)
4a
103
5.81 (d, J=47.4 Hz, 1H), 7.37–7.49 (m, 5H), 11.50 (br s, 1H)
2.30 (s, 3H), 5.70 (d, J=47.8 Hz, 1H), 7.14–7.22 (m, 4H)
C8H7FO2 (154.1)
C9H9FO2 (168.2)
C9H9FO2 (168.2)
62.34
62.52
64.28
64.27
64.28
64.44
48.25
48.25
4.58
4.68
5.39
5.53
5.39
5.45
3.04
3.07
–
–
–
4ca
4da
4eb
83–85.5
108–109
92–94
2.34 (s, 3H), 5.95 (d, J=47.7 Hz, 1H), 7.26–7.37 (m, 4H), 13.50 (br s,
1H)
6.27 (d, J=46.9 Hz, 1H), 7.72–8.33 (m, 4H)
C8H6FNO4
(199.1)
7.03
6.96
a 500 MHz spectra.
b In DMSO-d6.
3.4. Acid hydrolysis of 2-fluoronitriles 3 to the 2-
fluoroacetic acids 4; general procedure
2.3)/acetonitrile (70:30) and analyzed by HPLC (flow
1 mL/min, detection wavelength 210 nm).
A vigorously stirred emulsion of 3 in a 49% aqueous
solution of sulfuric acid (50 mL/g of 3) was heated at
80°C for at least 3 h. After being cooled to room
temperature, the aqueous phase was extracted twice
with the double volume of diethyl ether. Acids 4 were
extracted from the combined organic phases with a sat.
NaHCO3 solution (1/10 volume referred to the ethereal
phase). The aqueous phase was acidified to pH 4 and
extracted twice with the double volume of diethyl ether.
The combined extracts were dried (Na2SO4) and con-
centrated. The residue was recrystallized from chloro-
form/petroleum ether (Table 4).
3.6.2. Determination of enantiomeric excesses of com-
pounds 3–5. A 5 M aqueous Na2CO3 solution (250 mL)
was added to the remaining 5 mL of the sample. The
reaction mixture was extracted with diethyl ether. The
aqueous phase was separated, acidified with a 5 M HCl
solution (500 mL) and extracted with diethyl ether.
After esterification with diazomethane,22 the enan-
tiomeric excess of acid 4 was determined by gas
chromatography.
The ethereal phase was concentrated, and the mixture
of nitrile 3 and amide 5 was separated by chromatogra-
phy on a silica gel column (5×0.5 cm). Compound 3
was eluted with petroleum ether/ethyl acetate (90:10)
(3×5 mL) and the e.e. value was determined by gas
chromatography directly from the filtrate. Compound 5
was eluted with ethyl acetate (5 mL), concentrated, and
hydrolysed to the corresponding acid in half-conc. sul-
furic acid by heating at 60°C for 3 h. After extraction
with diethyl ether (5 mL) and esterification with dia-
zomethane,22 the enantiomeric excess was determined
by gas chromatography.
3.5. Crude extract preparation and enzymatic hydrolysis
of 2-fluoronitriles (RS)-3 using the nitrilase from
Arabidopsis thaliana; general procedure
Cells were separated from the nutrient medium by
centrifugation for 30 min at 4°C (5700×g) and washed
with Tris/HCl buffer (70 mM, pH 8.0–9.5). The pellet
was re-suspended in Tris/HCl buffer (100 mL/10 g wet
weight), sonicated with ice cooling (3×5 min), and
centrifuged for 40 min (186 000×g). The supernatant
containing the crude enzyme was diluted with Tris/HCl
buffer (to reach 15–40% conversion after 2–4 h), and a
1 M solution of 3 in methanol (10 mM end concentra-
tion) was added. After being stirred for 2–4 h, samples
of 6 mL volume were taken and analyzed as described
in Section 3.6.
3.7. Enzymatic hydrolysis of (RS)-3a,c and f using the
nitrilase of Arabidopsis thaliana on a preparative scale;
general procedure
To a solution of crude enzyme (156 U) in Tris/HCl
buffer (370 mL, 70 mM, pH 9) at 7°C was added 3a,c
or f (0.5 g each) as a 1 M solution in methanol, and the
reaction mixture was stirred for 9.5 h. Then it was
acidified to pH 4 with a 5 M HCl solution and
extracted with diethyl ether. In order to separate the
formed acid 4, the combined organic phases were
extracted twice with a 10% (v/v) sat. aqueous NaHCO3
solution. The organic phase, containing nitrile 3 and
amide 5, was dried (Na2SO4) and concentrated. The
mixture of 3 and 5 was separated by chromatography
on silica gel with petroleum ether/ethyl acetate (90:10)
(for 3), followed by ethyl acetate (for 5). After concen-
3.6. Analytical procedure for the determination of
conversion, acid/amide ratios and enantiomeric excesses
3.6.1. Ratio of 3:4:5 determined by HPLC. The sample
(1 mL) was acidified with a 5 M HCl solution (50 mL)
and extracted with diethyl ether (5 mL). After centrifu-
gation (2000×g) and cooling at −30°C for 30 min to
freeze the aqueous phase, the organic phase was
decanted and concentrated in vacuo. The residue was
taken up in 1 mL of phosphate buffer (50 mM, pH

284
F. Effenberger, S. Oßwald / Tetrahedron: Asymmetry 12 (2001) 279–285
tration, (R)-amides
petroleum ether/chloroform. The amides 5d,e, required
as reference compounds, were prepared analogously.
5
were recrystallized from
CHCl3), e.e.=98%. NMR data correspond to those of
racemic 4c.
3.8.3. (R)-2-Fluoro-2-(3-methoxyphenyl)acetic acid (R)-
4f. White needles; mp 67–69°C; [h]2D0=+154.0 (c 1.0,
3.7.1. (R)-2-Fluoro-2-phenylacetamide (R)-5a. White
needles; mp 144–145°C; [h]2D0=+122.3 (c 0.9, CHCl3),
e.e.=>99%. 1H NMR (500 MHz, DMSO-d6): l 5.76 (d,
J=48.4 Hz, 1H), 6.26, 6.47 (br s each, 2H), 7.46–7.48
(m, 5H). Anal. calcd for C8H8FNO (153.2): C, 62.74;
H, 5.26; N, 9.15. Found: C, 62.61; H, 5.27; N, 9.14%.
1
CHCl3), e.e.=>99%. H NMR (500 MHz): l 3.77 (s,
3H), 5.95 (d, J=47.6 Hz, 1H), 6.99–7.38 (m, 4H), 13.50
(br s, 1H). Anal. calcd for C9H9FO3 (184.2): C, 58.70;
H, 4.93. Found: C, 58.91; H, 4.98%.
3.7.2. (R)-2-Fluoro-2-(3-methylphenyl)acetamide (R)-5c.
White needles; mp 125–126°C; [h]2D0=+107.3 (c 0.7,
Acknowledgements
1
CHCl3), e.e.=97%. H NMR (500 MHz, DMSO-d6): l
2.32 (s, 3H), 5.73 (d, J=48.1 Hz, 1H), 7.21–7.31 (m,
4H), 7.56, 7.85 (br s each, 2H). Anal. calcd for
C9H10FNO (167.2): C, 64.66; H, 6.03; N, 8.38. Found:
C, 64.50; H, 6.00; N, 8.36%.
We would like to thank the Bundesministerium fu¨r
Bildung und Forschung (Zentrales Schwerpunktpro-
gramm Bioverfahrenstechnik, Stuttgart) and the Fonds
der Chemischen Industrie for financial support. We
acknowledge Dr. H. Wajant for cloning the enzyme,
Dr. S. Fo¨rster for fermentations, and Dr. A. Baro for
preparing the manuscript.
3.7.3. 2-Fluoro-2-(4-methylphenyl)acetamide 5d. Mp
1
140–141°C. H NMR (250 MHz, DMSO-d6): l 2.31 (s,
3H), 5.73 (d, J=48.2 Hz, 1H), 7.21–7.35 (m, 4H), 7.57,
7.86 (br s each, 2H). Anal. calcd for C9H10FNO (167.2):
C, 64.66; H, 6.03; N, 8.38. Found: C, 64.56; H, 6.07; N,
8.35%.
References
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3.7.4. 2-Fluoro-2-(4-nitrophenyl)acetamide 5e. Mp 111–
113°C. 1H NMR (500 MHz, DMSO-d6): l 5.73 (d,
J=48.2 Hz, 1H), 7.22–8.31 (m, 4H), 7.71, 8.02 (br s
each, 2H). HRMS calcd for C8H7FN2O3 (M+)
198.0441. Found: 198.0443.
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3.7.5. (R)-2-Fluoro-2-(3-methoxyphenyl)acetamide (R)-
5f. White needles; mp 89–90°C; [h]2D0=+141.8 (c 1.0,
1
CHCl3), e.e.=>99%. H NMR (250 MHz, DMSO-d6):
l 3.73 (s, 3H), 5.76 (d, J=48.0 Hz, 1H), 6.95–7.56 (m,
4H), 7.56, 7.86 (br s each, 2H). Anal. calcd for
C9H10FNO2 (183.2): C, 59.01; H, 5.50; N, 7.65. Found:
C, 59.10; H, 5.63; N, 7.45%.
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(R)-2-fluoroacetic acids 4a,c and f; general procedure
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Amide 5a,c or f (0.82–1.31 mmol) was suspended in a
10% solution of sulfuric acid (25 mL/100 mg 5). The
vigorously stirred reaction mixture was heated at 60°C
for 3 h, and extracted twice with the double volume of
diethyl ether. The corresponding acid 4 was extracted
from the organic phase with a sat. NaHCO3 solution
(1/10 volume of the organic phase). The aqueous phase
was acidified to pH 4 and extracted with the double
volume of diethyl ether. The combined extracts were
dried (Na2SO4) and concentrated. Acids 4 were recrys-
tallized from petroleum ether/chloroform.
3.8.1. (R)-2-Fluoro-2-phenylacetic acid (R)-4a. White
needles; mp 104°C; [h]D20=+150.3 (c 1.0, CHCl3), e.e.=
>99%. [h]2D0=+71.4 (c 1.25, CHCl3), e.e.=46%.19 NMR
data correspond to those of racemic 4a.
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White needles; mp 83–84°C; [h]2D0=+137.4 (c 1.0,
Eur. J. Biochem. 1992, 205, 417–424.
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.