(E)-Selective hydrolysis of (E,Z)-α,β-unsaturated nitriles by the recombinant nitrilase AtNIT1 from Arabidopsis thaliana

Article abstract of DOI:10.1016/S0957-4166(01)00449-9

From stereoisomeric α,β-unsaturated nitriles (E,Z)-1, the recombinant nitrilase AtNIT1 from Arabidopsis thaliana hydrolyses the (E)-isomers exclusively to the corresponding (E)-carboxylic acids (E)-2 with high specificity. The (E)-selectivity can also be utilised for the preparation of the isomerically pure nitriles (Z)-1. From (E,Z)-2-hydroxycinnamonitrile (E,Z)-3, the otherwise difficult obtainable (Z)-3 was prepared in 66% isolated yield. With β,γ-unsaturated (E,Z)-3-heptenenitrile (E,Z)-4, however, (E)-selectivity was not observed. AtNIT1 exhibits not only diastereoselectivity but also regioselectivity. From a mixture of the four isomers A-D of 3-(2-cyanocyclohex-3-enyl)propenenitrile 6, exclusively isomer D ((E)-cis-6) was hydrolysed to 3-(2-cyanocyclohex-3-enyl)propenoic acid (E)-cis-7, as stated by X-ray crystal structure. Only after complete conversion of D and high enzyme concentrations, isomer C ((E)-trans-6) was hydrolysed to a small extent.

Full text of DOI:10.1016/S0957-4166(01)00449-9

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 2581–2587
(E)-Selective hydrolysis of (E,Z)-a,b-unsaturated nitriles by the
recombinant nitrilase AtNIT1 from Arabidopsis thaliana†
Franz Effenberger* and Steffen Oßwald‡
Institut fu¨r Organische Chemie der Universita¨t Stuttgart, Pfaffenwaldring 55, D-70569 Stuttgart, Germany
Received 6 September 2001; accepted 12 October 2001
Abstract—From stereoisomeric a,b-unsaturated nitriles (E,Z)-1, the recombinant nitrilase AtNIT1 from Arabidopsis thaliana
hydrolyses the (E)-isomers exclusively to the corresponding (E)-carboxylic acids (E)-2 with high specificity. The (E)-selectivity can
also be utilised for the preparation of the isomerically pure nitriles (Z)-1. From (E,Z)-2-hydroxycinnamonitrile (E,Z)-3, the
otherwise difficult obtainable (Z)-3 was prepared in 66% isolated yield. With b,g-unsaturated (E,Z)-3-heptenenitrile (E,Z)-4,
however, (E)-selectivity was not observed. AtNIT1 exhibits not only diastereoselectivity but also regioselectivity. From a mixture
of the four isomers A–D of 3-(2-cyanocyclohex-3-enyl)propenenitrile 6, exclusively isomer D ((E)-cis-6) was hydrolysed to
3-(2-cyanocyclohex-3-enyl)propenoic acid (E)-cis-7, as stated by X-ray crystal structure. Only after complete conversion of D and
high enzyme concentrations, isomer C ((E)-trans-6) was hydrolysed to a small extent. © 2001 Elsevier Science Ltd. All rights
reserved.
1. Introduction
ple, is known to be 20 times as active as the corre-
sponding (E)-isomer.2 By addition of a lithium salt the
isomeric ratio in the Wittig reaction was improved to
4:1 in favor of the (Z)-isomer, but isolation of the pure
(Z)-isomer requires chromatographic purification steps
and crystallization.2
In general, a,b-unsaturated nitriles are prepared via
Wittig-type reactions or via Knoevenagel reaction from
cyanoacetates and aldehydes or ketones. Apart from a
few exceptions, complete control of the (E,Z)-selectivity
has not been possible. Due to the possibly different
biological activities of stereoisomers, methods for the
preparation of pure (E)- and (Z)-isomers are of partic-
ular interest, especially for the synthesis of pharmaceu-
ticals. The (Z)-isomer of the b-lactamase inhibitor
2b-acrylonitrile penam sulfone Ro 48-1220, for exam-
Enzymes are applied mainly for the preparation of pure
enantiomers by kinetic resolution and by enantioselec-
tive synthesis, respectively. On the contrary, only very
few examples of enzymatic (E,Z)-selectivity of a,b-
unsaturated carboxylic acid derivatives are published.
Scheme 1.
* Corresponding author. E-mail: franz.effenberger@po.uni-stuttgart.de
† See Ref. 1.
‡ Part of dissertation, Universita¨t Stuttgart, 2000.
0957-4166/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00449-9

2582
F. Effenberger, S. Oßwald / Tetrahedron: Asymmetry 12 (2001) 2581–2587
The only known nitrile-hydrolysing enzyme with (E)-
selectivity is the nitrilase from Rhodococcus ATCC
39484, which is capable of hydrolysing selectively (E)-3-
furylacrylic nitrile but not the corresponding (Z)-iso-
mer.3 The extremely small substrate range of this
enzyme, however, is disadvantageous for synthetic
applications. Even closely structurally related com-
pounds such as cinnamonitrile are not accepted as
substrates. Further examples of (E,Z)-selective enzymes
Since the nitrilase shows complete (E)-selectivity
already in the case of the small methyl substituent in
crotononitrile, cinnamonitrile 1b, 4-phenyl-2-pentene-
nitrile 1c,6 and 3-methoxypropenenitrile 1e were applied
as substrates (Scheme 1, Table 1).
As can be seen in Table 1, the (E)-isomers of nitriles
(E,Z)-1b–1e were exclusively hydrolysed to the corre-
sponding (E)-acids with high specificity. After workup,
in all cases only the (E)-acids 2b–2e were detected by
GC, which were identified unambiguously by coinjec-
include
a lipase from Candida antarctica, which
hydrolyses exclusively diethyl fumarate but not diethyl
maleate,4 and a porcine liver carboxylesterase convert-
ing different methyl 3-arylacrylates with poor (E,Z)-
selectivity.5
1
tion (for 2b) or by H NMR spectroscopy (for 2c–2e).
1-Cyano-1,3-butadiene (E,Z)-1d was prepared by reac-
tion of acetonitrile with acrolein following a literature
method.7 After elimination of the mesyloxy group from
intermediate 1-(cyanomethyl)prop-2-enyl methylsul-
fonate, (E,Z)-1d was obtained in a ratio of E:Z=68:32
(see Section 4).
In our investigations on the synthetic applications of
the recombinant nitrilase AtNIT1 from Arabidopsis
thaliana we also used a,b-unsaturated nitriles as sub-
strates for the enzyme-catalyzed hydrolysis. In the
present publication we report on the results obtained
with these compounds.
Besides the important (E)-selectivity of the AtNIT1-cat-
alyzed hydrolysis of isomeric mixtures of a,b-unsatu-
rated nitriles, in many cases chemical hydrolysis of
a,b-unsaturated nitriles is not possible at all. For exam-
ple, the enol ether (E,Z)-1e and the diene (E,Z)-1d,
which decompose under acid or base catalysis, were
hydrolysed by the nitrilase to (E)-2d and (E)-2e in good
yields. (E)-3-Methoxyacrylic acid (E)-2e, for example,
is used as the starting material for the preparation of
the antibiotic chlorothricin.8
2. Results and discussion
2.1. (E,Z)-Selectivity in the hydrolysis of a,b-unsatu-
rated nitriles 1
The (E)-selectivity in the hydrolysis of structurally very
different a,b-unsaturated nitriles with A. thaliana nitril-
ase AtNIT1 can be used advantageously for the prepa-
ration of isomerically pure (Z)-nitriles, as demonstrated
for the preparation of nitriles (Z)-1a and (Z)-1e. A
further interesting example in connection with this is
the preparation of (Z)-2-hydroxycinnamonitrile (Z)-3,
a precursor for the b-antagonists 1-(cyanovinylphen-
oxy)-3-(alkylamino)butan-2-ols A9 (Scheme 2).
The (E,Z)-selectivity of recombinant AtNIT1 was first
detected and investigated in detail on crotononitrile 1a,
which is commercially available only as an (E,Z)-iso-
meric mixture (Scheme 1, Table 1). The nitrilase
hydrolyses exclusively the minor isomer to crotonic acid
2a. The formed acid 2a (as methyl ester) was unambigu-
ously identified as (E)-crotonic acid by gas chromatog-
raphy and coinjection of pure (E)-2a. Additionally the
configuration of the double bond was determined by 1H
NMR spectroscopy. (E)-1a was hydrolysed completely
even at high concentrations of 0.25 M, giving (E)-2a in
27% isolated yield (referred to starting (E,Z)-1a 4:6)
and >99% GC purity. The corresponding (Z)-2a (detec-
tion limit <0.5% referred to (Z)-1a) could not be found
neither using higher enzyme concentrations nor by dou-
bling the reaction time. The remaining (Z)-1a was
isolated in isomerically pure form in 57% yield after
distillation (determined by GC and NMR).
(E,Z)-3 was prepared by Wittig reaction as outlined in
Scheme 2. The E:Z ratio could be improved in favor of
the (Z)-isomer to a ratio E:Z=19:81 by addition of
lithium perchlorate. After enzymatic hydrolysis, the
(E)-2-hydroxycinnamic acid was separated by extrac-
tion with sodium bicarbonate solution, and the nitrile
(Z)-3 was isolated in 66% total yield referred to starting
(E,Z)-3.
Table 1. (E)-Selective hydrolysis of a,b-unsaturated nitriles (E,Z)-1 catalyzed by Arabidopsis thaliana nitrilase
Substrate
Reaction time (h)
Rel. activity (%)a
Products
(E)-2
1
Ratio E:Z
(Z)-1
Yield (%)b
Yield (%)b
1a
1b
1c
1d
1e
4:6
6:4
3:1
68:32
37:63
3 (24)c
4
37
48
22
44
27
1a
1b
1c
1d
1e
51
–
–
–
47
2a
2b
2c
2d
2e
27
–
61
41
30
3 (21)c
2 (24)c
3 (48)c
a Rel. activity of the (E)-isomer.
b Isolated yield referred to total amount of starting (E,Z)-1.
c Reaction time of the preparative enzymatic hydrolysis at pH 8.5 and room temperature.

F. Effenberger, S. Oßwald / Tetrahedron: Asymmetry 12 (2001) 2581–2587
2583
Scheme 2.
pounds at higher temperature in acidic or basic
medium.11 Chemical hydrolysis of these compounds is
therefore accompanied by the formation of by-prod-
ucts. The A. thaliana nitrilase hydrolyses (E,Z)-4 with
high specific activity exclusively to (E)- and (Z)-3-hep-
tenoic acid 5 in 91% yield with an isomeric ratio of
E:Z=79:21, identical to that of the starting material
(E,Z)-4.
2.2. Investigation of E:Z selectivity in the hydrolysis of
the b,g-unsaturated nitrile (E,Z)-4
On the basis of the results described for a,b-unsatu-
rated nitriles, the scope of (E)-selectivity of recombi-
nant AtNIT1 was investigated with the b,g-unsaturated
nitrile (E,Z)-3-heptenenitrile 410 (Scheme 3, Fig. 1).
As can be seen from Fig. 1, the conversion rate of both
stereoisomers is very similar. Thus, the enrichment of
one isomer was not observed neither for the nitrile nor
the acid. Nevertheless, the enzymatic hydrolysis is of
synthetic interest, because b,g-unsaturated nitriles as
well as b,g-unsaturated acids tend to isomerize to the
thermodynamically more stable a,b-unsaturated com-
2.3. Regio- and stereoselective hydrolysis of 3-(2-
cyanocyclohex-3-enyl)propenenitriles 6
3-(2-Cyanocyclohex-3-enyl)propenenitrile 6, a cyclic
dinitrile with non-equivalent nitrile groups, was pre-
pared by Diels–Alder reaction of 1-cyano-1,3-butadiene
Scheme 3.
Figure 1. Course of conversion of (E,Z)-3-heptenenitrile, (E,Z)-4 (10 mM) at pH 8.0; (E)-isomer (ꢀ) and (Z)-isomer (ꢁ).

2584
F. Effenberger, S. Oßwald / Tetrahedron: Asymmetry 12 (2001) 2581–2587
Scheme 4.
1d (Scheme 4).12 After distillative and gas chromato-
3. Conclusion
graphic separation and enrichment, respectively, four
1
In summary, the extremely high selectivity in
hydrolysing only the dinitrile D from the mixture of the
four dinitriles A–D is due firstly to the high regioselec-
tivity of the A. thaliana nitrilase, preferring nitrile
groups unbranched in the a-position,1 secondly, due to
complete (E)/(Z) selectivity, hydrolysing exclusively the
isomer with the (E)-configurated exocyclic double bond
and thirdly, due to the very high cis/trans selectivity
(diastereoselectivity), favoring the cis-1,2-disubstituted
cyclohex-3-ene.
main products could be isolated and identified by H
NMR as E/Z (with respect to the exocyclic double
bond) and cis/trans (with respect to the ring sub-
stituents) isomers A–D12b (Scheme 4).
We have prepared the analytically pure compounds
A–D starting from (E,Z)-1d (E:Z=68:32) by heating at
160°C for 0.5 h in 69% yield with a purity of 95% for
the four isomers A–D, determined by gas chromatogra-
phy. The ratios A:B:C:D were 11:5:15:69. From this
isomeric mixture of dinitriles exclusively isomer D was
hydrolysed by the A. thaliana nitrilase. Only after com-
plete conversion of D and at higher enzyme concentra-
tions was isomer C also hydrolysed to a small extent.
The isomers A and B, however, remained totally
unchanged even at high enzyme concentrations and
long reaction times. The hydrolysis of both C and D
occurs exclusively on the alkylidene nitrile group yield-
ing the corresponding (E)-3-(2-cyanocyclohex-3-enyl)-
propenoic acids 7, as confirmed by NMR spectroscopy.
For the (E)-cyanocyclohexenylpropenoic acid (E)-cis-7
derived from isomer D the cis-configuration of the ring
substituents could unambiguously be assigned by X-ray
crystal structure (Fig. 2).13 Thus, isomer D has (E)-cis-
configuration ((E)-cis-6), while isomer C is assumed to
be (E)-trans-configurated [(E)-trans-6].
4. Experimental
4.1. Materials and methods
Melting points were determined on a Bu¨chi SMP-20
1
and are uncorrected. Unless otherwise stated, H and
13C NMR spectra were recorded on a Bruker AC 250 F
The different reaction rates for the enzyme-catalyzed
hydrolysis of (E)-trans-6 and (E)-cis-6 can be used to
separate these isomers. As shown in Scheme 4, the
enzymatic hydrolysis gave 97% (E)-cis-7 and 3% (E)-
trans-7 at 95% conversion referred to isomer D. After
recrystallization from petroleum ether/chloroform, (E)-
cis-7 was isolated in 84% yield (referred to D in the
starting isomeric mixture).
Figure 2. ORTEP view of (E)-cis-3-(2-cyanohex-3-enyl)-
propenoic acid 7 derived from isomer D.

F. Effenberger, S. Oßwald / Tetrahedron: Asymmetry 12 (2001) 2581–2587
2585
(250 MHz) and ARX 500 (500 MHz) in CDCl3 with
TMS as internal standard. Chromatography was per-
formed using silica gel S (Riedel-de Haen), grain size
0.032–0.063 mm. Gas chromatography separations
were conducted using capillary glass columns (20 m×
0.32 mm) with OV 1701, carrier gas hydrogen. All
solvents were dried and distilled. The recombinant
AtNIT1 was obtained by overexpression in E. coli as
described elsewhere.1
mer for 1b and 1c) was added (the concentration of the
stock solution must be as high as the added volume
should not exceed 100 mL/5 mL reaction solution).
After stirring for the time given in Table 1, samples of
1 mL volume were taken and analyzed by gas chro-
matography (see Section 4.4).
4.4. Determination of conversion by gas
chromatography
The sample taken was acidified with a 5 M HCl solu-
tion (50 mL) and extracted with diethyl ether (5 mL).
After centrifugation (2000×g) and cooling at −30°C for
30 min to freeze the aqueous layer, the organic layer
was decanted and a 0.2 M solution of diazomethane in
diethyl ether15 was added. The excess diazomethane and
diethyl ether was distilled off under vacuum. The
residue was taken up in diethyl ether (1 mL), and the
conversion was determined from the ratio area of
methyl ester to area of nitrile.
4.2. 1-Cyano-1,3-butadiene (E,Z)-1d
To a solution of BuLi in hexane (1.6 M, 131 mL, 210
mmol) and dry THF (160 mL) at −78°C under an inert
gas atmosphere was slowly added a solution of acetoni-
trile (8.21 g, 200 mmol) in THF (40 mL). After stirring
for 15 min, acrolein (13.45 g, 240 mmol) was added
over 5 min. The reaction mixture was warmed to −20°C
and methanesulfonyl chloride (27.49 g, 240 mmol) was
added over 5 min. After stirring for a further 1 h at
−20°C and 6 h at room temperature, the mixture was
poured into ice (250 mL)/satd NH4Cl solution (50 mL).
The layers were separated and the aqueous layer was
extracted with diethyl ether (2×100 mL). The combined
organic layers were washed with water until neutral,
dried (Na2SO4) and concentrated to give crude 1-
(cyanomethyl)prop-2-enyl methylsulfonate (39.13 g). To
a solution of crude intermediate (10 g, 57.1 mmol) in
THF (100 mL) was added dry sodium acetate (6.28 g,
76.5 mmol). After stirring at 40°C for 6 h, the mixture
was poured on ice (50 mL) and the aqueous layer was
extracted with diethyl ether (2×50 mL). The combined
organic layers were washed with water until neutral,
dried (Na2SO4) and concentrated. Distillation through
a Vigreux column under vacuum gave 2.40 g (53%) of
(E,Z)-1d, E:Z=68:32, bp 43°C/20 mbar. (E)-Isomer:
13C NMR (63 MHz): l 98.24 (CH), 116.01 (CN),
127.00, 132.74, 149.53 (CH, CH2ꢀ). (Z)-Isomer: 13C
NMR (63 MHz): l 99.90 (CH), 117.75 (CN), 126.77,
134.14, 150.62 (CH, CH2ꢀ). Anal. calcd for C5H5N
(79.1): C, 75.92; H, 6.37; N, 17.71. Found: C, 75.95; H,
6.43; N, 17.59%.
4.5. Enzymatic hydrolysis of nitriles (E,Z)-1 on a
preparative scale; general procedure
To a crude enzyme solution was added the respective
neat nitrile 1a, 1c–1e (Table 2), and the reaction mix-
ture was stirred for the time given in Table 1. Then the
mixture was acidified with 5 M HCl to pH 4.0 and
extracted with diethyl ether. The combined extracts
were washed twice with 10% (v/v) satd NaHCO3 solu-
tion, dried (Na2SO4) and concentrated. The combined
aqueous extracts were washed with a double volume of
diethyl ether, acidified to pH 4.0 and again extracted
with diethyl ether. The combined organic extracts were
dried (Na2SO4) and concentrated. The crude products
were purified as described.
4.5.1. (E)-4-Phenyl-2-pentenoic acid (E)-2c. Recrystal-
1
lization from benzene/chloroform, mp 171–172°C. H
NMR (250 MHz): l 1.44 (d, J=7.0 Hz, 3H, CH3), 3.64
(m, 1H, 4-CH), 5.81 (q, J1=15.6, J2=1.6 Hz, 1H,
2-CH), 7.18–7.42 (m, 6H, Ph, 3-CH). 13C NMR (63
MHz): l 20.09 (CH3), 42.15 (C4), 119.39 (C2), 126.87,
127.35, 128.76, 142.92 (Ph), 155.46 (C3), 172.03
(CO2H). Anal. calcd for C11H12O2 (176.2.): C, 74.98; H,
6.86. Found: C, 75.20; H, 6.86%.
4.3. Enzymatic hydrolysis of nitriles (E,Z)-1 on an
analytical scale; general procedure
A crude enzyme solution, prepared as previously
described,1,14 was diluted with Tris/HCl buffer, pH 8.5
(to reach 15–40% conversion after 2–4 h), and a solu-
tion of the respective nitrile in methanol (10 mM of the
(E)-isomer for 1a, 1d and 1e; 1.25 mM of the (E)-iso-
4.5.2. (E)-Penta-2,4-dienoic acid (E)-2d. Recrystalliza-
1
tion from petroleum ether, mp 34–37°C. H NMR (500
MHz): l 5.56 (d, J=11.2 Hz, 1H, CH2ꢀ), 5.67 (d,
J=16.8 Hz, 1H, CH2ꢀ), 5.91 (d, J=15.4 Hz, 1H,
Table 2. Preparative enzymatic hydrolysis of nitriles (E,Z)-1 at 100% conversion of the (E)-isomer
Substrate
g (mmol)
Nitrilase (Units)
Buffer (mL)
Products
(E)-2
1
(Z)-1
(g)
(g)
1a
1c
1d
1e
5.0 (74.5)
0.5 (3.18)
1.0 (12.6)
5.0 (60.2)
23.0
41.2
10.3
23.0
500
200
100
300
(Z)-1a
(Z)-1c
(Z)-1d
(Z)-1e
2.58
–
–
(E)-2a
(E)-2c
(E)-2d
(E)-2e
1.76
0.34
0.51
1.86
2.36

2586
F. Effenberger, S. Oßwald / Tetrahedron: Asymmetry 12 (2001) 2581–2587
2-CH), 6.49 (m, 1H, 4-CH), 7.36 (q, J=11.3 Hz, 1H,
3-CH), 11.30 (br s, 1H, CO2H). 13C NMR (126 MHz):
l 121.35, 126.84, 134.53, 147.05 (C2,3,4,5), 172.43
(CO2H). MS (EI, 70 eV) m/z (%): 220.1 (32), 205.2 (80),
98.1 (100) [M]+, 71.1 (70), 57.0 (99). HRMS (EI, 70 eV)
calcd for C5H6O2 (M+) 98.03666. Found: 98.03678.
4.7. Enzymatic hydrolysis of 3-(2-cyanocyclohex-3-
enyl)propenenitrile 6
4.7.1. (E)-cis-3-(2-Cyanocyclohex-3-enyl)propenoic acid
(E)-cis-7. An isomeric mixture of (E,Z)-612 (1 g, 6.32
mmol) was hydrolysed as described above with nitrilase
(34.3 U) in Tris/HCl buffer (250 mL, 70 mM, pH 8.5).
After 24 h at 95% conversion, the reaction was termi-
nated. Purification by recrystallization from petroleum
ether/chloroform gave 0.65 g (84%, referred to D in
4.5.3. (E)-3-Methoxypropenoic acid (E)-2e. Recrystal-
1
lization from petroleum ether/chloroform, mp 64°C. H
NMR (250 MHz): l 3.73 (s, 3H, CH3O), 5.19 (d,
J=12.6 Hz, 1H, 2-CH), 7.72 (d, 1H, 3-CH), 11.50 (br s,
1H, CO2H). 13C NMR (63 MHz): l 57.56 (CH3O),
95.40 (C2), 165.17 (C3), 173.72 (CO2H). Anal. calcd for
C4H6O3 (102.1): C, 47.06; H, 5.92. Found: C, 47.10; H,
5.94%.
1
starting (E,Z)-6) of (E)-cis-7, mp 81–82°C. H NMR
(500 MHz): l 1.81–1.90 (m, 2H, 6-CH2), 2.14–2.30 (m,
2H, 5-CH2), 2.67–2.72 (m, 1H, 1-CH), 3.35–3.39 (m,
1H, 2-CH), 5.67–5.73 (m, 1H, 3-CH), 5.95–6.06 (m, 2H,
4-,2%-CH), 7.14 (dd, J1=15.7, J2=7.8 Hz, 1H, 3%-CH).
13C NMR (126 MHz): l 24.07, 24.14 (C5,6), 31.30 (C2),
38.10 (C1), 118.32 (CN), 119.94, 122.62 (C3,4), 131.95
(C2%), 149.56 (C3%), 170.63 (CO2H). Anal. calcd for
C10H11NO2 (177.2): C, 67.78; H, 6.26; N, 7.90. Found:
C, 67.82; H, 6.31; N, 8.06%.
4.5.4. (Z)-3-Methoxypropenenitrile (Z)-1e. Distillation
through a Vigreux column under vacuum, bp 71°C/17
1
mbar. H NMR (250 MHz): l 3.92 (s, 3H, CH3O), 4.40
(d, J=6.4 Hz, 1H, 2-CH), 6.73 (d, 1H, 3-CH). 13C
NMR (63 MHz): l 61.79 (CH3O), 74.35 (C2), 115.34
(CN), 164.31 (C3). Anal. calcd for C4H5NO (83.1): C,
57.82; H, 6.07; N, 16.86. Found: C, 57.64; H, 6.10; N,
16.62%.
4.7.2.
(E)-trans-3-(2-Cyanocyclohex-3-enyl)propenoic
acid (E)-trans-7. The reisolated isomeric mixture of
(E,Z)-6 (see above) was hydrolysed with nitrilase (3.4
U) in Tris/HCl buffer (70 mM, pH 8.5). After 6 h at
complete conversion of (E)-cis-6, the reaction was ter-
minated. Unreacted nitrile 6 was re-isolated and again
hydrolysed with nitrilase (34.3 U) in Tris/HCl buffer
(70 mM, pH 8.5). After 48 h at 63% conversion of
(E)-trans-6, the reaction was terminated. Purification
by recrystallization from petroleum ether/chloroform
4.6. (Z)-3-(2-Hydroxyphenyl)propenenitrile (Z)-3
(a) To
a
stirred suspension of cyanomethyl-
triphenylphosphonium chloride (4.52 g, 13.4 mmol) and
lithium perchlorate (2.34 g, 22 mmol) in dry THF (50
mL) at −78°C under an inert gas atmosphere was added
a 1.6 M solution of BuLi in hexane (8.37 mL, 13.4
mmol). After stirring for 1 h, a solution of 2-acetoxy-
benzaldehyde (2 g, 12.2 mmol) in dry THF (5 mL) was
added. The mixture was stirred for a further 1 h at
−78°C, and was then allowed to warm to room temper-
ature within 6 h. The mixture was poured on ice (50
mL) and the layers were separated. The aqueous layer
was extracted with diethyl ether (2×100 mL). The com-
bined organic layers were washed with water until
neutral, dried (Na2SO4) and concentrated. The residue
was chromatographed on silica gel with petroleum
ether/ethyl acetate (80:20) to separate triphenylphos-
phine oxide. To a solution of satd Na2CO3 (50 mL),
water (350 mL) and methanol (50 mL) was added a
solution of the remaining intermediate in methanol (50
mL). After stirring for 1.5 h at room temperature, the
mixture was extracted with diethyl ether (3×150 mL).
The combined extracts were washed with water (3×50
mL), dried (Na2SO4) and concentrated to give crude
(E,Z)-3 in an E:Z ratio of 19:81.
1
gave (E)-trans-7, mp 75–77°C. H NMR (500 MHz): l
1.54–2.03 (m, 2H, 6-CH2), 2.16–2.21 (m, 2H, 5-CH2),
2.72–2.78 (m, 1H, 1-CH), 1.36–3.20 (m, 1H, 2-CH),
5.64–5.67 (m, 1H, 3-CH), 5.97–6.07 (m, 2H, 4-,2%-CH),
7.01 (dd, J1=15.7, J2=7.8 Hz, 1H, 3%-CH).
4.8. Crystallography
The isomer (E)-cis-7 was recrystallized from petroleum
ether/chloroform to obtain single crystals for X-ray
analysis. Intensity data were collected on a Nicolet P3
diffractometer with ꢀ-scan technique (graphite-
,
monochromated Mo Ka radiation, u=0.71073 A) at
293 K. The structure was solved by direct methods and
refined16 against F2. Crystal data: formula, C10H11NO2
(177.2); crystal size, 0.9×0.25×0.25 mm; F(000)=376;
crystal system, monoclinic, space group, P2(1)/n; Z=4;
,
a=5.9713(10), b=15.143(2), c=10.6172(14) A; i=
3
93.388(12)°; V=958.4(3) A ; Dcalcd=1.228 g/cm3; num-
,
ber of reflections=1849; number of independent
reflections (with 2q in the range of 2.4–25.0°)=1682;
number of reflections having I>2|(I)=1509; GooF=
1.161; R1=0.0545; wR2=0.116.
(b) To a solution of enzyme (123 U) in Tris/HCl buffer
(300 mL, 70 mM, pH 8.5) was added crude (E,Z)-3.
After complete conversion of (E)-3 (28 h), the mixture
was worked up as described (see Section 4.4) to give
1
1.17 g (66%) of (Z)-3, mp 63°C. H NMR (250 MHz):
Acknowledgements
l 6.09 (d, J=11.3 Hz, 1H, 2-CH), 6.77–7.28 (m, 5H,
Ph), 7.55 (d, 1H, 3-CH). 13C NMR (63 MHz): l 96.86
(C2), 116.89 (CN), 119.54, 121.27, 126.34, 129.86,
132.67 (Ph), 147.75 (C3), 155.89 (Ph). Anal. calcd for
C9H7NO (145.2): C, 74.47; H, 4.86; N, 9.65. Found: C,
74.20; H, 4.88; N, 9.69%.
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

F. Effenberger, S. Oßwald / Tetrahedron: Asymmetry 12 (2001) 2581–2587
9. Tucker, H. J. Med. Chem. 1981, 24, 1364–1368.
2587
acknowledge Dr. H. Wajant for cloning the enzyme
and Dr. A. Baro for preparing the manuscript.
10. Tsuji, Y.; Yamada, N.; Tanaka, S. J. Org. Chem. 1993,
58, 16–17.
11. Beyer, H.; Walter, W. Lehrbuch der Organischen Chemie,
22nd ed.; Hirzel Verlag: Stuttgart, 1991; p. 244.
12. (a) Snyder, H. R.; Poos, G. I. J. Am. Chem. Soc. 1949,
71, 1395–1396; (b) Weigert, J. F. J. Org. Chem. 1977, 42,
3859–3863.
13. Crystallographic data (excluding structure factors) for the
structure in this paper have been deposited with the
Cambridge Crystallographic Data Centre as supplemen-
tary publication number CCDC 168878. Copies of the
data can be obtained, free of charge, on application
to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK
[fax: +44(0)-1223-336033 or e-mail: deposit@ccdc.cam.ac.
uk].
14. Effenberger, F.; Oßwald, S. Synthesis 2001, 1866–1872.
15. deBoer, T. J.; Backer, H. J. Org. Synth., Coll. Vol. 4
1963, 250–253.
16. (a) Sheldrick, G. M. SHELXL-93. Program for Refining
Crystal Structures, University of Go¨ttingen, Germany,
1993; (b) Sheldrick, G. M. Acta Crystallogr., Sect. A
1990, 46, 467.
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