A simple separation method for (S)-hydroxynitrile lyase from cassava and its application in asymmetric cyanohydrination

Article abstract of DOI:10.1016/j.tetasy.2013.03.015

Using an acetone precipitation method, crude (S)-hydroxynitrile lyase [(S)-MeHNL] was separated from Munihot esculenta (cassava) leaves, and used directly as biocatalyst to catalyze asymmetric cyanohydrination and produce cyanohydrins with enantiomeric purities (≥90% ee) significantly greater than those previously reported. The use of a water/i-Pr2O system with an enzyme, NaCN, and appropriate amounts of acetic acid is crucial in improving the stereoselectivity of cyanohydrin formation by minimizing the non-enzymatic reaction and the racemization of the chiral products. The proposed isolation method for crude (S)-MeHNL has a high value because of its simplicity, and low cost as well as the high activity of the crude (S)-MeHNL.

Full text of DOI:10.1016/j.tetasy.2013.03.015

Tetrahedron: Asymmetry 24 (2013) 434–439
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
A simple separation method for (S)-hydroxynitrile lyase from cassava
and its application in asymmetric cyanohydrination
⇑
Zubiao Zheng, Yan Zi, Zhongzhou Li, Xinzhuo Zou
Department of Chemistry, East China Normal University, Shanghai 200062, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 January 2013
Accepted 11 March 2013
Using an acetone precipitation method, crude (S)-hydroxynitrile lyase [(S)-MeHNL] was separated from
Munihot esculenta (cassava) leaves, and used directly as biocatalyst to catalyze asymmetric cyanohydri-
nation and produce cyanohydrins with enantiomeric purities (P90% ee) significantly greater than those
previously reported. The use of a water/i-Pr2O system with an enzyme, NaCN, and appropriate amounts
of acetic acid is crucial in improving the stereoselectivity of cyanohydrin formation by minimizing the
non-enzymatic reaction and the racemization of the chiral products. The proposed isolation method
for crude (S)-MeHNL has a high value because of its simplicity, and low cost as well as the high activity
of the crude (S)-MeHNL.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
suitable for preparative purposes and to meet the demands of
industrial use, have stimulated much effort in the fields of cloning
Asymmetric cyanohydrination is an important process in organ-
ic synthesis. Cyanohydrins can be easily converted into a variety of
and genetic modification.21–23 Developing a simple and efficient
method for the large-scale extraction of (S)-MeHNL from cassava
is still a worthwhile endeavor, because cassava, as a valuable
renewable resource, is a widely grown crop all over the world.
Therefore, the use of extracts from cassava leaves for (S)-selective
HNL, instead of using isolated enzymes is worth noting.11,24 On the
other hand, the general use of the volatile and extremely toxic
HCN25–36 or acetone cyanohydrins37–40 as cyanide sources in asym-
metric cyanohydrination could result in safety concerns. The
development of non-volatile cyanide sources is also highly desir-
able.15,41 The simplest approach is to directly use a metal cyanide
salt as a substitute for HCN or acetone cyanohydrin9,42 (Fig. 1).
We are currently engaged in a search for a simple and accessible
separation method for crude (S)-MeHNL from cassava, and have
tested the capability of crude (S)-MeHNL to biocatalyze the addi-
tion of a nonvolatile cyanide source to aldehydes to produce the
corresponding cyanohydrins.
valuable synthetic intermediates, such as
a-hydroxyketones, a-
hydroxyacids, b-aminoalcohols, amino-nitriles, and so on, which
are important organic intermediates of academic and commercial
interest.1–7 Chiral catalysts, such as synthetic chiral8–10 and biolog-
ical catalysts,11–15 are generally required in asymmetric cyanohy-
drin synthesis. Due to the availability and the excellent
stereoselectivities obtained, the biocatalytic formation of asym-
metric cyanohydrins using enzymes has been widely investigated
and is considered to be a reliable method.16,17 However, the occur-
rence of (S)-hydroxynitrile lyases in Nature is not as widespread as
(R)-lyases, which are only present in a few cyanogenic plants such
as Manihot esculenta (MeHNL), Hevea brasiliensis (HbHNL), and Sor-
gum bicolor (SbHNL). The separation method for (S)-MeHNL from
cassava leaf tissue was reported by Effenberger et al., who com-
bined the application of anion exchange chromatography and gel
filtration; the (S)-MeHNL was capable of catalyzing the addition
of HCN to several aliphatic carbonyls in organic media, thus dem-
onstrating the potential usefulness of this enzyme in the stereose-
lective synthesis of cyanohydrins.18 By using ammonium sulfate
precipitation, the (S)-MeHNL was purified at 60–80% saturation
in the reports by Chulavatnatol et al.19 and White et al.20 Although
(S)-MeHNL could be isolated by the aforementioned methods from
cassava leaves, the quantities that can be obtained were limited.
Thus, attempts to acquire sufficient quantities of (S)-MeHNL
2. Results and discussion
In order to determine the tissue- and organ-specific localization
of HNL, total soluble proteins were extracted from leaves, stems,
and roots for the determination of HNL activity. In addition to
leaves, no detectable HNL activity was present in root and stem
crude extracts.20 Therefore, cassava leaves were chosen as raw
materials for the extraction of HNL. In an effort to optimize the
separation conditions with the cassava leaves as the source, we
found the total activity of lyase was determined by the separation
procedure. Generally, the higher the lyase purity, the lower the
⇑
Corresponding author. Tel.: +86 21 54340106.
E-mail address: xzzou@chem.ecnu.edu.cn (X. Zou).
0957-4166/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetasy.2013.03.015

Z. Zheng et al. / Tetrahedron: Asymmetry 24 (2013) 434–439
435
OAc
CN
O
O
OH
crude(S)- MeHNL
CN
(CH3CO)2O
H
H
NaCN, HOAc
pyridine,
30oC
2a
(S)-
(S)-3a
1a
OH
CN
H
O
1a
(S)-2a
= (S)-MeHNL
CN-
O
AcO-
+
HOAc
HCN
+
racemization
O-
OH
CN
H2O
CN
H
rac-2a
Figure 1. Competition of the enzymatic and non-enzymatic reactions.
stability of the lyase.43 Additionally, a more sophisticated isolation
method would result in a lower yield of the lyase and a higher pro-
duction cost.18 A one time acetone precipitation method was cho-
sen to separate the (S)-MeHNL from fresh cassava leaves because of
its simplicity. Therefore, the leaves were refrigerated and homoge-
nized by grinding in a mortar. The powder of the total soluble pro-
teins containing (S)-MeHNL was collected and used directly as a
catalyst in the asymmetric cyanohydrination, when 2.5 times the
volume of acetone was added to the extract at ꢀ20 °C, which
was prepared from cassava leaves with a solution of 5% ammonium
sulfate after filtration.
First, the acid inhibition of the nonenzymatic reaction was
investigated (Table 1). Racemic cyanohydrins were observed in
89% yield when 0.8 equiv of acetic acid were added into solution
(entry 1). Increasing the amount of acetic acid to 1.0 and 1.2 equiv
gave (S)-cyanohydrins with 55% yield, 94% ee and 61% yield, 97%
ee, respectively (entries 2 and 3). However, a further increase to
1.5 equiv led to no further improvement in the yield or ee value
(entry 4). These results showed that 1.2 equiv of acetic acid was
more conducive to this biocatalysis.
Table 2
Effect of water on the enzymatic reactiona
Entry
H2O/i-Pr2O (mL/mL)
Yieldb (%)
eec (%)
1
2
3
4
13:2
10:5
7.5:7.5
5:10
30
61
53
40
90
97
85
70
a
The reaction was performed with 3 mmol of benzaldehyde 1a, 4.0 g of crude
(S)-MeHNL, and 4.0 mmol of NaCN in ice-water bath for 48 h. The product of cya-
nohydrins 2a was then transformed into O-acetyl derivative 3a in 10 mmol of acetic
anhydride and 10 mmol of pyridine.
b
Isolated yield.
c
ee values were determined by chiral gas chromatography.
NaCN and catalyst mixtures (entry 1), resulting in only 30% yield
and 90% ee, respectively. By contrast, excessive water was benefi-
cial to the nonenzymatic reaction and racemization, which re-
sulted in a decrease in the enantiomeric excesses (entries 3 and
4). This phenomenon indicates that lyase and water are indispens-
able to the enantioselectivity of the enzymatic reaction.44–47
Next, the effect of crude (S)-MeHNL on the enzymatic reaction
was investigated (Table 3). In this system, when 2.0 g of crude
(S)-MeHNL was used, 3a was obtained with only 51% yield and
78% ee, whereas 4 g of crude (S)-MeHNL resulted in an increase
of the ee value of 3a to 97% (61% yield) (entries 1 and 2). However,
both the yield and the ee value of 3a decreased when a larger
Table 1
Effects of acid on the enzymatic reactiona
Entry
Acetic acid (mmol)
Yieldb (%)
eec (%)
1
2
3
4
3.2 (0.8 M)
4.0 (1.0 M)
5.0 (1.2 M)
6.0 (1.5 M)
89
55
61
49
0
94
97
95
Table 3
a
Effect of the catalyst separated by a one time acetone precipitation on the enzymatic
The reaction was performed with 3 mmol of benzaldehyde 1a, 4.0 g of crude
reactiona
(S)-MeHNL, 4.0 mmol of NaCN, and 5 mL of water in 10 mL of i-Pr2O in an ice-water
bath for 48 h. The product of cyanohydrins 2a was then transformed into O-acetyl
derivative 3a in 10 mmol of acetic anhydride and 10 mmol of pyridine.
Entry
Biocatalyst (g)
Yieldb (%)
eec (%)
b
Isolated yield.
1
2
3
4
2
4
6
8
51
61
32
35
78
97
93
90
c
ee values were determined by chiral gas chromatography.
Secondly, the effect of the volume ratio between water and i-
Pr2O was investigated (Table 2). Increasing the ratio of H2O/i-
Pr2O from 13:2 to 10:5 resulted in an evident increase in the ee va-
lue of 3a from 90% to 97% (entries 1 and 2). The addition of water
enhanced the homogeneity of the solid NaCN and crude lyase in
the reaction. Using not enough water was adverse to the solid
a
The reaction was performed with 3 mmol of benzaldehyde 1a, 4.0 mmol of
NaCN, and 5 mL of water in 10 mL of i-Pr2O in an ice-water bath for 48 h. Next, the
product of cyanohydrins 2a was transformed into O-acetyl derivative 3a in 10 mmol
of acetic anhydride and 10 mmol of pyridine.
b
Isolated yield.
c
ee values were determined by chiral gas chromatography.

436
Z. Zheng et al. / Tetrahedron: Asymmetry 24 (2013) 434–439
Table 4
amount of crude (S)-MeHNL (entries 3 and 4) was added to the
reaction mixture. The results indicate that crude (S)-MeHNL had
an affect on the enantiomeric purity of the product. The addition
of crude (S)-MeHNL with relatively high impurity content isolated
by one time acetone precipitation enhanced the unhomogeneity of
the solid NaCN and the catalyst in the reaction.
Although (S)-MeHNL could be isolated by a one time acetone
precipitation method from cassava leaves, (S)-MeHNL was only a
small part of the total proteins. Based on the relative cross-reactiv-
ity of the HNL present in leaves, Sayre et al. estimated that HNL
represents approximately 7% of the total leaf soluble protein.20
We also found that the crude (S)-MeHNL with a relatively higher
purity could be obtained by acetone fractional precipitation at
the ratio of 2.0/1–2.5/1 (Vacetone/Vextract). By contrast, the amount
of catalyst in the enzymatic reaction could be reduced from 4 to
0.8 g (Table 4, entry 2). However, the addition of a larger amount
of catalyst led to only slight improvements on the reaction (Table 4,
entries 3 and 4).
Effect of crude (S)-MeHNL separated by grade acetone precipitation on the enzymatic
reactiona
Entry
Crude (S)-MeHNL (g)
Yieldb (%)
eec (%)
1
2
3
4
0.4
0.8
1.2
1.6
51
65
67
67
78
97
97
96
a
The reaction was performed with 3 mmol of benzaldehyde 1a, 4.0 mmol of
NaCN, and 5 mL of water in 10 mL of i-Pr2O in an ice-water bath for 48 h. Next, the
product of cyanohydrins 2a was transformed into O-acetyl derivative 3a in 10 mmol
of acetic anhydride and 10 mmol of pyridine.
b
Isolated yield.
c
ee values were determined by chiral gas chromatography.
hyde, and aliphatic aldehydes. The results are listed in Table 5.
The yields and ee values of 3b, 3d–3f, with a strong electron-
donating group on the benzene ring and 3g, with a halogen atom
were obtained with P55% and P90%, respectively (Table 5, entries
2 and 4–7). The same process was used to prepare 3h from 1h with
two groups on the benzene ring; the yield obtained was 69% and
the ee value was 97%. Transformation of furan-2-yl carboxalde-
hyde into the corresponding cyanodydrin was accomplished in
medium yield and 90% ee (entry 9). Thien-2-yl cyanohydrin was
obtained in 75% yield and 94% ee (entry 10). The introduction of
The optimal reaction conditions in terms of yield required
3 mmol of 1a, 0.8 g of crude (S)-MeHNL separated by acetone frac-
tional precipitation, 4.0 mmol of NaCN, 4.8 mmol of acetic acid,
and 5 mL of water in 10 mL of i-Pr2O with an ice-water bath. In or-
der to examine the scope of this protocol, the optimized conditions
were then applied to the synthesis of a variety of substituted alde-
hydes, such as aromatic aldehydes, furfural, thienyl carboxalde-
Table 5
Synthesis of cyanohydrins by crude (S)-MeHNL catalyzed reactionsa
O
OH
OAc
(S)-MeHNL, H2O
(CH3CO)2O
pyridine, 30oC
NaCN, HOAc, i-Pr2O,
R
H
R
R
CN
CN
ice-water bath
1
2
3
Me
MeO
Me
OMe
S
MeO
Cl
1e
1a
1b
1c
1d
1f
1g
O
O
S
S
Me
Br
CHO
O
S
1m
1j
1k
1n
1l
1i
1h
Entry
Substrate
Product
Yieldb (%)
eec (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
98
67
54
60
72
55
61
69
54
75
40
40
90
85
97
91
85
90
96
91
96
97
90
94
96
86
88
87
a
The reaction was performed with 3 mmol of 1, 0.8 g of crude (S)-MeHNL, 4.0 mmol of NaCN, 4.8 mmol of acetic acid, and 5 mL of water in 10 mL of i-Pr2O in an ice-water
bath for 48 h, after which the products of cyanohydrins 2 were transformed into O-acetyl derivatives 3 in 10 mmol of acetic anhydride and 10 mmol of pyridine.
b
Isolated yield.
c
Determined by chiral GC on the O-protected derivatives.

Z. Zheng et al. / Tetrahedron: Asymmetry 24 (2013) 434–439
437
a methyl substituent at the 5-position of the thienyl ring (entry 11)
resulted in a decrease in the yield but not in the ee. Bromo substi-
tution at the 5-position (entry 12) gave a lower yield and ee of the
cyanohydrin compared with the non-substituted substrate. The
cyanohydrins of furan-2-yl carboxaldehyde and thien-2-yl carbox-
aldehydes had an (R)-configuration. Furthermore, an aliphatic
aldehyde, butyraldehyde 1n, was selected as a test substrate, and
(S)-(ꢀ)-pentanenitrile 3n was obtained in 85% yield and with
87% ee (entry 14).
move insoluble material. The crude extract was fractionated by
acetone precipitation at ꢀ20 °C. The precipitate at the ratio of
2.0/1–2.5/1 (Vacetone/Vextract) was collected by centrifugation
(4000 r/min) for 15 min at 4 °C and stored in a refrigerator for fur-
ther use.
4.4. General procedure for the synthesis of asymmetric O-acetyl
derivatives of cyanohydrins
A mixture of 0.8 g of crude (S)-MeHNL, 4.0 mmol of NaCN,
4.8 mmol of acetic acid, and 5 ml of water in 9.0 ml of i-Pr2O was
cooled in a 50 ml flask with an ice-water bath for 48 h, and then
3 mmol of substrate in 1.0 ml of i-Pr2O was added dropwise, and
the mixture was stirred 48 h. Afterward, the mixture was extracted
with acetic ether (10 ml ꢁ 3); the combined organic layer was
added to a mixture of 10 mmol of acetic anhydride and 10 mmol
of pyridine, and the mixture was stirred for 3 h at 30 °C. Next,
10 ml of water was added, and the organic layer was washed with
hydrochloric acid (10%, 10 mL ꢁ 1), brine (10 mL ꢁ 3), and dried
over anhydrous Na2SO4. After filtration, the solvent was removed
under reduced pressure, and the residue was purified by silica
gel chromatography (petroleum ether/ethyl acetate = 10/1,v/v) to
give product.
3. Conclusion
In conclusion, we have developed a simple two step separation
method in which crude (S)-MeHNL was obtained by grade acetone
precipitation with a ratio of 2.0/1–2.5/1 (Vacetone/Vextract). The crude
(S)-MeHNL catalyzed the asymmetric cyanohydrination to synthe-
size cyanohydrins from various substrates with high ee values and
medium yields in an ice-water bath. In the proposed system, the
non-enzymatic reaction of the substrate with a cyanide ion was re-
strained effectively by acetic acid in an appropriate water–i-Pr2O
solvent, and the enzymatic reaction was accelerated. The asym-
metric products were then transformed into O-acetyl derivatives
by a simple and quick method that avoided their racemization.
The process has many advantages such as the low price of the
raw materials, the wide range of substrates, the ease and safety
of the operation, and high enantioselectivity obtained, among oth-
ers, thus confirming its value.
4.4.1. (S)-(ꢀ)-1-Cyano-1-(phenyl) methyl acetate 3a
Yield 98%, 97% ee determined by GC on a CPcyclodextrin-b-
2,3,6-M-19 (Chrompack, ø 0.25 mm–25 m) column, temperature:
130 °C, flow rate: 1.0 mL/min, tR1 = 20.6 min (major), tR2 = 22.7 min
(minor); ½a 2D5
ꢂ
¼ ꢀ6:2 (c 0.10, CHCl3), {lit. ½a D25
¼ ꢀ6:2 (c 1, CHCl3)
ꢂ
for (S)-3a with 96% ee}.9 1H NMR (500 MHz, CDCl3) d 2.17 (s, 3H),
6.41 (s, 1H), 7.44–7.46 (m, 3H), 7.51–7.53 (m, 2H); 13C NMR
(125 MHz, CDCl3) d 20.4, 62.8, 116.1, 127.8, 129.2, 130.3, 131.6,
168.9.
4. Experimental
4.1. General
All required chemicals were used directly without separation
unless mentioned. The cassava (Munihot esculenta) leaves used
herein were obtained from local market. 1H NMR spectra were re-
corded on a Varian INOVA-500 (500 MHz) spectrometer with
CDCl3 as the solvent and TMS as the internal standard. Capillary
GC was performed with a CPcyclodextrin-b-2,3,6-M-19 (Chrom-
pack, ø 0.25 mm–25 m) column (Inj. 250 °C, Det. 260 °C). Optical
rotations were obtained on Pekin Eimer instruments Model 343
Polarimeter. Analytical thin-layer chromatography (TLC) was car-
ried out on precoated plates (silica gel 60 F254), and spots were
visualized with ultraviolet (UV) light. Flash column chromatogra-
phy was carried out with silica gel (300–350 mesh).
4.4.2. (S)-(+)-1-Cyano-1-(4-methylphenyl) methyl acetate 3b
Yield 67%, 91% ee determined by GC on a CPcyclodextrin-b-
2,3,6-M-19 (Chrompack, ø 0.25 mm–25 m) column, temperature:
130 °C, flow rate: 1.0 mL/min, tR1 = 30.9 min(major), tR2 = 34.6
min(minor); ½a 2D5
ꢂ
¼ þ6:4 (c 0.8, CHCl3), 1H NMR (500 MHz, CDCl3)
d 2.15 (s, 3H), 2.39 (s, 3H), 6.37 (s, 1H), 7.24–7.26 (d, J = 8.0 Hz, 2H),
7.39–7.41 (d, J = 8.0 Hz, 2H); 13C NMR (125 MHz, CDCl3) d 20.4,
21.2, 62.7, 116.3, 127.9, 128.9, 129.9, 140.6, 169.0.
4.4.3. (S)-(ꢀ)-1-Cyano-1-(3-methylphenyl) methyl acetate 3c
Yield 54%, 85% ee determined by GC on a CPcyclodextrin-b-
2,3,6-M-19 (Chrompack, ø 0.25 mm–25 m) column, temperature:
4.2. Preparation of racemic O-acetyl derivatives of cyanohydrins
130 °C, flow rate: 1.0 mL/min, tR1
= 27.4 min(major), tR2 =
29.7 min(minor); ½a D25
ꢂ
¼ ꢀ21:5 (c 0.5, CHCl3), 1H NMR (500 MHz,
A mixture of aldehyde (2 mmol), NaHSO3 (0.21 g, 2 mmol), tet-
rabutylammonium bromide (0.1 g, 0.3 mmol), cyclohexane (5 ml),
and water (1 ml) was stirred at room temperature for 1 h. To the
stirred mixture NaCN (0.2 g, 4 mmol) and pyridine (0.16 g, 2 mmol)
were added. The resultant mixture was stirred at 0–30 °C for 0.5 h,
and then acetic anhydride (0.4 g, 4 mmol) was added dropwise,
and the mixture was stirred at room temperature overnight. The
aqueous phase was separated and extracted with ethyl ether
(2 ꢁ 10 mL). The combined organic phase was washed with water
(10 mL) and dried over Na2SO4. Flash chromatography on silica gel
(eluent: petroleum ether/ethyl acetate = 10/1) gave the corre-
sponding racemic O-acetyl derivatives of cyanohydrins.
CDCl3) d 2.17 (s, 3H), 2.40 (s, 3H), 6.37 (s, 1H), 7.26–7.27 (m, 1H),
7.29–7.34 (m, 3H); 13C NMR (125 MHz, CDCl3) d 20.4, 21.3, 62.9,
116.2, 124.9, 128.4, 129.1, 131.1, 131.6, 139.2, 168.9.
4.4.4. (S)-(ꢀ)-1-Cyano-1-(2-methoxyphenyl) methyl acetate 3d
Yield 50%, 90% ee determined by GC on a CPcyclodextrin-b-
2,3,6-M-19 (Chrompack, ø 0.25 mm–25 m) column, temperature:
133 °C, flow rate: 1.0 mL/min, tR1
= 51.5 min (major), tR2 =
52.8 min (minor); ½a D25
ꢂ
¼ ꢀ18:4 (c 0.12, CHCl3), {lit. ½a D25
¼ ꢀ19:6
ꢂ
(c 1.6, CHCl3) for (S)-3d with 77% ee};48 1H NMR (500 MHz, CDCl3)
d 2.17 (s, 3H), 3.88 (s, 3H), 6.71 (s, 1H), 6.94 (d, J = 8.0 Hz, 1H), 7.04
(t, J = 8.0 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H); 13
C
NMR(125 MHz, CDCl3) d 20.5, 55.8, 58.2, 120.0, 111.1, 116.3, 121.0,
128.8, 131.9, 156.8, 169.0.
4.3. Crude enzyme preparation
Cassava leaves were chopped and homogenized in a blender
with 5% ammonium sulfate solution in a ratio of 3 mL to 1 g tissue
at ꢀ20 °C. The homogenate was filtered through filter cloth to re-
4.4.5. (S)-(ꢀ)-1-Cyano-1-(3-methoxyphenyl) methyl acetate 3e
Yield 72%, 96% ee determined by GC on a CPcyclodextrin-b-
2,3,6-M-19 (Chrompack, ø 0.25 mm–25 m) column, temperature:

438
Z. Zheng et al. / Tetrahedron: Asymmetry 24 (2013) 434–439
140 °C, flow rate: 1.0 mL/min, tR1 = 40.8 min (major), tR2 = 43.4 min
2.17 (s, 3H), 2.51 (s, 3H), 6.55 (s, 1H), 6.70 (d, 1H, J = 3.5 Hz), 7.14
(d, 1H, J = 3.5 Hz). 13C NMR (125 MHz, CDCl3) d 15.4, 20.5, 58.3,
115.5, 125.3, 129.8, 130.7, 144.3, 168.9.
(minor); ½a 2D5
ꢂ
¼ ꢀ5:1 (c 0.12, CHCl3), {lit. ½a D20
¼ þ4:8 (c 1.0, CHCl3)
ꢂ
for (R)-3e with 94% ee};49 1H NMR (500 MHz, CDCl3) d 2.18 (s, 3H),
3.84 (s, 3H), 6.38 (s, 1H), 6.99 (d, J = 7.5 Hz, 1H), 7.03 (bs, 1H),7.09
(d, J = 7.5 Hz, 1H), 7.36 (t, J = 8.1 Hz, 1H). 13C NMR (125 MHz,
CDCl3) d 20.5, 55.5, 62.8, 120.1, 113.3, 120.0, 130.42, 133.1,
160.2, 169.0.
4.4.12. (R)-(+)-1-Cyano-1-[2-(5-bromothienyl)] methyl acetate
3l
Yield 40%, 86% ee determined by GC on a CPcyclodextrin-b-
2,3,6-M-19 (Chrompack, ø 0.25 mm–25 m) column, temperature:
4.4.6. (S)-(+)-1-Cyano-1-(4-methoxyphenyl) methyl acetate 3f
Yield 55%, 91% ee determined by GC on a CPcyclodextrin-b-
2,3,6-M-19 (Chrompack, ø 0.25 mm–25 m) column, temperature:
140 °C, flow rate: 1.0 mL/min, tR1
= 37.8 min(major), tR2 =
41.7 min(minor); ½a D25
ꢂ
¼ þ15:7 (c 2.6, CHCl3), 1H NMR (500 Hz,
CDCl3) d 2.18 (s, 1H), 6.54 (s, 1H), 7.02 (d, J = 3.9 Hz, 1H), 7.13 (d,
J = 3.9 Hz, 1H). 13C NMR (125 MHz, CDCl3) d 20.4, 58.1, 114.9,
116.5, 130.0, 134.8, 168.7.
140 °C, flow rate: 1.0 mL/min, tR1 = 47.8 min (major), tR2
=
52.8 min (minor); ½a D25
ꢂ
¼ þ6:4 (c 0.08, CHCl3), (lit. ½a D26
¼ þ6:77
ꢂ
(c 0.69, CHCl3), for (S)-3f with 98% ee)50; 1H NMR (500 MHz, CDCl3)
d 2.14 (s, 3H), 3.84 (s, 3H), 6.36 (s, 1H), 6.95 (d, J = 8.8 Hz, 2H),
7.45(d, J = 8.8 Hz, 2H). 13C NMR (125 MHz, CDCl3) d 20.7, 55.7,
62.9, 116.6, 114.9, 124.2, 129.9, 161.5, 169.2.
4.4.13. (S)-(+)-1-Cyano-1-[2-(3-thienyl)] methyl acetate 3m
Yield 90%, 88% ee determined by GC on a CPcyclodextrin-b-
2,3,6-M-19 (Chrompack, ø 0.25 mm–25 m) column, temperature:
130 °C, flow rate: 1.0 mL/min, tR1 = 23.6 min(min), tR2 = 26.7 min
4.4.7. (S)-(+)-1-Cyano-1-(4-chlorophenyl) methyl acetate 3g
Yield 61%, 96% ee determined by GC on a CPcyclodextrin-b-
2,3,6-M-19 (Chrompack, ø 0.25 mm–25 m) column, temperature:
140 °C, flow rate: 1.0 mL/min, tR1 = 33.7 min (major), tR2 = 37.4 min
(major); ½a 2D5
ꢂ
¼ þ10:8 (c 0.1, CHCl3), 1H NMR (500 Hz, CDCl3) d
2.15 (s, 3H); 6.46 (s, 1H); 7.19 (dd, J = 5.1, 1.3 H, 1H); 7.40 (dd,
J = 5.1, 3.0 Hz, 1H); 7.58–7.64 (m, 1H); 13C NMR (125 MHz, CDCl3)
d 20.6, 58.5, 116.1, 126.5, 126.7, 128.0, 132.4, 169.1.
(minor); ½a 2D5
ꢂ
¼ þ10:7 (c 0.11, CHCl3), (lit. ½a D20
¼ þ8:3 (c 0.58,
ꢂ
CHCl3) for (S)-3g with 96% ee)50 1H NMR (500 MHz, CDCl3) d
;
2.17 (s, 3H), 6.38 (s, 1H), 7.40–7.50 (m, 4H); 13C NMR (125 MHz,
4.4.14. (S)-(ꢀ)-Pentanenitrile 3n
Yield 85%, 87% ee determined by GC on a CPcyclodextrin-b-
2,3,6-M-19 (Chrompack, ø 0.25 mm–25 m) column, temperature:
140 °C, flow rate: 1.0 mL/min, tR1 = 7.8 min(major), tR2 = 8.3 min(-
CDCl3) d 20.4, 61.1, 115.8, 122.2, 129.5, 130.2, 136.6, 168.6.
4.4.8. (S)-(+)-1-Cyano-1-[3,4-(methylenedioxy)-phenyl] methyl
acetate 3h
minor); ½a 2D5
ꢂ
¼ ꢀ56:8 (c 0.08, CHCl3), 1H NMR (500 Hz, CDCl3) d
1.0 (dd, J = 7.0, 7.5 Hz, 3H), 1.50–1.57 (m, 2H), 1.86–1.91 (m, 2H),
2.14 (s, 3H), 5.32 (dd, J = 6.0, 6.5 Hz, 1H). 13C NMR (125 MHz,
CDCl3) d 13.3, 17.9, 20.4, 34.2, 60.9, 116.9, 169.2.
Yield 69%, 97% ee determined by GC on a CPcyclodextrin-b-
2,3,6-M-19 (Chrompack, ø 0.25 mm–25 m) column, temperature:
160 °C, flow rate: 1.0 mL/min, tR1 = 37.3 min (major), tR2 = 39.2 min
(minor); ½a 2D5
ꢂ
¼ þ14:4 (c 0.14,CHCl3) {lit. ½a D24
¼ þ12:4 (c 0.70,
ꢂ
CHCl3) for (S)-3j with 91% ee);48 1H NMR (500 MHz, CDCl3) d
2.15 (s, 3H), 6.03 (s, 2H), 6.31 (s, 1H), 6.84 (dd, J = 0.7, 7.7 Hz,
1H), 6.98 (s, 1H), 7.0 (dd, J = 0.7, 7.7 Hz, 1H); 13C NMR (125 MHz,
CDCl3) d 20.3, 62.5, 101.7, 108.0, 108.4, 116.1, 122.2, 125.2,
148.2, 149.2, 168.8.
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ꢂ
¼ þ9:1 (c 0.09, CHCl3), {lit. ½a D20
¼ þ10:4 (c 0.3,
ꢂ
CHCl3), for (R)-3n with 99% ee);48 1H NMR (500 Hz, CDCl3) d 2.17
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3.6 Hz, 1H), 7.46 (dd, J = 1.0, 5.1 Hz, 1H); 13C NMR (125 MHz,
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