Chemoenzymatic Synthesis of α-Cyano Epoxides by a Tandem-Knoevenagel-Epoxidation Reaction

Article abstract of DOI:10.1002/ejoc.201501501

An efficient lipase-mediated synthesis of α-cyano epoxides through a tandem-Knoevenagel-epoxidation reaction was explored for the first time. This method provides a green, mild, and convenient route for the synthesis of α-cyano epoxides; moreover, the app

Full text of DOI:10.1002/ejoc.201501501

DOI: 10.1002/ejoc.201501501
Communication
Biocatalysis
Chemoenzymatic Synthesis of α-Cyano Epoxides by a Tandem-
Knoevenagel–Epoxidation Reaction
Fengjuan Yang,[a] Xiaowen Zhang,[a] Fengxi Li,[b] Zhi Wang,*[a] and Lei Wang*[a]
Abstract: An efficient lipase-mediated synthesis of α-cyano ep- mild, and convenient route for the synthesis of α-cyano epox-
oxides through a tandem-Knoevenagel–epoxidation reaction ides; moreover, the applicability of lipase in organic synthesis is
was explored for the first time. This method provides a green, further extended.
Introduction
chemoenzymatic synthesis of α-cyano epoxides has not yet
been reported.
Enzymes have been found to catalyze different types of reac-
tions than their physiological ones.[1] This phenomenon is
called catalytic promiscuity, and it has proven useful for the
development of organic chemistry.[2] Many elegant examples
of enzymatic promiscuity have been reported, such as aldol
additions, Michael additions, Mannich reactions, Henry reac-
tions, Knoevenagel condensations, and perhydrolysis.[3] Re-
cently, enzymatic tandem reactions have been reported to pro-
duce structurally complex molecules, and these investigations
have greatly widened the application of enzymes in organic
synthesis and have allowed the discovery of many new pharma-
ceutical structures.[4]
α-Cyano epoxides, important intermediates in organic syn-
thesis, have been used to synthesize a large variety of carbo-
cyclic, heterocyclic, and acyclic derivatives.[5] Generally, these
compounds can be synthesized by a two-step procedure involv-
ing the formation of an electron-deficient alkene by Knoeven-
agel condensation between an aldehyde and an ethanenitrile
derivative at elevated temperature followed by hypochloride-
mediated epoxidation.[6] However, this procedure usually suffers
from harsh reaction conditions or low yields. Therefore, the de-
velopment of a green and efficient method for the synthesis of
α-cyano epoxides has attracted great interest from many syn-
thetic chemists.
Scheme 1. Lipase-catalyzed synthesis of α-cyano epoxides through a tandem-
Knoevenagel–epoxidation reaction.
Results and Discussion
As a starting point, we chose benzaldehyde and malononitrile
as model substrates, and our results are shown in Table 1. The
corresponding α-cyano epoxide was obtained in high yield if
CalB (Candida antarctica lipase B) or Novozym 435 (a commer-
cial immobilized CalB) was used in the tandem-Knoevenagel–
epoxidation reaction. However, only Knoevenagel product 3
was detected if other lipases were used in the reaction. These
results are in accordance with previous work, for which it was
reported that PPL and PSL could not catalyze the epoxidation
of olefins.[7] Relative to that shown by non-immobilized CalB,
Novozym 435 exhibited higher catalytic performance in this re-
action, and this was attributed to the fact that the hydrophobic
support of Novozym 435 could keep the lipase in its active
As part of our studies towards the development of lipase
catalytic promiscuity, we aimed to synthesize α-cyano epoxides
4 through a tandem-Knoevenagel–epoxidation reaction cata-
lyzed by a lipase (Scheme 1). To the best of our knowledge, the
Table 1. Effect of enzyme sources for the synthesis of α-cyano epoxide 4.[a]
Entry
Catalyst
Yield[b] [%]
Yield[b] [%]
of 3
of 4
1
2
3
4
5
6
7
PPL (porcine pancreas lipase)
PSL (Pseudomonas sp. lipase)
CalB (Candida antarctica lipase B)
denatured CalB
Novozym 435
denatured Novozym 435
no enzyme
95
79
86
12
93
11
11
n.d.
n.d.
85
n.d.
91
[a] Key Laboratory of Molecular Enzymology and Engineering of Ministry of
Education, School of Life Sciences, Jilin University,
2699 Qianjin street, Changchun, P. R. China
E-mail: w_lei@jlu.edu.cn
n.d.
n.d.
www.mee.jlu.edu.cn
[b] School of Life Sciences, Jilin University,
2699 Qianjin street, Changchun, P. R. China
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/ejoc.201501501.
[a] Reaction conditions: malononitrile (1 mmol), benzaldehyde (1 mmol), DMF
(2 mL), enzyme (300 U), 50 °C, 12 h; then UHP (urea–hydrogen peroxide,
1.5 mmol), EtOAc (3 mL), 50 °C, 3 h. [b] n.d.: not detected.
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Communication
conformation and promote the enzymatic activity.[8] Upon us- the lipase was increased until the maximum value reached
ing the denatured lipase (Table 1, entries 4 and 6) as the cata- 300 U (the substrate was 1 mmol), and then the yield changed
lyst, the result was similar to that obtained with the control slightly. Considering the cost of the enzyme, 300 U was selected
(Table 1, entry 7), which suggested that a special active confor- as the optimal amount of lipase for this reaction.
mation of the enzyme played a crucial role in the Knoevenagel
condensation and epoxidation.
The lipase-mediated tandem-Knoevenagel–epoxidation reac-
tion was performed at various temperatures. As shown in Fig-
ure 1, the yield of the α-cyano epoxide increased as the temper-
ature was increased from 20 to 50 °C, and the yield did not
change markedly at higher temperatures. Therefore, 50 °C was
selected as the optimal temperature for this reaction.
Figure 2. Effect of the amount of enzyme on the synthesis of α-cyano epox-
ides. Reaction conditions: malononitrile (1 mmol), benzaldehyde (1 mmol),
DMF (2 mL), 50 °C, 12 h; then, UHP (1.5 mmol), EtOAc (3 mL), 50 °C, 3 h.
Subsequently, the substrate scope and the generality of the
lipase-mediated tandem reaction were investigated. Various ar-
omatic aldehydes and ethanenitrile derivatives were selected
for the synthesis of α-cyano epoxides 4 (Table 3), and all the
1
products were characterized by H NMR spectroscopy. A wide
range of substrates participated in the reaction, and the ob-
Figure 1. Effect of temperature on the synthesis of α-cyano epoxides. Reac-
tion conditions: malononitrile (1 mmol), benzaldehyde (1 mmol), DMF (2 mL),
tained yields were satisfactory. Our approach comprises two
sequential processes: (1) lipase-catalyzed Knoevenagel conden-
sation, (2) lipase-mediated epoxidation. It was found that prod-
uct 3 resulting from Knoevenagel condensation was easily ep-
oxidized (Table 3); thus, the yield of the α-cyano epoxide de-
pended on the efficiency of the Knoevenagel condensation.
Novozym 435 (300U), 12 h; then, UHP (1.5 mmol), EtOAc (3 mL), 3 h.
Various organic solvents with different polarities were se-
lected to investigate solvent effects. First, we screened solvents
for the first step of this tandem reaction upon using Nov-
ozym 435 as the catalyst. As shown in Table 2, the highest yield
of Knoevenagel product 3 was obtained if DMF was selected as
the solvent. Then, we continued to study the solvent effects in
the lipase-mediated epoxidation reaction. This step was very
efficient owing to the almost complete epoxidation of Knoeven-
agel product 3. Therefore, we selected DMF as the optimal sol-
vent for this tandem reaction.
Table 2. Effect of reaction media for the synthesis of α-cyano epoxides 4.
Solvent
Yield [%] of 3[a]
Yield [%] of 4[b]
MeCN
EtOAc
THF
1,4-dioxane
DMF
80
53
71
56
93
79
52
71
53
91
[a] Reaction conditions: ethanenitrile derivative (1 mmol), aromatic aldehyde
(1 mmol), solvent (2 mL), Novozym 435 (300 U), 50 °C, 12 h. [b] Reaction
conditions: ethanenitrile derivative (1 mmol), aromatic aldehyde (1 mmol),
solvent (2 mL), Novozym 435 (300 U), 50 °C, 12 h; then, UHP (1.5 mmol) and
EtOAc (3 mL), 50 °C, 3 h.
Figure 3. The reusability of Novozym 435 for the synthesis of α-cyano epox-
ides. Reaction conditions: malononitrile (1 mmol), benzaldehyde (1 mmol),
DMF (2 mL), Novozym 435 (300 U), 50 °C, 12 h; then, UHP (1.5 mmol), EtOAc
(3 mL), 50 °C, 3 h.
The effect of the amount of the enzyme on the yield of the
α-cyano epoxides was also investigated, and the results are
shown in Figure 2. The yield clearly increased as the amount of
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Table 3. Effect of substituents on the synthesis of α-cyano epoxides 4.
Generally, the immobilized enzyme had enhanced stability
relative to the free enzyme, and this helped to increase the
reusability of the enzyme and to make the enzymatic process
economically viable.[10] In this study, the reusability of Nov-
ozym 435 was investigated to assess the economic potential of
the process. About 90 % of the activity of the enzyme was re-
tained after five consecutive cycles, and then, the yields of the
α-cyano epoxide decreased dramatically. Thus, Novozym 435
has good reusability in this tandem reaction (Figure 3).
Conclusions
In summary, we disclosed a novel chemoenzymatic process for
the synthesis of α-cyano epoxides through
a tandem-
Knoevenagel–epoxidation reaction. All the products were ob-
tained in high yields (from 73 to 96 %) under the optimal reac-
tion conditions. Another advantage of this protocol is that the
immobilized lipase (Novozym 435) could be reused over five
cycles and retained its high activity. The simple, convenient,
and efficient strategy described herein provides another case
of catalytic promiscuity that expands the application of en-
zymes in organic synthesis.
Experimental Section
Materials: Porcine pancreas lipase (PPL, 5600 U g–1), Pseudomonas
sp. lipase (PSL, 8500 U g–1), Candida antarctica lipase B (CALB,
10000 U mL–1), and Novozym 435 (commercial immobilized CALB,
15000 U g–1) were purchased from Sigma (Beijing, China). One unit
of the enzyme activity was defined as the amount of enzyme re-
quired to hydrolyze 1 μmol of p-nitrophenyl acetate per minute at
30 °C. Aromatic aldehydes and ethanenitrile derivatives used in this
study were purchased from J&K Scientific (Beijing, China). All other
chemical reagents were purchased from Shanghai Chemical Rea-
gent Company (Shanghai, China). All commercially available rea-
gents and solvents were used without further purification. NMR
spectra were recorded with an Inova 500 (500 MHz) spectrometer.
General Procedure for the Lipase-Catalyzed Synthesis of α-
Cyano Epoxides:
A mixture of the ethanenitrile derivative
(1 mmol), the aromatic aldehyde (1 mmol), and Novozym 435
(300U) in DMF (2 mL) was stirred at 50 °C in a round-bottomed flask
for 12 h. The reaction was monitored by TLC. Then, UHP (1.5 mmol)
and EtOAc (3 mL) were added, and the mixture was stirred for 3 h.
Next, the mixture was filtered and the residue was washed with
EtOAc. The combined organic phase was dried with Na2SO4 (an-
hydrous) and concentrated under vacuum. The resulting residue
was purified by column chromatography (silica gel, EtOAc/hexane
1:6) to afford the desired α-cyano epoxide. All the isolated products
were well characterized by NMR spectroscopy and MS. Each experi-
ment was performed in triplicate, and all the data were obtained
on the basis of the average values.
[a] Reaction conditions: ethanenitrile derivative (1 mmol), aromatic aldehyde
(1 mmol), DMF (2 mL), Novozym 435 (300 U), 50 °C, 12 h. [b] Reaction condi-
tions: ethanenitrile derivative (1 mmol), aromatic aldehyde (1 mmol), DMF
(2 mL), Novozym 435 (300 U), 50 °C, 12 h; then, UHP (1.5 mmol), EtOAc (3 mL),
50 °C, 3 h.
Aromatic aldehydes with election-withdrawing groups gave
higher yields than those with electron-donating groups. Fur-
thermore, ortho-substituted benzaldehydes showed lower ac-
tivity than meta- and para-substituted benzaldehydes because
of steric hindrance. As for the ethanenitrile derivatives, malono-
nitrile showed the highest activity of all the derivatives. These
3-(2-Methoxyphenyl)oxirane-2,2-dicarbonitrile: White solid, Rf =
0.48 (EtOAc/hexane, 1:6). 1H NMR (500 MHz, CDCl3) 3.93 (s, 3 H),
4.94 (s, 1 H), 6.97 (d, J = 8.5 Hz, 1 H), 7.02 (t, J = 7.5 Hz, 1 H), 7.20
results are consistent with reports on Knoevenagel condensa- (d, J = 7.5 Hz, 1 H), 7.45 ppm (dd, J = 11.5, 4.5 Hz, 1 H). 13C NMR
tion reactions catalyzed by lipase.[9] In addition, lipase is respon-
(500 MHz, CDCl3) 41.3, 55.7, 62.8, 110.5, 110.6, 111.9, 116.5, 120.8,
126.3, 132.3, 158.6 ppm. MS (EI): m/z = 200 [M]+.
sible for the in situ formation of a peracid, and the peracid is
the oxidant in the epoxidation. Thus, no enantioselectivity was
found in this step.
3-(3-Chlorophenyl)oxirane-2,2-dicarbonitrile: White solid, Rf
0.62 (EtOAc/hexane, 1:6). H NMR (500 Hz, CDCl3) 4.68 (s, 1 H), 7.31
=
1
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Communication
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129.6, 130.5, 131.7, 135.4 ppm. MS (EI): m/z = 204 [M]+.
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Supporting Information (see footnote on the first page of this
article): Experimental details, characterization data, and copies of
1
the H NMR and 13C NMR spectra of all final products.
Acknowledgments
The authors gratefully acknowledge the National Natural Sci-
ence Foundation of China (NSFC) (grant number 21172093), the
Natural Science Foundation of Jilin Province of China (grant
number 20140101141JC), and the Graduate Innovation Fund of
Jilin University (grant number 2015023) for financial support.
Keywords: Domino reactions · Enzyme catalysis · Nucleophilic
addition · Epoxidation
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Received: December 22, 2015
Published Online: February 10, 2016
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