Synthesis of (E)-α,β-unsaturated carboxylic esters derivatives from cyanoacetic acid via promiscuous enzyme-promoted cascade esterification/Knoevenagel reaction

Article abstract of DOI:10.1016/j.bioorg.2019.02.041

A new enzymatic protocol based on lipase-catalyzed cascade toward (E)-α,β-unsaturated carboxylic esters is presented. The proposed methodology consists of elementary organic processes starting from acetals and cyanoacetic acid leading to the formation of desired products in a cascade sequence. The combination of enzyme promiscuous abilities gives a new opportunity to synthesize complex molecules in the one-pot procedure. Results of studies on the influence of an enzyme type, solvent, and temperature on the cascade reaction course are reported. The presented methodology provides meaningful qualities such as significantly simplified process, excellent E-selectivity of obtained products and recycling of a biocatalyst.

Full text of DOI:10.1016/j.bioorg.2019.02.041

BioorganicChemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis of (E)-α,β-unsaturated carboxylic esters derivatives from
cyanoacetic acid via promiscuous enzyme-promoted cascade esterification/
Knoevenagel reaction
Monika Wilk, Damian Trzepizur, Dominik Koszelewski, Anna Brodzka, Ryszard Ostaszewski⁎
Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new enzymatic protocol based on lipase-catalyzed cascade toward (E)-α,β-unsaturated carboxylic esters is
presented. The proposed methodology consists of elementary organic processes starting from acetals and cya-
noacetic acid leading to the formation of desired products in a cascade sequence. The combination of enzyme
promiscuous abilities gives a new opportunity to synthesize complex molecules in the one-pot procedure. Results
of studies on the influence of an enzyme type, solvent, and temperature on the cascade reaction course are
reported. The presented methodology provides meaningful qualities such as significantly simplified process,
excellent E-selectivity of obtained products and recycling of a biocatalyst.
Acetal
Cascade reaction
Esterification
Knoevenagel condensation
Lipase
Multi-promiscuity
1. Introduction
The main disadvantage is reversibility of every catalytic step what may
be responsible for low reaction yield or inescapable byproducts [9]. In
The formation of a carbon-carbon bond continues to be outstanding
importance in the way to build complex molecules. Among the syn-
thetic strategies, Knoevenagel condensation is one of the most efficient
methods, being commonly used in the chemical and pharmaceutical
industry [1]. Classically [2], the reaction between carbonyl and active
methylene compound occurs in the presence of an organic base [3] or
Lewis acid [4]. Literature data shows also other catalytic systems [5].
Moreover, there are some reports about the application of enzyme as a
mediator of Knoevenagel reaction and this phenomenon still is not well
recognized [6].
addition, products obtained through the Knoevenagel condensation are
a mixture of E/Z- isomers. The reaction proceeds smoothly just when a
highly reactive compound is used as a nucleophile (Path A, Scheme 1),
while less reactive carboxylic acids are more attractive substrates for
Knoevenagel reaction. It is worth to mention that cyanoacetic acid was
used as a substrate for Knoevenagel condensation. However, high
temperatures required for efficient synthesis and spontaneous dec-
arboxylation of products to α,β-unsaturated cyanates (Path B, Scheme
1) limit its application to Knoevenagel condensation [10].
The development of a cascade reaction is a key point for the efficient
application of difficult to handle substrates [11]. For example, com-
monly employed in standard procedure, highly volatile and irritant
acetaldehyde can be delivered by hydrolysis of vinyl acetate in the
chemoenzymatic tandem process consisting of hydrolysis/Passerini/
EKR reactions [12]. Cascade or tandem processes consist of sponta-
neous reaction sequence in which the bond-forming step takes place
under the same conditions [13]. This approach has many advantages.
The reaction proceeding without isolation of intermediates makes the
process cleaner and more economically friendly [14]. Cascade approach
allows to improve chemical transformation, provides superior effi-
ciency to obtain the desired product [15] and is intrinsically “green”
[16]. Unfortunately, overcoming the principal limitation to design an
elegant cascade with compatible conditions for all steps is still
The application of enzymes plays a key role in the modern synthesis
due to their high selectivity and promiscuous properties. Enzyme pro-
miscuity is defined as the capability of an enzyme to mediate reactions,
which are divergent from their natural role. Hult and Berglund classi-
fied different types of this unique feature. They pointed out several
examples of promiscuity regarding condition, substrate and catalytic
divergences of an enzyme [7]. Moreover, the significance of pro-
miscuous properties was established in Hantzsch-type reaction, perhy-
drolysis, Markovnikov and anti-Markovnikov addition, Henry reaction,
Morita–Baylis–Hillman reaction, Mannich reaction, aldol addition or
Michael addition [8].
Despite the common view of simplicity with proceeding
Knoevenagel condensation, this method suffers from some drawbacks.
⁎ Corresponding author.
E-mail address: ryszard.ostaszewski@icho.edu.pl (R. Ostaszewski).
https://doi.org/10.1016/j.bioorg.2019.02.041
Received 18 January 2019; Received in revised form 18 February 2019; Accepted 19 February 2019
0045-2068/©2019ElsevierInc.Allrightsreserved.
Pleasecitethisarticleas:MonikaWilk,etal.,BioorganicChemistry,https://doi.org/10.1016/j.bioorg.2019.02.041

M. Wilk, et al.
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Scheme 1. Methods used for the synthesis of α,β-unsaturated compounds through Knoevenagel condensation of cyanoacetic acid.
challenging [17].
methodology consists of elementary organic processes which can pro-
ceed simultaneously in a one-pot cascade fashion (Path E, Scheme 1).
Herein, arisen question, whether one enzyme can be used in the se-
quential process of esterification with simultaneous deacelization and
Knoevenagel reaction.
Recently, our group reported a tandem enzyme-catalyzed synthesis
of α,β-unsaturated compounds via hydrolysis of vinyl acetate, followed
by Knoevenagel condensation. Enzyme-mediated Knoevenagel reaction
of acetaldehyde is
a rare transformation in organic chemistry.
Interestingly, all conducted experiments were catalyzed by one enzyme
[18].
To the best of our knowledge, there is no precedence of using cya-
noacetic acid and acetal as substrates for Knoevenagel reaction in a one-
pot procedure. It may demonstrate the high synthetic potential of acetal
as a simultaneous precursor of aldehyde and alkoxy group donor for
esterification in the enzyme-promoted cascade. The successful im-
plementation of the proposed methodology is very promising since it
exploits enzyme multi-promiscuous ability in biomimetic cascades.
Herein, continuing our interest in the enzymatic formation of α,β-
unsaturated derivatives, we propose a new enzymatic protocol based on
lipase-catalyzed sequence for the formation of α,β-unsaturated car-
boxylic esters from cyanoacetic acid. This feature can be achieved by
esterification of cyanoacetic acid followed by Knoevenagel reaction. We
tested a general procedure involving the addition of an aldehyde to the
mixture of cyanoacetic acid in alcohol in the presence of an enzyme
(Path C+A, Scheme 1). However, we did not observe the formation of
desired α,β-unsaturated carboxylic ester. The utility of this reaction is
hindered by the reversible nature of direct enzymatic esterification with
alcohols. The way to circumvent existing limitations is the change of
the alkoxy group donor in the esterification step (Path D+A, Scheme
1). To the best of our knowledge acetals are excellent candidates, as
they are readily available and inexpensive substrates. Recently, we
proved that acetals can be successfully used as an alkoxy group donors,
for enzymatic esterification of carboxylic acid [19]. Moreover, they are
precursors of aldehydes. Therefore, we decide to use this feature
leading to obtaining α,β-unsaturated carboxylic esters. The proposed
2. Results and discussion
In the initial study on enzymatic cascade reaction, benzaldehyde
dimethyl acetal 1a (3 equiv) and cyanoacetic acid 2 (1 equiv) were used
as model substrates (Scheme 2). Solvent, the molar ratio of compounds
were chosen arbitrarily and the reaction mixture was incubated at 50 °C
for 4 days. The reaction with Novozym 435 gave ester 4a together with
the product of Knoevenagel condensation 5a with 43% yield.
In order to confirm that the enzyme takes part in every catalytic
step, we intended to conduct this cascade reaction in the absence of
enzyme (Table 1, entry 1). Neither the formation of methyl cyanoa-
cetate (4a) nor its subsequent condensation product 5a was observed.
Scheme 2. Cascade system toward α,β-unsaturated compounds.
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Table 1
Table 3
Enzymatic screeninga.
The influence of the solvent on the enzyme-catalyzed cascade reaction.
Enzyme
Yieldb (%)
Solvent
log P
Yielda (%)
1.
–
0
1.
Toluene
2.30
55
53
21
9
2.
Deactivated C. cylindracea l.c
Wheat germ l.
0
2.
Hexane
2.76
3.
21
26
27
22
24
32
34
50
34
43
22
23
40
43
3.
Chloroform
1.94
4.
Porcine pancreas l.
Rhizopus niveus l.
Hog pancreas l.
4.
Tetrahydrofuran
tert-Butyl alcohol
Cyclohexane
0.44
5.
5.
0.35
8
6.
6.
3.44
4
7.
Rhizomucor miehei l.
Lipozyme
7.
Diisopropyl ether
1,4-Dioxane
1.52
2
8.
8.
−0,27
−0.07
0.94
1
9.
Candida lypoliptica l.
Candida cylindracea l.
Aspergillus mellus acylase
Penicillum roqueforti l.
Mucor javanicus l.
Amano Lipase PS
Lipase acrylic resin C. Antarctica
Novozym 435
9.
Isopropyl alcohol
tert-Butyl methyl ether
Acetonitrile
< 1
< 1
< 1
0
10.
11.
12.
13.
14.
15.
16.
10.
11.
12.
13.
−0.33
−1.01
−1.40
N,N-Dimethyloformamide
Dimethyl sulfoxide
0
a
Determined by GC analysis. Reaction conditions: 0.125 mmol cyanoacetic
acid (2), 0.375 mmol benzaldehyde dimethyl acetal (1a), 0.5 mL solvent, 15 mg
CCL (4.01 U/mg), 4 days, 50 °C.
a
Reaction conditions: 0.5 mmol cyanoacetic acid (2), 1.5 mmol benzalde-
hyde dimethyl acetal (1a), 2 mL toluene, 30 mg enzyme, 4 days, 50 °C.
characterized by the impact of the quantitative solvents polarity, de-
noted as a logarithm of the partition coefficient (log P). Increasing the
value of log P results in decreasing polarity. Conducted experiments
revealed clearly a correlation between log P and reaction efficiency.
Selected solvents feature with the high value of the logarithm of the
partition coefficient. Surprisingly, compound 5a was formed in cyclo-
hexene with low yield (Table 3, entry 6), despite a high factor of log P.
Moreover, a similar result was obtained for diisopropyl ether. In spite of
the relatively high factor of log P, the product 5a was obtained with
only 2% yield (Table 3, entry 7). The influence of polarity of used
solvent strongly affects the reaction course, because of the well-known
fact that catalytic activity of an enzyme can be changed by the un-
favorable interaction between hydrophilic solvent and enzyme active
site [20].
b
c
All yields refer to product 5a isolated by column chromatography.
Deactivation by heating at 110 °C for 16 h.
This result showed that the enzyme is responsible and obligatory for the
formation of methyl 2-cyano-3-phenylprop-2-enoate (5a). Then, we
have tested more than thirty enzymes to find the most suitable bioca-
talyst. Among tested enzymes several leads to the formation of methyl
2-cyano-3-phenylprop-2-enoate (5a). Results are summarized in
Table 1.
Candida cylindracea lipase (CCL) was found to be the most efficient
biocatalyst resulted in the formation of product 5a with 50% yield.
(Table 1, entry 10). Thus this enzyme was used for further studies. The
result suggests, that the yield of each step of the cascade process is
higher than 80%. Furthermore, experiments are excellent examples of
enzyme multi-promiscuous activity in one cascade reaction Observed
types of promiscuity, are in full accordance with literature data [7].
In the next step, we tested the influence of the molar ratio of sub-
strates on the reaction course. Application of the equimolar amount of
substrates gave product 5a with 2% yield. (Table 2, entry 1). The same
result was obtained with a higher amount of acid 2 to acetal 1a
(Table 2, entry 2). Application of 2 equivalents of acetal 1a, allowed to
obtain product 5a with 15% yield (Table 2, entry 3). Initially used 1:3
ratio of acetal/acid resulted in 55% yield of desired product 5a
(Table 2, entry 4) and is the most profitable. The increasing amount of
acetal, caused in the decrease of reaction yield to 49% (Table 2, entry
5). All yields refer to GC analysis.
Prompted by the fact, that water has a crucial impact on the yield of
enzymatic reactions [21] we tested the influence of water on reaction
course. Reactions were conducted in toluene, using Candida cylindracea
lipase as a biocatalyst. The reaction with 0.5 vol% of water resulted in a
dramatic decrease of product yield to 8%, comparing to the reaction,
without extra addition of water (55%) (Table S1, Supporting informa-
tion).
Next, we investigated the influence of temperature. When the re-
action was conducted at room temperature, the formation of no product
was observed, even after the extension of reaction time to 7 days.
Reaction performed at 30 °C, gave the product 5a with 10% yield.
Increasing temperature in the range of 30–50 °C, resulted in increasing
reaction yield up to 55%. The model reaction carried out at 55 °C en-
tailed decreasing yield to 49%. The further experiments were per-
formed at 50 °C, which turned out to be the optimal temperature for
studied reaction.
The reaction media is the key factor affecting reaction efficiency.
Therefore, we tested the influence of the solvent on the reaction course.
Results are summarized in Table 3.
The obtained results showed a dramatic difference in the formation
of methyl 2-cyano-3-phenylprop-2-enoate (5a) regard to various or-
ganic solvents. Toluene and hexane (Table 3, entry 1–2) are the most
suitable medium for this reaction. Organic solvents can be
Due to the environmental aspects, we checked the reusability of a
catalytic system. During this study, we applied model substrates 1a and
2 at the previously optimized condition. The enzyme was recovered and
reused for up to five times leading to the formation product 5a with
47% yield in the last cycle. Candida cylindracea lipase can be recycled
after cascade reaction at least five times without significant loss of
activity (from 55 to 47%).
Table 2
Molar ratio influence on studied reaction.
In order to show the applicability of the proposed protocol, we
performed a series of experiments using various acetals. (Fig. 1) Sub-
strates were prepared according to the literature procedure [22].
The application of 4-methyl benzaldehyde dimethyl acetal (1b)
gave product 5b with 36% yield. Similar efficiency was obtained for
reaction providing product 5f and 5g. Interestingly, the application of
2-naphthylmethyl dimethyl acetal (1i) provided product 5i with 35%
yield, while employing benzaldehyde dimethyl acetal with benzyl
substituent (1j) did not give expected product 5j. The results showed
Acetal 1a
Acid 2
Yielda (%)
1.
2.
3.
4.
5.
1
1
2
3
4
1
2
1
1
1
2
2
15
55
49
a
Determined by GC analysis. Reaction conditions: cyanoacetic acid (2),
benzaldehyde dimethyl acetal (1a), 0.5 mL toluene, 15 mg CCL (4.01 U/mg),
4 days, 50 °C.
3

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Fig. 1. The scope and limitation of the cascade reaction. Reaction condition: typical experiment contained 0.5 mmol cyanoacetic acid (2), 1.5 mmol acetal (1) and
30 mg CCL in 1 mL of toluene. Reaction mixture was stirred for 4 days at 50 °C. All yields refer to isolated product 5.
that the enzyme active site is limited by substrates containing sterically
hindered substituent next to the reaction center. The remote of bulky
substituent effects on the reaction efficiency (1i-j), while there is no
significant difference caused by different nature of substituent at the
aromatic ring of benzaldehyde dimethyl acetal (1b-h).
to the formation only E-isomers of desired products 5, while employing
classical chemical methods, in almost all cases formation of E/Z isomers
mixture is observed. Moreover, the presented cascade consists of more
than one reaction running with yields up to 80% in each elementary
step. The protocol was limited to heterocyclic derivatives (1t-u), but the
application of other enzymes could expand the diversity of applied
substrates. Conducted experiments are compatible with metabolic
processes taking place in living cells, where not every substrate is sui-
table for every enzyme. Furthermore, (E)-α,β-unsaturated carboxylic
acid derivatives are valuable substances as they can act a significant
role in controlling the transport of mitochondrial pyruvate at living
cells. Their presence has an influence on the efficiency of alcohol fer-
mentation [23].
We investigated also the influence of the alkoxy group of benzal-
dehyde diacetal (1k-n). The application of benzaldehyde diethyl acetal
(1k) resulted in decreased reaction yield compared to the model reac-
tion occurring with benzaldehyde dimethyl acetal (1a). Benzaldehyde
dipropyl acetal (1l) gave the desired product 5l with 31% yield. Due to
the steric feature, benzaldehyde dibenzyl acetal (1n) lead to the for-
mation of product 5n with 5% yield.
A wide range of acetals can be successfully applied for presented
cascade reaction. The presented methodology is characterized by highly
selective properties of the examined enzyme, what is in accordance
with Nature strategy. Even more significant is the observation, that
under the conditions studied, the reactions are highly selective and lead
In order to compare the yield of product 5a obtained directly
through Knoevenagel condensation to the yield obtained via designed
cascade, we performed enzyme-catalyzed reaction using benzaldehyde
(3a) and methyl cyanoacetate (4) under studied condition. Surprisingly,
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by step leading to the production of additional quantities of aldehyde.
The Knoevenagel reaction occurs through a water-eliminating step. The
released water molecule undergoes reaction with the excess of acetal
resulting in an additional amount of aldehyde to Knoevenagel con-
densation. Moreover, the continuous consumption of byproduct makes
the system more efficient.
In order to confirm our assumption, we conducted additional ex-
periments that may evaluate the effect of water content in the hydro-
lysis of acetal. The mixture of 1 equivalent benzaldehyde dimethyl
acetal (1a) in toluene was incubated with 3 equivalents of water at
50 °C. The obtained results show a decreasing amount of acetal 1a
during the reaction course. Hydrolysis of acetal 1a provides benzalde-
hyde (3a), which is a suitable substrate to further Knoevenagel reac-
tion. The presence of the enzyme in the reaction mixture did not affect
significantly to the hydrolysis of acetal. (Table S2, Supporting in-
formation).
Nevertheless, we proved that the greater content of water in the
reaction mixture has an unfavorable influence on the reaction course.
Without the addition of water, in the presence of cyanoacetic acid (2),
acetal 1a reacted as an alkoxy group donor, leading to the formation of
ester 4a and aldehyde 3a. Then compound 6 is formed as a result of a
subsequent reaction between benzaldehyde (3a) and methyl cyanoa-
cetate (4a). The last transformation occurs through the water-elimina-
tion step to the desired product 5a. A released water molecule is con-
stantly used for hydrolysis of acetal 1a. According to the literature data,
acetals can be applied as a dehydrating agent [24], thus applying 3:1
ratio of the acetal (1a) to the cyanoacetic acid (2) is necessary to the
successful production of α, β-unsaturated carboxylic esters (5) in the
cascade system. Additional evidence proved that the water present in
the enzyme active site and released during the reaction course is suf-
ficient to shift the equilibrium of cascade reaction toward target methyl
2-cyano-3-phenylprop-2-enoate (5a) (Scheme 3).
Fig. 2. A kinetic curve of (E)-selective enzymatic cascade reaction.a 1a: ben-
zaldehyde dimethyl acetal; 3a: benzaldehyde; 4a: metyl cyanoacetate, 5a:
methyl 2-cyano-3-phenylprop-2-enoate.
the product 5a was obtained with 40% yield, while the cascade process
gave the desired product with 55% yield. This experiment proved the
advantage of cascade reaction over the classical approach and revealed
the significant role of esterification steps to obtain desired methyl 2-
cyano-3-phenylprop-2-enoate (5a).
These results encouraged us to study the possible mechanism of the
proposed cascade. For this purpose, we performed a series of experi-
ments in time with model substrates 1a and 2 under previously opti-
mized condition. The concentration of compounds was analyzed by GC.
Results are presented in Fig. 2.
The esterification step with acetal 1a proceeds smoothly during the
first 48 h of an experiment. After this time the obtained ester 4a un-
dergoes reaction with aldehyde 3a to form the desired product 5a. We
observed continuous consumption of methyl cyanoacetate (4a) with a
constant concentration of benzaldehyde (3a). Clearly, after 48 h, the
Knoevenagel condensation proceeds faster than the esterification step.
The higher concentration of product 5a was observed after 96 h. The
extension the reaction time did not affect to amount of 5a.
3. Conclusions
In summary, we designed a highly selective protocol for the
synthesis of (E)-α,β-unsaturated carboxylic esters using esterification-
Knoevenagel cascade reaction of cyanoacetic acid with acetals. After a
careful optimization of the reaction condition, target products were
isolated with yields up to 55% and excellent selectivity, while chemical
methods lead to the mixture of (E/Z)-isomers. All conducted
These results indicated that the proposed cascade must be enhanced
Scheme 3. Water-enhanced cascade system leading to (E)-α,β-unsaturated carboxylic esters.
5

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transformations were catalyzed by one enzyme, which can be recovered
and used for the next experiments without substantial loss of the ac-
tivity. We proved the ability of the enzyme to catalyze reactions di-
verged from their natural role. Moreover, the application of acetals as
precursors of aldehydes and alkoxy group donors for esterification of
cyanoacetic acid significantly simplifies the process and shows an ad-
vantage over classical approach leading to the formation of (E)-α,β-
unsaturated derivatives.
were consistent with reported values.[26]
4.2.3. 1-(dimethoxymethyl)-4-nitrobenzene (1d)
Pale yellow oil, Yield: 87%; 1H NMR (400 MHz, CDCl3) δ 8.19 (d,
J = 8.9 Hz, 2H), 7.62 (d, J = 8.4 Hz, 2H), 5.45 (s, 1H), 3.32 (s, 6H). 13
C
NMR (100 MHz, CDCl3) δ 148.0, 145.1, 129.8, 128.0, 127.8, 123.4,
101.6, 52.7. All the resonances of 1H and 13C NMR spectra were con-
sistent with reported values.[25]
4. Experimental section
4.2.4. 1-(dimethoxymethyl)-3-nitrobenzene (1e)
Pale yellow oil, Yield: 50%; 1H NMR (400 MHz, CDCl3) δ 8.34 (s,
1H), 8.19 (d, J = 7.4 Hz, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.55 (t,
J = 7.9 Hz, 1H), 5.47 (s, 1H), 3.35 (s, 6H). 13C NMR (100 MHz, CDCl3)
δ 140.4, 132.9, 129.2, 123.4, 122.1, 101.5, 52.7. All the resonances of
1H and 13C NMR spectra were consistent with reported values [27].
4.1. General information
All reactions were performed under
a normal atmosphere.
Experiments were monitored by TLC on silica gel 60 F254 aluminum
sheets using UV light as a visualizing agent. Products were purified on
column chromatography on Merck silica gel 60/230–400 mesh. As
solvent hexane: ethyl acetate was used. 1H- and 13C NMR spectra were
recorded in CDCl3 solution with TMS as an internal standard (0 ppm)
and are reported in parts per million. Enzymatic reactions were carried
in a vortex (Heidolph Promax 1020) equipped with an incubator (Hei-
dolph Inkubator 1000). GC analyses were performed on Clarus® 680 PE
AutoSystem GC with built-in-Autosampler (The PerkinElmer) equipped
with FID detector and Agilent J&W GC Column (VF-1701Ms, 30 m, 0.25
mmID, 0.25 µm df). The following temperature program was used: in-
itial temperature 70 °C (0 min), 10 °C/min to 200 °C (3 min), 10 °C/min
to 250 °C (5 min). Retention time for compound 1a: 7.22 min, 3a:
5.96 min, 4a:6.09 min and 5a: 15.77 min. Lipase from Candida cylin-
dracea was purchased from Fluka. Immobilized lipase from Candida
antarctica B (Novozym 435) from Novo Nordisk. The rest of the used
enzymes were purchased at Sigma-Aldrich. Solvents were of reagent
grade quality. Benzaldehyde, methyl cyanoacetate, and piperidine were
freshly distilled (commercially available reagents from Sigma-Aldrich).
Cyanoacetic acid was obtained commercially and used without addi-
tional purification. Benzaldehyde dimethyl acetal, acetaldehyde di-
methyl acetal, and acetone dimethyl ketone were purchased from
Sigma Aldrich and applied after distillation. Dimetoxymethane and
propionaldehyde dimethyl acetal obtained from TCI Chemicals and
used without additional purification. Benzaldehyde dibenzyl acetal
(1n), thiophene-2-carboxaldehyde dimethyl acetal (1t) and 2-fur-
aldehyde dimethyl acetal (1u) were kindly provided by Jakub
Grabowski from Institute of Organic Chemistry, Polish Academy of
Sciences, Warsaw, Poland.
4.2.5. 1-chloro-4-(dimethoxymethyl)benzene (1f)
Colourless oil; Yield: 81%; 1H NMR (400 MHz, CDCl3) δ 7.34 (dd,
J = 20.7, 8.6 Hz, 4H), 5.35 (s, 1H), 3.29 (s, 6H). 13C NMR (100 MHz,
CDCl3) δ 136.7, 134.3, 128.4, 128.2, 102.3, 52.6. All the resonances of
1H and 13C NMR spectra were consistent with reported values [25].
4.2.6. 1-chloro-2-(dimethoxymethyl)benzene (1g)
Colourless oil; Yield: 90%; 1H NMR (400 MHz, CDCl3) δ 7.62 (dd,
J = 7.0, 2.5 Hz, 1H), 7.36 (dd, J = 7.0, 2.3 Hz, 1H), 7.31–7.22 (m, 2H),
5.63 (s, 1H), 3.38 (s, 5H). 13C NMR (100 MHz, CDCl3) δ 135.4, 133.2,
129.7, 128.1, 126.5, 100.9, 53.8. All the resonances of 1H and 13C NMR
spectra were consistent with reported values [28].
4.2.7. 4-fluoro-2-(dimethoxymethyl)benzene (1h)
Colourless oil; Yield: 80%; 1H NMR (400 MHz, CDCl3) δ 7.42 (dd,
J = 8.7, 5.5 Hz, 2H), 7.04 (t, J = 8.7 Hz, 2H), 5.37 (s, 1H), 3.31 (s, 6H).
13C NMR (100 MHz, CDCl3) δ 164.0, 161.6, 134.0, 128.5, 115.1, 114.9,
102.5, 52.6. All the resonances of 1H and 13C NMR spectra were con-
sistent with reported values [25].
4.2.8. 2-(dimethoxymethyl)naphthalene (1i)
Pale yellow oil; Yield: 88%; 1H NMR (400 MHz, CDCl3) δ 8.34–8.26
(m, 2H), 7.87 (dd, J = 10.3, 8.5 Hz, 4H), 7.75 (d, J = 7.0 Hz, 2H),
7.58–7.44 (m, 6H), 5.94 (s, 2H), 3.40 (s, 11H). 13C NMR (100 MHz,
CDCl3) δ 133.9, 133.1, 130.8, 129.3, 128.5, 126.2, 125.7, 124.9, 124.2,
102.4, 77.4, 77.0, 76.7, 53.2. All the resonances of 1H and 13C NMR
spectra were consistent with reported values [29].
4.2. Synthesis and characterization of acetal 1b-j and 1o.
4.2.9. 2-benzyloxybenzaldehyde dimethyl acetal (1j)
Colourless oil; Yield: 70%; 1H NMR (400 MHz, CDCl3) δ 7.57 (dd,
J = 7.6, 1.7 Hz, 3H), 7.48–7.24 (m, 21H), 6.99 (ddd, J = 16.9, 11.9,
4.4 Hz, 7H), 5.74 (s, 3H), 5.13 (s, 6H), 3.40 (s, 16H). 13C NMR
(100 MHz, CDCl3) δ 156.4, 137.3, 129.8, 128.7, 128.7, 127.9, 127.4,
126.9, 120.8, 112.5, 99.6, 70.4, 54.0. All the resonances of 1H and 13C
NMR spectra were consistent with reported values [22].
To the mixture of proper derivatives of benzaldehyde (5 mmol) and
monohydrate of p-toluenesulfonic acid (0.25 mmol) in methanol
(3 mL), trimethyloxymethane (8 mL) was added in room temperature.
Then the solution was heated in an oil bath at 120° for 16 h. The re-
action was monitored by TLC chromatography. Then the rest of the
solvent was evaporated under reduced pressure and the product was
purified by column chromatography (hexane:ethyl acetate, 9:1) [22].
4.2.10. 3,3-dimethoxy-1-propenylbenzene (1o)
Pale yellow oil; Yield: 90%; 1H NMR (400 MHz, CDCl3) δ 7.45–7.27
(m, 5H), 6.73 (d, J = 16.2 Hz, 1H), 6.16 (dd, J = 16.2, 4.9 Hz, 1H),
4.96 (dd, J = 4.9, 1.0 Hz, 1H), 3.38 (s, 5H). 13C NMR (100 MHz, CDCl3)
δ 193.6, 152.7, 136.1, 133.6, 131.3, 129.1, 128.5, 128.1, 126.7, 125.7,
102.9, 52.7. All the resonances of 1H and 13C NMR spectra were con-
sistent with reported values [30].
4.2.1. 1-(dimethoxymethyl)-4-methylbenzene (1b)
Colourless oil; Yield: 75%;1H NMR (400 MHz, CDCl3) δ 7.33 (d,
J = 8.0 Hz, 2H), 7.18 (d, J = 7.9 Hz, 2H), 5.36 (s, 1H), 3.32 (s, 6H),
2.36 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 138.1, 135.2, 128.8, 126.6,
103.2, 52.6, 21.17. All the resonances of 1H and 13C NMR spectra were
consistent with reported values [25].
4.3. Synthesis and characterization of acetal 1k-m
4.2.2. 1-(dimethoxymethyl)-4-methoxybenzene (1c)
Colourless oil; Yield: 85%, 1H NMR (400 MHz, CDCl3) δ 7.36 (d,
J = 8.5 Hz, 6H), 6.89 (d, J = 8.8 Hz, 6H), 5.34 (s, 3H), 3.81 (s, 8H),
3.31 (s, 15H). 13C NMR (100 MHz, CDCl3) δ 131.1, 130.4, 127.9, 113.5,
103.1, 55.2, 53.4, 52.6. All the resonances of 1H and 13C NMR spectra
To the mixture of benzaldehyde (5 mmol) and monohydrate of p-
toluenesulfonic acid (0.25 mmol) in proper alcohol (3 mL), corre-
sponding orthoformate (8 mL) was added in room temperature. Then
the solution was heated in an oil bath at 120° for 16 h. The reaction was
6

M. Wilk, et al.
BioorganicChemistryxxx(xxxx)xxx–xxx
monitored by TLC chromatography. Then the rest of the solvent was
evaporated under reduced pressure and the product was purified by
column chromatography (hexane:ethyl acetate, 9:1).
8.22 (s, 1H), 7.90 (d, J = 8.1 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H), 3.92 (s,
3H), 2.43 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 163.3, 155.3, 144.8,
131.3, 130.1, 128.8, 115.7, 101.2, 53.3, 21.9. All the resonances of 1H,
13C NMR spectra and melting point were consistent with reported va-
lues [33].
4.3.1. (Diethoxymethyl)benzene (1 k)
Colourless oil; Yield: 90%; 1H NMR (400 MHz, CDCl3) δ 7.46 (ddd,
J = 7.5, 1.5, 0.6 Hz, 2H), 7.33 (dt, J = 19.5, 7.0 Hz, 3H), 5.49 (s, 1H),
3.57 (ddd, J = 32.5, 9.5, 7.1 Hz, 4H), 1.22 (t, J = 7.1 Hz, 6H). 13C NMR
(100 MHz, CDCl3) δ 139.1, 129.7, 128.9, 128.2, 126.6, 101.6, 61.0,
15.2. All the resonances of 1H and 13C NMR spectra were consistent
with reported values [22].
4.7.3. Methyl (E)- 2-cyano-3-(4-methoxyphenyl)prop-2-enoate (5c)
White solid; melting point 104 °C; 1H NMR (400 MHz, CDCl3) δ 8.10
(s, 1H), 7.96–7.89 (m, 2H), 6.92 (d, J = 8.9 Hz, 2H), 3.83 (d,
J = 8.8 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 163.9, 163.6, 154.6,
133.7, 124.3, 116.2, 114.8, 98.9, 55.6, 53.2, 29.7. All the resonances of
1H, 13C NMR spectra and melting point were consistent with reported
values [34].
4.3.2. Benzaldehyde di-n-propylacetal (1l)
Colourless oil; Yield: 70%; 1H NMR (400 MHz, CDCl3) δ 7.49 (d,
J = 7.1 Hz, 2H), 7.40–7.27 (m, 3H), 5.52 (s, 1H), 3.58–3.40 (m, 4H),
1.70–1.57 (m, 4H), 0.96 (t, J = 7.4 Hz, 6H). 13C NMR (100 MHz,
CDCl3) δ 139.2, 128.1, 126.7, 112.8, 101.6, 67.1, 65.6, 23.0, 22.8, 10.7.
HR ESI-MS: calcd for C13H21O2 [M+H+], 209.1595, found,
209.1590.
4.7.4. Methyl (E)-2-cyano-3-(4-nitrophenyl)prop-2-enoate (5d)
Yellow pale solid; melting point 175 °C; 1H NMR (400 MHz, CDCl3)
δ 8.37–8.27 (m, 3H), 8.13 (d, J = 8.8 Hz, 2H), 3.97 (s, 3H). 13C NMR
(100 MHz, CDCl3) δ 161.9, 151.9, 149.8, 136.8, 131.5, 124.3, 114.4,
106.9, 77.3, 77.0, 76.7, 53.8, 29.7. All the resonances of 1H, 13C NMR
spectra and melting point were consistent with reported values [35].
4.3.3. (Bis(allyloxy)methyl)benzene (1m)
Colourless oil; Yield: 60%; 1H NMR (400 MHz, CDCl3) δ 7.51–7.46
(m, 2H), 7.39–7.28 (m, 3H), 5.93 (ddt, J = 17.2, 10.4, 5.5 Hz, 2H), 5.62
(s, 1H), 5.30 (dq, J = 17.2, 1.7 Hz, 2H), 5.16 (ddd, J = 10.4, 3.1,
1.4 Hz, 2H), 4.05 (dt, J = 5.5, 1.5 Hz, 4H). 13C NMR (100 MHz, CDCl3)
δ 138.4, 134.5, 128.4, 128.2, 126.7, 116.7, 100.5, 66.2. All the re-
sonances of 1H and 13C NMR spectra were consistent with reported
values [31].
4.7.5. Methyl (E)-2-cyano-3-(3-nitrophenyl)prop-2-enoate (5e)
Yellow pale solid; melting point 135–137 °C; 1H NMR (400 MHz,
CDCl3) δ 8.70 (s, 3H), 8.40 (dd, J = 8.1, 1.8 Hz, 6H), 7.74 (d,
J = 8.0 Hz, 4H), 7.40 (d, J = 4.3 Hz, 4H), 3.97 (s, 9H). 13C NMR
(100 MHz, CDCl3) δ 152.1, 135.2, 132.8, 130.6, 128.8, 127.2, 125.9,
53.8. All the resonances of 1H, 13C NMR spectra and melting point were
consistent with reported values [36].
4.4. General procedure for synthesis of α,β- unsaturated compounds 5a-u
through cascade reaction
4.7.6. Methyl (E)-2-cyano-3-(4-chlorophenyl)prop-2-enoate (5f)
White solid; melting point 119–120 °C; 1H NMR (400 MHz, CDCl3) δ
8.19 (s, 1H), 7.95–7.87 (m, 2H), 7.50–7.42 (m, 2H), 3.92 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ 162.7, 153.6, 139.7, 132.2, 129.8, 115.2,
103.1, 53.5. Melting point: 119–120 °C. All the resonances of 1H, 13C
NMR spectra and melting point were consistent with reported values
[37].
An acetal 1a-u (1.5 mmol) was added to the suspension of enzyme
(30 mg) in toluene (1 mL), followed by addition of cyanoacetic acid
(0.5 mM). The reaction mixture was incubated at 150 rpm at 50° for
4 days. Then the enzyme was filtered off and toluene was evaporated in
vacuo. The resulting residue was purified by column chromatography
(silica gel, eluent ethyl acetate/hexanes) to obtain desired product 5a-u.
4.7.7. Methyl (E)-2-cyano-3-(2-chlorophenyl)prop-2-enoate (5g)
White solid; melting point 103 °C; 1H NMR (400 MHz, CDCl3) δ 8.70
(s, 1H), 8.24 (d, J = 7.7 Hz, 1H), 7.55–7.36 (m, 3H), 3.95 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ 162.3, 151.4, 136.5, 133.7, 130.4, 129.9,
127.5, 114.7, 105.8, 53.5. All the resonances of 1H, 13C NMR spectra
and melting point were consistent with reported values [37].
4.5. Procedure for the synthesis of product 5a with the addition of water
The procedure is the same as above, but the water (% v/v) is added
before the addition of the substrates.
4.6. Procedure for synthesis product 5a through classically enzyme-
promoted Knoevenagel reaction
4.7.8. Methyl (E)-2-cyano-3-(4-fluorophenyl)prop-2-enoate (5h)
White solid; melting point 92–93 °C; 1H NMR (400 MHz, CDCl3) δ
8.22 (s, 1H), 8.03 (dd, J = 8.7, 5.3 Hz, 2H), 7.19 (t, J = 8.5 Hz, 2H),
3.93 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 162.9, 153.7, 133.6, 127.8,
116.8, 116.6, 115.4, 53.4. All the resonances of 1H, 13C NMR spectra
and melting point were consistent with reported values [38].
The mixture of benzaldehyde (0.5 mmol) and methyl cyanoacetate
(0.5 mmol) in toluene 1 mL was incubated in the presence of Candida
cylindracea lipase (30 mg) at 50 °C for 4 days. The reaction was mon-
itored by TLC analysis. After this time enzyme was filtered out and the
solvent was evaporated in vacuo. The resulting residue was purified by
column chromatography (silica gel, eluent ethyl acetate/hexanes) to
obtain desired product 5a with 40% yield.
4.7.9. Methyl (E)-2-cyano-3-(1-naphthyl)prop-2-enoate (5i)
Pale yellow oil; 1H NMR (400 MHz, CDCl3) δ 9.10 (s, 4H), 8.31 (d,
J = 7.3 Hz, 4H), 8.03 (dd, J = 8.3, 3.8 Hz, 8H), 7.93–7.87 (m, 5H), 7.59
(ddd, J = 17.4, 11.2, 4.5 Hz, 14H), 3.97 (s, 11H). 13C NMR (100 MHz,
CDCl3) δ 162.9, 152.9, 133.5, 131.7, 129.2, 128.3, 127.9, 126.8, 125.4,
122.8, 115.3, 105.4, 53.5, 29.7. All the resonances of 1H and 13C NMR
spectra were consistent with reported values [39].
4.7. Characterization of obtained products 5a-u.
4.7.1. Methyl (E)-2-cyano-3-phenylprop-2-enoate; (5a)
White solid; melting point 89 °C; 1H NMR (400 MHz, CDCl3) δ 8.20
(s, 1H), 7.92 (dd, J = 7.1, 1.5 Hz, 2H), 7.53–7.40 (m, 3H), 3.87 (s, 3H).
13C NMR (100 MHz, CDCl3) δ 162.9, 155.3, 133.4, 131.4, 131.1, 129.3,
115.4, 102.6, 53.4. All the resonances of 1H, 13C NMR spectra and
melting point were consistent with reported values [32].
4.7.10. Ethyl (E)-2-cyano-3-phenyl-2-propenoate (5k)
White solid; melting point 47–48 °C; 1H NMR (400 MHz, CDCl3) δ
8.23 (s, 1H), 7.97 (d, J = 7.4 Hz, 2H), 7.70–7.36 (m, 3H), 4.37 (q,
J = 7.1 Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
162.4, 154.9, 133.3, 131.5, 131.0, 129.3, 115.4, 103.1, 62.7, 14.1. All
the resonances of 1H, 13C NMR spectra and melting point were con-
sistent with reported values [40].
4.7.2. Methyl (E)-2-cyano-3-(4-methylphenyl)prop-2-enoate (5b)
White solid; melting point 94–95 °C; 1H NMR (400 MHz, CDCl3) δ
7

M. Wilk, et al.
BioorganicChemistryxxx(xxxx)xxx–xxx
4.7.11. Propyl (E)-2-cyano-3-phenylprop-2-enoate (5l)
2311–2316;
Colourless oil; 1H NMR (400 MHz, CDCl3) δ 8.19 (s, 1H), 7.94 (d,
J = 7.7 Hz, 2H), 7.59–7.35 (m, 3H), 4.24 (t, J = 6.7 Hz, 2H), 1.75 (dd,
J = 14.2, 7.2 Hz, 2H), 0.99 (t, J = 7.5 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 162.4, 154.8, 133.2, 131.5, 131.0, 129.2, 115.4, 103.1, 68.1,
21.9, 10.3. All the resonances of 1H and 13C NMR spectra were con-
sistent with reported values [41].
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4.7.12. 2-Cyano-3 t-phenyl-acrylic acid allyl ester (5m)
Pale yellow oil; 1H NMR (400 MHz, CDCl3) δ 8.27 (s, 1H), 8.00 (d,
J = 7.4 Hz, 2H), 7.66–7.38 (m, 3H), 6.00 (ddd, J = 22.8, 10.9, 5.7 Hz,
1H), 5.51–5.27 (m, 2H), 4.82 (d, J = 5.7 Hz, 2H). 13C NMR (100 MHz,
CDCl3) δ 162.2, 155.3, 133.4, 131.5, 131.1, 129.3, 119.3, 102.8, 67.0.
HR ESI-MS: calcd for C13H11NO2 [M+H+], 214.0824, found,
214.0825.
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4.7.13. Benzyl (E)-2-cyano-3-phenylprop-2-enoate (5n)
Colourless oil; 1H NMR (400 MHz, CDCl3) δ 8.27 (s, 1H), 7.99 (d,
J = 7.3 Hz, 2H), 7.60–7.31 (m, 8H), 5.36 (s, 2H). 13C NMR (100 MHz,
CDCl3) δ 155.4, 134.9, 133.4, 131.1, 129.3, 128.7, 128.3, 68.1. HR ESI-
MS: calcd for C17H13NO2 [M+H+], 264.3024, found, 264.3025.
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4.7.14. (2-E,4-E)-2-cyano-5-phenyl-2,4-pentadienoic acid methyl ester
(5o)
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101–104;
Yellow pale solid; melting point 148 °C; 1H NMR (400 MHz, CDCl3)
δ 8.01 (dd, J = 6.8, 4.1 Hz, 2H), 7.59 (dd, J = 6.5, 2.9 Hz, 4H),
7.46–7.37 (m, 7H), 7.30–7.27 (m, 4H), 3.89 (s, 6H). 13C NMR
(100 MHz, CDCl3) δ 162.8, 155.6, 149.1, 134.6, 131.2, 129.2, 128.5,
127.1, 123.0, 104.1, 53.1. All the resonances of 1H, 13C NMR spectra
and melting point were consistent with reported values [41].
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4.7.15. Methyl 2-cyano-3-methylbut-2-enoate (5p)
Colourless oil; 1H NMR (400 MHz, CDCl3) δ 3.81 (s, 1H), 2.40 (s,
1H), 2.30 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 174.0, 162.3, 115.6,
104.7, 52.4, 27.3, 22.8. All the resonances of 1H and 13C NMR spectra
were consistent with reported values [42].
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4.7.16. Methyl 2-cyanobut-2-enoate (5q)
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Colourless oil; 1H NMR (400 MHz, CDCl3) δ 7.73 (q, J = 7.2 Hz,
1H), 3.86 (s, 3H), 2.22 (d, J = 7.2 Hz, 3H). 13C NMR (101 MHz, CDCl3)
δ 159.24 (s), 53.05 (s), 17.76 (s). 13C NMR (100 MHz, CDCl3) δ 159.3,
53.1, 29.7, 17.8. HR ESI-MS: calcd for C6H7NO2Na [M+Na+],
148.0595, found, 148.0580.
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Acknowledgements
We gratefully acknowledge the financial support from the Polish
State Committee for Scientific Research project OPUS No. 2016/B/ST5/
03307.
(e) R.V. Hangarge, S.A. Sonwane, D.V. Jarikote, M.S. Shingare, Green Chem. 3
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Appendix A. Supplementary material
(c) K.C. Nicolaou, J.S. Chen, Chem. Soc. Rev. 38 (2009) 2993–3009;
(d) J. Muschiol, C. Peters, N. Oberleitner, M.D. Mihovilic, U.T. Bornscheuer,
F. Rudroff, Chem. Com. 51 (2015) 5798–5811.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.02.041.
[12] A. Żądło- Dobrowolska, D. Koszelewski, D. Paprocki, A. Madej, M. Wilk,
R. Ostaszewski, ChemCatChem 9 (2017) 3047–3053.
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