Lipase-catalyzed Knoevenagel condensation between α,β- unsaturated aldehydes and active methylene compounds

Article abstract of DOI:10.1016/j.cclet.2014.03.036

A simple and efficient Knoevenagel condensation between α,β-unsaturated aldehydes and active methylene compounds is reported. Notably, this condensation can be catalyzed by PPL (lipase from porcine pancreas) with satisfied yields (49%-92%). Moreover, PPL induces moderate Z/E selectivity in the Knoevenagel condensation.

Full text of DOI:10.1016/j.cclet.2014.03.036

Accepted Manuscript
Title: Lipase-catalyzed Knoevenagel condensation between
α,␤-unsaturated aldehydes and active methylene compounds
Author: Zhi Wang Chun-Yu Wang Hao-Ran Wang Hong
Zhang Ya-Lun Su Teng-Fei Ji Lei Wang
PII:
S1001-8417(14)00138-7
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.cclet.2014.03.036
CCLET 2922
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
15-1-2014
7-3-2014
11-3-2014
Please cite this article as: Z. Wang, C.-Y. Wang, H.-R. Wang, H. Zhang, Y.-L. Su, T.-F.
Ji, L. Wang, Lipase-catalyzed Knoevenagel condensation between rmalpha,rmbeta-
unsaturated aldehydes and active methylene compounds, Chinese Chemical Letters
(2014), http://dx.doi.org/10.1016/j.cclet.2014.03.036
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Graphical Abstract
Lipase-catalyzed Knoevenagel condensation between α,β-unsaturated aldehydes and active methylene
compounds
，
，
Zhi Wang a #, Chun-Yu Wang c #, Hao-Ran Wang a, Hong Zhang a, Ya-Lun Su b, Teng-Fei Ji b,*, Lei
Wang a,*
a Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education, College of Life
Science, Jilin University, Changchun 130023, China
b State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia
Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050,
China
c State key Laborarory of Supramolecular Structure and Materials, Jilin University, Changchun 130023,
China
O
O
O
Lipase
+
CHO
O
A simple and efficient Knoevenagel condensation between α,β-unsaturated aldehydes and active
methylene compounds is reported. This reaction can be catalyzed by PPL (lipase from porcine pancreas)
with satisfied yields (49%-92%) and moderate Z/E selectivity.
Original article
Lipase-catalyzed Knoevenagel condensation between α,β-unsaturated aldehydes and active methylene
compounds
，
，
Zhi Wang a #, Chun-Yu Wang c #, Hao-Ran Wang a, Hong Zhang a, Ya-Lun Su b,
Teng-Fei Ji b,*, Lei Wang a,
a Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education, College of life
science, Jilin University, Changchun 130023, China;
b State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia
Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050,
China;
c State key laborarory of supramolecular structure and materials, Jilin University, Changchun 130023,
China;
———
Corresponding authors.
E-mail addresses: jitf@imm.ac.cn; w_lei@jlu.edu.cn
# These authors contributed equally to this work.
Page 1 of 6

ARTICLE INFO
ABSTRACT
Article history:
A simple and efficient Knoevenagel condensation between α,β-
unsaturated aldehydes and active methylene compounds is reported.
Notably, this condensation can be catalyzed by PPL (lipase from
porcine pancreas) with satisfied yields (49%-92%). Moreover, PPL
induces moderate Z/E selectivity in the Knoevenagel condensation.
Received 15 January 2014
Received in revised form 7 March
2014
Accepted 11 March 2014
Available online
Keywords:
Lipase
Knoevenagel condensation
α,β-Unsaturated aldehyde
Active methylene compound
1. Introduction
Enzyme catalytic promiscuity is a property shown by many enzymes [1]. It refers to the ability of an
enzyme of catalyzing more than one type of chemical transformation and has only recently been exploited
for synthetic applications [2-5]. As a part of our research to explore the applications in this new area, we
are focusing on the Knoevenagel condensation, which is a useful and widely employed method of
producing α,β-enones/enoesters in organic synthesis. The Knoevenagel condensation, discovered by E.
Knoevenagel in 1896, is widely used both in the academic environment and in the chemical
pharmaceutical and perfume industries [6-9]. Generally, these condensations can be carried out in the
presence of bases or Lewis acids [10-12]. In addition, some methods like electrochemistry, microwave and
ultrasound irradiation have also been reported for the Knoevenagel condensation [13-15]. However, these
methods suffer from numerous limitations such as hazardous solvents, high reaction temperatures or harsh
reaction conditions.
Recently, it has been found that hydrolases can catalyze the Knoevenagel condensation. For example,
lipase from porcine pancreas (PPL) was also used to catalyze the Knoevenagel condensation between
substituted benzaldehydes and methyl cyanoacetate in moderate yields (30%-85%), and PPL tolerated a
variety of functional groups on the aromatic ring [16]. However, much still remains to be done on the
applications of hydrolase-catalyzed Knoevenagel condensation. It is well known that the Knoevenagel
condensations are generally carried out with aromatic aldehydes as substrates. Only a limited number of
examples of α,β-unsaturated aldehydes as starting materials have been reported. Hu et al. reported that
papain could catalyze the Knoevenagel reactions in DMSO/water between the substituted
cinnamaldehydes and active methylene compounds (acetylacetone and ethyl acetoacetate). The products
were obtained in moderate yield (42%-86%) and low Z/E selectivity [17]. The application of lipase in this
type of Knoevenagel condensation has not been reported so far. This prompted us to further study the
Knoevenagel condensations catalyzed by lipase between α,β-unsaturated aldehydes and active methylene
compounds. In the current study, we investigated the lipase-catalyzed Knoevenagel condensation with
crotonaldehyde (1a) and acetoacetone (2a) as the substrates (Scheme 1).
2. Experimental
2.1 Materials
Porcine pancreas lipase (lipase type II) was purchased from Sigma (Beijing, China). C. rugosa lipase
(CRL) was purchased from Sigma (St Louis, MO). Pseudomonas sp. lipase (PSL) was purchased from
Amano Pharmaceutical Co. Ltd. (Japan). Candida antarctica lipase B (CAL-B) was kindly donated by
Page 2 of 6

Novo Nordisk Industries (China). Bacillus subtilis lipase (BSL2) was over expressed from Bacillus subtilis
strain IFFI10210 in our laboratory according to the method we have reported [18]. Aeropyrum pernix
esterase (APE1547) was produced from a hyperthermophilic archaeon strain, then purified and lyophilized
[19]. These enzymes were used after lyophilization for enzymatic reaction without further purification. 4-
Crotonaldehyde was purchased from Sigma. Other reagents were purchased from Shanghai Chemical
Reagent Company (Shanghai, China). Commercially available reagents and solvents were used without
further purification. NMR spectra were recorded on an Inova 500 (1H, 500 MHz) spectrometer. ESI-MS
was performed on an Agilent 1100 LC/MSD.
2.2 Lipase-catalyzed Knoevenagel condensation
o
The reaction was performed at 40 C for 120 h in a round bottom flask contained α,β-unsaturated
aldehyde (1 mmol), active methylene compound (1 mmol), tert-butyl alcohol (5 mL), deionized water
(20% v/v) and PPL powder (20 mg). After completion, the reaction mixture was evaporated and obtained
1
the crude product of condensation, then purified by silica gel chromatography, the data of H NMR and
ESI-MS of all products are given in Supporting information. All experiments were repeated three times
and the final results were based on the average value.
3. Results and discussion
It’s well known that the catalytic activity of a lipase depends mainly on the type and origin of the lipase
[20]. In this study, five kinds of lipases were screened to catalyze this Knoevenagel condensation. It could
be found (Table 1) that all the selected lipases can catalyze the condensation. Among the tested lipases,
PPL produced the highest yield (48%).
Many reports have shown that organic media is one of the most important factors influencing the enzyme
catalytic performance [21]. In this study, the effects of reaction solvents in the Knoevenagel condensation
between crotonaldehyde (1a) and acetoacetone (2a) were investigated in Table 2. The results clearly
indicated that the catalytic activity of PPL was significantly influenced by the solvent. It’s believed that
the solvent can lead to a conformational change of the enzyme and then affect the enzyme activity.
However, the relationship between the value of log P and enzyme activity is not clear so far and need to be
further investigated. According to the results, the highest yield was obtained while tert-Butyl alcohol was
selected as the reaction media.
It is known that the enzyme needs a certain amount of water to maintain its proper conformation [22].
Therefore, Water content strongly affects the catalytic behavior of an enzyme in non-aqueous media. As
shown in Fig. 1, the relationship between the yields of the condensation products and the water content
exhibited a bell-shaped curve and the highest yield (83%) was obtained when the water content was 20%
(v/v). Further increase or decrease of water content resulted in a decline of enzyme activity. When the
water content was low, the conformation of PPL was excessively rigid, which made the “induced-fit”
process of PPL difficult and led to the low enzyme activity. However, the enzyme conformation in organic
media with high water content may be too flexible, which may also decrease the activity of the enzyme
[23].
A set of reactions were carried out with a variety of active methylene compounds under the optimal
conditions. The two α,β-unsaturated aldehydes chosen were crotonaldehyde (1a) and cinnamaldehyde (1b),
respectively. As shown in Table 3, the yields of targeted products were lower when 1b was selected as the
substrate. It might be because cinnamaldehyde (1b) is more difficult to enter the active site of the enzyme
due to the steric hindrance. As for the active methylene compounds, methyl cyanoacetate exhibited the
highest activity in the condensation. It might be attributed to the specific substrate selectivity of enzyme
based on the structure geometry and the reactivity of substrate. Additionally, satisfactory Z/E selectivity
was obtained for the products from active methylene compounds (2b-2d). It is worth pointing out that PPL
exhibits a higher Z/E selectivity than previously reported papain in the Knoevenagel condensations [17].
Page 3 of 6

4. Conclusion
In conclusion, we have investigated the Knoevenagel condensation between α,β-unsaturated aldehydes
and active methylene compounds. Notably, this simple, efficient, and environmentally-benign method is
very clean and can give satisfied yields (49-92%). Moreover, PPL can produce moderate Z/E selectivity of
the products. With these significant advantages, this method has great potential for future applications.
This method represents a contribution to the realm of synthetic organic chemistry and might extend the
utility of enzyme promiscuity.
Acknowledgments
The authors are grateful for the financial support from the National Natural Science Foundation of
China (No. 21172093, 31070708, 21072075), the Natural Science Foundation of Jilin Province of China
(No. 201115039, 20140101141JC) and the Scientific Research Fund of Jilin University (No.
450060326007).
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Fig. 1 Effect of water content on the Knoevenagel reaction between crotonaldehyde and acetoacetone.
Reaction conditions: 1a (1mmol), 2a (1mmol), tert-butyl alcohol (5 mL), water content (0-30% v/v) and
PPL (20 mg) at 40 oC for 120 h.
Scheme 1 Knoevenagel condensation between crotonaldehyde (1a) and acetoacetone (2a)
Table 1 Effect of enzyme sources on the Knoevenagel reaction between crotonaldehyde (1a) and
acetoacetone (2a)a.
Entry
Lipase
Yield (%)
1
2
3
4
5
6
7
PPL
CaLB
BSL
PSL
CRL
45
32
31
15
22
APE1547
No
28
trace
enzyme
aReaction conditions: 1a (1 mmol), 2a (1 mmol), i-propanol (5 mL) and enzyme (20 mg) at 40 oC for 120 h.
Table 2 Effect of solvents on the Knoevenagel reaction between crotonaldehyde (1a) and acetoacetone (2a)
Entry Solvent
Log P
-0.07
0.58
Yield (%)
1
2
i-Propanol
tert-Butyl alcohol
N,N-
45
59
36
-1.0
Dimethylformamide
Dimethyl sulfoxide
Acetonitrile
3
4
-1.4
-0.33
51
24
Page 5 of 6

5
Chloroform
1.61
17
aReaction conditions: 1a (1 mmol), 2a (1 mmol), organic solvent (5 mL) and enzyme (20 mg) at 40 oC for
120 h.
Table 3 The PPL catalyzed Knoevenagel reactions between α,β-unsaturated aldehydes and active
methylene compounds. a
Entr Aldehyde
y
active
Yield (E/Z)
methylene
compound
O
O
1
2
3
3aa 83%
3ab 78%
1a
1b
1a
2a
2b
O
O
3ba
72%
O
(93:7)
3bb
(66:34)
3ca
(91:9)
3cb
(76:24)
3da
4
5
6
7
8
61%
51%
49%
92%
89%
1b
1a
1b
1a
1b
O
O
O
2c
O
NC
O
2d
(100:0)
3db
(100:0)
3ea 54%
3eb 49%
O
O
9
10
1a
1b
O
O
2e
aReaction conditions: α,β-unsaturated aldehyde (1 mmol), active methylene compound (1 mmol), tert-butyl
alcohol (5 mL), water content (20% v/v) and PPL (20 mg), at 40 oC for 120 h.
Page 6 of 6