Lipase-catalysed tandem Knoevenagel condensation and esterification with alcohol cosolvents

Article abstract of DOI:10.1039/c004547k

Six lipases were screened for their ability to catalyse the Knoevenagel condensation between benzaldehyde and methyl cyanoacetate. Lipase from porcine pancreas tolerated a variety of functional groups on the aromatic ring, produced the highest yields, and

Full text of DOI:10.1039/c004547k

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Lipase-catalysed tandem Knoevenagel condensation and esterification with
alcohol cosolvents†
Yi-Feng Lai,a,b Hui Zheng,b She-Jie Chai,b Peng-Fei Zhang*a,b and Xin-Zhi Chen*a
Received 22nd March 2010, Accepted 17th August 2010
DOI: 10.1039/c004547k
Six lipases were screened for their ability to catalyse
the Knoevenagel condensation between benzaldehyde and
methyl cyanoacetate. Lipase from porcine pancreas toler-
ated a variety of functional groups on the aromatic ring,
produced the highest yields, and also catalysed transes-
terification of the product in the presence of an alcoholic
cosolvent. We show that, in organic solvents, lipase from
porcine pancreas has higher activity for this “promiscuous”
reaction than for naturally occurring esterification catalysis.
ally occurs intramolecularly; for example, the formation of
phosphoenolpyruvate is catalysed from 2-phosphoglycerate by
hydrolyase. However, it has been reported that some enzymes
can catalyse carbon–carbon double bond formation between
two substrates in organic media. Hilvert and colleagues reported
that oxaloacetate undergoes decarboxylation, followed by aldol
condensation with aldehydes, in the presence of macrophomate
synthase.6 Ohta et al. demonstrated a decarboxylase-catalysed
intramolecular aldol reaction following a decarboxylation.7
However, large-scale application is restricted by atom economy
and the limited industrial outputs of these enzymes. It is
interesting, but challenging, to apply the promiscuity of easily
acquired porcine pancreatic lipase to these practical syntheses.
We initially began our work by screening a sample of
commercially available enzymes for their ability to catalyse the
Knoevenagel reaction. All of the enzymes were bought from
J&K Chemical Ltd. The reaction of benzaldehyde and methyl
cyanoacetate in the presence of enzymes in DMSO at 37 ◦C was
used as a model reaction. Reaction progress was monitored by
TLC and GC-MS. As shown in Table 1, porcine pancreatic lipase
from porcine pancreas produced the highest yield. Interestingly,
this result disagrees with a previous report.8 In our experiments,
no decarboxylative product was measured (0% versus 90% in
the previous report). We hypothesise that the different results
stem from the different specific activity exhibited by the natural
structure of different enzymes and from different reaction
conditions (they used amine as the additive).8 In addition, the
amylase from hog pancreas also showed high activity, while the
denatured enzyme, lipase AY30, bovine serum albumin (BSA)
and the control (in the absence of enzyme) demonstrated low
activity in this reaction. These results indicate that the enzymatic
tertiary structure plays a critical role, excluding the possibility
Research in the area of biocatalytic promiscuity has attracted
significant attention from chemists and biochemists in recent
years.1 The term “promiscuity” refers to secondary activities
of an enzyme in addition to its primary physiological activ-
ity. Enzyme promiscuity is hypothesised to contribute to the
natural evolution of enzymes and provides new possibilities
for exploiting enzymatic synthesis in organic chemistry.2 The
past few years have seen significant advances related to this
latent skill of certain enzymes. Berglund reported that wild-
type lipases3 and rationally designed mutants4 can catalyse
the addition of amines, thiols, and b-ketoesters to various
Michael acceptors, including acrylonitrile and a,b-unsaturated
carbonyl compounds. Additionally, Li et al.5 demonstrated that
lipase from porcine pancreas possesses the ability to catalyse
asymmetric aldol reactions between ketones and aldehydes.
Based on these reports, we conducted experiments to further
explore the variety of reactions catalysed by lipases. Here we
report that lipase from porcine pancreas is able to catalyse a
Knoevenagel condensation and esterification in one pot in the
presence of an alcohol cosolvent. Additionally, we found that
lipase from porcine pancreas has higher “promiscuous” activity
than “natural” activity in organic solvents.
The Knoevenagel condensation is the nucleophilic addition of
an active hydrogen compound to an aldehyde or ketone, often
followed by dehydration to form a carbon–carbon double bond.
Carbon–carbon double bond formation is one of the most useful
and fundamental reactions in synthetic organic chemistry—
particularly in the synthesis of complex natural products with
biological activity. In nature, enzymes catalyse many types of
reactions, but carbon–carbon double bond formation gener-
Table 1 The catalytic activities of the Knoevenagel reaction between
benzaldehyde and methyl cyanoacetatea
Entry
Catalyst
Yield (%)
1
2
3
4
5
6
7
Lipase (porcine pancreas)
Lipase A (Aspergillus niger)
Lipase M (Mucor javanicus)
Lipase AK (Pseudomonas fluorescens)
Lipase AY30 (Candida rugosa)
Lipase (recombinant, Candida Antarctica)
BSA
85
35
40
4
2
5
aCollege of Material Chemistry and Chemical Engineering, Zhejiang
University, Hangzhou, 310027, China
3
8
9
10
Amylase (hog pancreas)
Lipase (denatured, porcine pancreas)
Control (no enzyme)
74
3
3
bCollege of Material Chemistry and Chemical Engineering, Hangzhou
Normal University, Hangzhou, 310036, China.
E-mail: zpf100@163.com; Fax: +86 571 28862867; Tel: +86 571
28862867
a Reaction conditions: 50 mg enzyme, 1 mmol benzaldehyde, 1 mmol
methyl cyanoacetate, 5 mL DMSO, 37 ◦C, 150 rpm end-over-end
rotation for 12 h.
† Electronic supplementary information (ESI) available: Experimental
procedures and NMR data. See DOI: 10.1039/c004547k
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that it simply provides polymeric support. These results were
very encouraging and spurred us to further explore this reaction.
Because lipase is known to catalyse the transesterification
reaction in organic solvents,9 we selected ethanol and tert-
butanol as the substrates (and also solvents) to investigate
the possibility of a one-pot Knoevenagel condensation/ester-
exchange reaction. A protocol for one enzyme that catalyses two
reaction types sequentially would be of interest and, moreover,
studying the kinetics of the reaction may help us to better
understand the mechanism of the enzyme promiscuity.
Some substituted aromatic aldehydes and methyl cyanoac-
etate were tested to verify the catalytic effect of the enzyme
(Scheme 1). In a typical experiment, an aromatic aldehyde
(1 mmol) and methyl cyanoacetate (1 mmol) were added to
alcohol (5 mL) containing enzyme (50 mg) and then incubated
at 40 ◦C in an end-over-end rotator at 150 rpm for 12 h. The
isolated yield of 1, 2, and 3 were all greater than 75%, while the
yields of 4 through 12 ranged from 34% to 60%. The yields of
aldehydes with electron-withdrawing groups were higher than
those with electron-donating groups. The structure of products
Fig. 1 Kinetic curve of Knoevenagel condensation and esterification
of benzaldehyde and methyl cyanoacetate catalysed by lipase in ethanol.
hypothesised that observed promiscuity is not due to the
classical mechanism of hydrolytic enzymes, instead suggesting
that alternate-site enzyme promiscuity is at work. Although we
do not have direct evidence, our results are in line with their
conclusions.
In summary, our experiments suggest that several enzymes,
especially lipase from porcine pancreas, catalyse the Knoeve-
nagel condensation with esterification with high yields. This is
the first time that enzymes had been shown to catalyse two
different reactions sequentially to form complex compounds.
Although the details of the catalytic mechanism remain un-
clear, the present observations extend the utility of enzyme
promiscuity.
1
was confirmed by H NMR and MS. Both condensation and
transesterification proceeded cleanly in ethanol, and as expected,
the transesterification in tert-butanol was incomplete (less than
1%) because of steric hindrances. However, in tert-butanol,
the Knoevenagel condensation proceeded to nearly 100% (GC
yield). GC-MS was used to monitor the lipase-catalysed kinetics
of the reaction between benzaldehyde and methyl cyanoacetate
(Fig. 1). The initial reaction rate of the condensation was
0.54 mM h-1, whereas the rate of transesterification was 0.01
mM h-1; clearly, condensation proceeded much faster than the
esterification reaction.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (No. 21076052).
Notes and references
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Scheme 1 Knoevenagel condensation combined with esterification in
one pot in ethanol.
2 O. Khersonsky, C. Roodveldt and D. S. Tawfik, Curr. Opin. Chem.
We also varied temperature, catalyst concentration, and
reaction time. We found that the reactions were successful in
both aqueous and organic solvents when carried out for 64 h at
60 ◦C with 1% of the enzyme concentration. When carried out
in 1.5% water, the desired product (1) was obtained with a 90%
yield by GC.
A generally accepted catalytic mechanism for lipases is that
the active site for hydrolysis also contributes to promiscuous
catalysis.10 However; this mechanism cannot explain why some
lipases are active while others show no activity under the
same reaction conditions. This mechanism also cannot explain
our finding that hog pancreatic amylase can also catalyse this
reaction in organic solvents (Table 1). Reetz et al.11 recently
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1918 | Green Chem., 2010, 12, 1917–1918
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©