Simple, efficient, and green method for synthesis of trisubstituted electrophilic alkenes using lipase as a biocatalyst

Article abstract of DOI:10.1080/00397911.2010.525334

A simple and efficient Knoevenagel condensation method for the synthesis of trisubstituted electrophilic alkenes was developed using lipase as a biocatalyst. Knoevenagel condensation was performed using the conventional method and using lipases (Aspergillus oryzae or Rhizopus oryzae) as biocatalysts, and reaction time, reaction temperature, yield, and recyclability were compared. Using a lipase as a biocatalyst eliminated the need for bases such as piperidine and pyridine. A wide range of aromatic aldehydes and ketones readily undergo condensation with active methylene compounds. The workup procedure is also very simple, and yields of the reactions are in the range of 75%to 95%. Both the biocatalysts were effectively recycled four times with no major decrease in the yield of product. The remarkable catalytic activity and reusability of lipase widens its applicability in Knoevenagel condensation with good to excellent yields for synthesis of trisubstituted electrophilic alkenes.Copyright Taylor & Francis Group, LLC.

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Simple, Efficient, and Green Method for
Synthesis of Trisubstituted Electrophilic
Alkenes Using Lipase as a Biocatalyst
Bhushan Nanasaheb Borse a , Sanjeev Ramchandra Shukla a & Yogesh
Ashok Sonawane b
a Department of Fibres and Textile Processing Technology, Institute
of Chemical Technology, Mumbai, India
b Department of Dyestuff Technology, Institute of Chemical
Technology, Mumbai, India
Accepted author version posted online: 29 Jul 2011.Version of
record first published: 06 Oct 2011.
To cite this article: Bhushan Nanasaheb Borse , Sanjeev Ramchandra Shukla & Yogesh Ashok Sonawane
(2012): Simple, Efficient, and Green Method for Synthesis of Trisubstituted Electrophilic Alkenes Using
Lipase as a Biocatalyst, Synthetic Communications: An International Journal for Rapid Communication
of Synthetic Organic Chemistry, 42:3, 412-423
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Synthetic Communications1, 42: 412–423, 2012
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911.2010.525334
SIMPLE, EFFICIENT, AND GREEN METHOD FOR
SYNTHESIS OF TRISUBSTITUTED ELECTROPHILIC
ALKENES USING LIPASE AS A BIOCATALYST
Bhushan Nanasaheb Borse,1 Sanjeev Ramchandra Shukla,1 and
Yogesh Ashok Sonawane2
1Department of Fibres and Textile Processing Technology, Institute of
Chemical Technology, Mumbai, India
2Department of Dyestuff Technology, Institute of Chemical Technology,
Mumbai, India
GRAPHICAL ABSTRACT
Abstract A simple and efficient Knoevenagel condensation method for the synthesis of trisub-
stituted electrophilic alkenes was developed using lipase as a biocatalyst. Knoevenagel
condensation was performed using the conventional method and using lipases (Aspergillus
oryzae or Rhizopus oryzae) as biocatalysts, and reaction time, reaction temperature, yield,
and recyclability were compared. Using a lipase as a biocatalyst eliminated the need for bases
such as piperidine and pyridine. A wide range of aromatic aldehydes and ketones readily
undergo condensation with active methylene compounds. The workup procedure is also very
simple, and yields of the reactions are in the range of 75% to 95%. Both the biocatalysts were
effectively recycled four times with no major decrease in the yield of product. The remarkable
catalytic activity and reusability of lipase widens its applicability in Knoevenagel conden-
sation with good to excellent yields for synthesis of trisubstituted electrophilic alkenes.
Keywords Aromatic aldehyde; biocatalyst; Knoevenagel condensation; lipase
INTRODUCTION
The use of biocatalysis is an efficient and green tool for modern organic
synthesis because of its high selectivity and use of mild reaction conditions.[1] In
recent years, a new frontier in biocatalysis is catalytic promiscuity, which is mainly
exploring the catalytic activities of enzymes on unnatural substrates or alternative
reactions.[2] Biocatalytic promiscuity provides new tools for organic synthesis and
Received May 28, 2010.
Address correspondence to Sanjeev Ramchandra Shukla, Department of Fibres and Textile
Processing Technology, Institute of Chemical Technology, Mumbai 400 019, India. E-mail:
sanjeevrshukla@rediffmail.com
412

LIPASE-CATALYZED KNOEVENAGEL CONDENSATION
413
thus expands largely the application of enzymes. With increasing worldwide concern
about environmental and future resources, one of the challenges for chemists is to
identify new approaches that are less hazardous to human health and the environ-
ment.[3] Lipases can act as biocatalysts in aqueous media as well as in a wide range
of organic solvents.[4] They have high potential for organic synthesis because of their
stability, availability, and acceptability to a broad range of substrates.[5,6] The advan-
tages of performing enzymatic transformations in organic solvents are greater
stability of the enzymes and more facile product recovery.
Recent research has reported some biocatalytic basic reactions such as Aldol[7a,b]
and Mannich[7c] condensations, Michael[7d,e] and Markovnokov additions,[7f] forma-
tion of fluorophores,[7g] and decarboxylation[8] catalyzed by enzymes. Lipases have
the ability to catalyze various C-C, C-N, and C-S bond formations.[9] Although lipases
are well established for numerous applications in kinetic resolutions,[10] enantioselec-
tive syntheses based on meso compounds,[11] and amidation of racemic esters or
amines,[12,13] their application in Knoevenagel condensation for the synthesis of tri-
substituted alkenes has been limited. Nevertheless, limited industrial outputs of these
enzymes restrict their large-scale application.
Among the carbon–carbon bond-forming reactions, the Knoevenagel conden-
sation of aldehydes with active methylene compounds is one of the most important
synthetic methods for the preparation of electrophilic trisubstituted alkenes that
involves a simple operation process and mild conditions. It is widely employed to
produce important intermediates and end products for perfumes, pharmaceuticals,
and polymers.[14] The Knoevenagel condensation leads to key products, including
nitriles and double bonds. The nitriles are used in anionic polymerization, whereas
the unsaturated ester intermediates are employed in the synthesis of several thera-
peutic drugs (e.g., niphendipine and nitrendipine) and pharmacological products
(e.g., calcium channel blockers and antihypertensive).[15]
Knoevenagel condensation between carbonyl compound and active methylene
group has been explored traditionally by using various catalysts such as alkali metal
hydroxides, pyridine, and piperidine. Basic zeolites such as Cs-exchanged NaX
(CsNaX) and GeX, cesium and cesium-lanthanum impregnated mesoporous
MCM-4,[16,17] as well as alkali-exchanged zeolites[18] have been used. Amine and
ammonium groups are also effective homogeneous catalysts.[19] Many other solid base
catalysts have been studied, including ion-exchange resins; modified silica such as
xonotlite,[20] hydrotalcite,[21] and sepioloites;[22] and supports with surface oxyni-
trides.[23] In spite of their potential utility, some of these synthetic methods use
relatively harsh reaction conditions along with expensive and environmentally hazard-
ous reagents and solvents. Thus, there is an increasing interest in the development of
new catalysts working under mild reaction conditions with cleaner reaction profiles,
good yields, less catalyst load, and simple experimental and isolation procedures.
In recent years, chemists have paid much more attention to the clean synthesis
of trisubstituted electrophilic alkenes obtained using condensation reactions. There-
fore, the introduction of efficient and new methods based on green methodology is in
high demand. In consideration of green chemical methodology here, we focused our
research on the Knoevenagel condensation of aromatic aldehydes with active meth-
ylene groups for the synthesis of trisubstituted electrophilic alkenes using Rhizopus
oryzae and Aspergillus oryzae as biocatalysts.

414
B. N. BORSE, S. R. SHUKLA, AND Y. A. SONAWANE
The development of alternative methods from components that are inexpensive,
nontoxic to the environment, and biodegradable or obtainable from biodegradable
resources is therefore highly desirable to overcome these drawbacks. One of the fun-
damental challenges and ultimate goals for organic reactions is the ability to perform
the reaction in biocatalysts, because compared with toxic chemicals, biocatalysts are
safe and support environmentally friendly chemical processes.
EXPERIMENTAL
Material and Equipment
Commercial lipase produced from Thermomyces lanuginosus by submerged
fermentation of genetically modified Aspergillus oryzae (100 KLU=g) and Rhizopus
oryzae (41.6 U=mg) was used for the reactions. All the solvents and chemicals were pro-
cured from S.D. Fine Chemicals (India) and were used without further purification.
The reactions were monitored by thin-layer chromatography (TLC) using 0.25 mm
E-Merck silica gel 60 F254 precoated plates, which were visualized with ultraviolet
(UV) light. The 1H NMR spectra was recorded at 300 MHz on a Varian Mercury Plus
spectrometer. Chemical shifts are expressed in d ppm using tetramethylsilane (TMS) as
an internal standard. Mass spectral data were obtained with micromass–Q-ToF
(YA105) spectrometer. Elemental analysis was done on a Harieus rapid analyzer.
General Reaction Procedure
Lipase as biocatalyst. In a typical experimental procedure, absolute ethanol
(5 ml) was added to aromatic aldehydes (4.717 mmol) and active methylene compound
(4.952 mmol) with stirring. The ratio of aldehyde to active methylene compound (m=m)
was kept at 1:1.05 for monoaldehyde. The reaction mass was then cleared in ethanol
and was initiated by adding 10% (wt. by wt. of aldehyde) lipase. The reaction mixture
was agitated continuously at 55ꢀC. The reaction progress was monitored by using TLC
(mobile phase: 15% ethyl acetate in n-hexane). After completion of the reaction, it was
filtered to separate catalyst, and the reaction mass was then allowed to cool for precipi-
tation of product. The product was filtered and washed with a little chilled ethanol to
remove excess active methylene compound and unreacted aldehyde. The product was
recrystalized from benzene to afford the pure product.
Piperidine as conventional catalyst. The absolute ethanol (5ml) was added
to aromatic aldehydes (4.717mmol) and active methylene compound (4.952mmol) with
stirring. The ratio of aldehyde to active methylene compound (m=m) was kept at 1:1.05
for monoaldehyde. The reaction mass was stirred at reflux temperature for respective
period. The reaction was monitored using TLC. After completion of reaction, the reac-
tion mass was distilled, water was added and extracted with dichloromethane, and it
was dried on sodium sulfate followed by vacuum distillation to afford the product.
Recyclability Study
After completion of the reaction, catalyst was recovered by filtration and
washed with ethanol. The recovered catalyst was loaded in the same reactor, and fresh

LIPASE-CATALYZED KNOEVENAGEL CONDENSATION
415
reactants aldehyde and active methylene compound were added to ethanol. The
reaction was carried out at 55 ꢀC for 6 h. After completion of the reaction, the same
procedure was repeated for consecutive recycling of the catalyst.
Spectral Data for Selected Products
Ethyl 3-(4-chlorophenyl)-2-cyanoacrylate (Table 1, Entry 2). 1H NMR
(CDCl3, 300 MHz) d ¼ 1.4 (t, 3H, CH3), 4.4 (q, 2H, CH2), 7.49 (dd, J ¼ 6.96 Hz,
2H, aromatic), 7.94 (dd, J ¼ 8.58 Hz, 2H, aromatic), 8.2 (s, 1H, olefinic) ppm; IR
(neat): tmax=cmꢁ1 2915, 2223, 1720 and 1610 cmꢁ1; Anal. calcd. for C12H10NO2Cl
(235.62): C, 61.16; H, 4.28; N, 5.94. Found: C, 61.40; H, 4.13; N, 6.36.
Ethyl 2-cyano-3-(4-methoxyphenyl) acrylate (Table 1, Entry 3). 1H
NMR (CDCl3, 300 MHz) d ¼ 1.4 (t, J ¼ 7.81 Hz, 3 H, CH3), 3.85 (s, 3H, OCH3),
4.38 (q, 2H, OCH2), 7 (dd, J ¼ 8.74 Hz, 2H, aromatic), 8 (dd, J ¼ 8.97 Hz, 2H,
aromatic) and 8.2 (s, 1 H, olefinic) ppm; IR (neat): tmax=cmꢁ1 2930, 2216, 1714
and 1583 cmꢁ1; Anal. calcd. for C13H13NO3 (231.09): C, 67.52; H, 5.67; N, 6.06.
Found: C, 67.52; H, 5.55; N, 6.57.
Ethyl 2-cyano-3-(2-methoxyphenyl) acrylate (Table 1, Entry 4). 1H NMR
(CDCl3, 300 MHz) d ¼ 1.4 (t, 3H, CH3), 3.91 (s, 3H, CH3), 4.37 (q, 2H, CH2), 6.94
(dd, J ¼ 8.56 Hz, 1H, aromatic), 7.06(s, J ¼ 7.83 Hz, 1H, aromatic), 7.52 (t,
J ¼ 7.43 Hz, 1H, aromatic), 8.3 (dd, J ¼ 7.92 Hz, 1H, aromatic), 8.78 (s, 1H, olefinic
proton) ppm; IR (neat): tmax=cmꢁ1 2983, 2221, 1743 and 1588 cmꢁ1; Anal. calcd.
for C13H13NO3 (231.09): C, 67.52; H, 5.67; N, 6.06. Found: C, 67.608; H, 5.56 N, 6.51.
Ethyl 2-cyano-3-(4-fluorophenyl) acrylate (Table 1, Entry 5). 1H NMR
(CDCl3, 300 MHz): 1.36 (t, 3H, CH3), 4.35 (q, 2H, CH2), 8.18 (s, 1H, olefinic pro-
ton), 7.21 (d, J ¼ 7.45 Hz, 2H, aromatic), 8.04 (d, J ¼ 8.56 Hz, 2H, aromatic) ppm;
IR (neat): tmax=cmꢁ1 2997, 2225, 1714 and 1585 cmꢁ1; Anal. calcd. for C12H10FNO2
(219.2): C, 65.75; H, 4.6; N, 6.39. Found C, 65.96; H, 4.2; N, 6.73.
Ethyl 2-cyano-3-(3-nitrophenyl) acrylate (Table 1, Entry 7). 1H NMR
(CDCl3, 300 MHz) d ¼ 1.42 (t, 3H, CH3), 4.42 (q, 2H, CH2), 8.2 (s, 1H, olefinic
proton), 8.7 (s, 1H, aromatic), 8.42 (dd, J ¼ 8.43 Hz, 1H, aromatic), 7.64 (t, 1H,
aromatic), 8.4 (dd, J ¼ 8.06 Hz, 1H, aromatic) ppm; IR (neat): tmax=cmꢁ1 2962,
2225, 1716 and 1602 cmꢁ1. Anal. calcd. for C12H10N2O4 (246.02): C, 58.54; H,
4.09; N, 11.38. Found C, 58.3; H, 3.78; N, 11.78.
Ethyl
2-cyano-3-(4-(2-cyano-3-methox-3-oxoprop-1-enyl)
phenyl)
acrylate (Table 1, Entry 9). 1H NMR (CDCl3, 300 MHz): 1.41 (t, 6H, CH3), 4.4
(q, 4H, CH2), 8.09 (s, 4H, aromatic), 8.25 (s, 2H, olifinic proton) ppm; IR (neat):
t
max=cmꢁ1 2223, 2987, 1722 and 1588 cmꢁ1. Anal. calcd. for C18H16N2O4 (324.1):
C, 66.66; H, 4.97; N, 8.64. Found: C, 66.36; H, 4.6; N, 8.89.
Ethyl 2-cyano-3-(4-(diethylamino)-2-hydroxyphenyl) acrylate (Table 1,
Entry 11). 1H NMR (CDCl3, 300 MHz) d ¼ 1.3 (t, 3H, CH3), 1.6 (t, 6H, CH3),
3.4 (q, 4H, CH2), 4.3 (q, 2H, CH2), 8.4 (s, 1H, olefinic proton), 6.4 (d, J ¼ 7.17 Hz,
1H, aromatic), 6.6 (d, J ¼ 7.23 Hz, 1H, aromatic), 7.4 (s, 1H, aromatic) ppm;

416
B. N. BORSE, S. R. SHUKLA, AND Y. A. SONAWANE
IR(neat): tmax=cmꢁ1 3395, 2964, 2212 and 1685 cmꢁ1. Anal. calcd. for C16H20N2O3
(288.15): C, 66.65; H, 6.19; N, 9.72. Found: C, 65.89; H, 5.78; N 10.13.
Ethyl 2-cyano-3-(2-hydroxyphenyl) acrylate (Table 1, Entry 13).
1H NMR (CDCl3, 300 Hz) d ¼ 1.4 (t, 3H, CH3), 4.25 (q, 2H, CH2), 8.55 (s, 1H, olefinic
proton), 7.4 (t, 1H, aromatic), 7.65 (dd, J ¼ 8.13 Hz, 1H, aromatic), 7.3(dd,
J ¼ 7.63 Hz, 1H, aromatic) ppm; IR (neat): tmax=cmꢁ1 3390, 2962, 2206, 1681 and
1606 cmꢁ1. Anal. calcd. for C12H11NO3 (217.2): C, 66.35; H, 5.1; N, 6.45. Found:
C, 66.5; H, 5.2; N, 7.1.
Ethyl 2-cyano-3-(4-(dimethylamino) phenyl) acrylate (Table 1, Entry
15). 1H NMR (CDCl3, 300 MHz): 1.38 (t, 3H, CH3), 3.13 (s, 6H, CH3), 4.36
(q, 2H, CH2), 6.73 (d, J ¼ 7.10 Hz, 2H, aromatic), 7.97 (d, J ¼ 8.06 Hz, 2H,
aromatic), 8.09 (s, 1H, olefinic proton) ppm; IR (neat): tmax=cmꢁ1 3392, 1610,
1700, 2206 and 1610 cmꢁ1. Anal. calcd. for C14H16N2O2 (244.12): C, 68.83; H,
6.60; N, 11.47. Found: C, 68.413; H, 6.28; N, 11.46.
Ethyl 3-(4-acetamidophenyl)-2-cyanoacrylate (Table 1, Entry 23). 1H
NMR (CDCl3, 300 MHz): 1.41 (t, 3H, CH3), 4.4 (q, 2H, OCH2), 7.72 (d, J ¼ 7.67 Hz,
2H), 7.97 (d, J ¼ 7.89 Hz, 2H), 8.20 (s, 1H, olefinic proton), 1.71 (s, 3H, CH3CONH)
ppm; IR (neat): tmax=cmꢁ1 3382, 1600, 1710, 2216 and 1610 cmꢁ1. Anal. calcd.
for C14H14N2O3 (258.1): C, 65.11; H, 5.46; N, 10.85. Found C, 65.13; H, 5.1;
N, 10.89.
RESULTS AND DISCUSSION
In our efforts in the area of enzymatic synthetic methodologies, we report
herein the catalytic activity and recyclability of biocatalyst toward Knoevenagel
condensation of aromatic aldehydes with aliphatic and aromatic active methylene
compounds under milder reaction conditions (Scheme 1). Separation of the product
and recycling of the biocatalyst were easily achieved four additional times.
The biocatalyst lipases and conventional catalyst (piperidine) were employed in
the condensation of substituted aromatic aldehyde with a variety of active methylene
compounds such as malononitrile, ethyl cyano acetate, 5-fluoroindolin-2-one,
indolin-2-one, and 2-(benzo[d]thiazol-2-yl) acetonitrile to provide the corresponding
trisubstituted electrophilic alkenes. The first series of experiments was carried with
para-N,N-dimethyl benzaldehyde and ethyl cyanoacetate as a model reaction to opti-
mize the reaction parameters. The influence of solvent on the reaction conditions has
been evaluated with various organic solvents such as toluene, hexane, dichloromethane
Scheme 1. Reaction scheme of aromatic aldehyde with active methylene compound. Ar ¼ different
aromatic substituents, and EWG1, EWG2 ¼ different electron-donating or electron-withdrawing group.

LIPASE-CATALYZED KNOEVENAGEL CONDENSATION
417
(DCM), methanol, ethanol, and iso-propanol (IPA). It has been observed that the
polar solvents such as methanol, ethanol, and IPA give better yields of the product
than the nonpolar solvents. These results are summarized in Table 1.
To study the influence of the amount of catalyst on the product yield, it was
found that 10% (by wt. of aldehyde) concentration of lipase is optimum. With
increase in catalyst concentration, yield increases marginally, and 55 ꢀC reaction
temperature was found to be suitable for maximum yield and greater stability of
biocatalyst. Also, when the reaction mixture was stirred at 55 ꢀC for 6 h in the
absence of lipase, no formation of product was detected. The comparative results
of the three catalysts are summarized in Table 2.
Based on the remarkable results obtained using the reaction conditions and to
study generality of this process, a variety of substituted aromatic and heterocyclic
aldehydes carrying either electron-donating or electron-withdrawing substituents
were examined and found to give good yields of products. Several active methylene
compounds containing different active groups such as cyano, carbonyl, and ethoxy-
carbonyl groups could be successfully reacted with different substituted aldehydes.
Electron-deficient aldehydes gave relatively greater yields than their electron-rich
counterparts. Relatively lesser yields of product were observed when the reaction
was carried out with aromatic active methylene groups than aliphatic active methyl-
ene groups for their sterically hindered nature, and also absence of an electron-
withdrawing group decreased the labile nature of acidic proton in products 16, 17,
and 19. Another reason may be that the aromatic aldehyde used in these compounds
contains a dimethyl group at its para position, which helps to donate the electrons
and reduces electron deficiency to the aldehyde group. Heterocyclic aldehydes such
as pyridine 3-carboxyaldehyde (entry 10) gave comparatively greater yield than aryl
aldehydes because of the electron-withdrawing nature of the sp2 hybridized nitrogen
in the pyridine ring. a,b-Unsaturated aldehydes produced the corresponding product
without any difficulty (entry 14).
According to the literature, the formation of coumarin is achieved in a
major amount in the reaction of ortho hydroxy benzaldehyde with ethyl
cyanoacetate,[24,25] which is normally observed under strongly acidic or basic
conditions. It is noteworthy to mention here that in the proposed method only an
Table 1. Effect of solvent on the yield using lipases
Aspergillus
oryzae (%) yielda
Rhizopus
oryzae (%) yielda
No.
Solvent
1
2
3
4
5
6
Toluene
Hexane
DCMb
Methanol
IPA
0
15
50
93
90
95
0
12
45
90
88
91
Ethanol
Note. Reaction conditions: N,N-dimethyl benzaldehyde (4.71 mmol), ethyl
cyanoacetate (4.95 mmol), lipase (0.07 g), and solvent (5 ml) at 55 ꢀC.
aIsolated yields.
bReflux temperature.

418
B. N. BORSE, S. R. SHUKLA, AND Y. A. SONAWANE
Table 2. Lipase-catalyzed Knoevengel condensation
Yielda
(%)
Yieldb
Yieldc
(%)
(%)
[time
in h]
EWG1,
EWG2
[time
in h]
[time
in h]
Mp.
(ꢀC)
Entry
1
Ar
Product
85 [6]
80 [8]
95 [6]
85 [8]
90 [6]
81 [8]
50
88
2
3
87 [8]
83 [7]
95 [7]
91 [7]
90 [7]
86 [7]
86
70
4
5
6
7
78 [7]
83 [6]
73 [5]
87 [8]
95 [6]
85 [5]
81 [8]
88 [6]
78 [5]
97
169
76
8
9
73 [5]
77 [5]
72 [7]
67 [5]
82 [5]
88 [5]
87 [7]
87 [5]
74 [5]
80 [5]
79 [7]
78 [5]
194
207
80
10
11
183
12
13
85 [6]
80 [6]
91 [6]
90 [6]
87 [6]
85 [6]
168
177
(Continued )

LIPASE-CATALYZED KNOEVENAGEL CONDENSATION
419
Table 2. Continued
Yielda
(%)
Yieldb
(%)
Yieldc
(%)
EWG1,
EWG2
[time
in h]
[time
in h]
[time
in h]
Mp.
(ꢀC)
Entry
14
Ar
Product
67 [7]
90 [4]
82 [7]
95 [4]
75 [7]
91 [4]
113
142
15
16
55 [5]
79 [5]
73 [5]
229
17
18
57 [5]
78 [4]
80 [5]
89 [4]
76 [5]
83 [4]
243
151
19
80 [5]
88 [5]
82 [5]
>300
20
21
80 [4]
82 [4]
82 [4]
87 [4]
78 [4]
87 [4]
79
114
22
23
88 [4]
70 [5]
90 [4]
76 [5]
87 [4]
70 [5]
198
193
Note. The products were characterized by FTIR, mass, 1H NMR, and C, H, N analysis.
aIsolated yield by conventional Knoevenagel condensation in ethanol at reflux.
bIsolated yield by Aspergillus oryzae lipase.
cIsolated yield by Rhizopus oryzae lipase.

420
B. N. BORSE, S. R. SHUKLA, AND Y. A. SONAWANE
Scheme 2. Proposed mechanism for lipase-catalyzed Knoevenagel condensation.
olefin derivative is formed and no formation of coumarin product was observed in
compounds 11 and 13 (Table 2).
The proposed reaction mechanism involves sequential process of dehydration.
The active sites of lipase such as aspartate histidine dyad and oxyanion hole extract
the acidic proton of the active methylene group to form the nucleophile and then
attack the nucleophile to generate carbonyl carbon of aldehyde. Then sequential
dehydration takes place, which forms carbon–carbon double bonds (Scheme 2).
Table 3. Recyclability of lipase
Yield (%)a
Run
Aspergillus oryzae
Rhizopus oryzae
1
2
3
4
5
95
90
83
78
72
91
86
79
71
67
Note. Reaction conditions: para-N,N-dimethyl benzal-
dehyde (4.71 mmol), ethyl cyanoacetate (4.95 mmol), lipase
(0.07 g), and ethanol (5 ml) at 55ꢀC for 6 h.
aIsolated yields.

LIPASE-CATALYZED KNOEVENAGEL CONDENSATION
421
Formation of the enolate anion and complex are expected to be the key steps in this
enzymatic process.
To make the biocatalytic processes economical at a large scale, the recyclability
of both the lipases has to be taken into consideration. During this study, the lipase
was recycled for up to four cycles. There was no significant decrease in yield of pro-
duct during the second run whereas the yield declined up to 67% after the completion
of the fifth run as shown in Table 3.
CONCLUSION
In summary, we have described here a cleaner and efficient Knoevengel
condensation using lipase enzyme as a biocatalyst. The Knoevenagel condensation
using biocatalyst is an efficient and convenient method, minimizing the problems
of other toxic catalysts. This method offers marked improvements in terms of
simplicity, reaction conditions, general applicability, high isolated product yields,
and the use of environmentally benign procedures, which makes it highly suitable
for industrial condensation of aromatic aldehydes and active methylene compounds
for the synthesis of trisubstituted electrophilic alkenes. This method also eliminates
the use of toxic catalysts and thus provides a better and practical alternative to
existing procedures. It is easy to separate the catalyst and product after completion
of the reaction. Use of lipases as a biocatalyst provides a good alternative for
industrial synthesis as the reaction is readily scalable.
ACKNOWLEDGMENT
The authors express their thanks to the University Grants Commission,
New Delhi, India, for the financial support.
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