Biocatalysis and Biotransformation, MarchApril 2012; 30(2): 238244

ORIGINAL Article

Biocatalytic Knoevenagel reaction using alkaline protease from

Bacillus licheniformis

Bang-Hua Xie, Zhi Guan & Yan-Hong He

School of Chemistry and Chemical Engineering, Southwest University, Chongqing, China
Abstract
BLAP (alkaline protease from Bacillus licheniformis) was used as a biocatalyst in the Knoevenagel condensations of aromatic,
hetero-aromatic and a;b-unsaturated aldehydes with less reactive acetylacetone or ethyl acetoacetate. The reactions were
performed in organic solvent in the presence of water. The functionalized trisubstituted alkenes and a,b,g,d-unsaturated
carbonyl compounds could be obtained in moderate to good yields with E/Z selectivities up to 99:1. This biocatalytic
reaction provided an alternative pathway for Knoevenagel condensation, which also demonstrates a novel case of unnatural activity of existing enzymes.
Keywords: biocatalysis, Knoevenagel condensation, alkaline protease

Introduction
The concept of enzyme promiscuity, the ability of a
given enzyme to catalyse distinctly different chemical
transformations of natural or non-natural substrates,
has received more and more attention in recent years
(Busto etal. 2010; Wu etal. 2010; Humble & Berglund 2011; Bornscheuer & Kazlauskas. 2004). The
importance of the promiscuity concept in biocatalysis is noteworthy, since it not only highlights existing
catalysts, but may provide novel and practical synthetic pathways that are not currently available
(Kourist etal. 2008; Li etal. 2008; Xu etal. 2007).
A number of enzymes have been demonstrated that
are catalytically promiscuous in synthetic transformations such as Michael addition (Dhake et al.
2010; Cai etal. 2011; Xu etal. 2011), Henry (Fuhshuku & Asano 2011; Wang etal. 2010a), Mannich
(He etal. 2010), Markovnikov addition (Lou etal.
2009), domino reactions (Wang etal. 2010b), aldol
reactions (Li etal. 2010a, 2011) and so on.
The Knoevenagel condensation of aldehydes with
active methylene compounds is one of the most
important and widely used methods for carbon
carbon double bond formation in synthetic chemistry
(Sonawane etal. 2010; Xin etal. 2007; Jones. 1967;
Lorsbach & Kurth 1999). Since its discovery, the

Knoevenagel reaction has been widely employed in

organic synthesis to prepare coumarins and their
derivatives, which are important intermediates in the
synthesis of cosmetics, perfumes and pharmaceuticals. Knoevenagel products are also useful intermediates for further transformations, such as DielsAlder
and Michael additions (Li et al. 2010b; Mahendra
etal. 2010). In recent years, there has been a growing
interest in Knoevenagel products for their high biological activities (Lyall et al. 1989; Shiraishi et al.
1989). The classical Knoevenagel condensation has
been carried out in the presence of bases (Tietze &
Beifuss 1991; Texier-Boullet & Foucaud 1982), Lewis
acids (Lehnert 1970; Cabello et al. 1984; Rao &
Venkataratnam 1991; Prajapati & Sandhu 1993;
Bartoli etal. 2008; Barluenga etal. 2007; Yadav etal.
2009), the other modified inorganic solids (Reddy &
Varma 1997; Shanmuganathan et al. 2010), amino
acids (Hu etal. 2010a, 2010b), ionic liquids (Ranu
& Jana 2006) and nickel nanoparticles (Ghosh etal.
2011) as catalysts. Additionally, a novel light induced
Knoevenagel condensation in aqueous ethanol has
also been reported (Khurana & Vij 2011). However,
there are only a few reports of enzyme catalysed Knoevenagel reactions. The first example of an enzymatic
Knoevenagel reaction was reported in 2009 (Feng

Correspondence: Dr. Yan-Hong He, School of Chemistry and Chemical Engineering, Southwest University, Chongqing, 400715, China. Tel: +86-2368254091. Fax: +86-23-68254091. E-mail: heyh@swu.edu.cn
(Received 13 September 2011; revised 29 October 2011; accepted 30 January 2012)
ISSN 1024-2422 print/ISSN 1029-2446 online 2012 Informa UK, Ltd.
DOI: 10.3109/10242422.2012.662961


etal. 2009), in which CAL-B (acrylic resin immobilized Candida antarctica lipase B) was used to catalyse
decarboxylative Knoevenagel reaction of substituted
aromatic aldehydes and b-ketoesters in MeCN/H2O,
and a primary amine was used as an additive to form
a Schiff base in the course of the reaction. However,
very recently, the mechanism of the lipase-catalysed
Knoevenagel reaction has been challenged (Evitt &
Bornscheuer 2011). Lai etal. (2010) used lipase from
porcine pancreas to catalyse tandem Knoevenagel
condensation and esteriFB01cation with alcohol cosolvents. Chen etal. (2011) reported a tandem Aldol
condensation/dehydration to prepare Knoevenagel
products co-catalysed by acylase and N-heterocyclic
compounds in organic media. Since biocatalytic processes have often been proven to be ecologically
advantageous and more sustainable than current
chemical technologies because of the intrinsic advantages of biocatalysts in higher reaction selectivity,
milder reaction conditions and potential use of inexpensive regenerable resources (Fessner & Anthonsen
2009), further exploration of enzymatic Knoevenagel
reactions is useful. The protease from Bacillus licheniformis has been extensively investigated as a promiscuous catalyst to catalyse CC and CN bond
formation reactions (Lpez-Iglesias etal. 2011). Very
recently, our laboratory found that BLAP (alkaline
protease from Bacillus licheniformis No 2709) could
catalyse a domino Knoevenagel/intramolecular transesterification reaction to synthesize 2H-1-benzopyran-2-one derivatives (Wang etal. 2011). Herein, we
report BLAP catalysed Knoevenagel reaction of aromatic, hetero-aromatic and a,b-unsaturated aromatic
aldehydes with less reactive acetylacetone or ethyl
acetoacetate.
Experimental
General remarks
BLAP (200,000 U/g, one unit of activity is the
amount of enzyme that liberates 1.0 mg of tyrosine
from casein per minute at 40C and pH 10.5) was
purchased from Wuxi Xuemei Enzyme Co. Ltd.
(WuXi, China). Unless otherwise noted, all reagents
were obtained from commercial suppliers and were
used without further purification. 1H-NMR (300
MHz) spectra were recorded on a Bruker AV-300 in
CDCl3 at room temperature. Coupling constants (J)
were given in Hz. Melting points were determined
on an X-4 digital display micro melting point apparatus and were uncorrected. All reactions were monitored by thin-layer chromatography (TLC) with
Haiyang GF254 silica gel plates. Flash column chromatography was carried out using 100200 mesh
silica gel at increased pressure.

Biocatalytic Knoevenagel reaction 239

General procedure for the BLAP catalysed Knoevenagel
condensation (Table III, entries 119)
A mixture of aldehyde (0.5 mmol), acetylacetone or
ethyl acetoacetate (0.6 mmol), DMSO (0.95 ml),
deionized water (0.05 ml) and BLAP (70 mg) was
stirred at 45C. The progress of the reaction was
monitored by TLC. After complete conversion, the
enzyme was filtered, and ethyl acetate was used to
wash the filter paper and residue to assure that the
products were dissolved in the filtrate. The filtrate
was added with water and extracted with ethyl acetate. The combined organic extracts were dried over
anhydrous Na2SO4 and concentrated in vacuo. The
resulting product was directly charged onto a silica
gel column and eluted with a mixture of ethyl acetate/petroleum ether to afford pure products. Knoevenagel products Table III, entries 1, 3, 4, 5, 7, 8
(Hu et al. 2010); 2, 5, 6, 14, 15 (Barluenga et al.
2007); 9, 10 (Ranu & Jana 2006; Barluenga et al.
2007); 11, 12 (Deng & Xu 2002); 13, 16 (Yadav
etal. 2009) are all known compounds.

Results and discussion

Effect of solvents on the BLAP catalysed Knoevenagel
condensation and control experiments
Enzymes can not only work in organic media, but
also can acquire remarkable properties such as
enhanced stability, altered substrate specificities
and the ability to catalyse unusual reactions, which
are impossible in aqueous media (Klibanov 1989).
In this context, we have discovered that BLAP can
catalyse the Knoevenagel reaction. Initial investigations were undertaken using p-methoxy cinnamaldehyde and acetylacetone as a model reaction. A
preliminary solvent screen was performed to find
out the optimal solvent for this biocatalysis
(Table I). It was found that different solvents
affected the enzyme catalytic activity, with high
catalytic activity in polar solvents including DMSO
and DMF (Table I, entries 1 and 2) affording the
Knoevenagel product in good yields. In less polar
and non-polar solvents such as THF, TBME (tertbutyl methyl ether), CH2Cl2, CH3CN and cyclohexane (Table I, entries 37) the enzyme displayed
low activity giving the Knoevenagel product in low
yields. When the reaction was performed in deionized water, only a poor yield was obtained (Table I,
entry 8). This may be attributed to low solubility of
the substrates in water. We also performed the reaction under solvent-free conditions, which also gave
a low yield (Table I, entry 9). Thus, we chose
DMSO as the optimal solvent for BLAP catalysed
Knoevenagel condensation.

240 B.-H. Xie etal.

Table I. Solvent screening and control experiments.a

H3CO

BLAP

H3CO

O
O

Solvent / H2O
CHO
Solvent

Yield (%)b

DMSO
DMF
THF
TBME
CH2Cl2
MeCN
Cyclohexane
H2O
solvent-free
DMSO (no enzyme)
DMSO (BLAP denatured with EDTAc)
DMSO (BLAP inhibited with PMSFd)

66
60
22
14
14
22
18
12
16
13
15
14

Entry
1
2
3
4
5
6
7
8
9
10
11
12
aThe

reactions were carried out using p-methoxy cinnamaldehyde (81 mg,

0.5 mmol), acetylacetone (60 mg, 0.6 mmol), deionized water (0.1 ml), solvent (0.9
ml) and BLAP (alkaline protease from Bacillus licheniformis No 2709) (50 mg) at 35C
for 55 h. b Refers to yield of isolated product after flash chromatography. c Pre-treated
with EDTA at 25C for 24 h. d Pre-treated with PMSF at 25C for 24 h.

Effects of water content on the BLAP catalysed

Knoevenagel condensation
It is known that enzymes need essential bound water,
and enzymatic activity in organic solvents depends
on water content (Fan etal. 2003). Thus, we investigated the effect of water content, from 0 to 100%
(Vwater/VwaterDMSO), on the yield of the Knoevenagel condensation (Figure 1); the enzyme showed the
highest catalytic activity at 5% water content,
producing the Knoevenagel adduct in a yield of
68%. Above 40% water content, the yield decreased

significantly, possibly due to the insolubility of the

substrates. Therefore, a water content of 5% was
chosen as a suitable reaction condition.
Effects of temperature and enzyme loading on the
BLAP catalysed Knoevenagel condensation
The reaction rate increased with increasing temperature from 25C to 45C (Figure 2); however,
70
60
Yield (%)

In order to verify the specific catalytic effect of

BLAP on Knoevenagel condensation, some control
experiments were carried out. As expected, the Knoevenagel reaction of p-methoxy cinnamaldehyde and
acetylacetone in the absence of enzyme only gave
product in 13% yield after 55 h (Table I, entry 10).
Furthermore, complete inhibition of the catalytic
activity of BLAP in the Knoevenagel reaction was
observed using the serine protease inhibitor PMSF
(phenylmethanesulfonyl Fluoride), showing that it
did not arise from unspecific protein-derived activation (Table I, entry 12). In addition, since BLAP is
calcium-dependent, EDTA (ethylenediaminetetraacetic acid) inactivated enzyme was also examined
and shown to give a similar Knoevenagel condensation product yield as the blank, further demonstrating the role of enzyme catalytic activity (Table I,
entry 11).

50
40
30
20
10
-10

10

20

30 40 50 60
water content (%)

70

80

90 100

Figure 1. Effects of water content in DMSO on the BLAPcatalysed Knoevenagel condensation. Reaction conditions: BLAP
(50 mg), p-methoxy cinnamaldehyde (81 mg, 0.5 mmol),
acetylacetone (60 mg, 0.6 mmol), and VwaterVDMSO (1 ml) at
35C for 50 h. Deionized water was added from 0 to 100 %
(Vwater/VwaterDMSO). Yield refers to the isolated product after
flash chromatography.

Biocatalytic Knoevenagel reaction 241

Substrate range of the BLAP catalysed Knoevenagel
condensation

80

Yield (%)

70
60
50
40
30
20
20

30

40

50

60
T (C)

70

80

90

100

Figure 2. Effects of temperature on the BLAP-catalyzed

Knoevenagel condensation. All reactions were carried out using
p-methoxy cinnamaldehyde (81 mg, 0.5 mmol), acetylacetone
(60 mg, 0.6 mmol), deionized water (0.05 ml), DMSO (0.95 ml)
and BLAP (50 mg) at different temperature for 50 h. Yield refers
to the isolated product after flash chromatography.

the yield decreased significantly above 45C,

although the enzyme displayed good initial catalytic
activity at 4050C. At 100C, the enzyme appeared
to be denaturated, exhibiting no catalytic activity.
This further confirmed the need for a functional
active site for BLAP to catalyse the Knoevenagel
reaction. Based on the temperature screening, we
chose 45C as the optimum temperature for the
reaction.
To find the optimal loading of the enzyme,
1090 mg BLAP were screened for the model reaction (Table II). This indicated that 70 mg of BLAP
(200 U/mg) was the optimum quantity for the
Knoevenagel reaction between 0.5 mmol p-methoxy
cinnamaldehyde and 0.6 mmol acetylacetone in 1 ml
of DMSO/H2O (Table II, entry 3). Further increasing
the BLAP loading did not increase the reaction yield.
Thus, we chose 70 mg as the optimal amount of catalyst for further experiments.

Table II. Effects of BLAP (alkaline protease from Bacillus

licheniformis No 2709)loading on the yield of Knoevenagel
condensation.a
Entry
1
2
3
4
aAll

BLAP (mg)

Yield (%)b

10
50
70
90

25
65
82
75

reactions were carried out using p-methoxy cinnamaldehyde

(81 mg, 0.5 mmol), acetylacetone (60 mg, 0.6 mmol), deionized
water (0.05 ml), DMSO (0.95 ml) and BLAP (090 mg) at 45C
for 44 h. bYield of the isolated product after chromatography on
silica gel.

With the optimized catalytic system in hand, some

other aldehydes and active methylene compounds
were used to expand upon this BLAP-catalysed
Knoevenagel condensation to show the generality
and scope of the reaction (Table III). A wide range
of aldehydes, including a,b-unsaturated aromatic
aldehydes, heterocyclic aldehydes and aromatic
aldehydes, could be accepted by the enzyme to
react with acetylacetone and ethyl acetoacetate. In
general, both electron-donating and electronwithdrawing substituents were tolerateda;bUnsaturated aromatic aldehydes worked well and
gave functionalized a;b;g;d-unsaturated carbonyl
compounds in good yields within 2148 h (Table
III, entries 18). However, longer reaction times
(7296 h) were required for heterocyclic aldehydes
and aromatic aldehydes to give functionalized
trisubstituted alkenes in acceptable yields (Table
III, entries 1016), except for 2-furaldehyde, which
gave the best yield of 81% with acetylacetone after
29 h (Table III, entry 9). The a,b-unsaturated aromatic aldehydes exhibited better reactivity than
aromatic and hetero-aromatic aldehydes. This may
be ascribed to steric effects as the aldehyde (CHO)
groups in a,b-unsaturated aldehydes are less hindered than in aromatic and hetero-aromatic aldehydes. In addition, we attempted to extend this
biocatalysis to aliphatic aldehydes, but no products
were detected (Table III, entries 1719). This probably reflects the specific substrate selectivity of the
enzyme.
Reactions with ethyl acetoacetate could give
products as Z and E isomers according to the newly
formed double bonds. Z or E selectivities up to
99:1 were observed for the reactions, and their
ratios are given in Table III.
Conclusion
We have described a general method for Knoevenagel condensation catalysed by BLAP in a DMSO/
H2O system. Aromatic, hetero-aromatic and a;bunsaturated aromatic aldehydes worked well with
less reactive acetylacetone and ethyl acetoacetate,
and the functionalized trisubstituted alkenes and
a,b,g,d-unsaturated carbonyl compounds could be
obtained in acceptable yields. The influence of reaction conditions including solvents, water content,
loading of catalyst and temperature were investigated. The BLAP catalysed Knoevenagel condensation provides a novel case of unnatural activity in
existing enzymes and might be a useful synthetic
method.

242 B.-H. Xie etal.

Table III. Substrate scope of BLAP (alkaline protease from Bacillus licheniformis No 2709) catalysed
Knoevenagel condensation.a

EWG
R-CHO

BLAP

EWG

DMSO/H2O, 45 oC

EWG

+
EWG

b
a

Entry

b
O

CHO

Time (h)

Yield (%)b (E/Z)

44

82

24

80 (29:71)

48

70

24

76 (25:75)

24

76

32

67 (24:76)

21

80

48

68 (25:75)

29

81

72

63 (45:55)

96

25

96

42 (99:1)

96

64

72

73 (70:30)

72

40 (99:1)

96

24

H3CO
CHO

O
O

H3CO

CHO

H3C
CHO

O
O

H3C
5

CHO

CHO

O
O

O
O
O

CHO

Cl
CHO

O
O

Cl

10

11

12

CHO
CHO

13

CHO

14

CHO

O
O
O

CHO
CHO

O
O
O

O
CHO

15

O2N
16

CHO

(Continued)

Biocatalytic Knoevenagel reaction 243

Table III. (Continured).
Entry

17

CHO

18

CHO

O
O

O
O

Time (h)

Yield (%)b (E/Z)

96

No reaction

96

No reaction

96

No reaction

O
19

CHO

aReaction

conditions: a (0.5 mmol), b (0.6 mmol), deionized water (0.05 ml), DMSO (0.95 ml) and BLAP
(70 mg) at 45C. bYield of the isolated product after chromatography on silica gel and the Z or E
configuration was determined by 1H NMR in comparison with the known compounds.

Declaration of interest: The authors report no

conflicts of interest. The authors alone are responsible for the content and writing of the article. Financial support from Natural Science Foundation
Project of CQ CSTC (2009BA5051) is gratefully
acknowledged.
References
Barluenga J, Riesgo L, Vicente R, Luis AL, Toms M. 2007. Rearrangement of propargylic esters: metal-based stereospecific
synthesis of (E)- and (Z)-knoevenagel derivatives. J Am Chem
Soc 129:77727773.
Bartoli G, Bosco M, Carlone A, Dalpozzo R, Galzerano P,
Melchiorre P, Sambri L. 2008. Magnesium perchlorate as efficient Lewis acid for the Knoevenagel condensation between
b-diketones and aldehydes. Tetrahedron Lett 49:25552557.
Bornscheuer UT, Kazlauskas RJ. 2004. Catalytic promiscuity in
biocatalysis: using old enzymes to form new bonds and follow
new pathways. Angew Chem Int Ed 43:60326040.
Busto E, Gotor-Fernndez V, Gotor V. 2010. Hydrolases: catalytically promiscuous enzymes for non-conventional reactions
in organic synthesis. Chem Soc Rev 39:45044523.
Cabello JA, Campelo JM, Garcia A, Luna D, Marinas JM. 1984.
Knoevenagel condensation in the heterogeneous phase using
aluminum phosphate-aluminum oxide as a new catalyst. J Org
Chem 49:51955197.
Cai JF, Guan Z, He YH. 2011. The lipase-catalyzed asymmetric
CC Michael addition. J Mol Catal B Enzym 68:240244.
Chen X, Liu BK, Kang H, Lin XF. 2011. A tandem Aldol condensation/dehydration co-catalyzed by acylase and N-heterocyclic compounds in organic media. J Mol Catal B Enzym 68:
7176.
Deng L, Xu MX. 2002. Synthesis of 4-thienyl substituted dihydropyridines derivatives. Huaxi Yaoxue Zazhi 1:3738.
Dhake KP, Tambade PJ, Singhal RS, Bhanage BM. 2010. Promiscuous Candida antarctica lipase B-catalyzed synthesis of
b-amino esters via aza-Michael addition of amines to acrylates.
Tetrahedron Lett 51:44554458.
Evitt AS, Bornscheuer UT. 2011. Lipase CAL-B does not catalyze
a promiscuous decarboxylative aldol addition or Knoevenagel
reaction. Green Chem 13:11411142.
Fan H, Kitagawa M, Rakub T, Tokiwa Y. 2003. Role of water
activity on the synthesis of vinyl thymidine catalyzed by protease from Bacillus Subtilis. Macromol Biosci 3:420424.
Feng XW, Li C, Wang N, Li K, Zhang WW, Wang Z, Yu XQ. 2009.
Lipase-catalysed decarboxylative aldol reaction and decarboxylative Knoevenagel reaction. Green Chem 11:19331936.

Fessner WD, Anthonsen T. 2009. Modern biocatalysis: stereoselective and environmentally friendly reactions. New York: WileyVCH.
Fuhshuku K, Asano Y. 2011. Synthesis of (R)-b-nitro alcohols
catalyzed by R-selective hydroxynitrile lyase from Arabidopsis
thaliana in the aqueousorganic biphasic system. J Biotechnol
153:153159.
Ghosh S, Das J, Chattopadhyay S. 2011. A novel light induced
Knoevenagel condensation of Meldrums acid with aromatic
aldehydes in aqueous ethanol. Tetrahedron Lett 52:28692872.
He T, Li K, Wu MY, Feng XW, Wang N, Wang HY, Li C, Yu XQ.
2010. Utilization of biocatalytic promiscuity for direct Mannich
reaction. J Mol Catal B Enzym 67:189194.
Hu Y, Guan Z, He YH, Louwagie N, Yao MJ. 2010a. L-Arginine
as a cost-effective and recyclable catalyst for the synthesis of
a,b-unsaturated nitriles and ketones in an ionic liquid.
J Chem Res 34:2224.
Hu Y, He YH, Guan Z. 2010b. A simple method for the preparation of functionalized trisubstituted alkenes and a,b,g,d-unsaturated carbonyl compounds by using natural amino acid
l-tryptophan. Catal Commun 11:656659.
Humble MS, Berglund P. 2011. Biocatalytic promiscuity. Eur J
Org Chem 2011:33913401.
Jones G. 1967. Organic reactions. New York: Wiley.
Khurana JM, Vij K. 2011. Nickel nanoparticles catalyzed chemoselective Knoevenagel condensation of Meldrums acid and
tandem enol lactonizations via cascade cyclization sequence.
Tetrahedron Lett 52:36663669.
Kourist R, Bartsch S, Fransson L, Hult K, Bornscheuer UT.
2008. Understanding promiscuous amidase activity of an
esterase from Bacillus subtilis. ChemBioChem 9:6769.
Klibanov AM. 1989. Enzymatic catalysis in anhydrous organic
solvents. Trends Biochem Sci 14:141144.
Lai YF, Zheng H, Chai SJ, Zhang PF, Chen XZ. 2010. Lipasecatalysed tandem Knoevenagel condensation and esterification
with alcohol cosolvents. Green Chem 12:19171918.
Lehnert W. 1970. Verbesserte variante der knoevenagelkondensation mit TiCl4/THF/pyridin(I). Alkyliden- und Arylidenmalonester bei 025C. Tetrahedron Lett 11:47234724.
Li C, Hassler M, Bugg TDH. 2008. Catalytic promiscuity in the
a/b-hydrolase superfamily: hydroxamic acid formation, C-C
bond formation, ester and thioester hydrolysis in the C-C
hydrolase family. ChemBioChem 9:7176.
Li C, Zhou YJ, Wang N, Feng XW, Li K, Yu XQ. 2010a. Promiscuous protease-catalyzed aldol reactions: a facile biocatalytic
protocol for carboncarbon bond formation in aqueous media.
J Biotechnol 150:539545.
Li HH, He YH, Yuan Y, Guan Z. 2011. Nuclease p1: a new biocatalyst for direct asymmetric aldol reaction under solvent-free
conditions. Green Chem 13:185189.

244 B.-H. Xie etal.

Li JL, Kang TR, Zhou SL, Li R, Wu L, Chen YC. 2010b. Organocatalytic asymmetric inverse-electron-demand DielsAlder
reaction of electron-deficient dienes and crotonaldehyde.
Angew Chem Int Ed 49:64186420.
Lpez-Iglesias M, Busto E, Gotor V, Gotor-Fernndez V. 2011.
Use of protease from Bacillus licheniformis as promiscuous catalyst for organic synthesis: applications in C-C and C-N bond
formation reactions. Adv Synth Catal 353:23452353.
Lorsbach BA, Kurth MJ. 1999. Carboncarbon bond forming
solid-phase reactions. Chem Rev 99:15491582.
Lou FW, Liu BK, Wang JL, Pan Q, Lin XF. 2009. Controllable
enzymatic Markovnikov addition and acylation of thiols to vinyl
esters. J Mol Catal B Enzym 60:6468.
Lyall R, Zilberstein A, Gazit A, Gilon C, Levitzki A, Schlessinger
J. 1989. Tyrphostins inhibit epidermal growth factor (EGF)receptor tyrosine kinase activity in living cells and EGFstimulated cell proliferation. J Biol Chem 2649:1450314509.
Mahendra PP, Akhilesh KS, Raghavan BS. 2010. Importance of
the nature of a-substituents in pyrrolidine organocatalysts in
asymmetric michael additions. J Org Chem 75:73107321.
Prajapati D, Sandhu JS. 1993. ChemInform abstract: cadmium
iodide as a new catalyst for knoevenagel condensations. J Chem
Soc Perkin Trans 1:739740.
Ranu BC, Jana R. 2006. Ionic liquid as catalyst and reaction
medium -a simple, efficient and green procedure for Knoevenagel condensation of aliphatic and aromatic carbonyl compounds using a task-specific basic ionic liquid. Eur J Org Chem
16:37673770.
Rao PS, Venkataratnam RV. 1991. Zinc chloride as a new catalyst
for knoevenagel condensation. Tetrahedron Lett 32:5821
5822.
Reddy TI, Varma RS. 1997. Rare-earth (RE) exchanged NaY zeolite promoted knoevenagel condensation. Tetrahedron Lett
38:17211724.
Shanmuganathan S, Greiner L, Domnguez de Mara P. 2010. Silicaimmobilized piperazine: a sustainable organocatalyst for aldol and
Knoevenagel reactions. Tetrahedron Lett 51:66706672.
Shiraishi T, Owada MK, Tatsuki M, Yamashita Y, Kaun-aga T.
1989. Specific inhibitors of tyrosine-specific protein kinases:

Supplementary information available online

Supplementary information to be found online at
http://www.informahealthcare.com/bab/doi/10.3109/
10242422.2012.662961

properties of 4-hydroxycinnamamide derivatives in Vitro.

Cancer Res 49:23742378.
Sonawane YA, Phadtare SB, Borse BN, Jagtap Amit R, Shankarling GS. 2010. Synthesis of diphenylamine-based novel fluorescent styryl colorants by Knoevenagel condensation using a
conventional method, biocatalyst, and deep eutectic solvent.
Org Lett 12:14561459.
Texier-Boullet F, Foucaud A. 1982. Knoevenagel condensation
catalysed by aluminium oxide. Tetrahedron Lett 23:4927
4928.
Tietze LF, Beifuss U. 1991. In comprehensive organic synthesis.
Pergamon: Oxford.
Wang CH, Guan Z, He YH. 2011. Biocatalytic domino reaction:
synthesis of 2H-1-benzopyran-2-one derivatives using alkaline
protease from Bacillus licheniformis. Green Chem 13:2048
2054.
Wang JL, Li X, Xie HY, Liu BK, Lin XF. 2010a. Hydrolasecatalyzed fast Henry reaction of nitroalkanes and aldehydes in
organic media. J Biotechnol 145:240243.
Wang L, Li C, Wang N, Li K, Chen X, Yu XQ. 2010b. Enzymemediated domino synthesis of 2-alkylbenzimidazoles in solventfree system: a green route to heterocyclic compound. J Mol
Catal B Enzym 67:1620.
Wu Q, Liu BK, Lin XF. 2010. Enzymatic promiscuity for organic
synthesis and cascade process. Curr Org Chem 14:1966
1988.
Xin X, Guo X, Duan HF, Lin YJ, Sun H. 2007. Efficient Knoevenagel condensation catalyzed by cyclic guanidinium lactate
ionic liquid as medium. Catal Commun 8:115117.
Xu JM, Zhang F, Liu BK, Wu Q, Lin XF. 2007. Promiscuous
zinc-dependent acylase-mediated carboncarbon bond formation in organic media. Chem Commun 2007:20782080.
Xu KL, Guan Z, He YH. 2011. Acidic proteinase from Aspergillus
usamii catalyzed Michael addition of ketones to nitroolefins.
J Mol Catal B Enzym 71:108112.
Yadav JS, Bhunia DC, Singh VK, Srihari P. 2009. Solvent-free
NbCl5 catalyzed condensation of 1,3-dicarbonyl compounds
and aldehydes: a facile synthesis of trisubstituted alkenes.
Tetrahedron Lett 50:24702473.

Copyright of Biocatalysis & Biotransformation is the property of Taylor & Francis Ltd and its content may not
be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written
permission. However, users may print, download, or email articles for individual use.