Enzyme catalytic promiscuity: The papain-catalyzed Knoevenagel reaction

Article abstract of DOI:10.1016/j.biochi.2011.09.018

Papain as a sustainable and inexpensive biocatalyst was used for the first time to catalyze the Knoevenagel reactions in DMSO/water. A wide range of aromatic, hetero-aromatic and α,β-unsaturated aldehydes could react with less active methylene compounds acetylacetone and ethyl acetoacetate. The products were obtained in moderate to excellent yields with Z/E selectivities of up to 100:0. This case of biocatalytic promiscuity not only widens the application of papain to new chemical transformations, but also could be developed into a potentially valuable method for organic synthesis.

Full text of DOI:10.1016/j.biochi.2011.09.018

Biochimie 94 (2012) 656e661
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Research paper
Enzyme catalytic promiscuity: The papain-catalyzed Knoevenagel reaction
,
a
,
,
Wen Hua, Zhi Guana , Xiang Deng b, Yan-Hong Hea
*
*
a School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
b Department of Chemistry and Chemical Engineering, Sichuan University of Arts and Science, Sichuan Dazhou 635000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 May 2011
Accepted 19 September 2011
Available online 25 September 2011
Papain as a sustainable and inexpensive biocatalyst was used for the first time to catalyze the Knoeve-
nagel reactions in DMSO/water. A wide range of aromatic, hetero-aromatic and a,b-unsaturated alde-
hydes could react with less active methylene compounds acetylacetone and ethyl acetoacetate. The
products were obtained in moderate to excellent yields with Z/E selectivities of up to 100:0. This case of
biocatalytic promiscuity not only widens the application of papain to new chemical transformations, but
also could be developed into a potentially valuable method for organic synthesis.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Knoevenagel reaction
Papain
Enzyme promiscuity
Biocatalysis
1. Introduction
has been reported, which was the only example of enzymatic
Knoevenagel reaction [20]. CAL-B (acrylic resin immobilized
Knoevenagel reaction, as a facile and versatile method for the
formation of carbonecarbon bond [1], has been commonly applied in
the synthesis of chemicals and chemical intermediates such as
carbocyclic as well as heterocyclic compounds of biological signifi-
cance [2], coumarin derivatives, cosmetics, perfumes and pharma-
ceuticals [3]. Great efforts have been devoted to explore the effective
catalysts, and elegant works have been described with high effi-
ciency. Generally, these catalysts include bases [3], zeolites [4], ionic
liquids [5], amino acids [6,7] and some metal based Lewis acids
[8e10]. Moreover, the Knoevenagel reactions involving 1,3-diketones
have often been subjected to low yields ordrastic reaction conditions,
due to less activity of 1,3-diketones which tend to form stable six-
membered cyclic enols [11,12]. Therefore, the development of envi-
ronmentally benign and cost-efficient catalysts for the Knoevenagel
reactions of 1,3-diketones still maintains a significant challenge.
Over last three decades, enzymes as practical catalysts have been
increasingly exploited for organic synthesis for their simple pro-
cessing requirements, high selectivity and mild reaction conditions.
Enzyme promiscuity means, in the broadest terms, one single active
site of a given enzyme can catalyze different chemical trans-
formation of natural or non-natural substrates [13e16]. Although
the promiscuous behaviors were originally thought to be rare
events, a growing number of enzymes have been used to catalyze
the formation of carbonecarbon and carboneheteroatom bonds
through some classic and widely used organic reactions [17e19].
Recently a lipase-catalyzed decarboxylative Knoevenagel reaction
Candida antarctica lipase B) was used to catalyze decarboxylative
Knoevenagel reaction of substituted aromatic aldehydes and
b-ketoesters in CH3CN/H2O, and a primary amine was used as an
additive to form a Schiff base in the course of the reaction. But very
recently, the mechanism of lipase-catalyzed Knoevenagel reaction
has been challenged [21]. Herein, we wish to report a novel
discovery that papain could promote the direct Knoevenagel reac-
tion without using additives, and aromatic, hetero-aromatic and
a,b-unsaturated aldehydes could react with less active methylene
compounds acetylacetone and ethyl acetoacetate resulting in
moderate to good yields. Papain (EC 3.4.22.2) is a powerful proteo-
lytic enzyme belonging to the cysteine protease family. Its enzy-
matic and physiological properties have been extensively studied.
Papain is mainly produced from the extraction of Carica papaya
latex. Nowadays, some protocols for the cloning and overexpression
of papain using baculovirus/insect [22], yeast [23,24] and bacteria
[25e27] as expression host organisms have been reported. It is
possible to obtain recombinant papain in substantial quantities for
both basic research and industrial use. In this paper, papainwas used
for the first time to catalyze Knoevenagel reaction. It provides
a novel case of enzyme catalytic promiscuity and might be
a potential synthetic method for organic chemistry.
2. Material and methods
2.1. General information for the reagents
Papain (SigmaeAldrich) from C. papaya (catalog number: 76220,
ꢀ3 U/mg. 1 U corresponds to the amount of enzyme which
* Corresponding authors. Tel./fax: þ86 23 68254091.
E-mail addresses: guanzhi@swu.edu.cn (Z. Guan), heyh@swu.edu.cn (Y.-H. He).
0300-9084/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.biochi.2011.09.018

W. Hu et al. / Biochimie 94 (2012) 656e661
657
Table 1
hydrolyzes 1 mmol N-benzoyl-L-arginine ethyl ester (BAEE, Fluka
The catalytic activities of different proteinases in Knoevenagel reaction.a
No. 12880) per minute at pH 6.2 and 25 ꢁC) was purchased from
SigmaeAldrich. Papain (Pangbo) from the latex of C. papaya
(650 U/mg. One unit of activity was defined as the amount of the
enzyme to produce TCA-soluble hydrolysis products from casein,
O
CHO
enzyme
DMSO/water, 25 oC
O
O
+
which gives an absorbance value equivalent to 1 mg of tyrosine at
O
275 nm per minute at 37 ꢁC and pH 7.0), and bromelain from
pineapple peduncle (500 U/mg. One unit of activity was defined as
the amount of the enzyme to produce TCA-soluble hydrolysis
products from casein, which gives an absorbance value equivalent
Entry Enzyme
Time (h) Yield (%)b
1
2
3
No enzyme
110
72
72
6
9
Acidic proteinase from Aspergillus usamii No 537
Alkaline proteinase from Bacillus
licheniformis No 2709
19
to 1 m
g of tyrosine at 275 nm per minute at 37 ꢁC and pH 7.0) were
purchased from Guangxi Nanning Pangbo Biological Engineering
Co. Ltd. (Nanning, China). Alkaline proteinase from Bacillus lichen-
iformis No 2709 (200 U/mg. One unit of activity is the amount of
4
5
6
Neutral proteinase from Bacillus subtilis A.S.1.398 72
20
19
35
Bromelain from pineapple peduncle
Papain from the latex of Carica papaya (Pangbo)
72
72
a
enzyme that liberates 1 mg of tyrosine from casein per minute at
All reactions were carried out using benzaldehyde (212 mg, 2 mmol), acetyla-
40 ꢁC and pH 10.5), acidic proteinase from Aspergillus usamii No 537
cetone (240 mg, 2.4 mmol), enzyme (200 mg), deionized water (0.75 ml) and DMSO
(5 ml) at 25 ꢁC.
(50 U/mg. One unit of activity is the amount of enzyme that liber-
b
Yield of the isolated product after chromatography on silica gel.
ates 1
and neutral proteinase from Bacillus subtilis A.S.1.398 (130 U/mg.
One unit of activity is the amount of enzyme that liberates 1 g of
m
g of tyrosine from casein per minute at 40 ꢁC and pH 3.0),
3.2. The effect of solvents
m
tyrosine from casein per minute at 30 ꢁC and pH 7.5) were
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.
All reactions were monitored by thin layer chromatography (TLC)
with Haiyang GF254 silica gel plates. Flash column chromatography
was carried out using 100e200 mesh silica gel at increased
pressure.
Based on the concept that enzymes can work in organic media,
and they acquire remarkable properties such as enhanced stability,
altered substrate specificity, and the ability to catalyze unusual
reactions which was impossible in aqueous media [28], we investi-
gated the effect of different solvents on the model reaction. We found
that the catalytic activity of papain was remarkably influenced by
solvents (Fig. 1). The reaction in DMSO gave the best yield of 35%
while the reactions in water and toluene provided the yields of 28%
and 24% respectively. The other tested solvents including DMF,
chloroform, tert-butyl methyl ether (TBME), EtOH and THF gave the
low yields. Moreover, only 10% yield was obtained under solvent-free
conditions. No clear correlation between the solvent polarity and the
enzyme activity was observed. This result may be attributed to
specific interactions between the solvent and papain. Based on the
results of solvent screen, DMSO was chosen as the optimum solvent
for the papain-catalyzed Knoevenagel condensation.
2.2. Typical procedure for the papain-catalyzed Knoevenagel
condensation
Papain (150 mg) was added to a 10 ml round-bottom flask
containing benzaldehyde (212 mg, 2 mmol), acetylacetone (240 mg,
2.4 mmol), DMSO (3.75 ml) and deionized water (1.25 ml). The
resulting mixture was stirred at 60 ꢁC for specified time, and
monitored by thin layer chromatography (TLC). The reaction was
terminated by filtering the enzyme. The filtrate was diluted with
ethyl acetate (10 ml) and washed with water (5 ml ꢂ 2). The
aqueous phase was back-extracted with ethyl acetate (10 ml ꢂ 2).
Combined organic phase was washed with water and concentrated
in vacuo. The crude product was purified by flash column chro-
matography (ethyl acetate/petroleum ether).
3.3. The effect of water contents
Water plays a major role of “molecular lubricant” in enzyme
resulting in conformational flexibility of enzyme, and the increased
3. Results and discussion
3.1. The catalytic activities of different proteinases in Knoevenagel
reaction
Initial studies were undertaken using benzaldehyde and acety-
lacetone as a model reaction. We chose DMSO/water (Vwater
/
VDMSO ¼ 0.15) as the medium, and the reaction was performed at
25 ꢁC. The catalytic activities of 5 proteinases in Knoevenagel
reaction were investigated using the model reaction (Table 1). The
best result of 35% yield was achieved by using papain as the catalyst
after 72 h (Table 1, entry 6). Other proteinases also exhibited
varying degrees of catalytic activity in the experiment. It was worth
mentioning that not all proteinases could catalyze the Knoevenagel
reaction effectively such as acidic proteinase which only gave
a yield of 9% (Table 1, entry 2). Moreover, the blank experiment was
also performed under identical reaction conditions, and it only gave
the product in yield of 6% after 110 h (Table 1, entry 1). It was
obvious that papain was the most effective catalyst among the
tested enzymes. Therefore, we chose papain as the catalyst for
Knoevenagel condensation.
Fig. 1. The effect of solvents on the papain-catalyzed Knoevenagel reaction. Condi-
tions: benzaldehyde (212 mg, 2 mmol), papain (Pangbo) (200 mg), acetylacetone
(240 mg, 2.4 mmol), organic solvent (5 ml), deionized water (0.75 ml) at 25 ꢁC for 72 h.

658
W. Hu et al. / Biochimie 94 (2012) 656e661
hydration leads to enhanced activity in non-aqueous solvents
[29,30]. Thus, it is significant to confirm the optimal water content
in reaction system. We found that the activity of papain in Knoe-
venagel reaction could be evidently affected by the water content in
DMSO (Fig. 2). Generally, a hill-shaped curve could be used to
describe the results. When water content was lower than 25%
(water/water þ DMSO, v/v), the yield of the Knoevenagel reaction
could be enhanced by increasing the concentration of water. The
reaction reached the highest yield of 57% at 25% water content after
120 h. However, once the water content surpassed 30%, the yield
dropped sharply. All the results indicated that water is obviously
essential in the papain-catalyzed Knoevenagel reaction. Thus, we
chose 25% water content for the Knoevenagel reaction.
3.4. The effect of reaction temperature
Temperature also plays an important role in enzyme-catalyzed
reactions, due to its effects on enzyme stability and reaction rate.
Thus, a temperature screening was performed (Fig. 3). It was found
that the activity of papain in Knoevenagel reaction could be
increased by raising the temperature, and reached the best yield of
55% at 60 ꢁC after 81 h. However, once the temperature surpassed
60 ꢁC, the yield of the Knoevenagel product decreased sharply. In
order to verify whether or not the decrease of the yield was caused
by side-reactions, we performed the reactions at 70 ꢁC and 80 ꢁC
twice, and the reaction mixtures were processed very carefully. No
any by-product was detected, and the starting materials were left.
So the obvious decrease in the yields at temperatures higher than
60 ꢁC was probably due to the denaturation of papain caused by the
high temperature. On the other hand, the influence of temperature
implied that the catalytic behavior of papain was not simply caused
by the amino acid distribution on the protein surface. The specific
natural fold of papain was required for its ability to catalyze the
Knoevenagel reaction. Based on the temperature screening, we
chose 60 ꢁC as the optimum temperature for the reaction.
Fig. 3. The effect of temperature on the papain-catalyzed Knoevenagel reaction.
Conditions: benzaldehyde (212 mg, 2 mmol), papain (Pangbo) (200 mg), acetylacetone
(240 mg, 2.4 mmol), deionized water (1.25 ml), DMSO (3.75 ml) at specified temper-
ature for 81 h.
was the optimum quantity for the Knoevenagel reaction between
2 mmol of benzaldehyde and 2.4 mmol of acetylacetone in 5 ml of
DMSO/H2O. When higher papain loading was used, the reaction
yield became saturated.
3.6. The control experiments
To further demonstrate the specific catalytic effect of papain
under the optimized conditions, some control experiments have
been performed (Table 2). Because we used the papain purchased
from Pangbo Biological Engineering Co. Ltd without further puri-
fication, it was necessary to exclude the possibility that the reaction
was catalyzed by other proteins or impurities in the mixture
instead of papain itself. So the papain purchased from
SigmaeAldrich was also used to conduct the model reaction and
3.5. The effect of papain loading
Next, we investigated the effect of papain loading on the reac-
tion (Fig. 4). The results showed that 150 mg of papain (650 U/mg)
Fig. 2. The effect of water contents on the papain-catalyzed Knoevenagel reaction.
Conditions: benzaldehyde (212 mg, 2 mmol), papain (Pangbo) (200 mg), acetylacetone
(240 mg, 2.4 mmol) deionized water from 0% to 45% [water/(water þ DMSO), v/v] at
25 ꢁC for 120 h.
Fig. 4. The effect of papain loading on the yield of Knoevenagel reaction. Conditions:
benzaldehyde (212 mg, 2 mmol), acetylacetone (240 mg, 2.4 mmol), deionized water
(1.25 ml), DMSO (3.75 ml) and specified amount of papain (Pangbo) (650 U/mg) at
60 ꢁC for 120 h.

W. Hu et al. / Biochimie 94 (2012) 656e661
659
Table 2
condensation, which only gave a yield of 10% (Table 2, entry 10). The
facts that papain from different companies had the same effect on
the enzymatic Knoevenagel reaction, and the results from the
experiments using denatured and inhibited papain (from both
companies) indirectly excluded the possibility of other protein or
impurity catalysis. These results also indicated that the specific
natural fold and the native proteolytic active site of papain were
responsible for its activity in Knoevenagel condensation.
The control experiments.a
Entry
Catalyst
Yield (%)b
1
2
3
4
5
6
7
8
9
Papain (Pangbo)
Papain (SigmaeAldrich)
No enzyme
59
54
6
10
11
6
7
12
7
Papain (Pangbo) inhibited with MMTSc
Papain (SigmaeAldrich) inhibited with MMTSc
Papain (Pangbo) denatured by high temperatured
Papain (SigmaeAldrich) denatured by high temperatured
Papain (Pangbo) denatured with ureae
Papain (SigmaeAldrich) denatured with ureae
Albumin egg
3.7. Scope of the papain-catalyzed Knoevenagel reactions
10
10
Finally, with the optimized conditions in hand, some other
aldehydes and methylene compounds were used to expand upon the
papain-catalyzed Knoevenagel reaction to test the generality and
scope of this new enzymatic promiscuity. Aromatic and hetero-
aromatic aldehydes could react with acetylacetone and ethyl
acetoacetate smoothly to give the corresponding products under
optimized conditions (Table 3). In general, acetylacetone was more
reactive than ethyl acetoacetate under employed reaction conditions.
This observation was contrary to the reported amino acid catalyzed
Knoevenagel reactions [7], probably due to the special catalytic effect
of papain. For example, 4-nitrobenzaldehyde gave the best yield of
87% with acetylacetone (Table 3, entry 1), but it gave a very low yield
of 30% with ethyl acetoacetate and only Z stereoisomer was obtained
(Table 3, entry 2). In order to verify this low yield and the stereo-
selectivity of the products, the reaction of 4-nitrobenzaldehyde with
ethyl acetoacetate was repeated, and the reaction mixture was pro-
cessed carefully. Although there were two product spots observed on
TLC plate, one of them was too rare to be isolated. Only one product
was isolated which was determined as Z isomer after analysis of
spectroscopic data. Similarly, 2-chlorobenzaldehyde gave a good
yield of 71% with acetylacetone (Table 3, entry 7), but it gave a low
yield of 32% with ethyl acetoacetate (Table 3, entry 8). Moreover, both
electron-donating and electron-withdrawing substituents of
aromatic aldehydes were compatible (Table 3, entries 1e10), but
aromatic aldehydes containing electron-withdrawing substituent
generally gave better yields than those with electron-donating
substituent. For instance, the reactions of 4-chlorobenzaldehyde
with acetylacetone and ethyl acetoacetate gave good yields of 82%
a
Conditions: benzaldehyde (2 mmol), acetylacetone (2.4 mmol), deionized water
(1.25 ml), DMSO (3.75 ml) and enzyme (150 mg) at 60 ꢁC for 120 h.
b
Yield of the isolated product after chromatography on silica gel.
The mixture of papain (150 mg), DMSO (3.75 ml), deionized water (1.25 ml) and
c
MMTS (50 mL) was stirred at r.t. for 48 h before use.
d
The mixture of papain (150 mg), DMSO (3.75 ml) and deionized water (1.25 ml)
was stirred at 100 ꢁC for 48 h before use.
e
The mixture of papain (150 mg), DMSO (3.75 ml), deionized water (1.25 ml) and
urea (50 mg) was stirred at r.t. for 48 h before use.
the same set control experiments in Table 2. The Knoevenagel
condensation between benzaldehyde and acetylacetone under
optimized conditions gave satisfied yields of 59% (using papain
from Pangbo) (Table 2, entry 1) and 54% (using papain from
SigmaeAldrich) (Table 2, entry 2). The reaction in the absence of
enzyme under identical reaction conditions only gave the product
in yield of 6% (Table 2, entry 3). Furthermore, an almost complete
inhibition of the catalytic activity of papain in the Knoevenagel
condensation was observed by using serine protease inhibitor
methyl methanethiosulfonate (MMTS) [31], which gave the yields
of 10% (Pangbo) and 11% (SigmaeAldrich) (Table 2, entries 4 and 5).
Moreover, the high temperature (100 ꢁC) denatured papain (from
both companies) completely lost its activity in Knoevenagel
condensation giving the results equal to the blank (Table 2, entries
6 and 7), which was in agreement with the observation in the
temperature screening that high temperature could cause dena-
turation of papain under employed reaction conditions. Next, when
the reactants were incubated with urea-denatured papain, only
12% (Pangbo) and 7% (SigmaeAldrich) yields were obtained
(Table 2, entries 8 and 9). In addition, non-enzyme protein albumin
egg almost did not show catalytic activity for Knoevenagel
and 62% respectively (Table 3, entries
3 and 4). However,
4-methoxybenzaldehyde gave a low yield of 30% with acetylacetone
Table 3
The papain-catalyzed Knoevenagel reactions of aromatic and hetero-aromatic aldehydes with acetylacetone and ethyl acetoacetate.a
O
O
R1
O
O
papain
+
+
R1 CHO
R1
R
R
DMSO/water, 60 oC, 120 h
R
O
1
2
O
Entry
R
R1
Yield (%)b
[Z/E]c
1
2
3
4
5
6
7
8
Me
OEt
Me
OEt
Me
OEt
Me
OEt
Me
OEt
Me
OEt
Me
4-NO2C6H4
4eNO2C6H4
4-ClC6H4
4-ClC6H4
3-ClC6H4
3-ClC6H4
2-ClC6H4
2-ClC6H4
4-MeOC6H4
4-MeOC6H4
Furan-2-yl
Furan-2-yl
Thien-2-yl
87
30
82
62
84
60
71
32
30
25
77
70
51
100:0
72:28
75:25
90:10
100:0
18:82
9
10
11
12
13
a
Conditions: 1 (2 mmol), 2 (2.4 mmol), papain (Pangbo) (150 mg), deionized water (1.25 ml), DMSO (3.75 ml) at 60 ꢁC for 120 h.
Yield of the isolated product after chromatography on silica gel.
b
c
The Z/E geometry was determined by 1HNMR and the complete spectroscopic data is provided in the Supporting Information.

660
W. Hu et al. / Biochimie 94 (2012) 656e661
Table 4
The papain-catalyzed Knoevenagel reactions of
a
,b
-unsaturated aromatic aldehydes with acetylacetone and ethyl acetoacetate.a
O
R
O
O
O
O
papain
+
R2
+
R
R2
R2
CHO
R
DMSO/water, 60 oC, 120 h
3
2
O
Entry
R
R2
Yield (%)b
[Z/E]c
1
2
3
4
5
6
7
8
9
Me
4-CH3C6H4
4-CH3C6H4
4-MeOC6H4
4-MeOC6H4
Ph
76
68
81
86
70
42
74
62
66
OEt
Me
OEt
Me
OEt
Me
OEt
Me
55:45
56:44
58:42
51:49
Ph
4-ClC6H4
4-ClC6H4
4-FC6H4
a
Conditions: 2 (2.4 mmol), 3 (2 mmol), papain (Pangbo) (150 mg), deionized water (1.25 ml), DMSO (3.75 ml) at 60 ꢁC for 120 h.
Yield of the isolated product after chromatography on silica gel.
b
c
The Z/E geometry was determined by 1HNMR and the complete spectroscopic data is provided in the Supporting Information.
(Table 3, entry 9), while it gave a yield of 25% with ethyl acetoacetate
donating substituents gave higher yields than those containing
strong electron-withdrawing substituents. For instance, when
reacting with acetylacetone, 4-methoxy cinnamaldehyde gave the
(Table 3, entry 10) and only Z stereoisomer was obtained. We have
carefully checked the reaction of 4-methoxybenzaldehyde and ethyl
acetoacetate, and only one product spot could be observed on TLC
plate which was determined as Z isomer after isolation and analysis
of spectroscopic data. In addition, hetero-aromatic aldehydes
2-furaldehyde and 2-thienylaldehyde could react with acetylacetone
and ethyl acetoacetate to provide products in good yields (Table 3,
entries 11e13). Particularly noteworthy are the configurations of the
newly formed double bonds when ethyl acetoacetate was used as the
active methylene compound. This enzymatic reaction showed good
to excellent Z/E selectivities with the best Z/E ratios of up to 100:0.
The Z-selectivity was observed for aromatic aldehydes (Table 3,
entries 2, 4, 6, 8 and 10), however a good E-selectivity was obtained
for hetero-aromatic aldehyde (Table 3, entry 12).
product in
a good yield of 81% (Table 4, entry 3), but
4-fluorocinnamaldehyde only gave a yield of 66% (Table 4, entry 9).
Moreover, low Z/E selectivities were observed for the products from
a,
b-unsaturated aromatic aldehydes (Table 4, entries 2, 4, 6 and 8),
probably due tothe less steric hindrance of eCHO in -unsaturated
a,b
aromatic aldehydes than in aromatic aldehydes. Besides, when
aliphatic aldehydes such as isobutylaldehyde and 3-methyl butanal
were used to react with acetylacetone respectively, no Knoevenagel
adducts were detected under employed reaction conditions.
3.8. The proposed catalytic mechanism for the papain-catalyzed
Knoevenagel reaction
This biocatalytic transformation was also applicable to
a
,
b
-unsaturated aromatic aldehydes (Table 4). It was found that the
electronic effects of substituents had some impact on the reaction
yields. -Unsaturated aromatic aldehydes bearing strong electron-
Papain is a globular protein consisting of a single polypeptide
chain of 212 residues, folded to form two domains with a deep cleft
between them [32,33]. The active site cysteine residue (Cys-25) is
a,b
His-159
His-159
H
H
O
H
N
N
O
O
N
H
N
H
O
O
H
N
SH
O
H
H
O
O
N
H
Gln-19
H
Gln-19
NH2
Cys-25
NH2
Cys-25
Asn-175
Asn-175
His-159
His-159
His-159
O
H
N
H
N
R
N
O
R
H
H
N
H
N
H
R
N
H
H
-H2O
R
H
O
O
O
O
O
O
O
O
O
O
NH2
NH2
H
S
S
NH2
CH2
CH2
H
Asn-175
Asn-175
H
Asn-175
Cys-25
Cys-25
Gln-19
Cys-25
Scheme 1. The proposed catalytic mechanism for the papain-catalyzed Knoevenagel reaction.

W. Hu et al. / Biochimie 94 (2012) 656e661
661
functionalized trisubstituted alkenes via Knoevenagel condensation, Tetra-
hedron Lett. 47 (2006) 6501e6504.
[10] G. Bartoli, M. Bosco, A. Carlone, R. Dalpozzo, P. Galzerano, P. Melchiorre,
L. Sambri, Magnesium perchlorate as efficient Lewis acid for the Knoevenagel
part of the L1
histidine (His-159) is in a
a
-helix at the surface of the left domain, while the
b
sheet at the surface of the right domain of
the enzyme. From the control experiments using inhibited papain
and denatured papain, it was inferred that the catalysis of the
Knoevenagel reaction depends on the native proteolytic active site in
papain. Based on Hillier and co-worker’s predictions about the
catalytic mechanism of papain [34], we hypothesized the mechanism
of the papain-catalyzed Knoevenagel reaction (Scheme 1). Firstly, the
substrate acetylacetone was deprotonated by His-159 forming an
enolate anion, which was stabilized by the oxyanion hole formed by
the side chain of Gln-19 and the backbone NH of Cys-25 [35,36].
Secondly, another substrate aldehyde accepted the proton from the
imidazolium cation, and simultaneouslyconnected the enolate anion
with the formation of a carbonecarbon bond. Finally, the dehydra-
tion of the resulted adduct took place under the catalysis of Cys and
AsneHis dyad to gave the Knoevenagel product. Meanwhile, the
CyseHisionpairformedwhich wasstabilized byAsn-175via keeping
the imidazole ring of His-159 in a favorable orientation [34].
condensation between
b-diketones and aldehydes, Tetrahedron Lett. 49
(2008) 2555e2557.
[11] S. Kantevari, R. Bantu, L. Nagarapu, HClO4eSiO2 and PPAeSiO2 catalyzed
efficient one-pot Knoevenagel condensation, Michael addition and cyclo-
dehydration of dimedone and aldehydes in acetonitrile, aqueous and
solvent free conditions: scope and limitations, J. Mol. Catal. A: Chem. 269
(2007) 53e56.
[12] C. Su, Z.C. Chen, Q.G. Zheng, Organic reaction in ionic liquids: Knoevenagel
condensation catalyzed by ethylenendiammonium diacetate, Synthesis
4
(2003) 555e559.
[13] C. Carboni-Oerlemans, P. Dominguez de Maria, B. Tuin, G. Bargeman, A. van
der Meer, R. van Gemert, Hydrolase-catalysed synthesis of peroxycarboxylic
acids: biocatalytic promiscuity for practical applications, J. Biotechnol. 126
(2006) 140e151.
[14] A. Taglieber, H. Hobenreich, J.D. Carballeira, R.J.G. Mondiere, M.T. Reetz,
Alternate-site enzyme promiscuity, Angew. Chem. Int. Ed. 46 (2007)
8597e8600.
[15] R.J. Kazlauskas, Enhancing catalytic promiscuity for biocatalysis, Curr. Opin.
Chem. Biol. 9 (2005) 195e201.
[16] A. Babtie, N. Tokuriki, F. Hollfelder, What makes an enzyme promiscuous,
Curr. Opin. Chem. Biol. 14 (2010) 200e207.
[17] H.H. Li, Y.H. He, Y. Yuan, Z. Guan, Nuclease p1: a new biocatalyst for direct
asymmetric aldol reaction under solvent-free conditions, Green Chem. 13
(2011) 185e189.
[18] C. Branneby, P. Carlqvist, A. Magnusson, K. Hult, T. Brinck, P. Berglund, Carbon-
carbon bonds by hydrolytic enzymes, J. Am. Chem. Soc. 125 (2003) 874e875.
[19] C. Li, Y.J. Zhou, N. Wang, X.W. Feng, K. Li, X.Q. Yu, Promiscuous protease-
4. Conclusion
We describe here the first papain-catalyzed Knoevenagel reaction.
The catalyst, with a safe, economical and environmentally benign
quality, can catalyze the Knoevenagel reactions of a wide range of
catalyzed aldol reaction:
a facile biocatalytic protocol for carbon-carbon
aromatic, hetero-aromatic and
a,b-unsaturated aldehydes with less
bond formation in aqueous media, J. Biotechnol. 150 (2010) 539e545.
[20] X.W. Feng, C. Li, N. Wang, K. Li, W.W. Zhang, X.Q. Yu, Lipase-catalysed
decarboxylative aldol reaction and decarboxylative Knoevenagel reaction,
Green Chem. 11 (2009) 1933e1936.
[21] A.S. Evitt, U.T. Bornscheuer, Lipase CAL-B does not catalyze a promiscuous
decarboxylative aldol addition or Knoevenagel reaction, Green Chem. 13
(2011) 1141e1142.
[22] T. Vernet, D.C. Tessier, C. Richardson, F. Laliberté, H.E. Khouri, A.W. Bell,
A.C. Storer, D.Y. Thomas, Secretion of functional papain precursor from insect
cells, J. Biol. Chem. 265 (1990) 16661e16666.
active methylene compounds acetylacetone and ethyl acetoacetate to
give moderate to good yields. The influence of reaction conditions
including solvents, water content, temperature and enzyme loading
was investigated. The control experiments were also conducted to
demonstrate the specific catalysis of papain. This papain-catalyzed
Knoevenagel reaction provides a novel case of catalytic promiscuity
which widens the applicability of papain in organic synthesis.
[23] T. Vernet, J. Chatellier, D.C. Tessier, D.Y. Thomas, Expression of functional
papain precursor in Saccharomyces cerevisiae: rapid screening of mutants,
Protein Eng. 6 (1993) 213e219.
Acknowledgments
[24] M.K. Ramjee, J.R. Petithory, J. McElver, S.C. Weber, J.F. Kirsch,
A yeast
expression/secretion system for the recombinant plant thiol endoprotease
propapain, Protein Eng. 9 (1996) 1055e1061.
[25] L.W. Cohen, C. Fluharty, L.C. Dihel, Synthesis of papain in Escherichia coli, Gene
88 (1990) 263e267.
Financial support from the Natural Science Foundation Project
of CQ CSTC (2009BA5051) is gratefully acknowledged.
[26] M.A.J. Taylor, K.A. Pratt, D.F. Revell, K.C. Baker, I.G. Sumner, P.W. Goodenough,
Active papain renatured and processed from insoluble recombinant prop-
apain expressed in Escherichia coli, Protein Eng. 5 (1992) 455e459.
[27] D. Choudhury, S. Roy, C. Chakrabarti, S. Biswas, J.K. Dattagupta, Production and
recovery of recombinant propapain with high yield, Phytochemistry 70 (2009)
465e472.
Appendix. Supplementary material
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.biochi.2011.09.018.
[28] K. Griebenow, A.M. Klibanov, On protein denaturation in aqueouseorganic
mixtures but not in pure organic solvents, J. Am. Chem. Soc. 118 (1996)
11695e11700.
References
[1] S.V. Ryabukhin, A.S. Plaskon, D.M. Volochnyuk, S.E. Pipko, A.N. Shivanyuk,
[29] G.D. Yadav, P.S. Lathi, Synthesis of citronellol laurate in organic media cata-
lyzed by immobilized lipases: kinetic studies, J. Mol. Catal. B. Enzym. 27
(2004) 113e119.
A.A. Tolmachev, Combinatorial Knoevenagel reactions, J. Comb. Chem.
9
(2007) 1073e1078.
[30] A. Zaks, A.M. Klibanov, The effect of water on enzyme action in organic media,
J. Biol. Chem. 263 (1998) 8017e8021.
[2] L.F. Tietze, Domino reactions in organic synthesis, Chem. Rev. 96 (1996)
115e136.
[31] J. Drenth, K.H. Kalk, H.M. Swen, Binding of chloromethyl ketone substrate
analogues to crystalline papain, Biochemistry 15 (1976) 3731e3738.
[32] M. St-Vincent, M. Dickman, Chemical modification of papain and subtilisin: an
active site comparison. An undergraduate biochemistry experiment, J. Chem.
Educ. 81 (2004) 1048.
[33] I.G. Kamphuis, K.H. Kalk, M.B.A. Swarte, J. Drenth, Structure of papain refined
at 1.65 at 1.65 A resolution, J. Mol. Biol. 17 (1984) 233e256.
[34] M.J. Harrison, N.A. Burton, I.H. Hillier, Catalytic mechanism of enzyme papain:
[3] L.F. Tietze, U. Beifuss, in: B.M. Trost, I. Fleming, C.H. Heathcock (Eds.), Compre-
hensive Organic Synthesis, vol. 2, Pergamon Press, Oxford, 1991, pp. 341e394.
[4] T.I. Reddy, R.S. Verma, Rare earth-exchanged NaY zeolite-promoted Knoeve-
nagel condensation, Tetrahedron Lett. 38 (1997) 1721e1724.
[5] B.C. Ranu, R. Jana, 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 (2006) 3767e3770.
predictions with
a hybrid quantum mechanical/molecular mechanical
[6] Y. Hu, Z. Guan, Y.H. He, N. Louwagie, M.J. Yao, L-Arginine as a cost-effective
potential, J. Am. Chem. Soc. 119 (1997) 12285e12291.
[35] R. Menard, J. Carriere, P. Laflamme, C. Plouffe, H.E. Khouri, T. Vernet,
D.C. Tessier, D.Y. Thomas, A.C. Storer, Contribution of the glutamine 19 side
chain to transition state stabilization in the oxyanion hole of papain,
Biochemistry 30 (1991) 8924e8928.
[36] R. Menard, C. Plouffe, P. Laflamme, T. Vernet, D.C. Tessier, D.Y. Thomas,
A.C. Storer, Modification of the electrostatic environment is tolerated in the
oxyanion hole of the cysteine protease papain, Biochemistry 34 (1995)
464e471.
and recyclable catalyst for the synthesis of
ketones in an ionic liquid, J. Chem. Res. (2010) 22e24.
[7] Y. Hu, Y.H. He, Z. Guan, A simple method for the preparation of functionalized
a,b-unsaturated nitriles and
trisubstituted alkenes and a,b,g,d-unsaturated carbonyl compounds by using
natural amino acid l-tryptophan, Catal. Commun. 11 (2010) 656e659.
[8] P.S. Rao, R.V. Venkataratnam, Zinc chloride as a new catalyst for Knoevenagel
condensation, Tetrahedron Lett. 32 (1991) 5821e5822.
[9] G. Bartoli, R. Beleggia, S. Giuli, A. Giuliani, E. Marcantoni, M. Massaccesi,
M. Paoletti, The CeCl3$7H2O-NaI system as promoter in the synthesis of