Efficient promiscuous Knoevenagel condensation catalyzed by papain confined in Cu3(PO4)2 nanoflowers

Article abstract of DOI:10.1039/c7ra12940h

To develop an efficient and green immobilized biocatalyst for promiscuous catalysis which has a broad scope of applications, hybrid nanoflower (hNF) confined papain as a biocatalyst has been proposed and characterized in this study. hNFs were firstly prepared through mixing CuSO₄ aqueous solution with papain in phosphate saline (PBS) at room temperature. The resulting hNFs were characterized by SEM and verified through a hydrolysis reaction with N-benzoyl-dl-arginine amide as substrate. Under optimal conditions, this nano-biocatalyst demonstrated a 15-fold hydrolytic activity compared with papain of free form, along with better thermal stability. A series of reaction factors (reaction temperature, time, and solvent) have been investigated for Knoevenagel condensation reactions with hNFs as catalyst. At optimal conditions, product yield of the hNFs catalyzed reaction was 1.3 fold higher than that of the free enzyme with benzaldehyde and acetylacetone as substrates. A few aldehydes and methylene compounds have also been used to test the generality and scope of this new enzymatic promiscuity. To sum up, the obtained hNFs demonstrate better catalytic properties than free papain and the inorganic metal-salt crystal can function as both support and promotor in biocatalysis.

Full text of DOI:10.1039/c7ra12940h

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Efficient promiscuous Knoevenagel condensation
catalyzed by papain confined in Cu3(PO4)2
nanoflowers†
Cite this: RSC Adv., 2018, 8, 2357
a
a
a
Jianyun Yu,a Xinxin Chen,a Min Jiang,a Anming Wang,
Linlin Yang, Xiaolin Pei,
*
*
Pengfei Zhanga and Stephen Gang Wub
To develop an efficient and green immobilized biocatalyst for promiscuous catalysis which has a broad
scope of applications, hybrid nanoflower (hNF) confined papain as a biocatalyst has been proposed and
characterized in this study. hNFs were firstly prepared through mixing CuSO4 aqueous solution with
papain in phosphate saline (PBS) at room temperature. The resulting hNFs were characterized by SEM
and verified through a hydrolysis reaction with N-benzoyl-DL-arginine amide as substrate. Under optimal
conditions, this nano-biocatalyst demonstrated a 15-fold hydrolytic activity compared with papain of free
form, along with better thermal stability. A series of reaction factors (reaction temperature, time, and
solvent) have been investigated for Knoevenagel condensation reactions with hNFs as catalyst. At
optimal conditions, product yield of the hNFs catalyzed reaction was 1.3 fold higher than that of the free
Received 1st December 2017
Accepted 29th December 2017
enzyme with benzaldehyde and acetylacetone as substrates.
A few aldehydes and methylene
compounds have also been used to test the generality and scope of this new enzymatic promiscuity. To
sum up, the obtained hNFs demonstrate better catalytic properties than free papain and the inorganic
metal-salt crystal can function as both support and promotor in biocatalysis.
DOI: 10.1039/c7ra12940h
rsc.li/rsc-advances
Knoevenagel catalysts, for example, metal-salt, nanoparticle cata-
lyst, modied zeolites, ionic liquids or magnetic base
1. Introduction
analogues.6,10,11 Among them, supported metal and metal-salt
catalysts attract much attention because they can be easily recov-
ered from the reaction mixture and reused in the next cycle.12 This
recyclable heterogeneous Pd catalyst can partially resolve some
issues of homogeneous Pd ligands catalyst in Knoevenagel
condensation.13,14 However, as a transition metal element, Pd is
expensive and the preparation of this supported catalyst is time-
consuming and not environmentally friendly. In comparison,
copper is much more preferred in terms of cost and availability:
copper powder can also catalyze the condensation of cyanoacetate
and benzaldehyde under mild conditions.6 Further, copper salt
provides better catalytic activities compared metal form and is
considered as a green and effective catalyst for organic reactions.
For instance, Zhou et al. has developed a procedure for synthesis
of substituted benzimidazo[1,2-a] quinolones via Knoevenagel
condensation and intramolecular Ullmann-type coupling by using
CuI as catalyst, with a reaction condition of 5–7 hours at 90 ꢀC and
a yield of 84%.15 In general, most organic synthesis using metal or
metal salt as catalysts requires shorter reaction time at the cost of
higher reaction temperature.16–20
Knoevenagel condensation1,2 is a common and versatile method
for C–C bond formation and widely used in both academic
research and chemical industries for the preparation of ne
chemical intermediates, polymers, cosmetics, perfumes, and
therapeutic drugs such as antibody–drug conjugates (ADCs).3
This reaction can be catalyzed by organo-bases,4 such as pyridine
or piperidine.5 Homogeneous base catalysis is oen time-
consuming, because acid has to be added to neutralize the
waste aerwards. Further, undesired side-reactions such as olig-
omerization may occur with relatively low reaction temperatures,
and catalyst recovery is challenging.6 As reported, the Knoevena-
gel condensation reaction2,7,8 can take place on ethyl malonate (as
substrate) bound to the Wang resin, however, product cleavage
requires triuoroacetic acid, leading to excess pollution when it
afforded pure coumarin-3-carboxylic acid derivatives.9
To overcome those drawbacks mentioned above, numerous
efforts have been made to develop better heterogeneous
aCollege of Materials, Chemistry and Chemical Engineering, Hangzhou Normal
University, Hangzhou 310014, P. R. China. E-mail: waming@hznu.edu.cn; Fax: +86-
571-28865630; Tel: +86-571-28865978
In comparison, papain acting as a promiscuous biocatalyst has
been reported to catalyze Knoevenagel reactions in DMSO/water
and is considered as a sustainable and inexpensive option.21
Promiscuous catalysis is dened as catalyzing more than two
kinds reactions simultaneously, which is different from substrate
bDepartment of Energy, Environmental and Chemical Engineering, Washington
University, St. Louis, MO 63130, USA
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ra12940h
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 2357–2364 | 2357

View Article Online
RSC Advances
Paper
promiscuity. The difference lies in the fact that the latter requires ask. Subsequently, DMSO (3.75 mL), DI water (1.25 mL), and
stabilization of different transition state in the reaction papain-inorganic hybrid nanoower (containing 150 mg
process.22,23 Catalytic promiscuity has been widely employed in papain) were added in this sequence. This mixture was shaked
industrial biocatalysis and organic synthesis, because it expands at 150 round per minute and incubated at 60 ꢀC for 24 h (Fig. 1)
the genre of practicable reactions along with advantages of and monitored by thin chromatography (TLC). Aer that, the
enzymatic catalysis such as mild reaction conditions, simple reaction was terminated by ltering out enzyme. The ltrate was
separation, and high yields.24 Further, papain from carica papaya washed twice with DI water (5 mL) and diluted with ethyl acetate
has been reported to present catalytic promiscuity and catalyze (10 mL). The remaining aqueous phase was extracted with ethyl
the synthesis of triuoromethyl carbinol derivatives via aldol acetate until most organic matter was extracted from aqueous
reaction between a,a,a-triuoromethyl ketones and aliphatic phase. The solution was dried through adding anhydrous
ketones.22 Moreover, Wei and coworkers have studied the possible MgSO4 and the ltrate was obtained through ltering and
reaction pathway for papain-catalyzed hydrolysis of N-acetyl-Phe- puried via ash column chromatography (ethyl/petroleum
Gly 4-nitroanilide (APGNA).25 The whole hydrolysis process ether).
includes two stages of acylation and deacylation. However,
application of enzymatic promiscuity is challenged by low activity
of enzymes in promiscuous reactions. A type of enzyme catalyst
with high catalytic activity in enzymatic promiscuous reactions
would greatly expand the application spectrum of enzyme
2.3 Preparation and characterization of papain-inorganic
hybrid nanoowers
The papain-inorganic hybrid nanoowers were synthesized
following procedures reported:23,26 20 mL of CuSO4 solution (120
mM) was added to 3 mL of phosphate buffered saline (PBS)
promiscuity. It will be very much desirable if metal salt with
catalytic superiority can be combined with immobilized enzyme.
solution (10 mM) containing 0.25 mg mLÀ1 papain at pH 9.0
In this work, we propose to combine the advantages of
(Fig. 2). This mixture was then kept at room temperature (ꢁ25
enzyme promiscuity biocatalysis with the strengths of metal salt
ꢀC) for 3 days and centrifuged at 5000 rpm for 5 min (4 ꢀC). The
catalysis in Knoevenagel condensation. Inorganic Cu salt crystal
resulting precipitation was kept and washed with DI water for
has the potential to work as both chemical catalyst and support
three times.
simultaneously. For instance, promiscuous reactions can be
catalyzed by papain attached on inorganic Cu salt crystal.
2.4 Activity assay of papain preparations
Cu3(PO4)2$3H2O was rstly generated in papain aqueous solu-
tion, then papain was embedded to form papain-inorganic
hybrid nanoowers. Its catalytic properties were characterized
via Knoevenagel condensation under mild reaction conditions.
Further, we also determine its catalytic generality and scope
through a few aldehydes and methylene compounds.
Two methods were employed to test the activity of papain
preparations27,28 and to determine the catalytic properties of
hNFs. In this rst method, BAPNA was used as substrate,
enzyme liquor (100 mL) was rstly added into a 10 mL plastic
pipe and placed in water bath (40 ꢀC) for 5 min. Aer that,
BAPNA (0.4%, 2 mL) was added in and OD405 was determined
aer 10 minutes. In the second method, BAEE was used as the
substrate. Enzyme liquor (100 mL) was also added into 10 mL
2. Materials and methods
2.1 Materials
ꢀ
plastic pipe and placed in water bath at 37 C for 5 min. Aer-
Papain (activity $ 400 U mgÀ1) was purchased from Shanghai
Tripod Biological Technology Ltd. Dimethyl sulfoxide was
purchased from Sangon Biotech and acetaldehyde (40%) was
purchased from Tianjin Guangfu Chemical Industry Research
Institute. Phenyl acetaldehyde (95%), nitrobenzene formalde-
hyde (97%), 4-bromobenzaldehyde (99%), magnesium sulfate
anhydrous, N-alpha-benzoyl-L-arginine, and N-benzoyl-L-argi-
nine ethyl ester hydrochloride (BAEE) were purchased from
Aladdin (Shanghai). 4-Nitroaniline and N-benzoyl-DL-arginine
amide hydrochloride (BAPNA) were purchased from Sigma.
Benzoyl acetone was purchased from Alfa Aesar chemistry Ltd
(Tianjin). Ortho nitrobenzene (99%) was purchased from
Damas-beta Chemistry Ltd. 4-Chlorobenzene formaldehyde
(98.5%) was purchased from Acros Organics. All other chemicals
were provided by Sinopharm Chemical Reagent Ltd (Shanghai).
wards, BAEE (0.4%, 2 mL) was added in and mixed, OD253 was
examined aer 10 minutes. In both methods, substrate solu-
tions were used as blank control.
2.5 Characterization of products in promiscuous catalysis
1H NMR spectra and 13C NMR were recorded through a Brucker
DPX 300 spectrometer at 500 MHz and 126 MHz, respectively.
NMR experiment was performed in deuterated chloroform
(CDCl3) containing proper concentration of sample, a total 128
scans were recorded. Data was collected and analyzed through
MestReNova soware. Proton chemical shis were referenced
to tetramethylsilane (TMS) at 0.00 pm. Scanning electron
microscope (SEM) (S-4800, HITACHI) was employed to examine
2.2 General procedure of hybrid nanoowers-catalyzed
Knoevenagel condensation
Typically, benzaldehyde (212 mg, 2 mmol) and acetylacetone
Fig. 1 The Knoevenagel condensation reaction catalyzed by hNF (R1,
(240 mg, 2.4 mmol) were added to a 10 mL of round bottom phenyl; R2, methyl).
2358 | RSC Adv., 2018, 8, 2357–2364
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
hydrolysis reaction in the activity characterization, the prepared
hNFs demonstrate about 15 folds of activity higher than that of
free papain with either substrate. For kinetics property of the
enzyme preparations, the results were shown in Table 1. It was
found that the Km value of free papain was smaller than hNFs',
which mean that the hNFs had a better affinity for the corre-
sponding substrate. In addition, the Kcat/Km value of hNFs was
higher than that of free enzyme, which showed the reaction rate
of former enzyme preparation was better.
Fig. 2 Schematic forming process of hNFs.
During the formation process of hNFs, Cu2+ was proposed to
rstly react with phosphate to generate primary copper phos-
phate complexes. Subsequently, the amine groups from papain
backbone were coordinated with copper phosphate to initiate
the nucleation of nanocrystals. Large agglomeration of protein
molecules and primary crystals were formed aerwards. The
growth of copper phosphate crystals originated at the individual
Cu2+ binding sites on the surfaces of the agglomeration, leading
to the appearance of separate petals and nally the formation of
ower-like structure. The fact that papain can co-crystallize with
Cu3(PO4)2$xH2O was possibly driven by relatively high affinity
between amine groups of enzyme backbone and Cu2+. With the
growth of hybrid nanoowers, the morphology of hybrid
nanoowers also became complete and compact.
the morphology of hNFs and Gas Chromatography Mass Spec-
trometer (GC-MS, Agilent 5975) was used to measure the
molecular weight of products.
3. Results and discussions
3.1 Characterization of papain-inorganic hybrid nanoower
PBS solution of different concentrations (4, 20, 100, and 200
mM) and pHs (5.5, 7.0, and 9.0) to examine the hNFs
morphology and structure. As shown in Fig. 3 of SEM graphs of
papain-inorganic hybrid nanoowers, the size of hNF was
similar to those previously reported.29–31 From SEM images,
petal cannot grow when the concentration of buffer was low
(e.g., 4 or 20 mM), and was only observed on the surface of
Cu3(PO4)2 crystals at this condition. With the increase of PBS
concentration, nanoowers can be much more easily observed.
The size of ower synthesized at 100 mM PBS was larger than at
3.2 Effect of reaction temperature on 3-benzylidenepentane-
2,4-dione yield in promiscuous catalysis
200 mM (Fig. 3b and c). Petals cannot be observed only if the pH Fig. 4 shows the effect of reaction temperature on product yield
of PBS reached certain level (e.g., 7.0 and 9.0). For catalytic in promiscuous catalytic Knoevenagel condensation. The
Fig. 3 SEM image of papain nanoflowers synthesized with 0.25 mg mLÀ1 papain at different PBS; (a) PBS (200 mM, pH 5.5); (b) PBS (200 mM, pH
7.0); (c) PBS (200 mM, pH 9.0); (d) PBS (100 mM, pH 7.4).
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 2357–2364 | 2359

View Article Online
RSC Advances
Paper
Table 1 The kinetics parameter of free enzyme and hNFs
Km (mM)
Kcat (sÀ1
)
Kcat/Km (mMÀ1 sÀ1
)
Vmax (mM minÀ1
)
Papain
Papain-hNFs
1 Æ 0.35
2.67 Æ 0.55
1.03 Æ 0.04
2.67 Æ 0.59
3.06 Æ 0.1
12.17 Æ 0.93
15.27 Æ 0.23
0.34 Æ 0.02
product yield higher than hNFs, which can be explained by
improved mass-transfer limitations between enzyme and
substrate aer immobilization.32 It takes some time for hNFs to
achieve its maximal catalytic efficiency. The yield reached up to
32.67% aer 24 h with hNFs. Moreover, with additional reaction
time aer 24 h, the product yield will still increase, presenting
desiring thermostability. In a nutshell, the catalytic activity of
hNFs is better than papain in terms of product yield.
3.4 Effect of different media on 3-benzylidenepentane-2,4-
dione yield in promiscuous catalysis
Fig. 4 Effect of reaction temperature on 3-benzylidenepentane-2,4-
dione yield in promiscuous catalysis.
The effect of media on promiscuity catalysis was shown in
Fig. 6. Best yield was reached when DMSO was used as solvent,
which is associated with its good solubility. In addition, the
solvent may have an effect on the conformational of enzyme
which was likely to be the main inuence factor of catalytic
activity. The minimal changes of the enzyme structure and
optimum product yield reached 32.67% when hNFs was used as
catalyst, 1.25 folds of that was obtained when papain was used
as catalyst. Moreover, under other reaction temperatures,
product yields catalyzed by hNFs were also higher than that of
papain. In hNFs, papain was conned within Cu3(PO4)2$xH2O,
which stabilized enzyme protein under different reaction
temperatures. For copper phosphate, Cu2+ is able to stabilize
the enzyme structure by its complexation with the amine groups
in the backbone of papain and accelerate the reaction process in
promiscuous catalysis.11
3.3 Effect of reaction time on 3-benzylidenepentane-2,4-
dione yield in promiscuous catalysis
The effects of reaction time on the Knoevenagel condensation
were determined through reactions at 60 ꢀC for different
periods, ranging from 6 h to 24 h (Fig. 5). Aer 6 h, hNFs
catalyzed reaction demonstrated higher yields compared with
free papain. At 6 h, papain catalyzed reaction has a higher
Fig. 6 Effect of solvent on 3-benzylidenepentane-2,4-dione yield in
promiscuous catalysis.
Fig. 5 Effect of reaction time on 3-benzylidenepentane-2,4-dione Fig. 7 Water content on 3-benzylidenepentane-2,4-dione yield in
yield in promiscuous catalysis.
promiscuous catalysis.
2360 | RSC Adv., 2018, 8, 2357–2364
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
reaction rate. The hNFs of papain were shown to undergo
conformational changes, including rearrangement of surface
amino acids, stripping of water from the protein surface, and
weakening of hydrophobic interactions when exposed to
organic solvent.33 In our experiments, the result of catalysis
showed that the hNFs enzyme preparation was organic solvent-
tolerant. Using polar solvents such as DMSO and protic solvents
such as ethanol and methanol always lead to higher yields. In
contrary, less polar solvents such as toluene are not good media
for Knoevenagel condensation.21 Further, the effect of other
solvents was negligible compared with DMSO. For instance, the
yield was up to 32.67% when solvent was DMSO, however, the
yield only reached 1.86% when toluene was used as solvent for
hNFs. The results are consistent for either catalyst (hNFs or
papain). Moreover, the yield was unremarkable when hexane
and DMF were used as solvent.
Fig. 8 The recycle time on promiscuous catalysis.
conformation had an effect on the great change on the inter-
action between enzyme and substrate possibly, which conse-
quently result in sharply increasing or decreasing of the
Table 2 The hNFs-catalyzed promiscuous reaction of other aldehydes and methylenea
Number
1
R1
R2
Products
Yieldb (%)-papain
26
Yieldb (%)-hNFs
33
C6H4
Me
2
3
2-NO2C6H4
4-ClC6H4
Me
Me
55
8
53
9
4
CH3–C6H4
Me
Trace
46
5
6
7
–C6H4
–C6H4
–C6H4
–C6H4
3
21
50
9
2-NO2C6H4
4-BrC6H4
44
16
8
9
CH3–C6H4
–C6H4
39
47
Me
Et
Me
Me
9
15
47
10
39
a
Conditions: aldehyde (2 mmol), methylene (2.4 mmol), hNFs/papain (150 mg), DI water (1.25 mL), DMSO (3.75 mL) at 60 ꢀC for 24 h. b Yield of the
isolated product aer purication.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 2357–2364 | 2361

View Article Online
RSC Advances
Paper
of papain molecule was seem consist of two hemispheres. There
was a pretty deep groove between two hemispheres. The active
site was located at bottom of the groove, in which Cys-25 and
His-159 were on both side. Based on the predictions by Wen and
his coworkers regarding papain21 and the role of Cu(II) site
played in the enzyme biocatalysis proposed by Solomon,35 we
proposed a potential mechanism of hNFs-catalyzed Knoevena-
gel reaction in the presence of Cu2+ (Scheme 1). In the catalysis,
substrate molecule binds to the copper site as a monodentate
ligand via the carbonyl and then formed the Cu(II)–substrate
complex. At the beginning, the proton on the substrate acety-
lacetone is attacked by His-159 and the carbonyl oxygen on
acetylacetone attacked by carbon–oxygen double bond forming
an enolate anion, stabilized by the oxyanion hole formed by
Gln-19 and Cys-25 (ref. 16) and the His-159 is stabilized by Asn-
175. Subsequently, another substrate accepts the proton from
carbon–carbon double bond and simultaneously connected the
enolate anion with the formation of a carbon–carbon bond.
Lastly, the dehydration of the resulted compound took place
under the catalysis of Gln-19, Cys-25, and His-159, leading to
Knoevenagel product. Meanwhile, the Cys–His ion pair formed
which was stabilized by Cu2+ via keeping the imidazole ring of
His-159 in a favorable orientation.
3.5 Effect of water content and reuse cycle times on 3-
benzylidenepentane-2,4-dione yield in promiscuous catalysis
Fig. 7 displays the effect of water content in media on the yield
of promiscuous catalysis. The highest yield was reached when
the water content was 25%, whether catalyzed by free papain or
hNFs. This observation can be potentially explained by inter-
actions between water and solvent.21 There is a point that the
interaction between water and solvent contributed maximal
effects on promiscuous catalysis. Water can play a “lubricant”
role in keeping the conformation of enzymatic activity in
organic solvent, therefore, is required in promiscuous
catalysis.21
Fig. 8 shows the reuse cycle time in promiscuous catalytic
synthesis of 3-benzylidenepentane-2,4-dione in organic solvent
and the product 3-benzylidenepentane-2,4-dione yield every
time. The synthetic yield of this product at rst time was set as
100%. It was obvious that the stability of hNFs was good due to
the result that the yield was almost constant aer cycling for 3
times. The catalytic synthetic activity of the immobilized
enzyme preparation was almost invariable aer 36 h according
to the data from the Fig. 8. What' more, hNFs has the 87.8%
relative catalytic activity aer 6 times cycle. In the organic-water
medium, the hNFs enzyme preparation presents super stability
in the synthetic reaction.
When solely used in the chemical catalysis reaction, Cu2+ ion
oen plays an important role due to the coordination with N
atom. Jiang and his coworker36 have introduced Cu2+ to the p-
zazcrown[N,S,O]-styryl-modied boron-phenylpyrrin compound
synchronously possessing a diethylamino receptor (C-BPP-A).
Cu2+ could coordinated with the p-zazcrown[N,S,O] moiety of
C-BPP-A, forming the Cu2+-BPP-A complex and leading to the
signicant enhancement in uorescence emission at 621 nm.
Furthermore, if magnetic performance as report37 will be
3.6 Substrate scope of hNFs-catalyzed promiscuous
Knoevenagel condensation
The generality and scope of hNFs as catalyst for promiscuous
catalysis has been tested on a few aldehydes and methylene
compounds (Table 2). Catalytic synthetic activities were also
compared between hNFs and free papain under the same reac-
tion conditions and substrates. As shown in Table 1, the yield of
product 3 was 46% when catalyzed by hNFs, while nearly no
product can be detected when free papain as catalyst. The result
of product 4 was similar to product 3, which can be explained by
important roles of Cu2+ in promiscuous catalysis.11 The electronic
properties of substituents had some impacts on reaction yields. a-
b-Unsaturated aromatic aldehydes with strong electron-
withdrawing substituents demonstrated higher yields than
those containing strong electron-donating substituents, contra-
dicting to a reported papain-catalyzed Knoevenagel reactions.21
This inconsistence is possibly caused by special catalytic effect of
hNFs. The yield of product used papain as catalyst was better than
hNFs when the substituent group of unsaturated aromatic alde-
hydes was electrophilic and acetylacetone was substrate. The
yield of hNFs was better when the substrate was 3-phenyl-
azoacetylacetone. Control experiments have also been performed
with Cu3(PO4)2 without enzyme as catalyst. No product can be
detected, so we speculated that Cu2+ may concert catalysis with
papain.6,34
3.7 Proposed catalytic mechanism for the hNFs-catalyzed
Knoevenagel reaction
His-159, Gln-19, Asn-175 and Cys-25 all belong to single poly-
Scheme 1 Proposed mechanism for the hNFs-catalyzed Knoevenagel
peptide chain of papain that involved 212 residues. The shape condensation.
2362 | RSC Adv., 2018, 8, 2357–2364
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
Fig. 9 MS of 3-benzylidenepentane-2,4-dione catalyzed by hNFs.
Fig. 10 1H NMR of 3-benzylidenepentane-2,4-dione catalyzed by
hNFs.
Fig. 11 13C NMR of 3-benzylidenepentane-2,4-dione catalyzed by
hNFs.
with peak positions and assignments, was displayed in Fig. 10.
The data is: d 7.41 (s, 1H), 7.31 (m, 5H), 2.34 (s, 3H), 2.20 (s, 3H).
The 13C NMR spectrum of 3-benzylidenepentane-2,4-dione
catalyzed by hNFs in DMSO containing 25% water for 24 h,
along with peak positions and assignments, was displayed in
Fig. 11. The data is as following: d 204.57 (s), 195.53 (s), 141.77
(s), 138.82 (s), 131.87 (s), 129.65 (s), 128.68 (s), 128.02 (s), 30.62
(s), 25.47 (s).
attached to hNFs, this enzyme preparation would be applied
even more widely in the promiscuous catalysis.
3.8 MS and NMR characterization
MS, 1H NMR, and 13C NMR have been used to characterize the
obtained products, respectively. Fig. 9 shows the MS of 3-
benzylidenepentane-2,4-dione catalyzed by hNFs in DMSO
containing 25% water for 24 h. The molecular weight of the
product was 188.23 and the ion peak was 189.07 were indicated
4. Conclusion
in this gure. The ion peak is considered to be the M + H peak, In summary, we developed a simple method with mild condi-
consistent with molecular weight of the product.
tion to prepare a concerted papain@Cu3(PO4)2 catalyst for
The 1H NMR spectrum of 3-benzylidenepentane-2,4-dione Knoevenagel condensation. In this hybrid nanoowers catalyst,
catalyzed by hNFs (DMSO containing 25% water for 24 h), along Cu2+ from Cu3(PO4)2 crystals stabilize the structure of papain
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 2357–2364 | 2363

View Article Online
RSC Advances
Paper
protein and accelerates papain-catalyzed Knoevenagel conden- 14 N. Sharma, A. Sharma, A. Shard, R. Kumar, Saima and
sation. Moreover, the large size of hybrid nanoowers could be A. K. Sinha, Chem.–Eur. J., 2011, 17(37), 10350–10356.
easily recycled, avoiding tedious separation procedures and 15 H. W. Jin, B. W. Zhou, J. R. Gao, D. Jiang, J. H. Jia and
remained metal Cu2+ in liquid mixture. Further, metal Cu2+ in
Z. P. Yang, Synthesis, 2010, 2010(16), 2794–2798.
hNFs can also be recycled using NH3$H2O and phosphoric acid 16 X. Yu, X. Gao, R. Tao and L. Peng, Catalysts, 2017, 7(6), 182–
if hNFs lose its activity reaction, avoiding potential environment 191.
pollution. This facile preparation of hNFs green biocatalyst also 17 H. Nguyen, V. Nikolakis and D. G. Vlachos, ACS Catal., 2016,
implies simple applicability in organic synthesis by promis-
cuous catalysis.
6(3), 1497–1504.
18 T. Yang, B. Cai, X. Chang, H. Pan and Y. B. Huang,
ChemistrySelect, 2017, 2(13), 3775–3782.
19 H. Wang, K. Xu, Y. Cheng, Y. Pei, S. Yan, M. Qiao, Z. H. Li
and B. Zong, ChemCatChem, 2016, 8(24), 3701–3705.
20 S. H. Kim, G. H. Jeong, D. Choi, S. Yoon, H. B. Jeon, S. M. Lee
and S. W. Kim, J. Colloid Interface Sci., 2013, 389(1), 85–90.
21 W. Hu, Z. Guan, X. Deng and Y. H. He, Biochimie, 2012, 94(3),
656–661.
22 J. T. Guo, Y. Xiang, Z. Guan and Y. H. He, J. Mol. Catal. B:
Enzym., 2016, 131, 55–64.
23 X. Hua, Y. Xing and X. Zhang, ACS Appl. Mater. Interfaces,
2016, 8(25), 16257–16261.
24 R. Banerjee, Y. Proshlyakov, J. D. Lipscomb and
D. A. Proshlyakov, Nature, 2015, 518(7539), 431–434.
25 D. Wei, X. Huang, J. Liu, M. Tang and C.-G. Zhan,
Biochemistry, 2013, 52(30), 5145–5154.
26 J. Ge, J. Lei and R. N. Zare, Nat. Nanotechnol., 2012, 7(7), 428–
432.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
This study was supported by the National Science Foundation of
China (21576062), Natural Science Foundation of Zhejiang
Province (LY18B060009, LY15B060011), Technology Research
and Development Program of Hangzhou (20120232B13),
National Innovation and Entrepreneurship Training Program
for Undergraduates (201710346013), Scientic Research Inno-
vation Fund for Graduate Students of Hangzhou Normal
University, Research Plan for Sprout Talents in University in
Zhejiang Province (2017R423027), and the Program for Inno-
vative Research Team in Chinese University (IRT1231).
27 K. Meller, P. Pomastowski, D. Grzywinski, M. Szumski and
B. Buszewski, J. Chromatogr. A, 2016, 1440, 45–54.
28 S. Ganapathi-Desai, D. A. Buttereld and D. Bhattacharyya,
Biotechnol. Prog., 1998, 14, 865–873.
29 C. Altinkaynak, S. Tavlasoglu, N. Ozdemir and I. Ocsoy,
Enzyme Microb. Technol., 2016, 93–94, 105–112.
30 X. Hua, Y. Xing and X. Zhang, RSC Adv., 2016, 6(52), 46278–
46281.
31 Z. F. Wu, Z. Wang, Y. Zhang, Y. L. Ma, C. Y. He, H. Li,
L. Chen, Q. S. Huo, L. Wang and Z. Q. Li, Sci. Rep., 2016, 6,
22412.
32 J. Cui, Y. Zhao, R. Liu, C. Zhong and S. Jia, Sci. Rep., 2016, 6,
27928–27940.
References
1 F. Bigi and C. Quarantelli, Curr. Org. Synth., 2012, 9, 31–39.
2 F. Yang, Z. Wang, H. Wang, C. Wang and L. Wang, RSC Adv.,
2015, 5(70), 57122–57126.
3 R. A. Kudirka, R. M. Bareld, J. M. McFarland, P. M. Drake,
A. Carlson, S. Banas, W. Zmolek, A. W. Garofalo and
D. Rabuka, ACS Med. Chem. Lett., 2016, 7(11), 994–998.
4 E. Q. J. David, I. M. Ferreira, S. A. Yoshioka, L. P. Fonseca and
A. L. M. Porto, Curr. Microwave Chem., 2017, 4(2), 131–138.
5 R. H. Vekariya and H. D. Patel, Synth. Commun., 2014, 44(19),
2756–2788.
6 E. M. Schneider, M. Zeltner, N. Kranzlin, R. N. Grass and
W. J. Stark, Chem. Commun., 2015, 51(53), 10695–10698.
7 D. Koselewski, D. Paprocki, A. Madej, F. Borys, A. Brodzka
and R. Ostaszewski, Eur. J. Org. Chem., 2017, 31, 4572–4579.
8 F. Yang, H. Wang, L. Jiang, H. Yue, H. Zhang, Z. Wang and
L. Wang, RSC Adv., 2015, 5(7), 5213–5216.
9 B. T. Watson and G. E. Christiansen, Tetrahedron Lett., 1998,
39(33), 6087–6090.
10 Q. C. Li, X. Z. Wang, Y. Y. Yu, Y. Q. Chen and L. Y. Dai,
Tetrahedron, 2016, 72(50), 8358–8363.
33 V. Dachuri, J. Boyineni, S. Choi, H.-S. Chung, S.-H. Jang and
C. Lee, J. Mol. Catal. B: Enzym., 2016, 131, 73–78.
34 F. Farzaneh, M. K. maleki and M. Ghandi, Mech. Catal., 2015,
117(1), 87–101.
35 E. I. Solomon, D. E. Heppner, E. M. Johnston,
J. W. Ginsbach, J. Cirera, M. Qayyum, M. T. Kieber-
Emmons, C. H. Kjaergaard, R. G. Hadt and L. Tian, Chem.
Rev., 2014, 114(7), 3659–3853.
36 Y. Chen, H. Pan, K. Wang, D. Qi and J. Jiang, Eur. J. Inorg.
Chem., 2017, 44, 5254–5259.
37 M. C. Silva, J. A. Torres, F. G. E. Nogueira, T. S. Tavares,
11 B. Viswanadham, J. Pedada, H. B. Friedrich and S. Singh,
Catal. Lett., 2016, 146(8), 1470–1477.
ˆ
A. D. Correa, L. C. A. Oliveira and T. C. Ramalho, RSC Adv.,
¨
¨
12 H. Goksu and E. Gultekin, ChemistrySelect, 2017, 2(1), 458–
2016, 6(87), 83856–83863.
463.
13 A. Sharma, N. Sharma, R. Kumar, A. Shard and A. K. Sinha,
Chem. Commun., 2010, 46(19), 3283–3285.
2364 | RSC Adv., 2018, 8, 2357–2364
This journal is © The Royal Society of Chemistry 2018