Bovine serum albumin: An efficient and green biocatalyst for the one-pot four-component synthesis of pyrano[2,3-c]pyrazoles

Article abstract of DOI:10.1016/S1872-2067(15)61088-9

The use of biocatalysts is attracting an increasing amount of attention in chemical catalysis. Here, we have shown that bovine serum albumin (BSA), a ubiquitous, inexpensive, non-enzymatic transport protein, can serve as an efficient, retrievable catalyst

Full text of DOI:10.1016/S1872-2067(15)61088-9

Chinese Journal of Catalysis 37 (2016) 1461–1467
a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / c h n j c
Article
Bovine serum albumin: An efficient and green biocatalyst for the
one‐pot four‐component synthesis of pyrano[2,3‐c]pyrazoles
Xingtian Huanga, ZhipengLib, DongyangWangb, YiqunLia,b,
*
a College of Life Science and Technology, Jinan University, Guangzhou 510632, Guangdong, China
b Department of Chemistry, Jinan University, Guangzhou 510632, Guangdong, China
A R T I C L E I N F O
A B S T R A C T
Article history:
The use of biocatalysts is attracting an increasing amount of attention in chemical catalysis. Here,
we have shown that bovine serum albumin (BSA), a ubiquitous, inexpensive, non‐enzymatic
transport protein, can serve as an efficient, retrievable catalyst in the one‐pot four‐component reac‐
tion of aryl aldehydes, malononitrile, hydrazine hydrate, and ethyl acetoacetate for the synthesis of
pyrano[2,3‐c]pyrazoles under mild reaction conditions. The BSA biocatalyst also displayed a high
catalytic affinity for acyclic/cyclic ketones to yield the corresponding pyrano[2,3‐c]pyrazoles or
their spirocyclic variants. The BSA could be used for at least five cycles without serious loss of cata‐
lytic activity. This novel, efficient protocol has the merits of high yield, operational simplicity, and a
relatively benign environmental impact. Moreover, the method extends the promiscuity of BSA as a
biocatalyst.
Received 6 March 2016
Accepted 24 March 2016
Published 5 September 2016
Keywords:
Bovine serum albumin
Biocatalyst
Multicomponent reaction
Pyrano[2,3‐c]pyrazole
Biocatalytic promiscuity
© 2016, Dalian Institute of Chemical Physics, Chinese Academy of Sciences.
Published by Elsevier B.V. All rights reserved.
1. Introduction
have been widely explored as they form the essential core of
emerging drugs with diverse bioactivity, including a potential
Multicomponent reactions (MCRs) have emerged as power‐
ful and efficient tools for constructing various novel organic
molecules of potential interest. These reactions are particularly
useful in drug discovery owing to their flexibility and effective‐
ness compared with conventional multistep synthesis [1–3].
MCRs offer fast and elegant routes to target molecules via
one‐pot synthetic reaction schemes. These may be either sim‐
ultaneous reactions or sequential‐addition procedures that
have high atom economy, bond‐forming efficiency, and selec‐
tivity. Moreover, MCRs obviate the need for isolation and puri‐
fication of intermediates, thereby minimizing waste, saving
time, and reducing the cost of purification. Pyrano[2,3‐c]pyra‐
zoles are an important class of heterocyclic compounds that
inhibitor of human Chk1 kinase [4], as well as antitumor [5],
analgesic [6], antibacterial [7], and anti‐inflammatory [6–8]
agents. Various MCRs, including three‐component reactions
(3CRs), four‐component reactions (4CRs), and even five‐com‐
ponent reactions (5CRs), have been reported for the synthesis
of pyrano[2,3‐c]pyrazole derivatives. 3CRs of carbonyl com‐
pounds, malononitrile, and the corresponding pyrazolin‐5‐ones
using various basic catalysts, such as trimethylamine [9], tri‐
ethanolamine [10], piperidine [11], N‐methylmorpholine [12],
D/L‐proline [13], MgO [14], silica sodium carbonate [15], and
electrogenerated bases [16], have been reported. Furthermore,
4CRs have been developed for the reaction of aldehydes,
malononitrile, hydrazine hydrate, and ethylacetoacetate using
* Corresponding author. Tel/Fax: +86‐20‐85220223; E‐mail: tlyq@jnu.edu.cn
This work was supported by the National Natural Science Foundation of China (21372099, 21072077), and the the Natural Science Foundation of
Guangdong Province (10151063201000051, 8151063201000016).
DOI: 10.1016/S1872‐2067(15)61088‐9 | http://www.sciencedirect.com/science/journal/18722067 | Chin. J. Catal., Vol. 37, No. 9, September 2016

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Xingtian Huang et al. / Chinese Journal of Catalysis 37 (2016) 1461–1467
trimethylamine [17], Amberlyst A21 [18], cetyltrime‐
thylammonium chloride [19], molecular sieves [20], magnetic
Fe3O4 nanoparticles [21], SnO2 quantum dots [22], L‐proline
[23,24], glycine [25], meglumine [26], per‐6‐amino‐β‐cy‐
clodextrin [27], and lipase [28] as catalysts. More recently,
overall 5CRs have been established involving Suzuki coupling
of 4‐bromobenzaldehyde and arylboronic acids followed by
4CRs involving readily available ethyl acetoacetate, malono‐
nitrile, and hydrazine hydrate [29]. However, in spite of their
merits, most of these methods suffer from one or more draw‐
backs, such as harsh reaction conditions, expensive or unob‐
tainable reagents, stoichiometric amounts of catalyst, long re‐
action time, unsatisfactory product yields, high waste genera‐
tion, or laborious work‐up. Considering the importance of py‐
rano[2,3‐c]pyrazole derivatives, more convenient and practical
methodologies for their synthesis are still needed.
2. Experimental
2.1. Materials, chemicals, and characterization
Albumin bovine V from bovine serum (BSA), hemoglobin
from bovine erythrocytes (Lot# SG201402), egg white albumin,
lysozyme from chicken egg (activity ≥ 20000 U/mg, Lot#
KY201501), trypsin from porcine pancreas (activity ≥ 250 U/g,
Lot# KY201305), and lipase from porcine pancreas (activity ≥
30000 U/g, Lot# KY201312) were purchased from Sigma.
Alpha amylase from bacillus submerged fermentation (activity
≥ 3000 U/g, pH = 5.5–7.5) was purchased from Damao, and silk
fibroin was extracted from silk by a previously reported
method [44]. All chemicals were obtained from commercial
sources and used as received.
Fourier transform infrared (FT‐IR) spectra were recorded
In light of the above, we considered that biocatalysts might
be effective for this class of 4CRs owing to their promiscuous
activity. Exploiting the ability of a biocatalyst to achieve the
synthesis of pyrano[2,3‐c]pyrazole derivatives via 4CRs has
long been challenging. To date, only one previous study [28],
using lipase from Aspergillus niger, has reported the successful
use of a biocatalyst for this purpose.
on a Nicolet 6700 spectrometer in KBr pellets in the range
4000–400 cm−1. H NMR (300 MHz) and 13C NMR (75 MHz)
spectra were obtained on a Bruker AC‐300 using DMSO‐d6 as
the solvent and TMS as the internal standard. The melting
points were measured on an Electrothermal X6 microscopic
digital melting point apparatus.
1
In the past decade, considerable effort has been expended
on the development of biocatalysts, including proteins, en‐
zymes, and whole cells, for use in organic synthesis, owing to
their high catalytic activity and selectivity, as well as operation‐
al simplicity [30,31]. Among them, bovine serum albumin
(BSA), a ubiquitous, inexpensive, non‐enzymatic transport
protein derived from cows, with no natural catalytic function in
biological systems, has proven an efficient biocatalyst because
of its remarkable versatility for many kinds of catalytic activi‐
ties and biotransformations [32]. It is capable of binding or‐
ganic molecules by reversible non‐covalent complexation in its
hydrophobic pockets, thus providing a microenvironment for a
number of organic transformations including reduction [33],
Knoevenagel condensation [34,35], aldol condensation [34],
nitroaldol addition [36], Gewald reactions [37], Morita‐Baylis‐
Hillman reactions [38], Biginelli reactions [39], thio‐Michael
addition [40], oxidative coupling of thiols [41], thiosulfination
[42], and oxidation of tertiary amines to N‐oxides [43].
Inspired by these results, we tested the use of BSA as a new,
efficient and recyclable catalyst for the synthesis of pyra‐
no[2,3‐c]pyrazole derivatives via 4CRs (Scheme 1). To the best
of our knowledge, there has been no report of the use of BSA as
a biocatalyst for 4CRs of this kind to date. Thus, exploiting the
promiscuous activity of the abundant and readily available BSA
to catalyze MCRs should extend the synthetic utility of bio‐
catalysis.
2.2. Typical biocatalytic procedure for synthesis of
pyrano[2,3‐c]pyrazoles
A
round‐bottomed flask was charged with ethyl
acetoacetate (1 mmol), 80% hydrazine hydrate (V/V, 1 mmol),
malononitrile (1 mmol), the chosen carbonyl compound (1
mmol), BSA (50 mg), and 5 mL 90% aqueous ethanol solution
(V/V). The reaction was incubated at 45 C and stirred at 500
r/min for a specific time until completion. The progress was
tracked by thin‐layer chromatography. Upon completion, ethyl
acetate preheated to 60 C was added to quench the reaction.
The BSA was recovered by filtration, washed adequately with
ethanol and dried in preparation for the next run. The organic
fraction was successively washed with brine and saturated
sodium bisulfite solution and then concentrated on a rotary
evaporator to give the crude product. The product was further
purified by recrystallization with ethanol. The target products
were identified by FT‐IR, 1H NMR, 13C NMR, and melting point,
all of which were in agreement with the literature values.
3. Results and discussion
3.1. Screening of different biocatalysts
Initially, various biocatalysts, including enzymes and pro‐
teins, were screened using the 4CR of ethyl acetoacetate, hy‐
drazine hydrate, malononitrile, and p‐chlorobenzaldehyde as a
model reaction. The results are summarized in Table 1. Among
all the screened biocatalysts, BSA showed the most powerful
catalytic activity, achieving a rapid reaction time of 0.75 h and
affording the desired product in 95% yield (Table 1, entry 9).
The use of other enzymes or proteins also resulted in apprecia‐
bly greater yields (Table 1, entries 2–8). The lowest product
yield was obtained in the absence of any catalyst (Table 1, entry
Scheme 1. Synthesis of dihydropyrano[2,3‐c]pyrazole.

Xingtian Huang et al. / Chinese Journal of Catalysis 37 (2016) 1461–1467
1463
Table 1
Table 2
Screening of biocatalysts.
Optimization of reaction conditions.
Time Yielda
(h) (%)
Catalyst
(mg)
Temp.
(C)
Time
(h)
Yielda
(%)
Entry
1
Biocatalyst
None
Entry
Solvent
6
6
3
3
3
3
2
1
47
63
75
82
84
86
86
85
1
2
90% EtOH (V/V)
90% EtOH (V/V)
90% EtOH (V/V)
90% EtOH (V/V)
90% EtOH (V/V)
90% EtOH (V/V)
90% EtOH (V/V)
95% EtOH (V/V)
95% EtOH (V/V)
95% EtOH (V/V)
95% EtOH (V/V)
95% EtOH (V/V)
EtOH
0
45
45
45
45
45
45
45
6.0
1.5
47
80
87
91
95
93
93
2
3
4
5
6
7
8
9
Lysozyme from chicken egg
Alpha amylase from bacillus submerged fermentation
Hemoglobin from bovine erythrocytes
Silk fibroin
15
30
45
50
55
60
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
3
4
5
6
7
8
1.0
0.75
0.75
0.75
0.75
Trypsin from porcine pancreas
Egg white albumin
Lipase from porcine pancreas
BSA
25
35
55
65
75
45
45
45
45
45
45
45
45
45
45
2.0
1.5
0.5
0.4
0.25
0.75
0.75
1.0
1.5
3.0
1.0
1.0
1.5
1.5
3.0
85
88
87
88
88
88
86
84
35
38
55
42
63
58
15
0.75 95
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Reaction conditions: 4‐chlorobenzaldehyde (1 mmol), malononitrile (1
mmol), hydrazine hydrate (1 mmol), ethyl acetoacetate (1 mmol) and
biocatalyst (50 mg) in EtOH/water (9:1 V/V, 5 mL) at 45 °C.
a Isolated yield.
75% EtOH (V/V)
MeOH
1), showing that catalysis plays a key role in this transfor‐
mation. Encouraged by this result, we proceeded to explore in
detail the influence of the catalyst dose, reaction medium, and
reaction temperature on the model reaction using catalytic
amounts of BSA.
i‐PrOH
H2O
CH2Cl2
CH3CN
DMF
DMSO
None
3.2. Optimization of reaction conditions
Reaction conditions: 4‐chlorobenzaldehyde (1 mmol), malononitrile (1
mmol), hydrazine hydrate (1 mmol), and ethyl acetoacetate (1 mmol)
were stirred in the presence of BSA in the solvent (5 mL) at 45 C.
a Isolated yield.
The dosage of the biocatalyst was found to have an
appreciable effect on the product yields. The 4CR model was
studied with catalyst doses ranging from 0.0 to 60.0 mg of the
substrate (Table 2). The 4CR model was found to proceed
efficiently even with a low dose of BSA (15 mg in substrate),
affording an 80% product yield (Table 2, entry 2). As can be
seen from Table 2, with the catalyst dose increased from 15 to
50 mg, the apparent yield increased from 80% to 95% and the
reaction time decreased from 1.5 to 0.75 h (Table 2, entries
2–5). Further increasing the catalyst dose to 55 or even 60 mg
had no major effect on the product yield (Table 2, entries 6 and
7). Recall that, in the absence of BSA, the model reaction
proceeded with a prolonged reaction time and a lower yield
(Table 2, entry 1). In contrast, 50 mg of BSA biocatalyst was
sufficient to drive the reaction to completion in short time.
The reaction medium generally plays an important role in
biocatalysis. To identify the most suitable solvent, ten protic
and aprotic solvents with varying polarities or other properties
were screened. Several protic solvents, including EtOH,
aqueous EtOH, and MeOH, rendered high product yields (Table
2, entries 2–15), while in aprotic solvents, including CH2Cl2,
CH3CN, DMF, and DMSO, BSA exhibited low catalytic activity
(Table 2, entries 18–21). Poor yields were also obtained in H2O,
i‐PrOH, and the absence of solvent (Table 2, entries 16–17 and
22). Based on the screening results, 90% EtOH (V/V) was
chosen as the most suitable solvent for further study.
yield increased from 85% to 95% and the reaction time
decreased from 2 to 0.75 h when the temperature was raised
from 25 °C (room temperature) to 45 C (Table 2, entries 8, 9
and 5). However, temperatures over 45 C led to lower yields
(Table 2, entries 10–12), possibly because of the partial
deactivation of the biomolecular BSA at these temperatures,
indicating the importance of an appropriate operating
temperature to retain biocatalytic activity. Therefore, 45 C was
selected as the optimal temperature for the reaction.
3.3. Synthesis of pyrano[2,3‐c]pyrazoles
To obtain further insight into the catalytic possibilities of
BSA, we investigated the scope and generality of the 4CRs for
which it displays activity. A variety of substituted aryl alde‐
hydes carrying either electron‐withdrawing or electron‐donat‐
ing substituents on the aromatic ring were reacted with malo‐
nonitrile, hydrazine hydrate, and ethyl acetoacetate in 90%
aqueous ethanol in the presence of catalytic amounts of BSA
under the above‐mentioned optimal conditions. As shown in
Table 3, all of the reactions proceeded smoothly to produce the
expected pyrano[2,3‐c]pyrazoles with excellent yields of
73%–96%. With both electron‐donating and electron‐with‐
drawing substituents, at either the para or the meta position on
the aromatic ring of the aryl aldehyde, the reactions proceeded
without marked effects on their progress and with high yields.
Notably, the reactions of the aryl aldehydes bearing a substitu‐
ent at the ortho position afforded slightly lower yields owing to
Reaction temperature is an important parameter in
biocatalysis. To further investigate the optimum conditions for
the model MCR, we evaluated the effect of six different
temperatures in the range 25–78 C on the reaction yield and
reaction time. Both these variables were found to be highly
sensitive to temperature. As shown in Table 2, the product

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Xingtian Huang et al. / Chinese Journal of Catalysis 37 (2016) 1461–1467
Table 3
Synthesis of pyrano[2,3‐c]pyrazoles.
Carbonyl
compound
Time Yielda
(h) (%)
Carbonyl
compound
Time Yielda
Entry
1
Product
M.P. b (C)
Entry
12
Product
M.P. b (C)
(h)
(%)
Cl
232–234
(233–234 [45])
220–221
(223–224 [48])
0.75 95
0.75
91
CHO
Cl
242–243
(245–246 [26])
244–246
(245–246 [48])
CN
CN
2
3
1.5
1
87
90
13
14
0.75
0.75
95
89
N
N
N
H
N
H
O
1b
NH2
O
1m
NH2
OH
OMe
222–223
(229–230 [26])
233–235
(233–234 [48])
CHO
Br
179–180
(178–180 [46])
168–170
(167–169 [46])
4
0.75 91
15
0.75
88
CHO
Br
259–261
(259–261 [47])
175–177
(175 [49])
CN
5
6
2
84
86
16
17
6
8
85
73
N
N
H
O
1e
NH2
Br
221–223
(223–224 [48])
1.5
173–175
CN
N
N
H
O
1f
NH2
NO2
249–251
(249–250 [28])
176–178
(177–178 [18])
7
0.5
1
96
90
18
12
75
CN
N
N
H
O
NH2
1g
221–223
(220–222 [46])
245–246
(244–247 [7])
8
9
19
20
3
3
89
90
NO2
CHO
NO2
O
HN
NO2
O
195–196
(193–195 [46])
289–290
(286–287 [26])
CN
CN
0.75 92
0.75 94
O
N
N
N
H
NH2
N
H
O
1t
N
H
CHO
O
1i
NH2
204–205
(206–208 [46])
247–248
(245–246 [26])
10
11
21
3
1
88
95
H
N
H2N
NC
O
N
N
OMe
CHO
CHO
CHO
212–214
(212–213 [48])
> 300
(300 [50])
0.75 90
22c
NC
N
H
H2N
O
1v
Reaction conditions: carbonyl compound (1 mmol), hydrazine hydrate (1 mmol), malononitrile (1 mmol), and ethyl acetoacetate (1 mmol) were
stirred in the presence of BSA (50 mg) in 90% EtOH /water (V/V, 5 mL) at 45 C.
a Isolated yield.
b The data in parentheses are the melting points reported in the literature.
c Two equivalents of hydrazine hydrate, malononitrile, and ethyl acetoacetate were used.

Xingtian Huang et al. / Chinese Journal of Catalysis 37 (2016) 1461–1467
1465
the steric effect (Table 3, entries 2, 3, 5, and 8). Many sensitive
functional groups were compatible with the BSA biocatalyst,
such as methoxy, phenolic hydroxy, and N,N‐dimethylamino,
and gave high yields (Table 3, entries 11, 12, 14, and 15). Apart
from the aryl aldehydes, various ketones were also used as
components in the 4CR scheme, under similar conditions, to
produce the desired pyrano[2,3‐c]pyrazole derivatives in ex‐
cellent yields with slightly prolonged reaction time (Table 3,
entries 16–21). All of the products were fully characterized by
melting point and FT‐IR, 1H NMR, and 13C NMR spectroscopy.
0.75 h a
100
80
60
40
20
0
1.5 h
1 h
2 h
3 h
3.4. Catalyst reusability
For practical application, the reusability of a catalyst is a
crucial factor. To explore this aspect of BSA, catalytic recycling
experiments were performed using the 4CR of
4‐chlorobenzaldehyde, hydrazine hydrate, malononitrile and
ethyl acetoacetate as a model reaction. After completing the
reaction, the BSA could be conveniently and efficiently recov‐
ered from the reaction mixture by filtering, washing succes‐
sively with acetone three times, and then drying. The recovered
BSA was then reused directly in consecutive cycles of the reac‐
tion under the above mentioned optimized conditions. The
results (Fig. 1) show that only a slight loss of the catalytic activ‐
ity of BSA was observed after each cycle.
1
2
3
4
5
Recycle number
Fig. 1. Recyclability of BSA.a Reaction time.
materials, which make it a useful and practical process for the
synthesis of structurally diverse pyrano[2,3‐c]pyrazole deriva‐
tives.
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3.5. Proposed mechanism for synthesis of
pyrano[2,3‐c]pyrazoles
The protein BSA has no known natural catalytic function.
Structurally, BSA contains 60 Lys, 41 Asp and 58 Glu residues
[51] and, with an isoelectric point near pH = 4.5 [52], presents
as mildly basic at neutral pH. The catalytic activity of BSA is
determined by the basic character of the amino groups on the
side chains of certain residues, especially lysine. Therefore, we
propose the following mechanism for the synthesis of
pyrano[2,3‐c]pyrazoles via a tandem process, as depicted in
Scheme 2. First, an –NH2 group on BSA simultaneously
promotes both the condensation of ethyl acetoacetate with
hydrazine hydrate and the Knoevenagel condensation of the
aryl aldehyde with malononitrile to produce the 3‐methyl‐
1H‐pyrazol‐5(4H)‐one
A and the intermediate olefin B.
Subsequent Michael addition of A and B promoted by –NH2 on
BSA produces the intermediate C. Finally, we propose that C
undergoes intramolecular cyclization followed by tautomeri‐
zation to give the dihydropyrano[2,3‐c]pyrazole derivatives.
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4. Conclusions
We have developed a novel, efficient, and environmentally
friendly protocol for the synthesis of a diverse range of pyra‐
no[2,3‐c]pyrazole derivatives using BSA as a reusable biocata‐
lyst. This method offers the advantages of environmental com‐
patibility, mild reaction conditions, short reaction time, high
yields and operational simplicity. Moreover, the method is dis‐
tinguished by its multicomponent process and wide substrate
scope, accepting a variety of carbonyl compounds as starting
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Xingtian Huang et al. / Chinese Journal of Catalysis 37 (2016) 1461–1467
BSA
BSA
NH2
R
O
O H
O
N
H
O
H
HN
N
H
R
CN
CN
CN
N
HN
N
N
H
O
N
BSA
H3N
OEt
A
B
H2N NH2
-H2O
R
BSA
BSA
CN
NH
BSA
N
H
+
R
NH2
NH3
N
O
H
CN
N
+
NC H
NC H
NC
NC
O
R
NC
NC
OH
R
H
NH3
N
O
N
O
R
C
H
NH2
BSA
R
O
R
O
CN
NH2
CN
NH
N
N
N
N
H
H
Scheme 2. Proposed mechanism for synthesis of pyrano[2,3‐c]pyrazoles.
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Xingtian Huang et al. / Chinese Journal of Catalysis 37 (2016) 1461–1467
1467
Graphical Abstract
Chin. J. Catal., 2016, 37: 1461–1467 doi: 10.1016/S1872‐2067(15)61088‐9
Bovine serum albumin: An efficient and green biocatalyst
for the one‐pot four‐component synthesis of
pyrano[2,3‐c]pyrazoles
Xingtian Huang, Zhipeng Li, Dongyang Wang, Yiqun Li*
Jinan University
Bovine serum albumin has been used as an efficient and reusable
biocatalyst for the synthesis of pyrano[2,3‐c]pyrazoles under mild
and environmentally benign conditions.
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