Biocatalytic one-pot three-component synthesis of 3,3′-disubstituted oxindoles and spirooxindole pyrans using α-amylase from hog pancreas

Article abstract of DOI:10.1039/c4ra16825a

α-Amylase from hog pancreas displayed catalytic promiscuity in three-component reaction for the synthesis of 3,3′-disubstituted oxindoles and spirooxindole pyrans. The reactions between isatins, malononitrile and active methyl or active methylene compounds (acetone, nitromethane, indole, acetylacetone, 4-hydroxylcoumarin and dimedone) offered corresponding products via Knoevenagel/Michael reactions or Knoevenagel/Michael/cyclization reactions in one pot with high to excellent yields of up to 98% under mild reaction conditions. The α-amylase showed a broad spectrum of adaptability to various substrates. A possible mechanism of the α-amylase catalyzed three-component reaction was proposed. This journal is

Full text of DOI:10.1039/c4ra16825a

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PAPER
Biocatalytic one-pot three-component synthesis of
3,30-disubstituted oxindoles and spirooxindole
pyrans using a-amylase from hog pancreas†
Cite this: RSC Adv., 2015, 5, 37843
*
*
Tao He, Qing-Qing Zeng, Da-Cheng Yang, Yan-Hong He and Zhi Guan
a-Amylase from hog pancreas displayed catalytic promiscuity in three-component reaction for the
synthesis of 3,30-disubstituted oxindoles and spirooxindole pyrans. The reactions between isatins,
malononitrile and active methyl or active methylene compounds (acetone, nitromethane, indole,
acetylacetone, 4-hydroxylcoumarin and dimedone) offered corresponding products via Knoevenagel/
Michael reactions or Knoevenagel/Michael/cyclization reactions in one pot with high to excellent yields
of up to 98% under mild reaction conditions. The a-amylase showed a broad spectrum of adaptability to
various substrates. A possible mechanism of the a-amylase catalyzed three-component reaction was
proposed.
Received 22nd December 2014
Accepted 20th April 2015
DOI: 10.1039/c4ra16825a
www.rsc.org/advances
The oxindoles are the core structural motifs which are
present in many natural products and biologically active
compounds.33–37 A large number of compounds based on the
Introduction
As a green, economically feasible, environmentally friendly,
mild, and burgeoning approach in organic chemistry, bio-
structure of indoles show pharmaceutical activity and bioac-
tivity.38–40 3,30-Disubstituted oxindoles and spirooxindoles con-
taining a quaternary carbon center and diverse functional
groups are the powerful synthetic intermediates which provide a
shortcut to synthesize complex and potentially biologically active
compounds.40 Because of those promising advantages, more and
more chemists try to achieve the target of developing green and
easy methods to synthesize 3,30-disubstituted oxindole and spi-
rooxindole frameworks.41–50 Spirooxindole pyrans as a type of
heterocyclic spirooxindoles have attracted considerable atten-
tion, and some synthetic protocols have been reported for the
construction of these compounds. For example, the Michael/
cyclization reactions of isatylidenemalononitriles with a-keto
esters or 1,3-dicarbonyl compounds, or three-component
Knoevenagel/Michael/cyclization reactions of isatins, malono-
nitrile (cyanoacetic ester) and 1,3-dicarbonyl compounds were
conveniently employed to synthesize spirooxindole pyrans. The
rosin-derived tertiary amine–thiourea46 and cupreine48 were used
as catalysts to provide spirooxindole pyrans. L-proline51 as well as
an enzyme, lipase from porcine pancreas (PPL),47 were also
applied to catalyze this type of reactions, in which spirooxindole
pyrans were prepared as racemates. Moreover, some methods
have been developed for the enantioselective construction of
3,30-disubstituted oxindoles via the Michael reaction of ketones
to isatylidenemalononitriles catalyzed by a quinidine derived
primary amine with (R)-binol-phosphoric acid as a co-catalyst,52
catalysis demonstrates
a powerful potential in organic
synthesis.1 Enzymes, as typical representatives of biocatalysts,
are widely employed in organic synthesis as green catalysts and
have received more and more attention. However, there are
limited kinds of enzymes in nature and only some of them can
be available commercially.2 Thus, it is crucial to expand the
application scope of existing enzymes. Enzymatic promiscuity,
which means not only natural substrates but non-natural
substrates can be catalyzed by the single active site of a given
enzyme, has been investigated in the past decade,3–7 having
greatly enriched the reaction types of enzyme catalysis. Some
elegant works on the enzyme catalytic promiscuity have been
described.8,9 For example, a number of hydrolases were found to
have the ability to catalyze aldol,1,2,10–15 Michael,16–21 Henry,22–24
Mannich25 and Knoevenagel26–28 reactions, and even some
domino reactions.29 Nevertheless, promiscuous enzyme cata-
lyzed multicomponent reactions are still rather rare although
there are a few examples reported.30–32 Hence, in order to expand
the application of enzyme in multicomponent reactions, and to
further understand enzymatic promiscuity, it is still a formi-
dable and signicant task to discover enzyme-catalyzed multi-
component reactions.
Key Laboratory of Applied Chemistry of Chongqing Municipality, School of Chemistry
and Chemical Engineering, Southwest University, Chongqing, 400715, P. R. China.
E-mail: heyh@swu.edu.cn; guanzhi@swu.edu.cn; Fax: +86-23-68254091
or
a cinchona-based chiral primary amine with L-cam-
phorsulfonic acid as a co-catalyst.53 These 3,30-disubstituted
oxindoles could be further converted to spirooxindole dihy-
dropyrans by reduction/cyclization reactions.
† Electronic supplementary information (ESI) available. CCDC 1053771 and
1053772. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c4ra16825a
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Table 1 Solvent screeninga
Entry
Solvent
Time [d]
Yieldb [%]
1
2
3
4
5
6
7
8
Ethanol
DMF
DMSO
Methanol
1,4-Dioxane
Acetonitrile
THF
Methyl tert-butyl ether
Chloroform
Ethyl acetate
Butyl acetate
1,2-Dichloroethane
Toluene
2
2
2
2
2
4
4
4
4
4
4
4
4
55
54
53
53
52
43
29
27
24
21
18
18
8
9
10
11
12
13
a
Reaction conditions: isatin (0.25 mmol), malononitrile (0.25 mmol), acetone (2.5 mmol), a-amylase (738 U), solvent (0.90 mL), and deionized water
b
(0.10 mL) at 25 ꢀC. Yield of the isolated product.
Table 2 Control experimentsa
Entry
Catalyst
Time [d]
Yieldb [%]
Natural activityc [U mgꢁ1
]
1
2
3
4
5
6
7
8
9
a-Amylase
2
4
3
3
3
3
3
3
3
55
Trace
21
No reaction
Trace
No reaction
Trace
No reaction
33
24.6
—
4.9
—
1.1
—
1.1
—
None
a-Amylase (pretreated with urea)d
Urea (200 mg)
a-Amylase (pretreated with 250 mM Cu2+ e
CuSO4 (40 mg)
)
a-Amylase (pretreated with 250 mM Ag+)f
AgNO3 (42 mg)
a-Amylase (pretreated with miglitol)g
18.0
a
Unless otherwise noted, the reaction conditions: isatin (0.25 mmol), malononitrile (0.25 mmol), acetone (2.5 mmol), a-amylase (738 U), ethanol
(0.90 mL), and deionized water (0.10 mL) at 25 ꢀC. b Yield of the isolated product. c Unit denition (U mgꢁ1): one unit will liberate 1.0 mg of maltose
from starch in 3 min at pH 6.9 at 20 ꢀC. d a-Amylase (738 U) in urea solution (3.3 M) [urea (200 mg) in deionized water (1 mL)] was stirred at 25 ꢀC for
24 h, and then water was removed under reduced pressure before use. e a-Amylase (738 U) in Cu2+ solution (250 mM) [CuSO4 (40 mg) in deionized
water (1 mL)] was stirred at 25 ꢀC for 24 h, and then water was removed under reduced pressure before use. f a-Amylase (738 U) in Ag+ solution (250
mM) [AgNO3 (42 mg) in deionized water (1 mL)] was stirred at 25 ꢀC for 24 h, and then water was removed under reduced pressure before use. g a-
Amylase (738 U) in miglitol solution [miglitol (50 mg) in deionized water (1 mL)] was stirred at 25 ꢀC for 24 h, and then water was removed under
reduced pressure before use.
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Table 3 Substrate scope for the synthesis of 3,30-disubstituted oxindoles using acetonea
Entry
R
Product
Time [d]
Yieldb [%]
1
2
3
4
5
6
7
8
1-H
1-Me
4-Cl
4-Br
5-F
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
2
2
3
3
2
1
2
2
3
2
3
2
1
2
74
54
58
43
78
81
72
95
95
89
62
91
77
60
5-Cl
5-I
5-NO2
5-CF3O
5-Me
5-MeO
6-Br
9
10
11
12
13
14
7-Cl
4,6-2Br
a
Reaction conditions: isatin (0.25 mmol), malononitrile (0.25 mmol), acetone (7.5 mmol), a-amylase (246 U), ethanol (0.90 mL), and deionized
b
water (0.10 mL) at 25 ꢀC. Yield of the isolated product.
Herein we report a one-pot three-component reaction between
isatin, malononitrile and active methyl or active methylene
Results and discussion
compounds (acetone, nitromethane, indole, acetylacetone, 4-
hydroxylcoumarin and dimedone) for the synthesis of 3,30-disub-
stituted oxindoles and spirooxindole pyrans via Knoevenagel/
Michael reactions or Knoevenagel/Michael/cyclization reactions
using a-amylase from hog pancreas as a catalyst.
Initially, the three-component reaction of isatin, malononitrile
and acetone was used as a model reaction to investigate the
optimal conditions for the a-amylase catalyzed synthesis of 3,30-
disubstituted oxindole (4a). Since reaction medium is one of the
most important factors inuencing enzymatic reactions,
Table 4 Substrate scope for the synthesis of 3,30-disubstituted oxindoles using nitromethanea
Entry
R
Product
Time [d]
Yieldb [%]
1
2
3
4
5
6
1-Me
5-F
5-CF3O
5-Me
6-Br
6a
6b
6c
6d
6e
6f
2
4
4
3
3
3
91
90
88
96
56
89
7-Cl
a
Reaction conditions: isatin (0.25 mmol), malononitrile (0.25 mmol), nitromethane (7.5 mmol), a-amylase (246 U), ethanol (0.90 mL), and
deionized water (0.10 mL) at 25 ꢀC. b Yield of the isolated product.
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a-amylase lost its most natural activity (the activity reduced
from 24.6 U mgꢁ1 to 4.9 U mgꢁ1) (Table 2, entry 3). It indicated
that the urea treatment caused a great degree of denaturation of
the enzyme, which accordingly decreased its catalytic ability in
the unnatural reaction. The blank experiment suggested that
urea alone did not have any catalytic effect on the reaction
(Table 2, entry 4). Moreover, metal ions Cu2+ and Ag+ were used
as denaturation agents to pretreat the a-amylase, respectively,
and the reaction with the metal ion pretreated enzyme only gave
a trace amount of product detected on TLC (Table 2, entries 5
and 7). The enzymatic assay of a-amylase in starch hydrolyzing
reaction showed that the metal ion nearly completely disrupted
natural activity of the enzyme (Table 2, entries 5 and 7). The
product failed to form when using Cu2+ or Ag+ alone as catalyst
(Table 2, entries 6 and 8). The above control experiments with
denaturation agents, urea and metal ions, pretreated enzyme
suggested that the native fold of the enzyme was not only
responsible for its natural activity but also for its unnatural
activity. In addition, miglitol as a specic competitive inhibitor
of amylase was also used to pretreat the enzyme. The reaction
with miglitol pretreated a-amylase only gave the product in a
low yield of 33% aer 3 days (Table 2, entry 9), suggesting that
the active site of the enzyme has close relation with the
promiscuous reaction. Meanwhile, the activity of a-amylase in
starch hydrolyzing reaction was also obviously decreased aer
miglitol treatment (the activity reduced from 24.6 U mgꢁ1 to
18.0 U mgꢁ1) (Table 2, entry 9). The above results indicated that
the a-amylase promoted the observed three-component reac-
tion, and similar to the natural reaction, the unnatural reaction
may also take place on the active site of the enzyme.
Fig. 1 X-ray crystal structure of 6d.
different solvents were investigated to obtain the suitable
medium for this reaction (Table 1). Generally, the a-amylase-
catalyzed reaction in polar solvents (such as ethanol, meth-
anol, DMF, DMSO, 1,4-dioxane and acetonitrile) gave the higher
yields and the reaction proceeded faster (Table 1, entries 1–6)
than in the non- or low-polar solvents (such as methyl tert-butyl
ether, chloroform, ethyl acetate, butyl acetate, 1,2-dichloro-
ethane, toluene) (Table 1, entries 8–13). Based on the results we
obtained, ethanol was thought to be the best solvent for this
reaction, through a combination of environment protection,
ability to dissolve a large number of substrates and commercial
price.
Next, a series of control experiments to conrm the specic
catalytic effect of a-amylase were carried out. The reaction with
a-amylase gave the product in a yield of 55% aer 2 days (Table
2, entry 1). In the absence of a-amylase, only a trace amount of
product was observed even aer 4 days (Table 2, entry 2). The
urea pretreated a-amylase still promoted the reaction but the
yield was much lower, and only 21% yield was received aer 3
days (Table 2, entry 3). Meantime, to compare the natural
activity with the non-natural activity of the a-amylase, the
enzymatic assay of a-amylase in natural reaction (hydrolyzing
starch) was also conducted. It showed that the urea pretreated
Next, the effect of various parameters (mole ratio of
substrates, reaction temperature, enzyme loading, water
content in the system, and reaction time) on the a-amylase
catalyzed model three-component reaction was investigated (for
details, please see the ESI†). The optimized reaction conditions
Table 5 Substrate scope for the synthesis of 3,30-disubstituted oxindoles using indolea
Entry
R
Product
Time [d]
Yieldb [%]
1
2
3
4
5
1-H
5-I
5-NO2
5-CF3O
7-Cl
8a
8b
8c
8d
8e
5
5
5
5
5
90
83
97
92
95
a
Reaction conditions: isatin (0.25 mmol), malononitrile (0.25 mmol), indole (0.38 mmol), a-amylase (246 U), ethanol (0.90 mL), and deionized
b
water (0.10 mL) at 25 ꢀC. Yield of the isolated product.
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were found to consist of the following: mole ratio of substrates
(isatin: malononitrile: acetone) of 1 : 1 : 30, temperature of 25
ꢀC, enzyme loading of 246 U, water content in the system of 10%
[water/(water + ethanol), in vol] (%).
To verify the generality of the a-amylase catalyzed three-
component reaction for the synthesis of 3,30-disubstituted
oxindoles, various substituted isatins were used as substrates to
carry out the reaction. Basically, a wide range of substituted
isatins could effectively participate in the reaction with malo-
nonitrile and acetone to give the corresponding products
(Table 3). The product yields ranged from moderate to high
depending on the position and electronic property of substitu-
ents. 1-Methyl isatin was also able to be used as a substrate,
giving the desired product in a yield of 54% (Table 3, entry 2).
Isatins with 4-substituents showed low reactivity probably
attributed to the steric effects (Table 3, entries 3 and 4). The
electronic feature of 5-substituents on isatin had some effect on
the yield. The reactions using isatins with an electron-
withdrawing group in 5-position gave the products in good
yields up to 95% (Table 3, entry 8). On the contrary, the isatin
with an electron-donating group in 5-position provided product
in a lower yield (Table 3, entry 11). The 6- or 7-substituted isatins
performed well in the reaction giving products in good yields
(Table 3, entries 12 and 13). In addition, a 4,6-disubstituted
isatin was also used for the reaction, and a moderated yield was
obtained (Table 3, entry 14). Unfortunately, there was no
enantiomeric excess of the products observed by the chiral
HPLC analysis.
Scheme 1 Conversion of 3,30-disubstituted oxindoles (4) to spiroox-
indole dihydropyrans (9).
Isatin bearing either electron-withdrawing or electron-donating
groups performed very well except 6-Br substituted isatin.
Among the tested reactions, the best yield of 96% was obtained
(Table 4, entry 4). Unfortunately, there was no enantiomeric
excess of the products observed by the chiral HPLC analysis. To
the best of our knowledge, so far the products listed in Table 4
have not been reported yet; the products 6a–f were all new
compounds. To conrm the structure, product 6d as a repre-
sentative example was determined by single-crystal X-ray anal-
ysis (Fig. 1).
Indole could be used as a substrate to replace acetone in this
a-amylase catalyzed three-component reaction (Table 5). The
corresponding 3,30-disubstituted oxindoles were synthesized via
Knoevenagel reaction between isatin and malononitrile fol-
lowed by Friedel–Cras alkylation of indole in one pot. Excel-
lent yields up to 97% were obtained with various substituted
isatins (Table 5). To the best of our knowledge, a-amylase
catalyzed Friedel–Cras alkylation has not been reported yet.
The products 8a–e were all new compounds. No enantiomeric
excess of the products observed by the chiral HPLC analysis.
As described above, a-amylase catalyzed three-component
reactions of various isatins, malononitrile and the compounds
When nitromethane was used to replace the role of acetone,
the reaction could proceed smoothly to afford the desired
products in satised yields (Table 4). Generally, the reactions
with nitromethane gave higher yields than those with acetone.
Table 6 Substrate scope for the synthesis of spirooxindole pyrans using acetylacetonea
Entry
R
Product
Time [d]
Yieldb [%]
1
2
3
4
5
6
7
8
1-H
11a
11b
11c
11d
11e
11f
2
2
4
2
4
3
2
2
72
61
50
74
80
83
84
75
1-Me
5-NO2
5-Cl
5-I
5-Me
5-MeO
7-Cl
11g
11h
a
Reaction conditions: isatin (0.25 mmol), malononitrile (0.25 mmol), acetylacetone (7.5 mmol), a-amylase (246 U), ethanol (0.90 mL), and
deionized water (0.10 mL) at 25 ꢀC. b Yield of the isolated product.
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chiral HPLC analysis. To conrm the structure of the spiroox-
indole pyrans, product 11b as a representative example was
determined by single-crystal X-ray analysis (Fig. 2).
4-Hydroxylcoumarin could be used as a substrate in the a-
amylase catalyzed three-component reaction to react with
malononitrile and different substituted isatins (Table 7). In
general, the presence of substituents with different electronic
and steric properties in various positions of isatin did not have a
signicant effect on the formation of spirooxindole pyrans. All
provided good to excellent yields, however no enantiomeric
excess was observed by chiral HPLC analysis. The reactions with
4-hydroxylcoumarin were faster than those with acetylacetone
probably because the carbonyl of 4-hydroxylcoumarin can exist
in the form of an enol due to conjugation with the benzene ring
while the acetylacetone does not fully exist in the form of an
enol.
Another active methylene compound dimedone was also
used to explore the scope of the a-amylase catalyzed three-
component reaction for the synthesis of spirooxindole pyrans
(Table 8). To our surprise, all the reactions were completed
within 24 hours. The reactions with all the tested isatins, except
1-Me substituted isatin (Table 8, entry 2), gave products in good
to excellent yields efficiently. However, no enantiomeric excess
was observed by chiral HPLC analysis.
Fig. 2 X-ray crystal structure of 11b.
containing an active methyl or methylene group provided a
series of multi-functionalized 3,30-disubstituted oxindoles.
Among them, the products 4a–n could be easily converted to
spirooxindole dihydropyrans (9) by reduction/cyclization reac-
tions according to the literature54 (Scheme 1).
Moreover, several compounds which exist in the form of
enols such as acetylacetone, 4-hydroxylcoumarin and dimedone
were used to prepare spirooxindole pyrans. When acetylacetone
was used as a substrate to replace acetone in this a-amylase
catalyzed three-component reaction, highly functionalized spi-
rooxindole pyrans were prepared. The reaction with various
substituted isatins proceeded smoothly to form the products in
yields ranged from 50% to 84% (Table 6). The electronic feature
of substituents on the 5-position of isatin had some effect on
the yield. The isatins with electron-withdrawing group on the 5-
position afforded lower yields than those with electron-
donating group on the 5-position (Table 6, entries 3–7). There
was no enantiomeric excess of the products observed by the
Finally, in order to explore the mechanism of a-amylase
catalyzed three-component reaction, some comparison experi-
ments were conducted (Scheme 2). To verify if the rst-step
Knoevenagel reaction is spontaneous or not, the reaction of
isatin and malononitrile was carried out without catalyst, which
gave the Knoevenagel adduct (16) in 95% isolated yield aer 5
minutes. It showed that the reaction between isatin and
Table 7 Substrate scope for the synthesis of spirooxindole pyrans using 4-hydroxylcoumarina
Entry
R
Product
Time [d]
Yieldb [%]
1
2
3
4
5
6
7
8
9
10
1-H
13a
13b
13c
13d
13e
13f
13g
13h
13i
2
2
2
2
1
2
2
2
2
2
55
88
80
96
80
93
77
90
90
86
1-Me
5-NO2
5-F
5-Cl
5-I
5-Me
5-MeO
6-Br
7-Cl
13j
a
Reaction conditions: isatin (0.25 mmol), malononitrile (0.25 mmol), 4-hydroxylcoumarin (0.25 mmol), a-amylase (246 U), ethanol (0.90 mL), and
deionized water (0.10 mL) at 25 ꢀC. b Yield of the isolated product.
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Table 8 Substrate scope for the synthesis of spirooxindole pyrans using dimedonea
Entry
R
Product
Time [h]
Yieldb [%]
1
2
3
4
5
6
7
8
1-H
15a
15b
15c
15d
15e
15f
15g
15h
15i
3
3
18
18
3
18
3
3
98
47
63
82
92
88
81
75
85
76
87
1-Me
4-Cl
5-NO2
5-F
5-Cl
5-I
5-Me
5-MeO
6-Br
7-Cl
9
10
11
3
3
18
15j
15k
a
Reaction conditions: isatin (0.25 mmol), malononitrile (0.25 mmol), dimedone (0.25 mmol), a-amylase (246 U), ethanol (0.90 mL), and deionized
b
water (0.10 mL) at 25 ꢀC. Yield of the isolated product.
malononitrile spontaneously form the Knoevenagel adduct (16). place on the active site of the enzyme. Thus, we tried to spec-
Then, the Michael addition of intermediate (16) with acetone ulate the mechanism of a-amylase catalyzed three-component
was conducted for 2 days. In the absence of catalyst, the reaction reaction. The proposed mechanism for the synthesis of 3,30-
only gave a trace amount of product 4a, but in the presence of a- disubstituted oxindoles was demonstrated using the reaction of
amylase, the reaction gave product 4a in a good yield of 74%. isatin, malononitrile and acetone (Scheme 3). Firstly, Asp197 in
The results indicated that a-amylase catalyzed the Michael the a-amylase acts as a base to take off an acid proton from the
addition between Knoevenagel adduct (16) and acetone.
acetone, and Glu233 provides a proton to the carbonyl of
According to the literature,55–58 a-amylase from hog pancreas acetone to form the enol. Secondly, the enol reacts with the
contains 496 amino acid residues. The aspartic acid and glu- spontaneously formed Knoevenagel adduct via a Michael
tamic acid, as catalytic amino acid residues in the active site, are addition with the assistance of Asp197 and Glu233 affording the
located in 197 and 233 respectively. For the natural activity of 3,30-disubstituted oxindole. The proposed mechanism for the
the a-amylase, the Asp197 acts as a nucleophile and the Glu233 synthesis of spirooxindole pyrans was demonstrated using the
acts as a proton donor. Moreover, from the results of the control reaction of isatin, malononitrile and acetylacetone (Scheme 4).
experiments and the comparison of natural activity with the It includes the enolization of acetylacetone, the Michael addi-
unnatural reaction above, it could be inferred that similar to the tion of the enol to the Knoevenagel adduct, the cyclization and
natural reaction, the observed unnatural reaction may also took tautomerization.
Conclusion
a-Amylase from hog pancreas was found to display catalytic
promiscuity in three-component reaction for the synthesis of
3,30-disubstituted oxindoles and spirooxindole pyrans in
ethanol/water mixed solvents. The reaction conditions
including solvent, molar ratio of substrates, temperature,
enzyme loading, water content and reaction time were
investigated. The reactions between isatin, malononitrile and
active methyl or active methylene compounds (acetone,
nitromethane, indole, acetylacetone, 4-hydroxylcoumarin and
dimedone) offered corresponding products via Knoevenagel/
Michael reactions or Knoevenagel/Michael/cyclization reac-
Scheme 2 Comparison experiments.
tions in one pot with high to excellent yields of up to 98%
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Scheme 3 The speculated mechanism for the synthesis of 3,30-disubstituted oxindole.
under mild reaction conditions. The a-amylase showed a at 25 ꢀC, and monitored by thin-layer chromatography. The
broad spectrum of adaptability to various substrates. Some reaction was terminated by ltering off the enzyme. Ethyl acetate
control experiments were performed and the comparison of was employed to wash the residue on the lter paper to ensure
natural activity with the unnatural reaction was conducted, that the products were all dissolved in the ltrate. The ltrate was
suggesting this unnatural reaction may take place on the dried over anhydrous Na2SO4, and the organic solvents were then
active site of the a-amylase. A possible mechanism of the a- removed under reduced pressure. The crude products were
amylase catalyzed three-component reaction was speculated. puried by silica gel column chromatography with petroleum
ether/ethyl acetate as the eluent.
General method for the a-amylase
catalyzed synthesis of 3,30-
disubstituted oxindoles (products 4a–
n, 6a–f and 8a–e)
General method for the a-amylase
catalyzed synthesis of spirooxindole
pyrans (products 11a–h, 13a–j and
15a–k)
To a mixture of isatin (0.25 mmol), malononitrile (0.25 mmol), a-
amylase (246 U), acetone (or nitromethane) (7.5 mmol) [or indole To a mixture of isatin (0.25 mmol), malononitrile (0.25 mmol),
(0.38 mmol)] and deionized water (0.10 mL) was added ethanol a-amylase (246 U), acetylacetone (7.5 mmol) [or 4-hydroxyl-
(0.90 mL). The resultant mixture was stirred for the specied time coumarin (0.25 mmol) or dimedone (0.25 mmol)] and deionized
Scheme 4 The speculated mechanism for the synthesis of spirooxindole pyran.
37850 | RSC Adv., 2015, 5, 37843–37852
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water (0.10 mL) was added ethanol (0.90 mL). ꢀThe resultant 21 L.-H. Zhou, N. Wang, G.-N. Chen, Q. Yang, S.-Y. Yang,
mixture was stirred for the specied time at 25 C, and moni-
tored by thin-layer chromatography. Aer completion of the
W. Zhang, Y. Zhang and X.-Q. Yu, J. Mol. Catal. B: Enzym.,
2014, 109, 170–177.
reaction the precipitated product was ltered and washed with 22 R.-C. Tang, Z. Guan, Y.-H. He and W. Zhu, J. Mol. Catal. B:
water (5 mL ꢂ 4) and cooled EtOH (5 mL ꢂ 2) to afford the pure
Enzym., 2010, 63, 62–67.
23 N. Gao, Y.-L. Chen, Y.-H. He and Z. Guan, RSC Adv., 2013, 3,
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products.
24 J.-L. Wang, X. Li, H.-Y. Xie, B.-K. Liu and X.-F. Lin, J.
Biotechnol., 2010, 145, 240–243.
Acknowledgements
This work was nancially supported by the National Natural 25 Y. Xue, L.-P. Li, Y.-H. He and Z. Guan, Sci. Rep., 2012, 2, 761,
Science Foundation of China (no. 21276211 and no. 21472152).
DOI: 10.1038/srep00761.
We thank Dr Wen-Shan Ren for X-ray single crystal diffraction 26 W. Hu, Z. Guan, X. Deng and Y.-H. He, Biochimie, 2012, 94,
analysis.
656–661.
27 B.-H. Xie, Z. Guan and Y.-H. He, Biocatal. Biotransform., 2012,
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