Enzyme-catalyzed domino reaction: Efficient construction of spirocyclic oxindole skeleton using porcine pepsin

Article abstract of DOI:10.1039/c5cy00987a

Pepsin from porcine gastric mucosa was used as a sustainable and environmentally friendly biocatalyst in the domino Knoevenagel/Michael/Michael reaction for the synthesis of spirooxindole derivatives in methanol. A wide range of isatins and α,β-unsaturated ketones reacting with malononitrile provided the corresponding products in yields of up to 99% with diastereoselectivities of up to >99:1 dr. This pepsin-catalyzed domino reaction provided a novel case of enzyme catalytic promiscuity.

Full text of DOI:10.1039/c5cy00987a

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Enzyme-catalyzed domino reaction: Efficient constructionDoOfI: 10.1039/C5CY00987A
spirocyclic oxindole skeleton using porcine pepsin
Yan-Hong He*, Tao He, Jun-Tao Guo, Rui Li, Yang Xiang, Da-Cheng Yang, Zhi Guan*
Key Laboratory of Applied Chemistry of Chongqing Municipality, School of Chemistry and Chemical Engineering,
Southwest University, Chongqing 400715, PR China
Fax: +86-23-68254091; e-mails: heyh@swu.edu.cn (for Yan-Hong He); guanzhi@swu.edu.cn (for Zhi Guan)
Abstract: The pepsin from porcine gastric mucosa was used as a sustainable and
environmentally friendly biocatalyst in the domino Knoevenagel/Michael/Michael reaction for the
synthesis of spirooxindole derivatives in methanol. A wide range of isatins and α,β-unsaturated
ketones reacting with malononitrile provided the corresponding products in yields of up to 99%
with diastereoselectivity of up to >99:1 dr. This pepsin-catalyzed domino reaction provided a
novel case of enzyme catalytic promiscuity.
Key words: porcine pepsin; domino reaction; spirocyclic oxindole; biocatalysis; enzyme
promiscuity
1. Introduction
Enzyme catalysis is experiencing an unprecedented revolution since Klibalov’s early 1980s
discovery which showed that enzymatic activity can be stabilized in nearly anhydrous organic
solvents[1-3] allowing for the active site of an enzyme in the appropriate solvent environment[4] to
catalyze unnatural chemical reactions. This behavior is known as enzymatic promiscuity[5, 6]
.
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Research into enzymatic promiscuity has brought enzymatic organic reactions significantDaOtIt:e1n0.t1i0o3n9/C5CY00987A
and recognition from chemists and biochemists alike. Advantages including environmental
friendliness, easy operation and mild reaction conditions demonstrate a great potential for future
applications of enzyme catalysis in organic synthesis and significant advances have been made in
the field of enzymatic promiscuity[7, 8]. For instance, some elegant work on enzymatic promiscuity
have been reported including enzyme-catalyzed aldol[9-15], Michael[16-19], Mannich[20], Henry[21-23]
,
26]
Knoevenagel condensation[24], multi-component[25,
and domino reactions[27]. Among these
examples, multi-step reactions catalyzed by a single enzyme are relatively rare.
Cl
O
O
N
H
N
O
N
O
O
Et
O
N
O
HN
N
Cl
A
B
C
H
MDM2-p53 interaction inhibitor
B
spyrotryprostatin
gelsemine
NC
N
NC
O
O
N
N
H
H
E
D
receptor agonist
progesterone
Fig. 1 Example of some natural products and pharmaceutically relevant compounds containing
spriocyclic oxindoles
The spirocyclic oxindole skeletons are important structural motifs often found in natural products
as well as biologically active compounds[28-35] (Fig. 1). Due to these compounds’ unique
2

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architecture and important biological activity, methods for the synthesis of spirocyclic oxindoles
have aroused the attention and interest from chemists. Many excellent synthetic protocols from a
variety of starting materials have been developed to access the spirocyclic oxindole skeletons,
especially spiro[cyclohexanone-oxindole] backbones[29, 36-41]. Melchiorre and co-workers
disclosed an asymmetric synthesis of spirocyclic oxindoles with multiple stereocenters via a
tandem Michael/Michael addition in 2009[42]. Gong and co-workers reported an asymmetric
formal [4+2] cycloaddition reaction between Nazarov reagents and methyleneindolinone catalyzed
by bifunctional catalysts in 2010[34]. Wang and co-workers developed a cascade Michael-ketone
aldol-dehydration domino reaction involving 2-hydroxy-3-acetyl indoles and enones catalyzed by
Epi-Qui.-NH2 (a Cinchona-based primary catalyst) in 2010[43]. Wang and coworkers also reported
a method to access spirocyclic oxindoles via a tandem double Michael reaction starting from
oxindoles and dienones catalyzed by chiral primary amine in 2011[44]. Lan and coworkers used the
combination of a cinchona-based chiral primary amine and a BINOL-phosphoric acid as a
synergistic catalyst system for the double Michael addition of isatylidene malononitriles
(pre-generated from isatins and malononitrile) with α,β-unsaturated ketones to afford chiral spiro
[cyclohexane-1,3’-indoline]-2’,3-diones at 80 °C in 2011[45]. Hu and co-workers disclosed an
asymmetric synthesis of multistereogenic spirocyclic oxindoles via a Morita–Baylis–Hillman
reaction catalyzed by chiral phosphine in 2014[46]. Ramachary and co-workers developed a tandem
Michael/Michael process to access spiro[cyclohex[3]ene-1,3'-indoline]-2',5-dione derivatives
based on the reaction of unmodified ynones and isatylidene malononitriles[47]. Although many
elegant synthetic protocols have been reported in this field in recent years, the development of
novel catalysts for the synthesis of spirocyclic oxindoles that are environmentally friendly,
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operationally simple and cheap are still in great demand.
Very recently, we reported a one-pot three-component reaction to construct 3,3-disubstituted
oxindoles and spirooxindoles by using α-amylase from hog pancreas as a biocatalyst. Acetone,
nitromethane or indole could participate in the reaction with isatin and malononitrile to form
3,3-disubstituted oxindoles via Knoevenagel and Michael reaction, and spirooxindole pyrans
could be obtained via Knoevenagel, Michael and intramolecular cyclization reactions of
acetylacetone, 4-hydroxylcoumarin or dimedone with isatin and malononitrile[48]. To continue
exploring synthetic applications of enzymes, based on Lan and coworkers’ research[45], herein, we
report a novel discovery that pepsin from porcine gastric mucous could catalyze the domino
Knoevenagel/Michael/Michael reaction of isatins, malononitrile and α,β-unsaturated ketones in
methanol without any additives to afford spiro[cyclohexanone-oxindole] derivatives.
2. Results and discussion
Isatin, malononitrile and an α,β-unsaturated ketone were used as the substrates in this
pepsin-catalyzed domino reaction (Scheme 1). According to previous reports[48], isatin and
malononitrile can spontaneously form a Knoevenagel adduct (isatylidene malononitrile). We
speculated that the product spiro[cyclohexanone-oxindole] derivatives were formed via the
domino Michael/Michael reaction (the Michael addition of isatylidene malononitrile and
α,β-unsaturated ketone, followed by intramolecular Michael addition) (Scheme 1).
4

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DOI: 10.1039/C5CY00987A
NC
CN
O
O
reaction
domino
spontaneous
NC
CN
O
CN
O
O
N
N
CN
H
N
H
H
Michael
pepsin
reaction
reaction
pepsin
O
Michael
NC
CN
N
H
O O
Scheme 1 The sequence of pepsin-catalyzed domino reaction.
The reaction between isatin, malononitrile and benzalacetone was used as a model reaction.
Because reaction medium has obvious influence on enzyme activity, enzyme stability and the
ability to catalyze unnatural reactions[5], the effect of different organic solvents on the
pepsin-catalyzed model reaction was investigated (Table 1). As can be seen from the table,
reaction medium played a crucial role in the pepsin-catalyzed domino reaction. The highest yield
of 91% was obtained in methanol and a yield of 79% was achieved in ethanol after 5 days (Table
1, entries 1 and 2). The reaction in DMF, DMSO, THF and isopropanol provided the product in
yields of 20-40% (Table 1, entries 3-6). In other tested solvents very poor yields were received or
only trace amounts of product were observed (Table 1, entries 8-12). The observed results may be
attributed to both the solubility of substrates or intermediates and specific interactions between the
solvent and pepsin. Thus, methanol was chosen as the optimum solvent for the reaction in the
following studies.
Table 1 Solvent screening a
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O
O
O
pepsin
+
+
CN
O
NC
CN
solvent
CN
N
H
N
O
H
4a
1a
2
3a
Entry
1
Solvent
Yield (%) b
methanol
ethanol
91
2
79
3
DMF
40
4
DMSO
30
5
isopropanol
THF
20
6
20
7
1,4-dioxane
chloroform
water
16
8
6
9
4
10
11
12
butyl acetate
acetonitrile
hexane
trace
trace
trace
a Reaction conditions: isatin (0.25 mmol), malononitrile (0.25 mmol), benzalacetone (0.50 mmol), pepsin (9.5 kU),
solvent (0.90 mL), and deionized water (0.10 mL) at 30 °C for 5 days.
b Yield of the isolated product after purification by silica gel chromatography.
To demonstrate the specific catalytic effect of pepsin on the domino Michael/intramolecular
Michael sequence in methanol, a series of control experiments were carried out (Table 2). We
performed the model domino reaction in the absence of pepsin, and only a trace amount of desired
product was observed after 5 days (Table 2, entry 1). The pepsin-catalyzed model reaction gave
the product in a good yield of 91% (Table 2, entry 2), indicating that the pepsin preparation
catalyzed the reaction. Urea as a denaturation agent was employed to pretreat the pepsin, and the
urea-pretreated pepsin was then used to catalyze the model reaction which only gave the product
with a very low yield of 13% (Table 2, entry 4). At the same time, to compare the natural activity
with the non-natural activity of the both pretreated and untreated pepsin, an enzymatic assay of
pretreated and untreated pepsin in a natural reaction (hydrolyzing hemoglobin) was conducted.
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Results showed that the urea-pretreated pepsin lost most of its natural activity, with a DreOdI:u1c0.t1i0o3n9/C5CY00987A
from 106 U mg-1 to 42 U mg-1 (Table 2, entries 2 and 4). The urea treatment caused a great degree
of denaturation of the pepsin, which accordingly decreased its catalytic ability in the unnatural
reaction. In order to ascertain the effects of urea on the model reaction, urea alone was used to
catalyze the model reaction, and no catalytic effect was observed from urea (Table 2, entry 3).
Heavy metal ions can also be used to inactivate enzymes since they can react with some structural
groups (e.g. sulfhydryl groups) resulting in irreversible damage, or they can interact with some
amino acid residues causing changes in tertiary structure. Thus, Cu2+ and Ag+ were used to pretreat
the pepsin, separately. The metal ion-pretreated pepsin almost lost its ability to catalyze the model
reaction, and only 8% and 15% yields were obtained, respectively (Table 2, entries 6 and 8). The
metal ion treatment also caused a serious decrease on the natural activity of pepsin reducing
activity from 106 U mg-1 to 25 U mg-1 (Table 2, entries 2, 6 and 8). Blank reactions using Cu2+ or
Ag+ alone as the catalyst failed to give the product (Table 2, entries 5 and 7). The above control
experiments indicated that pepsin catalyzed the model domino reaction, and its specific
three-dimensional structure was crucial for this catalytic activity.
Moreover, we previously reported the addition of other nucleophiles such as active methyl or
active methylene compounds (acetone, nitromethane, indole, acetylacetone, 4-hydroxylcoumarin
and dimedone) to in situ generated isatylidenemalononitriles to form 3,3’-disubstituted oxindoles
and spirooxindole pyrans using α-amylase from hog pancreasas as a biocatalyst[48]. To verify if
α-amylase can also catalyze the present reaction, the α-amylase from hog pancreas was used as an
enzyme catalyst for the model reaction which only gave a low yield of 22% (Table 2, entry 9).
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The result showed that α-amylase has a certain degree of catalytic ability to this reactioDnO, Ib: u10t.1t0h3e9/C5CY00987A
catalytic activity is very low in comparison with the pepsin.
Table 2 Control experiments a
O
O
O
catalyst
+
+
CN
CN
O
NC
CN
methanol
N
N
O
H
H
1a
2
3a
4a
Entry
Catalyst
none
Yield (%) b
Natural activity (U mg-1 solid) c
1
2
3
4
5
6
7
8
9
trace
91
-
pepsin
106
-
urea (200 mg)
pepsin (pretreated with urea) d
trace
13
42
-
CuSO4 (39.9 mg)
trace
8
pepsin (pretreated with 250 mM Cu2+) e
AgNO3 (42 mg)
pepsin (pretreated with 250 mM Ag+) f
α-amylase from hog pancreas g
25
-
trace
15
25
--
22
a Reaction conditions: isatin (0.25 mmol), malononitrile (0.25 mmol), benzalacetone (0.50 mmol), pepsin (9.5 kU),
methanol (0.90 mL), deionized water (0.10 mL) at 30 ºC for 5 days.
b Yield of the isolated product after purification by silica gel chromatography.
c
Unit definition (U mg-1 solid), one unit will produce a change in A280 of 0.001 per minute at pH 2.0 at 37 ºC,
measured as TCA-soluble products using hemoglobin as substrate.
d Pepsin (9.5 kU) in urea solution (6.7 M) [urea (400 mg) in deionized water (1 mL)] was stirred at 25 ºC for 24 h,
and then water was removed by lyophilization before use.
e Pepsin (9.5 kU) in Cu2+ solution (250 mM) [CuSO4 (39.9 mg) in deionized water (1 mL)] was stirred at 25 ºC for
24 h, and then water was removed by lyophilization before use.
f Pepsin (9.5 kU) in Ag+ solution (250 mM) [AgNO3 (42 mg) in deionized water (1 mL)] was stirred at 25 ºC for 24
h, and then water was removed by lyophilization before use.
g α-Amylase from hog pancreas (30 mg, lyophilized powder) was used instead of pepsin.
Water content usually affects enzymatic reactions in organic solvents because water can affect the
conformational flexibility of an enzyme. To confirm the effect of water on this pepsin-catalyzed
domino reaction in methanol, the addition of water from 0 to 50% [Vwater/V(water+methanol)%] was
investigated (Table 3). The reaction yield significantly decreased with the addition of water into
8

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the reaction system, likely due to decreases in solubility of both substrates and intermediates as
water content increased. Thus, methanol without addition of water was chosen as the optimum
reaction medium. The methanol employed was A.R. grade, and it was used directly without drying
treatment.
Table 3 Effect of water addition on the pepsin-catalyzed domino reaction a
O
O
O
pepsin
+
+
NC
O
CN
CN
CN
N
methanol/water
H
N
O
4a
H
1a
Entry
2
3a
Addition of water [Vwater/V(water+methanol)%]
Yield (%) b
1
2
3
4
5
6
0
96
91
52
38
18
10
10
20
30
40
50
a Reaction conditions: isatin (0.25 mmol), malononitrile (0.25 mmol), benzalacetone (0.50 mmol), pepsin (9.5 kU),
V
(methanol+water) = 1.0 mL at 30 ºC for 5 days.
b Yield of the isolated product after purification by silica gel chromatography.
The effects of catalyst loading on the pepsin-catalyzed model domino reaction were examined
(Table 4). From the results we obtained, there was a linear relationship between enzyme loading
and the yield of the domino reaction when 1-9.5 kU of pepsin was used (Table 4). However, the
reaction with higher enzyme loading (11.7 kU) gave product in a slightly decreased yield (Table 4,
entry 6). Therefore, 9.5 kU of pepsin was chosen as the optimum enzyme loading for the reaction
of isatin (0.25 mmol), malononitrile (0.25 mmol) and benzalacetone (0.50 mmol) in methanol (1.0
mL).
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Table 4 Effect of enzyme loading on the pepsin-catalyzed domino reaction a
O
O
O
pepsin
+
+
O
CN
CN
NC
CN
N
methanol
H
N
O
4a
H
1a
2
3a
Entry
Pepsin (kU)
Yield (%) b
1
2
3
4
5
6
1
10
30
56
74
96
92
3.2
5.3
7.4
9.5
11.7
a Reaction conditions: isatin (0.25 mmol), malononitrile (0.25 mmol), benzalacetone (0.50 mmol), pepsin (1-11.7
kU) and methanol (1.0 mL) at 30 ºC for 5 days.
b Yield of the isolated product after purification by silica gel chromatography.
Temperature plays an important role in enzymatic reactions due to its effect on the reaction rate
and the stability of enzymes. Thus, the effect of temperature on the pepsin-catalyzed model
domino reaction was examined (Table 5). Rising temperature from 20 to 50 ºC led to a remarkable
increase of yield from 9 to 91% after reacting for 20 h (Table 5, entries 1-4). The reaction at 60 ºC
gave the product in an excellent yield of 96% after only 12 h (Table 5, entry 5). To exclude the
possibility that high temperature promoted the reaction instead of pepsin itself, a blank reaction
was conducted at 60 ºC and only 6% yield was obtained after 12 h (Table 5, entry 6). It was
proved that raising temperatures to 60 ºC improved the catalytic activity of pepsin in the model
domino reaction. However, further increasing the temperature to reflux temperature of the solvent
did not give better yield (Table 5, entry 7). Thus, 60 ºC was selected as the optimum temperature
for the reaction.
Table 5 Effect of temperature on the pepsin-catalyzed domino reaction a
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O
DOI: 10.1039/C5CY00987A
O
O
pepsin
CN
CN
+
O
+
NC
CN
N
methanol
H
N
O
4a
H
1a
2
3a
Entry
Temperature (ºC)
Time (h)
20
Yield (%) b
1
2
3
4
5
6
7
20
9
30
20
30
73
91
96
6
40
20
50
20
60
12
60 (without pepsin)
reflux
12
12
91
a Reaction conditions: isatin (0.25 mmol), malononitrile (0.25 mmol), benzalacetone (0.50 mmol), pepsin (9.5 kU),
methanol (1.0 mL).
b Yield of the isolated product after purification by silica gel chromatography.
The influence of the substrate molar ratio was also tested and the best yield could be achieved
with a molar ratio of 1:1:2 (isatin to malononitrile to benzalacetone).
Table 6 Influence of molar ratio of substrates on the pepsin-catalyzed domino reaction a
O
O
O
pepsin
+
+
O
CN
CN
NC
CN
methanol, 60 oC
N
H
N
O
4a
H
1a
2
3a
Entry
Molar ratio of 1a/2/3a
Yield (%) b
1
2
3
1:1:1
48
58
96
1:1:1.5
1:1:2
a Reaction conditions: isatin (0.25 mmol), malononitrile (0.25 mmol), benzalacetone (0.25-0.50 mmol), pepsin (9.5
kU), methanol (1.0 mL) at 60 °C for 12 h.
b Yield of the isolated product after purification by silica gel chromatography.
Reaction time of the pepsin-catalyzed model domino reaction was also investigated (Fig. 2).
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Product yield clearly increased in the first 4 h. The yield reached its limit of 95% after 12DhO.I: 10.1039/C5CY00987A
100
80
60
40
Yield
20
0
0
5
10
15
20
25
Time (h)
Fig. 2 Reaction time of the pepsin-catalyzed domino reaction.
The reactions were carried out with isatin (0.25 mmol), malononitrile (0.25 mmol) and benzalacetone (0.50 mmol)
in the presence of pepsin (9.5 kU) in methanol (1.0 mL) at 60 ºC for 4-24 h. Yield of the isolated product after
purification by silica gel chromatography.
With the optimized conditions in hand, we explored the scope of substrates and generality of the
pepsin catalyzed domino reaction (Table 7). A series of isatins containing various substituents
with different electronic properties were investigated (Table 7, entries 1-10). Reactions with
isatins bearing an electron-donating group in the 5-position gave better yields (Table 7, entries 3
and 4) than those bearing a strong electron-withdrawing group in the 5-position (Table 7, entries 5
and 6). The position of substituents also had effects on the reaction yield. The isatins with a
substituent in the 5-position gave higher yields than those with a substituent in the 1- or 7-position
(Table 7, entries 2, 4, 7 and 10). A series of α,β-unsaturated ketones were also used to examine the
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tolerance of pepsin (Table 7, entries 11-27). A wide range of substituted benzalacetones
containing either electron-donating or electron-withdrawing groups in the o, m, or p-position of
the benzene ring performed very well, affording the products in good to excellent yields (Table 7,
entries 11-21). The α,β-unsaturated ketones containing naphthyl, thienyl or furyl were also tested
and the products were obtained in satisfactory yields with excellent diastereoselectivity (Table 7,
entries 22-24). Besides aromatic α,β-unsaturated ketones, an aliphatic α,β-unsaturated ketone was
tested and gave a low yield of 32% with 99:1 dr (Table 7, entry 25). Not only methyl styryl
ketones reacted satisfactorily in this reaction, but also ethyl and propyl styryl ketones participated
in the reaction smoothly giving corresponding products in good yields with lower
diastereoselectivity after longer reaction time (Table 7, entries 26 and 27). In all circumstances,
the major products are 4 the minor products are 5 and the relative configuration are confirmed by
comparing with NMR recorded by literature[45]
.
Unfortunately, there was no enantiomeric excess of the products observed by the chiral HPLC
analysis. The products obtained were all racemic. Griengl and coworkers previously reported
lipase-catalyzed Michael-type carbon–carbon bond formations. The reactions proceeded without
enantioselectivity[49]. Moreover, Brinck and Berglund et al. explored the active-site of a rationally
redesigned lipase for catalysis of Michael-type additions, and the products were produced also
without enantioselectivity[50]. Enzyme induced enantioselectivity was not found in the present
work as well as these literatures indicating that the catalytic mechanism of enzyme promiscuity is
not as highly specific as its natural reaction.
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Table 7 Substrate scope of the pepsin-catalyzed domino reaction a
O
O
R3
R3
O
R2
CN
CN
R2
CN
CN
O
pepsin
methanol, 60 oC
R3
R1
R1
R1
+
+
+
R2
O
NC
CN
N
N
O
N
O
H
H
H
2
4
1
5
3
Entry
1
R1
R2
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
R3
Product
4a
4b
4c
Time (h)
12
15
17
16
16
16
17
19
16
12
15
12
19
15
12
19
15
17
12
18
18
15
17
17
17
48
60
Yield (%) b
96
Dr (4:5) c
97:3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Et
2
1-Me
5-MeO
5-Me
5-NO2
5-F
5-Cl
5-Br
5-I
7-Cl
H
84
95:5
3
99
>99:1
90:10
90:10
>99:1
88:12
95:5
4
99
4d
4e
5
75
6
72
4f
7
95
4g
4h
4i
8
92
9
99
87:13
88:12
82:18
>99:1
95:5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
73
4j
o-MeOC6H4
o-ClC6H4
m-MeOC6H4
m-ClC6H4
m-BrC6H4
p-MeC6H4
p-CF3C6H4
p-NO2C6H4
p-FC6H4
p-ClC6H4
p-BrC6H4
1-naphthyl
2-thienyl
2-furyl
85
4k
4l
H
99
H
95
4m
4n
4o
4p
4q
4r
H
86
>99:1
99:1
H
85
H
97
87:13
87:13
87:13
94:6
H
95
H
98
H
95
4s
H
81
94:6
4t
H
81
92:8
4u
4v
4w
4x
4y
4z
H
82
>99:1
>99:1
99:1
H
72
H
78
H
Me
32
99:1
H
Ph
89
58:42
73:27
H
Ph
71
4aa
a Reaction conditions: isatin (0.25 mmol), malononitrile (0.25 mmol), α,β-unsaturated ketones (0.50 mmol), pepsin
(9.5 kU), methanol (1.0 mL) at 60 °C.
b Yield of the isolated product after purification by silica gel chromatography.
c Determined by 1HNMR.
Furthermore, the kinetic parameters (Michaelis constant Km, catalytic constant Kcat and Kcat / Km)
of the pepsin-catalyzed domino reaction for each of the substrates in the model reaction were
14

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determined (Table 8). According to our previous research, isatylidene malononitrilDeOcI:a10n.10b3e9/C5CY00987A
formed via a spontaneous Knoevenagel reaction of isatin and malononitrile[48]. Thus based on Lan
and coworkers’ report[45], we speculated that the pepsin-catalyzed domino reaction actually is the
double Michael addition of Knoevenagel adduct with α,β-unsaturated ketone. Therefore, to
understand the reaction mechanism, the Knoevenagel adduct was also considered as a substrate
(Table 8). It can be seen that the Km for each substrate was generally quite high, showing that the
affinity of pepsin with non-native substrates was very poor. But the Km of the Knoevenagel adduct
was still lower than other substrates (Table 8, entry 4). Meanwhile, the Kcat of each substrate was
quite slow, indicating that the catalytic activity of pepsin for the promiscuous reaction was very
low. However, Kcat / Km of the Knoevenagel adduct was still higher than the other substrates,
suggesting that the efficiency of pepsin catalysis with the Knoevenagel adduct was higher than
with the other substrates. These results suggested that the pepsin-catalyzed domino reaction of
isatin, malononitrile and benzalacetone possibly takes place through the intermediate Knoevenagel
adduct.
Table 8 Kinetic parameters for the pepsin-catalyzed domino reaction a
O
+
O
NC
CN
O
N
H
O
1a
2
pepsin
CN
CN
+
or
methanol, 60 oC
N
O
NC
H
3a
CN
O
4a
N
H
knoevenagel adduct
Entry
1b
Substrate
Km (M)
0.0398
Kcat (h-1)
122.9
Kcat / Km (M-1h-1)
3088
Isatin (1a)
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DOI: 10.1039/C5CY00987A
2c
Malononitrile (2)
0.0619
33.2
536
3d
4e
Benzalacetone (3a)
0.1580
0.0083
82.9
29.4
525
Knoevenagel adduct
3555
a The reactions were carried out in methanol (1.0 mL) at 60 ºC for 1 h and the kinetic parameters were obtained at
the enzyme concentration of 0.463 mM. The experiments were based on HPLC determination of the product. b The
concentration of 1a varied from 0.03 M to 0.19 M, with 2 (0.25 M) and 3a (0.50 M). c The concentration of 2
varied from 0.03 M to 0.19 M, with 1a (0.25 M) and 3a (0.50 M). d The concentration of 3a varied from 0.06 M to
0.38 M, with 1a (0.25 M) and 2 (0.25 M). e The concentration of Knoevenagel adduct varied from 0.03 M to 0.13
M, with 3a (0.50 M).
According to the literatures[51, 52], pepsin contains 385 residues. The active site of pepsin from
porcine gastric mucosa contains Asp32 and Asp215 residues. The function of aspartic acid located
in 32 has been confirmed to act as a base. Based on the literatures, the control experiments and the
kinetic experiments, we speculated a tentative mechanism about the pepsin-catalyzed domino
reaction of isatin, malononitrile and benzalacetone (Scheme 2). First, the carbonyl of
benzalacetone is stabilized via a hydrogen bond with Asp215 in pepsin, and Asp32 acts as a base
to take away a proton from the benzalacetone forming the enol. Second, an intermolecular
Michael addition takes place through the enol attacking the isatylidene malononitrile which is
formed via a spontaneous Knoevenagel reaction of isatin and malononitrile[48]. Finally, the
intramolecular Michael addition occurs forming the spirocyclic oxindole skeleton.
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DOI: 10.1039/C5CY00987A
O
Asp215
Asp32
O
O
O
O
H
O
H
Asp32
Asp215
Asp215
O
O
HO
O
O
Asp32
O
O
O
N
O
O
H
H
O
OH
CN
CN
O
spontaneous
NC
CN
N
H
O
N
Asp215
H
NC
CN
O
O
H
Asp215
N
C
N
C
O
O
H
O
N
C
NC
N
H
O O
Asp32
O
H
O
O
N
H
N
C
N
C
N
H
O O
Scheme 2 The speculated mechanism of pepsin-catalyzed domino reaction.
3. Conclusion
Pepsin from porcine gastric mucosa was used as
a
biocatalyst in the domino
Knoevenagel/Michael/Michael reaction for the synthesis of spirooxindole derivatives in methanol.
A wide range of isatins and α,β-unsaturated ketones reacting with malononitrile provided their
corresponding products in yields of up to 99% with diastereoselectivity of up to >99:1 dr. The
effects of a series of parameters such as solvent, water content, enzyme loading, temperature,
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Catalysis Science & Technology
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molar ratio of substrates, and reaction time on the pepsin-catalyzed domino reactDioOnI: 10w.1e0r3e9/C5CY00987A
investigated. The control experiments suggested that the specific three-dimensional structure of
pepsin was responsible for the domino reaction, and a possible mechanism of the reaction was
speculated. Using enzymes as a regenerable and environmentally friendly catalyst for organic
synthesis meets the requirements of sustainable chemistry. This pepsin-catalyzed domino reaction
provides a novel case of enzyme catalytic promiscuity.
Materials
Pepsin from porcine gastric mucosa (EC number 3.4.23.1, CAS number: 9001-75-6, product
number: P7125-100G, ≥400 units/mg protein, 18% protein/mg) was purchased from
Sigma-Aldrich. One unit will produce a ΔA280 of 0.001 per min at pH 2.0 at 37 °C, measured as
TCA-soluble products using hemoglobin as substrate. Unless otherwise noted, all reagents were
purchased from commercial suppliers and used without further purification.
General procedure for the synthesis of substituted benzalacetones
A 50 mL round bottom flask was charged with aldehyde (20 mmol), acetone (5 mL) and H2O (20
mL), and then 5% NaOH (aq) (2 mL) was slowly added to the mixture at 40 oC. After completion
of the reaction (detected by TLC), acetone was removed under reduced pressure and ethyl acetate
(20 mL) was added. The mixture was extracted with ethyl acetate (3 × 20 mL) and the aqueous
phase was separated and discarded. The organic phase was concentrated in vacuum and the
residue was purified by chromatography on silica gel with petroleum ether/ethyl acetate as the
eluent to afford corresponding products.
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DOI: 10.1039/C5CY00987A
General procedure for the pepsin-catalyzed domino reaction
Pepsin (9.5 kU) was added to a mixture of isatin (0.25 mmol), malononitrile (0.25 mmol), an
α,β-unsaturated ketone (0.50 mmol), and methanol (1.0 mL). The resultant mixture was stirred for
the specified time at 60 ºC, and monitored by TLC. The reaction was terminated by filtering the
enzyme. Ethyl acetate was employed to wash the residue on filter paper to ensure that the products
obtained were all dissolved in the filtrate. The organic solvents were then removed under reduced
pressure. The crude products were purified by silica gel column chromatography with petroleum
ether/ethyl acetate as the eluent (VPE/VEA = 2:1 to 5:1).
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (No.
21276211 and No. 21472152).
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A table of contents entry
Pepsin from porcine gastric mucosa was used as
a
catalyst in the domino
Knoevenagel/Michael/Michael reaction for the synthesis of spirooxindoles.