Highly efficient and large-scalable glucoamylase-catalyzed Henry reactions

Article abstract of DOI:10.1039/c3ra41287c

Eco-friendly, highly efficient and large-scalable Henry reactions of aromatic aldehydes and nitroalkanes catalyzed by glucoamylase from Aspergillus niger (AnGA) are described. The reactions were carried out at 30 °C in the mixed solvents of ethanol and wa

Full text of DOI:10.1039/c3ra41287c

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Highly efficient and large-scalable glucoamylase-
catalyzed Henry reactions3
Cite this: RSC Advances, 2013, 3,
16850
Na Gao, Yan-Li Chen, Yan-Hong He* and Zhi Guan*
Eco-friendly, highly efficient and large-scalable Henry reactions of aromatic aldehydes and nitroalkanes
catalyzed by glucoamylase from Aspergillus niger (AnGA) are described. The reactions were carried out at
30 uC in the mixed solvents of ethanol and water, and the corresponding b-nitro alcohols were obtained in
yields of up to 99%. Only 3 mg of enzyme was sufficient to catalyze the reaction of 1 mmol aldehydes. The
natural activity and promiscuous activity of AnGA were compared under different conditions. Experiments
demonstrated that the product of the Henry reaction could inhibit AnGA at 80 uC. This enzymatic Henry
reaction showed a broad substrate scope, and could be facilely enlarged to gram scale. The possible
mechanism was also discussed.
Received 18th March 2013,
Accepted 11th July 2013
DOI: 10.1039/c3ra41287c
www.rsc.org/advances
as cross-linked enzyme aggregates (Alcalase-CLEA) catalyzed
the Henry reaction between 4-nitrobenzaldehyde and nitro-
1. Introduction
methane.28 Our group reported transglutaminase-catalyzed
Henry reactions in a biphasic system composed of water and
CH2Cl2.29
The Henry or nitroaldol reaction is a powerful reaction and an
important atom-economical methodology to furnish a b-nitro
alcohol by a carbonyl compound and an alkyl nitro compound
in organic synthesis.1–4 The resulting b-nitro alcohols, very
interesting functional compounds, have been used in various
beneficial organic transformations.5–8 Therefore, many cataly-
tic Henry reactions have been reported including metal9–11
and small-molecule catalysis.12 However, high-efficiency and
eco-friendly catalytic synthetic methods that satisfy increas-
ingly stringent environmental constraints are still in great
demand by the pharmaceutical and chemical industries. As a
kind of important green catalyst, enzymes have shown
immense advantages such as mild reaction conditions, high
yields, and good selectivities. In recent years, enzymatic
promiscuity has been paid much attention by chemists and
biochemists.13,14 Many enzyme promiscuous activities have
been revealed, such as aldol reactions,15–17 Michael addi-
tions,18,19 Mannich reactions,20,21 Baeyer–Villiger oxida-
tions,22,23 and so on. However, to date, only a few enzymatic
Henry reactions have been reported. Griengl and co-workers
reported the asymmetric enzymatic Henry reactions using
hydroxynitrile lyase from Hevea brasiliensis (HbHNL) (EC
4.1.2.39).24,25 Lin and co-workers reported the enzymatic
Henry reactions to form b-nitro alcohols followed by enzymatic
kinetic resolution of the stereoisomers,26 and D-aminoacylase-
catalyzed Henry reactions in DMSO.27 Gotor and co-workers
reported that protease from Bacillus licheniformis immobilized
Glucoamylase, also known as amyloglucosidase (1,4-a-D-
glucan glucohydrolase, EC 3.2.1.3), is an inverting exo-acting
hydrolase that catalyzes the hydrolysis of a-1,4 and a-1,6
glucosidic linkages to release b-D-glucose from the non-
reducing ends of starch and related saccharides.30,31
Glucoamylase is widely used in the food industry to produce
glucose syrup, high-fructose corn syrup and so on.32 Herein we
wish to report that glucoamylase from Aspergillus niger (AnGA)
is an efficient catalyst for Henry reactions of aromatic
aldehydes and nitroalkanes.
2. Results and discussion
Initially, the reaction of 4-cyanobenzaldehyde and nitro-
methane was used as a model reaction. In view of the fact
that the reaction medium is one of the most important factors
influencing the enzymatic reactions,33 different kinds of
solvents were screened (Table 1). Generally, the AnGA-
catalyzed model Henry reaction in polar solvents (such as
ethanol, acetonitrile, DMSO, water and polyethylene glycol)
gave products in higher yields (85–99%) in relatively shorter
reaction times (5–22 h) (Table 1, entries 1–3, 6 and 7) than in
the non- or low-polar solvents (such as butyl acetate, toluene,
cyclohexane and chloroform) (Table 1, entries 8, 9, 12 and 14).
The reaction in DMSO achieved an excellent yield of 94%
within only 5 h (Table 1, entry 3), while the reaction in water
gave a good yield of 89% after 22 h (Table 1, entry 6). Moreover,
under solvent-free conditions a good yield of 92% was
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
Electronic supplementary information (ESI) available. See DOI: 10.1039/
3
c3ra41287c
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Table 1 Solvent screening and control experimentsa
Entry
Solvent
Time [h]
Yield [%]b
1
2
3
4
5
6
7
8
Ethanol
Acetonitrile
DMSO
Solvent-free
Isopropyl ether
Water
Polyethylene glycol
Butyl acetate
Toluene
2-Propanol
THF
Cyclohexane
1,4-Dioxane
Chloroform
Ethanol (no enzyme)
Ethanol (bovine serum albumin)
Ethanol (egg white albumin)
Ethanol (purified AnGA)c
19
22
5
95
95
22
9
95
95
6.5
22
105
25
105
20
20
20
20
99
97
94
92
89
89
85
79
76
75
73
58
50
39
8
9
10
11
12
13
14
15
16
17
18
23
5
52
a
Reaction conditions: 4-cyanobenzaldehyde (1 mmol), nitromethane (10 mmol), AnGA (20 mg), solvent (0.90 mL), and deionized water (0.10
b
c
mL) at 30 uC. Yield of the isolated product after silica gel chromatography. Reaction conditions: 4-cyanobenzaldehyde (1 mmol),
nitromethane (5 mmol), purified AnGA (1 mg), ethanol (0.85 mL), and deionized water (0.15 mL) at 30 uC.
obtained after a long reaction time of 95 h (Table 1, entry 4).
Based on the results of solvent screening, in view of both
environmental protection and a good ability to dissolve a wide
scope of substrates, we chose ethanol as the optimum solvent
for further investigation.
activity may be due to the process of purification (in order to
rule out that buffer catalyzed the reaction, we did not use any
buffer during the whole process). The purified protein was
then used to catalyze the model Henry reaction, and 1 mg of
this protein as a catalyst gave the product in a yield of 52%
after 20 h (Table 1, entry 18). These experiments confirmed
that AnGA indeed catalyzed the Henry reaction.
Several control experiments were designed to verify the
specific catalytic effect of AnGA on the model Henry reaction in
ethanol. In the absence of AnGA, the reaction only gave the
product in a yield of 8% after 20 h (Table 1, entry 15). As a
comparison, some albumins instead of AnGA were used to
catalyze the reaction. The reaction with egg white albumin
only gave the product in a yield of 5%, similar to the blank
(Table 1, entry 17). However, the reaction with bovine serum
albumin gave the product in a yield of 23% (Table 1, entry 16),
suggesting that a protein without enzyme function was able to
catalyze the Henry reaction in a very unspecific way. On the
other hand, AnGA showed much higher activity than the
albumins indicating that some special group/s is/are respon-
sible for this high yield observed.
Moreover, in order to understand the relation between
natural and promiscuous activities of AnGA, we investigated
the catalytic activity of AnGA on the Henry reaction and natural
substrate (starch) under different conditions (Table 2). The
natural activity of the enzyme under normal conditions was 79
U mg21, but it was only 17 U mg21 when tested in mixed
solvents (water/(water + ethanol) = 0.15, v/v) (Table 2, entry 1).
Since metal ions can inactivate the enzyme by reacting with
some groups e.g. sulfhydryl groups and/or by disrupting the
bonds that hold the enzyme together to cause the enzyme to
undergo a conformational change, AnGA was pretreated with
Ag+ and Cu2+, respectively. It can be seen that the metal-
pretreated AnGA almost competely lost its catalytic activity on
the model Henry reaction (Table 2, entries 5 and 6). The blank
reactions were performed using the same amounts of Ag+ and
Cu2+ to catalyze the reaction, which only gave trace amounts of
the product proving that Ag+ or Cu2+ alone did not catalyze this
transformation (Table 2, entries 3 and 4). Another blank
reaction in which AnGA was pretreated in water at 30 uC
(Table 2, entry 2) showed that Ag+ and Cu2+ indeed inactivated
To rule out the possibility that another protein or inorganic
impurities catalyzed the reaction, the commercial preparation
of AnGA was checked with SDS-PAGE, and purified by
centrifugal ultrafiltration. (For the procedures of enzyme
purification and the SDS-PAGE of the commercial preparation,
please see the ESI .) The purified protein showed a glucoamy-
3
lase activity of 27 U mg21 (the glucoamylase activity of the
commercial preparation was 79 U mg21). The decrease of
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Table 2 Catalytic activities of AnGA on the Henry reactiona and natural substrateb
Entry
Catalyst
Yield [%]c
Natural activity [U mg21 d
]
1
2
3
4
5
6
7
8
AnGA
99
95
Trace
Trace
3
8
95
5
79 (17, in mixed solvents)e
AnGA (pretreated at 30 uC)f
AgNO3 (42 mg)
—
—
—
6
28
—
—
—
0
CuSO4 (39.9 mg)
AnGA (pretreated with 250 mM Ag+)g
AnGA (pretreated with 250 mM Cu2+ h
AnGA (pretreated at 100 uC)i
urea (100 mg)
)
9
guanidine hydrochloride (100 mg)
AnGA (pretreated with urea)j
AnGA (pretreated with guanidine hydrochloride)k
miglitol (100 mg)
6
10
11
12
13
63
70
51
70
0
—
30
AnGA (pretreated with miglitol)l
a
Reaction conditions: 4-cyanobenzaldehyde (1 mmol), nitromethane (10 mmol), AnGA or pretreated-AnGA (20 mg), ethanol (0.90 mL), and
deionized water (0.10 mL) at 30 uC for 20 h. Reaction conditions: AnGA or pretreated-AnGA dissolved in deionized water (0.01 mg mL21),
b
starch (1% W V21), and NaAc–HCl (50 mM, pH = 4.5), at 60 uC. Yield of the isolated product after silica gel chromatography. Unit
c
d
definition (U mg21): one unit corresponds to the amount of enzyme that liberates 1 mmol of glucose per minute at pH 4.5 and 60 uC. AnGA
e
dissolved in mixed solvents (water/(water + ethanol) = 0.15, v/v) to get a solution (0.01 mg mL21) used for the enzyme assay. AnGA (20 mg) in
f
deionized water (1 mL) was stirred at 30 uC for 24 h, and then water was removed under reduced pressure before use. AnGA (20 mg) in Ag+
g
solution (250 mM) [AgNO3 (42 mg) in deionized water (1 mL)] was stirred at 30 uC for 24 h, and then water was removed under reduced
pressure before use. AnGA (20 mg) in Cu2+ solution (250 mM) [CuSO4 (39.9 mg) in deionized water (1 mL)] was stirred at 30 uC for 24 h, and
h
i
then water was removed under reduced pressure before use. AnGA (20 mg) in deionized water (1 mL) was stirred at 100 uC for 24 h, and then
water was removed under reduced pressure before use. AnGA (20 mg) in urea solution (1.67 M) [urea (100 mg) in deionized water (1 mL)]
j
k
was stirred at 100 uC for 24 h, and then water was removed under reduced pressure before use. AnGA (20 mg) in guanidine hydrochloride
solution (1.04 M) [guanidine hydrochloride (100 mg) in deionized water (1 mL)] was stirred at 100 uC for 24 h, and then water was removed
l
under reduced pressure before use. AnGA (20 mg) in miglitol (0.97 M) [miglitol (100 mg) in deionized water (0.5 mL)] was stirred at 30 uC for
24 h, and then water was removed under reduced pressure before use.
the enzyme. At the same time, the metal-pretreated AnGA
displayed a much lower activty towards natural function
content of 0.15 was chosen as the optimal water content for
the reaction.
(Table 2, entries
5 and 6). Next, urea and guanidine
Then, the effect of the molar ratio of substrates on the
AnGA-catalyzed Henry reaction was investigated (Fig. 2). It can
be seen that an excellent yield of 99% was obtained when the
molar ratio of 4-cyanobenzaldehyde to nitromethane was 1 : 5.
hydrochloride were used to denature AnGA, which made the
enzyme lose its natural activity completely (Table 2, entries 10
and 11). In the meantime, urea and guanidine hydrochloride
decreased the enzyme activity towards the model Henry
reaction from 99% to 63% and 70%, respectively (Table 2,
entries 10 and 11). Finally, miglitol, as a competitive inhibitor
of amyloglucosidase, was used to pretreat the enzyme. The
blank reaction with miglitol gave the Henry product in a yield
of 51% (Table 2, entry 12), while the Henry reaction with
miglitol-pretreated AnGA only gave the product in a yield of
70% (Table 2, entry 13), indicating that miglitol strongly
inhibited the enzyme activity in the Henry reaction. Miglitol-
pretreated AnGA also showed a lower natural activity (Table 2,
entry 13). These data demonstrated that both the natural and
promiscuous activities of the enzyme were inhibited by
miglitol. Based on the above experiments, it can be inferred
that the natural active center of AnGA was responsible for its
activity in the Henry reaction.
Since a specific amount of water is essential to the enzymes
to enhance activity in non-aqueous solvents,21,34 the water
content [Vwater/V(water ethanol)] from 0 to 0.5 was screened
+
(Fig. 1). It can be seen that the yield was raised from 87% to
99% as the water content increased from 0 to 0.15. Continuing
to increase the water content to 0.40 did not cause any obvious
effects on the yield. Once the water content passed 0.40, the
yield of the Henry product began to decrease. Thus, the water
Fig. 1 Influence of water content on the AnGA-catalyzed Henry Reaction.
Reaction conditions: 4-cyanobenzaldehyde (1 mmol), nitromethane (10 mmol),
AnGA (20 mg), water content [0–0.5, water/(water + ethanol), v/v], water +
ethanol = 1 mL, at 30 uC for 20 h. Yield of the isolated product after silica gel
chromatography.
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Fig. 2 Influence of molar ratio of substrates on the AnGA-catalyzed Henry
reaction. Reaction conditions: 4-cyanobenzaldehyde (1 mmol), nitromethane
(1–10 mmol), AnGA (20 mg), ethanol (0.85 mL), and deionized water (0.15 mL)
at 30 uC for 19 h. Yield of the isolated product after silica gel chromatography.
Fig. 3 Influence of temperature on the AnGA-catalyzed Henry reaction[a] and
[a]
natural activity[b]
.
Reaction conditions: 4-cyanobenzaldehyde (1 mmol),
nitromethane (5 mmol), AnGA (3 mg), ethanol (0.85 mL), and deionized water
(0.15 mL) at 15–80 uC for 19 h. Yield of the isolated product after silica gel
[b]
chromatography.
Reaction conditions: AnGA (0.01 mg mL21) dissolved with
deionized water, starch (1% W V21), and NaAc–HCl (50 mM, pH = 4.5), at 15–60
uC.
Thus, the molar ratio of aromatic aldehyde to nitroalkane 1 : 5
was selected as the optimal ratio for further studies.
The effect of enzyme concentration on the model Henry
Reaction of 4-cyanobenzaldehyde (1 mmol) and nitromethane
(5 mmol) was examined (Table 3). AnGA displayed a prominent
catalytic activity on the model Henry reaction. The Henry
product could be furnished in a high yield of 95% with the
enzyme concentration of 1 mg mL21 (Table 3, entry 1). The
best yield of 99% was obtained with the enzyme concentration
of 3 mg mL21 (Table 3, entry 2). Thus, we chose 3 mg mL21 as
the optimal enzyme concentration for the AnGA-catalyzed
Henry reaction.
Temperature is another important factor for enzyme-
catalyzed reactions, due to its effect on the rate of the reaction
and the stability of the enzyme. Thus, a temperature screening
was performed (Fig. 3). Yields of ¢ 90% were obtained at a
temperature range from 15 to 50 uC. However, once the
temperature surpassed 50 uC, the yield decreased evidently,
and only 43% yield was gained at 80 uC. The best yield of 99%
was obtained at 30 uC. Thus, we chose 30 uC as the optimal
temperature for the AnGA-catalyzed Henry reaction.
The influence of temperature on the natural activity of
AnGA was also investigated (Fig. 3). The optimum temperature
for the promiscuous activity was 30 uC, while the optimum
temperature for the natural activity was 60 uC. However, both
promiscuous and natural activities decreased at 80 uC.
To explore the reasons for a low yield at a temperature of 80
uC, we designed and performed several experiments (Table 4).
Firstly, to verify whether AnGA could be inactivated in the
mixed solvents at 80 uC, AnGA was pretreated in the mixed
solvents of ethanol and deionized water at 80 uC for 19 h, and
then used to catalyze the model reaction, which gave the
product in a yield of 96%, demonstrating that the mixed
solvents at 80 uC caused no loss of the AnGA’s catalytic activity
(Table 4, entry 1). Secondly, we examined whether the
substrates could inactivate AnGA at 80 uC. AnGA, pretreated
with the substrates 4-cyanobenzaldehyde and nitromethane at
80 uC, respectively, was used to catalyze the model reaction,
and good yields of 99% and 92% were obtained in turn
(Table 4, entries 2 and 3). The results indicated that the
substrates did not cause the loss of AnGA’s catalytic activity.
The experiment (Table 4, entry 3) also demonstrated that the
possible volatilization of nitromethane was not the reason for
low yield at 80 uC. Thirdly, we determined whether the Henry
product 4-(1-hydroxy-2-nitroethyl)benzonitrile could inactivate
AnGA. AnGA, pretreated with Henry product at 30 uC and 80 uC
separately, was used to catalyze the model reaction, and the
yields of 90% and 27% were obtained, respectively (Table 4,
entries 4 and 5). The results clearly indicated that the Henry
product inactivated AnGA at a high temperature (80 uC);
however, it almost did not affect the activity of AnGA at low
temperature (30 uC). Therefore, we could conclude that the low
yield at a temperature of 80 uC is mainly due to the inactivation
Table 3 Effect of enzyme concentration on the AnGA-catalyzed Henry Reactiona
Entry
Enzyme concentration [mg mL21
]
Yield [%]b
1
2
3
4
5
1
3
5
10
20
95
99
97
96
99
a
Reaction conditions: 4-cyanobenzaldehyde (1 mmol), nitromethane
(5 mmol), AnGA (1–20 mg), ethanol (0.85 mL), and deionized water
b
(0.15 mL) at 30 uC for 19 h. Yield of the isolated product after
silica gel chromatography.
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Table 4 Exploring the reasons for low yield at 80 uCa
Entry
Pretreatment of AnGA
Yield [%]b
1
2
3
4
5
AnGA pretreated in the mixed solvents of ethanol and deionized water at 80 uCc
AnGA pretreated with substrate 4-cyanobenzaldehyde at 80 uCd
96
99
92
90
27
AnGA pretreated with substrate nitromethane at 80 uCe
AnGA pretreated with Henry product 4-(1-hydroxy-2-nitroethyl)benzonitrile at 30 uCf
AnGA pretreated with Henry product 4-(1-hydroxy-2-nitroethyl)benzonitrile at 80 uCf
a
Reaction conditions: 4-cyanobenzaldehyde (1 mmol), nitromethane (5 mmol), AnGA (3 mg), ethanol (0.85 mL), and deionized water (0.15
b
c
mL) at 30 uC for 19 h. Yield of the isolated product after silica gel chromatography. AnGA (3 mg) was pretreated in the mixed solvents of
d
ethanol (0.85 mL) and deionized water (0.15 mL) at 80 uC for 19 h. AnGA (3 mg) was pretreated with 4-cyanobenzaldehyde (1 mmol) in
mixed solvents of ethanol (0.85 mL) and deionized water (0.15 mL) at 80 uC for 19 h, and then nitromethane (5 mmol) was added and the
e
mixture was stirred at 30 uC for another 19 h. AnGA (3 mg) was pretreated with nitromethane (5 mmol) in mixed solvents of ethanol (0.85
mL) and deionized water (0.15 mL) at 80 uC for 19 h, and then 4-cyanobenzaldehyde (1 mmol) was added and the mixture was stirred at 30 uC
f
for another 19 h. AnGA (3 mg) was pretreated with Henry product 4-(1-hydroxy-2-nitroethyl)benzonitrile (85 mg) in mixed solvents of ethanol
(0.85 mL) and deionized water (0.15 mL) at 30 uC (entry 4) or 80 uC (entry 5) for 19 h, and then 4-cyanobenzaldehyde (1 mmol) and
nitromethane (5 mmol) were added and the mixture was stirred at 30 uC for another 19 h.
Table 5 Substrate scope of the AnGA-catalyzed Henry reactionsa
Entry
R1
R2
Product
Time [h]
Yield [%]b
dr [syn/anti]c
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
2-MeOC6H4
3-MeOC6H4
2-NO2C6H4
3-NO2C6H4
4-NO2C6H4
3-ClC6H4
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Me
Me
Et
Et
Et
Et
H
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
72
72
10
21
21
96
48
11
19
10
10
48
96
96
71
40
46
88
71
40
46
88
19
86
77
95
96
98
52
82
97
99
93
98
89
6
—
—
—
—
—
—
—
—
—
—
—
—
—
—
57 : 43
58 : 42
50 : 50
64 : 36
50 : 50
61 : 39
70 : 30
77 : 23
—
4-ClC6H4
3-CNC6H4
4-CNC6H4
2,6-Cl2C6H3
2,4-Cl2C6H3
4-BrC6H4
2-Furyl
2-Thienyl
2-MeOC6H4
4-NO2C6H4
4-CNC6H4
4-BrC6H4
2-MeOC6H4
4-NO2C6H4
4-CNC6H4
4-BrC6H4
8
72
87
89
65
61
87
87
74
95
17
18
19
20
21
22
3s
3t
3u
3v
3i
23 (large-scale)d
4-CNC6H4
a
Reaction conditions: aldehyde (1 mmol), nitroalkane (5 mmol), AnGA (3 mg), ethanol (0.85 mL), and deionized water (0.15 mL) at 30 uC.
b
c
Yield of the isolated product after silica gel chromatography. dr was determined by 1H NMR, and syn and anti isomers were assigned by
d
chemical shifts according to the literature. Reaction conditions: 4-cyanobenzaldehyde (20 mmol, 2.62 g), nitromethane (100 mmol, 6.10 g),
AnGA (60 mg), ethanol (17 mL), and deionized water (3 mL) at 30 uC.
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Scheme 1 Possible mechanism of the AnGA-catalyzed Henry reaction.
of AnGA caused by the Henry product that is formed during the
course of the reaction.
small scale reactions, and an excellent yield of 95% was
achieved after 19 h (Table 5, entry 23).
The Gotor group suggested an unspecific protein may
catalyze the Henry reaction.35 Based on the experiments with
denatured and inhibited AnGA, and the comparison of natural
activity and promiscuous activity, we determine that the
natural active center of AnGA is responsible for its activity in
the Henry reaction. Thus, we attempted to propose the
possible mechanism of the AnGA-catalyzed Henry Reaction
of aldehyde with nitroalkane (Scheme 1). According to
previous literature,36–38 the catalytic site of AnGA includes
the general acid and base catalysts Glu179 and Glu400 situated
at the bottom of a pocket. Based on the widely accepted
mechanism of hydrolysis of AnGA, we hypothesized the
possible mechanism. Glu400, as a base, deprotonates the
a-carbon of the nitroalkane providing intermediate I. At the
same time, Glu179, as an acid, donates a proton to the
carbonyl oxygen of the aldehyde generating intermediate II.
Then, the a-carbon of I, as a nucleophile, attacks the carbonyl
of II forming a new carbon-carbon bond. Finally, the product
(b-nitro alcohol) is released from the active site.
With the optimized conditions in hand, AnGA-catalyzed
Henry reactions of nitroalkanes with various aromatic and
hetero-aromatic aldehydes were investigated (Table 5). A wide
range of aromatic aldehydes can effectively participate in the
reactions with nitromethane, nitroethane and nitropropane.
This reaction tolerates both electron-donating and electron-
withdrawing substituents of aromatic aldehydes. In general,
the reactions with nitromethane gave better yields than those
with nitroethane and nitropropane. Electron-withdrawing
substituted aldehydes gave better yields than electron-donat-
ing ones. The best yield of 99% was achieved for the reaction
of 4-cyanobenzaldehyde with nitromethane (Table 5, entry 9).
Besides, disubstituted aromatic aldehydes such as 2,6- and 2,4-
dichlorobenzaldehyde could react with nitromethane effi-
ciently giving products in good yields (Table 5, entries 10
and 11). However, furfural and 2-thenaldehyde gave poor
yields of 6 and 8%, respectively (Table 5, entries 13 and 14). In
addition, when reacting with nitroethane and nitropropane,
AnGA displayed different degrees of diastereoselectivity for syn-
isomers. The best d.r. value of 77 : 23 (syn : anti) was obtained
(Table 5, entry 22). Unfortunately, no enantioselectivity was
observed.
3. Conclusions
We further performed a large-scale Henry reaction of
4-cyanobenzaldehyde (20 mmol, 2.62 g) and nitromethane
(100 mmol, 6.10 g) in ethanol (17 mL) and deionized water (3
mL) at 30 uC. Only 60 mg of AnGA was sufficient to catalyze this
large-scale reaction facilely using the same procedure as the
A facile, environmentally friendly and large-scalable synthetic
methodology of an AnGA-catalyzed Henry reaction for the
preparation of a-nitro alcohol derivatives was established. This
reaction can be carried out under mild conditions without the
need for additional cofactors or special equipment. The
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´
8 I. Kudyba, J. Raczko, Z. Urbanczyk-Lipkowska and
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remarkable catalytic activity of AnGA on the Henry reaction
was demonstrated adequately. Only 3 mg of AnGA is sufficient
to catalyze 1 mmol of aldehyde to give good yields in most
cases. A wide range of substrates were accepted by the enzyme
and yields of up to 99% were achieved. The control
experiments with the denatured and inhibited enzyme and
non-enzyme proteins indicated that the specific natural fold of
AnGA was responsible for its catalytic activity in the Henry
reaction. The influence of several factors, including solvent,
water content, molar ratio of substrates and temperature, was
investigated. It was found that the Henry product could
inactivate AnGA at high temperature (80 uC); however, it almost
did not affect the activity of AnGA at low temperature (30 uC).
This work broadens the scope of AnGA-catalyzed transforma-
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4. General procedure for the AnGA-
catalyzed Henry reactions (products 3a–v)
To a mixture of aldehyde (1.0 mmol), AnGA (3 mg), nitroalk-
anes (5.0 mmol) and ethanol (0.85 mL) was added deionized
water (0.15 mL). The resultant mixture was stirred for the
specified time at 30 uC, and monitored by thin-layer
chromatography. The reaction was terminated by filtering
the enzyme. Ethyl acetate was employed to wash the residue on
the filter paper to ensure that the products obtained were all
dissolved in the filtrate. The filtrate was dried over anhydrous
Na2SO4, and 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.
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Acknowledgements
Financial support from the National Natural Science
Foundation of China (No 21276211) and the Doctoral
Foundation of Southwest University (SWU112019) are grate-
fully acknowledged.
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