Biocatalytic Aza-Michael Addition of Aromatic Amines to Enone Using α-Amylase in Water

Article abstract of DOI:10.1002/adsc.201901254

The Michael addition of amines with enones for synthesizing β-amino carbonyls constitutes a valuable transformation in organic chemistry. While various catalyst have been made available for catalyzing the Michael addition of aromatic amines to enones but

Full text of DOI:10.1002/adsc.201901254

Title: Biocatalytic aza-Michael addition of aromatic amines to enone
using α-amylase in water
Authors: Sunil Dutt, Vanshita Goel, Neha Garg, Diptiman Choudhury,
Dibyendu Mallick, and Vikas Tyagi
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10.1002/adsc.201901254
Advanced Synthesis & Catalysis
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DOI: 10.1002/adsc.20101254
Biocatalytic aza-Michael addition of aromatic amines to enone
using α-amylase in water
Sunil Dutt,a Vanshita Goel,a Neha Garg,b Diptiman Choudhury,a Dibyendu Mallickc
and Vikas Tyagi*a
aSchool of Chemistry and Biochemistry, Thapar Institute of Engineering and Technology, Patiala-147004, Punjab, India
bSchool of Basic Sciences, Indian Institute of Technology, Mandi-175005, Himachal Pradesh, India
cDepartment of Chemistry, Presidency University, Kolkata-700073, West Bengal, India
*Corresponding author: vikas.tyagi@thapar.edu
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.((Please
delete if not appropriate))
Abstract. The Michael addition of amines with enones for Further, an application of α-amylase catalyzed aza-Michael
synthesizing ß-amino carbonyls constitutes a valuable addition in the cascade reactions has been exhibited by
transformation in organic chemistry. While various catalyst synthesizing biologically important 3-acetyl quinoline. In
have been made available for catalyzing the Michael addition addition, molecular docking and molecular dynamics (MD)
of aromatic amines to enones but there is no report of using α- simulation studies are carried out to get insight into the key
amylase enzyme to catalyze this transformation. The α- interactions of the substrates with the amino acid residues
amylase from Aspergillus oryzae was found to catalyze the near the active site and the probable reaction mechanism,
Michael addition of various aryl (hetero) amines to methyl which reveals Glu230 and Asn295 play a crucial role in the
vinyl ketone with high catalytic efficiency (63-83% yield). A substrate activation process.
hybrid of α-amylase with copper nanoparticle (α-
amylase@CuNPs) has been prepared and used to catalyze this
transformation as a reusable catalyst.
Keywords: Biocatalyst, α-amylase, aza-Michael addition,
C-N bond formation, enone.
boric acid in water has been proved to be valuable to
overcome the problems associated with conventional
catalysts.[7]
Introduction
The Michael addition of various nucleophiles to
electron deficient alkenes is one of the valuable
transformation in the area of synthetic organic
chemistry.[1] In particular, aza-Michael addition which
leads to the formation of a new carbon-nitrogen bond
to afford ß-amino carbonyl compounds has been
proved most important.[2] The ß-amino carbonyls are
imperative building blocks of various biologically
active compounds.[3] Furthermore, they constitute
versatile intermediates for the synthesis of ß-lactams,
amino alcohols, and ß-aminoacids.[4] Conventionally,
the aza-Michael addition reactions are carried out for
synthesizing ß-amino carbonyls under strong acidic or
basic conditions.[5] There are also reports where milder
Lewis acids have been used for this purpose.[6]
However, the conventional methods have certain
drawbacks such as requirement of high temperatures,
long reaction times, poor compatibility with various
substrate functional groups and generation of side
products. Moreover, novel catalysts such as ß-
cyclodextrin, bromo-dimethylsulfonium bromide,
Scheme 1. Aza-Michael addition of less nucleophilic
aromatic amines to α, β-unsaturated olefins
Despite this progress, these methods are limited only
to catalyse the aza-Michael addition of aliphatic
1
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10.1002/adsc.201901254
Advanced Synthesis & Catalysis
amines very efficiently. The development of a novel
catalyst for the addition of less nucleophilic aromatic
amines to electron deficient alkenes has seen
tremendous progress in the last few years. In this
context, early- and late-transition metals, ionic liquids
and molecular iodine have been reported as catalysts
for the Michael-addition of aromatic amines.[8] Very
recently, Lee’s group reported copper-catalyzed aza-
Michael addition of aromatic amines to α, β-
unsaturated olefins (Scheme 1a).[9] Although, this
method has a few shortcomings such as use of strong
bases, expensive and non-reusable ligands and
generation of toxic transition-metal waste.
we evaluated the effectiveness of different organic
solvents such as DMSO, THF and hexane in place of
water (Table 2). Among the various solvents tested for
this addition reaction water remains the best choice.
Also, we checked the different percentage of DMSO
in water as a solvent just to make both starting material
completely soluble during reaction (entry 5-7, Table 2).
We obtained maximum conversion when we used 10%
DMSO-water mixture (1:9 v/v) as a solvent (entry 6,
Table 2).
Table 1. Screening of different enzymes for Michael
addition of 2-bromoaniline to methyl vinyl ketonea
On the other hand, the use of enzymes for catalysing
the valuable organic transformations has exponentially
grown in the current times.[10] Further, a number of
enzymes either in the free form or in immobilized form
have been reported for catalysing the aza-Michael
addition reaction of amines with electron deficient
alkenes.[11] In this context, various lipase enzymes
have been used as a catalyst.[12] Lin’s group have been
reported alkaline protease or hydrolase enzymes to
catalysed the Michael addition of imidazole with
acrylates.[11b, 11c] Very recently, Chen et al. used lipase
enzyme to catalysed the aza-Michael addition of
amines to acrylates in supercritical carbon dioxide.[13]
Moreover, the α-amylase enzymes are known for
catalyzing the hydrolysis of α-glucosidic linkages of
polysaccharides such as starch or glycogen in
nature.[14] Over the years, α-amylase have been used to
catalyse various organic reactions.[15] Recently, Guan
and co-workers have reported a one-pot synthesis of
nitrocyclopropane using α-amylase enzyme as a
catalyst.[16] Additionally, Yu et al. developed α-
amylase catalyse synthesis of highly substituted
indoloquinolizines using tryptamines, β-ketoesters and
α, β-unsaturated aldehydes.[17] But, to the best of our
knowledge there is no previous report of using α-
amylase as a catalyst for the aza-Michael addition
reaction of amines to enones. Herein, we report α-
amylase from Aspergillus oryzae [E.C. 3.2.1.1] as an
efficient biocatalyst for catalysing the aza-Michael
addition of less nucleophilic aryl (hetero) amine
derivatives to enone (methyl vinyl ketone).
entry
1
enzymes
bconversion%
NR
Rhizomucor miehei lipase
(RML)
2
3
Pseudomonas stutzeri
NR
lipase (PSL)
Candida antarctica lipase B
trace
(CALB)
4
5
6
α-amylase from A. oryzae
α-amylase from A. oryzae
blank
28 %
c45 %
dNR
a)Reaction conditions: 1.0 equiv. of 2-bromoaniline (0.58
mmol) and 1.2 equiv. of methyl vinyl ketone (0.69 mmol),
1 mL of enzyme (1 mg/mL in H2O) and 1 mL of H2O were
added to a glass tube having a teflon cap and stirred at room
temperature for overnight. based on HPLC, temperature
40oC, d)Blank: conversion under same conditions without
enzyme.
b)
c)
Table 2. Screening of solvents for α-amylase-catalyzed
addition of 2-bromoaniline to methyl vinyl ketonea
Results and Discussion
We started our investigations by selecting 2-
bromoaniline and methyl vinyl ketone as the model
substrates and screened different enzymes to catalyze
the aza-Michael addition reaction of model substrates
(Table 1). To our surprise only α-amylase gave the
desired product (3h) in 28% conversion yield (entry 4,
Table 1). Further, this reaction gave 45% conversion
when performed at 40oC using α-amylase as catalyst in
water as a solvent (entry 5, Table 1). After having an
enzyme in hand for this addition reaction, we moved
to screen various reaction conditions such as solvents,
temperature and amount of reagents and catalyst to
improve the conversion of this transformation. First,
entry
solvents
H2O
bconversion
45%
1
2
3
DMSO
THF
42%
31%
2
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10.1002/adsc.201901254
Advanced Synthesis & Catalysis
4
5
Hexanes
15%
51%
In the final phase of optimization, we increased the
concentration of enzyme to improve the yield of this
transformation (Figure 1). To our delight, high yield
(89%) could be obtained even at low catalyst loading
(1.5 mg/mL); this undoubtedly proved the efficiency
of α-amylase as a catalyst for this addition reaction.
Figure 1. Optimization of the concentration of α-amylasea
5% DMSO-water
mixture
6
7
10% DMSO-water
mixture
65%
64%
20% DMSO-water
mixture
a)Reaction conditions: 1.0 equiv. of 2-bromoaniline (0.58
mmol) and 1.2 equiv. of methyl vinyl ketone (0.69 mmol),
1 mL of α-amylase (1 mg/mL in H2O) and 1 mL of solvent
were added to a glass tube having a teflon cap and stirred at
40oC for overnight, b)based on HPLC.
Further, we tested the effect of different molar ratios
of 2-bromoanilne and methyl vinyl ketone on the
conversion of this addition reaction (Table 3) and
found that 1:1.2 molar ratio of 2-bromo aniline and
methyl vinyl ketone remained the best choice to attain
the maximum conversion (entry 2, Table 3). We also
increased the temperature of this reaction upto 80oC
but found that 40oC was the optimum temperature for
getting the highest conversion.
a)Reaction conditions: 1.0 equiv. of 2-bromo aniline (0.58
mmol) and 1.2 equiv. of methyl vinyl ketone (0.69 mmol)
in 200 µL of DMSO, 1 mL of α-amylase in H2O (having 0.5,
1.0, 1.5 and 2.0 mg/mL conc. respectively) and 0.8 mL of
H2O were added to a glass tube having a teflon cap and
stirred at 40oC for overnight, b)based on HPLC, %error =
±4.7%.
Table 3. Optimization of the substrates molar ratio and
reaction temperaturea
After having the optimized conditions for the aza-
Michael addition of 2-bromo aniline (1 equiv.) and
methyl vinyl ketone (1.2 equiv.) using α-amylase (1.5
mg/mL) as catalyst in 2 mL of 10% DMSO-water
mixture as the solvent at 40oC for overnight, we
explored the scope of different substitution on
aryl(hetero) amines under the optimized reaction
conditions (Table 4). The reaction of aniline with
methyl vinyl ketone produced the Michael addition
product (3a) in 74% yield (entry 1, Table 4). Then, we
tested the effect of electron-donating groups such as -
Me and -OMe at the arene ring which allowed the
reaction to afford the products in good yields (entry 2-
3, Table 4). On the other hand, the presence of
electron-withdrawing group such as -NO2, -COCH3 at
the arene ring of aniline has significant effect on the
reactivity of the reaction and afforded the desired
products in slightly lower yield (entry 4-6, Table 4). A
much lower yield was obtained when –NO2 group was
at ortho-position (entry 4, Table 4). Moreover, the
entry
ratio of temperatur bconversion
reagent
s
e
(1a:2a)
1:1
1
2
3
4
5
6
7
40oC
40oC
40oC
40oC
50oC
60oC
80oC
54%
65%
64%
34%
63%
59%
43%
1:1.2
1:1.5
1:0.5
1:1.2
1:1.2
1:1.2
a)Reaction conditions: 2-bromoaniline (0.58 mmol) and
methyl vinyl ketone in 200 µL of DMSO, 1 mL of α-
amylase (1 mg/mL in H2O) and 0.8 mL of H2O were added
to a glass tube having a teflon cap and stirred for overnight,
b)based on HPLC.
3
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10.1002/adsc.201901254
Advanced Synthesis & Catalysis
8
82%
presence of halides such as -Cl, -Br and -F on different
positions of aryl amines slightly increased the yield of
the reaction (entry 7-9, Table 4). Gratifyingly, the
nitrogen containing heteroaromatic amines also
reacted well and gave the corresponding products in
very good yield (entry 10-12, Table 4).
1h
3h
3i
9
71%
81%
1i
Table 4. Scope of substrates for the α-amylase catalyzed
Michael addition reactiona
10
3j
1j
byield
74%
11
12
75%
69%
entr amines
y
1
products
3a
1k
3k
1a
1l
3l
2
81%
77%
1b
3b
3c
a)Reaction conditions:1.0 equiv. of substituted aromatic
amines and 1.2 equiv. of enones in 200 µL of DMSO, 1.5
mL of α-amylase (1 mg/mL in H2O) and 0.3 mL of H2O
were added to a glass tube having a teflon cap and stirred at
40oC for overnight, b)isolated yields.
3
1c
In the second phase of our endeavor, we tried to
isolate the α-amylase after the completion of the first
catalytic cycle for reusing it in further catalytic cycles,
but we were unsuccessful to isolate the α-amylase
from the reaction. Recently, there are various reports
for synthesizing a hybrid of enzyme with metal
nanoparticles which increase the operational stability
to use it in organic transformation as reusable
catalyst.[18] Very recently Bӓckvall’s group reported a
Pd(0)-CALB biohybrid catalyst and used it in a
cascade reaction.[19] Encouraged by previous reports,
we developed histidine protected biogenic copper
nanoparticles (CuNPs) and stabilized them on α-
amylase enzyme. The synthesized enzyme-
nanocluster is termed as "α-amylase@CuNPs".[20] The
characterization of the novel α-amylase@CuNPs was
done using various techniques such as UV-visible,
DLS, FT-IR, TEM and EDS (The details of synthetic
procedure and characterization is given in supporting
information).
4
53%
69%
1d
3d
3e
5
1e
6
63%
83%
3f
1f
7
3g
1g
4
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10.1002/adsc.201901254
Advanced Synthesis & Catalysis
tube having a teflon cap and stirred at 40oC for overnight,
bbased on HPLC, %error = ±6.2%
Next, we tested the hybrid catalyst α-amylase@CuNPs
for catalyzing the aza-Michael addition reaction of 2-
bromoaniline with methyl vinyl ketone (Table 5). To
our delight, we obtained very similar results when
comparing the catalytic efficiency of this hybrid with
the free enzyme (entry 1-2, Table 5). Subsequently, we
tested α-amylase@CuNPs for reusability and the
results are shown in Figure 2. These results indicated
that hybrid catalyst can be use many times with high
catalytic efficiency due to improved operational
stability under the optimized reaction conditions.
Table 5. Comparison of the catalytic activities between α-
amylase and α-amylase@CuNPsa
In addition, the tandem Michael addition/Aldol
condensation reaction to synthesize various
biologically important heterocyclic compounds has
gained significant attention over the years.[21] To
exhibit the application of α-amylase in the cascade
reaction consisting aza-Michael addition/Aldol
condensation, we started a reaction of 2-amino
benzaldehyde (1m) and methyl vinyl ketone (2) at
40oC. Gratifyingly, we obtained the desired product 3-
acetyl quinolone (3m) in 26% yield with ~93% purity
(Scheme 2, Figure S8). However, the conversion of
this reaction was lower and require additional
investigation to improve the yield.
entry
catalyst
bconversion
89%
91%
15%
trace
1
2
3
4
5
α-amylase
amylase@CuNPs
CuSO4
CuNPs
L-Histidine
no reaction
a)Reaction conditions: 1.0 equiv. of 2-bromoaniline (0.58
mmol) and 1.2 equiv. of methyl vinyl ketone (0.69 mmol)
in 200 µL of DMSO, 1.5 mL of catalyst (1 mg/mL in H2O)
and 0.3 mL of H2O were added to a glass tube having a
teflon cap and stirred at 40oC for overnight, b)based on
HPLC.
Scheme 2: Synthesis of 3-acetyl quinoline via α-amylase
catalyzed
aza-Michael
addition/Aldol
condensation/aromatization cascade reaction.
Table 6: Scale-up synthesis of ß-amino carbonyl (3h) and
calculation of green chemistry metricsa
Figure 2. Reusability test of amylase@Cu for the Michael
addition of 2-bromo aniline with vinyl ketonea
entry
metrics
results
1
isolated yield
selectivity
E-factor
87.1%
100%
0.16
1.16
100%
86.1%
2
3
4
5
6
PMI
atom-economy
reaction mass
efficiency
a)Reaction conditions: 2-bromoaniline (1.0 g, 5.81 mmol, 1
equiv.) and methyl vinyl ketone (0.426 g, 5.81 mmol, 1.0
equiv.) in 2.0 mL of DMSO, 15 mL of α-amylase (1 mg/mL
in H2O) and 3 mL of H2O were added and stirred the
resulting mixture at 40oC for overnight.
a)Reaction conditions: 1.0 equiv. of 2-bromoaniline (0.58
mmol) and 1.2 equiv. of methyl vinyl ketone (0.69 mmol)
in 200 µL of DMSO, 1.5 mL of α-amylase@CuNPs (1
mg/mL in H2O) and 0.3 mL of H2O were added to a glass
5
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10.1002/adsc.201901254
Advanced Synthesis & Catalysis
In order to assess the scalability of α-amylase-
strong H-bonding with the Glu230 increases the
nucleophilicity of the aniline, we have carried out a
Natural bond orbital (NBO)[25] charge analysis on the
model system comprised of aniline molecule H-
bonded to a glutamic acid residue using Density
Functional Theory (DFT). The structure of the model
is shown in Figure 4. We have observed a development
of a negative charge density (-0.055) on the H-bonded
aniline moiety suggesting an increase in electron
density on the aniline due to the H-bond formation
with glutamic acid residue. Hence, we can conclude
that H-bonding with Glu230 makes the aniline a better
nucleophile as compared to free aniline. A similar
trend is observed for H-bonded 2-bromoaniline moiety
for which even higher negative charge density (-0.062)
is formed on the 2-bromo aniline moiety (Figure S9).
catalyzed aza-Michael addition, the reaction of 2-
bromoaniline (1.0 g) and methyl vinyl ketone (0.426 g,
1.0 equiv.) to synthesize the aza-Michael product (3h)
using α-amylase (15 ml, 1mg/ml in H2O) was carried
out (Table 6). The successful isolation of 1.22 g of (3h)
in 95% purity from this reaction proved the synthetic
utility of α-amylase in the synthesis of ß-amino
carbonyl compounds in gram scales. Furthermore, we
calculated the green chemistry metrics for this reaction
such as E-factor, PMI, and atom-economy and reaction
mass efficiency to display the high greenness of this
protocol and compiled the results in Table 6.
Active site structure and proposed mechanism
A modeling of the interaction of the substrates with
the key amino acids of the α-amylase at the active site
was carried out to investigate the role play by the
enzyme in catalyzing the aza-Michael addition of
aromatic amines to enones. There have been several
experimental and computational reports on the binding
site of this particular strain. [22] These studies revealed
that a catalytic triad of Glu230, Asp297 and Asp206
constitutes the active site. Hence we started with the
crystal structure of the α-amylase from Aspergillus
oryzae (PDB id : 6TAA)22a for molecular modeling. To
explore the initial orientation of substrates inside the
binding site, first we have docked the substrates,
aniline and 2-butenone (methyl vinyl ketone), in the
active site by using AutoDock Vina software.[23] Then
we have carried out 50 ns Molecular Dynamics (MD)
simulation to get the most preferable orientation of the
substrates inside the binding site using GPU version64
of the AMBER 16 package.[24] The details of the
molecular docking and MD simulations are given in
supporting information. The orientation of the
substrate in the active site in one of the representative
snapshots from MD simulation and the key
interactions of the substrates with nearby amino acids
are shown in Figure 3. We have observed that the
nucleophile, aniline, forms a strong hydrogen bond
(1.85 Å) with the Glu230 and thus making it a stronger
nucleophile. On the other hand, the carbonyl group of
2-butenone forms a strong hydrogen bond with
Asn295 (1.92 Å) and hence becoming a better electron
acceptor in protein environment. Thus, based on the
substrate positioning in the active site we have
identified that the Glu230 and Asn295 play crucial
roles in activating the substrates for aza-Michael
addition reaction. In order to validate our claim that the
Figure 3: Overview of substrates (aniline and 2-
butenone) accommodated in the active site of α-
amylase. The representative snapshot was taken from
molecular dynamic (MD) simulation, revealing
substrates (aniline and 2-butenone) in the binding
pocket of α-amylase and the surrounding residues.
Figure 4: The optimized structure of model system
comprised of aniline H-bonded to a glutamic acid.
Relevent bond distances are given in Å.
6
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10.1002/adsc.201901254
Advanced Synthesis & Catalysis
On the basis of the active site structure, we have
aromatic amines to enone using α-amylase as a
catalyst. This strategy could be applied to a number of
substituted anilines and heteroaromatic amines in
combination with methyl vinyl ketone. A hybrid of α-
amylase with copper nanoparticles was synthesized
and used as a reusable catalyst in many catalytic cycles
with high efficiency. The scalability of this
biocatalytic transformation was exhibited by
synthesizing ß-amino carbonyl derivative (3h) in gram
scale and the green chemistry metrics for this reaction
such as E-factor, PMI, atom-economy and reaction
mass efficiency were calculated which displayed the
high greenness of this protocol. Moreover, the α-
amylase catalyzed aza-Michael addition was further
employed in a cascade reaction to synthesize
biologically important 3-acetyl quinoline. In addition,
an analysis of binding of substrates at the active site
using molecular docking and molecular dynamics
studies revealed that Glu230 acts as an acid/base
catalyst and Asn295 activates the enols through strong
hydrogen bonding. Finally, this work expands the
application of α-amylase to catalyze the valuable
transformations in the organic chemistry.
proposed a probable mechanism in which Glu230 acts
as an acid/base residue. The proposed mechanism is
shown in Scheme 3. In the first step of the reaction,
there will be a nucleophilic attack by the lone pair of
nitrogen of aniline to the C=C of the 2-butenone. This
nucleophilic attack step will be facilitated by Glu230,
which acts as a base, by accepting the proton from –
NH2 group of aniline. The intermediate (Int) generated
in this step will have higher electron density on the O-
atom of the carbonyl group of 2-butenone, which is
stabilized by the strong H-bonding with the Asn295.
In the subsequent step the product (P) will be formed
by rearrangement of electrons and transfer of proton
from Glu230. The transfer of proton can be assisted by
water molecule which is used as solvent as shown in
Scheme 3. The verification of the proposed
mechanism using Quantum Mechanical/ Molecular
Mechanical (QM/MM) approach would give us more
insights in terms of energetics but that would be a
study on its own and will be done in future.
Experimental Section
General information: All reagents and solvents were
purchased from commercial sources and used without
purification. The sterile distilled water was used for the
preparation of all aqueous solutions. The α-amylase from
Aspergillus oryzae (powder, ~30 U/mg) and other enzymes
were bought from Sigma-Aldrich. The NMR spectra were
recorded on a Jeol-400 MHz or Bruker-400 MHz
spectrometer in deuterated solvents with TMS as internal
reference (chemical shifts δ in ppm, coupling constant J in
Hz.). Multiplicities are reported as follows: singlet (s),
doublet (d), triplet (t), multiplet (m), and broad singlet (brs).
The reaction progress was monitored by thin layer
chromatography (TLC) on pre-coated silica gel plates.
General Procedure for the α-amylase catalyzed aza-
Michael Addition: To a 10 mL glass tube equipped with a
megnetic stirrer bar added 1.5 mL of α-amylase (1 mg/mL
in H2O) and 0.3 mL of H2O. Afterward, added aromatic
amines (100 mg, 1 equiv.) and methyl vinyl ketone (1.2
equiv.) after dissolving in 200 µL of DMSO and stirred the
reaction mixture at 40 oC for overnight. After completion of
the reaction as indicated by TLC, the resulting mixture was
filtered through a small pad of celite and washed the celite
two times with ethyl acetate. Further, the organic layer from
filterate was extracted using ethyl acetate and dried over
sodium sulfate. The volatiles were evaporated under
reduced pressure and the residue was purified by column
Scheme 3: Proposed mechanism for aza-Michael addition
of aniline to 2-butenone using α-amylase.
To get more evidence about the role of Glu230 in the
catalysis of aza-Michael addition, we have started a
control experiment in the presence of starch which has
been known as a natural substrate of α-amylase and
Glu230 plays a role in the hydrolysis of this.[26] The
results showed that the addition of starch decreased the
yield of the reaction from 88% to 50.9% (Scheme S1
& Figure S7) due to the competition reaction between
starch and aza-Michael addition substrates, this
indicates that Glu230 plays a role in the catalysis of
aza-Michael addition reaction of aromatic amines with
enones. However, further investigation such as
mutagenesis of active site are required to confirm the
role of Glu230 in the catalysis.
Conclusion
In summary, we have developed the first example of
biocatalytic aza-Michael addition of less nucleophilic
7
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10.1002/adsc.201901254
Advanced Synthesis & Catalysis
up research grant (SRG/2019/001461) for providing financial
support. NG is thankful to Mr. Kundlik Gadhave (PhD scholar at
IIT Mandi) for collection of TEM data and acknowledges to IIT
Mandi (BioX center and AMRC) for providing experimental
facilities and Ramanujan SERB grant, India (SB/S2/RJN-
072/2015).
chromatography on silica gel (eluent: hexane/ EtOAc)
affording the corresponding products 3a-3l in very good
yields.
Procedure for the α-amylase@CuNPs catalyzed aza-
Michael addition: To a 10 mL glass tube equipped with a
megnetic stirrer bar added 1.5 mL of hybrid catalyst α-
amylase@CuNPs (1.0 mg/ml in H2O) and 0.3 mL of H2O at
room temperature. After that, added 2-bromoaniline (0.58
mmol, 1 equiv.) and methyl vinyl ketones (0.69 mmol, 1.2
equiv.) after dissolving in 200 µL of DMSO and stirred the
reaction mixture at 40oC for overnight. After completion of
the reaction as indicated by TLC, the resulting mixtures was
transferred to the 10 mL tube and centrifuge for 10 min. @
10,000 RPM, the hybrid catalyst precipitate as a pellet
which was reused for further catalytic cycle and the
supernatant was used to extract crude product using ethyl
acetate affording the corresponding product (3h) in 91 %
conversion yield. To run the reusability experiment,
dissolved the pellets obtained from previous cycles into 1.8
mL of H2O and then followed the same prcodure.
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Acknowledgements
The financial support from Department of Science and Technology,
India (DST/INSPIRE/04/2017/000095) is extremely acknowledged.
VT and SD acknowledge to Dr. Amjad Ali (Head, School of
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FULL PAPER
Biocatalytic aza-Michael addition of aromatic
amines to enone using α-amylase in water
H O
2
Adv. Synth. Catal. 2019, Volume, Page – Page
α-amylase
Sunil Dutt,a Vanshita Goel,a Neha Garg,b Diptiman
Choudhury,a Dibyendu Mallickc and Vikas Tyagi*a
11
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