First biocatalytic Groebke-Blackburn-Bienaymé reaction to synthesize imidazo[1,2-a]pyridine derivatives using lipase enzyme

Article abstract of DOI:10.1016/j.tet.2020.131643

In this study, first biocatalytic synthesis of clinically important imidazo[1,2- a]pyridine based compounds has been achieved. The Candida antarctica lipase B (CALB) enzyme was found suitable to catalyze the Groebke-Blackburn-Bienaymé (GBB) multicomponent

Full text of DOI:10.1016/j.tet.2020.131643

Journal Pre-proof
First biocatalytic Groebke-Blackburn-Bienaymé reaction to synthesize imidazo[1,2-
a]pyridine derivatives using lipase enzyme
Meenakshi Budhiraja, Rajesh Kondabala, Amjad Ali, Vikas Tyagi
PII:
S0040-4020(20)30850-4
https://doi.org/10.1016/j.tet.2020.131643
TET 131643
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 26 April 2020
Revised Date: 27 August 2020
Accepted Date: 26 September 2020
Please cite this article as: Budhiraja M, Kondabala R, Ali A, Tyagi V, First biocatalytic Groebke-
Blackburn-Bienaymé reaction to synthesize imidazo[1,2-a]pyridine derivatives using lipase enzyme,
Tetrahedron, https://doi.org/10.1016/j.tet.2020.131643.
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First biocatalytic Groebke-Blackburn-Bienaymé reaction to synthesize
imidazo[1,2-a]pyridine derivatives using lipase enzyme
Meenakshi Budhiraja, Rajesh Kondabala, Amjad Ali* and Vikas Tyagi*
School of Chemistry and Biochemistry, Thapar Institute of Engineering and Technology,
Patiala-147004, Punjab, India
Email: vikas.tyagi@thapar.edu, amjadali@thapar.edu
Abstract: In this study, first biocatalytic synthesis of clinically important imidazo[1,2-
a]pyridine based compounds has been achieved. The Candida antarctica lipase B (CALB)
enzyme was found suitable to catalyze the Groebke-Blackburn-Bienaymé (GBB)
multicomponent reaction of substituted 2-aminopyridine, aryl benzaldehyde and
isocyanides to synthesize imidazo[1,2-a]pyridine derivatives in very good yields. Further,
CALB enzyme was immobilized on mesoporous silica and characterized using FT-IR,
XRD, SEM-EDS and HR-TEM to use as a reusable catalyst in this transformation. The
immobilized catalyst CALB@SiO displayed high catalytic efficiency up-to many cycles.
2
In addition, preliminary mechanistic studies such as molecular docking and molecular
dynamics (MD) simulation were performed which suggested that Thr40 and Ser105
residues are playing an important role in catalyzing the GBB-reaction.
Key
words:
Imidazo[1,2-a]pyridine,
Groebke-Blackburn-Bienaymé,
lipase,
immobilization, SiO molecular dynamics
2
,
Introduction: Imidazo[1,2-a]pyridine is a valuable framework found in a number of
nitrogen-bridgehead fused heterocycles having diverse biological activities such as
[
1]
antiviral, antifungal, antiparasitic, anti-inflammatory, and anti-cancer. Also, a number of
drugs containing imidazo[1,2-a]pyridine framework have been commercialized such as
hypnotic drug zolpidem, the anxiolytic drugs alpidem, saridipem or nicopedem, the
antiulcer agent zolmidine and PDE-3 inhibitor drug olprione for the treatment of heart and
[
2]
circulatory failures (Figure 1). Besides that, they have various applications in material
[
3]
and organometallic chemistry. The most common method for the synthesis of
imidazo[1,2- a]pyridine at the laboratory as well as at industrial level is the condensation
[3c, 4]
reaction of 2- aminopyridines with mono-α-halogenocarbonyl compounds.
However,
this method is not very suitable for the synthesis of diverse imidazo[1,2-a]pyridine
derivatives due to the shortage of commercially available monohalogenated compounds.
To overcome the shortcomings of conventional method a number of synthetic strategies
[
5]
have been developed over the years (Scheme 1). In this context, Groebke-Blackburn-
Bienaymé (GBB) reaction, which takes place between an aldehyde, 2-aminoazine, and an
isocyanide to synthesize imidazo[1,2-a]pyridine framework remains the most efficient and
[
6]
common method (Scheme 1f). In the last decades, numerous catalyst including Brønsted
acids, Lewis acids, solid-supported acids, organic bases and ionic liquids have been
[
7]
reported to catalyze this reaction. Besides, Song et al. reported microwave assisted
[
8]
GBB-multicomponent reaction for the synthesis of benzimidazole derivatives.
Recently, Gámez-Montaño’s group reported green synthesis of
carbazolylimidazo[1,2-a]pyridines via GBB- reaction using ultrasound.
[9]
1

On the other hand, the use of biomolecules to catalyze non-natural organic reactions
at laboratory as well as industrial level have seen exponential growth in last few
[10]
years.
Further, lipase enzymes have grabbed lot of attention to catalyze new to
nature organic reactions like multicomponent reactions due to their high operational
[11]
stability in the reaction conditions and easy availability in the market.
In this
context, Berlozecki and co-workers reported a lipase catalyzed Ugi-reaction to
synthesize dipeptides and this method had numerous advantages over the previous
[
12]
procedures.
Further, Wu et al. reported a lipase catalyzed five component
synthesis of spirooxazino derivatives using olefins, aldehydes, acetamides and
[
13]
cyclohexanone.
This group further reported the synthesis of spiroheterocycles
[14]
using isatin, 1,3-cyclohexanedione and pyrazoles.
Still, there is no biocatalyst
reported to catalyze the GBB-multicomponent reaction to synthesize imidazo[1,2-
a]pyridine framework. Encouraged by the biological importance of imidazo[1,2-
[15]
a]pyridines and our recent work in the area of biocatalysis herein, we are reporting
first biocatalytic Groebke-Blackburn-Bienaymé multicomponent reaction to
synthesize imidazo[1,2- a]pyridine derivatives using lipase enzyme as a catalyst.
Figure 1. Examples of imidazo[1,2-a]pyridine framework containing drugs
2

Scheme 1. Representative approaches for the synthesis of imidazo[1,2-a]pyridines
framework
Results and Discussion
We started our investigation by screening of various lipase enzymes to catalyze the
GBB-multicomponent reaction of model substrates 2-aminopyridine, benzaldehyde and
tert-butyl isocyanide (Table 1). Interestingly, the model reaction gave product (4a) in
29% yield in the absence of any catalyst (entry 1, Table 1), however, addition of
different lipases increased the yield of the reaction significantly (entries 2-5, Table 1).
Among different lipase enzymes, Candida antarctica lipase B (CALB) and Aspergillus
niger lipase were found best and provided product (4a) in 63% and 64% isolated yield
respectively (entry 3-4, Table 1). Although, we choose CALB enzyme for further
investigation in this study due to low cost and easy availability of CALB in comparison
of Aspergillus niger lipase enzyme.
3

a
Table 1. Screening of different lipases to catalyse the reaction of model substrates
b
entry
lipases
% yield
29
1
2
3
4
5
No enzyme
Rhizomucor mehei lipase (RML)
Candida antarctica lipase B (CALB)
Aspergillus niger lipase
Candida rugosa lipase
40
63
64
50
a
Reaction conditions: 2-aminopyridine (1a) (50 mg, 0.563 mmol), benzaldehyde (2a) (67.6 mg, 0.637 mmol,
1.2equiv.), tert-butyl isocyanide(3a) (72.5 mg, 0.637 mmol, 1.2 equiv.) and enzyme (20 mg) in 2 ml of ethanol
were taken in a glass tube with a teflon cap and stirred the reaction mixture at room temperature for overnight,
b
isolated yield.
Next, we tested different solvents along with the combination of solvents to improve
the yield of the model reaction (entries 1-10, Table 2). In case of H O and DMSO the
2
product (4a) formed in trace amount only however, no product formation was
observed in the case of DMF, THF and hexane (entries 1-5, Table 2). Further, reaction
gave product (4a) in 62% yield when MeOH was used as a solvent (entry 6, Table 2).
When we used phosphate buffer (pH=7) as a solvent, product (4a) was obtained in
4
8% yield (entry 10, Table 2). Besides, reaction gave no product when we used the
mixture of water-ethanol (1:1 v/v) or water-DMSO (1:1 v/v) as a solvent (entries 7 &
, Table 2). As a result, ethanol remained best solvent to attain the maximum
9
conversion in the model reaction (entry 8, Table 2).
Table 2. Screening of different solvents for CALB catalyze the reaction of model
a
substrates
b
entry
solvent
%yield
trace
trace
NR
1
2
3
4
5
6
7
8
9
H O
2
DMSO
DMF
THF
Hexane
Methanol
NR
NR
62%
NR
63%
NR
EtOH + H O (1:1 v/v)
2
Ethanol
DMSO + H O (1:1 v/v)
2
10
Phosphate buffer
48%
a
Reaction conditions: 2-aminopyridine (1a) (50 mg, 0.563 mmol) , benzaldehyde (2a) (67.6 mg, 0.637 mmol,
.2equiv.), tert-butyl isocyanide(3a) (72.5 mg, 0.637 mmol, 1.2 equiv.) and enzyme (20 mg) in 2 ml of
1
solvent were taken in a glass tube with a teflon cap and stirred the reaction mixture at room temperature for
b
overnight, isolated yield.
After choosing best biocatalyst (CALB) and solvent (EtOH), we investigated the effect
4

of the enzyme loading and substrate ratio on the conversion of the model reaction. As
shown in table 3, when loading of enzyme was decreased from 20 mg to 10 mg, the
reaction gave product (4a) in slightly lower yield (entries 1-2, Table 3). Next, we
increased the enzyme loading up-to 50 mg, however, maximum yield was observed in
the case of 40 mg of CALB enzyme (entries 2-5, Table 3). Further, the substrates
ratios were changed from 1:1.2:1.2 to 1:1:1 for model substrates, however, 1:1.2:1.2
remained the best substrate ratio in comparison to 1a:2a:3a to obtain the highest
conversion (entries 4 & 6, Table 3). After having the optimized reaction conditions in
hand, the effect of different substitutions including electron withdrawing and donating
groups on the aromatic ring of 2-aminopyridines and benzaldehydes was explored
(
Table 4).The reaction of unsubstituted 2-aminopyridine, benzaldehyde and tert-butyl
isocyanide furnished the product (4a) in 91% yield under the optimized reaction
conditions (entry 1, Table 4).Subsequently, we examined the effect of different
substitutions such as NO , -Cl, -Br and -OMe on the ortho- or para-position of
2
benzaldehyde.
a
Table 3. Optimization of the ratio of substrates and enzyme loading
b
entry
enzyme
loading
10
ratio of
1a:2a:3a
1:1.2:1.2
1:1.2:1.2
1:1.2:1.2
1:1.2:1.2
1:1.2:1.2
1:1:1
%yield
(4a)
55
1
2
3
4
5
6
20
63
30
80
40
91
50
80
40
58
a
Reaction conditions: 2-aminopyridine (1a) (50 mg, 0.563 mmol) , benzaldehyde (2a) (67.6 mg, 0.637 mmol,
1
.2equiv.), tert-butyl isocyanide(3a) (72.5 mg, 0.637 mmol, 1.2equiv.) and enzyme in 2 ml of ethanol were
taken in a glass tube with a teflon cap and stirred the reaction mixture at room temperature for overnight,
b
isolated yield.
Unexpectedly, in case of strong electron withdrawing group such as NO , reaction
2
gave product (4b) only in 55% yield (entry 2, Table 4), however, good yield was
observed in case of halides such as –Br, -Cl at the ortho-position of benzaldehyde
(
entries 3,4, Table 4). Moreover, when benzaldehyde having donating group such as
4
6
- OMe and 4-Me, reaction gave product (4e) and (4f) in slightly inferior yield i.e
1% and 63% respectively (entries 5 and 6, Table 4). Next, we investigated the
effect of different substitutions such as –CH , -Br and -NO at different positions of
3
2
2
-amino pyridine. In case of 4-CH substituted 2-aminopyridine, reaction gave
3
products (4g) in 80% yield (entries 7, Table 4), whereas, in case of 5-bromo
substitution reaction provided product (4h) in slightly inferior yield (entry 8, Table
4
). Further, we observed no product formation in the case of 5-NO at the aromatic
2
ring of 2-aminopyridine and it might be due to the strong decrement in the
5

nucleophilicity of 2-aminopyridine (entry 9, Table 4). Interestingly, when tert-butyl
isocyanide was replaced with cyclohexyl isocyanide, a slightly lower yield of
product (4i) was observed (entry 10, Table 4). Surprisingly, on taking –Br
substitution on pyridine as well as on benzaldehyde no product formation was
observed (entry 11, Table 4).
a
Table 4. Scope of substrates for the CALB-catalyzed GBB-reaction
b
entry
Pyridine (1)
Aldehyde (2)
Isocyanide (3)
Product (4), yield
1
1
a
3a
3a
2
2
a
4
a, 91%
b, 55%
2
3
4
1a
1a
1a
b
4
3a
3a
2
c
4
c, 72%
2
d
4
d, 70%
5
6
1a
1a
3a
3a
4
e,61%
2
e
4
f, 62%
2
f
7
2a
3a
4
g, 80%
8
9
2a
2a
2a
3a
3a
4
h, 70.4%
NR
O2N
N
NH2
1
0
1
a
3
b
4
i, , 80%
6

1
1
3a
NR
a
Reaction conditions: 2-aminopyridine (1 equiv.), benzaldehyde
(
1.2equiv.), isocyanide(1.2equiv.) and
enzyme (40 mg) in 2 ml of ethanol were taken in a glass tube with a teflon cap and stirred the reaction
b
mixture at room temperature for overnight, isolated yield.
In the next phase of our endeavor, we envisioned to reuse CALB enzyme after
completion of first catalytic cycle, however, it could not be possible with pure
enzyme. Previously, immobilization technique of confining naturally existing free
enzyme on
[15]
/
in different support has been useful to make enzyme reusable. There are plenty of
ways for immobilization of enzyme for example covalent binding, H-bonding,
[17]
adsorption, entrapment, cross linking etc.
In present work, we have used
entrapment method for immobilizing CALB on silica particles to make it reusable
under the optimized reaction conditions which could not be possible in the case of
pure CALB enzyme (The details of synthetic procedure, characterization and catalyst
loading optimization is given in supporting information). Next, we tested
CALB@SiO for the reusability using the GBB-multicomponent reaction of 2-
2
aminopyridine, benzaldehyde and tert-butyl isocyanide and compiled the results in
Figure 2. These results revealed that the immobilized enzyme i.e. CALB@SiO was
2
reusable up-to many catalytic cycles with high catalytic efficiency.
Figure 2: Reusability test of CALB@SiO2 for the reaction of 2-aminopyridine,
benzaldehyde and tert-butyl isocyanide
a
Reaction conditions: 2-aminopyridine (1a) (100 mg, 1.06mmol), benzaldehyde (2a) (135.2 mg, 1.27mmol,
1
.2equiv.), tert-butyl isocyanide(3a) (145 mg, 1.27 mmol,1.2equiv.) and CALB@SiO2 (50 mg) in 2 ml of
ethanol were taken in a glass tube with a teflon cap and stirred the reaction mixture at room temperature for
b
overnight, isolated yield.
Further, the synthetic utility of CALB catalyzed GBB-multicomponent reaction was
explored by increasing the concentration of model substrates up-to seven folds such as
2-amino pyridine (0.35 g, 3.71 mmol, 1 equiv.), benzaldehyde (0.45 g, 4.46 mmol,1.2
7

equiv.) and tert-butyl isocyanide (0.51 g, 4.45 mmol,1.2 equiv.) under the optimized
reaction conditions. The successful isolation of 0.903 g of product (4a) in 91.6% yield
from this reaction proved the synthetic utility and opens the door for the synthesis of
various drug precursors using lipase catalyzed GBB-multicomponent reaction.
Active site prediction and proposed mechanism: The molecular docking approach
was carried out for the prediction of model substrates orientation inside the active site
of the enzyme and to investigate the role of the active site residues in catalyzing the
Groebke-Blackburn-Bienaymé reaction. Previous studies have been revealed that
residues Thr40, Ser105, Gln106, Asp187, and His224 constitute the active site of
[
18]
Candida antarctica lipase B (CALB) enzyme. Hence, we started our molecular
docking studies with the X-ray crystal structure of CALB enzyme (PDB.ID: 4K6G).
[
19]
The GLIDE tool of Schrodinger software with the SP method was used to find the
positioning of 2-aminopyridine (1a) and benzaldehyde (2a) substrates inside the
active site of the enzyme (Figure 3a). Further, the general reaction mechanism of
GBB- reaction reported previously in literature suggested that 2-aminopyridine (1a)
and benzaldehyde (2a) substrates reacts under the catalytic conditions to form imine
intermediate (i) which further gives addition reaction with tert-butyl isocyanide to
produce subsequent intermediate (ii) which furnished the final product via [4+1]
[7f, 7i, 7j]
cycloaddition reaction followed by 1,3-proton transfer (Scheme S1).
Encouraged by previous reports, intermediates such as (Z)-N-benzylidenepyridin-2-
aminium (i) formed by the reaction of 2-aminopyridine (1a) and benzaldehyde (2a)
substrates and 2-methyl-N-(2-phenyl-2-(pyridin-2ylamino)ethylidyne) propan-2-
aminium (ii) formed by the addition of tert-butyl isocyanide on imine intermediate
during the GBB-reaction were also docked in the binding site of the protein using the
aforementioned method (Figure 3b & 3d). Further, the Molecular Dynamics (MD)
simulations were performed on the initially docked complexes of the enzyme with
substrates and intermediates for predicting the preferable orientation using GPU
[
20]
based DESMOND software.
The enzyme complex with substrates and intermediates were prepared by neutralizing
the system through adding ions, salt and the SPC solvent. The prepared system was
[21]
minimized with a 10ns time scale using OPLS2005 force field. Later relaxation
procedure NVT and NPT, each with 10ns time scale were carried out on the
minimized system. Finally, the MD simulations were performed with 50ns time
scale. (The detailed procedure of molecular docking and MD simulations has
discussed in the supporting information). From the computational studies, it was
observed that the 2-aminopyridine substrate forming a hydrogen bond with Thr40
(
2.51 Å) and Ser105 (2.59 Å) residues as a result getting a better orientation and
increasing nucleophilicity to react as a nucleophile in the protein environment
Figure 3a). The benzaldehyde substrate interacts with Ser105 (1.61 Å) residue
(
through a strong hydrogen bond and became a better electron accepter (Figure 3a)
whereas tert-butyl isocyanide did not show any hydrogen bonding (Figure 3c),
8

however, there is a possibility of forming π- cation interaction, which has a long bond
length than the threshold, therefore ignored. Interestingly, we found that the imine
intermediate (i) showed strong interaction with Thr40 (2.52 Å) and Ser105 (2.59 Å)
residues so becoming a good electrophile for the addition of tert-butyl isocyanide
(
(
Figure 3b). Further, the addition of tert- butyl isocyanide on the imine intermediate
i) altered the orientation of intermediate (ii), as a result, no interaction remained
between intermediate (ii) and Ser105 residue (Figure 3d). Based on these results, we
proposed a mechanism for the CALB catalyzed GBB- reaction (Scheme 3). The first
step is formation of imine intermediate (i) by the reaction of 2-aminopyridine and
benzaldehyde which was facilitated via Ser105 residue through a strong hydrogen
bond with carbonyl oxygen and nitrogen of 2- aminopyridine as a result making the
benzaldehyde a better electrophile and increasing
Figure 3: Orientation and interactions of substrates and intermediates in the binding site
of CALB enzyme (a) 2-aminopyridine & benzaldehyde substrates (b) imine intermediate
(i) formed by the reaction of 2-aminopyridine & benzaldehyde (c) Orientation of
isocyanide (d) 2-methyl-N-(2-phenyl-2-(pyridinylamino)ethylidyne)propan-2-aminium (ii)
formed by the addition of tert-butyl isocyanide on imine intermediate.
9

the nucleophilicity of 2-aminopyridine in protein environment. In the next step imine
intermediate (i) was attacked by tert-butyl isocyanide, this nucleophilic addition was
also assisted through Ser105 residues by making a strong hydrogen bond with the
imine nitrogen. The subsequent intermediate (ii) did not show any interaction with
the residues in the active site and provides the final product (4a) via [4+1]
cycloaddition followed by 1, 3-proton transfer. Moreover, the formation of final
product (4a) from intermediate (ii) may take place either in the active site of the
enzyme or outside of the active site.
Scheme 3: Plausible mechanism of CALB-catalyzed GBB-multicomponent reaction
To get more evidence about the role of Thr40 and Ser105 residues in this
transformation, we also investigated the substrates 5-bromo-2-aminopyridine and 2-
bromobenzaldehyde (Figure 4a) those did not show any reaction in previous
experiment under the optimized reaction conditions (entry 12, Table 4). We also
docked the imine intermediate formed by the reaction of these substrates (Figure 4b).
Interestingly, the imine intermediate formed by the reaction of 5-bromo-2-
aminopyridine and 2-bromobenzaldehyde substrates did not show any interactions
with Thr40 sand Ser105 residues as a result no GBB-reaction was occurred (Figure
4
b). This gave evidence that Thr40 and Ser105 residues play a crucial role in the
activation of imine intermediate to facilitate the addition of isocyanide.
1
0

Figure 4. Orientation and interactions of substrates and intermediates in the binding site of
CALB enzyme (a) Orientation of 5-bromo-2-aminopyridine and 2-bromobenzaldehyde
(b)orientation of imine intermediate formed by the reaction of 5-bromo-2-aminopyridine
&
2- bromo benzaldehyde.
Conclusion
In summary, we have developed first biocatalytic Groebke-Blackburn-Bienaymé
(
GBB) multicomponent reaction to synthesize fused
imidazo[1,2-a]pyridine
derivatives using Candida antarctica lipase B (CALB) enzyme. Also, various
substitutions were tolerated well during the enzymatic synthesis and provided
imidazo[1,2-a]pyridine derivatives in good to excellent yield. Further, CALB enzyme
was immobilized on mesoporous silica and used as reusable catalyst which displayed
high catalytic efficiency up-to many cycles. A preliminary mechanistic studies
including molecular docking and molecular dynamics (MD) simulation revealed that
Thr40 and Ser105 residues played a crucial role in catalyzing the GBB-
multicomponent reaction, however further studies such as Quantum Mechanical/
Molecular Mechanical (QM/MM) approach to get more insights about the mechanism
of this transformation are under progress in our lab. Finally, this work contributes to
expand the number of enzymatic transformations to synthesize clinically important
heterocycles.
Experimental Section
General information: All lipases, chemicals and solvents used in this project were
commercially purchased and used without extra purification. Lipases (EC 3.1.1.3) like
Candida antarctica lipase B (bought from Sigma-Aldrich, product number: L3170),
Rhizomucor miehei, Aspergillus niger, Candida rugosa, APTES (3-aminopropyl
triethoxysilane) and TEOS (tetraethoxyorthosilicate) were purchased from various
suppliers. The reaction progress was monitored using thin layer chromatography
(
TLC: thin silica layer coated on glass slide). The compounds were purified by column
chromatography using silica of mesh size 60-120 and ethyl acetate in hexane as
mobile phase. A JEOL NMR spectrometer was used to study proton and carbon NMR
spectrum of products at frequency 400 and 100 MHz using CDCl with TMS as
3
internal reference. The Coupling constant (J) is expressed in Hertz (Hz) and Chemical
shift (δ) is expressed in parts per million (ppm). Multiplicities are abbreviated as s:
singlet, d: doublet, dd: doublet of doublet, t: triplet, brs: broad singlet. The scanning
electron microscopy- electron diffraction X-ray analysis (SEM-EDS) of catalyst was
done using JEOL microscope. The X-ray analysis was done using X-ray
Diffractometer (PanAlytical). The Fourier transform infrared (FT-IR) spectrum was
collected using Perkin Elmer Spectrum version 10.4.00. High resolution Transmission
electron Microscope (HR-TEM) was done using JEOL microscope.
General procedure for the CALB-catalyzed GBB-reaction:
In a glass tube having a teflon cap and a stirrer bar added 2-aminopyridine (1a) (50
mg, 0.531 mmol, 1.0 equiv.), benzaldehyde (2a) (67.61 mg, 0.637 mmol, 1.2 equiv.)
11

and tert-butyl isocyanide (3a) (72.5 mg, 0.637 mmol, 1.2 equiv.) in 2 ml of ethanol as
a solvent. Thereafter, 40 mg of enzyme (CALB) was added and the reaction mixture
was stirred on a magnetic stirrer at room temperature for overnight and the progress of
reaction was monitored using TLC. After completion of the reaction as indicated by
TLC, the resulting mixture was filtered through a small pad of cellite followed by
washing of the cellite pad with ethyl acetate (2x5 ml). The solvent was evaporated
under reduced pressure and the residue was purified by column chromatography using
ethyl acetate in hexane as eluents affording the corresponding products 4a-4i in 55-
91% yields.
Procedure for the CALB@SiO catalyzed GBB-reaction:
2
In a glass tube having a teflon cap and a stirrer bar added 2-aminopyridine (1a) (100
mg, 1.06 mmol, 1.0 equiv.), benzaldehyde (2a) (129.7 mg, 1.27 mmol, 1.2 equiv.) and
tert-butyl isocyanide (3a) (144.8 mg, 1.27 mmol, 1.2 equiv.) in 2 ml of ethanol as a
solvent. Thereafter, 50 mg of CALB@SiO was added and the reaction mixture was
2
stirred on a magnetic stirrer at room temperature for overnight and the progress of
reaction was monitored using TLC. After completion of the reaction as indicated by
TLC, the resulting mixtures was transferred to the 10 ml tube and centrifuge for 20
min. @ 10,000 RPM, the CALB@SiO catalyst precipitate as a pellet which was
2
reused for further catalytic cycle and the supernatant was used to extract crude product
using ethyl acetate. Next, the solvent was evaporated under reduced pressure and the
residue was purified by column chromatography on silica gel (eluent: hexane/ EtOAc)
affording the corresponding product 4a in 90 % conversion yield (same procedure was
followed for reusability experiments).
Acknowledgements
The financial support from Department of Science
and
Technology,
India
(
DST/INSPIRE/04/2017/000095) is extremely acknowledged.
Conflicts of interest:
The authors declare no conflicts.
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16

Highlights:
‹
‹
‹
‹
First biocatalytic synthesis of clinically important imidazo[1,2-a]pyridine
CALB catalyzed Groebke-Blackburn-Bienaymé multicomponent reaction
Immobilization of CALB@SiO as reusable catalyst
2
Molecular docking and molecular dynamics (MD) simulation studies suggested Thr40
and Ser105 residues play an important role in catalysis.

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