A spectroscopic insight into the interaction of chromene 1,2,4-oxadiazole-based compounds with bovine serum albumin

Article abstract of DOI:10.1007/s11164-020-04323-4

Four synthesized 2H-chromene-based 1,2,4-oxadiazole compounds (6a-6d) were studied for binding with bovine serum albumin (BSA) by different spectroscopic and thermodynamic analyses. A molecular docking program was used to find out the possible binding sites and binding affinity of 2H-chromene based 1,2,4-oxadiazole compounds with bovine serum albumin (BSA). The intrinsic fluorescence of BSA was quenched by these compounds through static quenching mechanism, and the estimated binding constant (K_b) value was found to be 1.5 × 10³–10 × 10³. The conformational changes of BSA were monitored by circular dichroism analysis, and it was observed that BSA is structurally stable in the presence of these compounds. The thermodynamic results indicated that the interaction process is spontaneous and the binding between oxadiazole compounds and BSA is mainly driven by hydrogen bonding and van der Waals forces. This study would be helpful to understand the biological benefits of oxadiazole-based compounds as well as the nature of interactions with biomolecules. Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s11164-020-04323-4

Research on Chemical Intermediates
https://doi.org/10.1007/s11164-020-04323-4
A spectroscopic insight into the interaction of chromene
1,2,4‑oxadiazole‑based compounds with bovine serum
albumin
1
1
1
Nilima Priyadarsini Mishra · Lakoji Satish · Seetaram Mohapatra ·
1
2
Sabita Nayak · Harekrushna Sahoo
Received: 6 August 2020 / Accepted: 9 November 2020
Springer Nature B.V. 2020
©
Abstract
Four synthesized 2H-chromene-based 1,2,4-oxadiazole compounds (6a-6d) were
studied for binding with bovine serum albumin (BSA) by diꢀerent spectroscopic
and thermodynamic analyses. A molecular docking program was used to ꢁnd out
the possible binding sites and binding aꢂnity of 2H-chromene based 1,2,4-oxadia-
zole compounds with bovine serum albumin (BSA). The intrinsic ꢃuorescence of
BSA was quenched by these compounds through static quenching mechanism, and
3
3
the estimated binding constant (K ) value was found to be 1.5×10 –10×10 . The
b
conformational changes of BSA were monitored by circular dichroism analysis, and
it was observed that BSA is structurally stable in the presence of these compounds.
The thermodynamic results indicated that the interaction process is spontaneous and
the binding between oxadiazole compounds and BSA is mainly driven by hydrogen
bonding and van der Waals forces. This study would be helpful to understand the
biological beneꢁts of oxadiazole-based compounds as well as the nature of interac-
tions with biomolecules.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s1116
4
-020-04323-4) contains supplementary material, which is available to authorized users.
*
Seetaram Mohapatra
seetaram.mohapatra@gmail.com
1
2
Department of Chemistry, Ravenshaw University, Cuttack 753003, India
Department of Chemistry, NIT Rourkela, Rourkela 769008, India
Vol.:(012
1
3456789)
3

N. P. Mishra et al.
Graphic abstract
Keywords 1,2,4-oxadiazole · Microwave irradiation · Bovine serum albumin ·
Molecular docking · Circular dichroism
Introduction
Heterocyclic molecules implanted with nitrogen and oxygen atoms have been used
in diverse biological and pharmacological applications [1]. Among diꢀerent types of
nitrogen- and oxygen-containing heterocyclic compounds, ꢁve-membered heterocy-
clic compounds including azole, thiazole, oxadiazole, triazene, imidazole, purines,
pyrazole, etc., play a very important role in human life due to their medicinal action
against several diseases [2–9]. Speciꢁcally, oxadiazole-based compounds were found
to exhibit anti-inꢃammatory, antimicrobial, antitubercular, antidepressant, antima-
larial, anti-HIV and anticancer properties [10–19]. Due to their biological activities,
synthesis of novel oxadiazole compounds is the current research area of interest to
produce analgesics, antispasmodics and anti-inꢃammatories. Recently, our research
group reported microwave-assisted synthesis of chromene-based 1,2,4-oxadiazole
molecules having potent antibacterial activity [20]. Also, as reported earlier, these
oxadiazole nuclei contribute signiꢁcant bioactivity due to their lipophilic nature and
have ability to bind with the target protein through hydrogen bonding [21].
Protein molecules play a crucial role in eꢀective action of several drugs in liv-
ing beings [22]. The pharmacodynamics and pharmacokinetics of drugs generally
depend on the binding of these molecules with albumin proteins [23–25]. There-
fore, studying the binding mechanism responsible for the molecular interactions
between drug molecules and albumin could be helpful in designing new bioactive
1
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A spectroscopic insight into the interaction of chromene…
molecules. We selected bovine serum albumin (BSA) as a model protein for this
study as it is largely present in the blood plasma and plays a vital role in the
transportation of several endogenous and exogenous ligands (such as fatty acids,
amino acids, steroids, and various drugs) [26]. Also, BSA is structurally similar
(
around 76%) to human serum albumin (HSA). It consists of 583 amino acid resi-
dues with a molecular weight of 66 kDa. BSA structure can be divided into three
domains (I, II and III), and each domain further contains two subdomains (A and
B).
There were only few reports on interaction studies of oxadiazole compounds
with biomolecules that are described here. Laskar, K et al. reported the synthesis
of 1,3,4-oxadiazole compounds of fatty acids and evaluated the binding interac-
tion of (Z)-(2)-(heptadec-8′-enyl)-5-methyl-1,3,4-oxadiazole with serum albu-
min using diꢀerent spectrometers [27]. In another study by Anouar, E H et al.
several indole-based oxadiazole compounds were synthesized and screened for
their inhibitory potential against β-glucuronidase [28]. Also, they used molecular
docking to ꢁnd the interactions between these compounds and the active binding
site. Santosh, R et al. investigated the docking of several oxadiazole compounds
with DNA and performed binding studies using UV–Vis spectrometer [29].
In this study, we designed and synthesized four diꢀerent 2H-chromene-based
1
,2,4-oxadiazole compounds (Fig. 1). The main aim of our work is to understand
the binding mechanism involved in the interaction between the synthesized oxa-
diazole compounds and BSA. For that, we opted a universal strategy combining
both experimental and molecular docking methods. Diꢀerent spectrophotomet-
ric methods were employed to understand molecular-level interactions and the
eꢀect of oxadiazole compounds on the structure and stability of BSA. Also, we
used AutoDock Vina program to ꢁnd out the possible binding sites as well as
the binding aꢂnity between the oxadiazole compounds and BSA. This combining
approach will be helpful to study several protein-drug systems.
Fig. 1 1,2,4-oxadiazole compounds synthesized in this work. a 3-(2H-chromen-3-yl)-5-phenyl-1,2,4-
oxadiazole, b 3-(7-methoxy-2H-chromen-3-yl)-5-phenyl-1,2,4-oxadiazole, c 3-(2H-chromen-3-yl)-5-(4-
methoxyphenyl)-1,2,4-oxadiazole and d 3-(7-methoxy-2H-chromen-3-yl)-5-(4-nitrophenyl)-1,2,4-oxadi-
azole
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N. P. Mishra et al.
Experimental
Materials
BSA (Product No.- A2153) was purchased from Sigma-Aldrich and was used with-
out any further puriꢁcation. All samples are prepared using PBS buꢀer (0.01 M).
Milli-Q water (18 M cm, Millipore, Bedford, MA) was used to prepare PBS buꢀer.
The 2H-chromene-based 1,2,4-oxadiazole compounds (6a-6d) were synthesized
in our laboratory and puriꢁed by recrystallization method [20]. For synthesizing
these 1,2,4-oxadiazole compounds, all reagents and solvents were purchased from
commercial suppliers. Salicylaldehyde, acrylonitrile, DABCO, Et N, hydroxy-
3
lamine hydrochloride, EDCI, HOBT and benzoic acids were purchased from
Sigma-Aldrich. The progress of the reaction was monitored by TLC on silica gel 60
F
-coated TLC plates (Merck KGaA, Darmstadt, Germany).
45
2
Methods
Molecular docking
AutoDock Vina 1.5.4 program was used for the blind molecular docking between
BSA and oxadiazole compounds. The crystal structure of BSA (PDB-3v03) and opti-
mized chemical structure of chromene-based 1,2,4-oxadiazole compounds prepared
by ChemDraw ultra 8 were used in the docking. Polar hydrogens and partial atomic
Kollman charges were assigned to BSA using the default settings in auto-dock tools.
The center of the grid and grid dimension was set at (90.398×28.894×23.482) Å
and (84×56×82) Å, respectively. Out of the nine conformations produced, the low-
est energy conformation was taken to analyze the results. Discovery Studio visual-
izer software was used for visualization of the binding site residues.
Sample preparation for interaction studies
A stock solution of oxadiazole compounds was prepared by dissolving the required
amount of the compound in PBS buꢀer containing 1% ethanol. BSA–oxadiazole
solutions for the spectroscopic studies were prepared by adding a ꢁxed amount of
stock oxadiazole solution to the protein solution to obtain ꢁnal desired concentration
of compound. (The details of oxadiazole compound and BSA concentrations were
given in the respective section.) After addition of the compound, the solutions were
sonicated followed by incubation at room temperature for 2 h to attain equilibrium.
Fluorescence spectral measurements
The ꢃuorescence measurements were carried out with a Horiba Jvon Spectrom-
eter (Fluoromax-4P). The ꢃuorescence was collected as a function of wavelength
1
3

A spectroscopic insight into the interaction of chromene…
in the range of 305–450 nm. Excitation and emission slit widths were ꢁxed at
5
nm for each. The protein concentration was ꢁxed at 2 µM, and oxadiazole com-
pounds concentration was varied from 0 to 100 µM.
The ꢃuorescence intensities are corrected for absorption of the exciting light
and/or reabsorption of the emitted light by the molecules present in the solution
to eliminate the inner ꢁlter eꢀect using the relationship
(
Aex+Aem)∕2
Icor = Iobs
e
where I and I are the corrected and observed ꢃuorescence intensities, respec-
cor
obs
tively. A and A are the absorption values of oxadiazole compounds at the excita-
ex
em
tion and emission wavelengths, respectively.
Absorption measurements
The absorption spectra were performed on Cary-100UV-Vis spectrophotometer,
and 1.0 cm quartz cells are used for the measurements. The concentration of BSA
was 2 µM, and oxadiazole compounds were varied from 0 to 100 µM.
Circular dichroism spectroscopy
The CD spectra were obtained using a JASCO spectropolarimeter equipped with
a thermostat-controlled cell holder. The protein concentration was 2 µM, and
oxadiazole concentration was varied from 0 to 100 µM. The far-UV region was
scanned between 200 and 260 nm with an average of three scans and a bandwidth
of 1 nm.
Scheme 1. Synthesis of 1,2,4-Oxadiazole-based compounds 6a, 6b, 6c, 6d
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N. P. Mishra et al.
Synthesis of 1,2,4‑oxadiazole‑based compounds
The synthesis of chromene-based 1,2,4-oxadiazole compounds 6(a-d) was
described in our previous report [20]. In Scheme 1, substituted chromene-3-car-
bonitriles 3(a-b) were prepared by following one-pot two-component method by
using salicylaldehyde 1(a-b) and acrylonitrile 2(a-b) in the presence of DABCO
as catalyst under solvent-free condition at room temperature in good to high yield.
After that, chromene-3-carbonitriles 3(a-b) were treated with hydroxylamine
hydrochloride using Et N in ethanol for 2 h at 40 °C to obtain N-hydroxy-2H
3
chromene-3-carboximidamide 4(a-b) in to excellent yield. For the synthesis of
chromene-based 1,2,4-oxadiazole compounds 6(a-d), N-hydroxy-2H chromene-
3
-carboximidamide 4(a-b) and substituted benzoic acid 5 reacted in the presence
of EDCI/HOBT in DMF under microwave irradiation at 80 °C, 30 wt for 20 min
to produce good to excellent yield. The product 6(a-d) was puriꢁed by using
column chromatography with 92%, 90%, 85%, 80% of yield, respectively. The
structures of the synthesized chromene-based 1,2,4-oxadiazole compounds 6(a-d)
1
13
were conꢁrmed by H NMR, C NMR and by mass spectroscopy.
3
‑(2H‑chromen‑3‑yl)‑5‑phenyl‑1,2,4‑oxadiazole (6a). Yellowish-white solid; (92%).
m.p. 118–120 °C; 1 H-NMR(400 MHz, CDCl 3 ) δ (ppm): 8.17 (d, J =8.0 Hz,
2
2
1
H, Ar-H), 7.63–7.52 (m, 4 H, Ar-H), 7.25–7.18 (m, 2 H, Ar-H), 6.94–6.87 (m,
H, Ar-H), 5.25 (s, 2 H, CH 2 ); 13 C-NMR (100 MHz, CDCl 3 ): δ 175.3, 166.4,
54.7, 133.0, 131.1, 129.2, 128.4, 128.3, 124.1, 121.9, 121.5, 119.5, 116.2, 64.6;
ESIHRMS ( m/z ): Anal . Calcd for C 17 H 12 N 2 O 2 [M+H] + 277.0899; found:
2
77.0998.
3
‑(7‑methoxy‑2H‑chromen‑3‑yl)‑5‑phenyl‑1,2,4‑oxadiazole(6b). White solid; (90%)
m.p. 203–205 °C; 1 H NMR(CDCl 3 , 400 MH Z ) δ (ppm): 8.17 (d, J =8.0 Hz,
2
H, Ar-H), 7.62–7.53 (m, 4 H, Ar-H), 7.11(d, J =8.0 Hz, 1 H, Ar-H), 6.51 (d,
J =8.0 Hz, 1 H, Ar-H), 6.47 (d, J =2.0 Hz,1 H, Ar-H), 5.23 (s, 2 H, CH 2 ), 3.81
s, 3 H, OMe); C 13 NMR (CDCl 3 , 100 MHz) δ (ppm): 175.0, 166.4,162.1, 156.1,
32.8, 129.2, 129.1, 128.2, 128.1, 124.1, 116.1, 114.7, 108.0, 101.6, 64.7, 55.4; ESI-
(
1
HRMS ( m/z ): Anal. Calcd for C 18 H 14 N 2 O 3 [M+H] + 307. 1004 found:
3
07.1075.
3
‑(2H‑chromen‑3‑yl)‑5‑(4‑methoxyphenyl)‑1,2,4‑oxadiazole(6c). White solid; (85%)
m.p. 128–130 °C; 1 H-NMR (400 MHz, CDCl3): δ 8.10 (d, J =8 Hz, 2 H, Ar-H),
7
.53 (s, 1 H, Ar-H), 7.25–7.16 (m, 2 H, Ar-H), 7.02–7.00 (m, 2 H, Ar-H), 6.93–6.86
(
m, 2 H, Ar-H), 5.25 (s, 2 H, CH 2 ), 3.90 (s, 3 H, OMe); 13 C NMR (100 MHz,
CDCl 3 ): δ 175.2, 166.2, 163.4, 154.7, 131.0, 130.2, 128.3, 128.0, 121.9, 121.5,
1
1
19.6, 116.3, 116.1, 114.6, 64.6, 55.6; ESI-HRMS ( m/z ): Anal . Calcd for C 18 H
4 N 2 O 3 [M+H] + 307.1004; found: 307.1093.
3
‑(7‑methoxy‑2H‑chromen‑3‑yl)‑5‑(4‑nitrophenyl)‑1,2,4‑oxadiazole(6d). Yellow
solid; (80%) m.p.140–142 °C, 1 H NMR (CDCl 3 , 400 MHz) δ (ppm): 8.42–8.36
1
3

A spectroscopic insight into the interaction of chromene…
(
m, 4 H, Ar-H), 7.55 (s, 1 H, Ar-H), 7.12 (d, J =8.0 Hz, 1 H, Ar-H), 6.52 (dd,
J =4 Hz, 8 Hz, 1 H, Ar-H), 6.46 (d, J =4 Hz, 1 H, Ar-H), 5.22 (s, 1 H, CH 2 ), 3.81
s, 3 H, OMe).C 13 NMR (CDCl 3 , 100 MHz) δ (ppm): 172.9, 166.8, 162.4, 156.2,
50.2, 129.4, 129.3, 129.2, 128.9, 124.3, 115.4, 114.4, 108.2, 101.7, 64.5, 55.5.
(
1
Results and discussion
Molecular docking
To identify the possible binding sites of the oxadiazole molecule on BSA, AutoDock
Vina program was used. This enabled us to predict the binding of oxadiazole com-
pounds with BSA. It is noteworthy that all the oxadiazole compounds are bound at
the interface between two subdomains IIA and IIIA (Fig. 2 and Fig. S1). The inter-
active residues of BSA for the oxadiazole molecules are as follows. (i) For 6a, the
residues are Arg 458, Ile 455, Tyr 451, Arg 435, Lys 431, Ser 428 (H-bond), Arg
1
4
96, Ala 193, Ser 192 (ii) For 6b, the residues are Arg 458 (H-bond), Ile 455, Tyr
51, Arg 435, Lys 431, Ser 428 (H-bond), Ala 193, Ser 192, (iii) For 6c, the residues
are Arg 458, Ile 455, Tyr 451, Arg 435, Lys 431, Ser 428 (H-bond), Ala 193, Asp
1
08, and (iv) For 6d, the residues are Arg 458 (H-bond), Tyr 451, Arg 435, Lys 431,
Ser 192 (H-bond), Ala 193, Ser 428 (H-bond), His 145 (H-bond).
The binding free energies for oxadiazole derivatives (6a, 6b, 6c, and 6d) were
observed to be -9.2, −9.5, −9.7, −10.0 kcal/mol, respectively. Out of the four com-
pounds, 3-(2H-chromen-3-yl)-5-phenyl-1,2,4-oxadiazole (6a) exhibited lowest bind-
ing and 3-(7-methoxy-2H-chromen-3-yl)-5-(4-nitrophenyl)-1,2,4-oxadiazole (6d)
exhibited highest binding with BSA. Therefore, we selected these particular com-
pounds for further analysis to understand binding interactions with BSA. For sim-
plicity, we used only abbreviation for these compounds, 6a (lowest binding) and 6d
(
highest binding) in the whole work.
Fluorescence measurements
Intrinsic ꢃuorescence measurements can provide valuable information about the
change in structure and the microenvironment in the vicinity of the ꢃuorophores
during the interaction of the ligand molecules with the protein. BSA contains three
intrinsic ꢃuorophore residues (Trp, Tyr and Phe), out of which Trp has the highest
quantum yield and Phe has the lowest quantum yield. There are two Trp residues
(
Trp 134 and Trp212) in BSA. Trp 134 was present on surface in domain IB, and
Trp 212 was present in the hydrophobic pocket of domain IIA [30]. In order to get
valuable information about the change in the microenvironment of Trp residues after
interaction with oxadiazole derivatives, BSA was excited at 295 nm to avoid ꢃuores-
cence interference from Tyr and Phe residues. Two important ꢃuorescence param-
eters, namely maximum intensity (I ) and maximum emission wave length (λ ),
max
max
were explored for the BSA–oxadiazole systems. Figure 3 shows the ꢃuorescence
spectra of BSA in the presence of oxadiazole compounds (6a and 6d) with varying
1
3

N. P. Mishra et al.
Fig. 2 Molecular docking of 3-(2H-chromen-3-yl)-5-phenyl-1,2,4-oxadiazole (6a) (a) and 3-(7-methoxy-
2
H-chromen-3-yl)-5-(4-nitrophenyl)-1,2,4-oxadiazole (6d) (b) with BSA
1
3

A spectroscopic insight into the interaction of chromene…
Fig. 3 Representative ꢃuorescence emission spectra of BSA in the absence and presence of oxadiazole
compounds with increasing concentrations (0, 10, 25, 50, 75, 100 µM). a 6a and b 6d
concentration. The ꢃuorescence intensity of BSA decreases gradually with increase
in the concentration of oxadiazole derivatives. The λ
of BSA in the buꢀer solu-
max
tion is observed to be 344 nm. In the presence of oxadiazole compounds, it was
observed that there was no change in emission maxima in the case of “6a,” whereas
slight blueshift was noticed in the case of “6d” at higher concentration. Any changes
in emission maxima generally indicate the change in polarity around the Trp resi-
dues. This can be attributed to either (a) internalization of Trp into protein core dur-
ing protein folding which enhances the hydrophobicity or (b) replacement of polar
water molecules from the vicinity of Trp by less polar oxadiazole compounds. In our
case, the blueshift of λ implies a decrease in the polarity and thus an increase in
max
the hydrophobicity in the microenvironment of the Trp residues. Since the oxadia-
zole compound (6d) is found to be at the interface between subdomain IIA and IIIA
regions as observed from docking results, which is away from the Trp residues, the
blueshift of λ must be due to the moderate conformational changes of BSA in the
max
presence of this compound.
Fluorescence quenching happens with increase in the concentration of ligands by
various molecular interactions such as ground-state complex formation, excited state
reactions, energy transfer, molecular rearrangement and collision quenching. The
quenching process is applicable to understand binding thermodynamics of protein-
ligands interactions. The quenching process can be either static or dynamic. Static
quenching happens when there is ground-state complex formation between the ꢃuo-
rophore and quencher, and dynamic quenching happens by the collision between
ꢃuorophore and quencher at excited states. These two quenching mechanisms can
be diꢀerentiated by the Stern−Volmer plot using Eq. (1), which also provides the
quenching eꢂciency of oxadiazole compounds on BSA ꢃorescence [31].
F0
=
1 + K [Q] = 1 + kqꢀ [Q]
SV
(1)
0
F
where F and F are the ꢃuorescence intensities of BSA in the absence and pres-
0
ence of oxadiazole compounds, respectively. K is the Stern–Volmer quenching
SV
constant, τ is the ꢃuorescence lifetime of protein without any quencher, k is the
0
q
1
3

N. P. Mishra et al.
Table 1 Stern–Volmer constant
KSV (M−1
kq (M−1 s−1
)
r2
T (K)
98
)
(
(
K ) and quenching constant
SV
k ) for the interaction of 6a and
7.9×103
11
q
2
7.97×10
5.18×10
4.59×10
0.999
0.984
0.987
0.998
0.992
0.997
6
d with BSA at 298, 303 and
11
6
6
a
303
5.2×103
4.6×103
10.9×10
9.2×103
7.3×103
3
08 K
11
3
2
08
98
3
11
10.90×10
11
d
303
9.16×10
11
3
08
7.34×10
Fig. 4 Fluorescence quenching eꢂciency of oxadiazole compounds on BSA. Stern–Volmer plots of the
ꢃuorescence quenching of BSA by 6a (a) and 6d (b)
bimolecular quenching rate constant and [Q] is the concentration of oxadiazole com-
pounds. The calculated K and k values for BSA–oxadiazole complexes at diꢀer-
SV
q
ent temperatures are presented in Table 1. (Stern–Volmer plots are shown in Fig. 4.)
From Table 1, it is observed that calculated K values are decreased with increase
SV
in temperature. This suggests that the major quenching mechanisim is due to static
in nature.
Determination of binding parameters
We used the following equation considering the binding of oxadiazole molecules
independently to a set of equivalent sites on BSA to obtain the binding constant (K)
and a number of binding sites (n):
F0 − F
log
= log K + n log [Q]
b
(2)
F
where n is the number of binding sites and K is the binding constant. The values
b
of n and K were obtained from the slope and intercept of the plot of log [(F −F)/
b
0
F ] versus log [Q], respectively (Fig. S2). The calculated results for compounds (6a
0
and 6d) at diꢀerent temperatures (298, 303 and 308 K) are given in Table 2. The
value of n was found to be closer to unity indicating there was one independent class
3
of binding sites on BSA. K values for 6a and 6d were found to be 1.6×10 and
b
3
9
.0×10 , respectively, which are in agreement with the docking results where the
1
3

A spectroscopic insight into the interaction of chromene…
Table 2 Binding parameters (binding constant—K and binding sites—n) and thermodynamic param-
b
0
0
0
eters (Gibbs free energy—∆G , enthalpy—∆H and entropy—∆S ) for the interaction of 6a and 6d with
BSA at 298, 303 and 308 K
T (K) K (M−1) r2
n
−∆G (kJ mol−1) ∆H (kJ mol−1) ∆S (J mol K−1
)
0
0
0
−1
b
3
2
303
3
2
303
98
1.6×10
1.4×10
0.9×10
9.0×10
6.8×10
5.3×10
0.997 0.86 18.42
0.998 0.86 18.03
0.997 0.83 17.65
0.996 0.98 22.56
0.987 0.97 22.26
0.982 0.96 21.96
−41.59
−77.74
3
3
3
3
3
6
6
a
08
98
−40.58
−60.46
d
3
08
strongest binding was observed for 6d. The low values of binding constant reveal
that weak interactions exist between oxadiazole compounds and BSA. Gener-
5
8
ally, protein–ligand complexes exhibit binding constants in the range of 10 –10
−1
Lmol [32]. However, there were several reports which show lower binding aꢂnity
2
4
−1
(
10 –10 Lmol ) for several ligand–protein complexes [33, 34]. In one of the stud-
3
−1
ies, binding constant of 2.95×10 Lmol for 1,3,4-oxadiazole compounds of fatty
acid with human serum albumin (HSA) was evidenced by Laskar et al. [27]. Another
4
7
study by Rehman et al. reported binding constants in the range of 1.0×10 –3.0×10
−1
Lmol for diꢀerent 1,3,4-oxadiazole compounds with BSA [35].
Evaluation of thermodynamic parameters and mode of interaction
Several binding forces (viz. electrostatic forces, hydrogen bonding, hydrophobic
interactions and van der Waals interactions) are responsible for binding of ligands
with biomolecules. Thermodynamic parameters such as enthalpy change (ΔH),
entropy change (ΔS) and free energy change (ΔG) can be used to distinguish the
type of interaction involved in binding based on the signs and magnitude. The ther-
modynamic parameters can be obtained from the plot of lnK vs. temperature (Fig.
b
S3) according to the van’t Hoꢀ equation as follows:
ΔH ΔS
ln K = −
+
(3)
(4)
RT
R
ΔG = ΔH − TΔS
−
1
where, K is the equilibrium binding constant (L mol ), T is the temperature (K) and
−1
−1
R is the gas constant (8.314 J mol K ). According to Ross’s theory, if ΔH>0 and
ΔS>0, hydrophobic interactions play a major role; if ΔH<0 and ΔS<0, the major
binding forces involve hydrogen bonding or van der Waals forces, and if ΔH<0 and
ΔS>0, electrostatic forces play a major role. The calculated thermodynamic param-
eters for the interaction of oxadiazole compounds with BSA are given in Table 2.
The negative value of ∆G indicates that the interaction is spontaneous and the nega-
tive values of ΔH and ΔS suggest that the binding between oxadiazole compounds
and BSA is mainly driven by hydrogen bonding and van der Waals forces.
1
3

N. P. Mishra et al.
Fig. 5 Secondary structure and conformational stability of the BSA after interaction with oxadiazole
compounds. Far-UV CD spectra of BSA in the presence of 6a (a) and 6d (b) with concentrations (0, 10,
5
0, 100 µM) at 25 °C. Free oxadiazole solutions without protein are represented as 6a and 6d followed
by concentration
Table 3 α-helix percentage
of BSA in the absence and
presence of 6a and 6d
0
10 µM
50 µM
100 µM
6
6
a
d
61.2
61.2
60.4
61.1
59.1
59.1
58.7
59.9
Circular dichroism spectral analysis
CD spectroscopy has been employed to study the changes in the secondary struc-
ture of BSA after the interaction with 6a and 6d. The secondary structure of BSA
exhibits two characteristic negative bands in the far-UV region (190–250 nm).
The negative bands at 208 and 222 nm appear due to the π–π* and n–π* transi-
tions of peptide backbone in α-helices. Alterations in the band intensity and posi-
tion would indicate the conformational changes of BSA after interaction with 6a
and 6d. Figure 5 shows the secondary structure of BSA in the absence and pres-
ence of 6a and 6d. It was observed that the structure of BSA remains intact after
interaction with 6a and 6d. However, to investigate the slight changes induced by
the oxadiazole derivatives, we calculate the percentage of α-helix in BSA using
Eq. 5:
−
MRE208 − 4000
3000 − 4000
[where Cp is the molar concentration of the protein, n
∝
−helix (%) =
(5)
3
observed CD(mdeg)
where MRE =
C nl×10
p
is the number of amino acid residues (583 for BSA) and l is the path length
(
0.1 cm)].
Table 3 shows the percentage of α-helix in the presence of 6a and 6d. It was
observed that there was a slight decrease in the α-helicity of BSA after interaction
with 6a and 6d at higher concentration. Conformational changes of serum albumin
1
3

A spectroscopic insight into the interaction of chromene…
in the presence of 1,3,4-oxadiazole compounds with diꢀerent concentrations were
also reported in earlier studies [27].
Esterase‑like activity assay
The inꢃuence of the oxadiazole compounds on the activity of BSA was monitored
using p-nitrophenyl acetate (PNPA) substrate. The action of BSA on the substrate
was probed by measuring the absorbance of the liberated product, p-nitrophenol
(
λ
=400 nm). The catalytic activity of BSA is exhibited by the reactive residues
abs
Tyr-411, Lys-413 and 414, which readily react with substrate and acetylated [36].
The activity was provided in terms of units where one unit activity is equal to the
amount of enzyme (BSA) required to liberate 1 μM of p-nitrophenol from the sub-
strate (PNPA) per minute at 37 °C. For comparative study, BSA activity was repre-
sented in relative percentage where the activity of BSA in buꢀer medium was taken
as 100%. Figure 6 shows the relative esterase-like activity of BSA in the presence of
6
a and 6d with varying concentrations. It is clear from Fig. 6 that the esterase activ-
ity of BSA is not much inꢃuenced by the oxadiazole compounds. However, with an
increase in the concentration of the compounds, there was a slight loss of activity
observed, which indicates that BSA maintains the esterase-like activity (≈ 90%) in
the presence of both oxadiazole compounds (6a and 6d).
Conclusion
In this work, we have investigated the binding of oxadiazole compounds with BSA
and the eꢀect on the structural stability of BSA using diꢀerent spectroscopic meth-
ods including ꢃuorescence, UV-Vis and CD as well as molecular docking stud-
ies. Fluorescence results suggested that static quenching is the primary reason for
ꢃuorescence quenching of BSA by oxadiazole compounds. The negative value of
free energy change elucidated the spontaneous interaction process between BSA
and oxadiazole compounds. The negative values of enthalpy and entropy change
speciꢁed that the hydrogen bonding and van der Waals forces were involved in the
Fig. 6 Modulation in the
relative esterase-like activity of
BSA as a result of interaction
with the oxadiazole compounds
(
(
6a and 6d) with concentration
0, 10, 50, 100 µM). (Error bars
are found to be less than 6%)
1
3

N. P. Mishra et al.
binding of these compounds with BSA which were also supported by the molecular
docking results. Moreover, the structural stability of BSA in the presence of these
compounds was conꢁrmed by circular dichroism analysis and activity assays. The
results showed that BSA remains intact with overall secondary structure after inter-
action with these compounds retaining ≈ 90% esterase activity. Conclusively, this
study presents a comprehensive insight into the possible mechanism of interaction
between oxadiazole compounds and BSA that can be helpful for the development of
new 1,2,4-oxadiazole compounds.
Acknowledgements The author NPM acknowledges INSPIRE program (DST/INSPIRE/03/2015/004518,
New Delhi) for providing ꢁnancial support in the form of INSPIRE FELLOWSHIP. Author SRM thanks
the CSIR, New Delhi, No.-02(0381)/19/EMR-II for ꢁnancial support.
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