BSA binding and antibacterial studies of newly synthesized 5,6-Dihydroimidazo[2,1-b]thiazole-2-carbaldehyde

Article abstract of DOI:10.1016/j.saa.2019.117192

A new heterocyclic compound, 5,6-Dihydroimidazo[2,1-b]thiazole-2-carbaldehyde (ITC) was synthesized and its antibacterial activity and also its interaction with bovine serum albumin (BSA) were studied. The structure of the synthesized compound was confirmed by ¹H NMR, ¹³C NMR and IR spectroscopic techniques. The antibacterial activity was carried out by minimum inhibitory concentration (MIC) method. The compound showed a good antibacterial activity. The mechanism of interaction between the BSA and ITC under physiological conditions was investigated by various molecular spectroscopic techniques like, fluorescence, circular dichroism (CD), UV absorption and FT-IR. The interaction between ITC and BSA was followed by studying the quenching of intrinsic fluorescence of BSA upon the addition of ITC at three different temperatures. The binding constant (K), Stern-Volmer quenching constant (K_sv) and number of binding sites were determined. The separation distance between BSA and ITC was evaluated based on the fluorescence resonance energy transfer theory. The conformational changes in BSA upon binding of ITC were also confirmed. The interference of some metal ions on interaction was studies. The displacement studies with site specific markers confirm that the site III was the binding site for ITC on BSA.

Full text of DOI:10.1016/j.saa.2019.117192

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117192
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
BSA binding and antibacterial studies of newly
synthesized 5,6-Dihydroimidazo[2,1-b]thiazole-2-carbaldehyde
Basappa Chanabasappa Yallur a,1, Umesha Katrahalli b,1
,
a,
Panchangam Murali Krishna a, , Manjunatha Devagondahalli Hadagali
⁎
⁎
a
Department of Chemistry, MS Ramaiah Institute of Technology, Banagalore 560 054, India
b
PG Department of Chemistry, Vijaya College, Bangalore 560 004, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new heterocyclic compound, 5,6-Dihydroimidazo[2,1-b]thiazole-2-carbaldehyde (ITC) was synthesized and its
antibacterial activity and also its interaction with bovine serum albumin (BSA) were studied. The structure of the
synthesized compound was confirmed by 1H NMR, 13C NMR and IR spectroscopic techniques. The antibacterial
activity was carried out by minimum inhibitory concentration (MIC) method. The compound showed a good an-
tibacterial activity. The mechanism of interaction between the BSA and ITC under physiological conditions was
investigated by various molecular spectroscopic techniques like, fluorescence, circular dichroism (CD), UV ab-
sorption and FT-IR. The interaction between ITC and BSA was followed by studying the quenching of intrinsic
fluorescence of BSA upon the addition of ITC at three different temperatures. The binding constant (K), Stern-
Volmer quenching constant (Ksv) and number of binding sites were determined. The separation distance be-
tween BSA and ITC was evaluated based on the fluorescence resonance energy transfer theory. The conforma-
tional changes in BSA upon binding of ITC were also confirmed. The interference of some metal ions on
interaction was studies. The displacement studies with site specific markers confirm that the site III was the bind-
ing site for ITC on BSA.
Received 19 March 2019
Received in revised form 18 May 2019
Accepted 26 May 2019
Available online 28 May 2019
Keywords:
Imidazolidinethione
Antibacterial activity
Bovine serum albumin
Interaction studies
Multi-spectroscopy
© 2019 Elsevier B.V. All rights reserved.
1. Introduction
binds to IB, IIIA, IIIB whereas the aromatic as well as heterocyclic com-
pounds found to be binds to sub-domains IIA and IIIA of Site I and Site
Serum albumins are responsible for harmonizing the pH of the blood
in biological systems [1]. The blood plasma of bovine comprises about
60% of protein as bovine serum albumin (BSA) [2]. In vivo, the BSA
acts as a transporter for endogenous ligands and biologically active mol-
ecules. Binding of biologically active molecules to serum albumins is a
criterion for pharmacokinetics. The binding constant of drug to serum
albumin and availability of the free drug are inversely proportional.
That is, if the binding between drug and protein is stronger, availability
of the free drug in blood is lower. Similarly, if the binding between the
later and former is weaker, then the amount of free drug available in
the blood will be higher. BSA is a spherically structured protein
(Fig. 1) [3], comprises of 583 amino acids. The BSA consists of mainly
three domains, viz., domain I, domain II and domain III. Further each do-
main is sub-divided into two sub-domains, viz., sub-domain A and sub-
domain B. From the literature it was found that fatty acids prefers to
II [4–7]. BSA contains two tryptophan residues, tryptophan-134 and
tryptophan-213 which are mainly responsible for its intrinsic fluores-
cence [8]. In the present study we chose BSA over human serum albu-
min (HSA) because BSA bears a resemblance to HSA about 76% and
also it is low cost and easily available [9,10]. This prompted us to select
BSA over HSA.
The heterocyclic compounds are found to be very good biologically
active materials. From many decades the study of heterocyclic com-
pounds has been a fascinating field in medicinal chemistry. Literature
survey reveals that heterocyclic compounds and its derivatives bearing
nitrogen and sulphur atoms provides unique and versatile scaffolds in
drug development [11]. Amongst, imidazoles are very important het-
erocyclic molecules with wide range of pharmaceutical applications
such as anticancer, antihypertensive, anti-helminthic, anti-
inflammatory, analgesic, hypoglycemic agents and veterinary medicine
etc.
Some of the drugs like Tilomisole, an analog of levamisole which
contains the imidazothiazole moiety act as an active anthelminthic
agent [12], used in veterinary medicine to de-worm sheep, treat
human worm infestations, inflammatory conditions, cancer [13] and
as cocaine adulterant [14].
⁎
Corresponding authors.
E-mail addresses: muralikp@msrit.edu (P.M. Krishna), manjunathdh@msrit.edu
(M.D. Hadagali).
1
Contributed equally.
https://doi.org/10.1016/j.saa.2019.117192
1386-1425/© 2019 Elsevier B.V. All rights reserved.

2
B.C. Yallur et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117192
The elemental analyses of Carbon, Hydrogen and Nitrogen were car-
ried out on a Perkin Elmer CHN 2400 analyzer. The melting points of the
synthesized compounds were measured in evacuated capillary tubes.
Infrared spectra recorded between 4000 and 400 cm−1 range (KBr
disc) on a Nicolet protage 460 FT-IR spectrophotometer. Electronic
spectra recorded in DMF solvent on Elico SL 159 UV–Visible spectropho-
tometer. NMR spectra were recorded in d6-DMSO taking tetra methyl
silane as internal standard on Brukerat 400 and 100 MHz respectively
for 1H and 13C.
Hitachi spectrofluorimeter Model F-2700 equipped with 150 W
Xenon lamp was employed for the fluorescence measurements. Slit
width of the instrument was kept at 5 nm. JASCO-J-715 spectropolarim-
eter was used to record the CD graphs. The CD recordings were carried
out in 0.1 cm thickness cell and by keeping 0.2 nm intervals in the wave-
length range of 200–250 nm. The average of three scans was taken for
each CD spectrum. The absorption spectra were collected on a dual
beam Analytic Jena Specord 200 Plus UV–vis spectrophotometer oper-
ated with a 150 W Xenon lamp. A slit width of 5 nm was kept through-
out the absorption studies. FTIR spectra were registered on a Thermo
Nicolet-5700 FTIR spectrometer (Waltham, MA, USA) via the attenuated
total reflection with resolution of 4 cm−1 60 scans. Gel electrophoresis
experiments were carried out using UVITEC, Cambridge.
Fig. 1. Crystal Structure of BSA.
Nutlin 3, cis-imidazoline analogs are used in the better treatment of
cancer [15,16]. Similarly, 1H-4, 5-dihydroimidazoles are used as antihy-
pertensive [17], anti-helminthic [18,19] and analgesic agents [20]. Other
1H-4, 5-dihydroimidazole derivatives were studied as hypoglycemic
compounds [21]. In recent times, the importance of dihydroimidazole
moieties especially in biochemistry is increasing due to their biological
activities [22–26]. They are also used in organic synthesis as synthetic
intermediates [27,28], chiral auxiliaries [29,30] and chiral ligands
[31–33].
In the light of biological applications of sulphur and nitrogen con-
taining compounds, we synthesized the compound, hydro bromide
salt of 5,6-dihydroimidazo[2,1-b]thiazole-2-carbaldehyde (ITC)
(Fig. 2) and we studied its antibacterial activity and interaction with
BSA.
2.2. Methods
2.2.1. Synthesis of ITC
To a stirred solution of 2-imidazolidinethione (0.250 g, 0.0024 mol)
in ethanol was added an ethanolic solution of 2-bromomalonaldehyde
(0.370 g, 0.0024 mol) drop wise over a period of 15 min and stirred con-
stantly for an hour at room temperature and then at 80 °C for 2 h. The
yellow colored solid obtained was filtered and washed several times
with acetone, then dried under vacuum. Reaction completion was mon-
itored on TLC. Yield: 85% (0.320 g), MP: 205 °C. The Reaction Scheme for
the synthesis of ITC is given in Fig. 3. The supplementary data supports
the structure of the compound.
2. Experimental
2.1. Materials and equipment
2.2.2. Anti-bacterial activity
The reagents and solvents were purchased commercially and used
without further purification unless otherwise noted. 1, 1, 3, 3-
Tetramethoxy propane (99%), 2-Imidazolidinethione (98%) were pur-
chased from Sigma-Aldrich, Bangalore. All bacterial strains used were
received from Department of Biotechnology, MS Ramaiah Institute of
Technology and MS Ramaiah Medical College Bangalore. Bovine serum
albumin (BSA, Fraction V) was purchased from Sigma Chemical Com-
pany, St Louis, USA and pUC18 DNA from Genei, Bangalore. The com-
pound ITC was prepared and purified as per the procedure given and
the structure of the compound was confirmed by various spectroscopic
techniques (Supplementary data). A 0.1 M phosphate buffer of pH 7.4
containing 0.15 M NaCl was prepared and the solutions of ITC and BSA
were prepared in the phosphate buffer based on their molecular
weights. The other chemicals used were of analytical grade and bi-
distilled water was utilized for all the experiments.
A loop full of cultured bacterial cells from the nutrient agar plates
was incubated into a nutrient broth (50 ml) and incubated at 37 °C for
18 h. 100 μl of 18 h bacterial cultures were spread on nutrient agar
media using a sterile glass spreader. Agar dilution method was followed
to fix the Minimum Inhibitory Concentration (MIC) of the compound.
The bacterial strains were developed at 37 °C for 8-10 h and maintained
on nutrient agar bath. The ITC was dissolved in DMSO to get stock solu-
tion. Commercially available bactericide Ciprofloxacin was used as stan-
dard. Based on agar dilution method the concentrations of standard and
ITC were fixed at 9 mg/ml. The sterilized petri-dishes were used and
growth inhibition zones formed around disc were measured. DMSO
was used as control and showed no inhibitions in studies. The diameters
of inhibition zones are in mm.
2.2.3. ITC-BSA interactions
Preliminary experiments were carried out to fix the concentration of
BSA and found that 2.5 μM was optimal. The concentration of ITC was
kept wide-ranging from 0 to 100 μM. The fluorescence spectra were ac-
quired in the wavelength range of 290–500 nm, at three different tem-
peratures, 293 K, 300 K and 308 K. 296 nm was fixed as the excitation
wavelength for all the fluorescence measurements.
N
. HBr
S
N
O
2.2.3.1. The displacement experiment. The displacement experiments
were conducted by taking the site probes namely warfarin, ibuprofen
and digitoxin for sites I, II and III, respectively. The spectra were re-
corded at fixed concentrations of BSA and site probe (2.5 μM each).
The fluorescence quenching titrations were carried out as described in
the Section 2.2.3 at room temperature and the binding constants
H
Fig. 2. The Structure of ITC.

B.C. Yallur et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117192
3
Fig. 3. Reaction Scheme for the synthesis of 5,6-Dihydroimidazo[2,1-b]thiazole-2-carbaldehyde (ITC).
between ITC and BSA system was determined in the presence of above
mentioned site probes [34].
(2.92), N: 11.92 (11.57). All these obtained spectral data strongly agrees
the formation of the compound.
2.2.3.2. Effects of common ion. The fluorescence spectra of ITC (10–100
μM)-BSA (2.5 μM) systems were registered in the existence of some
common ions (2.5 μM) viz., K , Na , Fe2 , Ca2 , Ni2 , Zn2 , Mg2 , and Cu2
by following the procedure as mentioned in the Section 2.2.3.
3.2. Antibacterial studies
The bacterial study of ITC was determined according to the Kirby-
Bauer method [35]. The bacterial isolates were sub-cultured on nutrient
agar plates and incubated at 37 °C for 24 h. Bacteria used for these stud-
ies were gram positive bacteria (Staphylococus epidermidis, Bacillis
subtilis) and gram negative bacteria (Pseudomonas aeruginosa,
Escherichia coli). It was observed that ITC showed better inhibition activ-
ity towards gram negative bacteria, S. epidermidis than the other bacte-
rial stains. The results are given in Table 1.
2.2.3.3. Circular dichroism measurements. A stock solution of 250 μM BSA
(prepared in phosphate buffer) was used for the current study. In the
absence and presence of ITC, the CD measurements were carried out
in the wavelength range of 200–250 nm. The ratio of BSA to ITC was
kept at 1:0, 1:2, 1:4 and 1:6.
2.2.3.4. Absorption measurements. The UV–visible spectral data were
scanned in the range of 300–500 nm at 300 K. A concentration of 2.5
μM of BSA solution was used for the absorption studies whereas the
ITC concentration was kept wide-ranging from 0 to 100 μM.
3.3. BSA-binding studies
The intrinsic protein fluorescence is basically obtained from its
amino acid residues like Tryptophan, Tyrosine and Phenylalanine.
Amongst these, Tryptophan has the highest fluorescence intensity in
comparison to other two residues [36,37]. The fluorescence character
of Tryptophan is highly sensitive to its surrounding environment.
Hence, this behavior is being extensively utilized as endogenous fluo-
rescent probes for understanding the interactions of albumins with bio-
active molecules [38].
2.2.3.5. FTIR measurements. The FTIR spectra of BSA as well as BSA-ITC
system were registered at 300 K in the range of 1500–1700 cm−1
.
3. Results and discussion
3.1. Synthesis and characterization of ITC
Fig. 4 depicts the fluorescence spectra of all BSA upon the addition of
ITC. When the ITC concentration was increased gradually from 0 to 100
μM, it was found that the fluorescence intensity of BSA decreased regu-
larly. It was also observed that fluorescent intensity of BSA was de-
creased with blue shift of the λem (Δλ = 13 nm), which indicates ITC
can quench the fluorescence intensity of BSA and the surrounding envi-
ronment of Tryptophan was also altered.
Prepared compound (ITC) was stable at room temperature and its
structure was confirmed by the preliminary tests and also by instru-
mental methods- infrared, NMR and elemental analysis. The IR spectra
of ITC shown the strong stretching frequency bands at 1566 cm−1
,
,
1647 cm−1 for C_N and C_O groups, medium bands at 3163 cm−1
2947 cm−1, can be assigned for the C\\H for aromatic and aliphatic
stretching frequencies. The 1H NMR spectra of the compound shown
the resonance at δ 4.43–4.47(m; J = 8.4 Hz, 10 Hz, 2H), δ 4.57–4.61
(m; J = 10.4 Hz, 8.4 Hz, 2H), has been assigned to the non-aromatic
ring protons, at δ 8.37 (s;1H) can be assigned for aromatic ring proton
and at δ 9.67 (s; 1H) for carbonyl carbon proton. 13C NMR shown the
signals at 47.35 ppm and at 51.35 ppm for non-aromatic carbons,
127.70 ppm, 138.15 ppm, 170.62 ppm for the carbons of aromatic ring
and the single resonance at 182.69 has been assigned for carbonyl car-
bon. Elemental data: Found (Calculated) (%); C: 30.65 (30.12), H: 3.00
The mechanisms of quenching involved in fluorophore and
quencher interactions are of three different types viz., static, dynamic
Table 1
In vitro anti-bacterial activity of ITC.
Compound
Zone of inhibition (in mm)
B. subtilis
S. epidermidis
P. aeruginosa
E. coli
ITC
1
4
1
1
Ciprofloxacin
25
25
25
25

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B.C. Yallur et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117192
1200
1000
800
600
400
200
0
BSA
Addition of
ITC
290
310
330
350
370
390
410
430
Wavelength, nm
Fig. 4. Fluorescence spectra of BSA (2.5 μM) upon the addition of ITC from 10 to 100 μM at 300 K.
and combined static and dynamic quenching mechanisms [39–42]. The
static and dynamic quenching mechanisms can be attributed on the
basis of type of relationship between quenching constant (KSV) and
temperature or life time of the excited state [41,42]. That is, for static
quenching, the KSV and temperature values are inversely proportional,
where as they are directly proportional to each other for dynamic
quenching. Fluorescence intensity data of BSA upon the addition of ITC
at three different temperatures (293 K, 300 K and 300 K) were analyzed
to elucidate the quenching mechanism in the present study. For this
purpose, the following Stern-Volmer Eq. (1) [39] was used. The respec-
tive plots obtained are given in Fig. 5.
mechanism of quenching may be of dynamic type. But, the values of
kq ≫ 2.0 × 1010 M−1 s−1, which indicates mechanism of quenching is
not dynamic (the maximum dynamic quenching rate constant is 2.0
× 1010 M−1 s−1) [43]. Hence, it can be inferred that, the mechanism of in-
teraction of ITC with BSA is not a single quenching mechanism. It may be
static as well as dynamic quenching mechanism.
Further, the quenching data were examined using the modified
Stern-Volmer relation shown below [39,44]:
F0
1
1
¼
þ
ð2Þ
F0−F faKa½Qꢀ fa
F0
F
Where, F0 represents the fluorescence intensity of BSA in the ab-
sence of ITC, F represents the fluorescence intensity of BSA in the pres-
ence of ITC, Ka represents the Stern-Volmer quenching constant of the
accessible fraction, fa is the fraction of the initial fluorescence of BSA
that is accessible to ITC, [Q] is the concentration of the ITC. The graph
of F0/(F0\\F) vs 1/[Q] yielded f−a1 as the intercept on y-axis and (faKa)−1
at the slope and the plots are shown in Fig. 6. The average values of fa
for ITC-BSA system found to be 0.9 indicating that 110% of the initial
fluorescence of protein was available for ITC. The findings are tabulated
in Table 2.
¼ 1 þ kqτ0½Qꢀ ¼ 1 þ KSV½Qꢀ
ð1Þ
where, F0 is the fluorescence intensity of BSA alone, F is the fluorescence
intensity of BSA in the presence of ITC, Ksv is the Stern-Volmer
quenching constant, kq is the quenching rate constant, [Q] is the concen-
tration of ITC and τ0 is the average life-time of BSA in the absence of ITC
(τ0 = 10−8 s).
The KSV and kq values obtained are given in Table 2. They are calcu-
lated from the slope of regression equation. The results revealed that
the KSV values increased with temperature indicating that the
3.3.1. Analysis of binding and thermodynamic parameters
The fluorescence readings were exploited to calculate the binding
constant K and the number of binding sites n for ITC-BSA complex by
the following relation [45]:
3
2.6
2.2
logðF0−FÞ
¼ logK þ n log½Qꢀ
ð3Þ
F
1.8
308 K
Table 2
300 K
1.4
Stern-Volmer quenching constants (Ksv), correlation coefficient (R) and biomolecular
quenching rate constants (kq) for ITC-BSA system at different temperatures.
293 K
1
Temperature, K
Ksv (104/mol)
R2
kq (1012/mol)
fa
0.00E+00
4.00E-05
8.00E-05
1.20E-04
293
300
308
1.64 0.11
1.81 0.04
1.94 0.07
0.998
0.997
0.998
1.64 0.11
1.81 0.04
1.94 0.07
0.762 0.10
0.907 0.08
1.035 0.06
Concentration, [Q] (in µM)
Fig. 5. Stern-Volmer plots for quenching of BSA by ITC at different temperatures.

B.C. Yallur et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117192
5
8
7
6
5
4
3
2
1
Table 3
Relative thermodynamic parameters for ITC-BSA system at different temperatures.
Temperature, K
ΔG0
ΔH0
ΔS0
(104 kJ/mol)
(105 kJ/mol)
(102 kJ/mol)
293
300
308
−1.89 0.08
−2.24 0.10
−2.64 0.07
1.27 0.11
4.98 0.08
293 K
300 K
308 K
measured on the basis of fluorescence resonance energy transfer
(FRET) [48]. The separation space and the degree of spectral overlap es-
timate the amount of energy transfer. The overlap of protein fluores-
cence spectrum and ITC absorption spectrum is shown in Fig. 8. The
efficiency of energy transfer E is correlated to r as follows:
0
0.00E+00
3.00E+04
6.00E+04
9.00E+04
1.20E+05
1/[Q]
6
Fig. 6. Modified Stern-Volmer plots for quenching of BSA by ITC at different temperatures.
F
F0
R0
E ¼ 1−
¼
ð6Þ
6
R0 þ r6
We found a good correlation between log(F0\\F)/F and log[Q] for
the ITC-BSA complex and the values of K and n can be acquired from
the intercepts and slopes, respectively. The n values found to be almost
equal to one at all the three temperatures, suggesting that the ITC-BSA
complex forms 1:1 ratio. The K values were in the range of 2.33 × 103
to 2.95 × 104 M−1, indicating that the binding affinity of ITC with BSA
was normal [38]. But, the K values increased with increase in tempera-
ture, indicates an increase in the stability of the ITC-BSA complex at
higher temperatures.
where, R0 is the critical distance when the energy transfer efficiency is
50%, which can be calculated using the below mentioned equation.
2
R0 ¼ 8:8 ꢁ 10−25k η−4ΦJ
ð7Þ
6
where, k2 is the spatial direction factor of the dipole, η is the refractive
index of the medium, Φ is the fluorescence quantum yield of the BSA
and J is the overlap integral of the protein fluorescence spectrum and ac-
ceptor absorption spectrum. The value of J was calculated by the below
formula:
The following Van't-Hoff and thermodynamic Eqs. (4) and (5) are
used to analyze the thermodynamic parameters in the binding phe-
nomenon between ITC and BSA.
ΣFðλÞεðλÞλ4Δλ
J ¼
ð8Þ
ΔH0
ΔS0
ΣFðλÞΔλ
logK ¼ −
þ
ð4Þ
ð5Þ
2:303RT 2:303R
where, F(λ) represents fluorescence intensity of BSA of wavelength λ,
ε(λ) is the molar absorption coefficient of ITC at wavelength λ. In the
present case k2 = 2/3, η = 1.336 and Φ = 0.15 for BSA-ITC system
[49]. We calculated the following parameters by the Eqs. (6) to (8),
ΔG0 ¼ ΔH0−TΔS0
It is evident from the Fig. 7 that, there was a direct association be-
tween logK vs 1/T, which suggested that, the ΔH0 and ΔS0 can be seen
as a constant. The calculated ΔG0 values (Table 3) were found to be neg-
ative values signify that ITC spontaneously binds to BSA. In view of
water structure, frequently a positive ΔS0 is taken as support for hydro-
phobic interaction. The calculated values of ΔH0 and ΔS0 were 1.27
× 105 J mol−1 K−1 and 4.98 × 102 J mol−1 K−1 respectively, suggesting
that the binding of ITC to BSA is largely entropy motivated and the en-
thalpy is unfavorable for it, the hydrophobic forces playing a significant
part in the binding process [46,47]. The results are given in Table 3.
and the results were found to be J = 4.63 × 10−14 cm3 L mol−1, R0
=
2.41 nm, E = 0.19 and r = 3.10 nm. From these results it is evident
that the BSA to ITC space is b8 nm which revealed the energy transfer
from BSA to ITC takes place with high probability [50].
3.4. Study of conformational changes
3.4.1. UV spectral studies
UV absorption spectroscopy is a simple but valuable method used
for finding the complex formation between serum albumin and bioac-
tive molecule [51]. The absorbance spectra of BSA alone as well as
with increasing concentrations of ITC are depicted in Fig. 9. The peak
3.3.2. Energy transfer studies
The fluorescence spectral studies showed that BSA formed complex
with ITC. The distance r between residue of the protein and ITC was
⁎
at 280 nm obtained by the BSA represents the π π transition due to
amino acids of BSA. The absorbance of BSA increased after the addition
of ITC shows that the BSA molecules were coupled with ITC and ITC-BSA
complex was formed. Further the maximum absorbance peak slightly
shifted towards higher wavelength. These two observations, i.e., shift
in the maximum absorbance and increase in the absorption intensity to-
gether obviously inferred the presence of interaction between ITC and
BSA and also indicated the conformational changes in BSA [52].
4.8
4.5
4.2
3.9
3.6
3.3
3.0
3.4.2. Analysis of circular dichroism spectra
CD is a sensitized spectroscopic method used to analyze the second-
ary structure of protein [53]. The CD method was used in the current
work and the CD graphs of BSA in the absence as well as in the presence
of varied concentrations of ITC were registered (Fig. 10). The CD spectra
of BSA showed a peak at 208 nm and another peak at 222 nm. These two
minima are distinctive peaks of α-helical pattern of BSA. The negative
apexes at 208–209 nm and at 222–223 nm were recognized as n to π*
shift for the peptide bond of α-helix [54]. The helical content of bound
0.0032
0.00325
0.0033
0.00335
0.0034
0.00345
1/T
Fig. 7. Plot of 1/T versus log K.

6
B.C. Yallur et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117192
1200
0.12
0.09
0.06
0.03
0
1000
800
600
400
200
0
A
B
290
310
330
350
370
Wavelength, nm
390
410
430
450
Fig. 8. Spectral overlap fluorescence emission spectrum of BSA (2.5 μM) (A) with absorption spectrum of ITC (2.5 μM) (B).
and unbound protein was determined by MRE (Mean Residual
Elipticity) values at 208 nm from the following formula [55]:
3.4.3. Interpretation of FTIR spectra
It is well known that the FTIR spectra of proteins exhibit two bands,
namely amide I band between 1600 and 1700 cm−1 and amide II band in
the range of 1500–1600 cm−1. The amide I band is related to the second-
ary structure of protein mainly due to carbonyl (C=O) stretching vibra-
tions of amide moiety, whereas amide II band is mainly due to nitrile
(C\\N) stretch attached with N\\H bending frequency. The amide I
band is normally sensitive in comparison with amide II band due to
the alterations in secondary structure of the albumin [57]. The FTIR
spectra of BSA and its complex with ITC (Fig. 11) showed that, the loca-
tion of the peaks of amide I and amide II bands were moved from 1553.2
to 1549.3 cm−1 and from 1669.3 to 1652.1 cm−1. This indicated that, the
secondary structure of BSA was altered after the binding of ITC. The ITC
has interacted with carbonyl and nitrile moieties of amino acids of pro-
tein and induced reorganization of polypeptide carbonyl hydrogen
bonding networks [58].
ꢀ
ꢁ
θObs
Cpnl ꢁ 10
MRE ¼
in the units of deg ꢂ cm2dmol−1
ð9Þ
θObs represents observed CD (in milli degrees), Cp represents con-
centration BSA, n represents number of amino acids and l represents
path length of the cell in cm.
−MRE208−4000
33000−4000
α−Helixð%Þ ¼
ꢁ 100
ð10Þ
where, MRE208 represents experimental value of MRE at 208 nm, 4000
represents MRE of α form and random coil conformation cross at
208 nm and 33,000 represents value of MRE of a bare α-helix at 208 nm.
The α-helicity gradually declined from (58.6 0.9) % in BSA alone to
(42.7 0.5) % in bound BSA, there by revealing that ITC has changed the
hydrogen bonding system in BSA. It also confirms the alterations in the
secondary structure of BSA. The appearances of CD graphs of BSA in the
absence as well as in the presence of ITC were found to be identical re-
vealing that the configuration of BSA was considerably α-helical though
ITC was bound to it [56].
3.5. Selection of binding site
Reported studies revealed that, there are three main locations on
BSA for binding of different types of ligands, viz., Site I, Site II and Site
III [59,60]. Many ligands like Phenyl butazone, Oxyphen butazone, War-
farin attach to site I, whereas Diazepam, Ibuprofen, Indoxyl sulphate,
Diflunisal attach to site II [38] and Digitoxin binds to site III of the pro-
tein [61]. The displacement examination with site specific markers
0.8
0.6
5000
0
-5000
-10000
d
0.4
Concentration
-15000
-20000
of ITC
0.2
-25000
a
x
BSA
-30000
0
200
210
220
230
240
250
260
270
280
290
300
310
Wavelength, nm
Wavelength, nm
Fig. 10. CD spectra of BSA-ITC system. Concentration of BSA was kept constant at 2.5 μM
(a). In BSA-ITC system, the concentration of ITC was kept at 5 μM (b), 10 μM (c) and 15
μM (d).
Fig. 9. UV spectra of BSA (2.5 μM) in the absence and presence of ITC 10–100 μM. x is the
UV spectra of ITC (2.5 μM) alone at 300 K.

B.C. Yallur et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117192
7
Table 5
A
The binding constant values for BSA-ITC system in the absence and presence of some
metal ions at 300 K.
Systems
K (103/mol)
R2
BSA-ITC
BSA-ITC-Na
BSA-ITC-K
8.02 0.11
18.97 0.05
13.24 0.07
1.88 0.13
9.16 0.11
9.42 0.06
7.23 0.07
1.57 0.09
12.33 0.10
0.997
0.998
0.996
0.997
0.996
0.997
0.997
0.991
0.986
BSA-ITC-Cu2
BSA-ITC-Zn2
BSA-ITC-Ni2
BSA-ITC-Mg2
BSA-ITC-Ca2
BSA-ITC-Fe2
and Ca 2 was found to be decreased this might leads to the quick release
of the ITC from the blood [62]. This might direct to the requirement of
more dose of ITC to attain the therapeutic achievement of the ligand sig-
nificantly. But, the binding constant of ITC-BSA system was increased in
the presence of Fe , Na , and K there by allowing the ITC to be holding for
prolonged duration in the blood [63]. This would guide to requirement
of lower concentration of the ITC to achieve its therapeutic effect. Fur-
thermore, it was also observed that Zn 2, Ni2 and Mg2 would not inter-
fere in the binding of ITC to BSA as it is evident by their K values.
B
4. Conclusion
The biologically active compound, 5, 6-dihydroimidazo[2,1-b]thia-
zole-2-carbaldehyde was synthesized and its interaction behavior
with BSA was studied by multi-spectroscopic approach. The anti-
bacterial activity of ITC by MIC method showed that the compound
was anti-bacterial in nature and showed considerable response to
S. epidermidis in comparison with B. subtilis and E. coli. The study state
fluorescence spectral results revealed that the synthesized heterocyclic
compound, ITC quenched the fluorescence intensity of BSA and the
quenching mechanism was found to be the blend of both dynamic
and static type. From the thermodynamic data it was found that binding
of ITC to BSA is primarily entropy motivated and the enthalpy is inaus-
picious for it and also the hydrophobic interactions playing a predomi-
nant part in the binding process. It was also found that the ITC was
bound to Site III of BSA and BSA led to conformational changes upon
the interaction of ITC.
Fig. 11. FTIR spectra and difference spectra of BSA: the FTIR spectra of free BSA (A)
(subtracting the absorption of buffer solution from the absorption of protein solution)
and the FTIR difference spectra of BSA (B) (subtracting the absorption of ITC-free form
from the absorption of ITC-BSA bound form). The concentrations of BSA and ITC were
2.5 μM each.
like warfarin, ibuprofen and digitoxin were carried out to confirm the
binding location of ITC on BSA. The resultant binding constant, K values
in the presence of site specific probes were calculated using the Eq. (3)
and the results are tabulated in the Table 4. It is evident from the K
values that, Digitoxin and BSA complex is predominantly lesser than
that in the native BSA system, whereas K values in warfarin-BSA system
and ibuprofen-BSA complex has no considerable alteration. From these
results it can be concluded that Digitoxin is substituted from the
Digitoxin-BSA system by ITC. Hence, the binding location of ITC is as
similar as that of Digitoxin, i.e., the site III.
In summary we conclude that this work presented valuable informa-
tion on the binding and interaction studies of BSA with imidazolidine
thione derivative. Further studies need to be explored in concern with
mechanism of function, pharmacokinetics and other biological activities
of the compound.
3.6. Interference of metal ions
Appendix A. Supplementary data
The plasma contains few metal ions which influence the binding of
ligands to albumins. Hence, it is necessary to study their interference
in the binding studies. For these experiments, we have selected some
common metal ions like Na , K , Fe2 , Mg2 , Ni2 , Ca2 , Cu2 , and Zn2 . For
this study, concentrations of protein and metal ions were kept stable
at 2.5 μM, whereas the ITC concentration was kept wide-ranging from
10 to 100 μM. The corresponding binding constant values are tabulated
in Table 5. The binding constant value of ITC-BSA in the presence of Cu 2,
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.saa.2019.117192.
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