Deciphering binding mechanism between bovine serum albumin and new pyrazoline compound K4

Article abstract of DOI:10.1002/bio.3762

The binding mechanism of a new and possible drug candidate pyrazoline derivative compound K4 and bovine serum albumin (BSA) was investigated in buffer solution (pH 7.4) using ultraviolet–visible light absorption and steady-state and synchronous fluorescen

Full text of DOI:10.1002/bio.3762

Received: 2 September 2019
DOI: 10.1002/bio.3762
Revised: 2 November 2019
Accepted: 10 December 2019
R E S E A R C H A R T I C L E
Deciphering binding mechanism between bovine serum
albumin and new pyrazoline compound K4
Ebru Bozkurt1
| Halise Inci Gul2
1Programme of Occupational Health and
Safety, Erzurum Vocational Training School,
Ataturk University, Erzurum, Turkey
Abstract
The binding mechanism of a new and possible drug candidate pyrazoline derivative
compound K4 and bovine serum albumin (BSA) was investigated in buffer solution
(pH 7.4) using ultraviolet–visible light absorption and steady-state and synchronous
fluorescence techniques. The fluorescence intensity of BSA was quenched in the
presence of K4. The quenching process between BSA and K4 was examined at four
different temperatures. Decrease of the quenching constants calculated using the
Stern–Volmer equation and at increasing temperature suggested that the interaction
BSA–K4 was realized through a static quenching mechanism. Synchronous fluores-
cence measurements suggested that K4 bounded to BSA at the tryptophan region.
Fourier transform infrared spectroscopy results showed that there was no significant
change in polarity around the tryptophan residue The forces responsible for the
BSA–K4 interaction were examined using thermodynamic parameters. In this study,
the calculated negative value of ΔG, the negative value of ΔH and the positive value
of ΔS pointed to the interaction being through spontaneous and electrostatic interac-
tions that were dominant for our cases. This study provides a very useful in vitro
model to researchers by mimicking in vivo conditions to estimate interactions
between a possible drug candidate or a drug and body proteins.
2Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Ataturk University,
Erzurum, Turkey
Correspondence
Ebru Bozkurt, Program of Occupational Health
and Safety, Erzurum Vocational Training
School, Ataturk University, 25240 Erzurum,
Turkey.
Email: ebrubozkurt@atauni.edu.tr
K E Y W O R D S
bovine serum albumin, fluorescence quenching, FRET, pyrazoline
1
|
INTRODUCTION
steady-state and time-resolved fluorescence measurements are often
used to investigate the interactions between BSA or HSA and fluores-
Studies on the interaction between proteins and fluorescent probes
are very important as they act as models for biochemical applications
in cells.[1–3] Serum albumins are proteins that constitute approxi-
mately 60% of total plasma proteins. These proteins play an important
role in the transport of exogenous and endogenous substances such
as fatty acids, metal ions, drugs, and dyes. Bovine serum albumin
(BSA) and human serum albumin (HSA) are proteins that are fre-
quently used in biochemical and biophysical studies. BSA is the 76%
structural homologue of HSA. BSA is generally preferred as a model
protein due to its ease of use, low cost, stability, and unusual ligand
binding properties in laboratory experiments.[4] Spectroscopic tech-
niques such as ultraviolet–visible (UV–vis) light absorption, and
cence probes due to their ease of use, accuracy, high precision, and
rapid response.[5–7]
Pyrazoline derivatives are compounds with antimicrobial,[8]
antinociceptive,[9] anticancer,[10,11] antidepressant,[12] and anti-inflam-
matory[13] properties. In addition to the pharmacological importance
of these compounds, they are used as fluorescent compounds
because of their strong fluorescence properties and high quantum
yields. To understand the interaction of these bioactive fluorescent
compounds with protein molecules in the cell, studies in the labora-
tory environment with model systems constitute a marked research
area.[14,15] Therefore, it is very important to understand the interac-
tion mechanisms between new pyrazoline derivatives and BSA.
Luminescence. 2019;1–8.
wileyonlinelibrary.com/journal/bio
© 2019 John Wiley & Sons, Ltd.
1

2
BOZKURT AND GUL
This study aimed to investigate the binding mechanism between
absorption and the fluorescence spectra of the samples were
BSA and a new pyrazoline derivative 4-(3-(3,4,5-trimethoxyphenyl)-
3a,4-dihydroindeno[1,2-c]pyrazol-2(3H)-yl)benzenesulfonamide (K4)
(Scheme 1) in Tris–HCl buffer solution (pH 7.4) using UV–vis light
absorption, and steady-state and synchronous fluorescence measure-
ments. K4 was selected from a series of synthesized compounds
described in our previous study[16] because of its interesting cytotoxic
activity. Binding mechanism parameters such as binding constant,
binding mode, binding site, and binding distance between BSA and K4
were determined and will provide extra information on how the con-
formation and structure of BSA changes due to the binding to K4. The
results obtained from the presented study will explain the binding
mechanism between the possible drug candidate compound K4 and
BSA. Compound K4 and BSA constitute a model system that were
realized in vitro but mimic in vivo physiological conditions. This model
describes a very useful and practical approach to estimate the interac-
tion between a compound (K4), a possible drug candidate, and body
proteins.
recorded using a Perkin Elmer Lambda 35 UV/VIS spectrophotometer
and
a Shimadzu RF-5301PC spectrofluorophotometer, respec-
tively.[17] For the steady-state fluorescence measurements, sample
solutions were excited at 280 nm and fluorescence intensities were
recorded between 290 nm and 550 nm. Fluorescence spectra were
collected at Δλ = 15 nm and Δλ = 60 nm using synchronous fluores-
cence measurements.[18] Fourier transform infrared (FTIR) spectros-
copy measurements of the samples were recorded with a Vertex
80/80v FTIR spectrometer.
2.3
|
Synthesis of K4
The compounds were synthesized and characterized as described in
our previous study.[16] Synthesis of the compounds is summarized in
Scheme 2. An aqueous solution of sodium hydroxide (10% w/v, 10 ml)
was added into an ethanol (6 ml) solution of 1-indanone (20 mmol)
and 3,4,5-trimethoxybenzaldehyde (20 mmol) (Scheme 2). The mixture
was stirred overnight at room temperature and then poured onto ice-
water (100 ml) in a beaker. The mixture was neutralized with
hydrochloric acid (10% w/v, 10 ml). The coloured precipitate formed
was filtered and crystallized from the water–ethanol mixture to pre-
pare the chalcone compound, 4 (2-(3,4,5-trimethoxybenzylidene)-
2,3-dihydro-1H-inden-1-one). After that, a solution of 4 (1.00 mmol)
and 4-hydrazinobenzensulfonamide hydrochloride (1.10 mmol) in eth-
anol (50 ml) was heated at 100ꢀC, 200 W, 7 bar for 10 min and moni-
tored by thin layer chromatography (TLC). After the reaction was
stopped, the volume of the reaction mixture was concentrated to half
and the precipitate formed was filtered, washed with cold ethanol,
and the compounds were purified by crystallization from ethanol to
obtain K4. The chemical structure of the compound K4 was confirmed
by 1H nuclear magnetic resonance (NMR), 13C NMR, and high resolu-
tion mass spectrometry (HRMS). Melting point (M.p.) was
266–269ꢀC.[16]
2
|
EXPERIMENTAL
| Materials
2.1
BSA, molar mass ~66 kDa), ethanol (spectrophotometric grade),
DMSO (spectrophotometric grade), Trizma (Tris) and HCl (36.5%)
were obtained from Sigma.
2.2
|
Apparatus
1H and 13C nuclear magnetic resonance (NMR) spectra were recorded
on Varian 400 and Bruker 400 instruments in DMSO-d6. UV–vis light
2.4
|
Preparation of BSA, K4, and BSA–K4
solutions
Tris–HCl buffer solution consisting of 0.2 M Tris was adjusted to
SCHEME 1 Molecular structure of K4
pH 7.4 with 36.5% HCl. BSA concentration was kept constant at
SCHEME 2 Synthesis of K4

BOZKURT AND GUL
3
10 μM in Tris–HCl buffer solution (pH 7.4). A stock solution of K4
was prepared in ethanol at 1.0 × 10−3 M. Successive aliquots of K4
were added (final concentrations of 10; 20; 30; 40; 50; 70; 80 and
100 μM) using a 100 μl pipette into a vial. The solvent was evaporated
using argon gas purging and then the buffer solution containing
10 μM of BSA was added to the vial.[4] BSA concentration was kept
constant as 10 μM while the fluorescent probe (K4) concentration
was changed (10–100 μM) for all studies.
3
|
RESULTS AND DISCUSSION
3.1
K4
|
Fluorescence quenching process of BSA and
FIGURE 2 Absorption and fluorescence spectra of BSA (10 μM)
in Tris–HCl buffer solution (pH 7.4) (λexc 280 nm)
The K4 compound studied was synthesized as previously described[16]
and summarized in Scheme 2. Optical properties of K4 (100 μM) were
determined in Tris–HCl buffer solution (pH 7.4) using UV–vis light
absorption and fluorescence measurements (Figure 1). K4 exhibited
two absorption bands at 278 nm and 363 nm and produced a fluores-
cence band at 465 nm in Tris–HCl buffer solution (pH 7.4). The
absorption bands of K4 at 278 nm and 363 nm corresponded to n!π*
transitions and π!π* transitions, respectively, of the conjugated skele-
ton localized on the pyrazoline ring.[17]
(pH 7.4) was evaluated at room temperature. As can be seen in
Figure 3, the absorption band intensity of BSA increased with differ-
ent concentrations of K4 and in a concentration-dependent manner
due to the interaction between BSA and K4 in the ground state. BSA
itself gave only one absorption peak at 275 nm, but when K4 was
added to the solution, a new absorption band was observed at
~365 nm because of the interaction of BSA with K4. The peak seen at
~365 nm was attributed to the π!π* transitions of conjugated skele-
ton localized on the pyrazoline ring.[17] The intensity of the peak at
issue increased depending on the K4 concentration used.
UV–vis light absorption and fluorescence measurements were
also used to determine the optical properties of BSA (10 μM) in Tris–
HCl buffer solution (pH 7.4) (Figure 2). BSA exhibited an absorption
band at 275 nm and produced a fluorescence band at 337 nm in Tris–
HCl buffer solution (pH 7.4). It is known that the absorption and fluo-
rescence peaks of the BSA molecule are associated with the presence
of aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr) and
phenylalanine (Phe) in its structure.[19,20]
Fluorescence spectroscopy is the most effective and simple
method available to understand conformational changes in protein
and complex formation between a protein and a probe molecule
because of its sensitivity, accuracy, speed, and ease of use at low sam-
ple concentrations.[4,21] The fluorescence properties of BSA are
widely used to investigate binding mechanisms between BSA and
drugs. As mentioned previously, BSA contains Trp, Tyr, and Phe aro-
matic amino acids and the fluorescence properties of BSA come
UV–vis light absorption measurements are generally used to
understand a ground-state complex formation between a protein and
the fluorescent molecule.[21] To this aim, the effect of K4 concentra-
tion on the absorption spectra of BSA in Tris–HCl buffer solution
FIGURE 1 Absorption and fluorescence spectra of K4 (100 μM)
in Tris–HCl buffer solution (pH 7.4) (λexc 350 nm)
FIGURE 3 Absorption spectra of BSA with the increasing
concentration of K4 in Tris–HCl buffer solution (pH 7.4)

4
BOZKURT AND GUL
especially from Trp, as the fluorescence yield of Phe is very low and
Tyr has fluorescence quenching properties when ionized.[22] When
was BSA excited at 280 nm, it exhibited fluorescence because of the
available Tyr and Trp residues, but Trp was more dominant in the BSA
fluorescence because of the quenching properties of Tyr.[19]
concentration and KSV is the Stern–Volmer quenching constant. A
Stern–Volmer plot was drawn using the data obtained from fluores-
cence measurements for the interaction between BSA and K4 at
different temperatures (Figure 5). The slope shown in Figure 5 pro-
vided the Stern–Volmer quenching constants at various tempera-
tures (Table 1). As shown in Table 1, KSV values decreased when
the temperature was increased. These results indicated that a non-
fluorescent complex between the BSA and K4 in the ground state
had formed and that the quenching process was due to static
quenching.[21,23,25]
Investigation of the BSA fluorescence quenching process using
a quencher molecule can reveal the binding mechanism between
BSA and quencher. Fluorescence quenching corresponds to
a
decrease in the fluorescence intensity of a fluorescent probe by the
addition of a quencher. The quenching process includes two mecha-
nisms called dynamic and static quenching. If it occurs due to the
formation of a nonfluorescent ground-state complex between the
fluorophore and the quencher it is called static quenching. In
dynamic quenching, diffusive interactions are observed between
fluorophore and quencher during the lifetime of the excited state.
These two processes are distinguished using temperature, viscosity,
or lifetime measurements.[23]
The binding constant (Kb) and the number of binding site (n) were
determined for the interaction of K4 with BSA using data obtained
from fluorescence measurements at four different temperatures. Kb
and n values were calculated using eqn (2):
ðI0 −IÞ
log
= logKb + nlog½Qꢁ
ð2Þ
I
Changes in the fluorescence spectra of BSA (10 μM) dependent
on the K4 concentration in buffer solution (pH 7.4) are presented in
Figure 4. The fluorescence intensity of BSA decreased gradually and
the fluorescence band maxima of BSA displayed a hypsochromic shift
(~6 nm) with increasing concentrations of K4. This spectral shift dem-
onstrated the formation of a less polar environment around the Trp
and Tyr residues in the BSA structure in the presence of K4.[24] In
addition, the decrease in fluorescence intensity of BSA was due to the
fluorescence quenching process that resulted from the binding
between BSA and K4. The mechanism of this fluorescence quenching
process can be explained using the following Stern–Volmer equation
(eqn (1)):
where, I and I0 are fluorescence intensity of protein in presence or
absence of quencher, respectively. [Q] is the quencher concentra-
tion. The plot of log[(I0 − I)/I] versus log[Q] for the interaction of
K4 with BSA at four different temperatures are represented in
Figure 6. The number of binding sites (n) for K4 to BSA was deter-
mined from the slope of Figure 6 and n values are summarized in
Table 1. The values in Table 1 reflected that K4 bound to BSA only
on one site and that the molecular ratio of the complex between
BSA and K4 was 1:1. According to the data, Kb and n values
decreased when the temperature increased. This suggested that the
ground-state complex BSA–K4 forming the binding interaction was
unstable. However, the BSA–K4 complex was more stable when
tested at low temperatures and, when the temperature increased,
the structure of BSA changed. The fact that Kb values decreased
when the temperature increased reflected that the process of
quenching between BSA and K4 occurred via a static quenching
mechanism.
I0
= 1 + KSV½Qꢁ
ð1Þ
I
where I0 and I are fluorescence intensity in the absence or in the
presence of quencher, respectively. [Q] is the quencher
FIGURE 4 Fluorescence spectra of BSA with the increasing
concentration of K4 in Tris–HCl buffer solution (pH 7.4) (λexc 280 nm)
FIGURE 5 Stern–Volmer plot for steady-state BSA fluorescence
quenching by K4 at 288 K, 298 K, 308 K, and 318 K

BOZKURT AND GUL
5
TABLE 1 Stern–Volmer quenching constant and binding parameters for the interaction of BSA with K4 at different temperatures
Stern–Volmer quenching constant
Binding parameters
T (K)
288
298
308
318
KSV × 105 (M−1
)
r2
n
Kb × 103 (M−1
1.29
)
r2
1.35
1.04
0.98
0.93
0.9972
0.9986
0.9985
0.9990
1.01
0.99
0.93
0.78
0.9655
0.9444
0.9508
0.9716
0.91
0.56
0.19
where K is the Stern–Volmer quenching constants, R is the ideal gas
constant and T is the temperature (K). The plot of lnK versus 1/T was
drawn using the data obtained from fluorescence measurements at
different temperatures (Figure 7). ΔH (the slope of plot) and ΔS (the
intercept of plot) values were calculated from Figure 7. Conversely,
ΔG values were calculated using eqn (4) obtained with ΔH and ΔS
values. These values are presented in Table 2. The negative ΔG values
at the studied temperatures indicated that the bonding process was
spontaneous. The calculated ΔH (negative) and ΔS (positive) values
from Figure 7 indicated that the main force responsible for the bind-
ing interaction of K4 with BSA was electrostatic interaction.
3.3
|
Energy transfer from BSA to K4
FIGURE 6 Double logarithmic plots for fluorescence quenching
of BSA (10 μM) with different concentrations of K4 in Tris–HCl buffer
solution (pH 7.4) at different temperatures
Fluorescence resonance energy transfer (FRET) is used in many fields
such as photosensitization, ion sensors, and investigation of supramo-
lecular interactions. FRET is a process in which energy is transmitted
from one fluorescence molecule (donor = D) to another molecule
(acceptor = A). The transfer from donor molecule to acceptor mole-
cule takes place nonradiatively. The possibility of energy transfer
depends on parameters such as quantum yield of the donor, spectral
overlap between the donor emission and the acceptor absorption
spectra, relative orientation of the donor and acceptor transition
dipoles, and the distance between donor and acceptor transition
dipoles.[26]
3.2
|
Determination of interaction forces between
BSA–K4 using thermodynamic parameters
It is known that interactions between protein and fluorescent mole-
cule are caused by noncovalent interactions such as van der Waals
forces, hydrogen bonding interaction, hydrophobic interaction, and
electrostatic interaction. To understand the forces responsible for
interactions between protein and fluorophore, it is important to deter-
mine thermodynamic parameters such as the enthalpy change (ΔH),
entropy change (ΔS), and free energy change (ΔG). The type of possi-
ble interaction can be determined by considering the direction of ther-
modynamic parameters. For this, there are three possibility: (1) if ΔH
and ΔS values are both positive, a hydrophobic interaction is present;
(2) if van der Waals forces and/or hydrogen bonding interaction are
available, ΔH and ΔS values are both negative; and (3) if ΔH is nega-
tive and ΔS is positive, electrostatic interactions are present.[25] To
determine the type of interaction between K4 and BSA, the thermo-
dynamic parameters were calculated using the van ’t Hoff equation
eqn (3) and the thermodynamic equation eqn (4):
ΔH ΔS
lnK = −
+
ð3Þ
ð4Þ
RT
R
ΔG = ΔH−TΔS
FIGURE 7 van ’t Hoff plot for the interaction of K4 with BSA

6
BOZKURT AND GUL
TABLE 2 Thermodynamic parameters for the interaction of BSA
with K4 at different temperatures
where QD is the quantum yield of the donor in the absence of the
acceptor, η is the refractive index of the medium, κ2 (2/3) is the orien-
tation factor[26] η and QD were used as 1.3361 and 0.15, respectively
in this study.[27] J(λ) is the spectral overlap integral between the emis-
sion spectrum of the donor and the absorption spectrum of acceptor
and this value was calculated using eqn (7):
T (K)
288
298
308
318
ΔG (kjmol−1
−27.95
)
ΔH (kjmol−1
−6.35
)
ΔS (jmol−1 K−1
)
75.00
−28.70
−29.45
Ð
−30.20
∞FDðλÞεAðλÞλ4dλ
0
Ð
JðλÞ =
ð7Þ
∞FDðλÞdλ
0
The fluorescence measurements for BSA, K4 and the BSA–K4
pair were taken at the excitation wavelength of the donor
(λexc = 280 nm), to show that there may be energy transfer between
BSA and K4. The fluorescence spectrum of BSA–K4 pair gave two
peaks at 331 and 445 nm (Figure 8). As can be seen in Figure 8, the
fluorescence peak of the BSA–K4 pair at 331 nm had a lower inten-
sity than the peak from BSA alone; the fluorescence peak of the BSA–
K4 pair at 445 nm had higher intensity than the peak which belonged
to K4 alone. This result proved the possibility of energy transfer
between BSA and K4.[26]
where FD(λ) is the normalized fluorescence intensity of the donor in
the absence of acceptor and εA(λ) is the extinction coefficient of the
acceptor. The spectral overlap for BSA and K4 is presented in
Figure 9. J(λ) was calculated as 6.47 × 1012 using eqn (7). Moreover,
the values of E, R0 and r0 were 50.0%, 1.62 nm and 1.64 nm, respec-
tively. The indication of high probability energy transfer between the
donor (BSA) and the acceptor (K4) is that the distance between the
donor–acceptor is on the 2–8 nm scale with the rule
[28]
0.5R0 < r0 < 1.5R0.
The energy transfer efficiency (E) is one of the important parame-
ters for FRET. E was calculated using equation below (eqn (5))[23]
:
3.4
|
Investigation of changes in BSA conformation
IDA
R06
R60 + r60
with K4 binding
E = 1−
=
ð5Þ
ID
Synchronous fluorescence is one technique used to examine changes
in the conformation of a protein. BSA has fluorescence properties due
to the presence of the Trp, Tyr, and Phe residues. The polarity
changes around the Tyr or Trp residues are analyzed using Δλ (the dif-
ference between the excitation wavelength and the emission wave-
length) values. If Δλ is 15 nm, the synchronous fluorescence spectrum
of BSA reflects changes around the Tyr residue. However, if
Δλ = 60 nm, the synchronous fluorescence spectrum of BSA reflects
the changes around the Trp residue.[29] The changes in the BSA syn-
chronous fluorescence spectra with increasing K4 concentration are
given in Figure 10(a, b). When Δλ was 60 nm, the fluorescence inten-
sity of the Trp residue of BSA decreased regularly and the
where ID is the relative fluorescence intensity of the donor in the
absence of acceptor and IDA is the relative fluorescence intensity of
the donor in the presence of acceptor, r0 is the distance between
donor and acceptor and R0 is a distance at which energy transfer effi-
ciency is 50%. This distance can be calculated using the following
equation (eqn (6)):
ꢀ
ꢁ
1=6
R0 = 0:211 κ2η−4QDJðλÞ
ð6Þ
FIGURE 8 Fluorescence spectra of BSA (10 μM), K4 (100 μM)
and BSA-K4 in Tris–HCl buffer solution (pH 7.4) (λexc 280 nm)
FIGURE 9 Spectral overlap between BSA and K4

BOZKURT AND GUL
7
FIGURE 11 FTIR spectrum of BSA (10 μM) in the absence or
presence of K4 (100 μM)
4
|
CONCLUSION
This study focused on the interaction between BSA and K4, which is
a new pyrazoline derivative, using UV–vis light absorption, and
steady-state and synchronous fluorescence measurements. The
results showed that static quenching occurred between K4 and BSA
by means of ground-state complex formation. This result was con-
firmed by determining that the Stern–Volmer quenching constant
decreased with increasing temperature. The binding constant and
binding site values calculated at four different temperatures reflected
that BSA and K4 formed a complex in a 1:1 molecular ratio and that
their binding interactions were not stable. It was determined that the
binding process was spontaneous and the BSA–K4 interaction was
electrostatic according to the thermodynamic parameters calculated
using the van ’t Hoff equation. Energy transfer between BSA–K4 was
also examined. It was observed that there was a high rate of energy
transfer (50.0%) between two molecules. The changes in the confor-
mation of BSA via binding of K4 were discussed with synchronous
fluorescence measurements. The results revealed that K4 bound to
the BSA molecule from the Trp region. FTIR measurements were
recorded to investigate possible perturbations in the BSA structure
upon addition of a K4 solution. FTIR results showed that there was no
significant change in the polarity around the Trp residue. It is thought
that the determination of interactions between the possible drug can-
didate compound K4 and the model protein BSA will provide very
useful information for researchers for biophysical studies.
FIGURE 10 Synchronous fluorescence spectra of BSA (10 μM) at
pH 7.4 in the presence of different concentration of K4 at room
temperature Δλ 15 nm (a) and Δλ 60 nm (b)
fluorescence peak maxima of BSA were significantly blue shifted
(from 321 nm to 317 nm) with addition of K4. When Δλ is 15 nm, the
fluorescence intensity of the Tyr residue of BSA did not change regu-
larly with K4 concentration. These results suggested that the confor-
mation of the Trp residue was more affected than Tyr upon BSA–K4
interaction.[30]
To investigate possible perturbations in the BSA structure upon
addition of a K4 solution, we performed some experiments using FTIR
spectroscopy. The amide I and amide II bands of proteins in the
1600–1700 and 1500–1600 cm−1 region, respectively, provided use-
ful information about protein conformation in the native and unfolded
states. Figure 11 shows the FTIR spectra of BSA in the absence or
presence of K4. In the absence of K4, the infrared spectrum for BSA
presented two peaks at 1648 and 1555 cm−1 assigned to amide I and
amide II, respectively. When the FTIR spectrum was recorded in the
presence of K4, the amide I and amide II bands remained unchanged,
not only in their relative position, but also in relative intensity. These
results showed that there was no significant change in the polarity
around the Trp residue.[21]
ACKNOWLEDGEMENTS
Authors thank Mehtap Tugrak for her kind contributions during the
synthesis and NMR studies of this compound.
ORCID
Ebru Bozkurt
https://orcid.org/0000-0002-5345-9718

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BOZKURT AND GUL
[19] M. S. Ali, H. A. Al-Lohedan, J. Mol. Liq. 2017, 236, 232.
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