Crystal structure, Hirshfeld analysis and HSA interaction studies of N'-[(E)-(5-bromothiophen-2-yl)methylidene]-3-hydroxynaphthalene-2-carbohydrazide

Article abstract of DOI:10.1016/j.molstruc.2019.03.070

Synthesis of ONO tridentate imine based molecule N'-[(E)-(5-bromothiophen-2-yl)methylidene]-3-hydroxynaphthalene-2-carbohydrazide (HTp) is gained quick attention because of its operational simplicity carried by both conventional and microwave methods and structurally it was elucidated by analytical, spectral, X-ray crystallography and Hirshfeld surface techniques. Analysis from the X-ray study provided the details of molecular structure, packing and hydrogen bonding properties in the solid state. The molecule is crystallized in monoclinic P21/c space group. The apparent binding affinity for the coupling of human serum albumin (HSA) with HTp drug molecule was examined through in vitro spectroscopic methods and molecular docking studies. HTp strongly quenched the intrinsic fluorescence intensity of HSA in terms of a static mode. The thermal based binding parameters were quantified from the fluorescence quenching experimental data. The energy transfer efficiency determined according to F?rster's theory. Besides, the experimental results were in line with the molecular docking results.

Full text of DOI:10.1016/j.molstruc.2019.03.070

Accepted Manuscript
Crystal structure, Hirshfeld analysis and HSA interaction studies of N'-[(E)-(5-
bromothiophen-2-yl)methylidene]-3-hydroxynaphthalene-2-carbohydrazide
Savithri K, BC Vasantha Kumar, Chandra, H.D. Revanasiddappa
PII:
S0022-2860(19)30349-7
DOI:
https://doi.org/10.1016/j.molstruc.2019.03.070
MOLSTR 26339
Reference:
To appear in: Journal of Molecular Structure
Received Date: 8 November 2018
Revised Date: 16 March 2019
Accepted Date: 19 March 2019
Please cite this article as: S. K, B.V. Kumar, Chandra, H.D. Revanasiddappa, Crystal structure,
Hirshfeld analysis and HSA interaction studies of N'-[(E)-(5-bromothiophen-2-yl)methylidene]-3-
hydroxynaphthalene-2-carbohydrazide, Journal of Molecular Structure (2019), doi: https://
doi.org/10.1016/j.molstruc.2019.03.070.
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Title page
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Crystal structure, Hirshfeld analysis and HSA interaction studies of
N'-[(E)-(5-bromothiophen-2-yl)methylidene]-3-hydroxynaphthalene-2-
carbohydrazide
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Savithri K1, B C Vasantha Kumar1, Chandra2 and H D Revanasiddappa1*
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1Department of Studies in Chemistry, University of Mysore, Mysuru - 570 006, Karnataka, India
2Department of Physics, National Institute of Engineering (NIE), Mysuru- 570 008, India
*Corresponding author. Tel.: +91 9449271137, e-mail: hdrevanasiddappa@yahoo.com
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Abstract
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Synthesis of ONO tridentate imine based molecule N'-[(E)-(5-bromothiophen-2-
yl)methylidene]-3-hydroxynaphthalene-2-carbohydrazide (HTp) is gained quick attention
because of its operational simplicity carried by both conventional and microwave methods
and structurally it was elucidated by analytical, spectral, X-ray crystallography and
Hirshfeld surface techniques. Analysis from the X-ray study provided the details of
molecular structure, packing and hydrogen bonding properties in the solid state. The
molecule is crystallized in monoclinic P21/c space group. The apparent binding affinity for
the coupling of human serum albumin (HSA) with HTp drug molecule was examined
through in vitro spectroscopic methods and molecular docking studies. HTp strongly
quenched the intrinsic fluorescence intensity of HSA in terms of a static mode. The thermal
based binding parameters were quantified from the fluorescence quenching experimental
data. The energy transfer efficiency determined according to Förster's theory. Besides, the
experimental results were in line with the molecular docking results.
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Key points: Imine base; X-ray study; Hirshfeld analysis; Human serum albumin;
Molecular docking;
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Introduction
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1. INTRODUCTION
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It is well known that the acid hydrazines R-CO-N=N-NH2 and corresponding
hydrazones R-CO-NH-N=CH-R' have interesting and remarkable biological activity among
heterocyclic compounds due to their distinct physical, chemical and pharmaceutical
properties. Nowadays, the N-N linkage is employed in various bioactive agents as a key
structural motif [1]. It is well known that hydrazone compounds are a kind of distinct imine
bases, which possess versatile chemical and pharmacological properties owing to their
potential applications [2–8]. Aroyl hydrazones have stimulating ligation properties because
of their facile keto-enol tautomerization and availability of several coordination sites [9–
11]. The physiological effects of thiophene are similar to those of benzene (bioisostere),
with habitually superior pharmacodynamics and pharmacological properties [12].
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To accomplish the binding strategy of small molecules with the biomacromolecules
have been received great importance. Human serum albumin, the principal extracellular
ubiquitous blood serum constituent (~60 % of total plasma protein) serves as the major
transporter, play physiological role in bringing solutes into the bloodstream, deliver them to
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the target site and maintain the optimal pH range 4-9 as long as 10 h at 60 C [13-15]. A
multi-domain structure (Sudlow’s sites) of HSA accounts for high binding affinity to the
various chemical entities which is also a prerequisite factor for the application with the
biological drug molecules and make the protein a chief regulator of intercellular metabolic
and the pharmacokinetic behavior of many drugs in the body as well [16]. Hence, the
molecular structural features of HSA based drug delivery systems invariably provides
information on the therapeutic effect of a compound and have an immense interest in the
development of biologically active new compounds. Drug-protein interaction effects on the
drug absorption, circulation and abolition in the circulatory system. The binding efficiency
of the ligands to serum albumin is believed to affect their half-life [17]. Moreover, such
binding helps to increase drug solubility in plasma by preventing rapid exclusion of drugs
from the bloodstream, decreases its toxicity and prolong it’s in vivo half-time with the
protection from the oxidation [18].
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The exigency of the binding process of the imine based ligand (HTp) with HSA in
providing potential drugs is the target that the author had. This was achieved using
absorption, emission spectroscopy and molecular modeling under simulated physiological
conditions. The employed UV-Vis and fluorescence spectroscopic techniques were
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Introduction
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adequate to study the binding behavior of ligands with proteins due to its sensitivity,
reproducibility and convenience. The binding parameters, thermodynamic functions and
fluorescence resonance or Forster’s non-radiative energy transfer (FRET) on the protein
microenvironment were interpreted. Furthermore, it was curious to locate the binding sites
of ligands on HSA, and this was able to identify the pose of the molecules in the binding
sites by using docking tools and the binding score represents the strength of binding
affinity.
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2. EXPERIMENTAL
2.1. Instruments and operations
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All the used chemicals throughout the experiments were of reagent grade. A precision
digi melting point apparatus was used to determine the melting point of HTp in an open
capillary tube. For the structural identification, an Agilent NMR spectrometer was used to
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record H and C NMR spectra with TMS as a reference in DMSO-d6 solvent at ambient
temperature. LCMS (2010EV) Shimadzu spectrometer was employed to record the mass
spectrum. CHN microanalysis was performed with a Perkin-Elmer elemental analyzer. UV
measurements (200-800 nm) were performed with UV-Vis spectrophotometer (DU 730 M/S
Beckman coulter, ‘Life Science’ USA) using DMSO solvent. Vibrational spectra were
recorded via attenuated total reflection (ATR) on a Perkin-Elmer 783 spectrophotometer in
the range of 4000-400 cm-1.
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2.2.Synthetic procedure of compound (HTp):
N'-[(E)-(5-bromothiophen-2-yl)methylidene]-3-hydroxynaphthalene-2-carbohydrazide
OH
O
OH
O
+
O
CH3OH
S
HN NH2
Br
HN N
S
Br
HTp
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Scheme 1
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Classical method
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A methanolic solution of 5-Bromo-2-thiophenecarboxaldehyde was added to a well-
stirred methanolic solution of 2-amino-3-hydroxy-2-naphthoic acid hydrazide in an
equivalent ratio. The reactant mixture was continued to stir for an hour at room temperature
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(26 C), during which a creamy white precipitate was formed and TLC was utilized to
monitor the reaction. The solid product was recrystallized with the solvent system of
methanol and chloroform (3:7) by a slow evaporation method. Yield=83 %.
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Microwave irradiation method
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An equivalent reaction mixture of the 2-amino-3-hydroxy-2-naphthoic acid hydrazide
and 5-bromo-2-thiophenecarboxaldehyde was placed in a flask and subjected to microwave
power at 300 W with the temperature of 80 ᵒC for ~6 minutes. TLC monitored the completion
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of the reaction and the content was left for evaporation at ambient temperature (26 C). The
colorless crystalline product was formed from the recrystallization of methanol/chloroform
(3/7) system. Yield=89 %.
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Mp= 224-226 °C; Rf 0.55, Anal. (%) for C16H11BrN2O2S: Found (calc.): C 51.20 (51.21), H
2.92 (2.95), N 7.43(7.47); LCMS m/z 375.23; FT IR (υmax./cm-1); (HC=N) 1663, (υOH)
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3253; H NMR (DMSO-d6 400 MHz/295 K/δ in ppm) 9.776 (s, 1H, HC=N); 10.04 (s, 1H,
OH), 11.16 (s, 1H, NH), ppm 7.21-7.26 (m, 2H, Ar); 13C NMR (DMSO-d6 100 MHz)
154.333, 164.039, 143.132, 141.273, 136.257, 132.220, 131.810, 130.664, 129.070, 128.645,
127.219, 126.278, 124.236, 120.981, 115.509, 110.994.
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2.3. X-ray structure determination protocol
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The titled imine based molecule C16H11BrN2O2S has been synthesized; high-
resolution X-ray diffraction determines its crystal structure. The analysis provided the
details of the molecular packing and hydrogen bonding of HTp.
A single crystal of N'-[(E)-(5-bromothiophen-2-yl)methylidene]-3-hydroxynaphthalene-2-
carbohydrazide (HTp) with dimension 0.30× 0.25 ×0.20 mm was chosen for diffraction
studies. The crystallographic X-ray diffraction data were collected at 296 K on a Bruker
APEXII CCD detector with CuKα radiation (1.54178 Å). Processing and reduction of
crystal structure raw data were carried out using APEX2 and SAINT [19]. A crystal
structure of HTp was solved by direct methods; full-matrix least-squares refinement was
performed using SHELXS-97 and SHELXL-97 [20, 21] with a final residual value of
R1=0.045. The thermal ellipsoid plot [22] at 50% probability of the compound is publicized
in crystallographic figures.
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2.3. Hirshfeld surface analysis
Analysis of molecular crystal structure by Hirshfeld method to venture crystal
packing diagrams of various models, internuclear distances and angles, hydrogen bond
interactions and credentials of close contacts deemed to be significant given molecule as
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organic wholes; thereby the crystal lattice can be stabilized. Crystal Explorer 3.0 [23]
computational software was employed to perform Hirsh surface analysis.
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2.4. Preparation of HSA stock solution
Human serum albumin (HSA, >99% purity, essentially globulin free) was procured
from Sigma Aldrich Inc. An initial solution of HSA was prepared by dissolving and dialyzed
it in a buffer (Tris-HCl/NaCl at pH 7.4) prior to use, and it was kept at 4 °C for no longer
than two days. The standard working solutions of 0-25 µM were made the stock.
ꢀ%
∑
HSA concentration in buffer was determined by UV absorbance with ꢁꢂꢃꢄꢅ is 35700
M-1cm-1 [24].
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2.4.1. Electronic absorption and Fluorescence spectral measurements
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The UV-Visible spectra were recorded with a quartz cell of 1 cm path length.
Absorption spectra of HSA-HTp were scanned from 200 to 800 nm with concentration ranges
from 0-25 µM at ambient temperature.
Steady-state fluorescence spectral measurements were recorded on F-2000
spectrofluorometer, Hitachi equipped with a 150 W Xenon lamp at 298, 303 and 310 K. Very
dilute solutions of HSA (2×10-6 mol/L) and HTp (0-6×10-6 mol/L) were taken. The excitation
wavelength was made to fix at 295 nm, while the emission wavelength measured from 300 to
500 nm with a slit width of 5 nm. The Origin 7.0 software provided with Microcal was
employed to analyze the titration heat profiles to find binding parameters. Data well fitted to
a single set of identical binding parameter [25].
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The inner filter effect could stand as an error for any fluorescence measurement
particularly where an absorbing component is being titrated into the cuvette [26]. An
absorption spectrum of the ligand overlaps with the excitation and emission of HSA. Hence,
Inner filter effect and dilution effects were corrected for fluorescence intensities before
binding and quenching data analysis. Fluorescence intensity was corrected based on the Eq.
(1)
ꢏꢍ )/ꢑ
ꢆꢇꢈꢉꢉ = ꢆꢈꢊꢋ ꢌ(ꢍ
…(1)
ꢌꢎ
ꢌꢐ
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The corrected and observed fluorescence intensities are referred to as Fcorr and Fobs,
respectively and ꢍem and ꢍex are the absorption of HTp at the emission and excitation
wavelength of HSA. All the HTp solutions were prepared by weight/volume (w/v) basis.
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The fluorescence quenching phenomena are conventionally described in terms of Eq. (2)
given by Stern-Volmer [27].
ꢆ
ꢒ
ꢖ ꢘ
ꢖ ꢘ
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= ꢓ + ꢔꢋꢕ ꢗ = ꢓ + ꢔꢙƮꢈ ꢗ
… (2)
ꢆ
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where, the quenched fluorophore intensity and the steady-state fluorescence intensity are
represented by F and Fo, respectively. [Q] is the molar concentration of HTp, Stern-Volmer
quenching constant is given by Ksv and kq is the apparent biomolecular quenching rate
constant. Ʈo stands for the integral/average lifetime of protein fluorophore without quencher
and the fluorescence lifetime of the biomolecule is 10-8 s [28, 29].
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Energy transfer between HTp and HSA
Energy transfer between HTp and HSA gives valuable information about their
binding. Equimolar concentration (10-6 mol L-1) of the absorption spectra of ligands and the
fluorescence spectrum of HSA were recorded at 298 K with the range from 200 to 500 nm
(n=3 replicates). Then, the overlap of the UV absorption spectrum of HTp with the emission
spectrum of HSA was used to calculate the energy transfer and subtracted the buffer
background readings.
2.5. Molecular docking studies
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AutoDock was employed for docking simulation. The crystal structure of the N'-[(E)-
(5-bromothiophen-2-yl)methylidene]-3-hydroxynaphthalene-2-carbohydrazide (HTp) was
selected for docking study with receptor Human Serum Albumin target, obtained from the
protein data bank (PDB Code: 1H9Z). The AutoDock has to contain core technology, which
is used for simulations of bio-organics and macromolecules. Simulations are an essential tool
to validate HTp pose geometries, stability and the generation of macromolecule
conformations. For the docking methodology, Lamarckian genetic algorithm was used to
figure out the optimal docking position between small molecules and macromolecules. Also,
partial atomic charges were calculated using Gasteiger method. AutoDock tools (ADT) was
used to define the rotatable bonds in the ligand. In addition to that, both Kollman charges and
polar hydrogen bonds were assigned using ADT.
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3.
Results and Discussion
3.3. Chemistry
A potential imine-based molecule (HTp) was synthesized (Scheme 1) by two ways
where microwave assisted synthesis is more accessible, convenient and have short reaction
period. The obtained yield (89%) from this was found to be much more than the conventional
method (83%). The product HTp is stable at atmospheric conditions, non-hygroscopic and
moderate solubility in alcohol but highly soluble in hot alcohol and all common organic
solvents. Analytical data of the compound are reliable with the proposed formula and are
summarized in the above synthetic section.
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The 13C NMR spectrum of HTp is displayed in Fig. S1† shows a characteristic signal
at 143.132 ppm for the azomethine (HC=N) carbon. The chemical shift positions of other
carbons have appeared at 159.638, 158.848, 135.710, 124.494, 121.596, 115.471 and 109.150
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ppm. A singlet signal peaked at 9.77 ppm in H NMR spectrum of HTp is attributed to the
azomethine proton. The OH proton resonates as a singlet equivalent to single hydrogen at
10.04 ppm indicates that the OH proton is probably involved in the formation of
intramolecular hydrogen bonding. NH proton appears at11.16 ppm and aromatic protons are
resonated as multiplet in the range of 7.0-7.92 ppm. The molecular ion peak at m/z 375 [M]
and 376 [M+1] in the mass spectrum of HTp corresponding to its formulation and confirms
the presence of bromine in the molecule. The IR spectrum of the molecule exhibits a sharp
band at 1662 cm-1 due to the azomethine group (HC=N) vibration. Some significant
stretching bands associated with N-H, C=O, C=N and O-H were observed in the range 3339-
3175, 1630–1660, 1610-1635 and 3490-3515 cm-1, respectively [9,30]. Also, a new C–O
stretch has appeared in the range 1260–1285 cm−1. It suggests the occurrence of keto-imine
tautomerization of hydrazone groups. The electronic spectrum (Fig. S2†) of HTp show
intense bands in the range 320-385 nm attributed π →π*/n →π* transitions.
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3.3.1. Crystal features of HTp
The X-ray crystal and molecular structure analysis of the compound (HTp) suggested that the
compound crystallizes in monoclinic system under the space group P21/c, with cell
parameters a = 16.5155(19)Å, b = 11.4248(14)Å, c = 7.9982(10)Å, β = 92.993(4)Å and Z=4.
Table S1† detailed the crystal data and structure refinement for HTp. In the Oak Ridge
thermal ellipsoid plot (ORTEP) drawn for the HTp molecule (Fig. 1), the thermal ellipsoids
for naphthalene (C1—C10) and thiophene groups (S1—C13—C14—C15—C17) are almost
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collinear as appears from the relative dihedral angle of 1.70(12)° between the two groupings.
The hydroxyl group and bromine moiety are approximately coplanar with the naphthalene
and with atom O1 and Br1 deviating from the mean plane by -0.047 (2) Å and -0.019 (1) Å,
respectively. Also, carbohydrazide group is present in anti-periplanar conformation relating
to the naphthalene moiety, as indicated by the torsion angle value of -179.9 (2)° (N2—N1—
C11—C10). The crystal structure is stabilized by N(1)—H(1A)...O(1) intramolecular H-
bonds. The packing of the molecules is as shown in Fig.S3†.
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3.3.2. Hirshfeld surface analysis
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The molecular Hirshfeld surface (3D dnorm surface, shape index and curvedness) is
displayed in Fig.2. The deep red spot with large circular depressions visible on the
normalized contact distance (dnorm) surfaces with di and de indicates hydrogen bonding and
other weak contacts. The shape index displays red areas parallel to C-H-C (or C-H--π)
interactions, which coincide with similar regions in the dnorm surface. The van der Waals
(vdW) radii encodes particularly for intermolecular interactions [31]. The shape index is
extremely sensitive to subtle variations in the surface shape and is consistent with 2D
fingerprint plots. The curvedness is usually tending to divide the surface into patches,
indicates neighboring molecular interactions [32]. The two-dimensional fingerprint plots
further emphasized the significance of the H--H, C--H, O--H, S--H and H--Br interactions in
determining its arrangement in the solid state. The significant contribution obtained from the
interactions which make up C—H (15 %) and H—H (22.8 %) of the surface. Quantification
of different interactions and their relative contributions are displayed in Fig. 3.
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3.4. HSA Binding studies
UV–Vis spectral features
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The simple but effective method to study the HTp-HSA binding and to investigate the
structural modification of the aromatic protein residues upon drug accumulation is UV-
Vis/electronic absorption spectroscopy. As can be seen in Fig. 4, a sharp absorption peak at
~206 nm resulted by n to π* transition for the peptide bond of protein helix and a weak peak
at 278 nm is assigned to π to π* spin forbidden transition of the phenyl rings in aromatic
amino acids Trp, Tyr and Phe residues [33]. A hypochromic effect is observed (Fig. 4) in the
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absorption spectrum of HSA on gradual incremental addition of compound HTp (10 µM), the
reduced absorption intensity of HSA after the enhancement of HTp implied that the aromatic
amino acid residues of the protein in a hydrophobic cavity were exposed to an aqueous
environment upon complexation of HTp with HSA and could cause structural changes in the
protein.
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Fluorescence spectral analysis
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Fluorescence titration is a qualitative spectroscopic technique of the binding of the drug
to the proteins. Fluorescence titration is a qualitative spectroscopic technique to obtain the
details on the binding of the drug to the proteins, binding sites and intermolecular distances
exists between them. The fluorescence quenching is any process which diminishes the
fluorescence intensity of a compound. The quenching can be the consequence of a variety of
molecular interactions may include excited-state/ground state complex reactions, energy
transfer or collisional quenching. The fluorescence quenching of ligands and HSA is due to
the intrinsic tryptophan residue, which is used as an inherent probe to comprehend the
interaction of ligands with the protein as the other two aromatic fluors viz. tyrosine and
phenylalanine have meager quantum yield which undergoes ionization when they locates
close to carboxyl groups, so they get quenched completely [15].
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The fluorescence quenching of HTp and HSA is due to the intrinsic tryptophan residue,
which is used as a prime tool to understand the interaction of HTp with the protein (Fig. 5).
While titrating HTp with HSA, the maximum fluorescence intensity at 342 nm was decreased
with the addition of the compound continues, which leads to changes in the
microenvironment around tryptophan-214 when excited at 295 nm. The above data specifies
that the HTp binding site exists near to the tryptophan residue, which is located at the IIA
subdomain of HSA.
Diverse molecular interactions may results in fluorescence quenching of intrinsic
fluorophore due to energy transfer, molecular rearrangement and collision quenching. The
observed fluorescence quenching is likely related to collisional quenching by the ground state
complex formation when the inner filter effect is removed [34] Fluorescence quenching can
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be static or dynamic type according to their different temperature dependence. Hence,
fluorescence tests were performed at temperatures (298, 303 and 310 K) and the quenching
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data were analyzed using Stern-Volmer (SV) plot expressed in equation (2). SV quenching
plot of Fo/F v/s [Q] shown in Fig.5 helps to get the values for Kq and Ksv. The binding
parameters with a correlation coefficient (R2) at chosen temperatures are tabulated in Table
1. Ksv reflects the efficiency of fluorescence quenching which is given by the slope. The
linear regression of the SV plot proposes the binding process of HTp to HSA is indicative of
homogeneity of quenching and the phenomena found to be ‘static’ since the observed Ksv
values drastically decreased on raising the temperature with high magnitude of kq (1012 M-1S-
1) is found. The dynamic quenching occurs when the quencher and fluorophore approach
each other at excited state, while static quenching occurs when non-fluorescent complex
formed in the ground state.
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3.4.1. Analysis of binding equilibria
Fluorescence quenching data analysis afforded quantitative evaluation of the binding
constant (Kb) and binding stoichiometry (n) for the HSA-HTp interaction process and
analyzed according to modified Stern-Volmer Eq. (3)
log ꢚꢛ ꢝꢛꢞ = logKꢟ + n logꢖQꢘ …(3)
ꢜ
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ꢛ
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where, the quenched fluorophore intensity and the steady-state fluorescence intensity are
represented by F and Fo, respectively. The double logarithm regression plot of log(Fo-F)/F)
v/s log[Q] is shown in Fig. S4†, where slope and intercept gives Kb and n, the values of the
same at performed temperatures are reported in Table 1. It was observed that the binding
constant (Kb) decreases as temperature rises which indicates the formation of unstable HSA-
HTp complex.
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The values of n are observed to be close to unity indicating that there is one independent class
of binding site on HSA available for HTp. The thermodynamic parameters for the HSA-HTp
interaction process have also been estimated from the binding constant. The analysis of
binding isotherms gives the major binding parameters that fit as a function of the binding
constant K, ꢀSo (entropy of association) and ꢀHo (enthalpy of association) of the protein.
In general, the non-covalent nature of binding forces exists between quencher and
macromolecules. Hydrogen bonds, van der Waals force, hydrophobic force and electrostatic
forces are the four major molecular acting forces of interaction exists between HSA and HTp.
Thermodynamic analysis of protein-HTp binding shows the importance of the binding
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constant (K), entropy change (∆S), enthalpy change (∆H) and free energy change (∆G) in
determining the predominant type of interactions in HTp–protein complex system [35] and
were used to elucidate probable binding forces of drug-protein interactions and these can be
intended by Van’t Hoff and thermodynamic eq. (4) and (5) [36].
∆ꢥ°
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lnKꢟꢠꢡ = − ∆ꢢ°
+
…(4)
ꢣꢤ
ꢣ
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∆G° = ∆H° − T∆S° = −RTlnKꢟꢠꢡ …(5)
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in which, Kbin is apparent binding constant, T is the absolute temperature and R is gas
constant (1.987 cal/mol·K). Slope and intercept of the plot of logK v/s 1/T (Fig. S5†) gives
ꢀH° and ꢀS°, respectively. Gibbs-Helmholtz equation 5 helps to calculate ꢀG° and the
results are summarized in Table 2. A spontaneous and exergonic binding process observed
through the negative free energy change.
The obtained negative ꢀH° (-78.314 KJmol-1) and ꢀS° (-361.304 KJmol-1) indicate
predominant interactions in the binding process [37]. The compound HTp could actively
quench the intrinsic fluorescence of protein as the decreasing values of the binding constant
with an increase in temperature leads to the decrease in the stability of the HSA-HTp
complex which in turn suggests static quenching mechanism. Indeed, the unfavorable
enthalpy loss is canceled by the much larger entropic gain reveals hydrogen bonding and
weak van der Waals forces played a significant role in the interaction.
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3.4.2. Resonance energy transmission between HSA and HTp
The significance of the energy transfer in biochemistry is that the efficacy of
transmission can be used to measure the distance between the ligand and the tryptophan
residues in the HSA. To find out a distance between HTp-Trp protein, it is possible to study
Forster’s non-radiative process or fluorescence energy transfer (FRET) from donor (HSA) to
acceptor (HTp). FRET occurs when the fluorescence spectrum of HSA (fluorophore)
overlays on the UV absorption spectrum of HTp (acceptor) and is displayed in the Fig.S6†.
HSA has a solitary tryptophan residue (Trp 214) which gives a fluorescence emission at 300-
400 nm.
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As per the FRET theory [38], the energy transfer efficiency (E) is given by Eq. (6) and (7)
which is interrelated to the distance (r) from donor to acceptor and the critical energy transfer
distance when E is 50 % is known as Forster’s radius (Ro).
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ꢦ
ꢛ
ꢣ
ꢜ
1
E = 1 − ꢛ
=
ꢨ.…(6)
ꢏꢧ
ꢦ
ꢣ
ꢜ
ꢜ
ꢩ
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Rꢃ = 8.79kꢁNꢝꢪJφNꢝꢪ …(7)
where, the donor’s fluorescence quantum yield ΦD is 0.15, the orientation factor k2 =2/3,
and the refractive index (n=1.336) were obtained. The overlap integral of the absorption
spectrum of the HTp and the fluorescence emission spectrum of the HSA termed as J, and is
given by the Eq. (8)
ꢰ
J = Σꢫ (ꢭ)ꢮ (ꢭ)ꢭ ꢱꢭ …(8)
ꢬ
ꢯ
8
Σꢫ (ꢭ)ꢱꢭ
ꢬ
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where, the fluorescence intensity of the donor at wavelength λ is termed as FD(λ) and the
acceptor’s molar absorption coefficient at λ wavelength is ꢲꢳ(λ)
We observed a functional overlap between the emission spectrum of tryptophan and the
absorption spectrum of HTp (Fig. S5†). The values for J=4.67×10-14 cm3 Lmol-1, E=0.17,
Ro=2.49 nm and r=4.26 nm which were easy to obtain by using the equation 8.
These obtained data are consistent with a FRET mechanism being operative. i.e. The r value
is actually the average value between the bound drug and Trp214 (since HSA possess only
one tryptophan residue) and is found to be less than the standard value of 8 nm or on 2-8 nm
scale and 0.5 Ro < r < 1.5 Ro indicates a significant spectral overlap occurs between HSA-
ligands with high probability. Further, as per the prediction by Forster’s non-radiative energy
transfer theory, the more substantial value of r compared to that of Ro proves the static
quenching process [39, 40].
2.3.1. Molecular modeling study
The analysis was performed to decode the binding site of HTp against HSA. Crystal
structure of monomeric albumin revealed that the single polypeptide chain of heart-shaped
HSA consists three structurally homologous α-helical domains I(residues 1-195), II (196-33)
and III (34-585 amino acid residues) and each of the domain contains two subdomains A and
B, stabilized by 17 S-S bridges [41].
Using molecular docking simulations, the imine based ligand HTp was docked to the best
conformer of HSA and they generally found to bind within their hydrophobic pockets in
subdomains IIa and IIIA, such as site I and site II. The molecular docking analysis signified
the most critical regions of compound binding sites in the HSA. The ligand molecule (HTp)
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showed minimum binding energy of -7.5 kJ/mol with ligand efficiency -0.34. HTp molecule
shows hydrogen bond interaction with active site amino acid residues Arg257 and His288 at a
distance of 1.904 Å, 2.174 Å & 1.882 Å respectively. The molecular modeling results
revealed that the compound (HTp) have a good inhibition constant, vdW+Hbond+desol energy
with the best root mean square deviations. The best energy ranked details of the interaction
between the HTp and the receptor human serum albumin target (PDB Code: 1H9Z) in all the
runs of docking protocol have listed in Table S2†. Enfolding and H-bond interaction of
molecule (HTp) are in the active site pocket of serum albumin target (PDB Code: 1H9Z) as
shown in Fig. 6A and Fig. 6B.
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4.
CONCLUSION
2
3
We report the convenient synthesis of a tri-dentate imine based molecule (HTp) in
good yield. The molecular structure was well explored through crystallography, Hirshfeld
surface analysis and several spectroscopic techniques. As the key circulating protein in the
blood, albumin serves the excellent delivery platform for many exogenous and endogenous
compounds. The nature of the binding of synthesized HTp with HSA was studied using
fluorescence and UV-Vis absorption method under physiological conditions. The interactions
were confirmed through variations in the absorption spectra and intrinsic fluorescence
quenching of the HSA. Further study of the quenching process uses kq, ksv and kb parameters
and the higher values of Kb demonstrated strong interaction with HSA. The probable mode of
interaction strategy is found to be a static quenching process.
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FRET and fluorescence quenching model afforded the distance between the HSA and
HTp being the ratio 1:1 suggests that the high possibility of energy transfer has occurred.
HSA had a crucial effect on drug distribution and pharmacokinetics as the compound could
quench the protein fluorescence through static quenching phenomena. Quantitative assay for
the binding of HTp with HSA was proposed from multi-spectroscopic methods at different
temperature conditions and correlated by molecular docking technique. With the above data
provided, this will be the benchmark for the molecule HTp to get used for further clinical
applications in protein conjugation pharmacology.
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APPENDIX
CCDC 1548542 contains a supplement of crystallographic data for HTp.
ACKNOWLEDGMENTS
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One of the authors, Savithri K gratefully acknowledges the University Grants Commission,
New Delhi for the award of Senior Research Fellowship under RGNF Scheme.
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Fig.1. Ortep representation of HTp
de
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dnorm
d
i
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Shape index
Curvedness
Fig.2. 3dnorm surface, shape index and curvature mapped on Hirshfeld surface of HTp
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de
de
de
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
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3
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7
8
100%
di
O
H 5.2%
di
9
(Å)
0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4
S H 4.4%
di
(Å)
0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4
(Å)
0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4
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de
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de
de
2.4
2.2
2.0
1.8
1.6
1.4
1.2
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2.4
2.2
2.0
1.8
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1.4
1.2
1.0
0.8
0.6
H
H 22.8%
di
C H 14.4%
0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4
di
H Br 5.0%
0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4
di
(Å)
0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4
(Å)
(Å)
Fig.3. Fingerprint plot of the HTp.
Various contacts contributing to the total Hirshfeld surface of HTp molecule.
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Fig.4. UV absorption spectra of HSA in the absence and presence of varied concentrations of HTp
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2.15
A
B
1.75
1.35
0.95
298
303
310
0
1
2
[Q] e-6 M
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Fig.5.
a) Fluorescence spectra of HSA (5µM) in the presence of HTp
Arrow shows concentration ranges from 0-45 ꢁM at 298 K
b) SV curves for the binding of HTp-HSA at 298, 303 and 310 K at pH 7.4;
λex = 280 nm; λem = 340 nm;
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Fig.6. (A) Enfolding of molecule HTp in the active site pocket of Serum Albumin target (1H9Z)
(B) H-bond interaction of ligand molecule HTp with HSA.
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Table.1: The quenching constants (Ksv), binding constants (Kb), number of binding sites
(n) and corresponding correlation binding parameters for HSA-HTp interaction
system at different temperatures obtained from fluorescence spectrophotometry
Compound
Temp
(K)
Ksv ×104
(L/mol)
Kq ×1012
(L/mol/sec)
Kb ×105
(L/mol)
R2
*SD
n
HTp
298
303
310
2.8355 ±0.24 2.8355±0.21
44.452±0.47
37.132±0.35
13.135±0.22
0.9942 0.012
0.9883 0.021 1.0320
0.9801 0.024 1.1621
1.0461
2.7021±0.13
2.5801±0.57
2.7021±0.44
2.5801±0.32
5
6
R2 =Correlation coefficient for the Kb, *SD -standard deviation of the best fit
7
8
Table.2: Thermodynamic binding parameters for HSA-ligand interaction system at different
temperatures.
Compound Temp
(K)
Kb ×105
(L/mol)
∆Gº
(KJ/mol)
∆Hº
(KJ/mol)
∆Sº
(J/K/mol)
HTp
298
303
310
44.452±0.47
37.132±0.35
13.135±0.22
-33.518
-32.414
-29.186
-78.314
-361.304
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Fig.6. (A) Enfolding of molecule HTp in the active site pocket of Serum Albumin target (1H9Z)
(B) H-bond interaction of ligand molecule HTp with HSA.

ACCEPTED MANUSCRIPT
Highlights:
1. The tridentate imine based molecule (HTp) was synthesized conveniently both by
conventional and microwave methods.
2. Spectroscopic characterization, single x-ray crystallography and Hirshfeld surface
mapping analysis for its structural elucidation.
3. Biological assays (HSA binding studies) for the potential analysis of prepared compound.
4. Results revealed that the synthesized molecule is an efficient protein binding drug exhibit
higher binding affinity values was confirmed by both experimental (spectroscopic
methods) and computational molecular docking simulation.