4-Hydroxycoumarin Derivative : N-( diphenylmethyl )-2-[ ( 2-oxo-2 H-chromen-4-yl ) oxy ] acetamide Interaction with Human Serum Albumin

International Center for Young Scientists, National Institute for Materials Science, 1-2-1 Sengen, Tsukuba, Ibaraki 305-0047, Japan Department of Medical Physics, Anna University, Chennai 600025, India Centre for Nanoscience and Technology, Anna University, Chennai 600025, India Department of Physics, Periyar University, Salem 636011, India Nanotechnology Innovation Station, National Institute for Materials Science, 1-2-1 Sengen, Tsukuba, Ibaraki 305-0047, Japan


Introduction
Coumarin and its derivatives have been known for a long time, yet their vital role in plant and animal systems has not been thoroughly analyzed.Many coumarins and their derivatives exert antitumor, antiviral, anticoagulant, antiinflammatory, antioxidant, vasorelaxant, antimicrobial, and enzyme inhibition properties.
Serum albumins (Figure 1(a)) are the most fruitful proteins in the circulatory route of a wide diversity of various creatures and also blew the lid off the crucial first nature in the binding and transport of various endogenous and exogenous ligands.
e molecular interactions of proteins and lots of compounds like medicine and a few organic molecules are investigated successfully.However, the binding of 4-hydroxycoumarin products to serum albumins is now an emerging field to explore the role, in biological systems, and their mode of action in various diseases [14].
In the present study, synthesized N-(diphenylmethyl)-2-[(2-oxo-2H-chromen-4-yl)oxy]acetamide interaction with human serum albumin was carried out using various optical spectroscopy techniques.Also, computational techniques like molecular docking and density functional theory (DFT) were performed further to understand the hydrogen bonding, hydrophobic information with active site residues, internal stability, and charge distribution of the molecule at the atomic level [15].

Fluorescence Measurements.
e emission spectroscopy study was performed using a spectrofluorometer (ISA FluoroMax-2; Jobin Yvon-Spex, Edison, NJ); the spectral bandpass was fixed at 5 nm for monochromators of both wavelengths of excitation and emission, and the recorded wavelength region is 300-500 nm at 280 nm excitation.e lifetime fluorescence measurements of the coumarin derivative and human serum albumin complex were detected using Fluorolog (Horiba Jobin Yvon IBH, UK) with a fast response sensitive PMT detector (Hamamatsu Photonics, Japan) for an excitation wavelength of 280 nm to 450 nm. e decays were sliced and normalized each wavelength for HSA and HSA-coumarin complex.e excitation-emission matrix (EEM) was mapped with the following two conditions: the emission wavelength was recorded between 200 and 700 nm and the initial excitation wavelength was set to 200 nm with an increment of 10 nm.In EEM measurements, the spectral bandpasses were kept at 5 nm for both excitation and emission.In addition to that, the Forster resonance energy transfer analysis between the donor and acceptor and further single-point polarization studies between human serum albumin and the coumarin derivative complex were also carried out.

FTIR and CD Spectroscopy Measurements.
Fourier-transform infrared spectra (FTIR) of the human serum albumin solutions were administered at temperature by using an FTIR spectrometer (Interspec 2020; Estonia) in the attenuated total reflection (ATR) mode with a resolution of 4 cm −1 and 32 scans.e secondary structural analysis was carried out using circular dichroism (CD) for free human albumin and complex using a spectropolarimeter (Jasco J715; MD, USA) employing a quartz cell of 0.1 cm beam bath.

Docking Studies.
e human serum albumin structure was taken from the Protein Data Bank (PDB ID: 4L8U), and the coumarin derivative is drawn using the ChemDraw software.Proteins and small molecules are energetically minimized using the OPLS 2005 force field (Schrödinger LLC) used for molecular docking [15].

DFT Methods.
Energy minimization has been performed using the density functional theory (DFT) methodology with B3LYP functional and 6-311G * * basis sets for both coumarin molecule and cofactor with active site amino acids of HSA using the Gaussian 03 software.e electrostatic potential countermaps were pictured using the GView software [16].

Cell Viability Studies.
e cytotoxic assay for the drug was obtained using the Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Osaka, Japan).e cell density in the 96-well plate was 5000 cells/well, 24 hours later; the drug was added to the culture plate and incubated for another 24 h.After drug exposure, the cells were incubated with 10 µL CCK-8 for 2 h, and the absorbance at 450 nm was measured using a microplate reader (MTP-880Lab; Corona, Hitachinaka, Japan).
e cytotoxicity was represented as a percentage of untreated control cells.

Fluorescence Spectroscopy Studies.
e steady-state fluorescence spectroscopy is the most effective technique to understand the further binding mechanism of the HSAcoumarin derivative complex.In general, the human serum albumin emission will be at 350 nm due to the presence of tryptophan residue and other phenylalanines; tyrosine residues emission is considered to be less compared to tryptophan residues emission.
e emission spectrum of HSA (1 µM) and various concentrations of the coumarin derivative (0-3.6 µM) is shown in Figure 2(a), and it is indicated that there is a decrease without a shift in emission maximum of the HSA-coumarin derivative complex.Hence, we may conclude that fluorescence quenching occurs due to the presence of the coumarin derivative [17].
To know more about the type of quenching, we performed Stern-Volmer (Equation ( 1)) calculation at a different temperature as shown in Figure 2(b), and the calculated results of the quenching constant are tabulated in Table 1 [18,19]: Journal of Spectroscopy where F 0 and F is the free HSA and HSA complex (coumarin drug), k q is the bimolecular quenching constant (k q � K SV × τ −1 0 ), τ 0 is the lifetime of the fluorophore without the drug molecule (10 −8 s), K SV is the Stern-Volmer quenching constant (0.79 × 10 −5 , 1.00 × 10 −5 , and 1.33 × 10 −5 mol −1 •s −1 •L −1 ), and [Q] denotes the concentration of the drug.From Table 1, the calculation result of k q indicates that static quenching occurs due to the complex formation of the coumarin derivative.e binding constant and number of binding sites were calculated using the following equation [20]: where [K a ] and [Q] are the total concentration of HSA and coumarin drug, respectively, and the binding constant and number of binding sites were obtained and tabulated in Table 1 using the least-square fitting algorithm (Figure 2(c)).

ermodynamic Parameter Analysis.
e thermodynamic study is important for the drug-binding mechanism to calculate the binding stability of the drug with the macromolecule complex.ermodynamics parameters ΔH °, ΔS °, and ΔG °are calculated from the following equations: where R is the gas constant and T is the temperature, and here, three different temperatures are used: 293 K, 298 K, and 303 K.
According to Ross and Subramanian [17,21,22], the enthalpy change (ΔH °) and entropy change (ΔS °) tabulated in Table 1 indicate that (ΔH °< 0 and ΔS °> 0) type of interaction between HSA and the coumarin derivative complex is maybe due to the electrostatic interaction.

Time-Resolved Emission Spectroscopy Studies (TRES) and
Fluorescence Lifetime Studies.Time-resolved emission spectroscopy is one of the highly noninvasive techniques to understand the real-time dynamics of biomolecules.Figure 3(a) shows the emission decay profile of free HSA and HSA-coumarin derivative complex; each point consists of average photons of respective wavelengths.All the recorded emission profiles of HSA and HSA complex followed the multiexponential decay kinetics, and the average lifetime decay for free HSA was found to be 4.83 ns which is almost equal to previously reported values [21,23].However, in the case of HSA-coumarin derivative complex, the decay is slightly faster compared to that of free HSA, and the average estimation of the HSA complex is 4.67 ns (Table 2).From Figure 3(b), the observed result indicates that quenching occurs due to the binding of the coumarin derivative with HSA.It is important to mention that the emission decay profile of the HSA complex is faster than that of free HSA in all wavelengths due to carrying of the coumarin derivative.
is present result elucidates that protein unfolding is possible in the HSA complex because of the presence of the drug, and also, the tryptophan region gets more exposed to water.ese results suggest that free HSA is more labile and -6.6 -6.4 -6.2 -6.0 -5.8 -5.6 -5.4 Table 1: Fluorescence binding parameters for HSA-coumarin derivatives (pH 7.4). Compound Fluorescence decay-fitting parameters for free HSA Journal of Spectroscopy has faster hydration dynamics, and the similar process occurs in the HSA-coumarin derivative complex without any structural disturbance.Figure 3(b) represents the decay parameter-fitted values τ 0 of free HSA and HSA-coumarin derivative complex for easy understanding.

Excitation-Emission Matrix (EEM) Analysis.
e study is used to further understand the conformational change of HSA more carefully during the interaction with the coumarin derivative.Figure 4 shows the contour map of free HSA (a) and coumarin derivative-HSA complex (b) with the corresponding characteristic parameters presented.ere are two major fluorescence peaks observed in the figures: peak 1 and peak 2. Peak 1 (λ ex � 220 nm and λ em � 325 nm) is mainly due to polypeptide backbone structures, which correlated with the secondary structure of the protein, and peak 2 arises mainly (λ ex � 278 nm and λ em � 344 nm) due to  Journal of Spectroscopy the presence of tryptophan and tyrosine residues.From Figure 4, it is observed that there is a decrease in intensity maxima while adding the coumarin derivative into free HSA, and the quenching in both peak 1 and peak 2 may be due to the presence of the coumarin derivative, suggesting that the compound may be nearby the HSA chromophore.ese results conclude that there may be slight confirmation changes in HSA due to the presence of the coumarin derivative [23,24].

FTIR Spectroscopy Studies.
In general, the FTIR spectral technique is performed to understand the biochemical changes; here, the FTIR spectra of HSA were recorded to investigate the conformational changes to understand the binding mechanism of the coumarin derivative.Figure 5(a) shows the spectrum of free HSA and HSA-coumarin derivative complex, and the amide I peak of HSA is lying between 1600 and 1700 cm −1 and amide II region is almost in 1558 cm −1 , which is almost similar to the secondary  structure of proteins.Comparing these two bands, amide I is strong (1644 cm −1 ) as shown in Figure 5(a), and also while adding the coumarin derivative into free HSA, there is a decrease observed in the absorption of amide I maxima, which may be due to the coumarin derivative disturbance of the HSA amide region [21,23,24].

Circular Dichroism (CD) Spectral Analysis.
To understand the conformational changes of the protein secondary structure, CD spectral analysis was carried out.From Figure 5(b), CD spectra of HSA show that there are two negative bands in the ultraviolet region, at 208 nm and 222 nm.When the coumarin derivative is added into the free HSA molecule, there are notable increases observed in both 208 and 222 nm intensities while increasing the concentration and also particularly at 208 nm region of HSA (1 µM) and HSA-coumarin derivative (1.6 µM) show a slight shift towards the lower wavelength region, indicating that the α-helical structure may lose stability or rearrange the orientation of total structural confirmation due to presence of the coumarin derivative.
For the coumarin derivative at 1.6 µM concentration, the secondary structure parameters increased with respect to ∼58.1% α-helix, ∼24.7% β sheets, and 19.2% of the random coil, respectively.
ese results show that the secondary structure of HSA may be partially unfolded or reorientation occurs in the overall structure due to the presence of the coumarin derivative.

Molecular Docking Studies.
e molecular docking method is useful to understand the binding insight mechanism between the macromolecule and the small molecule at the atomic level [25,26].In this work, the molecular docking between HSA and coumarin derivative was performed using AutoDock 4.2 to clarify further the binding mode of coumarin derivative and HSA.Here, both small molecules and macromolecules are allowed for flexible docking, and the free energy values and hydrogen bonding and hydrophobic information results are shown in Table 3. Docking results show that the binding interaction between HSA and the coumarin derivative is in good agreement regarding energy and interaction.Figures 6(a) and 6(b) show the hydrogen bonding and hydrophobic information of the HSAcoumarin derivative complex further to understand more about internal stability of the coumarin derivative, and the charge distribution analysis was carried out at active residues of the HSA-coumarin derivative complex.e Mulliken atomic charges were analyzed for both coumarin derivative and cofactor with interaction amino acids of HSA. e MPA charges are presented in Table 4 (Figure 1(c)).e nitrogen atoms N 8 , N 10 , and N 11 in the amino acid arginine possess high negative charges; simultaneously, the oxygen atoms O 48 and O 56 possess low negative charges.Because of these nitrogen atoms, interaction occurs with O 48 and O 56 atoms (N 8 : −0.44e, N 10 : −0.45e, N 11 : −0.46e, O 48 : −0.16e, and O 56 : −0.38e), respectively; these charges are low compared with those in the cofactor.e oxygen atom O 69 in tyrosine has a very low negative charge, which interacts with O 91 .At the same time, the ligand contains nitrogen atom N 45 , which also has a low negative charge (N 45 : −0.39e, O 69 : −0.16e, and O 91 : −0.30e).From this, the MPA charges decreased while increasing the distance between the protein and ligand.e carbon C 9 atom possesses a high positive charge due to the carbon atom surrounded by nitrogen atoms [27].
e global reactivity descriptors [28][29][30][31][32][33][34] of the coumarin derivative and cofactor molecule such as electronegativity, electrophilicity, and chemical hardness have been calculated from HOMO and LUMO energies.e calculated values of ionization potential, electronegativity, and electron affinity of those molecules are 6.5/7.2,8.1/9.6, and 1.6/2.4eV (Table 5), and these values show that the coumarin derivative molecule has high tendency to accept electrons than the cofactor.e calculated electrophilicity of both molecules is 6.8/9.6 eV; this low value indicates that the molecule has low toxicity [35].
e chemical hardness of the molecule is 4.85/4.82eV, which reveals that the molecule exhibits high kinetic stability and reactivity [16,[36][37][38].e energy gap between the molecular orbitals is also calculated, and the HLG value is 4.86/4.82eV, which indicates that both molecules have high internal stability.Figure 7 shows the electrostatic potential countermap of the coumarin derivative and cofactor with active site amino acids of HSA.

Steady-State Fluorescence Anisotropy Studies.
Fluorescence anisotropy is widely used to measure the binding constants and kinetics of reactions that cause a change in the rotational time of the fluorescent molecules [24]. is technique will give information about changing the orientation of our coumarin derivative in human serum albumin.Here, the fluorescence anisotropy measurements were carried out between human serum albumin and a coumarin derivative of concentration ranging from 0 to 100 µM.Most reports say that anisotropy of small molecules may be zero and there is no rotation when it increases to the maximum value of 0.4.From Figure 8, coumarin derivative versus various increasing concentrations of human serum albumin shows that the anisotropy value of the coumarin derivative increases up to 20 µM and then reaches the steadystate maximum value (r � 0.13) after 40 µM of the HSA environment.is saturated value may be due to the coumarin derivative restriction in rotation motion of the HSA molecule, which also may be due to the formation of a hydrogen bond and hydrophobic interaction in active sites of HSA with the coumarin derivative.

Fluorescence Resonance Energy Transfer.
e Forster energy transfer method is used to understand the energy distance between the donor and acceptor of biomolecules.
e overlap absorbance spectrum of the drug and emission spectrum of human serum albumin are shown in Figure 9.
e energy transfer efficiency (E) is used to calculate the distance between the coumarin derivative and human serum albumin.e rate of energy transfer depends on the integral overlap (J) of the donor (HSA) and acceptor (a coumarin derivative).Transition dipoles and distance between the drug and protein molecules also play a role in deciding the rate of energy transfer [17,24].
e distance from the tryptophan residue which is located in the donor (HSA) and Journal of Spectroscopy bound drug acceptor (a coumarin derivative) in HSA can be calculated using Forster's theory.e energy transfer efficiency (E) was calculated using the following equation [39]: where r is the distance between the donor and acceptor and R 0 is the critical distance between the donor and acceptor when their transfer efficiency is 50%: where k 2 is the orientation factor of the dipole, φ D is the fluorescence quantum yield of the donor in the absence of the acceptor, and n is the refractive index of the complex medium: J is the integral overlap of the emission spectrum of the HSA (donor) with the absorption spectrum of the coumarin derivative (acceptor).F D (λ) is the fluorescence intensity of the donor at wavelength λ to λ + Δλ, with the total intensity normalized to unity, and ε A (λ) is the molar extinction coefficient of the acceptor at the wavelength (λ).It is possible for us to calculate the J value from the spectrum in which it is estimated that J � 1.3182 × 10 −20 M −1 •cm 3 .Under the experimental conditions, we have found R 0 � 2.5 nm from Equation (5).Using the following values, E � 0.018, k 2 � 2/3, N � 1.336 and ϕ D � 0.118, r � 4.88 nm has been calculated.Both R 0 and r values were found to be lower than the maximum values of R 0 (<10 nm), and the distance (r) between the donor and acceptor lies <7 nm.

Cytotoxicity of the Coumarin Derivative.
e cytotoxicity of a coumarin derivative, which was used to evaluate its biocompatibility, was studied using the HeLa cell as a model cell by the CCK-8.Figure 10 shows the cell viability test results conducted for the coumarin derivative up to 30 µg/mL.As shown in Figure 10, the amount of the coumarin derivative increases and the cell viability decreases in a tiny percentage even at the maximum level of concentration at 30 µg/mL.However, coumarin derivative is a safe molecule to bind with the serum albumin protein and deliver the targeted tissue region.

Conclusion
In this context, the synthesized coumarin derivative molecules have been subjected to various spectroscopy and computational techniques for understanding the interaction mechanism in the HSA molecule.From the UV-Vis   absorbance result, it can be seen that the HSA-coumarin derivative complex may be due to the ground state complex; further fluorescence technique reveals that the coumarin derivative in the HSA complex is in the static quenching mode, and also, the thermal parameters calculated show good binding results.Time-resolved emission spectroscopy results show that the HSA molecule could be able to carry the coumarin derivative, and the calculated average lifetime value found for free HSA is 4.83 ns and for the HSAcoumarin derivative complex is 4.67 ns.Energy transfer analysis also calculated between donor and acceptor and r values is found that 4.88 nm, which is in academic value.e anisotropy results conclude that the rotation motion of HSA is saturated at r � 0.13 after the concentration of HSA 40 µM which is maybe due to the presence of the coumarin derivative.e excitation-emission matrix results suggest that the confirmation of HSA changed due to the presence of the coumarin derivative, and following this, the FTIR and CD spectral analysis also confirmed those secondary structure changes.Computational analysis elucidate the interaction mechanism of the HSA-coumarin derivative complex at the atomic level and information on bonding and internal stability; from this overall result, the binding nature of coumarin derivatives with HSA is good, and it could carry the molecule to a particular target without cytotoxicity.

Figure 1 :Figure 2 :
Figure 1: (a) Crystal structure of human serum albumin (PDB ID: 4L8U).(b) Chemical structure of the coumarin derivative.(c) Pymol view of the energy-minimized structure of the coumarin derivative.

Figure 2 :
Figure 2: (a) Steady-state emission fluorescence spectroscopy studies of human serum albumin (1 µM) with the coumarin derivative complex (0-3.6 µM, 0.3 μM intervals).(b) e Stern-Volmer quenching constant of the HSA-coumarin derivative complex with different temperatures.(c) e double logarithmic plot of HSA with the coumarin derivative complex at different temperatures.

Figure 3 :
Figure 3: (a) Time-resolved emission spectroscopy (TRES) studies of HSA (1 µM) with the drug complex (1 µM) of different wavelength decay parameters.(b) Global fitted τ 0 value for each wavelength of the respective HSA and HSA-coumarin derivative.

Table 3 :
Binding energy and hydrogen bonding interaction of the coumarin derivative with HSA active site residues.

Figure 6 :
Figure 6: (a) Pymol view of HSA-coumarin derivative complex hydrogen bonding interaction with active site residues.(b) Charge distribution (DFT) analysis plot for the HSA-coumarin derivative complex of active site residues.

Table 4 :
e calculated Mulliken population analysis (MPA) of the coumarin molecule with active site amino acids of HSA.

Figure 7 :
Figure 7: e electrostatic potential countermap of the (a) coumarin derivative and (b) cofactor molecules with active site amino acids of HSA.

Figure 10 :
Figure 10: Cytotoxicity studies of the coumarin derivative.

Figure 8 :Figure 9 :
Figure 8: Anisotropy studies of the coumarin derivative with the HSA complex.

Table 2 :
Fluorescence lifetime profiles of HSA and increasing concentration of coumarin derivatives at different excitation wavelengths.

Table 5 :
e calculated global reactivity properties of the coumarin derivative molecule.