Oxoperoxovanadium Complexes of Hetero Ligands: X-Ray Crystal Structure, Density Functional Theory, and Investigations on DNA/BSA Interactions, Cytotoxic, and Molecular Docking Studies

Oxoperoxovanadium (V) complexes [VO (O)2 (nf) (bp)] (1) and [VO (O)2 (ox) (bp)] (2) based on 5-nitro-2-furoic acid (nf), oxine (ox) and 2, 2′ bipyridine (bp) bidentate ligands have been synthesized and characterized by FT-IR, UV-visible, mass, and NMR spectroscopic techniques. The structure of complex 2 shows distorted pentagonal-bipyramidal geometry, as confirmed by a single-crystal XRD diffraction study. The interactions of complexes with bovine serum albumin (BSA) and calf thymus DNA (CT-DNA) are investigated using UV-visible and fluorescence spectroscopic techniques. It has been observed that CT-DNA interacts with complexes through groove binding mode and the binding constants for complexes 1 and 2 are 8.7 × 103 M−1 and 8.6 × 103 M−1, respectively, and BSA quenching constants for complexes 1 and 2 are 0.0628 × 106 M−1 and 0.0163 × 106 M−1, respectively. The ability of complexes to cleave DNA is investigated using the gel electrophoresis method with pBR322 plasmid DNA. Furthermore, the cytotoxic effect of the complexes is evaluated against the HeLa cell line using an MTT assay. The complexes are subjected to density functional theory calculations to gain insight into their molecular geometries and are in accordance with the results of docking studies. Furthermore, based on molecular docking studies, the intermolecular interactions responsible for the stronger binding affinities between metal complexes and DNA are discussed.


Introduction
DNA interaction studies are the active research area at the interface of biology and chemistry and play a key role in the development of new anticancer medicines [1][2][3]. Small molecules bind through covalent and noncovalent modes of binding and bring about hydrolytic, oxidative, and photolytic cleavage of DNA. It interferes with the replication and transcription processes responsible for cell death [4]. Serum albumin is the main protein in the blood plasma and it plays a significant role in drug pharmacokinetics and pharmacodynamics [5,6]. Because of its capacity to bind reversibly to a wide range of molecules, it is considered the major transporter of regulatory mediators, metabolic products, nutrients, and fatty acids. rough hydrogen bonding, hydrophobic, and electrostatic interactions, serum albumin neutralizes endogenous and external poisons [7,8]. Several metal complexes are known to exert their anticancer action through effective binding with nucleases and proteins. In this context, vanadium complexes have been thoroughly studied for their binding efficacies with DNA and BSA. Vanadium forms complexes with many organic molecules and is advantageous over vanadium salts with lesser toxic effects and exerts greater biological efficacy at a very low dosage level. In bioinorganic chemistry, their compounds play a significant role in several enzyme-related biochemical responses like stimulating the functions of myosine ATPase, adenylate kinase, choline esterase, dynein, and phosphofructokinase and also inhibiting the functions of tyrosine phosphatase, glycogen synthase, lipoprotein lipase, and adenylate cyclase [9][10][11][12][13]. e inhibition of phosphatase enzymes instigated the interest in understanding the toxicity profile of vanadium in humans [14]. Ligands containing heteroatoms bind to vanadium and form diverse natures of vanadium species including peroxovanadate, polyoxovanadate monooxo, dioxo, and oxoperoxovanadium species [10].
Among these, peroxovanadium complexes represent an important class of compounds extensively explored for their wide spectrum of promising biological functions including antidiabetic, antitumor, and catalytic activities. ey also play an inhibitory role in the hydrolysis of phosphoproteins [15][16][17][18][19]. Oxoperoxovanadium (V) complexes mimic the catalytic action of vanadate-dependent haloperoxidases in oxidizing the halides in the presence of hydrogen peroxide. It also catalyzes the oxidation of hydrocarbons and organic sulfides [20][21][22]. Besides, peroxovanadium complexes also act as catalysts for many other organic reactions such as oxidation of methyl benzenes, alkenes, tertiary amines, thioanisole, alcohols, and olefin epoxidations [23][24][25]. Recently, quite a few studies are focused on their potential anticancer activity. ese compounds exert their action by generating ROS, interfering in cell cycle arrest, and inhibiting the enzymatic functions [26][27][28]. e redox behavior of vanadium (V) compounds in biological systems is quite interesting. ese compounds are reduced in the biological systems either enzymatically or nonenzymatically and capable of getting reoxidized by O 2 generating superoxide and peroxovanadyl moieties causing damage to cell organelles causing apoptosis [29].
In this work, we have designed two peroxovanadium (V) complexes based on two heteroligands, such as, 8-hydroxyquinoline (oxine) (ox) and 5-nitro-2-furoic acid (nf ) ligands with 2, 2′ bipyridine (bp) ancillary ligand. e ligands, by themselves, possess excellent cytotoxic and antitumor properties, apart from other applications [30][31][32][33][34]. Hence, it is expected that the metal and the ligand exert their action synergistically and bring about good therapeutic efficacy. e complexes are assessed for their DNA and BSA binding potentials in addition to the evaluation of their cytotoxic effect on HeLa cell lines. e geometry, bonding characteristics, and binding propensity with DNA of the metal complexes are evidenced through theoretical calculations and molecular docking studies.

Instrumentation.
A JASCO UV-VIS-NIR V-670 spectrophotometer was used to record electronic spectra. e Hitachi F-7000 FL spectrophotometer was used to record the fluorescence spectra. On a Shimadzu IR affinity-1CE model with resolution IV, FT-IR spectra were obtained using KBr pellets. e Waters Xevo G2-XS-QT high-resolution mass spectrometer was used to record mass spectra (HRMS). e Bruker (400 MHz) spectrometer was used to record NMR spectra with DMSO-d 6 solvent.

X-Ray Structure
Determination. e data collection for the compound 2 crystal was done on a Bruker D8 Venture equipped with a photon detector and graphite monochromated CuKα radiation (λ �1.54178Å).
e APEX2 program was used to reduce the data, and SADABS was used to compensate for absorption [35,36]. e crystal structures were solved using direct techniques in the SIR97 software and improved using full-matrix least-squares on F2 with anisotropic displacement parameters in the WINGX crystallographic tool [37,38]. Anisotropic temperature factors were applied to all atoms except hydrogen atoms, which are riding their parent atoms with an anisotropic temperature factor arbitrarily set to be 1.2 times that of the corresponding parent. Table 1 provides the final R (F), wR (F2), and goodness of fit agreement factors, as well as information on data collection and analysis. e crystallographic data for the structure presented in this work (excluding structural factors) have been deposited with the Cambridge Crystallographic Data Centre as supplemental publication nos. CCDC 1950180 for compound 2. Copies of the data are available for free upon request to the Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, U.K (Fax: +44-1223-335033; e-mail: deposit@ccdc.cam.ac.uk).

DNA Interaction Studies.
For all the studies, stock solutions have been prepared by diluting water: buffer 10 mM (Tris (hydroxymethyl) methylamine) HCl in a ratio of 1 : 10 at pH 7.2. e UV absorbance ratio (A260/ A280) of commercial calf thymus DNA in a buffer is roughly 1.9 : 1, showing that the DNA is adequately free of protein. e molar extinction coefficient of 6600 M −1 cm −1 at λ max 260 nm is used to calculate the concentration of DNA. e electronic spectra of the compounds (20 µM) in the presence of increasing CT-DNA concentrations (0-10 µl) were monitored for absorption spectral titration. e fluorescence experiment was carried out with an EtBr bound DNA reference solution and a complex concentration ranging from 0 to 100 µM. Viscosity experiments were carried out in an Ostwald viscometer at room temperature. e flow time was noted and replicated three times. DNA concentration (100 µM) was kept constant and varied complex concentration (20-200 µM).
e viscosity values were calculated using the following formula.
where t 0 represents the buffer alone flows time and t represents the DNA containing solution flow time. A graph was plotted (η/η 0 ) 1/3 versus (complex)/(DNA), where η 0 is the viscosity of DNA-complex and η is the viscosity of CT-DNA. e pBR322 plasmid was used in the agarose gel electrophoresis method. e samples were incubated for 2 h at 37°C. e samples with bromophenol blue were loaded onto the ethidium bromide-containing gel and run for 2 h, in 1X TBE buffer (pH 8) at 50 V. e DNA-complex cleavage bands were seen under the UV illuminator of the gel documentation system [39,40].

BSA Interaction Studies
2.6.1. Fluorescence Quenching Studies. A tryptophan emission quenching study was used to assess the interaction of complexes with BSA (bovine serum albumin). e concentration of BSA (2 μM) in PBS buffer was held constant, while the complex concentration (0-30 μM) increased at room temperature, and the quenching of emission signals at 345 nm (λ ex � 290 nm) was measured [41]. Bioinorganic Chemistry and Applications 3

UV-Visible Absorption Studies.
e absorption titration was carried out with an increasing amount of the complex concentration (0-10 µl) and the BSA (1 µM) concentration was kept constant.

DFT Study.
e quantum chemical calculations were carried out for complexes 1 and 2 by utilizing the density functional eory (DFT). e wB97XD hybrid functional in DFT was used for geometry optimization [42,43]. e 6-311++g (2d, 2p), split valence basis set, is combined with wB97XD hybrid functional for all the computational calculations [44,45]. e Gaussian 16 program was used for quantum chemical calculations [46].

Molecular Docking Study.
Molecular docking studies on synthetic metal complexes were carried out to get a thorough knowledge of the binding and orientation of metal complexes with DNA [47,48]. For docking investigations, the X-ray structure of E2 binding DNA (PDB ID:423D) provided by the Brookhaven Protein Data Bank was used [49]. By eliminating the water molecules and magnesium ions, the crystal structure was refined. Gasteiger-Marsili charges [50] hydrogen atoms were added by using AutoDockTools-1.5.6 [51]. AutoDock 4.2 was used for docking calculations, and AutoGrid was used to construct grid potential mappings between the DNA and different atom types. For docking calculations using default settings, a stochastic Lamarckian genetic algorithm approach was applied. During docking computations, AutoDock evaluates conformations using the AMBER force field. e binding energy is calculated using the following scoring function.
vdw stands for Van der Waal's, hbond for the hydrogen bonding, elec for the electrostatics (elec), ΔG tor for the rotation and translation, and ΔG desolv stands for the desolvation upon binding and the hydrophobic effect.

In Vitro Cytotoxic Activity.
To measure cell viability, the synthesized complexes were tested for cytotoxicity against cervical cancer cells-HeLa cell line-using the MTT method. e cell line was plated individually in 96-well plates at a concentration of 1 × 10 4 cells/well in DMEM medium with 1X antibiotic antimycotic solution and 10% fetal bovine serum (HiMedia, India) in a CO 2 incubator at 37°C with 5% CO 2 . e cells were rinsed with 200 μl of 1X PBS before being cultured for 24 h with various test concentrations of the chemical in a serum-free medium. After the treatment period, the media was aspirated from the cells. In a CO 2 incubator, 0.5 mg/ml MTT prepared in 1X PBS was added and incubated at 37°C for 4 h. Following the incubation time, the MTT-containing media was removed from the cells and washed with 200 μl of PBS. e produced crystals were properly mixed after being dissolved in 100 μl of DMSO. e formazan dye turns purple-blue, and the absorbance was measured at 570 nm with a microplate reader.

Spectral Characterization.
e electronic spectra of the complexes were recorded in acetonitrile solution. Both the complexes exhibited three bands. e spectrum showed a strong absorption band at 239, 279 nm and 241, 304 nm for complexes 1 and 2, respectively ( Figures S1 and S2). ese bands are assigned to π − π * intraligand transitions of the aromatic ring. e absorption bands at 382 and 371 nm were attributed to the presence of peroxo to vanadium (O₂ 2− ⟶V) ligand to metal charge transfer (LMCT) transition [52]. e spectrum was featureless beyond 400 nm attributed to its d 0 electronic configuration.

X-Ray Crystallography.
e MERCURY drawing of complex 2 is shown in Figure 1. e structure, bond distances, and angles are given in Tables 1-3, respectively. e mononuclear structure of the vanadium atom was surrounded by the two bidentate (N, N) (N, O) ligands such as bipyridine and oxine. e vanadium atom is coordinated to two oxygen atoms (O2 and O3) from the peroxo group and two bidentate ligands (N2, N3) and (O1, N1) of bipyridine and oxine ligand, respectively. e geometry of the complex is distorted pentagonal-bipyramidal. e crystal system of complex 2 is triclinic and the space group is P-1. e unit cell dimensions are as follows: a � 8.0268 (2)

Electronic Absorption Spectroscopy.
e electronic absorption spectral method is one of the best techniques for DNA binding studies [59,60]. e absorption spectra of the complexes in the presence and absence of the CT-DNA were recorded in Tris-HCl buffer (pH 7.2). e peroxocomplex 1 has shown two broad absorption bands at 235 and 278 nm, and complex 2 shows bands at 238 nm, assigned to the π − π * intraligand transitions. In general, any changes in the double helix structure of DNA on binding to complexes are correlated to hyperchromic or hypochromic effects. e complex concentration (20 µM) is kept constant and the concentration of DNA is varied. Upon the incremental addition of DNA concentration (0-10 µl) to the complexes, the intensity of absorbance increases showing a hyperchromic shift due to the interaction between the DNA base pairs and aromatic chromophore of vanadium complexes. e results reveal that the groove binding mode was observed for both the complexes (Figures 2 and 3). Besides, the spectrum for complex 2 has shown the isosbestic point at 246 nm, implying that the complex bound to DNA is homogeneous [61,62]. e intrinsic DNA binding constant (K b ) is calculated using the following equation: where (DNA) is the concentration of DNA in the base pairs, the absorption coefficients ε a , ε f , and ε b correspond to A obsd /[M], the extinction coefficient of the free compound, and the extinction coefficient of the compound when bound to the DNA, respectively. On plotting the values of (DNA)/(ε a −ε f ) vs. (DNA), the K b value is given by the ratio of slope to the intercept (Figure 4). e binding constants (K b ) of complexes 1 and 2 are 8.7 × 10 3 M −1 and 8.6 × 10 3 M −1 , respectively, and thus, complex 1 shows better binding affinity than complex 2.

Fluorescence Studies of Competitive Displacement
Assay with Ethidium Bromide (EtBr). e binding affinity of the complexes was examined using the fluorescence quenching method. Ethidium bromide (EtBr) is an organic cationic dye, a well-known DNA intercalator, and acts as a fluorescent tag. e displacement assay was done using EtBr bound CT-DNA solution (EtBr � 10 µM and DNA � 100 µM) acting as a probe. e EtBr emission intensity was increased,     90.66 (7) when it was intercalated into DNA base pairs and the emission wavelength was 601 nm ( Figure 5). In addition, by increasing the concentration of complexes (0-100 µM) gradually to EtBr-DNA, the fluorescent intensity decreased due to the displacement of EtBr by complexes [63][64][65]. e quantum yield of complexes 1 and 2 was 1.2 and 1.5, respectively. e fluorescence spectra are shown in Figures 6  and 7. e extent of binding is quantified by calculating the Stern-Volmer constant, K SV . e linear Stern-Volmer equation is where F and F 0 are the emission intensities in the presence and absence of the quencher and K SV is the Stern-Volmer constant. e Ksv is calculated from the slope of the plot F 0 /F versus Q (Figure 8). e K SV value of complexes 1 and 2 are 9.7 × 10 3 M −1 and 5.4 × 10 4 M −1 , respectively.

Viscosity Study.
e viscosity experiment is one of the effective methods to determine the binding mode between the DNA and complexes. Small molecules interact with DNA, and the intercalative binding mode changes the DNA conformation resulting in increasing the length of DNA and thereby increasing the relative viscosity of DNA. In contrast, electrostatic and groove binding modes would rather show no significant effect or less change in DNA viscosity [66,67]. Ethidium bromide (EtBr) with DNA and buffer shows a marked increase in viscosity inferring intercalative binding      mode; whereas, complexes 1 and 2 show only marginal changes in viscosity with an increase in concentration suggesting the complexes bind with DNA through groove binding mode (Figure 9).

Gel Electrophoresis Method.
e pBR322 plasmid DNA cleavage activity of peroxovanadium complexes was studied by using the agarose gel electrophoresis method. e molecules migrate in the gel as a function of their mass, charge, and shape. DNA cleavage shows supercoiled circular (form I) changed into nicked circular (form II) and linear (form III) [68][69][70]. e fastest migration was observed for form I and the slowest migration for form II, and the pace of migration for form III was in between form I and form II.
e results are shown with supercoiled circular, nicked, and linear forms without adding light or reducing agents. e complexes bring about pBR322 DNA cleavage hydrolytically. e gel picture of the complexes containing the bands is shown in Figure 10.

Fluorescence Quenching Studies.
Protein is one of the primary molecular targets of anticancer medicines. Fluorescence spectroscopy is the best technique to evaluate the interaction between BSA and metal complexes. e protein binding ability of complex can be studied using tryptophan fluorescence quenching experiments using BSA as the substrate in PBS (phosphate-buffered saline) (pH 7.4). Protein contains three aromatic amino acid residues such as tryptophan, tyrosine, and phenylalanine, but the fluorescence of BSA arises mainly due to two tryptophan residues, Trp-134 and Trp-212. Trp-212 is located within a hydrophobic binding pocket in subdomain IIA and Trp-134 is located on the surface of subdomain IB. e absorption and fluorescence emission wavelength maxima are observed at 280 nm and 345 nm, respectively. e fluorescence spectra of BSA were recorded in the absence and the presence of increasing concentrations of complexes. e fluorescence intensity of the protein, observed at around 345 nm, decreases as the complex concentration increases without any shifts towards lower or higher wavelengths [71] (0-30 µM) (Figures 11 and 12). is indicates that there is no alteration in the local dielectric environment of BSA, which would otherwise cause shifts in emission maxima. is quenching effect may be caused by subunit associations, protein conformational transitions, denaturation, or substrate binding.     (complex) (Figure 13) are created using corrected fluorescence data that takes dilution into account. e equation can be used to create a linear fit of the data.
where I and I 0 are the emission intensities of BSA in the presence and absence of a quencher of concentration (Q), respectively, which gave the quenching constant (K BSA ) using Origin Pro 8.5 software. τ 0 is the average lifetime of the tryptophan in BSA without quencher reported as 1 × 10 −8 s, and k q is the quenching rate constant. e mechanism of fluorescence quenching is classified into different types such as static quenching, dynamic quenching, and combined static and dynamic quenching. e formation of a fluorophore quencher complex is a part of the static quenching process. Dynamic quenching refers to the mechanism through which the quencher and fluorophore come into contact during the transient existence of the excited state. e k q quenching rate constants of the complexes 1 and 2, 6.28 M −1 S −1 and 1.6 M −1 S −1 , are higher than the maximum scattering collision quenching constant, 2 × 10 10 M −1 S −1 , suggesting a static fluorescence quenching mechanism. e binding propensity of the quenchers with respective serum proteins is expressed by the Scatchard equation [72,73]: For such static quenching interaction, the binding constant (K) and the number of binding sites (n) can be determined.        (Table 4).

UV-Visible Absorption
Studies. UV-Vis absorption method is a simple method to examine the possible quenching mechanisms. e spectrum was recorded in the absence and the presence of the increasing concentration of the vanadium complexes (Figures 15 and 16). e spectrum of BSA shows a band at 279 nm, due to the aromatic amino acid residues (Trp, Tyr, and Phe). Upon the addition of the complex concentration (0-10 µl) gradually to BSA, the absorbance value increases with a blue shift confirming the interaction between BSA and vanadium complexes. e results reconfirm the static quenching mechanism of the complexes, forming a complex-BSA in the ground state [74][75][76].       Figure 18: Frontier molecular orbitals density plots of complexes 1 (a) and 2 (b) obtained at wb97xd/6-311++g (2d, 2p) level of theory.

DFT Study.
e optimized geometry of complexes 1 and 2 was obtained at wb97xd/6-311++g (2d, 2p) level of theory as shown in Figure 17. e coordination bonding pattern and other geometrical parameters are depicted and are comparable with experimental observations (Table 5). e vanadium atom is coordinated with seven atoms and the geometry of the complex is distorted pentagonal-bipyramidal. e calculated bond lengths of V (1)-O (1), V (1)-O (2), and V (1)-O (3) are 2.03, 1.84, and 1.86, respectively. ere are only a few minor differences in the structural features of the complexes when the data obtained from DFT calculations and X-ray crystallography are compared [77]. e frontier molecular orbitals are the highest occupied molecular orbital (HOMO) associated with electron-donating potential and the lowest unoccupied molecular orbital (LUMO) related to electron affinity [78,79]. e frontier molecular orbital density plots shown in Figure 18 would dictate the reactivity and stability of metal complexes. In complex 1, HOMO is distributed over the oxygen atom and LUMO is distributed over the bipyridine molecule. In complex 2, HOMO is distributed over the oxine molecule and oxygen atom and LUMO is distributed over the bipyridine molecule.
e E HOMO-LUMO energy gaps of complexes 1 and 2 are calculated to be 0.253 eV and 0.242 eV, respectively.

Molecular Docking Studies on Metal Complexes
Binding to E2 Binding Region of DNA. Molecular docking calculations are carried out with both the major groove and the minor groove as binding sites for the metal complexes. It has been observed that the complexes bind to the major groove of double-stranded DNA. e binding (∆G BE ) and intermolecular energies (∆G intermol ) of complexes 1 and 2 are given in Table 6. e best-docked conformations of complexes 1 and 2 obtained by docking calculations are shown in Figure 19. e docked complexes of 1 and 2 possess binding energy in the range of−7.35 and−7.0 kcal/mol. is is due to the strong contribution of intermolecular van der Waals,  hydrogen bonding, and desolvation energies. It can also be observed that complex 1 forms three explicit hydrogen bonds with DG7, DA17, and DG19 residues, while complex 2 has only one hydrogen bond with DC18 residue which could be the reason for the stronger interaction observed in the case of complex 1 over complex 2. e binding ability of complexes to DNA is investigated through molecular docking procedures. Here, the E2 regulatory protein binding DNA targets (PDB ID: 423D) are considered for the calculations [80,81]. e regulatory signals of the virus are dependent on the nucleotides that are involved in protein binding along with the deformation ability of the corresponding target DNA region. erefore, it is proposed that blocking the above region through the introduction of inhibitory metal complexes that can strongly bind to the key oligonucleotides can consequently inhibit the regulatory signals of the DNA. e binding energy of complex 1 is greater as shown by its high binding constant value of 8.7 × 10 3 M −1 .

In Vitro Cytotoxic Activity.
e cytotoxicity of complexes 1 and 2 was tested against the HeLa cell line by using the MTT assay method. Cisplatin cytotoxic activity is the standard reference for the comparison purpose and the IC 50 value is 24 ± 1.46 µM. e plot of the percentage of the cell viability versus complex concentration is shown in Figure 20. e cell viability decreases with increased complex concentration indicating a dose-dependent growth inhibitory effect (Figures 21 and S12). Furthermore, the IC 50 value for complexes 1 and 2 against the HeLa cell line is calculated and is found to be 512 ± 4.27 µM and 788 ± 26.57 µM, respectively, showing a moderate cytotoxic effect. From the result, cisplatin shows higher cytotoxic activity, but the vanadium complexes exhibit a lower cytotoxic effect. Peroxovanadate complexes exert their probable mode of anticancer action through the inhibition of protein tyrosine phosphatase, lipoperoxidation, DNA cleavage, and strand breakage associated with ROS and act as potential cytotoxic agents [10].

Conclusion
e oxoperoxovanadium (V) complexes have been synthesized and characterized by spectral techniques and singlecrystal X-ray diffraction studies. e crystal system of complex 2 is triclinic and the geometry is found to be distorted pentagonal-bipyramidal. e complexes have shown groove binding mode with DNA and the binding constant values are assessed and supported by a molecular docking study. e strong binding interactions with BSA are evaluated, and the complexes have shown a moderate cytotoxic effect on HeLa cell lines. Based on the results obtained, it is observed that complex 1 shows stronger DNA/BSA binding ability than complex 2. Further studies are required to assess the probable mechanism through which the complexes exert their cytotoxic activity and are underway.
Data Availability e data on Figures S1-S12, UV-Visible, FT-IR, NMR, Mass spectra of the complexes supporting data are included within the supplementary materials.