In vitro DNA binding, pBR322 cleavage and molecular docking studies of 1,2-diaminobenzene, dichloro glycyl glycinate tin(IV) and zirconium(IV) complexes

Abstract De novo design and synthesis of complexes 1,2-diaminobenzene, dichloro glycyl glycinate tin(IV) and zirconium(IV), 1 and 2 as molecular drug entities were carried out. The structure elucidation of 1 and 2 was done by analytical techniques and spectroscopic methods viz. IR, UV–vis, 1H, 13C, 119Sn NMR, ESI–Mass and XRD techniques. In vitro DNA binding studies of 1 and 2 by various biophysical techniques viz electronic absorption, emission spectroscopy and circular dichroism measurements were carried out to evaluate their potential to act as chemotherapeutic candidates; furthermore, cleavage studies with pBR322plasmid DNA and computer-aided molecular docking studies were also done to study the mechanistic pathway and mode of binding at the molecular level. The observed results revealed that complex 1 exhibited greater DNA binding propensity in contrast to complex 2 primarily via electrostatic binding mode. The pBR322 DNA cleavage studies of both the complexes revealed the hydrolytic cleavage mechanism and DNA minor groove binding, which was ascertained by molecular docking studies of the drug candidate. Communicated by Ramaswamy H. Sarma


Introduction
Metal-based antitumor chemotherapeutic drugs have gained considerable interest after the serendipitous discovery of cisplatin, an archetypical metal complex in clinical use for treating solid malignancies (Kelland, 2007;Reedijk, 1996). Although the platinum drugs have witnessed spectacular success, nevertheless, the challenges of platinum drugs such as systemic toxicity, resistance and large number of phenotypes of cancers derived from multiple etiologies have motivated researchers to focus on other non-platinum drugs. A substantial investigation of other metals as antitumor chemotherapeutic agents which includes (transition metal ions Ti, Fe, Co, Cu and Zn; Zr, Ru, Rh, Pd, Ag, Au and non-transition metal ions, Sn and R 2 SnX 2 have been undertaken to overcome the problems of toxic side effects and resistance (Blower, 2004;van Rijt & Sadler, 2009). Tin(IV) complexes were proven as outstanding class of novel antitumor and antiproliferative agents; much of the earlier work has been carried out by Crowe (1989), Crowe and Fricker (1994), Gielen (1995) and Neboj sa et al. (2020) which has been published and also patented. Tin(IV) and organotin compounds are involved in cancer treatment via different mechanisms at the molecular level and most of the tin complexes are DNA target (Hong et al. 2013). The phosphate group of DNA sugar backbones usually acts as an anchoring site and nitrogen of DNA base binding is extremely effective, this often resulting in the stabilization of the tin center as an octahedral species. Literature revealed that tin-based complexes exhibit pronounced cytotoxicity against human cell lines (Waddhaah et al., 2018) viz., Hela cells, MCF-7 cells and human neuroblastoma cell line SY5Y (Chauhan et al., 2007) and further, it has also been demonstrated that these complexes induce apoptosis via mitochondrial genemediated pathway. Among the non-platinum chemotherapeutics, tin(IV) and by electronic analogy, zirconium(IV) complexes with amino acid scaffold and non-leaving diammine ligands are interesting as they could exhibit different mechanism of action than platinum complexes and relatively fewer toxic effects. Moreover, Zr(IV) is expected to act exclusively as a strong Lewis acid and Zr(IV) complexes catalyze the hydrolysis of phosphodiester and dinucleotides under weakly acidic conditions. Zirconium complexes reported in the literature (Zirconocene-diacido complexes) exhibit wide spectrum of antitumor activity against murine and human tumors with reduced toxicity in comparison with cisplatin (Kostova & Momekov, 2006). Among them, synthetic agents capable of hydrolytically cleaving nucleic acids are extremely important and have been attracting increasing attention in view of their wide applications. On the one hand, artificial hydrolytic catalysts may be employed as biomimetic systems in elucidating the mechanisms of the corresponding restriction enzymes and nucleases, and they could also be used as conformational probes in the determination of DNA structure, as customized DNA restriction agents in molecular biology, and as antibiotic and chemotherapeutic drugs.
It is well known that peptides and proteins are the naturally occurring multifunctional ligands and forms an excellent coordination platform because they contain a variety of potential donor centers such as N-terminal nitrogen atom (usually a primary-NH 2 ), carbonyl oxygen and the C-terminal carboxylate group of the peptide bond (Roy et al., 2014). The metallopeptides are considered unique among metal-based nucleic acid-binding agents due to their ability to recognize DNA or nucleotides. The nature of the peptide affects both the DNA binding affinity and the cross-linking efficiency of metallopeptides and have been widely used as synthetic hydrolyzes and chemical nucleases (Karidi et al., 2006;Sangeetha et al., 2014). Among the numerous metal complexes investigated, those containing amino acids and/or peptide ligands provide models of protein-DNA and metallopeptide-derived antitumor agent-DNA interaction (Chikira, 2008). Recently, a science daily has reported that peptides could target the protein-protein interface of a key enzyme in DNA synthesis crucial for cancer growth (Rosser, 2013). Therefore, metal-based peptide scaffolds could provide an excellent delivery system into the specific tumor target sites and could be less toxic to normal tissues. Previous studies reveals that interaction of peptides with organotin moieties is quite interesting since peptides can be very effective ligands and furthermore it has been demonstrated that these organotin dipeptide complexes shows modest activity against P388 lymphocytic leukemia cells. Moreover the conjugates of peptide nucleic acids and Zr(IV) complexes efficiently hydrolyze complementary single-stranded DNA in a sequence selective fashion (Arjmand & Jamsheera, 2011;. Herein, we report the synthesis and characterization of mixed ligand tin(IV) and zirconium(IV) complexes derived from 1,2-diaminobenzene and glycyl glycine ligand scaffold as potential chemo-therapeutic drug candidates. In vitro binding studies of these complexes with CT-DNA and nucleotide were explored by absorption and emission titration methods to elucidate their mode of binding and pharmacological potential. The pBR322 DNA cleavage studies of both the complexes revealed the hydrolytic cleavage mechanism and DNA minor groove binding. Furthermore, computer-aided molecular docking studies were carried out to visualize the binding mode of the drug candidate (minor groove binding) at the molecular level.

Reagents and materials
All reagents were commercially available and used as supplied without further purification for all syntheses and experiments. Glycyl glycine (Glygly), o-phenylenediamine (OPD), tris(hydroxymethyl)aminomethane or Tris buffer, agarose gel, ascorbic acid, sodium azide (NaN 3 ), DMSO, superoxide dismutase (SOD), methyl green, DAPI, mercaptopropionic acid (MPA) (Sigma-Aldrich), SnCl 4 .5H 2 O and ZrCl 4 (Fisher Scientific), 6X loading dye (Fermental Life Science), Human DNA topoisomerase I (Calbiochem) and Supercoiled pBR322 plasmid DNA (Genei) were utilized as received. Disodium salt of CT DNA was purchased from Sigma Chem. Co. and was stored at 4 C. DNA-melting experiments were carried out by monitoring the absorbance of CT DNA (1 Â 10 À4 M) at 260 nm with varying temperature in the absence and presence of complexes 1 and 2, in a 1:1 ratio of DNA to complex with a ramp rate of 0.1 ᭺ C min À1 in Tris buffer (pH 7.2) using a Peltier system attached to the UV-vis spectrophotometer.

Methods and instrumentation
Carbon, hydrogen and nitrogen contents were determined using CHNSO Elemental Analyzer Elementar Vario EL III model. Molar conductance was performed at room temperature on a Digisun electronic conductivity bridge. Fouriertransform IR (FTIR) spectra were recorded on an Interspec 2020 FTIR spectrometer. Electronic spectra were recorded on UV-vis 1700 PharmaSpec UV-vis spectrophotometer (Shimadzu). Data were reported in k max/nm . The 1 H and 13 C NMR spectra were obtained on a Bruker DRX-400 spectrometer operating at room temperature. XRD were recorded on Rikagu mini Flex II Instrument. Interaction of complex with calf thymus DNA was performed in 0.01 M buffer (pH 7.2). Solutions of CT DNA in buffer gave a ratio of absorbance at 260 nm and 280 nm of ca. 1.9 indicating that DNA was free from protein. The viscosity measurements were carried out using Oswald capillary viscometer maintained at 25 C. CD spectra were measured on Jasco J-815-CD spectropolarimeter at room temperature using a 1 cm quartz cuvette. Cleavage experiments were performed with the help of Axygen electrophoresis supported by a Genei power supply with a potential range of 50-500 V, visualized and photographed by Vilber INFINITY gel documentation system.

DNA binding and cleavage experiments
DNA binding experiments include absorption spectral traces, emission spectroscopy and circular dichroism conformed to the standard methods and practices previously adopted by our laboratory (Al-Asbahy & Manal, 2021). While measuring the absorption spectra an equal amount of DNA was added to the compound solution and the reference solution to eliminate the absorbance of the CT DNA itself, and the CD contribution by the CT DNA and Tris buffer was subtracted through base line correction. Flow time of viscosity experiments were carried out using Ostwald's viscometer at 30 ± 0.01 C with a digital stop-watch. Each sample was measured three times and an average flow time was calculated. Data were presented as (g/g o ) versus binding ratio ([Complex 1]/[DNA]), where g is a viscosity of CT-DNA in the presence of complex and g o is the viscosity of CT-DNA alone. Viscosity values were calculated from the observed flow time of DNA containing solution (t > 100 s) corrected for the flow time of the buffer alone (t o ), g ¼ t À t o (Farukh et al., 2015). The cleavage experiments of supercoiled pBR322 DNA (300 ng) by complexes 1 and 2 (10-30 mM) in Tris-HCl/NaCl (5:50 mM) buffer at pH 7.2 were carried out using agarose gel electrophoresis. The samples were incubated for 45 min at 310 K. A loading buffer containing 25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol was added and electrophoresis was carried out at 50 V for 1 h in Tris-HCl buffer using 1% agarose gel containing 1.0 mg/ml ethidium bromide. The DNA cleavage with added reductant was monitored as in case of cleavage experiment without added reductant using agarose gel electrophoresis.

Molecular docking
The rigid molecular docking studies were performed by using HEX 6.1 software (Al-Asbahy et al., 2016), which is an interactive molecular graphics program to understand the drug-DNA interactions. The structure of the complexes was sketched by CHEMSKETCH (http://www.acdlabs.com) and converted to pdb format from mol format by OPENBABEL (http://www.vcclab.org/lab/babel/). The structure of the B-DNA dodecamer (CGCGAATTCGCG)2 (PDB ID:1BNA) and human-DNA-Topo I complex (PDB ID: 1SC7) was downloaded from the protein data bank (http://www.rcsb.org./ pdb). All calculations were carried out on an Intel Pentium 4, 2.4 GHz based machine running MS Windows XP SP2 as operating system. Visualization of the docked pose has been done by using CHIMERA (http://www.cgl.ucsf.edu/chimera/) molecular graphics program.

Synthesis of C 10 H 22 N 4 O 7 Cl 2 Sn (1)
To a stirred methanolic solution (15 ml) of glycyl glycine (0.130 g, 1 mmol) was added a methanolic solution (15 ml) of SnCl 4 Á5H 2 O (0.343 g, 1 mmol). The solution was stirred for 24 h at room temperature. The completion of the reaction was monitored with the help of TLC. A solution of o-phenylenediamine (0.108 g. 1 mmol) in methanol was added slowly to the reaction mixture. The reaction mixture was refluxed at 65 C for 8 h, which yielded pale reddish orange colored precipitate. It was filtered, washed with cold MeOH and dried in vacuo over anhydrous CaCl 2 . Yield ¼ 70%. M.P. ¼ 120 ± 2 C. Anal. Calc. for C 10 H 22 N 4 O 7 Cl 2 Sn, (%) C,24.03 H,4.44;N,11.21,Found: C,22.19;H,4.

Synthesis and characterization
All the complexes are readily soluble in H 2 O solvent. In complexes 1 and 2, the coordination geometry of central metal atoms was found to be octahedral environment (Schemes 1 and 2), which was proposed on the basis of elemental analytical data and spectroscopic studies, IR, UV-vis, ESI-MS 1 H, 13 CNMR ( 119 Sn NMR in case of 1). These results are in agreement with the proposed 1:1:1 stoichiometry between the metal ion and ligands. The in vitro binding studies of 1 and 2 with CT DNA were carried out by using absorption, emission titrations and circular dichroic studies. Furthermore, the cleavage studies of 1 and 2 with pBR322 plasmid DNA and molecular docking with DNA by computational methods were carried out.

Infrared spectra
The IR spectra of complexes 1 and 2 agree with the corresponding formulae and the proposed structure (Scheme 1). The bands around 1678 and 1452-1492 cm À1 observed in the free amino acid corresponding to the anti-symmetric and symmetric COO À stretching vibrations were shifted in the complexes suggesting the coordination of carboxylate group of the amino acid to the metal ions through deprotonation (Yang et al., 2003). The bands around 1531 and 1269 cm À1 observed in the free dipeptide attributed to amide II and III bands [due to d(NH) þ (C-N)] in the compound containing neutral secondary peptide groups were replaced by a new absorption bands at 1349 and 1317 cm À1 , respectively in complexes 1 and 2. This is characteristic feature for a deprotonated secondary peptide complex, since on removal of peptide proton the band becomes a pure C-N stretch (Manessi-Zoupa et al., 1994). The D ¼ [ as (CO 2 ) À s (CO 2 )] value was used to determine the nature of binding of carboxylate to transition metal ion. In general, the difference in D between asymmetric as (CO 2) and symmetric s (CO 2 ) absorption frequencies below 200 cm À1 suggested the bidentate carboxylate moiety, while greater than 200 cm À1 implicates the unidentate carboxylation. In all cases, the D values were above 235 cm À1 suggestive of coordination of the carboxylate group in a monodendate fashion Tiliakos et al., 2003). The IR spectra of complexes 1 and 2 exhibited a very broad band at $3277-3408 cm À1 corresponding to the coordinated dipeptide of glycyl glycine and o-phenylenediamine, suggesting that the coordination of -NH 2 group of both dipeptide and o-phenylenediamine to the metal ions (de Sousa et al., 2006). Furthermore, the medium intensity bands at $440-452 and 527-583 cm À1 were attributed to stretching vibration of (Sn/Zr-N), (Sn/ Zr-O), respectively (Yin et al., 2004).

Nuclear magnetic resonance spectra
The 1 H and 13 C NMR spectra of complexes 1 and 2 were recorded in DMSO-d 6 solution and displayed signals for aliphatic and aromatic protons with chemical shift values in accordance with their proposed structure. In 1 H NMR spectrum of complex 1, signals due to free carboxylic (COOH) and amide linkage (-NH-) groups as expected in the range 12.0-10.0, 12.55 and 11.81 ppm, respectively were absent, which commensurate with the fact that coordination to the metal center takes place of these functional groups via deprotonation (Soliman, 2006;Yin et al., 2004). Additionally, the 1 H spectra exhibited characteristic resonance signals at 3.63-3.54 and 3.98-3.86 ppm corresponding to the NCH 2 and CH 2 proton of the peptide, the aromatic proton of o-phenylenediamine appeared as a multiplet at 6.84-7.40 ppm (Tan et al., 2007;Xu et al., 2003). The 13 C NMR spectra of complexes 1 and 2 were characterized by various resonances due to O-C ¼ O, CH 2 -C ¼ O, ArC-NH 2 , NH 2 -CH and -CH 2 carbons at 172. 84-170.78, 166.16, 40.93-38.7, 35.37-39.79 ppm, respectively of the coordinated dipeptide and o-phenylenediamine. Additionally, complexes 1 and 2 exhibited aromatic carbon signatures at 117-132 ppm. The 119 Sn chemical shift, d( 119 Sn) is sensitive to the chemical environment of the tin atom. The 119 Sn NMR of complex 1 displayed a single peak at À611 ppm indicating octahedral geometry of tin atom (Pettinari et al., 2001;Wolfe et al., 1987).

Mass spectroscopy
The formation of metal complexes and the speciation of various ionic forms in H 2 O solution were studied with ESI-MS. The spectra of complexes 1 and 2 displayed prominent peaks corresponding to the molecular ion fragment at 499.

Electronic spectra
The electronic absorption spectra of complexes 1 and 2 were obtained in the region 200-1100 nm at room temperature. The electronic spectrum of free dipeptide displayed intense absorption bands at 220 nm due to an n ! p Ã transition which was shifted to $230 nm upon coordination with metal ions, and has been assigned to a N À ! (Sn/Zr) ligand to metal charge transfer (LMCT) transition (Dehand et al., 1979). A broad band at 278 nm was attributed to p ! p Ã transitions of carboxylate group (COO À ) of dipeptide to the metal ion (charge transfer transition).

X-ray diffraction analysis
To confirm the structure of the metal complexes, X-ray powder diffraction studies of complexes 1 and 2 were performed as it was difficult to isolate single crystal suitable for singlecrystal X-ray crystallography. The diffractogram obtained for complexes 1 and 2 are given in Figure S1(a & b). The XRPD patterns indicate crystalline nature for complexes 1 and 2. A summary of the refined XRPD parameters of complexes is shown in Table 1.

Electronic absorption titration
Electronic absorption spectrum is one of the most commonly used tools for the study of the development of the binding of DNA with metal complexes. The absorption spectra of complexes 1 and 2 in the absence and presence of CT-DNA at constant concentration of complexes are shown in Figure 1(a, b). Complexes 1 and 2 exhibited intraligand absorption bands in the UV region at 230 and 278 nm, respectively. On addition of CT-DNA (increasing concentration [(0.066-0.333) Â 10 À4 M] to complexes 1 and 2, there is a sharp hyperchromism with a significant blue shift of $3-5 nmin the absorption band attributed to the electrostatic binding mode and stabilization of the complex-DNA adduct (Krishna et al., 2007;Pettinari et al., 2006). Moreover, 'hyperchromic' effect reflects the corresponding changes of DNA in its conformation and structure after the complex-DNA interaction has taken place due to (i) external contact (surface binding) with the duplex through hydrogen bonding interactions between coordinated -NH 2 and oxygen atoms of the ligands with the edge of DNA bases which are accessible both in the major and minor grooves, (ii) coordinate covalent binding to N7/N3 of the base pairs of DNA due to the replacement of the labile water molecule. Also, the presence of aromatic moieties could facilitate partial intercalation by insertion of the complexes into the adjacent base pairs of DNA.
To evaluate the binding ability of complexes 1 and 2 with CT-DNA, the intrinsic binding constants K b of the complexes were determined from Wolfe-Shimer Equation (1) by monitoring the changes in absorbance bands with increasing concentration of CT-DNA (Blackburn & Gait, 1996):   given by the ratio of the slope to the intercept. The binding constant obtained for 1 and 2 were found to be 5.17 Â 10 4 and 2.64 Â 10 4 M À1 , respectively. The K b values revealed that 1 exhibited greater propensity toward CT DNA than 2, thereby shows stronger binding affinity.
To further investigate the selective recognition and validate the binding mode of complexes 1 and 2 with CT-DNA via electrostatic binding, the interaction of complexes 1 and 2 was carried out with nucleotide 5 0 -GMP. The absorption spectra of complexes 1 and 2 in the presence of 5 0 -GMP are shown in Figure 2(a, b). On addition of 5 0 -GMP to complexes 1 and 2, there was a sharp increase 'hyperchromic' effect in the absorption bands at $278 nm with no shift in absorbance. This observation supports the binding of metal (Sn/Zr) complexes via electrostatic interaction mode as observed in case of CT DNA. The intrinsic binding constant K b values for 1and 2 were found to be 3.4 Â 10 4 and 1.77 Â 10 4 M À1 , respectively and the extent of binding of Sn(IV) 1 and Zr(IV) 2 was of the order 1 > 2, respectively which revealed higher preference of Sn(IV) complex to the 5 0 -GMP as compared to Zr(IV) complex.

Ethidium bromide displacement assay
In the absence of CT-DNA, no luminescence was observed for complexes 1 and 2 either in aqueous solution, in any organic solvent or in the presence of DNA. Hence, the binding of complexes 1 and 2 with CT-DNA cannot be directly predicted through the emission spectra. Therefore, interaction of complexes 1 and 2 with CT-DNA was carried out by a competitive binding experiment using ethidium bromide (EB) as a probe. EB (3,8-diamino-5-ethyl-6-phenylphenanthriumbromide), a phenanthridine fluorescence dye, is a typical probe for intercalation (Wilson et al., 1993) form soluble complexes with CT-DNA and emits intense fluorescence when intercalated into the base pairs of DNA (Reinhardt & Krugh, 1978). The addition of the complexes to DNA pretreated with EB ([DNA]/[EB] ¼ 1) solution caused an appreciable reduction in fluorescence intensity due to replacement of EB by the complexes (Lepecq & Paoletti, 1967). Complexes 1 and 2 showed decrease in the emission intensity of EB-DNA system Figure 3(a, b), which indicated that complexes 1 and 2 were bind to DNA via electrostatic interactions releasing EB molecules gradually from the EB-DNA system (Indumathy et al., 2007). The quenching efficiency for each complex was evaluated by using Stern-Volmer where I 0 and I represent the fluorescence intensities in the absence and presence of complexes 1 and 2, respectively; r is the concentration ratio of the complex to DNA, and K SV is used to evaluate the quenching efficiency and is obtained as the slope of I 0 /I vs. r (¼ [complex]/[DNA]). The K SV value for complexes 1 and 2 was found to be 2.25 and 1.51, respectively.
The quantitative assessment of the binding constants of 1 and 2 toward CT-DNA was ascertained by calculating intrinsic binding constant, K b values using the Scatchard equation (Healy, 2007). The intrinsic binding constant K b values for 1 and 2 with CT-DNA were found to be 6.89 Â 10 4 and 2.13 Â 10 4 M À1 , respectively, consistent with absorption spectral studies. The maximum value obtained for complex 1 is in agreement with its highest K b value, that is, complex 1 binds to CT-DNA very strongly, hence maximum quenching.

Viscosity measurements
In the absence of any crystallographic data, hydrodynamic studies such as viscometric measurements serve as an unambiguous tool to determine the binding mode of small molecules toward DNA. The interaction of the tin complex with CT-DNA was further explored by measuring the relative specific viscosity of DNA after the addition of varying concentrations of complexes 1 and 2. Classical intercalators like EB tend to increase the length of the DNA helix, due to the accommodation of the ligand in between the base pairs of CT-DNA resulting in an increase in the DNA viscosity. However, complexes that bind either electrostatically via sugar-phosphate backbone produce bends or kinks in the DNA helix reducing its effective length and its viscosity, while partial or non-classical intercalation reduces the effective DNA length as it produces kinks in the DNA helix, which results in a decrease in viscosity (Arno & Jessica, 2014). Plots of relative viscosity, (g/g o ) vs.
[complex]/[DNA] shown in Figure S2 demonstrated a decrease in DNA viscosity with increase in [compound]/[DNA] ratio, thereby implying that complexes 1 and 2 binds to CT-DNA via electrostatic mode.

Circular dichroism
The circular dichroism pattern observed for CT-DNA provide further and definitive conformational of the DNA binding event with complexes 1 and 2. The CD spectrum of CT-DNA exhibits a positive band at 278 nm (UV: k max , 260 nm) due to base stacking and a negative band at 240 nm due to the right-handed helicity of B-DNA form which are quite sensitive to the mode of DNA interactions with complexes. Groove binding and electrostatic interaction of the complexes with DNA are known to exhibit only marginal changes in the intensity of both negative band as well as the positive band of DNA. On the other hand, intercalators are known to enhance the intensities of both these bands (Grover et al., 1992). The CD spectrum of DNA exhibited decrease in intensity of both positive and negative bands when complexes 1 and 2 were incubated with CT-DNA (Figure 4) suggesting that the complexes could unwind the DNA helix and lead to loss of helicity. Incubation of B-DNA with complexes 1 and 2 induced marginal changes both in its positive and negative bands retaining the basic shape which suggested that the binding of the complexes retained B-conformational form of DNA (Yousuf et al., 2018) revealing the electrostatic binding mode and groove binding interactions consistent with the other DNA binding studies (see Figure 4) .

DNA cleavage without added reductant
In order to assess the chemical nuclease activity of complexes 1 and 2, agarose gel electrophoresis was performed using pBR322 plasmid DNA as a substrate in a medium of 5 mM Tris-HCl/50 mM NaCl buffer, at pH 7.2 without addition of any reductant. When circular plasmid DNA was subjected to gel electrophoretic mobility assays, relatively fast migration was observed for the intact supercoiled Form (I). However, if scission of DNA occurs atone strand (nicking), the supercoiled DNA converted into a slower moving open/ nicked circular Form (II). If both strands cleaved, a linear Form (III) was generated (Barton et al., 1984). A concentration dependent DNA cleavage by complexes 1 and 2 was first performed in which pBR322 DNA (300 ng) was incubated at 310 K for 45 min. As shown in Figure 5, with the increase of concentration of 1 and 2, DNA was converted from Form I (Supercoiled Form) to Form II (Nicked Circular Form) and then to Form III (Linear Form), which indicates that the complexes are capable of performing direct double strand scission of DNA.

In the presence of activators
The mechanistic pathway of DNA cleavage mediated by complexes was investigated by gel electrophoresis in the presence of various activators viz., glutathione (GSH), ascorbic acid (Asc), hydrogen peroxide (H 2 O 2 ) and mercaptopropionic acid (MPA) (Figure 5(a)). The cleavage activity of 1 and 2 was significantly enhanced by these activators and follows the order MPA > H 2 O 2 > Asc > GSH for 1, while 2 follows the order MPA > Asc > H 2 O 2 > GSH.

Effect of radical scavengers on DNA cleavage
In order to investigate the role of reactive oxygen species in the cleavage activity of DNA, comparative DNA cleavage experiments in the presence different common scavengers such as DMSO and ethyl alcohol (hydroxyl radical scavengers), NaN 3 (singlet oxygen scavenger) and SOD (superoxide oxygen scavenger) were used prior to the addition of 1 and 2 to DNA solution. As shown in Figure 5(b) the cleavage activity 1 and 2 was reduced dramatically in the presence of hydroxyl radical scavenger's ethyl alcohol and DMSO (Figure 6(a)), indicating that the freely diffusible hydroxyl radical is one of the intermediates involved in the DNA scission process. On the other hand, addition of NaN 3 and SOD (Lanes 2, 3 for 1 and Lanes 6, 7 for 2, 4 respectively), also do not attenuate the DNA strand scission and even in presence of SOD the cleavage reaction was quite enhanced. These results suggested that hydroxyl radicals, singlet oxygen and superoxide anion radicals are not involved in the DNA cleavage system. Since complexes 1 and 2 are able to cleave DNA in the absence of any reducing agent, it might be assumed that a discernible hydrolytic path might cleave DNA.

DNA cleavage in presence of recognition elements (groove binding)
DNA recognition elements, minor groove binding agent (DAPI) and major groove binding agent (Methyl Green) (Trotta et al., 2000;Wittung et al., 1996) were used to probe the potential interacting sites of complexes 1 and 2 with pBR322 DNA (Figure 6(b)). The cleavage patterns demonstrated that in the presence of methyl green DNA cleavage activity is affected significantly, suggesting that minor groove is involved in the complex-DNA interactions.

Molecular docking with DNA
Recently, molecular docking techniques are widely used to visualize experimental structures available for the drug-DNA interactions in rational drug design, as well as in the development of small molecule therapeutics. Targeting the minor groove of DNA through binding to a small molecule has long been considered an important tool in molecular recognition of a specific DNA sequence (De Castro & Zacharias, 2002). In order to get more insight into the mode of binding, complexes 1 and 2 were successively docked with DNA duplex of sequence d(CGCGAATTCGCG) 2 dodecamer (PDB ID:1BNA), and provide an energetically favorable docked pose that is shown in Figure  7(a, b). The results shows that complexes 1 and 2 interact with DNA via an electrostatic mode involving outside edge stacking interactions with the oxygen atom of the phosphate backbone of DNA. In this model, complexes 1 and 2 are snuggly fitted into the curve contour of DNA minor groove in the G-C ($13.2 Å) ones, and slightly bends the DNA in such a way that a part of the aromatic rings of o-phenylenediamine ligand makes more effective p-p stacking and p-cation interactions between DNA base pairs and lead to van der Waals interaction with the DNA functional groups which define the stability of groove. Moreover, the dipeptide ring of the complexes were arranged in a parallel fashion with respect to the deoxyribosegroove walls of the DNA and was stabilized by hydrogenbonding (2.8-3.8 Å) between the amino group and DC11 residue (3.11 Å), chlorine and DC16 residue (3.27 Å) for complex 1 whereas carbonyl group of peptide shows strong hydrogenbonding with the DC23 residue at a distance of 3.07 Å for complex 2 in the GC region of the minor groove. The resulting  relative binding energy of docked metal complexes 1 and 2 with DNA were found to be À260.6 and À218.8 kJ mol À1 , respectively. This value is consistent with the high binding constant obtained from spectroscopic titration and groove binding in the presence of minor groove binder DAPI and major groove binder MG from DNA cleavage studies (see Figure 8) .

Conclusion
We describe herein, the synthesis and characterization of new tin(IV) and zirconium(IV) complexes 1 and 2, respectively derived from the dipeptide (glygly) and o-phenylenediamine ligand scaffold. The tin(IV) complex 1 shows electronic analogy to zirconium(IV) complex 2, therefore their comparative DNA binding propensity was evaluated by employing  various biophysical techniques. The spectroscopic binding titrations show that 1 was more avid DNA binding agent as compared to 2. The cleavage efficiency of 1 and 2 was demonstrated with pBR322 plasmid DNA and it was observed that both the complexes undergo prominent double-strand DNA cleavage in a concentration dependent manner by the hydrolytic pathway. Both complexes are highly efficient nuclease agents due to its ability to perform direct double strand cleavage. Double-strand breaks in DNA duplex are thought to be more significant sources of cell death and genomic instability than single strand breaks, as they are less readily repaired by DNA repair mechanisms. These studies were additionally validated by computational docking technique with target molecules to examine its mode of DNA binding and the studies revealed the electrostatic interaction, in addition to selective binding toward the minor groove of DNA in G/C rich sequences.