Synthesis, structure elucidation and DFT studies of a new coumarin-derived Zn(II) complex: in vitro DNA/HSA binding profile and pBR322 cleavage pathway

Abstract

A new mononuclear zinc complex 1 bearing the bioactive coumarin scaffold 5,10-dioxo-5,10-dihydro-chromeno[5,4,3-cde]chromene-2,7-dicarboxylic acid was synthesized, characterized by elemental analysis and spectroscopic techniques, and further analyzed by single-crystal X-ray diffraction. Density functional theory studies were performed using an ab initio Gaussian 09 software package with a B3LYP/6-31+g(d,p) basis function. The in vitro DNA binding studies of complex 1 with calf-thymus DNA in Tris–HCl buffer was studied by various biophysical techniques, which revealed that 1 binds to calf-thymus DNA non-covalently via electrostatic interactions. Competitive binding experiments showed that 1 has the ability to displace DNA-bound ethidium bromide. Compound 1 exhibits efficient photoinduced DNA cleavage with supercoiled pBR322 involving the hydrolytic cleavage pathway due to the presence of coordinated water molecules. Such synthetic hydrolytic nucleases, in particular those that cleave DNA with sequence selectivity different from that of the natural enzymes, are gaining considerable attention owing to their importance in biotechnology and drug design.

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Introduction

Coumarins (both natural and synthetic) and their analogues are the key components of many bioactive compounds. Because of the broad spectrum of biological activities exhibited by coumarin and its derivatives, such as anticancer,¹ antioxidant,² anti-inflammatory,³ anti-HIV,⁴ antimicrobial,⁵ and anticoagulant⁶ activities, they are highly in demand for use as precursors in metal-based drug regimes. These coumarin compounds serve as unique, versatile ligand scaffolds for the development of newer drugs that may exhibit diverse mechanisms of action on a variety of biological targets for the development of therapeutic agents.⁷ Many biologically active compounds used as drugs possess modified pharmacological and toxicological properties when administered in the form of metal-based compounds.⁸ Zinc, an endogenously biocompatible metal ion exists as a divalent cation physiologically and it is actively involved in DNA synthesis, apoptosis, gene expression, and catalytic functions.⁹ Zinc complexes with diverse biological activities viz. antibacterial activity,¹⁰ anti-inflammatory activity,¹¹ treatment of Alzheimer's disease¹² and anti-proliferative and antitumor¹³ activities, have been reported. Besides this, Zn(II) complexes are regarded as some of the best-suited metal complexes for the development of artificial metallonucleases owing to their strong Lewis acidities, very rapid ligand-exchange abilities, low toxicities, redox inertness, and abilities to catalyze the hydrolytic cleavage of DNA.¹⁴

Since DNA is the primary intracellular target of anti-cancer drugs, the interaction between small molecules and DNA can cause DNA damage in cancer cells, hence blocking the division of cancer cells and resulting in cell death.¹⁵ Small molecules can interact with DNA through the following three non-covalent modes: intercalation, groove binding, and external static electronic effects. Recently, there has been great interest in the binding of small metal-based molecular entities that are particularly suited for DNA binding, because of their cationic character and three-dimensional structural profiles. They have a natural aptitude to interact with DNA and may give rise to cleavage reactions that are crucial for the development of new anti-cancer therapeutic agents.¹⁶ Similarly, investigations of the interactions of metallo-drugs with human serum albumin are of great importance—to understand the absorption, transport and distribution of these drugs in the body as well as to modulate their delivery to cells at specific sites—because serum protein binding can lead to premature decomposition of a metal-based agent through ligand exchange.¹⁷

Herein, we describe the synthesis, spectroscopic characterization and single-crystal X-ray studies of the Zn(II) complex (1) of the in situ-generated chromone scaffold 5,10-dioxo-5,10-dihydro-chromeno[5,4,3-cde]chromene-2,7-dicarboxylic acid (H₂L). An in vitro DNA-binding profile of 1 with calf-thymus DNA (CT-DNA) was carried out by UV-Vis, fluorescence and circular dichroism techniques in order to demonstrate their affinity towards the molecular drug target DNA. Studies of photo-induced cleavage of pBR322 DNA by 1 upon irradiation with monochromatic 365 nm wavelength UV-A light were carried out at different concentrations and in the presence of radical scavengers, to clarify the mechanistic pathway. Furthermore, the interaction of 1 with human serum albumin (HSA) was investigated, using fluorescence quenching, in order to understand the drug–protein interaction and the carrier role of HSA in the transport of the drug under physiological conditions in Tris–HCl buffer solution at pH 7.4.

Results and discussion

Compound 1 was synthesized, and characterized by analytical and spectroscopic methods. The expected formulation of the complex was confirmed by single-crystal X-ray diffraction and further supported by thermo-gravimetric analysis (TGA) and elemental analysis, while the phase purity of the bulk material was confirmed by powder X-ray diffraction (PXRD). Once isolated, it was found to be air-stable and soluble in DMSO.

Crystal structure description

Complex 1 was synthesized by the solvothermal reaction of biphenyl-2,4,6,2′,4′,6′-hexacarboxylic acid with Zn(NO₃)₂·6H₂O in mixed N,N-dimethylformamide and water solvent at 100 °C for 3 days to afford orange block-shaped crystals. A single-crystal X-ray structural study revealed that 1 crystallizes in the monoclinic space group P2₁. The asymmetric unit consists of one Zn(II) ion, two HL⁻ (5,10-dioxo-5,10-dihydro-chromeno[5,4,3-cde]chromene-2,7-dicarboxylic acid) ligand moieties and four coordinated water molecules (Fig. 1). The coordination environment around the Zn(II) ion can be portrayed as a slightly distorted octahedral geometry with ligation from the carboxylate groups of two ligand moieties in a monodentate fashion (Zn–O = 2.064(4)–2.101(4)) and the four coordinated water molecules (Zn–Ow = 2.075(5)–2.121(4)). The Zn–O bond distances are within the range reported for other Zn-based octahedral complexes.¹⁸ Here, both the carboxylate and carbonyl oxygen atoms of the ligand form extensive H-bonding with the coordinated water molecules (–C=O⋯OW = 2.073–2.215 Å, COOH⋯OW = 1.913–2.325 Å). Aromatic rings in translationally equivalent molecules lie in parallel planes and are involved in face-to-face π–π stacking interactions at a ring centroid distance of ∼3.39 Å (Fig. 2).¹⁹ These non-covalent interactions result in an overall 3D supramolecular architecture.

Fig. 1 Schematic ORTEP diagram (50% probability) of asymmetric unit in complex 1.

Fig. 2 Diagrammatic representation of (a) 3D packing (using a space-filling model), as well as (b) H-bonding and π⋯π interactions, in complex 1.

Theoretical calculations

In order to gain some insight into the molecular geometry, we performed quantum mechanical calculation of the free molecule. In Table S1,† selected geometrical parameters from the single-crystal X-ray structure are in excellent agreement with those obtained from first-principles density functional theory (DFT) calculations on complex 1. The optimized lattice parameters are a = 11.548 Å, b = 5.326 Å, c = 23.007 Å, α = γ = 90°, and β = 100.3°, which compare well with the experimental crystal parameters. The optimized structure is very similar to the X-ray crystal structure as depicted in Fig. 3. The Mulliken spin density on the metal centre is found to be zero. This is consistent with the +2 oxidation state of zinc (d¹⁰). The highest occupied molecular orbital (HOMO) is localised on the carboxylate groups of the ligand and metal centre while the lowest unoccupied molecular orbital (LUMO) is located on the metal centre. This distribution of electron density in the HOMO and LUMO (Fig. 4) implies that, during an absorption process, the charge is shifted from the HOMO to the LUMO. Hence, the electron density change between ligand and metal centre is ascribed to the ligand-to-metal charge-transfer (LMCT) process.

Fig. 3 The optimized geometrical structure of complex 1.

Fig. 4 The optimized structures showing the (a) HOMO and (b) LUMO frontier molecular orbitals for complex 1.

Thermal and powder X-ray studies

In order to examine the thermal stabilities of complex 1, thermal analysis was carried out in a N₂ atmosphere at a heating rate of 5 °C per minute. Complex 1 shows a weight loss of ∼9.25% (expected = 9.15%) between 70 and 120 °C that corresponds to the release of four coordinated water molecules. Decomposition of the complex is achieved at temperatures above 250 °C (Fig. S1†). The powder X-ray diffraction (PXRD) pattern of 1 matches well with the simulated patterns obtained from the single-crystal X-ray data, confirming that the crystals are representative of the bulk phase (Fig. S2†). The differences in intensity could be due to the different orientation of the powder sample.

DNA binding studies

The DNA-binding metal complexes have been extensively studied as DNA structural probes, DNA footprinting and sequence-specific cleaving agents, and potential anticancer drugs. Many compounds exert their antitumor effects by binding to DNA, thereby changing the replication of DNA and inhibiting the growth of the tumor cells. This approach is the basis of designing new and more efficient antitumor drugs, and their effectiveness depends on the mode and affinity of their binding to DNA.^20–22

Electronic absorption spectroscopy is one of the most frequently used techniques for the investigation of the binding mode of small molecules to DNA. We anticipate that 1—because of its structure, functional ligands and metal ion—can bind DNA in different ways, viz. groove binding and coordinate covalent linkage with nucleobase due to the Zn(II) metal ion. Moreover, the chromophore of the coumarin ligand could facilitate hydrophobic and H-bonding interactions. Upon addition of increasing amounts of CT-DNA to a fixed concentration of 1 in 5 mM Tris–HCl, 50 mM NaCl, pH 7.3 buffer, there is an increase in the absorption intensity of the intraligand absorption band. These spectral features (hyperchromism) indicate binding of 1 to the minor groove, unwinding of the DNA double helix, simultaneous exposure of the DNA bases, and damage to the DNA double helix (Fig. 5).²³ The results reveal that the complex interacts with DNA either by external contact (electrostatic interaction) with the phosphate backbone of the DNA double helix or by the formation of H-bonds between the carbonyl and carboxylate groups of 1 with suitable donors such as N₃ of the adjacent thymine base of DNA, supported by the favorable hydrophobic interaction of the coumarin ring with the surface of DNA, all of these interactions contribute to the overall hyperchromism. The intrinsic binding constant K_b for 1 is calculated from the plot of [DNA]/(ε_a − ε_f) versus [DNA], and is found to be 5.12 × 10⁴ M⁻¹. The K_b value of the complex is smaller than those observed for classical intercalators (e.g. ethidium bromide (EB) and [Ru(phen)₂(dppz)]₂²⁺ ∼ 10⁶ to 10⁷ M⁻¹) and similar to those of the corresponding Zn(II) complexes reported previously by our group.²⁴

Fig. 5 Absorption spectra of complex 1 in 5 mM Tris–HCl/50 mM NaCl buffer upon the addition of calf thymus DNA; inset: plots of [DNA]/ε_a − ε_f (m² cm) vs. [DNA] for the titration of CT DNA with complex ▲, experimental data points; full lines, linear fitting of the data. [Complex] = 6.67 × 10⁻⁶ M, [DNA] = (0–4.24) × 10⁻⁵ M. Arrow shows change in intensity with increasing concentration of DNA.

Complex 1 exhibits emissions at 331 and 360 nm in 0.01 M Tris–HCl/50 mM NaCl buffer when excited at 267 nm. Upon addition of increasing amounts of CT-DNA (0–0.66) × 10⁻⁵ M, to a fixed amount of 1, the emission intensities of both peaks increase (Fig. 6), indicating that 1 interacts with DNA. This is due to the hydrophobic environment inside the DNA helix reducing accessibility of the solvent molecules—as well as restricting the mobility of 1 at the binding site, which causes a reduction of the vibrational modes leading to higher emission intensity.²⁵ To determine the strength of the interaction of complex 1 with DNA, the value of the binding constant (K) was derived from a Scatchard equation, and was found to be 5.37 × 10⁴ M⁻¹, which is consistent with electronic absorption studies. To further investigate the interaction of 1 with the DNA, three-dimensional (3D) fluorescence spectroscopy was performed on 1 in the absence and presence of CT-DNA. As depicted in Fig. 7, two prominent peaks, peak A and peak B at λ_em = 333 and 369 nm respectively, are observed upon excitation at 270 nm. However, addition of DNA leads to a significant increase of the fluorescence intensity due to interaction of 1 with DNA.

Fig. 6 Emission spectra of complex 1 in Tris–HCl buffer (pH 7.3) in the presence and absence of CT DNA at room temperature. Arrow shows change in intensity with increasing concentration of DNA.

Fig. 7 3D fluorescence spectra and corresponding contour diagrams of (a) complex 1 and (b) complex 1–DNA system. The concentration of the complex is fixed at 1.46 mM and that of DNA is fixed at 11.5 mM. Data were collected at pH = 7.3, at room temperature.

A competitive ethidium bromide (EB) displacement assay was carried out to investigate the interaction of 1 with DNA. EB is a planar cationic dye that is widely used as a sensitive fluorescence probe and emits intense fluorescence in the presence of DNA due to its strong ability to intercalate between the adjacent DNA base pairs.²⁶ Upon addition of 1 to EB–DNA system, an appreciable reduction in the fluorescence intensity of about 84% (603 nm), with a blue shift, was observed either by replacing DNA-bound EB or by accepting an excited-state electron from EB and possibly deforming the secondary structure of DNA (Fig. 8).²⁷ Since EB is not completely displaced, an intercalative mode of binding in addition to the electrostatic interaction cannot be ruled out. The quenching efficiency K_sv evaluated from the slope of I₀/I vs. r = [complex]/[DNA] was found to be 0.42.

Fig. 8 Emission quenching spectra of CT DNA bound to ethidium bromide in the presence of complex 1 in a buffer of 5 mM Tris–HCl/50 mM NaCl, pH = 7.3 at 25 °C. Arrow shows change in intensity with increasing concentration of ethidium bromide.

To assess conformational changes in the DNA and complex 1, circular dichroism (CD) experiments were carried out. The CD spectra of CT-DNA exhibit a positive band at 275 nm (+0.51 mdeg) due to the base stacking and a negative band at 245 nm (−0.60 mdeg) due to the helicity, which are characteristics of the chiral B-DNA molecule. Upon addition of 1, the CD spectra show only a slight change in the ellipticity for both positive and negative bands revealing that the double helical structure is only slightly perturbed (Fig. 9). The results reveal that both the positive band at 274 nm (+0.28 mdeg) and negative band at 237 nm (−0.55 mdeg) display a decrease in the ellipticity and a slight blue-shift in the maximum wavelengths indicating conformational conversion from a B → Z helix, together with increased winding of the DNA helix through the rotation of the bases.

Fig. 9 CD spectra of CT-DNA (blue, 1 × 10⁻⁴ M) in the presence of complex 1 (green, 1 × 10⁻⁴ M), at [complex]/[DNA] molar ratios of 0.03.

HSA binding studies

Fluorescence quenching mechanism. An emission quenching experiment was carried out to study the interaction of complex 1 with HSA (Fig. 10). HSA shows a maximum emission wavelength at 327 nm, which is mainly due to the single tryptophan residue 214.²⁸ Molecules that bind to albumin particularly in the region containing this Trp residue can cause fluorescence quenching of albumin when excited at 295 nm. The fluorescence spectra of HSA in the absence and presence of 1 as a quencher in Tris–HCl buffer (pH 7.4) were monitored with an excitation wavelength of 295 nm. On addition of an increasing concentration of 1 (0.67 × 10⁻⁵ to 4.6 × 10⁻⁵ M) to a fixed amount of HSA, the intrinsic fluorescence intensity of HSA decreased gradually at 327 nm by 86%, accompanied by a slight blue-shift of 2 nm in the tryptophan emission maxima of HSA. The observed blue-shift is mainly due to the decrease in the polarity or an increase in the hydrophobicity of the microenvironment surrounding the fluorophore site.²⁹ These results indicate that the strong protein-binding ability of complex 1 leads to changes in protein secondary structure (affecting the tryptophan 214 residues of HSA) together with enhanced hydrophobicity.³⁰

Fig. 10 The fluorescence quenching spectra of HSA by different concentrations of complex 1 with the excitation wavelength at 293 nm in 5 mM Tris–HCl/50 mM NaCl buffer, pH 7.4, at room temperature: [HSA], 6.67 × 10⁻⁶ M; the concentration of complex 1 was 0.67 × 10⁻⁵ to 4.6 × 10⁻⁵ M. Arrow shows the intensity changes upon increasing concentration of the quencher.

In order to decipher a possible fluorescence quenching mechanism of HSA in the presence of 1, the Stern–Volmer equation is applied:

where F_o and F are the fluorescence intensities in the absence and presence of quencher, respectively, K_q, K_sv, τ_o and [Q] are the quenching rate constant of the biomolecules, the Stern–Volmer quenching constant, the average life-time of the molecule without quencher (τ_o = 10⁻⁸ s) and the concentration of the quencher, respectively. The Stern–Volmer plots of F_o/F versus [Q] for the quenching of HSA fluorescence by 1 is depicted in Fig. S3a† and the calculated K_SV and K_q values are found to be 3.24 × 10³ M⁻¹ and 3.24 × 10¹² M⁻¹ s⁻¹ respectively. The observed K_q value is larger than the limiting diffusion constant K_dif of the biomolecules (K_dif = 2.0 × 10¹⁰ M⁻¹ s⁻¹),³¹ indicating that the fluorescence quenching is mainly due to the specific interaction of 1 with HSA, consistent with the static quenching mechanism.³² For static quenching, the Scatchard equation is employed to calculate the binding constant and number of binding sites:³³where F_o and F are the fluorescence intensities of HSA in the absence and presence of quencher, and K and n are the binding constant and the number of binding sites, respectively. Thus, a plot of log[(F_o − F)/F] versus log[Q] is used to determine K (binding constant) from the intercept on the Y-axis and n (binding sites) from the slope (Fig. S3b†). From the corresponding Scatchard plot, the K and n values for 1 are calculated to be 0.526 and 1.06 respectively. The calculated K value of 1 is lower than the binding constant of one of the strongest known avidin–ligands interaction with HSA (K ≈ 10¹⁵ M⁻¹) suggesting a possible release of complex 1 from the serum albumin to the targeted cells.³⁴

In order to gain further insight in the conformational changes of HSA, 3D fluorescence spectroscopic studies were carried out in the presence and absence of complex 1. The 3D fluorescence spectra and contour diagrams of HSA and the 1–HSA complex are shown in Fig. 11. Peak A is the Rayleigh scattering peak (λ_ex = λ_em). Peak B (λ_ex = 295, λ_em = 332 nm) displays the dominating spectral behavior of the Trp residue, and the fluorescence intensity of this residue is associated with the polarity of the HSA micro-environment. The results reveal that fluorescence intensity of peak A increases and that of peak B (295, 332 nm, λ_ex, λ_em) decreases significantly, indicating quenching of fluorescence induced by Trp residues of HSA. However, the fluorescence intensity of peak B is strongly quenched, revealing that 1 binds to HSA near the tryptophan residues.³⁵ The results reveal that the interaction of 1 with HSA induced micro-environmental changes in the structure of HSA, which corroborates our spectroscopic results obtained from UV-vis and fluorescence measurements.

Fig. 11 3D fluorescence spectrum and corresponding contour diagrams of (a) HSA, and (b) complex 1–HSA system. The concentration of HSA was fixed at 1.6 mM and that of complex 1 was fixed at 13.5 mM. Data were collected at pH = 7.4, at room temperature.

Photoinduced DNA cleavage activity. The photoinduced cleavage of DNA by small molecules is considered to be the most suitable therapeutic approach as these molecules can be photoinduced to react locally, by irradiating the affected area, and thereby unwanted DNA damage can be avoided. DNA cleavage is promoted by relaxation of the supercoiled circular form of pBR322 DNA, resulting in nicked circular and/or linear forms. This type of nuclease can catalyze the hydrolytic cleavage of DNA due to its strong Lewis acid and inert redox nature. Note that it is imperative to study the effect of Zn(II) complex of bioactive scaffolds on pBR322 DNA in a concentration-dependent manner, and in the presence of reactive oxygen species and DNA recognition elements (binding groove).

The DNA cleavage activity of complex 1 was studied by UV irradiation of 1 and pBR322 DNA in 5 mM Tris–HCl/50 mM NaCl buffer (pH 7.3) in the absence of any external additives (Fig. 12). With increasing concentrations (Lanes 20–60 μM) of complex 1, the amount of Form I of pBR322 DNA diminishes gradually, whereas Form II increases gradually (Lanes 2–5) without the formation of Form III, suggesting cleavage of single-stranded DNA. However, at a concentration of 60 μM, the intensities of both forms apparently increase (Lane 6) with the maximum intensity of Form II indicating efficient cleavage activity; this may be due to the production of more hydroxyl radicals by the presence of water ligands, leading to the DNA hydrolysis. We therefore conclude that the cleavage activity was dependent on metal ion concentration and the presence of the active coumarin moiety, which acts as a photosensitizer and could be responsible for the higher photocleavage activity in UV light.³⁶

Fig. 12 Cleavage of pBR322 plasmid DNA (300 ng) by complex 1 in 50 mM Tris–HCl/NaCl buffer (pH 7.3) on photo-irradiation in monochromatic UV-A light at a wavelength of 365 nm after 40 min exposure time at different concentrations; Lane 1: DNA control; Lane 2: 20 μM 1 + DNA; Lane 3: 30 μM 1 + DNA; Lane 4: 40 μM 1 + DNA; Lane 5: 50 μM 1 + DNA; Lane 6: 60 μM 1 + DNA.

In order to determine the reactive species that are responsible for the photoactivated cleavage of the pBR322 DNA, cleavage experiments were carried out in presence of various additives such as hydroxyl radical scavengers (DMSO, EtOH), singlet oxygen quencher (NaN₃) and a superoxide scavenger (SOD) as shown in Fig. 13. Addition of DMSO and EtOH (hydroxyl radical scavengers) to the reaction mixture resulted in partial inhibition of the DNA photocleavage activity, suggesting the formation of a hydroxyl radical (˙OH) as the reactive oxygen species (ROS). Singlet oxygen scavengers, viz., sodium azide scarcely inhibited the DNA photocleavage activity. Furthermore, the addition of SOD does not show any effect on the DNA photocleavage activity suggesting no involvement of peroxide radicals. The results reveal that complex 1 is able to cleave DNA efficiently in the presence of hydroxyl radicals acting as the reactive oxygen species (ROS) responsible for the photo-damage of DNA, which implies that DNA might be cleaved by a discernible hydrolytic pathway. Hydrolytic pathways usually depend on the Lewis acidity of the central metal ion, which serves to activate the phospho-diester bonds towards nucleophilic attack via charge neutralization. The present complex contains coordinated water molecules, which facilitate the nucleophilic attack of water oxygen on phosphorus, followed by a five-coordinate phosphate intermediate and subsequent rearrangement of the phosphate, allowing the DNA to be cleaved readily (Scheme 2).

Fig. 13 Cleavage of pBR322 plasmid DNA (300 ng) by complex 1 in 50 mM Tris–HCl/NaCl buffer (pH 7.3) on photo-irradiation in monochromatic 365 nm-wavelength UV-A light after 40 min exposure time in the presence of different scavenging and groove-binding agents; Lane 1, DNA control; Lane 2, DNA + 1 + DMSO (0.4 mM); Lane 3, DNA + 1 + EtOH (0.4 mM); Lane 4, DNA + 1 + NaN₃ (0.4 mM); Lane 5, DNA + 1 + SOD (0.25 mM); Lane 6, DNA + 1 + MG (2.5 μL of a 0.01 mg mL⁻¹ solution); Lane 7, DNA + 1 + DAPI (8 μM).

Scheme 2 Proposed intermediate in the hydrolysis of DNA.

The propensity of the complexes to bind into the DNA groove was explored by using complex 1 in the presence of minor groove-binder DAPI, and major groove-binder methyl green (MG). Significant inhibition of DNA cleavage by 1 in the presence of DAPI indicates the binding of the complex to DNA through the minor groove.

Conclusion

In this work, we have designed and synthesized a new zinc(II) molecular entity (1) possessing the pharmacologically active chromone scaffold 5,10-dioxo-5,10-dihydro-chromeno[5,4,3-cde]chromene-2,7-dicarboxylic acid (H₂L), and have thoroughly characterized this complex by elemental and spectroscopic techniques in accordance with single-crystal X-ray crystallography. The DFT studies were performed by using an ab initio Gaussian 09 software package with a B3LYP/6-31+g(d,p) basis function and the HOMO–LUMO gap of 0.238 eV indicates the high stability of this molecule. The in vitro DNA-binding studies of complex 1 were carried out to examine its effect on DNA binding propensity, which reveals an electrostatic mode of binding, in addition to selective recognition for the minor groove of DNA. The complex 1 cleaves supercoiled plasmid pBR322 DNA through a hydrolytic mechanism. Furthermore, affinity of complex 1 for HSA was investigated in order to understand the carrier role of serum albumin for complex 1 in blood under physiological conditions, and this experiment demonstrated that complex 1 binds HSA with low affinity as compared to DNA, suggesting a possible release from the serum albumin to the DNA in the targeted cells.

Experimental

Materials

Reagent-grade 2-bromo-1,3,5-trimethyl-benzene and Zn(NO₃)₂·6H₂O were procured from Aldrich and calf thymus DNA (CT DNA), 6× loading dye (Fermental Life Science) and supercoiled plasmid DNA pBR322 (Genei) were used as received. HSA (fatty acid free, 99%) was purchased from Sigma and used without further purification.

Methods and instrumentation

Carbon, hydrogen and nitrogen contents were determined on Elementar Vario EL III model. Molar conductance was measured at room temperature on CON 510 Bench conductivity TDS Meter. An IR spectrum was recorded on Interspec 2020 FTIR spectrometer in KBr pellets from 400–4000 cm⁻¹. The NMR spectra were obtained on a Bruker DRX-400 spectrometer operating at room temperature. Electrospray mass spectra were recorded on a Micromass Quattro II mass spectrometer. XRD were recorded on Rikagu mini Flex II Instrument. Electronic spectrum was recorded on a UV-1700 PharmaSpec UV-vis spectrophotometer (Shimadzu) in DMSO cuvettes of 1 cm path length. Data were reported in λ_max/nm. Fluorescence measurements were determined on an RF-5301 PC spectrofluorophotometer (Schimadzu).

Synthesis of the ligand

The ligand was synthesized using a multi-step process. The precursor biphenyl-2,4,6,2′,4′,6′-hexacarboxylic acid was synthesized with slight modifications (Scheme 1), following an earlier reported procedure.³⁷

Scheme 1 Synthetic route to metal complex 1.

(1) Synthesis of trimethyl 2-bromobenzene-1,3,5-tricarboxylate (Triester). To a stirred mixture of 2-bromomesitylene (5.0 g, 0.025 mol) and aqueous NaOH (1.3 g, 0.0325 mol, 50 mL H₂O) under reflux, solid KMnO₄ (26.4 g, 0.167 mol) was added in portions during a period of 4 h. After an additional 4 h of heating, excess KMnO₄ was eliminated by addition of 25 mL methanol. The mixture was filtered and the separated MnO₂ was washed with hot water. The filtrate and washings were combined, concentrated to 30 mL and acidified to ∼pH 1 with concentrated HCl. The deposited triacid was isolated by filtration, washed with water and dried. Yield: 3.25 g (65.0%). Melting point: 291–294 °C. The crude acid (2.0 g, 6.92 mmol) was suspended in methanol (60 mL), concentrated sulfuric acid (5 mL) was added, and the mixture was heated to reflux overnight. The volume was reduced and poured in ice-cold water whereby a white precipitate was obtained, which was filtered, washed with water and dried. Yield: 1.72 g (86.0%). Melting point: 93–95 °C. ¹H-NMR (DMSO-d₆, 500 MHz), δ (ppm): 8.62 (s, 2H, H_Ar), 3.75 (s, 9H, CH₃). ¹³C-NMR (DMSO-d₆, 125 MHz), δ (ppm): 167.8, 149.8, 129.9, 129.4, 127.9, 55.2. ESI-MS: m/z [M + 1] 332.1071 (100%). Elemental analysis: calculated for C₁₂H₁₁BrO₆: C, 43.53; H, 3.35%; found: C, 43.65; H, 3.21% (Fig. S4–S6†).

(2) Synthesis of 1,1′-biphenyl-2,2′,4,4′,6,6′-hexacarboxylic acid (H₆L). Trimethyl 2-bromobenzene-1,3,5-tricarboxylate (1.7 g, 0.005 mol) was put in a round-bottomed flask containing dry xylene (40 mL) provided with a reflux condenser and a thermometer. The mixture was refluxed with vigorous stirring under argon atmosphere for 20 min. Copper powder (0.41 g, 0.006 mol) was added to the stirred mixture. The mixture was refluxed for another 90 min. After that, a second portion of copper powder (0.21 g) was added, and reflux was continued for an additional hour. The solution was filtered hot and washed extensively with dichloromethane to afford a white-colored filtrate. Complete removal of solvent under reduced pressure affords 1.4 g of crude product as a pale yellow powder. The crude product was recrystallized from methanol. This hexaester (1.2 g, 0.002 mol) was hydrolyzed by refluxing it with 6 N NaOH solution (20 mL) for 24 h. After cooling to 5 °C, the resulting solution was acidified with 6 N HCl to obtain a white precipitate. The precipitate was collected by filtration, washed thoroughly with water and dried in air. Yield: 1.02 g (85%). Melting point >370 °C. ¹H-NMR (DMSO-d₆, 500 MHz), δ (ppm): 8.67 (s, 4H; H_Ar). ¹³C-NMR (DMSO-d₆, 125 MHz), δ (ppm): 170.1, 167.0, 147.8, 138.9, 132.3, 129.8. ESI-MS: m/z [M − 1] 416.9977 (100%). Elemental analysis: calculated for C₁₈H₁₀O₁₂: C, 51.69; H, 2.41%. Found: C, 51.48; H, 2.34% (Fig. S7–S9†).

(3) Synthesis of [Zn(HL)₂·(H₂O)₄] (1). The ligand 5,10-dioxo-5,10-dihydro-chromeno[5,4,3-cde]chromene-2,7-dicarboxylic acid (H₂L) was achieved via in situ solvothermal synthesis. To the best of our knowledge, this is the first example wherein a chromene moiety is generated in situ by solvothermal synthesis. Biphenyl-2,4,6,2′,4′,6′-hexacarboxylic acid (40 mg, 0.096 mmol), Zn(NO₃)₂·6H₂O (60 mg, 0.19 mmol), 2 mL of DMF and 1 mL of H₂O were sealed in a Teflon-lined autoclave and heated under autogenous pressure to 100 °C for three days and then allowed to cool to room temperature at the rate of 1 °C per minute. Block-shaped orange crystals of 1 were collected in ∼54% yield. Melting point: 248 °C. Anal. calculated for C₃₂H₁₈O₂₀Zn: C, 48.79; H, 2.30%. Found: C, 48.71; H, 2.39%. IR (cm⁻¹): 3384(s), 1607(s), 1422(s), 1376(s), 1340(m), 1205(m), 940(m), 802(m), 776(s), 720(s). ESI-MS: m/z (100%) 788.83 [M + 1]⁺ (Fig. S10 and S11†).

Single-crystal X-ray studies

Single-crystal X-ray data of complex 1 were collected at 100 K on a Bruker SMART APEX CCD diffractometer using graphite monochromated MoK_α radiation (λ = 0.71073 Å). The linear absorption coefficients, scattering factors for the atoms and the anomalous dispersion corrections were referred from the International Tables for X-ray Crystallography.³⁸ The data integration and reduction were worked out with SAINT³⁹ software. An empirical absorption correction was applied to the collected reflections with SADABS,⁴⁰ and the space group was determined using XPREP.⁴¹ The structure was solved by direct methods using SHELXTL-97⁴² and refined against F² by full-matrix least-squares using the SHELXTL-97 programme⁴³ package. Only a few H atoms could be located in the difference Fourier maps in the structure. The rest were placed at calculated positions using idealized geometries (riding model) and assigned fixed isotropic displacement parameters. All non-H atoms were refined anisotropically. Atoms C16 and C32 were refined isotropically. Several DFIX commands were used for fixing some bond distances in complex 1. The crystal and refinement data are listed in Table 1. Selective bond distances and angles are given in Table S2.†

Table 1 Crystal and structure refinement data for 1

Parameters	1
a GOF is defined as{∑[w(Fo^2 − Fc^2)]/(n − p)}^1/2 where n is the number of data and p is the number of parameters.b R = {∑‖Fo|−|Fc‖/∑|Fo|, wR2 = {∑w(Fo^2 − Fc^2)^2/∑w(Fo^2)^2}^1/2.
Formula	C32H18O20Zn
Fw (g mol^−1)	787.83
Cyst syst.	Monoclinic
Space group	P21
a (Å)	11.5488(19)
b (Å)	5.3260(9)
c (Å)	23.007(4)
α (deg.)	90.00
β (deg.)	100.302(3)
γ (deg.)	90.00
U (Å^3)	1392.3(4)
Z	2
ρcalc (g cm^−3)	1.874
μ (mm^−1)	0.989
F (000)	796
Crystal size (mm)	0.21 × 0.16 × 0.13
Temp. (K)	100(2)
Measured reflns	7456
Unique reflns	4795
θ range (deg.)/completeness (%)	1.80 to 25.50/0.989
GOF^a	1.064
Final R^b indices	R1 = 0.0480
R^b indices	R1 = 0.0530
Largest diff. peak/hole (e Å^−3)	1.141/−0.550

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Theoretical calculations

Quantum chemical calculations were pursued at the level of density functional theory (DFT) to calculate the optimized geometry. The Gaussian 09 software package was used to find the optimized energies of frontier molecular orbitals (HOMO and LUMO) with B3LYP/6-31+g(d,p) basis function.⁴⁴ Optimizations were carried out employing X-ray coordinates using Becke's three-parameter hybrid exchange functional with the Lee–Yang–Parr correlation function (B3LYP).^45–47 The Hay and Wadt basis set LANL2DZ was used for the zinc metal centre while for rest of the atoms the 6-31G basis set was employed. Tight convergence criteria were used with the self-consistent field “tight” option in all calculations, in order to ensure sufficiently good convergence. The authenticity of the final optimized geometry was confirmed by the absence of any imaginary frequency values, and also the convergence in force and maximum displacement further established the occurrence of ground-state minima.

DNA-binding and cleavage experiments

DNA-binding experiments, including absorption spectral traces, emission spectroscopy and viscosity, conformed to the standard methods and practices previously adopted by our laboratory.^48–51 While measuring the absorption spectra, an equal amount of DNA was added to both the compound solution and the reference solution to eliminate the absorbance of the CT DNA itself, and Tris buffer was subtracted through baseline correction.

HSA-binding studies: sample preparation

Human serum albumin at a concentration of 1 × 10⁻³ M was prepared by dissolving the protein in Tris–HCl buffer solution at pH 7.4. The protein concentration was determined spectrophotometrically using an extinction coefficient of 35 219 M⁻¹ cm⁻¹ at 280 nm.⁵² A stock solution of complex 1 (1 × 10⁻³ M) was prepared by dissolving it in doubly distilled water. A NaCl (analytical grade, 1 M) solution was used to maintain the ionic strength of the buffer at 0.1 M, and the pH was adjusted to 7.4 by using HCl. Working standard solutions were obtained by appropriate dilutions of the stock solution.

Acknowledgements

We gratefully acknowledge the financial support received from the DST-PURSE, DST-FIST programme and DRS-1 (SAP) from UGC, New Delhi, India and Mohd Afzal and Mehvash Zaki are thankful for SRF from CSIR India. PKB gratefully acknowledge the financial support received from the DST-SERB program.

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Footnote

† Electronic supplementary information (ESI) available. CCDC 967235. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4ra05637j

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