Synthesis, crystal structure and biological activities of a Ag(I) complex based on the V-shaped ligand 1,3-bis(1-benzylbenzimidazol-2-yl)-2-thiapropane

2.1 Materials and physical measurements

Calf thymus DNA (CT-DNA) and ethidium bromide (EB) were obtained from Sigma-Aldrich. Tris-HCl buffer and EDTA-Fe(II) solution were prepared using bidistilled water. Silver crotonate was prepared from silver nitrate and sodium crotonate. Other reagents and solvents were reagent grade as obtained from commercial sources and used without further purification. The stock solution of the complex in DMF had the concentration 3×10⁻³ M. The stock solution of DNA (2.5×10⁻³ M) was prepared in 5 mM Tris-HCl/50 mM NaCl buffer (pH=7.2, stored at 4°C and used within 4 days). A solution of CT-DNA gave a ratio of UV absorbance at 260 and 280 nm of about 1.8–1.9, indicating that the CT-DNA was sufficiently free of protein [23].

C, H and N elemental analyses were determined using a Carlo Erba 1106 elemental analyzer. ¹H NMR spectra were recorded on a Varian VR400 MHz spectrometer with tetramethylsilane (TMS) as an internal standard. Melting points were determined on X-4 digital micro melting-point apparatus. IR spectra were recorded on a Nicolet FT-VERTEX 70 spectrometer in the range 4000–400 cm⁻¹ using KBr pellets. Electronic spectra were taken on a Lab-Tech UV Bluestar spectrophotometer. Fluorescence spectra were recorded on a Perkin Elmer LS-45 spectrofluorophotometer. The antioxidant activity against hydroxyl radicals was measured in a water bath with a Spectrumlab 722sp spectrophotometer. The synthetic route for the ligand bbbt is shown in Scheme 1.

Scheme 1:

Synthetic route for bbbt.

2.2 Preparation of bbbt

An amount of 5.88 g (0.02 mol) of 1,3-bis(benzimidazol-2-yl)-2-thiapropane (synthesized by the literature method [24]) was reacted with 1.56 g (0.04 mol) potassium in 150 mL distilled tetrahydrofuran, followed by the addition of 8.55 g (0.05 mol) benzyl bromide. The resulting solution was concentrated and the product recrystallized from methanol giving pale yellow block crystals of bbbt. Yield: 5.87 g (71%). M.p.: 140–142°C. – Elemental analysis for C₃₀H₂₆N₄S: calcd. C 75.92, H 5.52, N 11.80; found C 75.86, H 5.43, N 11.94. – IR (KBr): ν=1157 (C–N), 1454 (C=N), 1608 (C=C) cm⁻¹. – ¹H NMR ([D₆]DMSO, 400 MHz) δ: 4.2 (s, 4H, SCH₂), 5.5 (s, 4H, CH₂), 7.0–7.2 (m, 10H, phenyl), 7.4–7.9 (m, 8H, benzimidazolyl). – UV/Vis (DMF): λ=281, 289 nm.

2.3 Preparation of [Ag(bbbt)(crotonate)]·CH₃CH₂OH

To a stirred solution of ligand bbbt (0.0474 g, 0.10 mmol) in EtOH (5 mL) was added silver crotonate (0.0193 g, 0.1 mmol) in EtOH-CH₃CN (1:1, 6 mL). The resultant solution was allowed slowly to evaporate at room temperature in the dark. Block-shaped crystals suitable for X-ray diffraction studies were obtained after 2 weeks. Yield: 0.058 g (87%). – Elemental analysis for C₃₆H₃₇AgN₄O₃S: calcd. C 60.59, H 5.23, N 7.85; found C 60.96, H 4.79, N 7.83. – IR (KBr): ν=1160 (C–N), 1430 (C=N), 1630 (C=C) cm⁻¹. – UV/Vis (DMF): λ=280, 286 nm.

2.4 X-ray crystallography

A suitable single crystal was mounted on a glass fiber and the intensity data were collected with a Bruker APEX II area detector with graphite-monochromatized MoK_α radiation (λ=0.71073 Å) at 296 K. Data reduction and cell refinement were performed using Saint [25]. The absorption corrections were made by empirical methods. The structures were solved by Direct Methods and refined by full-matrix least squares against F² using the Shelxtl software [26]. The non-H atoms in the structure were subjected to anisotropic refinement. Hydrogen atoms were located geometrically and treated with the riding model. All H atoms were found in difference electron maps and subsequently refined in a riding model approximation with C–H distances ranging from 0.93 to 0.96 Å. Because of the disorder of the ethanol molecule and the crotonate carboxylate group, several restraints had to be applied. The crystal data and experimental parameters relevant to the structure determination are listed in Table 1. Selected bond lengths and angles are shown in Table 2.

CCDC 1509250 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

2.5 DNA-binding experiments

Absorption titration can monitor the interaction of a compound with DNA. A complex binding to DNA through intercalation is characterized by hypochromism in absorbance, as the intercalation involves a strong stacking interaction between the aromatic chromophore and the DNA base pairs. The obvious hypochromic and bathochromic shifts are usually characterized by noncovalent intercalative binding of a compound to the DNA helix. To compare quantitatively the affinity of the Ag(I) complex toward DNA, the intrinsic binding constants K_b of the three compounds to CT-DNA were determined by monitoring the changes of absorbance with increasing concentration of DNA. From the absorption titration data, the binding constant was determined using the following equation [27]:

[DNA]/(εa−εf)=[DNA]/(εb−εf)+1/Kb(εb−εf).

[DNA] is the concentration of base pairs, ε_a corresponds to the observed extinction coefficient (A_obsd/[M]), ε_f corresponds to the extinction coefficient of the free compound, ε_b is the extinction coefficient of the compound when fully bound to CT-DNA, and K_b is the intrinsic binding constant. The ratio of slope to intercept in the plot of [DNA]/(ε_a−ε_f) versus [DNA] gives the value of K_b.

The complex shows no luminescence at room temperature in aqueous solution, in the organic solvent examined and in the presence of calf thymus (CT-DNA). So in the emission spectra, the binding of the complex cannot be directly followed. Therefore, in order to examine the binding of the complex with DNA, EB binding studies were undertaken to gain support for the spectral result. In the presence of DNA, the enhanced fluorescence of EB can be quenched by the addition of a second molecule, affording a proof that the complex intercalates to base pairs of DNA [28], [29]. The fluorescence quenching of EB bound to CT-DNA by the complex is shown in Fig. 3. The quenching of EB bound to CT-DNA by the Ag(I) complex is in good agreement with the linear Stern–Volmer equation [30]:

I0/I=1+KSV[Q].

I₀ and I are the fluorescence intensities in the absence and presence of the quencher, respectively, and [Q] is the concentration of the complex, which provides further evidence that the Ag(I) complex binds to DNA and only one type of quenching process occurs.

Further clarification of the interactions between the metal complexes and DNA was reached by viscosity measurements. Viscosity experiments were conducted on an Ubbelohde viscometer, immersed in a water bath maintained at 25.0±0.1°C. Titrations were performed for the complex (1–10 μM), and the compound was introduced into the CT-DNA solution (50 μM) present in the viscometer. Viscosity values were calculated from the observed flow times of CT-DNA containing solutions corrected for the flow time of buffer alone (t₀), η=(t−t₀) [31]. Data were presented as (η/η₀)^1/3 versus the ratio of the concentration of the complex to CT-DNA, where η is the viscosity of CT-DNA in the presence of the complex and η₀ is the viscosity of CT-DNA alone.

2.6 Antioxidant study

Hydroxyl radicals were generated in aqueous media through the Fenton-type reaction [32], [33]. Aliquots of the reaction mixture (3 mL) contained 1 mL of 0.1 mmol aqueous safranin, 1 mL of 1.0 mmol aqueous EDTA-Fe(II), 1 mL of 3% aqueous H₂O₂ and a series of quantitative microadditions of solutions of the test compound. A sample without the tested compound was used as the control. The reaction mixtures were incubated at 37°C for 30 min in a water bath. The absorbance was then measured at 520 nm. All tests were run in triplicate and the results expressed as the mean and standard deviation. The scavenging effect for OH^· was calculated from the following expression [34]:

Scavenging ratio (%)=[(Ai–A0)/(Ac–A0)]×100%,

where A_i is the absorbance in the presence of the test compound, A₀ is the absorbance of the blank in the absence of the test compound and A_c is the absorbance in the absence of the test compound, EDTA-Fe(II) and H₂O₂.

3.1 IR and electronic spectra

The IR spectrum of the ligand bbbt shows characteristic absorption bands at 1157 and 1454 cm⁻¹ that can be assigned to ν_(C−N) and ν_(C=N), respectively [35]. The frequencies of the complex are slightly shifted by 3–15 cm⁻¹. These absorptions support the argument that the nitrogen atoms of the ligand are coordinated to the Ag(I) center [36].

The electronic spectra of bbbt and the complex were recorded in DMF solution at room temperature. The UV bands of bbbt (281, 289 nm) are slightly shifted by about 1–3 nm in the complex. These bands are assigned to n→π* and π→π* (imidazole) transitions, which is indicative of ligand coordination to the metal center [37].

3.2 X-ray structure of the complex

The complex crystallizes in the triclinic space group P1̅ with Z=2, and its structure along with the atomic numbering scheme is shown in Fig. 1. The mononuclear complex consists of a central metal Ag(I) atom, coordinated bbbt and crotonate ligands, and one uncoordinated ethanol molecule. The Ag(I) atom is coordinated by two oxygen atoms from a crotonate anion and two nitrogen atoms from a bbbt ligand. The coordination geometry is best described as distorted tetrahedral. Both Ag–N and Ag–O bond lengths are significantly unequal, respectively, particularly so the Ag–O bonds (Ag–O(1) 2.586(7) and Ag–O(2) 2.330(3) Å). The latter are somewhat obscured by the carboxylate disorder, however (see Table 2).

Fig. 1:

Molecular structure of the Ag(I) complex with displacement ellipsoids drawn at the 30% probability level; the solvent molecules and H atoms are omitted for clarity.

As one of the important supramolecular forces, π–π stacking maybe a specific structural requirement for the arrangement of complicated architectures or substrate recognition. As shown in Fig. 2, the centroid-to-centroid distance between π-stacked five-membered ligand rings of two neighboring complexes is 3.575 Å, indicating strong intermolecular interactions.

Fig. 2:

The structure of two Ag(I) complexes linked via a π–π stacking interaction.

3.3 DNA binding mode and affinity

3.3.1 Electronic absorption spectra

We first investigated the binding modes of DNA with bbbt and its Ag(I) complex through absorption titration experiments. The electronic absorption spectra of free bbbt and its Ag(I) complex in the absence and presence of CT-DNA are shown in Fig. 3. Upon addition of increasing CT-DNA concentrations, the bands exhibited hypochromism of 28.9% and 25.9%. From the electronic absorption spectroscopy experiments, K_b values of free bbbt and the complex were obtained as 4.07×10⁴ M⁻¹ (R²=0.9819) and 6.51×10⁴ M⁻¹ (R²=0.9898), respectively, compared with those of so-called DNA-intercalative complexes (K_b=2.9×10⁴–1.2×10⁵ M⁻¹) [38], [39], [40], [41], [42]. It may be concluded that both the ligand and the complex can bind to DNA by intercalation, with the binding affinity being stronger for the complex rather than for the free, uncomplexed ligand bbbt.

Fig. 3:

Electronic spectra of free bbbt and complex in tris-HCl buffer upon the addition of CT-DNA. The arrow shows that the emission intensity changes upon increasing DNA concentrations. [DNA]/(ε_a−ε_f) versus [DNA] for the titration of the ligand bbbt and the complex with CT-DNA.

The charge transfer of the coordinated bbbt ligand upon coordination to Ag(I) reduces the charge density of the benzimidazole, which is conducive to intercalation. In that way the affinity for DNA is stronger in the case of the Ag(I) complex as compared to the free ligand.

3.3.2 Fluorescence spectroscopy

No luminescence was observed for the complex at room temperature in any organic solvent or in the presence of CT-DNA. Competitive EB binding studies were undertaken in order to examine the binding of each compound with DNA. EB is a conjugated planar molecule. If a complex can replace EB from DNA-bound EB, the fluorescence of the solution will be quenched due to the fact that free EB molecules are readily quenched by the surrounding water molecules [43]. The Stern–Volmer plots (DNA–EB; Fig. 4) show that the quenching of EB bound to DNA by the two compounds follows a linear relationship, consistent with intercalation of the test compounds. The K_sv values for free bbbt and complex were 2.3×10³ M⁻¹ (R²=0.9972) and 4.41×10³ M⁻¹ (R²=0.9905), respectively, reflecting the higher quenching efficiency of the Ag(I) complex relative to that of bbbt. The phenomena suggest that the ligand and complex can compete for DNA-binding sites with EB and displace EB from the EB–DNA system [44], which is usually characteristic of the intercalative interaction of compounds with DNA [45].

Fig. 4:

Emission spectra of EB bound to CT-DNA in the presence of free bbbt and the Ag(I) complex. The arrows show that the intensity changes upon increasing concentrations of the complex. Fluorescence quenching curves of EB bound to CT-DNA by the free ligand and the complex. (Plots of I₀/I versus [complex].)

3.3.3 Viscosity experiment

We used viscosity measurements to probe the interactions of the two compounds with DNA. Hydrodynamic measurements that are sensitive to DNA length changes are regarded as the least ambiguous and most critical tests of a binding model in solution in the absence of crystallographic structural data [46]. Viscosity measurements were carried out on CT-DNA by varying the concentration of the complex. Figure 5 shows that the ligand and the complex cause an increase in the relative viscosity of DNA. This may be explained by the fact that the compounds can intercalate between adjacent DNA base pairs, leading to an increase in the separation of base pairs at intercalation sites and thus an increase in the overall DNA contour length. So the results demonstrate that the ligand and its complex bind to DNA by the intercalation mode.

Fig. 5:

Effect of increasing amounts of the compounds on the relative viscosity of DNA at 25.0±0.1°C.

3.4 Antioxidant activity

The complex has good hydroxyl radical scavenging activity. Figure 6 depicts the inhibitory effect of the compounds on OH^∙ radicals. The inhibitory activity of the compounds is marked, and the suppression ratio increases with increasing concentrations of the test compound. We compared the present compound with the well-known natural antioxidants mannitol and vitamin C, using the same method as reported in a previous paper [47]. The 50% inhibitory concentration (IC₅₀) values of mannitol and vitamin C are about 9.6 mM and 8.7 mM, respectively. According to the present experiments, the IC₅₀ of the complex is 42.65 μM (Fig. 6). The results imply that the complex has a higher ability to scavenge hydroxyl radicals OH^∙ than mannitol and vitamin C. As a result, the complex can be considered as a new potential antioxidant.

Fig. 6:

A plot of scavenging percentage (%) versus concentration of the Ag(I) complex on hydroxyl radicals.