DNA binding ability mode, spectroscopic studies, hydrophobicity, and in vitro antibacterial evaluation of some new Fe(II) complexes bearing ONO donors amino acid Schiff bases

2.5.1 Methodology for DNA binding using absorption spectroscopic studies

All the experiments involving the interaction of the complexes with DNA were carried out in Tris–HCl buffer (50 mM, pH 7.2). CT-DNA was purified by centrifugal dialysis before use. A solution of calf thymus DNA in the buffer gave a ratio of UV absorbance at 260 and 280 nm of about >1.86, indicating that the DNA was sufficiently free from protein contamination (Murmur, 1961; Raman et al., 2011; Abdel-Rahman et al., 2013a,b). The concentration of DNA was determined by monitoring the UV absorbance at 260 nm using ε₂₆₀ = 6600 mol⁻¹ cm². The stock solution was stored at 4 °C and used within only one day. Spectrophotometeric titration experiment was performed by maintaining the metal complex concentration constant while varying the nucleic acid concentration in the interaction medium. The absorption due to free CT-DNA was eliminated by adding an equimolar CT-DNA to pure buffer solution in the reference compartment and the resulting spectra were considered to result from the metal complexes and the DNA – metal complex aggregates. From the absorption data, the intrinsic binding constant (K_b) was determined by plotting [DNA]/(ε_a − ε_f) vs. [DNA] according to the following equation: $$\frac{[\text{DNA}]}{({\epsilon }_a-{\epsilon }_f)}=\frac{[\text{DNA}]}{({\epsilon }_b-{\epsilon }_f)}+\frac{1}{[K_b({\epsilon }_b-{\epsilon }_f)]}$$ Where, [DNA] is the concentration of DNA in base pairs, ε_a, ε_f and ε_b are the apparent, free and fully bound complex absorption coefficients, respectively. In particular, ε_f was determined from the calibration curve of the isolated metal complex; following the Beer’s law. ε_a was determined as the ratio between the measured absorbance and the metal(II) complex concentration, A_obs/[complex]. The data were fitted to the above equation with a slope equal to 1/(ε_b − ε_f) and y-intercept equal to 1/[K_b(ε_b − ε_f)] and K_b was obtained from the ratio of the slope to the intercept (Wolf et al., 1987). The standard Gibb’s free energy for DNA binding was calculated from the following relation (Sabolová et al., 2011): $$\mathrm{\Delta }{G}_b^{\circ }=-\mathit{RT}\ln K_b$$

2.5.2 Viscosity experiments for interaction of the prepared complexes with DNA

Viscosity measurements were carried out using an Oswald microviscometer, maintained at constant temperature at 25 ± 1 °C in thermostat. The flow times were recorded for different concentrations of the complex (10–250 μM), maintaining the concentration of DNA constant (250 μM). Mixing of the solution was achieved by bubbling the nitrogen gas through the viscometer. The average value of the three measurements was used to determine the viscosity of the samples. The buffer flow time in seconds was recorded as t°. The relative viscosities for DNA in the presence (η) and absence (η°) of the complex were calculated using the relation η = (t − t°)/t°. Where, t is the observed flow time in seconds and the values of the relative viscosity (η/η°) were plotted against 1/R (R = [DNA]/[Complex]) (Satyanarayana et al., 1992).

3.1.1 Melting points and decomposition temperature measurements

The prepared complexes are stable in the air. The decomposition temperature of all prepared complexes is higher than the reported melting points of the Schiff base ligands, so it is differing from all reagents used in the preparation (cf. Table 1). This can be used as an evidence for the higher thermal stability of reagents via complexation with the Fe(II) ion Banerjee et al., 1984 The formation of the investigated Schiff base amino acid ligands and their complexes is represented in Scheme 2.

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Scheme 2

HBrS = 5-bromosalicylaldehyde, AA = amino acid (ala, phala, aspa, his or arg), H₂L = Schiff base amino acid ligand, Fe(HL)₂ = Fe(II) Schiff base amino acid complexes (bsali, bsphali, bsasi, bshi or bsari).

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3.1.2 Microanalyses and molar conductance measurements

Microanalyses data of the prepared complexes are reported in Table 1 and suggested that the Schiff base ligands act as tridentate and form complexes in 1:2 ratio metal to ligand. Thus the general formula of the prepared complexes is suggested to be [Fe(HL)₂]·nH₂O. The presence of water as crystalline water is confirmed by heating the prepared complexes at 110 °C for about 2 h and the obtained weight loss percent data at 110 °C, for crystallization water, were consistent with the corresponding microanalytical data. The following results show a comparison between thermogaravimetric data at 110 °C and microanalytical data for crystallization water in the prepared complexes: {bsali: 5.5 (5.68)%; bsphali: 6.75 (6.72)%; bsasi: 2.51 (2.56)%; bshi: 6.85 (6.89)%; bsari: 4.49 (4.48)%}.

Consequently the divalent anions of the Schiff base amino acid ligand are coordinated to the central metal Fe(II) ion. The measured molar conductance values of 10⁻³–10⁻⁴ molar solutions of the prepared Fe(II) complexes in ethanol were found to be in the range 37–64 ohm⁻¹ cm² mol⁻¹ (cited in Table 1) which is in agreement with the non-electrolytic nature of the complexes (Abdel-Rahman et al., 2013a,b). The non electrolytic nature of the investigated complexes is matched with microanalytical data.

3.1.3 Magnetic moment measurements

In a magnetic field, the paramagnetic compounds will be attracted while the diamagnetic compounds repelled. For paramagnetic substances, the flux is greater within the substance than it would be in vacuum. Therefore, paramagnetic substances will have positive susceptibilities. Thus, the magnetic susceptibility measurements provide information regarding the geometric structure of the complexes. Magnetic susceptibility measurements showed that the prepared complexes have paramagnetic character and suggested high spin values (4.03–4.75 BM) i.e., the studied Schiff base amino acid ligands are so weak that they exhibited low t_2g and e_g d-splitting of the octahedral structures of the complexes (Canpolat and Kaya, 2002; El-Tabl et al., 2008; Abdel-Rahman et al., 2013a,b).

3.1.4 Infrared spectra of the prepared ligands and their Fe(II) complexes

The IR spectra provide valuable information regarding the nature of functional groups attached to the metal atom (Raman et al., 2007). The basic theory involved is that the stretching modes of the ligands changes upon complexation due to weakening/strengthening of the bonds involved in the bond formation resulting in subsequent change in the position of the bands appearing in the IR spectrum. To achieve an idea about the groups involved in the complex formation as well as the influence of the electrical field of the central metal ion on the charge distribution within the ligand, the difference between the spectrum of the free ligand and that of the complexes can full into three types:

1. Shift in band position

2. Intensity changes.

3. Band splitting and disappearance of bands and appearance of new bands.

The IR spectra of the investigated complexes are compared with those of the free ligands to determine the coordination sites which may be involved in chelation (cf. Table 2). The basic theory involved is that the stretching modes of the ligands change upon complexation due to weaking/strengthening of the bonds involved in the bond formation resulting in subsequent change in the position of the bands appearing in the IR spectrum. The spectrum of Schiff base ligands exhibits a characteristic band at 3396–3446 cm⁻¹ due to the stretching vibration of the OH group υ(OH). On the other hand, the recorded IR spectra of all the prepared complexes (cf. Table 2) show broad band at 3451–3414 cm⁻¹ which have been assigned to υ(OH) stretching vibration of water molecules, in accordance with the results of the elemental analysis listed in Table 1 (Shaker et al., 2003; Sharma and Dubey, 1994). The spectrum of the ligands show a sharp band at 1664–1638 cm⁻¹, due to an azomethine group (C⚌N) stretching vibration which indicates the presence of the imine structure (Begum et al., 2010). This band is shifted to lower wave numbers in the spectra of all complexes (1645–1619 cm⁻¹) indicating the involvement of the nitrogen atom of the azomethine group in the coordination to the metal ion (Thankamony and Mohanan, 2007; Garg et al., 2008). In the IR spectra of the Schiff bases, the two bands appeared at 1601–1577 and 1462–1406 cm⁻¹ are attributed to the asymmetric and symmetric stretching vibrations of the carboxylate group υ_as(COO) and υ_s(COO). These bands are shifted to lower wave number values upon complexation indicating that the oxygen atom of the carboxylate group is coordinated to the metal ion (cf. Table 2) Sharma and Dubey, 1994; Nakamoto, 1978. It is worth noting that the asymmetric vibration band of COO⁻ is obscured by the high intensity of band of the C⚌C stretching vibration (Teyssie and Charette, 1963; Abdel-Rahman et al., 2013b). All Schiff base ligands showed an absorption band in the region of 1374–1318 cm⁻¹ which can be attributed to the stretching vibration of the phenolic (C–O) group υ(CO)(phenolic). The shifting of these bands to lower wave number values upon complexation indicates that the oxygen atoms of the phenolic groups are coordinated to the metal ion (Sharma and Dubey, 1994). The bands observed in the regions 2940–2871 and 3054–3031 cm⁻¹ can be assigned to υ(C–H) aliphatic and υ(C–H) aromatic stretching vibrations, respectively (Abdel-Mawgoud, 1998). The IR bands found at 3359 and 3168 cm⁻¹ for the bsari complex which contains an arginine moiety could be assigned to the presence of the terminal guanidinium group (Ibrahim et al., 1999). Assignment of the proposed coordination sites is further supported by the appearance of two new medium bands at 553–540 and 478–450 cm⁻¹ which could be attributed to υ(Fe–N) and υ(Fe–O), respectively (Thomas et al., 1995; Abdel-Rahman et al., 2013a).

3.1.5 Electronic spectra

The electronic absorption spectra of ethanolic solutions of ligands and their complexes were recorded at the wavelength range 800–200 nm and at 298 K. The absorption region, the molar extinction coefficients and the band assignment of the different bands in the recorded spectra of the complexes were resulted from Figs. 1, 2). The ligand exhibits absorption bands in UV–Vis region around 380 and 410 nm which is assigned to n → π^∗ transition originating from the amide or imine function of the Schiff base ligand (Ahmad et al., 2002). Another band is observed below 306 nm, this band is assigned to π → π^∗ transition. The spectra of the complexes are dominated by intense intraligand bands centered at λ_max = 368–398 nm (ε_max = 2744–1310 mol⁻¹ cm²) and charge transfer bands centered at 428–440 nm (ε_max = 1518–824 mol⁻¹ cm²), since it is known that metal/metalloids are capable of forming dπ–pπ bonds with ligands containing nitrogen and oxygen as donor atoms (West and Nasar, 1999). Furthermore, the L → MCT band is followed by a long and a broad band lying in the region 458–494 nm (ε_max = 2650–900 mol⁻¹ cm²). This band could be mainly attributed to the d → d transition in the octahedral structure of the prepared complexes (Shaker et al., 2003; Lever, 1968; Abdel-Rahman et al., 2013b).

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Figure 1

Molecular electronic spectra of bsali complex and its components in ethanol at 298 K.

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Figure 2

Molecular electronic spectra of bsphali complex and its components in ethanol at 298 K.

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3.1.6 Determination of the stoichiometry of the investigated complexes

The spectrophotometric methods were utilized to establish the stoichiometry of various Schiff base amino acid complexes. The simplest spectrophotometric technique adopted to study the equilibria in solutions of complex compounds, is the molar ratio method (Yoe and Jones, 1944; El-Shiekh et al., 2011; Shah and Parmar, 2011) and the continuous variation method (Shah and Parmar, 2011; Job, 1928), as shown in Fig. 3. The results suggest the possible formation of 1:2 complexes as presented in Scheme 3. The curves of the continuous variation method (Fig. 3) displayed maximum absorbance at mole fraction X_ligand = 0.65–0.7 indicating the formation of complexes with metal ion to ligand ratio 1:2. Moreover, the data resulted from applying the molar ratio method support the same metal ion to ligand ratio of the prepared complexes (see supplementary material) (see Fig. 1).

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Figure 3

Continuous variation plots for the prepared complexes in aqueous-alcoholic mixture at [bsali] = 2 × 10⁻³ M, [bsphali] = 1 × 10⁻³ M, [bsasi] =5 × 10⁻³ M, [bshi] = 1 × 10⁻³ M, [bsari] = 1 × 10⁻³ M and 298 K.

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Scheme 3

The suggested structures of bsali, bsphali, bsasi, bshi complexes, bsali: n = 2, bsphali and bshi: n = 3, bsasi: n = 1 (R as shown in Scheme 1).

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3.1.6.1

3.1.6.1 Evaluation of the apparent formation constants of the synthesized complexes

The formation constants (K_f) of the studied Fe(II) Schiff base amino acid complexes formed in solution were obtained from the spectrophotometric measurements by applying the continuous variation method (Shaker et al., 2003; Issa et al., 2006) (cf. Table 3) according to the following relation: $$K_f=\frac{A/A_m}{4C^2{(1-A/A_m)}^3}$$ Where, A_m is the absorbance at the maximum formation of the complex, A is the arbitrary chosen absorbance values on either side of the absorbance mountain col (pass) and C is the initial concentration of the metal. As mentioned in Table 3, the obtained K_f values indicate the high stability of the prepared complexes. The values of K_f for the studied complexes increase in the following order: bsari > bsali > bsphali > bshi > bsasi. Moreover, the values of the stability constant (pK) and Gibbs free energy (ΔG^≠) of the investigated complexes are cited in Table 3. The negative values of Gibbs free energy mean that the reaction is spontaneous and favorable.

Table 3 The formation constant (K_f), stability constant (pK) and Gibbs free energy (ΔG^≠) values of the synthesized complexes in aqueous-ethanol at 298 K.

Complex	Type of complex	Kf	pK	ΔG≠ kJ mol−1
bsali	1:2	3.22 ± 0.02 × 109	9.98	−57.03
bsphali	1:2	3.67 ± 0.02 × 109	9.67	−54.56
bsasi	1:2	1.31 ± 0.02 × 109	8.86	−50.57
bshi	1:2	1.18 ± 0.02 × 1010	8.96	−51.13
bsari	1:2	3.15 ± 0.02 × 109	10.44	−59.55

The pH-profile (absorbance vs. pH) presented in Fig. 4 showed typical dissociation curves and a wide stability pH range (4–10) of the studied complexes. This means that the formation of the complex greatly stabilizes the Schiff base amino acid ligands. Consequently, the suitable pH range for the different applications of the prepared complexes is from pH = 4 to pH = 10.

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Figure 4

Dissociation curves of the prepared bromosalicylidene Schiff base amino acid Fe(II) complexes in aqueous alcoholic mixture at [complex] = 5 × 10⁻³ M and 298 K.

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Based on the results of elemental analysis, molar conductance, magnetic susceptibility, infrared and electronic spectra, the suggested structure of the complexes is shown in Scheme 3, 4 .

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Scheme 4

The suggested structure for bsari complex.

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3.1.7 Effect of hydrophilic/hydrophobic character of the investigated complexes on their solubilities

The hydrophobicity of the investigated Fe(II) Schiff base amino acid complexes can be rationalized to the low solubility of the investigated complexes in aqueous medium (cf. Tables 4). Moreover, the results of negative values transfer chemical potentials of CQ∗∗∗ the investigated Fe(II) Schiff base amino acid complexes in (cf. Table 5) can mainly be ascribed to of the hydrophobic nature of the investigated complexes. It observed that the hydrophobicity of the investigated complexes leads to stabilization of them with increasing solvent percent. The order of hydrophobicity can increase in the direction: $$\text{bsasi}<\text{bsali}<\text{bsari}<\text{bshi}<\text{bsphali}$$ Thus the results show preferential solvation by the solvent as molecule size increases. Also the order of hydrophobicity of bromosalicylidene Schiff base amino acid complexes could be attributed to the effect complexes.