Synthesis, Characterization, DNA, Fluorescence, Molecular Docking, and Antimicrobial Evaluation of Novel Pd(II) Complex Containing O, S Donor Schiff Base Ligand and Azole Derivative

Abstract

Pd(II) with the Schiff base ligand 2-Hydroxy-3-Methoxy Benzaldehyde-Thiosemicarbazone (HMBATSC) (L2) and 2-aminobenzothiazole (2-ABZ) (L1) was synthesized. The Schiff base ligand and the Palladium(II) complex were characterized by C.H.N.S, FT-IR, conductance studies, magnetic susceptibility, XRD, and TGA. From the elemental analysis and spectral data, the complex was proposed to have the formula [Pd(HMBATSC)(2-ABZ)H₂O]. The interaction between the Pd(II) complex and DNA was examined through various methods, including UV–Vis spectroscopy, fluorescence techniques, and DNA viscosity titrations. The findings provided strong evidence that the interaction between the Pd(II) complex and DNA occurs through the intercalation mode. The analysis yielded the following values: a Stern–Volmer quenching constant (k_sv) of 1.67 × 10⁴ M⁻¹, a quenching rate constant (k_q) of 8.35 × 10¹¹ M⁻¹ s⁻¹, a binding constant (k_b) of 5.20 × 10⁵ M⁻¹, and a number of binding the sites (n) of 1.392. DFT studies suggest that the azole derivative may act as an electron donor through pyridine nitrogen, while the Schiff base ligand may act as an electron donor via oxygen and sulfur atoms. TDDFT calculations indicate that the intramolecular charge transfer from the Schiff base to Pd(II) is responsible for the complex’s fluorescence quenching. The powder X-ray diffraction data revealed that the complex is arranged in a monoclinic system. The resulting Pd(II) complex was investigated for its antimicrobial activity and demonstrated antibacterial efficiency. Interestingly, it showed potent activity against E. coli and E. niger that was found to be more powerful than that recorded for Neomycin.

1. Introduction

The synthesis of transition metal complexes with 2-hydroxy-3-methoxy benzaldehyde-thiosemicarbazone (HMBATSC) ligands has received considerable attention due to the pharmacological properties of both ligands and coordination complexes [1,2,3,4,5]. Today, most 2-hydroxy-3-methoxy-benzaldehyde (ortho-vanillin) is used in the study of mutagenesis, and as a synthetic precursor for pharmaceuticals, for example, benafentrine, and an antiandrogen compound called Pentomone [6,7]. Additionally, it finds its major applications as an antitumor agent for bacterial infections, and as an antifungal and antibacterial [8]. Thiosemicarbazones are a class of organic compounds containing a central thiosemicarbazide functional group (-C(=S)N-NH₂). These compounds have been widely studied due to their diverse biological activities, including antifungal, antimicrobial, antiviral, and anticancer properties; for example, the ligands’ cytotoxic effects were investigated on four human cancer cells, and the greatest effect was on the breast MCF-7 cells. The human cancer cell lines {A-549 (lung), MCF-7 (breast), HEPG-2 (liver), and HCT-116 (colon)} and BHK (kidney) normal cells were provided from the American Type Culture Collection and maintained at the National Cancer Institute (Cairo, Egypt) [8,9,10]. Several substituted benzaldehyde thiosemicarbazone compounds were synthesized to act as inhibitors for xanthine oxidase (XO). The interaction between these compounds and XO was studied using UV–Vis spectroscopy [11]. Previously, 2-Hydroxy-3-methoxy benzaldehyde thiosemicarbazone was synthesized. Its absorption spectra were measured in the wavelength region of 320–600 nm. It was observed that changes in its absorption spectra in the presence of diverse ions within a tolerance limit of ±2% were noted [12]. 2-Aminobenzothiazole is a heterocyclic compound with a benzene ring fused to a thiazole ring and an amino group attached to the benzene ring. Heterocyclic compounds like benzothiazoles are known for their diverse biological activities and applications. Some potential areas of importance for 2-aminobenzothiazole are medicinal chemistry, material science, and drug development [13,14,15]. Schiff base ligands containing O, S, and N donors with Pd(II) complexes have been of research interest for many years because of the versatility of their steric effects, widely used in catalysis, organic synthesis, materials science, and electronic properties [16,17]. Furthermore, uses in the fields of chemistry, life science, and clinical medicine have primarily concentrated on investigating the interaction between small molecules, also known as metal complexes, or drugs, ligands, and DNA [8,15]. These studies are crucial for understanding disease prevention, enhancing drug effectiveness, and developing novel medicines [18,19]. Ethidium bromide (EB), a fluorescent compound commonly used as a probe, exhibits a higher affinity for DNA duplex compared to other complexes like acridine orange and methylene blue, making it a preferred choice for studying the DNA structure in interactions involving drugs and proteins [20]. The discovery of key elements governing complexation modes benefited greatly from DFT calculations. DFT techniques show the quenching property and the coordination modes for the most stable complexes [21]. In this present paper, 2-Hydroxy-3-Methoxy-Benzaldehyde-Thiosemicarbazone (HMBATSC) and 2-aminobenzothiazole (2-ABZ) combined with Palladium(II) and formed a reddish-brown-colored complex. The metal complex was characterized by elemental analysis, FT-IR, UV–Vis, magnetic susceptibility measurements, and molar conductance.

2. Results and Discussion

The novel Pd(II) compound was obtained by the reaction of Pd(II) acetate with 2-Hydroxy-3-Methoxy-Benzaldehyde-Thiosemicarbazone (HMBATSC) and 2-aminobenzothiazole (2-ABZ) (Figure 1) in a (1:1) molar ratio. The complex is air-stable and soluble in EtOH and DMSO.

2.1. FT-IR Spectral Studies

The FT-IR spectrum of the Pd(II) complex is listed in the KBr pellet and shown in Figure 2. The peaks shown at 3456 cm⁻¹ and 3340 cm⁻¹ may be due to the asymmetric and symmetric -N-H stretching frequency of the primary NH₂ group [22]. The peak noticed at 3200 cm⁻¹ may be accorded to the -OH stretching frequency of the phenolic group due to intramolecular hydrogen bonding [23]. The peak recorded at 3058 cm⁻¹ is listed as the Ar-H stretching frequency of the aromatic proton. The peak shown at 1612 cm⁻¹ is assigned to the -C=N stretching frequency of azometheine. The peaks noticed in the range 1526–1356 cm⁻¹ are characteristic of an aromatic ring stretching frequency. A peak shown at 952 cm⁻¹ is assigned to the C=S stretching frequency. Moreover, the stretching vibration of the NH₂ group in ABZ, observed at 3270 cm⁻¹, shifted to a lower wave number and appeared at 3160 cm⁻¹ in the complex, suggesting the coordination of the amino nitrogen to the metal(II) ion. The band at 3412 cm⁻¹ in the spectra of the complex was assigned to the νOH of the coordinated water molecule. M-oxygen and M-nitrogen bonding was manifested by the appearance of bands in the 552 and 438 cm⁻¹ regions, respectively [24] Figure 2.

2.2. UV–Vis Absorption and Fluorescence Spectra

In ethanol, the complex displays a pronounced and robust absorbance peak at 225 nm, linked to the spin-allowed S₀→S₂ transition. Two moderately intense bands appearing at a longer wavelength of 255 and 312 nm signify a spin-allowed S₀→S₁ transition [25]. Additionally, the bands with slightly reduced intensity at 320 nm are linked to the spin-forbidden S₀→T₁ transition [26]. The presence of the heavy Palladium atom connecting the two moieties (L1 and L2) enhances this transition, facilitating intersystem crossing, as shown in Figure 3.

Figure 4 shows that the absorption band peaks and intensity of 2-aminobenzothiazole (L1) and 2-Hydroxy-3-methoxybenzaldehyde thiosemicarbazone (L2) change when they form a complex through a Palladium bridge, suggesting alterations in their electronic structure. L1 exhibits a significant 6 nm blue shift in its absorption spectrum, accompanied by decreased absorption density in the S₀-S₁ transition, indicating changes in electronic energy levels. Similarly, L2 shows a 7 nm blue shift in the S₀-S₂ transition, with reduced absorption densities at lower-energy peaks, suggesting changes in electronic configuration and energy levels. These shifts and reductions serve as additional indicators of compositional changes induced by the Pd(II) complex association.

Figure 5 displays a substantial Stokes shift range of 109 nm for the Pd(II) complex in an ethanol solvent, suggesting excited-state solvation. This occurrence is attributed to the interaction between the electronic structure of the studied compound molecules and the polarity of the surrounding solvent molecules, indicating the possibility of an excited-state geometry different from the ground-state geometry. The absence of vibrational structure in the emission spectrum, in contrast to the absorption spectrum, along with a red shift indicating a higher ground-state solvation [25], as shown in Figure 4, supports this observation. Furthermore, this significant Stokes shift suggests a promising dye with a minimal re-absorption of photons, making it an efficient source of fluorescence emission, especially at high concentrations. Such a dye could have applications in optical devices. Figure 6 also demonstrates the excitation spectrum’s tendency to resemble the absorption spectrum and its relative coincidence with it, indicating the relative purity of the studied complex.

While examining the absorption peaks, we observe a 295 nm shoulder in L-1′s absorption spectrum. Upon the complex formation, this shoulder shifted to approximately 320 nm (see Figure 4), leading to the emergence of a complex-induced emission upon excitation of the complex at 320 nm as shown in Figure 7. This substantiates the formation of a superposition, particularly evident as the emission peak of the complex shifted towards longer wavelengths. The emission peak of ligand 1, initially at 402 nm, shifted to 420 nm by forming a Palladium complex. This red shift in the fluorescence spectrum is likely attributed to alterations in the excited-state dynamics of the complex [25]. The emission spectrum of the complex falling between the emission spectra of L1 and L2 is another indication of the formation of the Palladium complex.

The heavy Palladium(II) atom effect is responsible for the Schiff base (ligand2) emission intensity reduction from 100% to 40% at the 500 nm peak. Intersystem crossing (ISC) between the ligand’s singlet and triplet excited states is made easier by the Palladium(II) metal center, which improves the spin–orbit coupling in the Schiff base ligand. As seen in Figure 7, this transition reduces the fluorescence emission, promoting phosphorescence, which normally has a lower emission intensity than fluorescence.

The diamagnetic behavior of the Palladium(II) compound is consistent with a square planar geometry as shown in Figure 8. This square planar coordination with a Schiff base ligand can potentially lead to a decrease in the fluorescence intensity of the Schiff base, as the coordination can facilitate energy transfer processes and quench the Schiff base fluorescence. However, the specific relationship would require further investigation.

Moreover, the reduction in emission intensity of ligand 2 at the 500 nm peak, from 100% to 40%, is attributed to quenching due to complex formation. The diamagnetic behavior of the Pd(II) compound suggests square planar geometry. The suggested structures of the Pd(II) complex are shown in Figure 8.

2.3. Thermal Analysis of [Pd(HMBATSC)(2-ABZ)H₂O] Compound

Four decomposition steps were observed for the thermolysis curve of the Pd(II) compound. These occur in the temperature ranges 60–112, 114–262, 264–398, and 400–650 °C (Figure 9). The first mass loss corresponds well with the release of water molecules; the corresponding mass loss was found to be 3.25% (calc. 3.48%). For this stage, a D.T.G midpoint appears at 80 °C, with an endothermic peak in the D.T.A curve at 82 °C. The mass loss in the second stage agrees well with the expected loss of the 2-ABZ ligand (calc. 29.06%, found at 27.98%) (D.T.G peak at 235 °C). This stage is marked on the D.T.A curve as an exothermic peak at 237 °C. Then, decomposition products are produced in the rest of the third and fourth stages (DTG peak at 378 and 580 °C), for which exothermic peaks at 380 and 582 °C are recorded in the DTA trace. The residue was suggested to be PdO, based on mass loss consideration (calc. 23.68%, found 23.05%).

2.4. XRD Analysis of the Palladium(II) Compound

The XRD pattern of the prepared Pd(II) compound is shown in Figure 10. including Y obs and Y ca. The results revealed that the compound is crystalline. The crystal data for the Pd(II) compound belong to the monoclinic crystal system. The significant broadening of the peaks indicates that the particles are of nanometer (nm) dimensions. Scherrer’s equation was applied to evaluate the particle sizes of the complex. The XRD crystal data are presented in

Table 1. XRD data of Pd(II) complex.

Parameters	Pd(II) Complex
F.W	C16H20N5S2PdO4 516.83
Crystal System	Monoclinic
a (Å)	17.92
b (Å)	15.99
c (Å)	9.89
Alfa (°)	90.00
Beta (°)	90.00
Gamma (°)	90.00
Particle Size (nm)	18.7
V.U.C (Å3)	2836

Alfa, Beta, and Gamma are the angles of the crystal system; a, b, and c are the dimensions of the crystal system of the complex.

.

2.5. Antimicrobial Activity

The antimicrobial activity of the prepared Pd(II) complex was tested by the cup diffusion agar method. The four representative test microbes used were Staphylococcus aureus, Escherichia coli, Candida albicans, and Aspergillus niger. The inhibition zone diameters of the tested compound are shown in (

Table 2. The antimicrobial activity of the Pd(II) complex against different tests.

Sample Name	Clear Zone (mm)
	S. aureus	E. coli	C. albicans	A. niger
Pd(II) complex	28	27	26	28
Neomycin	31	25	29	0
Cyclohexamide	0	0	0	36

). Generally, the results revealed the observation that the most susceptible organisms were A. niger and E. Coli, exhibiting relatively more sensitivity to the Pd(II) complex. It was also obvious that the activity afforded for the metal complex was higher than that recorded for Neomycin, especially for E. coli and A.niger. (Figure 11, Figure 12, Figure 13 and Figure 14).

2.6. DNA Binding Studies

2.6.1. UV–Vis Spectral Study

The absorption spectra were used to investigate the mechanism of Pd(II) complex binding to DNA. Different DNA concentrations were included in the examination of the molecule, to assess the binding of the compound to DNA. Figure 15 illustrates the absorption spectra of the complex under study. Upon the addition of the DNA, a slight change is observed in the absorption spectra of the complex. Specifically, a redshift (bathochromic) of 3 nm is noticed from the absorption at 308 nm, and the absorption intensity decreases by 5.82% (hypochromic effect). These spectral characteristics suggest that the complex interacts with DNA through intercalative binding modes. It is worth noting that intercalation typically results in hypochromism and bathochromism in the electronic absorption spectra. The Benesi–Hildebrand equation was employed to calculate the binding constant (k_b) between the complex and DNA by analyzing the variations in absorbance (A) resulting from DNA intercalation [27]:

$$\frac{Ao}{Ao-A}=\frac{{\epsilon }_G}{{\epsilon }_{H-G}-{\epsilon }_G}+\frac{{\epsilon }_G}{{\epsilon }_{H-G}-{\epsilon }_G}\times \frac{1}{k_b}\frac{1}{C_{DNA}}$$

(1)

The k_b value was determined by analyzing the plot of A_o/A_o − A versus 1/C_DNA as shown in Figure 16, where A_o represents the initial absorbance and A represents the absorbance after DNA binding. The slope and intercept of the plot were used to calculate the k_b value. The calculated value for k_b was determined to be 5.20 × 10⁵ M⁻¹. The calculated value of k_b for the complex is within the range of classic intercalators like ethidium bromide and [Ru(phen) DPPZ, which typically fall between 1 × 10⁵ and 1 × 10⁶ M⁻¹ [28]. This suggests that the complex binds to the DNA through the intercalation mode. The computation of the standard Gibbs free change ΔG_o provides valuable insights into the degree of binding and the stability of the compound formed in the intercalation binding system. This information can be obtained through the utilization of the following equation:

$$\Delta G_o=-RT\ln k_b$$

(2)

Incorporating the gas constant (R) with a value of 8.31 J K⁻¹ mol⁻¹, the absolute temperature (T) set at 298.15 K, and the binding constant (k_b), the computation of ΔG_o reveals a negative value of −32.625 kJ/mol. This negative value signifies the favorable and spontaneous nature of the binding process between the complex and DNA.

2.6.2. Fluorescence Studies

In contemporary research, ethidium bromide has emerged as a prominent probe molecule for exploring the interaction between small molecules and DNA [29,30]. The binding of EB to DNA is predominantly recognized as intercalation binding, wherein it effectively inserts itself between adjacent base pairs within the DNA double helix. Notably, EB demonstrates exceptional sensitivity and selectivity, as evidenced by a substantial enhancement in fluorescence intensity upon its addition to a DNA solution. The interaction between the synthesized Pd(II) compound and DNA was scrutinized utilizing fluorescence quenching experiments employing EB. In a Tris buffer solution, both free EB and free DNA, alongside the complex, displayed extremely weak fluorescence emissions within the 520 to 680 nm range. In contrast, the amalgamation of DNA with EB exhibited a marked increase in fluorescence intensity, as depicted in Figure 17.

In Figure 18, the emission spectra of EB bound to DNA are presented, showcasing the effects of the presence or absence of the complex. Remarkably, the presence of the complex, which actively intercalates itself into the DNA base pair, leads to a significant 73.75% reduction in the fluorescence intensity of EB-DNA. This compelling evidence strongly suggests that the interaction between the Pd(II) compound and DNA occurs through the intercalation mode [31].

The assessment of fluorescence quenching efficiency involves the utilization of the Stern–Volmer constant, k_sv. This constant is derived by employing the classical Stern–Volmer equation, which allows for the quantification of the quenching process and its dependence on the concentration of the quencher [32]:

$$\frac{F_o}{F}=1+k_q{\tau }_o\left[Q\right]=1+k_{sv}\left[Q\right]$$

(3)

where F_o demonstrates the fluorescence intensity in the absence of the complex, while F represents the fluorescence intensity in its presence. The quenching rate constant for the interaction between DNA and EB is represented by k_q. Additionally, the average lifetime of the DNA-EB complex (τ_o) in the absence of the complex is determined to be 2 × 10⁻⁸ S when the DNA-to-EB ratio is 3 [19]. [Q] represents the concentration of the complex. k_sv, determined from the plot of F_o/F versus [Q] (Figure 19), is found to be 1.67 × 10⁴ M⁻¹. The quenching rate constant, k_q, is calculated as k_sv/τ_o, resulting in a value of 8.35 × 10¹¹ M⁻¹ s⁻¹. Interestingly, this value surpasses the maximum scatter collision quenching constant observed for various quenchers interacting with biomolecules, which is typically around 2.0 × 10¹⁰ M⁻¹ s⁻¹. Consequently, this finding strongly suggests that the interaction between MIS and DNA predominantly exhibits static quenching behavior [33].

The binding constant (k_b) and the binding site number (n) of the Pd(II) complex with the DNA-EB complex were evaluated using the following equation [34]:

$$\log \frac{F_o-F}{F}=\log k_b+n\log \left[Q\right]$$

(4)

A good linear correlation between log [(F_o − F)/F] and log [Q] (Figure 20) determined a binding constant (k_b) of 5.01 × 10⁵ M⁻¹ and a binding site number (n) of 1.392.

2.6.3. Viscosity Studies

In addition to the spectroscopic data, we performed viscosity experiments, which are more reliable and critical for confirming the DNA-binding model in solution. These experiments provide more compelling evidence in support of the intercalation mode [35] The classical intercalative mode of DNA binding leads to a significant increase in DNA viscosity as it elongates the DNA structure. On the other hand, partial or non-intercalative binding modes, such as electrostatic interactions, can cause a bend in the DNA helix, resulting in a decrease in viscosity. Additionally, the groove binding mode typically has little to no effect on the DNA viscosity [36]. Based on the results depicted in Figure 21, a notable increase in the relative viscosity of the DNA was observed upon the addition of the Pd(II) complex. This indicates that the binding between the DNA and the Pd(II) compound is consistent with the intercalation mode.

2.7. Computational Study of Ligands L2H2 and Deprotonated L2²⁻

As a first step, the electron donor characteristics of ligand L2H2 and its deprotonated derivative L2²⁻ were investigated. Srivani et al. [30] suggested that deprotonated L2²⁻ results from the dissociation of the hydroxyl proton and the SH proton. Figure 22 shows their optimal structures. The molecules L2H2 and L2²⁻ are nearly planar. From L2H2 to L2²⁻, the distances C-OH (1.34 Å) and NH-C (1.37 Å) are reduced to C=O (1.25 Å) and N=C (1.31 Å), while the distance S=O(1.66 Å) is increased to S-O (1.74 Å). The electronic structure of molecules L2H2 and L2²⁻ is analyzed using Gaussian software 09 [37]. The generated isosurfaces and electrostatic potential maps are depicted in Figure 23. The molecule’s HOMO orbitals mostly have the character p and are made up of the p-orbitals of S, O, N, and aromatic carbons. The electron density is highest on the sulfur and oxygen atoms.

According to the electron density distribution, the L2²⁻ ligand may operate as an electron donor via oxygen and sulfur atoms, as expected.

2.8. Computational Study of Ligand L1

Ligand L1′s electron donor characteristics were first examined. In Figure 24, the optimized structure is shown. It is a planar molecule. A Gaussian program was used to analyze the electronic structure of L1 [37]. Figure 25 displays the electrostatic potential maps and isosurfaces that were produced. Because they are made up primarily of the p-orbitals of S, N, and aromatic carbons, the molecule’s HOMO orbitals have a p-orbital nature. The nitrogen atoms have the highest electron density. The electron density distribution suggests that the L1 ligand could operate as an electron donor through pyridine nitrogen but also NH2 nitrogen.

2.9. Computational Study of Palladium(II) Complex

The ligand’s bonding with Pd is obvious with L2²⁻, as shown by the electron density of L2²⁻ (Figure 23) and suggested by Srivani et al. [30]. Various propositions were investigated to better understand how ligand L1 is linked to the Palladium. Figure 26 depicts the optimal molecular geometries of the complexes (a) Pd(L2)(^NL1), (b) Pd(L2)(^NH2L1), and Pd(L2)(^SL1). L1 is connected to the central atom via the pyridine nitrogen in Pd(L2)(^NL1). In Pd(L2)(^NH2L1), L2 is connected to the Palladium via the NH2 group nitrogen. The metal is bound to the sulfur atom in Pd(L2)(^SL1). The theoretical investigation demonstrated that the Pd(L2)(^NL1) complex is more stable in the gas phase and in ethanol than Pd(L2)(^NH2L1) and Pd(L2)(^SL1) (

Table 3. ΔE_gas, ΔE_solv, of studied complex.

Complex	ΔEgas (kcal/mol)	ΔEsolv Ethanol (kcal/mol)
Pd(L2)(NL1)	0	0
Pd(L2)(NH2L1)	6.1	7.5
Pd(L2)(SL1)	17.5	16.9

). Pd(L2)(^NL1) distances for Pd-S(L2) = 2.4 Å, Pd-O(L2) = 2.1 Å, Pd-N(L1) = 2.2 Å, and Pd-O(Et) = 2.2 Å. The electrostatic potential maps of the complex (Figure 27) reveal that the negative potential is primarily distributed on the nitrogen atoms in the middle of the L2 ligand, implying that this molecule can attract comparable molecules from the metal cation. Figure 28 shows the HOMO and LUMO orbitals of Pd(L2)(^NL1). The HOMO for the complex is primarily made up of L2²⁻ HOMO orbitals that overlap with one of the oxygen and nitrogen lobes and one of the Pd-d orbitals. As a result, the metal would connect with the ligand via Pd oxygen, sulfur, and nitrogen for L2²⁻. For L1, the metal would be poorly bound to the pyridine nitrogen because of the tiny overlap. The LUMO orbitals of Pd(L2)(^NL1) demonstrate that the sulfur-p orbital makes the largest contribution to the LUMO, followed by the Pd-d orbitals and the pyridine nitrogen orbital.

2.10. TDDFT Results

We estimated the potential transitions in these compounds using a time-dependent DFT. Figure 28 depicts the selected molecular orbitals of ligands L1 and L2H2, as well as the complex Pd(L2)(^NL1).

Table 4. TDDFT data of selected electronic transitions, their oscillator strengths, and their absorption energies.

Complex	Transition	λ (nm)	Oscillator Strength
L2H2	HOMO → LUMO	326.8	0.2939
L1	HOMO → LUMO	247.1	0.2445
Pd(L2)(NL1)	HOMO → LUMO	736.3	0.0304

contains TDDFT data concerning electronic transitions, oscillator strength, and wavelength. The electron density in ligand L2 is mostly found on the ring, oxygen, and sulfur atoms. In Pd(L2)(^NL1), the HOMO to LUMO transition provides an intramolecular charge transfer (ICT); the electron density in HOMO is primarily on the ring and oxygen atoms of the L2 molecule, whereas the electron density in the LUMO is primarily dispersed on the ligand and metal. This suggests that the quenching of fluorescence in this complex is caused by ICT from the L2 molecule to Pd(II). These findings are supported by the extremely low oscillator strength value for this transition.

3. Experimental

3.1. Materials

The chemicals used were of analytical reagent grade (AR) and the highest purity available. High-purity 2-hydroxy-3-methoxy benzaldehyde, thiosemicarbazide, 2-aminobenzothiazole and Palladium(II) acetate were supplied from the Sigma Aldrich company, St. Louis, MO, USA.

3.2. Physical Measurements

The chemical analyses; C. H. N. S were performed using the Analyischer Functions test Var. El Fab Nr. (11982027) Elemental Analyzer. The FT-IR was obtained using potassium bromide disks (400–4000 cm⁻¹) with an FT-IR spectrophotometer, and magnetic susceptibility measurements were conducted on a magnetic susceptibility balance of type MSB-Auto. Additionally, the conductance of the Pd(II) compound was measured using a conductivity meter model (4310, JENWAY, London, UK). Thermal studies of the complex were carried out in dynamic air on a Shimadzu (DTG 60-H) thermal analyzer (Kyoto, Japan) at a heating rate of 10 °C min⁻¹. The absorption spectra of the complex and ligand (5 × 10⁻⁴ M) were measured in ethanol solvent using a Shimadzu UV-1650PC spectrophotometer (200–800 nm) with a 1 cm quartz cell. Steady-state fluorescence dye measurements were performed on the compounds using a JASCO FP-8200 spectrometer (Tokyo, Japan). The XRD analysis was recorded using an XRD diffractometer (model PW 1710, Madrid, Spain) The anode material was Cu-Kα.

3.3. Synthesis of HMBATSC Ligand

The thiosemicarbazone was synthesized by condensing 2-hydroxy 3-methoxy benzaldehyde with thiosemicarbazide in an aqueous methanol medium, the formation of the product 2-hydroxy-3- methoxy benzaldehyde thiosemicarbazone [29].

3.4. Synthesis of [Pd(HMBATSC)(2-ABZ)H₂O] Complex

The Pd(II) complex was prepared by adding a solution of Palladium(II) acetate (0.8 g, 1.54 mmol), dissolved separately in (20 mL) acetonitrile, to a solution of (HMBATSC) ligand (0.8 g, 1.55 mmol), dissolved in (20 mL) (MeOH) in a round-bottomed flask. Then (ABZ) ligand (0.53 g, 1.03 mmol) was added to the mixture as a 15 mL solution in ethanol, with continuous stirring and refluxing for 3 h. The reddish-brown precipitated compound was washed with ethanol and dried in the air after cooling at room temperature. Anal. C/H/N/S values Calc. (%) for C₁₈H₂₁N₅S₂PdO₅: M.wt. 516.83, C, 37.18; H, 3.89; N, 13.55; S, 12.40. Found: C, 37.92; H, 3.68; N, 13.30; S, 12.06. FT-IR (νmax cm⁻¹, KBr): 3200, 2969, 3058, 1612, 1526, 1429, 1356, 952, 723, 680, 552, 515, 470, 438; UV–Vis spectra (DMSO) [λmax/nm]: 347, 296, 272; M.P.: 192 °C. Conductivity (10⁻³ M/DMSO): 21.95 (Ω ohm⁻¹ cm² mol⁻¹).

3.5. Antimicrobial Activity

3.5.1. Antibacterial Investigation

The antimicrobial activity of the prepared Pd(II) complex was tested by the cup diffusion agar method. The four representative test microbes used were Escherichia coli ATCC 25933 (G-ve), Staphylococcus aureus ATCC 6538-P (G+ve), Aspergillus niger NRRL-A326 (fungus), and Candida albicans ATCC 10231(yeast).

3.5.2. DNA-Binding Studies

Salmon double-strand DNA and ethidium bromide were obtained from Sigma without undergoing any additional purification steps. To prepare the stock solutions, the DNA was dissolved in deionized water. The concentration of DNA stock solution was determined by measuring its UV absorbance at 260 nm, using a molar extinction coefficient (ε) of 6600 M⁻¹cm⁻¹ [38]. The A₂₆₀/A₂₈₀ ratio of the DNA sample was found to be 1.89, exceeding the threshold of 1.8, confirming the absence of protein contamination. A 1mM solution of ethidium bromide was prepared in deionized water and stored in a container protected from light. The experiments involving the Pd(II) complex with DNA were conducted using a 10 mM Tris buffer solution prepared in deionized water. The pH of the solution was adjusted to 7.4 using HCl.

3.5.3. Viscosity Measurements

Viscosity was measured using a viscometer in a 25 °C water bath. Flow times were measured thrice and averaged. The flow rates of DNA (2.4 × 10⁻⁵ M) and DNA with various Pd(II) complex concentrations were determined. The Relative Specific Viscosity (g) was calculated as η = (t − t_o)/t_o, where t_o is the flow time for Tris–HCl buffer alone, and t is the observed flow time for DNA in the presence and absence of the Pd(II) compound. Data were plotted as (η/η_o)^1/3 vs. the complex-to-DNA concentration ratio (r), with η_o and η representing the DNA viscosity without and with the Pd(II) complex, respectively [39].

3.5.4. Theoretical Procedures

The computations were performed using a Gaussian program [40]. The complex and ligands underwent optimization at the B3LYP level, which includes Becke’s three-parameter hybrid functional [41] and the Lee, Yang, and Parr correlation functional [42]. The LanL2DZ basis set for Pd(II) was used, as well as the 6-31G(d) basis set for the remaining atoms. These early structures were created using the Gaussview application [43]. The ligand L2H2, deprotonated anion L2²⁻, and ligand L1 structures were optimized (Figure 22 and Figure 24). The solvent model (IEFPCM) [40] in ethanol was utilized to calculate the energies in solution and the excitation energies. They were computed in solvent using the functional PBE0 as a single-point calculation, using the resultin, equilibrium geometry of the complexes in the gas phase and a basis set of LanL2DZ for Pd(II) and 6-31+G(d) for the remaining elements. The Gaussview software 09 was used to generate molecular orbital isosurfaces and electrostatic potential maps [43].

4. Conclusions

The novel complex [Pd(HMBATSC)(2-ABZ)H₂O], bearing 2-Hydroxy-3-Methoxy-Benzaldehyde-Thiosemicarbazone(HMBATSC) and 2-aminobenzothiazole (2-ABZ) ligands, has been synthesized in the present work. A spectral analysis and solid-state X-ray confirmed the coordination mode of the ligands to the Palladium and revealed the tetrahedral structure around the Palladium(II) center. The antimicrobial study revealed that the Pd(II) compound prepared was more active against E. coli and E. niger than Neomycin. According to DFT calculations, the Schiff base ligand may operate as an electron donor via oxygen and sulfur atoms, and the azole derivative could operate as an electron donor through pyridine nitrogen. TDDFT calculations suggest that the quenching of fluorescence in the complex is caused by intramolecular charge transfer from the Schiff base to Pd(II). The Pad(II) complex binds to DNA through intercalation, exhibiting a strong binding affinity (k_sv) (1.67 × 10⁴ M⁻¹), a favorable and spontaneous binding process. The quenching rate constant (k_q) is 8.35 × 10¹¹ M⁻¹ s⁻¹, the binding constant (k_b) is 5.20 × 10⁵ M⁻¹, and the number of the binding sites (n) is 1.392.