Characterization and Biological Evaluation of a (Cp*)Rh(III) Complex Featuring Phosphonate-Modified Bipyridine Ligands

Characterization and Biological Evaluation of a (Cp*)Rh(III) Complex Featuring Phosphonate-Modified Bipyridine Ligands | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Characterization and Biological Evaluation of a (Cp*)Rh(III) Complex Featuring Phosphonate-Modified Bipyridine Ligands Thamilarasan Vijayan, Atifa Ashraf, Mohammad Azam, Jinheung Kim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6581437/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract We report the synthesis and biological activities of a (Cp*)Rh(III) complex containing 4,4’-bis(diethyl-methyl phosphonate)-2,2-bipyridine. The complex was characterized through various spectroscopic methods. Its DNA binding capacity was assessed using titration, viscosity measurements, and spectroscopic techniques including absorption, fluorescence, and circular dichroism (CD) spectroscopy. The findings revealed a positive absorption coefficient with a binding constant of 2.85 × 10 4 M − 1 . Additionally, the Rh complex showed a strong affinity for bovine serum albumin, with a binding constant that aligns with the observed DNA binding pattern. The cytotoxic effects of the complex were evaluated against selected cancer cell lines, such as MDA-MB-231 (human breast cancer) and A549 (human lung adenocarcinoma), as well as VERO (a normal human lung adenocarcinoma cell line) for comparison. The IC 50 values for MDA-MB-231 and A549 cells were determined to be 47.25 ± 0.05 µM and 48.75 ± 0.05 µM, respectively, indicating potent cytotoxicity at low micromolar concentrations. In contrast, the VERO normal cells exhibited significantly lower toxicity, with an IC 50 value of ≥ 1000 µM. Furthermore, the Rh complex was tested for its antimicrobial properties, demonstrating greater inhibitory activity compared to the free ligand. Rh(III) complex DNA binding protein binding anticancer activity cytotoxic study antimicrobial activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction In the realms of chemistry and pharmacology, the innovation of new chemical agents that can activate antibacterial and antibiotic properties is a critical concern. 1 The majority of antiviral and anti-inflammatory drugs developed over the past few decades have been centered on antibodies, natural toxins, and antagonistic drugs. 2 However, organometallic complexes of transition metals have garnered increasing interest for their potential to address these issues. Researchers are particularly drawn to organometallic complexes for their unique multi-modal reactivity, which is absent in purely organic drug molecules, offering a broader scope in the fight against antibiotic resistance. 3 Organometallic complexes have been extensively employed in recent years as promising candidates for antivirals, antimalarial agents, antibiotics, and diagnostic drugs. This is attributed to the diverse functionalities of both organic and metal complex moieties within biological systems. 4 The exploration of metals like ruthenium, rhodium, and iridium has intensified, driven by their superior stability, non-toxicity to biological entities, enhanced coordination capabilities, and chemical resilience. 5 Notably, rhodium(III) complexes are versatile in their ability to bind with a variety of ligand structures and interact with biomolecules, thereby disrupting cell metabolism and exerting antitumor effects. 6 – 7 The development of metal-based therapeutics has been bolstered by the effectiveness of cis -platin. However, due to its adverse side effects, there has been considerable research into complexes of other metals such as Ru, Rh, Ir, and Os. 8 – 10 In recent years, the formula [(η 5 -Cp*)/(η 6 -arene)M(L)Cl] 0/+ (where Cp* = C 5 (CH 3 ) 5 ; M = Pt group metals; L = a bidentate ligand) has garnered attention for its anti-cancer properties, attributed to its potent efficacy and specific mechanism of action against cancer cells. 11 Replacing the bidentate (L) ligand in half-sandwich metal complexes, a variety of ligands featuring N, O, P, and S donors have been synthesized and biologically evaluated. 12 These ligands demonstrate potential in catalysis, molecular devices, supramolecular assemblies, and exhibit antiviral, antibiotic, and antibacterial activities. For metal complexes to be effective as anticancer agents, favorable interactions with DNA are essential. Transition metal complexes have played a pivotal role in this regard, selectively targeting DNA sites. 13 The majority of tumor cells contain DNA biomolecules, which are considered a weak spot and thus a prime target for many anti-cancer drugs. These drugs and biomolecules typically interact with DNA in the β conformation through mechanisms of external attachment, binding, and intercalation between adjacent planar base pairs. Such interactions require specific features like aromatic geometry and the size of a flat stretch, or non-β motifs such as G-quadruplex DNA. 14 – 16 Pentamethylcyclopentadienyl rhodium and iridium complexes have been reported to be effective in infection prevention and exhibit high stability due to their chemical inertness. 17 Among these, the Rh(III) complex is inert and contributes to its potential inhibitory effect, while the Rh(II) complex is reactive and participates in ligand exchange processes, playing a significant role in biological functions. 18 Similarly, Rh(I) complexes, isoelectronic with Pt(II) and exhibiting square planar geometry, are anticipated to possess biochemical activities akin to cis -platin. This work aligns with recent advancements in rhodium(I) compounds within a biological context, highlighting how these molecules specifically target biomolecules for anticancer activities. Furthermore, rhodium complexes have shown significant pharmacological effects through their interactions with DNA, underscoring their utility in the biological sciences. 19 – 20 In this study, we present a pentamethylcyclopentadienyl Rh(III) complex coordinated with a 4,4-bis(diethyl-methyl phosphonate)-2,2-bipyridine ligand. We have characterized its affinity for calf-thymus DNA through titrations and a suite of analytical techniques, including spectroscopy, optical assays, and viscosity measurements. The interaction of the Rh complex with bovine serum albumin protein was also explored. Furthermore, we assessed the cytotoxicity of the Rh(III) complex against both cancerous and non-cancerous cell lines. The antimicrobial efficacy of the complex was evaluated against a panel of bacteria, encompassing two Gram-positive and two Gram-negative strains, as well as a viral pathogen. Experimental section Materials and Instrumentation . We employed analytical reagent-grade chemicals without further purification, and the solvents were treated according to standard procedures. The following reagents were used: calf thymus DNA (CT-DNA), Tris-HCl, Tris bases, NaCl, pentamethylcyclopentadienyl rhodium (III) chloride, triethyl phosphite, ethidium bromide (EB), and BSA (all from Sigma-Aldrich). Solutions were prepared using double-distilled water. Additional chemicals, including 4,4’-dicarboxy-2,2’-bipyridine, ammonium chloride, sodium borohydride, and magnesium sulfate, were sourced from TCI Chemicals. Spectroscopic analyses included UV-vis spectra recorded on a Shimadzu UV-3101PC spectrophotometer and FT-IR spectra obtained using a Bruker INVENIO-R spectrometer with potassium bromide pellets in the 4000 − 200 cm − 1 range. Conductivity measurements of the complex dissolved in DMF was conducted using the ELICO-SX80 model conductivity bridge. The 1 H-NMR spectra of compound 1 and complex 2 were recorded in CDCl 3 and DMSO- d 6 on a BRUKER 300 MHz FT-NMR system spectrometer at room temperature, with TMS as the internal reference. Elemental analysis was performed using an Elementarvario MACRO cube elemental analyzer. For biomolecular studies, we utilized an SHIMADZU-00774 spectrofluorometer (for fluorescence studies) and a JASCO J-810 spectropolarimeter (for circular dichroism studies). CT-DNA solutions were prepared in Tris-HCl/NaCl buffer (5 mM Tris-HCl, 50 mM NaCl, pH = 7.2) and stored at 4 ℃ for no more than one week. Protein-free DNA was quantified by UV absorbance at 260 and 280 nm, with the CT-DNA concentration determined based on its density at 260 nm and the molar extinction coefficient of 6600 M − 1 cm − 1 . Cytotoxicity testing was conducted using the MTT method at the BioMe Live Analytical Center in India. Synthesis of 4,4’-bis(diethylmethylphosphonate)-2,2’-bipyridine (1) . We synthesized ligand 4,4’-bis(diethylmethylphosphonate)-2,2’-bipyridine ( 1 ) from the precursor 4,4’-dicarboxy-2,2’-bipyridine using a four-step reaction method described in the literature (Scheme 1 ). 21 Synthesis of complex 2 . Complex 2 was prepared by reacting pentamethylcyclopentadienyl rhodium(III) chloride dimer (0.5 mmol) with 4,4-bis(diethyl-methyl phosphonate)-2,2-bipyridine (1.0 mmol) in 20 mL of ethanol. The mixture was stirred at room temperature, resulting in an orange-red liquid. After 6 h, the solvent was removed by rotary evaporation, yielding a crude dark orange precipitate (68%). This precipitate was subsequently washed with ethanol and diethyl ether at room temperature. Elemental anal. Calcd for C 30 H 46 ClN 2 O 6 P 2 Rh: C, 49.30; H, 6.36; N, 3.83. Found: C, 49.04; H, 6.41; N, 3.6. 1 H NMR (300 MHz, CDCl 3 , δ ) (Fig S1 ): 1.28(12H(CH 3 ), t); 1.76 (15H, cyclopentadienyl-CH 3 ), s; 3.62 (4H, CH 2 P-, d); 4.25 (8H, OCH 2 , quintet); 7.92 (2H, aryl H on C 5 and C 5 ’, m); 8.74 (2H, aryl H on C 3 and C 3’ , m); 8.98 (2H, aryl H on C 6 and C 6 ’, d, J = 5 Hz,). FT-IR (KBr, ν, cm − 1 , selected peaks) (Fig S2): 3340, 3070, 1643, 1500, 1475, 1455, 1440, 1394, 730, 318. [λ max /nm (ε max /mol − 1 ·cm − 1 )]: 229 (827), 252 (516), 304 (286), 315 (265), 360 (62). DNA binding studies. Absorbance spectral studies were conducted to investigate the binding of complex 2 with CT-DNA. The experiments were performed in Tris HCl/NaCl buffer (5.0 M/50 mM, pH = 7.2) at room temperature. A stable concentration of complex 2 (10 µM) was titrated with increasing DNA concentration. Intrinsic binding constants were determined from the absorption spectral data. For competitive binding experiments, we studied the relative binding of complex 2 to CT-DNA using a solution of [DNA/EB] complex (EB = ethidium bromide) in the same buffer. These experiments analyzed the effect of fluorescence quenching by recording fluorescence emission spectra at 510 nm excitation and 596–600 nm emission. Circular dichroic spectral studies were performed at 37 ºC using a 100 µM CT-DNA solution with 50 µM of complex 2 in Tris-HCl/NaCl buffer (5.0 mM/50 mM, pH 7.2). We collected three scans for each spectrum and subtracted the background using a reference solution without DNA. All circular dichroism (CD) experiments were conducted on a Jasco J-810 spectropolarimeter in the wavelength range of 320 − 220 nm at room temperature. To investigate DNA binding further, we measured viscosity. The viscometer was immersed in a water bath at 25°C ± 0.1°C. Viscosity measurements depend on the flow time through the capillary viscometer. We obtained relative viscosity using the equation: η = (t - t 0 )/t 0 , where t and t 0 represent the observed flow time of CT-DNA-containing solutions and buffer solution alone, respectively. The data were presented as (η/η 0 ) 1/3 vs the ratio of the concentration of complex 2 to that of CT-DNA ([complex]/[DNA]). 22 BSA binding studies . Absorption spectral titration experiments were conducted with fixed concentrations of BSA (14 µM) while manually varying the concentration of complex 2 using a micropipette. The change in λ max values at 280 nm, specific to the BSA molecule, was recorded after each addition of complex 2 . To determine the apparent association constant ( K app ) between the Rh complex and BSA, we employed the Benesi-Hildebrand equation: 23 1/( A obs – A 0 ) = 1/( A c – A 0 ) + 1/ K app ( A c – A 0 )[complex] Here, A obs represents the observed absorbance of the solution containing different concentrations of complex 2 at 280 nm, while A 0 and A c correspond to the absorbances of BSA and the Rh complex (at different concentrations) at 280 nm, respectively. The term K app denotes the apparent association constant. Additionally, we investigated protein binding by performing tryptophan fluorescence quenching experiments using BSA (3 µM). These experiments were conducted in the absence of 15 mM trisodium citrate and 150 mM NaCl, maintaining a pH of 7.0. We recorded the fluorescence spectrum from 300 to 500 nm with an excitation wavelength of 295 nm. Assessment of Cytotoxicity . Cytotoxicity studies were conducted by preparing serial dilutions ranging from 100 µM to 0 µM. These dilutions were used for treatment, and the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) method was employed (details provided in the supplementary file). Staining experiments were performed using acridine orange and ethidium bromide following standard protocols outlined in the supplementary materials. Antimicrobial Activity Assessment. We conducted in vitro antimicrobial screening of the 4,4-bis(diethyl-methylphosphonate)-2,2’-bipyridine ligand and complex 2 . The study evaluated their effects on specific human pathogenic bacteria and fungus using a diffusion method. Gram-positive bacteria ( Bacillus megaterium , B. subtilis ), Gram-negative bacteria ( Shigella dysenteriae , Klebsiella pneumoniae ), and Candida albicans were cultured in nutrient agar medium and incubated at 37 ℃ for 48 h, followed by frequent subculturing to fresh medium. Petri plates were inoculated with bacterial and fungal cultures, uniformly spread using a sterile glass spreader. The ligand and complex 2 were dissolved in DMSO solvent; 100 µl of each sample was added to separate wells, with 100 µl of DMSO as a negative control. Refluxin (5 mcg) served as the positive control. Incubation occurred at 37 ℃ (48 h for bacteria) and 27 ℃ (72 h for fungi). After incubation, inhibition zone diameters were measured and recorded. Each experiment was repeated three times, and average values were reported. Results and discussion Spectroscopic properties of complex 2 . In the absorption spectrum, the bands of complex 2 appear at 229, 252, 304, and 315 nm (Fig. 1 a). These bands derived from such organometallic complexes were mainly attributed to the transitions n → π *, π → π * and n → σ *. 42 Low-spin Ru(II), Rh(III), and Ir(III) complexes provide appropriately filled t 2g orbitals, which can interact with lower π* orbitals of ligands. 24 Interaction of complex 2 with CT-DNA . In our investigation, the interaction of complex 2 with calf-thymus DNA (CT-DNA) was examined via absorption spectroscopy (Fig. S3). The treatment induced a slight hypochromic effect and a marginal red shift near 260 nm. The isosbestic points observed at 231, 245, and 262 nm indicate a clean interaction between complex 2 and DNA. This hyperchromicity could be attributed to either electrostatic binding or a partial unwinding of the DNA helix, revealing more base pairs. The subtle hyperchromic shift points to a non-intercalative mode of binding, possibly due to hydrogen bonding between complex 2 and the DNA bases, or electrostatic interactions with the negatively charged phosphate groups of DNA. On the other hand, the bathochromic shift suggests an intercalative mode marked by strong π–π stacking interactions between the aromatic ligand of the metal complexes and the DNA base pairs. However, the extent of intercalation remains ambiguous, as the red shift is less pronounced than that reported in other studies. Additionally, the presence of isosebestic points at 245 and 262 nm indicates the formation of an equilibrium between two species in the solution. The absorption band originating from complex 2 at 231 nm exhibits significant shifts upon the addition of CT-DNA, indicative of a DNA-binding interaction (Fig. S3). The red shift observed further supports this interaction. The binding constant ( K b ) for complex 2 with DNA was determined by plotting the ratio of [DNA]/(ε a − ε f ) against [DNA], following the equation: [DNA]/ (ε a − ε f ) = [DNA]/(ε b − ε f ) + 1/ K b (ε b − ε f ), where ε a , ε f , and ε b represent the apparent, free, and bound complex extinction coefficients, respectively. Linear regression of these data sets yields a straight line, with the slope equal to 1/(ε b − ε f ) and the y-intercept corresponding to 1/ K b (ε b − ε f )) (Fig. 1 b). The calculated K b value is 2.85 × 10 4 M − 1 . Compared to literature values, classical intercalators exhibit K b values several orders of magnitude higher than that of complex 2 , suggesting that intercalation is not the predominant mode of DNA interaction. Conversely, the K b value is significantly higher than those reported for groove-binding agents, such as Cr(II) complexes and tris(1,10-phenanthroline)ruthenium(II), indicating a strong interaction with DNA. 25 – 26 Further experiments are necessary to elucidate the precise mode of binding. Competitive binding between EB and complex 2 . To further elucidate the interaction between complex 2 and DNA, emission experiments were conducted. Solutions containing EB-DNA conjugates (EB = ethidium bromide) exhibited a fluorescent emission peak at 596 nm (λ ex = 510 nm), characteristic of the intercalation of planar EB between DNA base pairs (Fig S4). The introduction of complex 2 that bind to DNA with an affinity equal to or greater than EB significantly alters the emission profile of DNA-EB. Notably, complex 2 itself is non-fluorescent. Upon titration of complex 2 into solutions of DNA-EB, a significant quenching of the 596 nm fluorescence was observed (Fig. S5). In the plot of [complex 2 ]/[DNA] against I 0 / I , K sv is calculated as 3.22 × 10 3 M − 1 by dividing the slope by the y-intercept (Fig S6). However, the extent of quenching was not as substantial when compared to other literature. 27 – 28 This attenuation in fluorescence intensity upon the addition of complex 2 suggests that it may displace EB from the DNA-EB complex or quench the fluorescence of EB intercalated within DNA. Circular dichroism analysis of DNA-conjugate interactions . Circular dichroism (CD) spectroscopy was utilized to assess potential conformational alterations in DNA upon interaction with complex 2 . CD spectra were recorded for CT-DNA in both the presence and absence of complex 2 . The CD spectra of a mixture of complex 2 with DNA in a Tris buffer, post a 2-h incubation, are displayed (Fig. S7). The molar ellipticities [θ] at the characteristic negative and positive bands of DNA remained consistent with those observed for DNA in isolation, and the absence of a negative CD band at 300 nm corroborates the lack of substantial evidence for the intercalation of complex 2 , in line with the findings from the above UV/vis spectroscopic studies. Viscosity measurement method . The change in viscosity of the EB intercalated DNA solution after the addition of the metal complex indicates the interaction of the complex with CT-DNA. The classic EB intercalator increases the relative viscosity of DNA by binding to the groove. Conversely, partial and non-classical bonds can bend or twist the DNA helix, reducing the length and viscosity of DNA. 29 – 30 Our results show that binding of adjacent DNA-pair grooves causes DNA helix elongation, which increases the viscosity of DNA bound with complex 2 (Fig. S8). BSA binding experiments . The electronic spectrum of pure BSA will show a strong absorption band at 280 nm from the aromatic residues of tryptophan and tyrosine, which may vary with the interaction of drug molecules or metal complexes. Absorption changes of BSA complexed with complex 2 (0–40 mM) showed a steady increase in BSA uptake as the concentration of the complex increased, indicating the formation of BSA-complex 2 (Fig S8). 31 The changes observed in this situation when there are different concentrations of the Rh complex indicate a normal interaction between complex 2 and BSA. A linear relationship between 1/(A obs -A 0 ) and the inverse of the concentration of complex 2 with a slope equal to 1/K app (A c -A 0 ) and an intercept equal to 1/(A c -A 0 ) (Fig. S9). The data indicates that complex 2 is strongly adsorbed on BSA surface. Fluorescence titration and interaction of complex 2 with BSA . In our study, we also investigated the interaction between complex 2 and BSA using fluorescence titration. BSA solutions exhibit strong fluorescence emission with a peak at 353 nm upon excitation at 295 nm, primarily due to tryptophan residues (Fig. S10). Complex 2 , introduced during the experiment, displayed a weaker emission peak at 328 nm. Upon adding complex 2 to BSA, we observed significant fluorescence quenching (up to 68% of initial fluorescence), as shown in Fig. S11. This quenching effect suggests that complex 2 binds to BSA, potentially altering the secondary protein structure. To analyze this interaction, we employed Stern-Volmer and Scatchard plots. The Stern-Volmer equation, I / I 0 = 1 + k q τ 0 [Q] = 1 + K sv [Q], allowed us to calculate the dynamic quenching constant ( K sv ) based on the fluorescence lifetime (τ 0 ) of tryptophan in BSA (approximately 1.0 × 10 − 8 s, Fig. S12). The calculated values for complex 2 were as follows: K sv = 2.39 × 10 4 M − 1 ; k q = 2.39 × 10 12 M − 1 S − 1 These data indicate that complex 2 exhibits strong quenching ability toward BSA and has a favorable binding propensity. Notably, the quenching rate constant (k q ) for complex 2 surpasses that of other known quenching agents, likely operating via a static quenching mechanism. 32 – 33 Furthermore, using Scatchard’s equation, Δ I / I 0 /[Q] = n K – K (ΔI/I 0 ), we determined that the number of binding sites per BSA molecule (n) for complex 2 is approximately 0.25 (Fig S12). The affinity constant ( K ) of complex 2 to BSA was calculated as 0.026 M − 1 , indicating a strong affinity. In summary, complex 2 demonstrates the remarkable binding affinity, as observed in our DNA binding studies. Evaluating cytotoxicity of complex 2 through in vitro assays . We assessed cytotoxic effects of complex 2 using in vitro anticancer assays on two malignant and one benign cell line via the MTT assay, as detailed in Table 1 and Fig. S13. Our findings revealed that antitumor potency of complex 2 varies across different cell types. Notably, increasing concentrations of complex 2 were associated with greater cellular membrane ion absorption, leading to enhanced penetration and more pronounced anticancer activity. Importantly, complex 2 demonstrated significant cytotoxicity towards MSA-MB-231 and A549 tumor cells, as evidenced by their reduced IC 50 values. However, it showed minimal toxicity towards normal VERO cells with an IC 50 value exceeding 1000 µM, indicating its selective action on malignant cells. Recent studies suggest that incorporating appropriate ligands into Rh complexes may amplify their targeting capabilities. 34 – 35 MDA-MB-231 cells were treated with varying concentrations (0–100 µM) of complex 2 or cis -platin. Cis-platin, a well-known potent cytotoxic agent, was used as a reference to compare and evaluate the cytotoxicity of the metal complexes, as described in previous studies. 36 The reduction in cell viability percentage confirmed the dose-dependent cytotoxic effects of the complexes on the cancer cell line (Fig. S13 and S14). The IC 50 value of cis-platin was determined to be 10.46 µM ± 0.05, which is consistent with previously reported literature data. 37 These findings further validate the reliability and reproducibility of the present experimental results. Table 1 In vitro cytotoxicity (IC 50 ) of complex 2 and cis -platin against cells . Cell line IC 50 (µM) complex 2 cis -platin MDA-MB-231 47.25 ± 0.05 10.46 ± 0.05 A549 48.75 ± 0.05 - VERO >= 1000 - Fluorescence microscopy assessment of apoptotic cell death . We also investigated the cell death patterns induced by complex 2 using a two-color staining method—acridine orange/ethidium bromide (AO/EB) - followed by fluorescence microscopy. 38 – 41 Both cancerous and non-cancerous cells were exposed to complex 2 for 24 h. Acridine orange permeates all cells, emitting bright green fluorescence upon binding to double-stranded DNA. In contrast, ethidium bromide selectively accumulates in non-viable cells, resulting in red fluorescence upon DNA intercalation. By analyzing fluorescent emissions and morphological features, we categorized cells into four distinct types: (1) Viable cells: These exhibit highly organized nuclei with intense green fluorescence. (2) Early apoptotic cells: Their green fluorescent nuclei show chromatin condensation and nuclear fragmentation. (3) Late apoptotic cells: Characterized by nuclei fluorescing orange to red, with chromatin condensation or fragmentation. (4) Necrotic cells: Display red fluorescence without chromatin disintegration. In summary, living cells display vivid green nuclear structures, apoptotic cells exhibit orange to red nuclei with condensed or fragmented chromatin, and necrotic cells show similarly colored nuclei with dense, unfragmented chromatin (Fig. 3 ). Our research reveals that complex 2 significantly induces apoptosis in cancer cells, corroborating the cytotoxicity data (Fig S15). The anticancer effects of complex 2 likely stem from its ability to bind to DNA, triggering cellular apoptosis. Notably, variations in lipophilicity and DNA affinity seem to govern complex 2 ’s oncogenic activity. These findings contribute to a deeper understanding of metal complexes’ anticancer properties and their interactions with proteins like BSA. Comprehensive clinical studies are crucial for unraveling the molecular mechanisms of cytotoxicity and determining optimal conditions for using metal complexes as therapeutic agents. Antimicrobial activity of complex 2: Insights from in vitro assays . In our study, we evaluated the antimicrobial properties of compound 1 , Rh-dimer (Cp*) 2 Rh 2 Cl 4 , ampicillin, and complex 2 using the diffusion agar method against Gram-positive bacteria ( Bacillus megaterium and Bacillus ), Gram-negative bacteria ( Shigella dysenteriae and Klebsiella pneumoniae ), and the fungus Candida albicans. Ampicillin served as the reference antibiotic for all tested microorganisms. Our analysis revealed that complex 2 exhibits potent antibacterial and antifungal effects (Fig. 4 ). Typically, bacterial growth appears as a turbid layer beyond regions where the antimicrobial agent surpasses the minimum inhibitory concentration (MIC), resulting in a clear zone of inhibition. The extent of this zone depends on factors such as medium composition, culture conditions, diffusion rates, and the concentration of the antimicrobial agent. The variability in complex 2 ’s efficacy against different bacteria may be attributed to unique permeability barriers in microbial cells or disparities in their ribosomal structures. The pronounced activity levels of complex 2 suggest a beneficial impact on both bacteria and fungi, potentially through direct interaction with these organisms. The inhibition zone values presented in Table 2 demonstrate the complex’s effectiveness against both Gram-positive bacteria ( Bacillus megaterium and Bacillus subtilis ) and Gram-negative bacteria ( Shigella dysenteriae and Klebsiella pneumoniae ), as well as fungi ( Candida albicans ). Complex 2 exhibited greater activity than compound 1 , attributed to its increased bactericidal effects. Notably, metal complexes’ toxicity is linked to enhanced lipophilicity resulting from chelation. According to Overtone’s concept of cell permeability, lipid membranes favor fat-soluble substances crucial for disease control. The heightened lipophilicity of complex 2 facilitates membrane penetration, blocking iron-binding sites in microbial enzymes. This disruption of cellular respiration limits bacterial growth and protein synthesis, ultimately leading to bacterial death. Among the tested microbes, Gram-positive Bacillus megaterium and Gram-negative Shigella dysenteriae were most susceptible, with inhibition zones measuring 25 ± 1 mm. Candida albicans also exhibited an inhibition zone of 24 ± 1 mm. Usually, Gram-positive bacteria, due to their thick peptidoglycan layer and outer membrane, are more easily targeted by antibiotics than Gram-negative bacteria. Interestingly, complex 2 exhibited a similar trend in its activity against both bacterial strains. Complex 2 ’s potent antibacterial properties make it a promising alternative to traditional antibiotics. Table 2 Comparison of antimicrobial activity (mm): compound 1 , rhodium dimer, complex 2 , and ampicillin. Species compound 1 Rh-dimer complex 2 ampicillin Bacillus megaterium 19 22 25 13 B. subtilis 17 20 24 10 Shigella dysenteriae 18 21 25 11 Klebsiella pneumoniae 17 21 24 8 Candida albicans 20 22 24 12 Conclusions A Rh(III) complex containing 4,4-bis(diethyl-methylphosphonate)-2,2’-bipyridine was synthesized to explore its biological activities. Various spectroscopic techniques were employed to evaluate the complex’s binding interactions with DNA and proteins. The Rh complex exhibited a binding constant ( K b ) of 2.85 × 10 4 M − 1 for CT-DNA, suggesting quite strong inbteraction to the DNA helix. Competitive studies with EB indicated that the complex can displace or quench EB from EB-DNA complexes, consistent with groove-binding interactions. Additionally, electronic and fluorescent spectral titration data revealed protein chain unwinding upon interaction with the Rh complex. The complex’s effect on tryptophan residues in an aqueous medium further characterized its binding behavior. Steady-state quenching experiments confirmed static binding to bovine serum albumin at a single site. Furthermore, in vitro cytotoxicity assays demonstrated that our Rh complex surpasses the currently used chemotherapeutic drug cis -platin in terms of cytotoxicity against cancer cell lines. Antibiotic testing revealed the complex’s antibacterial properties, proving its efficacy against Gram-positive and Gram-negative bacteria as well as fungi. This promising strategy could enhance safety and efficacy in future cancer therapies through in vivo studies. Declarations Acknowledgements This work is supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government(MSIT) (RS-2024-00397807) and the Researchers Supporting Project number (RSP2023R147), King Saud University, Riyadh, Saudi Arabia. A uthor contribution s Author statement: Jinheung Kim: Writing – review & editing, Writing – original draft, Methodology, Investigation, Data curation, Supervision, Resources, Project administration, Conceptualization, Funding acquisition. Thamilarasan Vijayan: Writing – review & editing, Writing – original draft, Methodology, Investigation, Data curation, Conceptualization. Atifa Ashraf: Methodology, Investigation, Data curation. Mohammad Azam: Methodology, Investigation, Funding acquisition, Conceptualization. Funding Not applicable. C ompeting interest The authors declare that they have no known financial conflicts of interest or personal relationships that could have influenced the work reported in this paper Ethical approval Not applicable. References H.W. Boucher, G.H. Talbot, J.S. Bradley, J.E. Edwards, D. Gilbert, L.B. Rice, M. Scheld, B. Spellberg, J. Bartlett, Bad bugs, no drugs: no ESKAPE! 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Dadashpour, Current developments of coumarin-based anti-cancer agents in medicinal chemistry, Eur J Med Chem. 102 (2015) 611–30. J.P. Mészáros, W. Kandioller, G. Spengler, A. Prado-Roller, B.K. Keppler, E.A. Enyedy, Half-sandwich rhodium complexes with releasable N-donor monodentate ligands: solution chemical properties and the possibility for acidosis activation, Pharmaceutics. 15 (2023) 356. M. Harlos, I. Ott, R. Gust, H. Alborzinia, S. Wölfl, A. Kromm, W.S. Sheldrick, Synthesis, Biological Activity, and Structure – Activity Relationships for Potent Cytotoxic Rhodium (III) Polypyridyl Complexes, J Med Chem. 51 (2008) 3924–33. A.C. Komor, C.J. Schneider, A.G. Weidmann, J.K. Barton, Cell-selective biological activity of rhodium metalloinsertors correlates with subcellular localization, J Am Chem Soc. 134 (2012) 19223–33. A.C. Lees, B. Evrard, T.E. Keyes, J.G. Vos, C.J. Kleverlaan, M. Alebbi, C.A. Bignozzi, Synthesis, Spectroscopy and Photophysical Properties of Ruthenium Triazole Complexes and Their Application as Dye-Molecules in Regenerative Solar Cells, Eur J Inorg Chem. 1999 (1999) 2309-17. S.J. Mehdi, A. Ahmad, M. Irshad, N. Manzoor, M.M. Rizvi, Cytotoxic effect of Carvacrol on human cervical cancer cells, Biol Med. 3 (2011) 307–12 C. Joel, S.T. David, R.B. Bennie, S.D. Abraham, S.I. Pillai, S.M. Sathyasheeli, Spectral, electrochemical and DNA binding properties of some macroacyclic transition metal complexes using diverse spectral methods, Int J Res Inorg Chem. 5 (2015) 14–20. C.G. Nongpiur, M.M. Ghate, D.K. Tripathi, K.M. Poluri, W. Kaminsky, M.R. Kollipara, Study of versatile coordination modes, antibacterial and radical scavenging activities of arene ruthenium, rhodium and iridium complexes containing fluorenone based thiosemicarbazones, J Organomet Chem. 957 (2022) 122148. R. Vijayalakshmi, M. Kanthimathi, V. Subramanian, B.U. Nair, Interaction of DNA with [Cr (Schiff base)(H2O) 2] ClO4, Biochim Biophys Acta (BBA)-Gen Subj. 1457 (2000) 157–62. M. Eriksson, M. Leijon, C. Hiort, B. Norden, A. Graeslund, Minor groove binding of [Ru (phen) 3] 2 + to [d (CGCGATCGCG)] 2 evidenced by two-dimensional NMR, J Am Chem Soc. 114 (1992) 4933–4. A. Bhattacharjee, S. Das, B. Das, P. Roy, Intercalative DNA binding, protein binding, antibacterial activities and cytotoxicity studies of a mononuclear copper (II) complex, Inorg Chim Acta. 514 (2021) 119961. L. Li, Q. Guo, J. Dong, T. Xu, J. Li, DNA binding, DNA cleavage and BSA interaction of a mixed-ligand copper (II) complex with taurine Schiff base and 1, 10-phenanthroline, J Photochem Photobiol B: Biol. 125 (2013) 56–62. E.L. Gilroy, M.R. Hicks, D.J. Smith, A. Rodger, Viscosity of aqueous DNA solutions determined using dynamic light scattering, Analyst. 136 (2011) 4159–63. A. Laesecke, J.L. Burger, Viscosity measurements of DNA solutions with and without condensing agents, Biorheology. 51 (2014) 15–29. A. Patra, B. Sen, S. Sarkar, A. Pandey, E. Zangrando, P. Chattopadhyay, Nickel (II) complexes with 2-(pyridin-3-ylmethylsulfanyl) phenylamine and halide/pseudohalides: Synthesis, structural characterisation, interaction with CT-DNA and bovine serum albumin, and antibacterial activity, Polyhedron. 51 (2013) 156–63. P. Tsiliki, F. Perdih, I. Turel, G. Psomas. Structure, DNA-and albumin-binding of the manganese (II) complex with the non-steroidal antiinflammatory drug niflumic acid, Polyhedron. 53 (2013) 215–22. K.A. Paterson, J. Arlt, A.C. Jones, Dynamic and static quenching of 2-aminopurine fluorescence by the natural DNA nucleotides in solution, Methods Appl Fluoresc. 8 (2020) 025002. W.Y. Zhang, H.E. Bridgewater, S. Banerjee, J.J. Soldevila-Barreda, G.J. Clarkson, H. Shi, C. Imberti, P.J. Sadler PJ. Ligand‐Controlled Reactivity and Cytotoxicity of Cyclometalated Rhodium (III) Complexes, Eur J Inorg chem. 2020 (2020) 1052-60. M. Sohrabi, M. Saeedi, B. Larijani, M. Mahdavi, Recent advances in biological activities of rhodium complexes: Their applications in drug discovery research, Eur J Med Chem. 216 (2021) 113308. A.M. Florea, D. Büsselberg, Cisplatin as an anti-tumor drug: cellular mechanisms of activity, drug resistance and induced side effects, Cancers. 3 (2011) 1351–71. N.A. Aziz, G.R. Froemming, S.H. Kadir, M.J. Ibahim, Apigenin increases cisplatin inhibitory effects on the telomerase activity of triple negative breast cancer cells, J Teknol. 80 (2018). D. Baskić, S. Popović, P. Ristić, N.N. Arsenijević, Analysis of cycloheximide-induced apoptosis in human leukocytes: Fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide, Cell biol int. 30 (2006) 924–32. K. Nagaraj, G. Velmurugan, S. Sakthinathan, P. Venuvanalingam, S. Arunachalam, Influence of self-assembly on intercalative DNA binding interaction of double-chain surfactant Co (iii) complexes containing imidazo [4, 5-f][1, 10] phenanthroline and dipyrido [3, 2-d: 2′-3′-f] quinoxaline ligands: experimental and theoretical study, Dalton Trans. 43 (2014) 18074–86. Y. Manojkumar, S. Ambika, R. Arulkumar, B. Gowdhami, P. Balaji, G.Vignesh, S. Arunachalam, P. Venuvanalingam, R. Thirumurugan, M.A. Akbarsha, Synthesis, DNA and BSA binding, in vitro anti-proliferative and in vivo anti-angiogenic properties of some cobalt (III) Schiff base complexes, New J Chem. 43 (2019) 11391–407. S. Ramakrishnan, V. Rajendiran, M. Palaniandavar, V.S. Periasamy, B.S. Srinag, H. Krishnamurthy, M.A. kbarsha, Induction of cell death by ternary copper (II) complexes of L-tyrosine and diimines: role of coligands on DNA binding and cleavage and anticancer activity, Inorg Chem. 48 (2009) 1309–22. Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files JBiolInorgChem2025ESI.doc Onlinefloatimage1.png Scheme 1. Synthesis of complex 2 from compound 1in EtOH. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 30 Sep, 2025 Reviews received at journal 30 Sep, 2025 Reviews received at journal 16 Sep, 2025 Reviewers agreed at journal 02 Sep, 2025 Reviewers agreed at journal 11 Aug, 2025 Reviews received at journal 08 Aug, 2025 Reviewers agreed at journal 05 Aug, 2025 Reviewers invited by journal 03 Aug, 2025 Editor assigned by journal 05 May, 2025 Submission checks completed at journal 05 May, 2025 First submitted to journal 02 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6581437","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":495802479,"identity":"0ab8860e-0f27-46d3-b9e4-32c38648df5d","order_by":0,"name":"Thamilarasan Vijayan","email":"","orcid":"","institution":"Ewha Womans University","correspondingAuthor":false,"prefix":"","firstName":"Thamilarasan","middleName":"","lastName":"Vijayan","suffix":""},{"id":495802481,"identity":"a14fe052-2677-44c4-b60a-ace4dc82e056","order_by":1,"name":"Atifa Ashraf","email":"","orcid":"","institution":"Ewha Womans University","correspondingAuthor":false,"prefix":"","firstName":"Atifa","middleName":"","lastName":"Ashraf","suffix":""},{"id":495802485,"identity":"0d4e0949-54d5-40dc-a66d-cac974057fd0","order_by":2,"name":"Mohammad Azam","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"","lastName":"Azam","suffix":""},{"id":495802488,"identity":"8359bc95-9c98-4dab-a3a5-6de41e14d342","order_by":3,"name":"Jinheung Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYBACxmYGNjCDnwHKYOAhVotkA7FaGGCGGxwgVgtzO++xBx/b7thtPt5j9uAHg508A8/ZBwQcxpduOLPtWfK2M2fMDXsYkg0beNsNCGjhMZPmbTucbHYjd5sEDwNzAgM/G36HgbX8BWoxnv92m+QfhnoitTC2HbYzkODdJs3DcDiBgbeNsBbJnnOHEyTO5H+TljE4btjGcwy/FsP+M2YSP8oO2/O3H0uTfFNRLc/Pk0ZASwOEToTQBvA0gBvIQ2l7QgpHwSgYBaNgBAMAQfo8c37gS7wAAAAASUVORK5CYII=","orcid":"","institution":"Ewha Womans University","correspondingAuthor":true,"prefix":"","firstName":"Jinheung","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2025-05-03 02:38:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6581437/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6581437/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88464694,"identity":"b6c050f1-6bd6-4e9b-bfea-a2b0a033a406","added_by":"auto","created_at":"2025-08-06 17:13:35","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":53350,"visible":true,"origin":"","legend":"\u003cp\u003e(a) UV-vis spectrum of complex \u003cstrong\u003e2 \u003c/strong\u003ein a 5 mM Tris buffer (pH 7.2). (b) Plot of [DNA]/(ε\u003csub\u003ea\u003c/sub\u003e-ε\u003csub\u003ef\u003c/sub\u003e) vs [DNA] for the titration of DNA with complex \u003cstrong\u003e2\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6581437/v1/85f6a27cffb5bf95bfe83645.jpg"},{"id":88465782,"identity":"2d447999-b02c-4720-bbe6-851accc71514","added_by":"auto","created_at":"2025-08-06 17:29:35","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":51820,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Effect of increasing amounts of EB, compound \u003cstrong\u003e1\u003c/strong\u003e, and complex \u003cstrong\u003e2\u003c/strong\u003e on the relative viscosity of CT-DNA at 25.0 ± 0.1 \u003csup\u003e0\u003c/sup\u003eC. (b) Fluorescence spectra of BSA in the presence of various concentrations of complex \u003cstrong\u003e2\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6581437/v1/1d9ec306e7eabc95b068c0aa.jpg"},{"id":88464699,"identity":"ebc0e6b1-aa89-4ff2-b548-a246aea19c45","added_by":"auto","created_at":"2025-08-06 17:13:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":220087,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs illustrating control and AO/EB-stained cancerous and non-cancerous cells after a 24-h incubation with complex \u003cstrong\u003e2\u003c/strong\u003e. Panels (a), (c), and (e) display untreated control cells from MDA-MB-231, A549, and VERO cell lines respectively. Panels (b), (d), and (f) show MDA-MB-231, A549, and VERO cells treated with complex \u003cstrong\u003e2\u003c/strong\u003e. Cell viability is indicated by light green staining; early apoptosis by bright green fluorescence; late apoptosis by red-orange fluorescence; and necrosis by red fluorescence.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6581437/v1/50637d84927cf6de81f5917f.jpg"},{"id":88465069,"identity":"8d9beb56-2184-4cd8-bacc-8be331370fcd","added_by":"auto","created_at":"2025-08-06 17:21:35","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":78963,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e antimicrobial activity of compound \u003cstrong\u003e1\u003c/strong\u003e, Rh dimer, complex \u003cstrong\u003e2\u003c/strong\u003e, and ampicillin.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6581437/v1/3e49bd4df89bef449dc0ad25.jpg"},{"id":88466114,"identity":"6eb1d1e4-d438-40dc-a9b0-fa49386bf09b","added_by":"auto","created_at":"2025-08-06 17:37:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1338712,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6581437/v1/013c969d-3181-4771-b0f6-7b30419b2b88.pdf"},{"id":88464715,"identity":"6577f919-b3d2-426d-8950-384f2eeeefc1","added_by":"auto","created_at":"2025-08-06 17:13:36","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9250304,"visible":true,"origin":"","legend":"","description":"","filename":"JBiolInorgChem2025ESI.doc","url":"https://assets-eu.researchsquare.com/files/rs-6581437/v1/cfadf29a165c7858ced70466.doc"},{"id":88464697,"identity":"284951c2-e24d-4a25-809d-220ec2eb4408","added_by":"auto","created_at":"2025-08-06 17:13:35","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":47542,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. \u003c/strong\u003eSynthesis of complex \u003cstrong\u003e2 \u003c/strong\u003efrom compound \u003cstrong\u003e1\u003c/strong\u003ein EtOH.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6581437/v1/3abc7d375c3b2365bc27f7a0.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characterization and Biological Evaluation of a (Cp*)Rh(III) Complex Featuring Phosphonate-Modified Bipyridine Ligands","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the realms of chemistry and pharmacology, the innovation of new chemical agents that can activate antibacterial and antibiotic properties is a critical concern.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e The majority of antiviral and anti-inflammatory drugs developed over the past few decades have been centered on antibodies, natural toxins, and antagonistic drugs.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e However, organometallic complexes of transition metals have garnered increasing interest for their potential to address these issues. Researchers are particularly drawn to organometallic complexes for their unique multi-modal reactivity, which is absent in purely organic drug molecules, offering a broader scope in the fight against antibiotic resistance.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Organometallic complexes have been extensively employed in recent years as promising candidates for antivirals, antimalarial agents, antibiotics, and diagnostic drugs. This is attributed to the diverse functionalities of both organic and metal complex moieties within biological systems.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e The exploration of metals like ruthenium, rhodium, and iridium has intensified, driven by their superior stability, non-toxicity to biological entities, enhanced coordination capabilities, and chemical resilience.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Notably, rhodium(III) complexes are versatile in their ability to bind with a variety of ligand structures and interact with biomolecules, thereby disrupting cell metabolism and exerting antitumor effects.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe development of metal-based therapeutics has been bolstered by the effectiveness of \u003cem\u003ecis\u003c/em\u003e-platin. However, due to its adverse side effects, there has been considerable research into complexes of other metals such as Ru, Rh, Ir, and Os.\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e In recent years, the formula [(η\u003csup\u003e5\u003c/sup\u003e-Cp*)/(η\u003csup\u003e6\u003c/sup\u003e-arene)M(L)Cl]\u003csup\u003e0/+\u003c/sup\u003e (where Cp* = C\u003csub\u003e5\u003c/sub\u003e(CH\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e5\u003c/sub\u003e; M\u0026thinsp;=\u0026thinsp;Pt group metals; L\u0026thinsp;=\u0026thinsp;a bidentate ligand) has garnered attention for its anti-cancer properties, attributed to its potent efficacy and specific mechanism of action against cancer cells.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Replacing the bidentate (L) ligand in half-sandwich metal complexes, a variety of ligands featuring N, O, P, and S donors have been synthesized and biologically evaluated.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e These ligands demonstrate potential in catalysis, molecular devices, supramolecular assemblies, and exhibit antiviral, antibiotic, and antibacterial activities.\u003c/p\u003e\u003cp\u003eFor metal complexes to be effective as anticancer agents, favorable interactions with DNA are essential. Transition metal complexes have played a pivotal role in this regard, selectively targeting DNA sites.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e The majority of tumor cells contain DNA biomolecules, which are considered a weak spot and thus a prime target for many anti-cancer drugs. These drugs and biomolecules typically interact with DNA in the β conformation through mechanisms of external attachment, binding, and intercalation between adjacent planar base pairs. Such interactions require specific features like aromatic geometry and the size of a flat stretch, or non-β motifs such as G-quadruplex DNA.\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003ePentamethylcyclopentadienyl rhodium and iridium complexes have been reported to be effective in infection prevention and exhibit high stability due to their chemical inertness.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Among these, the Rh(III) complex is inert and contributes to its potential inhibitory effect, while the Rh(II) complex is reactive and participates in ligand exchange processes, playing a significant role in biological functions.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Similarly, Rh(I) complexes, isoelectronic with Pt(II) and exhibiting square planar geometry, are anticipated to possess biochemical activities akin to \u003cem\u003ecis\u003c/em\u003e-platin. This work aligns with recent advancements in rhodium(I) compounds within a biological context, highlighting how these molecules specifically target biomolecules for anticancer activities. Furthermore, rhodium complexes have shown significant pharmacological effects through their interactions with DNA, underscoring their utility in the biological sciences.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eIn this study, we present a pentamethylcyclopentadienyl Rh(III) complex coordinated with a 4,4-bis(diethyl-methyl phosphonate)-2,2-bipyridine ligand. We have characterized its affinity for calf-thymus DNA through titrations and a suite of analytical techniques, including spectroscopy, optical assays, and viscosity measurements. The interaction of the Rh complex with bovine serum albumin protein was also explored. Furthermore, we assessed the cytotoxicity of the Rh(III) complex against both cancerous and non-cancerous cell lines. The antimicrobial efficacy of the complex was evaluated against a panel of bacteria, encompassing two Gram-positive and two Gram-negative strains, as well as a viral pathogen.\u003c/p\u003e"},{"header":"Experimental section","content":"\u003cp\u003e\u003cb\u003eMaterials and Instrumentation\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eWe employed analytical reagent-grade chemicals without further purification, and the solvents were treated according to standard procedures. The following reagents were used: calf thymus DNA (CT-DNA), Tris-HCl, Tris bases, NaCl, pentamethylcyclopentadienyl rhodium (III) chloride, triethyl phosphite, ethidium bromide (EB), and BSA (all from Sigma-Aldrich). Solutions were prepared using double-distilled water. Additional chemicals, including 4,4\u0026rsquo;-dicarboxy-2,2\u0026rsquo;-bipyridine, ammonium chloride, sodium borohydride, and magnesium sulfate, were sourced from TCI Chemicals.\u003c/p\u003e\u003cp\u003eSpectroscopic analyses included UV-vis spectra recorded on a Shimadzu UV-3101PC spectrophotometer and FT-IR spectra obtained using a Bruker INVENIO-R spectrometer with potassium bromide pellets in the 4000\u0026thinsp;\u0026minus;\u0026thinsp;200 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range. Conductivity measurements of the complex dissolved in DMF was conducted using the ELICO-SX80 model conductivity bridge. The \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-NMR spectra of compound \u003cb\u003e1\u003c/b\u003e and complex \u003cb\u003e2\u003c/b\u003e were recorded in CDCl\u003csub\u003e3\u003c/sub\u003e and DMSO-\u003cem\u003ed\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e on a BRUKER 300 MHz FT-NMR system spectrometer at room temperature, with TMS as the internal reference. Elemental analysis was performed using an Elementarvario MACRO cube elemental analyzer.\u003c/p\u003e\u003cp\u003eFor biomolecular studies, we utilized an SHIMADZU-00774 spectrofluorometer (for fluorescence studies) and a JASCO J-810 spectropolarimeter (for circular dichroism studies). CT-DNA solutions were prepared in Tris-HCl/NaCl buffer (5 mM Tris-HCl, 50 mM NaCl, pH\u0026thinsp;=\u0026thinsp;7.2) and stored at 4 ℃ for no more than one week. Protein-free DNA was quantified by UV absorbance at 260 and 280 nm, with the CT-DNA concentration determined based on its density at 260 nm and the molar extinction coefficient of 6600 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Cytotoxicity testing was conducted using the MTT method at the BioMe Live Analytical Center in India.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSynthesis of 4,4\u0026rsquo;-bis(diethylmethylphosphonate)-2,2\u0026rsquo;-bipyridine (1)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eWe synthesized ligand 4,4\u0026rsquo;-bis(diethylmethylphosphonate)-2,2\u0026rsquo;-bipyridine (\u003cb\u003e1\u003c/b\u003e) from the precursor 4,4\u0026rsquo;-dicarboxy-2,2\u0026rsquo;-bipyridine using a four-step reaction method described in the literature (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSynthesis of complex 2\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eComplex \u003cb\u003e2\u003c/b\u003e was prepared by reacting pentamethylcyclopentadienyl rhodium(III) chloride dimer (0.5 mmol) with 4,4-bis(diethyl-methyl phosphonate)-2,2-bipyridine (1.0 mmol) in 20 mL of ethanol. The mixture was stirred at room temperature, resulting in an orange-red liquid. After 6 h, the solvent was removed by rotary evaporation, yielding a crude dark orange precipitate (68%). This precipitate was subsequently washed with ethanol and diethyl ether at room temperature. Elemental anal. Calcd for C\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e46\u003c/sub\u003eClN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003eP\u003csub\u003e2\u003c/sub\u003eRh: C, 49.30; H, 6.36; N, 3.83. Found: C, 49.04; H, 6.41; N, 3.6. \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (300 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e, \u003cem\u003eδ\u003c/em\u003e) (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e): 1.28(12H(CH\u003csub\u003e3\u003c/sub\u003e), t); 1.76 (15H, cyclopentadienyl-CH\u003csub\u003e3\u003c/sub\u003e), s; 3.62 (4H, CH\u003csub\u003e2\u003c/sub\u003eP-, d); 4.25 (8H, OCH\u003csub\u003e2\u003c/sub\u003e, quintet); 7.92 (2H, aryl H on C\u003csub\u003e5\u003c/sub\u003e and C\u003csub\u003e5\u003c/sub\u003e\u0026rsquo;, m); 8.74 (2H, aryl H on C\u003csub\u003e3\u003c/sub\u003e and C\u003csub\u003e3\u0026rsquo;\u003c/sub\u003e, m); 8.98 (2H, aryl H on C\u003csub\u003e6\u003c/sub\u003e and C\u003csub\u003e6\u003c/sub\u003e\u0026rsquo;, d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5 Hz,). FT-IR (KBr, ν, cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, selected peaks) (Fig S2): 3340, 3070, 1643, 1500, 1475, 1455, 1440, 1394, 730, 318. [λ\u003csub\u003emax\u003c/sub\u003e/nm (ε\u003csub\u003emax\u003c/sub\u003e/mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)]: 229 (827), 252 (516), 304 (286), 315 (265), 360 (62).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDNA binding studies.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAbsorbance spectral studies were conducted to investigate the binding of complex \u003cb\u003e2\u003c/b\u003e with CT-DNA. The experiments were performed in Tris HCl/NaCl buffer (5.0 M/50 mM, pH\u0026thinsp;=\u0026thinsp;7.2) at room temperature. A stable concentration of complex \u003cb\u003e2\u003c/b\u003e (10 \u0026micro;M) was titrated with increasing DNA concentration. Intrinsic binding constants were determined from the absorption spectral data.\u003c/p\u003e\u003cp\u003eFor competitive binding experiments, we studied the relative binding of complex \u003cb\u003e2\u003c/b\u003e to CT-DNA using a solution of [DNA/EB] complex (EB\u0026thinsp;=\u0026thinsp;ethidium bromide) in the same buffer. These experiments analyzed the effect of fluorescence quenching by recording fluorescence emission spectra at 510 nm excitation and 596\u0026ndash;600 nm emission.\u003c/p\u003e\u003cp\u003eCircular dichroic spectral studies were performed at 37 \u0026ordm;C using a 100 \u0026micro;M CT-DNA solution with 50 \u0026micro;M of complex \u003cb\u003e2\u003c/b\u003e in Tris-HCl/NaCl buffer (5.0 mM/50 mM, pH 7.2). We collected three scans for each spectrum and subtracted the background using a reference solution without DNA. All circular dichroism (CD) experiments were conducted on a Jasco J-810 spectropolarimeter in the wavelength range of 320\u0026thinsp;\u0026minus;\u0026thinsp;220 nm at room temperature.\u003c/p\u003e\u003cp\u003eTo investigate DNA binding further, we measured viscosity. The viscometer was immersed in a water bath at 25\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u0026deg;C. Viscosity measurements depend on the flow time through the capillary viscometer. We obtained relative viscosity using the equation:\u003c/p\u003e\u003cp\u003eη = (t - t\u003csub\u003e0\u003c/sub\u003e)/t\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e, where t and t\u003csub\u003e0\u003c/sub\u003e represent the observed flow time of CT-DNA-containing solutions and buffer solution alone, respectively. The data were presented as (η/η\u003csub\u003e0\u003c/sub\u003e)\u003csup\u003e1/3\u003c/sup\u003e vs the ratio of the concentration of complex \u003cb\u003e2\u003c/b\u003e to that of CT-DNA ([complex]/[DNA]).\u003csup\u003e22\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eBSA binding studies\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eAbsorption spectral titration experiments were conducted with fixed concentrations of BSA (14 \u0026micro;M) while manually varying the concentration of complex \u003cb\u003e2\u003c/b\u003e using a micropipette. The change in λ\u003csub\u003emax\u003c/sub\u003e values at 280 nm, specific to the BSA molecule, was recorded after each addition of complex \u003cb\u003e2\u003c/b\u003e. To determine the apparent association constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eapp\u003c/sub\u003e) between the Rh complex and BSA, we employed the Benesi-Hildebrand equation:\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e1/(\u003cem\u003eA\u003c/em\u003e\u003csub\u003eobs\u003c/sub\u003e \u0026ndash; \u003cem\u003eA\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;1/(\u003cem\u003eA\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e \u0026ndash; \u003cem\u003eA\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)\u0026thinsp;+\u0026thinsp;1/ \u003cem\u003eK\u003c/em\u003e\u003csub\u003eapp\u003c/sub\u003e(\u003cem\u003eA\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e \u0026ndash; \u003cem\u003eA\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)[complex]\u003c/p\u003e\u003cp\u003eHere, \u003cem\u003eA\u003c/em\u003e\u003csub\u003eobs\u003c/sub\u003e represents the observed absorbance of the solution containing different concentrations of complex \u003cb\u003e2\u003c/b\u003e at 280 nm, while \u003cem\u003eA\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e and \u003cem\u003eA\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e correspond to the absorbances of BSA and the Rh complex (at different concentrations) at 280 nm, respectively. The term \u003cem\u003eK\u003c/em\u003e\u003csub\u003eapp\u003c/sub\u003e denotes the apparent association constant.\u003c/p\u003e\u003cp\u003eAdditionally, we investigated protein binding by performing tryptophan fluorescence quenching experiments using BSA (3 \u0026micro;M). These experiments were conducted in the absence of 15 mM trisodium citrate and 150 mM NaCl, maintaining a pH of 7.0. We recorded the fluorescence spectrum from 300 to 500 nm with an excitation wavelength of 295 nm.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAssessment of Cytotoxicity\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eCytotoxicity studies were conducted by preparing serial dilutions ranging from 100 \u0026micro;M to 0 \u0026micro;M. These dilutions were used for treatment, and the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) method was employed (details provided in the supplementary file). Staining experiments were performed using acridine orange and ethidium bromide following standard protocols outlined in the supplementary materials.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntimicrobial Activity Assessment.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe conducted \u003cem\u003ein vitro\u003c/em\u003e antimicrobial screening of the 4,4-bis(diethyl-methylphosphonate)-2,2\u0026rsquo;-bipyridine ligand and complex \u003cb\u003e2\u003c/b\u003e. The study evaluated their effects on specific human pathogenic bacteria and fungus using a diffusion method. Gram-positive bacteria (\u003cem\u003eBacillus megaterium\u003c/em\u003e, \u003cem\u003eB. subtilis\u003c/em\u003e), Gram-negative bacteria (\u003cem\u003eShigella dysenteriae\u003c/em\u003e, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e), and \u003cem\u003eCandida albicans\u003c/em\u003e were cultured in nutrient agar medium and incubated at 37 ℃ for 48 h, followed by frequent subculturing to fresh medium. Petri plates were inoculated with bacterial and fungal cultures, uniformly spread using a sterile glass spreader. The ligand and complex \u003cb\u003e2\u003c/b\u003e were dissolved in DMSO solvent; 100 \u0026micro;l of each sample was added to separate wells, with 100 \u0026micro;l of DMSO as a negative control. Refluxin (5 mcg) served as the positive control. Incubation occurred at 37 ℃ (48 h for bacteria) and 27 ℃ (72 h for fungi). After incubation, inhibition zone diameters were measured and recorded. Each experiment was repeated three times, and average values were reported.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cb\u003eSpectroscopic properties of complex 2\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eIn the absorption spectrum, the bands of complex \u003cb\u003e2\u003c/b\u003e appear at 229, 252, 304, and 315 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). These bands derived from such organometallic complexes were mainly attributed to the transitions n \u0026rarr; π *, π \u0026rarr; π * and n \u0026rarr; σ *.\u003csup\u003e42\u003c/sup\u003e Low-spin Ru(II), Rh(III), and Ir(III) complexes provide appropriately filled t\u003csub\u003e2g\u003c/sub\u003e orbitals, which can interact with lower π* orbitals of ligands.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eInteraction of complex 2 with CT-DNA\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eIn our investigation, the interaction of complex \u003cb\u003e2\u003c/b\u003e with calf-thymus DNA (CT-DNA) was examined via absorption spectroscopy (Fig. S3). The treatment induced a slight hypochromic effect and a marginal red shift near 260 nm. The isosbestic points observed at 231, 245, and 262 nm indicate a clean interaction between complex \u003cb\u003e2\u003c/b\u003e and DNA. This hyperchromicity could be attributed to either electrostatic binding or a partial unwinding of the DNA helix, revealing more base pairs. The subtle hyperchromic shift points to a non-intercalative mode of binding, possibly due to hydrogen bonding between complex \u003cb\u003e2\u003c/b\u003e and the DNA bases, or electrostatic interactions with the negatively charged phosphate groups of DNA. On the other hand, the bathochromic shift suggests an intercalative mode marked by strong π\u0026ndash;π stacking interactions between the aromatic ligand of the metal complexes and the DNA base pairs. However, the extent of intercalation remains ambiguous, as the red shift is less pronounced than that reported in other studies. Additionally, the presence of isosebestic points at 245 and 262 nm indicates the formation of an equilibrium between two species in the solution.\u003c/p\u003e\u003cp\u003eThe absorption band originating from complex \u003cb\u003e2\u003c/b\u003e at 231 nm exhibits significant shifts upon the addition of CT-DNA, indicative of a DNA-binding interaction (Fig. S3). The red shift observed further supports this interaction. The binding constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sub\u003e) for complex \u003cb\u003e2\u003c/b\u003e with DNA was determined by plotting the ratio of [DNA]/(ε\u003csub\u003ea\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;ε\u003csub\u003ef\u003c/sub\u003e) against [DNA], following the equation:\u003c/p\u003e\u003cp\u003e[DNA]/ (ε\u003csub\u003ea\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;ε\u003csub\u003ef\u003c/sub\u003e) = [DNA]/(ε\u003csub\u003eb\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;ε\u003csub\u003ef\u003c/sub\u003e)\u0026thinsp;+\u0026thinsp;1/\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sub\u003e (ε\u003csub\u003eb\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;ε\u003csub\u003ef\u003c/sub\u003e), where ε\u003csub\u003ea\u003c/sub\u003e, ε\u003csub\u003ef\u003c/sub\u003e, and ε\u003csub\u003eb\u003c/sub\u003e represent the apparent, free, and bound complex extinction coefficients, respectively. Linear regression of these data sets yields a straight line, with the slope equal to 1/(ε\u003csub\u003eb\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;ε\u003csub\u003ef\u003c/sub\u003e) and the y-intercept corresponding to 1/\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sub\u003e(ε\u003csub\u003eb\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;ε\u003csub\u003ef\u003c/sub\u003e)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The calculated \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sub\u003e value is 2.85 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Compared to literature values, classical intercalators exhibit \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sub\u003e values several orders of magnitude higher than that of complex \u003cb\u003e2\u003c/b\u003e, suggesting that intercalation is not the predominant mode of DNA interaction. Conversely, the \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sub\u003e value is significantly higher than those reported for groove-binding agents, such as Cr(II) complexes and tris(1,10-phenanthroline)ruthenium(II), indicating a strong interaction with DNA.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Further experiments are necessary to elucidate the precise mode of binding.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCompetitive binding between EB and complex 2\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eTo further elucidate the interaction between complex \u003cb\u003e2\u003c/b\u003e and DNA, emission experiments were conducted. Solutions containing EB-DNA conjugates (EB\u0026thinsp;=\u0026thinsp;ethidium bromide) exhibited a fluorescent emission peak at 596 nm (λ\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;510 nm), characteristic of the intercalation of planar EB between DNA base pairs (Fig S4). The introduction of complex \u003cb\u003e2\u003c/b\u003e that bind to DNA with an affinity equal to or greater than EB significantly alters the emission profile of DNA-EB. Notably, complex \u003cb\u003e2\u003c/b\u003e itself is non-fluorescent. Upon titration of complex \u003cb\u003e2\u003c/b\u003e into solutions of DNA-EB, a significant quenching of the 596 nm fluorescence was observed (Fig. S5). In the plot of [complex \u003cb\u003e2\u003c/b\u003e]/[DNA] against \u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e/\u003cem\u003eI\u003c/em\u003e, \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003esv\u003c/em\u003e\u003c/sub\u003e is calculated as 3.22 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e by dividing the slope by the y-intercept (Fig S6). However, the extent of quenching was not as substantial when compared to other literature.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e This attenuation in fluorescence intensity upon the addition of complex \u003cb\u003e2\u003c/b\u003e suggests that it may displace EB from the DNA-EB complex or quench the fluorescence of EB intercalated within DNA.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCircular dichroism analysis of DNA-conjugate interactions\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eCircular dichroism (CD) spectroscopy was utilized to assess potential conformational alterations in DNA upon interaction with complex \u003cb\u003e2\u003c/b\u003e. CD spectra were recorded for CT-DNA in both the presence and absence of complex \u003cb\u003e2\u003c/b\u003e. The CD spectra of a mixture of complex \u003cb\u003e2\u003c/b\u003e with DNA in a Tris buffer, post a 2-h incubation, are displayed (Fig. S7). The molar ellipticities [θ] at the characteristic negative and positive bands of DNA remained consistent with those observed for DNA in isolation, and the absence of a negative CD band at 300 nm corroborates the lack of substantial evidence for the intercalation of complex \u003cb\u003e2\u003c/b\u003e, in line with the findings from the above UV/vis spectroscopic studies.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eViscosity measurement method\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eThe change in viscosity of the EB intercalated DNA solution after the addition of the metal complex indicates the interaction of the complex with CT-DNA. The classic EB intercalator increases the relative viscosity of DNA by binding to the groove. Conversely, partial and non-classical bonds can bend or twist the DNA helix, reducing the length and viscosity of DNA.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Our results show that binding of adjacent DNA-pair grooves causes DNA helix elongation, which increases the viscosity of DNA bound with complex \u003cb\u003e2\u003c/b\u003e (Fig. S8).\u003c/p\u003e\u003cp\u003e\u003cb\u003eBSA binding experiments\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eThe electronic spectrum of pure BSA will show a strong absorption band at 280 nm from the aromatic residues of tryptophan and tyrosine, which may vary with the interaction of drug molecules or metal complexes. Absorption changes of BSA complexed with complex \u003cb\u003e2\u003c/b\u003e (0\u0026ndash;40 mM) showed a steady increase in BSA uptake as the concentration of the complex increased, indicating the formation of BSA-complex \u003cb\u003e2\u003c/b\u003e (Fig S8).\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e The changes observed in this situation when there are different concentrations of the Rh complex indicate a normal interaction between complex \u003cb\u003e2\u003c/b\u003e and BSA. A linear relationship between 1/(A\u003csub\u003eobs\u003c/sub\u003e-A\u003csub\u003e0\u003c/sub\u003e) and the inverse of the concentration of complex \u003cb\u003e2\u003c/b\u003e with a slope equal to 1/K\u003csub\u003eapp\u003c/sub\u003e(A\u003csub\u003ec\u003c/sub\u003e-A\u003csub\u003e0\u003c/sub\u003e) and an intercept equal to 1/(A\u003csub\u003ec\u003c/sub\u003e-A\u003csub\u003e0\u003c/sub\u003e) (Fig. S9). The data indicates that complex \u003cb\u003e2\u003c/b\u003e is strongly adsorbed on BSA surface.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFluorescence titration and interaction of complex 2 with BSA\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eIn our study, we also investigated the interaction between complex \u003cb\u003e2\u003c/b\u003e and BSA using fluorescence titration. BSA solutions exhibit strong fluorescence emission with a peak at 353 nm upon excitation at 295 nm, primarily due to tryptophan residues (Fig. S10). Complex \u003cb\u003e2\u003c/b\u003e, introduced during the experiment, displayed a weaker emission peak at 328 nm. Upon adding complex \u003cb\u003e2\u003c/b\u003e to BSA, we observed significant fluorescence quenching (up to 68% of initial fluorescence), as shown in Fig. S11. This quenching effect suggests that complex \u003cb\u003e2\u003c/b\u003e binds to BSA, potentially altering the secondary protein structure. To analyze this interaction, we employed Stern-Volmer and Scatchard plots. The Stern-Volmer equation, \u003cem\u003eI\u003c/em\u003e/\u003cem\u003eI\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1\u0026thinsp;+\u0026thinsp;k\u003csub\u003eq\u003c/sub\u003eτ\u003csub\u003e0\u003c/sub\u003e[Q]\u0026thinsp;=\u0026thinsp;1\u0026thinsp;+\u0026thinsp;\u003cem\u003eK\u003c/em\u003e\u003csub\u003esv\u003c/sub\u003e[Q], allowed us to calculate the dynamic quenching constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003esv\u003c/em\u003e\u003c/sub\u003e) based on the fluorescence lifetime (τ\u003csub\u003e0\u003c/sub\u003e) of tryptophan in BSA (approximately 1.0 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e s, Fig. S12).\u003c/p\u003e\u003cp\u003eThe calculated values for complex \u003cb\u003e2\u003c/b\u003e were as follows:\u003c/p\u003e\u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003esv\u003c/sub\u003e = 2.39 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; k\u003csub\u003eq\u003c/sub\u003e = 2.39 \u0026times; 10\u003csup\u003e12\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e S\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThese data indicate that complex \u003cb\u003e2\u003c/b\u003e exhibits strong quenching ability toward BSA and has a favorable binding propensity. Notably, the quenching rate constant (k\u003csub\u003eq\u003c/sub\u003e ) for complex \u003cb\u003e2\u003c/b\u003e surpasses that of other known quenching agents, likely operating via a static quenching mechanism.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eFurthermore, using Scatchard\u0026rsquo;s equation, Δ\u003cem\u003eI\u003c/em\u003e/\u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e/[Q]\u0026thinsp;=\u0026thinsp;n\u003cem\u003eK\u003c/em\u003e \u0026ndash; \u003cem\u003eK\u003c/em\u003e (ΔI/I\u003csub\u003e0\u003c/sub\u003e), we determined that the number of binding sites per BSA molecule (n) for complex \u003cb\u003e2\u003c/b\u003e is approximately 0.25 (Fig S12). The affinity constant (\u003cem\u003eK\u003c/em\u003e) of complex \u003cb\u003e2\u003c/b\u003e to BSA was calculated as 0.026 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, indicating a strong affinity. In summary, complex \u003cb\u003e2\u003c/b\u003e demonstrates the remarkable binding affinity, as observed in our DNA binding studies.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEvaluating cytotoxicity of complex 2 through\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eassays\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eWe assessed cytotoxic effects of complex \u003cb\u003e2\u003c/b\u003e using \u003cem\u003ein vitro\u003c/em\u003e anticancer assays on two malignant and one benign cell line via the MTT assay, as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig. S13. Our findings revealed that antitumor potency of complex \u003cb\u003e2\u003c/b\u003e varies across different cell types. Notably, increasing concentrations of complex \u003cb\u003e2\u003c/b\u003e were associated with greater cellular membrane ion absorption, leading to enhanced penetration and more pronounced anticancer activity. Importantly, complex \u003cb\u003e2\u003c/b\u003e demonstrated significant cytotoxicity towards MSA-MB-231 and A549 tumor cells, as evidenced by their reduced IC\u003csub\u003e50\u003c/sub\u003e values. However, it showed minimal toxicity towards normal VERO cells with an IC\u003csub\u003e50\u003c/sub\u003e value exceeding 1000 \u0026micro;M, indicating its selective action on malignant cells. Recent studies suggest that incorporating appropriate ligands into Rh complexes may amplify their targeting capabilities.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eMDA-MB-231 cells were treated with varying concentrations (0\u0026ndash;100 \u0026micro;M) of complex \u003cb\u003e2\u003c/b\u003e or \u003cem\u003ecis\u003c/em\u003e-platin. Cis-platin, a well-known potent cytotoxic agent, was used as a reference to compare and evaluate the cytotoxicity of the metal complexes, as described in previous studies.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e The reduction in cell viability percentage confirmed the dose-dependent cytotoxic effects of the complexes on the cancer cell line (Fig. S13 and S14). The IC\u003csub\u003e50\u003c/sub\u003e value of cis-platin was determined to be 10.46 \u0026micro;M\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, which is consistent with previously reported literature data.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e These findings further validate the reliability and reproducibility of the present experimental results.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e cytotoxicity (IC\u003csub\u003e50\u003c/sub\u003e) of complex \u003cb\u003e2\u003c/b\u003e and \u003cem\u003ecis\u003c/em\u003e-platin against cells .\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCell line\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;M)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ecomplex \u003cb\u003e2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ecis\u003c/em\u003e-platin\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMDA-MB-231\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e47.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA549\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e48.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVERO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;= 1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFluorescence microscopy assessment of apoptotic cell death\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eWe also investigated the cell death patterns induced by complex \u003cb\u003e2\u003c/b\u003e using a two-color staining method\u0026mdash;acridine orange/ethidium bromide (AO/EB) - followed by fluorescence microscopy.\u003csup\u003e\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e Both cancerous and non-cancerous cells were exposed to complex \u003cb\u003e2\u003c/b\u003e for 24 h. Acridine orange permeates all cells, emitting bright green fluorescence upon binding to double-stranded DNA. In contrast, ethidium bromide selectively accumulates in non-viable cells, resulting in red fluorescence upon DNA intercalation.\u003c/p\u003e\u003cp\u003eBy analyzing fluorescent emissions and morphological features, we categorized cells into four distinct types:\u003c/p\u003e\u003cp\u003e(1) Viable cells: These exhibit highly organized nuclei with intense green fluorescence.\u003c/p\u003e\u003cp\u003e(2) Early apoptotic cells: Their green fluorescent nuclei show chromatin condensation and nuclear fragmentation.\u003c/p\u003e\u003cp\u003e(3) Late apoptotic cells: Characterized by nuclei fluorescing orange to red, with chromatin condensation or fragmentation.\u003c/p\u003e\u003cp\u003e(4) Necrotic cells: Display red fluorescence without chromatin disintegration.\u003c/p\u003e\u003cp\u003eIn summary, living cells display vivid green nuclear structures, apoptotic cells exhibit orange to red nuclei with condensed or fragmented chromatin, and necrotic cells show similarly colored nuclei with dense, unfragmented chromatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOur research reveals that complex \u003cb\u003e2\u003c/b\u003e significantly induces apoptosis in cancer cells, corroborating the cytotoxicity data (Fig S15). The anticancer effects of complex \u003cb\u003e2\u003c/b\u003e likely stem from its ability to bind to DNA, triggering cellular apoptosis. Notably, variations in lipophilicity and DNA affinity seem to govern complex \u003cb\u003e2\u003c/b\u003e\u0026rsquo;s oncogenic activity. These findings contribute to a deeper understanding of metal complexes\u0026rsquo; anticancer properties and their interactions with proteins like BSA. Comprehensive clinical studies are crucial for unraveling the molecular mechanisms of cytotoxicity and determining optimal conditions for using metal complexes as therapeutic agents.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntimicrobial activity of complex 2: Insights from\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eassays\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eIn our study, we evaluated the antimicrobial properties of compound \u003cb\u003e1\u003c/b\u003e, Rh-dimer (Cp*)\u003csub\u003e2\u003c/sub\u003eRh\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e4\u003c/sub\u003e, ampicillin, and complex \u003cb\u003e2\u003c/b\u003e using the diffusion agar method against Gram-positive bacteria (\u003cem\u003eBacillus megaterium and Bacillus\u003c/em\u003e), Gram-negative bacteria (\u003cem\u003eShigella dysenteriae and Klebsiella pneumoniae\u003c/em\u003e), and the fungus Candida albicans. Ampicillin served as the reference antibiotic for all tested microorganisms. Our analysis revealed that complex \u003cb\u003e2\u003c/b\u003e exhibits potent antibacterial and antifungal effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Typically, bacterial growth appears as a turbid layer beyond regions where the antimicrobial agent surpasses the minimum inhibitory concentration (MIC), resulting in a clear zone of inhibition. The extent of this zone depends on factors such as medium composition, culture conditions, diffusion rates, and the concentration of the antimicrobial agent. The variability in complex \u003cb\u003e2\u003c/b\u003e\u0026rsquo;s efficacy against different bacteria may be attributed to unique permeability barriers in microbial cells or disparities in their ribosomal structures. The pronounced activity levels of complex \u003cb\u003e2\u003c/b\u003e suggest a beneficial impact on both bacteria and fungi, potentially through direct interaction with these organisms.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe inhibition zone values presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e demonstrate the complex\u0026rsquo;s effectiveness against both Gram-positive bacteria (\u003cem\u003eBacillus megaterium and Bacillus subtilis\u003c/em\u003e) and Gram-negative bacteria (\u003cem\u003eShigella dysenteriae and Klebsiella pneumoniae\u003c/em\u003e), as well as fungi (\u003cem\u003eCandida albicans\u003c/em\u003e). Complex \u003cb\u003e2\u003c/b\u003e exhibited greater activity than compound \u003cb\u003e1\u003c/b\u003e, attributed to its increased bactericidal effects. Notably, metal complexes\u0026rsquo; toxicity is linked to enhanced lipophilicity resulting from chelation. According to Overtone\u0026rsquo;s concept of cell permeability, lipid membranes favor fat-soluble substances crucial for disease control. The heightened lipophilicity of complex \u003cb\u003e2\u003c/b\u003e facilitates membrane penetration, blocking iron-binding sites in microbial enzymes. This disruption of cellular respiration limits bacterial growth and protein synthesis, ultimately leading to bacterial death. Among the tested microbes, Gram-positive \u003cem\u003eBacillus megaterium\u003c/em\u003e and Gram-negative \u003cem\u003eShigella dysenteriae\u003c/em\u003e were most susceptible, with inhibition zones measuring 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1 mm. Candida albicans also exhibited an inhibition zone of 24\u0026thinsp;\u0026plusmn;\u0026thinsp;1 mm. Usually, Gram-positive bacteria, due to their thick peptidoglycan layer and outer membrane, are more easily targeted by antibiotics than Gram-negative bacteria. Interestingly, complex \u003cb\u003e2\u003c/b\u003e exhibited a similar trend in its activity against both bacterial strains. Complex \u003cb\u003e2\u003c/b\u003e\u0026rsquo;s potent antibacterial properties make it a promising alternative to traditional antibiotics.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparison of antimicrobial activity (mm): compound \u003cb\u003e1\u003c/b\u003e, rhodium dimer, complex \u003cb\u003e2\u003c/b\u003e, and ampicillin.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ecompound 1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRh-dimer\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ecomplex 2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eampicillin\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus megaterium\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eB. subtilis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eShigella dysenteriae\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCandida albicans\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eA Rh(III) complex containing 4,4-bis(diethyl-methylphosphonate)-2,2\u0026rsquo;-bipyridine was synthesized to explore its biological activities. Various spectroscopic techniques were employed to evaluate the complex\u0026rsquo;s binding interactions with DNA and proteins. The Rh complex exhibited a binding constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sub\u003e) of 2.85 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for CT-DNA, suggesting quite strong inbteraction to the DNA helix. Competitive studies with EB indicated that the complex can displace or quench EB from EB-DNA complexes, consistent with groove-binding interactions. Additionally, electronic and fluorescent spectral titration data revealed protein chain unwinding upon interaction with the Rh complex. The complex\u0026rsquo;s effect on tryptophan residues in an aqueous medium further characterized its binding behavior. Steady-state quenching experiments confirmed static binding to bovine serum albumin at a single site. Furthermore, \u003cem\u003ein vitro\u003c/em\u003e cytotoxicity assays demonstrated that our Rh complex surpasses the currently used chemotherapeutic drug \u003cem\u003ecis\u003c/em\u003e-platin in terms of cytotoxicity against cancer cell lines. Antibiotic testing revealed the complex\u0026rsquo;s antibacterial properties, proving its efficacy against Gram-positive and Gram-negative bacteria as well as fungi. This promising strategy could enhance safety and efficacy in future cancer therapies through \u003cem\u003ein vivo\u003c/em\u003e studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government(MSIT) (RS-2024-00397807)\u0026nbsp;and the Researchers Supporting Project number (RSP2023R147), King Saud University, Riyadh, Saudi Arabia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003euthor contribution\u003c/strong\u003e\u003cstrong\u003es Author statement:\u0026nbsp;\u003c/strong\u003eJinheung Kim: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Methodology, Investigation, Data curation, Supervision, Resources, Project administration, Conceptualization, Funding acquisition.\u0026nbsp;Thamilarasan\u0026nbsp;Vijayan: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original\u0026nbsp;draft, Methodology, Investigation, Data curation, Conceptualization.\u0026nbsp;Atifa Ashraf:\u0026nbsp;Methodology, Investigation, Data curation.\u0026nbsp;Mohammad Azam:\u0026nbsp;Methodology, Investigation,\u0026nbsp;Funding acquisition,\u0026nbsp;Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003cstrong\u003eompeting interest\u003c/strong\u003eThe authors declare that they have no known financial conflicts of interest or personal relationships that could have influenced the work reported in this paper\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eH.W. 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Arsenijević, Analysis of cycloheximide-induced apoptosis in human leukocytes: Fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide, Cell biol int. 30 (2006) 924\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eK. Nagaraj, G. Velmurugan, S. Sakthinathan, P. Venuvanalingam, S. Arunachalam, Influence of self-assembly on intercalative DNA binding interaction of double-chain surfactant Co (iii) complexes containing imidazo [4, 5-f][1, 10] phenanthroline and dipyrido [3, 2-d: 2\u0026prime;-3\u0026prime;-f] quinoxaline ligands: experimental and theoretical study, Dalton Trans. 43 (2014) 18074\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eY. Manojkumar, S. Ambika, R. Arulkumar, B. Gowdhami, P. Balaji, G.Vignesh, S. Arunachalam, P. Venuvanalingam, R. Thirumurugan, M.A. Akbarsha, Synthesis, DNA and BSA binding, in vitro anti-proliferative and in vivo anti-angiogenic properties of some cobalt (III) Schiff base complexes, New J Chem. 43 (2019) 11391\u0026ndash;407.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eS. Ramakrishnan, V. Rajendiran, M. Palaniandavar, V.S. Periasamy, B.S. Srinag, H. Krishnamurthy, M.A. kbarsha, Induction of cell death by ternary copper (II) complexes of L-tyrosine and diimines: role of coligands on DNA binding and cleavage and anticancer activity, Inorg Chem. 48 (2009) 1309\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme ","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"jbic-journal-of-biological-inorganic-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [JBIC Journal of Biological Inorganic Chemistry](https://link.springer.com/journal/775)","snPcode":"775","submissionUrl":"https://submission.springernature.com/new-submission/775/3","title":"JBIC Journal of Biological Inorganic Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Rh(III) complex, DNA binding, protein binding, anticancer activity, cytotoxic study, antimicrobial activity","lastPublishedDoi":"10.21203/rs.3.rs-6581437/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6581437/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe report the synthesis and biological activities of a (Cp*)Rh(III) complex containing 4,4\u0026rsquo;-bis(diethyl-methyl phosphonate)-2,2-bipyridine. The complex was characterized through various spectroscopic methods. Its DNA binding capacity was assessed using titration, viscosity measurements, and spectroscopic techniques including absorption, fluorescence, and circular dichroism (CD) spectroscopy. The findings revealed a positive absorption coefficient with a binding constant of 2.85 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Additionally, the Rh complex showed a strong affinity for bovine serum albumin, with a binding constant that aligns with the observed DNA binding pattern. The cytotoxic effects of the complex were evaluated against selected cancer cell lines, such as MDA-MB-231 (human breast cancer) and A549 (human lung adenocarcinoma), as well as VERO (a normal human lung adenocarcinoma cell line) for comparison. The IC\u003csub\u003e50\u003c/sub\u003e values for MDA-MB-231 and A549 cells were determined to be 47.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u0026micro;M and 48.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u0026micro;M, respectively, indicating potent cytotoxicity at low micromolar concentrations. In contrast, the VERO normal cells exhibited significantly lower toxicity, with an IC\u003csub\u003e50\u003c/sub\u003e value of \u0026ge;\u0026thinsp;1000 \u0026micro;M. Furthermore, the Rh complex was tested for its antimicrobial properties, demonstrating greater inhibitory activity compared to the free ligand.\u003c/p\u003e","manuscriptTitle":"Characterization and Biological Evaluation of a (Cp*)Rh(III) Complex Featuring Phosphonate-Modified Bipyridine Ligands","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-06 17:13:31","doi":"10.21203/rs.3.rs-6581437/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-01T00:59:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-01T00:53:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-16T07:02:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60031877677396472187024427901118797735","date":"2025-09-02T05:00:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"160901096069586266257489485337209970043","date":"2025-08-11T08:54:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-08T08:06:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"151853295395437832576225653689314892825","date":"2025-08-05T06:32:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-03T22:16:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-05T23:41:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-05T23:40:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"JBIC Journal of Biological Inorganic Chemistry","date":"2025-05-03T02:22:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"jbic-journal-of-biological-inorganic-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [JBIC Journal of Biological Inorganic Chemistry](https://link.springer.com/journal/775)","snPcode":"775","submissionUrl":"https://submission.springernature.com/new-submission/775/3","title":"JBIC Journal of Biological Inorganic Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1eeb6a1e-7b5a-493a-8989-8f0493b3d5f7","owner":[],"postedDate":"August 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-21T22:23:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-06 17:13:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6581437","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6581437","identity":"rs-6581437","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}