Exploring the binding interaction of rupatadine with bovine serum albumin using multi-spectroscopic and molecular modeling approaches

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El-Messery, Fathalla Belal This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7321587/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Mar, 2026 Read the published version in BMC Chemistry → Version 1 posted 12 You are reading this latest preprint version Abstract Rupatadine (RUPA), a second-generation H 1 -receptor antagonist, is used to treat allergies with a further antagonistic action on platelet-activating factor. Here, RUPA and bovine serum albumin (BSA) binding interaction has been investigated via various approaches, including spectrofluorimetric techniques, thermodynamic studies, Fourier transform infrared spectroscopy (FTIR), ultraviolet, and molecular docking (MD). The spectrofluorimetric titration study was displayed at various temperatures, and the data revealed that the BSA native fluorescence is quenched by RUPA via a static process, which has been signified by UV absorption. The thermodynamic analysis revealed that the stoichiometry between RUPA and BSA is 1:1, and their binding affinity was weak to moderate. As revealed by the enthalpy change (ΔH) and entropy change (∆S) values of 32.84 kJ mol −1 and 0.18 kJ mol −1 , respectively, the hydrophobic forces are the main binding forces in the interaction between BSA and RUPA. The negative values of Gibbs free energy change (ΔG) indicate that the binding process between RUPA and BSA was spontaneous. Furthermore, results of the site marker technique and synchronous fluorescence measurements indicate that RUPA binding interaction occurs at site (I) on BSA in the vicinity of tryptophan residues, which was then confirmed by MD. Rupatadine Fluorescence Bovine serum albumin Thermodynamic Docking. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Investigation of drug-protein binding interactions helps in the discovery of protein binding sites for targeted therapies, and its evaluation has been essential for the development of innovative drugs as well as the advancement of knowledge regarding drug action mechanisms and metabolic processes [ 1 ]. The characteristics of the plasma protein albumin, such as its high binding capabilities for both hydrophobic and hydrophilic medicines, comparatively long half-life, ability to specifically target inflammatory sites, and essentially virtually low toxicity and immunogenicity, have drawn particular attention to it [ 2 ]. Serum albumin is the predominant circulating protein found in blood plasma, contributing good biocompatibility, biodegradability, and safety for its therapeutic use [ 3 ]. Its chemical structure and conformation enable interaction with a variety of medicines, which may shield them from metabolism and excretion in vivo and enhance their pharmacokinetic characteristics [ 4 ]. Because of the high solubility, low cost, and structural similarity between bovine serum albumin and human serum albumin (76%), it has been used extensively to investigate interactions between ligands and serum albumin. Three homologous domains (I, II, and III) and two subdomains (A and B) make up the single-chain polypeptide that makes up BSA [ 5 ]. It is crucial to delve deeper into the characteristics of the drug-protein interactions through diverse approaches using BSA as a protein model to study such interactions in order to minimize side effects and maximize the effectiveness of the medicines. El Gammal et al. [ 6 ] used such approaches to study similar binding interactions in vitro under the simulated physiological circumstances (pH 7.4) utilizing the quenching fluorescence technique, and the parameters of thermodynamics were applied in order to determine the binding constants at three temperatures. As well as synchronous fluorescence, Fourier transform infrared spectroscopy (FTIR), UV-visible spectroscopy, the site marker technique, and MD have been applied for exploring the site of reaction and the protein structure's alterations upon binding with RUPA [ 7 – 11 ]. Here, we studied how RUPA binds to BSA in order to elucidate the interaction mechanism of the binding utilizing several fluorescence spectroscopy techniques, Fourier transform infrared spectroscopy, and UV-visible spectroscopy. Furthermore, a detailed MD analysis was used to determine the most preferred binding site within BSA. Rupatadine (Fig. 1 ) chemically is 13-chloro-2-[1-[(5-methylpyridin-3-yl) methyl] piperidin-4-ylidene]-4-azatricyclo [9.4.0.03,8] pentadeca-1(11),3(8),4,6,12,14-hexaene [ 12 ]. Rupatadine is a second-generation antihistamine and antagonist of platelet-activating factor utilized for allergy treatment. It was discovered, manufactured, and commercialized as Rupafin 10mg and various other brand names in 2003. It is about 99% plasma protein bound, even though it is very well distributed to other tissues [ 13 ]. Deuster et al. [ 14 ] found in 2021 that RUPA targets the platelet-activating factor receptor to prevent ovarian cancer cells from proliferating and migrating in vitro. According to these results, RUPA could potentially have anticancer properties. As far as we are aware, we have evaluated the behavior of RUPA-BSA binding interaction for the first time. This evaluation assists in improving its pharmacological and clinical efficacy, ensuring effective therapeutic drug levels, and preventing any potential unwanted adverse events. 2. Experimental procedure 2.1 Chemicals Bovine serum albumin (standard grade, pH 7, Origin USA) was purchased from Cegrogen Biotech GmbH (Germany). The molar concentration of BSA was calculated using the assumption that its molecular weight was 66,500 Da. Rupatadine fumarate was kindly supplied by ATCO PHARMA for Pharmaceutical Industries (Cairo, Egypt). Diazepam (99.2% purity) was obtained from EIPICO (10th of Ramadan City, Egypt). Phenylbutazone (99.7% purity) was obtained from GLOBAL NAPI PHARMACEUTICALS (6th October City, Egypt). Tris hydrochloride (Tris–HCl) batch number 337012022 (purity > 99%) was purchased from Chemajet for Chemical and Pharmaceutical Industries, Alexandria, Egypt. The supplier of HPLC grade methanol was Sigma Aldrich (St. Louis, MO, USA). Double-distilled water was utilized. Analytical reagent-grade chemicals were all used. 2.2 Instrumentation Agilent Technologies' Cary Eclipse fluorescence spectrophotometer (Santa Clara, California, USA) was utilized. The quartz cuvette utilized was 1 cm. The slit width was 5 nm, and the smoothing factor was 19. The wavelengths were 280 and 340 nm, as excitation and emission, respectively. The voltage was adjusted to 600 V. Shimadzu UV-1601, a UV-visible double-beam spectrophotometer (Kyoto, Japan), was employed at a high scan speed. Bruker Tensor 27 FTIR spectrometer was used to scan the FTIR spectra under strictly constant conditions in the region of 400–5000 cm − 1 . All pH readings were taken using a Jenway 3510 pH meter (Jenway, Staffordshire, UK). 2.3 Software The software "Molecular Operating Environment (MOE) 2024.06 was utilized for both the docking of RUPA to examine how it binds and interacts with BSA and the surface mapping study of RUPA. 2.4 Preparation of solutions A stock solution of 200 µM bovine serum albumin was prepared using bidistilled water, and rupatadine (1000 µM) was freshly prepared using bidistilled water. The buffer solution (pH 7.4) of Tris-hydrochloride (20 mM) was freshly made using bidistilled water. Phenylbutazone and diazepam stock solutions (10 mM) were prepared in methanol. To prepare the working solutions, all stock solutions were diluted beforehand and stored at 277 K. 2.5 Spectroscopic measurements 2.5.1 Fluorescence measurements The fluorescence quenching of BSA by RUPA was examined at three different temperatures: 303, 310, and 318 K, along wavelengths (300/500 nm) following excitation at 279 nm. The concentration of BSA remained constant at 2 µM, whereas the concentration of RUPA was increased from 0 to 100 µM. 2.5.2 Synchronous fluorescence technique With increasing concentrations of RUPA (0–100 µM), by adjusting the Δλ at 60 nm for tryptophan (Trp) and Δλ at 15 nm for tyrosine (Tyr) residues at 303 K, synchronous fluorescence spectra of BSA were scanned in the 200/350 nm region. 2.5.3 FT-IR spectroscopy The FTIR spectra of the BSA solution (2 µM), both without and with RUPA (40 µM) in Tris-HCl buffer (20 mM) at pH 7.4, were scanned over the 400–5000 cm − 1 region. 2.5.4 UV-visible spectroscopic measurements The ultraviolet absorption spectra of the BSA solution (2 µM) with different amounts of RUPA (0-100 µM) in Tris-HCl buffer (20 mM) at pH 7.4 were measured over the 250–330 nm region in contrast to a reference solution that included all relevant system components except for BSA. The experiments were carried out at 303 K. 2.6 Competitive binding analysis using site probes Phenylbutazone and diazepam are the site probes for sites Ⅰ and Ⅱ, respectively. The BSA and site probe concentrations in this experiment were maintained at 2 µM and 10 µM, respectively. Rupatadine concentrations ranged from 10 to 100 µM. After excitation at 279 nm, fluorescence intensity measurements were made at 341 nm. At 303 K, measurements were made. The binding constants (K b ) of RUPA to diazepam-BSA, phenylbutazone-BSA, and BSA alone were compared. 2.7 Influence of metal ions on the RUPA-BSA binding interaction Bovine serum albumin and ion metal solutions were fixed at 2 µM (pH 7.4), and the concentration of RUPA was varied from 10 to 100 µM. Fluorescence intensity measurements were taken at 341 nm following excitation at 279 nm at 303 K. Comparisons were made between the equilibrium constants for RUPA binding (K b ) to BSA with each metal ion present and the equilibrium constant for RUPA binding (K b ) to BSA alone. 2.8 Molecular docking and Surface Mapping The binding behavior of BSA and RUPA was investigated using the Molecular Operating Environment (MOE) 2024.06. The structure of RUPA was initially drawn in the MOE. A conformational search was performed to obtain the most stable conformer using the systematic search method. The crystalline structure of bovine serum albumin was acquired from the Protein Data Bank (PDB) using the PDB code number 4f5s ( http://www.rcsb.org ). The docking procedure has been carried out between the binding pocket and the conformers with the lowest energy, ‘global minima’. Following preparation, both ligands and protein receptors were protonated, and the force field MMFF94X's default parameters were used to reduce energy. The parameters of MD utilized in the experiment have been employed with Triangle Matcher as a default. Along with 30 conformation generations to match the binding groove, rescoring functions 1 and 2 were set to London dG and GBVI/WSA dG, respectively. For additional investigation and assessment of the relationship between RUPA and BSA, the mdb output file was produced. Surface mapping of both the binding pocket and ligand was produced in a color-coded map where pink denotes hydrogen bond, blue denotes mild polar, and green denotes hydrophobic region [ 15 – 20 ]. 3. Results and discussion 3.1 Examination of quenching mechanism The presence of aromatic amino acid residues in the structure of BSA, such as tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) is what gives it its inherent fluorescence. Investigating protein conformational changes upon interactions with different drugs and providing precise information on ligand-protein interactions are two common uses for fluorescence spectroscopy [21]. In this experiment, we measured the decrease of the BSA intrinsic fluorescence due to quenching by RUPA (figure 2). Fluorescence quenching occurs through several mechanisms, including dynamic quenching, which arises from collisions between proteins and quenchers; static quenching, which results from ground state complex formation between proteins and quenchers; and the combined mechanism that combines static and dynamic quenching, resulting in both complex formation and collision with the same quencher. The process of fluorescence quenching was determined using the Stern-Volmer equation (equation 1): F o /F = 1+Kq τo [Q] = 1+Ksv [Q] (1) F and F 0 represent the emission intensity of BSA with and without RUPA, respectively. [Q] stands for the concentration of RUPA. K q , K SV , and τ 0 denote the quenching rate constant, the Stern–Volmer quenching constant, and the fluorescence lifespan without RUPA, which is equivalent to 10 −8 seconds, respectively. Generally, in the static quenching process, elevating the temperature causes the quenching constant to decrease, but in the dynamic mechanism, it induces an increase [22]. The K SV and K q values were determined by the graph of F 0 / F against [Q] (figure 3). Table (1) provides a summary of the K SV and K q values acquired at various temperatures. The data indicated that the K SV value decreased with rising temperature in the presence of RUPA, and the k q values exceeded the maximum K q for dynamic (2×10 10 L mol -1 s -1 ); hence, the quenching mechanism is a static process. Table. 1: RUPA-BSA interaction parameters at various temperatures. T(K) K sv (L.mol -1 ) K q (L.mol -1 .s -1 ) R 2 303 1.77×10 4 1.77×10 12 0.9972 310 1.52×10 4 1.52×10 12 0.9992 318 1.21×10 4 1.21×10 12 0.9992 3.2 Determinations of the binding constant and stoichiometric analysis Stoichiometric analysis is used to define the various equilibria for a macromolecule's binding of a ligand by binding sites (n) [23]. The number of binding sites (n) and the binding constant (K b ) were calculated using the modified Stern–Volmer equation (equation 2). Log F o −F / F = log K b + n log [Q] (2) Thus, by graphing log [(F 0 – F) / F] against log [Q], as illustrated in figure S1, we can obtain the number of binding sites (n) and the binding constant (K b ) for the interaction of RUPA and BSA. The values of log K b , and (n) as seen in Table 2, are obtained from the intercept and slope, respectively. Table. 2: RUPA-BSA complex binding characteristics at different temperatures. *SEM: Standard error of mean It was observed that upon rising temperatures, the binding constant (K b ) increased; this can be interpreted as the quenching being due to hydrophobic interactions rather than ionic interactions, as will be revealed in the next study. A weak to moderate binding affinity was indicated by the order of the binding constant (K b ), which ranged from 10 3 to 10 4 L mol -1 . The stoichiometry for RUPA-BSA binding is 1:1, which was indicated by the value of (n), which was close to 1. 3.3 Thermodynamic characteristics and nature of binding forces The thermodynamic characteristics of the binding reaction serve as the primary evidence for the binding force. Hydrogen bonds, hydrophobic forces, electrostatic interactions, van der Waals interactions, and other binding forces could all be present between small molecules and biomacromolecules [24]. The temperature dependence of the binding constant was thus measured for this purpose at three distinct temperatures: 303, 310, and 318 K (Figure 4). The acting forces in the binding process were described by the thermodynamic characteristics of the BSA-RUPA system, such as enthalpy change (ΔH), entropy change (ΔS), and the Gibbs free energy change (ΔG), as revealed in Table 3. The values of ΔH and ΔS derived from the van't Hoff equation (equation 3) are displayed in Figure 4. Ln K b = −ΔH/RT + ΔS/R (3) Gibbs–Helmholtz equation provided below was used to assess the free energy change (ΔG) value (equation 4) ΔG = ΔH – TΔS (4) Within the examined temperature range, the values of the enthalpy change (ΔH) and the entropy change (∆S) were 32.84 kJ mol−1 and 0.18 kJ mol−1, respectively. Thus, hydrophobic interactions appear to represent the main binding forces in the interaction between RUPA and BSA, as indicated by the positive values of ∆H and ∆S. Furthermore, the positive value of ΔH signified that the creation of the RUPA-BSA complex was an endothermic reaction. The findings indicated that the binding interaction between BSA and RUPA was spontaneous because the ΔG values were negative. Table. 3: Thermodynamic parameters of BSA-RUPA interaction at pH 7.4. T (K) ∆H o (KJ/mol) ∆G o (KJ/mol) SEM ∆S o (J mol -1 K -1 R 2 303 32.84 -55.08 0.79 181.91 0.9995 310 -56.35 318 -57.81 *SEM: Standard error of mean 3.4 Spectroscopic study of RUPA-BSA interaction 3.4.1 Synchronous fluorescence measurements Using synchronous fluorescence, the influence of has been thoroughly investigated [25]. The distinctive details regarding the protein's Tyr and Trp residues can be gained by fixing the scanning interval (∆λ) between the excitation and emission wavelengths at 15 and 60 nm, respectively. Figures S2(A) and S2(B) show the findings of the corresponding BSA fluorescence ligand binding on the polarity of the microenvironment surrounding fluorescent amino acid residues of the protein spectra as RUPA concentrations rise. Adding RUPA and monitoring at ∆λ=60 resulted in a minor red shift of ∆λ max of around 2 nm, while no shift was seen at ∆λ = 15. This indicates that RUPA binds to BSA at Trp residues, as confirmed by MD experiments that revealed drug binding near Trp residues. 3.4.2 FT-IR spectroscopy FTIR measurements have been performed to further explore the BSA conformational change that is caused by the interaction with RUPA. The spectrum in Figure S3 (a) represents free BSA, and Figure S3 (b) is the spectrum of the RUPA-BSA complex at pH 7.4 at 303 K. The C=O stretch is responsible for the amide I band, which is located between 1600 and 1700 cm -1 [26]. It is linked to the secondary structure of BSA and is more vulnerable to modifications in the secondary structure of proteins than amide II, which is located between 1500 and 1600 cm −1 . Following the RUPA reaction, the amide I peak's location shifted from 1646 to 1642 cm -1 , indicating that secondary protein structure changes during the RUPA-BSA interaction. 3.4.3 UV-visible spectroscopic measurements Ultraviolet-visible spectroscopy is an effective technique for identifying the conformational alterations of proteins mediated by drug molecules [27]. Alterations in the ultraviolet-visible spectra of the protein, whether in intensity or λ max , are primarily seen in the static process because a new ground-state complex has been established; conversely, dynamic quenching typically does not correlate with modifications in the protein's absorbance spectrum [28,29]. As shown in Figure 5, the UV absorption spectrum of BSA was captured both without and with RUPA added; it can be found that there were two absorption bands for all solutions of BSA. The band of absorption close to 220 nm associated with the α-helix indicates the structural shape of bovine serum albumin. A modest absorption band about 280 nm is attributed to the π–π* transitions of aromatic amino acids, including tryptophan, tyrosine, and phenylalanine residues. A notable escalation in intensity is observed with rising RUPA concentrations, accompanied by a red shift at the maximum wavelength near 220 nm. This indicates the involvement of static quenching and corroborates the earlier findings of the fluorescence quenching investigation. 3.5 Competitive binding analysis using site probes Two specific site probes, phenylbutazone for site I and diazepam for site II, were used as site probes in site marker competitive binding experiments to identify the preferential binding site of RUPA on BSA. For small ligands, BSA has two primary binding sites: site I, which is located in subdomain IIA's hydrophobic pocket, and site II, which is located in subdomain IIIA's hydrophobic cavity. Site I is comparatively bigger and mostly bound by hydrophobic interactions with neutral, bulky, and heterocyclic molecules. However, site II is smaller, and the interaction usually happens via a mix of electrostatic, hydrogen bonding, and hydrophobic forces [30-32]. The K b value for RUPA binding with BSA in the presence of phenylbutazone was found to be lower than that in the absence of the site marker, as shown in Table 4. However, there was no discernible variation in the K b value when diazepam was present. According to these findings, phenylbutazone and RUPA vie for the same binding site at BSA, which is site I. Table. 4: Binding constant of RUPA in absence and presence of site markers at 303 K. Site marker Log K b R 2 SEM Blank 3.84 0.9996 0.045 Phenylbutazone 2.99 0.9995 0.031 Diazepam 3.85 0.9997 0.086 *SEM: Standard error of mean 3.6 Influence of metal ions on RUPA-BSA interaction Metal ions interact with proteins, modifying their biological function and structure via coordination bond formation; therefore, they affect the interaction of the protein with the drugs and their binding behavior [33, 34]. Because proteins are used in many different industries and serve a variety of purposes in the human body, it is crucial to assess these alterations [35, 36]. For that, the influence of some metal ions (Na +1 , K +1 , Zn +2 , Ca +2 , Mg +2 , Fe +3 ) on the BSA-RUPA system was explored. The determined K b values of the RUPA-BSA complex in the presence of certain ions, such as Ca +2 , K +1 , and Fe +3 , were greater than those without them, as shown in Table 5. This may be explained by the metal ion-RUPA-BSA system formation. Other ions exhibited lower K b , which may be as a result of competitive binding with BSA. However, the change of K b in general could be attributed to changes in BSA confirmation by its interaction with metal ions at metal binding sites. More research is required in the future to try to understand the type, strength, and involvement of a protein's functional groups in metal-protein interactions. Table. 5: The K b values of RUPA-BSA system with and without metal ions. System Log K b K b (M -1 ) R 2 SEM BSA+RUPA 3.84 6.92×10 4 0.9996 0.045 BSA+RUPA+Na +1 3.69 4.90×10 4 0.9996 0.071 BSA+RUPA+K +1 4.85 7.80×10 4 0.9991 0.019 BSA+RUPA+Mg +2 4.30 2.00×10 4 0.9994 0.018 BSA+RUPA+Ca +2 4.85 7.10×10 4 0.9992 0.071 BSA+RUPA+Zn +2 4.09 1.23×10 4 0.9992 0.099 BSA+RUPA+Fe +3 3.89 7.76×10 4 0.9991 0.049 *SEM: Standard error of mean 3.7 Molecular docking analysis To provide a comprehensive understanding of the various binding patterns, molecular modelling is mandatory, so a docking study of the RUPA into the BSA protein was conducted. The most popular model serum albumin protein for researching these kinds of protein-ligand interactions is BSA [37]. To conduct the analysis, the protein structure (PDB code 4f5s) was selected from the Protein Data Bank using MOE 2024.06 [38]. Conformational Search: Conformational analysis of RUPA has been obtained for accurate further modeling studies, where its best and least energy conformer was produced by conformational search procedures that explore conformational space in torsional space, applying the multi-conformer approach. The results have been depicted in Figure S4. Molecular Docking and Surface Mapping As illustrated in figure S5 (a,b), the following amino acid residues were discovered to surround RUPA: Try149, Ser286, Arg256, Leu237, Ala290, Arg194, Arg217, Glu291, Lys187, and Asp450. The residues of the interacting amino acids are nearly identical with a small variation and are located in a hydrophobic cavity at site I. Since no hydrogen bonding contacts were found, it can be assumed that RUPA's interaction with Try149 and Arg217 is solely hydrophobic via arene cationic interaction. With a 6.41 kcal/mol calculated binding energy, which is in line with experimental results, it can be predicted that RUPA fits perfectly within site I, landing into the hydrophobic pocket of subdomain IIA's close to tryptophan residues. Numerous studies have demonstrated how hydrophobic interactions contribute to the stability of drug-serum albumin complexes based on [39], which was encouraging to conduct an in-depth surface mapping investigation. Further study about the relation between the hydrophobic character of RUPA and the existence of residues of hydrophobic amino acids at the BSA binding site has shown that this similarity may have helped to stabilize the RUPA-BSA system via hydrophobic forces; that was basically confirmed by both observing the 3D view of the overlay of RUPA into the binding pocket of BSA as revealed in Figure S5(b) and moreover, the surface map calculation of RUPA (Figure S6) showing a total green color confirming its hydrophobic character. The primary role of these hydrophobic forces is to energetically sustain RUPA at the BSA interface. 4. Conclusions In summary, fluorescence spectroscopy, ultraviolet-visible spectroscopy, FTIR, and docking approaches have all been utilised to investigate the biomolecular binding behavior of the RUPA-BSA interaction. According to the ultraviolet-visible spectra and the fluorescence study, BSA and RUPA can bind together to generate a ground-state complex through a static process. The determined order of the binding constant for the RUPA-BSA system ranges from 10 3 to 10 4 L mol - 1 , indicating poor to moderate affinity for binding. FTIR data obtained for the RUPA-BSA complex confirm that secondary protein structure alters during the interaction. According to the positive values of the thermodynamic characteristics (∆H° and ∆S°), hydrophobic forces stabilized the RUPA-BSA complex system. As well, the negative ΔG values in the temperature range under study suggest that the binding process occurs spontaneously. The docking analysis further corroborated the findings of the site probing experiment that site I is the binding site for RUPA at BSA, in the vicinity of tryptophan residues and primarily by hydrophobic interactions. The sub-domains IIA of site 1 were analyzed after docking with the best conformer obtained of RUPA, and the least possible BSA-RUPA complex energies were obtained, confirming the results of the site marker technique and synchronous fluorescence measurements. Declarations Author Contribution H.A.S wrote the main manuscript text, performed the experimental work, and data analysis.SH.M.E performed the Molecular Docking analysis, contributed to writing parts of the manuscript, and participated in manuscript revision.H.E and F.B designed the study, provided resources, reviewed the results of the manuscript. All authors have read and approved the final version of the manuscript. Acknowledgement For the gracious donation of the spectrofluorometer utilized in this study to one of the authors (FB), the authors express their appreciation to the Alexander Von Humboldt Foundation in Bonn, Germany. They also express their sincere thanks to the Department of Pharmaceutical Organic Chemistry at Mansoura University's Faculty, CCMML (Computational Chemistry and Molecular Modeling Lab), for their assistance with the molecular docking simulations. Conflict of interest declaration This article's authors declared that they had no competing interests when it was published. Funding declaration This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Data availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Di L. 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Dual quenching nature of bovine serum albumin and dopamine complex revealed using multi-spectral and docking studies. J Photochem Photobiol Chem. 2024. 451:115542.doi.org/10.1016/j.jphotochem.2024.115542 . Klotz IM, Hunston DL. Protein interactions with small molecules. Relationships between stoichiometric binding constants, site binding constants, and empirical binding parameters. J Biol Chem. 1975;250(8):3001–9. doi.org/10.1016/S0021-9258(19)41586-X . Ross PD, Subramanian S. Thermodynamics of protein association reactions: forces contributing to stability. Biochemistry. 1981;20(11):3096–102. 10.1021/bi00514a017 . Qi X, Xu D, Zhu J, Wang S, Peng J, Wei, Gao. Studying the interaction mechanism between bovine serum albumin and lutein dipalmitate: Multi-spectroscopic and molecular docking techniques. Food Hydrocolloids. 2021;113:106513. doi.org/10.1016/j.foodhyd.2020.106513 . Koenig JK, Tabb DL. Analytical Application of FT-IR to Molecular and Biological Systems. D. Reidel; Boston 1980. 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Elucidation of binding mechanism of dibutyl phthalate on bovine serum albumin by spectroscopic analysis and molecular docking method. Spectrochim Acta Mol Biomol Spectrosc. 2020. 230:118044.doi.org/10.1016/j.saa.2020.118044 . Bertozo LC, Tadeu HC, Sebastian A, Zieleniak MM, Samsonov SA, Ximenes VF. Role for Carboxylic Acid Moiety in NSAIDs: Favoring the Binding at Site II of Bovine Serum Albumin. Mol Pharm. 2024;21(5):2501–11. 10.1021/acs.molpharmaceut.4c00044 . Boroujeni ZA, Jahani S, Motlagh K, Kagan K, Aramesh N. Experimental and theoretical investigations of Dy (III) complex with 2,2′-bipyridine ligand: DNA and BSA interactions and antimicrobial activity study. J Biomol Struct Dyn. 2019;38(2):1–30. 10.1080/07391102.2019.1689170 . Witkowska D, Słowik J, Chilicka K. Heavy Metals and Human Health: Possible Exposure Pathways and the Competition for Protein Binding Sites. Molecules. 2021;26(19):6060. .doi.org/10.3390/molecules26196060 . Zhang Y, Zheng J. Bioinformatics of Metalloproteins and Metalloproteomes. Molecules. 2020;25(15):3366. 10.3390/molecules25153366 . Rodzik A, Pomastowski P, Sagandykova GN, Buszewski B. Interactions of Whey Proteins with Metal Ions. Int J Mol Sci. 2020;21(6):2156. 10.3390/ijms21062156 . Sugio S, Kashima A, Mochizuki S, Noda M, Kobayashi K. Crystal structure of human serum albumin at 2.5 Å resolution. Protein Eng. 1999;12:439. Bujacz A. Structures of bovine, equine and leporine serum albumin. Acta Crystallogr D Biol Crystallogr. 2012;68:1278–89. Ansari A, Shamsuzzaman. Decoding the binding interaction of steroidal pyridines with bovine serum albumin using spectroscopic and molecular docking techniques. Steroids. 2023;192:109156doi. 10.1016/j.steroids.2022.109156 . Additional Declarations No competing interests reported. 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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-7321587","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":506297853,"identity":"4f96321e-acb3-43e7-8ce6-8b5fe09a7119","order_by":0,"name":"Heba Abo Shamiya","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYBACAwYGNiAlAUJARgWQzczcQIqWMyAtjERpYYBoYWwDsQhoMWdvf/bgZ45FYv/s5mMPfs6rjeZvB2r5UbENpxbLnjPmhr3bJBJn3DmWDmQcz51xmLGBsefMbdwOu5HDJsEL1NJwI8cMyDiW2wDUwszYhk9L+jPJv0At82/kf5P8O+dY7nzCWhLMpEG2bABaJ83bUJO7gZAWoF/MpGW3SRhvvJFmbixz7EDuRqCWg/j8Agoxybfb6mTn3Uh+9vBNTV3uvPOHDz74UYFbCww4NkDow2DyAEH1QGAPpeuIUTwKRsEoGAUjDAAA4dRf96nVPrkAAAAASUVORK5CYII=","orcid":"","institution":"Mansoura University","correspondingAuthor":true,"prefix":"","firstName":"Heba","middleName":"Abo","lastName":"Shamiya","suffix":""},{"id":506297854,"identity":"892141f1-e4c3-4b88-8d9f-8c617e549583","order_by":1,"name":"Heba Elmansi","email":"","orcid":"","institution":"Mansoura University","correspondingAuthor":false,"prefix":"","firstName":"Heba","middleName":"","lastName":"Elmansi","suffix":""},{"id":506297856,"identity":"f9c9a7eb-a062-4b5e-a837-dbab1451fd0b","order_by":2,"name":"Shahenda M. El-Messery","email":"","orcid":"","institution":"Mansoura University","correspondingAuthor":false,"prefix":"","firstName":"Shahenda","middleName":"M.","lastName":"El-Messery","suffix":""},{"id":506297857,"identity":"e51aeb34-b3e4-418b-821d-0af095e0c258","order_by":3,"name":"Fathalla Belal","email":"","orcid":"","institution":"Mansoura University","correspondingAuthor":false,"prefix":"","firstName":"Fathalla","middleName":"","lastName":"Belal","suffix":""}],"badges":[],"createdAt":"2025-08-07 19:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7321587/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7321587/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13065-026-01769-2","type":"published","date":"2026-03-28T16:09:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90388316,"identity":"72264ada-bd0c-47e8-9b2b-d2a135bec26a","added_by":"auto","created_at":"2025-09-02 07:58:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":19464,"visible":true,"origin":"","legend":"\u003cp\u003eStructure of rupatadine.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7321587/v1/c506d6dff9feff50172bc834.png"},{"id":90388713,"identity":"9384a46c-b3c2-4515-be92-fe01444d43b0","added_by":"auto","created_at":"2025-09-02 08:06:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":89242,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence spectra of BSA (2μM) with growing amounts of RUPA from (0 to 100μM) at λ\u003csub\u003eex\u003c/sub\u003e =279 nm and λ\u003csub\u003eem\u003c/sub\u003e at 303 K.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7321587/v1/f605f9e0d7270de4e5eb1d77.png"},{"id":90388317,"identity":"29a0dac1-2af8-4950-8901-6167c064f476","added_by":"auto","created_at":"2025-09-02 07:58:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":34377,"visible":true,"origin":"","legend":"\u003cp\u003eStern-Volmer plot for determination of quenching rate constant of RUPA at various temperatures.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7321587/v1/e87610c7d52f25ed814637d2.png"},{"id":90388714,"identity":"acf6675c-7ea8-4403-9422-319b76d05406","added_by":"auto","created_at":"2025-09-02 08:06:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":24133,"visible":true,"origin":"","legend":"\u003cp\u003eThe van`t Hoff plot for BSA-RUPA binding.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7321587/v1/1222b80961d4869ce3b74f7c.png"},{"id":90388320,"identity":"55e351b4-72f0-49e8-bdd7-ebe5fa6754f4","added_by":"auto","created_at":"2025-09-02 07:58:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":92779,"visible":true,"origin":"","legend":"\u003cp\u003eUV absorption spectra of BSA (2µM) with different concentrations of RUPA (0-100 µM) at pH 7.4 at 303 K.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7321587/v1/931183cdb42e4d0717d38db2.png"},{"id":105755900,"identity":"dfe160eb-88de-41c4-81f1-666d8eb9a96e","added_by":"auto","created_at":"2026-03-30 16:32:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1368295,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7321587/v1/5c8d93e2-19e8-4081-a25b-4de3d9f859b5.pdf"},{"id":90388715,"identity":"6e0a54de-2fdc-4e9c-859f-ae661bc05f14","added_by":"auto","created_at":"2025-09-02 08:06:46","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2343999,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7321587/v1/136b9e6d73a6460d56cce1fc.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring the binding interaction of rupatadine with bovine serum albumin using multi-spectroscopic and molecular modeling approaches","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eInvestigation of drug-protein binding interactions helps in the discovery of protein binding sites for targeted therapies, and its evaluation has been essential for the development of innovative drugs as well as the advancement of knowledge regarding drug action mechanisms and metabolic processes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The characteristics of the plasma protein albumin, such as its high binding capabilities for both hydrophobic and hydrophilic medicines, comparatively long half-life, ability to specifically target inflammatory sites, and essentially virtually low toxicity and immunogenicity, have drawn particular attention to it [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSerum albumin is the predominant circulating protein found in blood plasma, contributing good biocompatibility, biodegradability, and safety for its therapeutic use [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Its chemical structure and conformation enable interaction with a variety of medicines, which may shield them from metabolism and excretion in vivo and enhance their pharmacokinetic characteristics [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBecause of the high solubility, low cost, and structural similarity between bovine serum albumin and human serum albumin (76%), it has been used extensively to investigate interactions between ligands and serum albumin. Three homologous domains (I, II, and III) and two subdomains (A and B) make up the single-chain polypeptide that makes up BSA [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIt is crucial to delve deeper into the characteristics of the drug-protein interactions through diverse approaches using BSA as a protein model to study such interactions in order to minimize side effects and maximize the effectiveness of the medicines. El Gammal et al. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] used such approaches to study similar binding interactions in vitro under the simulated physiological circumstances (pH 7.4) utilizing the quenching fluorescence technique, and the parameters of thermodynamics were applied in order to determine the binding constants at three temperatures.\u003c/p\u003e\u003cp\u003eAs well as synchronous fluorescence, Fourier transform infrared spectroscopy (FTIR), UV-visible spectroscopy, the site marker technique, and MD have been applied for exploring the site of reaction and the protein structure's alterations upon binding with RUPA [\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHere, we studied how RUPA binds to BSA in order to elucidate the interaction mechanism of the binding utilizing several fluorescence spectroscopy techniques, Fourier transform infrared spectroscopy, and UV-visible spectroscopy. Furthermore, a detailed MD analysis was used to determine the most preferred binding site within BSA.\u003c/p\u003e\u003cp\u003eRupatadine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) chemically is 13-chloro-2-[1-[(5-methylpyridin-3-yl) methyl] piperidin-4-ylidene]-4-azatricyclo [9.4.0.03,8] pentadeca-1(11),3(8),4,6,12,14-hexaene [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Rupatadine is a second-generation antihistamine and antagonist of platelet-activating factor utilized for allergy treatment. It was discovered, manufactured, and commercialized as Rupafin 10mg and various other brand names in 2003. It is about 99% plasma protein bound, even though it is very well distributed to other tissues [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDeuster et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] found in 2021 that RUPA targets the platelet-activating factor receptor to prevent ovarian cancer cells from proliferating and migrating in vitro. According to these results, RUPA could potentially have anticancer properties.\u003c/p\u003e\u003cp\u003eAs far as we are aware, we have evaluated the behavior of RUPA-BSA binding interaction for the first time. This evaluation assists in improving its pharmacological and clinical efficacy, ensuring effective therapeutic drug levels, and preventing any potential unwanted adverse events.\u003c/p\u003e"},{"header":"2. Experimental procedure","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Chemicals\u003c/h2\u003e\u003cp\u003eBovine serum albumin (standard grade, pH 7, Origin USA) was purchased from Cegrogen Biotech GmbH (Germany). The molar concentration of BSA was calculated using the assumption that its molecular weight was 66,500 Da. Rupatadine fumarate was kindly supplied by ATCO PHARMA for Pharmaceutical Industries (Cairo, Egypt). Diazepam (99.2% purity) was obtained from EIPICO (10th of Ramadan City, Egypt). Phenylbutazone (99.7% purity) was obtained from GLOBAL NAPI PHARMACEUTICALS (6th October City, Egypt). Tris hydrochloride (Tris\u0026ndash;HCl) batch number 337012022 (purity\u0026thinsp;\u0026gt;\u0026thinsp;99%) was purchased from Chemajet for Chemical and Pharmaceutical Industries, Alexandria, Egypt.\u003c/p\u003e\u003cp\u003eThe supplier of HPLC grade methanol was Sigma Aldrich (St. Louis, MO, USA). Double-distilled water was utilized. Analytical reagent-grade chemicals were all used.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Instrumentation\u003c/h2\u003e\u003cp\u003eAgilent Technologies' Cary Eclipse fluorescence spectrophotometer (Santa Clara, California, USA) was utilized. The quartz cuvette utilized was 1 cm. The slit width was 5 nm, and the smoothing factor was 19. The wavelengths were 280 and 340 nm, as excitation and emission, respectively. The voltage was adjusted to 600 V. Shimadzu UV-1601, a UV-visible double-beam spectrophotometer (Kyoto, Japan), was employed at a high scan speed. Bruker Tensor 27 FTIR spectrometer was used to scan the FTIR spectra under strictly constant conditions in the region of 400\u0026ndash;5000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. All pH readings were taken using a Jenway 3510 pH meter (Jenway, Staffordshire, UK).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Software\u003c/h2\u003e\u003cp\u003eThe software \"Molecular Operating Environment (MOE) 2024.06 was utilized for both the docking of RUPA to examine how it binds and interacts with BSA and the surface mapping study of RUPA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Preparation of solutions\u003c/h2\u003e\u003cp\u003eA stock solution of 200 \u0026micro;M bovine serum albumin was prepared using bidistilled water, and rupatadine (1000 \u0026micro;M) was freshly prepared using bidistilled water. The buffer solution (pH 7.4) of Tris-hydrochloride (20 mM) was freshly made using bidistilled water. Phenylbutazone and diazepam stock solutions (10 mM) were prepared in methanol. To prepare the working solutions, all stock solutions were diluted beforehand and stored at 277 K.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Spectroscopic measurements\u003c/h2\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.5.1 Fluorescence measurements\u003c/h2\u003e\u003cp\u003eThe fluorescence quenching of BSA by RUPA was examined at three different temperatures: 303, 310, and 318 K, along wavelengths (300/500 nm) following excitation at 279 nm. The concentration of BSA remained constant at 2 \u0026micro;M, whereas the concentration of RUPA was increased from 0 to 100 \u0026micro;M.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.5.2 Synchronous fluorescence technique\u003c/h2\u003e\u003cp\u003eWith increasing concentrations of RUPA (0\u0026ndash;100 \u0026micro;M), by adjusting the Δλ at 60 nm for tryptophan (Trp) and Δλ at 15 nm for tyrosine (Tyr) residues at 303 K, synchronous fluorescence spectra of BSA were scanned in the 200/350 nm region.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.5.3 FT-IR spectroscopy\u003c/h2\u003e\u003cp\u003eThe FTIR spectra of the BSA solution (2 \u0026micro;M), both without and with RUPA (40 \u0026micro;M) in Tris-HCl buffer (20 mM) at pH 7.4, were scanned over the 400\u0026ndash;5000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e region.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.5.4 UV-visible spectroscopic measurements\u003c/h2\u003e\u003cp\u003eThe ultraviolet absorption spectra of the BSA solution (2 \u0026micro;M) with different amounts of RUPA (0-100 \u0026micro;M) in Tris-HCl buffer (20 mM) at pH 7.4 were measured over the 250\u0026ndash;330 nm region in contrast to a reference solution that included all relevant system components except for BSA. The experiments were carried out at 303 K.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Competitive binding analysis using site probes\u003c/h2\u003e\u003cp\u003ePhenylbutazone and diazepam are the site probes for sites Ⅰ and Ⅱ, respectively. The BSA and site probe concentrations in this experiment were maintained at 2 \u0026micro;M and 10 \u0026micro;M, respectively. Rupatadine concentrations ranged from 10 to 100 \u0026micro;M. After excitation at 279 nm, fluorescence intensity measurements were made at 341 nm. At 303 K, measurements were made. The binding constants (K\u003csub\u003eb\u003c/sub\u003e) of RUPA to diazepam-BSA, phenylbutazone-BSA, and BSA alone were compared.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Influence of metal ions on the RUPA-BSA binding interaction\u003c/h2\u003e\u003cp\u003eBovine serum albumin and ion metal solutions were fixed at 2 \u0026micro;M (pH 7.4), and the concentration of RUPA was varied from 10 to 100 \u0026micro;M. Fluorescence intensity measurements were taken at 341 nm following excitation at 279 nm at 303 K. Comparisons were made between the equilibrium constants for RUPA binding (K\u003csub\u003eb\u003c/sub\u003e) to BSA with each metal ion present and the equilibrium constant for RUPA binding (K\u003csub\u003eb\u003c/sub\u003e) to BSA alone.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Molecular docking and Surface Mapping\u003c/h2\u003e\u003cp\u003eThe binding behavior of BSA and RUPA was investigated using the Molecular Operating Environment (MOE) 2024.06. The structure of RUPA was initially drawn in the MOE. A conformational search was performed to obtain the most stable conformer using the systematic search method. The crystalline structure of bovine serum albumin was acquired from the Protein Data Bank (PDB) using the PDB code number 4f5s (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.rcsb.org\u003c/span\u003e\u003cspan address=\"http://www.rcsb.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The docking procedure has been carried out between the binding pocket and the conformers with the lowest energy, \u0026lsquo;global minima\u0026rsquo;. Following preparation, both ligands and protein receptors were protonated, and the force field MMFF94X's default parameters were used to reduce energy.\u003c/p\u003e\u003cp\u003eThe parameters of MD utilized in the experiment have been employed with Triangle Matcher as a default. Along with 30 conformation generations to match the binding groove, rescoring functions 1 and 2 were set to London dG and GBVI/WSA dG, respectively. For additional investigation and assessment of the relationship between RUPA and BSA, the mdb output file was produced.\u003c/p\u003e\u003cp\u003eSurface mapping of both the binding pocket and ligand was produced in a color-coded map where pink denotes hydrogen bond, blue denotes mild polar, and green denotes hydrophobic region [\u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003e\u003cstrong\u003e3.1\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eExamination of quenching mechanism\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe presence of aromatic amino acid residues in the structure of BSA, such as tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) is what gives it its inherent fluorescence. Investigating protein conformational changes upon interactions with different drugs and providing precise information on ligand-protein interactions are two common uses for fluorescence spectroscopy [21]. In this experiment, we measured the decrease of the BSA intrinsic fluorescence due to quenching by RUPA (figure 2). Fluorescence quenching occurs through several mechanisms, including dynamic quenching, which arises from collisions between proteins and quenchers; static quenching, which results from ground state complex formation between proteins and quenchers; and the combined mechanism that combines static and dynamic quenching, resulting in both complex formation and collision with the same quencher.\u003c/p\u003e\n\u003cp\u003eThe process of fluorescence quenching was determined using the Stern-Volmer equation (equation 1):\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eF\u003csub\u003eo\u003c/sub\u003e/F = 1+Kq \u0026tau;o [Q] = 1+Ksv [Q] \u0026nbsp; \u0026nbsp;(1)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eF and F\u003csub\u003e0\u003c/sub\u003e represent the emission intensity of BSA with and without RUPA, respectively. [Q] stands for the concentration of RUPA. K\u003csub\u003eq\u003c/sub\u003e, K\u003csub\u003eSV\u003c/sub\u003e, and \u0026tau;\u003csub\u003e0\u0026nbsp;\u003c/sub\u003edenote the quenching rate constant, the Stern\u0026ndash;Volmer quenching constant, and the fluorescence lifespan without RUPA, which is equivalent to 10\u003csup\u003e\u0026minus;8\u003c/sup\u003e seconds, respectively.\u003c/p\u003e\n\u003cp\u003eGenerally,\u0026nbsp;in the static quenching process, elevating the temperature causes the quenching constant to decrease, but in the dynamic mechanism, it induces an increase [22]. The K\u003csub\u003eSV\u003c/sub\u003e and K\u003csub\u003eq\u003c/sub\u003e values were determined by the graph of F\u003csub\u003e0\u003c/sub\u003e / F against [Q] (figure 3). Table (1) provides a summary of the K\u003csub\u003eSV\u003c/sub\u003e and K\u003csub\u003eq\u003c/sub\u003e values acquired at various temperatures. The data indicated that the K\u003csub\u003eSV\u003c/sub\u003e value decreased with rising temperature in the presence of RUPA, and the k\u003csub\u003eq\u003c/sub\u003e values exceeded the maximum K\u003csub\u003eq\u003c/sub\u003e for dynamic (2\u0026times;10\u003csup\u003e10\u003c/sup\u003e L mol\u003csup\u003e-1\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e); hence, the quenching mechanism is a static process.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable. 1:\u0026nbsp;\u003c/strong\u003eRUPA-BSA interaction parameters at various temperatures.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"512\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eT(K)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eK\u003csub\u003esv\u0026nbsp;\u003c/sub\u003e(L.mol\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eK\u003csub\u003eq\u0026nbsp;\u003c/sub\u003e(L.mol\u003csup\u003e-1\u003c/sup\u003e.s\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e303\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e1.77\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e1.77\u0026times;10\u003csup\u003e12\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e0.9972\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e310\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e1.52\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e1.52\u0026times;10\u003csup\u003e12\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e0.9992\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e318\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003e1.21\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e1.21\u0026times;10\u003csup\u003e12\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e0.9992\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e3.2\u0026nbsp;Determinations of the binding constant and stoichiometric analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStoichiometric analysis is used to define the various equilibria for a macromolecule\u0026apos;s binding of a ligand by binding sites (n) [23]. The number of binding sites (n) and the binding constant (K\u003csub\u003eb\u003c/sub\u003e) were calculated using the modified Stern\u0026ndash;Volmer equation (equation 2).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLog F\u003csub\u003eo\u003c/sub\u003e\u0026minus;F / F = log K\u003csub\u003eb\u003c/sub\u003e + n log [Q] \u0026nbsp; \u0026nbsp; (2)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThus, by graphing log [(F\u003csub\u003e0\u003c/sub\u003e \u0026ndash; F) / F] against log [Q], as illustrated in figure S1, we can obtain the number of binding sites (n) and the binding constant (K\u003csub\u003eb\u003c/sub\u003e) for the interaction of RUPA and BSA. The values of log K\u003csub\u003eb\u003c/sub\u003e, and (n) as seen in Table 2, are obtained from the intercept and slope, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable. 2:\u0026nbsp;\u003c/strong\u003eRUPA-BSA complex binding characteristics at different temperatures.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" width=\"746\" height=\"192\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e*SEM: Standard error of mean\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt was observed that upon rising temperatures, the binding constant (K\u003csub\u003eb\u003c/sub\u003e) increased; this can be interpreted as the quenching being due to hydrophobic interactions rather than ionic interactions, as will be revealed in the next study. A weak to moderate binding affinity was indicated by the order of the binding constant (K\u003csub\u003eb\u003c/sub\u003e), which ranged from 10\u003csup\u003e3\u003c/sup\u003e to 10\u003csup\u003e4\u003c/sup\u003e L mol\u003csup\u003e-1\u003c/sup\u003e. The stoichiometry for RUPA-BSA binding is 1:1, which was indicated by the value of (n), which was close to 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Thermodynamic characteristics and nature of binding forces\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe thermodynamic characteristics of the binding reaction serve as the primary evidence for the binding force. Hydrogen bonds, hydrophobic forces, electrostatic interactions, van der Waals interactions, and other binding forces could all be present between small molecules and biomacromolecules [24]. The temperature dependence of the binding constant was thus measured for this purpose at three distinct temperatures: 303, 310, and 318 K (Figure 4). The acting forces in the binding process were described by the thermodynamic characteristics of the BSA-RUPA system, such as enthalpy change (\u0026Delta;H), entropy change (\u0026Delta;S), and the Gibbs free energy change (\u0026Delta;G), as revealed in Table 3. The values of \u0026Delta;H and \u0026Delta;S derived from the van\u0026apos;t Hoff equation (equation 3) are displayed in Figure 4.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cspan\u003eLn K\u003csub\u003eb\u003c/sub\u003e = \u0026minus;\u0026Delta;H/RT + \u0026Delta;S/R \u0026nbsp; \u0026nbsp;(3)\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGibbs\u0026ndash;Helmholtz equation provided below was used to assess the free energy change (\u0026Delta;G) value (equation \u0026nbsp;4)\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026Delta;G = \u0026Delta;H \u0026ndash; T\u0026Delta;S \u0026nbsp; \u0026nbsp; (4)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWithin the examined temperature range, the values of the enthalpy change (\u0026Delta;H) and the entropy change (∆S) were 32.84 kJ mol\u0026minus;1 and 0.18 kJ mol\u0026minus;1, respectively. Thus, hydrophobic interactions appear to represent the main binding forces in the interaction between RUPA and BSA, as indicated by the positive values of ∆H and ∆S. Furthermore, the positive value of \u0026Delta;H signified that the creation of the RUPA-BSA complex was an endothermic reaction. The findings indicated that the binding interaction between BSA and RUPA was spontaneous because the \u0026Delta;G values were negative.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable. 3:\u0026nbsp;\u003c/strong\u003eThermodynamic parameters of BSA-RUPA interaction at pH 7.4.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"673\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eT (K)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e∆H\u003csup\u003eo\u003c/sup\u003e(KJ/mol)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e∆G\u003csup\u003eo\u003c/sup\u003e(KJ/mol)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSEM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e∆S\u003csup\u003eo\u003c/sup\u003e(J mol\u003csup\u003e-1\u003c/sup\u003e K\u003csup\u003e-1\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e303\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e32.84\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e-55.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e181.91\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.9995\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e310\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e-56.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e318\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e-57.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e*SEM: Standard error of mean\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4\u0026nbsp; Spectroscopic study of RUPA-BSA interaction\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.1 \u0026nbsp; \u0026nbsp;Synchronous fluorescence measurements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing synchronous fluorescence, the influence of has been thoroughly investigated [25]. The distinctive details regarding the protein\u0026apos;s Tyr and Trp residues can be\u0026nbsp;gained by fixing the scanning interval (∆\u0026lambda;) between the excitation and emission wavelengths at 15 and 60 nm, respectively. Figures S2(A) and S2(B) show the findings of the corresponding BSA fluorescence ligand binding on the polarity of the microenvironment surrounding fluorescent amino acid residues of the protein spectra as RUPA concentrations rise. Adding RUPA and monitoring at ∆\u0026lambda;=60 resulted in a minor red shift of ∆\u0026lambda;\u003csub\u003emax\u003c/sub\u003e of around 2 nm, while no shift was seen at ∆\u0026lambda; = 15. This indicates that RUPA binds to BSA at Trp residues, as confirmed by MD experiments that revealed drug binding near Trp residues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.2 \u0026nbsp; \u0026nbsp;FT-IR spectroscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFTIR measurements have been performed to further explore the BSA conformational change that is caused by the interaction with RUPA. The spectrum in Figure S3 (a) represents free BSA, and Figure S3 (b) is the spectrum of the RUPA-BSA complex at pH 7.4 at 303 K. The C=O stretch is responsible for the amide I band, which is located between 1600 and 1700 cm\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003e[26]. It is linked to the secondary structure of BSA and is more vulnerable to modifications in the secondary structure of proteins than amide II, which is located between 1500 and 1600 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e. Following the RUPA reaction, the amide I peak\u0026apos;s location shifted from 1646 to 1642 cm\u003csup\u003e-1\u003c/sup\u003e, indicating that secondary protein structure changes during the RUPA-BSA interaction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.3 \u0026nbsp; \u0026nbsp;UV-visible spectroscopic measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUltraviolet-visible spectroscopy is an\u0026nbsp;effective technique for identifying the conformational alterations of proteins mediated by drug molecules [27]. Alterations in the ultraviolet-visible spectra of the protein, whether in intensity or \u0026lambda;\u003csub\u003emax\u003c/sub\u003e, are primarily seen in the static process because a new ground-state complex has been established; conversely, dynamic quenching typically does not correlate with modifications in the protein\u0026apos;s absorbance spectrum [28,29]. As shown in Figure 5, the UV absorption spectrum of BSA was captured both without and with RUPA added; it can be found that there were two absorption bands for all solutions of BSA. The band of absorption close to 220 nm associated with the \u0026alpha;-helix indicates the structural shape of bovine serum albumin. A modest absorption band about 280 nm is attributed to the \u0026pi;\u0026ndash;\u0026pi;* transitions of aromatic amino acids, including tryptophan, tyrosine, and phenylalanine residues. A notable escalation in intensity is observed with rising RUPA concentrations, accompanied by a red shift at the maximum wavelength near 220 nm. This indicates the involvement of static quenching and corroborates the earlier findings of the fluorescence quenching investigation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 \u0026nbsp;Competitive binding analysis using site probes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo specific site probes, phenylbutazone for site I and diazepam for site II, were used as site probes in site marker competitive binding experiments to identify the preferential binding site of RUPA on BSA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor small ligands, BSA has two primary binding sites: site I, which is located in subdomain IIA\u0026apos;s hydrophobic pocket, and site II, which is located in subdomain IIIA\u0026apos;s hydrophobic cavity. Site I is comparatively bigger and mostly bound by hydrophobic interactions with neutral, bulky, and heterocyclic molecules. However, site II is smaller, and the interaction usually happens via a mix of electrostatic, hydrogen bonding, and hydrophobic forces\u0026nbsp;[30-32]. The K\u003csub\u003eb\u003c/sub\u003e value for RUPA binding with BSA in the presence of phenylbutazone was found to be lower than that in the absence of the site marker, as shown in Table 4. However, there was no discernible variation in the K\u003csub\u003eb\u003c/sub\u003e value when diazepam was present. According to these findings, phenylbutazone and RUPA vie for the same binding site at BSA, which is site I.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable. 4: Binding constant of RUPA in absence and presence of site markers at 303 K.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"607\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 230px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSite marker\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLog K\u003csub\u003eb\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSEM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 230px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlank\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e3.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.9996\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.045\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 230px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhenylbutazone\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e2.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.9995\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.031\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 230px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDiazepam\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e3.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.9997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.086\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e*SEM: Standard error of mean\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 \u0026nbsp;Influence of metal ions on RUPA-BSA interaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMetal ions interact with proteins, modifying their biological function and structure via coordination bond formation; therefore, they affect the interaction of the protein with the drugs and their binding behavior [33, 34]. Because proteins are used in many different industries and serve a variety of purposes in the human body, it is crucial to assess these alterations [35, 36]. For that, the influence of some metal ions (Na\u003csup\u003e+1\u003c/sup\u003e, K\u003csup\u003e+1\u003c/sup\u003e, Zn\u003csup\u003e+2\u003c/sup\u003e, Ca\u003csup\u003e+2\u003c/sup\u003e, Mg\u003csup\u003e+2\u003c/sup\u003e, Fe\u003csup\u003e+3\u003c/sup\u003e) on the BSA-RUPA system was explored. The determined K\u003csub\u003eb\u003c/sub\u003e values of the RUPA-BSA complex in the presence of certain ions, such as Ca\u003csup\u003e+2\u003c/sup\u003e, K\u003csup\u003e+1\u003c/sup\u003e, and Fe\u003csup\u003e+3\u003c/sup\u003e, were greater than those without them, as shown in Table 5. This may be explained by the metal ion-RUPA-BSA system formation.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eOther ions exhibited lower K\u003csub\u003eb\u003c/sub\u003e, which\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003emay be as a result of competitive binding with BSA.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eHowever, the change of K\u003csub\u003eb\u003c/sub\u003e in general could be attributed to changes in BSA confirmation by its interaction with metal ions at metal binding sites. More research is required in the future to try to understand the type, strength, and involvement of a protein\u0026apos;s functional groups in metal-protein interactions.\u003c/p\u003e\n\u003cp\u003eTable. 5: The K\u003csub\u003eb\u003c/sub\u003e values of RUPA-BSA system with and without metal ions.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eSystem\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003eLog K\u003csub\u003eb\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003eK\u003csub\u003eb\u0026nbsp;\u003c/sub\u003e(M\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eBSA+RUPA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e3.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003e6.92\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.9996\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e0.045\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eBSA+RUPA+Na\u003csup\u003e+1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e3.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003e4.90\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.9996\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e0.071\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eBSA+RUPA+K\u003csup\u003e+1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e4.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003e7.80\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.9991\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e0.019\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eBSA+RUPA+Mg\u003csup\u003e+2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e4.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003e2.00\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.9994\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e0.018\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eBSA+RUPA+Ca\u003csup\u003e+2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e4.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003e7.10\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.9992\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e0.071\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eBSA+RUPA+Zn\u003csup\u003e+2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e4.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003e1.23\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.9992\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e0.099\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eBSA+RUPA+Fe\u003csup\u003e+3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e3.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 177px;\"\u003e\n \u003cp\u003e7.76\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.9991\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e0.049\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e*SEM: Standard error of mean\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7 \u0026nbsp;Molecular docking \u0026nbsp;analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo provide a comprehensive understanding of the various binding patterns, molecular modelling is mandatory, so a docking study of the RUPA into the BSA protein was conducted. The most popular model serum albumin protein for researching these kinds of protein-ligand interactions is BSA [37]. To conduct the analysis, the protein structure (PDB code 4f5s) was selected from the Protein Data Bank using MOE 2024.06\u0026nbsp;[38].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConformational Search:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConformational analysis of RUPA has been obtained for accurate further modeling studies, where its best and least energy conformer was produced by conformational search procedures that explore conformational space in torsional space, applying the multi-conformer approach. The results have been depicted in Figure S4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular Docking and Surface Mapping\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs illustrated in figure S5 (a,b), the following amino acid residues were discovered to surround RUPA: Try149, Ser286, Arg256, Leu237, Ala290, Arg194, Arg217, Glu291, Lys187, and Asp450. The residues of the interacting amino acids are nearly identical with a small variation and are located in a hydrophobic cavity at site I. Since no hydrogen bonding contacts were found, it can be assumed that RUPA\u0026apos;s interaction with Try149 and Arg217 is solely hydrophobic via arene cationic interaction. With a 6.41 kcal/mol calculated binding energy, which is in line with experimental results, it can be predicted that RUPA fits perfectly within site I, landing into the hydrophobic pocket of subdomain IIA\u0026apos;s close to tryptophan residues.\u003c/p\u003e\n\u003cp\u003eNumerous studies have demonstrated how hydrophobic interactions contribute to the stability of drug-serum albumin complexes based on [39], which was encouraging to conduct an in-depth surface mapping investigation.\u003c/p\u003e\n\u003cp\u003eFurther study about the relation between the hydrophobic character of RUPA and the existence of residues of hydrophobic amino acids at the BSA binding site has shown that this similarity may have helped to stabilize the RUPA-BSA system via hydrophobic forces; that was basically confirmed by both observing the 3D view of the overlay of RUPA into the binding pocket of BSA\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eas revealed in\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFigure S5(b)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eand moreover, the surface map calculation of RUPA (Figure S6) showing a total green color confirming its hydrophobic character. The primary role of these hydrophobic forces is to energetically sustain RUPA at the BSA interface.\u003c/p\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn summary, fluorescence spectroscopy, ultraviolet-visible spectroscopy, FTIR, and docking approaches have all been utilised to investigate the biomolecular binding behavior of the RUPA-BSA interaction. According to the ultraviolet-visible spectra and the fluorescence study, BSA and RUPA can bind together to generate a ground-state complex through a static process. The determined order of the binding constant for the RUPA-BSA system ranges from 10\u003csup\u003e3\u003c/sup\u003e to 10\u003csup\u003e4\u003c/sup\u003e L mol\u003csup\u003e- 1\u003c/sup\u003e, indicating poor to moderate affinity for binding. FTIR data obtained for the RUPA-BSA complex confirm that secondary protein structure alters during the interaction.\u0026nbsp;According to the positive values of the thermodynamic characteristics (∆H° and ∆S°), hydrophobic forces stabilized the RUPA-BSA complex system.\u0026nbsp;As well, the negative ΔG values in the temperature range under study suggest that the binding process occurs spontaneously. The docking analysis further corroborated the findings of the site probing experiment that site I is the binding site for RUPA at BSA,\u0026nbsp;in the vicinity of tryptophan residues and primarily by hydrophobic interactions. The sub-domains IIA of site 1 were analyzed after docking with the best conformer obtained of RUPA, and the least possible BSA-RUPA complex energies were obtained, confirming the\u0026nbsp;results of the site marker technique and synchronous fluorescence measurements.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eH.A.S wrote the main manuscript text, performed the experimental work, and data analysis.SH.M.E performed the Molecular Docking analysis, contributed to writing parts of the manuscript, and participated in manuscript revision.H.E and F.B designed the study, provided resources, reviewed the results of the manuscript. All authors have read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eFor the gracious donation of the spectrofluorometer utilized in this study to one of the authors (FB), the authors express their appreciation to the Alexander Von Humboldt Foundation in Bonn, Germany. They also express their sincere thanks to the Department of Pharmaceutical Organic Chemistry at Mansoura University's Faculty, CCMML (Computational Chemistry and Molecular Modeling Lab), for their assistance with the molecular docking simulations.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConflict of interest declaration \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article\u0026apos;s authors declared that they had no competing interests when it was published.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding declaration \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDi L. 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Steroids. 2023;192:109156doi. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.steroids.2022.109156\u003c/span\u003e\u003cspan address=\"10.1016/j.steroids.2022.109156\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"bmc-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccjo","sideBox":"Learn more about [BMC Chemistry](https://bmcchem.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ccjo/default.aspx","title":"BMC Chemistry","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Rupatadine, Fluorescence, Bovine serum albumin, Thermodynamic, Docking.","lastPublishedDoi":"10.21203/rs.3.rs-7321587/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7321587/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRupatadine (RUPA), a second-generation H\u003csub\u003e1\u003c/sub\u003e-receptor antagonist, is used to treat allergies with a further antagonistic action on platelet-activating factor. Here, RUPA and bovine serum albumin (BSA) binding interaction has been investigated via various approaches, including spectrofluorimetric techniques, thermodynamic studies, Fourier transform infrared spectroscopy (FTIR), ultraviolet, and molecular docking (MD). The spectrofluorimetric titration study was displayed at various temperatures, and the data revealed that the BSA native fluorescence is quenched by RUPA via a static process, which has been signified by UV absorption. The thermodynamic analysis revealed that the stoichiometry between RUPA and BSA is 1:1, and their binding affinity was weak to moderate. As revealed by the enthalpy change (ΔH) and entropy change (∆S) values of 32.84 kJ mol\u003csup\u003e−1\u003c/sup\u003e and 0.18 kJ mol\u003csup\u003e−1\u003c/sup\u003e, respectively, the hydrophobic forces are the main binding forces in the interaction between BSA and RUPA. The negative values of Gibbs free energy change (ΔG) indicate that the binding process between RUPA and BSA was spontaneous. Furthermore, results of the site marker technique and synchronous fluorescence measurements indicate that RUPA binding interaction occurs at site (I) on BSA in the vicinity of tryptophan residues, which was then confirmed by MD.\u003c/p\u003e","manuscriptTitle":"Exploring the binding interaction of rupatadine with bovine serum albumin using multi-spectroscopic and molecular modeling approaches","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-02 07:58:42","doi":"10.21203/rs.3.rs-7321587/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-19T12:32:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-15T16:27:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-15T06:52:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-03T17:31:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9451932620466945000925791271247734739","date":"2025-08-27T09:48:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"263803932255333095852373867738216969211","date":"2025-08-27T09:14:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"41230020153384094245919213195352764389","date":"2025-08-25T04:52:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-25T03:51:34+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-08T09:24:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-08T03:15:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-08T03:14:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Chemistry","date":"2025-08-07T19:30:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccjo","sideBox":"Learn more about [BMC Chemistry](https://bmcchem.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ccjo/default.aspx","title":"BMC Chemistry","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1cf00eb2-8858-4ce8-a1b3-c455f81676d6","owner":[],"postedDate":"September 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:26:10+00:00","versionOfRecord":{"articleIdentity":"rs-7321587","link":"https://doi.org/10.1186/s13065-026-01769-2","journal":{"identity":"bmc-chemistry","isVorOnly":false,"title":"BMC Chemistry"},"publishedOn":"2026-03-28 16:09:46","publishedOnDateReadable":"March 28th, 2026"},"versionCreatedAt":"2025-09-02 07:58:42","video":"","vorDoi":"10.1186/s13065-026-01769-2","vorDoiUrl":"https://doi.org/10.1186/s13065-026-01769-2","workflowStages":[]},"version":"v1","identity":"rs-7321587","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7321587","identity":"rs-7321587","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}