The examining on DNA binding activities of tetrakis-(4-tritylphenoxy)-phthalocyanine copper (II) phthalocyanine complex

The examining on DNA binding activities of tetrakis-(4-tritylphenoxy)-phthalocyanine copper (II) phthalocyanine complex | 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 The examining on DNA binding activities of tetrakis-(4-tritylphenoxy)-phthalocyanine copper (II) phthalocyanine complex Ali Arslantaş, Mehmet Salih Ağırtaş, Derya Güngördü Solğun This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4168319/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cu (II) phthalocyanine complex had been previously synthesized by reacting 4-(4-tritylphenoxy)phthalonitrile compound. The structure of obtained CuPc complex had been analyzed by absorption titration, infrared and NMR spectroscopies techniques. The DNA interacting property for the complex was analyzed in various concentration of CT-DNA utilizing elctronic absorption, emission spectroscopy, gel agarose electrophoresis and thermal melting procedures. In this report, the binding constant value for the complex was also estimated. Absorption and fluorescence spectroscopic processes proved that CuPc interacts by CT-DNA. At the the same time, the thermal melting and electrophoresis technics were practiced to analyze the intercating property of CuPc by DNA. The thermal melting and electrophoresis assays supported that CuPc combines with DNA via an intercalative interacting mechanism. The obtained findings demonstrated that the complex connects to DNA by an intercalative interacting mode. Therefore, CuPc complex may have potential cancer therapeutic agent. Copper (II) DNA interaction phthalocyanines absorption spectra Gel electrophoresis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Article Highlights • The syntheisi of Cu (II) phthalocyanine complex • Examining of DNA interaction activity Cu (II) phthalocyanine complex. • Identifying potential DNA binding property of the complex. 1 Introduction DNA molecule is one of the significant targets used in the treatment of many genetic diseases such as cancer. In recent years, it has been observed that there has been a significant increase in biological research due to the significant effects of transition metal-based compounds in cancer treatment [ 1 ]. The mechanism by which a drug binds to DNA provides very important preliminary information about the possibility of using the chemical compound as an anticancer drug in cancer treatment [ 2 ]. Within in this scope, the very broad spectral and electrochemical activities of transition metal-based compounds contribute significantly to the interaction activities of these compounds with DNA [ 3 , 4 ]. Under normal conditions, the presence of too much active oxygen radicals, which arise as a result of metabolic reactions, causes oxidative damage to biomolecules such as DNA, lipids and proteins. Consequently, it may increase the likelihood of some very critical diseases such as cancer, inflammation, cardiovascular and neurodegenerative diseases occurring very quickly. Depending on their chemical structural properties and the root of oxidative stress, metal-based compounds can serve as drugs [ 5 , 6 ]. Phthalocyanine compounds are highly functional molecules, and research on the derivatives of these compounds has been continuing for many years. Due to the thermal and chemical properties of phthalocyanines, they have applications in different fields such as sensors and photodynamic cancer therapy [ 7 ]. Additionally, due to the photosensitizing and antioxidant properties of phthalocyanine compounds, research is ongoing for photodynamic therapy in anticancer treatment [ 8 – 10 ]. Due to their ability to interact with DNA, phthalocyanine complexes have attracted great attention in the last decade for their application in cancer treatment approaches [ 11 – 17 ]. In studies published in the literature, different complexes of transition metal phthalocyanine compounds were synthesized to be used as chemotherapeutic active substances [ 18 ]. However, it is understood that copper phthalocyanine complexes still do not receive the expected importance in this research field. Encouraging the potential anticancer activities of phthalocyanines and their copper complexes, it reveals the importance of investigating the interactions of phthalocyanines and copper complexes with DNA. In the current study, the interaction activities of previously synthesized peripheral tetra-substituted Cu (II) phthalocyanine [ 19 ] with DNA were investigated using absorption spectroscopy, fluorescence, thermal melting point and gel electrophoresis techniques. 2 Experimental work 2.1. Chemicals and Techniques The structure and properties of the Cu (II) phthalocyanine complex containing tetrakis-(4-tritylphenoxy) group have been elucidated in a previous study [ 19 ]. All chemicals used as containing NaOH at pH 7.05 and stored in a cooler at 4 o C. In addition, the stock copper (II) phthalocyanine complex prepared by dissolving DMF, an organic solvent, for use in the study was kept at 25 o C. If needed, all solutions used in the study were prepared by diluting the desired samp consumables in this scientific research were obtained from Sigma-Aldrich company. DNA samples used as reagents in this study were prepared using Tris-HCl buffer solutionle volumes using Tris-HCl buffer. Instrumental devices such as UV/Vis, thermal melting point, emission, electrophoresis were used in this study to determine the interaction activities of the In addition to the above devices, Thermo Scientific Owl electrophoresis system was preferred in this study to perform gel electrophoresis tests. 2.2. Preparation of 4-(4-tritylphenoxy)phthalonitrile and Cu (II) phthalocyanine compound The 4-(4-tritylphenoxy) phthalonitrile compound was previously synthesized by our research group to form a phthalocyanine complex with copper (II), a transition metal [ 19 ]. The tetrakis-(4-tritylphenoxy)-phthalocyanine copper (II) reported in the existing literature has been isolated, synthesised and characterised corresponding to the earlier study of our research team [ 19 ]. 3 Results and discussion 3.1. Synthesis and analyzing Figure 1 shows the structure of CuPc phthalocyanine complex. The structural analysis of the Cu (II) phthalocyanine complex was done using a number of spectroscopic tools including IR, electronic absorption and NMR spectrometry [ 19 ]. The evidence gathered from IR, absorption spectra and NMR techniques supported the suggested structure of CuPc . Copper (II) phthalocyanine was dissolvable in solvents including CHCl 3 , toluene, CH 2 Cl 2 , DMSO, DMF and THF. For electronic absorption, (THF) ʎmax, nm (log Ɛ) for the complex CuPc were obtained as 674 (5.21), 608 (4.61), 346 (4.93), accordingly [ 19 ]. FT-IR spectrums were also acquired for Cu (II) phthalocyanine. IR spectrums (cm − 1 ) for CuPc were observed as 3053(C-H aromatic), 1598(C = C), 1490, 1447, 1442, 1230(Ar-O-Ar), 1085, 1050, 1033, 893, 825, 746, 700, 669, respectively [ 19 ]. All of this evidence was consistent with the proposed conformation of the complex. 3.2. DNA Binding Assays 3.2. 1. Electronic Absorption Titration Study for CuPc The binding of CuPc to the DNA has been explored by means of electronic absorption assays in order to comprehend their binding nature to the DNA. The samples of DNA had been made up in the buffer solution for this experiment. Electronic absorption spectra analysis of the stock solution of CT-DNA in a tris-HCl solution at pH 7.05 at 250 and 300 nm had been read and the data proved that the DNA sample did not possess protein. The quantity of DNA for every nucleotide phosphate group was assessed from the absorbance spectra at 260 nm, considering the value of the extinction coefficient [ 20 ]. The CT DNA specimens were preserved at 4 o C and used within a few days [ 20 , 21 ]. The stock specimen of CuPc complex was generated in DMF and diluted in the buffer solution. Electronic absorption titrations were done using a quartz sample holder and scanned in between the range of 250–900 nm at a pH of 7.05. Absorbance spectra titration tests were done for a fixed quantity of CuPc (20 µM) and variable quantities of DNA (0 to 15 µM) in increments of 5 µM. After each addition, the UV/Vis absorptions were monitored, and the results were recorded. The sample mixture of the complex and CT-DNA was run for 5 min at 25 o C and shifts in the absorbance spectra were observed. The UV/Vis absorption data were used to obtain the binding coefficient (K b ) for CuPc to DNA using the Wolfe-Schimer formula (1) [ 22 ]. $$\frac{\left[DNA\right]}{Ɛa-Ɛf}=\frac{\left[DNA\right]}{Ɛb-Ɛf}+\frac{1}{Kb\left(Ɛb-Ɛf\right)}$$ 1 The absorption titration of CT-DNA of CuPc is as presented in Fig. 2 . For CuPc complex, the intrinsic binding coefficient value was obtained, the Kb value for CuPc had been estimated to be 1.53 x 106 M − 1 by applying the Eq. ( 1 ) [ 22 ]. As the quantity of CT-DNA increases, CuPc complex produces hypochromism with a redshift band in the absorbance bands at around 674 and 346 nm as presented in Fig. 2 . The K b values of recognized intercalative anticancer medicine like doxorubicin and idrarubicin were calculated in published paper [ 23 , 24 ]. For this reason, by relating the K b value of CuPc to the tendency of the absorbance to alter when DNA is added, it can be assumed that CuPc reacts with DNA via an intercalative route. 3.2.2. Fluorescence Analysis of CuPc Complex for DNA Binding Emission spectrometry is a procedure frequently utilised to probe the bonds between the small molecular complex and DNA molecule. The advantages of fluorescence procedure over other approaches are its high precision, broad concentration spectrum and specificity [ 24 , 25 ]. The route of interaction of medicinal agents with DNA can be elucidated utilising emission spectrometry, and other procedures based on emission spectra can also offer further valuable insight. Emission experimentation yields detailed information on the localisation of agents and their attachment mechanisms to the DNA molecule [ 26 , 27 ]. The arrow marks the changes in intensity of the emission spectra with the increment of the quantity of CT-DNA. In moreover, to make clear the interaction process of CuPc complex with CT-DNA, the emission binding examinations were made to clarify the binding of medicinal drugs and DNA, by the fact that this method is a tender technique in the interaction examinations of medicinal drugs-DNA and it can yield further aid to the intercalating binding process of metal complexes. In absence of DNA, CuPc complex produces an emission spectrum as illustrated in Fig. 3 , with an emission peak at around 465 nm. In the presence of CT-DNA, the intensities of CuPc complex decline successively as the DNA is added. The decreasing intensities suggested that CuPc reacts with the DNA molecule by way of an intercalative binding pathway. These results apparently demonstrated that [tetrakis-(4-tritylphenoxy)-substituted Cu (II) phthalocyanine interacted with the DNA molecule by an intercalation pathway, consistent with the adsorption titration data. 3.2.3. Gel Electrophoresis Analysis of CuPc/DNA Interaction The interaction abilities of earlier synthesized CuPc to the DNA were assessed using gel electrophoresis by analyzing the influence of varying amounts of CuPc complex CT-DNA and its consequences obtained were illustrated in Fig. 4 . When comparison with the bands of standard DNA, the band intensities observed for the CuPc complex after interaction with the DNA were clearly reduced. The fall in the intensity of the band, recorded after the interaction of the phthalocyanine complex with the DNA, is linked to the breakdown of the double helix of the DNA molecule. The earlier reported research proposed that the damage to the DNA may have been caused by the splitting of the backbone due to a nucleophilic attack of the residues [ 27 ]. It has been suggested in the literature that the band intensities in agarose gel electrophoresis studies, which are visualized by the fluorescence emission spectra of ethidium bromide binding to DNA base pairs through the intercalative binding pathway, can be affected not only by the number of molecular bonds, but also by the length of the DNA molecule [ 28 ]. Consequently, overlapping of the metal complex during binding between the bases within the DNA helix or surface interaction at the reactive sites of the nucleophile on the DNA double helix may account for the decrease in electrophoretic band intensities of CT-DNA upon binding by the CuPc phthalocyanine complex. 3.2.4. Experiments on the Thermal Denaturation In this report, by analyzing the CT-DNA melting temperatures (T m ), the influence of the amount of adduct formation on the stabilisation of DNA supercoil has been studied. T m response of CT-DNA in presence of metal complexes can unveil their conformational structure variance with rising temperature, and assure insight into binding ability of tiny metal compounds to DNA molecule. Literature reports suggest that when metal compounds bind to the DNA double helix, molecular stability is promoted. In general, the T m of DNA rises when metal compounds react with the DNA molecule through an intercalative binding process, as the intercalative binding of metal compounds between the base pairs of DNA induces the stabilisation of the stacking of the base pairs of DNA and thus rises the T m of DNA. In most cases, electrostatic forces across the DNA phosphate backbone generate only a minor modification of the melting temperature, whereas an intercalative binding process induces a substantial rise in the melting point of the DNA due to the stabilisation of the base-pair duplex of the DNA [ 29 , 30 ]. The analysis of melting point of the DNA for current CuPc reveals an allowable favourable change in the T m of approximately 5 o C for CuPc complex. Figure 5 presents T m graphs of DNA in the absent and present of CuPc . In the absence of the complex, the melting temperature trials were done for the CT-DNA and T m of 69.6 o C was detected in the buffer at pH 7.05, and for the CuPc complex, T m of around 75.7 o C was determined. The substantial rise in the T m of the DNA along with CuPc complex is very similar to that reported for the standard intercalating substances [ 31 – 33 ]. 4 Conclusions The tetra-(4-tritylphenoxy)-substituted copper (II) phthalocyanine complex had been eralier produced by synthesis and characterised by UV/Vis, NMR and FTIR methodologies. Absorption titrations, emission spectra, gel electrophoresis and melting temperature assays were employed to examine the reaction behaviour of CuPc with CT-DNA. K b values generated by UV/Vis absorption spectrometry proved that the complex interfers with DNA by an intercalative bonding. The data derived from the emission titrations for the CuPc complex have revealed that the complex also has a binding to the DNA via an intercalative manner of interaction. The nature of the interaction of the complex with CT-DNA was proposed to be an intercalative bonding by all the data generated. In addition to the above examinations, the binding abilities of the CuPc complex were also tested on calf thymus DNA using agarose gel electrophoresis and melting temperature experimentation. The data generated by these procedures revealed that the complex intercalates with DNA molecule. The complex has the potential to be used for therapeutic medicine due to its bonding to DNA. Declarations Author contributions AA: supervises the experiments, planned, performed the experiments, sample preparation, data collection, analysis, interpretation of the results, and writing the manuscript. MSA: supervises the experiments, sample preparation and helps with sample characterization of the complex. DGS: contributed to the experiments and characterization of the compound. Funding The authors have not disclosed any funding. Competing interests The authors declare that they have no confict of interest. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. References Eshkourfu R, Cobeljic B, Vujcic M, Turel I, Pevec A, Sepcic K, Zec M, Radulovic S, Srdic-Radic T, Mitic D, Andjelkovic K, Sladic D. Synthesis, characterization, cytotoxic activity and DNA binding properties of the novel dinuclear cobalt(III) complex with the condensation product of acetylpyridine and malonic acid dihydrazide. J Inorg Biochem.2011; 105: 1196-1203. Li Y, Yang ZY, Wang MF. Synthesis, characterization, DNA binding properties and antioxidant activity of Ln(III) complexes with hesperetin-4-one-(benzoyl) hydrazone. Eur J Med Chem. 2009; 44: 4585-4595. Barton J. Metals and DNA: molecular left-handed complements. Science 1986; 233: 727-734. Sigman DS, Mazumder A, Perrin DM. Chemical nucleases, Chem Rev. 1993; 93: 2295 –2316. Özel A, Barut B, Demirbaş Ü, Biyiklioglu Z. Investigation of DNA binding, DNA photocleavage, topoisomerase I inhibition and antioxidant activities of water soluble titanium(IV) phthalocyanine compounds. J Photochem Photobiol B: Biol. 2016; 157: 32-38. Ağırtaş MS, Cabir B, Özdemir S. Novel metal (II) phthalocyanines with 3,4,5- trimethoxybenzyloxy-substituents: synthesis, characterization, aggregation behaviour and antioxidant activity. Dyes Pigm. 2013; 96: 152-157. Tretyakova IN, Chernii VY, Tomachynski LA, Volkov SV. Synthesis and luminescent properties of new zirconium (IV) and hafnium (IV) phthalocyanines with various carbonic acids as out-planed ligands. Dyes Pigm. 2007; 75: 67-72. Haimovici R, Ciulla TA, Miller JW, Hasan T, Flotte TJ, Kenney AG, Schomacker KT. Gragoudas E. Localization of rose bengal, aluminum phthalocyaninetetrasulfonate, and chlorin e(6) in the rabbit eye. Retina. 2002; 22: 65-74. Eldar M, Yerushalmi Y, Kessler E, Scheinowitz M, Goldbourt U, Ben-Hur E, Rosenthal I, Battler A. Preferential uptake of a water-soluble phthalocyanine by atherosclerotic plaques in rabbits. Atherosclerosis. 1990; 84: 135-139. Amaral GP, Puntel GO, Corte CLD, Dobrachinski F, Barcelos RP, Bastos LL, Avilla DS, Rocha JBT, da Silva EO, Puntel RL, Soares FAA. The antioxidant properties of different phthalocyanines. Toxicol in Vitro. 2012; 26: 125-132. Uslan C, Sesalan Ş. Synthesis of novel DNA-interacting phthalocyanines Dyes Pigm. 2012; 94: 127-135. Nishiyama N, Jang WD, Kataoka K. Supramolecular nanocarriers integrated with dendrimers encapsulating photosensitizers for effective photodynamic therapy and photochemical gene delivery. New J Chem. 2007; 31: 1074-1082. Tamaki Y. Prospects for nanomedicine in treating age-related macular degeneration. Nanomedicine.2009; 4: 341-354. Mao JF, Zhang Y, Zhu J, Zhang C, Guo Z. Molecular combo of photodynamictherapeutic agent silicon(IV) phthalocyanine and anticancer drug cisplatin. Chem Commun. 2009; 8: 908-910. Yabaş,E, Bağda E, Bağda E. The water soluble ball-type phthalocyanine as new potential anticancer drugs. Dyes Pigm. 2015; 120: 220-227. Rosenberg B, Camp LV, Trosko JE, Mansour, VH. Platinum compounds: a new class of potent antitumour agents, Nature. 1969; 222: 385-386. Banerjee SM, MacRobert AJ, Mosse CA, Periera B, Bown SG, Keshtgar MRS. Photodynamic therapy: Inception to application in breast cancer. Breast. 2017; 31: 105-113. Bas H, Biyiklioglu Z, Barut B, Yalçın CÖ, Ozel A. Highly water soluble axial disubstituted silicon(IV) phthalocyanine, naphthalocyanine: Synthesis, DNA interaction and anticancer effects against human lung (A549), liver (SNU-398), melanoma (SK-MEL128), prostate (DU-145), breast (BT-20) cell lines. Inorg Chem Commun. 2023; 156: 111139. Güngördü Solğun D, Horoz S, Ağırtaş MS. Synthesis of novel tetra (4-tritylphenoxy) substituted metallophthalocyanines and investigation of their aggregation, photovoltaic, solar cell properties. Inorg Nano-Met Chem. 2018; 48(10): 508-514. Marmur J. A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol. 1961; 3: 208-218. Amitha GS, Vasudevan S. DNA binding and cleavage studies of novel Betti base substituted quaternary Cu(II) and Zn(II) phthalocyanines. Polyhedron. 2020; 190: 114773. Wolfe A, Shimer GH, Meehan T. Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA. Biochemistry. 1987; 26: 6392-6396. Barut B, Sofuoglu A, Biyiklioglu Z, Özel A. The water soluble peripherally tetra-substituted zinc (II), manganese (III) and copper (II) phthalocyanines as new potential ant icancer agents. Dalton Trans. 2016; 45: 14301-14310. Esenpınar AA, Durmus M, Bulut M. Photophysical, photochemical and BSA binding/BQ quenching properties of quaternizable coumarin containing water soluble zinc phthalocyanine complexes. Spectrochim Acta Part A: Mol Biomol Spectrosc. 2011; 79: 608-617. Sirajuddin M, Ali S, Badshah A. Drug–DNA interactions and their study by UV–Visible, fluorescence spectroscopies and cyclic voltammetry. J Photochem Photobiol B: Biol. 2013; 124: 1-19. Gonzalez-Ruiz V, Olives AI, Martin MA, Ribelles P, Ramos MT, Menendez JC. An overview of analytical techniques employed to evidence drug–DNA interactions and applications to the Design of Genosensors. In TechOpen. 2011; 65: 3-664. Zipper H, Brunner H, Bernhagen J, Vitzthum F. Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications. Nucleic Acids Res. 2004; 32(12): e103. Umemura K, Nagami F, Okada T, Kuroda R. AFM characterization of single strand-specific endonuclease activity on linear DNA. Nucleic Acids Res. 2000; 28: e39. Mudasir M, Wahyuni ET, Tjahjono DH, Yoshioka N, Inoue H. Spectroscopic studies on the thermodynamic and thermal denaturation of the CT-DNA binding of methylene blue. Spectrochim Acta Part A: Mol Biomol Spectrosc. 2010; 77: 528-534. Norden B, Tjerneld F. Binding of inert metal complexes to deoxyribonucleic acid detected by linear dichroism. FEBS Lett. 1976; 67(3): 368-370. Waring MJ. Complex formation between ethidium bromide and nucleic acids. J Mol Biol. 1965; 13: 269-282. Neyhart GA, Grover N, Smith SR, Kalsbeck WA, Fairly TA, Cory M, Thorp HH. Binding and kinetics studies of oxidation of DNA by oxoruthenium (IV). J Am Chem Soc. 1993; 115: 4423-4428. Arslantas A, Agirtas MS. Investigation of DNA binding activities of peripherally 2,10,16,24-tetrakis dimethyl 5-(phenoxy)-isophthalate-substituted Ni(II) phthalocyanine complex. Chemistryselect. 2018; 3(11): 3155-3160. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4168319","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":285224399,"identity":"21ba3b94-1be3-4352-b20b-02ebc7ce5a25","order_by":0,"name":"Ali Arslantaş","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYNCCHzVyIOrAA6J1MPYcMwZrSSDeFjbmxAYQTZQW/tnHHz78wcOWPj/s8EOgLXZyug0EtEicyzE2kLCQyd14O80AqCXZ2OwAIWvO8LBJGPCw5W6cnQDSciBxGyEt8mfYn0kksDGnG85O/0CcFoMzDGYSB9iYE+Slc4i0xfAMj7FhY88xww3SOQUHEgyI8IvcGXZgiP2okZefnb75w4cKOznC3oe7EKzSgFjlICDfQIrqUTAKRsEoGFEAAKGnQyZvSL+YAAAAAElFTkSuQmCC","orcid":"","institution":"Bakırçay Üniversitesi","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"Arslantaş","suffix":""},{"id":285224400,"identity":"9cd7036c-40d2-4a70-9e05-fb626bcfa6a1","order_by":1,"name":"Mehmet Salih Ağırtaş","email":"","orcid":"","institution":"Van Yüzüncü Yıl University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mehmet","middleName":"Salih","lastName":"Ağırtaş","suffix":""},{"id":285224401,"identity":"8ba66360-a582-4b67-9986-5f40968c5bbc","order_by":2,"name":"Derya Güngördü Solğun","email":"","orcid":"","institution":"Van Yüzüncü Yıl University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Derya","middleName":"Güngördü","lastName":"Solğun","suffix":""}],"badges":[],"createdAt":"2024-03-26 08:37:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4168319/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4168319/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53836882,"identity":"1143909d-9a8a-400c-857c-9d6f205cdd3f","added_by":"auto","created_at":"2024-04-01 06:26:50","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":77677,"visible":true,"origin":"","legend":"\u003cp\u003eChemical formula of \u003cstrong\u003eCuPc \u003c/strong\u003ecomplex.\u003c/p\u003e","description":"","filename":"floatimage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4168319/v1/35d216225b0bde489066bf6c.jpg"},{"id":53836880,"identity":"c5e1420e-9101-4002-b92b-c09f83d655ae","added_by":"auto","created_at":"2024-04-01 06:26:50","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":204131,"visible":true,"origin":"","legend":"\u003cp\u003eSpectra of electronic titration of \u003cstrong\u003eCuPc \u003c/strong\u003e(20 µM) in enhancement amounts of CT-DNA (0-15 µM at pH 7.05 in tris-HCl buffer.\u003c/p\u003e","description":"","filename":"floatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4168319/v1/6d48da13bc765b1b7f52c3a1.jpg"},{"id":53836884,"identity":"29392826-e995-4062-af23-c35088352923","added_by":"auto","created_at":"2024-04-01 06:26:50","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":155141,"visible":true,"origin":"","legend":"\u003cp\u003eThe fluorescence titration spectrum of the \u003cstrong\u003eCuPc\u003c/strong\u003ecomplex with the DNA in the buffer.\u003c/p\u003e","description":"","filename":"floatimage3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4168319/v1/11e6ac44d5f058b021344290.jpg"},{"id":53836883,"identity":"75247148-9930-4be1-8e96-df0fee7e79d6","added_by":"auto","created_at":"2024-04-01 06:26:50","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":81614,"visible":true,"origin":"","legend":"\u003cp\u003eThe binding activities between the \u003cstrong\u003eCuPc\u003c/strong\u003ephthalocyanine complex and the DNA in the buffer solution are shown by gel electrophoresis of the DNA patterns. Lane M represents standard DNA ladder. Lanes (1-3) indicate (5, 10 and 15 µM CT-DNA) + 20 µM \u003cstrong\u003eCuPc\u003c/strong\u003e complex, respectively.\u003c/p\u003e","description":"","filename":"floatimage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4168319/v1/f89d2bb3eb23e76b51bf87da.jpg"},{"id":53836881,"identity":"8e726d9b-e14f-461b-b1ff-419305747f81","added_by":"auto","created_at":"2024-04-01 06:26:50","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":92620,"visible":true,"origin":"","legend":"\u003cp\u003eT\u003csub\u003em\u003c/sub\u003e graphs of the DNA in the lack and in the existence of \u003cstrong\u003eCuPc\u003c/strong\u003e, indicating the rise of the melting temperatures.\u003c/p\u003e","description":"","filename":"floatimage5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4168319/v1/6f741fc2ed281925a703762c.jpg"},{"id":55265357,"identity":"10506e7a-bde7-4f1d-937d-240d43e415bc","added_by":"auto","created_at":"2024-04-25 02:01:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":627101,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4168319/v1/f3af92cc-6b82-4555-a252-61641f7d9da2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The examining on DNA binding activities of tetrakis-(4-tritylphenoxy)-phthalocyanine copper (II) phthalocyanine complex","fulltext":[{"header":"Article Highlights ","content":"\u003cp\u003e\u0026bull; \u0026nbsp;The syntheisi of\u0026nbsp;Cu (II) phthalocyanine complex\u003c/p\u003e\n\u003cp\u003e\u0026bull; Examining of DNA interaction activity Cu (II) phthalocyanine complex.\u003c/p\u003e\n\u003cp\u003e\u0026bull; Identifying potential DNA binding property of the complex.\u003c/p\u003e"},{"header":"1 Introduction","content":"\u003cp\u003eDNA molecule is one of the significant targets used in the treatment of many genetic diseases such as cancer. In recent years, it has been observed that there has been a significant increase in biological research due to the significant effects of transition metal-based compounds in cancer treatment [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The mechanism by which a drug binds to DNA provides very important preliminary information about the possibility of using the chemical compound as an anticancer drug in cancer treatment [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Within in this scope, the very broad spectral and electrochemical activities of transition metal-based compounds contribute significantly to the interaction activities of these compounds with DNA [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnder normal conditions, the presence of too much active oxygen radicals, which arise as a result of metabolic reactions, causes oxidative damage to biomolecules such as DNA, lipids and proteins. Consequently, it may increase the likelihood of some very critical diseases such as cancer, inflammation, cardiovascular and neurodegenerative diseases occurring very quickly. Depending on their chemical structural properties and the root of oxidative stress, metal-based compounds can serve as drugs [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePhthalocyanine compounds are highly functional molecules, and research on the derivatives of these compounds has been continuing for many years. Due to the thermal and chemical properties of phthalocyanines, they have applications in different fields such as sensors and photodynamic cancer therapy [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, due to the photosensitizing and antioxidant properties of phthalocyanine compounds, research is ongoing for photodynamic therapy in anticancer treatment [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Due to their ability to interact with DNA, phthalocyanine complexes have attracted great attention in the last decade for their application in cancer treatment approaches [\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15 CR16\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In studies published in the literature, different complexes of transition metal phthalocyanine compounds were synthesized to be used as chemotherapeutic active substances [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, it is understood that copper phthalocyanine complexes still do not receive the expected importance in this research field. Encouraging the potential anticancer activities of phthalocyanines and their copper complexes, it reveals the importance of investigating the interactions of phthalocyanines and copper complexes with DNA. In the current study, the interaction activities of previously synthesized peripheral tetra-substituted Cu (II) phthalocyanine [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] with DNA were investigated using absorption spectroscopy, fluorescence, thermal melting point and gel electrophoresis techniques.\u003c/p\u003e"},{"header":"2 Experimental work","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals and Techniques\u003c/h2\u003e \u003cp\u003eThe structure and properties of the Cu (II) phthalocyanine complex containing tetrakis-(4-tritylphenoxy) group have been elucidated in a previous study [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. All chemicals used as containing NaOH at pH 7.05 and stored in a cooler at 4 \u003csup\u003eo\u003c/sup\u003eC. In addition, the stock copper (II) phthalocyanine complex prepared by dissolving DMF, an organic solvent, for use in the study was kept at 25 \u003csup\u003eo\u003c/sup\u003eC. If needed, all solutions used in the study were prepared by diluting the desired samp consumables in this scientific research were obtained from Sigma-Aldrich company. DNA samples used as reagents in this study were prepared using Tris-HCl buffer solutionle volumes using Tris-HCl buffer. Instrumental devices such as UV/Vis, thermal melting point, emission, electrophoresis were used in this study to determine the interaction activities of the In addition to the above devices, Thermo Scientific Owl electrophoresis system was preferred in this study to perform gel electrophoresis tests.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of 4-(4-tritylphenoxy)phthalonitrile and Cu (II) phthalocyanine compound\u003c/h2\u003e \u003cp\u003eThe 4-(4-tritylphenoxy) phthalonitrile compound was previously synthesized by our research group to form a phthalocyanine complex with copper (II), a transition metal [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The tetrakis-(4-tritylphenoxy)-phthalocyanine copper (II) reported in the existing literature has been isolated, synthesised and characterised corresponding to the earlier study of our research team [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and discussion","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Synthesis and analyzing\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the structure of \u003cb\u003eCuPc\u003c/b\u003e phthalocyanine complex. The structural analysis of the Cu (II) phthalocyanine complex was done using a number of spectroscopic tools including IR, electronic absorption and NMR spectrometry [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The evidence gathered from IR, absorption spectra and NMR techniques supported the suggested structure of \u003cb\u003eCuPc\u003c/b\u003e. Copper (II) phthalocyanine was dissolvable in solvents including CHCl\u003csub\u003e3\u003c/sub\u003e, toluene, CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e, DMSO, DMF and THF. For electronic absorption, (THF) ʎmax, nm (log Ɛ) for the complex \u003cb\u003eCuPc\u003c/b\u003e were obtained as 674 (5.21), 608 (4.61), 346 (4.93), accordingly [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. FT-IR spectrums were also acquired for Cu (II) phthalocyanine. IR spectrums (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for \u003cb\u003eCuPc\u003c/b\u003e were observed as 3053(C-H aromatic), 1598(C\u0026thinsp;=\u0026thinsp;C), 1490, 1447, 1442, 1230(Ar-O-Ar), 1085, 1050, 1033, 893, 825, 746, 700, 669, respectively [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. All of this evidence was consistent with the proposed conformation of the complex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.2. DNA Binding Assays\u003c/h2\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2. 1. Electronic Absorption Titration Study for CuPc\u003c/h2\u003e \u003cp\u003eThe binding of \u003cb\u003eCuPc\u003c/b\u003e to the DNA has been explored by means of electronic absorption assays in order to comprehend their binding nature to the DNA. The samples of DNA had been made up in the buffer solution for this experiment. Electronic absorption spectra analysis of the stock solution of CT-DNA in a tris-HCl solution at pH 7.05 at 250 and 300 nm had been read and the data proved that the DNA sample did not possess protein. The quantity of DNA for every nucleotide phosphate group was assessed from the absorbance spectra at 260 nm, considering the value of the extinction coefficient [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The CT DNA specimens were preserved at 4 \u003csup\u003eo\u003c/sup\u003eC and used within a few days [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The stock specimen of \u003cb\u003eCuPc\u003c/b\u003e complex was generated in DMF and diluted in the buffer solution. Electronic absorption titrations were done using a quartz sample holder and scanned in between the range of 250\u0026ndash;900 nm at a pH of 7.05. Absorbance spectra titration tests were done for a fixed quantity of \u003cb\u003eCuPc\u003c/b\u003e (20 \u0026micro;M) and variable quantities of DNA (0 to 15 \u0026micro;M) in increments of 5 \u0026micro;M. After each addition, the UV/Vis absorptions were monitored, and the results were recorded. The sample mixture of the complex and CT-DNA was run for 5 min at 25 \u003csup\u003eo\u003c/sup\u003eC and shifts in the absorbance spectra were observed. The UV/Vis absorption data were used to obtain the binding coefficient (K\u003csub\u003eb\u003c/sub\u003e) for \u003cb\u003eCuPc\u003c/b\u003e to DNA using the Wolfe-Schimer formula (1) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\frac{\\left[DNA\\right]}{Ɛa-Ɛf}=\\frac{\\left[DNA\\right]}{Ɛb-Ɛf}+\\frac{1}{Kb\\left(Ɛb-Ɛf\\right)}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe absorption titration of CT-DNA of \u003cb\u003eCuPc\u003c/b\u003e is as presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. For \u003cb\u003eCuPc\u003c/b\u003e complex, the intrinsic binding coefficient value was obtained, the Kb value for \u003cb\u003eCuPc\u003c/b\u003e had been estimated to be 1.53 x 106 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e by applying the Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. As the quantity of CT-DNA increases, \u003cb\u003eCuPc\u003c/b\u003e complex produces hypochromism with a redshift band in the absorbance bands at around 674 and 346 nm as presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The K\u003csub\u003eb\u003c/sub\u003e values of recognized intercalative anticancer medicine like doxorubicin and idrarubicin were calculated in published paper [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. For this reason, by relating the K\u003csub\u003eb\u003c/sub\u003e value of \u003cb\u003eCuPc\u003c/b\u003e to the tendency of the absorbance to alter when DNA is added, it can be assumed that \u003cb\u003eCuPc\u003c/b\u003e reacts with DNA via an intercalative route.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2. Fluorescence Analysis of CuPc Complex for DNA Binding\u003c/h2\u003e \u003cp\u003eEmission spectrometry is a procedure frequently utilised to probe the bonds between the small molecular complex and DNA molecule. The advantages of fluorescence procedure over other approaches are its high precision, broad concentration spectrum and specificity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The route of interaction of medicinal agents with DNA can be elucidated utilising emission spectrometry, and other procedures based on emission spectra can also offer further valuable insight. Emission experimentation yields detailed information on the localisation of agents and their attachment mechanisms to the DNA molecule [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The arrow marks the changes in intensity of the emission spectra with the increment of the quantity of CT-DNA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn moreover, to make clear the interaction process of \u003cb\u003eCuPc\u003c/b\u003e complex with CT-DNA, the emission binding examinations were made to clarify the binding of medicinal drugs and DNA, by the fact that this method is a tender technique in the interaction examinations of medicinal drugs-DNA and it can yield further aid to the intercalating binding process of metal complexes. In absence of DNA, \u003cb\u003eCuPc\u003c/b\u003e complex produces an emission spectrum as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, with an emission peak at around 465 nm. In the presence of CT-DNA, the intensities of \u003cb\u003eCuPc\u003c/b\u003e complex decline successively as the DNA is added. The decreasing intensities suggested that \u003cb\u003eCuPc\u003c/b\u003e reacts with the DNA molecule by way of an intercalative binding pathway. These results apparently demonstrated that [tetrakis-(4-tritylphenoxy)-substituted Cu (II) phthalocyanine interacted with the DNA molecule by an intercalation pathway, consistent with the adsorption titration data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3. Gel Electrophoresis Analysis of CuPc/DNA Interaction\u003c/h2\u003e \u003cp\u003eThe interaction abilities of earlier synthesized \u003cb\u003eCuPc\u003c/b\u003e to the DNA were assessed using gel electrophoresis by analyzing the influence of varying amounts of \u003cb\u003eCuPc\u003c/b\u003e complex CT-DNA and its consequences obtained were illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. When comparison with the bands of standard DNA, the band intensities observed for the \u003cb\u003eCuPc\u003c/b\u003e complex after interaction with the DNA were clearly reduced. The fall in the intensity of the band, recorded after the interaction of the phthalocyanine complex with the DNA, is linked to the breakdown of the double helix of the DNA molecule. The earlier reported research proposed that the damage to the DNA may have been caused by the splitting of the backbone due to a nucleophilic attack of the residues [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. It has been suggested in the literature that the band intensities in agarose gel electrophoresis studies, which are visualized by the fluorescence emission spectra of ethidium bromide binding to DNA base pairs through the intercalative binding pathway, can be affected not only by the number of molecular bonds, but also by the length of the DNA molecule [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Consequently, overlapping of the metal complex during binding between the bases within the DNA helix or surface interaction at the reactive sites of the nucleophile on the DNA double helix may account for the decrease in electrophoretic band intensities of CT-DNA upon binding by the \u003cb\u003eCuPc\u003c/b\u003e phthalocyanine complex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e3.2.4. Experiments on the Thermal Denaturation\u003c/h2\u003e \u003cp\u003eIn this report, by analyzing the CT-DNA melting temperatures (T\u003csub\u003em\u003c/sub\u003e), the influence of the amount of adduct formation on the stabilisation of DNA supercoil has been studied. T\u003csub\u003em\u003c/sub\u003e response of CT-DNA in presence of metal complexes can unveil their conformational structure variance with rising temperature, and assure insight into binding ability of tiny metal compounds to DNA molecule. Literature reports suggest that when metal compounds bind to the DNA double helix, molecular stability is promoted. In general, the T\u003csub\u003em\u003c/sub\u003e of DNA rises when metal compounds react with the DNA molecule through an intercalative binding process, as the intercalative binding of metal compounds between the base pairs of DNA induces the stabilisation of the stacking of the base pairs of DNA and thus rises the T\u003csub\u003em\u003c/sub\u003e of DNA. In most cases, electrostatic forces across the DNA phosphate backbone generate only a minor modification of the melting temperature, whereas an intercalative binding process induces a substantial rise in the melting point of the DNA due to the stabilisation of the base-pair duplex of the DNA [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe analysis of melting point of the DNA for current \u003cb\u003eCuPc\u003c/b\u003e reveals an allowable favourable change in the T\u003csub\u003em\u003c/sub\u003e of approximately 5 \u003csup\u003eo\u003c/sup\u003eC for \u003cb\u003eCuPc\u003c/b\u003e complex. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e presents T\u003csub\u003em\u003c/sub\u003e graphs of DNA in the absent and present of \u003cb\u003eCuPc\u003c/b\u003e. In the absence of the complex, the melting temperature trials were done for the CT-DNA and T\u003csub\u003em\u003c/sub\u003e of 69.6 \u003csup\u003eo\u003c/sup\u003eC was detected in the buffer at pH 7.05, and for the \u003cb\u003eCuPc\u003c/b\u003e complex, T\u003csub\u003em\u003c/sub\u003e of around 75.7 \u003csup\u003eo\u003c/sup\u003eC was determined. The substantial rise in the T\u003csub\u003em\u003c/sub\u003e of the DNA along with \u003cb\u003eCuPc\u003c/b\u003e complex is very similar to that reported for the standard intercalating substances [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4 Conclusions","content":"\u003cp\u003eThe tetra-(4-tritylphenoxy)-substituted copper (II) phthalocyanine complex had been eralier produced by synthesis and characterised by UV/Vis, NMR and FTIR methodologies. Absorption titrations, emission spectra, gel electrophoresis and melting temperature assays were employed to examine the reaction behaviour of \u003cb\u003eCuPc\u003c/b\u003e with CT-DNA. K\u003csub\u003eb\u003c/sub\u003e values generated by UV/Vis absorption spectrometry proved that the complex interfers with DNA by an intercalative bonding. The data derived from the emission titrations for the \u003cb\u003eCuPc\u003c/b\u003e complex have revealed that the complex also has a binding to the DNA via an intercalative manner of interaction. The nature of the interaction of the complex with CT-DNA was proposed to be an intercalative bonding by all the data generated. In addition to the above examinations, the binding abilities of the \u003cb\u003eCuPc\u003c/b\u003e complex were also tested on calf thymus DNA using agarose gel electrophoresis and melting temperature experimentation. The data generated by these procedures revealed that the complex intercalates with DNA molecule. The complex has the potential to be used for therapeutic medicine due to its bonding to DNA.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e AA: supervises the experiments, planned, performed the experiments, sample preparation, data collection, analysis, interpretation of the results, and writing the manuscript. MSA: supervises the experiments, sample preparation and helps with sample characterization of the complex. DGS: contributed to the experiments and characterization of the compound.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u0026nbsp;\u003c/strong\u003eThe authors have not disclosed any funding.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e The authors declare that they have no confict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen Access\u003c/strong\u003e This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEshkourfu R, Cobeljic B, Vujcic M, Turel I, Pevec A, Sepcic K, Zec M, Radulovic S, Srdic-Radic T, Mitic D, Andjelkovic K, Sladic D. Synthesis, characterization, cytotoxic activity and DNA binding properties of the novel dinuclear cobalt(III) complex with the condensation product of acetylpyridine and malonic acid dihydrazide. J Inorg Biochem.2011; 105: 1196-1203.\u003c/li\u003e\n\u003cli\u003eLi Y, Yang ZY, Wang MF. Synthesis, characterization, DNA binding properties and antioxidant activity of Ln(III) complexes with hesperetin-4-one-(benzoyl) hydrazone. Eur J Med Chem. 2009; 44: 4585-4595. \u003c/li\u003e\n\u003cli\u003eBarton J. Metals and DNA: molecular left-handed complements. Science 1986; 233: 727-734. \u003c/li\u003e\n\u003cli\u003eSigman DS, Mazumder A, Perrin DM. Chemical nucleases, Chem Rev. 1993; 93: 2295 \u0026ndash;2316. \u003c/li\u003e\n\u003cli\u003e\u0026Ouml;zel A, Barut B, Demirbaş \u0026Uuml;, Biyiklioglu Z. Investigation of DNA binding, DNA photocleavage, topoisomerase I inhibition and antioxidant activities of water soluble titanium(IV) phthalocyanine compounds. J Photochem Photobiol B: Biol. 2016; 157: 32-38. \u003c/li\u003e\n\u003cli\u003eAğırtaş MS, Cabir B, \u0026Ouml;zdemir S. Novel metal (II) phthalocyanines with 3,4,5- trimethoxybenzyloxy-substituents: synthesis, characterization, aggregation behaviour and antioxidant activity. Dyes Pigm. 2013; 96: 152-157. \u003c/li\u003e\n\u003cli\u003eTretyakova IN, Chernii VY, Tomachynski LA, Volkov SV. Synthesis and luminescent properties of new zirconium (IV) and hafnium (IV) phthalocyanines with various carbonic acids as out-planed ligands. Dyes Pigm. 2007; 75: 67-72. \u003c/li\u003e\n\u003cli\u003eHaimovici R, Ciulla TA, Miller JW, Hasan T, Flotte TJ, Kenney AG, Schomacker KT. Gragoudas E. Localization of rose bengal, aluminum phthalocyaninetetrasulfonate, and chlorin e(6) in the rabbit eye. Retina. 2002; 22: 65-74. \u003c/li\u003e\n\u003cli\u003eEldar M, Yerushalmi Y, Kessler E, Scheinowitz M, Goldbourt U, Ben-Hur E, Rosenthal I, Battler A. Preferential uptake of a water-soluble phthalocyanine by atherosclerotic plaques in rabbits. Atherosclerosis. 1990; 84: 135-139. \u003c/li\u003e\n\u003cli\u003eAmaral GP, Puntel GO, Corte CLD, Dobrachinski F, Barcelos RP, Bastos LL, Avilla DS, Rocha JBT, da Silva EO, Puntel RL, Soares FAA. The antioxidant properties of different phthalocyanines. Toxicol in Vitro. 2012; 26: 125-132. \u003c/li\u003e\n\u003cli\u003eUslan C, Sesalan Ş. Synthesis of novel DNA-interacting phthalocyanines Dyes Pigm. 2012; 94: 127-135. \u003c/li\u003e\n\u003cli\u003eNishiyama N, Jang WD, Kataoka K. Supramolecular nanocarriers integrated with dendrimers encapsulating photosensitizers for effective photodynamic therapy and photochemical gene delivery. New J Chem. 2007; 31: 1074-1082. \u003c/li\u003e\n\u003cli\u003eTamaki Y. Prospects for nanomedicine in treating age-related macular degeneration. Nanomedicine.2009; 4: 341-354. \u003c/li\u003e\n\u003cli\u003eMao JF, Zhang Y, Zhu J, Zhang C, Guo Z. Molecular combo of photodynamictherapeutic agent silicon(IV) phthalocyanine and anticancer drug cisplatin. Chem Commun. 2009; 8: 908-910. \u003c/li\u003e\n\u003cli\u003eYabaş,E, Bağda E, Bağda E. The water soluble ball-type phthalocyanine as new potential anticancer drugs. Dyes Pigm. 2015; 120: 220-227. \u003c/li\u003e\n\u003cli\u003eRosenberg B, Camp LV, Trosko JE, Mansour, VH. Platinum compounds: a new class of potent antitumour agents, Nature. 1969; 222: 385-386. \u003c/li\u003e\n\u003cli\u003eBanerjee SM, MacRobert AJ, Mosse CA, Periera B, Bown SG, Keshtgar MRS. Photodynamic therapy: Inception to application in breast cancer. Breast. 2017; 31: 105-113. \u003c/li\u003e\n\u003cli\u003eBas H, Biyiklioglu Z, Barut B, Yal\u0026ccedil;ın C\u0026Ouml;, Ozel A. Highly water soluble axial disubstituted silicon(IV) phthalocyanine, naphthalocyanine: Synthesis, DNA interaction and anticancer effects against human lung (A549), liver (SNU-398), melanoma (SK-MEL128), prostate (DU-145), breast (BT-20) cell lines. Inorg Chem Commun. 2023; 156: 111139. \u003c/li\u003e\n\u003cli\u003eG\u0026uuml;ng\u0026ouml;rd\u0026uuml; Solğun D, Horoz S, Ağırtaş MS. Synthesis of novel tetra (4-tritylphenoxy) substituted metallophthalocyanines and investigation of their aggregation, photovoltaic, solar cell properties. Inorg Nano-Met Chem. 2018; 48(10): 508-514. \u003c/li\u003e\n\u003cli\u003eMarmur J. A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol. 1961; 3: 208-218. \u003c/li\u003e\n\u003cli\u003eAmitha GS, Vasudevan S. DNA binding and cleavage studies of novel Betti base substituted quaternary Cu(II) and Zn(II) phthalocyanines. Polyhedron. 2020; 190: 114773. \u003c/li\u003e\n\u003cli\u003eWolfe A, Shimer GH, Meehan T. Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA. Biochemistry. 1987; 26: 6392-6396. \u003c/li\u003e\n\u003cli\u003eBarut B, Sofuoglu A, Biyiklioglu Z, \u0026Ouml;zel A. The water soluble peripherally tetra-substituted zinc (II), manganese (III) and copper (II) phthalocyanines as new potential ant icancer agents. Dalton Trans. 2016; 45: 14301-14310. \u003c/li\u003e\n\u003cli\u003eEsenpınar AA, Durmus M, Bulut M. Photophysical, photochemical and BSA binding/BQ quenching properties of quaternizable coumarin containing water soluble zinc phthalocyanine complexes. Spectrochim Acta Part A: Mol Biomol Spectrosc. 2011; 79: 608-617. \u003c/li\u003e\n\u003cli\u003eSirajuddin M, Ali S, Badshah A. Drug\u0026ndash;DNA interactions and their study by UV\u0026ndash;Visible, fluorescence spectroscopies and cyclic voltammetry. J Photochem Photobiol B: Biol. 2013; 124: 1-19. \u003c/li\u003e\n\u003cli\u003eGonzalez-Ruiz V, Olives AI, Martin MA, Ribelles P, Ramos MT, Menendez JC. An overview of analytical techniques employed to evidence drug\u0026ndash;DNA interactions and applications to the Design of Genosensors. In TechOpen. 2011; 65: 3-664. \u003c/li\u003e\n\u003cli\u003eZipper H, Brunner H, Bernhagen J, Vitzthum F. Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications. Nucleic Acids Res. 2004; 32(12): e103. \u003c/li\u003e\n\u003cli\u003eUmemura K, Nagami F, Okada T, Kuroda R. AFM characterization of single strand-specific endonuclease activity on linear DNA. Nucleic Acids Res. 2000; 28: e39. \u003c/li\u003e\n\u003cli\u003eMudasir M, Wahyuni ET, Tjahjono DH, Yoshioka N, Inoue H. Spectroscopic studies on the thermodynamic and thermal denaturation of the CT-DNA binding of methylene blue. Spectrochim Acta Part A: Mol Biomol Spectrosc. 2010; 77: 528-534. \u003c/li\u003e\n\u003cli\u003eNorden B, Tjerneld F. Binding of inert metal complexes to deoxyribonucleic acid detected by linear dichroism. FEBS Lett. 1976; 67(3): 368-370. \u003c/li\u003e\n\u003cli\u003eWaring MJ. Complex formation between ethidium bromide and nucleic acids. J Mol Biol. 1965; 13: 269-282. \u003c/li\u003e\n\u003cli\u003eNeyhart GA, Grover N, Smith SR, Kalsbeck WA, Fairly TA, Cory M, Thorp HH. Binding and kinetics studies of oxidation of DNA by oxoruthenium (IV). J Am Chem Soc. 1993; 115: 4423-4428. \u003c/li\u003e\n\u003cli\u003eArslantas A, Agirtas MS. Investigation of DNA binding activities of peripherally 2,10,16,24-tetrakis dimethyl 5-(phenoxy)-isophthalate-substituted Ni(II) phthalocyanine complex. Chemistryselect. 2018; 3(11): 3155-3160.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Copper (II), DNA interaction, phthalocyanines, absorption spectra, Gel electrophoresis","lastPublishedDoi":"10.21203/rs.3.rs-4168319/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4168319/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCu (II) phthalocyanine complex had been previously synthesized by reacting 4-(4-tritylphenoxy)phthalonitrile compound. The structure of obtained \u003cb\u003eCuPc\u003c/b\u003e complex had been analyzed by absorption titration, infrared and NMR spectroscopies techniques. The DNA interacting property for the complex was analyzed in various concentration of CT-DNA utilizing elctronic absorption, emission spectroscopy, gel agarose electrophoresis and thermal melting procedures. In this report, the binding constant value for the complex was also estimated. Absorption and fluorescence spectroscopic processes proved that \u003cb\u003eCuPc\u003c/b\u003e interacts by CT-DNA. At the the same time, the thermal melting and electrophoresis technics were practiced to analyze the intercating property of \u003cb\u003eCuPc\u003c/b\u003e by DNA. The thermal melting and electrophoresis assays supported that \u003cb\u003eCuPc\u003c/b\u003e combines with DNA via an intercalative interacting mechanism. The obtained findings demonstrated that the complex connects to DNA by an intercalative interacting mode. Therefore, \u003cb\u003eCuPc\u003c/b\u003e complex may have potential cancer therapeutic agent.\u003c/p\u003e","manuscriptTitle":"The examining on DNA binding activities of tetrakis-(4-tritylphenoxy)-phthalocyanine copper (II) phthalocyanine complex","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-01 06:26:45","doi":"10.21203/rs.3.rs-4168319/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8da2b3f6-1d80-4c8c-aac8-dc3870800190","owner":[],"postedDate":"April 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-24T12:38:11+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-01 06:26:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4168319","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4168319","identity":"rs-4168319","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}