Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 377–385

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Synthesis, characterization and multi-spectroscopic DNA interaction studies of a new platinum complex containing the drug metformin Nahid Shahabadi ⇑, Leila Heidari Department of Inorganic Chemistry, Faculty of Chemistry, Razi University, Kermanshah, Iran

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 A new platinum(II) complex

containing the drug metformin was synthesized.  This study is expected to provide greater insight into the use of other drugs like cis-platin as anticancer.  All the results showed that the complex was a DNA groove binder.

a r t i c l e

i n f o

Article history: Received 2 January 2014 Received in revised form 16 February 2014 Accepted 21 February 2014 Available online 12 March 2014 Keywords: DNA Metformin Platinum complex Spectroscopic methods Groove binding mode

a b s t r a c t A new platinum(II) complex; [Pt(Met)(DMSO)Cl]Cl in which Met = metformin and DMSO: dimethylsulfoxide, was synthesized and characterized by 1H NMR, IR, UV–Vis spectra, molar conductivity and computational methods. Binding interaction of this complex with calf thymus (CT) DNA has been investigated by using absorption, emission, circular dichroism, viscosity measurements, differential pulse voltammetry and cleavage studies by agarose gel electrophoresis. UV–Vis absorption studies showed hyperchromism. CD studies showed less perturbation on the base stacking and helicity bands in the CD spectrum of CT-DNA (B ? C structural transition). In fluorimeteric studies, the Pt(II) complex can bind with DNA–NR complex and forms a new non-fluorescence adduct. The anodic peak current in the differential pulse voltammogram of the Pt(II) complex decreased gradually with the addition of DNA. Cleavage experiments showed that the Pt(II) complex does not induce any cleavage under the experimental setup. Finally all results indicated that Pt(II) complex interact with DNA via groove binding mode. Ó 2014 Elsevier B.V. All rights reserved.

Introduction The involvement of some metal ions in regulation of physiological processes has stimulated the development of metal-based therapeutics. The pharmaceutical use of complexes arises from the fact that the positively charged metal centers are favored to bind to negatively charged biomolecules such as the amino acid ⇑ Corresponding author. Tel./fax: +98 831 8360795. E-mail address: [email protected] (N. Shahabadi). http://dx.doi.org/10.1016/j.saa.2014.02.167 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

residues as protein constituents, ATP and nucleic acids. Moreover, the ligands can interact with biomolecules through coordinative or hydrogen bonds as well as dipole dipole interactions [1]. Therefore, the use of the metal complexes as therapeutics has become increasingly important over the last years resulting in a variety of interesting drugs used in fields as cancer, arthritis, ulcer, diabetes, anemia and cardiovascular medicine [2]. Platinum-based drugs play an important role in clinical cancer chemotherapy. The first platinum antitumor drug introduced in the clinic was cis-diammine dichloro platinum(II). However, in

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spite of their high activity, the applications of cis-platin and similar compounds are limited by the side effects. Therefore, the search for soluble and less toxic analogues of cis-platin is one of the main goals in the synthesis of new platinum(II) complexes [3–5]. Recently, new active monofunctional platinum complexes with structural features that violate the ‘classical’ structure–activity relationships have been described [6–9]. Transition metal complexes can bind to DNA via covalent interactions, where a labile ligand of the complexes is replaced by a nitrogen base of DNA such as guanine N7, and/or non-covalent interactions which include intercalative, electrostatic and groove (surface) binding of metal complexes along outside of DNA helix, along major or minor groove [10]. These interactions have been shown to disrupt replication and/or transcription culminating in cellular death. It is well known that biguanide derivatives exhibit both biological and coordinative properties [11]. Interest in biguanide derivatives arises from their well acclaimed medicinal values as germicidal, bacteriostatic, hypoglycemic and anticarcinogenic agents [12]. Also, biguanide and biguanide derivatives are extremely powerful and formidable coordinating ligands which have played important roles in elucidating many interesting aspects of coordination chemistry [13,14]. Among biguanide drugs, the most widely prescribed type II diabetes medications is N,N-dimethylbiguanide known as Metformin [15]. Beside decreasing the glucose level, the N,N-dimethylbiguanide also acts as analgesic, antimalarial and antimetabolite for organisms that inhibit the metabolism of folic acid [16,17]. Recently, it was found that platinum(IV) complex with N,N-dimethylbiguanide shows antitumor activity [18]. Attempting to modulate the biological activity of this derivative it was demonstrated that some of their complexes possess antimicrobial activity and also have an interesting thermal behavior [19]. In addition, one approach to accelerate the availability of new drugs is to reposition drugs approved for other indications as anticancer agents. In our laboratory we investigated the interaction of known drugs with protein and DNA and the effects of metals on their interactions in order to study the anticancer ability of them and highlighted the role of metal centers on bioinorganic medicinal chemistry researches. Interestingly, the mechanism of action and toxic side effects of purely organic drugs can be modulated in the presence of metals. In this paper, we report the preparation, characterization and DNA binding studies of platinum(II) complex with metformin hydrochloride (Met) as ligand. The binding properties on Pt(II) complex to calf thymus DNA in physiological buffer (pH 7.4) was investigated by multi-spectroscopic methods. The results showed that spectroscopy techniques could provide a convenient way to characterize both the binding mode and the interaction mechanism of Pt(II) complex binding to DNA. We believe that the knowledge gained from this study will be helpful to further understand the binding mechanisms and can provide much fruitful information for designing a new type of highly effective anti-cancer drugs.

Experimental Materials and methods Commercial pure chemicals such as Metformin, Dipotassium tetrachloroplatinum(II), Methanol and DMSO were purchased from Merck and Tris–HCl, calf thymus DNA (CT-DNA), pUC18 and Neutral red (NR) were purchased from Sigma Co. Solutions were prepared with double distilled water. Experiments were carried out in Tris–HCl buffer at pH 7.0. The stock solution of CT-DNA was prepared by dissolving of DNA in

50 mM of the Tris–HCl buffer at pH 7.4 dialyzing exhaustively against the same buffer for 24 h. A solution of calf thymus DNA gave a ratio of UV absorbance at 260 and 280 nm more than 1.8, indicating that DNA was sufficiently free from protein. The stock solutions were stored at 4 °C and used over no more than 4 days. Synthesis of platinum complex Synthesis of cis-[Pt(DMSO)2Cl2] complex. The complex was prepared according to the following procedure and operated in dark. K2 [PtCl4] (0.415 g; 0.001 mol) was mixed with water 4 mL. DMSO (0.242 g; 0.003 mol) was then added to the red aqueous solution of tetrachloroplatinate(II). The resulting mixture was stirred for 12 h, at 20 °C. The yellow solid precipitate was filtered off, washed towice with water, ethanol and diethylether, and then air-dried at 20 °C to afford yellow crystals (Yield:84%). Synthesis of [Pt(Met)(DMSO)Cl]Cl. A suspension of cis-[PtCl2(DMSO)2] (0.222 g; 0.53 mmol) in MeOH (15 mL) was treated with the stoichiometric amount of metformin (0.09 g; 0.53 mmol) dissolved in 20 mL MeOH–Water (1:1) solution, stirred at room temperature for 1 day and filtered. The filterate was left for crystallization and after 3 days white crystals of the complex precipitated (Yield: 93.6%). Instrumentation The complex obtained was characterized by UV–Vis, FT-IR and H NMR spectroscopy. The NMR spectra were recorded with a Bruker Avance DPX 200 MHz (4.7 T-esla) spectrometer using d6-DMSO solvent. IR spectra were obtained with an ABB BOMEM MB 104 FTIR spectrometer using KBr discs for 400–4000 cm1 range. The elemental analysis was performed using a Heraeus CHN elemental analyzer. Absorbance spectra were recorded using an HP spectrophotometer (Agilent 8453) equipped with a thermostated bath (Huber polysat cc1). Absorbance experiments for the DNA interaction of Pt(II) complex were carried out by keeping the concentration of the complex constant (7.5  105 M) while varying DNA concentration from 0 to 1.05  104 M (ri = [DNA]/[complex] = 0, 0.2, 0.6, 0.8 and 1.4). Absorbance values were recorded after each successive addition of DNA solution, followed by an incubation period (2 h). The UV–Vis absorption spectra of DNA–NR complex and the mixture of different concentrations of Pt(II) complex and DNA– NR complex were measured under the pH 7.4, Tris–HCl buffer, respectively. The sample tube containing 2.0 mL mixture solution of 1.0  105 mol L1 NR and 6  105 mol L1 CT-DNA was allowed to stand for 5 min at room temperature. The mixture solution was titrated by successive additions of 1  103 mol L1 stock solutions of Pt(II) complex. For every addition, the mixture solution was shaken and allowed to stand for 5 min at room temperature. For viscosity measurements, a viscometer (SCHOT AVS 450) was used, kept at 25 °C by a constant temperature bath. Flow time was measured with a digital stopwatch; the mean values of two replicated measurements were used to evaluate the viscosity (g) of the samples. The data were reported as (g/g0) versus the [complex]/ [DNA] ratio, where g0 is the viscosity of the DNA solution alone. Viscosity values were calculated from the observed flow time of CT-DNA containing solution corrected from the flow time of buffer alone (t0) as g = (t  t0)/t0. CD measurements were recorded on a JASCO (J-810) spectropolarimeter by keeping the concentration of DNA constant (8  105 M) while varying the platinum complex concentration (ri = [complex]/[DNA] = 0.0, 0.2 and 0.6). 1

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Fluorescence measurements were carried out with a JASCO spectrofluorimeter (FP 6200). The competitive interaction between the neutral red and platinum complex with DNA was carried out as follows: fixed amounts of the neutral red and DNA were titrated by successive additions of 1  103 mol L1 stock solutions of Pt(II) complex. These samples were excited at 530 nm and emission was observed between 530 and 700 nm. The differential pulse voltammetry (DPV) measurements were performed using an AUTOLAB model (PG STAT C), with a threeelectrode system: a 0.10-cm-diameter Glassy carbon (GC) disc as working electrode, an Ag/AgCl electrode as reference electrode, and a Pt wire as counter electrode. Electrochemical experiments were carried out in a 25-mL voltammetric cell at room temperature. All potentials are referred to the Ag/AgCl reference. Their surfaces were freshly polished with 0.05 mm alumina prior to each experiment and were rinsed using double distilled water between each polishing step. The supporting electrolyte was 0.05 M of Tris– HCl buffer solution (pH 7.4) which was prepared with double distilled water. The current–potential curves and experimental data were recorded on software GPES. For the gel electrophoresis experiments, pUC18 plasmid DNA was treated with Pt complex in TAE buffer (pH = 8), and the contents were incubated for 2 h at 37 °C. The samples (ri = [Pt complex]/[DNA] = 0.0, 0.1, 0.3, 0.5, 0.7 and 0.9) were eletrophoresed for 1 h at 80 V on 0.8% agarose gel in TAE buffer. Gel was stained with ethidium bromide and photographed using UV illumination. For dialysis we used dialysis bag for each solutions of cis-PtDNA, Pt(II) com-DNA and Hoechst-DNA and then absorbance and emission values were recorded after 24 h for inner solutions dialysis bag.

Results and discussion Synthesis and characterization of platinum(II) complex The complex [Pt(Met)(DMSO)Cl]Cl (Scheme 1) was prepared by the reaction of cis-[PtCl2(DMSO)2] and Met in aqueous methanol. The computational methods, molar conductivity, elemental analysis, 1H NMR, FT-IR, UV–Vis spectra assignments confirm the preparation of the complex. The data of elemental analysis are shown in Table 1. In order to obtain information about the coordination mode of the ligand, FT-IR spectra of the complex and the free ligand were recorded and the data are shown in Table 2. In the complex spectrum a band at 1682 cm1, assigned to m(C@N), shifted to higher wave numbers as compare with ligand. These modifications indicate the involvement of the N2 and N4 atoms in coordination [20]. It is interesting to note that a band of medium intensity appears at 1300 cm1 in the platinum complex. This band is not present in the spectrum of the free metfromin. Hence, it could be

Scheme 1. The structure of [Pt(Met)(DMSO)Cl]Cl.

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Table 1 Elemental analysis data for platinum(II) complex. [Pt(Met)(DMSO)Cl]Cl

Theoretical calculation

Found

%C %N %H

15.22 14.79 3.59

15.38 14.91 3.64

tentatively attributed to the formation of the chelate ring on complexation or to the activation of some IR inactive band as a result of complexation [20]. The sharp absorption band at 1102 cm1 indicates S-bonded DMSO, as reported for this soft coordination center [21]. 1 H NMR spectra of Met and Pt(II) complex were recorded at room temperature in d6-DMSO as the solvent and the data are shown in Table 3. The 1H NMR spectra of all N,N-dimethylbiguanidium derivatives exhibit a pattern correlated with the isolated methyl protons in the region 2.9–3.1 ppm. Additional resonance arise from the amine and imines protons at d = 6.8 and d = 7.22 ppm, respectively. The 1H NMR spectrum of the complex exhibits a pattern assignable to methyl protons at 3.03 ppm. The shift of the broad peak of ligand assigned to imine protons from 7.2 ppm to downfield (7.53 and 9.76 ppm) in the spectrum indicates their coordination through N2 and N4 atoms. The peak of imine proton at 9.76 ppm assigned to a hydrogen bond with oxygen atom of DMSO group bonded to platinum. The peak at 2.54 assigned to methyl protons of coordinated DMSO in complex. Electronic spectra of the complex and the metformin ligand were recorded in Tris–HCl buffer with 104 M/L. Met exhibits an intense absorption band around 233 nm which is assigned to p ? p transition of drug while in the electronic absorption spectrum of platinum complex this band shifted to 228 nm. This band shows a blue shift and decreasing for the absorbance intensity in the complex (Fig. S1). Cis-[Pt(Cl)2(DMSO)2] exhibit an absorption band around 215 nm that absorption band of the platinum complex shifted to higher wavelength as compared with cis-platin (Fig. S2). New absorption band appeared around 316 nm can be attributed to the ligand-to-metal charge transfer bands from the electronic lone pairs of respective nitrogen to the metal ion [22]. The conductivity of metformin complex was measured in DMF solvent at room temperature. The complex showed the molar conductance value for 103 mol/L concentration equal to 65 X1 cm2 mol1. According to the literature data the complex is a 1:1 electrolyte as shown by its molar conductivity (KM) measurements in DMF [23]. The presence of chloride ion in metformin complex was confirmed by the addition an equimolar amount of silver nitrate to the complex (both 105 M) and leading to the appearance of white precipitate.

Computational method The calculations on the structure of the complex [C6H17ClN5OPtS]Cl (1) were performed by the appropriate theoretical method. The structure of [C6H17ClN5OPtS]Cl(1) was obtained by PM6 method [24]. The point group of [C6H17ClN5OPtS]Cl(1) is C1. The QM/ PM6 method was utilized, because of platinum at the structure of 1. Platinum was defined and identified by the Hamiltonians of PM6. The calculations have performed by Spartan ‘10 package [24]. The results of the calculations were demonstrated that the complex [C6H17ClN5OPtS]Cl (1) has a planar structure around the ‘‘Pt’’ (Fig. 1a). The DMSO molecule was oriented near to the N1AH17. An electrostatic attraction (hydrogen bond) was identified between oxygen in DMSO (O1) and H17 of the imine group (O1AH17). The heat of formation and the dipole moment of this

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Table 2 FT-IR data for free ligand and its Pt complex (in cm1). Compound

mas (NH2)

m (NH)

ms (NH2)

t (C@N)

m (chelate ring)

t (S-bonded DMSO)

Metformin Pt Complex

3372 3433

3299 3310

3162 3197

1626 1682

– 1300

– 1102

Table 3 Selected 1H NMR spectral data (in ppm). Compound

NH2

NH (amine)

NH (imine)

CH3 (Met)

CH3 (DMSO)

Metformin Pt Complex D

6.8 7.28 0.48

6.8 6.75 0.25

7.2 7.53, 9.76 0.33, 2.56

2.9 3.03 0.13

2.54

configuration were 276.309 kJ mol1 and 2.18 debye, respectively. The calculated IR spectrum for the complex [C6H17ClN5OPtS]Cl(1) was shown in Fig. 1b. The calculated IR data have shown very good agreement with the experimental results for the identified configuration. Study on the calculated vibration frequencies by PM6 method has shown that the signal of 2759 cm1 and 2683 cm1 were related to N2AH19 and N1AH17 stretching,

respectively. The signal of 1623 was related to the imine C3@N2 and C4@N1. The PM6 calculated signals at 322 and 792 cm1 have determined for the stretching vibrations of Pt1ACl1 and Pt1AS1, respectively. DNA binding studies Dialysis Transition metal complexes can bind to DNA via covalent interactions (irreversible binding), and/or non-covalent interactions (reversible binding) which include intercalative, electrostatic and groove (surface) binding of metal complexes along outside of DNA helix, along major or minor groove. So, at first we determined nature of the binding between Pt(II) complex and DNA. Competition dialysis is a powerful new tool for the discovery of ligands that

Fig. 1. (a) The most stable configuration of platinum complex. (b) The calculated IR spectrum for platinum complex.

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bind to nucleic acids. The method is based on firm thermodynamic principles and is simple to implement [25]. In this study, we used dialysis to discover the binding mode between the complex and DNA. If the nature of binding between DNA and platinum complex be covalent we expect that the absorbance or emission of the DNA– Pt complex does not show a lot of change before and after dialysis because the nature of the binding is irreversible and if the nature of the binding be non-covalent, the absorbance or emission of the DNA–Pt complex changes before and after dialysis according to separating Pt complex from DNA because the nature of the binding is reversible. For this aim, we studied absorbance/emission changes of cis-platin (cis-Pt(NH3)2Cl2) (covalent binder), Hoechst (groove binder) and metformin platinum complex in this way. Fig. S3 shows the absorbance of the cis-Pt(NH3)2Cl2–DNA complex does not change before and after dialysis. Fig. S4 shows that the emission of the Hoechst-DNA complex has a change after the dialysis according to separating of Hoechst from DNA, because emission of that after dialysis decreases, this shows that a lot of the Hoechst were separated from DNA. For the [Pt(Met)(DMSO)Cl]Cl complex (Fig. S5) absorbance of the platinum complex–DNA before and after dialysis show changes according to separating high amount of the platinum complex from DNA. So, the nature of the binding between the platinum complex and DNA is reversible and belongs to non-covalent interaction. UV–Vis absorption studies Monitoring the effect of adding increasing amounts of DNA on the absorption spectrum of a metal complex is one of the most widely used methods for determining overall binding constants. ‘‘Hyperchromic’’ and ‘‘hypochromic’’ effects are the spectra features of DNA with regard to its double helical structure. As shown in Fig. 2, upon adding CT DNA to [Pt(Met)(DMSO)Cl]Cl complex in Tris HCl buffer solution, the absorption band exhibits hyperchromism without shifts in band position. The Pt(II) complex can bind to the double-stranded DNA in different binding modes on the basis of their structure, charge and type of ligand. As DNA double helix possesses many hydrogen bonding sites which are accessible both in the minor and major grooves, it is likely that the NH groups of complex forms hydrogen bonds with N of adenine or O of thymine in the DNA, which may contribute to the hyperchromism observed in the absorption spectra. On the other hand, our Pt(II) complex which possesses methyl groups of the DMSO, can bind to DNA by van der Waals interaction between the methyl groups and the thymine methyl group [26]. The hyperchromic effect may also be due to the electrostatic interaction [27] between positively charged Pt(II)complex and the negatively charged phosphate backbone at the periphery of the double

Fig. 2. Absorption spectra of [Pt(Met)(DMSO)Cl]Cl complex (7.5  105 M) in the absence and presence of increasing amounts of CT-DNA: (ri = 0.0, 0.2, 0.6, 0.8 and 1.4).

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helix ct-DNA [28]. Also a coordination bond of DNA base with platinum can occur through replacement of the chloride ligand in the complex, probably through aquation as has been observed with cis-platin [29]. No significant shift in the UV absorption band was observed, which implies that there is no change of the coordination environment of the metal ion [30], covalent/coordinate linkage to the N(7) of nucleobase is strongly ruled out. The intrinsic binding constant, Kb, of Pt(II)complex with calf thymus DNA was calculated from Wolfe–Shimmer equation:

½DNA

ea  ef

¼

½DNA

eb  ef

þ

1 Kb

eb  ef




where [DNA] is the concentration of DNA in base pairs, the apparent absorption coefficient ea, ef and eb correspond to Aobsd/[Complex], the extinction coefficient for the free Pt(II) complex, for each addition of DNA to the Pt(II) complex and Pt(II) complex in the fully bound form, respectively. In plot of [DNA]/(ea  ef) versus [DNA], Kb (1.5  104 M1) is given by the ratio of slope to the intercept. The observed binding constant is more in keeping with groove binding, as observed in the literature [31–34]. Our results illustrate that platinum complex may bind to DNA via groove binding. Effect of ionic strength on the spectrum of Pt(II) complex–DNA Monitoring the change of ionic strength is an efficient method to recognize the binding modes between molecules and DNA. Increasing the concentration of cation will increase the complexation probability between the cation and DNA phosphate backbone. Due to a competition for phosphate anion, the addition of the cation will weaken the surface-binding interactions which include electrostatic interaction and hydrogen binding between DNA and molecules [35]. In order to prove whether Pt(II) complex provide an electrostatic binding or other kinds of binding with DNA, Pt(II) complex solutions was titrated with NaCl in the absence and presence of DNA (3  105 M). Pt(II) complex was titrated with NaCl with the increasing concentration of the salt from 5  107 up to 1.9  104 M1. As it is elucidated from Fig. 3, the slopes of the curves in the absence and presence of DNA are the same. It is deduced that NaCl has no effect on DNA binding with Pt(II) complex. Therefore, the results indicated that Pt(II) complex does not give an electrostatic or outside binding with DNA. Effect of Pt(II) complex on the absorption spectrum of NR–DNA system It has been shown that an absorption spectrum in the visible region of the NR solution is characterized by a band at 464 nm. This absorption gradually decreased with the increasing concentration of DNA, and a new band at 525 nm developed. An isosbestic point at 497 nm provided evidence of the new NR–DNA complex formation (Fig. 4). In general, hypochromism and red shift are commonly associated with molecular intercalation into the base stack of the DNA [36]. These two observed spectral effects were ascribed to a strong interaction between the electronic state of the intercalating

Fig. 3. Effect of ionic strength on the absorbance spectrum of the Pt(II) complex in the absence (j) and presence () of the DNA.

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Fig. 4. Absorption spectra of NR in the presence of DNA at different concentrations. Conditions: [NR]: 3  105 mol L1, [DNA] (105 mol L1): 0.0, 1.5 and 3.6.

chromophore and that of the DNA bases. The strength of this electronic interaction is expected to decrease as the cube of the distance between the chromophore and the DNA bases [36]. The observed large spectral hypochromism during the interaction of the NR dye with the DNA strongly proposed that the distance between the intercalated NR dye and the DNA bases was small. The effects of Pt(II) complex on the absorption spectrum of DNA–NR system were measured. As shown in Fig. 5, the absorption of DNA–NR complex decreases with the increasing concentration of Pt(II) complex. This indicates that the complex ion cannot exchange with the intercalated NR, because the spectrum of the free NR ligand is not restored.

Fig. 6. The changes of CD spectrum of 8  105 M DNA in the absence and presence of addition of Pt(II) complex.

including the conversion from a more B-like to a more C-like structure within the DNA molecule. These changes are indicative of a non-intercalative mode of binding of the complex and offer support to the groove binding nature [39].

Circular dichroism spectroscopy Circular dichroic spectral technique is useful in diagnosing changes in DNA morphology during drug–DNA interactions as the CD signals are quite sensitive to the mode of DNA interactions with target molecules. A solution of CT-DNA exhibits a positive band (275 nm) from base stacking interactions and a negative band (245 nm) from the right-handed helicity of DNA. These bands are sensitive towards binding of any small molecule or drug and hence changes in CD signals of DNA observed on interaction with drugs may often be assigned to the corresponding changes in DNA structure [37]. Simple groove binding and electrostatic interaction of small molecules show less or no perturbation on the base-stacking and helicity bands, while intercalation enhances the intensities of both the bands stabilizing the right-handed B conformation of CT DNA as observed for the classical intercalator methylene blue [38]. The effect of the Pt complex on DNA structure is evaluated by circular dichroism spectroscopy and the results were shown in Fig. 6. As it is observed the intensities of both the negative and positive bands decrease (shifting to zero levels). This suggests that the DNA binding of the complex induces conformational changes,

Fluorescence studies No luminescence is observed for the complex; it is hard to monitor the interaction of this complex with DNA by employing direct fluorescence emission methods but actually possible by using a fluorescence assay of the organic molecule probe. We used neutral red probe for the interaction of platinum complex with DNA. On addition of NR to DNA solution, it is found that the fluorescence intensity of the system is greatly enhanced. Thus, NR is a sensitive probe to study the interaction between DNA and drug molecules. When Pt(II) complex is added to the solution of DNA– NR complex, the fluorescence intensity of DNA–NR complex decrease with the increasing concentration of Pt(II) complex. The fluorescence spectra are shown in Fig. 7. There are three possible reasons considering the decrease of the fluorescence intensity for DNA–NR system upon addition of Pt(II) complex. Firstly, the binding between NR and Pt(II) complex might occur and the intensity of DNA–NR complex decreases. Secondly, Pt(II) complex competes against NR in binding with DNA and excludes the intercalated NR from the duplex, so the concentration of NR binding with DNA reduces. This competition binding generally occurs between the two intercalative binders. Thirdly, Pt(II) complex binding with DNA–NR forms a new non-fluorescence complex, DNA–NR–Pt(II) complex, which causes the fluorescence quenching of DNA–NR. In this

Fig. 5. Effect of the Pt(II) complex on the absorption spectrum of DNA–NR. [NR]: 3  105 mol L1; [DNA]:6  105 mol L1 [Pt(II) complex] (105 mol L1): 0.98, 1.45, 2.42, 4.28 and 7.8.

Fig. 7. Fluorescence spectra of the competition between Pt(II) complex and Neutral red. CPt(II) complex = 0, 3.37  105, 6.5  105, 1.11, 1.59, 2.15, 2.74 and 3.39  104 6 mol L1 for curves 1–8, CNuetral mol L1, and red = 5.0  10 CDNA = 4.97  105 mol L1.

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experiment, it is found that Pt(II) complex does not react with NR when DNA is absence. So, the first reason is not true of interpreting the decrease of DNA–NR intensity. In addition, the binding constant of NR with DNA is greater than that of the platinum complex. Pt(II) complex can hardly scramble for the binding sites on DNA with NR. Besides, in the UV–Vis experiment, it has been already indicated that the intercalated NR molecules are not restored with addition of Pt(II) complex to DNA–NR system. So, the second reason also seems not real reason. Then, the third reason above mentioned seems to be more reasonable, namely the decrease in the fluorescence of the system is due to the quenching of DNA–NR complex by the bound Pt(II) complex [40]. Quenching can occur by different mechanisms, which are usually classified as dynamic and static quenching. Dynamic quenching refers to a process in which the fluorophore and the quencher come into contact during the transient existence of the excited state, while static quenching refers to fluorophore–quencher complex formation. In general, dynamic and static quenching can be distinguished by their differing dependence on temperature and excited-state lifetime. Since in both cases the fluorescence intensity is related to the concentration of the quencher, the quenched fluorophore can serve as an indicator for the quenching agent [41]. Fluorescence quenching is described by the Stern–Volmer equation:

F0 ¼ 1 þ K q s0 ½Q ¼ 1 þ K sv ½Q  F where F0 and F represent the fluorescence intensities in the absence and in the presence of quencher, respectively. Kq is the fluorophore quenching rate constant, Ksv is quenching constant, s0 is the lifetime of the fluorophore in the absence of a quencher (s0 = 108), and [Q] is the concentration of quencher [42]. Dynamic and static quenching can be distinguished by their different dependence on temperature [43]. The results in Table 4 indicate that the probable quenching mechanism of Pt(II) complex by CT-DNA involves static quenching, because Ksv decreases with increasing temperature [44]. Binding constant and the number of binding sites. The binding constant (Kf) and the binding stoichiometry (n) for the complex formation between platinum complex with DNA were measured using the following equation [45]:

logðF 0  FÞ=F ¼ log K f þ n log½Q Here F0 and F are the fluorescence intensities of the fluorophore in the absence and in the presence of different concentrations of [Q], respectively. The values of Kf and n are shown in Table 5. Thermodynamic studies. The interaction forces between drug and biomolecule may involve hydrophobic forces, electrostatic interactions, van der Waals interactions, hydrogen bonds, etc. [46]. According to the data of enthalpy changes (DH) and entropy changes (DS), the model of interaction between drug and biomolecule can be concluded [47]: (1) DH > 0 and DS > 0, hydrophobic forces; (2) DH < 0 and DS < 0, van der Waals interactions and hydrogen bonds; (3) DH < 0 and DS > 0, electrostatic interactions [48]. When there is little change of temperature, the enthalpy change (DH) can be seen as a constant, and then its value and that

Table 4 The quenching constants of platinum complex by CT-DNA at different temperatures.

Table 5 Binding constants (Kf) and number of binding sites (n) of the DNA-complex system. T (K)

R2

n

log Kf

Kf

283 298 310

0.994 0.991 0.998

1.116 1.179 1.248

3.524 3.727 3.969

3.31  103 5.32  103 9.34  103

of entropy changes (DS) can be determined from the van’t Hoff equation:

Ln K ¼ 

DH DS þ RT R

where K is the binding constant at the corresponding temperature and R is gas constant. The values of DH and DS were obtained from the slope and intercept of the linear plot (van’t Hoff equation) based on ln K versus 1/T. The free energy change (DG) was estimated from following equation:

DG ¼ DH  T DS The values of DH, DS and DG between Pt(II) complex with DNA are listed in Table 6. It can be seen that the negative value of DG revealed the interaction process is spontaneous, the positive DH and DS values indicated that hydrophobic forces play main roles in the binding of Pt(II) complex to DNA. Viscosity measurements Optical photophysical probes generally provide necessary, but not sufficient clues to support binding mode. The hydrodynamic measurements are sensitive to the length change of DNA in absence and presence of foreign molecules. A classical intercalation model demands that the DNA helix must lengthen as base pairs are separated to accommodate the binding ligand, leading to increase in DNA viscosity [49]. In contrast, a partial and/or non-classical intercalation ligand could bend (or kink) the DNA helix, reduce its effective length and concomitantly its viscosity, while ligands that bind exclusively in the DNA grooves (e.g., netropsin, distamycin), under the same conditions, typically cause less pronounced changes (positive or negative) or no changes in DNA solution viscosity [50]. The values of relative specific viscosity (g/g0)1/3, here g0 and g are the specific viscosity contributions of DNA in the absence and in the presence of the Pt(II) complex were plotted against ri (ri = [complex]/[DNA]) (Fig. 8). For the platinum complex at lower loading, the viscosity increases and then fixes at higher loadings. In principle, this could be explained by changes in conformation, flexibility or solvation of the DNA molecule. The slow increase in viscosity is an indication of groove binding [51]. Cleavage of pUC18 plasmid DNA by metformin ligand and its platinum complex It is known that DNA cleavage is controlled by relaxation of supercoiled circular conformation of pUC18 DNA, nicked circular and/or linear conformations. When electrophoresis is applied to circular plasmid DNA, the relatively fast migration is related to the intact supercoiled form (Form I). If one strand is cleaved, the supercoil will be relaxed to produce a slower-moving nicked

Table 6 Thermodynamic parameters for the binding of Pt(II) complex to CT-DNA.

T (K)

R2

Ksv (L mol1)  103

Kq (L mol1)  1011

T (K)

DG (kJ mol1)

DH (kJ mol1)

DS (J mol1 K1)

283 298 310

0.980 0.981 0.987

1.463 1.445 1.381

1.463 1.445 1.381

283 298 310

18.99 21.46 23.42

27.33 27.33 27.33

163.71 163.71 163.71

384

N. Shahabadi, L. Heidari / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 377–385

[56]. The binding constant for the platinum complex estimated from equation below [57]:

log



   1 I ¼ log K þ log ½DNA I0  I

where K is the binding constant, I0 and I are the peak currents of the free guest and the complex, respectively. The binding constant with a value of 1.86  103 was obtained from the intercept of the plot of log (1/[DNA]) versus log (I/(I0  I)). Conclusions Fig. 8. Effect of increasing amounts of platinum complex on the viscosity of CT-DNA (5  105 M) in 50 mM in Tris–HCl buffer (ri = 0.0, 0.2, 0.4, 0.6, 0.9 and 1.0).

conformation (Form II). If both strands are cleaved, a linear conformation (Form III) will be generated which migrates in between [52,53]. The present study with agarose gel electrophoresis demonstrating that compared with DNA control (Lane 1), the patterns of Lanes 2–6, does not show any evidences of DNA cleavage (Fig. 9).

Differential pulse voltammetry (DPV) DPV technique provides higher sensitivity and better peak resolution compared to CV for studying the electrochemical behavior of biological systems [54]. Voltammogram of the platinum complex in the absence and presence of DNA is shown in Fig. 10. In the absence of CT-DNA, the voltammogram featured an anodic peak (Epa 1.125 V). In this experiment it can be seen that the anodic peak currents decreased gradually with the addition of DNA. The decrease in current may be attributed to the diffusion of the complex bound to the large, slowly diffusing DNA molecule. The decreases in the peak currents are ascribed to the stronger binding between the complexes and DNA [55]. Also during DNA addition, the anodic peak potential (Epa), showed positive shifts. These positive shifts are considered as evidences for hydrophobic interaction that this result is in agreement with fluorescence measurements

Fig. 9. Gel electrophoresis of pUC18 in the presence of increasing amounts of Pt(II) complex (ri = 0, 0.1, 0.3, 0.5,0.7 and 0.9).

Fig. 10. Differential pulse voltammogram of 1 mM platinum complex on a polished glassy carbon electrode in the absence and presence of different concentrations of DNA.

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