Voltammetric and spectroscopic investigation of binding in complexes of divalent metal ions with styrene–maleic acid-copolymer and monomeric analogues

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  Voltammetric and spectroscopic investigation of binding in complexes of divalent metal ions with styrene–maleic acid-copolymer and monomeric analogues
  Spectrochimica Acta Part A 75 (2010) 1082–1087 Contents lists available at ScienceDirect SpectrochimicaActaPartA:MolecularandBiomolecularSpectroscopy  journal homepage: www.elsevier.com/locate/saa Voltammetric and spectroscopic investigations of 4-nitrophenylferroceneinteracting with DNA Afzal Shah, Muhammad Zaheer, Rumana Qureshi ∗ , Zareen Akhter, Muhammad Faizan Nazar Department of Chemistry Quaid-i-Azam University, 45320 Islamabad, Pakistan a r t i c l e i n f o  Article history: Received 10 April 2009Received in revised form15 December 2009Accepted 18 December 2009 Keywords: 4-Nitrophenylferrocene (NFC)DNABinding constantDiffusion coefficientBinding site sizeBinding free energy a b s t r a c t Cyclic voltammetry (CV) coupled with UV–vis and fluorescence spectroscopy were used to probe theinteractionofpotentialanticancerdrug,4-nitrophenylferrocene(NFC)withDNA.Theelectrostaticinter-action of the positively charged NFC with the anionic phosphate of DNA was evidenced by the findingslike negative formal potential shift in CV, ionic strength effect, smaller bathochromic shift in UV–visspectroscopy, incomplete quenching in the emission spectra and decrease in viscosity. The diffusioncoefficients of the free and DNA bound forms of the drug were evaluated from Randles–Sevcik equation.The binding parameters like binding constant, ratio of binding constants ( K  red / K  ox ), binding site size andbindingfreeenergyweredeterminedfromvoltammetricdata.Thebindingconstantwasalsodeterminedfrom UV–vis and fluorescence spectroscopy with a value quite close to that obtained from CV. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Theclinicaluseof‘cisplatin’asantineoplasticagentanditsnega-tivesideeffectsstimulatedthechemiststoconcentratetheireffortsontheuseofalternativetransitionmetalbaseddrugsasanti-tumoragents with positive, low or no side effects [1]. In their strugglefor searching about such effective anticancer drugs, tremendousattention was paid to ferrocene and its derivatives in view of theirpotentialapplicationsinmedicalfield[2,3].Ithasbeenestablishedthat ferrocenes have appreciable antineoplastic activity [4–6] butthe mechanism of their effectiveness in eliciting an anti-tumoreffectisstilltobeexplored[7].ThemodeofinteractionandbindingextentofferroceneswithDNAaremattersofconsiderableinterestdue to the complexity of their sandwich like structures.Ferrocenes represent a hybrid area between organometallicchemistry and biochemistry. In these molecules, ferrocene moietyserves as spectroscopically active chromophore, biological markerand redox active site. They are the subject of intensive investi-gations due to their broad range properties such as variation inthe substituents at the cyclopentadienyl rings, accessible poten-tialrange,establishedantiproliferativeeffects,thermodynamicandkinetic characteristics [8,9].Drug-DNAinteractionshavebeenstudiedbyavarietyofanalyti-caltechniquessuchasluminescence[10],fluorescence[11],UV–vis ∗ Corresponding author. Tel.: +92 5190642142; fax: +92 512873869. E-mail address:  r qureshy@yahoo.com (R. Qureshi). spectroscopy [12], and voltammetric methods [13–15]. Among theseelectrochemicalmethodsarewidelyusedindrug–DNAbind-ing studies due to their advantages like high sensitivity, efficientselectivity, cost affectivity, more reliability, extensive versatilityand fast detection ability. The UV–vis spectroscopic technique isalsobestsuitedtoferrocenesowingtotheirintensecolors.Spurredby the clinical use of anticancer ferrocifen [16], we synthesized 4-nitrophenylferrocene (Scheme 1), and studied its interaction withchicken blood DNA by electrochemical, spectroscopic and visco-metrictechniques.Otherfactorsbehindtheselectionofthisspecificferrocenederivativewereitseasysyntheticroute,chemicalstabil-ity and attractive electrochemistry. The aim of the present studyis to provide useful insights in further understanding of the unre-solved mechanism of drug–DNA interactions. 2. Experimental  2.1. Materials 4-Nitrophenylferrocene(NFC)wassynthesizedaccordingtotheliteraturereportedmethod[17].Its10mMstocksolutionwaspre-pared in 10% aqueous ethanol (10% H 2 O:90% ethanol). The systemwas buffered at pH 6 by phosphate buffer (0.1M KH 2 PO 4 +0.1MNaOH) to avoid the decomposition of the ferrocenium state of thedruginabasicorevenneutralsolution[18]andprotonationoftheferrocenyl group in strongly acidic conditions [19]. Tetrabutylam-moniumperchlorate(TBAP)(Fluka,99%purity)wasfurtherpurifiedbyrecrystallizationusingmethanolasasolvent.DNAwasextracted 1386-1425/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2009.12.061   A. Shah et al. / Spectrochimica Acta Part A 75 (2010) 1082–1087  1083 Scheme 1.  Molecular structure of 4-nitrophenylferrocene (NFC). from chicken blood by the method mentioned in our previouspapers [20,21]. Its stock solution was prepared in doubly distilledwater and stored at 4 ◦ C. The concentration of the stock solution of CB-DNA(200  Minnucleotidephosphate,NP)wasdeterminedbyUVabsorbanceat260nmusingthemolarextinctioncoefficient( ε )of6600M − 1 cm − 1 [22].ForkeepingthesolventofthedrugandDNAthe same, the aqueous solution of DNA was diluted (with ethanol)to 10% aqueous ethanol.The nucleotide to protein (N/P) ratio of 1.85 was obtainedfrom the ratio of absorbance at 260 and 280nm (  A 260 /  A 280 =1.85),evidenced for protein free DNA [23]. All other reagents were of analytical grade. Doubly distilled water and absolute ethanol wereused for all solutions.All the experiments were conducted in 0.1M phosphate buffer(pH6)at25 ◦ C.Alltheresultsweretheaverageofthreeexperimen-tal measurements.  2.2. Apparatus and procedures Voltammetric experiments were performed using PGSTAT 302with Autolab GPES version 4.9 Eco Chemie, Utrecht, the Nether-lands. Measurements were carried out in a conventional threeelectrodecellwhichconsistedofsaturatedcalomelelectrode(SCE)from Fisher scientific company (cat no. 13-639-51) as a referenceelectrode,athinPtwireofthickness0.5mmwithanexposedendof 10mm as the counter electrode and a bare glassy carbon electrode(GCE)withageometricareaof0.071cm 2 astheworkingelectrode.Prior to experiments, the GCE was polished with 0.25(m diamondpaste on a nylon buffing pad. For electrochemical measurementsthe test solution was kept in an electrochemical cell (model K64PARC) connected to the circulating thermostat LAUDA model K-4R. The voltammogram of a known volume of the test solutionwas recorded in the absence of DNA after flushing out oxygen viapurgingargongasfor10min.Theprocedurewasthenrepeatedforsystems with constant concentration of the drug and varying con-centration of DNA. The working electrode was cleaned after everyelectrochemical assay.Absorption spectra were measured on a UV–vis spectrometer;Shimadzu 1601. For the extraction of DNA, Table Top Centrifuge,Model PLC-05 (Taiwan) was used.The electronic spectrum of a known concentration of the drugwasobtainedwithoutDNA.Thespectroscopicresponseofthesameamount of the drug was then monitored by the addition of smallaliquots of DNA solution. All of the samples were allowed to equi-librate for 5min prior to every spectroscopic measurement.The viscosity measurements were carried out by Anton PaarStabinger Viscometer SVM 3000. A series of solutions were madewith constant concentration of DNA and varying concentration of NFC. The values of relative specific viscosity (  /  0 ) were plotted versus [NFC]/[DNA]ratio,where  and  0  representtheviscosityof DNA solution with and without NFC.The steady-state fluorescence spectra were recorded using aPerkin Elmer LS 55 Luminescence Spectrometer, with an externaltemperature controlled cell holder at a temperature of 25 ± 1 ◦ C.The fluorescence emission spectrum of NFC (excitation at 328nm) Fig. 1.  Cyclic voltammograms of 3.25mM NFC on a polished GC electrode in theabsence (a) and presence of 30  M DNA (b) in 10% aqueous ethanol (10% H 2 O:90%ethanol).   : 100mVs − 1 , buffer: 0.1M phosphate buffer (pH 6), supporting elec-trolyte: 0.1M TBAP. was used to obtain the intensities of the peaks ( I  ). Good resolu-tion of the bands was obtained at the slit width (ex. 10.0nm; em.10.0nm). The scan range used was from 350 to 600nm. The PhotoMultipliertubevoltagewaskeptat665V.TheconcentrationofNFCwas 100  M. 3. Results and discussion  3.1. Voltammetric studies of NFC–DNA interaction Thecyclicvoltammetricbehaviorof3.25mMNFCintheabsenceand presence of 30  M DNA at bare GCE is shown in Fig. 1. Thevoltammogram without DNA (Fig. 1a) featured a couple of welldefined and stable redox peaks in the potential range of 0.0–1.0V.Theanodicandcathodicpeakswereappearedat0.614and0.429V versus  SCE with a formal potential ( E  0f   ) of 0.520V, while for sim-ple ferrocene, oxidation peak appeared at 0.518V under the sameconditions.Thedifferenceof96mVintheanodicpeakpotentialsof NFC and ferrocene is attributed to the electron withdrawing effectof p-nitrophenyl substituent attached to the cyclopentadienyl ringofferrocene,whichrendersitsoxidationdifficult.Theelectrochem-ical signal at 0.614V reflects the oxidation of the ferrocenyl groupofNFCtoferroceniumstate,whichgetsreducedtoitsneutralformuponscanreversal.Thelargepeaktopeakseparationmaybeduetokinetic complications. The anodic and cathodic peak current ratio( i pa / i pc ) of about 1 is suggestive of reversible electrochemical pro-cess.By the addition of 30  M DNA into 3.25mM drug (Fig. 1b) theanodic peak potential was shifted by 24.40mV in the negativegoing direction and  i pa  was dropped by 33.55%. The substantialdiminutioninpeakcurrentisattributedtotheformationofslowlydiffusingNFC–DNAsupramolecularcomplexduetowhichthecon-centration of the free drug (mainly responsible for the transfer of current) is lowered.Themodeofdrug–DNAinteractioncanbejudgedfromthevari-ationinformalpotential.Ingeneralthepositiveshift(anodicshift)in formal potential is caused by the intercalation of the drug intothe double helical structure of DNA [24], while negative shift isobserved for the electrostatic interaction of the cationic drug withtheanionicphosphateofDNAbackbone[25].Sotheobviousnega-tivepeakpotentialshift(cathodicshift)intheCVbehaviorofNFCbytheadditionofDNAisattributabletotheelectrostaticinteractionof the positively charged nitrogen and ferrocenium state of NFC withthepolyanionicDNA.Thecathodicpeakpotentialshiftfurtherindi-  1084  A. Shah et al. / Spectrochimica Acta Part A 75 (2010) 1082–1087  Scheme 2.  General redox process of the free and DNA bound NFC. cates that Fe (II) of NFC is easier to oxidize in the presence of DNAbecause its oxidized form is more strongly bound to DNA than itsreduced form (neutral form). For such a system, where both formsofthedruginteractwithDNA,Scheme2canbeapplied[26].Based upon the process discussed in Scheme 2, the following equation isobtained [27]: E  0b  − E  0f   = 0 . 059log  K  red K  ox   (1)where  E  0f   and  E  0b  are the formal potentials of the NFC (II)/NFC (III)couple in the free and bound forms respectively.For a shift of   − 14.5mV caused by the addition of 20  M DNAinto 3.25mM NFC (Fig. 2b) a ratio of   K  red / K  ox  was calculated as0.57,whichindicates1.75timesstrongerinteractionoftheoxidizedform of the drug with DNA than the reduced form.Based upon the decrease in peak current of NFC by the addi-tion of different concentration of DNA, ranging from 20 to 60  M(Fig. 2), the binding constant was calculated according to the fol-lowing equation [28]:1[DNA]  = K  (1 −  A )1 − ( i/i 0 )  − K   (2)where K  isthebindingconstant, i and i 0  arethepeakcurrentswithand without DNA and  A  is the proportionality constant.The plot of 1/[DNA]  versus  1/(1 − i / i 0 ) (Fig. 3) yielded K  =3.85 × 10 3 M − 1 , which is greater than the binding constant( K  =3.45 × 10 2 M − 1 ) of protonated ferrocene with DNA as reportedin our previous paper [29].Forthedeterminationofbindingsitesizethefollowingequationwas used [15]: C  b C  f  = K   [free base pairs] s   (3)where  s  is the binding site size in terms of base pairs. MeasuringtheconcentrationofDNAintermsof[NP],theconcentrationofthe Fig. 2.  Cyclic voltammograms of 3.25mM NFC in the absence of DNA (a) and pres-ence of 20  M (b), 30  M (c), 40  M (d), 50  M (e), and 60  M DNA (f). Fig. 3.  Plot of 1/1 − i / i 0  vs . 1/[DNA] for 3.25mM NFC with varying concentration of DNA ranging from 20 to 60  M in a medium buffered at pH 6, used to calculate thebinding constant of NFC–DNA adduct. base pairs can be expressed as [DNA]/2. So Eq. (3) can be writtenas: C  b C  f  = K   [DNA]2 s   (4) C  f   and  C  b  denote the concentration of the free and DNA-boundspecies respectively.The  C  b / C  f   ratio was determined by the equation given below[14]: C  b C  f  = i 0 − ii  (5)where  i  and  i 0  represent the peak currents of the drug in the pres-ence and absence of DNA.Putting the value of   K  =3.85 × 10 3 M − 1 as calculated accordingto Eq. (2), the binding site size of 0.9bp was obtained from theplot (Fig. 4) of   C  b / C  f   versus  [DNA]. The small value of s indicateselectrostaticinteractionofNFCwithDNA.SuchaninteractionmayinduceperturbationinthenormalfunctioningofDNAwhichcouldpresumablyculminateinthepreventionofreplicationandultimatecell death. 4. Effect of ionic strength on the interaction of NFC withDNA  The influence of the ionic strength on the binding properties of NFC with DNA was also assessed at various NaCl concentrations.Thepeakcurrentof3.25mMNFCinthepresenceof5  MDNAwas Fig. 4.  Plot of   C  b / C  f   vs . [DNA] for the determination of binding site size.   A. Shah et al. / Spectrochimica Acta Part A 75 (2010) 1082–1087  1085 Fig. 5.  Effect of ionic strength on NFC–DNA interaction, indicated by the decreasein  i pa  of 3.25mM NFC containing 5  M DNA with the increase in concentration of NaCl. gradually decreased with the increasing concentration (0–30mM)of NaCl (Fig. 5). This behavior signifies the electrostatically driveninteraction of NFC with DNA as the ionic environment can affectthe electrochemical interactions by ionic screening effect. At lowionic strength, where the ions are less effectively shielded, largerpeakcurrentsareexpected.Athighionicstrength,duetotheeffec-tive ionic shielding, the peak currents are expected to diminish.Intheelectrostaticmodeofinteraction,wherethedrugremainsincontactwiththemedium,theeffectoftheionicstrengthisconceiv-able.While,intheintercalativemodeofbinding(in-bindingmode),where the drug goes away from the solvent and inserts itself intothebasepairpockets,theionicstrengthofthemediumislesslikelyto affect the voltammetric current signals.To further ascertain the interaction of NFC with DNA,  i pa  wasplotted  versus   1/2 (Fig. 6) before and after the addition of DNA,using Randles–Sevcik expression [30]: i = 2 . 69 × 10 5 n 3 / 2  AC  ∗ 0 D 1 / 2  1 / 2 (6)where i isthepeakcurrent(A),  A isthesurfaceareaoftheelectrode(cm 2 ),  C  ∗ 0  is the bulk concentration (molcm − 3 ) of the electroactivespecies, D isthediffusioncoefficient(cm 2 s − 1 )and  isthescanrate(Vs − 1 ).The linear dependence of the peak currents of both NFC andNFC–DNA on the square root of the scan rate suggests thatthe redox process is kinetically controlled by the diffusion step.The diffusion coefficients of the free ( D f  =1.03 × 10 − 5 cm 2 s − 1 )and DNA bound drug ( D b =7.51 × 10 − 6 cm 2 s − 1 ) were determined Fig. 6.  i vs.   1/2 plots of 3.25mM NFC in the absence of DNA (a) and presence of 50  MDNA(b)atscanratesrangingfrom10to100mVs − 1 undertheexperimentalconditions of  Fig. 1. Fig. 7.  UV–vis absorption spectra of 30  M NFC in the absence of DNA (a) andpresence of 10–60  M DNA (b–g) in 10% aqueous ethanol buffered at pH 6. from the slopes of Randles–Sevcik plots. The lower diffusioncoefficient ( D f  =1.03 × 10 − 5 cm 2 s − 1 ) of the free NFC than the  D f  (1.88 × 10 − 5 cm 2 s − 1 ) of ferrocene [31] is attributed to its compar-atively high molecular weight. Furthermore, the smaller  i pa  versus  1/2 slopeofNFCinthepresenceofDNAascomparedtofreeNFCissuggestiveNFC–DNAadductformation.Thereasonforthedecreasein the apparent diffusion coefficient of NFC in the presence of DNAis the obviously large molecular weight of the adduct. 4.1. UV–vis absorption studies The interaction of NFC with DNA was also studied by UV–visabsorption titration for getting further clues about the mode of interaction and binding strength. The effect of different concen-tration of DNA (10–60  M) on the electronic absorption spectrumof 30  M NFC is shown in Fig. 7. The rationale behind the band inthe UV region (328nm) is the probable charge transfer betweenthe non-bonding or antibonding orbital of the cyclopentadienylring and the iron atom of NFC. The maximum absorption of thedrug at this wavelength exhibited slight bathochromic and pro-nouncehypochromicshiftsbytheincrementaladditionofDNA.Thebathochromic effect is associated with the decrease in the energygapbetweenthehighest(HUMO)andthelowestmolecularorbitals(LUMO)aftertheinteractionofNFCtoDNA.Thecompactnessinthestructureofeitherthedrugaloneand/orDNAaftertheformationof drug-DNA complex may result in hypochromism. Hypochromismdue to DNA contraction has also been reported by Li et al. [32].Based upon the decrease in absorbance, the binding constantwas calculated according to the following equation (7) [33]:  A 0  A −  A 0 = ε G ε H – G − ε G + ε G ε H – G − ε G .  1 K   [DNA] (7)where  K   is the binding constant,  A 0  and  A  are absorbance of thefree drug and the apparent one,  ε G  and  ε H–G  are their absorptioncoefficients respectively.The slope to intercept ratio of the plot between  A 0 /(  A −  A 0 )  ver-sus 1/[DNA]yieldedthebindingconstant, K  =2.02 × 10 3 M − 1 ,whichis close to the value of   K   (3.85 × 10 3 M − 1 ) obtained from CV. Themoderate binding constant is indicative of electrostatic interac-tion. The Gibbs energy change (  G =  − RT  ln K  ) of approximately − 20.45kJ/molat25 ◦ CsignifiesthespontaneityofNFC–DNAinter-action.  1086  A. Shah et al. / Spectrochimica Acta Part A 75 (2010) 1082–1087  Fig. 8.  Effect of increasing concentration of NFC on the relative viscosity of DNA at25 ◦ C. [DNA]=30  M and [NFC]=5–30  M. 4.2. Viscometric studies ViscometrictechniqueisaneffectivetoolinclarifyingthemodeofinteractionofsmallmoleculeswithDNA.Ingeneral,intercalation(in-binding mode) causes an increase in the viscosity of DNA solu-tionduetothelengtheningofDNAhelixasthebasepairpocketsarewidened to accommodate the binding molecule [34]. The reversecan be taken for electrostatic interaction (out-binding mode). Theplotofrelativespecificviscosity(  /  0 ) versus [NFC]/[DNA]isshownin Fig. 8. The plot reveals negative change in   /  0  with increasingconcentration of NFC. Such a behavior is suggestive of electro-staticinteraction,thatmaycausethecompactnessandaggregationof DNA. The aggregation reduces the number of independentlymoving DNA molecules which results in lowering of the solutionviscosity. 4.3. Steady-state fluorescence studies The interaction of NFC with DNA was also examined by fluo-rescence titration. The fluorescence emission spectra of NFC in theabsence and presence of different concentrations of DNA is showninFig.9.TheemissionmaximaofNFCarelocatedat412and440nm.The emission maxima were gradually decreased with the increasein concentration of DNA, indicating the quenching of fluorescenceintensity of NFC upon binding to DNA. The intercalative mode of binding is excluded as the NFC was not completely quenched evenwhen the concentration of DNA was up to 60  M. Thus, the outer-binding mode is further confirmed by the lower rate of quenching Fig.9.  Fluorescenceemissionspectraof100  MNFCintheabsence(a)andpresenceof 10–60  M DNA (b–g) in 10% aqueous ethanol. Fig. 10.  A Stern–Volmer quenching plot for the determination of binding constantof NFC with DNA. in the emission spectra of NFC by the addition of DNA. The bindingconstantwasevaluatedfromthedecreaseintheemissionmaximaat 440nm by the application of Stern–Volmer equation [35]: I  0 I   = 1 + K  [DNA] (8)where  I   and  I  0  are the fluorescence intensities of NFC with andwithout DNA.  K   is the Stern–Volmer binding constant, which isa measure of the efficiency of quenching by DNA. The plot of   I  0 / I versus [DNA]wasconstructed(Fig.10)usingthedatafromfluores-cence titration and a linear fitting of the data yielded the bindingconstant,4.1 × 10 3 M − 1 whichisingoodagreementwiththeresultobtained from cyclic voltammetry and UV–vis spectroscopy. 5. Conclusions The nature of NFC–DNA interaction was examined by CV baseduponthedifferenceintheredoxbehaviorofthedrugintheabsenceandpresenceofDNA,includingtheshiftsintheformalpotentialof the redox couple and the decrease of the peak current due to theremarkable decrease in the diffusion coefficient after binding toDNA. The results of CV and ionic strength effect indicated electro-staticinteractionofNFCwithDNAasthedominantmode.Thesamemode of interaction was also supported by the results obtainedfrom UV–vis spectroscopy, fluorescence and viscosity. The CV andspectroscopicdataconvenientlyallowedtheestimationofbindingparameters as required for the design of new anticancer drugs.  Acknowledgement We are highly grateful to Quaid-i-Azam University and HigherEducation Commission Islamabad, Pakistan for supporting thiswork. References [1] Z. Petrovski, R.P. Norton, S.S. Braga, C.L. Pereira, M.L. Matos, I.S. Goncalves, M.Pillinger, P.M. Alves, C.C. Romao, J. Organomet. Chem. 693 (2008) 675.[2] E.W. Neuse, J. Inorg. Organomet. Polym. Mater. 15 (2005) 3.[3] N.M. Nolte, Nachr. Chem. 54 (2006) 966.[4] R.F. Shago, J.C. Swarts, E. Kreft, C.E. Rensburg, Anticancer Res. 27 (2007) 3431.[5] R.Kovjazin,T.Eldar,M.Patya,A.Vanichkin,H.M.Lander,A.Novogrodsky,FASEB J. 17 (2003) 467.[6] M.D. Maree, E.W. Neuse, E. Erasmus, J.C. Swarts, Metal based Drugs, 2008, pp.10–20.[7] D. Osella, M. Ferrali, P. Zanello, F. Laschi, M. Fontani, C. Nervi, G. Cavigiolio,Inorg. Chim. Acta 306 (2004) 42.[8] E. Hillard, A. Vessieres, L. Thouin, G. Jaouen, C. Amatore, Angew. Chem. Int. Ed.45 (2006) 285.[9] P. James, J. Neudorfl, M. Eissmann, P. Jesse, A. Prokop, H.G. Schmalz, Org. Lett.8 (2006) 2763.[10] M.R. Arkin, E.D.A. Stemp, C. Turro, N.J. Turro, J.K. Barton, J. Am. Chem. Soc. 118(1996) 2267.
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