Electrochimica Acta 174 (2015) 155–163

Quadruplex-targeting anticancer drug BRACO-19 voltammetric andAFM characterization

Ana-Maria Chiorcea-Paquim, Ana Dora Rodrigues Pontinha, Ana Maria Oliveira-Brett*Department of Chemistry, Faculty of Sciences and Technology, University of Coimbra, 3004-535, Coimbra, Portugal

A R T I C L E I N F O

Article history:Received 8 April 2015Received in revised form 25 May 2015Accepted 25 May 2015Available online 29 May 2015

Keywords:BRACO-19telomerase inhibitorG-quadruplex ligandoxidationreduction

A B S T R A C T

The quadruplex-targeting anticancer drug BRACO-19 adsorption and redox behaviour were investigatedby atomic force microscopy (AFM) on a highly oriented pyrolytic graphite surface and by cyclic,differential pulse and square-wave voltammetry at a glassy carbon electrode. The AFM and voltammetricresults demonstrated that the BRACO-19 orientation and strong adsorption, with the acridine aromaticcore parallel or perpendicular to the carbon electrode surface depending on solution pH, directlyinfluences the peak potentials and redox behaviour. BRACO-19 oxidation was a complex, pH-dependent,four-step electrode process. The first oxidation step was reversible, the second, third and fourth oxidationsteps irreversible, and an electroactive irreversibly oxidized BRACO-19 oxidation product was formed.BRACO-19 reduction occurred in two irreversible, pH-independent steps. The proposed redoxmechanisms are related to the pyrrolidine and acridine moieties.

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1. Introduction

Cancer is a complex heterogeneous disease that represents thefirst leading cause of death worldwide, and the development ofdrugs targeting the telomeres and telomerase currently representthe first line fighting the disease. The telomerase reactivationrepresents a key step in human carcinogenesis, approximately 85–90% of all cancer tumours being telomerase positive [1]. Theproliferative capacity of normal human cells is controlled at thetelomere level; in contrast, most tumour cells have bypassed thetelomere maintenance system and acquired telomerase activity inorder to preserve their potential of unlimited growth. In thiscontext, much research have been focused on designing new andbetter drug molecules targeting the telomeric G-quadruplex DNA,in order to improve the selectivity and reduce the side effects ofsuch DNA-interactive drugs [2,3].

G-quadruplexes are alternative four-stranded secondary struc-tures adopted by G-rich DNA molecules [2–11], which consist ofplanar association of four guanine bases held together byHoogsteen hydrogen bonds, stack on top of each other by p-phydrophobic interactions and stabilised by the presence ofmonovalent cations, such as sodium and potassium. The anticancer

* Corresponding author at: Department of Chemistry, Faculty of Sciences andTechnology, University of Coimbra, 3004�535, Coimbra, Portugal. Tel. fax: +351 239835295

E-mail address: brett@ci.uc.pt (A.M. Oliveira-Brett).

http://dx.doi.org/10.1016/j.electacta.2015.05.1460013-4686/ã 2015 Elsevier Ltd. All rights reserved.

effect of G-quadruplex formation in telomeres and promoterregions of several oncogenes makes them attractive targets incancer research [12–17].

The G-quadruplex ligands in telomeres promote G-quadruplexformation, and prevent the telomeric DNA from unwinding andopening to telomerase, thus indirectly targeting the telomeraseand inhibiting its catalytic activity. Recently, remarkable progresshas been made in the development of selective G-quadruplexligands; some entered in clinical trials for cancer therapy,presenting significant telomerase inhibition or suppression ofthe transcription activity of oncogenes [14–18].

A prototype of a new generation of aminoacridine based anti-cancer drugs that act as telomerase inhibitors by G-quadruplexstabilization in telomeres, BRACO-19, (9-[4-(N,N-dimethylamino)phenylamino]-3,6-bis(3-pyrrolidino-propionamido) acridine(Scheme 1A), was developed [2,15,16,19,20]. BRACO-19 has beenshown to produce cell growth arrest, end-to-end chromosomalfusion and anticancer activity in tumour xenografts [2,19–23].However, its characterisation has been limited to pharmacologicalparameters, such as cellular uptake or membrane permeability [24].

Voltammetric methods present high sensitivity and have beensuccessfully used for the detection and determination of manybiological compounds, because they can mimic in vitro the redoxmechanisms of organisms [25–30]. Many BRACO-19 metabolicprocesses in the cell involve the transfer of electrons, and themechanisms by which the transfer between BRACO-19 and othermacromolecules occurs is essential for the correct evaluation of thedrug pharmacological action on the telomeric DNA. The

Scheme 1. BRACO-19: (A) chemical structure and (B, C) 3D representations.

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investigation of the BRACO-19 redox behaviour by electrochemicaltechniques has the potential for providing valuable insights into itsredox reaction mechanisms. Moreover, the morphological char-acteristics of DNA molecules in G-quadruplex configurations havebeen recently investigated at carbon electrodes [31–36].

The present study is concerned with the investigation ofBRACO-19 adsorption process and oxidation and reductionmechanisms, over a wide pH range, using atomic force microscopyon the surface of highly oriented pyrolytic graphite and cyclic,differential pulse and square-wave voltammetry at a glassy carbonelectrode.

2. Experimental

2.1. Materials and Reagents

BRACO-19 was purchased from Sigma and used without furtherpurification. Stock solutions of 200 mM BRACO-19 were prepared.Solutions of different concentrations of BRACO-19 were obtainedby dilution of the appropriate volume in supporting electrolyte.

All supporting electrolyte solutions [37]: HAc + NaAc pH's 3.4,4.3 and 5.4; NaH2PO4 + Na2HPO4 pH's 6.1, 7.0 and 8.0; NH3+ NH4ClpH 9.2; were prepared using analytical grade reagents and purifiedwater from Millipore Milli-Q system, with conductivity �0.1 mScm�1.

Microvolumes were measured using EP-10 and EP-100 PlusMotorized Microliter Pipettes (Rainin Instruments Co. Inc. Woburn,USA). The pH measurements were carried out with a GLP 21CrisonpH meter. All experiments were done at room temperature(25 �1 �C).

Nitrogen saturated solutions were obtained by bubbling highpurity N2 for a minimum of 10 min in the solution and continuing

with a flow of pure gas over the solution during voltammetricexperiments.

2.2. Atomic force microscopy

Atomic force microscopy (AFM) was performed in the acousticAC (AAC) mode, with a PicoScan controller and a CS AFM S scannerwith a scan range of 6 mm in x-y and 2 mm in z, from AgilentTechnologies, USA. AppNano type FORT of 225 mm length,3.0 N m�1 spring constants and 47–76 kHz resonant frequenciesin air (Applied NanoStructures, Inc. USA) were used. All AFMimages were topographical and were taken with 512 samples/linex 512 lines and scan rates of 0.8–2.5 lines s�1. When necessary,the AFM images were processed by flattening in order to removethe background slope and the contrast and brightness wereadjusted.

Highly oriented pyrolytic graphite (HOPG), grade ZYB of15 �15 � 2 mm3 dimensions, from Advanced Ceramics Co., USA,was used as a substrate in the AFM study, because is atomically flat,with less than 0.06 nm of root-mean-square (r.m.s.) roughness for a1000 � 1000 nm2 surface area. The GCE used for the voltammetriccharacterization was much rougher, with 2.1 nm r.m.s. roughnessfor the same surface area, and therefore unsuitable for AFM surfacecharacterization. Furthermore, the experiments using glassycarbon and HOPG showed similar electrochemical behaviour.

The HOPG was freshly cleaved with adhesive tape prior to eachexperiment and imaged by AFM in order to establish itscleanliness.

For the AFM study, solutions of 20 mM BRACO-19 in 0.1 Macetate buffer pH = 4.5 and 0.1 phosphate buffer pH = 7.0 were used.HOPG surfaces modified by BRACO-19 molecules were obtained byspontaneous adsorption, by depositing 100 mL of the appropriate

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BRACO-19 solutions onto the freshly cleaved HOPG surface, over aperiod of 3 min. The excess of solution was gently cleaned with a jetof Millipore Milli-Q water, and the HOPG with adsorbed moleculeswas then dried in a sterile atmosphere and imaged by AAC ModeAFM in air.

2.3. Voltammetric parameters and electrochemical cells

Voltammetric experiments were carried out using a mAutolabrunning with GPES 4.9 software, Eco-Chemie, The Netherlands. Themeasurements were carried out using a three-electrode system ina 2 mL one-compartment electrochemical cell (DAQ, Europe), a

Fig. 1. AFM images of 20 mM BRACO-19: (A, B) pH = 5.4 and (C, D) pH = 7.0. (E, F) C

glassy carbon (GCE, d = 1.5 mm) working electrode, a Pt wirecounter electrode and a Ag/AgCl (3 M KCl) reference electrode.

The experimental conditions were for cyclic voltammetry (CV)scan rate 100 mV s�1, for differential pulse (DP) voltammetry pulseamplitude 50 mV, pulse width 70 ms and scan rate 5 mV s�1, andfor square wave (SW) voltammetry a frequency of 50 Hz and apotential increment of 2 mV, corresponding to an effective scanrate of 100 mV s�1.

Before each electrochemical experiment, the GCE was polishedusing diamond spray, particle size 3 mm (Kemet, UK) and rinsedthoroughly with Milli-Q water. Following this mechanical treat-ment, the GCE was placed in supporting electrolyte and DPvoltammograms were recorded until a steady state baseline

ross-section profiles through the white lines in the AFM images (C) and (D).

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voltammogram was obtained. This procedure ensured veryreproducible experimental results.

2.4. Acquisition and presentation of voltammetric data

The DP voltammograms were baseline corrected using themoving average with a step window of 2 mV included in the GPES(version 4.9) software. This mathematical treatment improves thevisualization and identification of peaks over the baseline withoutintroducing any artefact, although the peak intensity is, in somecases, reduced (<10%) relative to that of the untreated curve.Nevertheless, the values for peak current presented in all plotswere determined from the original untreated voltammogramsafter subtraction of the baseline.

3. Results and Discussion

3.1. Atomic force microscopy characterisation

The capacity of BRACO-19 to interact and adsorb on the HOPGsurface, forming different morphological films, depending on thesolution pH, was investigated using AFM in air. The HOPG wasmodified by BRACO-19 films obtained by spontaneous adsorptionduring 3 min, from solutions of 20 mM BRACO-19 in buffersupporting electrolyte pH = 5.4 and pH = 7.0.

AFM images of BRACO-19 in pH = 5.4 showed the moleculespredisposition to adsorb close to each other, forming a compactfilm of densely packed filaments, that covers the HOPG surfacealmost completely (Fig. 1A,B). The film height of 0.82 � 0.09 nmwas comparable to the expected dimensions of BRACO-19 perpen-dicular to the acridine aromatic rings, of approximately 0.9 nm.This means that BRACO-19 formed a monolayer, with the BRACO-19 planar acridine aromatic rings lying flat on the surface, bondingby p-p hydrophobic interactions with the hydrophobic HOPG(Scheme 1C), and with the non-planar pyrrolidine groups formingadditional hydrophobic interactions with the surface.

The structure of the HOPG surface had a strong influence on theBRACO-19 self-assembling proprieties at pH = 5.4. HOPG is apolycrystalline material, consisting of individual crystallinedomains, which are rotated against each other. The cleavingprocess of the HOPG electrode with adhesive tape revealed many

Fig. 2. CVs in a N2 saturated solution of 100 mM BRACO-19 in pH = 7.0: ( ) first and( ) second scan; n = 100 m V s�1.

different atomically flat terraces on the basal plane of graphite.BRACO-19 interactions with flat HOPG terraces consist of BRACO-19 packed filaments oriented mainly along three directions, at 60�

and 120� to each other, imposed by the threefold symmetry of thegraphite substrate (Fig. 1A, B). The formation of these elongatedlattices, running in the same direction as the underlying graphitesurface symmetry, was observed consistently in many indepen-dent experiments run under the same experimental conditions.The BRACO-19 chains were not oriented at a fixed angle relative tothe direction of the step edges of the HOPG substrate, indicatingthat the orientation of the filaments was not induced by particularsurface defects on the HOPG step edges, but induced by the BRACO-19 epitaxial growth on the perfectly flat HOPG terraces.

AFM images of BRACO-19 in pH = 7.0 (Fig. 1C,D) showed theformation of a less tight network film, and decreased adsorptionextent when compared with pH = 5.4 (Fig. 1A,D) was observed. TheBRACO-19 network film presented two distinct areas of differentheight (delimited by red line in Fig. 1C): the A1 area of0.89 � 0.07 nm height, corresponding to a monolayer of BRACO-19 adsorbed in planar orientation, with the acridine moieties lyingflat on the surface and bonding by p-p hydrophobic interactions

Fig. 3. DP voltammograms ( ) first and ( ) second scan baseline-corrected in asolution of 20 mM BRACO-19: (A - red line) pH = 4.3, (A - black line) pH = 5.4 and (B -black line) pH = 7.0, and of (B, blue line) BRACO-19 adsorbed onto GCE in bufferelectrolyte pH = 7.0.

Fig. 4. DP voltammograms baseline-corrected in a solution of 20 mM BRACO-19 inpH = 4.3, ( ) first and ( ) second scans.

Fig. 5. Plots of (A) Epa and (B, C) Ipa vs. pH of the supporting electrolyte, for oxidationpeaks (&) 1a, (~) 2a, (*) 3a, ( ) 4a and ( ) PBRACO-19.

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with the HOPG (Scheme 1C), and the A2 area of 1.7 � 0.34 nmheight, consistent with a BRACO-19 monolayer adsorbed in avertical orientation, with the pyrrolidine groups bounding to theHOPG and the acridine aromatic rings perpendicular to the surface(Scheme 1B). The cross-section profiles through the white lines inthe AFM images (Fig.1E and F) confirm the BRACO-19 adsorption inpH = 7.0 in a planar and a perpendicular orientation to the HOPGsurface.

3.2. Voltammetric characterisation

The electrochemical redox behaviour of BRACO-19 at a GCE wasinvestigated by CV, DP and SW voltammetry, and showed that theoxidation and the reduction processes occurred independently ofeach other and they were investigated separately (Fig. 2).

3.2.1. Oxidation

3.2.1.1. Cyclic voltammetry. The voltammetric behaviour ofBRACO-19 was first investigated by CV at a GCE, in 100 mMBRACO-19 in phosphate buffer pH = 7.0, at a scan ratev = 100 mV s�1. During the voltammetric measurements aconstant flux of N2 was kept over the solution surface in orderto avoid the diffusion of atmospheric oxygen into the BRACO-19 solution. The CVs showed that BRACO-19 undergoes a complexoxidation mechanism and different potential windows wereinvestigated (Fig. 2).

In the first experiment the CV of BRACO-19 in pH = 7.0, started at0.0 V (Fig. 2A ), and five anodic peaks, peak 1a', at E1a0 = + 0.16 V,peak 1a, at E1a = + 0.26 V, peak 2a, at E2a = + 0.72 V, peak 3a, atE3a = + 0.85 V, and a small peak 4a, at E4a� + 1.02 V, were observed.The scan was reversed at + 1.10 V, and on the first negative-goingscan of the first voltammogram, three cathodic peaks, peak 1c, atE1c= + 0.05 V, peak 1c', at E1c0 = + 0.23 V, and a small peak 5c, atE5c = – 0.96 V, occurred. The scan was again reversed at – 1.00 V andin the second scan all the oxidation peak currents decreased(Fig. 2 ).

Table 1BRACO-19 pH dependent species.

pH

Specie 1 (%) Specie 2 (%) Specie 3 (%) Specie 4 (%) Specie 5 (%) Specie 6 (%)

3.4 0 0 0 1 9 904.3 0 0 3 6 40 514.4 0 0 4 7 45 455.0 0 0 20 9 57 145.4 0 0 41 7 47 56.1 0 0 79 3 18 07.0 0 1 96 0 3 08.0 0 8 92 0 0 09.2 16 48 36 0 0 0

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In another experiment, at the same pH, the CV was started at0.0 V and reversed at + 0.70 V, immediately after the occurrence ofpeaks 1a' and 1a, and before peak 2a, showing the cathodic peaks1c, 1c' and reduction peak 5c (Fig. 2A ), and demonstrating peaks1a' and 1a reversibility.

3.2.1.2. Differential pulse voltammetry and pH effect. DPvoltammetry is more sensitive to quantify faradaic oxidationcurrents, with a lower capacitive current contribution, thanconventional CV [38], therefore a systematic DP voltammetricstudy was performed in 20 mM BRACO-19 in buffer supportingelectrolytes over a wide pH range from 3.4 to 9.2.

The first DP voltammogram, recorded in a solution of BRACO-19 in 0.1 M acetate buffer pH = 5.4, showed the occurrence of thefour oxidation peaks (Fig. 3A ): peak 1a, at E1a = + 0.24 V, peak 2a,at E2a = + 0.71 V, peak 3a, at E3a = + 0.81 V, and peak 4a, atE4a = + 1.07 V. On the second scan obtained in the same conditionsand without cleaning the electrode surface (Fig. 2A ), theoxidation peaks 1a, 3a and 4a current decreased and the peak2a disappeared, due to the adsorption of the BRACO-19 oxidationproducts on the GCE surface, and a new peak, designated as PBRACO-19, due to the oxidation of a BRACO-19 oxidation product, atEPBRACO-19 = + 0.45 V, occurred.

Similar results were obtained for BRACO-19 in 0.1 M acetatebuffer pH = 4.3, but the peaks 1a, 2a, 3a, 4a and PBRACO-19 were allshifted to more positive potentials (Fig. 3A first and secondscans).

The first DP voltammogram recorded at a clean GCE in BRACO-19, in 0.1 M phosphate buffer pH = 7.0, showed the occurrence offive oxidation peaks (Fig. 3B ). A new anodic peak 1a', atE1a0 = + 0.11 V, peak 1a, at E1a = + 0.20 V, peak 2a, at E2a = + 0.68 V,small peak 3a, at E3a = + 0.80 V, and peak 4a, at E4a = + 1.04 V. On thesecond scan the 1a and 3a oxidation peaks current decreased, thepeaks 1a' and 2a disappeared, and no new oxidation peaks occurred(Fig. 3B ). On the third scan the peak 3a also disappeared.

The adsorption of BRACO-19 at the GCE surface was alsostudied. The GCE was held during 3 min in a solution of 20 mMBRACO-19, in 0.1 M phosphate buffer pH = 7.0, then the electrodewas washed with a jet of Milli Q water and placed in supportingelectrolyte in the electrochemical cell. The DP voltammograms(Fig. 3B ) showed all oxidation peaks 1a', 1a, 2a, 3a and 4a withsignificantly higher peak currents than in the solution of BRACO-19(Fig. 3B ), demonstrating that BRACO-19 adsorbed strongly ontothe GCE.

However, the peak 1a' was observed in BRACO-19 in only pH �7.0. For pH < 6.0 and pH > 8.0, only peak 1a was observed and this

was explained considering the different orientations of BRACO-19 molecules parallel or perpendicular on the carbon electrodesurface as already observed by AFM (Fig. 1).

At pH = 5.4, the AFM image showed the adsorption of BRACO-19 molecules only lying flat, with the acridine aromatic ringsparallel to the surface (Fig. 1A, B and Scheme 1C), and in the DPvoltammogram peak 1a was observed (Fig. 3A ).

At pH = 7.0, the AFM image showed BRACO-19 moleculesadsorbed in two different orientations: planar, with the acridinemoiety lying flat on the surface (A1 area in Fig. 1C, D, E and F,and Scheme 1C), and vertical, with the pyrrolidine groups nearthe carbon surface and the acridine ring perpendicular to thesurface (A2 area in Fig. 1C, D, E and F, and Scheme 1B). Theoccurrence of both peaks 1a and 1a' in the DP voltammogram(Fig. 3B ), was explained because BRACO-19 vertical orientation,but after scanning the potential, BRACO-19 molecules wererearranged on the electrode surface, and on the second scan(Fig. 3B ) and successive DP voltammograms recorded under thesame conditions, peak 1a' disappeared and only peak 1a wasobserved.

In order to clarify which BRACO-19 oxidation peaks 1a to 4a ledto the oxidation product PBRACO-19, four independent experimentswere carried out (Fig. 4). Each experiment consisted in recordingtwo successive DP voltammograms in solutions of 20 mM BRACO-19 in 0.1 M acetate buffer pH = 4.3, starting at 0.0 V using differentpotential windows. For each experiment, the first DP voltammo-gram was recorded at a clean GCE, then the second scan wasrecorded without cleaning the GCE, and the absence/presence ofthe oxidation peak PBRACO-19, due to the oxidation of the productPBRACO-19, was evaluated.

In the first experiment the DP voltammogram stopped at +0.65 V, the potential after the occurrence of peak 1a and beforepeak 2a, and in the second scan no peak PBRACO-19 occurred. In asecond experiment the DP voltammogram stopped at + 0.88 V,after the peak 2a but before peak 3a, and in the second scan nopeak PBRACO-19 occurred. In a third experiment the DP voltammo-gram stopped at + 1.05 V, after the peak 3a but before peak 4a, andin the second scan no peak PBRACO-19 occurred.

Finally, in a fourth experiment the DP voltammogram stoppedat + 1.25 V, after the peak 4a and in the second scan peak PBRACO-19occurred. This last experiment clearly demonstrated that theoxidation peak PBRACO-19 is due to the oxidation of the BRACO-19 oxidation product formed in peak 4a, at a higher potential.

The pH effect was investigated in solutions of BRACO-19 over awide pH range from 3.4 to 9.2. The four oxidation peaks occurredfor all pHs, their potential was shifted to less positive values with

Fig. 6. SW voltammograms baseline-corrected in a solution of 20 mM BRACO-19 in(A) pH = 4.3 and (B) pH = 7.0; f = 50 Hz, DEs = 2 mV, pulse amplitude 100 mV,neff = 50 mV s-1; ( ) It – total current, ( ) If – forward and Ib – backward currents.

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increasing supporting electrolyte pH, and the relationships werelinear (Fig. 5).

The oxidation peak 1a followed the relationship E1a (V) = 0.59–0.06 pH (Fig. 5A). The slope of � 60 mV per pH unit was inagreement with the transfer of the same number of electrons andprotons [38]. The width at half-height, W1/2 � 86 mV, close to thetheoretical value of W1/2 = 90 mV, indicated that the oxidationmechanism involved the transfer of one electron and one proton[38]. However, for pH = 7.0, oxidation peak 1a', due to BRACO-19 parallel and perpendicular orientations on the GCE surface, wasfound.

The oxidation peak 2a followed the relationship Ep2a (V) = 1.07–0.060 pH (Fig. 5A), the slope of – 60 mV per pH unit and W1/2 �77 mV, indicated that the oxidation involved the transfer of oneelectron and one proton. The oxidation peak 3a followed therelationship Ep3a (V) = 1.17–0.06 pH (Fig. 5A), the slope of – 60 mVper pH unit and W1/2 � 86 mV, indicated that the oxidation alsoinvolved the transfer of one electron and one proton. The oxidationpeak 4a followed the relationship Ep4a (V) = 1.42–0.06 pH (Fig. 5A),the slope of – 60 mV per pH unit and W1/2 � 58 mV, indicated thatthe oxidation involved the transfer of two electrons and twoprotons.

The oxidation peak PBRACO-19 occurred only for pH < 7.0(Figs. 4 and 5), the oxidation PBRACO-19 peak potential was pH-independent, and W1/2 � 55 mV, indicated that the oxidationinvolved the transfer of two electrons. For 7.0 < pH < 9.0, theoxidation peak PBRACO-19 cannot be detected by DP voltammetrybecause the peak 4a also presented a very small oxidation current.

All oxidation peaks 1a, 2a, 3a and 4a currents showed maximumvalues at approximately pH = 5.6 (Fig. 5B and C), and the BRACO-19 acridine ring pKa is � 5.62.

BRACO-19 has four pKa values: the acridine moiety, at pKa �5.62, the dimethylaniline moiety, at pKa � 4.34, and the twopyrrolidine moieties, at pKa� 9.68 and 9.08 (Scheme 1A), resultingon the existence of six pH-dependent BRACO-19 species (Table 1).At pH = 4.3 BRACO-19 species 5 and 6 prevail, while atpH = 5.4 species 3 and 5 are found (Table 1), which may influencethe adsorption and oxidation at the carbon electrode surfaces. Theprotonation of the acridine and dimethylaniline moieties mayberesponsible for the difference observed in the oxidation peakcurrents at pHs 5.4 and 4.3. Due to the existence of these differentpH-dependent species in solution, there is no evidence thatBRACO-19 diffusion coefficient has the same value at pH = 5.4 and4.3; moreover the standard rate constant, ko, may also be differentat different pH values.

The molecular fragments identified in the structure of BRACO-19, namely acridine, pyrrolidine, dimethylaminobenzene and thegroup amide, have been studied by electrochemistry some in non-aqueous, in acetonitrile, and others in aqueous supportingelectrolytes, making the comparison of the oxidation potentialsdifficult.

Nevertheless, electrochemical studies reported that anodicoxidation of acridine in acetonitrile (vs. Ag/AgClO4) showed twoanodic peaks: one due to oxidation of acridine, at Ep = + 1.35, andthe other due to protonated acridine, at Ep = + 2.0 V [39]. Anodicoxidation of acridine derivatives in acetonitrile (vs. Ag/AgCl)showed anodic peaks due to the oxidation of the acridine moiety atthe NH+ position, at potentials over Ep = + 0.9 V, [40]. The oxidationin 0.1 M phosphate buffer pH = 7.0 (vs. Ag/AgCl) of two disubsti-tuted triazole-linked acridine compounds occur for GL15, atEp = + 0.89 V, and for GL7, at Ep = + 0.76 V and + 0.88 V [41]. Anodicoxidation of pyrrolidine in aqueous solution occurred at Ep = +1.26 V and in acetonitrile (vs. AgQRE) at Ep = + 1.09 V [42].

A proposed BRACO-19 oxidation mechanism has to take intoconsideration the BRACO-19 different orientations, parallel and

perpendicular, on the GCE surface, and the oxidation of thepyrrolidine and acridine moieties.

3.2.1.3. Square Wave Voltammetry. SW voltammetry ischaracterised by greater speed of analysis, lower consumption ofthe electroactive species when compared to differential pulsevoltammetry, and by reduced difficulties regarding the electrodesurface blocking by the electroactive species [23]. Furthermore,SW voltammetry enables to determine during only one scan if theelectron transfer reaction is reversible or not. Since the current issampled in both positive and negative-going pulses, the oxidationand reduction peaks are both obtained in the same experiment[38].

Fig. 7. N2 saturated solution of 100 mM BRACO-19 in pH = 7.0: (A) CVs atn = 100 mV s�1, ( ) first and ( ) second scans, and (B) SW voltammogrambaseline-corrected at f = 50 Hz, DEs = 2 mV, pulse amplitude 100 mV, neff = 100 mVs�1, ( ) It – total current, ( ) If – forward and Ib – backward currents.

Fig. 8. DP voltammograms baseline-corrected in N2 saturated solutions of 20 mMBRACO-19 in ( ) pH = 4.3, ( ) pH = 5.3 and ( ) pH = 7.0.

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Successive SW voltammograms in 20 mM BRACO-19, in 3.5 <

pH < 9.0, showed similar features to the DP voltammograms.Plotting the forward and backward currents components of thetotal current in BRACO-19 in pH = 4.5 (Fig. 6A) and pH = 7.0 (Fig. 6B),the reversibility of peaks 1a/1c and 1a'/1c', and the irreversibility ofpeaks 2a, 3a and 4a was observed.

3.2.2. Reduction

3.2.2.1. Cyclic voltammetry. The reduction of BRACO-19 at a GCEwas investigated by cyclic voltammetry, in a N2 saturated solutionof 100 mM BRACO-19 in 0.1 M phosphate buffer pH = 7.0, at a scanrate n = 100 mV s�1 (Fig. 7A).

In the first experiment the cyclic voltammogram started at 0.0 Vtill – 1.10 V (Fig. 7A ) and the reduction peak 5c, at E5c = –0.96 V,occurred. Reversing the scan direction, on the positive-going scanof the first voltammogram till 0.0 V, no oxidation peak wasobserved, showing that the reduction of BRACO-19 is anirreversible process. The decrease of the peak 5c current occurredwith successive scans, due to the adsorption of BRACO-19 and/orits non-electroactive oxidation products on the GCE surface.

In another experiment the cyclic voltammogram started at 0.0 V till– 1.10 V (Fig. 7B ) and the reduction peak 5c, at E5c = –0.96 V,occurred. Reversing the scan direction, on the positive-going scantill + 1.10 V, all the oxidation peaks appeared.

3.2.2.2. Square Wave Voltammetry. Successive SWvoltammograms recorded in a N2 saturated solution of 20 mMBRACO-19 in 0.1 M phosphate buffer pH = 7.0, showed similarfeatures to the cyclic voltammograms. The first SW voltammogramshowed the BRACO-19 reduction peak 5c, at E5c = – 1.11 V, and thepeak irreversibility was confirmed by plotting the forward andbackward components of the total current (Fig. 7B).

3.2.2.3. Differential pulse voltammetry and pH effect. The pH studyofBRACO-19 reductionprocesswascarriedout byDPvoltammetry inN2 saturated solutions of 20 mM BRACO-19 in 3.4 < pH < 9.2 buffersupporting electrolytes, starting at 0.0 V till – 1.2 V.

The first DP voltammogram recorded in BRACO-19 in 0.1 Mphosphate buffer pH = 7.0, showed the reduction peak 5c, at E5c = –

0.90 V, and the occurrence of a new cathodic peak 6c, at E6c = –

1.00 V (Fig. 8). On the second scan obtained in the same conditionswithout cleaning the electrode surface, a decrease of 5c and 6cpeak currents was observed, due to the adsorption of the BRACO-19 reduction products on the GCE surface.

The reduction peaks 5c and 6c were observed only for3.4 < pH < 7.0, and their potentials were pH-independent. TheW1/2� 100 mV for peaks 5c and 6c, indicating that the peaks 5c and7c reduction processes occurred with the transfer of one electroneach. A reduction of BRACO-19 acridine group is proposed.

4. Conclusions

The adsorption and redox behaviour of the quadruplex-targetinganticancer drug BRACO-19 were studied by AFM onto HOPG and byvoltammetry at a GCE in electrolytes with 3.5 < pH < 9.0.

The AFM and voltammetric results demonstrated that in neutralelectrolytes, the oxidation processes are influenced by the BRACO-19 strong adsorption onto the carbon electrode surface, andorientation of the acridine aromatic core parallel or perpendicularto the electrode surface.

The oxidation of BRACO-19 occurs in a complex, pH-dependent,four-step mechanism. The first step is a reversible process, the

A.-M. Chiorcea-Paquim et al. / Electrochimica Acta 174 (2015) 155–163 163

second, third and fourth oxidation steps are irreversible, giving riseto an electroactive oxidation product, PBRACO-19, presenting anirreversible, pH-independent oxidation. The reduction of BRACO-19 is an irreversible process.

The BRACO-19 proposed electron transfer mechanism followedan oxidation at the pyrrolidine and acridine moieties and areduction also at the acridine aromatic group.

Acknowledgements

Financial support from Fundação para a Ciência e Tecnologia(FCT), Grant SFRH/BPD/92726/2013 (A.-M. Chiorcea-Paquim),Project Grant (A.D.R. Pontinha), projects PTDC/SAU-BMA/118531/2010, PTDC/QEQ-MED/0586/2012, PEst-C/EME /UI0285/2013 andCENTRO-07-0224-FEDER-002001 (MT4MOBI) (co-financed by theEuropean Community Fund FEDER), FEDER funds through theprogram COMPETE – Programa Operacional Factores de Compet-itividade is gratefully acknowledged.

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