Inorganica Chimica Acta 399 (2013) 177–184

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Inorganica Chimica Acta

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Effect of the chelate ring size on the cleavage activity of DNA by copper(II)complexes containing pyridyl groups

Salah S. Massoud a,⇑, Richard S. Perkins a, Kathleen D. Knierim a, Sean P. Comiskey a, Kara H. Otero a,Corey L. Michel a, Wesley M. Juneau a, Jörg H. Albering b, Franz A. Mautner c, Wu Xu a,⇑a Department of Chemistry, University of Louisiana at Lafayette, Lafayette, LA 70504, USAb Institut für Physikalische and Theoretische Chemie, Technische Universität Graz, A-8010 Graz, Austriac Institut für Chemische Technologie von Materialien, Technische Universität Graz, A-8010 Graz, Austria

a r t i c l e i n f o

Article history:Received 20 August 2012Received in revised form 26 November 2012Accepted 20 January 2013Available online 1 February 2013

Keywords:DNA cleavageHydrolytic cleavageOxidative cleavageCopperPolypyridylCrystal structure

0020-1693/$ - see front matter Published by Elsevierhttp://dx.doi.org/10.1016/j.ica.2013.01.020

Abbreviations: TPA, tris(2-pyridylmethyl)amine;2-(2-pyridylethyl)- amine; pmap, bis(2-(2-pyridylethtepa, tris (2-(2-pyridyl)ethyl)amine; pzdpy, 1,4-bis(2-(hpzdpy, 1,4-bis(2-(2-pyridylmethyl))homopiperazine.⇑ Corresponding authors. Tel.: +1 337 482 5672;

Massoud). Tel.: +1 337 482 5684; fax: +1 337 482 56E-mail addresses: ssmassoud@louisiana.edu (

louisiana.edu (W. Xu).

a b s t r a c t

Square pyramidal five-coordinate copper(II) complexes of the general formula [Cu(N4)ClO4]ClO4, N4 rep-resents a tetradentate ligand where N4 = pzdpy (1,4-bis(2-pyridylmethy)piperazine), 1; hpzpy (1,4-bis(2-pyridylmethyl)homopiperazine), 2; pmap, (bis(2-(2-pyridylethyl))-(2-pyridylmethyl)-amine), 4; and[Cu(N4)Cl]ClO4 with N4 = pmea (bis(2-pyridylmethyl)-2-(2-pyridylethyl)amine), 3; pmap, 4a; tepa (tris(2-(2-pyridyl)ethyl)amine), 5 were structurally characterized. The single crystal X-ray crystallographyof 3 was determined. The molar conductivity studies of the complexes in H2O reveal the presence of[Cu(N4)(H2O)]2+ as the reactive species in the aqueous solutions. The synthesized complexes were usedto study the DNA cleavage activity at pH 7.0 and 37 �C. Under pseudo Michaelis–Menten conditions, theconstant for the catalytic cleavage of DNA, kcat decreases in the following order: 1 > 3 > 2 > 4� 5. Com-plex 1 showed very high nuclease activity with a rate enhancement of 25-million-fold over the non-cat-alyzed DNA. The results demonstrated that an increase in the number of six-membered rings in thecomplexes suppresses the cleavage process. Although the mechanistic studies of DNA cleavage by thecomplexes in presence of oxidative scavengers indicate that the mechanism of the cleavage in complexes2–4 is most likely hydrolytic in nature, an oxidative mechanism via hydroxyl radical was revealed withcomplex 1.

Published by Elsevier B.V.

1. Introduction

The phosphodiester bonds in DNA have remarkable stability to-ward cleavage under physiological conditions (kuncat = 3.6 � 10�8

h�1, t1/2 � 130,000 years). This stability is considered to be one ofthe essential requirements for the survival and maintenance of lifeand may also explain why nature chose DNA for this mission [1].However, in vivo nature has developed certain enzymes such asrestriction enzymes and EcoRI endonuclease which efficientlyand rapidly catalyze the hydrolytic cleavage of P–O bonds inDNA. A variety of transition metal complexes have been launchedas ‘‘artificial nucleases’’ to catalyze the DNA cleavage under the

B.V.

pmea, bis(2-pyridylmethyl)-yl))-(2-pyridylmethyl)amine;2-pyridylmethyl))piperazine;

fax: +1 337 482 5670 (S.S.70 (W. Xu).S.S. Massoud), wxx6941@

physiological conditions but many of these compounds cannot becompare to the enzymatic cleavage [2–4].

In general, two mechanisms have been reported for DNA cleav-age by artificial nucleases: the oxidative cleavage and the hydro-lytic cleavage. In the former mechanistic pathway, reactiveoxygen species, ROS (singlet molecular oxygen, 1O2

��; hydroxylradical, HO�; superoxide radical, O2

�–) which are generated duringthe cleavage process and these non-natural fragments cause dam-age to the deoxyribose sugar or the nucleic base moieties [5,6].These species hamper their use in vivo. In the hydrolytic mecha-nism, the hydrolytic cleavage agents do not induce this problemand hence the product can be enzymatically ligated. This hydro-lytic feature makes these compounds function by a mechanismthat is similar to the natural enzymatic reactions but also provesuseful in elucidating the precise role of metal ions in this enzy-matic catalytic process [2–4].

In the search for artificial nucleases that effectively catalyzeDNA cleavage many divalent mono- and dinuclear-copper(II), co-balt(II) and zinc(II) as well as trivalent cobalt(III) complexes de-rived from a wide range of ligands of varied skeletal structuresand geometrical environments have been extensively investigated

N

N N

N

N

N

N

N

N

N

N

N

NN

N

N

N

N

N

N

N

N N

N

TPA pmea pmap tepapzdpy hpzdpy

Scheme 1. Structural formulas of some poly-pyridyl amine ligands used in this study.

178 S.S. Massoud et al. / Inorganica Chimica Acta 399 (2013) 177–184

[7–40]. Although some of these compounds, especially those de-rived from Cu(II) and Co(II), have been reported to reveal high cat-alytic activity [8,13,14,30,33,35–38], the DNA cleavage mechanismwas found to be oxidative in nature [18–24]. Therefore in order todesign artificial nucleases that efficiently and hydrolytically cata-lyze DNA cleavage, a systematic study should be undertaken to ex-plore the factors that might affect the cleavage of DNA such as thenature of the central metal ion [8–11] and structural features of theligand. In an effort to understand how the structural parametersincorporated in the ligand skeleton (ring size, chelate ring) coordi-nating a metal ion affect the DNA cleavage process, a series ofmononuclear copper(II) complexes with tetradentate amine li-gands with different pyridyl arm lengths, and 1,4-piperazine and1,5-homopiperazine with different ring sizes have been synthe-sized and their cleavage activities with DNA were investigated at37 �C and pH 7.0. The ligand skeletons used in this study are illus-trated in Scheme 1.

2. Experimental

2.1. Materials and physical measurements

2-Picolylchloride hydrochloride and 2-pyridylmethylamine, 2-vinylpyridine, piperazine and homopiprazine were purchased fromAldrich Chemical Company, USA, whereas dipicolylamine was ob-tained from TCI-America. All other materials were reagent gradequality. 2-Vinylpyridine was distilled and purified by column chro-matography using alumina and eluted with a 90/10 (v/v) mixtureof ethyl acetate/MeOH. All other materials were reagent gradequality. Infrared spectra were recorded on a JASCO FT/IR-480 plusspectrometer as KBr pellets. Electronic spectra were recorded on anAgilent 8453 HP diode UV–VIS spectrophotometer. 1H and 13CNMR spectra were obtained at room temperature on a Varian400 NMR spectrometer operating at 400 MHz (1H) and 100 MHz(13C). 1H and 13C NMR chemical shifts (d) are reported in ppmand were referenced internally to residual solvent resonances(DMSO-d6: dH = 2.49, dC = 39.4 ppm). Mass spectra were obtainedon Agilent 7890 AGC coupled to an Agilent 5975C Mass SelectiveDetector (MSD). Electrical conductivity measurements were thesame as reported elsewhere [8]. The molar conductivity of a solu-tion sample was determined from KM = (1.0 � 103 j)/M, wherej = cell constant and M is the molar concentration of the complex.Elemental microanalyses were performed at the Atlantic Microlab-oratory, Norcross, Georgia USA. The pUC19 Plasmids DNA purifica-tion, determination of concentrations and plasmid DNA cleavage aswell as gel electrophoresis procedures were similar to those whichhave been recently reported [7,8]. The intensity of the differentforms of plasmid DNA was quantified using Quantity One software(Bio-Rad Laboratories, Hercules, CA 94547).

Caution: Salts of perchlorate and their metal complexes arepotentially explosive and should be handled with great care andin small quantities.

2.1.1. Synthesis of the ligandsThe syntheses of bis(2-pyridylmethyl)-2-(2-pyridylethyl)amine

(pmea) [41,42], bis(2-(2-pyridyl)ethyl)-(2-pyridylmethyl)amine(pmap) [42,43], tris(2-(2-pyridyl)ethyl)amine (tepa) [43], 1,4-bis(2-(2-pyridylmethyl))piperazine (pzdpy), and 1,4-bis(2-(2-pyri-dylmethyl))homopiperazine (hpzdpy) were synthesized andcharacterized following the published procedures [44].

2.1.2. Synthesis of [Cu(N4)X]ClO4 complexesThe perchlorato complexes [Cu(N4)ClO4]ClO4 (1, N4 = pzpdy; 2,

N4 = hpzdpy [44]; 4, N4 = pmap; 5, N4 = tepa) were obtained bytreating Cu(ClO4)2�6H2O dissolved in MeOH with the appropriateligand, followed by heating on a steam-bath for 5 min. Complexesobtained were recrystallized from H2O (yield 70–80%). The chlorocomplex [Cu(pmea)Cl]ClO4�H2O (3) was synthesized by the addi-tion of 1 mL of saturated solution of NaClO4 to a hot mixture con-taining CuCl2�2H2O (0.170 g, 1 mmol) and an equimolar amount ofbis(2-pyridylmethyl)-2-(2-pyridylethyl)amine (pmea) in MeOH(20 mL). The resulting blue precipitate was collected by filtrationand recrystallized from H2O (yield: 70–80%). Single crystals of 3suitable for X-ray were obtained from dilute aqueous solutions.Characterization of the complexes:

[Cu(pzdp)ClO4]ClO4 (1): Anal. Calc. for C16H20N4Cl2CuO8

(MM = 530.81 g/mol): C, 36.20; H, 3.80; N, 10.56%. Found: C,36.22; H, 3.84; N, 10.47%. KM (25� C) = 188 and 300 O�1 cm2 mol�1

in H2O and CH3CN, respectively. UV–Vis (kmax in nm, e in M�1cm�1)in CH3CN: 645 (310) and in H2O: 647 (278).

[Cu(hpzdp)(ClO4)]ClO4 (2): Anal. Calc. for C17H22N4Cl2CuO8

(MM = 544.84 g/mol): C, 37.48; H, 4.07; N, 10.28%. Found: C,37.36; H, 3.99; N, 10.22%. KM (25� C) = 203 and KM 289 O�1 cm2

mol�1 in H2O and CH3CN, respectively. UV–Vis (kmax in nm, e inM�1cm�1) in CH3CN: 621 (320); in H2O: 622 (215). The UV–VISin CH3CN and the solid IR spectra of the complexes 1 and 2 werein complete agreement with that reported by Halfen et al. [44].

[Cu(pmea)Cl]ClO4�H2O (3): Anal. Calc. for C19H22N4Cl2CuO5

(MM = 520.85 g/mol): C, 43.81; H, 4.26; N, 10.76%. Found: C,43.76; H, 4.24; N, 10.81%. KM (H2O, 25� C) = 172 O�1 cm2 mol�1.UV–Vis (kmax in nm, e in M�1 cm�1) in CH3CN: 669 (139); in H2O:670 (112). Selected IR bands (KBr, cm�1): 3516 (m, br), 1608 (s),1571 (m), 1485 (m), 1442 (s), 1092 (vs) cm�1.

[Cu(pmap)ClO4]ClO4 (4): Anal. Calc. for C20H22N4Cl2CuO8

(MM = 580.87 g/mol): C, 41.36; H, 3.82; N, 9.65%. Found: C,41.43; H, 3.68; N, 9.54%. KM (H2O, 25� C) = 200 O�1 cm2 mol�1.UV–Vis (kmax in nm, e in M�1cm�1) in CH3CN: 643 (186); in H2O:637 (130). Selected IR bands (KBr, cm�1): 1607 (s), 1572 (m),1486 (m), 1442 (s), 1090 (vs) cm�1.

[Cu(pmap)Cl]ClO4 (4a): Anal. Calc. for C20H22N4Cl2CuO4

(MM = 516.87 g/mol): C, 46.48; H, 4.29; N, 10.84%. Found: C,46.36; H, 4.31; N, 11.02%. KM (25� C) = 172 and 139 O�1 cm2 mol�1

in H2O and CH3CN, respectively, UV–Vis (kmax in nm, e in M�1cm�1)in CH3CN: 643 (186); in H2O: 637 (130). Selected IR bands (KBr,cm�1): 1607 (s), 1572 (m), 1486 (m), 1442 (s), 1090 (vs) cm�1.

Fig. 1. Perspective view and atom numbering scheme for 3.

Fig. 2. Packing view of 3.

S.S. Massoud et al. / Inorganica Chimica Acta 399 (2013) 177–184 179

[Cu(tepa)Cl]ClO4 (5): Anal. Calc. for C21H24N4Cl2CuO4

(MM = 530.90 g/mol): C, 47.51; H, 4.56; N, 10.55%. Found: C,47.75; H, 4.62; N, 10.47%. KM (25� C) = 204 and 181 O�1 cm2 mol�1

in H2O and CH3CN, respectively. UV–Vis (kmax in nm, e in M�1cm�1)in CH3CN: 664 (190); in H2O: 657 (123).

2.2. Crystal structure analysis of [Cu(pmea)Cl]ClO4�H2O (3)

The X-ray single-crystal data of compound 3 were collected on aBruker-AXS APEX II CCD diffractometer at 100(2) K. The crystallo-graphic data, conditions retained for the intensity data collectionand some features of the structure refinements are listed inTable S1. The intensities were collected with Mo Ka radiation(k = 0.71073 Å). Data processing, Lorentz-polarization and absorp-tion corrections were performed using APEX, SAINT and the SADABS

computer programs [45]. The structure was solved by direct meth-ods and refined by full-matrix least-squares methods on F2, usingthe SHELXTL [46] program package. All non-hydrogen atoms were re-fined anisotropically. The hydrogen atoms were located from dif-ference Fourier maps, assigned with isotropic displacementfactors and included in the final refinement cycles by use of geo-metrical constraints. Split occupancy of the counter perchlorateion was applied for disordered oxygen atoms O(12A) and O(12B).Selected bond parameters are listed in Table S2, hydrogen bondsare summarized in Table S3.

2.3. Kinetic measurements for the DNA cleavage by Cu(II) complexes

DNA cleavage rates at varying concentration of Cu(II) complexes{0 (control), 50, 100, 150, 300 and 450 lM} were determined in1.0 mM Tris–Cl buffer, pH 7.0, at 37 �C for different intervals oftime according to previously published procedures [7,8]. The per-centages of form II quantified by Quantity One software were plot-ted against time for each catalyst concentration. The observed rateconstants, kobs were extracted from the single-exponential curve(pseudo first-order kinetics) using Eq. (1) [47]:

y ¼ ð100� yoÞð1� exp ð�kobstÞÞ ð1Þ

where y is the percentage of a specific form of DNA at any time t andyo is the percentage of that form at t = 0. The values of kobs are thenplotted vs. the Cu(II) concentrations (catalyst = metal(II) complex;pseudo Michaelis–Menten analysis) (Eq. (2)) [7,8,14,29,31,47] todetermine the catalytic rate constant, kcat and affinity constant, KM:

Kobs ¼ kcat½complex�=ðKM þ ½complex�Þ ð2aÞ

This equation can be rearranged to the straight line Eq. (2b).

1=kobs ¼ ðKM=kcatÞð1=½complex�Þ þ 1=kcat ð2bÞ

3. Results and discussion

3.1. Description of the structure of [Cu(pmea)Cl]ClO4�H2O (3)

Single crystal structure determination of 3 revealed a triclinicunit cell with V = 4257.6(2) Å3 and Z = 8. Karlin and his coworkershave reported a smaller triclinic unit cell with V = 2162.4(7) Å3 andZ = 4 for the same complex [41]. A perspective view of the asym-metric unit together with the partial atom numbering scheme for3 is presented in Fig. 1. The structure consists of monomeric com-plex [Cu(pmea)Cl]+ cations and ClO4

� counter ions and latticewater molecules. The Cu(II) centers of the complex cations are pen-ta-coordinated by 4 N donor atoms of the pmea ligand and one ter-minal chloro ligand. The geometry of the CuClN4 chromophoresmay be described as distorted SP [s-values: 0.15 (Cu1); 0.15(Cu2); 0.12 (Cu3) and 0.12 (Cu4)] [48]. The basal sites of each SP

are ligated by the three N(py) of the tetradentate blocker pmeaand the chloro ligand, whereas the axial sites are occupied by theN(amine) of pmea. The basal Cu–N(py) bond lengths are in therange from 1.9851(18) to 2.0690(17) Å, the axial Cu–N(amine) varyfrom 2.2374(17) to 2.2965(17) Å, and the Cu–Cl bond distancesrange from 2.2676(5) to 2.2833(6) Å (Table S2). These values agreewell with those reported by Karlin’s group [41]. Hydrogen bonds oftype O–H� � �O are observed between lattice water molecules andperchlorato oxygen atoms (Table S3). A packing plot with thehydrogen bond system is presented in Fig 2.

3.2. Syntheses and characterization of the Cu(II) complexes

The IR spectra of synthesized complexes [Cu(N4)ClO4]ClO4 (1,N4 = pzpdy; 2, N4 = hpzdpy [44]; 4, N4 = pmap; 5, N4 = tepa) and[Cu(pmea)Cl]ClO4�H2O (3) have some common features. The com-plexes display a strong absorption band around 1090 cm�1 attrib-utable to m(Cl–O) of the perchlorate and a series of bands over1440–1610 cm�1 region characteristic of the pyridyl groups. Thebroad band detected around 3516 cm�1 in 3 is assigned to thestretching vibration m(O–H) of the lattice water.

The visible spectral data for the complexes under investigationtogether with [Cu(TPA)Cl]ClO4 (6) are collected in Table 1. Theelectronic spectra of the complexes 1–5 in CH3CN display abroad absorption band in the 620–670 nm region. This feature is

Table 1Characterization of the Cu(II)–pyridyl complexes.

Complex UV–Vis kmax, nm (e, M�1 cm�1) KM (O�1 cm2 mol�1)

CH3CN H2O H2O

[Cu(pzdp)ClO4]ClO4 (1) 645 (310)a 647 (278) 188[Cu(hpzdp)ClO4]ClO4 (2) 621 (320)a 622 (215) 203[Cu(pmea)Cl]ClO4 (3) 669 (139) 670 (112, b) 172[Cu(pmap)ClO4]ClO4 (4) 643 (186) 639 (130) 200[Cu(tepa)ClO4]ClO4.½H2O (5) 664 (190) 657 (123) 204[Cu(TPA)Cl]ClO4.½H2Oa) (6) 730 (sh), 950 (209) 177

a) Ref. [44].

180 S.S. Massoud et al. / Inorganica Chimica Acta 399 (2013) 177–184

characteristic for five-coordinate Cu(II) complexes with a squarepyramidal (SP) geometry and the observed band originates fromthe 2B1 2E transition [42,49]. The presence of a single d–d bandat k > 800 nm (dxy, dx2 � y2 ? dz2) with a high-energy shoulder(spin forbidden, dxz, dyz ? dz2) is typical for trigonal bipyramidal(TBP) stereochemistry as in the case of complex 6 [49]. The disso-lution of the complexes in H2O was associated with significant de-crease in the molar absorptivity which is most likely attributed tothe formation of the aqua species, [Cu(N4)(H2O)]2+. This processtakes place with no stereochemical changes, i.e. the complexes stillretain their SP geometries. The geometrical SP finding about theCu(II) ion in solution is consistent with the X-ray structural datafor 1, 2 [44], 3, 4 and 5 [41,43].

As indicated above, in aqueous solution the perchlorato or thechloro complex ions [Cu(N4)ClO4/Cl]+ undergo instantaneous par-tial aquation with the formation of a small amount of [Cu(N4)(H2-

O)]2+ ion in equilibrium with the undissociated [Cu(N4)ClO4/Cl]+

species. To clarify this process, the molar conductivity, KM of thecomplexes 1–5 were measured in CH3CN and in H2O. While themolar conductivity of the complexes 3–5, measured in CH3CN,gave KM values within the range of 140–150 O�1cm2mol�1 whichare typical for a 1:1 electrolyte, the complexes 1 and 2 gave valuesof 300 and 289 O�1cm2mol�1, respectively, suggesting the pres-ence of a 1:2 electrolyte and in accordance with the complete dis-sociation of the complexes to the corresponding acetonitrilecomplex ions, [Cu(pzdp)(CH3CN)]2+ and [Cu(hpzdp)(CH3CN)]2+

(Eq. (3)). The results of the conductivity measurements in com-plexes 1 and 2 are supported by the isolation of the structurallycharacterized [Cu(N4)(CH3CN)](PF6)2 complexes, where N4 = pzdpand hpzdp in which the axial coordination site of each central Cu2+

ion is occupied by an acetonitrile molecule [44].

½CuIIðN4ÞClO4�ClO4 þ CH3CNðN4¼pzdp;1;hpzdp; 2Þ

� ½CuIIðN4ÞðCH3CNÞ�2þ þ 2ClO�4 ð3Þ

The molar conductivity measurements of the complexes in H2O(Table 1) reveal KM = 172–204 O�1 cm2 mol�1 which are higherthan a 1:1 electrolyte and much smaller than one would predictbased on a 1:2 electrolyte. These results indicate that in aqueoussolution, the dissociation of the coordinated ClO4

� or Cl� ion in[Cu(N4)ClO4/Cl]+ is not complete and an equilibrium of the typerepresented by Eq. (4) is taking place where the aqua species isconsidered to be the reactive species in aqueous medium.

½CuIIðN4ÞClO4=Cl�þH2O� ½CuIIðN4ÞðH2OÞ�2þ þ ClO�4 =Cl� ð4Þ

3.3. DNA cleavage study

3.3.1. Cleavage of plasmid DNA by [Cu(N4)X]ClO4 complexes (X = Cl orClO4

�)The synthesized square pyramidal five-coordinate copper(II)

complexes [Cu(N4)ClO4/Cl]ClO4 (1–5), in which the tetradentateamine ligands, N4 are showing systematic structural changes ongoing from pzdpy to hpzdpy and from TPA to tepa (Scheme 1) have

been used to study the DNA cleavage at pH 7.0, 1.0 mM Tris–Cl andat 37 �C. The incorporation of a methylene group(s) into the skele-ton of piperazine and into pyridyl arms of TPA should allow us toevaluate how the ring size and chelate rings (five vs. six) might af-fect the catalytic cleavage of DNA by the complexes. Among thefive complexes used in this study, the complexes 1–4 showed sig-nificant DNA cleavage where the supercoiled DNA (form I) wascleaved to the relaxed open circular DNA (form II) over a periodof 20–24 h as illustrated in Fig. 3. During this incubation period,complex 1 showed pronounced capability to further cleave formII to the linear DNA (form III) and continued cleavage of form IIIto shorter DNA fragments as observed by DNA smear (Fig. 4). Suchfurther cleavages of DNA to form III were not observed in com-plexes 2–5. Inspection of Fig. 3 reveals that although high DNAcleavage activity was clearly observed in complex 1, moderatecleavages were obtained in complexes 2, 3 and 4. In contrast, com-plex [Cu(tepa)Cl]ClO4 (5) did not exhibit obvious DNA cleavage asindicated by the fact that the percentages of forms I and II were al-most unchanged compared with the control over a period of 24 h(Fig. 3). Therefore, no more study was performed on this complexbut the complexes 1–4 were the subject of further detailed andmechanistic kinetics studies.

Since the five coordinate complexes 1–5 used in the DNA cleav-age reactions contain either the perchlorato or the chloro ligandsoccupying the fifth coordination site, in the square pyramidalgeometry of the complexes, it was important to examine what dif-ference these groups would make on the DNA cleavage activity. Toaddress this question, the DNA cleavage reactions were investi-gated using the complexes [Cu(pmap)ClO4]ClO4 (4) and[Cu(pmap)Cl]ClO4 (4a). Under similar conditions, the results whichare represented in Fig. 5 did not show any obvious difference in theDNA cleavage activity (Fig 5). Therefore, it was concluded that thecoordinated perchlorate and the chloride ligands would have thesame catalytic efficiency on the DNA cleavage; both lead to thegeneration of the same reactive aqua species, [Cu(pmap)(H2O)]2+

and probably the same concentration from the two complexes.

3.3.2. Kinetics of plasmid DNA cleavageThe kinetics studies of DNA cleavage by the complexes

[Cu(N4)ClO4/Cl]ClO4 (1–4) were carried out by following the con-version of form I to form II over various concentrations of the com-plexes (50–450 lM) and at constant pUC19 DNA concentration(76.8 lM) at different time intervals. A representative set of datafor the plots of the percentages of form II vs. time resulting fromDNA cleavage by 1 is shown in Fig. 6. Each of the curves followsa pseudo-first-order kinetic profile and was fitted to a single-exponential function using Eq. (1). From these curve-fitting results,the cleavage rate constant, kobs at each complex concentration wascalculated. The values of kcat and KM were evaluated from linearleast squares analyses of the plots of 1/kobs vs. 1/[Cu(II)] concentra-tions (Eq. (2b)) allowing a pseudo Michaelis–Menten analysis. Thisis illustrated in Fig. 7 for the DNA cleavage by complex 1. Underthese experimental conditions, values of kcat and KM were obtained

Form IIForm I

20 h

Form IIForm I Form III

24 h

Form IIForm I

24 h

Form IIForm I

24 h

Form IIForm I

24 h

(a)

(c)

(b)

(d)

(e)

Fig. 3. Electrophoretic separations of pUC19 plasmid DNA cleavage by Cu(II) complexes [Cu(N4)ClO4/Cl]ClO4: (a) 1, N4 = pzdpy; (b) 2, N4 = hpzdpy; (c) 3, pmea; (d) 4,N4 = pmap; (e) 5, N4 = tepa in 0.1 mM Tris–Cl buffer pH 7.0 and at 37 �C. The incubation time of plasmid DNA with a variety of Cu(II) complexes (0, 50, 100, 150, 300 and450 lM from left lane to the right lane) is indicated in each panel.

10 min 20 min 40 min

Form II Form I

60 min 90 min 120 min

Form II Form I

4 h 8 h

Form II Form I Form III

Fig. 4. The cleavage profile of pUC19 plasmid DNA by complex 1. The forms I, II and III as resolved by gel electrophoresis. DNA (76.8 lM) was incubated with varyingconcentrations of 1 (0, 50, 100, 150, 300 and 450 lM) in 0.1 mM Tris–Cl buffer pH 7.0 at 37 �C.

S.S. Massoud et al. / Inorganica Chimica Acta 399 (2013) 177–184 181

and data are collected in Table 2. Probably, we should add that theconsecutive conversion of DNA form II to form III by complex 1 is aslow process and no interference was observed during the forma-tion of form II.

The DNA cleavage activity by 1 is about one order of magnitudegreater than 2. Under similar conditions (pH 7.0 and 37 �C), the

constant for the catalytic cleavage of DNA (kcat) by the complexesunder investigation decreases in the following order: 1 > 3 >2 > 4� 5. With the exception of complex 5, the data shown inTable 2 clearly reveals the efficiency of these complexes to cleavethe double-stranded DNA. This reactivity trend indicates thatincreasing the number of the six-membered chelate rings in the

Form II Form I

[Cu(pmap)ClO4]ClO4 [Cu(pmap)Cl]ClO4

Form II Form I

(a) (b)

Fig. 5. Electrophoretic separations of pUC19 plasmid DNA cleavage by [Cu(pmap)ClO4/Cl]ClO4 (4/4a) complexes in 0.1 mM Tris–Cl buffer pH 7.0, and 37 �C for 20 h incubationtime. (a) [Cu(pmap)Cl]ClO4 and (4) and (b) [Cu(pmap)ClO4]ClO4 (4a).

DNA Cleavage Profile of Complex 1

0 20 40 60 80 100 1200

20

40

60

80

50 µM100 µM150 µM300 µM450 M

Time (min)

Perc

enta

ge o

f For

m II

µ

Fig. 6. The quantified percentages of form II vs. time for the cleavage of pUC19plasmid DNA by complex 1 at pH 7.0 and 37 �C. DNA (76.8 lM) was incubated withvarying concentrations of 1 (0, 50, 100, 150, 300 and 450 lM). The data was fittedby one phase exponential decay by prizm.

0.000 0.005 0.010 0.015 0.020 0.0250

10000

20000

30000

40000

1/[Cu]

1/k'

obs

Fig. 7. Pseudo first-order Plot of 1/kobs vs. 1/[1] for the DNA ([DNA] = 76.8 lM)cleavage by 1 (Eq. (2b)).

Table 2Pseudo Michaelis–Menten Kinetic data for the cleavage of pUC19 plasmidDNA(76.8 lM) by [Cu(N4)ClO4/Cl]ClO4 complexes (1–4).

Complex [Cu(II)] (lM) kobs (s�1) a kcat (h�1) KM (M)

1 50 3.17 � 10�5 8.89 � 10�1 3.35 � 10�4

100 6.08 � 10�5

150 7.36 � 10�5

300 1.24 � 10�4

450 1.28 � 10�4

2 50 5.75 � 10�6 9.79 � 10�2 1.96 � 10�4

100 8.22 � 10�6

150 1.06 � 10�5

300 1.77 � 10�5

450 2.32 � 10�5

3 50 1.24 � 10�5 1.47 � 10�1 1.93 � 10�4

100 1.58 � 10�5

150 1.78 � 10�5

300 2.04 � 10�5

450 2.30 � 10�5

4a 50 1.14 � 10�5 3.74 � 10�2 3.51 � 10�5

100 1.72 � 10�5

150 1.90 � 10�5

300 2.27 � 10�5

450 2.48 � 10�5

a For fitting purpose a linear form of Eq. (1) was used with the natural logarithmof the faction of form I plotted as a function of time and straight line fit used toobtain a slope of �kobs.

Con

trol

1 DM

SO

1+

DM

SO

KI 1+

KI

NaN

3

1+

NaN

3Form IIForm I

DM

SO

Fig. 8. Electrophoretic separations of pUC19 plasmid DNA cleavage by complex 1 inthe presence and absence of oxidative scavengers. Plasmid DNA (76.8 lM) wasincubated with 450 lM 1 in the presence of 0.4 M DMSO, 500 lM KI and 500 lMNaN3 at 37 �C for 2 h.

182 S.S. Massoud et al. / Inorganica Chimica Acta 399 (2013) 177–184

complexes by incorporating a methylene group into the piperazinering (hpzdpy) or into the TPA pyridyl arms (pmea, pmap and tepa)(Scheme 1) tends to suppress their DNA cleavage activity.

Con

trol

2 DM

SO

2+

DM

SO

KI 2+

KI

NaN

3

2+

NaN

3

Form II

Form I

Fig. 9. Electrophoretic separations of pUC19 plasmid DNA cleavage by complex 2 inthe presence and absence of oxidative scavengers. Plasmid DNA (76.8 lM) wasincubated with 150 lM 2 in the presence of 0.4 M DMSO, 500 lM KI and 500 lMNaN3 at 37 �C for 12 h.

3.3.3. DNA cleavage mechanismIn order to clarify the DNA cleavage mechanism of complexes

1–4 (oxidative vs. hydrolytic cleavage), cleavage of DNA was fur-ther investigated in presence and absence of oxidative scavengers:DMSO (OH�), NaN3 (1O2) and KI (O2

��). It is well established thatreactive oxygen species (ROS) such as singlet oxygen (1O2), hydro-gen peroxide (H2O2) or hydroxyl radical (HO�) cause damage to thesugar and/or base moieties of DNA [30]. For complex 1, three inde-pendent experiments demonstrated that NaN3 and KI scavengersshowed very little effect on the DNA cleavage while DMSO scaven-ger significantly inhibits the DNA cleavage activity (Fig. 8). These

Table 3Pseudo Michaelis–Menten kinetic data for the cleavage of DNA by some Cu(II)–pyridylcomplexes (1–5) at pH 7.0 and 37 �C.

Complex kcat (h�1) KM (M) Enhancementa

[Cu(pzdp)ClO4]ClO4 (1) 0.889 3.35 � 10�4 2.5 � 107

[Cu(hpzdp)ClO4]ClO4

(2)0.0979 1.96 � 10�4 2.7 � 106

[Cu(pmea)Cl]ClO4 (3) 0.147 1.93 � 10�4 4.1 � 106

[Cu(pmap)Cl]ClO4 (4a) 0.0374 3.51 � 10�5 1.0 � 106

[Cu(tepa)Cl]ClO4 (5) Almost nocleavage

a Rate enhancement over the non-catalyzed DNA (k = 3.6 � 10�8 h�1 at 37 �C andpH 7.0).

S.S. Massoud et al. / Inorganica Chimica Acta 399 (2013) 177–184 183

results suggest that the DNA cleavage by 1 most likely involves oxi-dative cleavage by hydroxyl radical species. For complexes 2, 3 and4, none of the ROS species were involved in the cleavage process;no inhibition of DNA cleavage was observed in the presence ofoxidative scavengers. The DNA cleavage by 2 in the presence andabsence of oxidative scavengers is shown in Fig 9. Therefore, it isconcluded that the DNA cleavage by these complexes preferablyundergoes a hydrolytic cleavage process.

4. Conclusion

In this study, we demonstrated that under pseudo Michaelis–Menten conditions the DNA cleavage activity (pH 7.0 and 37 �C)by the chloro or perchlorato complexes 1–5 decreases withincreasing the number of six-membered chelate rings; the catalyticrate constant, kcat decreases in the series 1 > 2 and 3 P 4� 5.These data are summarized in Table 3 together with the enhance-ments over the non-catalyzed DNA (k = 3.6 � 10�8 h�1 at 37 �C [1]).The increased reactivity of Cu(II) complexes with five-memberedchelate ring sizes, most likely is not only attributed to the in-creased stability associated with these chelate rings compared tothe corresponding six-membered chelate rings [50], but also the in-creased size of the chelate rings may suppress the approach of thecomplexes to the DNA and hence inhibit its cleavage.

The rate enhancement obtained here with 1, which correspondsto 25-million-fold over the non-catalyzed DNA reveals the effi-ciency of the complex to cleave the ds DNA and puts the complexas one of the most effective artificial nucleases. Although all com-plexes 2–4 cleave form I DNA to form II, complex 1 showed bothsingle and double stranded DNA cleavage activity. The operatingmechanism in the latter complex is oxidative via the hydroxyl rad-ical species, whereas the DNA cleavage by the complexes 2–4 ismost likely proceeding via the hydrolytic mechanism. There is nodirect evidence to link the catalytic oxidative cleavage of DNA by1 to its high artificial nuclease activity.

Acknowledgments

We thank the Department of Chemistry (UL Lafayette) forfinancing this work, and the students Nicole M. Leger and BrittanyA. Burke for conducting some of the DNA cleavage experiments.

Appendix A. Supplementary data

CCDC 896681 contains the supplementary crystallographic datafor this paper. These data can be obtained free of charge from TheCambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif. Supplementary data associated withthis article can be found, in the online version, at http://dx.doi.org/10.1016/j.ica.2013.01.020.

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