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

Inorganica Chimica Acta 399 (2013) 177–184

Contents lists available at SciVerse ScienceDirect

Inorganica Chimica Acta journal homepage: www.elsevier.com/locate/ica

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 b c

Department of Chemistry, University of Louisiana at Lafayette, Lafayette, LA 70504, USA Institut für Physikalische and Theoretische Chemie, Technische Universität Graz, A-8010 Graz, Austria 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 2012 Received in revised form 26 November 2012 Accepted 20 January 2013 Available online 1 February 2013 Keywords: DNA cleavage Hydrolytic cleavage Oxidative cleavage Copper Polypyridyl Crystal structure

a b s t r a c t Square pyramidal five-coordinate copper(II) complexes of the general formula [Cu(N4)ClO4]ClO4, N4 represents a tetradentate ligand where N4 = pzdpy (1,4-bis(2-pyridylmethy)piperazine), 1; hpzpy (1,4-bis(2pyridylmethyl)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 crystallography of 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 used to study the DNA cleavage activity at pH 7.0 and 37 °C. Under pseudo Michaelis–Menten conditions, the constant for the catalytic cleavage of DNA, kcat decreases in the following order: 1 > 3 > 2 > 4  5. Complex 1 showed very high nuclease activity with a rate enhancement of 25-million-fold over the non-catalyzed DNA. The results demonstrated that an increase in the number of six-membered rings in the complexes suppresses the cleavage process. Although the mechanistic studies of DNA cleavage by the complexes in presence of oxidative scavengers indicate that the mechanism of the cleavage in complexes 2–4 is most likely hydrolytic in nature, an oxidative mechanism via hydroxyl radical was revealed with complex 1. Published by Elsevier B.V.

1. Introduction The phosphodiester bonds in DNA have remarkable stability toward cleavage under physiological conditions (kuncat = 3.6  108 h1, t1/2  130,000 years). This stability is considered to be one of the essential requirements for the survival and maintenance of life and may also explain why nature chose DNA for this mission [1]. However, in vivo nature has developed certain enzymes such as restriction enzymes and EcoRI endonuclease which efficiently and rapidly catalyze the hydrolytic cleavage of P–O bonds in DNA. A variety of transition metal complexes have been launched as ‘‘artificial nucleases’’ to catalyze the DNA cleavage under the

Abbreviations: TPA, tris(2-pyridylmethyl)amine; pmea, bis(2-pyridylmethyl)2-(2-pyridylethyl)- amine; pmap, bis(2-(2-pyridylethyl))-(2-pyridylmethyl)amine; tepa, tris (2-(2-pyridyl)ethyl)amine; pzdpy, 1,4-bis(2-(2-pyridylmethyl))piperazine; hpzdpy, 1,4-bis(2-(2-pyridylmethyl))homopiperazine. ⇑ Corresponding authors. Tel.: +1 337 482 5672; fax: +1 337 482 5670 (S.S. Massoud). Tel.: +1 337 482 5684; fax: +1 337 482 5670 (W. Xu). E-mail addresses: [email protected] (S.S. Massoud), wxx6941@ louisiana.edu (W. Xu). 0020-1693/$ - see front matter Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ica.2013.01.020

physiological conditions but many of these compounds cannot be compare to the enzymatic cleavage [2–4]. In general, two mechanisms have been reported for DNA cleavage by artificial nucleases: the oxidative cleavage and the hydrolytic cleavage. In the former mechanistic pathway, reactive oxygen species, ROS (singlet molecular oxygen, 1O2; hydroxyl radical, HO; superoxide radical, O2–) which are generated during the cleavage process and these non-natural fragments cause damage to the deoxyribose sugar or the nucleic base moieties [5,6]. These species hamper their use in vivo. In the hydrolytic mechanism, the hydrolytic cleavage agents do not induce this problem and hence the product can be enzymatically ligated. This hydrolytic feature makes these compounds function by a mechanism that is similar to the natural enzymatic reactions but also proves useful in elucidating the precise role of metal ions in this enzymatic catalytic process [2–4]. In the search for artificial nucleases that effectively catalyze DNA cleavage many divalent mono- and dinuclear-copper(II), cobalt(II) and zinc(II) as well as trivalent cobalt(III) complexes derived from a wide range of ligands of varied skeletal structures and geometrical environments have been extensively investigated

178

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

N N

N

N N

N N

pzdpy

N

N N

N

hpzdpy

N N

N

TPA

N

N N

N

pmea

N

N N

N

pmap

N N

tepa

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

[7–40]. Although some of these compounds, especially those derived from Cu(II) and Co(II), have been reported to reveal high catalytic activity [8,13,14,30,33,35–38], the DNA cleavage mechanism was found to be oxidative in nature [18–24]. Therefore in order to design artificial nucleases that efficiently and hydrolytically catalyze DNA cleavage, a systematic study should be undertaken to explore the factors that might affect the cleavage of DNA such as the nature of the central metal ion [8–11] and structural features of the ligand. In an effort to understand how the structural parameters incorporated in the ligand skeleton (ring size, chelate ring) coordinating a metal ion affect the DNA cleavage process, a series of mononuclear copper(II) complexes with tetradentate amine ligands with different pyridyl arm lengths, and 1,4-piperazine and 1,5-homopiperazine with different ring sizes have been synthesized and their cleavage activities with DNA were investigated at 37 °C and pH 7.0. The ligand skeletons used in this study are illustrated in Scheme 1.

2. Experimental 2.1. Materials and physical measurements 2-Picolylchloride hydrochloride and 2-pyridylmethylamine, 2vinylpyridine, piperazine and homopiprazine were purchased from Aldrich Chemical Company, USA, whereas dipicolylamine was obtained from TCI-America. All other materials were reagent grade quality. 2-Vinylpyridine was distilled and purified by column chromatography using alumina and eluted with a 90/10 (v/v) mixture of ethyl acetate/MeOH. All other materials were reagent grade quality. Infrared spectra were recorded on a JASCO FT/IR-480 plus spectrometer as KBr pellets. Electronic spectra were recorded on an Agilent 8453 HP diode UV–VIS spectrophotometer. 1H and 13C NMR spectra were obtained at room temperature on a Varian 400 NMR spectrometer operating at 400 MHz (1H) and 100 MHz (13C). 1H and 13C NMR chemical shifts (d) are reported in ppm and were referenced internally to residual solvent resonances (DMSO-d6: dH = 2.49, dC = 39.4 ppm). Mass spectra were obtained on Agilent 7890 AGC coupled to an Agilent 5975C Mass Selective Detector (MSD). Electrical conductivity measurements were the same as reported elsewhere [8]. The molar conductivity of a solution sample was determined from KM = (1.0  103 j)/M, where j = cell constant and M is the molar concentration of the complex. Elemental microanalyses were performed at the Atlantic Microlaboratory, Norcross, Georgia USA. The pUC19 Plasmids DNA purification, determination of concentrations and plasmid DNA cleavage as well as gel electrophoresis procedures were similar to those which have been recently reported [7,8]. The intensity of the different forms of plasmid DNA was quantified using Quantity One software (Bio-Rad Laboratories, Hercules, CA 94547). Caution: Salts of perchlorate and their metal complexes are potentially explosive and should be handled with great care and in small quantities.

2.1.1. Synthesis of the ligands The 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], 1,4bis(2-(2-pyridylmethyl))piperazine (pzdpy), and 1,4-bis(2-(2-pyridylmethyl))homopiperazine (hpzdpy) were synthesized and characterized following the published procedures [44].

2.1.2. Synthesis of [Cu(N4)X]ClO4 complexes The perchlorato complexes [Cu(N4)ClO4]ClO4 (1, N4 = pzpdy; 2, N4 = hpzdpy [44]; 4, N4 = pmap; 5, N4 = tepa) were obtained by treating Cu(ClO4)26H2O dissolved in MeOH with the appropriate ligand, followed by heating on a steam-bath for 5 min. Complexes obtained were recrystallized from H2O (yield 70–80%). The chloro complex [Cu(pmea)Cl]ClO4H2O (3) was synthesized by the addition of 1 mL of saturated solution of NaClO4 to a hot mixture containing CuCl22H2O (0.170 g, 1 mmol) and an equimolar amount of bis(2-pyridylmethyl)-2-(2-pyridylethyl)amine (pmea) in MeOH (20 mL). The resulting blue precipitate was collected by filtration and recrystallized from H2O (yield: 70–80%). Single crystals of 3 suitable 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 O1 cm2 mol1 in H2O and CH3CN, respectively. UV–Vis (kmax in nm, e in M1cm1) 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 O1 cm2 mol1 in H2O and CH3CN, respectively. UV–Vis (kmax in nm, e in M1cm1) in CH3CN: 621 (320); in H2O: 622 (215). The UV–VIS in CH3CN and the solid IR spectra of the complexes 1 and 2 were in complete agreement with that reported by Halfen et al. [44]. [Cu(pmea)Cl]ClO4H2O (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 O1 cm2 mol1. UV–Vis (kmax in nm, e in M1 cm1) in CH3CN: 669 (139); in H2O: 670 (112). Selected IR bands (KBr, cm1): 3516 (m, br), 1608 (s), 1571 (m), 1485 (m), 1442 (s), 1092 (vs) cm1. [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 O1 cm2 mol1. UV–Vis (kmax in nm, e in M1cm1) in CH3CN: 643 (186); in H2O: 637 (130). Selected IR bands (KBr, cm1): 1607 (s), 1572 (m), 1486 (m), 1442 (s), 1090 (vs) cm1. [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 O1 cm2 mol1 in H2O and CH3CN, respectively, UV–Vis (kmax in nm, e in M1cm1) in CH3CN: 643 (186); in H2O: 637 (130). Selected IR bands (KBr, cm1): 1607 (s), 1572 (m), 1486 (m), 1442 (s), 1090 (vs) cm1.

179

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

[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 O1 cm2 mol1 in H2O and CH3CN, respectively. UV–Vis (kmax in nm, e in M1cm1) in CH3CN: 664 (190); in H2O: 657 (123). 2.2. Crystal structure analysis of [Cu(pmea)Cl]ClO4H2O (3) The X-ray single-crystal data of compound 3 were collected on a Bruker-AXS APEX II CCD diffractometer at 100(2) K. The crystallographic data, conditions retained for the intensity data collection and some features of the structure refinements are listed in Table S1. The intensities were collected with Mo Ka radiation (k = 0.71073 Å). Data processing, Lorentz-polarization and absorption corrections were performed using APEX, SAINT and the SADABS computer programs [45]. The structure was solved by direct methods and refined by full-matrix least-squares methods on F2, using the SHELXTL [46] program package. All non-hydrogen atoms were refined anisotropically. The hydrogen atoms were located from difference Fourier maps, assigned with isotropic displacement factors and included in the final refinement cycles by use of geometrical constraints. Split occupancy of the counter perchlorate ion was applied for disordered oxygen atoms O(12A) and O(12B). Selected bond parameters are listed in Table S2, hydrogen bonds are summarized in Table S3.

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

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 in 1.0 mM Tris–Cl buffer, pH 7.0, at 37 °C for different intervals of time according to previously published procedures [7,8]. The percentages of form II quantified by Quantity One software were plotted against time for each catalyst concentration. The observed rate constants, kobs were extracted from the single-exponential curve (pseudo first-order kinetics) using Eq. (1) [47]:

y ¼ ð100  yo Þð1  exp ðkobs tÞÞ

ð1Þ

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

Fig. 2. Packing view of 3.

3. Results and discussion

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

3.1. Description of the structure of [Cu(pmea)Cl]ClO4H2O (3)

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

Single crystal structure determination of 3 revealed a triclinic unit cell with V = 4257.6(2) Å3 and Z = 8. Karlin and his coworkers have reported a smaller triclinic unit cell with V = 2162.4(7) Å3 and Z = 4 for the same complex [41]. A perspective view of the asymmetric unit together with the partial atom numbering scheme for 3 is presented in Fig. 1. The structure consists of monomeric complex [Cu(pmea)Cl]+ cations and ClO4 counter ions and lattice water molecules. The Cu(II) centers of the complex cations are penta-coordinated by 4 N donor atoms of the pmea ligand and one terminal chloro ligand. The geometry of the CuClN4 chromophores may 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

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]ClO4H2O (3) have some common features. The complexes display a strong absorption band around 1090 cm1 attributable to m(Cl–O) of the perchlorate and a series of bands over 1440–1610 cm1 region characteristic of the pyridyl groups. The broad band detected around 3516 cm1 in 3 is assigned to the stretching vibration m(O–H) of the lattice water. The visible spectral data for the complexes under investigation together with [Cu(TPA)Cl]ClO4 (6) are collected in Table 1. The electronic spectra of the complexes 1–5 in CH3CN display a broad absorption band in the 620–670 nm region. This feature is

K obs ¼ kcat ½complex=ðK M þ ½complexÞ

ð2aÞ

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

1=kobs ¼ ðK M =kcat Þð1=½complexÞ þ 1=kcat

ð2bÞ

180

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

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

[Cu(pzdp)ClO4]ClO4 [Cu(hpzdp)ClO4]ClO4 [Cu(pmea)Cl]ClO4 [Cu(pmap)ClO4]ClO4 [Cu(tepa)ClO4]ClO4.½H2O [Cu(TPA)Cl]ClO4.½H2Oa) a)

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

UV–Vis kmax,

nm (e, M1 cm1)

KM (O1 cm2 mol1)

CH3CN

H2O

H2O

645 621 669 643 664 730

(310)a (320)a (139) (186) (190) (sh), 950 (209)

647 622 670 639 657

(278) (215) (112, b) (130) (123)

188 203 172 200 204 177

Ref. [44].

characteristic for five-coordinate Cu(II) complexes with a square pyramidal (SP) geometry and the observed band originates from 2 the 2B1 E transition [42,49]. The presence of a single d–d band at 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 dissolution of the complexes in H2O was associated with significant decrease in the molar absorptivity which is most likely attributed to the formation of the aqua species, [Cu(N4)(H2O)]2+. This process takes place with no stereochemical changes, i.e. the complexes still retain their SP geometries. The geometrical SP finding about the Cu(II) ion in solution is consistent with the X-ray structural data for 1, 2 [44], 3, 4 and 5 [41,43]. As indicated above, in aqueous solution the perchlorato or the chloro complex ions [Cu(N4)ClO4/Cl]+ undergo instantaneous partial aquation with the formation of a small amount of [Cu(N4)(H2O)]2+ ion in equilibrium with the undissociated [Cu(N4)ClO4/Cl]+ species. To clarify this process, the molar conductivity, KM of the complexes 1–5 were measured in CH3CN and in H2O. While the molar conductivity of the complexes 3–5, measured in CH3CN, gave KM values within the range of 140–150 O1cm2mol1 which are typical for a 1:1 electrolyte, the complexes 1 and 2 gave values of 300 and 289 O1cm2mol1, respectively, suggesting the presence of a 1:2 electrolyte and in accordance with the complete dissociation of the complexes to the corresponding acetonitrile complex ions, [Cu(pzdp)(CH3CN)]2+ and [Cu(hpzdp)(CH3CN)]2+ (Eq. (3)). The results of the conductivity measurements in complexes 1 and 2 are supported by the isolation of the structurally characterized [Cu(N4)(CH3CN)](PF6)2 complexes, where N4 = pzdp and hpzdp in which the axial coordination site of each central Cu2+ ion is occupied by an acetonitrile molecule [44]. 

½CuII ðN4ÞClO4 ClO4 þ CH3 CN ½CuII ðN4ÞðCH3 CNÞ2þ þ 2ClO4

ð3Þ

ðN4¼pzdp;1;hpzdp; 2Þ

The molar conductivity measurements of the complexes in H2O (Table 1) reveal KM = 172–204 O1 cm2 mol1 which are higher than a 1:1 electrolyte and much smaller than one would predict based on a 1:2 electrolyte. These results indicate that in aqueous solution, the dissociation of the coordinated ClO4 or Cl ion in [Cu(N4)ClO4/Cl]+ is not complete and an equilibrium of the type represented by Eq. (4) is taking place where the aqua species is considered to be the reactive species in aqueous medium. 



½CuII ðN4ÞClO4 =Clþ H2 O ½CuII ðN4ÞðH2 OÞ2þ þ ClO4 =Cl

ð4Þ

3.3. DNA cleavage study 3.3.1. Cleavage of plasmid DNA by [Cu(N4)X]ClO4 complexes (X = Cl or ClO4) The synthesized square pyramidal five-coordinate copper(II) complexes [Cu(N4)ClO4/Cl]ClO4 (1–5), in which the tetradentate amine ligands, N4 are showing systematic structural changes on going 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 and at 37 °C. The incorporation of a methylene group(s) into the skeleton of piperazine and into pyridyl arms of TPA should allow us to evaluate how the ring size and chelate rings (five vs. six) might affect the catalytic cleavage of DNA by the complexes. Among the five complexes used in this study, the complexes 1–4 showed significant DNA cleavage where the supercoiled DNA (form I) was cleaved to the relaxed open circular DNA (form II) over a period of 20–24 h as illustrated in Fig. 3. During this incubation period, complex 1 showed pronounced capability to further cleave form II to the linear DNA (form III) and continued cleavage of form III to shorter DNA fragments as observed by DNA smear (Fig. 4). Such further cleavages of DNA to form III were not observed in complexes 2–5. Inspection of Fig. 3 reveals that although high DNA cleavage activity was clearly observed in complex 1, moderate cleavages were obtained in complexes 2, 3 and 4. In contrast, complex [Cu(tepa)Cl]ClO4 (5) did not exhibit obvious DNA cleavage as indicated by the fact that the percentages of forms I and II were almost unchanged compared with the control over a period of 24 h (Fig. 3). Therefore, no more study was performed on this complex but the complexes 1–4 were the subject of further detailed and mechanistic kinetics studies. Since the five coordinate complexes 1–5 used in the DNA cleavage reactions contain either the perchlorato or the chloro ligands occupying the fifth coordination site, in the square pyramidal geometry of the complexes, it was important to examine what difference these groups would make on the DNA cleavage activity. To address this question, the DNA cleavage reactions were investigated using the complexes [Cu(pmap)ClO4]ClO4 (4) and [Cu(pmap)Cl]ClO4 (4a). Under similar conditions, the results which are represented in Fig. 5 did not show any obvious difference in the DNA cleavage activity (Fig 5). Therefore, it was concluded that the coordinated perchlorate and the chloride ligands would have the same catalytic efficiency on the DNA cleavage; both lead to the generation 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 cleavage The kinetics studies of DNA cleavage by the complexes [Cu(N4)ClO4/Cl]ClO4 (1–4) were carried out by following the conversion of form I to form II over various concentrations of the complexes (50–450 lM) and at constant pUC19 DNA concentration (76.8 lM) at different time intervals. A representative set of data for the plots of the percentages of form II vs. time resulting from DNA cleavage by 1 is shown in Fig. 6. Each of the curves follows a pseudo-first-order kinetic profile and was fitted to a singleexponential function using Eq. (1). From these curve-fitting results, the cleavage rate constant, kobs at each complex concentration was calculated. The values of kcat and KM were evaluated from linear least squares analyses of the plots of 1/kobs vs. 1/[Cu(II)] concentrations (Eq. (2b)) allowing a pseudo Michaelis–Menten analysis. This is illustrated in Fig. 7 for the DNA cleavage by complex 1. Under these experimental conditions, values of kcat and KM were obtained

181

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

(a)

(b)

Form II Form I

Form III Form II Form I 24 h

20 h

(c)

(d)

Form II Form I

Form II Form I 24 h

24 h

(e) Form II Form I 24 h 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 and 450 lM from left lane to the right lane) is indicated in each panel.

Form II Form I 10 min

20 min

40 min

60 min

90 min

120 min

Form II Form I

Form II Form III

Form I

4h

8h

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 varying concentrations of 1 (0, 50, 100, 150, 300 and 450 lM) in 0.1 mM Tris–Cl buffer pH 7.0 at 37 °C.

and data are collected in Table 2. Probably, we should add that the consecutive conversion of DNA form II to form III by complex 1 is a slow process and no interference was observed during the formation of form II. The DNA cleavage activity by 1 is about one order of magnitude greater than 2. Under similar conditions (pH 7.0 and 37 °C), the

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

182

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

(a)

(b)

Form II

Form II

Form I

Form I

[Cu(pmap)ClO4]ClO4

[Cu(pmap)Cl]ClO4

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 incubation time. (a) [Cu(pmap)Cl]ClO4 and (4) and (b) [Cu(pmap)ClO4]ClO4 (4a).

DNA Cleavage Profile of Complex 1

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

60 50 µM 100 µM 150 µM 300 µM 450 µM

40

20

0

20

40

60

80

100

120

40000

KM (M)

3.17  10 6.08  105 7.36  105 1.24  104 1.28  104

8.89  101

3.35  104

2

50 100 150 300 450

5.75  106 8.22  106 1.06  105 1.77  105 2.32  105

9.79  102

1.96  104

3

50 100 150 300 450

1.24  105 1.58  105 1.78  105 2.04  105 2.30  105

1.47  101

1.93  104

4a

50 100 150 300 450

1.14  105 1.72  105 1.90  105 2.27  105 2.48  105

3.74  102

3.51  105

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

30000

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

complexes by incorporating a methylene group into the piperazine ring (hpzdpy) or into the TPA pyridyl arms (pmea, pmap and tepa) (Scheme 1) tends to suppress their DNA cleavage activity. 3.3.3. DNA cleavage mechanism In order to clarify the DNA cleavage mechanism of complexes 1–4 (oxidative vs. hydrolytic cleavage), cleavage of DNA was further investigated in presence and absence of oxidative scavengers: DMSO (OH), NaN3 (1O2) and KI (O2). It is well established that reactive oxygen species (ROS) such as singlet oxygen (1O2), hydrogen peroxide (H2O2) or hydroxyl radical (HO) cause damage to the sugar and/or base moieties of DNA [30]. For complex 1, three independent experiments demonstrated that NaN3 and KI scavengers showed very little effect on the DNA cleavage while DMSO scavenger significantly inhibits the DNA cleavage activity (Fig. 8). These

1 + NaN3

NaN3

1 + KI

KI

1 + DMSO

Form II Form I Fig. 8. Electrophoretic separations of pUC19 plasmid DNA cleavage by complex 1 in the presence and absence of oxidative scavengers. Plasmid DNA (76.8 lM) was incubated with 450 lM 1 in the presence of 0.4 M DMSO, 500 lM KI and 500 lM NaN3 at 37 °C for 2 h.

2 + NaN3

0.025

NaN3

0.020

2 + KI

0.015

1/[Cu]

KI

0.010

2 + DMSO

0.005

Control

0 0.000

Control

10000

DMSO

20000

DMSO

1/k'obs

kcat (h1)

50 100 150 300 450

Time (min) Fig. 6. The quantified percentages of form II vs. time for the cleavage of pUC19 plasmid DNA by complex 1 at pH 7.0 and 37 °C. DNA (76.8 lM) was incubated with varying concentrations of 1 (0, 50, 100, 150, 300 and 450 lM). The data was fitted by one phase exponential decay by prizm.

a 5

1

1

0

kobs (s1)

[Cu(II)] (lM)

2

Percentage of Form II

80

Form II Form I

Fig. 9. Electrophoretic separations of pUC19 plasmid DNA cleavage by complex 2 in the presence and absence of oxidative scavengers. Plasmid DNA (76.8 lM) was incubated with 150 lM 2 in the presence of 0.4 M DMSO, 500 lM KI and 500 lM NaN3 at 37 °C for 12 h.

S.S. Massoud et al. / Inorganica Chimica Acta 399 (2013) 177–184 Table 3 Pseudo Michaelis–Menten kinetic data for the cleavage of DNA by some Cu(II)–pyridyl complexes (1–5) at pH 7.0 and 37 °C. Complex

kcat (h1)

KM (M)

Enhancementa

[Cu(pzdp)ClO4]ClO4 (1) [Cu(hpzdp)ClO4]ClO4 (2) [Cu(pmea)Cl]ClO4 (3) [Cu(pmap)Cl]ClO4 (4a) [Cu(tepa)Cl]ClO4 (5)

0.889 0.0979

3.35  104 1.96  104

2.5  107 2.7  106

0.147 0.0374 Almost no cleavage

1.93  104 3.51  105

4.1  106 1.0  106

a Rate enhancement over the non-catalyzed DNA (k = 3.6  108 h1 at 37 °C and pH 7.0).

results suggest that the DNA cleavage by 1 most likely involves oxidative cleavage by hydroxyl radical species. For complexes 2, 3 and 4, none of the ROS species were involved in the cleavage process; no inhibition of DNA cleavage was observed in the presence of oxidative scavengers. The DNA cleavage by 2 in the presence and absence of oxidative scavengers is shown in Fig 9. Therefore, it is concluded that the DNA cleavage by these complexes preferably undergoes 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 with increasing the number of six-membered chelate rings; the catalytic rate constant, kcat decreases in the series 1 > 2 and 3 P 4  5. These data are summarized in Table 3 together with the enhancements over the non-catalyzed DNA (k = 3.6  108 h1 at 37 °C [1]). The increased reactivity of Cu(II) complexes with five-membered chelate ring sizes, most likely is not only attributed to the increased stability associated with these chelate rings compared to the corresponding six-membered chelate rings [50], but also the increased size of the chelate rings may suppress the approach of the complexes to the DNA and hence inhibit its cleavage. The rate enhancement obtained here with 1, which corresponds to 25-million-fold over the non-catalyzed DNA reveals the efficiency of the complex to cleave the ds DNA and puts the complex as one of the most effective artificial nucleases. Although all complexes 2–4 cleave form I DNA to form II, complex 1 showed both single and double stranded DNA cleavage activity. The operating mechanism in the latter complex is oxidative via the hydroxyl radical species, whereas the DNA cleavage by the complexes 2–4 is most likely proceeding via the hydrolytic mechanism. There is no direct evidence to link the catalytic oxidative cleavage of DNA by 1 to its high artificial nuclease activity. Acknowledgments We thank the Department of Chemistry (UL Lafayette) for financing this work, and the students Nicole M. Leger and Brittany A. Burke for conducting some of the DNA cleavage experiments. Appendix A. Supplementary data CCDC 896681 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via http://www.ccdc. cam.ac.uk/data_request/cif. Supplementary data associated with this article can be found, in the online version, at http:// dx.doi.org/10.1016/j.ica.2013.01.020.

183

References [1] G.K. Schroeder, C. Lad, P. Wyman, N.H. Williams, R. Wolfenden, Proc. Natl. Acad. Sci. U.S.A. 103 (2006) 4052. [2] J.A. Cowan, Nucl. Acids Mol. Biol. 14 (2004) 339. [3] C. Liu, M. Wang, T. Zhang, H. Sun, Coord. Chem. Rev. 248 (2004) 147. [4] A. Sreedhara, J.A. Cowan, J. Biol. Inorg. Chem. 6 (2001) 337. [5] M. Pitié, C. Boldron, G. Pratviel, in: R. van Eldik, J. Reedijk (Eds.), Advances in Inorganic Chemistry 58 (2006) 71. [6] K.J. Humphreys, K.D. Karlin, S.E. Rokita, J. Am. Chem. Soc. 124 (2002) 6009. [7] S.S. Massoud, F.R. Louka, W. Xu, R. Perkins, R. Vicente, J.H. Albering, F.A. Mautner, Eur. J. Inorg. Chem. (2011) 3469. [8] W. Xu, J.A. Craft, P.R. Fontenot, B. Marion, K.D. Knierim, J.H. Albering, F.A. Mautner, S.S. Massoud, Inorg. Chim. Acta 373 (2011) 159. [9] J. Hong, Y. Jiao, J. Yan, W. He, Z. Guo, L. Xhu, J. Zhang, Inorg. Chim. Acta 363 (2010) 793. [10] X.-Y. Wang, J. Zhang, K. Li, N. Jiang, S.-Y. Chen, H.-H. Lin, Y. Huang, L.-J. Ma, X.Q. Yu, J. Bioorg, Med. Chem. 14 (2006) 6745. [11] Y. Jin, M.A. Lewis, N.H. Gokhale, E.C. Long, J.A. Cowan, J. Am. Chem. Soc. 129 (2007) 8353. [12] J.L. Gracía-Giménez, G. Alzuet, M. Gonzáles-Álvarez, A. Castiñeiras, M. Gonzáles-Álvarez, J. Borrás, Inorg. Chem. 46 (2007) 7178. [13] J. Qian, X. Ma, J. Tian, W. Gu, J. Shang, X. Liu, S.-P. Yan, J. Inorg. Biochem. 104 (2010) 993. [14] Y. An, S.-D. Liu, S.-Y. Deng, L.-N. Ji, Z.-W. Mao, J. Inorg. Biochem. 100 (2006) 1586. [15] Q.-X. Xiang, J. Zhang, P.-Y. Liu, C.-Q. Xia, Z.-Y. Zhou, R.-G. Xie, X.-Q. Yu, J. Inorg. Biochem. 99 (2005) 1661. [16] L.M. Rossi, A. Neves, R. Hörner, H. Terenzi, B. Szpoganicz, J. Sugai, Inorg. Chim. Acta 337 (2002) 366. [17] D.-D. Li, J.-L. Tian, W. Gu, X. Liu, H.-H. Zeng, S.-P. Yan, J. Inorg. Biochem. 105 (2011) 894. [18] D.-D. Li, F.-P. Huang, G.-J. Chen, C.-Y. Gao, J.-L. Tian, W. Gu, X. Liu, S.-P. Yan, J. Inorg. Biochem. 104 (2010) 431. [19] H. Prakash, A. Shodal, H. Yasui, H. Sakurai, S. Hirota, Inorg. Chem. 47 (2008) 5045. [20] V. Uma, M. Kanthimathi, T. Weyhermuller, B.U. Nair, J. Inorg. Biochem. 99 (2005) 2299. [21] (a) K. Ghosh, P. Kumar, N. Tyagi, U.P. Singh, Inorg. Chem. 49 (2010) 7614; (b) K. Ghosh, P. Kumar, N. Tyagi, U.P. Singh, N. Goel, Inorg. Chem. Commun. 14 (2011) 489. [22] Y. Zhao, J. Zhu, W. He, Z. Yang, Z. Zhu, Y. Li, J. Zhang, Z. Guo, Chem. Eur. J. 12 (2006) 6621. [23] F.B.A. El Amrani, L. Perelló, J.A. Real, M. Gonzáles-Álvarez, G. Alzuet, J. Borrás, S. Gracía-Granda, J. Montejo-Bernardo, J. Inorg. Biochem. 100 (2006) 1208. [24] R. Indumathy, T. Weyhermuller, B.U. Nair, Dalton Trans. 39 (2010) 2087. [25] L. Tjioe, A. Meininger, T. Joshi, L. Spiccia, B. Graham, Inorg. Chem. 50 (2011) 4327. [26] L. Tjioe, T.T. Joshi, J. Brugger, B. Graham, L. Spiccia, Inorg. Chem. 50 (2011) 621. [27] J. Qian, L. Wang, W. Gu, X. Liu, J. Tian, S.-P. Yan, Dalton Trans. 40 (2011) 5617. [28] X. Dong, X. Wang, M. Lin, X. Yang, Z. Guo, Inorg. Chem. 49 (2010) 2541. [29] D.-D. Li, J. Inorg. Biochem. 104 (2010) 171. [30] J.-T. Wang, Q. Xia, X.-H. Zheng, H.-Y. Chen, H. Chao, Z.-W. Mao, L.-N. Ji, Dalton Trans. 39 (2010) 2128. [31] M.J. Belousoff, M.B. Duriska, B. Graham, S.R. Batten, B. Moubaraki, K.S. Murray, L. Spiccia, Inorg. Chem. 45 (2006) 3746. [32] Q.-L. Li, J. Huang, Q. Wang, N. Jiang, C.-Q. Xia, H.-H. Lin, J. Wu, X.-Q. Yu, Bioorg. Med. Chem. 14 (2006) 4151. [33] V. Rajendiran, M. Karthik, M. Palaniandavar, V.S. Periasamy, M.A. Akbarsha, B.S. Srinag, H. Krishnamurthy, Inorg. Chem. 46 (2007) 8208. [34] J. Yan, J.A. Cowan, J. Am. Chem. Soc. 127 (2005) 8408. [35] Y. An, Y. M.L. Tong, L.N. Ji, Z.W. Mao, Dalton Trans. (2006) 2066. [36] S. Dhar, P.A.N. Reddy, A.R. Chakravarty, Dalton Trans. (2004) 697. [37] J. He, J. Sun, Z.W. Mao, L.N. Ji, H.Z. Sun, J. Inorg. Biochem. 103 (2009) 851. [38] T. Itoh, H. Hisada, T. Sumiya, T. Hosono, Y. Usui, Y. Fujii, Chem. Commun. (1997) 677. [39] W. Xu, F.R. Louka, P.E. Doulain, C.A. Landry, F.A. Mautner, S.S. Massoud, Polyhedron 28 (2009) 1221. [40] R. Hettich, H.-J. Schneider, J. Am. Chem. Soc. 119 (1997) 5638. [41] M. Schatz, M. Becker, F. Thaler, F. Hampel, S. Schindler, R.R. Jacobson, Z. Tyeklár, N.N. Murthy, P. Ghosh, Q. Chen, J. Zubieta, K.D. Karlin, Inorg. Chem. 49 (2001) 2312. [42] (a) R. Vicente, E. Ruiz, J. Cano, S.S. Massoud, F.A. Mautner, Inorg. Chem. 47 (2008) 4648; (b) F.A. Mautner, F.R. Louka, T. LeGuet, S.S. Massoud, J. Mol. Struct. 919 (2009) 196. [43] Z. Tyeklár, R.R. Jacobson, N. Wei, N.N. Murthy, J. Zubieta, K.D. Karlin, J. Am. Chem. Soc. 115 (1993) 2677. [44] J.A. Halfen, J.M. Uhan, D.C. Fox, M.P. Mehn, L. Que Jr., Inorg. Chem. 39 (2000) 4913. [45] (a) Bruker, SAINT, v. 7.23, Bruker AXS Inc., Madison, Wisconsin, USA, 2005.; (b) Bruker, APEX 2, v. 2.0-2, Bruker AXS Inc., Madison, Wisconsin, USA, 2006.; (c) G.M. Sheldrick, SADABS, v. 2, University of Goettingen, Germany, 2001. [46] G.M. Sheldrick, Acta Crystallogr. A64 (2008) 112. [47] A. Sreedhara, J.D. Freed, J.A. Cowan, J. Am. Chem. Soc. 122 (2000) 8814.

184

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

[48] A.W. Addison, T.N. Rao, J. Reedijk, J.V. Rijin, G.C. Verschoor, J. Chem. Soc., Dalton Trans. (1984) 1349. [49] (a)B.J. Hathaway, G. Wilkinson, R.D. Gillard, J.A. McCleverty (Eds.), Comprehensive Coordination Chemistry, vol. 5, Pergamon Press, Oxford, England, 1987, p. 533;

(b) M. Duggan, N. Ray, B. Hathaway, G. Tomlinson, P. Brint, K. Pelin, J. Chem. Soc., Dalton Trans. (1980) 1342. [50] P. Atkins, T. Overton, J. Rourke, M. Weller, F. Armstrong, M. Hagerman, Inorganic Chemistry, fifth ed., W.H. Freeman and Company, New York, 2010.