Efficient hydrolytic cleavage of plasmid DNA by chloro-cobalt(II) complexes based on sterically hindered pyridyl tripod tetraamine ligands: synthesis, crystal structure and DNA cleavage.

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Efficient hydrolytic cleavage of plasmid DNA by chloro-cobalt(II) complexes based on sterically hindered pyridyl tripod tetraamine ligands: synthesis, crystal structure and DNA cleavage† Salah S. Massoud,*a Richard S. Perkins,a Febee R. Louka,a Wu Xu,a Anne Le Roux,a Quentin Dutercq,a Roland C. Fischer,b Franz A. Mautner,c Makoto Handa,d Yuya Hiraoka,d Gabriel L. Kreft,e Tiago Bortolottoe and Hernán Terenzi*e Four new cobalt(II) complexes [Co(6-MeTPA)Cl]ClO4/PF6 (2/2a), [Co(6-Me2TPA)Cl]ClO4/PF6 (3/3a), [Co(BPQA)Cl]ClO4/PF6 (4/4a) and [Co(BQPA)Cl]ClO4/PF6 (5/5a) as well as [Co(TPA)Cl]ClO4 (1) where TPA = tris(2-pyridylmethyl)amine, 6-MeTPA = ((6-methyl-2-pyridyl)methyl)bis(2-pyridylmethyl)amine, 6-Me2TPA = bis(6-methyl-2-pyridyl)methyl)-(2-pyridylmethyl)amine, BPQA = bis(2-pyridylmethyl)-(2quinolylmethyl)-amine and BQPA = bis(2-quinolylmethyl)-(2-pyridylmethyl)amine were synthesized and structurally characterized. Single crystal X-ray crystallography confirmed the distorted trigonal bipyramidal geometries of complexes 2a–5a. Spectrophotometric titrations and conductivity measurements of the complexes in the CH3CN–H2O mixture showed that the chloro complexes exist in equilibrium with the corresponding hydrolyzed aqua species, [Co(L)(H2O)]2+. The pKa values of the coordinated H2O in aqua complexes vary from 8.4 to 8.7 (37 °C). The interactions of the complexes (1–5) with DNA have been investigated at pH = 7.0 and 9.0 (10 mM Tris-HCl buffer) and 37 °C where very high catalytic cleavage was observed. Under pseudo Michaelis–Menten kinetic conditions, the catalytic rate constants, kcat, decrease in the order 4 > 2 > 5 > 1 > 3. At pH 7.0 (10 mM Tris-HCl buffer) and 37 °C, the kcat value for complex 4 (6.02 h−1), where [Co(BPQA)(H2O)]2+ is the major species, corresponds to 170 million rate enhancement over the non-catalyzed DNA. Electrophoretic experiments conducted in the presence and absence of radical scavengers (DMSO, KI, NaN3) ruled out the oxidative mechanistic pathway of the reaction and

Received 28th February 2014, Accepted 1st May 2014 DOI: 10.1039/c4dt00615a www.rsc.org/dalton

suggested that the hydrolytic mechanism is the preferred one. This finding was in agreement with the observed increase in the kcat values at pH 9.0 compared to the corresponding values at pH 7.0 as a result of the increased concentration of the reactive hydroxo species, [Co(L)(OH)]+. The reactivity of the synthesized complexes in catalyzing the DNA cleavage is discussed in relation to the steric effect imposed by the coordinated pyridyl ligand around the central cobalt(II) center.

a Department of Chemistry, University of Louisiana at Lafayette, P.O. Box 44370 Lafayette, LA 70504, USA. E-mail: [email protected]; Fax: +1 337-482-5676; Tel: +1 337-482-5672 b Institut für Anorganische Chemische, Technische Universität Graz, Stremayrgasse 9/V, A-8010 Graz, Austria c Institut für Physikalische and Theoretische Chemie, Technische Universität Graz, Stremayrgasse 9/II, A-8010 Graz, Austria d Department of Chemistry, Interdisciplinary Graduate School of Science and Engineering, Shimane University, Nishikawatsu, Matsue, Japan e Centro de Biologia Molecular Estrutural, Departamento de Bioquímica, Universidade Federal de Santa Catarina, SC, 88040900 Florianópolis, Brasil. E-mail: [email protected]; Fax: +55 48 3721 9672; Tel: +55 48 3721 6426

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† Electronic supplementary information (ESI) available: Potentiometric pH titration of [Co(BQPA)(H2O)]2+ is shown in Fig. S1. Agarose gel electrophoresis and pseudo-Michaelis–Menten kinetics for the cleavage of DNA by complexes 1 and 2 ( pH 7.00) are shown in Fig. S2 and S3, respectively. Agarose gel electrophoresis and pseudo-Michaelis–Menten kinetics for the cleavage of DNA by complexes 2, 3 and 5 ( pH 9.00) are shown in Fig. S4–S6. DNA cleavage by complexes 1, 2 and 5 in the presence and absence of ROS is shown in Fig. S7–S9. Fig. S10 illustrates the cleavage of Oligo1 (49-mer) oligonucleotide by complex ions [Co(6-MeTPA)Cl]+ and [Co(BPQA)Cl]+. Fig. S11–S13 show the cleavage of DNA by complexes 1–3 in the presence of DNA groove binders. Pseudo-Michaelis–Menten kinetic data for the cleavage of DNA by complexes 1–5 at pH = 9.00 is given in Table S1. The CCDC 988526, 988527, 988529 and 988528 contain the crystallographic data in CIF format for complexes 2a–5a, respectively. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4dt00615a

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Introduction Phosphodiester bonds make up the backbone of DNA and RNA strands. The remarkable stability associated with the P–O bonds in the phosphodiester compounds is an essential requirement for survival and maintenance of life.1 This stability of the P–O bond towards hydrolysis results from the repulsion between the negatively charged phosphate diester bonds which reduce the rate of nucleophilic attack on the DNA backbone and hence resist its hydrolytic cleavage.1,2 Under physiological conditions, the half-life time t1/2 for the hydrolysis of DNA was estimated to be ∼130 000 years.1 However, nature has developed a number of hydrolytic metalloenzymes that efficiently catalyze the hydrolysis of P–O bonds of the DNA phosphate backbone.1 These metalloenzymes contain metal ions in their active sites. Modeling these hydrolytic enzymes is a fundamental step in understanding the “role of metal ion in these metalloenzymes” and in designing “artificial nucleases” capable of competing with natural ones.3–7 In the last two decades divalent 3d and 4d metal complexes derived from a wide range of ligands of varied skeletal structures and geometrical environments have been utilized as catalysts for phosphodiester hydrolysis8–16 and for DNA cleavage.16–46 Although it has been reported that multinuclear metal complexes are in general more efficient in hydrolyzing the phosphate ester and in DNA cleavage reactions than their corresponding mononuclear analogues,4,6,13,14,18,47–51 due to the potential cooperativity between the metal centers, still many mononuclear Cu(II), Zn(II) and Co(II) complexes show high catalytic activity.26,29,30,38–43 The catalytic efficiency of the currently available chemical nucleases cannot put them at the same level as that of the natural enzymes. The cleavage of DNA by small metal complexes is known to occur via two mechanisms, oxidative cleavage and hydrolytic cleavage. The former mechanism requires the addition of external agents such as light and oxidative and/or reductive species to initiate the cleavage. Oxidative cleavage results in the formation of reactive oxygen species, ROS: reactive singlet oxygen (1O2), superoxide (O2−) or hydroxyl radical (OH•) species.5,32–35 These generated fragments are not suitable for further enzymatic manipulation as they damage the ribose sugar and/or nucleic bases of the DNA and this hampers their use in vivo.41,52 In contrast, hydrolytic cleavage does not suffer from such drawbacks because the DNA products generated by this mechanism can be enzymatically relegated.35–43 Effective cleavage of the phosphate ester bond by the hydrolytic mech-

Chart 1

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anism requires a cis-nucleophile activation.6,31,47 Thus, small metal complexes that promote the hydrolytic cleavage of DNA under physiological conditions could be useful in elucidating the precise role of metal ions not only in enzymatic catalysis but also in molecular biology and drug design technology. The design and synthesis of inexpensive small-molecule catalysts that function as “artificial nucleases” for highly effective hydrolytic cleavage of the P–O bond in DNA is becoming a crucial tool in biotechnology. In this regard and to generate agents with improved reactivity, the cleavage of DNA was investigated by a series of dinuclear copper(II) complexes derived from tetrakis(2-pyridylmethyl)benzene-1,x-diamine, where x = 2, 3 or 4. Interestingly, this study not only showed significant dependence on the position of the substituents, but also the cleavage proceeded with different cleavage mechanisms.34 Recently, in a systematic study of DNA cleavage using a series of tripodal poly-pyridyl compounds, we demonstrated that complexes with five-membered chelate ring sizes are more efficient in catalyzing the DNA cleavage than the corresponding six-membered chelate rings.39 Also, to explore the influence of metal ions, the DNA cleavage was examined by a series of [MII(TPA)Cl]ClO4 complexes (M = Co, Cu, Zn; TPA = tris(2-pyridylmethyl)amine). It has been shown that [Co(TPA)Cl]ClO4 (1) resulted in an unusual catalytic enhancement and the compound promoted the cleavage of DNA through the hydrolytic pathway mechanism.40 This result motivated us to synthesize a series of TPA derivatives with the hope of increasing the steric hindrance around the five-coordinate Co(II) center, thus enhancing the DNA hydrolytic cleavage activities. Therefore, the ligands ((6-methyl-2-pyridyl)methyl)bis(2-pyridylmethyl)amine (6-MeTPA), bis(6-methyl-2-pyridyl)methyl)-(2pyridylmethyl)amine (6-Me2TPA), bis(2-pyridylmethyl)-(2quinolylmethyl)-amine (BPQA), and bis(2-quinolylmethyl)-(2pyridylmethyl)amine (BQPA) were synthesized (Chart 1). The corresponding cobalt(II) complexes of these ligands were also synthesized and their DNA cleavage activities were investigated in order to evaluate their catalytic efficiencies.

Results and discussion Syntheses Although the tripod amine based pyridyl ligands 6-MeTPA, 6-Me2-TPA, BPQA and BQPA were previously prepared by Mota et al., Nagao et al., Que’s and Karlin’s groups,53 we prepared them by various easy and efficient methods (yield: 50–85%).

TPA and its derivatives employed in this study.


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Fig. 1 Perspective views of complexes (a) [Co(6-MeTPA)Cl]PF6 (2a), (b) [Co(6-Me2TPA)Cl]PF6 (3a), (c) [Co(BPQA)Cl]PF6 (4a) and (d) [Co(BQPA)Cl]PF6 (5a) together with their atom-labeling schemes.

The compounds were characterized by 1H and 13C NMR, IR and by ESI-MS in some cases as indicated above. The ligands obtained were sufficiently pure to be used for the synthesis of their cobalt(II) complexes. Syntheses of the green cobalt(II) complexes [Co(L)Cl]X (L = TPA, 6-MeTPA, 6-Me2-TPA, BPQA; X = ClO4−, 1–5; X = PF6−, 2a–5a) were straightforward by reacting equimolar amounts of a methanolic solution containing CoCl2·6H2O and the corresponding ligand followed by the addition of NaClO4 or NH4PF6. Single crystals suitable for X-ray crystallography were obtained from dilute methanolic solutions containing hexafluorophosphate upon standing at room temperature or recrystallization from CH3CN. The very low solubility of [Co(L)Cl]PF6 compounds (2a–5a) in H2O prohibits their use for the DNA cleavage study and instead the corresponding [Co(L)Cl]ClO4 (1–5), which are relatively more soluble in aqueous medium, were used for the DNA cleavage studies. The synthesized complexes were characterized by IR and UV-Vis spectroscopy, elemental microanalyses, ESI-MS and by single crystal X-ray crystallography for the 2a–5a series. Crystal structures of complexes (2a–5a) Perspective views together with the partial atom numbering scheme for 2a–5a are presented in Fig. 1, and selected bond parameters are given in Table 1. The structures consist of monomeric complex [Co(L)Cl]+ cations and PF6− counterions. Complex 4a crystallizes with a lattice water molecule having a partial occupancy of 0.20. The Co(II) centers of the complex cations are penta-coordinated by 4 N donor atoms of the

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coligand L and one terminal chloro ligand. The geometry of the CoClN4 chromophore may be described as distorted TBP [τ-values: 0.77 (2a); 0.84 (3a); 0.81 (4a) and 0.70 (5a)].54 The equatorial sites of each TBP are ligated by the three N( py) of the tetradentate amine L, whereas the trans-axial sites are occupied by the N(amine) of L and the Cl(1) atom of the terminal chloro ligand. The equatorial Co–N bond lengths are in the range from 2.082(3) to 2.1366(8) Å, the axial Co–N(amine) vary from 2.162(3) to 2.1974(17) Å, and the Co–Cl(1) bond distances range from 2.2843(8) to 2.3057(9) Å (Table 1). Characterization of the complexes The IR spectra of the complexes under investigation showed a series of bands over the frequency region 1610–1440 cm−1 due to the pyridyl groups. The complexes [Co(L)Cl]ClO4 displayed a strong single band around 1090 cm−1 in the mono-substituted pyridyl compounds (2: L = 6-Me-TPA, 4: L = BPQA) attributable to the stretching ν(O–Cl) frequency of the ClO4− ion. However, this band was split in 3 (L = 6-Me2-TPA) and broad in 5 (L = BQPA), respectively. The split and/or broadening of the perchlorate band in these complexes may result from the reduction in the symmetry of the ClO4− ion to C3v or C2v. The corresponding hexafluorophosphate compounds (2a–5a) revealed a very strong band around 840 cm−1 attributable to the ν(P–F) stretching frequency of the PF6− counterion. The UV-Vis spectral data of all complexes recorded in CH3CN and in H2O are summarized in Table 2. The complexes display a general characteristic pattern in the two solvents


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Table 1

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Selected bond lengths (Å) and angles (°) for compounds 2a–5a

Compound 2a Co(1)–N(1) Co(1)–N(2) Co(1)–Cl(1) N(1)–Co(1)–N(2) N(1)–Co(1)–N(3) N(1)–Co(1)–N(4) N(1)–Co(1)–Cl(1) N(2)–Co(1)–N(3)

2.086(3) 2.082(3) 2.3057(9) 121.09(10) 76.63(10) 113.60(10) 95.62(8) 77.76(10)

Co(1)–N(3) Co(1)–N(4)

2.193(3) 2.106(3)

N(2)–Co(1)–N(4) N(2)–Co(1)–Cl(1) N(3)–Co(1)–N(4) N(3)–Co(1)–Cl(1) N(4)–Co(1)–Cl(1)

111.45(10) 98.84(8) 77.97(10) 167.81(7) 114.01(7)


2.099(3) 2.121(3) 2.2900(12) 111.87(13) 76.58(14) 120.91(13) 97.07(11) 76.56(12)

Co(1)–N(3) Co(1)–N(4)

2.162(3) 2.134(3)


110.64(13) 111.95(9) 75.54(13) 171.04(9) 103.02(10)


2.0841(18) 2.0821(18) 2.2977(6) 119.73(7) 77.66(7) 109.85(7) 99.16(5) 76.63(8)

Co(1)–N(3) Co(1)–N(4)

2.1974(17) 2.1046(17)


116.16(7) 95.65(5) 77.48(7) 168.31(5) 114.06(5)


2.1628(8) 2.1366(8) 2.2843(8) 78.68(3) 75.97(3) 76.66(3) 166.15(2) 106.64(3)

Co(1)–N(3) Co(1)–N(4)

2.1361(8) 2.1348(9)


114.33(3) 114.89(2) 124.12(3) 101.44(2) 94.25(2)

regardless of the differences in the ligand skeleton. In CH3CN, the complexes reveal the presence of two d–d absorption bands in the 450–650 nm region: around 490 and 630, whereas in H2O the former band is shifted to ∼470 nm. The dissolution of the complexes in H2O is instantaneously accompanied by the formation of light purple colors. The blue shift observed in this process when the [Co(L)Cl]ClO4/PF6 complexes dissolve in

H2O is most likely associated with chloride displacement and the formation of the aqua species, [Co(L)(H2O)]2+; the ligand field of H2O is stronger than the Cl− ligand. These spectral features can be attributed to formation of the aqua five-coordinate Co(II) complexes with increased distortion towards tetrahedra where the coordinated H2O is weakly bound to the central Co2+ ion.55 Spectrophotometric titration of acetonitrile solution containing 2.00 × 10−3 M [Co(L)Cl]ClO4 with increasing amounts of H2O shows a decrease of the intensity of the two maxima observed at 490 and 630 nm with the shift of the former band to ∼470 nm due to the increased amount of [Co(L)(H2O)]2+ species. Typical examples for the spectrophotometric studies are illustrated in Fig. 2 and 3 for complexes [Co(6-MeTPA)Cl]ClO4 (2) and [Co(BQPA)Cl]ClO4 (5), respectively. These spectral changes clearly reveal the presence of an equilibrium between the green complex ion [Co(L)Cl]+ and the corresponding light purple aqua species, [Co(L)(H2O)]2+. This equilibrium is represented as follows: ½CoðLÞClþ þ H2 O Ð ½CoðLÞðH2 OÞ2þ þ Cl

ð1Þ

To shed light on the nature of the above equilibrium, the molar conductivity, ΛM, of the complexes was measured in CH3CN and in CH3CN–H2O (1 : 1 by volume) solutions. In the CH3CN solution, ΛM values for [Co(L)Cl]ClO4/PF6 which vary from 130 to 150 Ω−1 cm2 mol−1 (Table 2) correspond to 1 : 1 electrolytic behavior.56 However, the conductivity values determined in CH3CN–H2O solution (1 : 1 by volume) for the complexes [Co(L)Cl]ClO4 were in the range 170–190 Ω−1 cm2 mol−1, intermediate conductivity values which are larger than that expected for 1 : 1 electrolyte and smaller than what were predicted based on 1 : 2 electrolytic solutions.56 These results clearly indicate the generation of certain amounts of 1 : 2 electrolyte, which can be explained by the formation of [Co(L)(H2O)]2+ species. This result is in complete agreement with the equilibrium represented by eqn (2) where under these conditions the equilibrium does not go to completion. The acid dissociation constants, pKa, of the coordinated H2O in [Co(L)(H2O)]2+ (eqn (2)) were determined at 37 °C by potentiometric pH titration of the generated complex [Co(L)-

Table 2 Electronic and molar conductivity data for chloro-tripod pyridyl-cobalt(II), [Co(L)Cl]ClO4/PF6 complexes in CH3CN, H2O and in 50% (by volume) H2O–CH3CN mixture

λmax (εmax, M−1 cm−1)

ΛM (Ω−1 cm2 mol−1)

Complex

CH3CN

H2Oa

CH3CN

[Co(TPA)Cl]ClO4 c (1) [Co(6-Me-TPA)Cl]ClO4 (2) [Co(6-Me-TPA)Cl]PF6 (2a) [Co(6-Me2-TPA)Cl]ClO4 (3) [Co(6-Me2-TPA)Cl]PF6 (3a) [Co(BPQA)Cl]ClO4 (4) [Co(BPQA)Cl]PF6 (4a) [Co(BQPA)Cl]ClO4 (5) [Co(BQPA)Cl]PF6 (5a)

486 (178), 617 (139) 490 (171), 632 (113) 486 (175), 631 (175) 484 (111), 631 (88) 485 (125), 632 (87) 489 (191), 630 (107) 490 (188), 630 (106) 489 (191), 630 (107) 486 (161), 635 (83)

∼638 (sh) ∼470 (b) ∼480 (b) ∼467 (b) ∼475 (b) ∼477 (b) ∼474 (b) ∼477 (b) ∼475 (b)

145 140 130 174 141 187 135 142

a

CH3CN/H2Ob 194d 189 173 178 173

Saturated solution. b In CH3CN–H2O mixture (1 : 1 by volume). c Ref. 40. d Measured in H2O.


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Fig. 2 Spectral changes of [Co(6-MeTPA)Cl]ClO4 (2) (2.00 × 10−3 M ) in acetonitrile solution with increasing amounts of H2O (v/v ratio of H2O/ CH3CN): (1) 0.0/10, (2) 0.20/9.8, (3) 0.50/9.5, (4) 1.0/9.0, (5) 2.0/8.0 and (6) 4.0/6.0.

Fig. 3 Spectral changes of [Co(BQPA)Cl]ClO4 (5) (2.00 × 10−3 M) solution in acetonitrile with increasing amounts of H2O (v/v ratio of H2O/CH3CN): (1) 0.0/10, (2) 0.20/9.8, (3) 0.50/9.5, (4) 1.0/9.0 and (5) 2.0/8.0.

the volume of added hydroxide is 1/2 of the volume to the equivalence point where pH = pKa. These values (average of three determinations) are as follows: 8.6 ± 0.1, 8.7 ± 0.1, 8.6 ± 0.1, 8.5 ± 0.1 and 8.4 ± 0.2 for L = TPA, 6-MeTPA, 6-Me2TPA, BPQA, and BQPA, respectively. The low solubility of the chloro complex [Co(BQPA)Cl]ClO4 in H2O prohibits the possibility of obtaining an accurate pKa value for its aqua complex. Thus, it is obvious that since the pKa values of all complexes are ≥8.4, one can conclude that the major active species around pH 7.0, which is used for the DNA cleavage study, would be the aqua species, [Co(L)(H2O)]2+.

Fig. 4 Potentiometric pH titration of [Co(6-MeTPA)(H2O)]2+ (6.0 × 10−3 M) with the standard 0.05 M NaOH at 37 °C.

(H2O)]2+ with NaOH. A typical potentiometric pH titration curve is shown in Fig. 4 for [Co(6-MeTPA)(H2O)]2+ and Fig. S1† for [Co(BQPA)(H2O)]2+ (ESI†). The titration data were analyzed for the acid–base equilibrium constant Ka (eqn (3)) and the values of pKa were determined from the titration curves when

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½CoðLÞðH2 OÞ2þ þ H2 O Ð ½CoðLÞðOHÞþ þ H3 Oþ

ð2Þ

K a ¼ ½CoðLÞðOHÞþ ½H3 Oþ =½CoðLÞðH2 OÞ2þ

ð3Þ

Cleavage of plasmid DNA by [Co(L)Cl]ClO4 complexes 1–5. The plasmid DNA cleavage by the complexes [Co(L)Cl]ClO4, where L = TPA (1), 6-MeTPA (2), 6-Me2-TPA (3), BPQA (4) and BQPA (5), was investigated at pH 7.0 and 9.0 in 10 mM Tris-HCl buffer and at 37 °C. The activity of the complexes towards the cleavage of DNA was initially observed as a function of different complex concentrations over a fixed incubation time period of the reaction (2 h). All the complexes


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Fig. 5 (a) Agarose gel electrophoresis pattern and the corresponding plots for the cleavage of pBSK II plasmid DNA (∼25 μM) by complex 2 at different complex concentrations (50–450 μM) at pH 7.0 Tris-HCl buffer (10 mM) and 37 °C; t = 2 h. Results are expressed as mean standard deviation (n = 3). (b) Pseudo Michaelis–Menten kinetics of the cleavage of pBSK II plasmid DNA (∼25 μM), [2] = 50–900 μM, pH 7.0 Tris-HCl buffer (10 mM) and 37 °C.


assayed show a concentration-dependent activity, converting the supercoiled form of plasmid DNA (Form I) to the relaxed open circular DNA (Form II) as represented in Fig. 5–7 for complexes 2–4 and Fig. S2 and S3† for complexes 1 and 5, respectively, at pH = 7.0. Under these reaction conditions, none of the complexes completely cleaved the intact form of plasmid. However, further cleavage of double-strand DNA into the linear Form III was observed at higher complex concentrations in 1, 2 and 4 as illustrated in Fig. S2, 5 and 7,† respectively. The increase of complex concentrations affects the complex activity in different ways. Complexes 1 to 4 tend to have an increase of their activities on going from 50 to 450 μM, while complex 5 reached the maximum activity with a much lower concen-


tration (100 μM). After reaching the highest DNA cleavage, an increase of the complex concentration does not improve the cleavage of DNA. This effect had a strong influence on the kinetic profile of the cleavage process and will be discussed later. As indicated above, DNA cleavage by complexes 1–5 was also conducted at pH = 9.0 and a similar set of data is represented in Fig. 8 and 9 for complexes 1 and 4, respectively, and Fig. S4, S5 and S6 (ESI†) for complexes 2, 3 and 5, respectively. The general features of these plots are similar to those obtained above at pH 7.0 except that no Form III was observed with complexes 1 and 3 (Fig. 8 and S5†) and very small amounts were detected with the rest of the compounds at the

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Fig. 8 (a) Agarose gel electrophoresis pattern and the corresponding plots for the cleavage of pBSK II plasmid DNA (∼25 μM) by complex 1 at different complex concentrations (5–100 μM) at pH 9.0 Tris-HCl buffer (10 mM) and 37 °C; t = 15 min. Results are expressed as mean standard deviation (n = 3). (b) Pseudo Michaelis–Menten kinetics of the cleavage of pBSK II plasmid DNA (∼25 μM), [1] = 5–150 μM, pH 9.0 Tris-HCl buffer (10 mM) and 37 °C.

highest complex concentrations used under our experimental conditions (Fig. S4, 9 and S5†). Kinetics of plasmid DNA cleavage. The kinetics of cleavage of plasmid DNA was conducted following the decay of conversion of the supercoiled Form I to a singly nicked relaxed form (Form II) over time under pseudo-first order conditions where the apparent rate constants, kobs, were evaluated (see the Experimental section). These values were then plotted vs. complex concentrations, and from the curve fit of eqn (4) (Experimental section), the catalytic rate constant, kcat (= Vmax at saturation), and affinity constant, KM, were obtained.57 In all

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measurements, the DNA concentrations were kept constant at approximately 25 μM in 10 mM Tris-HCl buffer at pH 7.0 or pH 9.0 and 37 °C. The detailed data measurements are collected in Table 3 and typical plots of the saturation kinetic profiles are represented in Fig. 5b–7b at pH 7.0 and Fig. 8b and 9b ( pH 9.0), as well as in Fig. S2 and S3† at pH = 7.0 and Fig. S4–S6† at pH 9.0. The apparent rate constants at different complex concentrations and the pseudo-Michaelis–Menten first order kinetic parameters at pH 9.0 are collected in Table S1† and these parameters are summarized in Table 4. It should be mentioned that although the kinetic parameters of


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Fig. 9 (a) Agarose gel electrophoresis pattern and the corresponding plots for the cleavage of pBSK II plasmid DNA (∼25 μM) by complex 4 at different complex concentrations (50–450 μM) at pH 9.0 Tris-HCl buffer (10 mM) and 37 °C; t = 15 min. Results are expressed as mean standard deviation (n = 3). (b) Pseudo Michaelis–Menten kinetics of the cleavage of pBSK II plasmid DNA (∼25 μM), [4] = 50–900 μM, pH 9.0 Tris-HCl buffer (10 mM) and 37 °C.

Table 3 Pseudo-Michaelis–Menten kinetics of pBSK II plasmid DNA cleavage by chloro-cobalt(II) complexes 1–5 at different complex concentrations. Apparent first-order rate constants, kobs, and kinetic parameters (kcat and KM). Reaction conditions: [DNA] ≈ 25 μM, [buffer] = 10 mM Tris-HCl buffer pH 7.0 and 37 °C

Complex [Co(TPA)Cl]ClO4 (1)

[Co(6-MeTPA)Cl]ClO4 (2)

[Co(6-Me2TPA)Cl]ClO4 (3)

[Co(BPQA)Cl]ClO4 (4)

[Co(BQPA)Cl]ClO4 (5)

a

a

[Co(II)] (μM)

kobs (h−1)

kcat (h−1)

KM (M)

50 100 150 300 450 600 900 50 100 150 300 450 600 900 50 100 150 300 450 600 900 50 100 150 300 450 600 900 5 10 25 50 100 150 300

0.195 0.245 0.394 0.474 0.719 0.824 0.775 0.170 0.213 0.350 0.684 0.810 0.961 0.990 0.115 0.181 0.333 0.553 0.672 0.706 0.695 0.347 0.572 1.14 2.82 3.06 3.59 3.56 0.0864 0.227 0.374 0.698 0.764 0.958 0.821

1.11

2.97 × 10−4

Complex

kcat (h−1)

KM (M)

[Co(TPA)Cl]ClO4 (1) [Co(6-MeTPA)Cl]ClO4 (2) [Co(6-Me2TPA)Cl]ClO4 (3) [Co(BPQA)Cl]ClO4 (4) [Co(BQPA)Cl]ClO4 (5)

3.02 10.1 1.99 16.8 5.92

3.64 × 10−5 3.47 × 10−4 2.40 × 10−4 3.64 × 10−4 1.37 × 10−4

Table 5 Pseudo-Michaelis–Menten first-order kinetic data for DNA cleavage by chloro-cobalt(II) complexes 1–5 at pH 7.0 and 37 °C

1.58

1.02

4.49 × 10−4

3.01 × 10−4

Complex

kcat (h−1)

KM (M)

Enhancementa


1.11 1.58 1.02 6.02 0.98

2.97 × 10−4 4.49 × 10−4 3.02 × 10−4 4.81 × 10−4 3.03 × 10−5

3.08 × 107 4.39 × 107 2.83 × 107 1.67 × 108 2.72 × 107

a

6.02

4.81 × 10−4

0.980

3.03 × 10−5

kcat represents the maximum rate of cleavage at the saturation (Vmax = kcat).


Table 4 Pseudo-Michaelis–Menten first-order kinetic parameters for DNA cleavage by chloro-cobalt(II) complexes 1–5 at pH 9.0 and 37 °C

Uncatalyzed ds DNA where k = 3.6 × 10−8 h−1 at 37 °C and pH 7.0.

DNA cleavage by [Co(TPA)Cl]ClO4 (1) were previously determined under slightly different conditions (low buffer concentration: 1 mM Tris-HCl buffer),40 we decided to repeat its measurements under the kinetic conditions indicated in this work and data are incorporated in Table 4. We believe that this would ensure a fair comparison between the five systems. Under pseudo-Michaelis–Menten first-order kinetics at pH 7.0, the catalytic rate constants, kcat (Table 5), reveal that the five complexes under investigation clearly show very high catalytic DNA activity as reflected in the kcat values that range from 0.98 to 6.0 h−1. The enhanced rates of DNA cleavage by these complexes are 30–40 million (complexes 1, 2, 3 and 5) to 170 million (complex 4) fold more reactive than the

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un-catalyzed reaction; the reactivity decreases in the order: 4 > 2 > 1 ≥ 3–5 (Table 5). The DNA binding affinities, KM, of the complexes varied from 0.30 × 10−4 to 4.8 × 10−4 M but they did not show any correlation with the corresponding kcat values. However, this result seems to be linked to saturation effects of complex concentration (as observed in Fig. 5–9 and S2–S6†) since the complex that reached maximum activity (in terms of cleavage yield) at lowest concentrations (5) has also the lowest KM value. The observed trend of reactivity for the DNA cleavage by the five complexes was not fully discriminative between the reactivity of the complexes 1–3 and 5 of the complexes with regard to the steric structural environment around the central Co(II) centers. This could be attributed to the fact that all the aqua complex ions, [Co(L)(H2O)]2+, are having very close pKa values and hence the major components around pH 7.0 are the aqua species. To explore the role of the steric effect, the cleavage of DNA was also conducted at pH 9.0 where substantial amounts of the hydroxo species, [Co(L)(OH)]+, are formed ( pKa of the complexes [Co(L)(H2O)]2+ ≥ 8.4). The extent of plasmid DNA cleavage by complexes 1–5 was found to vary with pH and complex concentration (Table S1†) in a fashion that is similar to those observed at pH 7.0. A summary of the catalytic rate constants, kcat, for the DNA cleavage by the complexes [Co(L)Cl]ClO4 1–5 at pH 7.0 and 9.0 together with the pKa values of the corresponding aqua species are tabulated in Table 6. Inspection of the data at pH 9.0 reveals that the catalytic rate constants kcat decrease in the order 4 > 2 > 5 > 1 > 3. Also, it shows that the complexes display 2–6 fold more DNA cleavage activity compared to those reported at pH 7.0 (Table 6), with the largest increase being for complex 2. The increased DNA catalytic activity of the complexes at pH 9.0 compared to pH 7.0 is explained by the deprotonation of the aqua complexes and the increased concentration of the reactive hydroxo species, [Co(L)(OH)]+. The coordinated OH− ligand is a good nucleophile compared to H2O making the aqua species least active and as a result, reduced DNA cleavage is observed at lower pH. Since the acid dissociation constants, pKa, of the aqua complex ions under investigation [Co(L)(H2O)] are not significantly different from each other ( pKa = 8.4–8.7) (Table 6) as well as the geometrical coordination environment around the central cobalt(II) ion in these aqua species as was supported by X-ray structural determination of their parent chloro compounds [Co(L)Cl]+ (155 and 2a–5a, all display distorted TBP geometry with τ = 0.96–0.70), one can conclude that the activity order observed here for DNA cleavage by the mono-

nuclear complexes 1–5 is most likely attributed to the variation in the ligand skeleton (see the next section). Effect of ROS scavengers on DNA cleavage. To clarify the mechanism of the DNA cleavage process (oxidative cleavage vs. hydrolytic cleavage) by the complexes under investigation, a series of ROS scavengers were employed. This includes DMSO which is known as a hydroxyl radical (OH•) scavenger,58 KI which acts as a peroxide (O22−) scavenger,59 and NaN3 which acts as a singlet (1O2) scavenger.60 Involvement of these species in nuclease activity could be diagnosed by monitoring the quenching of DNA cleavage in the presence of radical scavengers in solution. Addition of DMSO, KI or NaN3 did not affect the DNA cleavage efficiency as demonstrated by Fig. 10 and 11 for complexes 3 and 4, respectively. Similar figures are also given in the ESI† (S7–S9† for complexes 1, 2 and 5, respectively). The fact that none of the ROS scavengers used were able to inhibit the cleavage of DNA by the title complexes suggests ruling out the oxidative mechanism and the preference of the hydrolytic mechanism over the oxidative one. Even with the evidence that none of the ROS scavengers were able to inhibit the DNA cleavage promoted by the title complexes, an oxidative mechanism cannot be completely ruled out. Therefore, further assays were performed under an

Fig. 10 Cleavage of pBSK II plasmid DNA (∼25 μM) by 3 (450 μM) in the presence and absence of ROS scavengers. Reaction conditions: [Buffer] = 10 mM Tris-HCl pH 7.0, 37 °C, t = 30 min, [DMSO] = 0.5 M, [KI] = 0.4 mM, [NaN3] = 0.5 mM. Results are expressed as mean ± standard deviation (n = 3).

Table 6 The first-order catalytic rate constants for DNA cleavage by chloro-cobalt(II) complexes 1–5 at pH 7.0 and 9.0, and the pKa values for the corresponding aqua species at 37 °C

[Co(L)Cl]ClO4

pH = 7.0 kcat (h−1)

pH = 9.0 kcat (h−1)

pKa of [Co(L)(H2O)]2+


1.11 1.58 1.02 6.02 0.98

3.02 10.1 1.99 16.8 5.92

8.6 ± 0.1 8.7 ± 0.1 8.6 ± 0.1 8.5 ± 0.1 8.4 ± 0.2

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Fig. 11 Cleavage of pBSK II plasmid DNA (∼25 μM) by 4 (450 μM) in the presence and absence of ROS scavengers. Reaction conditions: [Buffer] = 10 mM Tris-HCl pH 7.0, 37 °C, t = 30 min., [DMSO] = 0.5 M, [KI] = 0.4 mM, [NaN3] = 0.5 mM. Results are expressed as mean ± standard deviation (n = 3).

argon atmosphere where oxygen is absent, hence preventing the formation of ROS. A comparison between the activity of the complexes in the presence and absence of oxygen is shown in Fig. 12 (A and B, respectively). The positive control of oxygen presence (Fe-EDTA/DTT) successfully demonstrated that under an argon atmosphere the ROS dependent cleavage of DNA was strongly inhibited. The title complexes, on the other hand, did not suffer significant inhibition in the absence of oxygen, indicating that the cleavage mechanism of

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DNA strand scission might occur in an oxygen-independent pathway. This evidence clearly reinforces that the cleavage mechanism should be hydrolytic rather than oxidative. Oligonucleotide cleavage assays. To search for complexmediated cleavages at preferential nucleotide sequences, a 49mer 5′-FAM-labeled (FAM = 6-fluorescein) self-complementary oligonucleotide (Oligo1) was used as a DNA substrate. The sequence of Oligo1 and its probable plain structure is embedded in Fig. S10.† Oligo1 has two different sequence regions (10 bp each): an AT-rich site, close to the strands termini, and a GC-rich site, close to the hairpin forming sequence. These distinguishable regions might assist in discriminating the complex-mediated cleavage sites (in terms of nucleotide preferences) with a single DNA substrate. Only two Co(II) compounds [Co(6-MeTPA)Cl] (2) and [Co(BPQA)Cl] (4) were able to cleave Oligo1, but under severe reaction conditions (24 h at 50 °C) (Fig. S10†). An increase of reaction time or complex concentration did not improve the activity of complexes 1, 3 and 5 (data not shown). The active complexes induced the fragmentation of Oligo1 in all possible nucleotides, i.e. without sequence preference. In addition, the complex-mediated fragments migrate in the same manner as the G + A ladder, which indicates that the cleavage products might have the same termini as those generated by piperidine treatment (3′-phosphate).61 This evidence is reinforced when the cleavage products of DNAse I (3′-OH termini)62 were compared to the G + A ladder. The fragments with 3′-OH ends are clearly distinguishable, since they migrate faster than those having 3′-phosphate ends. These findings strongly indicate that the complex-mediated Oligo1 cleavage products have 3′-phosphate ends. Furthermore, there is no evidence of formation of fragments containing 3′-phosphoglycolate termini

Fig. 12 Cleavage of pBSK II plasmid DNA in the presence (a) and absence (b) of oxygen. System Fe/EDTA (100/200 μM) in the presence of 10 mM DDT. Reaction conditions: [DNA] = ∼25 μM; [Buffer] = 10 mM Tris-HCl pH 7.0; [l, 2, 3 and 4] = 450 μM and [5] = 150 μM; temperature = 37 °C, t = 3 h. Results are expressed as mean ± standard deviation (n = 3).


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Fig. 13 Cleavage of pBSK II plasmid DNA (∼25 μM) by complexes 4 (a) and 5 (b) ([Co] = 450 μM) in the presence of DNA groove binders distamycin and methyl green (MG) ([Distamycin or MG] = 50 μM). Reaction conditions: [Buffer] = 10 mM Tris-HCl pH 7.0, 37 °C, t = 30 min. Results are expressed as mean ± standard deviation (n = 3).

which is only produced by the oxidation at C4 of 2-deoxyribose.63 Thus, the lack of ROS involvement and oxygen-independent strand scission in addition to the absence of oxidative generated DNA fragments support the suggestion that the title complexes might cleave the DNA strands by a hydrolytic mechanism. Effect of DNA groove binders. The chemical nuclease activity of the complexes has been studied using the supercoiled pBSK II plasmid DNA at pH 7.0 (Tris-HCl buffer) and 37 °C in the presence of two different DNA groove binders: distamycin, which binds to the minor groove, and methyl green (MG), which binds to the major groove.64 None of these typical DNA groove binders distamycin or methyl green could effectively block the cleavage process mediated by complexes 1–5 as illustrated in the representative Fig. 13 for complexes 4 and 5 and in Fig. S11–S13 (ESI†) for complexes 1, 2 and 5. In the presence of methyl green, the cleavage of DNA is only slightly diminished for complexes 1–5, which indicates some dependence of major groove binding. The lack of strict dependence of groove binding could indicate that none of the complexes are sequence specific, since many compounds that have this property interact with the DNA bases through groove binding (most likely through the minor groove). Based on the results shown above, we propose the mechanism represented in Scheme 1 for the hydrolytic cleavage of DNA by the investigated complexes 1–5. The first step in this mechanism involves the formation of the aqua species [Co(L)(H2O)]2+ where the corresponding chloro complex [Co(L)Cl]ClO4 is instantaneously hydrolyzed as soon as it dissolves in aqueous medium. The aqua species undergoes an acid–base equilibrium with the most reactive hydroxo species, [Co(L)(OH)]+. Upon the addition of DNA to the aqua/hydroxo species,

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the Co(II) center is bound to one of the oxygen atoms of the exposed phosphate ester linkers in DNA expanding its coordination number to 6. The tripodal skeleton of the ligands used in this study enforces the formation of complexes of cis-geometry where the coordinated H2O (or the OH− ion depending on pH) is positioned cis for proper intra-nucleophilic attack at the phosphorus atom,2,5,65 hence leading to the hydrolytic cleavage of the P–O bond in DNA. A tentative mechanism for the formation of the intermediate which is leading to the hydrolysis of DNA is illustrated in Scheme 1. The large enhancement of the cleavage rates observed in complexes 2 and 4 on going from un-substituted pyridyl groups in TPA to 6-MeTPA to BPQA (Chart 1) can be explained by the increase of steric effects, which is known to accelerate the rate of dissociative-type reactions. However, a large increase of the steric crowding around the central Co2+ ion, as in complexes [Co(6-Me2TPA)Cl]ClO4 (3) and [Co(BQPA)Cl]ClO4 (5), may suppress the approach of the complexes to DNA and hence reduce the cleavage rates.

Experimental Materials and physical measurements The compounds bis(2-pyridylmethyl)amine (DPA), 2-aminomethylpyridine and (2-chloromethyl)quinoline hydrochloride were purchased from TCI-America, whereas 6-methyl-2-pyridylmethanol was obtained from Aldrich Chemical Company, USA. All other chemicals were commercially available and used without further purification. 6-Methyl-2-pyridylmethanol was converted to 6-methyl-2-pyridylmethyl chloride hydrochloride by the reaction with excess SOCl2 in CHCl3, followed by recrystallization from ethanol.


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Scheme 1

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A plausible mechanism for the hydrolytic cleavage of DNA by the [Co(L)Cl]+ ion, where L represents a tripod tetraamine ligand.

Infrared spectra were recorded on a JASCO FT/IR-480 plus spectrometer as KBr pellets. Electronic spectra were recorded using an Agilent 8453 HP diode UV-Vis spectrophotometer. 1 H 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 (δ) are reported in ppm and were referenced internally to residual solvent resonances (DMSO-d6: δH = 2.49, δC = 39.4 ppm). ESI-MS were measured on an LC-MS Varian Saturn 2200 Spectrometer. The acid dissociation constants, Ka, of the generated aqua complexes [Co(L)(H2O)]2+ (obtained by dissolving an appropriate concentration of the corresponding chloro complex and allowing the solution to equilibrate at room temperature for 1 h before the measurement) were evaluated at 37 °C by potentiometric pH titration of an aqueous solution containing [Co(L)Cl]ClO4 {4.0–6.0 × 10−3 M} vs. standard 0.050 M NaOH. The pH was determined using a Delta 320 pH meter – Mettler Toledo upon the addition of aliquots of NaOH to well-stirred thermostated solutions. The molar conductivity of a solution sample was determined from ΛM = (1.0 × 103κ)/M, where κ is the cell constant and M is the molar concentration of the complex. The measurements were performed using a Mettler Toledo Seven Easy conductometer and the cell constant was determined with the aid of the 1413 μS cm−1 conductivity standard. Elemental analyses were carried out by the


Atlantic Microlaboratory, Norcross, Georgia, USA. The purification of pBSK II DNA was performed with a commercial kit (from Oiagen). Caution: Salts of perchlorate and their metal complexes are potentially explosive and should be handled with great care and in small quantities. Syntheses The ligands 6-MeTPA, 6-Me2TPA, BPQA and BQPA were previously prepared using different synthetic routes.53 An easy effective method for synthesizing these ligands is described here, for example the synthesis of 6-MeTPA and 6-Me2TPA depends on refluxing a mixture of bis(2-pyridylmethyl)amine (DPA) or 2-aminomethylpyridine and 6-methyl-2-pyridylmethylchloride hydrochloride in a 1 : 1 or 1 : 2 molar ratio, respectively, in anhydrous CH3CN and in the presence of anhydrous K2CO3 as a base. A similar approach was also used for the synthesis of BPQA and BQPA. (6-Methyl-2-pyridyl)methyl)bis(2-pyridylmethyl)amine (6MeTPA). To 6-methyl-2-pyridylmethylchloride hydrochloride (0.79 g, 5 mmol) suspended in anhydrous CH3CN (50 mL), bis(2-pyridylmethyl)amine (1.00 g, 5 mmol) was added. The mixture was treated with anhydrous K2CO3 (2.10 g, 15 mmol) and magnetically stirred under gentle reflux for 48 h, during which the color turned light yellow and a white precipitate was

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formed. The resulting mixture was cooled in the refrigerator and then filtered off to remove KCl and unreacted K2CO3. The solvent was removed with a rotary evaporator under reduced pressure and the resulting residue was crystallized from Et2O with the aid of activated charcoal. Several recrystallizations from Et2O afforded the desired product as a light yellow solid (yield: 1.30 g, 85%). Characterization: mp = 82–84 °C, selected IR (KBr disc, cm−1): 3072 (m), 3010 (m) ( pyridyl C–H stretching), 2918 (w), 2871 (w), 2819 (s) (aliphatic C–H stretching), 1589 (vs), 1568 (s), 1469 (s), 1436 (s) (CvC, CvN pyridyl ring stretching), 794 (s), 759 (vs) (C–H out of plane bending). ESI-MS in MeOH (100%) = 305.177 (Calcd for [M + H]+ = 305.178). 1H NMR (DMSO-d6, 400 MHz, δ in ppm): δ = 2.42 (3H, s, –CH3), 3.73 (2H, s, –CH2), 3.77 (4H, s, –CH2), 7.08 (1H, d, py-H), 7.23 (2H, m, py-H), 7.39 (1H, d, py-H), 7.57 (1H, m, py-H), 7.63 (2H, d, py-H), 7.76 (2H, m, py-H), 8.47 (2H, d, pyH). 13C NMR: (DMSO-d6, 100 MHz) δ = 24.0 (CH3), 59.4, 59.4 (CH2), 119.2, 121.3, 122.1, 122.5, 136.6, 136.8, 148.8, 157.0, 158.3, 159.0 ( pyridyl carbons). Bis((6-methyl-2-pyridyl)methyl)-(2-pyridylmethyl)amine (6Me2TPA). This compound was synthesized using a procedure that is essentially similar to that described above for the MeTPA. In a typical experiment, 6-methyl-2-pyridylmethylchloride hydrochloride (1.78 g, 10. mmol), 2-aminomethylpyridine (0.54 g, 5.0 mmol), and anhydrous K2CO3 (2.40 g, 17 mmol) in CH3CN (50 mL) were used. On recrystallization from Et2O (four times), the compound was isolated as a pale yellow solid (yield: 1.29 g, 81%). Characterization: mp = 78–80 °C. Selected IR (KBr, cm−1): 3067 (w), 2996 (w) ( pyridyl C–H stretching), 2925 (w), 2871 (w), 2818 (s) (aliphatic C–H stretching), 1591 (vs), 1577 (s), 1466 (vs), 1435 (s) (CvC, CvN pyridyl ring stretching), 800 (s), 760 (C–H out of plane bending). ESI-MS in MeOH: m/z = 319.192 (100%) (Calcd for [M + H]+ = 319.197). 1H NMR (DMSO-d6, 400 MHz, δ in ppm): δ = 2.40 (6H, s, –CH3), 3.71 (4H, s, –CH2), 3.74 (2H, s, –CH2), 7.07 (2H, d, py-H), 7.22 (1H, m, py-H), 7.39 (2H, d, py-H), 7.59 (2H, m, py-H), 7.73 (1H, d, py-H), 7.77 (1H, m, py-H), 8.46 (1H, d, py-H). 13C NMR: (DMSO-d6, 100 MHz) δ = 24.0 (CH3), 59.4, 59.5 (CH2), 119.2, 121.3, 122.1, 122.5, 136.6, 136.8, 148.8, 157.0, 158.4, 159.0 ( pyridyl carbons). Bis(2-pyridylmethyl)-(2-quinolylmethyl)amine (BPQA). A procedure was used similar to that described for MeTPA except that 2-chloromethylquinoline hydrochloride was used instead of 6-methyl-2-chloromethylpyridine. The compound was isolated as a yellowish orange solid after several crystallizations from Et2O and activated charcoal (yield: 56%). Characterization: mp = 45–47 °C. Selected IR (KBr, cm−1): 3061 (w), 3014 (w) ( pyridyl C–H stretching), 2928 (w), 2875 (w), 2814 (s) (aliphatic C–H stretching), 1590 (vs), 1568 (s), 1503 (vs), 1474 (s), 1438 (s) (CvC, CvN pyridyl and quinolyl ring stretching), 830 (s), 775 (s), 762 (s) (C–H out of plane bending). ESI-MS in MeOH m/z = 341.177 (100%) (Calcd for [M + H]+ = 341.438). 1H NMR (DMSO-d6, 400 MHz, δ in ppm): δ = 3.79 (4H, s, CH2-py), 3.95 (2H, s, CH2-quinolyl), 7.21 (2H, t), 7.53 (1H, m), 7.58 (1H, d), 7.73–7.80 (5H, m), 7.92 (2H, m), 8.30 (1H, m), 8.49 (2H, d, py6-H). 13C NMR: (DMSO-d6, 100 MHz) δ = 59.5 (CH2-py), 60.1

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(CH2-quinolyl), 120.8, 122.2, 122.7, 126.2, 126.9, 127.8, 128.5, 129.5, 136.5, 136.6, 147.0, 148.9, 158.8, 160.0 ( pyridyl and quinolyl carbons). Bis(2-quinolylmethyl)-(2-pyridylmethyl)amine (BQPA). The compound was obtained by method A and is similar to that described for BPQA using 2-chloromethylquinoline hydrochloride (4.28 g, 20.0 mmol) and 2-aminomethylpyridine (1.08 g, 10.0 mmol) instead of DPA in CH3CN (80 mL) and in the presence of K2CO3 (6.9 g, 50 mmol). The crude product was recrystallized from Et2O to give a light yellowish-orange solid (yield: 3.40 g, 87%). Characterization: mp = 86–88 °C. Selected IR (KBr, cm−1): 3057 (m), 3009 (w) ( pyridyl C–H stretching), 2974 (w), 2924 (w), 2882 (w), 2819 (m) (aliphatic C–H stretching), 1618 (s), 1601 (vs), 1590 (m), 1568 (s), 1504 (vs), 1473 (m), 1426 (s) (CvC, CvN pyridyl and quinolyl ring stretching), 826 (vs), 757 (C–H out of plane bending). ESI-MS in MeOH m/z = 391.191 (100%) (Calcd for [M + H]+ = 391.498. 1H NMR (DMSO-d6, 400 MHz, δ in ppm): δ = 3.84 (2H, s, CH2-py), 3.98 (4H, s, CH2-quinolyl), 7.23 (1H, t), 7.52–7.63 (4H, m), 7.73–7.80 (5H, m), 7.90–7.96 (3H, m), 8.32 (2H, d), 8.48 (1H, d, py6-H). 13 C NMR: (DMSO-d6, 100 MHz) δ = 59.6 (CH2-py), 60.3 (CH2quinolyl), 120.9, 122.2, 122.8, 126.2, 127.0, 127.8, 128.5, 129.5, 136.5, 136.6, 147.0, 148.9, 158.7, 159.9 ( pyridyl and quinolyl carbons). [Co(L)Cl]ClO4/PF6 (1–5/2a–5a). The complex [Co(TPA)Cl]ClO4 (1) was synthesized according to literature methods,40,54 whereas a general method was used to synthesize the rest of the complexes ( perchlorate complexes, 2–5, and hexafluorophosphate complexes, 2a–5a). In a typical experiment NaClO4 (0.122 g, 1 mmol) or NH4PF6 (0.085 g, 0.50 mmol) was added to a warm solution containing CoCl2·6H2O (0.129 g, 0.5 mmol) and the corresponding ligand (0.5 mmol) was dissolved in MeOH (15 mL). The resulting green solution was further heated for another 5 min, filtered through celite and then allowed to stand at room temperature. The precipitate obtained over a period of 1–3 days was collected by filtration, washed with propan-2-ol and Et2O and then recrystallized from MeOH or CH3CN. Single crystals suitable for X-ray structure determination were obtained from dilute solutions in the case of hexaflurophosphate complexes (2a–5a). [Co(6-MeTPA)Cl]ClO4/PF6 (2/2a). Characterization for 2 (yield based on 0.5 mmol of CoCl2·6H2O: 0.184 g, 74%): Found: C, 45.74; H, 3.95; N, 11.22%. Calcd for C19H20Cl2CoN4O4 (MM = 498.01 g mol−1): C, 45.82; H, 4.05; N, 11.25%. Selected IR bands (cm−1): 1609 (vs) ν(CvC); 1572 (m), 1469 (m), 1442 (s) ν(CvN); 1090 (vs) ν(Cl–O). UV-Visible spectrum {λmax, nm (ε, M−1 cm−1)} in CH3CN: 490 (171), 632 (113); in H2O: ∼470 (b). ESI-MS in CH3CN: m/z = 398.070 (88.1%) (Calcd for [M + H-ClO4]2+ = 398.56), m/z = 98.949 (100%) [ClO4− = 99.453]. Molar conductivity in CH3CN, ΛM = 145 Ω−1 cm2 mol−1. Characterization for 2a (yield based on 0.5 mmol of CoCl2·6H2O: 0.209 g, 84%): Found: C, 42.24; H, 3.68; N, 10.37%. Calcd for C19H20ClCoN4PF6 (MM = 543.54 g mol−1): C, 41.99; H, 3.71; N, 10.31%. Selected IR bands (cm−1): 1611 (vs) ν(CvC); 1575 (m), 1470 (m), 1443 (s) ν(CvN); 839 (vs) ν(P–F). UV-Visible spectrum {λmax, nm (ε, M−1 cm−1)} in CH3CN: 486


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(175), 631 (120), 804 (32,b); in H2O (saturated): ∼480 (b); ΛM = 140 Ω−1 cm2 mol−1 (CH3CN). [Co(6-Me2TPA)Cl]ClO4/PF6 (3/3a). Characterization for 3 (yield based on 0.5 mmol of CoCl2·6H2O: 0.164 g, 64%): Found: C, 46.81; H, 4.29; N, 11.21%. Calcd for C20H22Cl2CoN4O4 (MM = 512.26 g mol−1): C, 46.89; H, 4.33; N, 10.94%. Selected IR bands (cm−1): 1609 (s) ν(CvC); 1468 (m) ν(CvN); 1146 (s), 1120 (s), 1090 (s) ν(Cl–O). UV-Visible spectrum {λmax, nm (ε, M−1 cm−1)} in CH3CN: 484 (111), 631 (88); in H2O (saturated): ∼470 (b). ESI-MS in CH3CN: m/z = 412.09 (37.93%) (Calcd for [M–ClO4]+ = 412.81), m/z = 98.946 (100%) [ClO4− = 99.453]. ΛM = 130 Ω−1 cm2 mol−1 (CH3CN). Characterization for 3a (yield based on 0.5 mmol of CoCl2·6H2O: 0.243 g, 87%): Found: C, 42.94; H, 3.69; N, 10.27%. Calcd for C20H22ClCoN4PF6 (MM = 557.54 g mol−1): C, 43.09; H, 3.80; N, 10.05%. Selected IR bands (cm−1): 1610 (s) ν(CvC); 1577 (m), 1469 (s), 1445 (s) ν(CvN); 845 (vs) ν(P–F). UV-Visible spectrum {λmax, nm (ε, M−1 cm−1)} in CH3CN: 485 (125), 632 (87), 807 (23,b); in H2O (saturated): ∼475 (b); ΛM = 174 Ω−1 cm2 mol−1 (CH3CN). [Co(BPQA)Cl]ClO4 (4) and [Co(BPQA)Cl]PF6·0.2H2O (4a·0.2H2O). Characterization for 4 (yield based on 0.5 mmol of CoCl2·6H2O: 0.163 g, 61%): Found: C, 49.42; H, 3.68; N, 10.51%. Calcd for C22H20Cl2CoN4O4 (MM = 534.02 g mol−1): C, 49.48; H, 3.78; N, 10.49%. Selected IR bands (cm−1): 1604 (s) ν(CvC); 1573 (w), 1515 (m), 1468 (m), 1436 (s) ν(CvN); 1087 (vs) ν(Cl–O). UV-Visible spectrum {λmax, nm (ε, M−1 cm−1)} in CH3CN: 489 (191), 630 (107), 795 (32); in H2O (saturated): ∼477 (b), 738 (w), 976. ESI-MS in CH3CN: m/z = 434.071 (100%) (Calcd for [M–ClO4]+ = 434.567), m/z = 98.949 (100%) [ClO4− = 99.453). ΛM = 141 Ω−1 cm2 mol−1 (CH3CN). Characterization for 4a·0.2H2O (yield based on 0.5 mmol of CoCl2·6H2O: 0.187 g, 64%): Found: C, 45.63; H, 3.62; N, 9.67%. Calcd for C22H20.40ClCoF6N4O0.20P (MM = 583.15 g mol−1): C, 45.31, H,

Table 7

3.53; N, 9.61%. Selected IR bands (cm−1): 1607 (vs) ν(CvC); 1572 (m), 1518 (s), 1483 (m), 1443 (vs) ν(CvN); 836 (vs) ν(P–F). UV-Visible spectrum {λmax, nm (ε, M−1 cm−1)} in CH3CN: 490 (188), 630 (106), ∼800 (31,b); in H2O: ∼474 (b); ΛM = 187 Ω−1 cm2 mol−1 (CH3CN). [Co(BQPA)Cl]ClO4/PF6 (5/5a). Characterization for 5 (yield based on 0.5 mmol of CoCl2·6H2O: 0.251 g, 86%) Found: C, 53.18; H, 3.83; N 9.46%. Calcd for C26H22Cl2CoN4O4 (MM = 584.11 g mol−1): C, 53.46; H, 3.86; N 9.59%. Selected IR bands (cm−1): 1604 (s) ν(CvC); 1571 (m), 1516 (s), 1478 (m), 1435 (s) ν(CvN); 1108 (vs, centered) ν(Cl–O). UV-Visible spectrum {λmax, nm (ε, M−1 cm−1)} in CH3CN: 489 (163), 633 (109), ∼800 (35); in H2O: ∼470 (b); ΛM = 135 Ω−1 cm2 mol−1 (CH3CN). Characterization for 5a (yield based on 0.5 mmol of CoCl2·6H2O: 0.296 g, 94%) Found: C, 49.56; H, 3.44; N, 8.90%. Calcd for C26H22ClCoF6N4P (MM = 629.63 g mol−1): C, 49.60; H, 3.52; N 8.90%. Selected IR bands (cm−1): 1607 (s) ν(CvC); 1572 (m), 1517 (m), 1478 (w), 1435 (s) ν(CvN); 839 (vs) ν(P–F). UV-Visible spectrum {λmax, nm (ε, M−1 cm−1)} in CH3CN: 486 (161), 635 (83); in H2O: ∼475 (b); ΛM = 142 Ω−1 cm2 mol−1 (CH3CN). X-Ray structure determination and refinement The X-ray single-crystal data of the four compounds 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 7. The intensities were collected with Mo-Kα radiation (λ = 0.71073 Å). Data processing, Lorentzpolarization and absorption corrections were performed using APEX, SAINT and the SADABS computer programs.66 The structures were solved by direct methods and refined by full-matrix least-squares methods on F2, using the SHELXTL program package.67 All non-hydrogen atoms were refined anisotropi-

Crystallographic data and processing parameters for complexes 2a–5aa

Compound

[Co(6-MeTPA)Cl]PF6 (2a)

[Co(6-Me2TPA)Cl] PF6 (3a)

[Co(BPQA)Cl]PF6- (H2O)0.20 (4a)

[Co(BQPA)Cl]PF6 (5a)

Empirical formula Formula mass System Space group a (Å) b (Å) c (Å) β (°) V (Å3) Z μ (mm−1) Dcalc (Mg m−3) Crystal size (mm) θ max (°) Data collected Unique refl./Rint Parameters Goodness-of-fit on F2 R1/wR2 (all data) Residual extrema (e Å−3)

C19H20ClCoF6N4P 543.74 Monoclinic P21/n 13.6304(13) 13.4681(13) 13.6781(14) 119.219(14) 2191.5(5) 4 1.043 1.648 0.28 × 0.21 × 0.12 26.36 17 023 4449/0.0253 290 1.109 0.0506/0.1167 2.00/−0.99

C20H22ClCoF6N4P 557.77 Monoclinic P21/c 14.0290(12) 14.0550(13) 12.5854(14) 109.197(14) 2343.6(4) 4 0.978 1.581 0.28 × 0.16 × 0.12 25.30 16 550 4261/0.1008 222 1.060 0.0604/0.1507 1.02/−0.52

C22H20.40ClCoF6N4O0.20P 583.37 Monoclinic P21/n 13.9800(12) 13.6286(14) 14.0348(15) 117.119(14) 2380.0(5) 4 0.968 1.628 0.30 × 0.22 × 0.17 26.34 18 052 4839/0.0227 326 1.060 0.0369/0.0981 0.68/−0.28

C26H22ClCoF6N4P 629.83 Monoclinic C2/c 33.652(2) 9.7685(6) 15.7094(9) 90.830(2) 5163.6(5) 8 0.898 1.620 0.34 × 0.25 × 0.19 32.55 61 695 9347/0.0243 352 1.038 0.0252/0.0682 0.53/−0.45

a

The CCDC 988526, 988527, 988529 and 988528 contain the crystallographic data in the CIF format for the complexes 2a–5a, respectively.


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cally. The hydrogen atoms were located from difference Fourier maps, assigned with isotropic displacement factors and included in the final refinement cycles by the use of geometrical constraints. In the case of 4a partial occupancy of 0.20 was applied for lattice water molecules.


Plasmid DNA cleavage by Co(II) complexes and agarose gel electrophoresis In general, 330 ng of pBSK II DNA (∼25 μM bp) in 10 mM TrisHCl ( pH 7.0 and 9.0) buffer were treated with different concentrations of the title complexes for 2 h at 37 °C in a final volume of 20 μL. All assays were conducted including a reaction control (without complex) to serve as a reference for spontaneous plasmid DNA fragmentation. Thereafter, each reaction was quenched by adding 5 μL of a loading buffer solution (50% glycerol, 250 mM EDTA and 0.01% bromophenol blue) and then subjected to electrophoresis on a 1.0% agarose gel containing 0.3 μg mL−1 of ethidium bromide in 0.5 × TBE buffer (44.5 mM Tris pH 8.0, 44.5 mM boric acid, and 1 mM EDTA) at 90 V for 100 min. The resulting gels were visualized and digitized using a DigiDoc-It gel documentation system (UVP, USA). The proportion of plasmid DNA in each band was quantified using KODAK Molecular 3 Imaging Software 5.0 (Carestream Health, USA). The quantification of supercoiled DNA (Form I) was corrected by a factor of 1.47, since the ability of ethidium bromide to intercalate into this DNA topoisomeric form is decreased relative to open circular and linear DNA.68 Kinetics of DNA cleavage The kinetics of plasmid DNA cleavage by the complexes was evaluated following the loss of supercoiled DNA fraction under pseudo-first-order conditions. At different time intervals (the range was different for each complex), an aliquot of reacted DNA (20 μL) was taken and the reaction stopped with loading buffer (5 μL). The apparent plasmid DNA cleavage rates, kobs, for the conversion of form I to form II were obtained from the slope of the linear plot of ln[supercoiled DNA (%)] vs. t at each complex concentration. The reaction conditions were identical to those described above. The values of kobs for a given system were then plotted vs. the concentrations of cobalt(II) complex (catalyst = cobalt(II) complex) and fitted to eqn (4). This allows a pseudo-first order Michaelis–Menten kinetics and the extraction of the corresponding kinetic parameters: the pseudo-first order catalytic rate constant, kcat, which is interpreted as the maximum rate of cleavage at complex saturation (Vmax = kcat) and the affinity constant, KM.7,17,20b,22a,32,39,40,57,69 kobs ¼ V max ½complex=ðK M þ ½complexÞ

ð4Þ

Effect of ROS scavengers on DNA cleavage To evaluate the mechanism of plasmid DNA cleavage performed by the complexes, different ROS scavengers [DMSO (0.5 M), KI (0.4 mM) and NaN3 (0.5 mM)] were added to the reaction media in the presence and absence of the complexes.

10100 | Dalton Trans., 2014, 43, 10086–10103

Thereafter, the cleavage assays were conducted as described above or as stated in figure legends. DNA cleavage under an argon atmosphere To clarify the mechanistic pathway of DNA cleavage by the title complexes (oxygen-dependent, oxidative with ROS or oxygenindependent, hydrolytic cleavage), several reactions were performed under an argon atmosphere, i.e. in the absence of oxygen.70 To achieve these conditions, the assays were fully performed within sealed glove-bags filled with argon. All solutions used were previously degassed using a vacuum pump and purged with argon. To guarantee the absence of oxygen during the assays, Fe-EDTA (100 μM/200 μM) plus DTT (10 mM), which are known to cleave the DNA via ROS formation in the presence of oxygen, was used as a “positive control”. In general, the assays were performed with 450 μM of complexes 1–4 and 150 μM for complex 5 for 3 h at 37 °C at pH 7.0. Oligonucleotide cleavage assays A 49-mer 5′-FAM-labeled (FAM = 6-fluorescein) self-complementary oligonucleotide Oligo1 ( purchased from Integrated DNA Technologies – Coralville, IA) was used as a DNA substrate to search for complex-mediated cleavages at preferential nucleotide sequences. Reaction assays were performed in a final volume of 10 μL in which the Oligo1 (50 pmol) was treated with the complexes (450 μM) for 24 h at 50 °C in 10 mM Tris-HCl buffer ( pH 9.0). The reactions were quenched with 10 μL formamide loading buffer (80% formamide, 10 mM NaOH, 1 mM EDTA and 0.05% bromophenol blue). The samples were heated at 95 °C for 5 min and 4 μL (Oligo1, ∼10 pmol) were loaded onto 16% denaturing PAGE (29 : 1) containing 7 M urea to achieve single-nucleotide fragment resolution. Electrophoresis was conducted at 50 W (constant), ∼55 °C for 150 min and the gel was scanned on a FLA 9000 (Fuji, Japan) Imaging System set for fluorescence detection of the FAM dye. A Maxam-Gilbert G + A ladder61 was prepared as previously described.71 In addition, a ladder containing DNAse I treated Oligo1 fragments (with 3′-OH termini) was prepared as described72 with minor modifications: 25 pmol Oligo1 were treated with 0.03 U DNAse I in 10 mM HEPES buffer ( pH 8.0) for 90 s. The reaction was quenched with 1 volume of sodium acetate (3 M, pH 5.2), glycogen (20 mg mL−1) and EDTA (50 mM) followed by ethanol precipitation. Effect of DNA groove binders For a better understanding of the interaction of complexes with the DNA, additional assays were performed in the presence of two different DNA groove binders: distamycin, which binds to the minor groove, and methyl green (MG), which binds to the major groove. The plasmid DNA was pre-treated with the groove binders for 30 min to reach equilibrium and then reacted with the title complexes as described in figure legends.


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Conclusions Cobalt(II) complexes (1–5 and 2a–5a) of the tripod pyridylbased ligands have been synthesized and characterized. Single crystal X-ray crystallography of [Co(L)Cl]PF6, 2a–5a, confirmed the distorted TBP geometry of the complexes. In aqueous solution, the chloro complexes are instantaneously hydrolyzed to their corresponding aqua species as soon as they dissolve. The DNA cleavage results clearly reveal the efficiency of the complexes, 1–5, to cleave double-strand DNA. The catalytic rate constant kcat for the complexes under investigation decreases in the order [Co(BPQA)Cl]ClO4 (4) > [Co(6-MeTPA)Cl]ClO4 (2) > [Co(BQPA)Cl]ClO4 (5) > [Co(TPA)Cl]ClO4 (1) > [Co(6-Me2TPA)Cl]ClO4 (3). The enhancement of reactivity order observed in complex 4 compared to 2 and complex 5 compared to 1 and 3 could be attributed to the increase of π–π-stacking interactions of the quinolyl groups, which are incorporated into the ligand skeletons of BQPA and BPQA, with the purine or pyrimidine moieties of DNA73 which may facilitate the approach of complexes 4 and 5 to DNA. In contrast, a large increase in the steric environment around the central metal ion may lead to an opposite effect as was clearly demonstrated in complex 3 when compared to 1 and 2. Delicate tuning of steric effect is necessary to ensure enhanced reactivity. Another interesting feature in the DNA cleavage by the addressed complexes is the hydrolytic cleavage nature of the reactions which is mechanistically similar to the DNA cleavage by natural enzymes. The compounds 1–5 utilize very efficient catalysts for DNA cleavage and this puts them at the level of “most efficient artificial nucleases”.21a,30,34b,38,41–43 Under physiological conditions ( pH 7.0 and 37 °C), the complex [Co(BPQA)Cl]ClO4 (4) is 170 million fold more reactive than the un-catalyzed reaction. It is interesting to compare the DNA cleavage activity of [Co(BPQA)Cl]ClO4 (4) with the most highly effective artificial nucleases which were derived from Cu(II) complexes.19,21a,26,30,34b,38b,41–43 Under comparable conditions, the catalytic enhancement revealed by complex 4 is very close (1.6 ± 0.10 × 108) to those reported for mono- and poly-nuclear Cu(II) complexes: [Cu(dpq)2(H2O)]2+, [Cu(tdp)(dpq)]+ and [Cu3(L1)(H2O)4]6+ where dpq = dipyrioquinoxaline, tdp = 2-[2(2-hydroxyethyl-amino)-(ethylimino)methyl]phenolate anion and L1 = N,N,N′,N′-tetra(2-pyridylmethyl)-5,5′-bis(aminomethyl2,2′-dipyridyl, respectively.19,30,42 At the same time, the catalytic efficiencies of the above mentioned complexes are still lower than those determined in [Cu(L)Cl2] complexes (L = 5,5′di[N1-thyminylmethyl]-2,2′-bipyridine or 5,5′-di[N9-adenylmethyl]-2,2′-bipyridine).41 On the other hand, the enhancement observed by complex 4 is about 17 times greater than the bridged dihydroxo-Co(III), [Co2(L2)2(μ-OH)2]2+ (L2 = 1,4,7-triazacyclononane-N-acetate anion), which is considered to be the only cobalt complex among the most efficient artificial DNA cleavage catalysts.74 Future designing of new efficient artificial nucleases should take into consideration the following strategies: (1) Synthesis of coordinatively unsaturated five-coordinate metal(II) complexes which can expand their coordination number to 6 (Co2+,


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Cu2+ and Zn2+) when binding the exposed phosphate group of DNA. (2) Using tripod tetraamine ligands to ensure the formation of complexes with cis-geometry. (3) Synthesizing chloro complexes which rapidly hydrolyze when dissolved in water or aqua complexes. (4) The aqua complexes, [M(L)(H2O)]2+, should have pKa < 7, a property which will allow the formation of substantial amounts of the more reactive hydroxo species, [Co(L)(OH)]+, and hence produce an increase in the DNA cleavage activity. This requires modification of the ligand skeleton and/or incorporating electron withdrawing groups into its structure. (5) A careful increase of the steric crowding around the central metal ion. These strategies are under investigation to generate a new series of metal(II) complexes with superior DNA cleavage efficiency.

Acknowledgements This research was financially supported by the Department of Chemistry-University of Louisiana at Lafayette. F. A. M. thanks Dr J. Baumgartner (TU-Graz) for assistance.

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Dalton Trans., 2014, 43, 10086–10103 | 10103

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