Synthesis, crystal structure, magnetic property and oxidative DNA cleavage activity of an octanuclear copper(II) complex showing water–perchlorate helical network

Synthesis, crystal structure, magnetic property and oxidative DNA cleavage activity of an octanuclear copper(II) complex showing water–perchlorate helical network

JOURNAL OF Inorganic Biochemistry Journal of Inorganic Biochemistry 101 (2007) 95–103 www.elsevier.com/locate/jinorgbio Synthesis, crystal structure...

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JOURNAL OF

Inorganic Biochemistry Journal of Inorganic Biochemistry 101 (2007) 95–103 www.elsevier.com/locate/jinorgbio

Synthesis, crystal structure, magnetic property and oxidative DNA cleavage activity of an octanuclear copper(II) complex showing water–perchlorate helical network Koushik Dhara a, Jagnyeswar Ratha b, Mario Manassero c, Xin-Yi Wang d, Song Gao d, Pradyot Banerjee a,* a

Department of Inorganic Chemistry, Indian Association for the Cultivation of Science, 2A, 2B Raja Subodh Mullick Road, Jadavpur, Kolkata, West Bengal 700 032, India b Cellular Biochemistry Division, Indian Institute of Chemical Biology, Jadavpur, Kolkata, West Bengal 700 032, India c Dipartimento di Chimica Strutturale e Stereochimica Inorganica dell’Universita` di Milano, Via G. Venezian 21, 20133 Milano, Italy d College of Chemistry and Molecular Engineering, State Key Laboratory of Rare Earth Materials Chemistry and Applications, Department of Chemistry, Peking University, Beijing 100 871, PR China Received 28 March 2006; received in revised form 14 August 2006; accepted 17 August 2006 Available online 3 September 2006

Abstract A new octanuclear copper(II) complex has been synthesized and structurally characterized by X-ray crystallography: [Cu8(HL)4(OH)4(H2O)2(ClO4)2] Æ (ClO4)2 Æ 2H2O (1) (H3L = 2,6-bis(hydroxyethyliminoethyl)-4-methyl phenol). The complex is formed by the linkage of two terminal bimetallic cationic units and a tetranuclear l3-hydroxo bridged dicubane core by a very short intramolecular ˚ and the angle 175). The coordination sphere of the terminal copper atoms is square pyramidal, hydrogen bond (O–H    O, 1.48(3) A the apical positions being occupied by water and a perchlorate ion. Complex 1 self-assembles to form a new type of water–perchlorate helical network [(H2O)2(ClO4)]1 involving oxygen atoms of coordinated perchlorate ion and the two lattice water molecules through hydrogen-bonding interaction. The variable temperature-dependent susceptibility measurement (2–300 K) of 1 reveals a strong antiferromagnetic coupling, J1 = 220 cm1 and J2 = 98 cm1 (J1 and J2 representing the exchange constant within [Cu2+]4 and [Cu2+]2 units, respectively). The complex binds to double-stranded supercoiled plasmid DNA giving a Kapp value of 1.2 · 107 M1 and displays efficient oxidative cleavage of supercoiled DNA in the presence of H2O2 following a hydroxyl radical pathway.  2006 Elsevier Inc. All rights reserved. Keywords: Octanuclear Cu(II) complex; N2O3-donor Schiff base ligand; [(H2O)2(ClO4)]1 helical supramolecular network; DNA binding; Oxidative DNA cleavage

1. Introduction Identifying molecules that intercalate into DNA helices has attracted considerable interest over the last few decades. Intercalation was first proposed by Lerman and is defined as insertion between base pairs [1]. The cleavage of nucleic acids may be considered as an enzymatic reac-

*

Corresponding author. Tel.: +91 33 2473 4971; fax: +91 33 2473 2805. E-mail address: [email protected] (P. Banerjee).

0162-0134/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2006.08.010

tion which comprises of various biological processes as well as the biotechnological manipulation of genetic material. The application of artificial DNA cleaving agents is manifold: biotechnology, structural studies of nucleic acids, or development of new drug [2–10]. Compounds showing the property of effective binding as well as cleaving double stranded DNA under physiological conditions are of importance since these could be used as diagnostic agents in medicinal and genomic research [11– 34]. Noting the very sensitive nature of DNA towards oxidative cleavage, the majority of the studies on artificial

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DNAs have been centered around molecules capable of cleaving DNA with an oxidative mechanism [10]. Several efficient cleaving agents have been developed in course of time. These involve reactive oxygen species (ROS) or free radicals that are able to induce an oxidative pathway. The antitumor antibiotic leinamycin and its analogues have been shown to play the role of ‘‘chemical nuclease’’ exhibiting reduction of molecular oxygen to form reactive hydroxyl species [35,36].The glycopeptide antitumor antibiotic, Bleomycins (BLMs), cleaves DNA in an oxidative manner requiring the presence of Fe(II), hydrogen peroxide or molecular oxygen and non diffusible metal-oxene species are thought to be involved in the process [22,37]. Alternatively, in the case of a commonly used system, Fe(EDTA)2, diffusible hydroxyl radicals produced via Fenton reaction, in the presence of hydrogen peroxide or molecular oxygen, take part in the DNA cleavage [22]. A similar pathway apparently operates also in the case of bis(1,10-phenanthroline)copper(I) complex [22]. Transition metal complexes have been extensively studied for their nuclease-like activity [17]. In this regard polynuclear copper complexes have attracted a special attention with respect to the biological role played by them. The driving force for further investigation of complexes containing three copper atoms lies in the recognition that the perticulate methane monooxygenase (pMMO) from Methylococcus capsulates (both) is constituted of 15 copper atom active center which is further organized into two types of five trinuclear aggregates of yet unknown structure [38,39]. The molecule counts in this pMMO are 12.8 copper and 0.9 iron atoms. There are some distinct advantages of chemical nucleases over conventional enzymatic nucleases: the former are smaller in size rendering quick stretching in the hindered sites of a macromolecule. In oxidizing DNA, the redox properties of the metal and dioxygen are being utilized in many cases leading to direct strand scission or base modification [21,22]. Both the reactions are exhibited by copper complexes although none of these exhibit notable nucleotide sequence specificity. Large attention on multinuclear Cu(II) complexes owes their origin to their potentiality for efficient intramolecular activation of bound O2 and to selective binding to a particular nucleic acid conformation. Studies with polynuclear copper complexes have been amply reported by Karlin et al. [40–43]. Also Cu(II) complexes with linear [44] or macrocyclic polyamines [45–48], aminoglycosides [49,50], and even with simple histidine [51] show a good nuclease activity. The most effective Cu(II) complexes are kanamycin, neamine [49,50] and cis-1,3,5-triaminocyclohexane (TACH) [46]. Herein, we report the synthesis, crystal structure, magnetic property of a new octanuclear copper(II) complex, [Cu8(HL)4(OH)4(H2O)2(ClO4)2] Æ (ClO4)2 Æ 2H2O (1) (H3L = 2,6-bis(hydroxyethyliminoethyl)-4-methyl phenol), which shows the formation of a new type of water–perchlorate, [(H2O)2(ClO4)]1, helical supramolecular network by the self-assembly process in which the lattice water and perchlorate molecules are involved through hydrogen-bonding

interaction. Complex 1 binds to double-stranded DNA in the minor groove and efficiently cleaves supercoiled plasmid DNA in presence of hydrogen peroxide in the oxidative manner. 2. Experimental 2.1. Materials and physical methods All reagents and chemicals were purchased from commercial sources and used without further purification. The Schiff base ligand 2,6-bis(hydroxyethyliminoethyl)-4methyl phenol (H3L) was prepared by a reported method [52]. The supercoiled (SC) pUC19 DNA (caesium chloride purified) was purchased from Bangalore Genie (India). The calf thymus (CT) DNA, agarose (molecular biology grade), distamycin and ethidium bromide (EB) were obtained from Sigma. Tris(hydroxymethyl)-aminomethane–HCl (Tris– HCl) buffer was prepared using deionized and sonicated triple distilled water. Solvents used for spectroscopic studies were purified and dried by standard procedures before use [53]. FT-IR spectra were obtained on a Nicolet MAGNA-IR 750 spectrometer with samples prepared as KBr pellets. Elemental analysis was carried out in a 2400 Series-II CHN analyzer, Perkin–Elmer, USA. Variable temperature magnetic susceptibility and field dependence of magnetization measurements were performed on a Quantum Design MPMS XL-5 SQUID magnetometer. Absorption and luminescence spectra were studied on Shimadzu UV2100 UV–VIS recording spectrophotometer and Perkin–Elmer LS 55 Luminescence Spectrometer, respectively. Caution: Perchlorate salts of metal complexes with organic ligands are potentially explosive. Only a small amount of material should be prepared and handled with caution. 2.2. Synthesis of [Cu8(HL)4(OH)4(H2O)2(ClO4)2] Æ (ClO4)2 Æ 2H2O (1) A 20 mL methanolic solution of Cu(ClO4)2 Æ 6H2O (0.30 g, 0.81 mmol) was added slowly to a magnetically stirred 15 mL methanolic solution of the ligand (H3L) (0.09 g, 0.36 mmol). The mixture was stirred in air for 45 min by adding NEt3 (0.05 mL, 0.36 mmol) whereby a deep green solution was formed. A 10 mL aqueous solution of KOH (0.05 g, 0.89 mmol) was added and the mixture was refluxed for 2 h. The solution was cooled to an ambient temperature. It was filtered and kept in air. Single crystals of 1 for X-ray crystallographic were obtained on slow evaporation of the filtrate at ambient temperature after a few days. (Yield: 65%). Anal. Calc. for C52H76N8O36Cl4Cu8: C, 30.59; H, 3.73; N, 5.49. Found: C, 30.65; H, 3.47; N, 5.27%. FT-IR (KBr phase) (cm1): 3433br, 3215br, 1647vs, 1556vs, 1452m, 1323m, 1238w, 1113vs, 625s, 489w (br, broad; w, weak; m, medium; s, strong; vs, very strong).

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2.3. X-ray crystallography Crystal data for 1, C52H76Cl4Cu8N8 O36, M = 2039.34, monoclinic, space group P21/n (No. 14), a = 11.103(1), ˚, b = 14.960(1), c = 22.104(2) A b = 96.12(1), U= 3 ˚ 3650.6(6) A , Z = 2, Dc = 1.855 g cm3, F(0 0 0) = 2064,. Reflections measured 56 087, independent 10 463 with Rint = 0.030. Empirical absorption correction, SADABS (Tmax = 1.00, Tmin = 0.798). Final R2 (F2, all reflections) = 0.052, R2w = 0.079, conventional R1 = 0.031 for 555 parameters, GoF. = 1.09. Bruker SMART CCD area˚ ), T = 150 K, x detector, Mo-Ka radiation (k = 0.71073 A scan mode, hmin = 3, hmax = 28.00. The structure was solved by direct methods (SHELXS) and refined by fullmatrix least squares with anisotropic thermal parameters for non-hydrogen atoms. Hydrogen atoms were partly refined with isotropic thermal parameters, and partly placed in calculated position. The program used was Personal SDP on a Pentium III PC. 2.4. DNA-binding and cleavage experiments The concentration of CT DNA was determined from the absorption intensity at 260 nm with a known e value of 6600 M1 cm1 [54]. The binding of 1 to CT DNA has been studied by fluorescence spectral method using the emission intensity of EB. The apparent binding constant, Kapp, for 1 was estimated by the equation: KEB[EB] = Kapp[Complex], where KEB is 1.0 · 107 M1 [55]. The absorption titration of 1 binding to DNA was performed by monitoring the absorption spectra of the 1 (50 lM) in Tris–HCl buffer (50 mM, pH 7.2) containing NaCl (50 mM) in the presence of increasing amount of CT DNA. The DNA cleavage experiment was done by agarose gel electrophoresis. Supercoiled pUC19 DNA (2.5 lL, 0.5 lg) in Tris–HCl buffer (50 mM, pH 7.2) containing NaCl (50 mM) was treated with the complex 1 in the presence or absence of additives. The oxidative DNA cleavage by 1 (10–40 lM) was studied in the presence of H2O2 (60– 90 lM). The experimental samples were incubated first for 60 min at 37 C and after that loading buffer (25% bromophenol, 30% glycerol (3 lL), 0.25% xylene cyanol) was added. It was finally loaded on 1% agarose gel containing 1.0 lg mL1 EB. Electrophoresis was carried out at 40 V for 2.0 h in Tris-acetate EDTA (TAE) buffer. The cleavage activity was measured by determining the ability of 1 in relaxing the SC DNA to its nicked circular (NC) form. Bands were visualized by UV light and photographed. The proportion of DNA in the SC and NC forms after electrophoresis was estimated quantitatively from the intensities of the bands by using a UVITEC Gel Documentation System with due correction of the low level of NC present in the original sample and the low affinity of EB binding to SC compared to NC and linear forms of DNA [56]. Control experiments were done using different reagents such as DMSO (4 lL), distamycin (100 lM), 3mercaptopropionic acid (MPA, 5 mM) and dithiothreitol

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(DTT, 5 mM) added to SC DNA prior to the addition of complex 1. 3. Results and discussion 3.1. Crystal structure Complex 1 crystallizes in the monoclinic space group P21/n (No. 14) from methanol–water mixture. The cluster is formed by the linkage of two bimetallic cationic units and a tetranuclear dicubane core [57]. The metal centers in tetranuclear core are coordinated by a l2-alkoxo oxygen atom and a l2-phenoxo oxygen atom of the ligand H3L and one l3-hydroxo oxygen atom to form a dicubane-like core. In dicubane core the copper centres are in square˚ pyramidal geometry with bridging Cu–O (2.4472(12) A ˚ and 2.2641(12) A) bond being the axial group. The octanuclear cationic entity is shown in Fig. 1. The shortest Cu–Cu ˚ . The dicubane distances are 2.99 and 3.11, 3.23 A core and the two bimetallic cationic units are linked by a very short and ‘‘strong’’ intramolecular hydrogen bond ˚ and the angle 175(3)) [58] (O7–H3  O1, 1.48(3) A

Fig. 1. A perspective view of the cationic entity in [Cu8(HL)4(OH)4(H2O)2 (ClO4)2] Æ (ClO4)2 Æ 2H2O showing the short intramolecular hydrogen bond (indicated by the blue dotted line) between terminal [Cu2+]2 and central dicubane [Cu2+]4 core. Copper ; Oxygen ; Nitrogen ; Chlorine ; Carbon ; Hydrogen . (For interpretation of the references to colours in this figure legend, the reader is referred to the web version of this paper.)

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through anionic alkoxo-oxygen atom of the Cu4 core and the hydrogen atom of nonionic alkoxo group of terminal metal Cu2 unit. Selected bond distances and angles are given in Table 1. This type of strong hydrogen bonds is quasi-covalent [58] in nature. Besides this, there are two ‘‘moderate’’ [58] intramolecular hydrogen bonds operating ˚ and the angle 171(3)) between O4–H2  O8, (1.98(3) A ˚ and O3–H1  O5, (1.56(4) A and the angle 162(4)). The copper atoms in the bimetallic unit have two different environments in the coordination sphere. The fragmented octanuclear entity of 1 with labeling scheme is shown in Fig. 2 showing the strong hydrogen bonds. The geometry of each copper ion is square pyramidal with one water molecule ˚) occupying the axial position (Cu–O distance 2.253(2) A and for other metal centre, perchlorate ion [59] is an axial ˚ ). The Cu–O bond disgroup (Cu–O distance 2.5473(17) A tances observed for 1 are comparable to those observed previously [60,61]. That all copper atoms in 1 are in Cu2+ state was confirmed by magnetic susceptibility measurements. The most interesting feature of complex 1 is that it self assembles to form a new type of water–perchlorate helical supramolecular structure as shown in Fig. 3 involving O(16) and O(17) of coordinated perchlorate ion and two water molecules through hydrogen bonding interactions. Hydrogen-bond distances and bond angles associated with the helical chain are given in Table 2. Atoms Table 1 ˚ ) and bond angles () for [Cu8(HL)4(OH)4 Selected bond distances (A (H2O)2(ClO4)2] Æ (ClO4)2 Æ 2H2O (1) Cu1–O1 Cu1–O2 Cu1–O4 Cu1–N1 Cu1–O4a Cu2–O2 Cu2–O3 Cu2–O4 Cu2–N2 Cu2–O1a O1–Cu1–O2 O1–Cu1–O4 O1–Cu1–N1 O1–Cu1–O4a O2–Cu1–O4 O2–Cu1–N1 O2–Cu1–O4a O4–Cu1–N1 O4–Cu1–O4a O4a–Cu1–N1 O2–Cu2–O3 O2–Cu2–O4 O2–Cu2–N2 O1a–Cu2–O2 O3–Cu2–O4 O3–Cu2–N2 O1a–Cu2–O3 O4–Cu2–N2 O1a–Cu2–O4 O1a–Cu2–N2 a

1.9633(13) 1.9789(13) 1.9472(12) 1.9226(18) 2.2641(12) 1.9801(13) 1.9423(14) 1.9264(13) 1.9277(19) 2.4472(12) 174.06(5) 101.80(5) 86.58(7) 84.63(5) 80.12(5) 90.66(7) 101.18(5) 167.49(7) 84.78(5) 105.43(6) 166.91(5) 80.60(5) 90.23(6) 95.91(5) 102.81(5) 85.11(6) 97.12(5) 169.65(7) 80.54(5) 105.34(6)

Symmetry codes: 1  x, y, z.

Cu3–O15 Cu3–N3 Cu3–O6 Cu3–O8 Cu3–O5 Cu4–O6 Cu4–O7 Cu4–N4 Cu4–O8 Cu4–O9 O5–Cu3–N3 O6–Cu3–O8 O6–Cu3–O15 O6–Cu3–N3 O8–Cu3–O15 O8–Cu3–N3 O15–Cu3–N3 O5–Cu3–O15 O5–Cu3–O6 O5–Cu3–O8 O6–Cu4–O8 O7–Cu4–O8 O6–Cu4–O9 O6–Cu4–N4 O6–Cu4–O7 O9–Cu4–N4 O8–Cu4–N4 O7–Cu4–O9 O7–Cu4–N4 O8–Cu4–O9

2.5473(17) 1.9139(19) 1.9401(13) 1.9414(13) 1.9212(13) 1.9441(13) 1.9552(17) 1.926(2) 1.9540(13) 2.253(2) 86.16(7) 80.56(5) 99.09(6) 91.81(7) 90.57(6) 171.38(7) 94.62(7) 92.12(6) 168.74(6) 100.54(5) 80.15(5) 100.81(7) 104.15(6) 91.64(7) 169.02(7) 101.42(8) 167.73(7) 86.82(7) 85.53(8) 89.49(6)

Fig. 2. Fragmented [Cu8(HL)4(OH)4(H2O)2 (ClO4)2] Æ (ClO4)2.2H2O showing the bridging atoms with labeling (blue dotted lines indicate the intramolecular hydrogen-bonding). (For interpretation of the references to colours in this figure legend, the reader is referred to the web version of this paper.)

Ow(10) and Ow(9) of the water molecules are hydrogen ˚ . The Ow(10) is bonded giving O–O distance 2.77 A hydrogen bonded to O(17) of perchlorate from another ˚ leading asymmetric unit giving O–O distance of 2.90 A to formation of the helical network in which the metalorganic building block acts as a template. The angles O(9)–O(10)–O(17) and O(10)–O(9)–O(16) in the helix are 141.73 and 106.91, respectively. The unit cell-packing diagram shows the presence of four helices located at the half of the centre of the c-axes having half site occupancy. Each of the water and perchlorate molecules is covalently linked with copper ions. Right handed helical strand is connected with the left handed one through the two terminal ends of the copper complex (Fig. 4). To the best of our knowledge, this is the first example of a supramolecular water–perchlorate, [(H2O)2(ClO4)]1, helical network. 3.2. Magnetic property The temperature-dependent susceptibility of 1 was measured under an external field of 5 kOe from 2 to 300 K and

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Fig. 3. The perspective view of the packing of 1 showing the water–perchlorate helical network along b-axis and metal-organic network which acts as a template for the formation of helix. Color codes are the same as those in Fig. 1. (For interpretation of the references to colours in this figure legend, the reader is referred to the web version of this paper.)

Table 2 ˚ ) and bond angles () associated with the water– Selected bond distances (A perchlorate helical network ˚) ˚) ˚) D–H  A D–H (A H  A (A D  A (A D–H  A () O9–H11  O10 O9–H12  O16 O10–H14  O17

0.82(2) 0.81(3) 1.06(4)

1.96(2) 2.35(3) 1.91(4)

2.770(3) 2.882(3) 2.902(3)

177.5(18) 124(3) 153(4)

the result is shown in Fig. 5(a) (vMT(T) and vM(T)). vM decreases with the decreasing temperature from 0.0031 cm3 mol1 at 300 K to 0.0009 cm3 mol1 at 50 K, and then increases with a paramagnetic impurity tail. The absence of a broad hump even at 300 K reveals a very strong AF coupling between the Cu2+ ions through the l-O bridges. The observed vMT at 300 K is only 0.92 cm3 mol1 K, far below the spinonly value (1.5 cm3 mol1 K) expected for four isolated Cu2+ ions. Considering the structure which is composed by an approximately tetranuclear [Cu2+]4 plus one [Cu2+]2 dimer, we can fit our data using the Hamiltonian H = 2J1(S1 Æ S2 + S2 Æ S3 + S3 Æ S4 + S4 Æ S1)2J2S5 Æ S6, with J1 representing the exchange constant within the [Cu2+]4 and J2 for the [Cu2+]2 units. The best fit gives J1 = 220 cm1 and J2 = 98 cm1 (with g = 2.2), indicating a very strong antiferromagnetic coupling between the Cu2+ ions. The field dependent magnetization at 2 K up to 50 kOe was also measured (Fig. 5(b)), but the behavior is somewhat dominated by the paramagnetic impurity, with the M = 0.045 lB at 50 kOe, far away from the Ms = 4 lB for four isolated Cu2+ ions confirming the strong antiferromagnetic coupling. 3.3. DNA-binding and cleavage studies The binding of 1 to CT DNA has been studied by fluorescence spectral method using the emission intensity of

Fig. 4. A space-filling view of the right and left-handed helices of water– perchlorate in the octanuclear Cu(II) network along b-axis. Color codes are the same as those in Fig. 1. (For interpretation of the references to colours in this figure legend, the reader is referred to the web version of this paper.)

EB. EB does not show any emission in buffer medium due to fluorescence quenching by the solvent molecules [62]. In presence of CT DNA, it shows emission due to its intercalative binding to DNA. Addition of 1 results in

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H / kOe Fig. 5. (a) Temperature-dependent susceptibility of 1 at external field of 5 kOe from 2 to 300 K. (b) The field dependent magnetization at 2 K up to 50 kOe.

the competitive binding to DNA causing reduction of the emission intensity. This is due to the displacement of the EB from the DNA bound state to the free state and copper(II) complex from free state to DNA bound state. We have measured the reduction of emission intensity of EB at different concentration of 1 shown in Fig. 6(a). The apparent binding constant (Kapp) has been calculated from the equation: KEB[EB] = Kapp[Complex], (Kapp(EB) = 1.0 · 107 M1) and the concentration of EB is 50 lM. The concentration of the complex is taken for observing 50% reduction of the emission intensity of EB [55]. The Kapp value for the complex 1 is estimated as 1.2 · 107 M1 and comparable to reported values [63]. The complex 1 is emissive at 527 nm (390 nm excitation) in Tris–HCl/NaCl buffer (50 mM, pH 7.2). The emission spectral changes of 1 in presence of CT DNA have been studied. Interestingly after addition of DNA, the emission at 515 nm is observed first, and the intensity is found to be somewhat less than for the free state of the complex. With increasing time it shows a red shift (12 nm) leading to the enhancement of emission intensity keeping the emission maximum (527 nm) fixed so as to reach a constant value after ca. 30 min (Fig. 6(b)). This result indicates the immediate formation of 1-DNA combined complex corresponding to the emission at 515 nm which is reflected with

0 500

550

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Wavelength (nm) Fig. 6. The effect of addition of complex 1 (j) on the emission intensity of 50 lM CT DNA-bound ethidium bromide in Tris–HCl/NaCl buffer (50 mM, pH 7.2) at 25C; (h) on the emission intensity of the ethidium bromide in absence of CT DNA but at different concentrations of 1. (b) Emission spectra of complex 1 (10 lM) in the presence of CT DNA (430 lM) showing initially the emission intensity at 515 nm leading to final enhancement of intensity at 527 nm (excitation wavelength: 390 nm) with time in Tris–HCl/NaCl buffer (50 mM, pH 7.2). Green line indicates the emission of 1. (For interpretation of the references to colours in this figure legend, the reader is referred to the web version of this paper.)

quenching in intensity. The corresponding enhancement (at 527 nm) at longer times is due to significant intercalative binding. The binding phenomenon is further studied by electronic absorption spectral technique. The UV–visible absorption spectrum of 1 shows the absorption maximum at 378 nm. Binding of a complex to DNA usually results in hypochromism and red shift (bathochromic shift) due to the intercalative mode. The extent of the hypochromism in the charge transfer band is generally consistent with the strength of intercalative binding/interaction [64–67]. Fig. 7 shows the changes in absorption for 1 (20 lM) upon the addition of the CT DNA (0–700 lM). There is an increase in absorption intensity initially indicated by the red lines. On a further increase in DNA concentration, a large (20 nm) red shift is observed (green lines) commensurate with significant intercalative binding.

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Wavelength (nm) Fig. 7. Absorption spectral changes on addition of CT DNA to the complex 1 in Tris–HCl/NaCl buffer (50 mM, pH 7.2). (shown by arrow). The inset shows a plot of the absorbance at 378 nm versus the amount of DNA added.

A question hangs about the exact nature of the complex species involved in binding. We are unaware of any references encompassing the results of a macromolecular bulky complex involved in DNA binding. The diffuse reflectance spectrum of 1 at 650 nm characteristic of a CuO4N core with the copper ion in a square pyramidal environment. In the working buffer medium, [Tris–HCl/NaCl (50 mM, pH 7.2)], the electronic spectrum shows a blue shift of ca. 25 nm (Figure S1). This is quite significant and indicates the modification of the coordination sphere of the metal ion during intercalative interaction. It is quite logical that this large complex disintegrates into lower nuclearity so as to fit between the two consecutive base pairs of DNA. The electronspray mass spectrum of 1 was obtained in water. The spectrum shows peaks at 1134.4, 1010.6 and 910.7 that can be assigned to the tetranuclear species (Figure S2) derived from the octanuclear complex. The ability of the complex 1 to perform DNA cleavage has been studied by gel electrophoresis using supercoiled (SC) pUC 19 DNA in Tris–HCl/NaCl buffer (50 mM, pH 7.2). The octanuclear complex on reaction with DNA in presence of H2O2 as oxidizing agent presents high nuclease activity (Fig. 8). In presence of reducing agents, the com-

Fig. 8. Agarose gel electrophoresis diagram showing the cleavage of SC pUC19 DNA (0.5 lg) by complex 1 treated with various concentration of H2O2 in Tris–HCl/NaCl buffer (50 mM, pH 7.2) in dark. Incubation time: 1 h (37 C). Lane 1: DNA control; lane 2: DNA + H2O2 (80 lM); lane 3: DNA + H2O2 (80 lM) + 1 (10 lM); lane 4: DNA + H2O2 (60 lM) + 1 (20 lM); lane 5: DNA + H2O2 (65 lM) + 1 (35 lM); lane 6: DNA + H2O2 (65 lM) + 1 (40 lM); lane 7: DNA + 1 (40 lM); lane 8: DNA + H2O2 (60 lM) + 1 (30 lM).

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pound is inactive towards any cleavage. A 30 lM solution of 1 cleaves SC DNA (0.5 lg) to the extent of 94% on treatment with 60 lM H2O2. A significant cleavage (77%) is also observed at 20 lM complex concentration using 60 lM H2O2. In the lanes 5 and 6 using 35 lM and 40 lM of 1, respectively with 65 lM H2O2, bands (Fig. 8) could not be identified due to smearing. This is caused by the increase of the concentrations of 1. At higher concentrations the nicked DNA degraded completely into small pieces and only smear was present and it could not induce the conversion of SC to linear form at the tested concentration. DNA cleavage data of SC pUC19 DNA by complex 1 on chemical oxidation by hydrogen peroxide are given in Table 3. In fact a comparison of lane 3 and 4 [DNA + H2O2 (60 lM) + CuCl2 Æ 2H2O (30 lM); DNA + H2O2 (60 lM) + CuCl2 Æ 2 H2O (40 lM), respectively] with lane 7 [DNA + H2O2 (60 lM) + 1 (30 lM)] shows that the complex 1 cleaves completely the SC DNA to NC (nicked circular) DNA while Cu(II) salt shows only 7% cleavage (Fig. 9). The control experiments, under aerobic conditions, show that H2O2 (80 lM) or 1 (40 lM) alone is cleavage inactive in dark. DNA cleavage databy complex 1 in presence of potential inhibitors and the copper(II) salt Table 3 DNA cleavage dataa of SC pUC19 (0.5 lg) DNA by 1 on chemical oxidation by hydrogen peroxide No. Chemical nuclease data

Reaction condition

[H2O2] (lM)

[1] (lM)

DNA cleavage % NC

1 2 3 4 5 6 7 8

DNA control DNA + H2O2 DNA + H2O2 + 1 DNA + H2O2 + 1 DNA + H2O2 + 1 DNA + H2O2 + 1 DNA + 1 DNA + H2O2 + 1

– 80 80 60 65 65 – 60

– – 10 20 35 40 40 30

2 3 18 77

a b

b b

3 94

Incubation time 1 h in dark. It could not be determined by smearing.

Fig. 9. Agarose gel electrophoresis diagram showing the cleavage of SC pUC19 DNA (0.5 lg) by complex 1 treated with H2O2and potential inhibitors in Tris–HCl/NaCl buffer (50 mM, pH 7.2) in dark. Incubation time: 1 h (37 C). Lane 1: DNA control; lane 2: DNA + H2O2 (80 lM); lane 3: DNA + H2O2 (60 lM) + CuCl2.2 H2O (30 lM); lane 4: DNA + H2O2 (60 lM) + CuCl2 Æ 2H2O (40 lM); lane 5: DNA + dithiothreitol (DTT, 5 mM) + 1 (50 lM); lane 6: DNA + 3-mercaptopropionic acid (MPA, 5 mM) + 1 (50 lM); lane 7: DNA + H2O2 (60 lM) + 1 (30 lM); lane 8: DNA + H2O2 (60 lM) + 1 (30 lM) + distamycin (100 lM); lane 9: DNA + H2O2 (60 lM) + 1 (30 lM) + DMSO (4 lL).

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Table 4 DNA cleavage dataa of SC pUC19 (0.5 lg) DNA by 1 in presence of potential inhibitors and the copper(II) salt No. Chemical nuclease data

Reaction condition

[H2O2] (lM)

[1] (lM)

[Cu(II)] (lM)

DNA cleavage % NC

1 2 3 4 5 6 7 8 9

DNA control DNA + H2O2 DNA + H2O2 + CuCl2  2H2O DNA + H2O2 + CuCl2  2H2O DNA + dithiothreitol (DTT, 5 mM) + 1 (50 lM) DNA + 3-mercaptopropionic acid (MPA, 5 mM) + 1 DNA + H2O2 + 1 DNA + H2O2 + 1 + distamycin (100 lM) DNA + H2O2 + 1 + DMSO (4 lL)

– 80 60 60 – – 60 60 60

– – – – 50 50 30 30 30

– – 30 40 – – – – –

2 3 4 7 2 3 94 2 3

a

Incubation time 1 h in dark.

are given in Table 4. Control experiments also show that the addition of distamycin completely inhibits the cleavage of SC DNA which indirectly supports the minor groove binding of 1. The existence of diffusible radical species in the nuclease mechanism can be inferred by monitoring quenching of DNA cleavage (Fig. 9) in the presence of DMSO which suggests the possibility of formation of HO radicals as the reactive species. Moreover, since the complex is not able per se to induce any effect on double stranded DNA (lane 7, Fig. 8), it appears that an oxidizing agent is required for producing the cleavage process. On comparison of the cleavage property with some reported results [68,69] of Cu(II) complex, we note that less amount of oxidizing agents (here hydrogen peroxide) and complex are required in our case for maximum cleavage of DNA. This is well reflected from the cleavage data which show more than 75% of cleavage occurs with a very less amount of complex concentration (20 lM). 4. Conclusion An octanuclear antiferromagnetic copper(II) metallointercalator (1) having a N2O3-donor Schiff base ligand (H3L) is prepared and structurally characterized by X-ray crystallography. A very short and strong intramolecular hydrogen bond is linked to the two bimetallic unit [Cu2+]2 with a dicubane [Cu2+]4 core. The supramolecular structure shows the presence of a new type of water–perchlorate, [(H2O)2(ClO4)]1, helical network through hydrogen bonding interaction. Complex 1 binds to double-stranded DNA possibly in the minor groove and displays efficient oxidative cleavage of supercoiled DNA in the presence of H2O2 following a hydroxyl radical pathway. The results are of significance in designing a new transition metal complex based on simple Schiff base ligand for the inorganic complex nucleases. 5. Abbreviations ROS BLMs pMMO

reactive oxygen species bleomycins perticulate methane monooxygenase

TACH CT DNA EDTA EB TAE Tris SC NC DMSO DTT MPA

cis-1,3,5-triaminocyclohexane calf-thymus DNA ethylenediamine-N,N,N 0 ,N 0 -tetraacetate ethidium bromide tris-acetate EDTA tris-(hydroxymethyl) aminomethane supercoiled nicked circular dimethyl sulfoxide dithiothreitol mercaptopropionic acid

Acknowledgements K.D. acknowledges the Council of Scientific and Industrial Research, New Delhi, India, for financial support. S.G. acknowledges NSFC (20125104, 20490210). Appendix A. Supplementary data Figures S1 and S2. Full details of the crystal structure analyses of 1 have been deposited with the Cambridge Crystallographic Data Center, (CCDC Ref. no. 288181). Copies may be obtained free of charge on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (Fax: Int. Code +44(0)1223/336-033, e-mail: [email protected]). Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jinorgbio.2006.08.010. References [1] L.S. Lerman, J. Mol. Biol. 3 (1961) 18–30. [2] E.L. Hegg, J.N. Burstyn, Coord. Chem. Rev. 173 (1998) 133–165. [3] M. Komiyama, J. Sumaoka, Curr. Opin. Chem. Biol. 2 (1998) 751– 757. [4] M. Komiyama, N. Takeda, H. Shigekawa, Chem. Commun. (1999) 1443–1451. [5] R. Ott, R. Kra¨mer, Appl. Microbiol. Biotechnol. 52 (1999) 761–767. [6] J.A. Cowan, Curr. Opin. Chem. Biol. 5 (2001) 634–642. [7] A. Sreedhara, J.A. Cowan, J. Biol. Inorg. Chem. 6 (2001) 337–347. [8] S.J. Franklin, Curr. Opin. Chem. Biol. 5 (2001) 201–208. [9] C. Liu, M. Wang, T. Zhang, H. Sun, Coord. Chem. Rev. 248 (2004) 147–168.

K. Dhara et al. / Journal of Inorganic Biochemistry 101 (2007) 95–103 [10] B.N. Trawick, A.T. Daniher, J.K. Bashkin, Chem. Rev. 98 (1998) 939–960. [11] A.M. Pyle, J.K. Barton, Prog. Inorg. Chem. 38 (1990) 413–475. [12] K.E. Erkkila, D.T. Odom, J.K. Barton, Chem. Rev. 99 (1999) 2777– 2796. [13] J.K. Barton, Science 233 (1986) 727–734. [14] J.K. Barton, A.L. Raphael, Proc. Natl. Acad. Sci. USA 82 (1985) 6460–6464. [15] B. Meunier, Chem. Rev. 92 (1992) 1411–1456. [16] G. Pratviel, J. Bernadou, B. Meunier, Adv. Inorg. Chem. 45 (1998) 251–312. [17] G. Pratviel, J. Bernadou, B. Meunier, Angew. Chem., Int. Ed. Engl. 34 (1995) 746–769. [18] B. Armitage, Chem. Rev. 98 (1998) 1171–1200. [19] D.R. McMillin, K.M. McNett, Chem. Rev. 98 (1998) 1201–1220. [20] C. Metcalfe, J.A. Thomas, Chem. Soc. Rev. 32 (2003) 215–224. [21] C.J. Burrows, J.G. Muller, Chem. Rev. 98 (1998) 1109–1152. [22] W.K. Pogozelski, T.D. Tullius, Chem. Rev. 98 (1998) 1089–1108. [23] O. Zelenko, J. Gallagher, D.S. Sigman, Angew. Chem., Int. Ed. Engl. 36 (1997) 2776–2778. [24] D.S. Sigman, T.W. Bruice, A. Mazumder, C.L. Sutton, Acc. Chem Res. 26 (1993) 98–104. [25] D.S. Sigman, A. Mazumder, D.M. Perrin, Chem. Rev. 93 (1993) 2295–2316. [26] D.S. Sigman, Acc. Chem. Res. 19 (1986) 180–186. [27] D.S. Sigman, Biochemistry 29 (1990) 9097–9105. [28] L.E. Marshall, D.R. Graham, K.A. Reich, D.S. Sigman, Biochemistry 20 (1981) 244–250. [29] D.S. Sigman, D.R. Graham, V. D’Aurora, A.M. Stern, J. Biol. Chem. 254 (1979) 12269–12272. [30] H. Ali, J.E. Van Lier, Chem. Rev. 99 (1999) 2379–2450. [31] J. Reedijk, J. Inorg. Biochem. 86 (2001) 89. [32] S.E. Wolkenberg, D.L. Boger, Chem. Rev. 102 (2002) 2477–2496. [33] Y. Jung, S.J. Lippard, Biochemistry 42 (2003) 2664–2671. [34] E.R. Jamieson, S.J. Lippard, Chem. Rev. 99 (1999) 2467–2498. [35] S.J. Behroozi, W. Kim, J. Dannaldson, K.S. Gates, Biochemistry 35 (1996) 1768–1774. [36] K. Mitra, W. Kim, J.S. Daniels, K.S. Gates, J. Am. Chem. Soc. 119 (1997) 11691–11692. [37] R.M. Burger, Chem. Rev. 98 (1998) 1153–1170. [38] E.I. Solomon, U.M. Sundaram, T.E. Machonkin, Chem. Rev. 96 (1996) 2563–2606. [39] S.J. Elliott, D.W. Randall, R.D. Britt, S.I. Chan, J. Am. Chem. Soc. 120 (1998) 3247–3248. [40] S.T. Frey, H.H.J. Sun, N.N. Murthy, K.D. Karlin, Inorg. Chim. Acta 242 (1996) 329–338. [41] K.J. Humphreys, K.D. Karlin, S.E. Rokita, J. Am. Chem. Soc. 123 (2001) 5588–5589. [42] K.J. Humphreys, K.D. Karlin, S.E. Rokita, J. Am. Chem. Soc. 124 (2002) 6009–6019. [43] K.J. Humphreys, A.E. Johnson, K.D. Karlin, S.E. Rokita, J. Biol. Inorg. Chem. 7 (2002) 835–842.

103

[44] M. Scarpellini, A. Neves, R. Holrner, A.J. Bortoluzzi, B. Szpoganics, C. Zucco, R.A. Nome Silva, V. Drago, A.S. Mangrich, W.A. Ortiz, W.A.C. Passos, M.C.B. de Oliveira, H. Terenzi, Inorg. Chem. 42 (2003) 8353–8365. [45] E.L. Hegg, J.N. Burstyn, Inorg. Chem. 35 (1996) 7474–7481. [46] T. Itoh, H. Hisada, T. Sumiya, M. Hosono, Y. Usui, Y. Fujii, Chem. Commun. (1997) 677–678. [47] D.K. Chand, H.-J. Schneider, A. Bencini, A. Bianchi, C. Giorgi, S. Ciattini, B. Valtancoli, Chem. Eur. J. 6 (2000) 4001–4008. [48] K.M. Deck, T.A. Tseng, J.N. Burstyn, Inorg. Chem. 41 (2002) 669– 677. [49] A. Sreedhara, J.A. Cowan, Chem. Commun. (1998) 1737–1738. [50] A. Sreedhara, J.D. Freed, J.A. Cowan, J. Am. Chem. Soc. 122 (2000) 8814–8824. [51] R. Ren, P. Yang, W. Zheng, Z. Hua, Inorg. Chem. 39 (2000) 5454– 5463. [52] W.-X. Zhang, C.-Q. Ma, X.-N. Wang, Z.-G. Yu, Q.-J. Lin, D.-H. Jiang, Chin. J. Chem. 13 (1995) 497–503. [53] D.D. Perrin, W.L.F. Armarego, D.R. Perrin, Purification of Laboratory Chemicals, Pergamon Press, Oxford, 1980. [54] M.E. Reichmann, S.A. Rice, C.A. Thomas, P. Doty, J. Am. Chem. Soc. 76 (1954) 047–3053. [55] M. Lee, A.L. Rhodes, M.D. Wyatt, S. Forrow, J.A. Hartley, Biochemistry 32 (1993) 4237–4245. [56] J. Bernadou, G. Pratviel, F. Bennis, M. Girardet, B. Meunier, Biochemistry 28 (1989) 7268–7275. [57] S. Wo¨rl, H. Pritzkow, I.O. Fritsky, R. Kra¨mer, Dalton Trans. (2005) 27–29. [58] T. Steiner, Angew. Chem., Int. Ed. 41 (2002) 48–76. [59] T. Gajda, A. Jancso´, S. Mikkola, H. Lo¨nnberg, H. Sirges, J. Chem. Soc. Dalton Trans. (2002) 1757–1763. [60] V.A. Milway, V. Niel, T.S.M. Abedin, Z. Xu, L.K. Thompson, H. Grove, D.O. Miller, S.R. Parsons, Inorg. Chem. 43 (2004) 1874– 1884. [61] T. Kajiwara, N. Kon, S. Yokozawa, T. Ito, N. Iki, S. Miyano, J. Am. Chem. Soc. 124 (2002) 11274–11275. [62] M.J. Waring, J. Mol. Biol. 13 (1965) 269–282. [63] Y. Zhao, J. Zhu, W. He, Z. Yang, Y. Zhu, Y. Li, J. Zhang, Z. Guo, Chem. Eur. J. 12 (2006) 6621–6629. [64] A. Ambroise, B.G. Maiya, Inorg. Chem. 39 (2000) 4264–4272. [65] S.A. Tysoe, R.J. Morgan, A.D. Baker, T.C. Strekas, J. Phys. Chem. 97 (1993) 1707–1711. [66] J.K. Barton, A.T. Danishefsky, J. Goldberg, J. Am. Chem. Soc. 106 (1984) 2172–2176. [67] J.M. Kelly, A.B. Tossi, D.J. McConnell, C. OhUigin, Nucleic Acid Res. 13 (1985) 6017–6034. ´ lvarez, J.L. Garcı´a-Gimenez, [68] R. Cejudo, G. Alzuet, M. Gonza´lez-A J. Borra´s, M. Liu-Gonza´lez, J. Inorg. Biochem. 100 (2006) 70– 79. ´ lvarez, G. Alzuet, J. Borra´s, B. Macı´as, A. Castin˜eiras, [69] M. Gonza´lez-A Inorg. Chem. 42 (2003) 2992–2998.