Journal of Inorganic Biochemistry 145 (2015) 19–29

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Bio-relevant cobalt(II) complexes with compartmental polyquinoline ligand: Synthesis, crystal structures and biological activities Jun-Ling Li a,b,d, Lin Jiang a,b,c, Bi-Wei Wang a,b,c, Jin-Lei Tian a,b,c,d,⁎, Wen Gu a,b, Xin Liu a,b,c,⁎⁎, Shi-Ping Yan a,b a

Department of Chemistry, Nankai University, Tianjin 300071, People's Republic of China Tianjin Key Laboratory of Metal and Molecule Based Material Chemistry, Tianjin 300071, People's Republic of China Key Laboratory of Advanced Energy Materials Chemistry (MOE), Tianjin 300071, People's Republic of China d Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), People's Republic of China b c

a r t i c l e

i n f o

Article history: Received 6 June 2014 Received in revised form 11 November 2014 Accepted 12 November 2014 Available online 20 November 2014 Keywords: Co(II) complexes ‘end-off’ type ligand Crystal structures DNA cleavage Protein binding

a b s t r a c t Three new Co(II) complexes, [Co4(L)2(μ3-CrO4)2](ClO4)2 · 2CH3CN (1), [Co2(L)(μ2-na)(H2O)](ClO4)2 (2) and [Co2(L)(μ2-ba)](ClO4)2 · 0.5CH3CN (3) (Hna = nicotinic acid, Hba = benzoic acid, HL = N,N,N′,N′-tetrakis (2quinolylmethyl)-1,3-diaminopropan-2-ol), have been synthesized and characterized by various physicochemical techniques. The Co(II) centers are connected by endogenous alkoxy bridge from L− and various extrinsic auxiliary linkers, some of which display coordination number asymmetry (5, 6-coordinated for 1 and 2; 5, 5coordinated for 3). It is worth mentioning that complex 1 contains two rare reported μ3-η1, η1, η1-CrO2− 4 moieties. Susceptibility data of three complexes indicated intramolecular antiferromagnetic coupling of high-spin Co(II) atoms with exchange integral values (J) −14.94 cm−1, −11.26 cm−1 and −13.66 cm−1 for 1, 2 and 3, respectively. Interaction of compounds with calf thymus DNA (CT-DNA) have been investigated by absorption spectral titration, ethidium bromide (EB) displacement assay and viscosity measurement, which revealed that compounds bound to CT-DNA with a moderate intercalative mode, accompanied the affinities order: 1 N 2 ≈ 3. Three complexes exhibit oxidative cleavage of pBR322 plasmid DNA including a reliance on H2O2 as the activator. Compound 1 demonstrates an increased DNA cleavage activity as compared with 2 and 3, which could degrade super coiled DNA (SC DNA) into nicked coiled DNA (NC DNA) in lower concentration (5 μM). Moreover, all compounds could quench the intrinsic fluorescence of bovine serum albumin (BSA) in a static quenching process. Complex 1 also shows higher anticancer activity than cisplatin with lower IC50 value of incubation for both 24 h and 48 h. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Design of effective anticancer drugs is greatly driven in order to overcome one of the biggest headaches, cancer. Much attention has been focused on designing variety of metal compounds for mediation strand scissions of duplex DNA, for their potential applications in gene engineering and related drug development [1–3]. Cisplatin and its derivatives, the successful and well-known anticancer drugs, have been widely used in clinical cancer therapy, which act by binding covalently to the purine base of DNA [4]. However, inherent limitations such as side effects, general toxicity (like nephrotoxicity, emetogenesis and neurotoxicity) and intrinsic acquired resistance greatly limited further application of platinum drugs [5–8]. To overcome the drawbacks, much effort should be undertaken

⁎ Correspondence to: J.-L. Tian, Department of Chemistry, Nankai University, Tianjin 300071, People's Republic of China. ⁎⁎ Correspondence to: X. Liu, Tianjin Key Laboratory of Metal and Molecule Based Material Chemistry, Tianjin 300071, People's Republic of China. E-mail addresses: [email protected] (J.-L. Tian), [email protected] (X. Liu).

http://dx.doi.org/10.1016/j.jinorgbio.2014.11.003 0162-0134/© 2014 Elsevier Inc. All rights reserved.

to design an idle agent. Recently, some non-platinum compounds have been designed, which also demonstrates some good properties: possessing various coordination numbers and geometries, better solubility and so on [9,10]. For example, some ruthenium complexes have been reported for their potential ability as anticancer drugs [11–13]. In recent years, numerous transitional metal compounds, such as Cu(II) [14–17], Zn(II) [18–20], Ni(II) [21,22], and VO(II) [23–25] complexes, have been synthesized and tested for their effective cleavage of DNA. Compared with above metals, the paramagnetic 3d7-Co(II), is an essential trace element for humans, which can substitute for Zn(II) in many metalloenzyme catalyzed reactions in vitro and shows similar charge density and ionization potential [26,27]. Moreover, the active site for EcMetAP (human methionine aminopeptidase from Escherichia coli) was proved to be binuclear cobalt centers [28]; many Co(II) complexes have been reported for their effective DNA binding and cleavage activity [29–32]. As a result, we choose Co(II) as the metal center in our work. Since active sites of many natural nucleases have more than one metal ion, dinuclear and multinuclear metallonucleases have attracted great interests; especially since Karlin et al have shown that nuclearity play an important role in the mechanism of oxidative DNA cleavage

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with possible synergy between metal ions [33,34]. Moreover, Sheng et al also showed metal ions is a crucial parameter in the hydrolytic DNA cleavage within polynuclear Zn(II) compounds [35]. Some polynuclear complexes have already been proved as good DNA cleavage agents [36–38]. For example, a noncatena “1 + 2 + 1” tetranuclear Cu(II) compounds, [Cu4(atc)2(dien)4(ClO4)2](ClO4)2 · 2H2O (H2atc = 5-aminol,2,4-triazole-3-carboxylic acid and dien = diethylenetriamine), displayed efficient oxidative cleavage of DNA in the presence of ascorbate [39]. So dinuclear and multinuclear system attracted our attention. For the purpose of exploring chemical nuclease of polynuclear Co(II) complexes, in this work, we have synthesized three new Co(II) compounds with one reported ‘end-off’ type ligand, N,N,N′,N′-tetrakis (2quinolylmethyl)-1,3-diaminopropan-2-ol) (HL) [40]. Recently, the tetranuclear Mn(II,III,III,II) and dinuclear Zn(II) compounds of HL have been structurally characterized [40,41]. However, authors mainly focused on their properties of magnetics or optics, whereas the potential bio-relevant application prospects have not been investigated. HL is a typical dinucleating compartmental ligand, which could provide μalkoxo dimetal cores relevant to active sites of some bimetallic enzymes [42]. Moreover, incorporation of a 2-propanol linker in analogues of HL has been proved as a good strategy for effective cooperation two metal ions with binding and hydrolysis of phosphate diesters by different authors [43–46]. For example, many transitional metal complexes have been synthesized with polybenzimidazole dinucleating ligand (HL1 = N,N,N′,N′-tetrakis (2-benzimidazolylmethyl)-1,3-diaminopropan-2-ol) [47–49]. Previously, our group has synthesized and characterized one new binuclear Co(II) complex of [Co2L1(μ2-Cl)](ClO4)2, which exhibits effective DNA hydrolysis activity [50]. However, with 2-propanol linker, quinoline ring was incorporated instead of benzimidazole in this work, which could be due to better planarity, stronger hydrophobic DNA interaction [51]. Moreover, the quinoline skeleton, isolated from Camptotheca acuminate, shows important biological activities [52–54]. In our work, with Co(II) ion, as well as different auxiliary bridges, L forms structural difference complexes: two dinuclear compounds and one tetranuclear compound. To our knowledge, the tetranuclear Co(II) compound is the first novel type, which not only contains two moieties but also bears coordination number asymmetry μ3-CrO2− 4 for Co(II) ions. It is expected that designing of different coordination entity with ‘end-off’ type ligand could fit well in DNA binding, cleavage as well as interactions with some protein. We explored some biological activities of three Co(II) compounds. As expected, three compounds, besides showing efficient DNA binding and cleavage properties, also exhibit good binding abilities with bovine serum albumin (BSA). To our surprise, complex 1 also shows potential anticancer activity, with lower IC50 value, compared with cisplatin. 2. Experimental CAUTION Perchlorate salts of metal complexes are potentially explosive and therefore should be prepared in small quantities. 2.1. Materials and measurements All reagents and chemicals were purchased from commercial sources and used as received. EB, pBR322 plasmid DNA, agarose, BSA and calf thymus (CT-DNA) were obtained from Sigma. Stock solutions of Co(II) complexes (1.0 × 10−3 M in DMF (N,N-dimethyformamide)) were stored at 4 °C and prepared to required concentrations for all experiments. Ultrapure MilliQ water (18.24 MΩ · cm) was used in our experiments. Tris–HCl and phosphate buffer solution were prepared using ultrapure water. Elemental analyses for C, H and N were obtained on a Perkin-Elmer analyzer model 240. Infrared spectroscopy on KBr pellets were performed on a Bio-Rad FTS 6000 spectrophotometer in the 4000– 400 cm−1 regions. Electrospray ionization mass spectrometry (ESI-MS) was obtained on Agilent 6520 Q-TOF LC/MS (top of flight liquid

chromatography/mass spectrometry). The magnetic susceptibility measurements of the samples were measured over the temperature range of 2–300 K with a Quantum Design MPMS-XL7 SQUID magnetometer using an applied magnetic field of 1000 Oe. Electronic spectra were measured on a JASCO V-570 spectrophotometer. Fluorescence spectral data were obtained on a MPF-4 fluorescence spectrophotometer at room temperature. Viscosity measurements were carried out on an Ubbelodhe viscometer maintained at a constant temperature (37.0 ± 0.1 °C) in a thermostatic water-bath. The Gel Imaging and documentation DigiDoc-It System were assessed using Labworks Imaging and Analysis Software (UVI, UK). 2.2. Synthesis of ligand L and corresponding Co(II) complex (1–3) 2.2.1. Preparation of ligand The HL (Scheme 1) was prepared according to a procedure described in the literature[40]. A mixture of 2-chloromethylquinoline hydrochloride (1.82 g, 8.4 mmol), 1, 3-diamino-2-propanol (191 mg, 2.1 mmol), and potassium carbonate (3.5 g, 26 mmol) in acetonitrile (30 mL) was refluxed for 48 h. After the solvent was removed under reduced pressure, the residue was separated by chloroform/water and organic phase was dried, evaporated, and the residue was washed with ethanol to give HL as pale yellow powder. 2.2.2. Synthesis of [Co4(L)2(μ3-CrO4)2](ClO4)2 · 2CH3CN (1) To an ethanol solution (10 ml) of HL (0.0657 g, 0.1 mmol) and triethylamine (0.1 mmol), an aqueous solution (5 mL) of Co(ClO4)2 · 6H2O (0.0744 g, 0.2 mmol) was added. The resulting mixture was stirred for 2 h at room temperature. Then an aqueous solution of K2CrO4 (0.0195 g, 0.1 mmol) was added to the reaction mixture. Plenty of precipitates appeared immediately and then filtered. The obtained precipitates were dissolved in a mixed solvent of acetonitrile (5 ml) and ethanol (10 ml). Green block crystals suitable for x-ray diffraction were obtained by slow evaporation of solution after fifteen days, which were collected by filtration, washed with diethyl ether and dried in air. Yield: 0.033 g, 32%. Elemental analysis (%): calc. for C90H80Cl2Co4Cr2N14O18: C, 52.27; H, 3.92; N, 9.54. Found: C, 52.36; H, 3.69; N, 9.59; Selected IR data (KBr, ν, cm− 1): 3059(m, ν C–H), 1147(m, ν C–O), 1094(m, ν Cl–O), 880 (m, ν Cr–O). ESI-MS (m/z = 1964.01, [Co4L2(CrO4)2(ClO4)(DMF)(H2O)]+; m/z = 861.16, [Co2L(C2H5O)2]+). 2.2.3. Synthesis of [Co2(L)(μ2-na)(H2O)](ClO4)2 (2) Complex 2 was prepared by a procedure similar to that given in the case of 1, using nicotinic acid (Hna) (0.0123 g, 0.1 mmol) in place of K2CrO4 to the reaction mixture. Three hours later, after filtration, magenta sediments were obtained. Magenta block crystals suitable for x-ray diffraction were obtained by slow evaporation of solution after ten days, which were collected by filtration, washed with diethyl ether and dried in air. Yield: 0.04 g, 36%. Elemental analysis (%): calc. for C49H42O12N7Cl2Co2: C, 53.04; H, 3.81; N, 8.84. Found: C, 53.12; H, 3.61; N, 8.91. Selected IR data (KBr, ν, cm− 1): 3607(m, ν free O–H), 3062(m, ν C–H), 1707(w, ν C_O), 1091(s, ν Cl–O). ESI-MS m/z = 861.16, [Co2L(C2H5O)2]+). 2.2.4. Synthesis of 2{[Co2(L)(μ2-ba)](ClO4)2} · CH3CN (3) Complex 3 was prepared by a procedure similar to that given in the case of 1, using benzoic acid (Hba) (0.0122 g, 0.1 mmol) in place of K2CrO4 to the reaction mixture. Fifteen minutes later, amount of magenta sediments was obtained. Then sediments were dissolved in a mixed solvent of acetonitrile (5 ml) and ethanol (10 ml). Magenta needleshaped crystals suitable for x-ray diffraction were obtained by slow evaporation of solution after two days, which were collected by filtration, washed with diethyl ether and dried in air. Yield: 0.05 g, 45%. Elemental analysis (%): calc. For C102H87O22N13Cl4Co4: C, 55.08; H, 3.94; N, 8.19. Found: C, 54.14; H, 4.10; N, 7.98. Selected IR data (KBr, ν, cm−1):

J.-L. Li et al. / Journal of Inorganic Biochemistry 145 (2015) 19–29

21

Scheme 1. Schematic structures of HL1(left) and HL(right).

3065(m, ν C–H), 1710(w, ν C_O), 1144(m, ν C–O), 1090(s, ν Cl–O). ESI-MS m/z = 861.16, [Co2L(C2H5O)2]+). 2.3. X-ray crystallographic studies Single-crystal X-ray diffraction data of compounds were collected on a Bruker Smart 1000 CCD diffractometer using Mo-Kα radiation (λ = 0.71073 Å) with the ω scan technique. The structures were solved by direct methods (SHELXS-97 and refined with full-matrix leastsquares technique on F2 using the SHELXL-97 [55]. The hydrogen atoms were added theoretically, and riding on the concerned atoms and refined with fixed thermal factors. The details of crystallographic data and structure refinement parameters are summarized in Table 1, and selected bond angles and distances are listed in Table 2. Crystallographic data for complex 1–3 have been deposited with the Cambridge Crystallographic Data Centre with the corresponding CCDC reference numbers of 946521(for 1), 970644(for 2) and 970645(for 3), respectively. 2.4. DNA binding and cleavage activity studies The UV absorbance at 260 nm and 280 nm of the CT-DNA solution in Tris–HCl buffer (5 mM Tris, 50 mM NaCl pH = 7.2) gives a ratio of 1.8–1.9, indicating that CT-DNA was sufficiently free of protein [56]. The concentration of CT-DNA was determined from its absorption intensity at 260 nm with a molar extinction coefficient of

6600 M−1 cm−1 [57]. The absorption spectra of complex 1–3 binding to DNA were performed by increasing amounts of CT-DNA to complex in Tris–HCl buffer. The relative binding abilities of complexes to CT-DNA were studied with an EB-bound CT-DNA solution in Tris–HCl (50 mM Tris, 18 mM NaCl pH = 7.2). The fluorescence spectra were recorded at room temperature with excitation at 510 nm and emission at about 602 nm. The experiments were carried out by adding a certain volume of a stock of complex into EB-DNA solution (2.4 μM EB and 48 μM CTDNA). The system was allowed to equilibrate for 5 min, and then spectra were recorded. The DNA cleavage experiments were carried out by agarose gel electrophoresis, which was performed by incubation at 37 °C as follows: pBR322 DNA (0.1 μg/μL) in 50 mM Tris–HCl /18 mM NaCl buffer (pH = 7.2) was treated with complex 1–3. The samples were incubated for 3 h, and then loading buffer was added. Then samples were electrophoresed for 2 h at 0.9% agarose gel using Tris–boric acid–EDTA buffer. After electrophoresis, bands were visualized by UV light and photographed. DNA cleavage mechanism of complex was investigated in the presence of some radical scavengers and reaction inhibitors. The reactions were conducted by adding standard radical scavengers of KI, L-Histidine, EDTA and groove binding agent Methyl Green (for major groove), SYBR Green(for minor groove) to pBR322 DNA prior to addition of complex. Cleavage experiment was quenched with 2 μL of loading buffer. Further analysis was carried out by the above standard methods.

Table 1 Crystallographic data for complex 1–3. Compound

1

2

3

Formula Mr Crystal system Space group

C90H80Cl2Co4Cr2N14O18 2056.30 Triclinic

C49H42Cl2Co2N7O12 1109.66 Triclinic

P1 293(2) 12.400(3) 13.350(3) 14.770(3) 103.16(3) 103.20(3) 101.74(3) 2232.2(10) 1 1.530 1.098 32,397 7849 0.0454 0.0479 0.1236 0.0600 0.1324 1.075

P1 293(2) 10.853(2) 13.269(3) 17.095(3) 103.29(3) 94.54(3) 90.82(3) 2387.0(8) 2 1.544 0.879 14,265 8275 0.0558 0.0753 0.1639 0.1254 0.1889 1.081

C102H87Cl4Co4N13O22 2224.36 Monoclinic C2/c

T/K a/Å b/Å c/Å α/° β/° γ/° V/Å3 Z D/g/cm3 μ/mm−1 Reflections measured Independent reflections Rint R1 (I N2σ(I)) wR(F2) (I N2σ(I)) R1 (all data) wR(F2) (all data) Goodness of fit on F2

293(2) 24.871(5) 21.993(4) 21.232(4) 90.00 110.04(3) 90.00 10,910(4) 4 1.354 0.768 31,486 9600 0.0617 0.0658 0.1623 0.0930 0.1782 1.075

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2.6. In vitro cell assay

Table 2 Selected bond lengths and angels for complex 1–3. Complex 1 Co(1)–O(2) Co(1)–O(1) Co(1)–O(4)#1 Co(1)–N(4) Co(1)–N(6) Co(1)–N(5) Co(2)–O(3) Co(2)–O(1) O(2)–Co(1)–O(1) O(2)–Co(1)–O(4)#1 O(1)–Co(1)–O(4)#1 O(2)–Co(1)–N(4) O(1)–Co(1)–N(4) O(4)#1–Co(1)–N(4) O(2)–Co(1)–N(6) O(1)–Co(1)–N(6) O(4)#1–Co(1)–N(6) N(4)–Co(1)–N(6) O(2)–Co(1)–N(5) O(1)–Co(1)–N(5) O(4)#1–Co(1)–N(5)

2.018(3) 2.059(3) 2.140(3) 2.148(3) 2.226(3) 2.234(3) 1.963(3) 1.969(3) 97.58(10) 86.61(10) 91.32(11) 176.29(11) 81.08(11) 89.96(11) 104.42(11) 158.00(11) 90.07(11) 76.96(12) 103.08(11) 88.50(11) 170.25(11)

Co(2)–N(1) Co(2)–N(3) Co(2)–N(2) Cr(1)–O(5) Cr(1)–O(4) Cr(1)–O(2) Cr(1)–O(3)

2.097(3) 2.150(3) 2.174(3) 1.622(3) 1.635(3) 1.663(2) 1.673(3)

N(4)–Co(1)–N(5) N(6)–Co(1)–N(5) O(3)–Co(2)–O(1) O(3)–Co(2)–N(1) O(1)–Co(2)–N(1) O(3)–Co(2)–N(3) O(1)–Co(2)–N(3) N(1)–Co(2)–N(3) O(3)–Co(2)–N(2) O(1)–Co(2)–N(2) N(1)–Co(2)–N(2) N(3)–Co(2)–N(2) Co(2)–O(1)–Co(1)

80.38(11) 86.49(11) 95.58(11) 112.77(13) 107.55(12) 99.07(12) 146.03(12) 94.73(13) 166.66(12) 81.61(12) 80.43(14) 77.11(13) 133.96(13)

Complex 2 Co(1)–O(1) Co(1)–O(3) Co(1)–N(5) Co(1)–O(13) Co(1)–N(4) Co(1)–N(6) O(1)–Co(1)–O(3) O(1)–Co(1)–N(5) O(3)–Co(1)–N(5) O(1)–Co(1)–O(13) O(3)–Co(1)–O(13) N(5)–Co(1)–O(13) O(1)–Co(1)–N(4) O(3)–Co(1)–N(4) N(5)–Co(1)–N(4) O(13)–Co(1)–N(4) O(1)–Co(1)–N(6) O(3)–Co(1)–N(6) N(5)–Co(1)–N(6)

2.017(4) 2.074(4) 2.138(4) 2.153(5) 2.203(5) 2.215(5) 94.15(15) 82.14(16) 174.91(16) 85.43(18) 87.08(16) 89.15(18) 98.72(16) 103.43(16) 80.66(17) 168.30(18) 158.26(15) 104.76(16) 78.28(17)

Co(2)–O(1) Co(2)–O(2) Co(2)–N(3) Co(2)–N(1) Co(2)–N(2)

1.954(4) 2.017(4) 2.127(5) 2.149(5) 2.163(5)

O(13)–Co(1)–N(6) N(4)–Co(1)–N(6) O(1)–Co(2)–O(2) O(1)–Co(2)–N(3) O(2)–Co(2)–N(3) O(1)–Co(2)–N(1) O(2)–Co(2)–N(1) N(3)–Co(2)–N(1) O(1)–Co(2)–N(2) O(2)–Co(2)–N(2) N(3)–Co(2)–N(2) N(1)–Co(2)–N(2) Co(2)–O(1)–Co(1)

84.93(19) 87.38(18) 93.84(15) 100.90(18) 109.22(17) 151.78(18) 101.85(18) 95.90(19) 82.83(17) 170.63(18) 80.06(19) 77.91(19) 131.52(17)

Complex 3 Co(1)–O(1) Co(1)–O(3) Co(1)–N(4) Co(1)–N(5) Co(1)–N(6) O(1)–Co(1)–O(3) O(1)–Co(1)–N(4) O(3)–Co(1)–N(4) O(1)–Co(1)–N(5) O(3)–Co(1)–N(5) N(4)–Co(1)–N(5) O(1)–Co(1)–N(6) O(3)–Co(1)–N(6) N(4)–Co(1)–N(6) N(5)–Co(1)–N(6) O(1)–Co(2)–O(2)

1.964(3) 1.992(3) 2.132(4) 2.141(4) 2.153(4) 95.85(12) 128.28(14) 100.54(14) 111.01(13) 107.64(14) 109.92(14) 81.51(13) 174.03(14) 77.21(15) 78.32(16) 95.01(12)

Co(2)–O(1) Co(2)–O(2) Co(2)–N(2) Co(2)–N(1) Co(2)–N(3) O(1)–Co(2)–N(2) O(2)–Co(2)–N(2) O(1)–Co(2)–N(1) O(2)–Co(2)–N(1) N(2)–Co(2)–N(1) O(1)–Co(2)–N(3) O(2)–Co(2)–N(3) N(2)–Co(2)–N(3) N(1)–Co(2)–N(3) Co(1)–O(1)–Co(2)

1.982(3) 1.986(3) 2.122(4) 2.127(4) 2.136(3) 132.45(14) 100.54(13) 113.62(14) 109.23(14) 103.07(14) 81.41(13) 171.11(13) 76.41(14) 79.65(14) 128.98(15)

a

Symmetry code: #1 for 1: 1–x,–y, 1–z.

2.5. Protein binding studies Fluorescence quenching experiments have been carried out to investigated interaction between BSA and complexes, by using bovine serum albumin stock solution (BSA, 1.5 mM) in 10 mM phosphate buffer (pH = 7.0). The fluorescence spectra were recorded at room temperature with excitation wavelength of BSA at 280 nm and the emission at 342 nm by keeping the concentration of BSA constant (29.4 μM) while varying the complex concentration from 0 to 20 μM for 1, from 0 to 40 μM for 2 and 0 to 35 μM for 3.

The cytotoxicity of complex 1–3 against cervical carcinoma cells (HeLa) was evaluated by a CCK-8 kit assay. CCK-8 assay is a wellestablished method for determination of cell viability in cell proliferation and cytotoxicity assays, which utilizes Dojindo's highly watersoluble tetrazolium salt, WST-8, to produce water-soluble fornazan dye on reduction in living cells to determine the number of living cells. HeLa cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (5% CO2, 37 °C). Cells were seeded into 96-well plates at a density of 104 cell / well and incubated for 24 h and 48 h to allow cell attachment. Then, cells were washed with PBS, and the medium was replaced with a fresh medium containing indicated concentrations of complex. The cell without treatment was used as control. After a period of incubation, cells were washed with PBS and incubated in DMEM/F12 with 10% WST-8 solution for another 2 h. The absorbance of each well was measured at a wavelength of 450 nm with a plate reader. The results were expressed as the mean values of three measurements. The cell viability was calculated as follows cell viabilityð%Þ ¼

Asample −Ablank  100 Acontrol −Ablank

where Asample, Acontrol, and Ablank represent the absorbance intensity at 450 nm determined for cells treated with different samples, for control cells (nontreated), and for blank wells without cells (the same amounts of the CCK-8 solution and the sample solution as the sample wells were added to the blank wells), respectively. 3. Results and discussion 3.1. Description of the crystal structure Complex 1–3 have been structurally characterized by x-ray crystallography (Fig. 1), details of data collection conditions and parameters of refinement process are given in Table 1, and the selected bond lengths and angles are given in Table 2, respectively. Compound 1 was a centrosymmetric tetranuclear Co(II) complex. Two dinuclear Co(II) units bridged by the alkoxo group of L− are further bridging groups, resulting in a linked by two extrinsic μ3-CrO2− 4 tetranuclear entity (dimer-of-dimer). From Fig. 1A, two types of Co(II) centers can be distinguished based on their coordination environment: (i) Co2 shows pentacoordinated geometry with a N3O2 donor set derived from one tertiary amine N, two quinoline N atoms, one bridged anion. The τ value of O atom from L− and one O atom from a CrO2− 4 Co2 is calculated to be 0.34, which can be described as a distorted squarepyramidal configuration [58]. (ii) Co1, exhibiting hexacoordinated geometry with a N3O3 donor set, is constituted by one tertiary amine N, two quinolyl-N atoms and one alkyl O atom from L−, two O atoms anions, which shows a highly distorted octahefrom two distinct CrO2− 4 dral environment. In 1, each HL is deprotonated and acts as a dinucleating compartmental ligand, which provides a μ-alkoxo dimetal core of [Co2(μ2L)]3+ with the Co1…Co2 distance of 3.707 Å. Two dinuclear units are moieties to confurther connected by two distinct μ3-η1, η1, η1-CrO2− 4 struct the Co4Cr2 entity with the Co1…Co2# distance of 5.048 Å. For each dinuclear unit, the distance between two metal ions is larger than 2 (3.622 Å) and 3 (3.561 Å), which may be due to the coordinated CrO2− 4 moieties. The chromate anion has been usually used as a bridging ligand to bind to main group, transition, and lanthanide elements to form binuclear compounds or polymeric materials [59]. Usually, μ2-CrO2− 4 ligand is found in the heterometallic Cr(VI) complexes [59–61]. However, a rare example of heterometallic Cr-oxo cluster featuring two μ3chromate ligands was found in our work, although Cu(II)Cr(VI) complex [62] and a molecular Bi(III)Cr(VI) cluster [63]: Bi4Cr4O16, have also been found out containing μ3-CrO2− 4 moieties. The coordination geometry of

J.-L. Li et al. / Journal of Inorganic Biochemistry 145 (2015) 19–29

Fig. 1. Molecular structures of complexes; A for complex 1 [Co4], B for complex 2 [Co2], C for complex 3 [Co2]; Hydrogen atoms, perchlorate ions and some acetonitrile molecules have been omitted for clarity.

chromate shows a slightly distorted tetrahedron with the O–Cr–O bond angles in the range 108.67(13)–110.76(13)°. The Cr–O bonds where O is also coordinated to Co(II) [1.635(3) Å, 1.663(2) Å, 1.673(3) Å for Co1#,

23

Co1, and Co2, respectively] are slightly longer than the “uncoordinated” Cr–O bond [1.605(4) Å] [62]. Moreover, the Cr–O(5) bond is also affected by coordination between chromate anion and Co(II) atoms [1.622(3) Å]. An interesting aspect of structure 1 (Fig. 2) is that three metalcontaining ring systems (two six-membered Co1–O2–Cr1–O3–Co2– O1 and one eight-membered Co1–O2–Cr1–O4–Co1#–O2#–Cr1#– O4#) of coordinated atoms are generated as a result of the introduction ions. Another interesting aspect is that each ring contains of two CrO2− 4 two Co(II) atoms and the eight-membered ring is similar to the chair conformations of hexane. Compound 2 crystallizes in a triclinic cell with P-1 space group. From Fig. 1B, two Co(II) atoms also display coordination number asymmetry. Each of them is coordinated to two N atoms of quinoline moiety, one tertiary amine N atom, a bridging alkoxo-O atom and one O atom from the carboxyl of nicotinic acid. However, one Co(II) center is only five-coordinate, the other is six-coordinate where a water molecule occupies the sixth coordination position. The structure of 2 is similar to the active site for Escherichia coli (EcMetAP) [64]. The five-coordinate Co(II) atom is in a distorted square-pyramidal coordination environment with τ value of 0.31, which is apparently lower than the pentacoordinated Co(II) in MPA (methionine aminopeptidase) from EcMetAP(τ = 0.83) [65]. The six-coordinate Co(II) atom is in a distorted octahedral arrangement. The distance between two Co(II) atoms is 3.622 Å, which is a little far compared to several similar reported cobalt complexes [65,66], while the Co–μ(O)–Co angle amounts to ca. 131°. For the binuclear sites, their potential coordination environments available have been classified [67]: (i) symmetric, (ii) donor asymmetry, (iii) geometric asymmetry and (iv) coordination number asymmetry. The binuclear unit of 1 and compound 2 could be classified to the last one, in which an unequal number of donor atoms are coordinated to each metal atom. Several examples of bimetallic Co(II) complexes bearing five- and six-coordinate sites have been reported in the literature [65,68], although the use of unsymmetrical ligands have been always required to generate two different coordination environments [69]. In conclusion, we have synthesized the first novel example of a tetracobalt complex, compound 1, which not only contains two μ3-CrO2− 4 moieties, but also bears five- and six-coordinate sites by using a symmetrical polydentate ligand. As for compound 3, Co(II) atom exhibits only one coordinated model: five-coordinated environment (Fig. 1C). It is crystallized in monoclinic C2/c space group; each Co(II) atom of 3 was coordinated with two quinoline N atoms, one tertiary amine N atom, and a bridging alkoxo-O atom and one O atom from carboxyl of benzoate. The fivecoordinate Co(II) atoms are found in a distorted trigonal–bipyramidal coordination environment, with τ value of 0.77 (Co1) and 0.64 (Co2). The distance between two metal ions is 3.561 Å, which is nearer than several reported five-coordinate complexes [70], but farer than our previously reported Co(II) complexes [Co2L1(μ2-Cl)](ClO4)2 and [Co2L1(μ2DPP)]2+(DPP = diphenylphosphinate) [71].

3.2. IR data analysis

Fig. 2. View of the CrO2− moieties with the metal environment of [Co4]. Symmetry code, 4 #: 1 − x, −y, 1 − z.

The FT-IR spectra of three complexes are shown in Fig. S1. For three compounds, C–H stretching vibrations of aromatic rings were found at about 3060 cm−1 and the ring C–C stretching vibrations occur in the region 1600–1400 cm−1 [72]. The C–H stretching vibrations of –CH3 and – CH2 of corresponding complexes are in the range of 2860–2960 cm−1. The deformation band of –CH3 were found at 1373, 1468 cm−1 (for 1) and 1374, 1469 cm−1(for 3). The ClO− 4 vibrations for three compounds were found at 1090 cm−1, which is similar with the literature [73]. The band 1707 (for 2) and 1710 cm−1 (for 3) should be C_O stretching vibrations. For 1, the strong band at about 880 cm− 1 is due to the stretching Cr–O vibrations [60]. The absorption at 3607 cm− 1 is the free O–H stretching vibrations of water molecule in 2.

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J.-L. Li et al. / Journal of Inorganic Biochemistry 145 (2015) 19–29

Fig. 3. Experimental (○) and calculated (solid line) χMT vs T curve for 1.

3.3. Magnetic properties The magnetic behavior of complex 1 is shown in Fig. 3 as temperature dependence of χMT where χM is the molar magnetic susceptibility (2 and 3 in Fig. S2). Magnetic susceptibility measurements have been investigated in the temperature range of 2–300 K. Co(II) ions in our complexes belong to 3d7 high spin states. The magnetic data have been treated based on dinuclear Co model: (i) in order to keep correspondence with follow treatment of χM; (ii) high-spin 3d7 center metal in the different coordination number asymmetry in some of our complexes. At room temperature, χMT for 1 (per two Co atoms), 2 and 3 is 4.16, 4.07 and 3.84 cm3 · K · mol−1, respectively; which is higher than high spin only value of 3.75 cm3 K mol−1 for two uncoupled spins (g = 2.00 S = 3/2). Moreover, μeff values were 5.77 μB (1, based on dinuclear unit), 5.54 μB (2) and slightly higher than 3 (5.48 μB) which is approximated with spin-only value of two Co(II) atoms (5.47 μB). Higher μeff and χMT values of 1 and 2 may be due to the orbital angular momentum typical for 4 T1 ground state [74,75]. There is no maximum in the curve but it shows strong temperature dependence. The decrease of χMT is generated by combined effect of intramolecular antiferromagnetic interactions between metal centers and intrinsic spin-orbit coupling of high-spin Co(II) metal ions [70]. The precise calculation of moment not only requires reliable values of ligand-field and spin-orbit coupling parameters, but also of the degree of electron delocalization, so some literatures [71,76], treat such cases merely as an empirical term be estimated. For 1, the susceptibility was calculated as dinuclear unit: Karl Wieghardt [77] et al has investigated that removal of the μ-oxo bridge and substitution by a third bridge 2− or CrO2− bridge as in complex leads to dramatically as HPO2− 4 , HAsO4 4 reduced spin-exchange, which is clearly for two complexes, [L′Fe(μ2CrO4)3,FeL′] · H2O and [L′2Fe2(μ-O)(μ-CrO4)2] · 4H2O (L′ = 1,4,7trimethyl-1,4,7-triaza-cyclonon), with the J value ranging from − 7.5 moieties in 1 are the poor mediator to −124 cm−1. So two μ3-CrO2− 4 for super-exchange mechanism and their interactions between two units are treated as intermolecular interaction. In this case, the molar paramagnetic susceptibility data are fit the expression [78], which is based on the general isotropic exchange Hamiltonian, Ĥ = −2JŜ1 · Ŝ2, with J = magnetic exchange coupling constant and S1 = S2 = 3/2: χM ¼

0

χM

The best fit of experimental data using above equation yielded values: g = 2.45, J = − 14.94 cm−1, zJ′ = − 3.05 cm− 1 for 1, g = 2.26, J = − 11.26 cm−1 for 2; g = 2.23, J = − 13.66 cm−1 for 3. The data indicated that antiferromagnetic interaction exists between the neighboring Co(II) atoms of three compounds. For 1, the value of zJ′ is much smaller than J, which verified our previous treatments. Larger g values for 1 and 2 could be attributed to spin-orbit coupling in the distorted octahedral coordinated Co(II) atoms [79]. The g value of compound 3 is similar with our previously reported five-coordinated complexes [Co2L1(μ2-Cl)](ClO4)2 (2.22) and [Co2L1(μ2-DPP)]2+(2.22) [71]. Several literatures [75,80,81] suggested that Co(II)-X-Co(II) bond angles around 96° in certain complexes give rise to ferromagnetic coupling through orthogonal magnetic orbitals. So, antiferomagnetic coupling happen in our compound due to larger angles in our compounds (133.96°, 131.52° and 128.98° for 1, 2 and 3). With same coordination environments, Co(II)–X–Co(II) bridge angles appear to have more influence on magnitude and sign of J than Co(II)–X bond distances, Co(II)… Co(II) distance, and the extent of distortion around Co(II) center [81]. Several reported literature also have suggested the influence of Co(II)– X–Co(II) angle on the value of J [75,80,82]. As a result, the larger angles of Co(II)–X–Co(II) in our compounds may play some roles in both larger values of |J| and antiferromagnetic coupling between Co(II) ions. 3.4. DNA-binding modes and affinities studies 3.4.1. Absorption spectroscopy studies The binding of metal complex to DNA is considered to be an important step in DNA cleavage. After interacting the base pairs of DNA [83], the π* orbital of the intercalated ligand can couple with π orbital of the base pairs, thus decreasing the π–π* transition energy and resulting in bathochromism [84]. On the other hand, the coupling π orbital is partially filled by electrons, thus decreasing the transition probabilities and concomitantly resulting in hypochromism. CT-DNA binding ability of complex 1–3 has been ascertained on the UV absorption. Absorption titration experiments were performed by recording UV absorbance of complex with increasing concentration of CT-DNA stock solution. As shown in Fig. 4 (Fig. S3), upon addition of CT-DNA to the solution of complex, dramatic decrease of absorption intensities appeared. In the spectra of respective complexes around 306 nm are assigned to ligand centered π–π* absorption, and the observed trend in hypochromism is 1 (~39.4%), 2 (~33.1%) and 3 (~33.3%). Such a marked change in the intensity of spectral bands is expected an intimate association of compound with CT-DNA, and it is also likely that Co(II) complex bind to

2Ng 2 β2 14e12 J=kT þ 5e6 J=kT þ e2 J=kT  12 J=kT kT 7e þ 5e6 J=kT þ 3e2 J=kT þ 1

χM
 ¼ : 1− 2z J 0 =Ng 2 β2 χ M

Fig. 4. Absorption spectra of complex 1 (25 μM) in the absence (black line) and presence (colored line) of increasing amounts of CT-DNA (9.3, 18.6, 27.9, 37.2, 46.5, 55.8, 65.1, 74.4, 83.7 μM) in 5 mM Tris–HCl/50 mM NaCl buffer (pH = 7.2). The insert shows the leastsquares fit of (εa − εf)/(εb − εf) vs [DNA] for complex 1.

J.-L. Li et al. / Journal of Inorganic Biochemistry 145 (2015) 19–29

25

Table 3 Ligand (L) binding and fluorescence spectral properties of complex 1–3 bound to CT–DNA. Complex

Kb (M−1)

s

Δε (%)

Kapp (M−1)

Ref

1 2 3 [Co2L1(μ2-Cl)](ClO4)2 [Co2L22(μ-OH)2](ClO4)2 [Co2L3(μ-OH)(H2O)4] [{(phen)Co}2(μ-dtdp)2] [{(dpq)Co}2(μ-dtdp)2] [{(dppz)Co}2(μ-dtdp)2]

1.45 × 106 7.78 × 105 7.90 × 105 unknown 1.16 × 104 1.22 × 103 4.3(±1.0) × 105 3.9(±0.9) × 106 4.0(±0.5) × 106

0.84 0.44 0.46 unknown unknown unknown 1.3 1.4 0.9

39.4 33.1 33.3 35.1 29 unknown ~40 ~40 ~40

9.08 × 106 2.58 × 106 2.85 × 106 1.90 × 10 4 4.9 × 104 unknown unknown unknown unknown

This work This work This work 50 87 88 89

L1: N,N,N′,N′ -Tetrakis (2-benzimidazolylmethyl) -1,3-diaminopropan-2-ol). L2: 1,4,7-triazacyclononane-N-acetate monoanion. L3: dinuclear ligand based on μ-1,2 diazine bridging phen: 1,10-phenanthroline; dpq: dipyrido[3,2-d:2′3′-f]quinoxaline; dppz: dipyrido[3,2-a:2′3′-c]phenazine; dtdp: 3,3-dithiodipropionic acid. (Kb: intrinsic equilibrium DNA binding constant; s: binding site size value; Δε: the trend in hypochromism; Kapp: apparent DNA binding constant; estimated errors for the constants are ±5%).

CT-DNA helix via intercalation [83]. In order to determine the binding strength of complex with CT-DNA, intrinsic binding constants Kb were calculated from a nonlinear fitting according to the equation [85]. 1=2 .       2 2 ε a −ε f = ε b −ε f ¼ b− b −2K b C t ½DNA=s 2K b C t

ð1aÞ

b ¼ 1 þ K b C t þ K b ½DNA=2s

ð1bÞ

The intrinsic equilibrium DNA binding constant (Kb) values of complex, along with binding site size (s), are given in Table 3. Kb values of complex (~105 M−1) follow the order: 1 N 2 ≈ 3; the s values obtained from the fitting, which shows the number of DNA bases associated with single molecular of complex, also follow the similar order: 1(0.84) N2(0.44) ≈ 3(0.46). The low s value (s b 1) was attributed to the aggregation of hydrophobic molecule on the DNA surface [86]. Moreover, Kb and s values of 2 (7.78 × 105 M−1, 0.44) and 3 (7.90 × 105 M−1, 0.46) are almost the same, which is expected due to their very similar dinuclear unit. According to the electronic absorption studies, binding constants indicate medium binding strength of three complexes with CT-DNA. The Kb values of current dinuclear Co(II) complexes are higher than once previously reported complexes [87, 88] given in Table 3, which may be due to the planarity and strong hydrophobicity of L. In addition, 1 exhibits similar DNA binding ability with Co(II) complex [89], which also indicates the importance of planarity in DNA binding ability of complex.

Fig. 5. Emission spectra of EB bound to CT-DNA in the absence (dashed line) and presence (solid lines) of complex 1 (0–5 μM) in 50 mM Tris–HCl/18 mM NaCl buffer (pH = 7.2). Inset: the plot of I0/I versus complex concentration.

3.4.2. Competitive DNA binding assay As a means for further investigating the interaction between test complexes and DNA, fluorescence spectral titration measurement has been carried out. EB, as a planar cationic dye, can intercalate strongly between the adjacent DNA base pairs [90]. So it could enhance the emission intensity when it interacts with DNA. However, addition a second DNA intercalate molecule could replace EB from DNA-bound EB system, fluorescence of system will be quenched as the fact that free EB molecules are readily quenched by surrounding water molecules [91]. The competitive binding studies were carried out by adding a certain volume of a stock of complex into EB-DNA system and the emission intensities were recorded. The fluorescence quenching spectra of DNAbound EB is shown in Fig. 5 and Fig. S4. With the increasing concentrations of tested compound, 602 nm emission band exhibited dramatic hypochromism effect. The quenching plot illustrates that quenching of EB system by complex is in agreement with the classical linear Stern– Volmer equation [92], I0 /I = 1 + Ksv[Q], where I0 and I represent the fluorescence intensities in the absence and presence of quencher and [Q] is the concentration of quencher. In the linear fit plot of I0 /I versus [Q], Ksv is the Stern–Volmer dynamic quenching constant. The apparent DNA binding constant (Kapp), which could express the degree of affinity toward DNA, was calculated on the basis of equation: K EB ½EB ¼ K app ½complex: Here KEB = 1.0 × 107 M−1, ([EB] = 2.4 μM). KEB is the binding constant of EB to DNA, and [complex] is the concentration value of complex at a 50% reduction of fluorescence intensity of EB. As expected, the competitive binding of three complexes to DNA could result in displacement of EB and could cause dramatic decrease of emission intensity. The DNA binding constant Kapp values of complex is ~ 106 M− 1 (9.08 × 106 M− 1for 1, 2.58 × 106 M−1 for 2, 2.85 × 106 M− 1 for 3), with the order 1 N 2 ≈ 3. Compared with several reported dinuclear Co(II) complexes listed in Table 3, Kapp values of our current complexes are higher, especially with [Co2L1(μ2-Cl)](ClO4)2 (Kapp = 1.90 × 104 M− 1) [50], which may verify the importance of planarity and hydrophobicity in DNA binding ability of metal complex [51,93]. 3.4.3. Viscosity studies Viscosity measurements have been carried out to examine the effect of complex on specific relative viscosity of DNA. The classical DNA intercalator like EB causes a significant increase in viscosity of DNA solutions due to the increase in separation of base pairs at intercalation sites and hence an increase in overall DNA contour length [94]. However, a partial or non-interaction of complex could result in a less pronounced effect on the viscosity [94]. The plots of relative specific viscosity (η/η0)1/3 versus [complex]/[DNA] ratio for 1, 2 and 3 are given in Fig. 6.

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J.-L. Li et al. / Journal of Inorganic Biochemistry 145 (2015) 19–29

Fig. 6. Effects of increasing amount of complex on relative viscosity of CT-DNA at 37 ± 0.1 °C. Conditions: [DNA] = 1 mM, [complex] = 0 − 0.1 mM.

With increasing amounts of complex, the relative viscosity of DNA increases efficiently. The result suggests complex could bind to DNA by intercalation, which rules out the electrostatic or groove binding interactions between complex and DNA. The increase in relative viscosity is expected to correlate with compound's DNA intercalating potential, and followed the order 1 N 2 ≈ 3 [9]. 3.5. DNA cleavage activity 3.5.1. Cleavage of pBR322 DNA by complex DNA-cleavage is controlled by relaxation of supercoiled circular (SC) form of plasmid DNA into nicked circular (NC) and/or linear forms. When pBR322 DNA is subjected to electrophoresis, there may be three phenomena in cleavage reaction: (i) the fastest migration will be observed for supercoiled form (Form I); (ii) If scission occurs on one strand (nicking), a slower-moving nicked form (Form II) will be generated; (iii) if both strands are cleaved, a linear form (Form III) will be generated that migrates in between. The cleavage activity of complex 1 to 3 have

Fig. 7. (a–c) Cleavage of plasmid pBR322 DNA (0.1 μg /μL) at different concentrations of complex after 3 h incubation at 37 °C; Line 0: DNA control; Line 1: DNA + 0.25 mM H2O2; (a) Line 2–5: DNA + 0.25 mM H2O2+ complex 1(0.5, 2, 3.5, 5 μM); (b) Line 2–5: DNA + 0.25 mM H2O2+ complex 2 (5, 10, 15, 20 μM); (c) Line 2–5: DNA + 0.25 mM H2O2 + complex 3 (5, 10, 15, 20 μM).

been investigated by gel electrophoresis using supercoiled pBR322 plasmid DNA as a substrate in a medium of 50 mM Tris–HCl/18 mM NaCl buffer (pH = 7.2). Concentration-dependent DNA cleavage of three complexes was first performed, keeping the concentration of DNA constant, and then different concentration of complexes was added. As Fig. S5 shows, in the absence of external redox agent, almost no cleavage was found even at the maximum concentration of complex. So the activator H2O2 was incorporated in the following experiment on the assumption that some redox agents may play some roles in the process. From Fig. 7, in the presence of H2O2, three complexes show efficient concentrationdependent DNA cleavage activity under physiological conditions. Complex 1 shows efficient cleavage ability even at 5 μM concentration, the conversion of DNA from Form I to Form II is almost completed (ca. 100%). Cleavage activity of 1 is similar with one tetranuclear copper complex [39], indicating that 1 is indeed an efficient chemical nuclease. However, for 2 and 3, when concentration is 5 μM, the conversion of DNA Form I to Form II is only about 50%. As increasing concentrations of 2 and 3, the amount of Form II increases and reaches the maximum (complete conversion) at nearly 20 μM. The result demonstrated that the cleavage of pBR322 DNA is highly dependent on the concentration of complex. 3.5.2. DNA cleavage mechanism In order to identify mechanism of oxidative cleavage of pBR322 by 1–3, experiments with different scavenging agents were carried out and shown in Fig. 8 and Fig. S6. The involvement of ROS was investigated using agents like KI, DMSO as hydroxyl radical scavengers; LHistidine, NaN3 as a singlet oxygen (1O2) quencher. The experiment was also carried out in the presence of major groove binder agent, methyl green; minor groove binder, SYBR green; and metal ion chelating agent, EDTA. When ˙OH scavenger KI is added, effective inhibition of DNA cleavage is observed, suggesting that cleavage reaction involves ˙OH radicals. The addition of L-Histidine does not prevent the cleavage, indicating singlet oxygen was ruled out. Moreover, methyl green and EDTA efficiently inhibit the cleavage, which implies that complex may bind to DNA in the major groove and the metal ion may participate in the process. In conclusion, cleavage process was effectively affected by KI, methyl green, and EDTA, which suggested that: (i) the hydroxyl radical may be the effective ROS agent; (ii) the complexes may bind to DNA in the major groove; (iii) metal ions play a very important role in the cleavage process.

Fig. 8. Bar diagram shows the effect of ROS scavengers on the cleavage of supercoiled DNA by complex 1–3 (1 green, 2 red, 3 blue) in the presence of H2O2.

J.-L. Li et al. / Journal of Inorganic Biochemistry 145 (2015) 19–29

27

Scheme 2. Proposed mechanism for DNA cleavage by CoII complexes in the presence of H2O2.

Recently, many cobalt complexes have been investigated due to their good properties in binding with DNA and catalyzing DNA cleavage. Some of them were investigated as photodynamic therapeutic (PDT) agents [29,95,96], some mediate DNA oxidation activity in the presence of oxidants or reductants [31,95]. These oxidative cleavage agents are highly efficient in inducing strand scissions of DNA. The ROS play an important role in the oxidative cleavage [1]. Here, Co(II) species is easily to be oxidized to Co(III) species in the presence of some oxidative agents, so we calculated that a possible oxidative cleavage mechanism of our complexes may be initiated via a Fenton-type reaction, which is shown in Scheme 2 [97]. In the presence of oxidative agent H2O2, Co(II) species were oxidized to Co(III) species. ˙OH radicals were generated in the process, resulting in DNA cleavage. Then Co(III) species obtained e− from H2O2 and were reduced to Co(II) species to fulfill the cycle. The mechanistic studies on DNA cleavage show that ˙OH radicals are involved in DNA cleavage reaction. 3.6. Protein binding activities BSA constitutes ~55% of the total protein in blood plasma and acts as an important role in drug transport and drug metabolism [98]. Fluorescence quenching measurements have been widely used to study the interaction of metal complexes or small molecules with proteins [99]. So interaction between our complexes with BSA has been investigated from concentration dependence of the change in fluorescence intensity of protein upon addition of complex. In order to explore interaction between our complexes and BSA, a solution of BSA (29.4 μM) was titrated with varying concentrations of complexes and fluorescence intensity was recorded in the range of 290–450 nm upon excitation at 280 nm. Fig. 9 shows the effect of complex 1 on fluorescence intensity of BSA at room temperature. Upon increasing concentration of 1, a gradual decrease in fluorescence intensity has been observed. Following the same

Fig. 10. A: The plot of I0 /I versus increasing concentrations of complex 1–3. B: Plot of log (I0 − I)/I vs. log [Q] for BSA in the presence of complex 1–3.

procedure, the fluorescence titration experiments of BSA with 2 and 3 have been executed (Fig. S7). The fluorescence intensity of BSA at 345 nm was decreased about 61.28% (complex 1), 39.65% (complex 2), 41.91% (complex 3) from the initial fluorescence intensity of BSA, which suggested a definite interaction of compounds with BSA [100]. To study quenching process further, fluorescence quenching data were analyzed with Stern–Volmer equation and Scatchard equation. The quenching constant Ksv could be calculated by Stern–Volmer equation: I0/I = 1 + Ksv[Q] = Kqτ0[Q], where I0 and I are the fluorescence intensities of the fluorophore in the absence and presence of quencher, respectively, Kq is the quenching rate constant, τ0 the average life-time of the biomolecule without quencher (about 10−8 s), and Ksv is observed by using the plot of I0/I versus [Q] (Fig. 10). Moreover, based on the Scachard equation: log½ðI 0 −IÞ=I  ¼ logK þ n log ½Q  Table 4 The quenching constant, binding constant and number of binding sites for the interactions of complex 1–3 with BSA. Complex 1 2 3

Fig. 9. Fluorescence emission spectra of the BSA (29.4 μM) system in the absence (dashed line) and presence (solid lines) of complex 1 (2.5–20 μM, respectively).

Ksv(M−1)

Kq (M−1.s−1) 4

5.56 × 10 1.63 × 104 2.04 × 104

12

7.64 × 10 1.63 × 1012 2.04 × 1012

K (M−1)

n 5

3.63 × 10 1.07 × 104 1.00 × 104

1.15 0.96 0.90

(Ksv: quenching constant; Kq: quenching rate constant; K: binding constant of compound with DNA; n: the number of binding sites; estimated errors for the constants are ±5%).

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J.-L. Li et al. / Journal of Inorganic Biochemistry 145 (2015) 19–29

where K is the binding constant of compound with DNA and n is the number of binding sites. The number of binding sites (n) and binding constant (K) have been obtained from the plot of log(I0 − I) / I versus log[Q]. The calculated Ksv, K, n, Kq values are given in Table 4. The value of n is around 1, which indicated just a single binding site in BSA for complex [14]. The binding constant values (K) for complex were calculated to be 3.63 × 105 M−1 (1), 1.07 × 104 M−1 (2), 1.00 × 104 M−1 (3), which suggests that complex 1 interacts more strongly with BSA protein compared with 2 and 3. Fluorescence quenching could occur through two different mechanisms, which are classified as dynamic quenching and static quenching; dynamic quenching refers to a process in which the fluorophore and quencher come into contact during the transient existence of excited state. In addition, static quenching refers to fluorophore-quencher complex formation in the ground state. Electronic spectroscopy is one of the sample methods to explore quenching type, and the result was shown in Fig. S8. After adding complex into BSA solution, absorption intensity of BSA was enhanced. It is well known that dynamic quenching did not change the absorption spectrum, because it only affected the excited state of fluorophore. As a result, the change in absorption spectrum may be induced by the static quenching process, accompanied the formation of a non-fluorescence ground state. 3.7. In vitro cell cytotoxicity The in vitro cytotoxicity against human cervical cancer cells (HeLa) of complex 1–3 have been investigated by CCK-8 assay. The results have been analyzed by means of cell inhibition expressed as IC50 values and shown in Table 5. Compared with cisplatin, complex 2 and 3 shows approximately cytotoxicity against HeLa cells after incubation for 48 h. however, it is noted that complex 1 shows potent cytotoxicity with even lower IC50 value (3.73 and 2.33 μM, for 24 h and 48 h, respectively) than cisplatin [101,102]. The incubation time dependence for 1 may play less influence than for 2 and 3, which shows markedly increased cytotoxic potencies compared with 24 h incubation and reveal time dependence. In conclusion, our complexes demonstrate the potential as new metal-based anticancer drugs. However, more cancer cell lines and investigations should be carried out.

these results our complexes appear to be regard as promising candidates for new DNA cleavage reagents even as anticancer drugs. Abbreviations

CT-DNA EB SC DNA NC DNA BSA Hna Hba ROS PBS

Acknowledgments This work was supported by the National Natural Science Foundation of China (21171101, 21371103 and 21471085), Tianjin Science Foundation (No. 12JCYBJC13600), NFFTBS (No. J1103306) and MOE Innovation Team (IRT13022) of China. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jinorgbio.2014.11.003. References [1] [2] [3] [4] [5] [6] [7]

4. Conclusion

[8]

With the ‘end-off’ type polyquinoline ligand (HL), three new cobalt complexes have been synthesized with one tetra-nuclear cobalt complex and two binuclear Co(II) complexes. To our knowledge, complex 1 is the first tetranuclear Co(II) complex, which not only contains two moieties, but also bears five- and six-coordinate sites preμ3-CrO2− 4 pared by using a symmetrical polydentate ligand. Three compounds bind to CT-DNA preferentially through intercalative interaction. Moreover, complexes are observed to display concentration dependent DNA cleavage and complex 1 shows the highest activity. With activator H2O2, 1–3 facilitates generation of ROS by the Co(II) center leading to better relaxation of SC DNA to its NC form. In addition, their abilities binding with protein have also been investigated, which is useful in the development of new therapeutic agents for some certain diseases. In addition, our complexes show cytotoxicity against HeLa cell, especially 1 demonstrates higher anticancer activity than cisplatin. Based on

Table 5 IC50values (μM) of complex 1\3 of Hela cell line with different incubation time. Complex(μM) / incubation time (h)

24

48

1 2 3 Cisplatin

3.73 ± 0.04 45.08 ± 3.22 42.68 ± 4.25 8 [102]

2.33 ± 0.09 18.3 ± 1.02 19.75 ± 2.10 15 [101]

IC50 value: drug concentrations necessary for 50 % inhibit ion of cell viability.

Calf thymus DNA Ethidium bromide Super coiled DNA Nicked coiled DNA Bovine serum albumin Nicotinic acid Benzoic acid Reactive oxygen species Phosphate buffered saline

[9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26]

Q. Jiang, N. Xiao, P. Shi, Y. Zhu, Z.J. Guo, Coord. Chem. Rev. 251 (2007) 1951–1972. J. Steinreiber, T.R. Ward, Coord. Chem. Rev. 252 (2008) 751–766. F. Mancin, P. Scrimin, P. Tecilla, Chem. Commun. 48 (2012) 5545–5559. C.Y. Gao, X. Qiao, Z.Y. Ma, Z.G. Wang, J. Lu, J.L. Tian, J.Y. Xu, S.P. Yan, Dalton Trans. 41 (2012) 12220–12232. A.V. Klein, T.W. Hambley, Chem. Rev. 109 (2009) 4911–4920. E.J. Gao, K.H. Wang, X.F. Gu, Y. Yu, Y.G. Sun, W.Z. Zhang, H.X. Yin, Q. Wu, M.C. Zhu, X.M. Yan, J. Inorg. Biochem. 101 (2007) 1404–1409. Y.Z. Liu, J. Vinje, C. Pacifico, G. Natile, E. Sletten, J. Am. Chem. Soc. 124 (2002) 12854–12862. R. Gust, W. Beck, G. Jaouen, H. Schönenberger, Coord. Chem. Rev. 253 (2009) 2742–2759. S. Anbu, S. Kamalraj, B. Varghese, J. Muthumary, M. Kandaswamy, Inorg. Chem. 51 (2012) 5580–5592. A. Djeković, B. Petrović, Ž.D. Bugarčić, R. Puchta, R. van Eldik, Dalton Trans. 41 (2012) 3633–3641. L. Xu, N.J. Zhong, Y.Y. Xie, H.L. Huang, G.B. Jiang, Y.J. Liu, C.L. Bai, PLoS One 8 (2014) e96082. G.B. Jiang, J.H. Yao, J. Wang, W. Li, B.J. Han, Y.Y. Xie, G.J. Lin, H.L. Huang, Y.J. Liu, New J. Chem. 38 (2014) 2554–2563. Y.Y. Xie, H.L. Huang, J.H. Yao, G.J. Lin, G.B. Jiang, Y.J. Liu, Eur. J. Med. Chem. 63 (2013) 603–610. D.S. Raja, N.S.P. Bhuvanesh, K. Natarajan, Inorg. Chem. 50 (2011) 12852–12866. M.C.B. Oliveira, D. Mazera, M. Scarpellini, P.C. Severino, A. Neves, H. Terenzi, Inorg. Chem. 48 (2009) 2711–2713. A. Prisecaru, M. Devereux, N. Barron, M. McCann, J. Colleran, A. Casey, V. McKee, A. Kellett, Chem. Commun. 48 (2012) 6906–6908. C. Santini, M. Pellei, V. Gandin, M. Porchia, F. Tisato, C. Marzano, Chem. Rev. 114 (2014) 815–862. A. Panja, T. Matsuo, S. Nagao, S. Hirota, Inorg. Chem. 50 (2011) 11437–11445. S. Anbu, R. Ravishankaran, A.A. Karande, M. Kandaswamy, Dalton Trans. 41 (2012) 12970–12983. J. He, J. Sun, Z.W. Mao, L.N. Ji, H. Sun, J. Inorg. Biochem. 103 (2009) 851–858. S. Anbu, M. Kandaswamy, B. Varghese, Dalton Trans. 39 (2010) 3823–3832. F. Bisceglie, R. Alinovi, S. Pinelli, M. Goldoni, A. Buschini, S. Franzoni, A. Mutti, P. Tarasconi, G. Pelosi, Metallomics 5 (2013) 1510–1518. P.K. Sasmal, A.K. Patra, M. Nethaji, A.R. Chakravarty, Inorg. Chem. 46 (2007) 11112–11121. P.K. Sasmal, S. Saha, R. Majumdar, R.R. Dighe, A.R. Chakravarty, Inorg. Chem. 49 (2010) 849–859. I.E. Leon, A.L. Di Virgilio, V. Porro, C.I. Muglia, L.G. Naso, P.A.M. Williams, M.B. Fogolin, S.B. Etcheverry, Dalton Trans. 42 (2013) 11868–11880. J. Hernández-Gil, S. Ferrer, E. Salvador, J. Calvo, E. Garcia-España, J.C. MarequeRivas, Chem. Commun. 49 (2013) 3655–3657.

J.-L. Li et al. / Journal of Inorganic Biochemistry 145 (2015) 19–29 [27] W. Maret, a.B.L. Vallee, Methods Enzymol. 226 (1993). [28] W.T. Lowther, A.M. Orville, D.T. Madden, D.H.R. Sejin Lim, B.W. Matthews, Biochemistry 38 (1999) 7678–7688. [29] M. Roy, B. Pathak, A.K. Patra, E.D. Jemmis, M. Nethaji, A.R. Chakravarty, Inorg. Chem. 46 (2007) 11122–11132. [30] S. Yellappa, J. Seetharamappa, L.M. Rogers, R. Chitta, R.P. Singhal, D'Souza, Bioconjug. Chem. 17 (2006) 1418–1425. [31] M.H. Hou, W.J. Lu, C.Y. Huang, R.J. Fan, J.M.P. Yuann, Biochemistry 48 (2009) 4691–4698. [32] G. Psomas, D.P. Kessissoglou, Dalton Trans. 42 (2013) 6252–6276. [33] K.J. Humphreys, K.D. Karlin, S.E. Rokita, J. Am. Chem. Soc. 124 (2002) 8055–8066. [34] L. Li, K.D. Karlin, S.E. Rokita, J. Am. Chem. Soc. 127 (2005) 520–521. [35] X. Sheng, X. Guo, X.M. Lu, G.Y. Lu, Y. Shao, F. Liu, Q. Xu, Bioconjug. Chem. 19 (2008) 490–498. [36] Y. Zhao, J. Zhu, W. He, Z. Yang, Y. Zhu, Y. Li, J. Zhang, Z.J. Guo, Chem. Eur. J. 12 (2006) 6621–6629. [37] M. González-Älvarez, J.B.G. Alzuet, B. Macías, A. Castiñeiras, Inorg. Chem. 42 (2003) 2992–2998. [38] C. Tu, Y. Shao, N. Gan, Q. Xu, Z.J. Guo, Inorg. Chem. 43 (2004) 4761–4766. [39] J. Hernández-Gil, S. Ferrer, A. Castiñeiras, F. Lloret, Inorg. Chem. 51 (2012) 9809–9819. [40] Y. Mikata, M. Wakamatsu, H. So, Y. Abe, M. Mikuriya, K. Fukui, S. Yano, Inorg. Chem. 44 (2005) 7268–7270. [41] Y. Mikata, A. Ugai, R. Ohnishi, H. Konno, Inorg. Chem. 52 (2013) 10223–10225. [42] M. Suzuki, H. Furutachi, H. Okawa, Coord. Chem. Rev. 200–202 (2000) 105–129. [43] E. Kinoshita, M. Takahashi, H. Takeda, M. Shiro, T. Korke, Dalton Trans. (2004) 1189–1193. [44] M. Yashiro, H. Kaneiwa, K. Onakab, M. Komiyamab, Dalton Trans. (2004) 605–610. [45] M. Yashiro, A. lshikubo, M. Komiyama, J. Chem. Soc. Chem. Commun. (1995) 1973–1974. [46] H. Linjalahti, G.Q. Feng, J.C. Mareque-Rivas, S. Mikkola, N.H. Williams, J. Am. Chem. Soc. 130 (2008) 4432–4433. [47] W.F. Zeng, C.P. Cheng, S.M. Wang, Inorg. Chem. 34 (1995) 728–736. [48] P. Chaudhuri, M. Winter, K. Wieghardt, S. Gehring, W. Haase, B. Nuber, J. Weisss, Inorg. Chem. 27 (1988) 1564–1569. [49] W.F. Zeng, C.P. Cheng, S.M. Wang, G.H. Lee, Inorg. Chem. 35 (1996) 2259–2267. [50] J.L. Tian, L. Feng, W. Gu, G.J. Xu, S.P. Yan, D.Z. Liao, Z.H. Jiang, P. Cheng, J. Inorg. Biochem. 101 (2007) 196–202. [51] C. Rajarajeswari, R. Loganathan, M. Palaniandavar, E. Suresh, A. Riyasdeen, M.A. Akbarsha, Dalton Trans. 42 (2013) 8347–8363. [52] G.P. Volynets, M.O. Chekanov, A.R. Synyugin, A.G. Golub, O.P. Kukharenko, V.G. Bdzhola, S.M. Yarmoluk, J. Med. Chem. 54 (2011) 2680–2686. [53] M. Özyanik, S. Demirci, H. Bektas, N. Demirbas, A. Demirbas, S.A. Karaõlu, Turk. J. Chem. 36 (2012) 233–246. [54] V.R. Solomon, H. Lee, Curr. Med. Chem. 18 (2011) 1488–1508. [55] G.M. Sheldrick, Acta Crystallogr. A 64 (2008) 112–122. [56] J. Marmur, J. Mol. Biol. 2 (1961) 208. [57] Y. Gultneh, A.R. Khan, D. Blaise, S. Chaudhry, B. Ahvazi, B.B. Marvey, R.J. Butcher, J. Inorg. Biochem. 75 (1999) 7–18. [58] W. Addison, T. Nageswara-Rao, J. Reedijk, J. Rijn, G.C. Verschoor, J. Chem. Soc. Dalton Trans. (1984) 1349–1356. [59] P. Gili, P.A. Lorenzo-Luis, Coord. Chem. Rev. 193–195 (1999) 747–768. [60] R.E. Sykora, S.M. McDaniel, D.M. Wells, T.E. Albrecht-Schmitt, Inorg. Chem. 41 (2002) 5126–5132. [61] C. Yuste, L. Cañadillas-Delgado, C. Ruiz-Pérez, F. Lloret, M. Julve, Dalton Trans. 39 (2010) 167–179. [62] L.H. Yin, W.H. Gu, P. Cheng, L.Z. Zhang, D.Z. Liao, J. Li, X.L. Yang, X.Q. Fu, S.P. Yan, Z.H. Jiang, Eur. J. Inorg. Chem. (2002) 1937–1940. [63] G.C. Wang, X.Y. Yi, I.D. Williams, W.H. Leung, Inorg. Chem. 50 (2011) 9141–9146. [64] J.A. Larrabee, C.H. Leung, R.L. Moore, T. Thamrong-nawasawat, B.S.H. Wessler, J. Am. Chem. Soc. 126 (2004) 12316–12324. [65] L. Vaiana, C. Platas-Iglesias, D. Esteban-Gómez, F. Avecilla, J.M. Clemente-Juan, J.A. Real, A. de Blas, T. Rodríguez-Blas, Dalton Trans. (2005) 2031–2037. [66] C. Incarvito, A.L. Rheingold, A.L. Gavrilova, C.J. Qin, B. Bosnich, Inorg. Chem. 40 (2001) 4101–4108.

29

[67] J.H. Satcher, M.W. Droege, T.J.R. Weakley, R.T. Taylor, Inorg. Chem. 34 (1995) 3317–3328. [68] A.L. Gavrilova, C.J. Qin, R.D. Sommer, A.L. Rheingold, B. Bosnich, J. Am. Chem. Soc. 124 (2002) 1714–1722. [69] C.E. Fenton, H. Okawa, Chem. Ber./Rec. 130 (1997) 433. [70] J. Olguin, M. Kalisz, R. Clerac, S. Brooker, Inorg. Chem. 51 (2012) 5058–5069. [71] J.L. Tian, W. Gu, S.P. Yan, D.Z. Liao, Z.H. Jiang, Z. Anorg. Allg. Chem. 634 (2008) 1775–1779. [72] M. Kumru, V. Küçük, M. Kocademir, H.M. Alfanda, A. Altun, L. Sarı, Spectrochim. Acta A 134 (2015) 81–89. [73] D. Das, Z. Dai, A. Holmes, J.W. Canary, Chirality 20 (2008) 585–591. [74] S.G. Telfer, T. Sato, R. Kuroda, J. Lefebvre, D.B. Leznoff, Inorg. Chem. 43 (2004) 421–429. [75] O. Fabelo, L.C. Delgado, J. Pasan, F.S. Delgado, F. Lloret, J. Cano, M. Julve, C. RuizPerez, Inorg. Chem. 48 (2009) 11342–11351. [76] D. Black, A.J. Blake, K.P. Dancey, A. Harrison, M. McPartlin, S. Parsons, P.A. Tasker, G. Whittaker, M. Schröder, Dalton Trans. (1998) 3953–3960. [77] S. Driieke, K. Wieghardt, J.B. Nuber, J. Weiss, H.P. Fleischhauer, S. Gehring, W. Haase, J. Am. Chem. Soc. 111 (1989) 8622–8631. [78] P.W. Ball, A.B. Blake, J. Chem. Soc. Dalton Trans. (1974) 852–859. [79] H. Wang, D. Zhang, D. Sun, Y. Chen, L.F. Zhang, L. Tian, J. Jiang, Z.H. Ni, Cryst. Growth Des. 9 (2009) 5273–5282. [80] Z. Tomkowicz, S. Ostrovsky, S. Foro, V. Calvo-Perez, W. Haase, Inorg. Chem. 51 (2012) 6046–6055. [81] L.J. Daumann, P. Comba, J.A. Larrabee, G. Schenk, R. Stranger, G. Cavigliasso, L.R. Gahan, Inorg. Chem. 52 (2013) 2029–2043. [82] F.B. Johansson, A.D. Bond, U.G. Nielsen, B. Moubaraki, K.S. Murray, K.J. Berry, J.A. Larrabee, C.J. McKenzie, Inorg. Chem. 47 (2008) 5079–5092. [83] F. Gao, H. Chao, F. Zhou, Y.X. Yuan, B. Peng, L.N. Ji, J. Inorg. Biochem. 100 (2006) 1487–1494. [84] V. Uma, M. Kanthimathi, T. Weyhermuller, B.U. Nair, J. Inorg. Biochem. 99 (2005) 2299–2307. [85] M.T. Carter, M. Rodriguez, A.J. Bard, J. Am. Chem. Soc. 111 (1989) 8901–8911. [86] S. Saha, R. Majumdar, M. Roy, R.R. Dighe, A.R. Chakravart, Inorg. Chem. 48 (2009) 2652–2663. [87] J. Qian, X.F. Ma, J.L. Tian, W. Gu, J. Shang, X. Liu, S.P. Yan, J. Inorg. Biochem. 104 (2010) 993–999. [88] A. Kamath, K. Naik, S.P. Netalkar, D.G. Kokare, V.K. Revankar, Med. Chem. Res. 22 (2013) 1948–1956. [89] D. Lahiri, T. Bhowmick, B. Banik, R. Railkar, S. Ramakumar, R.R. Dighe, A.R. Chakravarty, Polyhedron 29 (2010) 2417–2425. [90] F.J. Meyer-Almes, D. Porschke, Biochemistry 32 (1993) 4246–4253. [91] R.F. Pasternack, M. Caccam, B. Keogh, T.A. Stephenson, A.P. Williams, E.J. Gibbst, J. Am. Chem. Soc. 113 (1991) 6835–6840. [92] J.R. Lakowicz, G. Weber, Biochemistry 12 (1973) 4161–4170. [93] R. Loganathan, S. Ramakrishnan, E. Suresh, A. Riyasdeen, M.A. Akbarsha, M. Palaniandavar, Inorg. Chem. 51 (2012) 5512–5532. [94] J.M. Veal, R.L. Rill, Biochemistry 30 (1991) 1132–1140. [95] S. Roy, S. Roy, S. Saha, R. Majumdar, R.R. Dighe, E.D. Jemmis, A.R. Chakravarty, Dalton Trans. 40 (2011) 1233–1242. [96] S. Roy, A.K. Patra, S. Dhar, A.R. Chakravarty, Inorg. Chem. 47 (2008) 5625–5633. [97] W.J. Pogozelski, T.J. McNeese, T.D. Tullius, J. Am. Chem. Soc. 117 (1995) 6428–6433. [98] A. Patra, T.K. Sen, A. Ghorai, G.T. Musie, S.K. Mandal, U. Ghosh, M. Bera, Inorg. Chem. 52 (2013) 2880–2890. [99] S.M.T. Shaikh, J. Seetharamappa, S. Ashoka, P.B. Kandagal, Dyes Pigments 73 (2007) 211–216. [100] D.S. Raja, G. Paramaguru, N.S.P. Bhuvanesh, J.H. Reibenspies, R. Renganathan, K. Natarajan, Dalton Trans. 40 (2011) 4548–4559. [101] R.W.Y. Sun, A.L.F. Chow, X.H. Li, J.J. Yan, S.S.Y. Chui, C.M. Che, Chem. Sci. 2 (2011) 728–736. [102] S. Ray, R. Mohan, J.K. Singh, M.K. Samantaray, M.M. Shaikh, D. Panda, P. Ghos, J. Am. Chem. Soc. 129 (2007) 15042–15053.