N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267 257one-pot-reaction. Complex 1 possesses 5-hydroxy-[5,5′]-bipyrimidinyl- 2.4. Synthesis of complex 12,4,6,2′,4′,6′-hexaone (5-hydroxy-hydurilic acid) whereas the othercomplex, 2 contains 4-hydroxy-2,5-dioxo-imidazolidine-4-carboxylic A methanolic solution (10 mL) of Cu(bpy)(NO3)2·2H2O(0.361 g,acid (alloxanic acid) as co-ligand. Since none of the complexes bear the 1 mmol) was added drop wise to a solution of LH4 (0.374 g, 1 mmol) inframework of LH4, the original ligand must have been transformed 5 mLdimethylformamide(DMF).Thereactionmixturewasthenreﬂuxedduring the reaction. In order to explore the effect of 2,2′-bipyridyl groups for 2 h. The resulting solution was kept at room temperature for slowon the transformation of LH4, a reaction between LH4 and copper nitrate evaporation. After 5–6 days, the dark green coloured crystals weresalt was also carried out. This reaction provided complex 3 of type [Cu obtained. These crystals were washed with methanol and dried in air.(LH3)2(H2O)2], which retained the original ligand framework. Yield: 54%, M.P. 220 °C, elemental analysis calculated for C18H11CuN6O10 In view of the aforementioned reports and owing to the biological (%): C, 40.44; H, 2.05; and N, 15.73. Found (%): C, 41.24; H, 2.17; andsigniﬁcance of Cu(II) ions , the nuclease property of the novel N, 16.21. UV–vis absorptions: λmax (DMSO, 10−4 M), nm (ε/104 M−1complexes was studied. In addition, the present article embodies the cm−1) 266 (4.0), 312 (2.922) and 620 (0.023). IR (KBr): νmax, cm−1spectroscopic and single crystal characterization of the newly synthesized 3436 (OH, H2O), 3201 (NH), 3086 (CH, Ph), 1695 (CO), and 1603 (2,2′-bpy).complexes. The DNA binding and cleavage properties of copper(II)complexes 1 and 3 have been studied (complex 2 is insoluble in common 2.5. Synthesis of complex 2organic solvents and was not investigated further). The cytotoxic effects ofcomplexes 1 and 3 against Daltons lymphoma cell lines are also reported. After isolation of complex 1, the ﬁltrate thus obtained provided a bluish brown solid product after two weeks. It was then redissolved in2. Experimental section MeOH and left for slow evaporation at room temperature. After 24 h, block shaped blue colour crystals were formed which were found2.1. Materials insoluble in all common organic solvents. The crystals were then washed with diethyl ether and dried in air. Yield: 25%, M.P. N250 °C, Barbituric acid, 2,2′-bipyridine and salicylaldehyde were purchased elemental analysis calculated for C14H10CuN4O6 (%): C, 42.74; H, 2.54;from Sigma Aldrich Chem. Co and copper(II) nitrate dihydrate was and N, 14.24. Found (%): C, 42.85; H, 3.08; and N, 14.84. IR (KBr): νmax,purchased from S.D. Fine Chemicals, India and used as received. Solvents cm−1 3303 (NH), 1731 and 1656 (CO), 3037 (CH, 2,2′-bpy), 2929were purchased from E. Merk and were freshly distilled prior to their (CH), 1266, 1024 (C–O–C), and 3378 (OH, water).use. The barbiturate ligand (LH4) was synthesized using slightmodiﬁcation of the reported procedure . Calf thymus (CT) DNA 2.6. Synthesis of complex 3and supercoiled (SC) plasmid DNA pBR322 (as a solution in Tris bufferand cesium chloride puriﬁed), with a length of 4361 base pairs were A solution of Cu(NO3)2·2H2O (0.241 g, 1 mmol) in MeOH (10 mL)purchased from Bangalore Genei, India. pUC19 plasmid DNA with a was added drop wise to a solution of LH4 (0.374 g, 1 mmol) in DMFlength of 2686 base pairs was purchased from Fermentas. Restriction (5 mL). The reaction mixture after stirring for 5–6 h at roomenzymes were purchased from New England Biolabs and DNA temperature was left for slow evaporation. Fluorescent block shapedoligonucleotide primers were purchased from Sigma Aldrich Chem. Co. green colour crystals were grown in solution after 4–5 days. The crystals were washed with MeOH followed by diethyl ether and then dried2.2. Physical measurements in air. Yield: 72%, M.P. N250 °C, elemental analysis calculated for C30H38CuN8O22 (%): C, 38.87; H, 4.10; and N, 12.09. Found (%): C, 39.20; IR (KBr disc, 400–4000 cm−1) spectra were recorded on a Varian FTIR H, 4.76; and N, 12.98. UV–vis absorptions: λmax (DMSO, 10−4 M), nm3100 spectrometer; elemental analysis was done on Carbo-Erba 1108 (ε/104 M−1 cm−1) 329 (4.059), 379 (0.088) and 408 (0.056). IR (KBr):elemental analyzer, UV-visible (UV-vis) spectra were recorded on a νmax, cm−1 3225 (NH), 1705 and 1658 (CO), 3020 (CH, Ph), 2937 (CH),Shimadzu UV-1601 spectrometer while TGA plots were taken on a DU- 1266, 1039 (C–O–C), and 3409 (OH, water).PONT9900thermalanalyzingsystem(heatingrate10 °C/min)upto400 °C.Cyclic voltammetric measurements were performed on a CHI 620c 2.7. X-ray structural studiesElectrochemical Analyzer using glassy carbon as working electrode, aplatinum wire auxiliary electrode, and Ag/Ag+ reference electrode in a Single crystal X-ray diffraction data for the complexes were collectedstandard three-electrode conﬁguration. Tetrabutylammonium perchlo- in the temperature range of 100(2) K to 293(2) K on an Enraf Noniusrate(TBAP)wasusedasthesupportingelectrolyte,andtheconcentrationof MACH 3 diffractometer using graphite monochromatized Mo KαsolutionsofthecomplexesinDMSOwasmaintainedas10−3 M.ESRspectra radiation (λ = 0.71073 ) from block shaped crystals in the ω–2θ scanwere recorded at 273 K and 77 K on a Varian E-line Century Series ESR mode for complexes 1, 2 and 3. Intensities of these reﬂections werespectrometer equipped with a dual cavity and operating at X-band of measured periodically to monitor crystal decay. The crystal structures100 kHz modulation frequency. Tetracyanoethylenewas used asthe ﬁeld were solved by direct methods and reﬁned by full matrix least squaresmarker (g = 2.00277). The CD measurements of DNA with and without (SHELX-97) . Due to high degree of hydration, thermal motion andcomplexeswerecarriedoutwithaJascoJ500spectropolarimetercalibrated disorder, hydrogen atoms of water of crystallization could not bewithammonium(+)-10-camphorsulfonate. located. Drawings were carried out using MERCURY  and special computations were carried out with PLATON . The crystal2.3. Equipments used for DNA cleavage studies reﬁnement data are collected in Table 1 while selected bond distances and bond angles are reported in Table 2. PCR ampliﬁcation was performed on an Eppendorf Mastercycler epgradient S. Polyacrylamide gel electrophoresis was carried out with 2.8. Interaction of complexes 1 and 3 with DNA20× 30 cm self-cast denaturing polyacrylamide gels (5–20% acrylamide,7 M urea, 1× TBE (89 mM Tris, 89 mM boric acid, and 2 mM EDTA, pH 2.8.1. Absorption titration8.3) on CBS Scientiﬁc DNA sequencing systems using PowerPac HV The binding of complexes 1 and 3 with DNA was measured in a Na-power supply from Biorad. Gels were dried on a Whatman 3MM ﬁlter phosphate buffer solution (pH 7.2). The absorption ratio at 260 nmpaper using a gel dryer model 583 from Biorad at 80 °C for 30 min. and 280 nm of calf thymus DNA (CT DNA) solutions was found asPhosphorimaging was performed with a Storm 820 Phosphorimager 1.9:1, demonstrating that DNA is sufﬁciently free of protein. Thefrom GE Healthcare. concentration of DNA was then determined by UV-visible absorbance
258 N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267Table 1Crystal data for 1, 2 and 3. Compound 1 2 3 Chemical formula C18H11N6O10Cu C14H10N4O6Cu C30H38N8O22Cu Formula weight 534.87 393.80 926.22 Temperature 150(2) K 273(2) K 150(2) K Wavelength 0.71073 A 0.71073 A 0.71073 A Crystal system Triclinic Monoclinic Triclinic Space group P-1 P 1 21/n 1 (n = 14) P-1 a(Å) 9.201(2) 13.5470(11) 7.7024(2) b(Å) 9.201(2) 7.2157(6) 8.9975(4) c(Å) 14.331(4) 14.8634(12) 13.9532(6) α(°) 72.31(2) 90.00 99.533(4) β(°) 72.31(2) 90.187(3) 97.280(3) γ(°) 73.159(2) 90.00 104.157(3) Volume(Å3) 1074.9(4) 1452.9(2) 910.42(6) Z 2 18 2 Absorption coefﬁcient 1.086 mm−1 6.497 mm−1 0.705 mm−1 F(000) 540 918 479 Theta range for data collection 2.98 to 25.00° 2.03 to 23.74° 2.91 to 25.00° Reﬂections collected/unique 10974/3727 [R(int) = 0.0859] 11283/1578 [R(int) = 0.0576] 8621/3192 [R(int) = 0.0207] Completeness to theta 98.4% 71.2% 99.7% Goodness-of-ﬁt on F2 1.021 0.92 1.021 Final R indices [I N 2σ(I)] R1 = 0.0638, wR2 = 0.1613 R1 = 0.0472, wR2 = 0.1213 R1 = 0.0262, wR2 = 0.0653 R indices (all data) R1 = 0.1058, wR2 = 0.1818 R1 = 0.0795, wR2 = 0.1409 R1 = 0.0329, wR2 = 0.0686 Largest diff. peak and hole 2.003 and −0.959 e. Å3 0.347 and −0.470 e. Å3 0.296 and −0.356 e. Å3using the molar absorptivity (6600 M−1 cm−1) at 260 nm . The [Complex], the extinction coefﬁcient for the free copper(II) complexabsorption titration of 1 and 3 (100 μM) in Na-phosphate buffer (pH initially, after sequential addition of DNA and extinction coefﬁcient for7.2) with 10% DMSO against CT DNA were performed by monitoring the copper(II) complex in the fully bound form, respectively . Kb isthe changes in absorption spectra. The titration experiments were the ratio of slope to the intercept.performed by maintaining the concentration of metal complexesconstant at 100 μM while the concentration of CT DNA was varied 2.8.2. Competitive binding studieswithin 25–225 μM. An equal quantity of CT DNA was also added to the Relative binding of the copper complexes to CT DNA was studied byreference solution to eliminate the absorption by DNA. From the ﬂuorescence spectroscopy using ethidium bromide (EB) bound to CTabsorption data, the intrinsic binding constant Kb was calculated from DNA in a Na phosphate buffer solution (pH 7.2). In a typical experiment,a plot of [DNA] / (εa − εf) vs. [DNA] using the equation: 20 μL of CT-DNA solution (A260 = 2.0) was added to 2.0 mL of EB buffer solution (pH 7.2) and the ﬂuorescence intensity was measured upon½DNA = ðεa −εf Þ = ½DNA = ðεb −εf Þ + ½Kb ðεb −εf Þ −1 excitation at 510 nm; maximum emission was observed at 600 nm. The complex concentration was increased by addition of aliquots from a 0.1 mM stock solution until the ﬂuorescence intensity did not decreasewhere [DNA] represents the concentration of DNA in base pairs. The any further. Stern–Volmer quenching constants were calculated usingapparent absorption coefﬁcients εa, εf and εb correspond to Aobsd/ the following equation , Iο = I = 1 + Ksv r;Table 2Selected bond lengths (Å) and angles (°). where Iο and I are the ﬂuorescence intensities in absence and Complex 1 presence of complexes, respectively, Ksv is a linear Stern–Volmer Cu1–O2 1.873(9) O2–Cu1–O1 97.27(18) quenching constant and r is the ratio of the total concentration of Cu1–O1 1.877(19) O2–Cu1–N1 89.43(19) complex to that of DNA. The value of Ksv is given by the ratio of slope Cu1–N1 1.985(24) O1–Cu1–N1 169.87(20) Cu1–N2 1.987(7) O2–Cu1–N2 168.93(22) to intercept in a plot of Iο/I vs. [Complex]/[DNA]. O1–C14 1.418(15) O1–Cu1–N2 92.3(2) O2–C18 1.288(17) N1–Cu1–N2 81.86(21) 2.8.3. DNA cleavage study The nuclease activity of the copper(II) complexes was studied Complex 2 using supercoiled pBR322 and pUC19 plasmid DNA. Electrophoresis in Cu1–O1 1.881(5) N2–Cu1–O4 95.66(23) native agarose gel was used to quantify the unwinding of plasmid Cu1–N2 1.971(6) O1–Cu1–N1 94.58(21) DNA induced by copper(II) complexes. The cleavage reactions on Cu1–O4 1.973(6) N2–Cu1–N1 81.58(23) Cu1–N1 1.998(6) O4–Cu1–N1 165.84(23) pBR322 were carried out for 24 h at 37 °C in a total volume of 25 μL Cu1–O6i 2.303(5) O1–Cu1–O6i 95.08(19) containing 0.5 μg pBR322 DNA and different concentrations of O6–Cu1ii 2.303(5) N2–Cu1–O6i 93.07(21) complexes (ranging from 10 to 500 μM) in 5 mM Tris–HCl buffer O1–Cu1–N2 171.70(21) O4–Cu1–O6i 87.72(19) (pH 7.2), 50 mM NaCl and 10% DMSO. The samples were analyzed by O1–Cu1–O4 86.27(21) N1–Cu1–O6i 106.25(22) electrophoresis for 3 h at 50 V on 1% agarose gel in 1× TAE buffer Complex 3 (40 mM Tris acetate and 1 mM EDTA) pH 8.3. The gel was stained with a 0.5 μg/ml ethidium bromide and visualized by UV light and then Cu1–O1 1.963(3) O1–Cu1–O1i 179.99(5) Cu1–O1i 1.963(3) O1–Cu1–O8i 90.55(6) photographed for analysis. The extent of cleavage was determined Cu1–O8i 1.976(2) O1i–Cu1–O8i 89.45(6) from the intensities of the bands using the AlphaImager 2200 Cu1–O8 1.976(2) O8i–Cu1–O8 179.99(6) software . However, cleavage study on pUC19 was carried in a Cu1–O7i 2.415(7) O1–Cu1–O7i 95.43(5) total reaction volume of 10 μL, containing 100 ng (1 μL) of pUC19 Cu1–O7 2.415(7) O8i–Cu1–O7i 89.11(6) DNA, and different concentrations of complexes 1 and 3 in 5 mM Tris–
N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267 259HCl buffer (pH 7.2) containing 25% DMSO for 24 h at 37 °C. The triphosphate) mix (2 mM), 1 μL Taq buffer (10 mM Tris–HCl, 50 mMsamples were analyzed by electrophoresis for 1.3 h at 75 V on 1% KCl, and 1.5 mM MgCl2) pH 8.3 and 0.5 μL Taq DNA polymerase (5 U/μL)agarose gel in 1× TAE buffer. The gel was stained with 1:20000 stain G, in a ﬁnal volume of 10 μL. The primer extension reactions were runvisualized by UV light, and photographed for analysis. under PCR conditions with temperature cycling (30 cycles of denatur- ation at 94 °C (30 s), annealing at 50 °C (30 s), and extension at 72 °C2.8.4. Determination of site of DNA cleavage (30 s), followed by a ﬁnal extension at 72 °C for 5 min). After the For this study pUC19 plasmid DNA was used. The linearization of completion of PCR reaction, 3 μL of stop solution was added to both thepUC19 with complexes was studied ﬁrst and then primer extension tubes and heated at 90 °C for 2 min. The tubes were then cooled in an icereactions were carried out to locate the probable site of cleavage. The bath for 2 min and the samples were loaded on a 10% denaturingdetails of expected extended product and primers and restriction polyacrylamide gel. The gel was run for 1.5 h at 35 W. The gel wasenzyme combinations used are given in Table 3. soaked on a ﬁlter paper and then dried in a gel dryer (80 °C for 30 min) and exposed overnight to a phosphor screen. The screen was scanned to184.108.40.206. Restriction digestion of pUC19 visualize the DNA bands. 220.127.116.11.1. Eco-RI restriction digestion. In a reaction tube, 100 ng(1 μL) of pUC19 DNA, 1 μL of Eco buffer (50 mM Tris–HCl pH 7.5, 2.8.5. In vitro cytotoxicity assay10 mM MgCl2, 100 mM NaCl, 0.02% Triton X-100 and 0.1 mg/mL BSA), The DL (Daltons lymphoma: a transplantable T cell lymphoma) cells0.5 μL of EcoRI restriction enzyme (10 U/μL) and 7.5 μL of deionized were collected from the mouse ascite. The viable DL cells, determined bywater were mixed together. Then the tube was incubated at 37 °C for trypan blue exclusion test, were seeded onto 96 well plates in 100 μL of1 h. This reaction mixture was used for a control lane for visualizing the RPMI-1640 culture medium supplemented with 10% fetal bovinelinear pUC19 DNA. serum, penicillin G(100 U/mL), and streptomycin(100 μg/mL). The cells 18.104.22.168.2. PvuII restriction digestion. 50 ng of pUC19 (1 μL), 1 μL of were then allowed to grow in a CO2 incubator with 5% CO2 at 37 °C. Afterbuffer G (10 mM Tris–HCl pH 7.5, 10 mM MgCl2, 50 mM NaCl, 0.1 mg/ 24 h incubation, different concentrations (10−15 to 10−8 M) of theml bovine serum albumine), 0.5 μL of PvuII restriction enzyme (10 copper (II) complexes, made by serial dilutions in the culture medium,units/μL) and 7.5 μL of deionized water were incubated for 1 h at were added and the plates were incubated for another 24 h. Cell viability37 °C. This PvuII digested DNA was used as template in control primer was determined by using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-extension reactions. diphenyltetrazolium bromide) assay, which is based on the ability of the viable cells to reduce a soluble yellow tetrazolium salt to blue22.214.171.124. pUC19 linearization with complex 1. 50 ng of pUC19 was formazan crystals . Brieﬂy, after 24 h of the treatment, the MTT dyeincubated with 100 μM of complex 1 at 37 °C for 24 h in 5 mM Tris– (10 μL/100 μL of medium), prepared in phosphate buffered saline (PBS),HCl buffer (pH 7.2), 25% DMSO. This linearized DNA was used as was added to all the wells. The plates were then incubated for 4 h attemplate in primer extension reactions. 37 °C, the medium was discarded and 100 μL of DMSO was added to each well. Optical density was measured at 570 nm. As described in our126.96.36.199. 5′-32P-Labeling of primers with T4 polynucleotide kinase (PNK). previous report  the percentage of viable cells was determined byIn a reaction tube, 25 pmol (0.25 μL) of the primer, 1 μL of PNK buffer taking the cell counts in the untreated sets as 100%. The semi logarithmic(70 mM Tris–HCl, 10 mM MgCl2 and 5 mM dithiothreitol) pH 7.6, dose–response plots, constructed using the Graphpad Prism5 software0.5 μL of [γ-32P] ATP (10 mCi/mL) of speciﬁc activity 3000 Ci/mmol, , were used to determine the IC50 values as the complex0.5 μL of PNK enzyme (10 U/μL) and 7.75 μL of deionized water were concentrations that inhibited DL cell growth by 50%.added together. After incubation for 1 h at 37 °C, stop solution (10 μL)containing 95% formamide, 1 mg/ml bromophenol blue and 1 mg/ml 3. Results and discussionxylene cyanol was added. The enzyme was deactivated by incubationat 90 °C for 2 min and the reaction mixture was loaded on 12% 3.1. Synthesis and characterizationdenaturing polyacrylamide gel. The gel was run at 35 W for 1 h. Thelabeled DNA was extracted by crush-and-soak using TEN buffer In our earlier study it has been reported that LH4 reacts with Zn(bpy)(10 mM Tris–HCl pH 8.0, 1 mM EDTA, and 300 mM NaCl) and then (NO3)2 2H2O, and provides a supramolecular structure consisting of twoprecipitated using three volumes of cold absolute ethanol. LH− anion and one [Zn(bpy)2·2H2O]2+ cation together with seven co- 3 crystallized water molecules . Enthused by this study, a reaction of188.8.131.52. Primer extension studies. In a typical primer extension LH4 was carried with another metal precursor 2,2′-bipyridyl-dinitrato-experiment, two reactions were performed in parallel using Taq DNA copper(II)-dihydrate in anticipation that the Cu(II) ion, due to itspolymerase and linearized pUC19 DNAs as templates. For the control distorted conﬁguration and its redox active nature, may interact withreaction, pUC19 was linearized with PvuII restriction enzyme, whereas the ligand LH4 in a different way compared to Zn(II) ion. The reactionthe reaction product from incubation of pUC19 with complex 1 was used between Cu(bpy)(NO3)2·2H2O and LH4 in DMF containing MeOHfor locating the cleavage site. Primer extensions were run with both resulted initially in a dark green solution, from which two productstemplates using 1 μL 32P-labeled primer, 2 μL dNTP (deoxynucleotide crystallized, a dark green complex 1 and a blue coloured complex 2.Table 3Primers of pUC19 and restriction enzymes used for primer extension studies. Primer 5′–3′ Sequence Comment Restriction enzyme used Length of extended to prepare template DNA product (bp) P1 GTAAAACGACGGCCAGT M13/pUC fwd 379–395 PvuII 249 P2 AACAGCTATGACCATC M13/pUC rev 476–461 PvuII 170 P3 GGAGACGGTCACAGC pUC19 fwd 50–64 PvuII 256 P4 TCGGAACAGGAGAGC pUC19 rev 1000–986 PvuII 372 P5 GGTACCTGTCCGCC pUC19 fwd 1016–1029 BSaI 750 P6 AAGCATCTTACGGATG pUC19 rev 2162–2147 BSaI 396 P7 CAATAACCCTGATAAATGC pUC19 rev 2531–2513 ScaI 354 P8 CACATTTCCCCGAAAAGT pUC19 fwd 2592–2610 PvuII 400
260 N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267 These complexes were characterized initially by their IR spectra.Complex 1 showed sharp peaks at 1695 and 1602 cm−1, whereascomplex 2 showed a distinct peak at 1731 cm−1 in addition to othermajor peaks at 1665 and 1611 cm−1. Thus, the IR spectra suggestedthat both complexes 1 and 2 contain ligands of different constitution.This was conﬁrmed by solving their X-ray crystal structure, whichdemonstrated that the original framework of LH4 was no longerpresent. The ligand LH4 was transformed into two different com-pounds which in situ coordinated with Cu(II)-2,2′-bipyridine to givetwo new complexes (Scheme 1). In addition, LH4 was allowed to react with copper nitrate in theabsence of the bipyridyl ligand. This resulted in formation of greencoloured complex 3, which was characterized by the presence of twomolecules of the original barbiturate derivative LH4 acting as Cu(II)ligands.3.2. Structural description of complexes Complex 1 consists of a tetra coordinated Cu(II) ion having aN2O2 coordination core (Fig. 1a), involving 2 nitrogen atoms from 2,2′-bipyridine and 2 oxygen atoms from 5-hydroxy-hydurilic acid. Itcrystallizes into a triclinic P-1 space group and Cu–N and Cu–O distancesare lying in the reported range (Table 2). It has a Kitaigorodskii PackingIndex (KPI) of 70.2% which shows compact packing with few solvent Fig. 1. (a) Molecular structure of 1 (30% probability ellipsoid), hydrogen atoms areaccessible voids . Several H-donor and acceptor functional groups omitted for clarity and (b) a perspective of water clusters in crystal lattice of 1.present on the skeleton of the ligand form ten hydrogen bonds (S-1).The formation of six non-conventional hydrogen bonds involves C–H asH-donor and oxygen as H-acceptor whereas four conventional H-bondsare formed using N–H as donors and O as acceptors. The co-crystallized Like complex 1, complex 3 also crystallizes in a triclinic P-1 spacewater molecules are arranged in a C3 chain water cluster in packing group. Its Cu(II) ion is surrounded by 4 oxygen atoms, two originatediagram forming a water hexamer (Fig. 1b). from two monodentate ligands as LH−, whereas two other oxygen 3 Complex 2 is monoclinic with P-1 space group. It consists of a penta- atoms are from two coordinated water molecules (Fig. 3a). Complex 3coordinated Cu(II) ion with N2O3 coordination core from 2,2′-bipyridyl exhibits square planar geometry and also contains four co-crystallized(2N) and alloxanic acid (3O) (Fig. 2a). The Cu–O and Cu–N bond water molecules (Fig. 3b) which stabilize the structure by formationdistances (Table 2) are found in range as reported for other penta- of hydrogen bonds (S-2).coordinated Cu(II) complexes . The assembly of monomeric unitleads to a helical 1D polymeric framework (Fig. 2b). The study of weak 3.3. UV–vis spectroscopy and electrochemical studiesinteractions using PLATON indicates the presence of seven hydrogenbonds in crystal packing of complex 2. Five non-conventional hydrogen The complexes were characterized by UV–vis spectroscopy andbonds involve C–H donor groups, and two conventional hydrogen bonds their electrochemical properties were determined. The paramagneticinvolve O–H as donors (Fig. 2c). copper(II) complexes 1 and 3 in solution (10−4 M in DMSO) exhibited O O HN NH O HN NH H O O N N O Cu(bpy)(NO3)2.2H2O O O O O Cu O N DMF, MeOH O O N Cu N O HN N HN N O O N O H O 1 2 Cu(NO3)2.2H2O O O NH DMF, MeOH HN NH H2O HN O O O Cu O O O H2O HN NH O O NH N H O O 3 Scheme 1. Synthetic strategy for complexes 1-3.
N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267 261Fig. 2. (a) Molecular structure of a single unit of 2 (30% probability ellipsoid), hydrogen atoms are omitted for clarity, (b) zigzag polymeric structure of 2 and (c) conventionalhydrogen bonds in crystal lattice of 2.a broad d–d band in the range of 590–690 nm with a molar extinction complex 2 in powder displayed well resolved four lines at liquid N2coefﬁcient of 145–235 M−1 cm−1. However, intense absorption bands temperature. The axial g and A tensor values with g∥ N g⊥ suggest thatare observed at 260–270 nm, which are attributed to π–π* transitions. dx2–y2 is a ground state while g0 values are calculated using theAbsorption bands observed in the region of 300–400 nm are assigned relationship g0 = (g∥ + 2g⊥) . The values of calculated ESRto n–π* transition overlapping with ligand to metal charge transfer parameters are shown in Table S-6. Although the ratio g∥/|A∥| is(LMCT) transition between the heterocyclic base and the metal ion. normally taken as an indication of the stereochemistry of the copperSince, the DNA binding and cleavage studies were carried out in (II) complexes, yet it is suggested that this ratio may be an empiricalaqueous medium, UV–vis spectra of complexes 1 and 3 were also indication of the tetrahedral distortion of a square planar geometryrecorded in DMSO/water (v/v, 1:10) mixture. It showed (S-3) that . The values of hyperﬁne splitting lower than 135 cm−1 arecomplexes retain their structures in DMSO as well as in DMSO/water observed for square planar structures and those higher than 150 cm−1mixture. for tetrahedrally distorted complexes. The data shown in Table S-6 are The complexes display a quasi-reversible cyclic voltammetric found in consistence with earlier reports as well as structure observedresponse in the range of 0.2 to 0.5 V (vs. silver reference electrode) in from their X-ray diffraction studies.DMSO (10−4 M). The redox peak is assigned to Cu(II)/Cu(III) couple inview of reported redox potential data (S-4). 3.5. Thermo-gravimetric studies3.4. Electron spin resonance Thermo gravimetric analysis (TGA) (S-7) of the complexes showed that the loss of crystallized water molecules starts at ~90 °C in each The ESR spectra of complexes 1 and 3 in DMSO at 66 K displayed complex. The weight loss continues up to 165 °C in complex 1 and thethe typical four-line pattern as expected from 63Cu nucleus (S-5). magnitude of the weight loss (%) corresponds to three waterThree parallel hyperﬁne lines were well resolved in both complexes molecules (observed 11.1, calculated 10.4). In complex 2 weightwhile the fourth line overlapped with g⊥ signal. The spectrum of loss of 4.2% corresponds to removal of one water molecule (calcd.
262 N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267 Fig. 3. (a) Molecular structure of 3 (30% probability ellipsoid), hydrogen atoms are omitted for clarity and (b) a perspective of water clusters in crystal lattice of 3.3.8%) and it continues up to 150 °C. However, in complex 3 six watermolecules (two coordinated and four co-crystallized) are lostbetween 90 and 130 °C (observed weight loss 12.3%, calculatedweight loss 11.6%). The TGA data thus showed that the watermolecules are bound weakly in the lattice of complex 3 as comparedto complexes 1 and 2 .3.6. DNA binding studies In general, intercalation of a complex into DNA results in ahypochromic red shift of its absorption band. This may occur due tostrong stacking interactions between the aromatic chromophore ofthe complex and the base pairs of the DNA. On increasing theconcentration of CT DNA, the hypochromicity increased in the ligand-centred (LC) band of complex 1. In contrast, a hyperchromic effectwas observed in LC band of complex 3 (Fig. 4). The copper(II)complexes can bind to the double-stranded DNA in different modeson the basis of their structure, charge and type of ligands. As DNAdouble helix possesses many hydrogen bonding ligands accessibleboth in the minor and major grooves, it is likely that the N–H group ofbarbiturate ligand might be forming hydrogen bonds with DNA.Hence, it may contribute to the hyperchromic shift in its absorptionspectrum. In order to compare the binding strength of the complexeswith CT DNA, the intrinsic binding constants Kb were obtained fromthe ratio of slope to the intercept from the plots of [DNA] / (εa − εf) vs.[DNA]. The calculated Kb values of 1.9 × 106 M−1 and 1.7 × 105 M−1 forcomplexes 1 and 3 respectively show that DNA binds complex 1stronger than complex 3.3.7. Competitive binding with ethidium bromide The ability of a complex to affect the ﬂuorescence intensity of EB-DNA adduct is a reliable tool for the measurement of its afﬁnitytowards DNA. Intense ﬂuorescent light is emitted from EB in presenceof DNA owing to its strong intercalation between adjacent DNA basepairs. A complex binds with DNA by the displacement of EB bound toDNA. Consequently, the intensity of emission is reduced as emissionfrom free EB is readily quenched by surrounding water molecule . Fig. 4. UV–vis absorption spectra of (a) [complex 1] = 25 μM in the absence and inThe emission quenching from DNA bound ethidium bromide is due to presence of increasing amounts of DNA = 0–225 μM and (b) [complex 3] = 25 μM in thedisplacement of ethidium bromide from the DNA helix. The emission absence and in presence of increasing amounts of DNA = 0–225 μM. Arrow shows thespectra of EB-DNA system in the presence and absence of copper absorbance changes upon increasing DNA concentrations.
N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267 263Fig. 5. Emission spectra of EB bound to DNA in the absence (—) and in the presence of, [complex 1] 0–4 μM, [EB] 10 μM, [DNA] 10 μM. Arrow shows changes in the emission intensityupon addition of increasing concentration of the complex.complexes 1 and 3 are shown in Figs. 5 and 6. The addition of shows two conservative CD bands in the UV region, a positive band atcomplexes to DNA pretreated with EB shows appreciable reductions 278 nm due to base stacking and a negative band at 246 nm due toin emission intensity. On the addition of 4 μM of complex 1 to 10 μM of poly nucleotide helicity . The changes in CD pattern of DNACT DNA pretreated with EB, ~ 80% displacement of ethidium bromide observed after interaction with these complexes is considered towas observed. This suggests that complex 1 is a good intercalator. assign the corresponding changes in structure of DNA . SimpleHowever, complex 3 brings about only ~ 40% displacement of groove binding and electrostatic interaction of small molecules showethidium bromide at the same concentrations of both CT DNA and less or no perturbation on the base-stacking and helicity bands, whilethe complex. The quenching plots of Iο/I vs. [Complex]/[DNA] (insets intercalation enhances the intensities of both bands and stabilizes thein Figs. 5 and 6) are in good agreement with the linear Stern–Volmer right-handed B conformation of CT DNA, as observed for classicalequation. Stern–Volmer quenching constants (Ksv) were calculated to intercalator methylene blue .be 3.8 and 1.2 for complex 1 and complex 3 respectively. CD spectral variations of calf thymus DNA (50.0 μM, in 0.1 mM Na- phosphate buffer (pH = 7.4), were recorded in the presence of3.8. CD spectral studies increasing amounts of complexes 1 and 3 until [complex]/[DNA] molar ratios approached approximately 0.4. By addition of complex 1, Circular dichroism measurements were conducted in order to a blue shift of the positive CD band of DNA was observed (Fig. 7a).determine the extent of changes which occur in DNA conformation These ﬁndings indicate that a subtle change of the DNA double helixupon binding of complexes 1 or 3. The B form conformation of DNA occurs owing to the interaction of the metal complex with DNA .Fig. 6. Emission spectra of EB bound to DNA in the absence (—) and in the presence of, [complex 3] 0–4 μM, [EB] 10 μM, [DNA] 10 μM. Arrow shows changes in the emission intensityupon addition of increasing concentration of the complex.
264 N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267 Fig. 8. (a) Gel electrophoresis diagram showing the cleavage of SC pBR322 DNA (0.5 μg) by complex 1 on 12 h of incubation in 50 mM Tris–HCL buffer (pH 7.2): lane 1, DNA control; lane 2, DNA + 10 μM; lane 3, DNA + 25 μM; lane 4, DNA + 50 μM; lane 5, DNA + 100 μM; and lane 6, DNA + 500 μM, (b) cleavage of supercoiled pBR322 DNA showing the decrease in SC DNA and the formation of NC DNA with increasing concentration of complex 1. activity in the physiological pH range. Though, future experiments will be needed to characterize the cleavage mechanism in detail. However, a preliminary experiment showed that neither Cu(bpy) (NO3)2·2H2O nor LH4 separately caused DNA cleavage (S-8). 3.10. Determination of site of DNA cleavage The gel electrophoretic separation of plasmid pUC19 DNA inducedFig. 7. Circular dichroism spectra of CT-DNA (50 μM) in the absence (—) and presence of by complexes 1 and 3 and EcoRI is shown in Figs. 10 and 11.complex 1 (a) and complex 3 (b) in 0.1 mM Na-phosphate buffer. Complexes 1 and 3 linearize pUC19 at concentrations of 25 μM and 10 μM respectively on incubation for 24 h in a medium of Tris–HCl/ NaCl pH 7.2 containing 25% DMSO. The intensity of linear formTherefore, it can be inferred that complex 1 tightly binds to DNA. increases with the increase in the concentration of complexes.However, binding of DNA with complex 3 induces a decrease in the Primer extension reactions were performed to assay the site ofintensity of both positive and negative bands with a red shift in the DNA cleavage by complex 1. In these experiments, 5′-32P-labeledposition of the band . primers annealed to template DNA are extended from their 3′-end with Taq DNA polymerase until the 5′-end of the template DNA is3.9. DNA cleavage study reached. This “end” in the template DNA can either be generated by a restriction enzyme (linearization of the plasmid DNA) or by Cu- The DNA-cleaving ability of the copper(II) complexes has been complex catalyzed cleavage of the DNA. A set of 8 primers instudied by the relaxation of supercoiled pBR322 DNA to the nicked combination with three restriction enzymes was used to probe the fullcircular DNA. When circular plasmid DNA is subjected to electropho- length of the 2686 base-pair long pUC19 plasmid (Fig. 12). Primerresis, relatively fast migration is observed for intact supercoiled form extension of 32P-labeled primer 8 from the DNA template that had(S form). However, if scission of DNA occurs at one strand (nicking), been treated with complex 1 yielded an extension product that wasthe supercoiled DNA will relax to generate a slower-moving open/ much shorter than the product generated from a control reaction withnicked circular (NC form). If both strands are cleaved, a linear form (L) PvuII-digested DNA (S-9). No stop was observed on DNA treated withwill be generated . The gel electrophoretic mobility assay (Figs. 8 complex 1 in other primer extension reactions using the primers ofand 9) showed that both copper(II) complexes convert supercoiled Table 3. A likely explanation for this observation is that complex 1(SC) plasmid pBR322 DNA into nicked circular (NC) DNA after cleaves pUC19 speciﬁcally within the ca 400 bp region between theincubation at 37 °C for 24 h in a medium of Tris–HCl/NaCl pH 7.2. primer 8 binding site and the ﬁrst PvuII cleavage site (position 306,Complex 1 converts more than 90% of SC form into NC form at a see Fig. 12).concentration of 100 μM, whereas, for a similar level of conversion,only 50 μM of complex 3 is required. Thus, both complexes show 3.11. Evaluation of cytotoxicity in vitronuclease activity without addition of any external oxidizing orreducing agent unlike most of the other Cu(II)-based nucleases. In presence of copper(II) complexes, IC50 values were determinedThese results suggest that the copper(II) complexes show nuclease against Daltons lymphoma (DL) cell lines. The MTT assay measures
N. Dixit et al. / Journal of Inorganic Biochemistry 105 (2011) 256–267 265 Fig. 11. Gel electrophoresis diagram showing the cleavage of pUC19 DNA (0.1 μg) by complex 3 on 24 h of incubation in 5 mM Tris–HCL buffer (pH 7.2): lane 1, marker; lane 2, DNA control; lane 3, EcoRI treated DNA; lane 4, DMSO control; lane 5, DNA + 10 μM; lane 6, DNA + 25 μM; lane 7, DNA + 50 μM.Fig. 9. Gel electrophoresis diagram showing the cleavage of SC pBR322 DNA (0.5 μg) by and the IC50 values are determined using the GraphPad Prism5complex 3 on 12 h of incubation in 50 mM Tris–HCL buffer (pH 7.2): lane 1, DNA software.control; lane 2, DNA + 10 μM; lane 3, DNA + 25 μM; lane 4, DNA + 50 μM; and lane 5, The results thus obtained suggested that after 24 h of incubation,DNA + 100 μM, (b) cleavage of supercoiled pBR322 DNA showing the decrease in SCDNA and the formation of NC DNA with increasing concentration of complex 3. the copper(II) complexes are cytotoxic against DL cells with an IC50 values ~9.0 nm and 0.6 nm for complexes 1 and 3 respectively. Copper (II) complexes decreased viability of DL cells in a concentration-mitochondrial dehydrogenase activity as an indication of cell viability. dependent manner (with increasing concentration from 10−15 M toIt has been carried out with the copper complexes using murine 10−8 M). A ~40% decrease in cell viability is observed in the presenceDaltons lymphoma cells which are T cell lymphoma of spontaneous of Cu(II) complexes as compared to control. The values of IC50origin in the thymus. Daltons lymphoma cells have often been indicate that complex 3 is a stronger cytotoxic agent than complex 1successfully used to identify the anticancer potential of newly when tested against DL cell (S-10). These values are found to besynthesized compounds both in vitro and in vivo . Hence, the signiﬁcantly higher than the IC50 value of cisplatin against DL celleffect of Cu(II) complexes on the viability of DL cell lines has been lines .measured after 24 h of treatment as a function of concentration. Theexperiments have been performed in triplicates for all the complexes 4. Conclusion Three new copper(II) complexes of different geometry were prepared and characterized. Complexes 1 and 2 bearing 2,2′-bipyridyl as terminal ligand were isolated in a one pot synthesis as a result of transformation of the original barbiturate ligand LH4 in the presence of Cu(bpy)(NO3)2·H2O. The complexes contain a signiﬁcant number of co-crystallized water molecules in their crystal lattice which stabilize the corresponding supramolecular structures through H-bonds. Complexes 1 and 3 bind with the calf thymus DNA strongly though the binding constant for complex 1 is little higher than that of complex 3. These complexes also transform supercoiled DNA to nicked and linear forms under physiological conditions and possess considerable chemical nuclease activity. In contrast to DNA binding results, DNA cleavage studies indicated that complex 3 is a better nuclease in comparison to complex 1. The better binding afﬁnity of complex 1 with DNA could be due to the presence of 2,2′-bipyridine ligand which reportedly intercalates well with DNA. However, the better nuclease property of complex 3 could be attributed to the presence of ligand LH4 bearing various H donor and acceptor functionalities in its structure. The ﬁndings also suggest that the DNA cleavage property of the described complexes is region-speciﬁc. Such molecules may offer new prospects for controlled manipulationFig. 10. Gel electrophoresis diagram showing the cleavage of pUC19 DNA (0.1 μg) bycomplex 1 on 24 h of incubation in 5 mM Tris–HCL buffer (pH 7.2): lane 1, marker; lane of the genome and therefore, can be of great interest in biotechnology2, DNA control; lane 3, EcoRI treated DNA; lane 4, DMSO control; lane 5, DNA + 10 μM; and therapeutics. Both complexes 1 and 3 are also active againstlane 6, DNA + 25 μM; lane 7, DNA + 50 μM. Daltons lymphoma cell lines at nano-molar concentrations.
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