Acknowledgments

1. DNA binding, protein interaction and nuclease activity of a new Cu(II)-N-substituted sulfonamide complex Tamara Liana Topală,a Alejandro Pascual-Álvarez,b Alfonso Castiñeiras,c Andreea Bodoki,a Luminiţa Oprean,a Radu Oprean,d Gloria Alzuet-Piñab a) Department of General and Inorganic Chemistry, Faculty of Pharmacy, UMF “Iuliu Haţieganu” Cluj-Napoca, Romania; b) Department of Inorganic Chemistry, Faculty of Pharmacy, Universitat de Valencia, Spain; c) Department of Inorganic Chemistry, University of Santiago de Compostela, Santiago de Compostela, Spain; d) Department of Analytical Chemistry and Instrumental Analysis, Faculty of Pharmacy, UMF “Iuliu Haţieganu” Cluj-Napoca, Romania topala.liana@umfcluj.ro Introduction [Cu(L)Cl]n crystal structure The ample potential applications of DNA cleavage agents in molecular biology, biotechnology, and medicine [1] sparked interest in designing new molecules with this property. Studying the binding of molecules to albumins is a very pertinent research area, taking into consideration their crucial roles in drug pharmacodynamics and pharmacokinetics [2]. Since the discovery of the first chemical nuclease [3], increasing attention was focused on metal complexes as nuclease mimics because of their advantages in electronic and structural diversities [4]. The objectives of the present work were to synthesize and characterize a new copper(II) complex with N-(pyridin-2-ylmethyl)quinoline-8-sulfonamide (HL) and explore its ability to bind to biomolecules (DNA and bovine serum albumin, BSA), as well as its nuclease activity. Interaction with biomacromolecules DNA interaction Interaction with the EtBr-DNA system Selected bonds and angles Cu(1)-N(18) 1.9817(13) Cu(1)-N(11) 2.0282(13) Cu(1)-N(27) 2.0494(13) Cu(1)-Cl(1) 2.2571(4) Cu(1)-O(11) 2.3431(11) Cu(1)-O(11)#1 2.8882(11) Cu(1)-Cu(1)#1 5.0738(3) N(18)-Cu(1)-N(11) 82.37(5) N(18)-Cu(1)-N(27) 88.81(5) N(11)-Cu(1)-N(27) 170.88(5) N(18)-Cu(1)-Cl(1) 161.82(4) N(11)-Cu(1)-Cl(1) 95.53(4) N(27)-Cu(1)-Cl(1) 93.58(4) N(18)-Cu(1)-O(11) 101.84(5) N(11)-Cu(1)-O(11) 93.61(5) N(27)-Cu(1)-O(11) 85.87(5) Cl(1)-Cu(1)-O(11) 96.31(3) τ= 0.151 T5 = 0.887  slightly distorted square pyramidal geometry DNA thermal denaturation [Cu(L)Cl]n :  21 % [Cu(L)Cl]n DNA cleavage Photocleavage [DNA] = 100 µM [Complex] = 50 µM 5 % DMF ΔTm = 7 °C Agarose gel electrophoresis of pUC18 plasmid treated with the complex; buffer: cacodylate 0.1 M, pH = 6; photoactivation: λ = 300-400 nm. [DNA] = 50 µM [EtBr] = 50 µM 5 % DMF Thermal denaturation of CT-DNA in the absence and presence of [Cu(L)Cl]n Emission spectrum of EtBr bound to DNA in the absence and presence of increasing concentrations of [Cu(L)Cl]n. The arrow shows the effect of the complex on the emission intensity of the DNA-EtBr aduct 20 25 30 40 50 20 25 30 40 50 µM The Cu(II) complex stabilizes the DNA structure, increasing its melting point. SC DNA CuCl2 [Cu(L)Cl]n λDNA/EcoRI + HindIII SC DNA (non-irradiated) The decrease in the emission intensity of the EtBr-DNA system indicates that the complex binds to DNA. The difference between the nuclease activity shown by the complex and that of the Cu(II) salt is observed at lower concentrations (20-25 µM). Nuclease activity in the presence of ascorbic acid Bovine serum albumin interaction Agarose gel electrophoresis of pUC18 plasmid treated with the complex; buffer: cacodylate 0.1 M, pH = 6; ascorbic acid: 1x.  40 % [Cu(L)Cl]n / [BSA] : KSV = 2.6 x 105 M-1 Kb = 1.4 x 105 M-1 n = 0.95 The decrease in the fluorescence emission intensity is due to changes in the environment of the tryptophan moieties in the flourophore caused by the binding of [Cu(L)Cl]n. 10 15 20 25 30 10 15 20 25 30 µM CuCl2 [Cu(L)Cl]n SC DNA + ascorbic acid λDNA/EcoRI + HindIII SC DNA There is a significant difference between the nuclease activity shown by the complex and that of the Cu(II) salt in the presence of ascorbic acid. [BSA] = 1.3 µM 5 % DMF Fluorescence quenching spectrum of BSA as a function of concentration of [Cu(L)Cl]n. The arrow shows the effect of the complex on the emission intensity of BSA. Acknowledgments T.L.T., A.B., L.O. and R.O. acknowledge the financial support offered by research Project POSDRU 107/1.5/S/78702. A.P.A and G.A.P. acknowledge financial support from the Spanish Comisión Interministerial de Ciencia y Tecnología (CTQ2007–63690/BQU). References [1] C.–L. Liu, M. Wang, T.–L. Zhang, H.–Z. Sun, Coord. Chem. Rev. 248 (2004) 147–168; [2] P. Sathyadevi, P. Krishnamoorthy, E. Jayanthi, R.R. Butorac, A.H. Cowley, N. Dharmaraj. Inorganica Chimica Acta 384 (2012) 83-96. [3] D.S. Sigman, D.R. Graham, V. Daurora, A.M. Stern, J. Biol. Chem. 254 (1979) 12269–12272; [4] M. Oivanen, S. Kuusela, H. Lonnberg, Chem. Rev. 98 (1998) 961–990. Open circular DNA Supercoiled DNA Conclusions The newly synthesized Cu(II) complex shows a relatively high affinity towards biomacromolecules, as shown by the fluorescence studies, and a significant nuclease activity in the presence of ascorbic acid as well as upon irradiation with ultra-violet light.