UNIVERSIDADE DO ALGARVE INTERACTION OF VANADIUM COMPOUNDS WITH DNA Nataliya Butenko Dissertação para obtenção do Grau de Doutor em Química Trabalho efetuado sob a orientação de: Prof. Doutora Isabel Maria Palma Antunes Cavaco 2013 Interaction of Vanadium Compounds with DNA Declaração de autoria de trabalho Declaro ser a autora deste trabalho, que é original e inédito. Autores e trabalhos consultados estão devidamente citados no texto e constam da listagem de referências incluída: Copyright por Nataliya Butenko, estudante do Universidade do Algarve. A Universidade do Algarve tem o direito, perpétuo e sem limites geográficos, de arquivar e publicitar este trabalho através de exemplares impressos reproduzidos em papel ou de forma digital, ou por qualquer outro meio conhecido ou que venha a ser inventado, de o divulgar através de repositórios científicos e de admitir a sua cópia e distribuição com objetivos educacionais ou de investigação, não comerciais, desde que seja dado crédito ao autor e editor. "Whatever you are, or whatever has happened, just be glad. Be glad because you are here. You are here in a beautiful world; and all that is beautiful may be found in this world... Just be glad, and you always will be glad. You will always have better reason to be glad. You will have more and more things to make you glad. For great is the power of sunshine, especially human sunshine. It can change anything, transform anything, remake anything, and cause anything to be become as beautiful as itself. Just be glad and your fate will change; a new life will begin and a new future will dawn for you". Cristian D. Larson Acknowledgements I would like to express my profound gratitude to my advisor Prof. Dr. Isabel Cavaco for her excellent supervision, knowledge, all practical teachings, especially in agarose gel electrophoresis, immense help, continuous encouragement and support during my PhD work. I wish to acknowledge several people who contributed to this work directly and indirectly: - Professor João Costa Pessoa for giving me the opportunity to work in Centro de Química Estrutural, for welcoming and accepting me into his group, for keen interest and valuable suggestions; - Professor José Paulo Pinheiro for teachings in voltammetry, constant support and willingness to help; - Professor Vera Ribeiro for sharing her knowledge on plasmid DNA preparation and the opportunity to work in her laboratory; - Professor Susana Etcheverry and her PhD student Ignacio León for collaboration, for kind and positive attitude in work; - Doctor Isabel Tomaz for practical teachings and help in conducting Circular Dichroism and UV-Vis experiments as well as in synthesizing vanadium complexes, for her sincere interest and impressive dedication to work; - Doctor Isabel Correia for practical teachings in NMR, for her boundless support, encouragement and friendship along these years; - A big group in Centro de Química Estrutural, namely, Sérgio Marques, Sofia Gama, Gisela Gonçalves, Pedro Adão and many others for a very enjoyable work environment and the help to easily find my way around the laboratory; - Professor Igor Khmelinskii and Professor Jorge Martins for allowing to use spectrofluorometry equipment; - Miguel Manuel for practical teaching in Fluorescence; - José Paulo da Silva for mass spectrometry experiments and corresponding discussions; iv - Doctor Rosário Lopes, Doctor Hélio Martins and Engineer Catarina Pires for the constant help in organizing my working process and friendly work environment; - My lab mates, Ana Luísa Ribeiro, Ana Catarina, Jorge Correia, Ana Guerreiro, Ana Margarida Eufigénia, and Rute Félix, for creating an excellent atmosphere in the laboratory and constant help; - A great team from International mobility office (namely, Mercês Covas, Paula Simões, Marleni Azevedo, Celia Oliveira, Sofia Nunes) and Igor Khmelinskii for taking care of organizational and paper work, and amicable relationships; - My family, especially my mother, for being always there for me; - All my friends for unconditional acceptance and boundless support, for being the sourse of continuous joy and laughter. With deep respect and gratitude, in memory of Professor Lidiia Svirenko (1939-2011) who was my teacher and supervisor at the National Academy of Municipal Economy, Kharkiv (Ukraine). She was the first to introduce me to research and helped to carry out my first investigation. Special thanks to my teacher Yuri Vergeles. If it were not for him, I would probably not have learnt about the Erasmus Mundus program and the great opportunity to study in Portugal. I am also very grateful to my teacher Oleksandr Spirin who keeps my spirit high, encourages my personal development, and is always helpful in many aspects. This work was financially supported by the Erasmus Mundus External Cooperation Window Lot 6 and Fundação para a Ciência e Tecnologia (SFRH/BD/69444/2010). Thanks to their granting, my work was successfully completed. I would like to acknowledge Portuguese people for their hospitality and cordiality, willingness to help and create a delightful environment for foreigners. THANK YOU. v Abstract The DNA cleavage activity of several vanadium complexes (VC) was studied. The focus IV was on vanadium acetylacetonate, V O(acac)2, 1, and several β-diketonate IV IV oxidovanadium(IV/V) derivatives: V O(hd)2 (2, hd = 3,5-heptanedione), V O(Cl-acac) 2, (3, IV Cl-acac = 3-chloro-2,4-pentanedione), V O(Et-acac)2 (4, Et-acac = 3-ethyl- 2,4- IV pentanedione) and V O(Me-acac)2 (5, Me-acac = 3-methyl-2,4-pentanedione), V V V 2O4(acac)2 (6), V O2(acac)(phen) (7, phen = 1,10-phenanthroline) and VVO(OH)(OMe)(acac) (8). The nuclease activity of 28 additional vanadium, copper and nickel complexes was also analysed and compared. The experimental techniques involved digestion of plasmid DNA (pDNA) followed by agarose gel electrophoreses to evaluate nuclease efficiency. pDNA was prepared and purified to assure the absence of interference from EDTA (ethylenediaminetetraacetic acid) and Tris present in commercial pDNA. The stability of complexes in aqueous solutions was studied by UV/vis spectroscopy and electroanalytical techniques, cyclic (CV) and square wave voltammetry (SWV). The nature of DNA cleavage mechanisms was assayed using fluorescent probes and 1H and 51V NMR spectroscopy. IV V O(acac)2 was found to be efficient in cleaving pDNA. The extent of this pDNA cleavage is dependent on buffer media. In organic buffers (Tris, HEPES MOPS) no significant changes are observed, whereas in phosphate medium the nuclease activity is remarkable. The activity of the different complexes follows the order 3>1≥2>>4~5. The V(V) derivatives, 6-8, do not show any significant activity, except in the presence of an oxidant activating agent. CV results show that 1-3 have a quasi-reversible electrochemical behaviour, while of 4 and 5 have an irreversible one, similar to 6. The DNA cleavage by these complexes takes place through an oxidative mechanism. Complex 1 also cleaves DNA hydrolytically, however the reaction is too slow to compete with the radical mechanism. IV In conclusion, phosphate buffer potentiates the DNA cleavage by V O(acac)2 derivatives through a species which presents a quasi-reversible redox behaviour and facilitates the formation of ROS, probably a mixed V(IV)-V(V)-acac-phosphate complex. Keywords: Inorganic nucleases, DNA cleavage, Vanadyl acetylacetonate, Oxidovanadium (IV/V) complexes, Phosphate, ROS, Agarose Gel Electrophoresis. vi Resumo Este trabalho estuda a interação de complexos de vanádio com ADN, em particular a IV atividade nuclease do acetilacetonato de oxovánadio(IV), V O(acac)2, 1, e alguns dos seus derivados: VO(hd)2 (2, hd = 3,5-heptanodiona), VO(Cl-acac)2, (3, Cl-acac = 3-cloro-2,4- pentanodiona), VO(Et-acac)2 (4, Et-acac = 3-etil-2,4-pentanodiona) e VO(Me-acac)2 (5, Me- acac = 3-metil-2,4-pentanodiona), V2O4(acac)2 (6), VO2(acac)(phen) (7, phen = 1,10- fenantrolina) and VO(OH)(OMe)(acac) (8). O objetivo inicial foi identificar a(s) espécie(s) metálicas presentes em solução responsáveis pela degradação do ADN. A atividade nuclease de seis compostos de vanádio e três compostos de cobre contendo aminoácidos e 1,10-fenantrolina e/ou 2,2‟-bipiridina como co-ligandos, 10-36, foi também analisada. A eficiência da clivagem do ADN pelos complexos metálicos em soluções tamponizadas a pH 7 foi determinada por electroforese em gel de agarose. Comparou-se o efeito de diferentes tampões de pH sobre a atividade. A estabilidade dos complexos em solução aquosa foi estudada por espectroscopia de absorção molecular no UV-Vis e por técnicas electroanalíticas (voltametria cíclica e voltametria de onda quadrada). A natureza radicalar ou hidrolítica dos mecanismos de clivagem foi averiguada recorrendo a compostos modelo e usando técnicas de fluorescência molecular e de ressonância magnética nuclear. O VO(acac)2 mostrou ser extremamente eficiente na clivagem de ADN (pADN), em particular em soluções tamponizadas com fosfato. Não requer agentes ativadores, ar ou fotoirradiação para degradar o pADN. A clivagem de uma cadeia simples (“nicking”) observa-se na presença de concentrações de metal na ordem dos 1,2 µM (correspondendo a ri = 0,08, onde ri é a razão da concentração de metal para a concentração do ADN, em pares de bases). A extensão da clivagem aumenta à medida que a concentração de 1 aumenta, e para concentrações de metal da ordem dos 10 µM (ri = 0,7) observa-se a clivagem da dupla cadeia. Quando ri = 1,7 observa-se a degradação completa da forma superenrolada (Sc) do pADN. viii Todos os complexos estudados apresentam atividade nuclease, dependente da concentração. O complexo 2 é uma nuclease um pouco menos eficiente do que 1, atingindo a degradação completa da forma Sc para ri = 3,3. O complexo 3 é muito pouco solúvel em solução aquosa, mas ainda assim demonstra uma atividade nuclease razoável, sugerindo ser mais eficiente do que os anteriores.