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Photinus Pyralis) Simone Marin Marques Doctoral Program in Chemistry Department of Chemistry and Biochemistry 2014

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Photinus Pyralis) Simone Marin Marques Doctoral Program in Chemistry Department of Chemistry and Biochemistry 2014 Development and application of bioanalytical methodologies based on firefly luciferase D (Photinus pyralis) Simone Marin Marques Doctoral Program in Chemistry Department of Chemistry and Biochemistry 2014 Supervisor Joaquim Carlos Gomes Esteves da Silva Associate Professor with Aggregation, Faculty of Sciences This work was supported by a Ph.D. scholarship with the reference SFRH/BD/65109/2009, co-funded by the European Social Fund (Fundo Social Europeu, FSE), through Programa Operacional Potencial Humano - Quadro de Referência Estratégico Nacional (POPH - QREN), and by national funds from the Ministry of Education and Science through the Portuguese Fundação para a Ciência e a Tecnologia, I.P. (FCT, I.P.). Dedicated to everyone who contributed to it Acknowledgements | VII Acknowledgements To my scientific supervisor, Professor Joaquim C.G. Esteves da Silva, without whom none of this work would exist. To the Evaluation Panel of the Chemistry scientific area of the Fundação para a Ciência e a Tecnologia, I.P. (FCT, I.P.) Ph.D. Scholarship Call 2009. They didn’t know me, I didn’t know them, yet they changed my life. To FCT, I.P., for granting the funding for my Ph.D. project. To my host institution, Chemistry Research Center of the University of Porto (Centro de Investigação em Química da Universidade do Porto, CIQ-UP), represented by its scientific coordinator, Professor Manuel A.V. Ribeiro da Silva, for receiving me as a student and collaborator, and for providing me the conditions to perform my work. To the Doctoral Program in Chemistry scientific commission, Professor Manuel A.V. Ribeiro da Silva, Professor Maria João Ramos and Professor Paula Gomes. To Professor Gomes my special gratitude for always endure my countless e-mails. To my schoolfellows in the Program. To the teaching staff from the Department of Chemistry and Biochemistry and the invited lecturers who gave their time and their knowledge to enrich the curricular part of the Doctoral Program. To the Scientific Council of Faculty of Sciences, for accepting me as a student and carefully processing all my bureaucracy over these four years with competence and attention. To Doctor Valentin N. Petushkov, for the amazing opportunity of taking part in the elucidation of Fridericia heliota’s bioluminescent system. VIII | Acknowledgements To Zélia Azevedo, for the able assistance in liquid chromatography-mass spectrometry (LC-MS) analysis. To Mariana Andrade, from Materials Center of the University of Porto (Centro de Materiais da Universidade do Porto, CEMUP), for all the help with the nuclear magnetic resonance (NMR) software. Always available, always so nice. To the Department of Chemistry and Biochemistry secretariat and the Post-Graduate students office staff, for all the patience and professionalism to solve all my issues. To the lab managers Aurora Leal, Maria José Vasconcelos, Maria Arminda Silva, Maria de Fátima Carvalho and Moisés Xavier, for doing a discrete yet important job. To my lab buddies Margarida Miranda, Joel Santos, Diana Gomes, Conceição Mendonça, Helena Gonçalves, Abel ‘MacGyver’ Duarte, Emanuel Ferreira, Ana Reis and Marcela Oliveira, as well as my adoptive lab buddies Natércia Teixeira, Sónia Salomé and Isabel Tavares – always keeping up the fun. To Sport Lisboa e Benfica, for the supreme joy and fun it gave me in the last few weeks of the 2012-2013 soccer season. To all my family. To little misses Carolina and Catarina big kisses and hugs. To the new generation of Ph.D. students at lab 2.30 of the Chemometric Research of Chemical, Environmental, Forensic and Biological Systems group, Eliana Simões, Luís Silva, Bruno Campos and Dilson Pereira – may the success be with you. R e s u m o | IX Resumo Os recentes progressos da química bioanalítica conduziram a diversas aplicações de uso generalizado. Neste âmbito destacam-se as técnicas bioluminescentes, com franco desenvolvimento nos últimos anos, nas quais as luciferases, denominação genérica de enzimas que, agindo sobre o seu substrato natural, luciferina, promovem uma reacção bioquímica na qual ocorre libertação de fotões de luz visível, têm um papel dominante. Este projecto teve como objectivo o desenvolvimento de métodos bioanalíticos visando determinar espécies de interesse biológico, farmacêutico e ambiental baseando-se na luciferase do pirilampo norte-americano Photinus pyralis (EC 1.13.12.7) e aplicando metodologias de desenho experimental estatístico. Os analitos escolhidos foram os pesticidas organofosforados, sulfato inorgânico, óxido nítrico (•NO) e ácidos gordos livres. Em paralelo procedeu-se à análise estrutural de compostos semelhantes à luciferina de Fridericia heliota, uma minhoca siberiana cujas características bioluminescentes foram recentemente descobertas e que pode constituir um novo sistema bioanalítico de interesse. O método bioluminescente acoplado para pesticidas organofosforados apresenta uma gama linear entre 2,5-15 M, com limite de detecção (LD) de 1,5 M e limite de quantificação (LQ) de 5,0 M, e foi testado em águas de poços domésticos. O método bioluminescente para sulfato inorgânico, por sua vez, apresenta uma gama linear entre 14-134 mg·mL-1, com LD de 10 mg·mL-1 e LQ de 34 mg·mL-1, e foi testado também em águas de poços domésticos. Relativamente ao •NO, o método desenvolvido apresenta uma gama linear entre 10-100 nM, LD de 4 nM e LQ de 15 nM, e foi testado em saliva humana e meio de cultura de microalgas. Por fim, o método bioluminescente para ácidos gordos livres apresenta uma gama linear entre 1-20 M, LD de 1,3 e LQ de 4,5 M, e foi testado em plasma sanguíneo. Devido à reduzida quantidade de luciferina de Fridericia heliota que se consegue extrair, não foi ainda possível estabelecer a sua estrutura química. No entanto, foram realizados estudos utilizando-se compostos semelhantes à luciferina, que serviram como modelos. Estes compostos foram informalmente denominados CompostoX (CompX) e AsLn (acompanhante similar à luciferina). AsLn parece estar intimamente relacionado com a verdadeira luciferina, como um sub-produto ou um intermediário na sua biossíntese, enquanto CompX é um fragmento de AsLn. X | P a l a v r a s - c h a v e Palavras-chave Química bioanalítica Métodos bioluminescentes Ensaio enzimático Bioluminescência Método de vias enzimáticas acopladas com detecção por bioluminescência Luminometria Luciferase de pirilampo Photinus pyralis Desenho experimental Pesticidas organofosforados 2− Sulfato inorgânico (SO4 ) Óxido nítrico (•NO) Ácidos gordos livres Limite de detecção Limite de quantificação Gama linear Curva de calibração Método das adições de padrão Enquitreídeo Fridericia heliota Luciferina de Fridericia heliota Compostos relacionados com a luciferina CompX AsLn(2) Determinação da estrutura química Cromatografia líquida de alto desempenho Espectrometria de massa Ressonância magnética nuclear A b s t r a c t | XI Abstract Recent advances in bioanalytical chemistry led to several general purpose applications. In this context the focus is on the bioluminescent techniques, with a fast development in recent years, in which luciferases, generic name for enzymes that, by acting on its natural substrate, luciferin, promote a biochemical reaction which release photons of visible light, have a dominant role. The purpose of this project was the development of bioanalytical methods to determine species of biological, pharmaceutical and environmental interest based on luciferase from the North-American firefly Photinus pyralis (EC 1.13.12.7) and applying statistical experimental design methodologies. The chosen analytes were organophosphorus pesticides, inorganic sulfate, nitric oxide (•NO) and free fatty acids. In parallel proceeded the structural analysis of luciferin-related compounds from Fridericia heliota, a Siberian earthworm whose bioluminescent features were recently discovered and which may constitute a new interesting bioanalytical system. The coupled bioluminescent method for organophosphorus pesticides has a linear range between 2.5-15 M, with a limit of detection (LOD) of 1.5 M and a limit of quantitation (LOQ) of 5.0 M, and has been tested in water from domestic wells. The bioluminescent method for inorganic sulfate, in turn, shows a linear range between 14 to 134 mg·mL-1, LOD 10 mg·mL-1 and LOQ 34 mg·mL-1 and was also tested in domestic wells’ water. Regarding •NO, the developed method has a linear range of 10-100 nM, LOD 4 nM and LOQ 15 nM and was tested in human saliva and microalgae culture medium. Finally, the bioluminescent method for free fatty acid has a linear range between 1-20 M, LOD 1.3 M and LOQ 4.5 M and was tested in blood plasma. Due to the reduced amount of Fridericia heliota luciferin obtained in extracts, it was not yet possible to establish its chemical structure. However, studies were performed using compounds similar to the luciferin, which served as models. These compounds were informally called CompoundX (CompX) and AsLn (accompanying similar to luciferin). AsLn appears to be closely related to the true luciferin, as either a by-product or an intermediate in its biosynthesis, whereas CompX is a fragment of AsLn. XII | K e y w o r d s Keywords Bioanalytical chemistry Bioluminescent methods Enzymatic assay Bioluminescence Coupled bioluminescent assay Luminometry Firefly luciferase Photinus pyralis Experimental design Organophosphorus pesticides 2− Inorganic sulfate (SO4 ) Nitric oxide (•NO) Free fatty acids Limit of detection Limit of quantitation Linear range Calibration curve
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