Síntese, caraterização espetroscópica de novos corantes esquarílicos e sua avaliação como sondas fluorescentes para deteção de proteínas
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2018-02-26
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A utilização da fluorescência como técnica analítica tem sido vastamente utilizada em vários campos científicos. Desde a sua descoberta, por Sir George Stokes em 1852, até hoje, a fluorescência tem sido usada como uma ferramenta qualitativa e quantitativa, estando envolvida em várias análises laboratoriais. Determinadas análises efetuadas com base na fluorescência necessitam da adição de um composto (fluoróforo) que interage com o analito emitindo fluorescência ou alterando as suas propriedades. Neste trabalho é apresentada a síntese de várias cianinas esquarílicas, assim como dos seus precursores, com o propósito da sua aplicação como sondas fluorescentes (fluoróforo) para deteção e quantificação da proteína BSA (bovine serum albumin). Para tal, foram sintetizadas várias cianinas esquarílicas simétricas e assimétricas contendo no anel central de quatro membros grupos dicianometileno, grupos amino e átomos de oxigénio ou átomos de enxofre. A grande parte das cianinas são constituídas por um heterociclo de indolenina e, por outro heterociclo de oxazole, benzotiazole, benzoselenazole e quinaldina possuindo grupos N-alquilo com número diferente de átomos de carbono. Esta dissertação é constituída por quatro capítulos, consistindo o primeiro numa revisão bibliográfica sobre o conceito de fluorescência e a utilização de cianinas esquarílicas como potenciais fluoróforos. No segundo capítulo encontram-se discutidos os resultados experimentais da síntese dos vários compostos e a sua caraterização espetroscópica. Esta caraterização estrutural baseia-se na medição do ponto de fusão (p.f.), espetroscopia no infravermelho (IV), ressonância magnética nuclear (RMN) de 1H e de 13C e espetrometria de massa de alta resolução (EMAR). Ainda neste segundo capítulo é discutido o estudo fotofísico das cianinas e seus precursores sintetizados, o qual se baseou nos espetros no ultravioletavisível (UV-Vis), Visível (Vis) nos solventes dimetilsulfóxido e clorofórmio que, permitiram estudar propriedades como a absortividade molar, o solvatocromismo, e, a determinação do rendimento quântico de fluorescência. No final deste capítulo, são apresentados e discutidos os resultados da interação entre algumas cianinas selecionadas e a proteína BSA. No terceiro capítulo, é apresentada uma descrição detalhada da síntese efetuada para a obtenção dos compostos e, a sua caracterização espetroscópica. São também descritas as técnicas utilizadas para a determinação do rendimento quântico de fluorescência e o protocolo utilizado para o estudo da interação corante-proteína. Por fim, o quarto capítulo apresenta as referências bibliográficas consultadas nesta dissertação.
The use of fluorescence as an analytical technique has been widely used in several scientific fields. Since its discovery by Sir George Stokes in 1852 to date, fluorescence has been used as a qualitative and quantitative method and has been used in many laboratory analysis. Some analysis performed on the basis of fluorescence require the addition of a compound (fluorophore) that interacts with the analyte by emitting fluorescence or by modifying its properties. In this work the synthesis of several cyanines, as well as their precursors, with the purpose of their application as fluorescent probes (fluorophore) for the detection and quantification of BSA (bovine serum albumin) protein is presented. For that, a number of symmetrical and unsymmetrical squarylium cyanines dyes containing in the fourmembered central ring, dicyanomethylene group, amino groups, and oxygen atoms or sulfur atoms were synthesized. Most of the cyanines possessing an indolenine heterocyclic and oxazole, benzothiazole, benzoselenazole and quinaldine heterocyclic moities, having N-alkyl groups with a different number of carbon atoms. This thesis consists in four chapters, being the first a bibliographical review on the concept of fluorescence and the use of squarylium cyanines dyes as potential fluorophores. In the second chapter we discuss the experimental results of the synthesis of the various compounds and their spectroscopic characterization. This structural characterization is based on the measurement of the melting point (m.p.), infrared spectroscopy (IR), 1H and 13C nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HRMS). Also in this second chapter we discuss the photophysical study of the dyes and their synthesized precursors, which was based on the UV-Vis, Visible (Vis) spectra in the solvents dimethylsulfoxide and chloroform that allowed us to study properties such as molar absorptivity, solvatochromism and the determination of quantum fluorescence yield. At the end of this chapter, the results of the interaction between selected dyes and the BSA protein are presented and discussed. In the third chapter, a detailed description of the synthesized compounds and their spectroscopic characterization is presented. Also described the techniques used to determine the fluorescence quantum yield and the protocol used for the study of the dyeprotein interaction. Finally, the fourth chapter presents the bibliographical references used in this dissertation.
The use of fluorescence as an analytical technique has been widely used in several scientific fields. Since its discovery by Sir George Stokes in 1852 to date, fluorescence has been used as a qualitative and quantitative method and has been used in many laboratory analysis. Some analysis performed on the basis of fluorescence require the addition of a compound (fluorophore) that interacts with the analyte by emitting fluorescence or by modifying its properties. In this work the synthesis of several cyanines, as well as their precursors, with the purpose of their application as fluorescent probes (fluorophore) for the detection and quantification of BSA (bovine serum albumin) protein is presented. For that, a number of symmetrical and unsymmetrical squarylium cyanines dyes containing in the fourmembered central ring, dicyanomethylene group, amino groups, and oxygen atoms or sulfur atoms were synthesized. Most of the cyanines possessing an indolenine heterocyclic and oxazole, benzothiazole, benzoselenazole and quinaldine heterocyclic moities, having N-alkyl groups with a different number of carbon atoms. This thesis consists in four chapters, being the first a bibliographical review on the concept of fluorescence and the use of squarylium cyanines dyes as potential fluorophores. In the second chapter we discuss the experimental results of the synthesis of the various compounds and their spectroscopic characterization. This structural characterization is based on the measurement of the melting point (m.p.), infrared spectroscopy (IR), 1H and 13C nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HRMS). Also in this second chapter we discuss the photophysical study of the dyes and their synthesized precursors, which was based on the UV-Vis, Visible (Vis) spectra in the solvents dimethylsulfoxide and chloroform that allowed us to study properties such as molar absorptivity, solvatochromism and the determination of quantum fluorescence yield. At the end of this chapter, the results of the interaction between selected dyes and the BSA protein are presented and discussed. In the third chapter, a detailed description of the synthesized compounds and their spectroscopic characterization is presented. Also described the techniques used to determine the fluorescence quantum yield and the protocol used for the study of the dyeprotein interaction. Finally, the fourth chapter presents the bibliographical references used in this dissertation.
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cianinas esquarílicas , fluorescência , rendimento quântico de fluorescência , interação cianina-BSA