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Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

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A presente tese descreve o estudo cinético e mecanístico da desativação do estado singlete- e triplete-excitado de flavinas por esteróis (colesterol e ergosterol), vitamina D, coenzima Q10, vitamina K, ácidos graxos e metabólitos da cafeína. Através da análise de Stern-Volmer da supressão de fluorescência da riboflavina pelo colesterol, ergosterol, vitamina D, vitamina K, e pela coenzima Q10 observa-se que o estado singlete excitado da riboflavina é desativado com constante de velocidade superior ao limite da difusão. No entanto, a presença de colesterol, ergosterol, vitamina D, vitamina K e coenzima Q10 não afeta o tempo de vida do estado singlete excitado da riboflavina como demonstrado por experimentos de contagem de fótons resolvido no tempo sugerindo a formação de um complexo precursor [riboflavina...substrato]. O complexo 1:1 formado entre a riboflavina e a vitamina D apresenta Ka = 4 x 104 ± 3 mol⋅L-1 a 25 0C com ΔH0 = -36 ± 7 kJ⋅mol-1 e ΔS0 = -5 ± 3 J⋅mol-1 ⋅K-1. O complexo 1:1 entre a riboflavina e a vitamina K (vit K) e a riboflavina com a coenzima Q10 (CoQ10) apresentam Ka = 1 x 103 ± 1 mol⋅L-1 com ΔH0 = -110 ± 22 kJ⋅mol-1 e ΔS0 = 51 ± 9 J ⋅mol-1⋅K-1 para a vit K e Ka = 4 x 102 ± 1 mol⋅L-1, ΔH0 = -120 ± 27 kJ ⋅mol-1 e ΔS0 = 41 ± 7 J ⋅mol-1⋅K-1 para a CoQ10 a 25 0C. Para a desativação do estado triplete da riboflavina, foram obtidas constantes bimoleculares de velocidade variando de 1,4 x 108 L ⋅mol-1⋅s-1 (vit D) a 1,4 x 109 L ⋅mol-1⋅s-1 (CoQ10). Observa-se supressão da emissão de fluorescência da riboflavina na presença de metabolitos da cafeína, no entanto, sem alterar o tempo de vida do estado singlete excitado da riboflavina sugerindo a formação de um complexo precursor [riboflavina...substrato]. O complexo formado entre a riboflavina e os metabólitos da cafeína apresentam Ka = 295 ± 1 mol⋅L-1 com ΔH0 = -45 ± 8 kJ⋅mol-1 e ΔS0 = 12 ± 1 J⋅mol-1⋅K-1 para o ácido 1,7-dimetil úrico, Ka = 289 ± 1 mol⋅L-1 com ΔH0 = -38 ± 5 kJ ⋅mol-1 e ΔS0 = 9 ± 2 J ⋅mol-1⋅K-1 para o ácido 1-metil úrico e Ka = 275 ± 1 mol⋅L-1 com ΔH0 = 16 ± 3 kJ ⋅mol-1 e ΔS0 = 6 ± 1J ⋅mol-1⋅K-1 para a 1,7-dimetilxantina a 25 0C. Para a desativação do estado triplete da riboflavina, foram obtidas constantes de velocidade de 3kq = 4,2 x 108 L.mol-1.s-1 para a 1,7-dimetilxantina, 3kq = 1,0 x 108 L.mol-1.s-1 para o ácido 1,7-dimetil úrico e 3kq = 1,4 x 108 L.mol-1.s-1 para o ácido 1-metil úrico. Os ésteres metílicos (oleato de metila, ácido linoléico conjugado (CLA), linoleato de metila, linolenato de metila, araquidonato de metila, eicosapentanoato de metila, e docosahexanoato de metila) não desativam o estado singlete-excitado da riboflavina. Entretanto, os ésteres metílicos se mostraram reativos frente ao estado triplete da riboflavina com constantes de velocidades 3kq variando de 8,4 x 105 a 3,3 x 107 L⋅smol-1 ⋅s-1 com uma dependência linear do número de hidrogênios bis-alílicos com excessão do CLA. / The present thesis describes the kinetic and mechanistic studies of flavin singlet- and triplet-excited states deactivation by sterols (ergosterol and cholesterol), vitamin D, coenzyme Q10, vitamin K, fatty acids, and caffeine metabolites. From the Stern-Volmer analysis of the riboflavin fluorescence quenching by cholesterol, ergosterol, vitamin D, vitamin K and coenzyme Q10, it is noted that the singlet-excited state of riboflavin is deactivated with a rate constant exceeding the diffusion limit. However, the presence of cholesterol, ergosterol, vitamin D, vitamin K, coenzyme Q10 did not affect the lifetime of singlet-excited riboflavin as probed by single photon counting experiments suggesting the formation of a ground-state precursor complex [riboflavin ...substrate]. The 1:1 complex formed between riboflavin and vitamin D showed Ka = 4 x 104 ± 3 mol⋅L-1 a 25 0C with ΔH0 = -36 ± 7 kJ⋅mol-1 and ΔS0 = -5 ± 3 J⋅mol-1 ⋅K-1. The 1:1 complex formed between riboflavin and vitamin K (vit K) and riboflavin with coenzyme Q10 (CoQ10) showed Ka = 1 x 103 ± 1 mol⋅L-1 with ΔH0 = -110 ± 22 kJ⋅mol-1 and ΔS0 = 51 ± 9 J ⋅mol-1⋅K-1 for vit K e Ka =4 x 102 ± 1 mol⋅L-1, ΔH0 = -120 ± 27 kJ ⋅mol-1 and ΔS0 = 41 ± 7 J ⋅mol-1⋅K-1 for CoQ10 a 25 0C. For the deactivation of triplet riboflavin, bimolecular rate constants were found to vary from 1,4 x 108 L ⋅mol-1⋅s-1 (vit D) a 1,4 x 109 L ⋅mol-1⋅s-1 (CoQ10). The caffeine metabolites quench fluorescence emission of riboflavin however, without affecting the lifetime of the singlet-excited state, suggesting the formation of a ground state precursor complex [riboflavin ... substrate]. The complex formed between riboflavin and caffeine metabolites showed Ka = 295 ± 1 mol⋅L-1 with ΔH0 = -45 ± 8 kJ⋅mol-1 and ΔS0 = 12 ± 1 J⋅mol-1⋅K-1for 1,7-dimetyl uric acid, Ka = 289 ± 1 mol⋅L-1 with ΔH0 = -38 ± 5 kJ ⋅mol-1 and ΔS0 = 9 ± 2 J ⋅mol-1⋅K-1 for 1-methyl uric acid and Ka = 275 ± 1 mol⋅L-1 with ΔH0 = 16 ± 3 kJ ⋅mol-1 and ΔS0 = 6 ± 1J ⋅mol-1⋅K-1 for 1,7-dimethylxanthine at 25 0C. For the deactivation of triplet riboflavin, rate constant were obtained with 3kq = 4,2 x 108 L.mol-1.s-1 para a 1,7-dimethylxanthine, 3kq = 1,0 x 108 L.mol-1.s-1 for 1,7-dimethyl uric acid, and 3kq = 1,4 x 108 L.mol-1.s-1 for 1-methyl uric acid. The methyl esters (methyl oleate, conjugated linoleic acid (CLA), methyl linoleate, methyl linoleate, methyl arachidonate, methyl eicosapentanoate, and methyl docosahexanoate) did not quench the singlet-excited riboflavin. However, the methyl esters were shown to be reactive towards triplet riboflavin with rate constants ranging from 8,4 x 105 a 3,3 x 107 L⋅mol-1 ⋅s-1and depending linearly with the number of bis-allylic hydrogens with exception to CLA.

This thesis focuses on investigations of transient dark states of fluorescentmolecules using spectroscopic techniques. The main purpose is to show andconvince the reader that transient dark states are not always a nuisance, butalso represent an additional source of information. Several studies with fluorescencecorrelation spectroscopy were performed, all related to non-fluorescentstates such as triplet state or isomerized states.Photobleaching is one of the main problems in virtually all of the fluorescencetechniques. In this thesis, mechanisms that retard photobleaching arecharacterized. Several compounds, antioxidants and triplet state quenchers,which decrease photobleaching, are studied, and guidelines for achieving optimalfluorescence brightness using these compounds are presented.Triplet state quenching by several compounds was studied. Detailed investigationsof the fluorescence quencher potassium iodide demonstratedthat for some of fluorophores, except of quenching, there is fluorescence enhancementmechanism present. In agreement with the first publication inthis thesis, antioxidative properties were found to play an important role inthe fluorescence enhancement. Quenching of the triplet state is proposedas a tool for monitoring diffusion mediated reactions over a wide range offrequencies.Specially designed fluorophores combining high triplet yields with reasonablefluorescence brightness and photostability were characterized forpossible applications in novel super-resolution imaging techniques based onfluorescence photoswitching. Except of benefits for imaging techniques, photoinducedswitching to non-fluorescent states could be used for monitoringmolecular diffusion, which was also demonstrated in this thesis.Studies of the triplet state kinetics of fluorophores close to dielectric interfaceswere performed using fluorescence spectroscopy. The analysis of thetriplet state kinetic can provide information about the local microenvironmentand electrostatic interactions near dielectric interfaces. / QC 20100414

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Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 81-86).

published_or_final_version / Chemistry / Master / Master of Philosophy