This master thesis is focused on theoretical study of the Cy3 and Cy5 dyes and their interactions with DNA. The main aim was to find the mot populated conformations of the Cy3-DNA and Cy5-DNA complexes. A comparison with the experimental structure was also done and the influence of the cyanine dyes on the conformational changes of the DNA chain was evaluated.

Near-infrared (NIR) fluorescence imaging has emerged as an attractive non-invasive approach for direct visualization of diseases which depends on the development of stable, highly specific and sensitive optical probes. The NIR region of the electronic spectrum offers a reduction in the background autofluorescence and an increase in the tissue penetration depth. Cyanine dyes have often been considered promising contrast optic agents owing to their photophysical properties. Herein the synthesis of various penta- and heptamethine benzothiazole cyanine dyes has been described and their in vivo imaging efficacy was determined. Varying functionalities on the benzothiazole aromatic ring and changing substituents on the benzothiazolium nitrogen atom reflected subsequent changes in the imaging pattern and have resulted in the development of promising brain targeting agents.

The use of fluorescent imaging probes for the real time detection of cellular malfunctions, such as enzyme over expression has shown promise. Fluorescent dyes with absorption and emission values below 600 nm are limited in their in vivo applications due to high background auto-fluorescence and low resolution images. Employing near infrared (NIR) fluorophores such as cyanine dyes can overcome this disadvantage. Cyanine dyes can be synthesised using solution or solid-phase techniques with the use of solution phase chemistry allowing for larger scale and higher yielding reactions. Utilising a selection of functional groups and varying polymethine chain lengths a cyanine dye library with tuneable absorption and emission wavelengths was synthesised. This thesis gives the first detailed examples of how modifications on heptamethine cyanine dyes alter their cellular uptake and cellular toxicity. Furthermore, a NIR fluorescent microsphere is reported as well as NIR functionalised microspheres with the ability to be tracked within cells. Additional lines of work involved the synthesis of a fluorescent sensor for the visualisation of bacteria. Aminopeptidases are present within the peptidoglycan cell wall of Gram negative bacteria and therefore can be targeted for real time detection of bacteria to aid in the detection of infectious diseases. A coumarin based probe is reported which detects aminopeptidase in gram negative bacteria in vitro.

Near-infrared (NIR) fluorescence imaging has emerged as an attractive non-invasive approach for direct visualization of diseases which depends on the development of stable, highly specific and sensitive optical probes. The NIR region of the electronic spectrum offers a reduction in the background autofluorescence and an increase in the tissue penetration depth. Cyanine dyes have often been considered promising contrast optic agents owing to their photophysical properties. Herein the synthesis of various penta- and heptamethine benzothiazole cyanine dyes has been described and their in vivo imaging efficacy was determined. Varying functionalities on the benzothiazole aromatic ring and changing substituents on the benzothiazolium nitrogen atom reflected subsequent changes in the imaging pattern and have resulted in the development of promising brain targeting agents.

This thesis reports the synthesis of new near infrared dyes in three chapters. The first two chapters outline the synthetic procedure for synthesizing mono- and pentamethine cyanine dyes. The initial chapter encompasses the synthesis of asymmetric monomethine dyes with red-shifted optical properties. The second chapter involves the synthesis and assessment of new symmetrical quinolin-4-yl and phenanthridin-6-yl pentamethine dyes as potential oxidative DNA cleavage agents. The last chapter of the thesis details the synthesis and evaluation of new phenoxizinum dyes as contrast agents for insulunomia, a pancreatic cancer. Furthermore, all new compounds were characterized via NMR and their coherent optical properties were obtained.

Understanding the properties of electronically interacting molecular chromophores, which involve internally coupled electronic-vibrational motions, is important to the spectroscopy of many biological systems. Here we apply linear absorption, circular dichroism, and two-dimensional fluorescence spectroscopy to study the local structure and excited state dynamics of excitonically coupled cyanine dimers that are rigidly positioned within the sugar-phosphate backbones of the DNA. Dimer probes were positioned within the double-stranded DNA duplex and at the single-strand/double-stranded DNA junction to examine the positional dependence of the structural variation and fluctuations. We interpret spectroscopic measurements in terms of the Holstein vibronic dimer model, from which we obtain information about the local conformation of the dimer probe locally within their respective DNA environments. We show that the exciton-coupling strength of the dimer-DNA construct can be systematically varied with temperature below the double-stranded – single-strand DNA denaturation transition. Using time-resolve 2DFS measurements we observed long lived vibronic coherences in the system. The properties of the cyanine DNA construct we determine suggest that it may be employed as a useful model system to test fundamental concepts of protein DNA interactions and the role of electronic-vibrational coherence in electronic energy migration within exciton-coupled biomolecular arrays. This dissertation contains previously published and unpublished co-authored material.

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A terapia fotodinâmica (PDT) consiste em introduzir no organismo um composto fotoativo (fotossensibilizador) e irradiar o local a ser tratado com a luz visível. Este procedimento permite obter a(s) forma(s) ativa(s) do composto que tem a capacidade de matar células neoplásicas, bem como fungos e bactérias. Algumas desvantagens dos fotossensibilizadores atualmente aplicados na clínica estimulam a busca de novos compostos mais adequados. Os corantes ciânicos com dois cromóforos (BCD) possuem características espectrais promissoras para aplicação em PDT. Isso estimulou a realização deste trabalho que teve como finalidade estudar as características espectrais, energéticas e de fotodecomposição dos BCD: (i) em diferentes solventes (escolhidos pela constante dielétrica $\\xi$ e capacidade de formar ligação hidrogênio), (ii) na interação com micelas, e (iii) na interação com DNA. Foram estudados $4$ isômeros de corantes ciânicos com dois cromóforos (BCD) com estrutras iguais. Estes isômeros diferem pela estrutura da parte central da molécula. Foram utilizados métodos espectroscópicos estáticos e com resolução temporal tais como absorção ótica, flash-fotólise, fluorescência, anisotropia, dicroísmo circular e calorimetria fotoacústica. Os resultados experimentais foram analisados em comparação com resultados obtidos do cálculo computacional em que foi feita a otimização geométrica das moléculas, análise de orbitais eletrônicos, dos níveis de energia e das probabilidades de transição entre estes. A partir destes dados foram construídos os espectros de absorção S-S dos corantes. Os cálculos confirmaram que a probabilidade de transição entre os níveis de energia dos BCD depende do ângulo entre os cromóforos que é determinado pela estrutura da parte central da molécula. Para um ângulo de $180^{0}$ somente uma banda de absorção está presente no espectro e para o de $90^{0}$ aparecem duas bandas de intensidades iguais. Foi mostrado que no intervalo de temperaturas $<100^{0}$ as conformações dos cromóforos de BCD no estado fundamental são trans-trans, exceto para o BCD com ângulo de $120^{0}$ entre os cromóforos em que no intervalo $0^{0}$ a $100^{0}$ ocorre a mistura das conformações trans-trans e cis-trans. Os resultados experimentais mostraram que em solventes orgânicos os BCD não formam agregados. Como suas características espectroscópicas não seguem a equação de Lippert concluímos que sua solvatação está determinada tanto pelo mecanismo não específico como específico devido as ligações hidrogênio. Os tempos de vida ($\\tau_{S1},\\tau{}_{T1}$) e rendimentos quânticos ($\\phi_{S1},\\phi{}_{T1}$) dos estados excitados singleto e tripleto dos BCD diminuíram com o aumento da constante dielétrica $\\xi$ do solvente. Entretanto o aumento da viscosidade do solvente aumentou estes parâmetros. A presença de água nos solventes orgânicos promove a formação dos agregados de BCD. Os valores de $\\tau_{S1},\\tau\\,{}_{T1}$ e $\\phi_{S1},\\phi\\,{}_{T1}$ diminuíram, provavelmente, devido a dois efeitos: diminuição da microviscosidade ao redor da molécula de BCD e agregação do BCD estimulada pela presença da água. A interação dos BCD com micelas depende da carga dos tensoativos. Para uma carga negativa houve a presença de $3$ espécies do corante dependendo da concentração das micelas: os monômeros livres, os monômeros ligados à micela e os agregados ligados a micela. O tensoativo zwiteriônico gerou duas espécies: os monômeros livres e os monômeros ligados à micela. As constantes de ligação dos BCD com micelas de LPC são da ordem $10^{7}M^{-1}$. Não houve interação com os tensoativos positivos. A interação com DNA estimula a agregação de BCD. Na presença de DNA em solução existem três espécies de BCD: os monômeros livres e ligados com DNA e os agregados de BCD na superfície do DNA. O conteúdo relativo dessas espécies depende da razão {[}BCD]/{[}DNA]. As constantes de ligação dos monômeros de todos BCD com DNA foram da ordem de E7M^{-1}$. Os valores de tau_{S1},\\tau{}_{T1}$ e $\\phi_{S1},\\phi{}_{T1}$ aumentaram com a ligação dos BCD ao DNA, o que significa que a contribuição dos processos não radiativos diminuiu em comparação a água e aos solventes orgânicos. Os BCD sofrem fotodecomposição e esta depende da presença de oxigênio. Na fotodecomposição do BCD ocorre destruição do sistema de conjugação-pi num dos cromóforos pela ligação do oxigênio singlete, formado devido a transferência de energia do estado tripleto do próprio BCD para oxigênio molecular. Os resultados obtidos (alta afinidade com sistemas biológicos e modelos, altos rendimentos quânticos do estado tripleto na interação com estes sistemas, formação de oxigênio singleto) mostram que os BCD apresentam características vantajosas para suas aplicações como fotossensibilizadores em PDT, motivo pelo qual o estudo da sua fototoxicidade e testes in vivo deve prosseguir. / Photodynamic therapy (PDT) consists in the administration of photoactive compounds (photosensitizers) in an organism with subsequent application of visible light to the area to be treated. This procedure leads to the activation of the compound which kills neoplasic cells, as well as fungus and bacteria. The photosensitizers used nowadays show some disadvantages, thus motivating the search for more effective compounds. Bichromophoric cyanine dyes (BCD) exhibit good spectral characteristics to be used in PDT, encouraging the development of this study. This work aimed at studying spectral, energetic and photodecomposition characteristics of BCD: (i) in different solvents (choosen accordingly to their dieletric constant $\\xi$ and the capacity to form hydrogen bonds), (ii) at their interaction with micelles and (iii) at their interactions with DNA. Four BCD isomers with equal chromophores and different structure of the central part of the molecule were studied. Static and time resolved spectroscopic methods such as optical absorption, fluorescence, anisotropy, circular dicroism and photoacustic calorimetry were used. Experimental results were compared with computational ones, based on geometry optimization and analysis of electronic orbital densities, energy levels and transition probabilities. This data allowed to create the BCD singlet absorption spectrum. The computer modelling confirmed that the transition probabilities depended on the angle between chromophores determined by the central structure. The $180^{0}$ BCD showed a single absorption band, while the $90^{0}$ BCD showed two bands of equal intensities. It was demonstrated that at $<100^{0}C$ BCD chromophores are present in the trans-trans form, except for the BCD with $120^{0}$ where the mixture of trans-trans and cis-trans forms was present in the $0^{0}-100^{0}C$ temperature interval. Experimental results have shown that BCD did not form aggregates in organic solvents. The BCD spectral characteristics did not followed the Lippert equation, which means that both non-specific and specific mechanisms, such as the hydrogen bond formation determined the BCD solvatation The life times ($\\tau_{S1},\\tau\\,{}_{T1}$) and quantum yields ($\\phi_{S1},\\phi\\,{}_{T1}$) of singlet and triplet excited states decreased with increase of the solvent dielectric constant $\\xi$. Though, the solvent viscosity increment caused the increase of these parameters. The addition of water to organic solvents led to BCD aggregation. The $\\tau_{S1},\\tau\\,{}_{T1}$ and $\\phi_{S1},\\phi\\,{}_{T1}$ values decreased, probably due to two effects: the decrease of microviscosity around the molecules and aggregation of BCD due to interaction with water. The interaction of BCD with micelles depended on the micelle electric charge. In the presence of negatively charged ones three species were produced in the solution: BCD free monomers, BCD aggregates bound to micelles and BCD monomers bound to micelles. Zwiterionic micelles produced two species: BCD free monomers and monomers bound to micelles. The binding constants with zwiterionic LPC micelles were $\\sim10^{6}M^{-1}$. Positively charged micelles did not interact with BCD. Interaction with DNA stimulated the BCD aggregation. Three species were produced in the solution: BCD free monomers and monomers bound to DNA and BCD aggregates bound to DNA. The relative content of these species depended on the {[}BCD]/{[}DNA] ratio. The BCD monomer to DNA binding constants were $\\sim10^{7}M^{-1}$. The $\\tau_{S1},\\tau{}_{T1}$ and $\\phi_{S1},\\phi{}_{T1}$ values increased with BCD binding to DNA, indicating that non radioactive processes decreased in comparison with pure water and homogeneous organic solvents. The BCD photodecomposition depended on the oxygen presence. This fotodecomposition occured when singlet oxygen (generated due to the energy transfer from the BCD molecule in its triplet state to molecular oxygen) interacted with one of the BCD chromophores destroying its $\\pi$-conjugation system. The obtained results (high affinity to biological and model systems, high triplet state quantum yields at interaction with these systems, singlet oxygen formation) showed that BCD possess promising characteristics for application as photosensitizers in PDT, encouraging future studies on their phototoxicity and \\textit{in vivo} tests.

O câncer é uma patologia responsável por um grande número de óbitos. Os tratamentos convencionais, quimioterapia e radioterapia, nem sempre são eficientes e, além disso, os efeitos colaterais são intrínsecos às terapias. A partir da década de 70 surgiu uma nova forma de terapia chamada de “terapia fotodinâmica" (“Photodynamic Therapy", PDT) que apresenta poucos efeitos colaterais quando comparada com as terapias convencionais. O princípio da PDT é introduzir no organismo um composto fotoativo e irradiar com luz visível o local a ser tratado, obtendo assim a forma ativa do composto que induz a morte das células cancerígenas. Os compostos atualmente usados em PDT possuem algumas desvantagens na sua aplicação que estimula a busca de outros compostos mais satisfatórios. Neste projeto foram estudados corantes ciânicos com dois cromóforos (BCD) que possuem boas vantagem para uso em PDT. O trabalho foi focado no estudo das características espectrais e energéticas dos BCD na interação com DNA, e o efeito da força iônica nessa interação para sugerir seu uso em fotoquimioterapia, principalmente em PDT. Foram estudados quatro BCD que se diferenciam pelo ângulo entre os cromóforos. As análises experimentais foram feitas através de métodos espectroscópicos de absorção ótica, fluorescência e dicroísmo circular, e as teóricas através de programas computacionais comerciais e um programa desenvolvido neste trabalho. Os resultados obtidos mostram que os BCD possuem alta afinidade com as moléculas de DNA, formando diferentes espécies que afetam nas características dos seus estados excitados. A presença de outros íons influencia as características dos corantes e a sua interação com DNA. Pelos resultados obtidos podemos considerar que os BCD apresentam boas características para aplicação em PDT e que estes resultados dão suporte para estudos futuros da interação destes corantes com sistemas naturais (células) e para fazer testes clínicos em animais. / Cancer is a pathology responsible for a great number of deaths. The conventional methods of treatment, chemotherapy and radiotherapy, are not always efficient, and multiple side effects are intrinsic for these therapies. In early 70’s appeared a new method called “Photodynamic Therapy" (PDT), that shows few side effects as compared with conventional therapies. The main idea of PDT is to introduce a fotoactive compound into the organism and then irradiate with visible light the site to be treated, producing the active form of the compound that induces the death of cancers cells. The compounds used for this purpose have some disadvantages in their application, which stimulates the search for best compounds. In this work were studied several cyanine dyes with two interacting chromophores (BCD), which seem advantageous for use in PDT. The aim is to study spectral and energetic features of BCD in the presence of DNA, and at various ionic strengths, to suggest their use in cancer photochemotherapy. Four BCD studied in this work were characterized by different angles between chromophores. The experimental analysis was made using various spectroscopic methods such as optical absorption, fluorescence and circular dichroism, and the theoretical treatment was based on computational programs both conventional and created in this work. The results show that BCD has high affinity with DNA molecules, forming various species thus changing the BCD excited state features. The presence of ions modifies the characteristics of dyes and their interaction with DNA. Analyzing the results we can conclude that the characteristics of BCD are promising for their application in PDT, and this makes reasonable future studies of the interaction of these dyes with natural systems such as cells culture and tumors in laboratory animals.

Heptamethine cyanine dyes are attractive compounds for imaging purposes in biomedical applications because of their chemical and photophysical properties exhibited in the near-infrared region. A series of meso amino-substituted heptamethine cyanine dyes with indolenine, benz[e]indolenine and benz[c,d]indolenine heterocyclic moieties were synthesized and their spectral properties including fluorescence quntum yield were investigated in ethanol and ethanol/water mixture. Upon substitution with amines, the absorption maxima of the dyes shifted to the lower wavelength region (~600 nm), showed larger Stokes shifts and stronger fluorescence which can be attributed to an excited state intramolecular charge transfer (ICT). High quantum yields were observed for primary amine derivatives and lower quantum yields were observed for secondary amine derivatives. Fluorescence quantum yields are greater for dyes with 3H-indolenine terminal moieties than for dyes with benz[e]indolenine end groups. Benz[c,d]indolenine based heptamethine cyanine dyes exhibited the lowest quantum yield due to aggregation in solution. In general, the benz[e]indolenine hepatemethine cyanines showed high Stokes shifts compared to indolenine dyes. For the meso-chloro dyes, the absorption maxima for the dyes shifted bathochromically in the order of indolenine, benz[e]indolenine and benz[c,d]indolenine.