Atualmente, grande parte dos fármacos desenvolvidos com propriedades antitumorais tem como alvo molecular o DNA. Estas moléculas são desenvolvidas na maioria das vezes para, a despeito da grande variedade de sequências e conformações possíveis para uma molécula de DNA, interagirem específicamente com uma dada região e/ou sequência do DNA. Um dos objetivos mais desafiadores para o desenvolvimento destes fármacos é compreender como estas moléculas interagem especificamente com seu alvo biológico. Uma vez que estes processos ocorrem em escalas de tempo que ainda não podem ser alcançadas por técnicas de simulação clássicas, como a dinâmica molecular, novas metodologias foram propostas. Um destes métodos foi o proposto por Laio e Parrinello, em 2002, e denominado metadinâmica. A metadinâmica usa um potencial adaptativo em função de uma coordenada relevante ao sistema, permitindo que o sistema evolua para regiões diferentes do mínimo local da superfície de energia livre. Com isso, este trabalho teve como objetivo verificar se a metadinâmica descreveria de forma satisfatória o processo de dissociação e associação da Netropsina, usando como variável coletiva a distância entre o centro de massas destes ligantes ao eixo principal formado pelo DNA. Objetivou-se também verificar se a metadinâmica é capaz de fornecer a variação de energia livre de dissociação do Hoechst 33258 para diferentes sequências de DNA. Os resultados obtidos neste trabalho para a dissociação e associação da Netropsina revelaram uma boa concordancia entre os dados de energia livre experimentais com os obtidos pelo emprego da metadinâmica. O mecanismo de dissociação e associação da Netropsina obtido por metadinâmica revelou grande coerência com o modelo mecanistico proposto na literatura. Uma vez que não foi possível encontrarmos um conjunto de parâmetros adequados para a as simulações de metadinâmica para a dissociação da molécula do Hoechst 33258, não foi possível verificarmos o comportamento da metadinâmica em relação à variação da sequência do DNA. / Nowadays, most of drugs which are developed with antitumoral properties have as their molecular target the DNA molecule. Despite of the wide variety of conformational and sequence possibilities of the DNA molecule, these molecules are developed for interacting specifically with a particular region and/or DNA sequence. One of the most challenging objectives in the development of these drugs is to understand how these molecules interact specifically with the biological target. Since these processes take place at time scales larger than those we can reach with classical simulation techniques, such as Molecular Dynamics, new methodologies have been proposed to work with this processes. One of these methods, called Metadynamics, was proposed by Laio e Parrinello, at 2002. Metadinamics uses an non-Markovian potential dependent of a coordinate which is relevant to systen, also named collective variable, allowing the system to evolve through regions on the free energy surface which are different of the local minimum. This study aimed to verify if the Metadinamics methodology will describe satisfactorily the process of dissociation and association of Netropsin, using as a collective variable the distance between the center of mass of this molecule to the principal axis of the DNA molecule. This work was also aimed to determine whether Metadinamics is able to provide the variation of free energy for dissociation of Hoechst 33258 molecule from different DNA sequences. The results obtained in this work for dissociation and association of Netropsin molecule showed a good agreement between the experimental free energy data with those obtained by the use of Metadynamics simulations.The dissociation and association mechanism of Netropsin obtained by Metadinamics showed great coherence with the mechanistic model proposed in the literature. However, it was not possible to find a suitable set of parameters for the Metadynamics simulations for the process of Hoechst 33258 dissociation, therefore, we could not evaluate in this work the behavior of Metadynamics against different DNA sequences.

Atualmente, grande parte dos fármacos desenvolvidos com propriedades antitumorais tem como alvo molecular o DNA. Estas moléculas são desenvolvidas na maioria das vezes para, a despeito da grande variedade de sequências e conformações possíveis para uma molécula de DNA, interagirem específicamente com uma dada região e/ou sequência do DNA. Um dos objetivos mais desafiadores para o desenvolvimento destes fármacos é compreender como estas moléculas interagem especificamente com seu alvo biológico. Uma vez que estes processos ocorrem em escalas de tempo que ainda não podem ser alcançadas por técnicas de simulação clássicas, como a dinâmica molecular, novas metodologias foram propostas. Um destes métodos foi o proposto por Laio e Parrinello, em 2002, e denominado metadinâmica. A metadinâmica usa um potencial adaptativo em função de uma coordenada relevante ao sistema, permitindo que o sistema evolua para regiões diferentes do mínimo local da superfície de energia livre. Com isso, este trabalho teve como objetivo verificar se a metadinâmica descreveria de forma satisfatória o processo de dissociação e associação da Netropsina, usando como variável coletiva a distância entre o centro de massas destes ligantes ao eixo principal formado pelo DNA. Objetivou-se também verificar se a metadinâmica é capaz de fornecer a variação de energia livre de dissociação do Hoechst 33258 para diferentes sequências de DNA. Os resultados obtidos neste trabalho para a dissociação e associação da Netropsina revelaram uma boa concordancia entre os dados de energia livre experimentais com os obtidos pelo emprego da metadinâmica. O mecanismo de dissociação e associação da Netropsina obtido por metadinâmica revelou grande coerência com o modelo mecanistico proposto na literatura. Uma vez que não foi possível encontrarmos um conjunto de parâmetros adequados para a as simulações de metadinâmica para a dissociação da molécula do Hoechst 33258, não foi possível verificarmos o comportamento da metadinâmica em relação à variação da sequência do DNA. / Nowadays, most of drugs which are developed with antitumoral properties have as their molecular target the DNA molecule. Despite of the wide variety of conformational and sequence possibilities of the DNA molecule, these molecules are developed for interacting specifically with a particular region and/or DNA sequence. One of the most challenging objectives in the development of these drugs is to understand how these molecules interact specifically with the biological target. Since these processes take place at time scales larger than those we can reach with classical simulation techniques, such as Molecular Dynamics, new methodologies have been proposed to work with this processes. One of these methods, called Metadynamics, was proposed by Laio e Parrinello, at 2002. Metadinamics uses an non-Markovian potential dependent of a coordinate which is relevant to systen, also named collective variable, allowing the system to evolve through regions on the free energy surface which are different of the local minimum. This study aimed to verify if the Metadinamics methodology will describe satisfactorily the process of dissociation and association of Netropsin, using as a collective variable the distance between the center of mass of this molecule to the principal axis of the DNA molecule. This work was also aimed to determine whether Metadinamics is able to provide the variation of free energy for dissociation of Hoechst 33258 molecule from different DNA sequences. The results obtained in this work for dissociation and association of Netropsin molecule showed a good agreement between the experimental free energy data with those obtained by the use of Metadynamics simulations.The dissociation and association mechanism of Netropsin obtained by Metadinamics showed great coherence with the mechanistic model proposed in the literature. However, it was not possible to find a suitable set of parameters for the Metadynamics simulations for the process of Hoechst 33258 dissociation, therefore, we could not evaluate in this work the behavior of Metadynamics against different DNA sequences.

Atualmente, grande parte dos fármacos desenvolvidos com propriedades antitumorais tem como alvo molecular o DNA. Estas moléculas são desenvolvidas na maioria das vezes para, a despeito da grande variedade de sequências e conformações possíveis para uma molécula de DNA, interagirem específicamente com uma dada região e/ou sequência do DNA. Um dos objetivos mais desafiadores para o desenvolvimento destes fármacos é compreender como estas moléculas interagem especificamente com seu alvo biológico. Uma vez que estes processos ocorrem em escalas de tempo que ainda não podem ser alcançadas por técnicas de simulação clássicas, como a dinâmica molecular, novas metodologias foram propostas. Um destes métodos foi o proposto por Laio e Parrinello, em 2002, e denominado metadinâmica. A metadinâmica usa um potencial adaptativo em função de uma coordenada relevante ao sistema, permitindo que o sistema evolua para regiões diferentes do mínimo local da superfície de energia livre. Com isso, este trabalho teve como objetivo verificar se a metadinâmica descreveria de forma satisfatória o processo de dissociação e associação da Netropsina, usando como variável coletiva a distância entre o centro de massas destes ligantes ao eixo principal formado pelo DNA. Objetivou-se também verificar se a metadinâmica é capaz de fornecer a variação de energia livre de dissociação do Hoechst 33258 para diferentes sequências de DNA. Os resultados obtidos neste trabalho para a dissociação e associação da Netropsina revelaram uma boa concordancia entre os dados de energia livre experimentais com os obtidos pelo emprego da metadinâmica. O mecanismo de dissociação e associação da Netropsina obtido por metadinâmica revelou grande coerência com o modelo mecanistico proposto na literatura. Uma vez que não foi possível encontrarmos um conjunto de parâmetros adequados para a as simulações de metadinâmica para a dissociação da molécula do Hoechst 33258, não foi possível verificarmos o comportamento da metadinâmica em relação à variação da sequência do DNA. / Nowadays, most of drugs which are developed with antitumoral properties have as their molecular target the DNA molecule. Despite of the wide variety of conformational and sequence possibilities of the DNA molecule, these molecules are developed for interacting specifically with a particular region and/or DNA sequence. One of the most challenging objectives in the development of these drugs is to understand how these molecules interact specifically with the biological target. Since these processes take place at time scales larger than those we can reach with classical simulation techniques, such as Molecular Dynamics, new methodologies have been proposed to work with this processes. One of these methods, called Metadynamics, was proposed by Laio e Parrinello, at 2002. Metadinamics uses an non-Markovian potential dependent of a coordinate which is relevant to systen, also named collective variable, allowing the system to evolve through regions on the free energy surface which are different of the local minimum. This study aimed to verify if the Metadinamics methodology will describe satisfactorily the process of dissociation and association of Netropsin, using as a collective variable the distance between the center of mass of this molecule to the principal axis of the DNA molecule. This work was also aimed to determine whether Metadinamics is able to provide the variation of free energy for dissociation of Hoechst 33258 molecule from different DNA sequences. The results obtained in this work for dissociation and association of Netropsin molecule showed a good agreement between the experimental free energy data with those obtained by the use of Metadynamics simulations.The dissociation and association mechanism of Netropsin obtained by Metadinamics showed great coherence with the mechanistic model proposed in the literature. However, it was not possible to find a suitable set of parameters for the Metadynamics simulations for the process of Hoechst 33258 dissociation, therefore, we could not evaluate in this work the behavior of Metadynamics against different DNA sequences.

Le développement de nouvelles molécules capables d’intéragir avec l’ADN, leur mode d’intéraction et leur affinité est un domaine de recherche particulièrement important. Dans ce travail nous avons étudié les interactions avec l’ADN de la molécule Hoechst 33258 connu pour être un ligand du petit sillon ainsi que plusieurs de ces analogues.Dans un premier temps nous avons étudié la stabilité des complexes ADN-Hoechst 33258 en solution avec et sans DMSO comme co-solvant. Deuxièmement, les affinités de dérivés nouvellement conçus et synthétisés du Hoechst 33258 vis-à-vis de l'ADN ont été évaluées. Enfin, nous avons étudiés la capacité d’induction d’excimers en présence d’ADN de différentes molécules pyrénylées. Ces études ont été effectuées par différentes méthodes spectroscopiques, telles que l'absorbance UV-visible, la fluorescence, le dichroïsme circulaire, la spectroscopie de masse ESI et de la modélisation moléculaire. / The development of new DNA binders and the evaluation of their affinity toward DNA as well as their mode of binding is an area of research of prime importance. In this thesis we studied the interactions of Hoechst 33258, a well-known groove binder, as well as some of its newly synthesized derivatives with DNA. The stability of DNA-Hoechst 33258 complex in solution with and without DMSO as a co-solvent was evaluated.Secondly, the affinities of newly designed and synthesized derivatives of Hoechst 33258 toward DNA were evaluated. Finally, a set of pyrene derivatives able to induced excimer formation upon binding to DNA were studied. Different spectroscopic methods, such as UV-vis absorbance, fluorescence, circular dichroism, ESI mass spectroscopy and molecular docking were applied for the complete evaluation of the affinity of these ligands toward DNA.

This thesis consists of two major chapters, each with its own introduction, experimental section and discussion. The TG4T/daunomycin and G4/daunomycin complexes described in Chapter One are two out of only five crystallographic quadruplex/ligand structures reported to date. In both structures daunomycin molecules stack onto a terminal G quartet preventing the G4 quadruplex from destacking and unwinding. The number of interacting ligand molecules depends on the quadruplex structure itself. The G4 quadruplex can accommodate four daunomycin molecules within one layer, while the TG4T tetraplex only accommodates three. In both structures daunosamine moieties form hydrogen bonds with the quadruplex but only daunosamine moieties from the TG4T/daunomycin structure make slight incursions into the quadruplex grooves. Both structures are stabilised by π-π interactions, hydrogen bonds, Van der Waals contacts and electrostatic interactions. The daunomycin/TG4T complex is the first ever reported and the only structure where a ligand interacts directly with the quadruplex groove. Chapter Two describes nine crystal structures of Hoechst 33258 analogues with d(CGCAAATTTGCG)2 and d(CGCGAATTCGCG)2 oligonucleotides, and is divided into two sections. Section A includes seven structures with Halogenated Hoechst 33258 analogues that are potential agents in radiotherapy, phototherapy, radioimmunotherapy or photoimmunotherapy, and the structure of the precursor. In all of the examined complexes the ligand binds to the minor groove but not all halogen substituents refine to 100% occupancy. The refined occupancies of the halogen atoms reveal that the degree of carbon-halogen cleavage is highest for ortho and lowest for para substitution. Among meta substituents pointing outside the minor groove, bromine atoms had a higher occupancy than the larger iodines. The position of the halogen atom in the minor groove is influenced by additional substituents on the phenyl ring. In most cases the bulky halogen atom is facing outside of the minor groove. Only in the 3-iodo-5-isopropylHoechst complex is iodine positioned towards the floor of the groove allowing the big isopropyl group to face outside. Section B describes the structure of a carborane-containing ligand (JW-B) bound to the minor groove of d(CGCAAATTTGCG)2. The analysis shows that is possible to position boron-rich moieties close to the cell nucleus, and JW-B may have potential in Boron Neutron Capture Therapy. / Data file restricted at the request of the author, but available by individual request, use the feedback form to request access.

This thesis consists of two major chapters, each with its own introduction, experimental section and discussion. The TG4T/daunomycin and G4/daunomycin complexes described in Chapter One are two out of only five crystallographic quadruplex/ligand structures reported to date. In both structures daunomycin molecules stack onto a terminal G quartet preventing the G4 quadruplex from destacking and unwinding. The number of interacting ligand molecules depends on the quadruplex structure itself. The G4 quadruplex can accommodate four daunomycin molecules within one layer, while the TG4T tetraplex only accommodates three. In both structures daunosamine moieties form hydrogen bonds with the quadruplex but only daunosamine moieties from the TG4T/daunomycin structure make slight incursions into the quadruplex grooves. Both structures are stabilised by π-π interactions, hydrogen bonds, Van der Waals contacts and electrostatic interactions. The daunomycin/TG4T complex is the first ever reported and the only structure where a ligand interacts directly with the quadruplex groove. Chapter Two describes nine crystal structures of Hoechst 33258 analogues with d(CGCAAATTTGCG)2 and d(CGCGAATTCGCG)2 oligonucleotides, and is divided into two sections. Section A includes seven structures with Halogenated Hoechst 33258 analogues that are potential agents in radiotherapy, phototherapy, radioimmunotherapy or photoimmunotherapy, and the structure of the precursor. In all of the examined complexes the ligand binds to the minor groove but not all halogen substituents refine to 100% occupancy. The refined occupancies of the halogen atoms reveal that the degree of carbon-halogen cleavage is highest for ortho and lowest for para substitution. Among meta substituents pointing outside the minor groove, bromine atoms had a higher occupancy than the larger iodines. The position of the halogen atom in the minor groove is influenced by additional substituents on the phenyl ring. In most cases the bulky halogen atom is facing outside of the minor groove. Only in the 3-iodo-5-isopropylHoechst complex is iodine positioned towards the floor of the groove allowing the big isopropyl group to face outside. Section B describes the structure of a carborane-containing ligand (JW-B) bound to the minor groove of d(CGCAAATTTGCG)2. The analysis shows that is possible to position boron-rich moieties close to the cell nucleus, and JW-B may have potential in Boron Neutron Capture Therapy. / Data file restricted at the request of the author, but available by individual request, use the feedback form to request access.

This thesis consists of two major chapters, each with its own introduction, experimental section and discussion. The TG4T/daunomycin and G4/daunomycin complexes described in Chapter One are two out of only five crystallographic quadruplex/ligand structures reported to date. In both structures daunomycin molecules stack onto a terminal G quartet preventing the G4 quadruplex from destacking and unwinding. The number of interacting ligand molecules depends on the quadruplex structure itself. The G4 quadruplex can accommodate four daunomycin molecules within one layer, while the TG4T tetraplex only accommodates three. In both structures daunosamine moieties form hydrogen bonds with the quadruplex but only daunosamine moieties from the TG4T/daunomycin structure make slight incursions into the quadruplex grooves. Both structures are stabilised by π-π interactions, hydrogen bonds, Van der Waals contacts and electrostatic interactions. The daunomycin/TG4T complex is the first ever reported and the only structure where a ligand interacts directly with the quadruplex groove. Chapter Two describes nine crystal structures of Hoechst 33258 analogues with d(CGCAAATTTGCG)2 and d(CGCGAATTCGCG)2 oligonucleotides, and is divided into two sections. Section A includes seven structures with Halogenated Hoechst 33258 analogues that are potential agents in radiotherapy, phototherapy, radioimmunotherapy or photoimmunotherapy, and the structure of the precursor. In all of the examined complexes the ligand binds to the minor groove but not all halogen substituents refine to 100% occupancy. The refined occupancies of the halogen atoms reveal that the degree of carbon-halogen cleavage is highest for ortho and lowest for para substitution. Among meta substituents pointing outside the minor groove, bromine atoms had a higher occupancy than the larger iodines. The position of the halogen atom in the minor groove is influenced by additional substituents on the phenyl ring. In most cases the bulky halogen atom is facing outside of the minor groove. Only in the 3-iodo-5-isopropylHoechst complex is iodine positioned towards the floor of the groove allowing the big isopropyl group to face outside. Section B describes the structure of a carborane-containing ligand (JW-B) bound to the minor groove of d(CGCAAATTTGCG)2. The analysis shows that is possible to position boron-rich moieties close to the cell nucleus, and JW-B may have potential in Boron Neutron Capture Therapy. / Data file restricted at the request of the author, but available by individual request, use the feedback form to request access.

This thesis consists of two major chapters, each with its own introduction, experimental section and discussion. The TG4T/daunomycin and G4/daunomycin complexes described in Chapter One are two out of only five crystallographic quadruplex/ligand structures reported to date. In both structures daunomycin molecules stack onto a terminal G quartet preventing the G4 quadruplex from destacking and unwinding. The number of interacting ligand molecules depends on the quadruplex structure itself. The G4 quadruplex can accommodate four daunomycin molecules within one layer, while the TG4T tetraplex only accommodates three. In both structures daunosamine moieties form hydrogen bonds with the quadruplex but only daunosamine moieties from the TG4T/daunomycin structure make slight incursions into the quadruplex grooves. Both structures are stabilised by π-π interactions, hydrogen bonds, Van der Waals contacts and electrostatic interactions. The daunomycin/TG4T complex is the first ever reported and the only structure where a ligand interacts directly with the quadruplex groove. Chapter Two describes nine crystal structures of Hoechst 33258 analogues with d(CGCAAATTTGCG)2 and d(CGCGAATTCGCG)2 oligonucleotides, and is divided into two sections. Section A includes seven structures with Halogenated Hoechst 33258 analogues that are potential agents in radiotherapy, phototherapy, radioimmunotherapy or photoimmunotherapy, and the structure of the precursor. In all of the examined complexes the ligand binds to the minor groove but not all halogen substituents refine to 100% occupancy. The refined occupancies of the halogen atoms reveal that the degree of carbon-halogen cleavage is highest for ortho and lowest for para substitution. Among meta substituents pointing outside the minor groove, bromine atoms had a higher occupancy than the larger iodines. The position of the halogen atom in the minor groove is influenced by additional substituents on the phenyl ring. In most cases the bulky halogen atom is facing outside of the minor groove. Only in the 3-iodo-5-isopropylHoechst complex is iodine positioned towards the floor of the groove allowing the big isopropyl group to face outside. Section B describes the structure of a carborane-containing ligand (JW-B) bound to the minor groove of d(CGCAAATTTGCG)2. The analysis shows that is possible to position boron-rich moieties close to the cell nucleus, and JW-B may have potential in Boron Neutron Capture Therapy. / Data file restricted at the request of the author, but available by individual request, use the feedback form to request access.