This dissertation describes our study of a photophysical process that leads to ultrafast generation of triplet excitons following photoexcitation, singlet exciton fission, in three different acene dimers and diradicaloids. In pentacene and tetracene dimers, we investigate their mechanism of singlet fission. In a series of diradicaloids, we study the relation between molecular structure, diradical character and the suitability for singlet fission. In the first two chapters we explore singlet fission in pentacene dimer. We demonstrate fast and highly efficient intramolecular singlet fission, consisting of two covalently attached pentacene units. The singlet fission pathway is governed by the energy gap between singlet and charge-transfer states, which change dynamically with molecular geometry but are primarily set by the side group. The process exhibits a sensitivity to solvent polarity and competes with geometric relaxation in the singlet state, while subsequent triplet decay is strongly dependent on conformational freedom. The near orthogonal arrangement of the pentacene units is unlike any structure currently proposed for efficient singlet fission and points toward new molecular design rules. Furthermore, these results are the first to demonstrate the role of charge-transfer states in singlet fission and highlight the importance of solubilising groups to optimise excited-state photophysics. In the next chapter, we examine singlet fission in tetracene dimer, where singlet fission is energetically unfavourable. We demonstrate triplet yield as high as 190% can be achieved via fission from higher singlet excited states mediated by charge-transfer states. The outcomes of this study provide deeper insight into the role of hot singlet states in singlet fission and point toward less stringent molecular design rules. In the last chapter, we shift our focus on a new class of molecules, diradicaloid molecules. We explore a family of zethrene molecules, with tuneable diradical character, and demonstrate their general ability to undergo rapid singlet fission via spin-entangled and emissive triplet-pair state TT. A wide range of zethrene molecules are found to be suitable for singlet fission, with additional benefits of high absorption coefficients and photo-/chemical stability.

In our efforts to engineer a DNA binding and cleaving protein with greater sequence discrimination, we have designed dimeric proteins derived from engrailed homeodomain and calmodulin. Previous research by our group has shown that a hydrolytically active lanthanide binding site can be incorporated into a DNA binding motif. To understand protein-DNA interaction and improve the sequence selectivity of the chimeric complex, two lanthanide-binding homodimers were designed and expressed. One of the dimers, F2, is coupled together by a flexible polypeptide linker and the other, R7C, is a disulfide cross-linked cysteine mutant at the N-terminus. Studies of fluorescence of tryptophan residues document that the overall affinity for lanthanide and calcium is similar to traditional EF-hand peptides (1-10 μM). Metal titrations monitored by circular dichroism (CD) revealed that the secondary structures of the dimers contained a lower degree of -helicity than the designed monomeric protein due to additional modifications, but because of their flexibility and their two active-site domain, hydrolytic activity was several folds faster than our previously designed proteins and peptides. Unlike earlier reports on our chimeras, F2 also demonstrated the capability to hydrolyze DNA in the presence of some biological relevant metal ions suggesting different cleavage mechanisms were carried out. Extensive DNA sequencing studies on cleavage patterns with oligonucleotide duplexes confirmed the unique sequence selectivity and kinetic properties of F2. Two engrailed homeodomain target sites, TAATTA, were favored for hydrolytic activity corresponding to one domain acting as a DNA anchor on the first target site while the other was an "opportunist" at recognizing the second site. Nonetheless, the hydrolytic behavior at the phosphodiester bond on a specific dsDNA sequence is in good agreement with the behavior of restriction endonucleases. Unlike restriction enzymes, metallated F2 has not only demonstrated the ability to cleave DNA plasmid, but it also excises the entire nucleotide on a selected sequence. This homodimer is the first example of an active and selective hydrolytic artificial nuclease based on the modular turn substitution design approach that can be a potential template for genomic modification.

Our interest in the red alga Delisea pulchra (=D.fimbriata) (Greville) Montagne 1844 (Rhodophyceae, Bonnemaisoniales, Bonnemaisoniaceace) was stimulated by its activity in the biosssays done at Wyeth Pharmaceuticals. Halogenated compounds from D. pulchra interfere with Gram-negative bacterial signaling systems, affect the growth of Gram-positive bacteria, inhibit quorum sensing and swarming motility of marine bacteria (inhibit bacterial communication). They also inhibit surface colonization in marine bacteria and exhibit antifouling properties against barnacle larvae and macroalgal gametes. Chemical investigation of D.pulchra collected near Palmer Station, Antarctica yielded three new dimeric halogenated furanones, pulchralide A-C (41-43), along with previously reported fimbrolide (21), acetoxyfimbrolide (22), hydroxyfimbrolide (23) and halogenated ketone 40. The reported Compounds were characterized by comparison of their 1H and 13C NMR data with that previously published. Pulchralide A-C were characterized by both 1D (1H NMR, 13C NMR, DEPT, 1H-1H COSY) and 2D (gHMQC, gHMBC) NMR techniques, supported by HREIMS/HRESIMS data. The absolute stereochemistry of Pulchralide A was determined by a single crystal X-ray analysis. Significant antimicrobial activity was observed in acetoxyfimbrolide (22) and hydroxyfimbrolide (23), where as pulcharlide A (41) and fimbrolide (21) were weakly active.

Grb2, a modular protein comprised of a central SH2 domain flanked between a N-terminal SH3 (nSH3) domain and a C-terminal SH3 (cSH3) domain, is a component of cell signaling networks involved in the transmission of extracellular information in the form of growth factors and cytokines to downstream targets such as transcription factors within the nucleus. The Grb2-Sos1 interaction is mediated through the combinatorial binding of nSH3 and cSH3 domains of Grb2 to various sites - designated S1, S2, S3, and S4 - containing PXpsiPXR motifs within Sos1. Here, using a diverse array of biophysical techniques, including in particular isothermal titration calorimetry coupled with molecular modeling and semi-empirical analysis, I provide new insights into the Grb2-Sos1 interaction in thermodynamic and structural terms. My data show that Grb2 exists in monomer-dimer equilibrium in solution and that the dissociation of dimer into monomers is entropically-driven. The heat capacity change observed was much smaller than that expected from the rather large molecular surfaces becoming solvent-occluded upon dimerization, implying that monomers undergo conformational rearrangement upon dimerization. 3D structural models suggest strongly that such conformational rearrangement may arise from domain swapping. I further show that the nSH3 domain of Grb2 binds to the S1 site containing the proline-rich consensus motif PXpsiPXR with an affinity that is nearly three-fold greater than that observed for the binding of the cSH3 domain. It is also demonstrated that such differential binding of the nSH3 domain relative to the cSH3 domain is largely due to the requirement of a specific acidic residue, in the RT loop, to engage in the formation of a salt bridge with the arginine residue in the consensus motif PXpsiPXR. The data further reveal that, while binding of both SH3 domains to Sos1 is under enthalpic control, the nSH3 binding suffers from entropic penalty in contrast to entropic gain accompanying the binding of cSH3, implying that the two domains employ differential thermodynamic mechanisms for Sos1 recognition. Additionally, my data reveal that while the nSH3 domain of Grb2 binds with affinities in the physiological range to all four sites S1-S4, the cSH3 domain can only do so at the S1 site. Further scrutiny of these sites yields rationale for the recognition of various PXpsiPXR motifs by the SH3 domains in a discriminate manner. Unlike the PXpsiPXR motifs at S2, S3 and S4 sites, the PXpsiPXR motif at S1 site is flanked at its C-terminus with two additional arginine residues that are absolutely required for high-affinity binding of the cSH3 domain. In contrast, these two additional arginine residues augment the binding of the nSH3 domain to the S1 site but their role is not critical for the recognition of S2, S3 and S4 sites. Molecular modeling is employed to rationalize my new findings in structural terms. Taken together, this thesis provides novel insights into the physicochemical basis of a key protein-protein interaction pertinent to cellular signaling and cancer. My studies bear the potential for the development of novel therapies with less toxicity but more effectiveness for the treatment of disease.

In nature, transition metal containing enzymes display many biologically important, attractive and efficient catalytic oxidation reactions. Many transition metal catalysts have been designed to mimic the predominant oxidation catalysts in nature, namely, the cytochrome P450 enzymes. Ruthenium porphyrin complexes have been the center of this research and have successfully been utilized, as catalysts, in major oxidation reactions, such as the hydroxylation of alkanes. The present work focuses on photocatalytic studies of aerobic oxidation reactions with well characterized ruthenium porphyrin complexes. The photocatalytic studies of aerobic oxidation reactions of hydrocarbons The photocatalytic studies of aerobic oxidation reactions of hydrocarbons catalyzed by a bis-porphyrin-ruthenium(IV) μ-oxo dimer using atmospheric oxygen as the oxygen source in the absence of co-reductants were investigated. The ruthenium(IV) μ-oxo bisporphyrin (3a-d) was found to catalyze aerobic oxidation of a variety of organic substrates efficiently. By comparison, 3d was found to be a more efficient photocatalyst than the well-known 3a under identical conditions. A KIE at 298K was found to be larger than those observed in autoxidation processes, suggesting a nonradical mechanism that involved the intermediacy of ruthenium(V)-oxo species as postulated. The reactivity order in the series of ruthenium(IV) μ-oxo bisporphyrin complexes follows TPFPP>4- CF3TPP>TPP, and is consistent with expectations based on the electrophilic nature of the ruthenium(IV) μ-oxo bisporphyrin species. The trans-dioxoruthenium(VI) porphyrins have been among the best characterized metal-oxo intermediates and their involvement as the active oxidant in the hydrocarbon oxidation have been extensively studied. In addition to the well-known chemical methods, we developed a novel approach for generation of trans-dioxoruthenium( VI) porphyrins with visible light by extension of the known photoinduced ligand cleavage reactions. A series of trans-dioxoruthenium(VI) porphyrin complexes (6a-d) were photochemically synthesized and spectroscopically characterized by UV-vis, and 1H-NMR.

Understanding the intermolecular forces and dynamics of insulin self-assembly is crucial for devising formulations for the treatment of insulin-dependent diabetes. Insulin must dissociate from its hexameric storage form, through an intermediate dimer form, to the bioactive monomer before receptor binding. Specifically, the dimer dissociation is a pivotal step to control insulin dynamics and self-assembly. Steered molecular dynamics simulations were performed on native insulin to provide molecular insight into the insulin dissociation force spectroscopy experiment. Our simulation results of force-induced dimer dissociation revealed that the dimer dissociation occurs near the limit of extensibility of the B-chain with significant conformational changes to the monomer(s). These long-range interactions, consistent with our experiments, are due to stronger inter-monomer interactions across the anti-parallel β-sheet interface than any other intra-monomer interaction. Novel atomistic data played an important role in detailed structural characterization of multiple unfolding and dissociation pathways that depend on the relative strength of the inter-monomer interactions and the intra-monomer interactions. Comparative simulations of two rapid-acting insulin analogues (LysB28ProB29, AspB28) to native insulin were performed to investigate the effect of sequence on the dimer dissociation. The hypothesis is that site-specific alterations to the dimer-forming surface of two rapid-acting analogues will result in a weakening of the inter-monomer interactions, which would be reflected during force-induced dimer dissociation. The results revealed that these analogues dissociates with lower probability of long-range interactions and a corresponding reduction in B-chain extension. B-chain extensibility is thus a characteristic marker of inter-monomer interactions and multiple unfolding pathways. These data agree with the design strategies of sequence modifications to the weakened inter-monomer interface applied to the synthesis of rapid-acting insulin analogues. In contrast, the ligand-induced alteration to the strengthened inter-monomer interactions through a specific GluB13s-zinc bridge contributed to the unique unfolding force curves, so it can be applicable as design strategy to the development of a novel long-acting analogue. Overall, our force spectroscopy studies on insulin native and analogues have successfully provided atomistic insights into the dimer dissociation characteristics and control strategies of self-assembly. In addition, this study would provide a framework for the structure-dynamics-function relationships of insulin-insulin receptor binding.

No description available.

Understanding the intermolecular forces and dynamics of insulin self-assembly is crucial for devising formulations for the treatment of insulin-dependent diabetes. Insulin must dissociate from its hexameric storage form, through an intermediate dimer form, to the bioactive monomer before receptor binding. Specifically, the dimer dissociation is a pivotal step to control insulin dynamics and self-assembly. Steered molecular dynamics simulations were performed on native insulin to provide molecular insight into the insulin dissociation force spectroscopy experiment. Our simulation results of force-induced dimer dissociation revealed that the dimer dissociation occurs near the limit of extensibility of the B-chain with significant conformational changes to the monomer(s). These long-range interactions, consistent with our experiments, are due to stronger inter-monomer interactions across the anti-parallel β-sheet interface than any other intra-monomer interaction. Novel atomistic data played an important role in detailed structural characterization of multiple unfolding and dissociation pathways that depend on the relative strength of the inter-monomer interactions and the intra-monomer interactions. Comparative simulations of two rapid-acting insulin analogues (LysB28ProB29, AspB28) to native insulin were performed to investigate the effect of sequence on the dimer dissociation. The hypothesis is that site-specific alterations to the dimer-forming surface of two rapid-acting analogues will result in a weakening of the inter-monomer interactions, which would be reflected during force-induced dimer dissociation. The results revealed that these analogues dissociates with lower probability of long-range interactions and a corresponding reduction in B-chain extension. B-chain extensibility is thus a characteristic marker of inter-monomer interactions and multiple unfolding pathways. These data agree with the design strategies of sequence modifications to the weakened inter-monomer interface applied to the synthesis of rapid-acting insulin analogues. In contrast, the ligand-induced alteration to the strengthened inter-monomer interactions through a specific GluB13s-zinc bridge contributed to the unique unfolding force curves, so it can be applicable as design strategy to the development of a novel long-acting analogue. Overall, our force spectroscopy studies on insulin native and analogues have successfully provided atomistic insights into the dimer dissociation characteristics and control strategies of self-assembly. In addition, this study would provide a framework for the structure-dynamics-function relationships of insulin-insulin receptor binding.

The mitochondrial F-ATPases make about 90% of cellular ATP. They are multi-protein assemblies with a membrane extrinsic catalytic domain attached to a membrane embedded sector. They operate by a mechanical rotary mechanism powered by an electro-chemical gradient, generated across the inner mitochondrial membrane by respiration. A detailed molecular description has been provided by X-ray crystallographic studies and "single molecule" observations of the mechanism of the F1 catalytic domain. Details are known also of the architecture of the peripheral stalk of part of the stator and the membrane embedded region of the rotor. However, knowledge of the detailed structure of the rest of the membrane domain, and the detailed mechanism of generation of rotation is lacking. Recently, studies of the intact mitochondrial F-ATPases, determined by cryo-electron microscopy (cryo-em), have provided structural information at intermediate levels of resolution. Whilst these structures have given insights into the mechanism of generation of rotation, the information required for a molecular understanding of this mechanism is still lacking. Moreover, the locations and roles of six supernumerary membrane subunits are unclear. Some of them are likely to be involved in the formation of dimers of the enzyme which line the edges of mitochondrial cristae. Therefore, in this thesis, a procedure is described for the purification of dimers of the bovine and yeast F-ATPases. The structure of the bovine dimer has been determined by cryo-em at a resolution of ca. 6.9 Angstrom. This structure confirms features concerning the trans-membrane spans of the a-, A6L- and b-subunits observed in the monomeric complex. In addition, the single trans-membrane a-helix of the f-subunit has been located, and the subunit appears to mediate dimer formation. The structure of A6L has been extended, and the a-helices of subunits e- and g- have been located. Another novel feature has been assigned to the DAPIT subunit, and may provide links between dimers in forming larger oligomers. Further improvement in the resolution of the structure is hampered by the extreme conformational heterogeneity of the F-ATPase. To this end, the simpler Fo membrane domain has been isolated and characterized initially by electron microscopy in negative stain.

A água, conhecida como solvente universal, participa da maioria das reações químicas e composições biológicas, tem importância vital em nosso planeta, tornando a vida como conhecemos inconcebível sem a sua presença. Tais propriedades advêm da forma como as moléculas interagem entre si, e a principal ligação química existente é a ligação de hidrogênio. Portanto, compreender como ela acontece e ser capaz de manipulá-la pode ser considerada uma importante técnica na busca de novos comportamentos. Esta dissertação visa compreender como o campo elétrico nas suas variantes: direção, sentido e intensidade podem afetar a água. Sendo tal estudo realizado através de cálculos utilizando métodos de química quântica: Teoria do Funcional da Densidade e a Teoria de Perturbação Moller-Plesset de segunda ordem. Como resultado obteve-se que quando o campo elétrico é aplicado em uma direção e sentido fora do plano da ligação de hidrogênio, a estrutura inicial tende a se rearranjar produzindo uma estrutura mais fracamente ligada do que a ordinária. Quando aplicamos o campo no plano da ligação de hidrogênio, temos resultados mais pronunciados, sendo que, se o sentido for : Doador -> Receptor a estrutura resultante estará mais fracamente ligada e se o sentido for Receptor -> Doador a estrutura produzida estará mais fortemente ligada. Os resultados obtidos mostram também que tais mudanças acontecem principalmente devido a alterações eletrostáticas, como mudança na densidade de carga da ligação de hidrogênio. Também confirmou-se um ponto crítico no campo 0,15 V/Å, como sugerido na literatura. Esta singularidade foi atribuída neste trabalho à uma transição estrutural. O campo também modificou a importância das interações secundárias, principalmente entre os átomos de oxigênio, e ao desligá-lo, o sistema tende a voltar ao estado inicial. / The water is know as the universal solvent, part of most chemical reactions and biological compositions, it is vitally important to our planet, making life as we know it inconceivable without its presence. These properties arise from how the molecules interact, and the main existing chemical link is the hydrogen bond. Therefore, to understand how this happens and be able to manipulate it can be considered an important technique in the search for new behaviors. Thus, this paper aims to understand how the electric field in its variants: direction, sense and intensity can affect the water. This study is undertaken through calculations using quantum chemistry methods: Density Functional Theory and Moller-Plesset Perturbation Theory to Second Order. As a result was found that when the electric field is applied in direction and sense out of the plane of the hydrogen bond, the initial structure tends to rearrange itself to produce a more loosely bound than the ordinary. When the electric field is applied in the hydrogen bond plane, the have results obtained are more pronounced. Sense is: Donor -> Receiver initial structure will be more loosely connected and that when the sense is Receiver -> Donor initial structure will be tightly linked. Our results also show that such changes occur mainly due to electrostatic changes, mainly in the change in the charge density of hydrogen bond. It was also confirmed a critical point in the field 0, 15 V / Å, as suggested by Vegiri. This was attributed to a structural transition. The field also changed the importance of secondary interactions, especially between the oxygen atoms. And when the field is shutdown the system tends back to its to initial structure.