Fluorescent protein labelling is a powerful tool for the sensitive visualization of proteins in living cells, allowing the elucidation of their localization, trafficking and ultimately their cellular function. We have developed a novel labelling technique based on the genetic fusion of a protein of interest to a small helical peptide sequence containing two Cys residues (dC10). This tag can undergo an efficient reaction with small fluorogenic labelling agents composed of a fluorophore and a dimaleimide core (dM10) that confers high reaction specificity, and quenches the latent fluorescence through photo-induced electron transfer, until both of its maleimide groups have formed robust covalent bonds with the tag Cys thiol groups. Our initial efforts at intracellular protein labelling demonstrated the importance of the selectivity of the labelling reaction, which is dependent on the reactivity of the dC10 tag. To that end, we re-engineered the dC10 tag through rational protein design. Mutant libraries were prepared through combinatorial mutation at specific positions of the helical tag sequence, and screened for their fluorogenic reactivity. In this way, we identified a novel sequence for a next-generation dC10 tag that confers 10-fold greater selectivity that we then applied to in cellulo labelling. Subsequent mechanistic studies revealed the basis for this dramatic increase in reactivity. Current applications of this powerful labelling technique, including the site-specific chelation of lanthanide ions for NMR spectroscopy and site-specific covalent heavy-atom labelling for X-ray crystallography, will also be discussed.

No description available.

Ferroelectric materials have been an area of keen interest for researchers due to their useful electro-mechanical coupling properties for a range of modern applications, such as sensing, precision actuation, or energy harvesting. The distribution of the piezoelectric coefficients, which corresponds to the piezoelectric properties, in traditional crystalline ferroelectric materials are determined by their inherent crystalline structure. This restriction limits the tunability of their piezoelectric properties. In the present work, ferroelectric lattice materials capable of a wide range of rationally designed piezoelectric coefficients are achieved through lattice micro-architecture design. The piezoelectric coefficients of several lattice designs are analyzed and predicted using an analytical volume-averaging approach. Finite element models were used to verify the analytical predictions and strong agreement between the two sets of results were found. Select lattice designs were additively manufactured using projection microstereolithography from a PZT-polymer composite and their piezoelectric coefficients experimentally verified and also found to be in agreement with the analytical and numerical predictions. The results show that the use of lattice micro-architecture successfully decouples the dependency of the piezoelectric properties on the material's crystalline structure, giving the user a means to tune the piezoelectric properties of the lattice materials. Real-world application of a ferroelectric lattice structure is demonstrated through application as a multi-directional stress sensor. / Master of Science / Ferroelectric materials have been an area of keen interest for researchers due to their useful electro-mechanical coupling properties for a range of modern applications, such as sensing, precision actuation, or energy harvesting. However, the piezoelectric properties of traditional materials are not easily augmented due to their dependency on material crystalline structure. In the present work, material architecture is investigated as a means for designing new piezoelectric materials with tunable sets of piezoelectric properties. Analytical predictions of the properties are first obtained and then verified using finite element models and experimental data from additively manufactured samples. The results indicate that the piezoelectric properties of a material can in fact be tuned by varying material architecture. Following this, real-world application of a ferroelectric lattice structure is demonstrated through application as a multi-directional stress sensor.

Para os ingredientes farmacêuticos ativos (APIs) que exibem baixa solubilidade e/ou estabilidade e/ou perfis de dissolução, dentre outros problemas capazes de afetar a sua eficácia terapêutica, as abordagens da Engenharia de Cristais têm se destacado nas últimas décadas como uma solução satisfatória. No estado sólido, os APIs podem apresentar polimorfos, sais, solvatos, co-cristais, amorfos e combinações dos mesmos. Associadas a cada estado sólido estão propriedades físicas e químicas, que podem ou não variar em relação ao API de referência. Neste contexto, este projeto de pesquisa visou o planejamento racional, síntese e caracterização de novas formas sólidas do pró-fármaco 5-fluorocitosina (5-FC) e do fármaco antineoplásico 5-fluorouracila (5-FU), ambos exibindo problemas fisicoquimicos que dificultam sua aplicação em formas de dosagem sólidas. Apesar de ser usado como fungicida, recentemente o 5-FC se tornou um dos pró-fármacos mais utilizados na terapia antineoplásica por meio de terapia dirigida por gene-enzima-pró-fármaco, uma vez que na presença da enzima citosina-desaminase o 5-FC é convertido em 5-FU dentro das células cancerosas. Para esta finalidade, coformadores adequados foram pre-selecionados a partir de análises estatísticas, realizadas utilizando o banco de dados estruturais da Cambridge, com base na competição entre sintons. Na sequencia, protocolos de cristalização foram desenvolvidos de acordo com duas técnicas: evaporação a partir de solvente e mecanoquímica, em particular a moagem com gota-solvente (SDG). Nove estruturas foram obtidas com 5-FC. Todas foram analisadas por microscopia óptica de luz polarizada e por difração de raios X por monocristal. Seis foram caracterizadas como co-cristais e três como sais. Sais farmacêuticos são preferidos nas formulações porque melhoram consideravelmente a solubilidade dos APIs. Co-cristais farmacêuticos têm sido cada vez mais explorados porque promovem igualmente melhorias nas propriedades físico-químicas dos APIs sem alterar a natureza dos mesmos. Os sais de 5-FC foram obtidos com os ácidos fumárico, oxálico e maleico e os cocristais com os ácidos adípico, tereftálico, málico, succínico e benzóico. Além disso, obteve-se um co-cristal multi-API, de 5-FC e 5-FU. Estes resultados, obtidos por meio do desenho racional de novas formas sólidas, concordaram bem com a regra de três, implementada para variações nos valores pKa (pKabase - pKaácido). Esta regra permite estimar a formação sais/co-cristais durante o processo de cristalização. Os sais de 5-FC foram analisados estrutural e termicamente. Os cocristais de 5-FC foram estruturalmente avaliados. O co-cristal multi-API também foi sintetizado por mecanoquímica, como parte dos esforços direcionados à aplicação dos princípios da Química Verde para a produção em larga escala de fármacos. Os resultados obtidos nesta tese oferecem um API como forte candidato a coformador, a saber a 5-FC, e introduz um co-cristal multi-API como potencial candidato para a terapia antineoplásica. / For active pharmaceutical ingredients (APIs) exhibiting low solubility and/or stability and/or dissolution profiles, among other problems capable of affecting their therapeutic efficacy, Crystal Engineering approaches have been highlighted in recent decades as a satisfactory solution. In the solid state, APIs may exhibit polymorphs, salts, solvates, hydrates, cocrystals, amorphous and combinations of them. Associated to each solid form are physical and chemical properties which may or not vary in relation to the reference API. In this context, this research project aimed to rationally design, synthesize and characterize new solid forms of the prodrug 5-fluorocytosine (5-FC) and the antineoplastic drug 5-fluorouracil (5-FU), by considering that both exhibit physicochemical issues that difficult their application in solid dosage forms. Although used as a fungicide, 5-FC has become one of the most used prodrugs for cancer treatment by gene-directed enzyme prodrug therapy, as in the presence of the enzyme cytosine deaminase, 5-FC is converted into 5-FU inside cancer cells. For this purpose, suitable coformers were pre-selected from statistical analyses performed on the Cambridge Structural Database, based on the synthon competition approach. Briefly, crystallization protocols were designed following two techniques: slow evaporation from solution and mechanochemistry, in particular the solvent-drop grinding (SDG). Nine structures were obtained with 5-FC. All were analyzed by polarized light optical microscopy and by single-crystal X-ray crystallography. Six were characterized as cocrystals and three as salts. Pharmaceutical salts are preferred in formulations because they considerably improve the solubility/stability of APIs. Cocrystals have been increasingly explored because they promote improvements in the physicochemical properties of the API while not altering the API´s nature. 5-FC salts were obtained with fumaric, oxalic, and maleic acids, and 5-FC cocrystals with adipic, terephthalic, malic, succinic, and benzoic acids. In addition, a multi-API cocrystal of 5-FC and 5-FU was obtained. These results, derived from the rational design of new solid forms, agreed well with the rule of three, implemented to variations in pKa values (pKabase - pKaácido). This rule allows for estimation of the salts/cocrystals formation during the crystallization process. The 5-FC salts were structurally and thermally analyzed. The 5-FC´s cocrystals were structurally evaluated. The multi-API co-crystal was also synthesized by SDG as part of the ongoing efforts toward Green Chemistry application for drug scale-up production. The results obtained in this thesis offer a strong API candidate to be used as a coformer, namely the 5-FC, and introduce a multi-API co-crystal as a potential candidate for anticancer therapy.

Para os ingredientes farmacêuticos ativos (APIs) que exibem baixa solubilidade e/ou estabilidade e/ou perfis de dissolução, dentre outros problemas capazes de afetar a sua eficácia terapêutica, as abordagens da Engenharia de Cristais têm se destacado nas últimas décadas como uma solução satisfatória. No estado sólido, os APIs podem apresentar polimorfos, sais, solvatos, co-cristais, amorfos e combinações dos mesmos. Associadas a cada estado sólido estão propriedades físicas e químicas, que podem ou não variar em relação ao API de referência. Neste contexto, este projeto de pesquisa visou o planejamento racional, síntese e caracterização de novas formas sólidas do pró-fármaco 5-fluorocitosina (5-FC) e do fármaco antineoplásico 5-fluorouracila (5-FU), ambos exibindo problemas fisicoquimicos que dificultam sua aplicação em formas de dosagem sólidas. Apesar de ser usado como fungicida, recentemente o 5-FC se tornou um dos pró-fármacos mais utilizados na terapia antineoplásica por meio de terapia dirigida por gene-enzima-pró-fármaco, uma vez que na presença da enzima citosina-desaminase o 5-FC é convertido em 5-FU dentro das células cancerosas. Para esta finalidade, coformadores adequados foram pre-selecionados a partir de análises estatísticas, realizadas utilizando o banco de dados estruturais da Cambridge, com base na competição entre sintons. Na sequencia, protocolos de cristalização foram desenvolvidos de acordo com duas técnicas: evaporação a partir de solvente e mecanoquímica, em particular a moagem com gota-solvente (SDG). Nove estruturas foram obtidas com 5-FC. Todas foram analisadas por microscopia óptica de luz polarizada e por difração de raios X por monocristal. Seis foram caracterizadas como co-cristais e três como sais. Sais farmacêuticos são preferidos nas formulações porque melhoram consideravelmente a solubilidade dos APIs. Co-cristais farmacêuticos têm sido cada vez mais explorados porque promovem igualmente melhorias nas propriedades físico-químicas dos APIs sem alterar a natureza dos mesmos. Os sais de 5-FC foram obtidos com os ácidos fumárico, oxálico e maleico e os cocristais com os ácidos adípico, tereftálico, málico, succínico e benzóico. Além disso, obteve-se um co-cristal multi-API, de 5-FC e 5-FU. Estes resultados, obtidos por meio do desenho racional de novas formas sólidas, concordaram bem com a regra de três, implementada para variações nos valores pKa (pKabase - pKaácido). Esta regra permite estimar a formação sais/co-cristais durante o processo de cristalização. Os sais de 5-FC foram analisados estrutural e termicamente. Os cocristais de 5-FC foram estruturalmente avaliados. O co-cristal multi-API também foi sintetizado por mecanoquímica, como parte dos esforços direcionados à aplicação dos princípios da Química Verde para a produção em larga escala de fármacos. Os resultados obtidos nesta tese oferecem um API como forte candidato a coformador, a saber a 5-FC, e introduz um co-cristal multi-API como potencial candidato para a terapia antineoplásica. / For active pharmaceutical ingredients (APIs) exhibiting low solubility and/or stability and/or dissolution profiles, among other problems capable of affecting their therapeutic efficacy, Crystal Engineering approaches have been highlighted in recent decades as a satisfactory solution. In the solid state, APIs may exhibit polymorphs, salts, solvates, hydrates, cocrystals, amorphous and combinations of them. Associated to each solid form are physical and chemical properties which may or not vary in relation to the reference API. In this context, this research project aimed to rationally design, synthesize and characterize new solid forms of the prodrug 5-fluorocytosine (5-FC) and the antineoplastic drug 5-fluorouracil (5-FU), by considering that both exhibit physicochemical issues that difficult their application in solid dosage forms. Although used as a fungicide, 5-FC has become one of the most used prodrugs for cancer treatment by gene-directed enzyme prodrug therapy, as in the presence of the enzyme cytosine deaminase, 5-FC is converted into 5-FU inside cancer cells. For this purpose, suitable coformers were pre-selected from statistical analyses performed on the Cambridge Structural Database, based on the synthon competition approach. Briefly, crystallization protocols were designed following two techniques: slow evaporation from solution and mechanochemistry, in particular the solvent-drop grinding (SDG). Nine structures were obtained with 5-FC. All were analyzed by polarized light optical microscopy and by single-crystal X-ray crystallography. Six were characterized as cocrystals and three as salts. Pharmaceutical salts are preferred in formulations because they considerably improve the solubility/stability of APIs. Cocrystals have been increasingly explored because they promote improvements in the physicochemical properties of the API while not altering the API´s nature. 5-FC salts were obtained with fumaric, oxalic, and maleic acids, and 5-FC cocrystals with adipic, terephthalic, malic, succinic, and benzoic acids. In addition, a multi-API cocrystal of 5-FC and 5-FU was obtained. These results, derived from the rational design of new solid forms, agreed well with the rule of three, implemented to variations in pKa values (pKabase - pKaácido). This rule allows for estimation of the salts/cocrystals formation during the crystallization process. The 5-FC salts were structurally and thermally analyzed. The 5-FC´s cocrystals were structurally evaluated. The multi-API co-crystal was also synthesized by SDG as part of the ongoing efforts toward Green Chemistry application for drug scale-up production. The results obtained in this thesis offer a strong API candidate to be used as a coformer, namely the 5-FC, and introduce a multi-API co-crystal as a potential candidate for anticancer therapy.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23213号 / 工博第4857号 / 新制||工||1758(附属図書館) / 京都大学大学院工学研究科材料化学専攻 / (主査)教授 松原 誠二郎, 教授 杉野目 道紀, 教授 中尾 佳亮 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

No description available.

The creation of new materials is absolutely essential for developing new technologies. However, experimental efforts toward the material discovery are usually based on trial-and-error approach and thus require a huge amount of time and money. Alternatively, computational predictions can now provide a more systematic, rapid, inexpensive, and reliable method for the design of novel materials with properties suitable for new technologies. This dissertation describes the technique of theoretical predictions and presents the results on the successfully predicted and already produced (in some cases) unusual molecules, clusters, nanoparticles, and solids. The major part of scientific efforts in this dissertation was devoted to rationalizing of size- and composition-dependent properties of the materials based on understanding of their electronic structure and chemical bonding. It was shown that understanding relations between bonding and geometric structure, bonding and stability, and bonding and reactivity is an important step toward rational design of new, yet unknown materials with unusual properties. Our findings led to the discovery of the first simplest inorganic double helix structures, which can be used in the design of novel molecular devices. A significant part of this work also deals with the pseudo John-Teller effect, which potentially can be a powerful tool for rationalizing and predicting molecular and solid state structures, their deformations, transformations, and properties. Therefore, the works on the pseudo Jahn-Teller effect presented in this dissertation can be considered the steps toward further generalization and elevation of the pseudo Jahn-Teller effect to a higher level of understanding of the origin of molecular and solid state properties.

Inhibition of angiogenesis is an effective and low toxic therapeutic avenue for the treatment of cancer patients in addition to traditional interventions. Majority of current available angiogenesis inhibitors for cancer therapies are growth factor inhibitors and small molecule tyrosine kinase inhibitors. A number of endogenous proteins and/or proteolytic fragments of extracellular matrix proteins are shown to have the activity of inhibition of angiogenesis by directly targeting endothelial cells. Structural analyses have indicated that a common structure of anti-parallel β-sheet with a highly positively charged surface presents in many of those inhibitors. This common structural feature is critical for the maintenance of their anti-angiogenic function. With this structural information, we have designed and developed a new class of anti-angiogenic proteins by integrating the short anti-parallel β-sheet forming sequences of endogenous anti-angiogenic proteins into a stable host protein, the extracellular domain-1 of cluster of differentiation 2 molecule (CD2D1). 1D 1H NMR spectra analyses indicated that the designed anti-angiogenic protein (ref to as ProAgio) folded as a β-sheet structure similar to that of the parental protein, CD2D1. ProAgio inhibited the growth of human umbilical vein cells (HUVECs) without affecting the growth of epithelial cells, suggesting a specific effect to endothelial cells. ProAgio effectively reduced endothelial tubules formed by the co-culture of HUVECs and PC3 cells on matrix gel in vitro. The designed anti-angiogenic protein was further site-specifically PEGylated in order to improve PK/PD properties and reduce immunogenicity. Examinations with PC3 xenografts showed that both ProAgio and the PEGylated ProAgio dramatically inhibited tumor growth. Immunofluorescence staining analyses of the endothelial marker CD31 indicated dramatic decreases in tumor vessels in lengths and branching points. Histological and immunofluorescence staining analyses of tissue slices of major organs indicated that there were no pathological damages to the tissue structure or disruption of normal vessels associated with the treatment of our designed anti-angiogenic agent. Overall, our studies developed a novel anti-angiogenesis agent that may have great clinical potentials. Our concept of protein design can be extended to the development of other novel protein drugs.

Enzyme structure dynamics has recently been revealed to be essential for structure-function relationship. Among various structure dynamics analysis platforms, hydrogen deuterium exchange mass spectrometry stands as an efficient and high-throughput way to analyze protein dynamics upon ligand binding, protein folding, and enzyme catalysis. HDX-MS can be used to study the regional dynamics of proteins based on the m/z value or percentage of deuterium incorporation for the digested peptides in the HDX experiments. Various software packages have been developed to analyze HDX-MS data. However, for the accurate, enhanced, and explicit statistical analysis of HDX-MS data statistical analysis of software was developed as HDXanalyzer. The capability of HDX-MS analysis for the identification of enzyme structure dynamics was tested by using model catalysis endoxylanase A (XYN I) from Trichoderma longibrachiatum. The HDX data of XYN I revealed a highly dynamic personality of XYN I through the interaction with two substrates. The dynamic data which certainly restricts the targeted regions for the protein engineering efforts provided useful knowledge about the essential structural modifications for the catalysis of XYN I. The obtained knowledge was then employed for the engineering studies in order to improve the certain characteristics of XYN I protein. The high level stabilization of XYN I protein was gathered and the two highly active and moderately thermostable XYN I recombinants were developed based on the HDX-MS data which further confirmed the efficiency of the current strategy for the rational designs of catalytic proteins. A differential dynamics analysis of the two structurally similar catalysts was also performed through HDX-MS. The functionally and sequentially different but structurally highly similar XYN I and endoglucanase (Eg1A) enzymes revealed distinct structure dynamic characteristics. Compared to XYN I, Eg1A from Aspergillus niger indicated quite restricted structural motions. The data clearly postulated that the intrinsic dynamic modifications of during the enzymatic catalysis may not be the only driving force in all cases. In summary, the integration of the structure dynamics knowledge to the current biochemical and biophysical data of catalysts may provide novel insights to further enzyme improvement applications.