Techn. Hochsch., Diss., 2002--Aachen.

Universiẗat, Diss., 2003--Kiel.

Orientadores: Sonia Maria Alves Bueno, Everson Alves Miranda / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-14T01:27:15Z (GMT). No. of bitstreams: 1 Bresolin_IaraRochaAntunesPereira_M.pdf: 5778906 bytes, checksum: fdc379e439c53f0a541cc811f65e2be1 (MD5) Previous issue date: 2009 / Resumo: Nos últimos tempos têm-se estudado plantas como biorreatores para a produção de proteínas recombinantes de interesse industrial e farmacêutico. Quando comparadas a outros sistemas de expressão, as plantas apresentam algumas vantagens, como a realização de modificações pós traducionais, baixo custo de produção, baixos riscos de contaminação por patógenos humanos, possibilidade das proteínas serem acumuladas em órgãos específicos que podem gerar maior estabilidade das mesmas. Visando a obtenção de conhecimento de base para posterior aplicação na purificação de proteínas recombinantes produzidas em plantas transgênicas, realizaram-se estudos de purificação por cromatografia negativa em Sepharose-TREN de proteínas contendo ampla faixa de pI (IgG humana), pI ácidos (HSA e BSA) e pI alcalinos (aprotinina e lisozima), adicionadas artificialmente ("spiking") a extrato de soja. Na cromatografia negativa, a proteína alvo é recuperada na fração não retida, enquanto as proteínas do extrato de soja permanecem adsorvidas na matriz. Visando maximizar a adsorção de proteínas do extrato de soja, avaliaram-se diferentes sistemas tamponantes, sendo que 2,8% das proteínas alimentadas foram detectadas na etapa de lavagem, quando se utilizou MES 25 mmol/L pH 6,5. Adicionandose IgG humana na forma de "spiking" ao extrato de soja na concentração de 1,0 mg/mL, recuperou-se 38% na lavagem com pureza de 86% e fator de purificação de 4,6. Por meio de experimentos de curva de ruptura, determinou-se a capacidade dinâmica de Sepharose- TREN como sendo 25,4 mg/mL de gel a uma vazão de 0,5 mL/min. Para proteínas de valores de pI ácidos, houve adsorção total das proteínas alimentadas enquanto que proteínas de pI alcalinos foram parcialmente adsorvidas, indicando que as interações que predominam entre o adsorvente Sepharose-TREN e proteínas são de natureza eletrostática. Para efeitos de comparação foram realizados experimentos de IMAC com íons Ni(II) e Cu(II) imobilizados em Sepharose-TREN alimentando-se extrato de soja com "spiking" de IgG humana. Resultados mostraram que a IgG apresentou pureza similar, porém com menor recuperação que em cromatografia negativa em Sepharose-TREN. Experimentos em Sepharose-DEAE foram realizados alimentando-se extrato de soja com "spiking" de IgG humana, possibilitando a adsorção de proteínas do extrato de soja e a purificação de IgG, porém com menor recuperação de IgG na lavagem quando comparado a Sepharose-TREN. / Abstract: In recent years, plants have been studied as bioreactors for the production of recombinant proteins with industrial and pharmaceutical interest. When compared to other expression systems, plants have several advantages, such as the possibility of posttranlationals modification, low production cost, low risk of contamination by human pathogens, and the accumulation of proteins in specific organs that can generate greater protein stability. Bioseparation is a key step in the bioprocessing of plant material used as biorreactors and chromatography a key unit operation in the bioseparation train. Negative chromatography - a chromatography in which the target protein is recovered in the nonretained fractions while impurities remain adsorbed in the matrix - allows product recovery in one step. Aiming to obtain the basic knowledge for further application in the purification of recombinant proteins produced in transgenic plants, in this work we evaluated the use of negative chromatography on Sepharose-TREN with spiking of proteins of wide pI range (human IgG), high pI (HSA and BSA) and low pI (aprotinin and lysozyme) added in soybean extract. Experiments using MES 25 mmol/L pH 6.5 as adsorption buffer, 2.8% of the total protein fed was found in flowthrough fractions. Adding 1.0 mg/mL of human IgG in soybean extracts, 38% of total protein was recovered in the washing step with 86% purity and purification factor of 4.6. Breakthrough curves showed that Sepharose-TREN presented a dynamic capacity of 25.4 mg of total protein per milliliter of gel at a flow rate of 0.5 mL/min. Total protein adsorption was achieved when proteins of low pI were used while proteins of high pI were partially adsorbed, showing that the interactions between the adsorbent and TREN-Sepharose proteins are electrostatic. Sepharose-TREN was used as chelating ligand in IMAC with immobilized Ni(II) and Cu(II). Experiments with soybean extract spiked with IgG as feedstream solution resulted in similar purity but a lower IgG recovery than those with Sepharose-TREN in negative chromatography. Classic ion exchanger Sepharose-DEAE adsorbed the native soybean proteins while IgG was recovered in nonretained fractions with similar purity but lower recovery than when using Sepharose-TREN. / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

Nesse trabalho foi desenvolvido um método de síntese quimioenzimática de organo-seleno aminas (1-((2, 3 ou 4 selenocianato)fenil)etanonas) e amidas (N-(1-(2, 3 ou 4-(etilseleno)fenil)etil)acetamida) enantiomericamente enriquecidas. Inicialmente, as organo-seleno aminas, na forma racêmica, foram sintetizadas a partir das orto-, meta- e para- aminoacetofenonas. A incorporação do átomo de selênio nas cetonas aromáticas foi realizada através da reação de selenocianato de potássio com sais de diazônio, preparados a partir das aminoacetofenonas, para levar as o, m ou p-selenocianato acetofenonas (28-65 %). Reações desses compostos com NaBH4, formaram os intermediários organo-selenoboro, que foram posteriormente alquilados com haletos de alquila de modo a formar as organo-seleno acetofenonas (1-(2, 3 ou 4-(etilseleno)fenil)etanona) (63-78 %). As Organo-seleno aminas racêmicas foram preparadas por aminação redutiva das cetonas correspondentes (39-73 %). Após desenvolvido o protocolo de síntese das organo-seleno aminas, nós estudamos a resolução cinética desses compostos através de reação de acetilação mediada por lipases. Um estudo inicial foi conduzido com a amina para substituído, como substrato modelo, de modo a buscar a lipase, solvente, temperatura, razão lipase/substrato e acilante apropriados para a resolução cinética. De acordo com os resultados obtidos, as condições ideais para se conduzir a resolução cinética foi CAL-B como biocatalisador, hexano como solvente e acetato de etila ou metóxi-acetato de etila como acilante a 30°C. Utilizando esse protocolo, as organo-seleno amidas foram preparadas com excelentes excessos enantioméricos (99 %). / In this work, we have developed a chemoenzymatic method to enantiomerically synthesize enriched organoselenium amines (1-(2, 3 or 4 -(ethylselanyl)phenyl)ethanamine) and amides (N-(1-(2, 3 or 4-(ethylselanyl)phenyl)ethyl)acetamide). Initially, the organoselenium amines, in the racemic form, were synthesized from ortho-, meta- and para- aminoacetophenones. The incorporation of the selenium atom into the aromatic ketones was achieved by the use of reaction of potassium selenocyanate and diazonium salts, prepared from aminoacetophenones, to afford selenocyanate acetophenones (28-65 %). These compounds were alkylated with alkyl halide to yield the organoselenium acetophenones (1-(2, 3 or 4-(ethylselanyl)phenyl)ethanone) (63-78 %) which were converted into their corresponding racemic organoselenium amines by reductive amination (39-73 %). After developing the protocol for the synthesis of racemic organoselenium amines, we studied the kinetic resolution of these compounds by their acetylation mediated by lipases. An initial study was carried out with the organoselenium amine para substituted, as a model substrate, in order to screen for appropriate lipase, solvent, temperature, lipase/substrate ratio and acylant. This study showed that the ideal condition to conduct the kinetic resolution was CAL-B as biocatalyst, hexane as solvent and ethyl acetate or ethyl methoxyacetate as acylant at 30°C. By using this protocol, the organoselenium amides were prepared in excellent enantiomeric excess (99 %).

Inhibiting the formation of ice is an essential process commercially, industrially, and medically. Compounds that work to stop the formation of ice have historically possessed drawbacks such as toxicity or prohibitively high active concentrations. One class of molecules, ice recrystallization inhibitors, work to reduce the damage caused by the combination of small ice crystals into larger ones. Recent advances made by the Ben lab have identified small molecule carbohydrate analogues that are highly active in the field of ice recrystallization and have potential in the cryopreservation of living tissue. A similar class of molecules, kinetic hydrate inhibitors, work to prevent the formation of another type of ice – gas hydrate. Gas hydrates are formed by the encapsulation of a molecule of a hydrocarbon inside a growing ice crystal. These compounds become problematic in high pressure and low temperature areas where methane is present - such as an oil pipeline. A recent study has highlighted the effects of antifreeze glycoprotein, a biological ice recrystallization inhibitor, in the inhibition of methane clathrates. Connecting these two fields through the synthesis and testing of small molecule ice recrystallization inhibitors in the inhibition of methane hydrates is unprecedented and may lead to a novel class of compounds.

<p>Since their discovery by Burg and Schlesinger in 1937, amine-boranes have enjoyed a rich preparative history and have experienced reinvigorated interest as valuable reagents for organic syntheses. Previously, the Herbert C. Brown Center for Borane Research has reported their synthesis from NaBH₄ and amines via the intermediacy of (NH₄)₂SO4 or NaHCO₃. Described herein is a CO₂-mediated amine-borane synthesis that accommodates all classes of amines, particularly long-chain trialkyl- and pyridine-like heteroarylamines.</p>

Biocatalysis is considered a green and environmentally friendly technology. Therefore, novel enzymes and enzymatic systems, together with cascades and protein engineering approaches, are in high demand. Here, three very different biocatalytic approaches have been studied. First, the richness of enzymes in the Red Sea brine pools has been assessed, and the discovery and characterization of a novel halophilic γ-carbonic anhydrase is described, together with the protein engineering approach, which boosted the initial catalytic activity of the γ- carbonic anhydrase. The understanding of polyextremophilicity principles from enzymes from the Red Sea brine pool, contributes to the bioengineering effort of turning mesophilic enzymes into more stable variants. Next, focus is given to the use of amine-transaminases in cascades for chiral amine synthesis. This resulted in the development of a self-sufficient sustainable cascade for chiral and non-chiral amine synthesis. This cascade was achieved by combining a lysine decarboxylase with an amine-transaminase to generate a cheap amino donor source for a more sustainable reaction economy. Finally, gas vesicle nanoparticles are functionalized by various engineering principles to create floating platforms for the immobilization of enzymes. The proof-of-concept was achieved by anchoring a phytase via anchoring peptides on the gas vesicle nanoparticles surface. These bioengineering approaches contributed to the effort of generating first principles for protein engineering.

The objective of this work was to synthesize functionalized mesoporous silsesquioxanes with high concentrations of amine groups. During typical sol-gel syntheses, these materials are obtained by co-condensation of organic precursors with suitable linkers, such as tetraethoxysilane, necessary to prevent the mesoporous structure from collapsing. Thus, concentrations of amine groups in organosilicas usually do not exceed 2.7-3.4 mmol g -1. The use of bridged bis-trimethoxysilanes, however, allowed formation of mesoporous materials with no linker. Polycondensation of bis-trimethoxysilanes containing amine groups was conducted in acidic, neutral and basic media, resulting in high yields of solid bridged silsesquioxanes. Gelation occurred quickly if no acid or base was added to the reaction mixture. The hybrid organic/ inorganic nature of obtained materials was confirmed by FT-IR and MAS CP NMR spectroscopy. Elemental analysis showed that amino group concentration in the products was 3.3-4.1 mmol g -1. Measurement of particle size distribution confirmed that choice of reaction media significantly affects particle sizes and agglomeration degrees, with the largest agglomerates (up to 50 μm) formed in basic media. A morphology study, using smallangle X-Ray scattering, displayed two-level fractal structures composed of aggregated 6.5-10.5 nm particles. Reactions in the presence of a surfactant resulted in formation of mesoporous structures. Furthermore, the obtained bridged silsesquioxanes were thermally stable down to 260 °C, but could reversibly absorb water and CO 2 at temperatures below 120 °C. Thus, condensation of the bridged precursor without a linker resulted in formation of a highly functionalized mesoporous material.

Thesis advisor: Masayuki Wasa / This dissertation describes the development of cooperative catalyst systems for the functionalization of monocarbonyl compounds and stereoselective transformations of alpha-C–H bonds of N-alkylamines, inspired by the concepts of frustrated Lewis pairs (FLPs). Prior to this dissertation research, practical and broadly applicable C–C and C–heteroatom bond forming reactions involving the FLP complexes that provide synthetically desirable products with high enantioselectivity remained to be developed. Chapter 1 of this dissertation describes the recent advances in the transformations involving FLPs and B(C₆F₅)₃-catalyzed reactions. Inspired by the unique capability of FLP catalysts to activate otherwise unreactive molecules, and circumvent undesirable acid–base complexation, we have developed potent cooperative acid/base catalysts for C–C bond forming reactions of various monocarbonyl compounds and an appropriate electrophile, which will be discussed in Chapter 2. Another reactivity of FLPs to be explored has to do with the catalytic and enantioselective reactions of N-alkylamines, where two Lewis acid catalysts with potentially overlapping functions, work cooperatively to activate alpha-amino C–H bonds and promote the enantioselective C–C bond forming reaction between N-alkylamines and a nucleophilic species. In Chapter 3, B(C₆F₅)₃-catalyzed union of N-alkylamines and silicon enolates followed by the enantioselective B(C₆F₅)₃/Mg–PyBOX-catalyzed alpha-alkylation of N-alkylamines and alpha,beta-unsaturated compounds to form beta-amino carbonyl compounds will be described. In Chapter 4, B(C₆F₅)₃/Cu–PyBOX-catalyzed alpha-C–H alkynylation of N-alkylamines and the applications in late-stage functionalization and stereoselective synthesis will be discussed. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

1-Decene is a valuable product in linear alpha olefins plants that is contaminated with 2-EHA (2-ethyl hexyl amine). Using organic solvent nanofiltration membranes for this separation is quite challengeable. A membrane has to be a chemically stable in this environment with reasonable and stable separation factor. This paper shows that Teflon AF 2400 and cellulose acetate produced interesting results in 1-decene/2-EHA separation. The separation factor of Teflon AF 2400 is 3 with a stable permeance of 1.1x10-2 L/(m2·h·bar). Likewise, cellulose acetate gave 2-EHA/1-decene separation factor of 2 with a lower permeance of 3.67x10-3 L/(m2·h·bar). A series of hydrophilic membranes were tested but they did not give any separation due to high degree of swelling of 2-EHA with these polymers. The large swelling causes the membrane to lose its diffusivity selectivity because of an increase in the polymer's chain mobility.