The protein alpha-synuclein (α-synuclein) accumulates in the brain in disorders such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). It is believed that the monomeric form of α-synuclein can adopt a partially folded structure and start to aggregate and form intermediately sized oligomers or protofibrils. The aggregation process can continue with the formation of insoluble fibrils, which are deposited as Lewy bodies. The oligomers/protofibrils have been shown to be toxic to neurons and are therefore believed to be involved in the pathogenesis of the actual diseases.       The overall aims of this thesis were to investigate the properties of α-synuclein oligomers and to generate and characterize antibodies against these species. In addition, the potential for immunotherapy of the α-synuclein oligomer-selective antibodies were evaluated in a transgenic mouse model with α-synuclein pathology. Stable, β-sheet rich α-synuclein oligomers were induced by incubation with either one of the reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). The oligomers exhibited distinct morphological properties, although both types were toxic when added to a neuroblastoma cell line. The seeding effects of ONE-induced oligomers were studied in vitro and in vivo. The oligomers induced seeding of monomeric α-synuclein in a fibrillization assay but not in a cell model or when injected intracerebrally in transgenic mice. It seemed, however, as if the oligomers affected α-synuclein turnover in the cell model. By immunizing mice with HNE-induced oligomers antibody producing hybridomas were generated. Three monoclonal antibodies were found to have strong selectivity for α-synuclein oligomers. These antibodies recognized Lewy body pathology in brains from patients with PD and DLB as well as inclusions in the brain from young α-synuclein transgenic mice, but did not bind to other amyloidogenic proteins. Finally, immunotherapy with one of the oligomer/protofibril selective antibodies resulted in lower levels of such α-synuclein species in the spinal cord of α-synuclein transgenic mice. To conclude, this thesis has focused on characterizing properties of α-synuclein oligomers. In particular, antibodies selectively targeting such neurotoxic forms were generated and evaluated for passive immunization in a transgenic mouse model. Such immunotherapy may represent a future treatment strategy against Lewy body disorders.

Direct generation of enantioenriched mono-α-alkylated aldehydes by intermolecular nucleophilic substitution is a general and long-standing problem in synthesis, and is of importance due to the diverse reactions such aldehydes undergo for introducing asymmetry into molecules. The work described in this thesis initially details the development of the first lithium amide capable of efficiently converting terminal epoxide into enamine functionality, where the latter also demonstrates effective C-alkylation activity. Not only addition to Michael acceptors, but more notably substitution using activated organohalides (α-bromoacetates, benzyl, allyl and propargyl bromide) gave the corresponding α-substituted aldehydes in good to excellent yields. Alkylation with propargyl bromide yielded only the propargyl-substituted aldehyde with none of the corresponding allene observed; this result shows that N-alkylation followed by [3,3] sigmatropic rearrangement is not occuring. Importantly, a range of short-, longer-chain and secondary unactivated alkyl iodides also proved viable. Significantly, with chiral lithium amides, the corresponding chiral enamines could be alkylated with strongly electrophilic benzyl, allyl and propargyl (no allene seen) bromides in very good yields, and with short chain alkyl iodides – MeI and EtI in satisfactory yields, to provide the first direct access to α-alkylated aldehydes with high asymmetric induction by intermolecular nucleophilic substitution.

Globalmente, os problemas de poluição são mais severos em regiões densamente povoadas ou industrializadas. A Região Metropolitana de São Paulo (RMSP) é um exemplo de megalópole com um conjunto único de emissões, insolação e condições meteorológicas que determinam sua elevada poluição atmosférica. Entre os compostos mais tóxicos presentes nesta atmosfera estão os aldeídos, moléculas de grande potencial mutagênico e carcinogênico que podem ser originados da oxidação de combustíveis fósseis e etanol, além de possuir fontes endógenas. Sua adição covalente em biomoléculas como DNA e proteínas é estudada como mecanismo de sua ação mutagênica e carcinogênica. Desenvolvemos um método ultrassensível de HPLC acoplado à espectrometria de massas para análise simultânea de vários adutos de DNA com aldeídos presentes na atmosfera urbana como formaldeído, acetaldeído, crotonaldeído e acroleína, além de modificações oxidativas. Foram utilizados para validação da metodologia células modelo de Anemia Fanconi e tecido de músculo de ratos modelo para ELA (Esclerose Lateral Amiotrófica), os quais apresentaram níveis basais dos adutos de formaldeído, acetaldeído, acroleína e crotonaldeído. Outros modelos de exposição aos aldeídos foram utilizados na validação desta metodologia, como a fumaça de cigarro de tabaco e de cannabis sendo que a formação desses adutos também foi verificada em DNA de timo de bezerro. Pela primeira vez, foi mostrada a formação inequívoca do aduto de acetaldeído (1,N2-propanodGuo) em pulmões e cérebros de ratos Wistar expostos à 10 ppb de acetaldeído isotopicamente marcado e sua estrutura confirmada por espectrometria de massas de alta resolução. Esse resultado comprova o mecanismo de formação do aduto por meio da adição de duas moléculas de acetaldeído in vivo por inalação e em baixa concentração, sendo crucial para formulação de políticas públicas por agências ambientais. / Globally, air pollution problems are more severe in densely populated or industrialized regions. The Metropolitan Region of São Paulo (RMSP) is an example of a megalopolis with a unique set of emissions, insolation and meteorological conditions that determinates its high atmospheric pollution. Among the most toxic compounds present in this atmosphere are aldehydes, molecules of great mutagenic and carcinogenic potential that can be originated from the oxidation of fossil fuels and ethanol, besides having endogenous sources. Its covalent addition in biomolecules like DNA and proteins is studied as a mechanism of its mutagenic and carcinogenic action. We developed an ultra-sensitive HPLC method coupled with mass spectrometry for the simultaneous analysis of several DNA adducts with aldehydes present in the urban atmosphere as formaldehyde, acetaldehyde, crotonaldehyde and acrolein and oxidative lesions as well. To validate the method, we used a cell model of Fanconi Anemia and muscle tissue from a rat model for ALS (Amyotrophic Lateral Sclerosis) which presented basal levels of the adducts of formaldehyde, acetaldehyde, acrolein and crotonaldehyde. Other models of exposure to aldehydes were used to validate the method, such as tobacco and cannabis smoke and the formation of these adducts was also verified in Calf Thymus DNA. For the first time, the unequivocal formation of the acetaldehyde (1, N2-propanodGuo) adduct was shown in lungs and brains of Wistar rats exposed to 10 ppb of isotopically labeled acetaldehyde and its structure was confirmed by high resolution mass spectrometry. This result confirms the mechanism of adduct formation by the addition of two molecules of acetaldehyde in vivo by inhalation of a low concentration of acetaldehyde, being crucial for the formulation of public policies by environmental agencies.

A importância que a catálise representa para a sociedade pode ser vista em números: 90% dos processos da indústria química e mais de 20% de todos os produtos industriais comercializados no mundo utilizam uma ou mais etapas catalíticas. Assim, desenvolver catalisadores eficientes, ativos e seletivos é a solução para criar tecnologias mais limpas e sustentáveis. Além disso, reações químicas que geram novas ligações C-C estão entre as transformações mais relevantes na química orgânica e são a base desse trabalho. Os catalisadores de ródio apresentados aqui fazem parte de um trabalho minucioso de desenvolvimento, síntese e caracterização de nanopartículas e suportes magnéticos funcionais que foram utilizados em transformações de diversas moléculas. O estudo inicial com nanopartículas de ródio suportadas, em reações de hidrogenação do cicloexeno, serviu para a compreensão de como se comportam essas nanoestruturas e da influência que diferentes ligantes orgânicos e estabilizantes podem ter em uma aplicação catalítica bastante conhecida. O sistema catalítico mostrou-se bastante ativo e reutilizável,despertando o nosso interesse ao seu aperfeiçoamento para aplicação em reações de hidroformilação. Antes da síntese de catalisadores suportados, estudos com nanopartículasnão-suportadas mostraram que um sistema modificado pela adição de fosfinas era necessário para ativação do catalisador e que o estabilizante utilizado afetava a atividade catalítica. Assim, para possibilitar o ancoramento eficiente das espécies ativas, uma modificação da superfície do suporte magnético com a metildifenilfosfina foi realizada. A fosfina funcionalizada sobre o suporte viabilizou sua interação com as espécies ativas e evitou a sua lixiviação, possibilitando o reuso do catalisador. A reação de hidroformilação do oct-1-eno atingiu 96% de conversão e 82% de seletividade para aldeídos, em 6 horas a 80°C. A carga metálica do catalisador foi de apenas 0,2%. Buscando aumentar a eficiência na etapa de imobilização do metal e uma melhor atividade catalítica que possibilitasse o uso de substratos mais complexos, o suporte magnético foi modificado com um polímero hiper-ramificado. Essa modificação possibilitou aumentar a quantidade de grupos fosfinas sobre o suporte, assim como levou a um significativo aumento na carga de metal. A reação de hidroformilação de produtos naturais foi possível e, com o composto estragol, conversões de 100% foram alcançadas em 6 horas, com seletividade de 70% para aldeídos. Mesmo com evidências que sugerem a formação de espécies ativas moleculares, o suporte modificado possibilitou que o catalisador mantivesse sua atividade e seletividade por pelo menos seis reações sucessivas. Os materiais desenvolvidos apresentaram estabilidade quando manuseados ao ar, sem prejudicar sua vida útil e fácil separação. / The importance of catalysis to society may be seen in numbers: 90% of chemical production processes and more than 20% of all industrial products sold in the world use one or more catalytic steps. Thus, the development of efficient, active, and selective catalysts is crucial for creating cleaner and sustainable technologies. In addition, chemical reactions that generate new C-C bonds are among the most important transformations in organic chemistry and are the basis of this work. Rhodium catalysts presented herein are part of a careful investigation, which included the development, synthesis and characterization of metal nanoparticles and magnetic functional supports for use in the transformation of various molecules. The initial study of supported rhodium nanoparticles in cyclohexene hydrogenation reactions has driven our understanding of the behavior of these nanostructures, and the influence that different ligands and stabilizers may have in a well-known catalytic application. The identification of a highly active and recyclable catalytic system aroused our interest for its improvement for application in hydroformylation reactions. Prior to the synthesis of supported catalysts, studies with non-supported nanoparticles revealed that a modified system with the addition of phosphines was required for activation of the catalyst and the stabilizer used affected the catalytic activity. Thus, to enable efficient immobilization of the active species, the surface of the magnetic support was modified with methyldiphenylphosphine. The catalyst preparation removed, at least partially, the stabilizer adsorbed on the nanoparticles surfaces. The phosphine-functionalized support anchored the active species and avoided their leaching, allowing the reuse of the catalyst. The hydroformylation reaction of oct-1-ene reached 96% of conversion and 82% of selectivity to aldehydes, in 6 hours at 80°C. The metal loading of the catalyst was only 0.2%. Seeking to increase the efficiency in metal immobilization step and a better catalytic activity that would enable the use of more complex substrates, the magnetic support was modified with a hyperbranched polymer, which allowed an increase in the amount of external phosphines, as well as a significant increase in metal loading on the support. The hydroformylation reaction of natural products was possible and, with the estragole compound, 100% of conversion was achieved in 6 hours with 70% of selectivity to aldehydes. Despite evidence that suggests the formation of active molecular species, the modified support has enabled the catalyst to retain its activity and selectivity for at least six successive reactions. The materials developed could be handled in air without damaging their catalytic activity, durability and separation properties.

As reações de adição aldólica entre a cetimina 1 e aldeídos aromáticos foram inicialmente efetuadas à temperatura ambiente, em sistema bifásico constituído por uma fase aquosa básica (KOH 10% ou NaOH 5% m/v) e por uma fase orgânica (aldeído), na ausência de solventes e de catalisadores, observando-se baixa conversão em produto. Porém, quando se utilizou o catalisador aliquat®-336, foi possível reduzir a concentração da base (NaOH 1%), com conversão total da imina em produto que, na maioria dos casos, era uma mistura de duas oxazolidinas isoméricas de estereoquímica cis e trans. Esses compostos puderam ser isolados e purificados por recristalização de etanol ou metanol. Em todas as reações efetuadas com benzaldeído, m-clorobenzaldeído e p-nitrobenzaldeído, não se observou excesso diastereomérico significativo. No entanto, as reações com p-clorobenzaldeído mostraram-se diastereosseletivas, conduzindo, à temperatura ambiente, quase que exclusivamente à oxazolidina de estereoquímica cis. A comparação entre o resultado de reações efetuadas a curto e longo tempo de reação, ou em diferentes temperaturas, permitiu concluir que o aldol de estereoquímica anti é o produto cinético, o qual se transforma lentamente na oxazolidina cis. O produto termodinâmico (aldol syn) cicliza rapidamente, não sendo observado nos espectros de RMN de H dos produtos brutos de reação, mas sim seu produto ciclizado, a oxazolidina trans. Tentativas de obter os produtos de reação com excesso enantiomérico, pelo emprego de catalisadores de transferência de fase assimétricos, não foram bem sucedidas. / The aldol addition reactions of ketimine 1 with four aromatic aldehydes could be performed at room temperature, in the presence of an alkaline aqueous solution (KOH 10% or NaOH 5% m/v), and in the absence of organic solvents or catalysts. Under this conditions, conversion to product was often incomplete. However, when the reaction was performed in the presence of aliquat®-336, the base concentration could be reduced (1% m/v), and the ketimine was completely converted into a mixture of diastereomeric oxazolidines, that could be isolated and purified by crystallization from ethanol or methanol. Although no significative diastereomeric excess was observed for the reactions of 1 with benzaldehyde, m-chlorobenzaldehyde or p-nitrobenzaldehyde, for reactions with p-chlorobenzaldehyde the cis oxazolidine was almost exclusively formed. The comparison of diastereomeric ratio at shorter or longer reaction times seems to indicate that the aldol anti adduct is the kinetic product and that cyclization to the corresponding cis oxazolidine is slower than the retro-aldol reaction. On the other hand, in lieu of the thermodynamic product (aldol syn), only the corresponding cyclized product (oxazolidine trans) could be visualized in the H NMR spectra of the crude product, as a result of a fast cyclization step. Attempts to produce enantiomeric enriched oxazolidines by performing the aldol reactions in the presence of chiral catalysts were unsuccessful.

The market of medicinal plants and herbs such as cilantro (Coriandrum sativum) and stevia (Stevia rebaudiana) has been growing due to the demand of the food, chemical and pharmaceutical industries. Leaves of C. sativum are used by many countries in the preparation of food and in folk medicine. The essential oil from the leaves has potential for use in the food industry as an antimicrobial agent and as a condiment to mask undesirable odors and flavors. Studies were conducted to evaluate the activity of the essential oil of cilantro against Leishmaniasis, and the results showed that the essential oil and its major compounds inhibited the growth of L. donovani in its different forms, indicating potential use of this plant for the production of new drugs against Leishmaniasis. From these results, another study was conducted to evaluate five materials of cilantro, a cultivar and four germplasm accessions from the North American Department of Agriculture regarding the quality of the raw material for the food and pharmaceutical industries. The materials with the highest potential to use were AMES 18596 and PI193770 for producing larger amount of dry biomass and the highest production of major compounds, which are desirable by industries. Stevia is a species with high potential to be consumed by diabetics and obese, it is a source of diterpene glycosides used as natural sweeteners. Sweeteners derived from stevia are ingredients in the production of foods, juices and soft drinks in Asia, Europe and in the Americas. The compounds responsible for the sweetness of stevia leaves are well characterized in the literature. However, good agronomic practices are still poorly studied. Some countries have worked on improving this culture and have achieved cultivars with higher yield. In the area of plant nutrition, calcium is one of the most important nutrients for the production of biomass and stevioside in stevia plants. Thus, a preliminary study was conducted to test the effect of calcium applied in nutrient solution in stevia yield, and stevioside and rebaudioside in hydroponic system. The leaf, stem and shoot biomass yield, by stevia plants were influenced by Ca rates in nutrient solutions and were maximal with 7.0, 5.7, 6.2 mmol L-1 of Ca, respectively. The production of stevioside and rebaudioside A was maximal with addition of 4.0 mmol of Ca L-1. / O mercado de plantas medicinais, condimentares e aromáticas tais como coentro (Coriandrum sativum) e estévia (Stevia rebaudiana) vêm crescendo devido à demanda das indústrias alimentícias, químicas e farmacêuticas. As folhas C. sativum são utilizadas por diversos países no preparo de alimentos e na medicina popular. O óleo essencial das folhas tem potencial para utilização na indústria de alimentos como agente antimicrobiano e como condimento para mascarar cheiros e sabores indesejáveis. Estudos foram realizados para avaliar a atividade do óleo essencial de coentro contra a leishmaniose, e os resultados revelaram que o óleo essencial e seus compostos majoritários inibiram o crescimento L. donovani em suas diferentes formas, indicando potencial de uso dessa planta para a produção de novos medicamentos contra Leishmaniose. A partir desses resultados, outro estudo foi realizado com o objetivo de avaliar cinco materiais de coentro, uma cultivar e quatro acessos do germoplasma do Departamento de Agricultura norte americano quanto à qualidade da matéria prima para as indústrias de alimentos e farmacêuticas. Os materiais variaram entre si quanto à produção de biomassa, acúmulo de nutrientes e produção de aldeídos alifáticos tais como (E)- 2-decenal, (E)- 2-dodecenal. Os materiais que apresentaram maior potencial de uso foram AMES 18596 e PI193770, pois, produziram maior quantidade de massa seca e produção de compostos majoritários, que são os desejáveis pelas indústrias. Quanto à estévia, essa espécie apresenta elevado potencial para ser usado por pessoas diabéticas e obesas, pois é fonte de diterpenos glicosídicos usados como adoçantes naturais. Os adoçantes derivados de estévia são usados na produção de alimentos, sucos e refrigerantes em diversos países asiáticos, europeus e no continente americano. Os compostos responsáveis pelo dulçor das folhas de estévia são bem caracterizados pela literatura, entretanto, as boas práticas agronômicas ainda são pouco estudadas. Alguns países trabalharam no melhoramento dessa cultura e conseguiram alcançar cultivares mais produtivas. Na área de nutrição, o cálcio está entre os nutrientes mais importantes para a produção de esteviosídeo e produção de biomassa de estévia. Assim, um estudo preliminar foi conduzido com o objetivo de estudar o efeito do cálcio aplicado em solução nutritiva na produção de estévia, e de esteviosídeos e rebaudiosídeos, em sistema hidropônico. A produção de folhas, caule e parte aérea por plantas de estévia foi influenciada por níveis de Ca na solução nutritiva e foram máximos com as doses de 7,0, 5,7, 6,2 mmol L-1 de Ca, respectivamente. A produção de esteviosídeo e rebaudiosídeo A foi máxima com a adição de 4,0 mmol L-1 de Ca.

O colesterol é um importante componente das membranas celulares em eucariotos superiores, desempenhando papéis estruturais e funcionais. O colesterol possui uma insaturação em sua estrutura sendo, portanto, alvo de oxidação mediada por espécies reativas de oxigênio e/ou nitrogênio. A oxidação não enzimática do colesterol gera, como produtos primários, os hidroperóxidos de colesterol. Tais moléculas, por sua vez, são altamente reativas e podem reagir com metais livres e/ou metaloproteínas, trazendo consequências à celula. Neste sentido, o primeiro capítulo deste trabalho tem como objetivo estudar a reação dos hidroperóxidos de colesterol (ChOOH) com o citocromo c (citc), uma heme proteína envolvida no transporte de elétrons na mitocôndria. Análises de espectroscopia no UV-Vis mostraram que o ChOOH promove o bleaching da banda Soret do citc de uma maneira dose-dependente. Mais ainda, esta reação leva à formação de radicais centrados em carbono tanto na proteína como no lipídeo, sugerindo uma redução homolítica do ChOOH. Como consequências, pode-se observar a oligomerização do citc, um processo que pode influenciar no transporte de elétrons bem como na sinalização para a apoptose. A partir da reação do citc com ChOOH podem surgir, direta ou indiretamente, outras espécies reativas, como aldeídos, cetonas e epóxidos. Dentre estas, destacam-se os aldeídos de colesterol, em particular o colesterol secoaldeído (CSec) e o carboxialdeído (ChAld), uma vez que foram encontrados elevados em placas ateroscleróticas e em tecidos cerebrais de pacientes com doenças neurodegenerativas. Tais espécies podem reagir com resíduos de aminoácidos provocando alterações estruturais e funcionais em proteínas. Neste sentido, o segundo capítulo deste trabalho tem como objetivo estudar a reação do ChAld com citc. Usando modelos mimétivos de membrana e espectrometria de massas, foi mostrado que o ChAld modifica covalentemente o citc por um mecanismo consistente com a formação de bases de Schiff. Tal modificação ocorre preferencialmente em resíduos de lisina que interagem com a membrana. Estas modificações influenciam na afinidade do citc pela membrana, aumentando sua aderência, o que pode ter influência no transporte de elétrons e sinalização para a apoptose. No terceiro e último capítulo deste trabalho nós buscamos uma ferramente analítica que permitisse analisar modificação de proteínas promovidas por produtos de oxidação de colesterol e outros esteróis. Em um estudo realizado em colaboração com o grupo do professor Porter na Universidade de Vanderbilt, utilizamos ensaios baseados em click chemistry para buscar proteínas modificadas. Para isso, foram sintetizados derivados de colesterol e 7-deidrocolesterol (7-DHC, precursor imediato do colesterol) contendo um grupo alquinil na sua cadeia lateral. Este grupo pode ser ligado a um grupo azida por meio de uma reação de cicloadição, em um processo conhecido como click chemistry. Após a síntese e caracterização dos derivados lipídicos contendo o grupo alquinil na cadeia lateral, células Neuro2a foram tratadas com o alquinil-7-DHC e o alquinil-colesterol para averiguar seu metabolismo. Análises por HPLC-MS/MS mostraram que ambos derivados contendo o grupo alquinil foram metabolisados e convertdos nos respectivos ésteres. Usando um modelo celular para a doença conhecida como Sindrome de Smith-Lemli-Opitz (SLOS), doença caracterizada pela deficiência na enzima 7-deidrocolesterol redutase, foi mostrado que o acúmulo característico de 7-DHC nos pacientes pode levar a uma maior modificação de proteínas promovidas por seus derivados, o que pode contribuir para o desenvolvimento da doença. / Cholesterol is an important component of eukaryotic cellular membranes, where it has an influence in the fluidity and stability. Due to the presence of a double bond in its structure, cholesterol can be oxidized by reactive oxygen and nitrogen species. This non-enzymatic oxidation generates, as primary products, cholesterol hydroperoxides. Such molecules, in turn, are highly reactive and can react with free metal ions and/or metalloproteins, affecting cell metabolism. Therefore, the first chapter of the present study aims to investigate the reaction of cholesterol hydroperoxides (ChOOH) with cytochrome c (cytc), a heme protein involved in the mitochondrial electron transport. Spectroscopic analyses in the UV-Vis region showed that ChOOH induces a dose-dependent bleaching of cytc\'s Soret band. In addition, this reaction leads to the formation of carbon-centered radicals on both protein and lipid, suggesting a homolytic reduction of ChOOH. As consequences, cytc undergoes oligomerization, a process that can influence electron transport and apoptosis signaling. The reaction of cytc and ChOOH can produce, directly or indirectly, reactive species such as epoxides, aldehydes and ketones. Among them, cholesterol aldehydes, such as cholesterol secoaldehyde (CSec) and cholesterol carboxyaldehyde (ChAld), are of particular interest, since they were previously found elevated in atherosclerotic plaques and brain tissue of patients bearing neurodegenerative diseases. These species can also react with amino acid residues leading to protein denaturation and malfunction. With that in mind, the second chapter of this study aims to investigate the reaction of ChAld and cytc. Using mimetic membrane models and mass spectrometry analyses, we showed that ChAld covalently modifies cytc through a mechanism consistent with the formation of Schiff base adducts. Such modification occurs mostly at lysine residues that are known to interact with the membrane. The modifications have an influence in the affinity of cytc to the membrane, where they increase its binding to the membrane, a process that could affect the electron transport and apoptosis signaling. In the last and third chapter of this study we wanted an analytical tool that allowed the investigation of protein adduction promoted by cholesterol and other sterols-derived oxidation products. In a study performed in collaboration with the Porter group from Vanderbilt University, we used analyses based on click chemistry to search for protein adduction. To address that, we first synthesized derivatives of cholesterol and 7-dehydrocholesterol (7-DHC, the immediate precursor of cholesterol) containing an alkynyl group in the side chain. The alkynyl group can be ligated to an azide group through a cycloaddition reaction, in a process known as click chemistry. After the synthesis and characterization of alkynyl derivatives, Neuro2a cells were treated with alkynyl-7-DHC and alkynyl-cholesterol to check their metabolism. HPLC-MS/MS analyses showed that both alkynyl derivatives are metabolized and converted into their respective esters. In addition, using a cell model for Smith-Lemli-Optiz Syndrome (SLOS), a disease characterized by the deficiency in the dehydrocholesterol reductase 7, we showed that the characteristic accumulation of 7-DHC in SLOS patients might be associated with protein adduction promoted by its oxidation products, which might contribute to the development of the disease.

本論文主要探討銠卟啉和銥卟啉絡合物，分別與酮類與醛類進行的鍵活化化學。 / 第一部分主要介紹由β-乙基羥基銠卟啉絡合物(Rh{U+1D35}{U+1D35}{U+1D35}(ttp)CH₂CH₂OH)與酮類進行的羰基碳及α-碳(C(CO)-C(α)) 鍵活化(下稱碳碳鍵活化)。於室溫至50ºC時，在非溶劑的條件下，Rh{U+1D35}{U+1D35}{U+1D35}(ttp)CH₂CH₂OH選擇性地斷裂芳香酮和脂肪酮類的C(CO)-C(α)鍵，生成相對應的銠卟啉酰基絡合物(Rh{U+1D35}{U+1D35}{U+1D35}(ttp)COR, R = 烷基或芳基)，產率最高可達80%。作為銠卟啉羥基絡合物(Rh{U+1D35}{U+1D35}{U+1D35}(ttp)OH)的前體，Rh{U+1D35}{U+1D35}{U+1D35}(ttp)CH₂CH₂OH的活性展示出Rh{U+1D35}{U+1D35}{U+1D35}(ttp)OH是碳碳鍵活化的重要中間體。 / 第二部分主要介紹由β-乙基羥基銥卟啉絡合物(Ir{U+1D35}{U+1D35}{U+1D35}(ttp)CH₂CH₂OH)與芳香醛類進行，具選擇性的醛碳氫鍵活化。在160ºC和非溶劑的條件下，Ir{U+1D35}{U+1D35}{U+1D35}(ttp)CH₂CH₂OH與芳香醛類反應，生成相對應的銥卟啉酰基絡合物(Ir{U+1D35}{U+1D35}{U+1D35}(ttp)COAr)作為碳氫鍵活化產物，產率最高可達72%。銥卟啉羥基絡合物(Ir{U+1D35}{U+1D35}{U+1D35}(ttp)OH)和乙烯配位銥卟啉絡合正離子((CH₂=CH₂)Ir{U+1D35}{U+1D35}{U+1D35}(ttp)⁺)被推斷為醛碳氫鍵活化的可能中間體。 / This research focuses on the bond activation chemistry by rhodium and iridium porphyrin complexes with ketones and aldehyde respectively. / Part 1 describes the C(CO)-C(α) bond activation (CCA) of ketones by Rh{U+1D35}{U+1D35}{U+1D35}(ttp)CH₂CH₂OH (ttp = 5,10,15,20-tetratolylporphyrinato dianion). Rh{U+1D35}{U+1D35}{U+1D35}(ttp)- CH₂CH₂OH selectively cleaved the C(CO)-C(α) bond of aromatic and aliphatic ketones in solvent-free conditions at room temperature to 50ºC, giving the corresponding rhodium(III) porphyrin acyls (Rh{U+1D35}{U+1D35}{U+1D35}(ttp)COR, R = alkyl or aryl) up to 80% yield. The activity of the Rh{U+1D35}{U+1D35}{U+1D35}(ttp)OH precursor, Rh{U+1D35}{U+1D35}{U+1D35}(ttp)CH₂CH₂OH, demonstrates Rh{U+1D35}{U+1D35}{U+1D35}(ttp)OH as the key intermediate in the CCA of ketones. / [With images]. / Part 2 describes the selective aldehydic carbon-hydrogen bond activation (CHA) of aryl aldehydes by Ir{U+1D35}{U+1D35}{U+1D35}(ttp)CH₂CH₂OH. Ir{U+1D35}{U+1D35}{U+1D35}(ttp)CH₂CH₂OH reacted with aryl aldehydes in solvent-free conditions at 160ºC to give the corresponding iridium(III) porphyrin acyls (Ir{U+1D35}{U+1D35}{U+1D35}(ttp)COAr) as the CHA products up to 72% yield. Ir{U+1D35}{U+1D35}{U+1D35}(ttp)OH and (CH₂=CH₂)Ir{U+1D35}{U+1D35}{U+1D35}(ttp)⁺ were proposed as the possible intermediate for the CHA reaction. / [With images]. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chan, Chung Sum. / "November 2012." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references. / Abstracts also in Chinese. / Abstract --- p.i / Acknowledgement --- p.iii / Table of Contents --- p.iv / Abbreviations --- p.vii / Structural Abbreviations of Porphyrin --- p.viii / Chapter Part 1 --- Carbon-Carbon Bond Activation of Ketones with Rhodium(III) Porphyrin β-Hydroxyethyl --- p.1 / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Properties of Ketones --- p.1 / Chapter 1.2 --- Carbon(CO)-Carbon(α) Bond Activation (CCA) of Ketones --- p.2 / Chapter 1.2.1 --- CCA of Ketones by Transition Metal Complexes --- p.2 / Chapter 1.2.2 --- CCA of Ketones by Metalloporphyrins --- p.5 / Chapter 1.3 --- Porphyrin Ligands and Rhodium(III) Porphyrins --- p.7 / Chapter 1.3.1 --- Porphyrin Ligands --- p.7 / Chapter 1.3.2 --- Rhodium(III) Porphyrins --- p.8 / Chapter 1.4 --- Rhodium(III) Porphyrin Hydroxide --- p.10 / Chapter 1.4.1 --- Nature of Bonding in Late Transition Metal Hydroxides --- p.10 / Chapter 1.4.1.1 --- Hard-Soft Acid-Base principle --- p.11 / Chapter 1.4.1.2 --- dπ-pπ Interaction Model --- p.11 / Chapter 1.4.1.3 --- E-C Model --- p.12 / Chapter 1.4.2 --- Attempted Preparation of Rhodium(III) Porphyrin Hydroxides --- p.13 / Chapter 1.4.3 --- Chemistry of Rhodium(III) Porphyrin Hydroxides --- p.15 / Chapter 1.5 --- Rhodium(III) Porphyrin β-hydroxyethyl as Rhodium(III) Hydroxide Precursor --- p.18 / Chapter 1.6 --- Objective --- p.20 / Chapter Chapter 2 --- Carbon-Carbon Bond Activation of Ketones with Rhodium(III) Porphyrin β-Hydroxyethyl --- p.21 / Chapter 2.1 --- Preparation of Starting Materials --- p.21 / Chapter 2.1.1 --- Synthesis of Porphyrin --- p.21 / Chapter 2.1.2 --- Synthesis of Rhodium(III) Porphyrins --- p.21 / Chapter 2.2 --- CCA of Diisopropyl Ketone by Rh{U+1D35}{U+1D35}{U+1D35}(ttp)CH₂CH₂OH --- p.22 / Chapter 2.3 --- Optimization of Reaction Conditions --- p.22 / Chapter 2.3.1 --- Atmosphere Effect --- p.22 / Chapter 2.3.2 --- PPh3 Effect --- p.23 / Chapter 2.3.3 --- Solvent Effect --- p.24 / Chapter 2.4 --- Substrate Scope --- p.26 / Chapter 2.4.1 --- CCA of Isopropyl Ketones --- p.26 / Chapter 2.4.2 --- CCA of Non-Isopropyl Ketones --- p.28 / Chapter 2.5 --- Proposed Mechanism --- p.29 / Chapter 2.6 --- Comparison on CCA of Ketones by Different Rh{U+1D35}{U+1D35}{U+1D35}(por)OH Sources --- p.31 / Chapter 2.6.1 --- Reaction Conditions --- p.31 / Chapter 2.6.2 --- Substrate Scope --- p.32 / Chapter 2.6.3 --- Regioselectivity --- p.33 / Chapter 2.7 --- Comparison on Bond Activation of Carbonyl Compounds by Rhodium Porphyrin β-Hydroxyethyl --- p.34 / Chapter 2.8 --- CCA of Ketones with Ir{U+1D35}{U+1D35}{U+1D35}(ttp)CH₂CH₂OH --- p.36 / Chapter 2.9 --- Conclusion --- p.37 / Chapter Chapter 3 --- Experimental Sections --- p.39 / References --- p.54 / List of Spectra I --- p.59 / Spectra --- p.60 / Chapter Part 2 --- Aldehydic Carbon-Hydrogen Bond Activation with Iridium(III) Porphyrin β-Hydroxyethyl --- p.63 / Chapter Chapter 1 --- Introduction --- p.63 / Chapter 1.1 --- Properties of Aldehydes --- p.63 / Chapter 1.2 --- Carbon-Hydrogen Bond Activation (CHA) of Aldehydes --- p.64 / Chapter 1.2.1 --- CHA of Aldehydes by Transition Metal Complexes --- p.64 / Chapter 1.2.2 --- Aldehydic CHA by Metalloporphyrins --- p.74 / Chapter 1.3 --- Iridium(III) Porphyrins --- p.77 / Chapter 1.4 --- Iridium(III) Porphyrin Hydroxide --- p.78 / Chapter 1.4.1 --- Attempted Preparation of Iridium(III) Porphyrin Hydroxides --- p.78 / Chapter 1.4.2 --- Chemistry of Iridium(III) Porphyrin Hydroxides --- p.81 / Chapter 1.5 --- Iridium(III) Porphyrin β-hydroxyethyl as Iridium(III) Hydroxide Precursor --- p.83 / Chapter 1.6 --- Objective --- p.85 / Chapter Chapter 2 --- Aldehydic Carbon-Hydrogen Bond Activation with Iridium(III) Porphyrin β-Hydroxyethyl --- p.86 / Chapter 2.1 --- Preparation of Iridium(III) Porphyrins --- p.86 / Chapter 2.2 --- Aldehydic CHA of Benzaldehyde by Ir{U+1D35}{U+1D35}{U+1D35}(ttp)CH₂CH₂OH --- p.87 / Chapter 2.3 --- Optimization of Reaction Conditions --- p.87 / Chapter 2.3.1 --- Temperature Effect --- p.87 / Chapter 2.3.2 --- Solvent Effect --- p.88 / Chapter 2.3.3 --- PPh₃ Effect --- p.90 / Chapter 2.4 --- Substrate Scope --- p.93 / Chapter 2.5 --- Proposed Mechanism --- p.94 / Chapter 2.6 --- Conclusion --- p.96 / Chapter Chapter 3 --- Experimental Sections --- p.97 / References --- p.108 / List of Spectra II --- p.112 / Spectra --- p.112

Intracellular polymerization of deoxygenated sickle hemoglobin (Hb S) remains the principal cause of the pathophysiology associated with sickle cell disease (SCD). Naturally occurring and synthetic allosteric effectors of hemoglobin (AEH) have been investigated as potential therapeutic agents for the treatment of SCD. Several aromatic aldehydes, including vanillin, have been studied previously as AEHs for their antisickling activities. Specifically, these compounds form Schiff- base adduct with Hb to stabilize the oxygenated (R) state, increase Hb affinity for O2 and concomitantly prevent the hypoxia-induced primary pathophysiology of Hb S polymerization and RBC sickling, in turn, ameliorating several of the cascading secondary adverse effects. These compounds, however, undergo significant metabolism leading to suboptimal pharmacokinetic properties, e.g. short duration of pharmacologic action and low bioavailability. These drawbacks have severely hampered the use of aromatic aldehydes as AEHs to treat SCD. To counter these challenges, we designed and synthesized 14 novel compounds (PP- compounds) based on previously studied pyridyl derivative of vanillin. These modifications were expected to increase binding interactions with the protein and thus stabilize the Schiff-base adduct, as well as lead to perturbation of the surface-located F-helix that would stereospecifically destabilize polymer contacts. We investigated the in vitro pharmacokinetic/pharmacodynamic properties of these newly synthesized compounds to ascertain sustained binding and modification of normal human Hb. Subsequently, we conducted in vitro screening assays to test for inhibition of sickling, modification of Hb to the high-affinity form, as well as for a direct left-shift in oxygen equilibrium curves (OEC). Three selected compounds, PP6, PP10, and PP14, that demonstrated not only high antisickling activity but also showed sustained pharmacologic action were chosen for preliminary in vivo studies. Our results showed maximal levels of Hb modification, which were sustained for the entire 24 h experimental period. In contrast, TD-7 after reaching maximum effect at 1 h gradually decreased in potency and at 24 h has lost 45% of its activity, consistent with the low bioavailability of this compound. These findings suggested that our modifications appeared to successfully limit drug metabolism in red blood cells. Most of these compounds showed almost complete inhibition of sickling at 2 mM concentration; with significant modification of Hb to a higher affinity Hb as well as an increase in O2 affinity of Hb. Interestingly, while TD-7 demonstrated a clear linear correlation between its ability to increase Hb-O2 affinity and antisickling activity, PP2, PP3, PP6, PP9, PP10, and PP14, showed a weak correlation between these parameters. In fact, these compounds demonstrated high antisickling effect despite only marginally increasing Hb affinity for O2. This observation indicated that these compounds possibly exhibit the dual mechanism of antisickling activity. We hypothesize that their secondary mechanism of action is due to interactions with the surface located αF-helix that would lead to direct or stereospecific inhibition of polymer formation. To establish the mode of interaction with Hb, we further conducted x-ray crystallography studies and co-crystallized PP2, PP6, PP9 and PP11 with CO-liganded Hb. Our studies showed that these compounds bind in symmetry-related fashion at the α-cleft of Hb to form Schiff-base adducts with the N-terminal Val1 and showed direct interactions with the F-helix which overall enhanced interactions with Hb. PP6, PP10, and PP14 demonstrated significant in vivo modification of intracellular Hb in mice after IP administration, with increasing levels from 1 h to the 6 h experimental period. They also showed corresponding changes in O2 affinity observed at 3 h and 6 h, compared to 0 h pre-treatment samples in vivo. Thus, our results establish these compounds as a novel, promising group of potent anti-sickling agents, demonstrate their proposed mechanism of action and provide proof-of-concept justifications for our structure-based approach to developing potent therapeutics for SCD.

<p>For many years, primates have been considered to be animals with a poorly developed sense of smell. However, in recent years several studies have shown that at least some primate species have a high olfactory sensitivity for a variety of odorants. The present study used a two-choice instrumental conditioning paradigm to test the olfactory sensitivity for six aromatic aldehydes in four spider monkeys (Ateles geoffroyi). With helional, cyclamal,canthoxal and lilial all animals discriminated concentrations below 1 ppm from the odorless solvent, with single individuals even scoring better. With 3-phenyl-propionic aldehyde all animals detected concentrations below 2 ppb, and with bourgeonal even below 0.3 ppb. The detection thresholds of the odorants changed systematically with molecular structure. Addition of a dioxo or methoxy group to the benzene ring led to an increase in threshold values,while the absence of a methyl group close to the aldehyde moiety was linked to a low threshold value for the odorant. The study shows that spider monkeys have a well developed olfactory sensitivity for aromatic aldehydes.</p>