Óxido nítrico (NO) é um segundo mensageiro biosintetizado a partir de L-Arginina e envolvido em sinalização celular por diferentes mecanismos: ativação da produção de cGMP pela Guanilil Ciclase; regulação de enzimas pela interação com seus centros metálicos; ou pela S-nitrosilação de cisteína e nitração de tirosina, modificações pós-traducionais, capazes de modular a atividade de diversas proteínas. Neste trabalho buscou-se investigar se a interação de Trypanosoma cruzi, o agente etiológico da doença de Chagas, com a matriz extracelular (ECM) era capaz de modular a sinalização por NO em T. cruzi. Tripomastigotas de T. cruzi incubados com ECM apresentaram diminuição na atividade de NOS e menor produção de NO. Da mesma maneira, observou-se, por imunofluorescência indireta e imunoblotting, uma diminuição no padrão geral de S-nitrosilação e nitração de proteínas do parasita incubado com ECM. Além disto, os perfis de proteínas S-nitrosiladas e nitradas foram modificados, predominando a denitrosilação e denitração (de 40 para 22 proteínas nitradas após a adesão a ECM), embora em alguns casos tenha sido observado um aumento de nitração, como em proteínas de citoesqueleto (de 2,5% para 9,1% após adesão). O mesmo padrão foi observado em relação a proteínas nitradas, com diminuição de 48 para 20 proteínas após adesão a ECM e novamente com modificação no percentual de proteínas nitradas pertencentes a processos biológicos distintos, como proteínas relacionadas à síntese proteica (35,4% das proteínas nitradas no grupo controle e apenas 5,0% no grupo incubado com ECM). Apesar do perfil de denitração, algumas classes de proteínas têm aumento no número de alvos nitrados, como proteínas relacionadas a metabolismo (de 18,8% para 35,0%), além de alguns alvos específicos que têm aumento na nitração, como enolase. Em suma, os resultados sugerem que a sinalização intracelular por NO em tripomastigotas de T. cruzi é modulada durante a adesão do parasita a componentes da matrix extracelular, tanto através da via clássica de produção de óxido nítrico, quanto por modificações pós-traducionais induzidas por NO. / Nitric oxide (NO) is a second messenger biosynthesized from L-Arginine and involved in cell signaling by different mechanisms: activation of cGMP production by guanilyl cyclase; regulation of enzymes by interaction with their metallic centers; or by S-nitrosylation of cysteine and nitration of tyrosine, posttranslational modifications capable of modulating the activity of several proteins. In this work, we sought to investigate whether the interaction between extracellular matrix (ECM) and Trypanosoma cruzi, the etiological agent of Chagas\' disease, was capable of modulating NO signaling in the parasite. Trypomastigotes incubated with ECM presented a decrease in NOS activity and NO production. Accordingly, a decrease in S-nitrosylation and tyrosine nitration of proteins from ECM-incubated parasites was also observed, as evidenced by indirect immunofluorescence and immunoblotting. In addition, S-nitrosylated and tyrosine nitrated proteins profiles were modified in ECM-incubated parasites, with an enhancement in protein denytrosylation and denitration. A decrease from 40 to 22 of S-nitrosylated proteins was detected after parasite adhesion to ECM, more evident in some protein groups (as for example 52.5% hypothetical proteins modified in the control group against 36.4% after adhesion). On the other hand, an increase of S-nitrosylation was detected in other groups of proteins, such as cytoskeleton proteins (from 2.5% of total S-nitrosylated proteins to 9.1% after adhesion). The same general pattern was observed in relation to tyrosine-nitrated proteins, with a decrease in the number of modified proteins from 48 to 20 after incubation with ECM, exemplified by those related to protein synthesis, with a contribution of 35.4% in the control group versus 5.0% after treatment with ECM. Despite this general denitration profile, some protein classes have an increase in nitration, such as metabolic proteins (from 18.8% to 35.0%), in addition to some specific targets, such as enolase. Taken together, the results suggest that NO signaling is modulated during adhesion of T. cruzi to components of the extracellular matrix, probably by the classical nitric oxide pathway and by NO-induced post translational modifications.

Doxorubicin (DOX)-induced cardiotoxicity is thought to be mediated by the generation of superoxide anion radicals (superoxide) from redox cycling of DOX in cardiomyocyte mitochondria. Reduction of superoxide generates H2O2, which diffuses throughout the cell and potentially contributes to oxidant-mediated cardiac injury. The mitochondrial and cytosolic glutathione peroxidase 1 (Gpx1) primarily functions to eradicate H2O2. In this study, we hypothesize that Gpx1 plays a pivotal role in the clearance of H2O2 generated by DOX. To test this hypothesis, we compared DOX-induced cardiac dysfunction, mitochondrial injury, protein nitration, and apoptosis in Gpx1-deficient and wild type mouse hearts. The Gpx1-deficient hearts showed increased susceptibility to DOX-induced acute functional derangements than wild type hearts, including impaired contractility and diastolic properties, decreased coronary flow rate, and reduced heart rate. In addition, DOX treatment impaired the mitochondrial function of Gpx1-deficient hearts. Specifically, Gpx1-deficient hearts treated with DOX demonstrated an increased rate of NAD-linked state 4 respiration and a decline in the P/O ratio relative to wild type hearts, suggesting that DOX uncouples the electron transfer chain and oxidative phosphorylation in Gpx1-deficient hearts. Finally, apoptosis and protein nitration were significantly increased in Gpx1-deficient mouse hearts compared to wild type hearts. These studies suggest that Gpx1 plays significant roles in protecting DOX-induced mitochondrial impairment and cardiac dysfunction in the acute phase.

Proteína dissulfeto isomerase (PDI) é uma ditiol-dissulfeto óxido redutase ubíqua que é responsável por uma série de funções celulares, inclusive na sinalização celular e nas respostas a eventos que causam dano celular. Entretanto, a PDI pode se tornar disfuncional através das modificações pós-traducionais, incluindo as promovidas por oxidantes biológicos. Estes oxidantes são provavelmente os responsáveis pelas modificações oxidativas pós-traducionais da PDI que foram detectadas em várias condições associadas ao estresse oxidativo, levando à disfunção da proteína. Devido a falta de estudos cinéticos com a PDI nativa e a falta de caracterização dos produtos dessas reações, investigamos se a diminuição da fluorescência da PDI nativa pode ser empregada para estudos da cinética de oxidação com peróxido de hidrogênio. Posteriormente, investigamos a cinética e os produtos da reação entre PDI e peroxinitrito. Nossos experimentos mostraram que a oxidação por excesso de peróxido de hidrogênio levava a uma diminuição da fluorescência de forma dependente do tempo e da concentração do oxidante, permitindo a determinação da constante de velocidade de segunda ordem (k = (17,3±1,3) M-1 s-1, pH 7,4, 25 ºC). Relevantemente, mostramos que o processo era totalmente revertido por DDT, mostrando que o peróxido de hidrogênio oxida quase que exclusivamente os grupos ditióis da PDI (Cys53 e Cys56 e Cys397 e Cys400). Utilizando a mesma abordagem para estudar a oxidação da PDI por peroxinitrito, notamos que o decréscimo da fluorescência intrínseca da PDI nativa e a velocidade só era proporcional à concentrações sub-estequiométricas ou estequiométricas do oxidante em relação aos tióis reativos da PDI. Somente nessas condições o processo se mostrava reversível por DDT, indicando que os ditióis da PDI eram o alvo preferencial do peroxinitrito mas que a oxidação de outros resíduos também ocorria. A reação dos tióis reativos da PDI com peroxinitrito foi considerada relativamente rápida (6,9 ± 0,6 × 104 M-1 s-1, pH 7,4, 25 °C), e os resíduos de Cys reativos dos domínios a e a\' aparentam reagir com constantes de velocidade similares. Experimentos de proteólise limitada, simulações cinética e análises de MS e MS/MS confirmaram que o peroxinitrito oxida preferencialmente os tióis redox ativos da PDI para os ácidos sulfênicos correspondentes, que, subsequentemente, reagem com os tióis vizinhos, produzindo dissulfetos (Cys53- Cys56 e Cys397- Cys400). Entretanto, uma fração de peroxinitrito decai para radicais levando à hidroxilação e nitração de outros resíduos próximos ao sítio redox ativo (Trp52 Trp396 e Tyr393). Assim, investigamos também a oxidação da PDI por excesso de peroxinitrito em relação aos grupos tióis reativos por diferentes metodologias. Experimentos de SDS-PAGE, western-blot e atividade redutase mostraram que o peroxinitrito promove inativação, nitração e agregação da PDI de forma dependente da concentração de peroxinitrito. Análises de MS e MS/MS mostraram que, em excesso, o peroxinitrito promove nitração (Tyr43, Tyr49, Tyr196, Tyr393, Trp52, Trp396) e hidroxilação (Trp52, Trp396) da PDI. Em síntese, nossos estudos contribuem para melhor compreensão da oxidação da PDI por peroxinitrito e de suas possíveis consequências biológicas. / Protein disulfide isomerase (PDI) is a ubiquitous dithiol-disulfide oxidoreductase that performs an array of cellular functions, including in cellular signaling and responses to cell-damaging events. Nevertheless, PDI can become dysfunctional by post-translational modifications, including those promoted by biological oxidants. These oxidants are likely responsible for the oxidative post-translational modifications of PDI, which have detected under various conditions associated with oxidative stress, leading to protein dysfunction. However, the kinetics of the reactions of PDI with biological oxidants received limited studies and the products of these reactions were not characterized. Here, we examined whether the decrease in PDI fluorescence can be employed to follow the kinetics of the reaction of the full-length protein with biological oxidants. Also, we investigated the kinetics and products of the reaction between PDI and peroxynitrite. Our experiments showed that oxidation by excess hydrogen peroxide led to a decrease of PDI intrinsic fluorescence in a time- and concentration-dependent manner , permitting the determination of the second-order rate constant of the reaction (k = (17.3 ± 1.3 ) M1 s-1, pH 7.4, 25 ° C). The oxidation was reversed by DDT, indicating that hydrogen peroxide oxidizes mainly PDI dithiols (Cys53 and Cys56 and Cys397 and Cys400). Using the same approach to study PDI oxidation by peroxynitrite we noted that the decrease of the native PDI fluorescence was proportional to sub-stoichiometric or stoichiometric concentrations of the oxidant relative to that of PDI reactive thiols. Only under these conditions, PDI oxidation was reversed by DDT, indicating that PDI dithiols were the preferred target of peroxynitrite but that oxidation of other residues also occurred. The reaction of the active redox thiols of the PDI with peroxynitrite can be considered relatively fast (6.9 ± 0.6 × 104 M-1 s-1, pH 7.4, 25 ° C), and the reactive Cys residues of domains a and a\' were kinetically indistinguishable. Limited proteolysis experiments, kinetic simulations, and MS and MS/MS analyses confirmed that peroxynitrite preferentially oxidizes the redox-active Cys residues of PDI to the corresponding sulfenic acids, which subsequently react with the resolving thiols to produce disulfides (Cys53-Cys56 and Cys397-Cys400). However, a fraction of peroxynitrite decays to radicals leading to hydroxylation and nitration to other residues located close to the active site (Trp52 Trp396 and Tyr393). SDS-PAGE, western blotting and inhibition of the reductase activity experiments confirmed that excess peroxynitrite promotes further PDI oxidation, nitration, inactivation and aggregation in a concentration-dependent manner. MS and MS/MS analyzes showed that peroxynitrite in a ten times excess relative to PDI reactive thiols promote PDI nitration (Tyr43, Tyr49, Tyr196, Tyr393, Trp52, Trp396) and hydroxylation (Trp52, Trp396). In conclusion, our studies contribute to a better understanding of PDI oxidation by peroxynitrite and its possible biological consequences

The first part of the thesis presents the KMnO4/ carboxylic acid/ organic solvent which is a powerful reagent for C-C bond formation, aryl coupling reactions and enone oxidation. The a&cent / -acetoxylation of enones and the a-acetoxylation of aromatic ketones were carried out with potassium permanganate and acetic acid, in which acetoxylation products were obtained in 74-96% yields. The same reaction was carried out with carboxylic acids other than acetic acid, which furnished corresponding acyloxy ketones with the same regioselectivity. For the first time, formyloxylation products were synthesized in a 61-85% yield by using formic acid. The potassium permanganate and acetic acid method was also used for aryl coupling reactions. The reaction of arylboronic acids and aryl hydrazines in benzene with potassium permanganate and acetic acid in turn furnished biaryls in a 85-96% yield. We showed that potassium permanganate/carboxylic acid/organic solvent behaves as manganese(III) acetate. In the second part of the thesis, ipso-nitration of arylboronic acids with AgNO2/ TMSCl was performed. Nitration of aromatic compounds is one of the most extensively studied reactions, and nitroaryl moieties play key roles in the physical and chemical properties of many target molecules in organic synthesis. For electrophilic nitration of aromatic compounds, a wide variety of reagents are available to date. Most of them are very strong nitrating agents and often lead to further nitration and mixture of isomers. Since most nitrating agents are oxidants, oxidation of other functional groups can also occur, giving a mixture of products. Thus, a search for milder and selective nitrating agents is a good research goal. In this work, we aimed to apply AgNO2/ TMSCl system to ipso nitration of arylboronic acids.

Im Nitratbericht 2005 werden die Nitratuntersuchungen auf Dauertestflächen sowie die Bewertung verschiedener Einflussgrössen dargestellt.

[Truncated abstract. Please see the pdf format for the complete text.] Interest in the role of peroxynitrite in the pathogenesis of atherosclerosis has increased due to many in vitro studies which have demonstrated its potent oxidising and nitrating capability and immunohistochemical staining studies which demonstrate nitration of tyrosine in vivo. It is frequently suggested that the production of nitric oxide and superoxide at sites of inflammation implicates peroxynitrite as the major damaging reactive nitrogen species in vivo. Evidence for a role for peroxynitrite is often demonstrated by measurement of 3-nitrotyrosine yet even this cannot distinguish peroxynitrite from other nitrating species. Clearly, however, if peroxynitrite is important in atherogenesis, then identification of mechanisms for its detoxification could provide a means of preventing such effects. Therefore, this Thesis has sought to determine whether phenolic compounds of dietary origin can be preferentially nitrated by reactive nitrogen species thereby protecting endogenous structures, such as low density lipoproteins, from atherogenic modifications. This Thesis focuses upon phenolic acids as they have received relatively less attention than other classes of phenolic compounds, such as flavonoids, yet they are quite abundant in socially important beverages such as red wine. In order to complete the required analyses, the development of methods to detect phenolic acids and their nitration products together with 3-nitrotyrosine, dityrosine and 5-nitro-γ-tocopherol was necessary. The initial in vitro experiments described herein sought to determine the products of reaction of peroxynitrite with phenolic acids of the 4-hydroxy and 3,4-dihydroxy type and then to examine whether these products could account for a protective effect upon tyrosine, lipids and endogenous anti-oxidants, if any was observed, when isolated LDL was treated with SIN-1, which releases peroxynitrite through the simultaneous generation of nitric oxide and superoxide. A concurrent minor focus was to examine the relationship between structure and activity of these phenolic acids under various regimes of oxidative insult. These experiments indicate that, at least in this in vitro model, oxidation is a dominant mechanism over nitration. Peroxynitrite was shown to nitrate coumaric acid in moderate yields but exclusive oxidation of caffeic acid appeared to occur. Although a potential role for γ-tocopherol as an anti-nitration agent was inferred, all types of chemical treatment of LDL in the presence of phenolic acids yielded oxidation as the primary end point. In fact, nitration of tyrosine was not detected and nitration of coumaric acid was at the limit of detection. Since nitration of tyrosine is generally regarded as important in many disease states, a more physiological nitrating mechanism involving artificially stimulated neutrophils was used. This system demonstrated that although physiologically relevant reactive nitrogen species can result in nitration of phenolic compounds, in a complex system including biological structures (LDL) and phenolic compounds, oxidation but not nitration of all species appears to occur. As a consequence of the results above, an examination of carotid plaque was undertaken to determine to what extent nitration occurred relative to oxidation in atherosclerotic tissue. These studies applied methods developed herein to detect 3-nitrotyrosine and dityrosine in complex biological matrices as markers of nitration and oxidation respectively. The data obtained demonstrated that nitration was a minor modification of protein (0.01%) compared to oxidation (0.3%) even in a highly diseased tissue such as carotid artery plaque. A secondary study examining plasma revealed that dityrosine, which has been implicated in irreversible albumin aggregation in chronic renal failure and more recently in heart disease, is elevated in chronic renal failure subjects compared to well matched controls. A separate examination of plasma from healthy subjects revealed that in both the fasting and post prandial state 3-nitrotyrosine could not be detected and, in fact, interfering species could be problematic in the GC-MS analysis of 3-nitrotyrosine. The lack of nitration of any substrate observed in vitro using reactive nitrogen species generated in the aqueous phase, the relative lack of nitration of tyrosine in plaque proteins and the lipophilicity of nitric oxide, the precursor of all reactive nitrogen species, suggested that nitration could be more closely associated with lipid structures. The known ability of γ-tocopherol to form 5-nitro-γ-tocopherol was used to probe this concept. The 5-nitro-γ-tocopherol content of lipid extracts obtained from carotid artery plaques was very high (30%). This indicated that nitration is predominantly a lipid phase phenomenon and that nitrating species are present in much greater abundance than oxidising species in vivo.

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 Aug 16.

Alcohol liver disease (ALD) is a major health concern throughout the world. Currently, in the United States, 17 million people suffer from alcoholism, of which 1.4 million people are receiving treatment [1, 2]. The link between ethanol metabolism, reactive oxygen species (ROS) and liver injury in ALD has been well characterized over the last couple decades [3-10]. Ethanol metabolism relies on the availability of the cofactor NAD+ for the oxidation of ethanol into acetate, consequently causing alterations in redox potential. Redox dysfunction within the mitochondria can affect multiple pathways important in maintaining cellular homeostasis. Chapter 1 provides an introduction to the role of ethanol metabolism in oxidative stress and alcohol liver injury (ALI). During ethanol metabolism, both the cytochrome bc1 and NADH dehydrogenase complexes within the mitochondria have been demonstrated to be major contributors to ROS formation and “leak” free radicals [11-13]. As a result, the free radicals superoxide (O2-) and hydrogen peroxide (H2O2) is diffused into the cytoplasm where they can react with other molecules, proteins and DNA and cause tissue injury [4, 14]. Chapter 1 aims to introduce the link between ethanol metabolism and histone post-translational modifications (PTM) such as tyrosine nitration and lysine acetylation using proteomics techniques. Chapter 2 uses a global proteomic study to identify links between gender and ALI. A 10-day chronic-binge mouse model was employed in order to identify gender-specific proteins that may influence the development of ALD. It has previously been established that females are more susceptible to developing ALD, however, the cause is still unknown. This study identifies gender differences in the family of cytochrome P450 proteins using a mouse model for chronic-binge alcohol exposure. The cytochrome P450 family of proteins are important in the metabolism of toxic compounds, such as acetaldehyde, a byproduct of ethanol metabolism. Interestingly, I also identified that female mice expressed naturally higher levels of histone acetylation prior to alcohol exposure when compared to males. Following alcohol exposure, the female mice did not show much change in acetylation, whereas male acetylation levels were raised to similar levels of the female mice. These acetylation changes raised the question, how does alcohol influence epigenetic marks on histone proteins? Recently, new evidence has emerged that supports the role of epigenetics in the pathophysiology of ALD [4, 14-27]. Ethanol metabolism will promote shifts in redox potential and mitochondrial dysfunction, the result is the formation of reactive oxygen and/or nitrogen species (ROS/RNS) [4, 5, 7, 10, 14, 28]. As ethanol is metabolized, the accumulation of ROS/RNS species such as NO- and O2- can induce the post-translational modification nitrotyrosine. Shifts in redox potential will cause the electron transport chain to “leak” the free radical O2-. Another free radical known as nitric oxide (NO-) has been shown to be elevated during times of ethanol consumption [29, 30]. Traditionally, NO has a protective role within the cell at low concentrations, however, in surplus can lead to tissue damage. Ethanol-induced increases in NO- and O2- can instigate to peroxynitrite (ONOO-) formation; a potent oxidant and nitrating agent of tyrosine residues [29, 31-34]. Chapter 3 examines the indirect effect of alcohol metabolism and ROS/RNS formation on histone tyrosine nitration. This project used mass-spectrometry to identify novel targets of histone tyrosine nitration using a mouse-model of chronic-binge alcohol exposure. Interestingly, histone H3 was found to be nitrated on the hinge-region of the N-terminal tail at tyrosine 41. Molecular dynamics of the nitrated and unmodified proteoforms revealed that the DNA prefers a change in conformation upon H3Y41 nitration. Further studies using an antibody synthesized against the nitrated H3y41 region of the protein revealed potential targets within the genome important in fatty acid synthesis and metabolism. Chapter 4 looks at the direct influence of alcohol metabolism and its contribution to histone acetylation via acetate production and acetyl-CoA. Alcohol metabolism has traditionally been thought influence acetylation through the sirtuin family of deacetylase proteins. Sirtuin deacetylases are NAD+-dependent and have been shown to be a regulate protein acetylation within the mitochondria, cytoplasm, and nucleus during times of ethanol exposure [35-37]. Shifts in redox potential attributed to ethanol metabolism can inhibit sirtuin deacetylase activity by out-competing the enzymes for available NAD+, ultimately leading to mitochondrial and nuclear hyperacetylation [17, 28, 38-42]. Currently, there is evidence that ethanol increases acetylation of histone 3 lysine 9, which then targets activation of the alcohol dehydrogenase gene (ADH) [17, 18, 43]. Moreover, Shukla et.al. (2008) support the idea that ethanol can alter epigenetic transcriptional activation based on which modification is selected for a site during times of stress when it can be occupied by more than one modification [22]. Chapter 4 demonstrates the use of mass-spectrometry to metabolically trace 13C2-labeled ethanol in vivo. These new data show clear evidence of 13C2 heavy-labeled ethanol being incorporated into known sites of acetylation on the N-terminal tails of histone H3 and H4. Incorporation of heavy-label was calculated using extracted ion chromatograms (XIC) for the double and singly acetylated and unmodified peptides belonging to H3K9-R17 and H3K18-R23. Total change in acetylation was also assessed for each peptide using the ratio of ratios of total acetylation to unmodified peptide over the fold change in ethanol- to control-fed groups. An interesting observation was observed in that the incorporation of heavy-label suggests site-selectivity of lysine residues over time. Histone 4 contains multiple sites of acetylation on the peptide H4K5-R17, making it hard to quantify manually. MaxQuant evidence files in conjunction with R were used to calculate the 13C2 incorporation on the multiple H4 acetyl-sites over 24-hours. Ethanol-heavy label incorporation at multiple acetyl-sites occurred as a mixture suggesting a role in transcriptional regulation. These new data establish a link between alcohol metabolism and known epigenetic marks on histone proteins. These studies have now established that alcohol metabolism is indirectly linked to histone tyrosine nitration through increased ROS/RNS and directly through acetate production. Understanding how these epigenetic marks fluctuate as ALD progresses will provide potential targets for the development of new drug therapies. The epigenetic marks identified in these studies have previously been established to be important activators in transcription. These data provide novel techniques using proteomics-based metabolic tracing in vivo. Future studies will assess how these marks change after chronic ethanol exposure and whether the changes in epigenetics are heritable. Understanding hereditary of alcoholism will provide insight to those predisposed to the disease.

Orientador: Prof. Dr. Anderson Orzari Ribeiro / Dissertação (mestrado) - Universidade Federal do ABC. Programa de Pós-Graduação em Ciência e Tecnologia/Química, 2015. / Neste trabalho foram obtidos dez compostos derivados de fenóis, sendo eles o 2-hidroxi-5-nitro-benzaldeído, (AQ-1), 2-hidroxi-3-nitro-benzaldeído, (AQ-2), 1-(2,4-dihidroxi-3-nitro-fenil)-etanona (HMQ-1), 1-(2,4-dihidroxi-3,5-dinitro-fenil)-etanona (HMQ-2), 2-(3-metilbut-2-eniloxi)-benzaldeído, (MP-1), 1-(3-metilbut-2-eniloxi)-2-(2-nitrovinil)-benzeno, (MP-2), 2-[2-(3-metil-but-2-eniloxi)-benzilideno]-malononitrila, (MP-3), 2-(furan-2-carbonil)-3-[2-(3-metil-but-2-eniloxi)-fenil]-acrilonitrila, (AQ-12), 2-(3-metil-but-2-eniloxi)-5-nitro-benzaldeído, (AQ-3) e 1-(3-metil-but-2-eniloxi)-4-nitro-2-(2-nitro-vinil)-benzeno, (AQ-6). Para a obtenção dos compostos foram empregados diversos métodos sintéticos. Destaca-se a obtenção dos compostos AQ-1, AQ-2 e HMQ-1 pelo método de nitração direta sem a utilização de catalisadores, do composto HMQ-2 que possui dois substituintes nitro no anel aromático, dos compostos MP-1 e MP-2 obtidos por micro-ondas o que diminui drasticamente o tempo de obtenção destas moléculas e do composto AQ-6, análogo estrutural do MP-2 que possui um substituinte nitro no anel aromático, o que confere a molécula características eletrônicas diferentes ao do seu antecessor. Os produtos obtidos foram caracterizados mediantes diferentes métodos de caracterização, entre eles a espectroscopia de absorção infravermelho, massa, RMN-1H e 13C, DEPT e de duas dimensões 1H,1H COSY, 1H,1H NOESY e 1H,13C espectro combinatório (HSQC, HMBC). O composto MP-2, obtido a partir da síntese Knoevenagel do composto MP-1 com nitrometano, apresentou significativa atividade antitumoral seletiva, sendo 4,64 vezes mais ativo frente as células tumorais do que as células sadias e ação antimetastática, inibindo por completo o processo de migração das células tumorais nas diferentes concentrações testadas. / In this study were obtained ten phenol derivatives, 2-hydroxy-5-nitrobenzaldehyde, (AQ-1), 2-hydroxy-3-nitrobenzaldehyde (AQ-2), 1-(2,4-dihydroxy-3-nitro-phenyl)-ethanone (HMQ-1), 1- (2,4-dihydroxy-3,5-dinitro-phenyl)-ethanone (HMQ-2) 2-(3-methylbut-2-enyloxy)benzaldehyde, (MP-1), 1-(3-methylbut-2-enyloxy)-2-(2-nitrovinyl)benzene, (MP-2), 2-[2-(3-methyl-but-2-enyloxy)benzylidene]-malononitrila (MP-3), 2-(furan-2-carbonyl)-3-[2-(3-methyl-but-2-enyloxy)-phenyl]-acrilonitrila, (AQ-12), 2-(3-methyl-but-2-enyloxy)-5-nitrobenzaldehyde (AQ-3) and 1- (3-methyl-but-2-enyloxy)-4-nitro-2-(2-nitro-vinyl)benzene (AQ-6). Various synthetic methods were employed to obtain the compounds, as such, ultrasound bath, microwave reactor and agitation plate. Noteworthy is the achievement of AQ-1 compounds, AQ-2 and HMQ-1 by direct nitration method without the use of catalysts, the HMQ-2 compound having two nitro substituents on the aromatic ring, the MP-1 compounds and MP-2 using microwave-oven which dramatically reduces the reaction time for obtaining these molecules and AQ-6 compound structural analogue of the MP-2, but having a nitro substituent on the aromatic ring, which gives the molecule different electronic characteristics to that of its predecessor. The products obtained were characterized by the use of different spectroscopic methods in different centers, highlighting the characterization carried out in Germany Rostock. The employer spectroscopic methods were: infrared absorption spectroscopy, mass spectrometry, 1H-NMR and 13C-NMR spectroscopy, DEPT and two-dimensional 1H, 1H COSY, 1H, 1H NOESY and 1H, 13C combinatorial spectra (HSQC, HMBC). The MP-2 compound obtained from the Knoevenagel synthesis of MP-1 compound and nitromethane, showed significant selective antitumor activity and antimetastatic action of tumor cells.

Proteína dissulfeto isomerase (PDI) é uma ditiol-dissulfeto óxido redutase ubíqua que é responsável por uma série de funções celulares, inclusive na sinalização celular e nas respostas a eventos que causam dano celular. Entretanto, a PDI pode se tornar disfuncional através das modificações pós-traducionais, incluindo as promovidas por oxidantes biológicos. Estes oxidantes são provavelmente os responsáveis pelas modificações oxidativas pós-traducionais da PDI que foram detectadas em várias condições associadas ao estresse oxidativo, levando à disfunção da proteína. Devido a falta de estudos cinéticos com a PDI nativa e a falta de caracterização dos produtos dessas reações, investigamos se a diminuição da fluorescência da PDI nativa pode ser empregada para estudos da cinética de oxidação com peróxido de hidrogênio. Posteriormente, investigamos a cinética e os produtos da reação entre PDI e peroxinitrito. Nossos experimentos mostraram que a oxidação por excesso de peróxido de hidrogênio levava a uma diminuição da fluorescência de forma dependente do tempo e da concentração do oxidante, permitindo a determinação da constante de velocidade de segunda ordem (k = (17,3±1,3) M-1 s-1, pH 7,4, 25 ºC). Relevantemente, mostramos que o processo era totalmente revertido por DDT, mostrando que o peróxido de hidrogênio oxida quase que exclusivamente os grupos ditióis da PDI (Cys53 e Cys56 e Cys397 e Cys400). Utilizando a mesma abordagem para estudar a oxidação da PDI por peroxinitrito, notamos que o decréscimo da fluorescência intrínseca da PDI nativa e a velocidade só era proporcional à concentrações sub-estequiométricas ou estequiométricas do oxidante em relação aos tióis reativos da PDI. Somente nessas condições o processo se mostrava reversível por DDT, indicando que os ditióis da PDI eram o alvo preferencial do peroxinitrito mas que a oxidação de outros resíduos também ocorria. A reação dos tióis reativos da PDI com peroxinitrito foi considerada relativamente rápida (6,9 ± 0,6 × 104 M-1 s-1, pH 7,4, 25 °C), e os resíduos de Cys reativos dos domínios a e a\' aparentam reagir com constantes de velocidade similares. Experimentos de proteólise limitada, simulações cinética e análises de MS e MS/MS confirmaram que o peroxinitrito oxida preferencialmente os tióis redox ativos da PDI para os ácidos sulfênicos correspondentes, que, subsequentemente, reagem com os tióis vizinhos, produzindo dissulfetos (Cys53- Cys56 e Cys397- Cys400). Entretanto, uma fração de peroxinitrito decai para radicais levando à hidroxilação e nitração de outros resíduos próximos ao sítio redox ativo (Trp52 Trp396 e Tyr393). Assim, investigamos também a oxidação da PDI por excesso de peroxinitrito em relação aos grupos tióis reativos por diferentes metodologias. Experimentos de SDS-PAGE, western-blot e atividade redutase mostraram que o peroxinitrito promove inativação, nitração e agregação da PDI de forma dependente da concentração de peroxinitrito. Análises de MS e MS/MS mostraram que, em excesso, o peroxinitrito promove nitração (Tyr43, Tyr49, Tyr196, Tyr393, Trp52, Trp396) e hidroxilação (Trp52, Trp396) da PDI. Em síntese, nossos estudos contribuem para melhor compreensão da oxidação da PDI por peroxinitrito e de suas possíveis consequências biológicas. / Protein disulfide isomerase (PDI) is a ubiquitous dithiol-disulfide oxidoreductase that performs an array of cellular functions, including in cellular signaling and responses to cell-damaging events. Nevertheless, PDI can become dysfunctional by post-translational modifications, including those promoted by biological oxidants. These oxidants are likely responsible for the oxidative post-translational modifications of PDI, which have detected under various conditions associated with oxidative stress, leading to protein dysfunction. However, the kinetics of the reactions of PDI with biological oxidants received limited studies and the products of these reactions were not characterized. Here, we examined whether the decrease in PDI fluorescence can be employed to follow the kinetics of the reaction of the full-length protein with biological oxidants. Also, we investigated the kinetics and products of the reaction between PDI and peroxynitrite. Our experiments showed that oxidation by excess hydrogen peroxide led to a decrease of PDI intrinsic fluorescence in a time- and concentration-dependent manner , permitting the determination of the second-order rate constant of the reaction (k = (17.3 ± 1.3 ) M1 s-1, pH 7.4, 25 ° C). The oxidation was reversed by DDT, indicating that hydrogen peroxide oxidizes mainly PDI dithiols (Cys53 and Cys56 and Cys397 and Cys400). Using the same approach to study PDI oxidation by peroxynitrite we noted that the decrease of the native PDI fluorescence was proportional to sub-stoichiometric or stoichiometric concentrations of the oxidant relative to that of PDI reactive thiols. Only under these conditions, PDI oxidation was reversed by DDT, indicating that PDI dithiols were the preferred target of peroxynitrite but that oxidation of other residues also occurred. The reaction of the active redox thiols of the PDI with peroxynitrite can be considered relatively fast (6.9 ± 0.6 × 104 M-1 s-1, pH 7.4, 25 ° C), and the reactive Cys residues of domains a and a\' were kinetically indistinguishable. Limited proteolysis experiments, kinetic simulations, and MS and MS/MS analyses confirmed that peroxynitrite preferentially oxidizes the redox-active Cys residues of PDI to the corresponding sulfenic acids, which subsequently react with the resolving thiols to produce disulfides (Cys53-Cys56 and Cys397-Cys400). However, a fraction of peroxynitrite decays to radicals leading to hydroxylation and nitration to other residues located close to the active site (Trp52 Trp396 and Tyr393). SDS-PAGE, western blotting and inhibition of the reductase activity experiments confirmed that excess peroxynitrite promotes further PDI oxidation, nitration, inactivation and aggregation in a concentration-dependent manner. MS and MS/MS analyzes showed that peroxynitrite in a ten times excess relative to PDI reactive thiols promote PDI nitration (Tyr43, Tyr49, Tyr196, Tyr393, Trp52, Trp396) and hydroxylation (Trp52, Trp396). In conclusion, our studies contribute to a better understanding of PDI oxidation by peroxynitrite and its possible biological consequences