As propriedades da molécula de nitroxil livre e do ligante nitroxil em tetraamminas de Ru(II) (trans-[Ru(NH3)4(nitroxyl)n(L)]2+n, n = carga do nitroxil e L = NH3, py, P(OEt)3, H2O, Cl- and Br-) foram estudadas utilizando-se a teoria do funcional da densidade (DFT). De acordo com as energias calculadas para as conformações dos complexos de HNO, esses são mais estáveis do que os análogos desprotonados e a configuração singleto (trans-1[Ru(NH3)4(L)HNO]2+) é de menor energia em relação a correspondente tripleto <br /> (trans-3[Ru(NH3)4(L)HNO]2. A avaliação dos componentes &sigma; e &pi; na ligação <br /> L-Ru-HNO sugere que o aumento da estabilidade destes orbitais juntamente com o aumento das contribuições dos orbitais HNO estão correlacionacionado a menores valores de distâncias Ru-N(H)O e freqüências inferiores para os estiramentos &nu;NO. A estabilidade da ligação Ru-HNO também foi avaliada através de um estudo cinético teórico da dissociação de HNO a partir de <br /> trans-1[Ru(NH3)4(L)HNO]2+. De acordo com a ordem de estabilidade de ligação Ru-HNO em trans-1[Ru(NH3)4(L)HNO]2+ observa-se a seguinte ordem em função de L: H2O &gt; Cl- ~ Br- &gt; NH3 &gt; py &gt; P(OEt)3, que corresponde à ordem do efeito trans e influência trans medida experimentalmente para a série de L em complexos octaédricos. A mesma tendência foi também observada utilizando o modelo explícito solvente, considerando-se, assim, a presença de uma molécula de H2O juntamente com uma molécula de HNO no estado de transição. Para esta série, as energias calculadas para a ligação Ru-HNO estão no intervalo de 21,4 - 41,5 kcal.mol-1. Observou-se uma boa concordância entre os valores calculados de &Delta;G&dagger; HNO para substituição por H2O a partir do complexo trans-1[Ru(NH3)4(P(OEt)3HNO]2+ (28.5 kcal mol-1) e os dados experimentais disponíveis para reações de substituição em sistemas trans-[Ru(NH3)4(POEt)3(Lx)]2+ (19.4 e 24.0 kcal.mol-1 para Lx = isn e P(OET)3, respectivamente). Experimentos de voltametria cíclica foram realizados observando os processos de redução do ligante nitrosônio gerando nitroxil e podendo gerar amônia, o que foi também observado por cálculos envolvendo efeito de uma primeira esfera de solvatação. / The properties of free nitroxyl molecule and the nitroxyl ligand in Ru(II) tetraammines (trans-[Ru(NH3)4(nitroxyl)n(L)]2+n, n = nitroxyl charge and L = NH3, py, P(OEt)3, H2O, Cl- and Br-) were studied trough density functional theory (DFT). According to the calculated conformation energies the HNO complexes are more stable than the deprotonated analogues and the singlet configuration (trans-1[Ru(NH3)4(L)HNO]2+) is lower energy the than the corresponding triplet (trans-3[Ru(NH3)4(L)HNO]2+). Evaluation of the &sigma; and &pi; components in the L-Ru-HNO bond suggest that increased stability of these orbitals and enhanced contributions from the HNO orbitals correlate to shorter Ru-N(H)O distances and lower &nu;NO stretching frequencies. The stability of the Ru-HNO bond was also evaluated through a theoretical kinetic study on HNO dissociation from trans-1[Ru(NH3)4(L)HNO]2+. Accordingly, the order of the Ru-HNO bonding stability in trans-1[Ru(NH3)4(L)HNO]2+ as a function of L is: H2O &gt; Cl- ~ Br- &gt; NH3 &gt; py &gt; P(OEt)3, which parallels the order of the trans effect and trans influence series experimentally measured for L in octahedral complexes. The same trend was also observed using the explicit solvent model, thus considering the presence of both the HNO and H2O molecules in the transition state. For this series, the calculated bond energies for the Ru-HNO bond are in the range 21.4 to 41.5 kcal.mol-1. Good agreement was observed between the calculated &Delta;G&dagger; values for HNO substitution by H2O from the complex trans-1[Ru(NH3)4(P(OEt)3HNO]2+ (28.5 kcal mol-1) and the available experimental data for substitution reactions of trans-[Ru(NH3)4(POEt)3(Lx)]2+ (19.4 to 24.0 kcal.mol-1 for Lx = isn and P(OET)3, respectively). Cyclic voltammetry experiments were carried out observing the reduction processes of the nitrosonium ligand generating nitroxil and could generate ammonia, which was also observed by calculations involving the effect of a first sphere of solvation.

As propriedades da molécula de nitroxil livre e do ligante nitroxil em tetraamminas de Ru(II) (trans-[Ru(NH3)4(nitroxyl)n(L)]2+n, n = carga do nitroxil e L = NH3, py, P(OEt)3, H2O, Cl- and Br-) foram estudadas utilizando-se a teoria do funcional da densidade (DFT). De acordo com as energias calculadas para as conformações dos complexos de HNO, esses são mais estáveis do que os análogos desprotonados e a configuração singleto (trans-1[Ru(NH3)4(L)HNO]2+) é de menor energia em relação a correspondente tripleto <br /> (trans-3[Ru(NH3)4(L)HNO]2. A avaliação dos componentes &sigma; e &pi; na ligação <br /> L-Ru-HNO sugere que o aumento da estabilidade destes orbitais juntamente com o aumento das contribuições dos orbitais HNO estão correlacionacionado a menores valores de distâncias Ru-N(H)O e freqüências inferiores para os estiramentos &nu;NO. A estabilidade da ligação Ru-HNO também foi avaliada através de um estudo cinético teórico da dissociação de HNO a partir de <br /> trans-1[Ru(NH3)4(L)HNO]2+. De acordo com a ordem de estabilidade de ligação Ru-HNO em trans-1[Ru(NH3)4(L)HNO]2+ observa-se a seguinte ordem em função de L: H2O &gt; Cl- ~ Br- &gt; NH3 &gt; py &gt; P(OEt)3, que corresponde à ordem do efeito trans e influência trans medida experimentalmente para a série de L em complexos octaédricos. A mesma tendência foi também observada utilizando o modelo explícito solvente, considerando-se, assim, a presença de uma molécula de H2O juntamente com uma molécula de HNO no estado de transição. Para esta série, as energias calculadas para a ligação Ru-HNO estão no intervalo de 21,4 - 41,5 kcal.mol-1. Observou-se uma boa concordância entre os valores calculados de &Delta;G&dagger; HNO para substituição por H2O a partir do complexo trans-1[Ru(NH3)4(P(OEt)3HNO]2+ (28.5 kcal mol-1) e os dados experimentais disponíveis para reações de substituição em sistemas trans-[Ru(NH3)4(POEt)3(Lx)]2+ (19.4 e 24.0 kcal.mol-1 para Lx = isn e P(OET)3, respectivamente). Experimentos de voltametria cíclica foram realizados observando os processos de redução do ligante nitrosônio gerando nitroxil e podendo gerar amônia, o que foi também observado por cálculos envolvendo efeito de uma primeira esfera de solvatação. / The properties of free nitroxyl molecule and the nitroxyl ligand in Ru(II) tetraammines (trans-[Ru(NH3)4(nitroxyl)n(L)]2+n, n = nitroxyl charge and L = NH3, py, P(OEt)3, H2O, Cl- and Br-) were studied trough density functional theory (DFT). According to the calculated conformation energies the HNO complexes are more stable than the deprotonated analogues and the singlet configuration (trans-1[Ru(NH3)4(L)HNO]2+) is lower energy the than the corresponding triplet (trans-3[Ru(NH3)4(L)HNO]2+). Evaluation of the &sigma; and &pi; components in the L-Ru-HNO bond suggest that increased stability of these orbitals and enhanced contributions from the HNO orbitals correlate to shorter Ru-N(H)O distances and lower &nu;NO stretching frequencies. The stability of the Ru-HNO bond was also evaluated through a theoretical kinetic study on HNO dissociation from trans-1[Ru(NH3)4(L)HNO]2+. Accordingly, the order of the Ru-HNO bonding stability in trans-1[Ru(NH3)4(L)HNO]2+ as a function of L is: H2O &gt; Cl- ~ Br- &gt; NH3 &gt; py &gt; P(OEt)3, which parallels the order of the trans effect and trans influence series experimentally measured for L in octahedral complexes. The same trend was also observed using the explicit solvent model, thus considering the presence of both the HNO and H2O molecules in the transition state. For this series, the calculated bond energies for the Ru-HNO bond are in the range 21.4 to 41.5 kcal.mol-1. Good agreement was observed between the calculated &Delta;G&dagger; values for HNO substitution by H2O from the complex trans-1[Ru(NH3)4(P(OEt)3HNO]2+ (28.5 kcal mol-1) and the available experimental data for substitution reactions of trans-[Ru(NH3)4(POEt)3(Lx)]2+ (19.4 to 24.0 kcal.mol-1 for Lx = isn and P(OET)3, respectively). Cyclic voltammetry experiments were carried out observing the reduction processes of the nitrosonium ligand generating nitroxil and could generate ammonia, which was also observed by calculations involving the effect of a first sphere of solvation.

Nitric oxide synthases (NOS) are responsible for the production of nitric oxide (NO), an essential cell-signaling molecule, in mammals. There are three isoforms of NOS with widely different tissue distribution. The overproduction of NO is marked in many human disease states and cancers, however due to the similarities of the enzyme isoforms, targeting NOS for inhibition has proven challenging. Endogenously, the methylated arginines, N[superscript ω]-monomethyl-L-arginine (NMMA) and asymmetric N[superscript ω], N[superscript ω]-dimethyl-L-arginine (ADMA), inhibit NOS. N[superscript ω], N[superscript ω]-Dimethylarginine dimethylaminohydrolase (DDAH1) metabolizes these methylated arginines and thus relieves NOS inhibition. The role of DDAH1 in the regulation of diseases such as cancer and septic shock is still being elucidated. It is thought that targeting DDAH1 for inhibition rather than NOS may circumvent many of the current problems with the treatment of NO overproduction such as isoform selectivity. My PhD studies focus on the synthesis of a series of irreversible inhibitors of DDAH1, an extensive study of their in vitro mode of inhibition, a comparison of analytical fitting methods, and the viability and efficacy of the inactivators in a human cell line. I also studied a potential endogenous inactivator of DDAH1, nitroxyl (HNO), a one-electron reduction product of NO. / text

New polymer cure chemistry is described, wherein the onset of free radical cross-linking is delayed without compromising cure yields. The addition of an acrylate-functionalized nitroxyl, 4-acryloyloxy-2,2,6,6-tetramethylpiperidine-N-oxyl (AOTEMPO), to a peroxide-cure formulation quenches free radical activity during the initial stages of the cross-linking process, trapping alkyl radicals as alkoxyamines that bear acrylate functionality. Polymer cross-linking by macro-radical combination is suppressed until all nitroxyl is consumed, at which point radical oligomerization of polymer-bound acrylate groups generates the desired covalent network. As a result, cross-link density losses incurred during radical trapping are recovered during the oligomerization phase of the process. The effectiveness of this approach is demonstrated for a range of polymers, peroxide initiators, reaction temperatures and reagent loadings. Furthermore, AOTEMPO formulations are compared directly to other delayed-onset additives that are used in commercial practice. / Thesis (Master, Chemical Engineering) -- Queen's University, 2012-06-11 10:09:20.848

This thesis describes the synthesis of derivatives of 2'-ethynyl-2'-deoxynucleosides, and their conversion into phosphoramidite building blocks for DNA synthesis. Herein is also outlined the preparation of azide-bearing nitroxyl radicals. After site-selective incorporation of 2'-alkynylnucleosides into DNA using a standard solid-supported phosphoramidite protocol, the modified oligonucleotides were spin labelled with the azide-bearing nitroxyl spin labels via the Cu(I)-catalysed azide-alkyne [3+2] Huisgen cycloaddition (CuAAC or 'click' reaction). The destabilising effect of the spin labelling was determined by UV denaturation studies and circular dichroism, and the spin labelled DNA was investigated by EPR spectroscopy. It was found that this novel site-directed spin labelling strategy afforded conformationally restricted systems, and that the structure of the spin label exerts a significant influence on the results.

Foram testados complexos de rutênio trans-[RuNO(NH3)4L](X)3 (X = BF4- ou PF6- e L = imN, 4-pic, isn, py, pz, L - hist, nic, imC, P(OEt)3, SO3-2) e [Ru(NO)Hedta)] como agentes antiproliferativos contra o parasito Leishamania major. Os complexos onde L = imN, pz, 4-pic, py, isn, e P(OEt)3 exibem IC50pro na faixa de 36 (L = imN) a 280&mu;M (L= isn). A curva de crescimento das formas promastigotas, incubadas com os compostos mais promissores, trans-[RuNO(NH3)4L](BF4)3 (L= imN, pz, py, 4-pic), foi avaliada por meio de contagem de células viáveis e por ensaio colorimétrico (MTT). Uma relação entre os valores de k-NO e efeito antipromastigota segue a seguinte ordem: imN>4-pic>pz>py. Foi observado um aumento na inibição do crescimento das formas promastigotas incubadas com o complexo trans-[RuNO(NH3)4P(OEt)3](PF6)3 na presença de ácido ascórbico, confirmando o mecanismo de nitrosilação dos complexos utilizados. O complexo trans-[RuNO(NH3)4imN](BF4)3 e o sal de Angeli (Na2N2O3) exibiram efeitos similares frente as formas amastigotas intracelulares. O composto trans-[RuNO(NH3)4imN](BF4)3 apresentou efeito antileishmania em experimentos in vivo. A reação entre trans-[RuNO(NH3)4L](X)3 (X = BF4- or PF6- and L = imN, 4-pic, py, isn, P(OEt3)) e excesso de L-cisteína foi investigada e os seus produtos analisados. HNO e NO0 foram encontrados como produtos da redução do ligante nitrosônio. Esta reação ocorre em varias etapas com a formação dos complexos trans-[Ru(NH3)4LN(O)SR]n-1 e trans-[Ru(NH3)4LN(O)(SR)2]n-2 cujo as constantes de formação k1 (1,7×107 - 2,2×104 mol-1Ls-1) e k2 (3,3×104 - 4,9×101mol-1Ls-1) foram calculados para L = P(OEt)3, 4-pic, py e isn). Estas espécies podem sofrer reações subseqüentes com dissociação de HNO e NO. Exceto para L = P(OEt3), a reação não ocorre quando CH+>= 1×10-6 mol L-1. Em solução onde CH+ = 4×108 mol L-1, a reação ocorre principalmente segundo: trans-[RuNO(NH3)4L]+3 + 2RS- + H2O &rarr; trans-[Ru(NH3)4L(H2O)]2+ + RSSR + NO- Para L = P(OEt3), e em meio ácido, NO0 é preferencialmente o produto da redução do ligante NO+: trans-[RuNO(NH3)4P(OEt)3]+3 + RSH + H2O &rarr; trans-[Ru(NH3)4P(OEt)3(H2O)]2+ + &frac12; RSSR + NO0 + H+ Os resultados experimentais em solução sugerem que ambas as espécies NO e HNO podem ser responsáveis pelo efeito antiparasitário dos nitrosilos de rutênio aqui estudados. A relação entre a concentração das espécies NO/HNO será determinada pelas condições de pH do meio e concentração do redutor. / Ruthenium complexes type trans-[RuNO(NH3)4L](X)3 (X = BF4- or PF6- and L = imN, 4-pic, isn, py, pz, L- hist, nic, imC, P(OEt)3, SO32-), and [RuNO(Hedta)] were tested as antiproliferative agents against the Leishmania major parasite. The complexes where L = imN, pz, 4-pic, py, isn, and P(OEt)3 exhibited IC50pro ranging from 36 for trans-[RuNO(NH3)4imN](BF4)3 to 280 &mu;M for trans-[RuNO(NH3)4isn](BF4)3. The growth rate of the promastigote forms incubated with the most promising compounds, trans-[RuNO(NH3)4L](BF4)3 (L= imN, pz, py, 4-pic) had been evaluated trough motile cell counting and colorimetric assay (MTT). The trend between k-NO values and the antipromastigote effect follows the order: imN>4-pic>pz>py. An increase on the inhibition of the promastigote forms growth by the trans-[RuNO(NH3)4P(OEt)3](PF6)3 and trans-[RuNO(NH3)4imN](BF4)3 species was observed when the ruthenium complexes and ascorbic acid were simultaneously incubated, confirming the nitrosylation behavior of these complexes. Inhibitory effects of the trans-[RuNO(NH3)4imN](BF4)3 and of the Angeli\'s salt (Na2N2O3) were similar on the intramacrophage amastigote form grown. Encouraged by the antileishmanial effect of the complex trans-[RuNO(NH3)4imN](BF4)3 observed in vitro, in vivo experiments were carried out in infected BALB/c mice. The reaction between trans-[RuNO(NH3)4L](X)3 (X = BF4- or PF6- and L = imN, 4-pic, isn, P(OEt)3) and excess of L-cysteine was investigated and the products analysed. HNO and NO are the products of nitrosonium ligand reduction. This reaction yields the complexes trans-[Ru(NH3)4LN(O)SR]n-1 and trans-[Ru(NH3)4LN(O)(SR)2]n-2 which present k1 (1,7×107 - 2,2×104 mol-1Ls-1) and k2 (3,3×104 - 4,9×101mol-1Ls-1) for L = P(OEt)3, 4-pic, py e isn). This species react with RS- producing HNO and/or NO. Exception for L = P(OEt)3, this reaction does not occurs when CH+>= 1×10-6 mol L-1. For solution in which CH+ = 4×10-8 mol L-1, the reaction occur mainly trough: trans-[RuNO(NH3)4L]+3 + 2RS- + H2O &rarr; trans-[Ru(NH3)4L(H2O)]2+ + RSSR + NO- For L = P(OEt)3, and in acid media, NO0 is preferentially the reduction product of NO+ ligand. trans-[RuNO(NH3)4P(OEt)3]+3 + RSH + H2O &rarr; trans-[Ru(NH3)4P(OEt)3(H2O)]2+ + &frac12;RSSR + NO0 + H+ The experimental results from solution studies suggest that both NO and HNO could be responsible for the antiparasitaric effect of these ruthenium nitrosyl. The concentration ratio of NO/HNO could be dictated by the local conditions of pH.

Within the PhD. thesis the problems of H-transfer from selected types of phenols, aromatic secondary amines and compounds with acidic CH bond were investigated. This process was initiated by the action of redox agents, mostly PbO2, in nonpolar solvents. The abstraction of hydrogen atom results in the formation of radical intermediates, which were detected by EPR spectroscopy. Because in most cases, highly unstable radicals were produced, it was neccessary to apply the technique of indirect detection, so called spin trapping. This method enables to interpret the character of generated radicals on the basis of EPR parameters of radical adducts, which are formed by their reaction with suitably chosen compounds, spin traps, which are added to the reaction system. As spin traps, the aromatic nitrosocompounds were preferentially used. Due to the fact that EPR spectra of radical adducts were in most cases very complex, their interpretation was possible only using the computer simulation. Based on this approach, new information about the character of addition of phenoxyl tradicals to aromatic nitrosocompounds, stability of alkyl substituents in methyl substituted phenols and character of C-radicals, generated from substituted coumarines and beta-diketones, was obtained. Simultaneously, the small ability of nitrogen radicals, primary products of the splitting of NH bond, to enter the reaction with spin traps was proved by aromatic secondary amines.

In the frame of submitted diploma thesis dealing with the radical products of the decomposition of different X-H bonds, primary attention was focused on the detection of radical intermediates from decomposed N-H bonds. This access was realized with secondary amines of N-alkylaniline group, as well as with other structures R1-NH-R2. The aim was to confirm the formation of unstable aminyl radicals R1-N•-R2, which is possible by using spin-trapping method. In the series of experiments, where the compounds of lead and cobalt, as well as stable radical DPPH were applied as initiators of the decomposition, the unambiguous evidence for aminyl radicals was found only in the case of N-alkylanilines. With other secondary amines the detection of aminyl radicals is an open problem, because their adducts with nitrosobenzene are in very low concentration. Besides the study of the decomposition of N-H bonds also the products of the decomposition of phenolic O-H bonds, as well as products of the decomposition of C-H bonds in alkylsubstituents in phenols was studied.

O Nitroxil (HNO), forma reduzida em um elétron e protonada do óxido nítrico (NO), apresenta características químicas diferentes do seu congênere redox, com ações farmacológicas distintas e vantagens terapêuticas. Em conjunto, NO e HNO parecem ter um papel fundamental no controle do tônus vascular. A produção e/ou biodisponibilidade do HNO deve estar preservada durante o estresse oxidativo, ao contrário do que acontece com o NO. O HNO também apresenta potencial atividade antioxidante, atuando assim como citoprotetor e exibindo características desejáveis no tratamento de doenças cardiovasculares. O presente trabalho teve como objetivo estudar o efeito relaxante do nitroxil liberado do composto sal de Angeli (SA) e investigar os mecanismos celulares envolvidos nesse efeito em veia cava de ratos. Verificamos que o SA aumentou a concentração citosólica de HNO, medida pela sonda fluorescente DAF-2DA, por citometria de fluxo em células de veia umbilical humana (HUVECs). O aumento na intensidade de fluorescência foi abolido pelo sequestrador de HNO (L-cisteína), mas não foi alterado pelas espécies reativas de oxigênio (EROs). O SA promoveu relaxamento dependente da concentração em aorta e veia cava de ratos, com endotélio. Entretanto, o relaxamento induzido em veia cava foi menor que o relaxamento máximo (Emáx) em aorta. Analisando o tempo necessário para o composto induzir o Emáx, observamos que na aorta de ratos, o tempo máximo foi de 50 seg e na veia cava foi de 20 min. A conversão de HNO a NO pela superóxido dismutase (SOD) não foi necessária para a ativação da via de sinalização, uma vez que o relaxamento foi reduzido na presença de L-cisteína, mas não foi alterado em presença do inibidor da SOD (DDC) e do sequestrador de NO (Hidroxicobalamina). O relaxamento estimulado com o SA também foi inibido pelo L-NAME e ODQ, indicando a participação das enzimas NO-sintase e guanilil-ciclase solúvel (GCs), respectivamente. O bloqueador não seletivo de canais para K+ (TEA) e o sequestrador de ânion superóxido (O2¯) (Tiron) não modificaram o relaxamento para o SA quando realizada a curva concentração-efeito. Porém, ambos inibiram o relaxamento quando estudamos o efeito temporal do composto. Na presença do inibidor da NADPH oxidase (Apocinina), o relaxamento também foi reduzido. Deste modo, canais para K+, O2¯ e a NADPH oxidase parecem contribuir para o relaxamento induzido pelo SA. Aparentemente, não há participação da enzima proteína quinase G (GK), Ca2+-ATPase reticular (SERCA) ou de canais para Ca2+ dependentes de voltagem na via de sinalização. Com relação ao potencial antioxidante, em menor concentração (0,1 mmol/L) o SA apresentou efeito antioxidante, enquanto que em altas concentrações (1 mmol/L) ele atuou como um pró-oxidante. A principal espécie envolvida no efeito pró-oxidante do SA é o O2¯. Este é produzido, pelo menos em parte, pela ação da NADPH oxidase, uma vez que o aumento da produção de EROs estimulado pelo SA foi inibido em pelo Tiron e reduzido em presença de Apocinina. Esses dados foram obtidos por fluorescência da sonda DHE por citometria de fluxo em HUVECs. Apesar da produção de EROs em altas concentrações, o composto não apresentou toxicidade. / Nitroxyl (HNO), the one electron reduced and protonated form of nitric oxide (NO), displays different chemical characteristics compared to its redox sibling, with different pharmacological actions and therapeutic benefits. Together, NO and HNO seem to have an integral role in the control of vascular tone. The production and/or bioavailability of HNO must be preserved during oxidative stress, different from what happens to NO. In addition, HNO also has a potential antioxidant activity, thus acting as a cytoprotector and displaying desirable characteristics in the treatment of cardiovascular diseases. The present study aimed to study the relaxing effect of HNO released from Angeli\'s salt (AS) and to investigate the cellular mechanisms involved in this effect in rat vena cava. We found that AS increased cytosolic concentration of HNO, measured by fluorescent probe DAF-2DA by flow cytometry in human umbilical vein cells (HUVECs). HNO increase was abolished by HNO scavenger (L-cysteine), but it did not change in presence of the reactive oxygen species (ROS). AS promoted concentration-dependent relaxation in aorta and vena cava of rats with endothelium. However, the relaxation induced in vena cava was lower than the maximum relaxation (ME) induced in aorta. Analyzing the time necessary for the compound to induce ME, we observed that the effect in function of time was also significantly different between the vessels. In rat aorta, the maximum time was 50 seconds and in vena cava was 20 minutes. HNO conversion to NO by superoxide dismutase (SOD) was not required for signaling pathway activation, since AS relaxation was reduced in presence of L-cysteine, but it was not modified in presence of SOD inhibitor (DDC) or NO scavenger (Hydroxocobalamin). SA-induced relaxation was reduced by L-NAME and ODQ, indicating the involvement of NO synthase and soluble guanylyl cyclase (sGC), in the relaxation. Non-selective blocker of K+ channels (TEA) and superoxide anion (O2¯) scavenger (Tiron) did not modify the relaxation induced by AS when concentration-effect curve was performed. However, both inhibited relaxation when we performed the temporal effect compound study. Moreover, in the presence of NADPH oxidase inhibitor (Apocynin) relaxation was also reduced. Thus, K+ channels, O2¯ and NADPH oxidase seem to contribute to AS relaxation. Apparently there is no participation of protein kinase G (GK), sarcoplasmic reticulum Ca2+ ATPase (SERCA) or voltage-dependent Ca2+ channels in relaxation signaling pathway. Also, low concentrations (0.1 mmol/L) of AS presented antioxidant effect, whereas in high concentrations (1 mmol/L) it acted as a prooxidant. The main ROS involved in AS prooxidant effect was O2¯, which was produced, at least in part, by the action of NADPH oxidase, since the increase of ROS production by AS was inhibited by Tiron and reduced in the presence of Apocynin. These data were obtained by fluorescence DHE probe by flow cytometry in HUVECs. Despite the production of ROS in high concentrations, the compound did not show toxicity.

O Nitroxil (HNO), forma reduzida em um elétron e protonada do óxido nítrico (NO), apresenta características químicas diferentes do seu congênere redox, com ações farmacológicas distintas e vantagens terapêuticas. Em conjunto, NO e HNO parecem ter um papel fundamental no controle do tônus vascular. A produção e/ou biodisponibilidade do HNO deve estar preservada durante o estresse oxidativo, ao contrário do que acontece com o NO. O HNO também apresenta potencial atividade antioxidante, atuando assim como citoprotetor e exibindo características desejáveis no tratamento de doenças cardiovasculares. O presente trabalho teve como objetivo estudar o efeito relaxante do nitroxil liberado do composto sal de Angeli (SA) e investigar os mecanismos celulares envolvidos nesse efeito em veia cava de ratos. Verificamos que o SA aumentou a concentração citosólica de HNO, medida pela sonda fluorescente DAF-2DA, por citometria de fluxo em células de veia umbilical humana (HUVECs). O aumento na intensidade de fluorescência foi abolido pelo sequestrador de HNO (L-cisteína), mas não foi alterado pelas espécies reativas de oxigênio (EROs). O SA promoveu relaxamento dependente da concentração em aorta e veia cava de ratos, com endotélio. Entretanto, o relaxamento induzido em veia cava foi menor que o relaxamento máximo (Emáx) em aorta. Analisando o tempo necessário para o composto induzir o Emáx, observamos que na aorta de ratos, o tempo máximo foi de 50 seg e na veia cava foi de 20 min. A conversão de HNO a NO pela superóxido dismutase (SOD) não foi necessária para a ativação da via de sinalização, uma vez que o relaxamento foi reduzido na presença de L-cisteína, mas não foi alterado em presença do inibidor da SOD (DDC) e do sequestrador de NO (Hidroxicobalamina). O relaxamento estimulado com o SA também foi inibido pelo L-NAME e ODQ, indicando a participação das enzimas NO-sintase e guanilil-ciclase solúvel (GCs), respectivamente. O bloqueador não seletivo de canais para K+ (TEA) e o sequestrador de ânion superóxido (O2¯) (Tiron) não modificaram o relaxamento para o SA quando realizada a curva concentração-efeito. Porém, ambos inibiram o relaxamento quando estudamos o efeito temporal do composto. Na presença do inibidor da NADPH oxidase (Apocinina), o relaxamento também foi reduzido. Deste modo, canais para K+, O2¯ e a NADPH oxidase parecem contribuir para o relaxamento induzido pelo SA. Aparentemente, não há participação da enzima proteína quinase G (GK), Ca2+-ATPase reticular (SERCA) ou de canais para Ca2+ dependentes de voltagem na via de sinalização. Com relação ao potencial antioxidante, em menor concentração (0,1 mmol/L) o SA apresentou efeito antioxidante, enquanto que em altas concentrações (1 mmol/L) ele atuou como um pró-oxidante. A principal espécie envolvida no efeito pró-oxidante do SA é o O2¯. Este é produzido, pelo menos em parte, pela ação da NADPH oxidase, uma vez que o aumento da produção de EROs estimulado pelo SA foi inibido em pelo Tiron e reduzido em presença de Apocinina. Esses dados foram obtidos por fluorescência da sonda DHE por citometria de fluxo em HUVECs. Apesar da produção de EROs em altas concentrações, o composto não apresentou toxicidade. / Nitroxyl (HNO), the one electron reduced and protonated form of nitric oxide (NO), displays different chemical characteristics compared to its redox sibling, with different pharmacological actions and therapeutic benefits. Together, NO and HNO seem to have an integral role in the control of vascular tone. The production and/or bioavailability of HNO must be preserved during oxidative stress, different from what happens to NO. In addition, HNO also has a potential antioxidant activity, thus acting as a cytoprotector and displaying desirable characteristics in the treatment of cardiovascular diseases. The present study aimed to study the relaxing effect of HNO released from Angeli\'s salt (AS) and to investigate the cellular mechanisms involved in this effect in rat vena cava. We found that AS increased cytosolic concentration of HNO, measured by fluorescent probe DAF-2DA by flow cytometry in human umbilical vein cells (HUVECs). HNO increase was abolished by HNO scavenger (L-cysteine), but it did not change in presence of the reactive oxygen species (ROS). AS promoted concentration-dependent relaxation in aorta and vena cava of rats with endothelium. However, the relaxation induced in vena cava was lower than the maximum relaxation (ME) induced in aorta. Analyzing the time necessary for the compound to induce ME, we observed that the effect in function of time was also significantly different between the vessels. In rat aorta, the maximum time was 50 seconds and in vena cava was 20 minutes. HNO conversion to NO by superoxide dismutase (SOD) was not required for signaling pathway activation, since AS relaxation was reduced in presence of L-cysteine, but it was not modified in presence of SOD inhibitor (DDC) or NO scavenger (Hydroxocobalamin). SA-induced relaxation was reduced by L-NAME and ODQ, indicating the involvement of NO synthase and soluble guanylyl cyclase (sGC), in the relaxation. Non-selective blocker of K+ channels (TEA) and superoxide anion (O2¯) scavenger (Tiron) did not modify the relaxation induced by AS when concentration-effect curve was performed. However, both inhibited relaxation when we performed the temporal effect compound study. Moreover, in the presence of NADPH oxidase inhibitor (Apocynin) relaxation was also reduced. Thus, K+ channels, O2¯ and NADPH oxidase seem to contribute to AS relaxation. Apparently there is no participation of protein kinase G (GK), sarcoplasmic reticulum Ca2+ ATPase (SERCA) or voltage-dependent Ca2+ channels in relaxation signaling pathway. Also, low concentrations (0.1 mmol/L) of AS presented antioxidant effect, whereas in high concentrations (1 mmol/L) it acted as a prooxidant. The main ROS involved in AS prooxidant effect was O2¯, which was produced, at least in part, by the action of NADPH oxidase, since the increase of ROS production by AS was inhibited by Tiron and reduced in the presence of Apocynin. These data were obtained by fluorescence DHE probe by flow cytometry in HUVECs. Despite the production of ROS in high concentrations, the compound did not show toxicity.