Identification of pentosidine as a native structure for advanced glycation end products in beta-2-microglobulin-containing amyloid fibrils in patients with dialysis-related amyloidosis.

beta-2-Microglobulin (beta-2m) is a major constituent of amyloid fibrils in patients with dialysis-related amyloidosis (DRA). Recently, we found that the pigmented and fluorescent adducts formed nonenzymatically between sugar and protein, known as advanced glycation end products (AGEs), were present in beta-2m-containing amyloid fibrils, suggesting the possible involvement of AGE-modified beta-2m in bone and joint destruction in DRA. As an extension of our search for the native structure of AGEs in beta-2m of patients with DRA, the present study focused on pentosidine, a fluorescent cross-linked glycoxidation product. Determination by both HPLC assay and competitive ELISA demonstrated a significant amount of pentosidine in amyloid-fibril beta-2m from long-term hemodialysis patients with DRA, and the acidic isoform of beta-2m in the serum and urine of hemodialysis patients. A further immunohistochemical study revealed the positive immunostaining for pentosidine and immunoreactive AGEs and beta-2m in macrophage-infiltrated amyloid deposits of long-term hemodialysis patients with DRA. These findings implicate a potential link of glycoxidation products in long-lived beta-2m-containing amyloid fibrils to the pathogenesis of DRA.

Antimycobacterial activities of antisense oligodeoxynucleotide phosphorothioates in drug-resistant strains.

Strains of Mycobacterium smegmatis, a mycobacterium which shares genetic sequences, grows more rapidly, and is nonpathogenic in man as compared with Mycobacterium tuberculosis, were utilized for the initial development of new antimycobacterial therapy. Drug-resistant strains of M. smegmatis which are known to arise in a manner identical to the emergence of drug-resistant strains of M. tuberculosis were isolated and utilized as models for the antimycobacterial activities of modified and unmodified oligodeoxynucleotide phosphorothioates in broth cultures. Under normal conditions, oligodeoxynucleotide phosphorothioates do not enter mycobacteria, and several strategies were successfully utilized to afford entry of oligonucleotides into the mycobacterial cells. One involved the presence of very low levels of ethambutol, which enables the entry of oligonucleotides into mycobacteria because of its induced alterations in the cell wall, and another involved the utilization of oligonucleotides covalently attached to a D-cycloserine molecule, whereby entry into the mycobacterial cell is achieved by a receptor-mediated process. Another low molecular weight, covalently attached ligand that enabled the entry and subsequent antimycobacterial activities of oligodeoxynucleotide phosphorothioates in the absence of a cell wall modifying reagent was biotin. Significant sequence-specific growth inhibition of wild-type, as well as of drug-resistant, M. smegmatis was obtained by modified oligonucleotides complementary in sequence to a specific region of the mycobacterium aspartokinase (ask) gene when utilized in combinations with ethambutol (as compared to ethambutol alone) or as D-cycloserine or biotin covalent adducts without the presence of any other cytotoxic or cytostatic agent.

Stimulation-dependent I kappa B alpha phosphorylation marks the NF-kappa B inhibitor for degradation via the ubiquitin-proteasome pathway.

The nuclear translocation of NF-kappa B follows the degradation of its inhibitor, I kappa B alpha, an event coupled with stimulation-dependent inhibitor phosphorylation. Prevention of the stimulation-dependent phosphorylation of I kappa B alpha, either by treating cells with various reagents or by mutagenesis of certain putative I kappa B alpha phosphorylation sites, abolishes the inducible degradation of I kappa B alpha. Yet, the mechanism coupling the stimulation-induced phosphorylation with the degradation has not been resolved. Recent reports suggest a role for the proteasome in I kappa B alpha degradation, but the mode of substrate recognition and the involvement of ubiquitin conjugation as a targeting signal have not been addressed. We show that of the two forms of I kappa B alpha recovered from stimulated cells in a complex with RelA and p50, only the newly phosphorylated form, pI kappa B alpha, is a substrate for an in vitro reconstituted ubiquitin-proteasome system. Proteolysis requires ATP, ubiquitin, a specific ubiquitin-conjugating enzyme, and other ubiquitin-proteasome components. In vivo, inducible I kappa B alpha degradation requires a functional ubiquitin-activating enzyme and is associated with the appearance of high molecular weight adducts of I kappa B alpha. Ubiquitin-mediated protein degradation may, therefore, constitute an integral step of a signal transduction process.

Fotofísica de enodionas

Em 1906, Albrecht obteve a primeira enodiona conhecida, ao reagir benzoquinona com ciclopentadieno. A estrutura desta enodiona somente foi elucidada em 1928, por Diels e Alder que, entretanto, perceberam ser possív.el existirem dois diferentes isômeros para o composto em questão: endo e exo. A descoberta, em 1958, da reação de fotocilização deste aduto de Diels-Alder, por Cookson e colaboradores, demonstrou que se tratava do isômero endo e esta reação passou a ser utilizada para se comprovar a configuração endo de vários compostos de estrutura análoga. Porém, apesar de sua importância histórica como método para comprovar a configuração dos adutos de Diels-Alder, os dados disponíveis até hoje são insuficientes para o esclarecimento dos processos subjacentes à fotociclização desses compostos, especialmente porque hoje sabemos que existem adutos de configuração endo que não fotociclizam. Visando aumentar o conhecimento sobre o comportamento destes adutos frente à reação de fotociclização, determinaram-se seus espectros de absorção (na região do UVNisível) e de emissão de luminescência e concluiu-se, com base em nossos resultados e daqueles relatados na literatura, que o fato de não ocorrer a fotociclização de alguns adutos de configuração endo não depende exclusivamente da estabilidade dos estados excitados envolvidos, ainda que essa característica e a natureza destes certamente afete os rendimentos quânticos da reação. Antes, parece-nos que estruturas que permitam deslocalizar um dos elétrons do birradical intermediariamente formado neste processo propiciam a reversão deste intermediário ao reagente, ao invés de se formar o produto de fotociclização. Alem disso, reinvestigamos a reação de fotoisomerização endo → exo do aduto de benzoquinona e ciclopentadieno, por irradiação em etanol/trietil-amina, relatada por Pandey e colaboradores em 1990, tendo verificado que tal isomerização não ocorre nas condições descritas na literatura, obtendo-se, ao invés disso, principalmente o tautômero aromático da enodiona e seu produto de fotociclização. Por outro lado, descobrimos que, deixar em repouso, no escuro, o aduto de benzoquinona e ciclopentadieno em etanol/trietil-amina conduz,a um dímero deste aduto, não descrito previamente.

Novel Photochemical Methodologies For Diversity Oriented Synthesis And Screening Of Combinatorial Libraries

The main goal of this project was to develop an efficient methodology allowing rapid access to structurally diverse scaffolds decorated with various functional groups. Initially, we discovered and subsequently developed an experimentally straightforward, high-yielding photoinduced conversion of readily accessible diverse starting materials into polycyclic aldehydes and their (hemi)acetals decorated by various pendants. The two step sequence, involving the Diels-Alder addition of heterocyclic chalcones and other benzoyl ethylenes to a variety of dienes, followed by the Paternò-Büchi reaction, was described as an alkene-carbonyl oxametathesis. This methodology offers a rapid increase in molecular complexity and diversity of the target scaffolds. To develop this novel methodology further and explore its generality, we directed our attention to the Diels-Alder adducts based on various chromones. We discovered that the Diels-Alder adducts of chromones are capable of photoinduced alkene-arene [2+2] cycloaddition producing different dienes, which can either dimerize or be introduced into a double-tandem [4π+2π]·[2π+2π]·[4π+2π]·[2π+2π] synthetic sequence, followed by an acid-catalyzed oxametathesis, leading to a rapid expansion of molecular complexity over a few experimentally simple steps. In view of the fact that oxametathesis previously was primarily observed in aromatic oxetanes, we decided to prepare model aliphatic oxetanes with a conformationally unconstrained or "flexible" methyl group based on the Diels-Alder adducts of cyclohexadiene or cyclopentadiene with methyl vinyl ketone. Upon addition of an acid, the expected oxametathesis occurred with results similar to those observed in the aromatic series proving the generality of this approach. Also we synthesized polycyclic oxetanes resulting from the Diels-Alder adducts of cyclic ketones. This not only gave us access to remarkably strained oxetane systems, but also the mechanism for their protolytic ring opening provided a great deal of insight to how the strain affects the reactivity. Additionally, we discovered that although the model Hetero-Diels-Alder adducts did not undergo [2+2] cycloaddition, both exo- and endo-Sulfa-Diels-Alder products, nonetheless, were photochemically active and various products with defined stereochemistry could be produced upon photolysis. In conclusion, we have developed an approach to the encoding and screening of solution phase libraries based on the photorelease of externally sensitized photolabile tags. The encoding tags can be released into solution only when a binding event occurs between the ligand and the receptor, equipped with an electron transfer sensitizer. The released tags are analyzed in solution revealing the identity of the lead ligand or narrowing the range of potential leads.

Lesões em DNA promovidas por produtos de oxidação do β-caroteno: possíveis implicações biológicas

Apesar de diversos estudos in vitro e em populações indicarem um efeito protetor do β-caroteno em sistemas biológicos, estudos epidemiológicos como o \"The Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study\" (ATBC) e o \"The Beta-Carotene and Retinol Efficacy Trial\" (CARET) mostraram um aumento na incidência de câncer pulmonar em indivíduos fumantes suplementados com β-caroteno. Essa ação contraditória tem sido chamada na literatura de \"Paradoxo do β-Caroteno\". Sabe-se que este carotenóide sob altas pressões de oxigênio ou na presença de peróxidos pode sofrer oxidação e levar a formação de compostos como aldeídos, epóxidos, etc, que são capazes de se adicionarem covalentemente ao DNA. Estudos, in vitro e in vivo têm demonstrado a possibilidade de os metabólitos do β-caroteno agirem como agentes pró-carcinogênicos. Estes agentes quando ativados quimicamente podem levar à formação de adutos de DNA. Já se sabe que alguns desses adutos encontramse em níveis aumentados em diversas situações de risco de câncer. Diversos grupos, incluindo o nosso, têm demonstrado a formação de lesões em DNA a partir de aldeídos e epóxidos exógenos ou gerados endogenamente. O presente trabalho mostra que a reação do β-caroteno e dois de seus produtos de oxidação, retinal e β-apo-8\'-carotenal, com 2\'-desoxiguanosina e DNA leva à formação de adutos. Dentre os adutos formados, foi caracterizado o aduto 1,N2eteno-2\'-desoxiguanosina (1 ,N2-εdGuo). Os níveis de outro aduto de DNA, a 8-oxo-7,8-dihidro-2\'-deoxiguanosina (8-oxodGuo), também foram monitoradas para estudo comparativo. A formação dos adutos também foi verificada em fibroblastos normais de pulmão humano (linhagem IMR-90) expostos ao β-caroteno e aos seus produtos de oxidação. Experimentos com ratos suplementados com β-caroteno e expostos à fumaça de cigarro em períodos de 7, 30 e 180 dias, mostraram níveis aumentados de 1,N2-εdGuo nos animais suplementados com o carotenóide comparado ao grupo veículo. Aumento no nível de 8-oxodGuo também foi verificado nos tratamentos de 7 e 180 dias. Um aumento significativo no nível do eteno aduto também foi verificado nos animais suplementados com β-caroteno e expostos à fumaça de cigarro, comparado ao grupo apenas exposto à fumaça após 7 e 180 dias de exposição. Nestes mesmos grupos, o aumento do 8-oxodGuo só foi observado no tratamento por 180 dias. Sabendo que estas lesões são comprovadamente mutagênicas, nossos estudos podem contribuir para o esclarecimento dos mecanismos envolvidos na formação de câncer em fumantes suplementados ou não com β-caroteno.

Enantioselective alkylation of β-keto esters promoted by dimeric Cinchona-derived ammonium salts as recoverable organocatalysts

Dimeric anthracenyldimethyl-derived Cinchona ammonium salts are used as chiral organocatalysts in 5 mol% for the phase-transfer enantioselective alkylation reaction of 2-alkoxycarbonyl-1-indanones with activated bromides. The corresponding adducts bearing a new all-carbon quaternary center are obtained usually in high yield and with moderate and opposite enantioselectivity (up to 55%) when using ammonium salts derived from quinidine and its pseudoenantiomer quinine as organocatalysts. These catalysts can be almost quantitatively recovered by precipitation in ether and reused.

Enantioselective Michael addition of isobutyraldehyde to nitroalkenes organocatalyzed by chiral primary amine-guanidines

Primary amine-guanidines derived from trans-cyclohexane-1,2-diamines are used as organocatalysts for the enantioselective conjugate addition of isobutyraldehyde to arylated and heteroarylated nitroalkenes. The reaction was performed in the presence of imidazole as the additive in aqueous DMF as the solvent at 0 °C. The corresponding Michael adducts bearing a new stereocenter were obtained in high yields and with enantioselectivities of up to 80%. Theoretical calculations are used to justify the observed sense of the stereoinduction.

Enantioselective Michael addition of aldehydes to maleimides organocatalysed by chiral 1,2-diamines: an experimental and theoretical study

Simple and commercially available chiral 1,2-diamines were used as organocatalysts for the enantioselective conjugate addition of aldehydes, including α,α-disubstituted, to maleimides. The reaction was carried out in the presence of hexanedioic acid as an additive in aqueous solvents at room temperature. By employing (1S,2S)- and (1R,2R)-cyclohexane-1,2-diamine as organocatalysts, the corresponding Michael adducts bearing new stereocenters were obtained in high or quantitative yields with enantioselectivities of up to 92%, whereas the use of (1S,2S)-1,2-diphenylethane-1,2-diamine gave a much lower ee. Theoretical calculations were used to justify the observed sense of the stereoinduction.

Synthesis of α,β-diamino acid derivatives via asymmetric Mannich reactions of glycine imino esters catalyzed by a chiral phosphoramidite·silver complex

AgOTf·phosphoramidite complexes efficiently catalyze the enantioselective Mannich-type reaction between benzophenone-imine glycine methyl ester and N-tosyl aldimines in the absence of a base. The corresponding syn-adducts, which are the direct precursors of α,β-diamino acids, are obtained with moderate to good syn-diastereoselectivities (up to 9:1) and high enantioselectivities (up to 99% ee).

Solvent-dependent enantioswitching in the Michael addition of α,α-disubstituted aldehydes to maleimides organocatalyzed by mono-N-Boc-protected cyclohexa-1,2-diamines

Enantiomerically pure mono-N-Boc-protected trans-cyclohexa-1,2-diamines are used as organocatalysts for the enantioselective conjugate addition of α,α-disubstituted aldehydes to maleimides. Using a single enantiomer of the organocatalyst, both enantiomeric forms of the resulting Michael adducts bearing a new quaternary stereocenter are obtained in high yields, by only changing the reaction solvent from chloroform (up to 86% ee) to aqueous DMF (up to 84% ee).

Regio and diastereoselective multicomponent 1,3-dipolar cycloadditions between prolinate hydrochlorides, aldehydes and dipolarophiles for the direct synthesis of pyrrolizidines

A general synthesis of highly substituted pyrrolizidines can be performed by a multicomponent 1,3-dipolar cycloaddition using proline ester hydrochlorides, aldehydes and dipolarophiles, at room temperature without catalysts or in the presence of AgOAc (5 mol %). In the case of (2S,4R)-4-hydroxyproline derivatives it is possible to obtain enantioenriched pyrrolizidines with high control of the regio- and diastereoselectivity affording the adducts 2,4-trans-2,5-trans according to an endo-approach and a S-dipole geometry of the in situ generated azomethine ylide. For proline esters a similar regioselectivity and endo-diastereoselectivity are observed when the dipole promotes an α-attack. However, when ethyl glyoxylate is used as aldehyde component the γ-attack of the S-ylide takes place preferentially giving rise the opposite regioselectivity for acrylic dipolarophiles, being crucial the role of silver acetate. In this case, the exo-adducts with a 2,3-cis-2,5-trans relative configuration are diastereoselectively obtained. In addition, computational studies have also been carried out to shed light on the origins of the diastereo- and regioselectivity observed for the described 1,3-dipolar cycloadditions.

Solvent-Induced Reversal of Enantioselectivity in the Synthesis of Succinimides by the Addition of Aldehydes to Maleimides Catalysed by Carbamate-Monoprotected 1,2-Diamines

A simple change in the polarity of the solvent allows both enantiomers of substituted succinimides to be obtained in the enantioselective conjugate addition reaction of aldehydes, mainly α,α-disubstituted, to maleimides catalysed by chiral carbamate-monoprotected trans-cyclohexane-1,2-diamines. Using a single enantiomer of the organocatalyst, both enantiomers of the resulting Michael adducts are obtained in high yields by simply changing the reaction solvent from aqueous DMF (up to 84 % ee) to chloroform (up to 86 % ee). Theoretical calculations are used to explain this uncommon reversal of the enantioselectivity; two transition state orientations of different polarities are differently favoured in polar or nonpolar solvents.

Synthesis of xanthone-1,2,3-triazole dyads

Xanthones and 1,2,3-triazoles are known to exhibit several biological, pharmacological and biocidal properties[1]. The potential applications of these two classes of heterocycles led us to develop new strategies to synthesize xanthone-1,2,3-triazole dyads, aiming to get potentially improved therapeutic agents[2]. With this rational in mind we designed and synthesized novel chromone derivatives 1a-d to be used as building motifs and to explore the reactivity of the two unsaturated systems (the diene and the alkyne). In the present communication we will present a new synthetic route towards the synthesis of xanthone-1,2,3-triazole dyads 7a-d using consecutively the azide-alkyne Huisgen 1,3-dipolar cycloaddition and Diels-Alder reaction. Our approach involves the synthesis chromone-triazole derivatives 2a-d using the reaction of 1a-d with sodium azide, followed by the methylation of the NH of the triazole moiety. The methylation afforded three isomers 3a-d, 4a-d and 5a-d, as expected. The major isomers 3a-d were used in the Diels-Alder reaction with N-methylmaleimide, and the adducts obtained 6a-d were oxidized to afford the xanthone-1,2,3-triazole dyads 7a-d. All the synthetic details as well as the structural characterization (by 1D and 2D NMR studies) of the new synthesised compounds will be presented and discussed.

Déficience dans la réparation par excision de nucléotides en phase S induite par la séquestration du facteur de réplication RPA

Introduction Les lésions induites par les rayons UV peuvent causer des blocages dans la réplication de l'ADN. Ces dommages sont éliminés par le processus moléculaire très conservé de réparation par excision de nucléotides (NER). Nous avons précédemment démontré que la protéine ATR, une kinase majeure impliquée dans le stress réplicatif, est requise pour une NER efficace, et ce exclusivement durant la phase S. Des résultats subséquents ont suggéré que ce prérequis n’était pas lié à la réponse induite par ATR, mais plutôt d’une conséquence globale causée par la présence de stress réplicatif. En ce sens, nous mettons l’emphase qu’après irradiation UV, le complexe RPA joue un rôle crucial dans l'activation des mécanismes de NER ainsi que dans le redémarrage des fourches de réplication bloquées. Hypothèses: En général, les mutations qui confèrent une augmentation du stress réplicatif engendrent une séquestration excessive du facteur RPA aux fourches de réplication bloquées ce qui réduit son accessibilité pour le NER. Méthodes et résultats: Le modèle de la levure a été choisi pour vérifier cette hypothèse. Nous avons développé un essai de NER spécifique à chacune des phases du cycle cellulaire pour démontrer que les cellules déficientes en Mec1, l’homologue d’ATR, sont défectives dans la réparation par excision de nucléotides spécifiquement en phase S. De plus, plusieurs autres mutants de levure, caractérisés par un niveau de dommages spontanés élevé, ont aussi exhibé un défaut similaire. Ces mutants ont démontré une fréquence et une intensité de formation de foyers de RPA plus élevée. Finalement, une diminution partielle de RPA dans les levures a induit un défaut significatif dans le NER spécifiquement durant la phase S. Conclusion: Nos résultats supportent la notion que la séquestration de RPA aux fourches de réplication endommagées durant la phase S prévient son utilisation pour la réparation par excision de nucléotides ce qui inhibe fortement l'efficacité de réparation. Cette étude chez la levure facilite l’élucidation du phénomène analogue chez l’humain et, ultimement, comprend des implications majeures dans la compréhension du mécanisme de développement des cancers UV-dépendants.