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<p>An abstract of a thesis devoted to using helix-coil models to study unfolded states.\\</p><p>Research on polypeptide unfolded states has received much more attention in the last decade or so than it has in the past. Unfolded states are thought to be implicated in various</p><p>misfolding diseases and likely play crucial roles in protein folding equilibria and folding rates. Structural characterization of unfolded states has proven to be</p><p>much more difficult than the now well established practice of determining the structures of folded proteins. This is largely because many core assumptions underlying</p><p>folded structure determination methods are invalid for unfolded states. This has led to a dearth of knowledge concerning the nature of unfolded state conformational</p><p>distributions. While many aspects of unfolded state structure are not well known, there does exist a significant body of work stretching back half a century that</p><p>has been focused on structural characterization of marginally stable polypeptide systems. This body of work represents an extensive collection of experimental</p><p>data and biophysical models associated with describing helix-coil equilibria in polypeptide systems. Much of the work on unfolded states in the last decade has not been devoted</p><p>specifically to the improvement of our understanding of helix-coil equilibria, which arguably is the most well characterized of the various conformational equilibria</p><p>that likely contribute to unfolded state conformational distributions. This thesis seeks to provide a deeper investigation of helix-coil equilibria using modern</p><p>statistical data analysis and biophysical modeling techniques. The studies contained within seek to provide deeper insights and new perspectives on what we presumably</p><p>know very well about protein unfolded states. \\</p><p>Chapter 1 gives an overview of recent and historical work on studying protein unfolded states. The study of helix-coil equilibria is placed in the context</p><p>of the general field of unfolded state research and the basics of helix-coil models are introduced.\\</p><p>Chapter 2 introduces the newest incarnation of a sophisticated helix-coil model. State of the art modern statistical techniques are employed to estimate the energies</p><p>of various physical interactions that serve to influence helix-coil equilibria. A new Bayesian model selection approach is utilized to test many long-standing </p><p>hypotheses concerning the physical nature of the helix-coil transition. Some assumptions made in previous models are shown to be invalid and the new model </p><p>exhibits greatly improved predictive performance relative to its predecessor. \\</p><p>Chapter 3 introduces a new statistical model that can be used to interpret amide exchange measurements. As amide exchange can serve as a probe for residue-specific</p><p>properties of helix-coil ensembles, the new model provides a novel and robust method to use these types of measurements to characterize helix-coil ensembles experimentally</p><p>and test the position-specific predictions of helix-coil models. The statistical model is shown to perform exceedingly better than the most commonly used </p><p>method for interpreting amide exchange data. The estimates of the model obtained from amide exchange measurements on an example helical peptide </p><p>also show a remarkable consistency with the predictions of the helix-coil model. \\</p><p>Chapter 4 involves a study of helix-coil ensembles through the enumeration of helix-coil configurations. Aside from providing new insights into helix-coil ensembles,</p><p>this chapter also introduces a new method by which helix-coil models can be extended to calculate new types of observables. Future work on this approach could potentially</p><p>allow helix-coil models to move into use domains that were previously inaccessible and reserved for other types of unfolded state models that were introduced in chapter 1.</p> / Dissertation

Les MICI (maladies inflammatoires chroniques de l'intestin) invalident 200 000 personnes en France. La région Nord-Pas-de-Calais est particulièrement touchée par ces affections et les traitements disponibles pour ces pathologies demeurent coûteux et peu nombreux. Des études récentes ont suggéré que le système endocannabinoïde, exprimé au seing du tractus gastro-intestinal, est une cible thérapeutique prometteuse pour le traitement des MICI. Ce système se compose des récepteurs aux cannabinoïdes CB1 et CB2, des ligands endogènes de ces récepteurs, notamment l'anandamide et le 2-arachidonoylglycérol et des protéines impliquées dans l'anabolisme et le catabolisme des ligands. L'anandamide a présenté des capacités à prévenir la colite induite par le TNBS à des rongeurs. Toutefois, in vivo ce composé possède un temps de demi-vie court et est rapidement dégradé par une amidase à sérine, la FAAH (Fatty Acid Amide Hydrolase). Nous avons ainsi envisagé la conception, la synthèse et l'évaluation pharmacologique de nouveau inhibiteurs de la FAAH. L'une de nos molécules, le composé 95, présentant une CI50 sur l'enzyme de 88 nM a ensuite été injectée par voie intrapéritonéale à des souris dont la colite a été induite trois jours plus tard par l'injection intrarectale de TNBS. L'évaluation des scores macroscopiques et microscopiques des dommages causés sur le côlon par l'agent irritant a ensuite été effectuée. L'inflammation du côlon a été significativement réduite chez le groupe de souris ayant été traité par le composé 95, montrant que l'inhibition de la FAAH est une stratégie thérapeutique efficace dans le traitement des MICI. / IBD (Inflammatory Bowel Diseases) invalidate 200 000 persons in France. Nord-Pas-de-Calais region is particularly touched by these diseases and the available treatments for these pathologies are few and expensive. Recent studies have suggested that endocannabinoid system expressed in the gastrointestinal tract, was a promising therapeutic target for the IBD treatment. This system is made up of cannabinoids receptors CB1 and CB2, endogenous ligands of these receptors, notably anandamide and 2-arachidonoylglycerol, and proteins involved in ligands metabolism. Anandamide has shown properties to prevent TNBS-induced colitis in mice. However, in vivo, anandamide possesses a short life time and is rapidly hydrolyzed by a serine amidase, the FAAH (Fatty Acid Amide Hydrolase). In this context, we have considered the design, the synthesis and the pharmacological evaluation of new FAAH inhibitors. One of our molecules, compound 95, inhibiting the enzyme with an IC50 value of 88 nM has been injected intraperitonally to mice which the colitis was induced three days later by intrarectal TNBS-administration. The assessment of macroscopic and microscopic scores of colonic damages was undergone. Colonic inflammation was significatively reduced in the group of mice which has been treated by 95, showing evidence that FAAH inhibition was an effective therapeutic target for the treatment of IBD.

This thesis is concerned with the development of methodology for the regioselective ring-opening of aziridines and aziridinium intermediates and its subsequent application to the asymmetric synthesis of &beta;-substituted-&alpha;-amino acids and 1,2,3,4-tetrahydroisoquinolines. Chapter 1 introduces methods for the formation of aziridines and aziridinium ions and focusses on their utility as intermediates in synthesis. Chapter 2 describes studies into the synthesis of aziridines from enantiopure &alpha;-hydroxy-&beta;-amino esters and their subsequent conversion to the corresponding &beta;-hydroxy-&alpha;-amino acids via either a regioselective ring-opening with Cl₃CCO₂H, or a rearrangement promoted by Cl3CCO2H. Application of this procedure to both syn- and anti-configured substrates enabled the syntheses of (S,S)-allo-threonine, (2R,3S)-threonine, (R,R)-3-hydroxyphenylalanine and (2S,3R)-3-hydroxyphenylalanine. Chapter 3 details attempts to truncate the synthesis described in Chapter 2 by investigating the synthesis of enantiopure anti-&beta;-hydroxy-&alpha;-amino acids via the intermediacy of aziridinium ions. These studies culminated in the development of a regioselective and stereospecific one-pot aziridinium formation and ring-opening protocol, leading to the synthesis of a range of C(3)-aryl and C(3)-alkyl substituted anti-&beta;-hydroxy-&alpha;-amino acids. Chapter 4 discusses the conversion of enantiopure anti-&alpha;-hydroxy-&beta;-amino esters to anti-&beta;-fluoro-&alpha;-amino esters via the regioselective and stereospecific ring-opening of an aziridinium intermediates in situ. The subsequent development of a one-pot deprotection strategy leads to a concise and expedient synthesis of anti-&beta;-fluorophenylalanines. The extension of this methodology to access a representative anti-&alpha;,&beta;-diamino acid is also demonstrated. Chapter 5 describes the development of a one-pot diastereoselective rearrangement of enantiopure &alpha;-hydroxy-&beta;-amino esters to 1,2,3,4-tetrahydroisoquinolines. The substrate scope of this reaction manifold is examined and application to the asymmetric synthesis of enantiopure 1,2,3,4-tetrahydroisoquinolines also discussed. Chapter 6 contains full experimental procedures and characterisation data for all compounds synthesised in Chapters 2, 3, 4 and 5.

Foram estudados os produtos da interação entre o acetato e o trifluoroacetato de ródio (respectivamente, Rh2(OAc)4 e Rh2(TFA)4) com amidas (formamida, FA; acetamida, AA; N-metil-acetamida, MA; benzamida, BA; N-fenil-acetamida, NFAA; trifluoroacetamida, TMA; ciclofosfamida, CFA), objetivando caracterização química e avaliação do potencial anti-tumor. Foram sintetizados por refluxo em clorofórmio dois adutos de Rh2(OAc)4 (Rh2(OAc)4-2FA e Rh2(OAc)4-2AA) e sete adutos inéditos de Rh2(TFA)4, de fórmula geral Rh2(TFA)4-2L (L = AA, BA, CFA, FA, MA, NFAA e TMA). Também obtivemos o novo amidato de Rh(II), Rh2(CF3CONH)4(CF3CONH2)2 (Rh2(TFACAM)4-2TMA), por fusão de Rh2(OAc)4 com TMA. Através da fusão de Rh2(TFA)4 com MA, obtivemos um composto que formulamos como Rh2(CF3COO)2(CH3CONCH3)2. Os resultados de análise elementar foram satisfatórios. Os adutos apresentaram bandas de absorção características dos carboxilatos de ródio (~ em 600, 450, 250 - ombro - e 220 nm). O modo de coordenação do ligante axial, pelo átomo de oxigênio da amida, foi determinado pela diminuição da freqüência de estiramento C-O na região do infravermelho (IV). Os amidatos apresentaram apenas uma banda na região do visível, e na região do IV os valores de?estiramentos característicos dos principais grupos orgânicos. Estudos comparativos dos espectros Raman mostraram que a freqüência Rh-Rh diminui ao se passar de um carboxilato para um amidato. Medidas de susceptibilidade magnética atestam o diamagnetismo de todas as moléculas (ligação Rh-Rh simples). O mecanismo de termodecomposição do Rh2(TFA)4 e dos seus adutos com amidas envolve mais de uma etapa, sendo que os intermediários podem apresentar estruturas do tipo Rh2(CF3COO)4-x(L)n (x e n = 1 ou 2). Avaliação do potencial citostático (frente a células U937, K562 e ascite de Ehrlich) e da DL50 (camundongos Balb-c) do complexo Rh2(TFACAM)4-2TMA mostraram atividade in vitro superior e toxicidade in vivo semelhante às da cisplatina. / We studied the interaction products of two Rh carboxylates (acetate, AC; and trifluoroacetate, TFA) with amides (formamide, FA; acetamide, AA; N-methyl-acetamide, MA; benzamide, BA; N-phenyl-acetamide, NFAA; trifluoroacetamide, TMA; cyclophosphamide, CFA). Two adducts of AC (AC-2FA e AC-2AA) and seven new adducts of TFA (TFA-2FA, TFA-2AA, TFA-2MA, TFA-2BA, TFA-2NFAA, TFA-2TMA, TFA-2CFA) were synthesized by reflux in CHCl3 solution. We obtained also the new Rh(II) amidates Rh2(CF3CONH)4(CF3CONH2)2 (TFACAM-2TMA) and a compound formulated as Rh2(CF3COO)2(CH3CONCH3)2 (\"Semi-MACAM\"), by fusion of the appropriate reagents. Elemental microanalysis results were satisfactory. Thermal decomposition mechanism of TFA and its adducts involves more than one step, and the intermediates can exhibit structures as Rh2(CF3COO)4-x(L)x (x = 0 or 1). Citostatic potential evaluation (towards U937, K562 and Ehrlich ascites cells) and of LD50 (Balb-c mice) of the compound TFACAM-2TMA showed superior in vitro activity and similar in vivo toxicity when compared with cisplatin.

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

Foram estudados os produtos da interação entre o acetato e o trifluoroacetato de ródio (respectivamente, Rh2(OAc)4 e Rh2(TFA)4) com amidas (formamida, FA; acetamida, AA; N-metil-acetamida, MA; benzamida, BA; N-fenil-acetamida, NFAA; trifluoroacetamida, TMA; ciclofosfamida, CFA), objetivando caracterização química e avaliação do potencial anti-tumor. Foram sintetizados por refluxo em clorofórmio dois adutos de Rh2(OAc)4 (Rh2(OAc)4-2FA e Rh2(OAc)4-2AA) e sete adutos inéditos de Rh2(TFA)4, de fórmula geral Rh2(TFA)4-2L (L = AA, BA, CFA, FA, MA, NFAA e TMA). Também obtivemos o novo amidato de Rh(II), Rh2(CF3CONH)4(CF3CONH2)2 (Rh2(TFACAM)4-2TMA), por fusão de Rh2(OAc)4 com TMA. Através da fusão de Rh2(TFA)4 com MA, obtivemos um composto que formulamos como Rh2(CF3COO)2(CH3CONCH3)2. Os resultados de análise elementar foram satisfatórios. Os adutos apresentaram bandas de absorção características dos carboxilatos de ródio (~ em 600, 450, 250 - ombro - e 220 nm). O modo de coordenação do ligante axial, pelo átomo de oxigênio da amida, foi determinado pela diminuição da freqüência de estiramento C-O na região do infravermelho (IV). Os amidatos apresentaram apenas uma banda na região do visível, e na região do IV os valores de?estiramentos característicos dos principais grupos orgânicos. Estudos comparativos dos espectros Raman mostraram que a freqüência Rh-Rh diminui ao se passar de um carboxilato para um amidato. Medidas de susceptibilidade magnética atestam o diamagnetismo de todas as moléculas (ligação Rh-Rh simples). O mecanismo de termodecomposição do Rh2(TFA)4 e dos seus adutos com amidas envolve mais de uma etapa, sendo que os intermediários podem apresentar estruturas do tipo Rh2(CF3COO)4-x(L)n (x e n = 1 ou 2). Avaliação do potencial citostático (frente a células U937, K562 e ascite de Ehrlich) e da DL50 (camundongos Balb-c) do complexo Rh2(TFACAM)4-2TMA mostraram atividade in vitro superior e toxicidade in vivo semelhante às da cisplatina. / We studied the interaction products of two Rh carboxylates (acetate, AC; and trifluoroacetate, TFA) with amides (formamide, FA; acetamide, AA; N-methyl-acetamide, MA; benzamide, BA; N-phenyl-acetamide, NFAA; trifluoroacetamide, TMA; cyclophosphamide, CFA). Two adducts of AC (AC-2FA e AC-2AA) and seven new adducts of TFA (TFA-2FA, TFA-2AA, TFA-2MA, TFA-2BA, TFA-2NFAA, TFA-2TMA, TFA-2CFA) were synthesized by reflux in CHCl3 solution. We obtained also the new Rh(II) amidates Rh2(CF3CONH)4(CF3CONH2)2 (TFACAM-2TMA) and a compound formulated as Rh2(CF3COO)2(CH3CONCH3)2 (\"Semi-MACAM\"), by fusion of the appropriate reagents. Elemental microanalysis results were satisfactory. Thermal decomposition mechanism of TFA and its adducts involves more than one step, and the intermediates can exhibit structures as Rh2(CF3COO)4-x(L)x (x = 0 or 1). Citostatic potential evaluation (towards U937, K562 and Ehrlich ascites cells) and of LD50 (Balb-c mice) of the compound TFACAM-2TMA showed superior in vitro activity and similar in vivo toxicity when compared with cisplatin.

N-Acylethanolamines (NAEs) are bioactive lipids derived from the hydrolysis of the membrane phospholipid N-acylphosphatidylethanolamine (NAPE). In animal systems this reaction is part of the “endocannabinoid” signaling pathway, which regulates a variety of physiological processes. The signaling function of NAE is terminated by fatty acid amide hydrolase (FAAH), which hydrolyzes NAE to ethanolamine and free fatty acid. Our previous work in Arabidopsis thaliana showed that overexpression of AtFAAH (At5g64440) lowered endogenous levels of NAEs in seeds, consistent with its role in NAE signal termination. Reduced NAE levels were accompanied by an accelerated growth phenotype, increased sensitivity to abscisic acid (ABA), enhanced susceptibility to bacterial pathogens, and early flowering. Here we investigated the nature of the early flowering phenotype of AtFAAH overexpression. AtFAAH overexpressors flowered several days earlier than wild type and AtFAAH knockouts under both non-inductive short day (SD) and inductive long day (LD) conditions. Microarray analysis revealed that the FLOWERING LOCUS T (FT) gene, which plays a major role in regulating flowering time, and one target MADS box transcription factor, SEPATALLA3 (SEP3), were elevated in AtFAAH overexpressors. Furthermore, AtFAAH overexpressors, with the early flowering phenotype had lower endogenous NAE levels in leaves compared to wild type prior to flowering. Exogenous application of NAE 12:0, which was reduced by up to 30% in AtFAAH overexpressors, delayed the onset of flowering in wild type plants. We conclude that the early flowering phenotype of AtFAAH overexpressors is, in part, explained by elevated FT gene expression resulting from the enhanced NAE hydrolase activity of AtFAAH, suggesting that NAE metabolism may participate in floral signaling pathways.

The iron-molybdenum cofactor (FeMo cofactor) is the catalytic center of nitrogen fixation in molybdenum-dependent nitrognease enzymes. The resting state cofactor is a complex [MoFe7S9X] cluster, in which the central ligand X is a central hexacoordinated monoatomic light atom (2p), and the exact identity of X is uncertain. The heteroligated, nitrogen-containing core environment of the cofactor cluster may also be relevant to active states, as several mechanistic proposals for cofactor catalysis incorporate substrate-derived nitrogenous moeities into the cluster core during turnover. To this end, we have explored synthetic pathways to the dinuclear and tetranuclear nitrogen-containing iron-sufur clusters, which may mimic the heteroligated core environment of the cofactor. Dinuclear iron-amide clusters Fe2(μ-NHtBu)2[N(SiMe3)2]2 (46) and Fe2(μ-NHtBu)2(μ-S)[N(SiMe3)2]2 (47) are useful precursors for the preparation of [Fe4(NtBu)n(S)4-nCl4]z cubane complexes that span all mixed imide/sulfide core compositions between the classic [Fe4S4] and the more recently reported [Fe4(NtBu)4] homoleptic motifs. The [Fe4NS3] core of the n = 1 cluster is particularly noteworthy in being essentially isometric with the analogous [Fe4S3X] subunit of the FeMo cofactor structure. Synthetic compounds are characterized by single crystal X-ray crystallography, cyclic voltammetry, and UV-Vis, 1H NMR spectroscopies.

Zugl.: Oldenburg, Univ., Diss., 2009