Les peptides sont des molécules pouvant posséder une activité biologique intéressante (antibiotique, anti-oxydante, antivirale, anti-hypertensive…). Ce sont cependant des molécules difficiles à utiliser car elles possèdent un faible temps de demi-vie in vivo et sont peu bio-disponibles. Le greffage d’un acide gras permet de les protéger et d’accroître leur potentiel d’action. Cette réaction appelée acylation peut être catalysée par des enzymes. A l’heure actuelle, peu de recherches sont faites sur l’acylation de peptides par voie enzymatique et sur la recherche de nouveaux biocatalyseurs adaptés pour cette réaction. Les objectifs de cette thèse ont été, dans un premier temps, de comprendre les mécanismes de la sélectivité d’acylation de peptides de la lipase B de Candida antarctica par une approche de modélisation moléculaire combinant docking et dynamique moléculaire, couplée à une approche expérimentale. Cette étude a permis d’identifier des interactions enzyme-substrats impliquées dans la sélectivité enzymatique et a permis de construire un modèle expliquant la régio- et chimio-sélectivité de l’acylation peptidique catalysée par cette enzyme. Dans un deuxième temps, une étude préliminaire a été menée afin d’identifier de nouvelles enzymes de type acylases présentes dans des surnageants de culture de différentes espèces de Streptomyces. Ces enzymes sont capables de catalyser des réactions d’acylation de peptides en milieux aqueux. Une méthode de semi-purification a été établie et une étude comparative a été menée sur la sélectivité d’acylation de la lipase B de C. antarctica et celle de nouvelles enzymes de type aminoacylases présentes dans un extrait protéique de surnageant de culture de Streptomyces ambofaciens. Ces nouvelles enzymes présentent une spécificité différente de celle de la lipase B de C. antarctica, permettant notamment, une acylation des acides aminés sur leur fonction amine en position α. Une caractérisation partielle des activités amino-acylase du surnageant de culture de S. ambofaciens a été réalisée. Dans une troisième et dernière partie, une comparaison des séquences génétiques a été réalisée entre treptomyces mobaraensis et S. ambofaciens afin d’identifier les gènes codant pour les acylases découvertes chez S. ambofaciens. Des mutants de S. ambofaciens délétés pour ces gènes ont été construits et la fonctionnalité des enzymes codées par ces gènes a été vérifiée ; enfin, une expression hétérologue de l’ε-lysine acylase a été initiée / Peptides exhibit various beneficial effects such as antioxidant, anti-hypertensive, neuroprotective, antiviral or antimicrobial activities. However, their use can be limited by their short half-life and their low biological availability. One solution to overcome these drawbacks is the acylation of peptides with fatty acids. This reaction called acylation can be catalyzed using enzymes. To date, very few studies focus on enzymatic acylation of peptides and on finding new enzymes catalyzing this reaction. The objectives of this work were, in a first time, to understand the selectivity mechanisms of the lipase B of Candida antarctica for peptides acylation combining experimental and molecular modeling approaches. This study highlighted enzyme/substrate interactions involved in the enzymatic selectivity and a modelexplaining the chemo- and regio-selectivity of this enzyme for peptide acylation reactions was built. In a second time, a preliminary study was carried out in order to identify new aminoacylase enzymes produced in the culture supernatant of various species of Streptomyces. These enzymes are able to catalyze acylation of peptides in aqueous media. A partial purification method was set and a comparative study was performed on the selectivity of C. antarctica lipase Band that of the new aminoacylases discovered in the culture supernatant of Streptomyces ambofaciens ATCC 23877. These enzymes presented a selectivity different from C. antarctica lipase B allowing the acylation of the N-terminal amino group of amino acids or peptides. A partial description of the aminoacylase activity of the supernatant crude extract of S. ambofaciens was performed. In a third and final part, a comparison of sequences of aminoacylases from Streptomyces mobaraensis with the genome of S.s ambofaciens ATCC 23877 was performed in order to identify genetic sequences encoding the new discovered aminoacylases from S. ambofaciens ATCC 23877. Each identified gene was deleted to correlate it with the aminoacylase activity observed in the crude extract of S. ambofaciens. Lastly, a heterologous expression of the ε-lysine acylase was initiated

L’acylation de Friedel-Crafts est parmi les réactions les plus fondamentales et les plus utiles pour former des cétones aromatiques. Bien que la mise en oeuvre de ces réactions soit habituellement pénalisante en termes de sous produits formés, de rendement atomique et de quantité de catalyseur utilisée, cette voie est encore largement utilisée dans l’industrie de la chimie fine. Ces dernières années, la prise de conscience de l’impact des activités industrielles sur l’environnement a mené les chimistes à travailler sur de nouvelles voies, moins dangereuses et plus respectueuses de l’environnement.Nous avons considéré ici l’acylation d’un dérivé du benzofurane par un chlorure d’acyle, correspondant à une étape intermédiaire pour la fabrication d’un principe actif pharmaceutique. Dans cette étude, la première proposition a consisté à remplacer les catalyseurs classiques (FeCl3 ou AlCl3) par des catalyseurs solides réutilisables. Dans ce travail, plusieurs catalyseurs (zéolites, résines échangeuses d’ions...) ont d’abord été testés dans des conditions « conventionnelles » , c’est-à-dire en utilisant un solvant organique, afin de déterminer le plus performant, en termes de réactivité, de durée de vie et de réutilisabilité. Une zéolite Y s’est avéré la plus adaptée. Cependant, l’utilisation d’un solvant organique (ici le 1,2-dichlorobenzène) reste discutable et l’utilisation du CO2 supercritique comme solvant a donc été envisagée. Pour cela, nous avons imaginé et dimensionné un réacteur tubulaire à lit fixe de catalyseur fonctionnant sous pression et en continu. Les résultats présentés montrent la faisabilité d’un tel procédé et pose les jalons pour arriver à une ou des solutions pour une meilleure mise en oeuvre industrielle des réactions de Friedel-Crafts. / Friedel-Crafts acylation is among the most fundamental and useful reactions to yield aromatic ketones, but it is one of the less acceptable in terms of unwanted polluting by-products or atom economy because of overconsumption of catalyst which is used in stoichiometric quantities in the conventional process. This route is nevertheless still widely used in the fine chemicals industry. In recent years, awareness of the impact of industrial activities on the environment has lead chemists to work on new chemical routes, less dangerous and more environmentally friendly. We considered here the acylation of a benzofurane derivative by an acid chloride, as an intermediary step for a pharmaceutical product. In this study, one of the first alternative was to replace conventional catalysts (FeCl3 or AlCl3), by reusable solid catalysts. In this work, different catalysts (zeolites, ion-exchange resins...) were first tested in "conventional" conditions, i.e., using an organic solvent (1,2-dichlorobenzene in our case), to determine the best one, in terms of reactivity, lifetime and reusability. The zeolite Y was found the most appropriate. However, the use of an organic solvent still remains questionable and the use of supercritical carbon dioxide as the solvent was also considered. Its inherent properties include non-flammability, mild critical conditions, tuneable solubility near to the critical point and very low environmental impact. The reaction was operated using a specifically designed continuous high pressure fixed bed and results concerning yield and selectivity are presented. These results demonstrate the feasibility of such an approach, which would ultimately yield to better industrial operation of Friedel-Crafts reactions.

Approche multi-échelle pour l’étude de la réaction de N-acylation enzymatique d’acides aminés La réaction de N-acylation d’acides aminés ou de peptides permet l’obtention de dérivés de ces molécules présentant des propriétés bioactives et/ou techno-fonctionnelles, avec une biodisponibilité, une hydrophobie et une stabilité accrue. Les acides aminés acylés ont été largement décrits comme constituant une classe d'agents tensioactifs avec d'excellentes propriétés de surface, des activités biologiques intéressantes, un faible potentiel de toxicité et un faible impact environnemental. Actuellement réalisée de manière chimique à l’échelle industrielle, l’acylation de ces acides aminés ou peptides présente des contraintes en termes de sélectivité réactionnelle et d’innocuité vis-à-vis de l’environnement ainsi qu’en termes de coût de retraitement des effluents polluants. Une alternative à cette voie chimique est l’utilisation d’enzymes capables de catalyser ces réactions d’acylation. Dans la littérature, différents couples d’enzymes et de solvants ont déjà été décrits. Néanmoins, les performances réactionnelles de ces systèmes demeurent parfois limitées. L’objectif de cette thèse a donc été l’amélioration du procédé d’acylation par une approche à différentes échelles. À l’échelle moléculaire, une étude a été réalisée avec la lipase B de Candida antarctica (CALB). Une approche de modélisation moléculaire a été utilisée afin de mettre au point une méthodologie associant des simulations de docking et des calculs d’interaction permettant d’améliorer la compréhension et permettre la prédiction de la régiosélectivité de CALB lors de l’acylation de la lysine par différents acides gras. Des études ont également été conduites à l’échelle réactionnelle, notamment avec la recherche de nouveaux biocatalyseurs de type aminoacylases dans l’extrait brut de Streptomyces ambofaciens. La régiosélectivité et les performances de la réaction catalysée par ces enzymes ont été comparés à celles de CALB. Les résultats ont mis en évidence un potentiel très prometteur des aminoacylases de S. ambofaciens concernant la synthèse d’acide aminés/peptides acylés. En effet, en plus de leur aptitude à réaliser la réaction d’acylation en milieu aqueux, ces enzymes possèdent une régio-sélectivité qui diffère de celle de CALB. Cette régio-sélectivité orientée vers les groupements N-terminaux est un atout très peu décrit à ce jour, car elle permet d’acyler ces molécules sans modifier les chaînes latérales des acides aminés ou des peptides et donc leurs fonctionnalités. Dans la dernière partie de ces travaux, des études à l’échelle procédé ont été menées. Tout d’abord, l’immobilisation des aminoacylases sur des matériaux mésoporeux silicatés a été réalisée et différentes méthodes d’immobilisation ont pu être comparées. Cette étude a permis de proposer une méthode d’immobilisation des aminoacylases de S. ambofaciens par physisorption, permettant de conserver l’activité spécifique pendant au moins 3 cycles. Puis, dans une dernière partie, l’intensification de la réaction d’acylation en réacteur micro-ondes ou microstructurés a été abordée. Les expérimentations réalisées dans un réacteur chauffé par irradiation micro-onde ont montré que ce type de réacteur était adapté à la réaction d’acylation catalysé par CALB sous sa forme immobilisée commerciale (Novozym435®) en solvant organique, ce qui n’est pas le cas avec des aminoacylases de S. ambofaciens libres, en milieux aqueux. Pour cette réaction, d’autres méthodes d’intensification ont été envisagées, notamment en réacteur microstructuré de type microfluidique. L’efficacité du mélange étant primordiale notamment en milieu biphasique, celle-ci a pu être améliorée avec un taux de conversion supérieur dans ce réacteur comparativement à un réacteur classique agité mécaniquement / N-acylation of amino acids or peptides results in bioactive and/or functional molecules showing increased bioavailability, hydrophobicity and stability. Acylated amino acids have been broadly described as being a kind of surfactant with great surface chemistry properties, interesting biological activities, weak toxicity and low environmental impact. Acylation of amino acids or peptides is being performed chemically at industrial scale. It creates constraints in term of reaction selectivity, environmental safety and cost of polluted wastewater treatment. Enzymatic catalysis is an alternative to chemical acylation reaction. Several enzyme/solvent pairs have already been described in the literature. Their performance are however somewhat limited. The objective of this thesis work was thus to improve the capacity of acylation processes at different scales. At the molecular scale, a study was performed using Candida antarctica’s (CALB) lipase B. Molecular modeling was used to create a methodology coupling docking simulation and interaction calculus that would allow for a better understanding of CALB regioselectivity during lysine acylation by different fatty acids. Studies were also conducted at the reaction level, especially by searching for new aminoacylase-type of biocatalysts in Streptomyces ambofaciens raw extract. Regioselectivity and performance of these enzyme’s catalytic reactions were compared to those of CALB. Results brought into light a promising potential from S. ambofaciens’ aminoacylases in synthesizing acylated amino acids/peptides. Indeed, on top of their ability to catalyse acylation reaction in aqueous solution, these enzymes have a different regioselectivity compared to CALB’s. Regioselectivity targeting N-terminal groups is a rarely researched phenomenon allowing acylation to be performed without modifying amino acids or peptides lateral chains and hence their functionality. In the last part part of this work, studies at process scale were performed. Aminoacylase were first immobilized on mesoporous silicates and several immobilisation methods were compared. Using physisorption, a method for the immobilisation of S. ambofaciens’ aminoacylases was developed to reach a conserved specific activity during 3 cycles. Finally, intensification of acylation reaction was examined in microwave or microstructured reactors. First, an experimental set up was performed in an heated reactor using microwaves irradiation. This kind of reactor was demonstrated as being adapted to acylation reaction using a commercial immobilized form of CALB (Novozym435®) as catalyst in organic solvent. The microwave reactor was however not suited for free S. ambofaciens aminoacylase in aqueous solution. For that latter reaction, intensification had to be approached through other aspects of the process. Hydrodynamic appeared indeed as an important aspect for this reaction occurring in a biphasic medium composed of fatty acids and aqueous solution. A microstructured microfluidic reactor was hence tested. Conversion yield were increased with this system. This study demonstrated how mixing quality was an important factor for acylation reaction and could be a way to intensify the enzymatic process at larger scale

Ce travail a pour objectif d'étudier les perfomances et la régiosélectivité de deux lipases lors de l'acylation de flavonoïdes en milieu organique. Cette étude a permis de montrer que la solubilité dans l'acétonitrile, l'acétone et le tert-amyl alcool dépend de la nature du flavonoïde. La solubilité la plus élevée a été obtenue avec la naringénine et l'hepéritine et la plus faible pour la rutine et l'isoquercitrine. Les propriétés thermophysiques sont également affectées par la nature du flavonoïde. Ainsi, les flavonoïdes glycosylés possèdent un point de fusion moins élevé et une enthalpie de fusion plus élevée que ceux des aglycones. Du point de vue cinétique d'acylation, des rendements de conversion de 99% ont été obtenus avec la quercétine. Ces rendements varient en fonction de la nature du flavonoïde et du donneur d'acyle, du rapport molaire (vinyle acétate/ flavonoïde) et de la nature du solvant. Le rendement le plus faible a été obtenu avec l'hespéritine. La modélisation moléculaire de flavonoïdes dans le vide et dans des solvants a permis d'étudier le rôle de la conformation sur la solubilité et de dégager des relations structure-activité pour un certain nombre de descripteurs moléculaires. Enfin des modèles OPLS tout atomes ont été construits pour étudier par dynamique moléculaire la quercétine dans des phases condensées de solvants organiques / This work aims to study the performances and the regioselcetivity of two lipases throughout the acylation of flavonoids in organic medium. This study showed that the solubility in acetonitrile, acetone and tert-amyl alcohol depends on the nature of the flavonoid. The highest solubility, in actonitrile, was obtained with the naringenin and hesperitin and the lowest with rutin and isoquercitrin. The thermophysical properties are also affected by the nature of flavonoids. Thus glycosylated flavonoids are characterized by a low melting point and a high enthalpy of fusion compared to the aglycon ones. From the kinetic acylation data, the highest conversion yields of 99% were obtained with quercetin. These conversion yields vary according to the nature of the flavonoid and the acyl donor, the molar ratio (vinyl acetate/flavonoid) and the nature of the solvent. The lowest conversion yield was obtained with hesperitin. Molecular modeling of flavonoids in vacuum and solvents allows to study the role of conformation structure on solubility and to release the structure-activity relationship with many electronic descriptors. Finally, OPLS all atoms were built to study, by molecular dynamics, quercetin in condensed phases of organic solvents.

Platelets affect vascular integrity by secreting a host of molecules that promote hemostasis and its sequela. Given its importance, it is critical to understand how platelet exocytosis is controlled. Post-translational modifications, such as phosphorylation and acylation, have been shown to affect signaling pathways and platelet function. In this dissertation, I focus on how these modifications affect the t-SNARE proteins, SNAP-23 and syntaxin-11, which are both required for platelet secretion. SNAP-23 is regulated by phosphorylation. Using a proteoliposome fusion assay, I demonstrate that purified IκB Kinase (IKK) phosphorylated SNAP-23, which increased the initial rates of SNARE-mediated liposome fusion. SNAP-23 mutants containing phosphomimetics showed enhanced initial fusion rates. These results, combined with previous work in vivo, confirm that SNAP-23 phosphorylation is involved in regulating membrane fusion, and that IKK-mediated signaling contributes to platelet exocytosis. To address the role(s) of acylation, I sought to determine how syntaxin-11 and SNAP-23 are associated with plasma membrane. Using metabolic labeling, I showed that both proteins contain thioester-linked acyl groups which turn over in resting cells. Mass spectrometry mapping showed that syntaxin-11 is modified on C275, 279, 280, 282, 283 and 285, while SNAP-23 is modified on C79, 80, 83, 85, and 87. To probe the effects of acylation, I measured ADP/ATP release from platelets treated with the acyl-transferase inhibitor, cerulenin, or the thioesterase inhibitor, palmostatin B. Cerulenin pretreatment inhibited t-SNARE acylation and platelet function while palmostatin B had no effect. Interestingly, pretreatment with palmostatin B blocked the inhibitory effects of cerulenin suggesting that maintaining the acylation state of platelet proteins is important for their function. Thus my work indicates that the enzymes controlling protein acylation could be valuable targets for modulating platelet exocytosis in vivo.

Carbon nano-onions (CNOs) discovered by Ugarte in 1992 are multi-layered fullerenes that are spherical analogs of multi-walled carbon nanotubes with diameters varying from 6 nm to 30 nm. Among the various methods of synthesis, CNOs prepared by graphitization of nanodiamonds (N-CNOs) and underwater electric arc of graphite rods (A-CNOs) are the subject of our research. N-CNOs are considered as more reactive than A-CNOs due to their smaller size, high curvature and surface defects. This dissertation focuses on structural analysis and surface functionalization of N- CNOs with diameters ranging from 6—10 nm. Synthetic approaches such as oleum- assisted oxidation, Freidel-Crafts acylation and Billups reductive alkylation were used to functionalize N-CNOs to improve their dispersion properties in aqueous and organic solvents. Functionalized N-CNOs were characterized using various techniques such as TGA, TG-MS, Raman spectroscopy and pH-titrimetry. We designed an experimental method to isolate polycyclic aromatic adsorbates formed on the surface of oleum oxidized N-CNOs (ON-CNOs) and characterized them. A-CNOs, on the other hand are bigger than N-CNOs with diameters ranging from 20—40 nm. In this dissertation, we discuss the preparation of graphene structures by unzipping of A-CNOs using KMnO4 as oxidizing agent. These graphene structures were characterized using powder X-ray diffraction, TGA, BET nitrogen adsorption/desorption studies and compressed powder conductivity. This dissertation also focuses on lithiation/delithiation studies of N-CNOs, A- CNOs and A-CNO-derived graphene structures to use them as negative electrode materials in lithium-ion batteries. The cycling performances of these materials at a charge/discharge rate of C/10 were discussed. The cycling performance of N-CNOs was tested at faster charge/discharge rate of C.

The intramolecular Friedel-Crafts acylation of aromatics with Meldrum?s acid derivatives catalyzed by metal trifluoromethanesulfonates and other Lewis acids is reported. Meldrum?s acids are easily prepared, functionalized, handled, and purified. The synthesis of polysubstituted 1-indanones from benzyl Meldrum's acids was investigated thoroughly, and it was shown that a variety of catalysts were effective, whilst accommodating a diversity of functional groups under mild conditions. The scope, limitations, and functional group tolerance (terminal alkene and alkyne, ketal, dialkyl ether, dialkyl thioether, aryl methyl ether, aryl TIPS and TBDPS ethers, nitrile- and nitro-substituted aryls, alkyl and aryl halides) for a variety of 5-benzyl (enolizable Meldrum?s acids) and 5-benzyl-5-substituted Meldrum?s acids (quaternarized Meldrum?s acids), forming 1-indanones and 2-substituted-1-indanones respectively, are delineated. <br ><br /> This method was further applied to the synthesis of 1-tetralones, 1-benzosuberones, and the potent acetylcholinesterase inhibitor donepezil. <br ><br /> Mechanistic investigations were undertaken to determine the rate-determining step in the acylation sequence using Meldrum?s acid, as well as to examine the role of the Lewis acid catalyst. Enolizable Meldrum?s acid derivatives can react via an acyl ketene intermediate under thermal conditions, while quaternarized Meldrum?s acid derivatives are thermally stable and only act as effective Friedel-Crafts acylating agents in the presence of a Lewis acid catalyst. <br ><br /> The total synthesis of (??)-Taiwaniaquinol B was completed. This natural product was the first ever isolated containing an unusual 6-5-6 fused ring system, and it also contains a hexasubstituted aromatic ring, and two all-carbon quaternary centers. This synthesis was accomplished via an intramolecular Friedel-Crafts acylation/carbonyl a-<em>tert</em>-alkylation reaction that exploits the unique chemistry of Meldrum?s acid. This novel methodology can be used to access a variety of highly substituted fused ring systems of various sizes.

O objetivo deste trabalho é estudar a reatividade de acilação de celulose por anidridos de ácidos carboxílicos sob condições homogêneas em solventes apróticos dipolares (SAD), incluindo LiCl/N,N-dimetilacetamida (DMAC) e líquidos iônicos (LIs)/SAD. Os factores que contribuem para a reatividade foram quantificados através do estudo da dependência das constantes de velocidade e parâmetros de ativação sobre a composição do solvente. Após estabelecer que a condutividade é uma técnica experimental adequada para calcular as constantes de velocidade, foi estudada a acilação não catalisada e catalisada de celulose microcristalina, MCC. Foram empregados anidridos de ácidos carboxílicos com diferentes grupos acila (acetil a hexanoil; Nc = 2 a 6) nos seguintes sistemas de solventes: LiCl/DMAC, misturas de LI cloreto de 1-alil-3-metilimidazólio ( AlMeImCl ) e acetonitrila (MeCN), DMAC , dimetilsulfóxido (DMSO ) e sulfolano. Na celulose, a unidade anidra de glucose possui um grupo hidroxila primário e dois hidroxilas secundários. Usamos ciclohexilmetanol, CHM, e trans-1 ,2- ciclo-hexanodiol, CHD , como compostos modelo para os grupos (OH) primário e secundários, respectivamente. As razões das constantes de velocidade de acilação dos compostos modelo (CHM; Prim-OH) e (CHD; SEC-OH) foram empregados, após correção, a fim de dividir as constantes de velocidade global da reação de MCC em contribuições dos grupos (OH) presentes. Para os compostos modelo, verificou-se que k3 (Prim-OH) /k3 (Sec-OH) > 1, semelhante as reações de celulose sob condições heterogéneas; esta relação aumenta como uma função do aumento da Nc. As constantes de velocidade globais e parciais de acilação de MCC diminuim de anidrido etanóico a butanóico e, em seguida, aumentam para anidrido pentanóico e hexanóico, devido a mudanças sutis em - e compensações da entalpia e entropia de ativação. As constantes de velocidade para a acetilação de MCC, por anidrido etanóico na presença de concentrações crescentes do LI em DMAC, MeCN, DMSO e sulfolano foram calculados a partir de dados de condutividade. As constantes de velocidade de terceira ordem mostraram dependência linear sobre [LI]. Estes resultados foram explicados assumindo que o reagente é celulose ligado ao LI por ligação de hidrogénio. Isto foi confirmado pelos dados cinéticos da acetilação de CHM, espectroscopia de IV do último composto, e de celobiose nas misturas de LI/SAD e condutividade das misturas de solventes binários, na ausência e presença de MCC. A acetilação de celulose é mais rápida nas misturas de em LI com DMAC e DMSO do que com MeCN e sulfolano. Esta diferença é explicada, em parte, com base na alta viscosidade das soluções de biopolímeros em LI/sulfolano. Obteve-se mais informações sobre os efeitos do solvente molecular a prtir das propriedades microscópicas dos solventes e simulações por dinâmica molecular, DM. Os dados solvatocrômicos (polaridade empírica e basicidade) têm mostrado a importância da basicidade do solvente; solventes mais básicos formam ligações de hidrogênio mais fortes com os grupos (OH) da celulose, aumentando sua acessibilidade e, consequentemente sua reatividade. Este é o caso de DMAC e DMSO. Os resultados das simulações por DM indicaram a formação de ligações de hidrogénio, entre os grupos (OH) da unidade de glucose anidra do MCC, (Cl-) de LI, e o dipolo do DMAC e DMSO . Observamos que a acilação de celulose em LiCl/DMAC é eficientemente catalisada por imidazol, mas não pelo cloreto de tosila. Resultados de IV de FT e RMN de 1H indicaram a formação de N-acilimidazol que é o agente de acilação. As constantes globais e parciais de velocidade de acilação do MCC diminuiram de anidirido etanóico a butanóico e depois aumentou para anidrido pentanóico e hexanóico, devido a mudanças sutis em- e compensações da entalpia e entropia de ativação. / The objective of this work is to study the reactivity in cellulose acylation by carboxylic acid anhydrides under homogeneous conditions in dipolar aprotic solvents (DAS), including LiCl/ N,N-dimethylacetamide (DMAC) and ionic liquids (ILs)/DAS. Factors that contribute to reactivity were quantified by studying the dependence of reaction rates on temperature and solvent composition. After establishing that conductivity is an appropriate experimental technique to calculate the rate constants, we studied the kinetics of the homogeneous uncatalyzed and catalyzed acylation of microcrystalline cellulose, MCC, with carboxylic acid anhydrides with different acyl chain-length (Nc; ethanoic to hexanoic) in the following solvent systems: LiCl/DMAC; mixtures of the IL, 1-allyl-3-methylimidazolium chloride, (AlMeImCl) and acetonitrile (MeCN), DMAC, dimethyl sulfoxide (DMSO) and sulfolane. The anhydroglucose unit of cellulose carries one primary- and two secondary hydroxyl groups. We used cyclohexylmethanol, CHM, and trans-1,2-cyclohexanediol, CHD, as model compounds for the hydroxyl groups of the anhydroglucose unit of cellulose. The ratios of rate constants of acylation of primary (CHM; Prim-OH) and secondary (CHD; Sec-OH) groups were employed, after correction, in order to split the overall rate constants of the reaction of MCC into contributions from the discrete OH groups. For the model compounds, we have found that k3 (Prim-OH)/k3 (Sec-OH) > 1, akin to reactions of cellulose under heterogeneous conditions; this ratio increases as a function of increasing Nc. The overall and partial rate constants of the acylation of MCC decrease from ethanoic- to butanoic anhydride and then increase for pentanoic- and hexanoic anhydride, due to subtle changes in- and compensations of the enthalpy and entropy of activation. Rate constants for the acetylation of MCC, by ethanoic anhydride in the presence of increasing concentrations of the ionic liquid, IL, 1-allyl-3-methylimidazolium chloride in dipolar aprotic solvents, DAS, N,N-dimethylacetamide, DMAC, acetonitrile, MeCN, dimethylsulfoxide, DMSO and sulfolane, have been calculated from conductivity data. The third order rate constants showed a linear dependence on [IL]. These results have been explained by assuming that the reactant is cellulose hydrogen-bonded to the IL. This is corroborated by kinetic data of the acetylation of cyclohexyl methanol, FTIR spectroscopy of the latter compound, and cellobiose in mixtures of IL/DAS, and conductivity of the binary solvent mixtures in absence, and presence of MCC. Cellulose acetylation is faster in IL/DMAC and IL/DMSO than in IL/MeCN and IL/Sulfolane. This difference is explained, in part, based the high viscosity of the biopolymer solutions in IL-Sulfolane. Additional explanation came from microscopic solvents properties and molecular dynamics, MD simulations. The solvatochromic data (empirical polarity and basicity) have shown the importance of solvent basicity; basic solvents hydrogen-bond to the hydroxyl groups of cellulose increasing its accessibility, hence its reactivity. This is the case of DMAC and DMSO. Results of MD simulations indicated hydrogen-bond formation between the hydroxyl groups of the anhydroglucose unit of MCC, (Cl-) of the IL, and the dipole of the DMAC and DMSO. It has been observed that cellulose acylation in LiCl/DMAC is efficiently catalyzed by imidazole, but not by p-tosyl chloride. FTIR and 1H NMR have indicated the formation of N-acylimidazole which is the acylating agent. The overall and partial rate constants of the acylation of MCC decreased from ethanoic- to butanoic-anhydride and then increased for pentanoic- and hexanoic anhydride, due to subtle changes in- and compensations of the enthalpy and entropy of activation.

Croton é o segundo maior gênero de Euphorbiaceae, com aproximadamente 1.300 espécies, sendo 300 delas existentes no Brasil em diversos biomas. Várias espécies apresentam um característico látex vermelho-sangue, chamado \"sangue-de-dragão\", sendo usadas mundialmente na medicina tradicional. Estudos químicos indicam a presença de múltiplas classes de compostos, sendo as principais: diterpenos (clerodanos, labdanos, kauranos e traquilobanos), óleos voláteis, esteroides e triterpenoides, alcaloides, proantocianidinas e flavonoides. Estes últimos são metabólitos secundários com grande variedade estrutural, possuindo atividades biológicas reconhecidas e de potencial interesse medicinal. Croton floribundus Spreng. e Croton urucurana Baill., por apresentarem várias atividades biológicas de interesse medicinal, são utilizadas na medicina tradicional. Entretanto, poucos estudos têm sido desenvolvidos no sentido de se conhecer melhor aquela classe de substâncias fenólicas. Um dos objetivos deste trabalho corresponde ao isolamento e identificação de flavonoides de folhas de Croton floribundus e Croton urucurana. O material pulverizado foi extraído por refluxo com metanol 80% e concentrado. O extrato seco foi tratado com tolueno e diclorometano. A fase metanólica resultante foi fracionada e subfracionada por meio de cromatografia em coluna de PVPP e Sephadex LH-20, e cromatografia em papel. Depois de fracionadas, as amostras foram analisadas por cromatografia líquida de alta eficiência (CLAE) e os compostos identificados por ressonância magnética nuclear de hidrogênio (1H) e carbono (13C) ou por CLAE com co-injeção de amostras autênticas. Em ambas as espécies foi identificado o flavonoide acilado tilirosídeo (5). Este parece ser um flavonoide característico do gênero. É um derivado acilado de kaempferol, isolado de um grande número de espécies de Croton. Em C.floribundus foram identificados três triglicosídeos: alcesefolisídeo (1), mauritianina (2), e isoramnetina-3-O-(2,6-di-ramnosil)-galactosídeo (3). Foram identificados também: quercetina-3-O-glucosídeo (4), helicrisosídeo-3\'-metil-éter (6), kaempferol (7), isoramnetina (8) 3-O-metil-kaempferol (9) e 3-O-metil isoramnetina (10). Em C.urucurana, foram identificados: orientina (11), rutina (12), vitexina (13), quercetina-7-O-ramnosídeo (14), ramnetina-3-O-ramnosídeo (15) e quercetina (16). Os flavonoides 1, 3, 14 mostraram-se inéditos, tanto para o gênero, quanto para a família. O flavonoide 15 foi inédito para o gênero, tendo sido encontrado na família, porém, na subfamília Euphorbioideae. Com a detecção do tilirosídeo, foi verificada a possibilidade de se utilizar os extratos proteicos de folhas jovens dessas duas espécies para acilar enzimaticamente flavonoides de interesse, pois estudos na literatura indicam que a acilação pode aumentar a estabilidade e biodisponibilidade de flavonoides, e também melhorar suas atividades biológicas. Para isso, folhas jovens foram coletadas e mantidas em N2 líquido, trituradas e extraídas com tampão de extração. O extrato obtido foi concentrado, sua concentração proteica foi determinada e, posteriormente, foi utilizado para acilar os substratos quercetina-3-O-glucosídeo, kaempferol-3-O-glucosídeo, quercetina-3-O-galactosídeo e quercetagetina-7-O-glucosídeo. p-Cumaroil-CoA e uma mistura de ácido p-cumárico e Coenzima A, foram utilizados como agentes acilantes. Foi verificado que o rendimento do extrato proteico de C.floribundus foi menor que o de C.urucurana. Enquanto o extrato de C.urucurana acilou os substratos glicosilados na posição 3, (kaemferol-3-O-glucosídeo, quercetina-3-O-glucosídeo e quercetina-3-Ogalactosídeo), não acilou o mono-glicosilado na posição 7 (quercetagetina-7-Oglucosídeo), indicando que as aciltransferases dessa espécie são regiosseletivas quanto à posição do resíduo de açúcar. Também demonstrou capacidade de acilação usando como agentes acilantes p-cumaroil-CoA e a combinação ácido p-cumárico + CoA + ATP. O extrato proteico de C.floribundus, talvez por ter apresentado um menor rendimento de extração, acilou apenas o kaempferol-3-O-glucosídeo, resultando no tilirosídeo, o flavonoide acilado característico do gênero / Croton is the second largest genus of Euphorbiaceae, with approximately 1,300 species, 300 among them native from Brazilian biomes. Several species of the genus, used worldly in traditional medicine, possess a characteristic blood colored latex, called \"dragon\'s blood\". Chemical studies about Croton species have uncovered multiple classes of secondary metabolites, such as diterpenes (clerodanes, labdanes, kauranes and trachylobanes), volatile oils, steroids and triterpenoids, alkaloids, proanthocyanidins and flavonoids. The latter are secondary metabolites with high structural diversity and recognized as having biological activities with medicinal potential. Croton floribundus Spreng. and C. urucurana Baill. have shown several medicinally promising biological activities and are used in traditional medicine. However, few investigations have been performed aiming the flavonoid chemistry of any of the two species. One of the objectives of the present study is the isolation and identification of flavonoids from leaves of C. floribundus and C. urucurana. Powdered material from both species was extracted by reflux with 80% methanol. The dry extracts were treated with toluene and dichloromethane, lyophilized and solubilized in methanol. The methanol solution was analyzed by polyvinylpolypyrrolidone column chromatography (PVPP-CC). The fractions obtained were further analyzed by PVPP-CC, Sephadex LH-20 column chromatography and paper chromatography. The fractions and isolated compounds obtained were analyzed by HPLC. Isolated compounds were identified by 1H and 13C NMR and HPLC co-chromatography with authentic samples. The acylated flavonol tiliroside (5) was obtained from extracts of both species. It seems to be a characteristic marker of the genus, having been reported for a high number of Croton species. From the leaf extract of C. floribundus three triglycosides were obtained in the present work: alcesefoliside (1), mauritianin (2) and isorhamnetin-3-O-(2,6-dirhamnosyl)-galactoside (3). Other glycosides identified were quercetin-3-O-glucoside (4), 3\'-helichrysoside-3-O-methyl ether (6), kaempferol (7), isorhamnetin (8), 3-O-methyl-kaempferol (9) and 3-O-methyl-isorhamnetin (10). The following compounds were obtained from C. urucurana: orientin (11), rutin (12), vitexin (13), quercetin-7-O-rhamnoside (14), rhamnetin-3-O-rhamnoside (15) and quercetin (16). Flavonoids 1, 3 and 14 are new regarding genus Croton and family Euphorbiaceae. Flavonoid 15 was previously found in subfamily Euphorbioideae and is now reported for the first time in Croton. Taking into account the detection of tiliroside in the material analyzed, and that acylation increases both stability and bioavailability of flavonoids, while enhancing their biological activity, an approach was planned to use protein extracts of young leaves of both species aiming the enzymatic acylation of several flavonoids. Young leaves were maintained in liquid N2, ground and treated with extraction buffer. The extract obtained was concentrated and mixed with p-coumaroyl-CoA and a mix of p-coumaric acid and Coenzyme A. The extract was used in assays aiming the acylation of quercetin-3-O-glucoside, kaempferol-3-O-glucoside, quercetin-3-O-galactoside and quercetagetin-7- O-glucoside. The concentration of the protein extract from C. floribundus was lower than that of C. urucurana. The extract from C. urucurana acylated the 3-O-glycosilated substrata kaempferol-glucoside, quercetin-glucoside and quercetin-galactoside, but was ineffective toward quercetagetin-7-O-glucoside. These results suggest that acyltransferases in the extract are regioselective about the position of attachment of the sugar moiety. They were shown to be effective using either p-coumaroyl or the combination p-coumaroyl-CoA + ATP. C. floribundus protein extract acylated only kaempferol-3-O-glucoside, yielding tiliroside, the characteristic acylated flavonoid of Croton

O objetivo deste trabalho é estudar a reatividade de acilação de celulose por anidridos de ácidos carboxílicos sob condições homogêneas em solventes apróticos dipolares (SAD), incluindo LiCl/N,N-dimetilacetamida (DMAC) e líquidos iônicos (LIs)/SAD. Os factores que contribuem para a reatividade foram quantificados através do estudo da dependência das constantes de velocidade e parâmetros de ativação sobre a composição do solvente. Após estabelecer que a condutividade é uma técnica experimental adequada para calcular as constantes de velocidade, foi estudada a acilação não catalisada e catalisada de celulose microcristalina, MCC. Foram empregados anidridos de ácidos carboxílicos com diferentes grupos acila (acetil a hexanoil; Nc = 2 a 6) nos seguintes sistemas de solventes: LiCl/DMAC, misturas de LI cloreto de 1-alil-3-metilimidazólio ( AlMeImCl ) e acetonitrila (MeCN), DMAC , dimetilsulfóxido (DMSO ) e sulfolano. Na celulose, a unidade anidra de glucose possui um grupo hidroxila primário e dois hidroxilas secundários. Usamos ciclohexilmetanol, CHM, e trans-1 ,2- ciclo-hexanodiol, CHD , como compostos modelo para os grupos (OH) primário e secundários, respectivamente. As razões das constantes de velocidade de acilação dos compostos modelo (CHM; Prim-OH) e (CHD; SEC-OH) foram empregados, após correção, a fim de dividir as constantes de velocidade global da reação de MCC em contribuições dos grupos (OH) presentes. Para os compostos modelo, verificou-se que k3 (Prim-OH) /k3 (Sec-OH) > 1, semelhante as reações de celulose sob condições heterogéneas; esta relação aumenta como uma função do aumento da Nc. As constantes de velocidade globais e parciais de acilação de MCC diminuim de anidrido etanóico a butanóico e, em seguida, aumentam para anidrido pentanóico e hexanóico, devido a mudanças sutis em - e compensações da entalpia e entropia de ativação. As constantes de velocidade para a acetilação de MCC, por anidrido etanóico na presença de concentrações crescentes do LI em DMAC, MeCN, DMSO e sulfolano foram calculados a partir de dados de condutividade. As constantes de velocidade de terceira ordem mostraram dependência linear sobre [LI]. Estes resultados foram explicados assumindo que o reagente é celulose ligado ao LI por ligação de hidrogénio. Isto foi confirmado pelos dados cinéticos da acetilação de CHM, espectroscopia de IV do último composto, e de celobiose nas misturas de LI/SAD e condutividade das misturas de solventes binários, na ausência e presença de MCC. A acetilação de celulose é mais rápida nas misturas de em LI com DMAC e DMSO do que com MeCN e sulfolano. Esta diferença é explicada, em parte, com base na alta viscosidade das soluções de biopolímeros em LI/sulfolano. Obteve-se mais informações sobre os efeitos do solvente molecular a prtir das propriedades microscópicas dos solventes e simulações por dinâmica molecular, DM. Os dados solvatocrômicos (polaridade empírica e basicidade) têm mostrado a importância da basicidade do solvente; solventes mais básicos formam ligações de hidrogênio mais fortes com os grupos (OH) da celulose, aumentando sua acessibilidade e, consequentemente sua reatividade. Este é o caso de DMAC e DMSO. Os resultados das simulações por DM indicaram a formação de ligações de hidrogénio, entre os grupos (OH) da unidade de glucose anidra do MCC, (Cl-) de LI, e o dipolo do DMAC e DMSO . Observamos que a acilação de celulose em LiCl/DMAC é eficientemente catalisada por imidazol, mas não pelo cloreto de tosila. Resultados de IV de FT e RMN de 1H indicaram a formação de N-acilimidazol que é o agente de acilação. As constantes globais e parciais de velocidade de acilação do MCC diminuiram de anidirido etanóico a butanóico e depois aumentou para anidrido pentanóico e hexanóico, devido a mudanças sutis em- e compensações da entalpia e entropia de ativação. / The objective of this work is to study the reactivity in cellulose acylation by carboxylic acid anhydrides under homogeneous conditions in dipolar aprotic solvents (DAS), including LiCl/ N,N-dimethylacetamide (DMAC) and ionic liquids (ILs)/DAS. Factors that contribute to reactivity were quantified by studying the dependence of reaction rates on temperature and solvent composition. After establishing that conductivity is an appropriate experimental technique to calculate the rate constants, we studied the kinetics of the homogeneous uncatalyzed and catalyzed acylation of microcrystalline cellulose, MCC, with carboxylic acid anhydrides with different acyl chain-length (Nc; ethanoic to hexanoic) in the following solvent systems: LiCl/DMAC; mixtures of the IL, 1-allyl-3-methylimidazolium chloride, (AlMeImCl) and acetonitrile (MeCN), DMAC, dimethyl sulfoxide (DMSO) and sulfolane. The anhydroglucose unit of cellulose carries one primary- and two secondary hydroxyl groups. We used cyclohexylmethanol, CHM, and trans-1,2-cyclohexanediol, CHD, as model compounds for the hydroxyl groups of the anhydroglucose unit of cellulose. The ratios of rate constants of acylation of primary (CHM; Prim-OH) and secondary (CHD; Sec-OH) groups were employed, after correction, in order to split the overall rate constants of the reaction of MCC into contributions from the discrete OH groups. For the model compounds, we have found that k3 (Prim-OH)/k3 (Sec-OH) > 1, akin to reactions of cellulose under heterogeneous conditions; this ratio increases as a function of increasing Nc. The overall and partial rate constants of the acylation of MCC decrease from ethanoic- to butanoic anhydride and then increase for pentanoic- and hexanoic anhydride, due to subtle changes in- and compensations of the enthalpy and entropy of activation. Rate constants for the acetylation of MCC, by ethanoic anhydride in the presence of increasing concentrations of the ionic liquid, IL, 1-allyl-3-methylimidazolium chloride in dipolar aprotic solvents, DAS, N,N-dimethylacetamide, DMAC, acetonitrile, MeCN, dimethylsulfoxide, DMSO and sulfolane, have been calculated from conductivity data. The third order rate constants showed a linear dependence on [IL]. These results have been explained by assuming that the reactant is cellulose hydrogen-bonded to the IL. This is corroborated by kinetic data of the acetylation of cyclohexyl methanol, FTIR spectroscopy of the latter compound, and cellobiose in mixtures of IL/DAS, and conductivity of the binary solvent mixtures in absence, and presence of MCC. Cellulose acetylation is faster in IL/DMAC and IL/DMSO than in IL/MeCN and IL/Sulfolane. This difference is explained, in part, based the high viscosity of the biopolymer solutions in IL-Sulfolane. Additional explanation came from microscopic solvents properties and molecular dynamics, MD simulations. The solvatochromic data (empirical polarity and basicity) have shown the importance of solvent basicity; basic solvents hydrogen-bond to the hydroxyl groups of cellulose increasing its accessibility, hence its reactivity. This is the case of DMAC and DMSO. Results of MD simulations indicated hydrogen-bond formation between the hydroxyl groups of the anhydroglucose unit of MCC, (Cl-) of the IL, and the dipole of the DMAC and DMSO. It has been observed that cellulose acylation in LiCl/DMAC is efficiently catalyzed by imidazole, but not by p-tosyl chloride. FTIR and 1H NMR have indicated the formation of N-acylimidazole which is the acylating agent. The overall and partial rate constants of the acylation of MCC decreased from ethanoic- to butanoic-anhydride and then increased for pentanoic- and hexanoic anhydride, due to subtle changes in- and compensations of the enthalpy and entropy of activation.