Glicoconjugados são abundantes na superfície de Trypanosoma cruzi e têm sido bastante estudados por diferentes grupos. A degradação dessas moléculas, no entanto, tem sido alvo de pouco interesse. O objetivo deste trabalho foi determinar a atividade de alfa-L-fucosidase em T.cruzi uma vez que trabalhos anteriores haviam concluído que várias hidrolases, entre elas a alfa-L-fucosidase estavam ausentes em epimastigota (AVILA, et al., 1979). Empregando-se p-nitrofenilfucopiranosídeo como substrato e extrato de formas epimastigotas, verificou-se que a enzima apresenta praticamente a mesma atividade em um intervalo de pH entre 6,0 e 7,5, caindo drasticamente em pHs mais ácidos. A incubação prévia da enzima a 28°C em pH 7,0 leva à perda de aproximadamente 30% de sua atividade após 1h 30mim e à perda de 100% após 4 horas de incubação. O efeito de íons na atividade da enzima foi estudado,verificando-se que Zn +2 inibe 90% sua atividade, enquanto que outros, como o Ca +2 praticamente não tem efeito. A enzima é parcialmente encontrada na fração particulada, podendo ser solubilizada parcialmente com 1% de Triton X-100 ou com NaCl 1 M. As tentativas feitas de purificar a enzima foram infrutíferas, uma vez que não se encontraram condições para manter a proteína ativa por longos períodos de tempo. A alfa-L-fucosidase está presente não só em pimastigotas, mas também em tripomastigotas, embora parentemente com diferentes atividades específicas, sendo maior em epimastigotas. Mesmo nos epimastigotas, grandes variações de atividade específica foram detectadas ao longo deste trabalho (de 0,03 a 0,23 unidades). Anticorpos preparados contra alfa-L-fucosidase comercial de epidídimo bovino imunoprecipitaram de extratos de epimastigotas previamente marcados com 35 S-metionina, um polipeptídeo em torno de 50 kDa após eletroforese em gel desnaturante e uma banda de 130-150 kDa em gel não desnaturante, sugerindo que a enzima em T.cruzi pode ser dimérica, a exemplo de outras alfa-L-fucosidases descritas na literatura. A imunoprecipitação de extrato de epimastigotas marcados com 35 S-metionina na presença de tunicamicina, com o anticorpo anti-alfa-L-fucosidase revelou um polipeptídeo de 45 kDa, mostrando que a enzima é glicosilada. A glicosilação daenzima também foi observada pelo emprego de corantes comerciais. Além disso, os anticorpos anti-alfa-L-fucosidase imunoprecipitam moléculas com atividade de alfa-L-fucosidase,embora não se tenha observado aumento da atividade, possivelmente devido à perda de atividade da enzima nas condições empregadas durante a imunoprecipitação. Os anticorpos anti-alfa-L-fucosidase reconhecem, por imunofluorescência indireta, tanto as formas epimastigotas como tripomastigotas de cultura de tecido. A análise por microscopia de transmissão mostra a reatividade intensa do anticorpo com uma região membranar localizada na região posterior do epimastigota. No caso do tripomastigota, a reatividade é menos pronunciada mostrando uma leve marcação no interior do parasita. / Alpha-L-fucose is a component of glycoproteins, inc1uding glycoproteins isolated from Tcruzi. a-L fucosidases have been isolated from different sources, but earlier studies were unable to detect this enzyme in T. cruzi epimastigotes (AVILA et al., 1979). In this work immunocytochemical and biochemical techniques have been used to localize and characterize a membrane-associated, neutral-pH-optimum alpha-L fucosidase from Trypanosoma cruzi epimastigotes. Light and electron microscopy specifically localized the alpha-L fucosidase on membranes in the posterior region of the epimastigotes and on the parasite surface. Immunoreactivity for alpha-L-fucosidase, a1though less intense, was also detected on the surface of trypomastigotes. Fractionation of epimastigotes homogenates indicated that over 50% of the a-Lfucosidase activity was associated with the 80 000 g pellet. This pellet-associated activity could be solubilized with 1 M NaCl or with 1% Triton X-I 00, suggesting that alpha-L-fucosidase is peripherally associated with membranes. Analysis of alpha-L-fucosidase on epimastigote extracts indicated that the enzyme had a pH-activity curve (with an optimum near 7) which was comparable to other alpha-L-fucosidases reported in the literature. A higher specific activity (in units/mg) was found in epimastigotes as compared to the other differentiation stages of the parasite: 0.028 for epimastigotes, 0.002 for metacyc1ic trypomastigotes and 0.015 for tissue - cultured trypomastigotes. SDS/PAGE and Westem blotting analysis indicated that epimastigotes have a protein band of 50 kDa which was immunoreactive with anti-alpha-L-fucosidase antibodies.

Les iminosucres sont des composés azotés polyhydroxylés mono- (pyrrolidine, piperidine, azepane) ou bicycliques (pyrrolizidine, indolizidine, nortropane) démontrant une forte activité inhibitrice envers les glycosidases, enzymes catalysant l'hydrolyse des liaisons glycosidiques des glycoconjugués. Le développement de nouveaux dérivés d'iminosucres est essentiel afin d'obtenir de nouveaux traitements contre des maladies comme le diabète de type II, la mucoviscidose ou les troubles du stockage lysosomale (maladies de Gaucher ou de Fabry par exemple). Des études récentes ont démontré que l'utilisation de systèmes multivalents d'iminosucres peut amener à des inhibitions plus fortes et plus sélectives envers les glycosidases comparés aux inhibiteurs monovalents. Cependant, une grande majorité de ces systèmes multivalents, incluant des systèmes multivalents basés sur une plateforme de type calixarène synthétisés au début de cette thèse, sont obtenus grâce à la cyclo-addition azoture alcyne catalysée par le cuivre(CuAAC). Malheureusement, cette réaction puissante mène à la contamination des systèmes multivalents par des quantités non négligeables d'ions cuivre toxiques. C'est pour cela que le principal but de ce doctorat a été de développer de nouvelles méthodes de ligations afin de former des architectures multivalentes d'iminosucres sans utiliser de catalyseurs métalliques toxiques.Premièrement, des ligations déjà exploitées pour la préparation de sucres multivalents comme l'addition radicalaire photoinduite d'un thiol sur un alcène terminal (couplage thiol-ène) et la ligation oxime ont été appliqués aux iminosucres avec succès. Ces approches ont alors permis de synthétiser des systèmes multivalents basés respectivement sur des plateformes de type calixarènes ou peptides cycliques.Dans un second temps, une nouvelle approche vers des systèmes multivalents de sucres et d'iminosucres a été développée en exploitant les remarquables stabilité et réactivité des fluorures de sulfonyle. Le couplage de ces derniers avec des partenaires portant une amine primaire a permis d'obtenir des clusters de sucres et d'iminosucres liés par une fonction sulfonamide avec de très bons rendements.Parallèlement, le couplage thiol-ène a permis la préparation simple et rapide de pseudo-disaccharides d'iminosucres, une nouvelle classe d'inhibiteur de glycosidases exhibant de meilleures activités et sélectivités que les iminosucres monosaccharidiques correspondants. Ce comportement est probablement du à la présence de l'unité saccharidique qui améliore l'analogie entre l'inhibiteur et les oligosaccharides naturels, substrats des glycosidases. / Iminosugars are naturally occurring, polyhydroxylated monocyclic (pyrrolidine, piperidine, azepane) and bicyclic (pyrrolizidine, indolizidine, nortropane) nitrogenated compounds endowed with strong inhibition activity against glycosidases, the enzymes that catalyse the cleavage of the glycosidic bonds in glycoconjugates. The development of new iminosugar derivatives is essential to obtain new treatments against diseases such as type II diabetes, cystic fibrosis or lysosomal storage disorders (Gaucher and Fabry diseases). Although the development of glycosidase inhibitors based on iminosugar clusters was not explored for a long period of time, recent studies have demonstrated that multivalent iminosugars are stronger and more selective inhibitors than the corresponding monovalent compounds. However, nearly two thirds of all the di- and multivalent iminosugars known to date, including the calixarene-based iminosugar clusters synthesized at the beginning of the thesis work, were obtained by means of the copper-mediated azide-alkyne cycloaddition (CuAAC). Unfortunately, this highly efficient reaction leads to the contamination of the multivalent compounds by significant amounts of noxious copper ions. Thus, the main aim of the present PhD research was the development of new ligation tools for the synthesis of multivalent iminosugars in the absence of metal catalysts. First, the ligations already exploited for the preparation of multivalent sugars, such as the photoinduced radical addition of thiol to terminal akenes (thiol-ene coupling) and the oxime ligation, were successfully applied to the iminosugars. Both approaches allowed the synthesis of iminosugar clusters based on calixarene and cyclopeptide scaffolds, respectively. Then, an unprecedented approach to multivalent sugars and iminosugars was developed taking advantage of the uncommon stability and reactivity of the sulfonyl fluoride moieties. The coupling of the latter with partners bearing a primary amine group afforded the corresponding sulfonamide-linked sugar and iminosugar clusters in high yield. Finally, the above-mentioned thiol-ene coupling also allowed the straightforward preparation of new iminosugar pseudo-disaccharides, a class of inhibitors endowed with higher glycosidase selectivity than the corresponding monosaccharidic iminosugar. This feature is due to the presence of the sugar unit which improves the analogy with the natural oligosaccharidic substrates of the glycosidases.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

It is widely known that fossil fuels are limited; consequently, the generation of new sources of energy in a clean and environmentally friendly manner is a research priority. Bioethanol appears to be one potential solution, especially second-generation production from renewable biomass. In order to use lignocellulosic feedstock to produce bioethanol, its polysaccharide components, cellulose and hemicellulose, must be hydrolysed into soluble sugars, which can then be converted into ethanol by fermentative microorganisms such as Geobacillus thermoglucosidasius TM242 used by the company ReBio Technologies Ltd. To date, the cost of commercial enzymes used during the hydrolysis process remains a major economic consideration in the production of second-generation bioethanol as an alternative fuel. The research project presented in this thesis aims to improve this rate-limiting step of microbial bioethanol production through an investigation of the different enzymes associated with hemicellulose hydrolysis. Firstly, the TM242 genome sequence revealed a number of genes encoding glycoside-hydrolases. Six of these genes were cloned and expressed in E. coli and the recombinant enzymes characterised; three of them, two β-xylosidases and an α arabinofuranosidase, are relevant to xylan hydrolysis, and were found to be highly active and thermostable. Crystallisation of one of the β-xylosidases permitted the determination of a high-resolution (1.7 Å) structure of the apo-enzyme along with a lower resolution (2.6 Å) structure of the enzyme-substrate complex, resulting in the first reported structure of a GH52 family member (Espina et al., 2014). Secondly, as the TM242 microorganism lacks xylanase enzymes, four genes encoding xylanases from closely-related Geobacillus strains were cloned and expressed in E. coli, with one of them being also successfully cloned and expressed in G. thermoglucosidasius TM242. This heterologous xylanase was secreted in active form representing an enhanced biomass utilisation by TM242. In conclusion, it is felt that the findings presented here have the potential to make a valuable contribution towards second-generation bioethanol production.

Glicosídeos essenciais a vida podem ser sintetizados por métodos enzimáticos com altíssima especificidade. O mecanismo de reação de β-glicosidases GH1 por dupla-substituição envolve a formação de intermediário covalente (glicosil-enzima) que pode seguir duas rotas. Uma envolve sua hidrólise na ligação β-glicosídica entre glicone e aglicone do substrato, liberando o monossacarídeo da extremidade não-redutora; enquanto na outra rota, transglicosilação ou síntese por controle cinético, o intermediário é atacado por um aceptor glicosídico (2ª molécula de substrato), gerando um novo glicosídeo. BglB possui resíduos (W e H) que formam um \"canal\" por onde a água ataca o intermediário covalente no sítio ativo, sendo alvos de potenciais mutações que alteram o balanço entre as rotas e ampliando a eficiência de transglicosilação. Para caracterizar as bases moleculares da razão entre essas duas rotas catalíticas, β-glicosidases da família GH1 Spodoptera frugiperda (Sfβglu; AF052729) e Tenebrio molitor (Tmβglu; AF312017) foram produzidas como enzimas recombinantes em leveduras Pichia pastoris GS115, concentradas com 90% (p/v) de sulfato de amônio e diálise reversa com PEG 10000, e dialisadas em tampão CP 100 mM pH 6. A pureza foi confirmada por banda única com tamanho semelhante a 50 kDa (Sfβgly) e 56 kDa (Tmβgly) em SDS-PAGE. A atividade recuperada após purificação é 152% (Sfβgly) e 171% (Tmβgly) e a concentração protéica de 0,134 µg/µL (Sfβgly) e 0,217 µg/µL (Tmβgly). A razão entre as reações de hidrólise e transglicosilação (vH/vT) foi analisada utilizando os substratos pNPβ-gluco (0,1 mM a 8 mM) e MUβ-gluco (0,1 mM a 40 mM), a partir da velocidade de formação dos produtos pNP ou MU e glicose, respectivamente. Tmβgly catalisa reações de transglicosilação com ambos, mas vH/vT depende da [S] variando de +∞ (vH>>vT) a 1,5 para pNPβ-gluco e +∞ a 1 para MUβ-gluco. Sfβgly, ao contrário, não transglicosila com substrato MUβ-gluco. Além disso, vH/vT para pNPβ-gluco é 2 e independe da [S]. Já Sfβgly K201F conjuga propriedades de ambas, pois não transglicosila com MUβ-gluco, mas para pNPβ-gluco há ocorrência de transglicosilação dependente da [S], variando de +∞ (vH>>vT) a 1,25. Os parâmetros cinéticos (Vmax e Km) foram ajustados no Enzifitter e por simulação numérica na equação do modelo cinético ping-pong para dois substratos, e mostram maior (Km2) em Tmβgly e Sfβgly K201F do que em Sfβgly. Também foi avaliado o efeito da adição de aceptores alternativos que substituem a água em ensaios com pNPβ-gluco 4mM na presença de alcóois. Para as três enzimas a adição de metanol torna a vglc menor do que de vpNP, indicando ocorrência de transglicosilação. Com adição de n-propanol vH/vT diminui cerca de 7 vezes em Sfβgly e 2 vezes em Tmβgly. Os efeitos destes alcoóis sobre Sfβgly são maiores do que para Tmβgly e Sfβgly K201F, sugerindo acesso mais fácil destes ao intermediário covalente em Sfβgly, indicando diferenças entre as enzimas na configuração desta região. Portanto, notamos que vH/vT não está ligada à afinidade pela segunda molécula de substrato, podendo ser modulada por mutação do resíduo K201 de Sfβgly, posição relacionada ao acesso da água ou aceptor alternativo ao sítio ativo. / Glycosides are essential to life and can be synthesized by enzymatic methods with high specificity. The reaction mechanism of GH1 β-glucosidases by double-substitution involves the formation of covalent intermediate (glycosyl-enzyme) which may follow two routes. One of them involves hydrolysis in β-glycosidic bond between aglycone glucone and the substrate, releasing a monosaccharide from the non-reducing end, whereas in the other route, transglycosylation or synthesis by kinetic control, the intermediate is attacked by a glucosyl acceptor (second substrate molecule), generating a new glucoside. BglB has two residues (W and H) that form a \"channel\" through which water attacks the covalent intermediate on the active site. Thus, the residues are potential targets for mutations that alter the balance between routes, increasing the efficiency of transglycosylation. To characterize the molecular basis of the ratio between these two catalytic routes, two β-glycosidases from GH1 family, Spodoptera frugiperda (Sfβgly; AF052729) and Tenebrio molitor (Tmβgly; AF312017) were produced as recombinant enzymes in Pichia pastoris GS115, concentrated using 90% (w/v) ammonium sulfate and reverse dialysis with PEG 10000, and dialyzed in 100 mM CP buffer pH 6. SDS-PAGE confirmed that Sfβgly (~50 kDa) and Tmβgly (~56 kDa) were pure after that procedure. The activity recovered after purification were 152% (Sfβgly) and 171% (Tmβgly) and protein concentration were 0.134 mg/mL (Sfβgly) and 0.217 mg/mL (Tmβgly). The ratio between the hydrolysis and transglycosylation (vH/vT) was analyzed using pNPβ-gluco substrates (0.1 mM to 8 mM) and MUβ-gluco (0.1 mM to 40 mM), the rate of formation of pNP or MU and glucose. Tmβgly catalyzes transglycosylation reactions with both substrates, but vH/vT depends on [S] ranging from +∞ (vH>>vT) to 1.5 for pNPβ-gluco and + ∞ to 1 for MUβ-gluco. In contrast, Sfβgly did not catalyses transglycosilation with MUβ-gluco. Moreover, vH/vT for pNPβ-gluco-is 2 and is independent of [S]. Sfβgly K201F combines properties of both, because it does not catalyse transglycosylation with MUβ-gluco, but for pNPβ-gluco the occurrence of transglycosylation is dependent on [S], ranging from + ∞ (vH>>vT) to 1.25. The kinetic parameters (Vmax e Km) were adjusted in Enzfitter and numerical simulation showing that Km2is higher for Tmβgly and Sfβgly K201F than Sfβgly. We also evaluated the effect of adding alternative acceptors that replace the water in pNPβ gluco-4 mM. For the three enzymes the addition of methanol makes vglc less than pNP, indicating the occurrence of transglycosylation. Addition of n-propanol decreases vH/vT about 7 times in Sfβgly and 2 times Tmβgly. The effects on Sfβgly are higher than on Tmβgly and Sfβgly K201F, suggesting easier access to the covalent intermediate in Sfβgly. We observe that vH/vT is not related to affinity for the second substrate molecule and may be modulated by mutation of residue K201 Sfβgly, position related to the access of water or alternative acceptor to the active site.

No description available.

Glicosídeos essenciais a vida podem ser sintetizados por métodos enzimáticos com altíssima especificidade. O mecanismo de reação de β-glicosidases GH1 por dupla-substituição envolve a formação de intermediário covalente (glicosil-enzima) que pode seguir duas rotas. Uma envolve sua hidrólise na ligação β-glicosídica entre glicone e aglicone do substrato, liberando o monossacarídeo da extremidade não-redutora; enquanto na outra rota, transglicosilação ou síntese por controle cinético, o intermediário é atacado por um aceptor glicosídico (2ª molécula de substrato), gerando um novo glicosídeo. BglB possui resíduos (W e H) que formam um \"canal\" por onde a água ataca o intermediário covalente no sítio ativo, sendo alvos de potenciais mutações que alteram o balanço entre as rotas e ampliando a eficiência de transglicosilação. Para caracterizar as bases moleculares da razão entre essas duas rotas catalíticas, β-glicosidases da família GH1 Spodoptera frugiperda (Sfβglu; AF052729) e Tenebrio molitor (Tmβglu; AF312017) foram produzidas como enzimas recombinantes em leveduras Pichia pastoris GS115, concentradas com 90% (p/v) de sulfato de amônio e diálise reversa com PEG 10000, e dialisadas em tampão CP 100 mM pH 6. A pureza foi confirmada por banda única com tamanho semelhante a 50 kDa (Sfβgly) e 56 kDa (Tmβgly) em SDS-PAGE. A atividade recuperada após purificação é 152% (Sfβgly) e 171% (Tmβgly) e a concentração protéica de 0,134 µg/µL (Sfβgly) e 0,217 µg/µL (Tmβgly). A razão entre as reações de hidrólise e transglicosilação (vH/vT) foi analisada utilizando os substratos pNPβ-gluco (0,1 mM a 8 mM) e MUβ-gluco (0,1 mM a 40 mM), a partir da velocidade de formação dos produtos pNP ou MU e glicose, respectivamente. Tmβgly catalisa reações de transglicosilação com ambos, mas vH/vT depende da [S] variando de +∞ (vH>>vT) a 1,5 para pNPβ-gluco e +∞ a 1 para MUβ-gluco. Sfβgly, ao contrário, não transglicosila com substrato MUβ-gluco. Além disso, vH/vT para pNPβ-gluco é 2 e independe da [S]. Já Sfβgly K201F conjuga propriedades de ambas, pois não transglicosila com MUβ-gluco, mas para pNPβ-gluco há ocorrência de transglicosilação dependente da [S], variando de +∞ (vH>>vT) a 1,25. Os parâmetros cinéticos (Vmax e Km) foram ajustados no Enzifitter e por simulação numérica na equação do modelo cinético ping-pong para dois substratos, e mostram maior (Km2) em Tmβgly e Sfβgly K201F do que em Sfβgly. Também foi avaliado o efeito da adição de aceptores alternativos que substituem a água em ensaios com pNPβ-gluco 4mM na presença de alcóois. Para as três enzimas a adição de metanol torna a vglc menor do que de vpNP, indicando ocorrência de transglicosilação. Com adição de n-propanol vH/vT diminui cerca de 7 vezes em Sfβgly e 2 vezes em Tmβgly. Os efeitos destes alcoóis sobre Sfβgly são maiores do que para Tmβgly e Sfβgly K201F, sugerindo acesso mais fácil destes ao intermediário covalente em Sfβgly, indicando diferenças entre as enzimas na configuração desta região. Portanto, notamos que vH/vT não está ligada à afinidade pela segunda molécula de substrato, podendo ser modulada por mutação do resíduo K201 de Sfβgly, posição relacionada ao acesso da água ou aceptor alternativo ao sítio ativo. / Glycosides are essential to life and can be synthesized by enzymatic methods with high specificity. The reaction mechanism of GH1 β-glucosidases by double-substitution involves the formation of covalent intermediate (glycosyl-enzyme) which may follow two routes. One of them involves hydrolysis in β-glycosidic bond between aglycone glucone and the substrate, releasing a monosaccharide from the non-reducing end, whereas in the other route, transglycosylation or synthesis by kinetic control, the intermediate is attacked by a glucosyl acceptor (second substrate molecule), generating a new glucoside. BglB has two residues (W and H) that form a \"channel\" through which water attacks the covalent intermediate on the active site. Thus, the residues are potential targets for mutations that alter the balance between routes, increasing the efficiency of transglycosylation. To characterize the molecular basis of the ratio between these two catalytic routes, two β-glycosidases from GH1 family, Spodoptera frugiperda (Sfβgly; AF052729) and Tenebrio molitor (Tmβgly; AF312017) were produced as recombinant enzymes in Pichia pastoris GS115, concentrated using 90% (w/v) ammonium sulfate and reverse dialysis with PEG 10000, and dialyzed in 100 mM CP buffer pH 6. SDS-PAGE confirmed that Sfβgly (~50 kDa) and Tmβgly (~56 kDa) were pure after that procedure. The activity recovered after purification were 152% (Sfβgly) and 171% (Tmβgly) and protein concentration were 0.134 mg/mL (Sfβgly) and 0.217 mg/mL (Tmβgly). The ratio between the hydrolysis and transglycosylation (vH/vT) was analyzed using pNPβ-gluco substrates (0.1 mM to 8 mM) and MUβ-gluco (0.1 mM to 40 mM), the rate of formation of pNP or MU and glucose. Tmβgly catalyzes transglycosylation reactions with both substrates, but vH/vT depends on [S] ranging from +∞ (vH>>vT) to 1.5 for pNPβ-gluco and + ∞ to 1 for MUβ-gluco. In contrast, Sfβgly did not catalyses transglycosilation with MUβ-gluco. Moreover, vH/vT for pNPβ-gluco-is 2 and is independent of [S]. Sfβgly K201F combines properties of both, because it does not catalyse transglycosylation with MUβ-gluco, but for pNPβ-gluco the occurrence of transglycosylation is dependent on [S], ranging from + ∞ (vH>>vT) to 1.25. The kinetic parameters (Vmax e Km) were adjusted in Enzfitter and numerical simulation showing that Km2is higher for Tmβgly and Sfβgly K201F than Sfβgly. We also evaluated the effect of adding alternative acceptors that replace the water in pNPβ gluco-4 mM. For the three enzymes the addition of methanol makes vglc less than pNP, indicating the occurrence of transglycosylation. Addition of n-propanol decreases vH/vT about 7 times in Sfβgly and 2 times Tmβgly. The effects on Sfβgly are higher than on Tmβgly and Sfβgly K201F, suggesting easier access to the covalent intermediate in Sfβgly. We observe that vH/vT is not related to affinity for the second substrate molecule and may be modulated by mutation of residue K201 Sfβgly, position related to the access of water or alternative acceptor to the active site.

A notable need exists for noninvasive tools to increase our mechanistic understanding of disease progression at a cellular and molecular level. Studying the functions of proteins in their innate in vivo tissue environment can provide useful information about pathology enabling appropriate treatment and early diagnosis. Chemical exchange saturation transfer MRI contrast provides real-time functional characterization of the biological landscape and can be used to detect multiple enzyme biomarker activities. A dual-enzyme catalyCEST contrast agent was developed as a proof-of-concept to demonstrate the potential of using a salicylic acid scaffold and control the CEST signal through enzyme activation. In addition, a straightforward route was designed to synthesize a diamagnetic catalyCEST MRI agent that is a substrate for β-galactosidase and β-glucuronidase enzymes. The synthesized agents generated two peaks in the CEST spectrum, at 4.25 ppm corresponding to a carbamate moiety and at 9.25 ppm corresponding to the salicylic acid moiety. Chemical exchange rates of liable protons were determined from a QUESP Hanes-Woolf plot. In the presence of the corresponding enzymes, the catalyCEST agent was activated via saccharide hydrolysis followed by a spontaneous disassembly to produce 4-aminosalicylic acid. This reaction converted the carbamate moiety into a free primary amine, and caused a loss of CEST signal at 4.25 ppm. The CEST signal at 9.25 ppm was unaffected by the enzyme catalysis, and therefore used as an internal control signal. Michaelis-Menten enzyme kinetics studies were performed with CEST MRI to verify that catalyCEST MRI could truly detect enzyme activity. The Michaelis-Menten kinetics constants from MRI studies were compared to the kinetics constants measured with UVvis results from the same contrast agent, demonstrating the quantitative potential of catalyCEST MRI with both contrast agents. These findings demonstrate that the newly synthesized modular agents have the potential to become reliable catalyCEST MRI imaging probes. In addition, the modular design of these agents facilitates the conjugation of other enzyme substrates to the carbamate spacer, so that this approach constitutes a platform technology for the detection of enzyme activity.

Cette thèse s'inscrit dans le cadre du programme PentoRaf, dans la thématique « Transformation par voie chimique d'agromolécules en produits bioactifs ». Le travail de ce projet porte sur la synthèse de nouveaux azépanes tétrahydroxylés comportant un bras anomérique fonctionnel et sur l'évaluation des propriétés inhibitrices sur des glycosidases. La séquence réactionnelle a la particularité de valoriser les co produits issus du son et de la paille de blé.Dans une première partie, l'accès à un synthon polyfonctionnel, développé à partir des travaux précédents, a été réalisé avec des groupements protecteurs pivaloylés et méthoxyméthylés sur les hydroxyles communs aux sucres de départ (D-xylose et L-arabinose).La deuxième partie de ce travail consiste d'abord à introduire les différents groupements électroattracteurs : CO2Et, CN, PO(OEt)2, (CO2Et)PO(OEt)2 via une réaction de Wittig-Horner-Emmons. Les problèmes rencontrés lors de l'addition de l'allylamine ont clôturé les travaux sur la préparation d'azépanes avec un substituant phosphonate. Seuls les azépanes insaturés, comportant les fonctions : ester et nitrile, sont isolés.Une stratégie visant à obtenir les différentes distributions des hydroxyles (cis et trans) sur l'insaturation des azépanes obtenus est établie, dans une troisième partie, sur les composés fonctionnalisés avec un ester. Une réaction de déprotection finale donne ainsi accès à une famille de diastéréoisomères d'azépanes comportant un bras anomérique avec une fonction acide carboxylique, dont un composé a montré une activité biologique intéressante. Cette stratégie a été reproduite sur les azépanes comportant un nitrile jusqu'aux tétrahydroxyazépanes cis protégés. / This thesis is part of the PentoRaf program, in the theme “Chemical Transformation of agromolecules into bioactive products”. The aim of this project is the synthesis of new tetrahydroxylated azepanes containing a functional anomeric arm and the evaluation of their inhibitive properties on glycosidases. The reactive sequence advantageously values the co-products obtained from wheat bran and wheat straw.In the first part, the access to a polyfunctional synthon, developed from previous research, is carried out by protecting the common hydroxyl groups of the starting sugars (D-xylose and L-arabinose) with pivaloyl and methoxymethyl groups.The second part of this work is to introduce the various electroattracting groups: CO2Et, CN, PO(OEt)2, (CO2Et)PO(OEt)2 via a Wittig-Horner-Emmons reaction. The problems encountered during the addition of the allylamine ended our approach to prepare azepanes with a phosphonate substituent. Only the unsaturated azepanes containing ester and nitrile functions are isolated.A strategy is developed in the third part in order to achieve a specific distribution (cis and trans) of the hydroxyl groups on the unsaturation of the resulting azepanes by functionalizing these compounds with an ester. A final deprotection reaction leads to a family of azepane diastereoisomers containing an anomeric arm with a carboxylic acid function, among which one compound showed an interesting biological activity. This strategy was applied to azepanes containing a nitrile and lead to cis-protected tetrahydroxyazepanes.

Des résultats récents ont montré les premières preuves d’un effet multivalent puissant des iminosucres sur l’inhibition des glycosidases, avec des gains d’affinité allant jusqu’à 10000. Afin de comprendre les différentes caractéristiques de cet « effet de cluster » et d’en poursuivre l’optimisation, de nouvelles charpentes doivent être conçues. Le premier axe de recherche a donc consisté en la mise au point d’un ensemble de techniques d’ingénierie « click » pour la synthèse de systèmes multivalents, avec le développement d’une stratégie Sweet LEGO®. Cette approche permettrait un accès simple à une grande variété de néocyclodextrines préfonctionnalisées. Le second axe a consisté en une étude de relation structure-activité autour de charpentes moléculaires polyalcynes utilisées pour préparer de nouveaux clusters par chimie « click ». Une partie des clusters a été préparée en utilisant de nouvelles charpentes cyclopeptoïdes. Ils ont permis de poursuivre l’optimisation de l’effet multivalent des iminosucres sur l’inhibition de glycosidases. En particulier, un composé portant 30 ligands a montré le meilleur effet multivalent connu sur une enzyme modèle. / Recent reports have demonstrated the first pieces of evidence of a strong multivalent effect in glycosidase inhibition by iminosugars, with affinity enhancements close to 10000. In order to understand the different parameters of this “cluster effect” and to continue its optimization, new scaffolds must be designed. The first research topic was thus to develop a set of « Click » Chemistry engineering techniques for the synthesis of multivalent systems, with the development of a Sweet LEGO® strategy. In the end, it would allow an easy access to a broad range of prefunctionalized neocyclodextrins. The second research topic consisted in a structure-activity relationship study by varying the molecular polyalkyne scaffold used for the preparation of new clusters by way of « Click » Chemistry. They allowed to investigate the specific features of the iminosugar cluster effect in the inhibition of glycosidases. In particular, a compound of unprecedented valency bearing 30 iminosugar units demonstrated an unprecedented dramatic affinity enhancement for the inhibition of a model enzyme (Jack bean alpha-mannosidase).