Ice recrystallization during cryopreservation results in cell death and decreased cell viabilities due to cellular damage. This is a significant problem particularly in regenerative medicine where decreased cell viabilities post-thaw affect the success of the therapy. Given the success of these therapies to treat various diseases, the development of novel cryprotectants which have the ability to inhibit ice recrystallization during freezing and thawing are urgently required. Current cryoprotectant such as dimethyl sulfoxide, is associated with cytotoxicity in the clinical settings and thus are not optimal cryoprotectants. Our laboratory is interested in the rational synthesis of non-cytotoxic small molecules which possess the property of ice recrystallization inhibition (IRI) activity. Previously, the Ben laboratory has demonstrated that simple monosaccharides possess moderate ice recrystallization inhibition activity and that this activity is linked to hydration. The “compatibility” of the carbohydrate within the three-dimensional hydrogen bonded network of water is inversely proportional to its IRI activity. Hydration has previously been directly linked to the stereochemical relationship of individual hydroxyl groups on the carbohydrate. Additionally, it has been proposed that intramolecular hydrogen bond formation and hydrogen bonding cooperativity has a large effect on the water structure thus impacting hydration. Structure-function work has suggested that the presence of an amine as a hydrogen donor at the endocyclic position within the pyranose ring maybe beneficial to IRI activity. Thus, the first part of this thesis describes the synthesis and IRI activity of D-glucose and D-galactose based azasugars and its analogues. These azasugars have replaced the endocyclic ring oxygen with an amine. These azasugars and their analogues were found to possess moderate to potent IRI activity suggesting that hydrogen bond donation may be important for hydration and thus, IRI activity at the endocyclic ring oxygen. During the development of these azasugars, the Ben laboratory developed carbohydrate-based surfactants and hydrogelators possessing unprecedented IRI activity. A potential use of molecules possessing IRI activity is towards the inhibition of gas hydrate formation. Gas hydrates are ice-like solids containing gases within a highly ordered network of water molecules. These gas hydrates tend to accumulate in oil and gas pipelines posing significant dangers as the build-up of solid material leads to blockages in the pipeline reducing flow. Previous work had demonstrated the use of antifreeze proteins possessing potent IRI activity in inhibiting gas hydrate formation. However, their complex structure limits commercial use. Thus, the second part of the thesis describes the use of the azasugars, carbohydrate-based surfactants and hydrogelators in inhibiting gas hydrate formation. The effectiveness of the small molecules is compared to a commercial inhibitor PVP 10. Some of these small molecules were significantly better inhibitors of gas hydrate formation than the currently utilized inhibitor PVP 10. The low molecular weights of these small molecules, easy synthesis and potency make them excellent alternatives to PVP 10. However it was found that while some of the structural features in the small molecules may be amenable to both activities, it seems that the ability to inhibit ice recrystallization is not a good indicator of a compounds ability to inhibit gas hydrate formation. In a continuing effort to develop novel small molecule IRIs, the Ben laboratory has develop three classes of compounds. These include: carbohydrate-based surfactants and hydrogelators, lysine-based surfactants and truncated C-linked glycopeptides. Structure-function work utilizing these compounds revealed that presence of long alkyl chains, an amide linkage and the presence of an open-alditol chain are all important to IRI activity. However, the surfactant-like nature limits their use in cryopreservation and thus prompted the discovery of phenoxyglycosides as IRI active molecules. The structural features of these recently developed small molecules were combined to generate novel small molecule IRIs which do not resemble surfactants. These novel small molecules included “disaccharides” which possessed an aryl group at the anomeric position of a pyranose ring and an open-alditol chain linked via an amide bond. Additionally, N-cycloalkyl-D-aldonamides and N-phenyl-D-aldonamides were also synthesized. Of these novel small molecules, two very potent IRI active molecules were discovered: a “disaccharide” possessing an aryl group at the anomeric position with the open-alditol chain of D-galactose linked via an amide bond at C3 and N-phenyl-D-arbonamide. Both of these small molecules were assessed for their ability to cryopreserve hematopoietic stem cells. Unfortunately, the additional of these compounds failed to improved percent cell viabilities as compared to DMSO.

L’objectif de mon projet de thèse était de synthétiser différentes familles d’iminosucres comme chaperons pharmacologiques potentiels de la B-galactocérébrosidase (GALC). Cette glycosidase lysosomale est responsable de la maladie de Krabbe, une maladie rare et héréditaire qui ne dispose actuellement pas de traitement. La thérapie chaperon est une nouvelle stratégie consistant à administrer, à des concentrations très faibles, une petite molécule ayant des interactions fortes avec l'enzyme. La plupart du temps, les molécules à effet chaperon les plus efficaces sont de puissants inhibiteurs de l'enzyme et les iminosucres sont connus pour inhiber fortement les glycosidases. La conception de ces iminosucres a été dictée par la structure de l'état de transition de la réaction catalysée par la GALC et l'expérience de notre groupe dans la synthèse d’iminosucres en tant que chaperons pharmacologiques pour la maladie de Gaucher. Dans un premier temps, la synthèse d’imino-L-arabinitols a été réalisée en utilisant une méthodologie d’élongation de chaîne en C1. Puis la même stratégie a été appliquée à la synthèse d’imino-Dgalactitols C-alkylés. Des difficultés rencontrées avec cette méthodologie nous ont alors conduits à développer une autre voie d’accès à ces composés, via un allongement de chaîne en C6. Enfin une dernière famille de composés de type galacto-isofagomine a été préparée. Ces nouveaux composés ont été testés comme inhibiteurs de deux galactosidases lysosomales, afin d'évaluer leur sélectivité et leur potentiel en tant que chaperons pharmacologiques pour la maladie de Krabbe. / The objective of my PhD was to synthesize different families of iminosugars as potential pharmacological chaperones of B-galactocerebrosidase (GALC). This lysosomal glycosidase is responsible for Krabbe disease, a rare inherited disease that currently has no treatment. Chaperone therapy is a new strategy that consists in the administration, at very low concentrations, of small molecules having strong interactions with the enzyme. Most of the time, the most effective chaperones are potent inhibitors of the enzyme and iminosugars are known to strongly inhibit glycosidases. The design of these iminosugars was dictated by the transition state structure of the reaction catalyzed by GALC and the experience of our group in the synthesis of iminosugars as pharmacological chaperones for Gaucher disease. As a first stage, the synthesis of imino-L-arabinitols was conducted using a C1 chain extension methodology. Then the same strategy was applied to the synthesis of 1-C-alkyl-imino-D-galactitols. The difficulties encountered with this methodology led us then to develop another approach to these compounds, by way of a C6 chain extension. Finally a last family of galacto-isofagomine derivatives was prepared. These new compounds have been evaluated as inhibitors of two lysosomal galactosidases, to assess their selectivity and their potential as pharmacological chaperones for Krabbe disease.

Les iminosucres, analogues de sucres dont l'oxygène intracyclique a été remplacé par un azote, constituent une classe importante de mimes de sucres. Introduire une chaîne alkyle sur le carbone pseudo-anomérique donne accès à une classe importante d'iminosucres, les iminosucres C-glycosides, qui peuvent être des inhibiteurs de glycosidases puissants et sélectifs.Le principal défi associé à la synthèse d'iminosucres C-glycosides est la mise au point de voies de synthèse efficaces et applicables à tous types de sucres permettant ainsi d'accéder à une grande diversité de synthons et d'accélérer la découverte de molécules d'intérêt biologique.Ce travail de thèse a consisté dans un premier temps à élaborer une synthèse efficace et convergente d'iminosucres C-glycosides à six et sept chaînons, à partir d'un précurseur commun, le 6-azido-6-désoxy-2,3,4-tri-O-benzyl-D-glucopyranose. Cette nouvelle méthode implique une réaction tandem Staudinger/Aza-Wittig ainsi qu'une isomérisation de cycle d'azépanes stéréocontrolée.La deuxième partie de ce manuscrit traite de l'extension de cette méthodologie à la synthèse de nouveaux D- et L-iminosucres C-glycosides à six chainons mimes de la N-acétyle-D-glucosamine.La dernière partie de ce travail a été consacrée à la synthèse d'iminosucres-aza-couronnes, qui constituent un nouveau type de récepteur moléculaire. L'étude de leur capacité à complexer des métaux a été effectuée par des techniques de RMN ou fluorimétrie et a donné des résultats prometteurs. / Iminosugars, sugar analogs in which the endocyclic oxygen has been replaced by a nitrogen, constitute a major class of sugar mimetics. Introducing an alkyl chain at the pseudoanomeric carbon position leads to another class of important iminosugars, the iminosugars C-glycosides that can be potent and selective glycosidase inhibitors. The main challenge associated with iminosugars C-glycosides synthesis is currently the design of efficient and general routes applicable to any starting sugar and enabling introduction of structural diversity from advanced synthons to accelerate the discovery of biologically relevant molecules.The first part of this work focused on the development of an efficient and convergent synthesis of six and seven membered iminosugars C-glycosides from a common 6-azido-6-deoxy-2,3,4-tri-O-benzyl-D-glucopyranose precursor. This new methodology involves a highly diastereoselective tandem ring enlargement/alkylation and a stereocontrolled ring contraction.The second part of the thesis delt with the extension of the methodology to access six-membered D- and L-iminosugars C-glycosides derived from N-acetyl-D-glucosamine.The third part of this work was devoted to the synthesis of iminosugar-aza-crowns, which constitute a new type of molecular receptors, using the synthetic route developed in the first part. The ability of these compounds to complex metals was studied either by NMR or fluorimetric techniques and showed promising results.

Les glycoprotéines localisées à la surface des cellules jouent un rôle dans les phénomènes inflammatoires, les infections virales, la reconnaissance cellule/hôte ou l'adhésion cellulaire. Parmi les enzymes responsables de la biosynthèse de ces glycoprotéines, l'alpha-L-fucosidase est impliquée dans un grand nombre de phénomènes biologiques, parfois liés à de sévères pathologies. Des études récentes ont montré que l'activité de la fucosidase est sur-exprimée chez les patients atteints de certains types de cancers. Ces travaux présentent dans un premier temps la synthèse totale et l'évaluation biologique de puissants inhibiteurs de l'alpha-L-fucosidase de type pyrrolidine polyhydroxylée comme outil pour mieux appréhender le rôle de cette enzyme. Une première série est diversement susbtituée par un groupement lipophile apportant de fortes interactions avec l'enzyme alors qu'une deuxième série de molécules dimériques permet d'étudier l'effet de la multivalence sur l'inhibition de l'alpha-L-fucosidase. Dans un deuxième temps, des molécules hybrides de types ferrocényl-iminosucres comme nouvelle classe d'agents anticancéreux sont étudiées. Dans cette approche, l'alpha-L-fucosidase est envisagée comme récepteur potentiel pour cibler les tissus cancéreux. La conjugaison d'une pyrrolidine, affichant une forte affinité pour la fucosidase, à un groupement cytotoxique comme le ferrocène pourrait permettre d'acheminer une molécule cytotoxique sélectivement vers les tissus cancéreux. La synthèse, la puissance d'inhibition, l'activité antiproliférative et l'analyse cristallographique de ces conjugués organométalliques sont présentées dans ces travaux. / Glycoproteins located on cells surface are involved in a number of biological processes including cell adhesion, cell/host recognition, inflammation and viral infections. The biosynthesis of these glycoproteins is assumed in part by alpha-L-fucosidase. Many biological phenomena are related with this enzyme, sometimes associated with severe disease. Recent studies show that fucosylation activities are markedly enhanced in several types of cancer cell lines. On the one hand, this work presents the synthesis and the biological evaluation of polyhydroxylated pyrrolidines as potent alpha-L-fucosidase inhibitors. A series of iminosugars that incorporate a hydrophobic subtituent at the pseudoanomeric position should provide strong interaction in the active site and could serve as a tool to better understand the role of this enzyme. A second series of dimeric molecules allows the study of multivalency effect on inhibition. On the other hand, ferrocenyl-iminosugars are studied as a new class of anticancer agents. In this approach, we explore the possibility of using the fucose binding protein AFU as a target for the selective delivery of a cytotoxic molecule towards cancer cells. A panel of conjugates was prepared, composed of a pyrrolidine moiety, showing a high affinity for the fucosidase, linked to a ferrocenyl moiety, displaying antitumoral properties by reactive oxygen species production. The synthesis of these hybrids, the biological results and the structure/activity relationships are presented.

Les iminosucres, analogues de sucres dans lesquels l'oxygène endocyclique a été remplacé par un atome d'azote, constitue une classe importante de mimes de sucres. Aujourd'hui, leurs applications se limitent au domaine biologique car ces composés ont montré un potentiel thérapeutique prometteur. Il serait intéressant d'élargir le domaine d'application de ces iminosucres, et la combinaison de ces structures présentant un azote endocyclique pourrait conduire à de nouveaux macrocycles inédits présentant des propriétés de chélation innovantes. Pour cela, l'introduction d'une chaîne alkyle en position pseudoanomérique donne accès à une nouvelle classe de composés, les iminosucres C-glycosides dont la fonctionnalisation en positions C-5 et C-1 est nécessaire.La première partie de ce travail se focalise sur le développement d'une voie de synthèse rapide et efficace d'iminosucres C-glycosides à six et sept chaînons au moyen d'une réaction tandem de Staudinger aza-Wittig. Pour accéder à ces composés de choix, notre stratégie se base sur des réactions de fonctionnalisation diastéréosélectives et stéréocontrollées. La seconde partie de cette thèse a été consacrée à la synthèse d'iminosucres aza-couronnes, dont les structures constituent un nouveau type de récepteurs moléculaires. L'étude des propriétés de chélation de ces nouveaux macrocycles des cations métalliques a montré des premiers résultats prometteurs et encourageants grâce à des analyses par RMN et par fluorimétrie. / Iminosugars, sugar analogs in which the endocyclic oxygen has been replaced by nitrogen, constitute a major class of sugar mimetics. Their application has been limited to the biological field so far as these compounds have shown promising therapeutic properties[1]. Interestingly, their structural analogy with sugars combined with the presence of an endocyclic nitrogen atom could deliver innovative macrocycles that could display chelation properties as well as catalytic potential when bound to metals and associated as duplex or higher multiplicity scaffolds. For this purpose, efficient introduction of an alkyl chain at the pseudoanomeric position of the iminosugar to yield an iminosugar C-glycoside[2] displaying two arms at C-5 and C-1 position is necessary. The first part of this work focused on the development of an efficient and convergent synthesis of seven and six membered iminosugars C-glycosides using a highly diastereoselective tandem Staudinger-Aza-Wittig reaction is presented. To access to these new compounds, our strategy is based on a highly diastereoselective and stereocontrolled functionalization. The second part of this work is based on the use of these structures to build up unprecedented iminosugar-aza-crowns, a new type of molecular receptors, using the strategy way developed in the first part. These news sweet aza-crowns displaying with various linkages showed promising results through their preliminary chelation properties by NMR and fluorimetric techniques.

Iminosugars are carbohydrate mimics, where the endocyclic ring oxygen has been replaced by nitrogen. This substitution affords these compounds their inhibitory activity towards sugar-processing enzymes (glycosidases) and, as a consequence, their chemotherapeutic potential in the treatment of a broad range of diseases. Several iminosugars are currently in clinical trials or have entered the market as approved drugs. This has consequently led to increasing levels of research into their synthesis and application, both in terms of the development of efficient methodology to access naturally occurring examples, and also to elaborate novel scaffolds. The presence of multiple chiral centres within iminosugars provides a considerable challenge in accessing these targets by asymmetric means, whereas carbohydrates pose a more attractive chiral pool. As such the majority of literature methods have employed this latter method. The focus of the thesis is on the elaboration of robust methodologies to access both naturally occurring and novel iminosugars, and their precursors, from readily available carbohydrate starting materials. Chapter 1 presents an introduction to iminosugars, including an overview of glycosidase inhibition by this class of sugar-mimic, their historical medical usage and the basis for their potential employment in treating diabetes, lysosomal storage disorders (LSDs) and cancer. This chapter also gives a general review of the methods employed in the literature for the assembly of iminosugar scaffolds. Chapter 2 is concerned with the synthesis of iminosugars from the carbohydrate glucuronolactone. This versatile chiron has previously allowed for access to many homochiral targets, and in this thesis is used to access DGJNAc on a gram-scale. This iminosugar has been shown to be a potent α-N-acetylgalactosaminidase inhibitor and is potentially extremely valuable in the treatment of late-stage cancer. Both enantiomers of glucuronolactone are also utilised in the divergent synthesis of every stereoisomer of two classes of five-membered iminosugars; the pyrrolidines (including DMDP), and the proline amides. These compounds demonstrate remarkable biological activity against a panel of glycosidases and hexosaminidases, allowing for the analysis of the structure-activity relationship between these compounds and the target enzymes. Chapter 3 describes the development of a novel, one-pot methodology - a tandem Strecker reaction and iminocyclisation - for the assembly of trihydroxy piperidine α-iminonitriles from a range of unbranched and branched pentose monosaccharides. These piperidine α-iminonitriles are precursors to pipecolic acids which may also be potentially valuable targets in the treatment of cancer.

Au cours de leur métabolisme, les céramides, produits de novo au niveau du réticulum endoplasmique, sont transportés vers l'appareil de Golgi pour être convertis en sphingomyéline. Le mode principal de ce transport implique la protéine cytosolique CERT (CERamide Transfer). La surexpression de CERT, responsable d'un abaissement du taux intracellulaire en céramide pro-apoptotique, a été associée au phénomène de résistance aux agents chimiothérapeutiques de plusieurs lignées de cellules tumorales. L'inhibition de CERT permet de resensibiliser ces lignées cellulaires aux agents anti-cancéreux. Cependant, une seule famille d'inhibiteurs de CERT est connue à ce jour : les HPAs. A l'extrémité C-terminale de la protéine, le domaine START contient le site de liaison du céramide nécessaire à l'activité de transport de CERT. A partir de structures cristallographiques, une méthode d'identification de nouveaux ligands, combinant des outils in silico et in vitro, a été développée. La jaspine B, des analogues HPAs et des iminosucres ont été mis à jour en tant qu'antagonistes potentiels de CERT par cette méthode. Certains des composés identifiés ont été synthétisés et évalués in vitro. Des sondes fluorescentes de la jaspine B ont été conçues afin d'approfondir la compréhension de son mécanisme d'inhibition. En parallèle, un test de liaison in vitro HTR-FRET a été développé, permettant le criblage haut-débit de la Chimiothèque Nationale Essentielle. / During its metabolism, ceramides, produced de novo in the endoplasmic reticulum, are transported to the Golgi complex to be converted into sphingomyelin. The main way of this transport involves the cytosolic CERT protein (Ceramide Transfer). Overexpression of CERT, responsible for a diminution of intracellular level of proapoptotic ceramide, is associated with the phenomenon of resistance to chemotherapeutic agents in several tumor cell lines. The CERT inhibition allows to resensitize these cell lines to anticancer drugs. Yet, only a single family of inhibitors is known to date: HPAs. Located at the C-terminal region of the protein, the START domain contains the binding site of ceramide necessary for the transport activity of CERT. Based on crystallographic structures, a method for the identification of new CERT ligands, combining in silico and in vitro tools, was developed. Jaspine B, HPAs analogs and iminosugars were identified as potential antagonists using this method. Some of these compounds were synthesized and evaluated in vitro. Fluorescent probes of jaspine B were designed for a better understanding of it mechanism of action. In parallel, an in vitro HTR-FRET binding assay was developed, allowing the high-throughput screening of the National Essential Compound Library.

No description available.

Les premiers iminosucres multivalents rapportés dans la littérature datent de 1999. Depuis, c’est plus d’une centaine de clusters de ce type qui ont été synthétisés et décrits une quarantaine de publications. L’obtention d’un premier effet multivalent fort sur une glycosidase, en 2010, a initié de nouveaux travaux visant à étudier et à comprendre son mécanisme et ses limites. Dans cette optique le présent travail de thèse a exploité plusieurs approches. La première partie décrit la synthèse de dendrons "cliquables" permettant de multiplier par trois ou par neuf la valence initiale des plateformes utilisées. La deuxième est une étude de la synthèse d’espaceurs rigides. La troisième est la préparation de plateformes modulables, des neo-cyclodextrines, pour obtenir un contrôle plus fin de la topologie des clusters. L’association de dendrons "cliquables" et de plateformes cyclopeptoïdes de dimensions contrôlées a abouti à un résultat sans précèdent en termes d’effet multivalent. Ainsi, le cluster 36-valent à ligands DNJ est un inhibiteur 170 000 fois plus fort que l’analogue monovalent correspondant sur l’alpha-mannosidase des pois sabre blanc (Jack Bean). / The first multivalent iminosugars were published in 1999. From this date, it’s more than a hundred of clusters that were synthesized and presented in about forty publications. In 2010, the first strong multivalent effect in glycosidase inhibition was obtained and prompted further studies of its mechanism and its limits. To reach these goals, this PhD work has developed different strategies. The first was to synthesize "clickable" dendrons which can lead to a multiplication of the initial valency of our scaffolds by three or by nine. The second approach was a study to obtain rigid linkers. The third one was the preparation of modular scaffolds, neo-cyclodextrins, in order to finely tune the topology of the resulting clusters. The combination of our "clickable" dendrons with cyclopeptoid scaffolds gave an unprecedented multivalent effect on glycosidase inhibition. The 36-valent DNJ-based cluster is indeed a 170 000-fold more potent inhibitor than the corresponding monovalent control for Jack Bean alpha-mannosidase.

Parmi les analogues de sucres, les iminosucres constituent la classe la plus prometteuse au niveau biologique. En effet, leur structure, dans laquelle l'atome d'oxygène intracyclique est remplacé par un atome d'azote, leur confère des propriétés uniques d'inhibition de glycosidases et/ou glycosyltransférases, et en fait donc de très bons candidats thérapeutiques. L'introduction d'un substituant pseudo-anomérique carboné permet de mimer la partie aglycone du substrat de l'enzyme et d'accéder aux iminosucres C-glycosides, des composés chimiquement stables qui sont des inhibiteurs sélectifs et puissants des glycosidases.Afin d'accélérer la découverte de molécules d'intérêt thérapeutique, il est nécessaire de trouver des voies de synthèses conduisant à une plus grande diversité structurale. La méthodologie que nous avons mise au point est basée sur la débenzylation régiosélective de la position 2 de C-allyl glycosides exploitant une iodocyclisation. Cette dernière a été appliquée avec succès à la C-allyl-1-déoxynojirimycine puis étendue à plusieurs iminosucres de configurations variées en séries pipéridine et pyrrolidine.L'introduction de diversité moléculaire a été réalisée à partir de la C-allyl-1-déoxynojirimycine O-débenzylée régiosélectivement en position 2. Nous avons ainsi obtenu plusieurs iminosucres de configurations D-gluco- et D-manno- portant différentes fonctionnalités en position 2. La mise au point de cette synthèse a donc permis d'accéder à une grande variété de C-allyl iminosucres à partir d'un synthon unique. L'accès à des iminosucres bicycliques de structures inédites a également été possible en exploitant un C-allyl 2-céto iminosucre obtenu par notre méthodologie de débenzylation régiosélective. / Iminosugars constitute undoubtedly the most promising class of sugar analogues, their unique glycosidase and/or glycosyltransferase inhibition profile making them promising therapeutics. To generate more potent and selective inhibitors called C-glycoside iminosugars, introduction of a stable pseudoanomeric substituent is usually performed, the improved efficacy being attributed in part to the information brought by the aglycon moiety.The main challenge associated with this class of iminosugars C-glycosides is currently the design of efficient and general routes enabling introduction of structural diversity at a late stage from advanced synthons to accelerate the discovery of biologically relevant molecules. In this context, we have explored a strategy based on a regioselective debenzylation at C-2 and a stereocontrolled nucleophilic substitution assisted by the N-benzyl group. We have successfully applied this methodology on the C-allyl-1-deoxynojirimycin and extended it to several iminosugars in the piperidine and pyrrolidine series.The introduction of molecular diversity was performed from the C-allyl-1-deoxynojirimycin selectively O-debenzylated at position 2. We obtained several iminosugars in the D-gluco- and D-manno- series bearing various functionalities at position 2. This strategy allowed us to access a wide range of C-allyl iminosugars from one single synthon. We have also described the access to unknown bicyclic iminosugars starting from a C-allyl 2-keto iminosugar obtained by our regioselective debenzylative methodology.