Glycosylation may be a general strategy for the transport of biologically active neuro(glyco)peptides into the brain. With that in mind, a series of modified DAMGO analogues were synthesized and subjected to conformational analysis, and in vitro and in vivo studies related to opioid analgesia. Those studies will help to determine the balance of carbohydrate and peptide, to reach maximum BBB transport; in other words this is a study to test the biousian hypothesis. 1) The μ-agonist DAMGO was altered by incorporating moieties of increasing water solubility into the C-terminus, including carboxamide and simple glycosides. The hydrophilic C-terminal moieties were varied from glycinol in DAMGO to L-serine amide (LYM100), L-serine amide β-D-xyloside (LYM50), L-serine amide β-Dglucoside (LYM110), L-serine amide β-lactoside (LYM147). Two phosphopeptides LYM1311 and LYM1312 were synthesized with the phosphate group attached to Lserine amide at the C-terminus. Conformational analysis experiments included: 1HNMR, diffusion, variable temperature experiments to find the temperature coefficient, circular dichroism, 2DNMR noesy and tocsy, and molecular modeling. The peptides associate with SDS micelles with a strong electrostatic component. The SDS micelles stabilized the β-turn that is nascent in water. CSI (chemical shift indexes), temperature coefficients and circular dichroism do not give much insight into the structural conformation. 2D NMR analysis followed by molecular modeling confirmed a β-turn preferred conformation. No specific type of β-turn could be assigned to the DAMGO analogs. 2) Antinociceptive mouse tail-flick studies were performed, and opioid binding was determined. Analgesic potency (i.v.) increased, passing through a maximum (A₅₀ ≈ 0.2 μmol/Kg) for LYM100 & LYM50 as membrane affinity vs. water solubility became optimal, and then dropped off (A₅₀ ≈ 1.0 μmol/Kg) for LYM110 & LYM147 as water-solubility dominated the molecular behavior. Correlation of i.v. A₅₀ values with estimated hydrodynamic values (glucose units) for the glycoside moieties, or the hydrophilic/hydrophobic Connolly surface areas (A₅₀ vs e^(-Awater/Alipid)), provided U-shaped or V-shaped curves, as predicted by the “biousian hypothesis.” The μ-selective opioid agonism was maintained upon modifications at the C-terminus. The optimal “degree of glycosylation” that achieves the maximum degree of transport for the DAMGO peptide message seems to be between the peptide with the carboxamide C-terminal group and the xyloside.

No description available.

<p>The glycopeptide antibiotic vancomycin is used as front line treatment for serious Gram-positive infections and resistance to this drug is widespread. Three genes are essential for resistance, <em>vanHAX</em>, which are controlled by a two-component regulatory system VanR and VanS. Here, glycopeptide resistance is found to be ancient and diverse in the environment. A <em>vanA</em> open reading frame from 30 000 yr old DNA was identified and the enzyme was shown to be as functional as comparable to modern day VanA homologs. In the environment resistance is found to be diverse and widespread. For example, the organism <em>Desulfitobacterium hafniense</em> Y51 VanH was shown as non-essential in conferring inducible resistance. Furthermore in the glycopeptide producer <em>Amycolatopsis balhimycina</em> harboring the classic <em>vanHAX,</em> a functional VanA homolog is described as an orphan gene outside of any recognizable gene cassette .</p> <p>Glycopeptides are natural products made by members of the Actinomycete family and are modified by different types of tailoring enzymes. Of particular interest is the glycopeptide A47934, which is ‘aglyco’, and sulfated. The sulfotransferase StaL will transfer not only a sulfate group to A47934, but a sulfamide and fluorosulfonate group. Focusing on additional tailoring enzymes, the biosynthetic cluster of the sulfated glycopeptide UK68597 was sequenced. This cluster has provided a resource for glycopeptide tailoring enzymes for use to modify the A47934 backbone. Sulfation was the first focus and the substrate promiscuity of StaL was explored to expand the chemical diversity A47934 and vancomycin. This work has led to the discovery that glycopeptide sulfation will antagonize the activation and expression of <em>vanHAX</em>. A new sulfated vancomycin derivative was created with this antagonizing activity in the clinical pathogen <em>Enterococcus faecium</em> of the VanB phenotype. Implications of these results and the further use of tailoring enzymes to modify glycopeptides to antagonize resistance will be discussed.</p> / Doctor of Philosophy (PhD)

Ce travail de thèse a pour objectif la simplification de la préparation d’un nouveau type d’inhibiteur d’entrée du VIH conçu, synthétisé et validé dans le cadre d’une collaboration entre l’équipe de Glycochimie Moléculaire et Macromoléculaire dont je dépends, l’Institut de Biologie Structurale de Grenoble et l’Institut Pasteur de Paris. Ce prototype est constitué d’un mime fonctionnel de CD4 lié de façon covalente à un fragment dodécasaccharidique d’Héparane Sulfate dont la synthèse est complexe. Nous avons donc proposé de préparer des oligomaltosides sulfatés afin de déterminer s’ils pouvaient se comporter comme des mimes d’Héparane Sulfate.Dans un premier temps, nous avons mis au point la synthèse, en huit étapes et 38 % de rendement global, d’un précurseur trisaccharidique oligomérisable à partir de maltotriose, un trisaccharide biosourcé commercial. Au cours de ce travail, nous avons résolu trois points particulièrement délicats : l’allylation de l’extrémité réductrice du maltotriose, l’installation d’un groupement paraméthoxybenzylidène en position O-4III et O-6III et la protection sélective des positions O-6I et O-6II par un groupement silylé. Les optimisations menées nous ont permis de limiter la formation de produits secondaires, d’augmenter le rendement de chaque étape et de pouvoir mener sans problème cette synthèse sur une échelle d’une dizaine de grammes. Dans un deuxième temps, le précurseur trisaccharidique a été transformé en différents accepteurs et donneurs de glycosyle dont les comportements dans différentes conditions de glycosylation ont été étudiés. Nous avons ainsi pu démontrer qu’une activation des donneurs sous forme de trichloroacetimidate conduisait à des rendements faibles de part la formation d’une quantité importante des produits de réarrangement en trichloroacétamides anomériques. Une activation sous forme de N-Phényltrifluroacétimidate a permis de résoudre ce problème, sans toutefois que les rendements en soient toujours augmentés. En effet, nous avons pu montrer que la nature du groupement protecteur en O-6I du donneur a une influence déterminante sur l’issue de la réaction de glycosylation, tant au niveau de sa stéréosélectivité que de son rendement. Un groupement encombré ou un ester en O-6I du donneur est ainsi indispensable pour avoir une bonne stéréosélectivité alpha. Le meilleur rendement obtenu est, pour le moment, de 56 %. Des optimisations en cours permettront d’augmenter le rendement et de préparer les oligomaltosides sulfatés visés dans un avenir proche afin de tester leur activité biologique. / This work aims at simplifying the preparation of a new type of HIV entry inhibitor, conceived, synthesized and validated within a collaboration between our group, the "Institut de Biologie Structurale" (Grenoble) and the Institut Pasteur (Paris). This prototype is composed of a CD4 functional mimetic linked to a dodecasaccharide fragment of Heparan Sulfate, whose synthesis is complex. In order to determine if Heparan Sulfate may be replaced by simpler sulfated oligosaccharides, we decided to prepare a set of sulfated oligomaltosides.To this goal, we first optimized the synthesis of an oligomerizable maltotrioside building block in eight steps and 38% global yield from maltotriose, a commercial and biosourced trisaccharide. In this work, we had to address three major points: the allylation of the reducing end of maltotriose, the introduction of a paramethoxybenzylidene group between positions O-4III and O-6III and the selective protection of the remaining primary positions O-6I and O-6II by a silylated protecting group. Each step has been optimized to minimize the amount of secondary products and thus to enhance its yield. The resulting synthesis was thus shown to be highly reproducible up to ten grams scale.Then, glycoside acceptors and donors were prepared from the oligomerizable maltotrioside building block and we studied their behaviors in glycosylation reactions. We found that trichloroacetimidate activation led to poor glycosylation yields, due to the competitive formation of trichloroacetamidyl glycoside rearrangement product. Gratifyingly, N-phenyltrifluroacetimidate activation solved the rearrangement problem, but yields sometimes remained low. Indeed, we were able to demonstrate that the nature of the protecting group in position O-6I of the donor strongly influenced both the stereoselectivities and yields of the glycosylations: a bulky or ester group is needed in this position to obtain a full alpha stereoselecticity. To date, the highest yield obtained is 56 %.Ongoing optimizations will allow us to enhance the yields and to prepare the targeted sulfated oligomaltosides in a near future in order to test their biological activity.

Ce travail de thèse a pour objectif la simplification de la préparation d’un nouveau type d’inhibiteur d’entrée du VIH conçu, synthétisé et validé dans le cadre d’une collaboration entre le laboratoire, l’Institut de Biologie Structurale de Grenoble et l’Institut Pasteur de Paris. Ce prototype est constitué d’un mime fonctionnel de CD4 lié de façon covalente à un fragment dodécasaccharidique d’Héparane Sulfate dont la synthèse est complexe. Nous avons donc proposé de préparer des oligomaltosides sulfatés afin de déterminer si ils pouvaient se comporter comme des mimes d’Héparane Sulfate. Dans un premier temps, nous avons mis au point la synthèse d’un précurseur trisaccharidique oligomérisable à partir de maltotriose, un trisaccharide biosourcé commercial. Au cours de ce travail, nous avons résolu trois points particulièrement délicats : l’allylation de l’extrémité réductrice du maltotriose, l’installation d’un groupement paraméthoxybenzylidène en position O-4ᴵᴵᴵ et O-6ᴵᴵᴵ et la protection sélective des positions O-6ᴵ et O-6ᴵᴵ par un groupement silylé. Les optimisations menées nous ont permis de limiter la formation de produits secondaires, d’augmenter le rendement de chaque étape. La synthèse définitive du trisaccharide oligomérisable comprend 8 étapes, a un bon rendement global de 44 % et peut être menée sans problème à des échelles allant jusqu’à 10 g. Dans un deuxième temps, nous avons étudié les réactions de glycosylation α(1→4) visant à oligomériser la brique maltotrioside : nous avons constaté qu'une activation des donneurs sous forme de N-phényltrifluoroacétimidate (PTFA) est un meilleur choix que sous forme de trichloroacétimidate (TCA). En effet, ces derniers subissent une réaction parasite du réarrangement de l’imidate en trichloroacétamide. Nous avons aussi observé que le groupement benzoyle (Bz) en position O-6ᴵᴵᴵ du donneur est préférable à l’acétyle (Ac) qui est moins stable dans les conditions acides et basiques. Ensuite, nous avons synthétisé différents donneurs PTFA possédant différents groupements protecteurs en position O-6ᴵ (TBDPS, Bn, pNO2Bz, Piv, Ac et Bz). Nos études de glycosylation avec ces différents donneurs nous ont permis de conclure qu'un groupement TBDPS en position O-6ᴵ permet d'obtenir de hauts rendements et une totale stéréosélectivité 1,2-cis dans les réactions de glycosylation et, de plus, est parfaitement indiqué comme groupement protecteur dans les étapes de diversifications ultérieures. Pour finir, nous avons optimisé différents paramètres comme : le promoteur, la température, l’ordre d’addition des réactifs, le type de tamis moléculaire, l’effet de solvant, le rapport donneur/accepteur, la concentration de l’accepteur et l’échelle de la réaction. Les conditions optimisées nous ont permis d’accéder efficacement, avec de bons rendements et une stéréosélectivité α totale, aux hexa, nona et dodecasaccharide protégés ciblés. Dans un troisième temps, nous avons réussi à réaliser les étapes de déprotection et fonctionnalisation des oligomaltosides synthétisés : débenzoylation et désilylation chimiosélective (hexa et nonasaccharide), sulfatations (hexa et nonasaccharides), puis déprotection des groupements protecteurs résiduels (hexasaccharides). La finalisation de ce projet impliquera de sulfater les nonasaccharides et de déprotéger les groupements protecteurs résiduels, puis d'introduire un espaceur fonctionnalisé sur tous les membres de la chimiothèque ainsi obtenue (hexa et nonasaccharides) et enfin de réaliser l'hydrogénolyse finale des groupements benzyles pour conduire aux oligomaltosides sulfatés libres dont les activités biologiques seront déterminées en collaboration avec nos partenaires. / This work aims at simplifying the preparation of a new type of HIV entry inhibitor, conceived, synthesized and validated within a collaboration between our group, the "Institut de Biologie Structurale" (Grenoble) and the Institut Pasteur (Paris). This prototype is composed of a CD4 functional mimetic linked to a dodecasaccharide fragment of Heparan Sulfate, whose synthesis is complex. In order to determine if Heparan Sulfate may be replaced by simpler sulfated oligosaccharides, we decided to prepare a set of sulfated oligomaltosides. To this goal, we first optimized the synthesis of an oligomerizable maltotrioside building block in eight steps and 44% global yield from maltotriose, a commercial and biosourced trisaccharide. In this work, we had to address three major points: the allylation of the reducing end of maltotiose, the introduction of a paramethoxybenzylidene group between positions O-4ᴵᴵᴵ and O-6ᴵᴵᴵ and the selective protection of the remaining primary positions O-6ᴵ and O-6ᴵᴵ by a silylated protecting group. Each step has been optimized to minimize the amount of secondary products and thus to enhance its yield. The resulting synthesis was thus shown to be highly reproducible up to ten grams scale. Then, glycoside acceptors and donors were prepared from the oligomerizable maltotrioside building block and we studied their behaviors in glycosylation reactions. We found that trichloroacetimidate activation led to poor glycosylation yields, due to the competitive formation of trichloroacetamidyl glycoside rearrangement product. Gratifyingly, N-phenyltrifluroacetimidate activation solved the rearrangement problem. We demonstrate that benzoyl group (Bz) at position O-6ᴵᴵᴵ in the donor should be preferred to acetyl (Ac) which is less stable both in acidic or basic conditions. Then, we synthesized various PTFA donors bearing different protecting groups at O-6ᴵ position (TBDPS, Bn, pNO2Bz, Piv, Ac et Bz) in order to study their influence on the yields and stereochemical outcome of the glycosylation reactions. We concluded that TBDPS represent the best compromise for efficient glycosylation and later protecting group manipulation strategies. Then we optimized various reaction parameters: promotor nature, temperature, reagents addition order, molecular sieve type, solvent nature, donor/acceptor ratio, concentrations and scale of the reaction. The optimized conditions allowed efficient access, in high yields and full α stereoselectivity, to the targeted protected hexa, nona and dodecasaccharides. Then we validated the deprotection and functionnalization steps: chimioselective debenzoylation and desilylation (hexa and nonasaccharide), sulfations (hexa and nonasaccharides), the final deprotection of the residual protecting groups (hexasaccharides).

One of the largest problems in current medicine is the shortage of organs for transplant due to technological limitations in the storage of organs for any length of time. A possible solution to this problem would involve cryopreservation. However, current cryopreservatives such as sucrose or DMSO have concerning cytotoxic issues that limit their possible applications. A major cause of cryoinjury is the uncontrolled recrystallization of inter and intra-cellular ice crystals that occurs during the thawing process leading to mechanical damage and dehydration. The Ben lab has thus been interested in the design of compounds that are capable of inhibiting this process but do not possess other undesirable properties found in the native compounds. These synthetic analogues have been shown to increase cellular viability post-thaw. A series of mixed α/β glycopeptides are prepared and analyzed for antifreeze properties. The results of this study imply that it is not the gross conformation of the glycopeptide that is responsible for activity, but rather that intramolecular relationships may be responsible for disrupting the reorganization of ice. A technique was devised for the incorporation of triazoles into the analogues to investigate the importance of the linker and to greatly simplify the synthesis of a library of glycoconjugates. It was found that the IRI activity of glycopeptides is very sensitive to the distance between carbohydrate and peptide backbone. The electron density at the anomeric oxygen is an important parameter with respect to intramolecular networks. A series of substituted galactosides is presented that modify the electronics of the anomeric oxygen. The results demonstrate that decreasing electron density at this position appears to improve IRI activity in a predictable manner. To better understand the remarkable IRI activity of a key analogue, it was systematically truncated. This study led to the serendipitous discovery of a series of very highly IRI active analogues that do not contain a peptide backbone. These compounds represent the first non-glycopeptides that can show very significant IRI activity even at very low concentrations. The final portion of the thesis reports the efforts towards the preparation of a carbasugar analogue of AFGP-8.

One of the largest problems in current medicine is the shortage of organs for transplant due to technological limitations in the storage of organs for any length of time. A possible solution to this problem would involve cryopreservation. However, current cryopreservatives such as sucrose or DMSO have concerning cytotoxic issues that limit their possible applications. A major cause of cryoinjury is the uncontrolled recrystallization of inter and intra-cellular ice crystals that occurs during the thawing process leading to mechanical damage and dehydration. The Ben lab has thus been interested in the design of compounds that are capable of inhibiting this process but do not possess other undesirable properties found in the native compounds. These synthetic analogues have been shown to increase cellular viability post-thaw. A series of mixed α/β glycopeptides are prepared and analyzed for antifreeze properties. The results of this study imply that it is not the gross conformation of the glycopeptide that is responsible for activity, but rather that intramolecular relationships may be responsible for disrupting the reorganization of ice. A technique was devised for the incorporation of triazoles into the analogues to investigate the importance of the linker and to greatly simplify the synthesis of a library of glycoconjugates. It was found that the IRI activity of glycopeptides is very sensitive to the distance between carbohydrate and peptide backbone. The electron density at the anomeric oxygen is an important parameter with respect to intramolecular networks. A series of substituted galactosides is presented that modify the electronics of the anomeric oxygen. The results demonstrate that decreasing electron density at this position appears to improve IRI activity in a predictable manner. To better understand the remarkable IRI activity of a key analogue, it was systematically truncated. This study led to the serendipitous discovery of a series of very highly IRI active analogues that do not contain a peptide backbone. These compounds represent the first non-glycopeptides that can show very significant IRI activity even at very low concentrations. The final portion of the thesis reports the efforts towards the preparation of a carbasugar analogue of AFGP-8.

Drug resistant bacteria pathogen poses a severe threat to human health. Bacterial drug efflux pumps are transporter proteins involved in the export of antibiotics out of cells. Efflux by transporters is one of the major drug resistant mechanisms. Multidrug efflux pumps can transport multiple classes of antibiotics and are associated with bacteria multiple drug resistance (MDR). Overproduction of these pumps reduces susceptibility of bacteria to a variety of antibiotics. MDR regulators are cytoplasmic proteins that control the expression level of MDR transporters in response to the cellular concentration of antibiotics. This thesis research focuses on three main directions in the area of bacteria drug resistance: the structural and functional study of a MDR transporter, the characterization of a novel MDR regulator protein, and the development of a sensing method for the detection of glycopeptide antibiotics. Acriflavine resistance protein B (AcrB) in Escherichia coli belongs to resistance nodulation division (RND) superfamily of efflux transporters. It plays an important role in confering multidrug resistance in Gram-negative bacteria. The functional unit of AcrB is a trimer in vivo. However, the relationship between AcrB trimer stability and functionality remains elusive. In chapter 2, a residue that is critical for AcrB trimerization, Pro 223, was identified. The replacement of Pro 223 by other residues destabilized AcrB trimer, and thus decreased its activity. The loss of transport activity could be partially recovered when the AcrB trimer was stabilized by the introduction of a pair of inter-subunit disulfide bond. In chapter 3, a systematically alanine-scanning study of the producing loop (amino acid residues 211-240) was conducted. Five residues in the loop were found to be important for AcrB activity. These residues form a collar or belt in the loop close to the tip. These mutation studies revealed new insight into the conformation of the loop during AcrB trimerization. In chapter 4, residue Arg 780 was identified to be crucial for the pump function of AcrB. The study results indicated that Pro 223 serves as a “wedge” and Arg 780 as a “lock” via hydrogen bonding between the backbone carbonyl oxygen of Pro 223 and side chain of Arg780. Similar as Pro 223, replacement of Arg 780 by other residues drastically decreased the activity of AcrB. Dissociation of the AcrB trimer also contributed to the decrease of activity. However, the introduction of inter-subunit disulfide bond could not restore the function of the mutant, indicating that Arg 780 plays multiples roles in the operation of AcrB. In chapter 5, a MDR regulator ST1710 from the archaeon Sulfolobus tokodaii, homologous to the multiple-antibiotic resistance repressor (MarR) family bacterial regulators, was characterized in vitro. The binding affinities of ligands and double strand (ds) DNA for ST1710 were measured. The presence of substrates suppressed the interaction between ST1710 and dsDNA, which indicated that ST1710 functioned as a repressor in vivo. Finally, in chapter 6, a direct fluorescence polarization based method for the detection of glycopeptide antibiotics is developed. Briefly, the acetylated tripeptide L-Lys-D-Ala-D-Ala was labeled with a fluorophore (fluorescein isothiocyanate or AlexaFluor 680) to create a peptide probe. The fluorescence polarization signal of the peptide probe increased upon binding with glycopeptide antibiotics in a concentration dependent manner. The detection is highly selective toward glycopeptide antibiotics. The designed method is expected it to have broad applications in both research and clinical settings.

Synthetic glycopeptides have attracted much interest in the biomedical field due to their structural similarities to the natural glycopeptides or glycoproteins. It is still difficult to synthesize glycopeptides with greater efficiency and ring opening polymerization remains an effective way to do so. Proteoglycans are a special class of glycoproteins with glycosaminoglycan chains. In this study, I tried to do controlled ring opening polymerization of Hyaluronic acid derivatives with smaller to higher molecular weight while avoiding side reactions. It is challenging to work with higher molecular weight molecules and do a click reaction in water effectively. Making nanopolymers with a desired size, studies of the characteristics, and how to build nanocarriers for drug delivery application was the focus of this work. Polymeric characteristics, e.g., modification and polymer formation were studied by nuclear magnetic resonance technique; Particle size was studied by dynamic light scattering and the loading of rhodamine B encapsulated into the polymer was measured by confocal imaging technique.

One of the largest problems in current medicine is the shortage of organs for transplant due to technological limitations in the storage of organs for any length of time. A possible solution to this problem would involve cryopreservation. However, current cryopreservatives such as sucrose or DMSO have concerning cytotoxic issues that limit their possible applications. A major cause of cryoinjury is the uncontrolled recrystallization of inter and intra-cellular ice crystals that occurs during the thawing process leading to mechanical damage and dehydration. The Ben lab has thus been interested in the design of compounds that are capable of inhibiting this process but do not possess other undesirable properties found in the native compounds. These synthetic analogues have been shown to increase cellular viability post-thaw. A series of mixed α/β glycopeptides are prepared and analyzed for antifreeze properties. The results of this study imply that it is not the gross conformation of the glycopeptide that is responsible for activity, but rather that intramolecular relationships may be responsible for disrupting the reorganization of ice. A technique was devised for the incorporation of triazoles into the analogues to investigate the importance of the linker and to greatly simplify the synthesis of a library of glycoconjugates. It was found that the IRI activity of glycopeptides is very sensitive to the distance between carbohydrate and peptide backbone. The electron density at the anomeric oxygen is an important parameter with respect to intramolecular networks. A series of substituted galactosides is presented that modify the electronics of the anomeric oxygen. The results demonstrate that decreasing electron density at this position appears to improve IRI activity in a predictable manner. To better understand the remarkable IRI activity of a key analogue, it was systematically truncated. This study led to the serendipitous discovery of a series of very highly IRI active analogues that do not contain a peptide backbone. These compounds represent the first non-glycopeptides that can show very significant IRI activity even at very low concentrations. The final portion of the thesis reports the efforts towards the preparation of a carbasugar analogue of AFGP-8.