[pt] Nanopartículas de ouro dispersas em água (AuNPs-H) e nanopartículas de ouro na presença dos surfactantes brometo de cetiltrimetilamônio (AuNPs-CTAB) e brometo de didecildimetilamônio (AuNPs-C10DAB) foram sintetizadas e caracterizadas por diversas técnicas. As variações nos perfis espectrais no UV-Vis dessas nanopartículas foram estudadas levando-se em consideração a intensidade e o comprimento de onda máximo da banda de ressonância plasmônica de superfície localizada (LSPR); foram acompanhadas em função do tempo para nanopartículas estocadas sob refrigeração (4 graus celsius) com as mantidas na temperatura ambiente (27 graus celsius). Um comportamento anômalo (mais instável) foi observado para AuNPs-C10DAB na concentração 1,0 ×10-4 mol L-1. As suas atividades catalíticas também foram avaliadas na presença de 4-nitrofenol e verificou-se que a cinética mais efetiva foi do sistema sem surfactante. Finalmente, um estudo de interação com aminoglicosídeos (AMG) foi feito visando o uso das nanopartículas como sonda analítica. Todavia, somente AuNPs-H apresentaram resultados satisfatórios, o que indica que o surfactante impede que o AMG se aproxime da superfície das nanopartículas, visto que, em concentrações mais elevadas de CTAB ou C10DAB, não há variação significativa no sinal original da sonda quando o AMG está presente. Um método analítico foi desenvolvido baseado na interação AuNPs-H-gentamicina e AuNPs-H-neomicina a partir da otimização dos parâmetros: concentração de AuNPs-H, tampão, tempo de medição e faixa de concentração de AMG. Na condição ajustada para sondagem quantitativa, cada dispersão de trabalho foi constituída por 40 porcento v/v (1,2 × 10-9 mol L-1) de AuNPs-H 1,0 mL de tampão citrato pH 4,0 (1,0 × 10-2 mol L-1) e volume final de 5,0 mL ajustado pela adição água ultrapura, após micro-volumes apropriados de soluçãoestoque de AMG (exceto na dispersão branco) ou de amostras adicionadas. O monitoramento da variação dos perfis espectrais foi feito em 511 e em 681 nm, habilitando a construção de curvas analíticas em 681, e na razão (681/511). Para gentamicina, a faixa linear variou de 0,6 a 600 microgramas L-1 com o limite de detecção (LD) de 0,06 microgramas L-1. Para neomicina, faixa linear foi de 7,3 a 550 microgramas L-1, tendo LD de 6,2 microgramas L-1. A aplicação do método foi feita determinando-se gentamicina (fortificação) em leite integral, e neomicina em solução controle (amostra aquosa simulada), medicamento e em saliva. Para habilitar a seletividade do método foi necessária a utilização de extração em fase sólida (SPE) em cartuchos comercias empacotados com polímero de impressão molecular de AMG. Em amostras de leite (40 microlitros), após limpeza e SPE, foi possível quantificar 1,72 mais ou menos 0,03 microgramas de gentamicina, o equivale a 39,1 mais ou menos 0,2 porcento. As recuperações para neomicina em amostras de medicamento foram de aproximadamente 45 mais ou menos 3,0 porcento (após SPE), indicando interferências de outros componentes. Neomicina também foi recuperada em amostras de saliva após uso do medicamento obtendo valores próximos a 0,36 mais ou menos 0,02 microgramas. Em contraste, as recuperações em amostras simuladas chegaram até 102,6 mais-menos 1,3 porcento (sem necessidade de SPE). Por fim foi feita uma avaliação comparativa de interação de AuNPs-H e nanopartículas de ouro sintetizadas com redução com citrato (AuNPs-citrato) que indicou diferença de comportamento na interação com AMG. / [en] Water-dispersed gold nanoparticles (AuNPs-H) and gold nanoparticles in the presence of surfactants cetyltrimethylammonium bromide (AuNPs-CTAB) and didecyldimethylammonium bromide (AuNPs-C10DAB) were synthesized and characterized using several techniques. The differences in the UV-Vis spectral profiles of these nanoparticles were studied monitoring the intensity and the localized surface plasmon resonance band (LSPR) maximum wavelength; these were monitored as a function of time taking into account storage conditions: under refrigeration (4 Celsius degrees) and at room-temperature (27 Celsius degrees). Anomalous (more unstable) profile was observed for AuNPs-C10DAB at 1.0 × 10-4 mol L-1. Their catalytic activities were also evaluated in the presence of 4-nitrophenol and it was shown that the most effective kinetics was observed for the system without surfactant. Finally, an interaction study with aminoglycosides (AMG) was conducted aiming the application of nanoparticles as analytical probe. Only AuNPs-H produced adequate results, indicating that the surfactant prevents interaction between AMG and the surface of the nanoparticles since at higher concentrations of CTAB or C10DAB there is no significant variation in the original probe signal in presence of AMG. An analytical method was developed based on the AuNPs-H-gentamicin and AuNPs-H-neomycin interaction taking into consideration the optimization of the parameters: AuNPs-H concentration, buffer, signal stabilization time and the AMG concentration range. In the adjusted condition for quantitative probing, each dispersion consisted of 40 percent v/v (1.2 × 10-9 mol L-1) of AuNPs-H, 1.0 mL of citrate buffer pH 4.0 (1.0 × 10-2 mol L-1) and final volume of 5.0 mL adjusted with ultrapure water addition, after appropriate micro-volumes of a stock solution of AMG (except in blank dispersion) or sample added. The spectral profiles were monitored at 511 and 681 nm, allowing theconstruction of analytical curves at 681, and at the ratio (681/511). For gentamicin, the linear range reached from 0.6 to 600 microgram L-1 with the limit of detection (LD) of 0.06 microgram L-1 . For neomycin, the linear range was 7.3 to 550 microgram L-1, with LD of 6.2 microgram L-1. The application of the method was made by determining gentamicin(fortification) in whole milk, and neomycin in control solution (simulated aqueous sample), pharmaceutical samples and saliva. In order to enable selectivity of the method it was necessary to use solid phase extraction (SPE) using a commercial SPE cartridge packed with an AMG molecular imprinted polymer. In milk samples (40 microlitre), after clean up and SPE, it was possible to quantify 1,72 plus-minus 0,03 microgram gentamicin, which is equivalent to 39,1 plus-minus 0,2 percent). Recoveries for neomycin in drug samples were approximately 45 plus-minus 3.0 percent (after SPE), indicating interferences of other components. Neomycin was also recovered in saliva samples after drug use obtaining values close to 0.36 plus-minus 0.02 microgram. In contrast, the recoveries in simulated samples reached up to 102.6 plus-minus 1.3 percent (no need for SPE). Finally, a comparative evaluation of the interaction of AuNPs-H and gold nanoparticles synthesized with reduction with citrate (AuNPs-citrato) was performed, indicating differences in terms of the interaction with AMG.

Les antibiotiques aminoglycosidiques sont des agents bactéricides de grande valeur et d’efficacité à large spectre contre les pathogènes Gram-positifs et Gram-négatifs, dont plusieurs membres naturels et semisynthétiques sont importants dans l’histoire clinique depuis 1950. Des travaux crystallographiques sur le ribosome, récompensés par le prix Nobel, ont démontré comment leurs diverses structures polyaminées sont adaptées pour cibler une hélice d’ARN dans le centre de codage de la sous-unité 30S du ribosome bactérien. Leur interférence avec l’affinité et la cinétique des étapes de sélection et vérification des tARN induit la synthèse de protéines à basse fidélité, et l’inhibition de la translocation, établissant un cercle vicieux d’accumulation d’antibiotique et de stress sur la membrane. En réponse à ces pressions, les pathogènes bactériens ont évolué et disséminé une panoplie de mécanismes de résistance enzymatiques et d’expulsion : tels que les N acétyltransférases, les O phosphotransférases et les O nucleotidyltransférases qui ciblent les groupements hydroxyle et amino sur le coeur des aminoglycosides; des méthyl-transférases, qui ciblent le site de liaison ribosomale; et des pompes d’expulsion actives pour l’élimination sélective des aminoglycosides, qui sont utilisés par les souches Gram-négatives. Les pathogènes les plus problématiques, qui présentent aujourd’hui une forte résilience envers la majorité des classes d’antibiotiques sur le bord de la pan-résistance ont été nommés des bactéries ESKAPE, une mnémonique pour Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa et Enterobacteriaceae. La distribution globale des souches avec des mécanismes de résistance envers les standards cliniques aminoglycosides, tels que la tobramycine, l’amikacine et la gentamicine, est comprise entre 20 et 60% des isolées cliniques. Ainsi, les aminoglycosides du type 4,6-disubstitués-2-deoxystreptamine sont inadéquats comme thérapies anti-infectieuses à large spectre. Cependant, la famille des aminoglycosides 4,5-disubstitués, incluant la butirosine, la neomycine et la paromomycine, dont la structure plus complexe, pourrait constituter une alternative. Des collègues dans le groupe Hanessian et collaborateurs d’Achaogen Inc. ont démontré que certains analogues de la paraomomycine et neomycine, modifiés par désoxygénation sur les positions 3’ et 4’, et par substitution avec la chaîne N1-α-hydroxy-γ-aminobutyramide (HABA) provenant de la butirosine, pourrait produire des antibiotiques très prometteurs. Le Chapitre 4 de cette dissertation présente la conception et le développement d’une stratégie semi-synthétique pour produire des nouveaux aminoglycosides améliorés du type 4,5 disubstitués, inspiré par des modifications biosynthétiques de la sisomicine, qui frustrent les mécanismes de résistance bactérienne distribuées globalement. Cette voie de synthèse dépend d’une réaction d’hydrogénolyse de type Tsuji catalysée par palladium, d’abord développée sur des modèles monosaccharides puis subséquemment appliquée pour générer un ensemble d’aminoglycosides hybrides entre la neomycine et la sisomicine. Les études structure-activité des divers analogues de cette nouvelle classe ont été évaluées sur une gamme de 26 souches bactériennes exprimant des mécanismes de résistance enzymatique et d’expulsion qui englobe l’ensemble des pathogènes ESKAPE. Deux des antibiotiques hybrides ont une couverture antibacterienne excellente, et cette étude a mis en évidence des candidats prometteurs pour le développement préclinique. La thérapie avec les antibiotiques aminoglycosidiques est toujours associée à une probabilité de complications néphrotoxiques. Le potentiel de toxicité de chaque aminoglycoside peut être largement corrélé avec le nombre de groupements amino et de désoxygénations. Une hypothèse de longue date dans le domaine indique que les interactions principales sont effectuées par des sels des groupements ammonium, donc l’ajustement des paramètres de pKa pourrait provoquer une dissociation plus rapide avec leurs cibles, une clairance plus efficace et globalement des analogues moins néphrotoxiques. Le Chapitre 5 de cette dissertation présente la conception et la synthèse asymétrique de chaînes N1 HABA β substitutées par mono- et bis-fluoration. Des chaînes qui possèdent des γ-N pKa dans l’intervalle entre 10 et 7.5 ont été appliquées sur une neomycine tétra-désoxygénée pour produire des antibiotiques avancés. Malgré la réduction considérable du γ N pKa, le large spectre bactéricide n’a pas été significativement affecté pour les analogues fluorés isosteriques. De plus, des études structure-toxicité évaluées avec une analyse d’apoptose propriétaire d’Achaogen ont démontré que la nouvelle chaîne β,β difluoro-N1-HABA est moins nocive sur un modèle de cellules de rein humain HK2 et elle est prometteuse pour le développement d’antibiotiques du type neomycine avec des propriétés thérapeutiques améliorées. Le chapitre final de cette dissertation présente la proposition et validation d’une synthèse biomimétique par assemblage spontané du aminoglycoside 66-40C, un dimère C2 symétrique bis-imine macrocyclique à 16 membres. La structure proposée du macrocycle a été affinée par spectroscopie nucléaire à un système trans,trans-bis-azadiène anti-parallèle. Des calculs indiquent que l’effet anomérique de la liaison α glycosidique entre les anneaux A et B fournit la pré-organisation pour le monomère 6’ aldéhydo sisomicine et favorise le produit macrocyclique observé. L’assemblage spontané dans l’eau a été étudié par la dimérisation de trois divers analogues et par des expériences d’entre croisement qui ont démontré la généralité et la stabilité du motif macrocyclique de l'aminoglycoside 66-40C. / Aminoglycosides are valuable and effective broad-spectrum bactericidal antibiotics against Gram-positive and Gram-negative pathogens, with several members of natural and semisynthetic origin occupying prominent roles in clinical practice since 1950. Nobel-prize winning crystallographic studies on the ribosome have revealed how their diverse polyaminated sugar framework is tailored to target a RNA helix within the decoding centre of the bacterial 30S subunit. By interfering with the affinity and kinetics of the tRNA selection and proof-reading steps, they induce error-prone protein synthesis, and translocation inhibition and lead to a lethal cycle of antibiotic uptake and membrane stress. In retaliation, bacterial pathogens have evolved and disseminated a number of enzymatic and efflux resistance mechanisms. These include N acetyl-transferases, O phosphotransferases and O nucleotidyltransferases, which target the core hydroxyl and amino groups of aminoglycosides promiscuously; methyltransferases, which target the ribosomal binding-site; and energy-dependent drug efflux pumps for aminoglycoside-selective elimination, in Gram-negative pathogens. The most problematic infectious pathogens which are currently resilient to most unrelated antibiotic classes and in the verge of pan-resistance have been defined ‘ESKAPE’ bacteria, a mnemonic for Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacteriaceae. The world-wide spread of aminoglycoside resistance to current clinical standards, such as tobramycin, amikacin and gentamicin, ranges from 20 to 60% of clinical isolates. Hence, the contemporary 4,6-disubstituted-2-deoxystreptamine aminoglycosides are currently inadequate as broad-spectrum anti-infective therapies. The 4,5-disubstituted class of aminoglycosides are a challenging framework for medicinal chemistry, which includes butirosin, neomycin and paromomycin. Exploring the potential of these alternatives, colleagues in the Hanessian group and collaborators of Achaogen Inc. have demonstrated that paromomycin and neomycin analogs modified by deoxygenation of positions 3' and 4', as well as N1-substituted analogs possesing the α hydroxy-γ-aminobutyryl amide (HABA) chain of butirosin, could produce promising antibiotics. Chapter 4 of this dissertation features the conception and development of an expedient semi-synthetic strategy to access novel aminoglycosides of the 4,5 disubstituted class, inspired from biosynthetic modifications of the sisomicin subfamily, that surmount the wide-spread bacterial resistance mechanisms. This synthetic methodology relies on a novel Tsuji palladium-catalyzed hydrogenolysis developed on model monosaccharides, which was applied to generate a library of aminoglycosides comprising ring A hybrids of the neomycin and sisomicin families. The structure-activity relationships of this new class were assessed against a panel of 26 bacterial strains expressing modifying enzymes and efflux systems to provide an overview of ESKAPE pathogens. Two novel hybrid aminoglycoside analogs exhibited excellent antibacterial coverage, and may be promising candidates for preclinical development. Aminoglycoside therapy is also invariably associated with a probability of nephrotoxic complications. Aminoglycoside toxicity has been largely correlated with the number of amino groups, and more loosely with the extent of deoxygenation. A long standing hypothesis in the field states that because the foremost interactions are effected by ammonium group salts, the tuning of pKa parameters could provide a higher target dissociation rate, more effective clearance and overall less nephrotoxic analogs. Chapter 5 in this dissertation features the conception and asymmetric synthesis of isosteric β substituted N1 HABA chains, modified by mono- and bis-fluorination. These chains covering a range of γ-N pKa values from 10 to 7.5 were applied to advanced tetra-deoxygenated neomycin antibiotics. In spite of the important reduction in γ N pKa, broad spectrum antimicrobial activity was not significantly disrupted for isosteric fluorinated analogs. Furthermore, structure-toxicity relationships, assessed by Achaogen’s proprietary luciferase-coupled apoptosis assay, revealed that the novel β,β difluoro-N1-HABA chain is less harmful in a Human Kidney 2 cell-line model and promising for the development as new generation neomycin antibiotics with improved therapeutic properties. The final chapter in this dissertation features the proposal and validation of the concise biomimetic synthesis and self-assembly of aminoglycoside 66-40C, a remarkable C2-symmetric 16 membered macrocyclic bis-imine dimer. The proposed structure was spectroscopically characterized as an anti-parallel s-trans-bis-azadiene macrocyclic system. Calculations indicate the anomeric effect of the α glycosidic bond between rings A and B is important for pre-organization of the monomeric sisomicin 6' aldehyde and favors the observed macrocycle product. Self-assembly in aqueous solutions was studied through the dimerization of three diverse analogs and cross-over experiments, which demonstrated the generality and stability of the macrocyclic motif of aminoglycoside 66-40C.

Plusieurs aminoglycosides font partie d’une famille d’antibiotiques à large spectre d’action. Les aminoglycosides ayant une activité antibiotique viennent interférer dans la synthèse protéique effectuée par les bactéries. Les protéines mal codées entraineront la mort cellulaire. Au fil des années, de nombreux cas de résistance ont émergé après une utilisation soutenue des aminoglycosides. De nombreux aminoglycosides semi-synthétiques ont été synthétisés avec comme objectif de restaurer leur activité antimicrobienne. Parmi les modifications ayant connu du succès, notons la didésoxygénation d’un diol et l’introduction de la chaine latérale HABA. Des études précédentes ont montré l’efficacité de ces modifications sur les aminoglycosides. Les présents travaux portent sur l’installation de la chaine latérale HABA et la didésoxygénation d’un diol sur la paromomycine et la néomycine. La didésoxygénation sélective des diols a été effectuée en utilisant la méthodologie développée par Garegg et Samuelsson, une variation de la réaction de Tipson-Cohen. Cette méthode a permis l’obtention du motif didésoxygéné sur les cycles A et D dans des rendements jamais égalés pour ce motif synthétique. La chaîne latérale a été introduite en tirant profit de la réactivité et de la sélectivité d’un carbamate cyclique. Ces méthodes combinées ont permis la synthèse efficace de nombreux analogues semi-synthétiques nouveaux. La 3',4'-didéhydro-N-1-HABA-néomycine et la 3',4',3''',4'''-tétradésoxy-N-1-HABA-néomycine montrent une activité impressionnante contre des souches de bactéries résistantes aux aminoglycosides. Des tests de toxicité effectués en collaboration avec Achaogen Inc. ont démontré que ces composés sont relativement toxiques sur les cellules rénales de type H2K, ce qui réduit de façon importante leur index thérapeutique. Afin d’abaisser la toxicité des composés, la relation entre toxicité et basicité a été explorée. Des substitutions de l’amine en 6''' ont été effectuées afin d’abaisser la basicité de l’amine. Les résultats de toxicité et d’activité antimicrobienne démontrent une corrélation importante entre la basicité des amines et la toxicité/activité des aminoglycosides antibiotiques. L’effet d’une modulation du pKa a aussi été exploré en installant des chaines fluorées sur l’amine en 6''' de la paromomycine et de la néomycine. Une séquence synthtétique pour isoler l’amine en 6''' de la néomycine a aussi été développée. / Some aminoglycosides are part of a broad-spectrum family of antibiotics used in the clinic. They interfere in protein synthesis in bacterium cell by interfering with the transcription of proteins leading to cellular death. After an intense usage of aminoglycosides in the clinic, numerous cases of resistance have been encountered which render aminoglycosides less effective. Semi-synthetic aminoglycosides have been synthesized with the objective of restoring their original antimicrobial activity. Deoxygenation of the diol on ring A and introduction of the lateral chain HABA at N-1 had a significant impact on their antimicrobial activity against resistant strains. The present work will focus on deoxygenation of the diol at 3', 4' and on the introduction of the lateral HABA chain on aminoglycoside, more specificaly on paromomycin and neomycin. The selective dideoxygenation of the A ring diol was done using a methodology developed by Garegg and Samuelsson, which is a modification of the original Tipson-Cohen reaction. This method allows the dideoxygenation on ring A and D with unprecedented yields. The lateral HABA chain was introduced via the ring opening of a cyclic carbamate. These methods were combined to produce very potent analogs such as 3',4'-didehydro-N-1-HABA-neomycin and 3',4',3''',4'''-tetradeoxy-N-1-HABA-neomycin. Toxicity tests done in collaboration with Achaogen Inc. showed that these analogs were toxic to H2K renal cells, which reduced significantly their therapeutic index. In order to lower the toxicity of those compounds, the relation between toxicity and basicity was explored. Substitution of the amine at 6''' was done in order to lower the basicity of this amine. The results showed a strong correlation beetween the basicity of this amine and toxicity/activity. The pKa of this amine was modulated by installing fluorinated alkyl chain on the amine at 6''' in order to see the effect of the pKa on the activity/toxicity on paromomycin and neomycin. A synthetic sequence was also developed to allow the 6''' amine on neomycin to be modified selectively.

Les antibiotiques aminoglycosidiques sont des agents bactéricides de grande valeur et d’efficacité à large spectre contre les pathogènes Gram-positifs et Gram-négatifs, dont plusieurs membres naturels et semisynthétiques sont importants dans l’histoire clinique depuis 1950. Des travaux crystallographiques sur le ribosome, récompensés par le prix Nobel, ont démontré comment leurs diverses structures polyaminées sont adaptées pour cibler une hélice d’ARN dans le centre de codage de la sous-unité 30S du ribosome bactérien. Leur interférence avec l’affinité et la cinétique des étapes de sélection et vérification des tARN induit la synthèse de protéines à basse fidélité, et l’inhibition de la translocation, établissant un cercle vicieux d’accumulation d’antibiotique et de stress sur la membrane. En réponse à ces pressions, les pathogènes bactériens ont évolué et disséminé une panoplie de mécanismes de résistance enzymatiques et d’expulsion : tels que les N acétyltransférases, les O phosphotransférases et les O nucleotidyltransférases qui ciblent les groupements hydroxyle et amino sur le coeur des aminoglycosides; des méthyl-transférases, qui ciblent le site de liaison ribosomale; et des pompes d’expulsion actives pour l’élimination sélective des aminoglycosides, qui sont utilisés par les souches Gram-négatives. Les pathogènes les plus problématiques, qui présentent aujourd’hui une forte résilience envers la majorité des classes d’antibiotiques sur le bord de la pan-résistance ont été nommés des bactéries ESKAPE, une mnémonique pour Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa et Enterobacteriaceae. La distribution globale des souches avec des mécanismes de résistance envers les standards cliniques aminoglycosides, tels que la tobramycine, l’amikacine et la gentamicine, est comprise entre 20 et 60% des isolées cliniques. Ainsi, les aminoglycosides du type 4,6-disubstitués-2-deoxystreptamine sont inadéquats comme thérapies anti-infectieuses à large spectre. Cependant, la famille des aminoglycosides 4,5-disubstitués, incluant la butirosine, la neomycine et la paromomycine, dont la structure plus complexe, pourrait constituter une alternative. Des collègues dans le groupe Hanessian et collaborateurs d’Achaogen Inc. ont démontré que certains analogues de la paraomomycine et neomycine, modifiés par désoxygénation sur les positions 3’ et 4’, et par substitution avec la chaîne N1-α-hydroxy-γ-aminobutyramide (HABA) provenant de la butirosine, pourrait produire des antibiotiques très prometteurs. Le Chapitre 4 de cette dissertation présente la conception et le développement d’une stratégie semi-synthétique pour produire des nouveaux aminoglycosides améliorés du type 4,5 disubstitués, inspiré par des modifications biosynthétiques de la sisomicine, qui frustrent les mécanismes de résistance bactérienne distribuées globalement. Cette voie de synthèse dépend d’une réaction d’hydrogénolyse de type Tsuji catalysée par palladium, d’abord développée sur des modèles monosaccharides puis subséquemment appliquée pour générer un ensemble d’aminoglycosides hybrides entre la neomycine et la sisomicine. Les études structure-activité des divers analogues de cette nouvelle classe ont été évaluées sur une gamme de 26 souches bactériennes exprimant des mécanismes de résistance enzymatique et d’expulsion qui englobe l’ensemble des pathogènes ESKAPE. Deux des antibiotiques hybrides ont une couverture antibacterienne excellente, et cette étude a mis en évidence des candidats prometteurs pour le développement préclinique. La thérapie avec les antibiotiques aminoglycosidiques est toujours associée à une probabilité de complications néphrotoxiques. Le potentiel de toxicité de chaque aminoglycoside peut être largement corrélé avec le nombre de groupements amino et de désoxygénations. Une hypothèse de longue date dans le domaine indique que les interactions principales sont effectuées par des sels des groupements ammonium, donc l’ajustement des paramètres de pKa pourrait provoquer une dissociation plus rapide avec leurs cibles, une clairance plus efficace et globalement des analogues moins néphrotoxiques. Le Chapitre 5 de cette dissertation présente la conception et la synthèse asymétrique de chaînes N1 HABA β substitutées par mono- et bis-fluoration. Des chaînes qui possèdent des γ-N pKa dans l’intervalle entre 10 et 7.5 ont été appliquées sur une neomycine tétra-désoxygénée pour produire des antibiotiques avancés. Malgré la réduction considérable du γ N pKa, le large spectre bactéricide n’a pas été significativement affecté pour les analogues fluorés isosteriques. De plus, des études structure-toxicité évaluées avec une analyse d’apoptose propriétaire d’Achaogen ont démontré que la nouvelle chaîne β,β difluoro-N1-HABA est moins nocive sur un modèle de cellules de rein humain HK2 et elle est prometteuse pour le développement d’antibiotiques du type neomycine avec des propriétés thérapeutiques améliorées. Le chapitre final de cette dissertation présente la proposition et validation d’une synthèse biomimétique par assemblage spontané du aminoglycoside 66-40C, un dimère C2 symétrique bis-imine macrocyclique à 16 membres. La structure proposée du macrocycle a été affinée par spectroscopie nucléaire à un système trans,trans-bis-azadiène anti-parallèle. Des calculs indiquent que l’effet anomérique de la liaison α glycosidique entre les anneaux A et B fournit la pré-organisation pour le monomère 6’ aldéhydo sisomicine et favorise le produit macrocyclique observé. L’assemblage spontané dans l’eau a été étudié par la dimérisation de trois divers analogues et par des expériences d’entre croisement qui ont démontré la généralité et la stabilité du motif macrocyclique de l'aminoglycoside 66-40C. / Aminoglycosides are valuable and effective broad-spectrum bactericidal antibiotics against Gram-positive and Gram-negative pathogens, with several members of natural and semisynthetic origin occupying prominent roles in clinical practice since 1950. Nobel-prize winning crystallographic studies on the ribosome have revealed how their diverse polyaminated sugar framework is tailored to target a RNA helix within the decoding centre of the bacterial 30S subunit. By interfering with the affinity and kinetics of the tRNA selection and proof-reading steps, they induce error-prone protein synthesis, and translocation inhibition and lead to a lethal cycle of antibiotic uptake and membrane stress. In retaliation, bacterial pathogens have evolved and disseminated a number of enzymatic and efflux resistance mechanisms. These include N acetyl-transferases, O phosphotransferases and O nucleotidyltransferases, which target the core hydroxyl and amino groups of aminoglycosides promiscuously; methyltransferases, which target the ribosomal binding-site; and energy-dependent drug efflux pumps for aminoglycoside-selective elimination, in Gram-negative pathogens. The most problematic infectious pathogens which are currently resilient to most unrelated antibiotic classes and in the verge of pan-resistance have been defined ‘ESKAPE’ bacteria, a mnemonic for Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacteriaceae. The world-wide spread of aminoglycoside resistance to current clinical standards, such as tobramycin, amikacin and gentamicin, ranges from 20 to 60% of clinical isolates. Hence, the contemporary 4,6-disubstituted-2-deoxystreptamine aminoglycosides are currently inadequate as broad-spectrum anti-infective therapies. The 4,5-disubstituted class of aminoglycosides are a challenging framework for medicinal chemistry, which includes butirosin, neomycin and paromomycin. Exploring the potential of these alternatives, colleagues in the Hanessian group and collaborators of Achaogen Inc. have demonstrated that paromomycin and neomycin analogs modified by deoxygenation of positions 3' and 4', as well as N1-substituted analogs possesing the α hydroxy-γ-aminobutyryl amide (HABA) chain of butirosin, could produce promising antibiotics. Chapter 4 of this dissertation features the conception and development of an expedient semi-synthetic strategy to access novel aminoglycosides of the 4,5 disubstituted class, inspired from biosynthetic modifications of the sisomicin subfamily, that surmount the wide-spread bacterial resistance mechanisms. This synthetic methodology relies on a novel Tsuji palladium-catalyzed hydrogenolysis developed on model monosaccharides, which was applied to generate a library of aminoglycosides comprising ring A hybrids of the neomycin and sisomicin families. The structure-activity relationships of this new class were assessed against a panel of 26 bacterial strains expressing modifying enzymes and efflux systems to provide an overview of ESKAPE pathogens. Two novel hybrid aminoglycoside analogs exhibited excellent antibacterial coverage, and may be promising candidates for preclinical development. Aminoglycoside therapy is also invariably associated with a probability of nephrotoxic complications. Aminoglycoside toxicity has been largely correlated with the number of amino groups, and more loosely with the extent of deoxygenation. A long standing hypothesis in the field states that because the foremost interactions are effected by ammonium group salts, the tuning of pKa parameters could provide a higher target dissociation rate, more effective clearance and overall less nephrotoxic analogs. Chapter 5 in this dissertation features the conception and asymmetric synthesis of isosteric β substituted N1 HABA chains, modified by mono- and bis-fluorination. These chains covering a range of γ-N pKa values from 10 to 7.5 were applied to advanced tetra-deoxygenated neomycin antibiotics. In spite of the important reduction in γ N pKa, broad spectrum antimicrobial activity was not significantly disrupted for isosteric fluorinated analogs. Furthermore, structure-toxicity relationships, assessed by Achaogen’s proprietary luciferase-coupled apoptosis assay, revealed that the novel β,β difluoro-N1-HABA chain is less harmful in a Human Kidney 2 cell-line model and promising for the development as new generation neomycin antibiotics with improved therapeutic properties. The final chapter in this dissertation features the proposal and validation of the concise biomimetic synthesis and self-assembly of aminoglycoside 66-40C, a remarkable C2-symmetric 16 membered macrocyclic bis-imine dimer. The proposed structure was spectroscopically characterized as an anti-parallel s-trans-bis-azadiene macrocyclic system. Calculations indicate the anomeric effect of the α glycosidic bond between rings A and B is important for pre-organization of the monomeric sisomicin 6' aldehyde and favors the observed macrocycle product. Self-assembly in aqueous solutions was studied through the dimerization of three diverse analogs and cross-over experiments, which demonstrated the generality and stability of the macrocyclic motif of aminoglycoside 66-40C.

Plusieurs aminoglycosides font partie d’une famille d’antibiotiques à large spectre d’action. Les aminoglycosides ayant une activité antibiotique viennent interférer dans la synthèse protéique effectuée par les bactéries. Les protéines mal codées entraineront la mort cellulaire. Au fil des années, de nombreux cas de résistance ont émergé après une utilisation soutenue des aminoglycosides. De nombreux aminoglycosides semi-synthétiques ont été synthétisés avec comme objectif de restaurer leur activité antimicrobienne. Parmi les modifications ayant connu du succès, notons la didésoxygénation d’un diol et l’introduction de la chaine latérale HABA. Des études précédentes ont montré l’efficacité de ces modifications sur les aminoglycosides. Les présents travaux portent sur l’installation de la chaine latérale HABA et la didésoxygénation d’un diol sur la paromomycine et la néomycine. La didésoxygénation sélective des diols a été effectuée en utilisant la méthodologie développée par Garegg et Samuelsson, une variation de la réaction de Tipson-Cohen. Cette méthode a permis l’obtention du motif didésoxygéné sur les cycles A et D dans des rendements jamais égalés pour ce motif synthétique. La chaîne latérale a été introduite en tirant profit de la réactivité et de la sélectivité d’un carbamate cyclique. Ces méthodes combinées ont permis la synthèse efficace de nombreux analogues semi-synthétiques nouveaux. La 3',4'-didéhydro-N-1-HABA-néomycine et la 3',4',3''',4'''-tétradésoxy-N-1-HABA-néomycine montrent une activité impressionnante contre des souches de bactéries résistantes aux aminoglycosides. Des tests de toxicité effectués en collaboration avec Achaogen Inc. ont démontré que ces composés sont relativement toxiques sur les cellules rénales de type H2K, ce qui réduit de façon importante leur index thérapeutique. Afin d’abaisser la toxicité des composés, la relation entre toxicité et basicité a été explorée. Des substitutions de l’amine en 6''' ont été effectuées afin d’abaisser la basicité de l’amine. Les résultats de toxicité et d’activité antimicrobienne démontrent une corrélation importante entre la basicité des amines et la toxicité/activité des aminoglycosides antibiotiques. L’effet d’une modulation du pKa a aussi été exploré en installant des chaines fluorées sur l’amine en 6''' de la paromomycine et de la néomycine. Une séquence synthtétique pour isoler l’amine en 6''' de la néomycine a aussi été développée. / Some aminoglycosides are part of a broad-spectrum family of antibiotics used in the clinic. They interfere in protein synthesis in bacterium cell by interfering with the transcription of proteins leading to cellular death. After an intense usage of aminoglycosides in the clinic, numerous cases of resistance have been encountered which render aminoglycosides less effective. Semi-synthetic aminoglycosides have been synthesized with the objective of restoring their original antimicrobial activity. Deoxygenation of the diol on ring A and introduction of the lateral chain HABA at N-1 had a significant impact on their antimicrobial activity against resistant strains. The present work will focus on deoxygenation of the diol at 3', 4' and on the introduction of the lateral HABA chain on aminoglycoside, more specificaly on paromomycin and neomycin. The selective dideoxygenation of the A ring diol was done using a methodology developed by Garegg and Samuelsson, which is a modification of the original Tipson-Cohen reaction. This method allows the dideoxygenation on ring A and D with unprecedented yields. The lateral HABA chain was introduced via the ring opening of a cyclic carbamate. These methods were combined to produce very potent analogs such as 3',4'-didehydro-N-1-HABA-neomycin and 3',4',3''',4'''-tetradeoxy-N-1-HABA-neomycin. Toxicity tests done in collaboration with Achaogen Inc. showed that these analogs were toxic to H2K renal cells, which reduced significantly their therapeutic index. In order to lower the toxicity of those compounds, the relation between toxicity and basicity was explored. Substitution of the amine at 6''' was done in order to lower the basicity of this amine. The results showed a strong correlation beetween the basicity of this amine and toxicity/activity. The pKa of this amine was modulated by installing fluorinated alkyl chain on the amine at 6''' in order to see the effect of the pKa on the activity/toxicity on paromomycin and neomycin. A synthetic sequence was also developed to allow the 6''' amine on neomycin to be modified selectively.

HIV-1 has evolved with a great deal of functional complexity contained within a very small genome by encoding small, but critical viral proteins within larger viral genes and dividing the replication cycle into early and late phases to differentially produce all proteins leading to efficient replication and virion release. Early replication is restricted by the host spliceosome that processes HIV-1 vRNA transcripts so only the small intragenomic proteins are produced, one of which is Rev (Regulator of Virion Expression). Rev in turn governs the transition from early to late replication by interacting with a highly structured region of vRNA termed the Rev Response Element (RRE). The binding of Rev to the RRE is believed to cause a change in the vRNA tertiary structure and inhibition of splicing of the vRNA. Once, a Rev:RRE complex is formed, a nuclear export signal within Rev facilitates the export of partially spliced and unspliced vRNA to the cytoplasm. During late replication the partially spliced and unspliced vRNA is translated to polyproteins and is packaged into a budding virion where the viral aspartyl protease (HIV-1 PR) autocatalytically excises itself from the larger polyprotein and processes the remaining polyproteins to release all viral structural and functional proteins to form a mature and infectious virion. Since the vRNA salvaged by Rev is translated to the polyproteins containing HIV-1 PR, the inhibition of Rev function will reduce the amount of HIV-1 PR available and thereby reduce the amount of HIV-1 PR therapeutics required to elicit a clinical effect. Therfore a combination approach to HIV-1 treatment using suitably developed therapeutics that inhibit Rev and HIV-1 PR function represents an attractive synergistic approach to treating HIV-1 infection in vivo. The work of this thesis was divided into two parts, the first part was concerned with HIV-1 PR structural biology and addressing problems encountered with inhibitor design. A bicyclic peptide (based on inhibitors of analogous structure) was co-crystallised with active HIV-1 PR to develop an enzyme-product (E-P) complex and with a catalytically inactive mutant HIV-1 PR to provide an analogy to the enzyme-substrate (E-S) complex. Both structures of the E-P and E-S complexes were solved to 1.6Å resolution and were compared to a hydroxyethylamine isostere enzyme-inhibitor complex (E-I), highlighting the similarity of binding mode for all ligands. The inhibitor in the E-I complex was translated towards the S1 - S3 pockets of the substrate binding cleft relative to the substrate in the E-S complex due to the increased length of the hydroxylethylamine isostere compared to the peptide backbone, although the inhibitor "puckered" the isostere linkage and maintains a binding mode similar to the substrate with very little overall differences in the position of the ligands and surrounding protein. The similarity of the E-S, E-I and E-P complexes was attributed to the macrocyclic ligands ordering the surrounding protein environment, especially the protein -strand "flap" structures that form a roof over the ligands in the active site but were not found to close more tightly in any of the trapped catalytic states. The new structures allowed refinement of details of the mechanism of peptide hydrolysis. The mechanism relies on the optimal nucleophilic attack of a water molecule on the scissile amide bond with concerted acid-base catalysis of the active site aspartyl residues intitiated by D125. The alignment and intrinsic position of the N-terminus of the bicyclic substrate was interpreted as being critical to facilitate efficient electron transfer with the bicyclic substrate. An N-terminal cyclic inhibitor, similar to the N-terminal portion of the bicyclic substrate, was used to address a major problem in HIV-1 PR drug design termed "cooperativity," where the sequential optimisation of an inhibitor (or substrate) to individual pockets of the substrate binding cleft, can negatively impact on adjacent and downfield subsites and thereby alter the binding mode of the "optimised" inhibitor. The technique referred to here as "templating" uses the N-terminal cycle to lock the binding mode into a known conformation, probing the S1' and S2' pockets. The structure activity relationship suggested that by viewing the S1' - S3' pockets as a single trough, bulky aromatic groups attached to an N-alkyl sulfonamide could be directed along the line of the trough without adverse interactions with the tops of the S1' and S3' pockets, providing very potent inhibitors. It was also found that specificity and potency of an inhibitor can be maintained with smaller functionalities that carry their bulk low and close to the inhibitor backbone in the S2' pocket, making the P2 functionalities more substrate-like. The second part of the thesis was concerned with establishing suitable surface plasmon resonance assays for testing potential inhibitors of Rev function. Recombinant Rev and its minimal RNA aptamer target (stem loop II of the RRE termed RBE3), were expressed, purified, and used to develop BIAcore-based assays and test potential inhibitors of their interaction. The system was applied to screening of aminoglycoside antibiotics and other small molecules in a competitive assay, and also to quantitative assay of Neomycin and moderate sized analytes: Rev and three peptidic analogues of the high-affinity binding site of Rev - the native peptide, succinylated form of the peptide and a form incorporating a novel helix-inducing cap. The peptide and protein assay was undertaken to test the proposition that helix induction of the high-affinity binding site of Rev can increase affinity for the biologically important RNA target and thereby form the basis of a new class of inhibitors. The screen of small molecule antagonists found that Neomycin was the best inhibitor of the Rev:RBE3 interaction and that efficacy of other aminoglycosides was due to the neamine-base structure presenting charge to bind to the RNA and blocking interaction with Rev. The quantitative assay was optimised to reduce non-specific interactions of Rev protein to allow reliable studies of the analytes with RBE3 by the sytematic testing of buffers and modifiers. It was found that mutliple analytes bound to the RBE3 aptamer and a comparison of the KD values found that the native and capped peptides had similar affinity for RBE3 RNA (native slightly greater at 21 ± 7nM cf capped 41 ± 10nM) that was greater than the Rev protein (101 ± 19nM), however the succinylated peptide exhibited stronger binding with a KD ≤8nM and Neomycin had the lowest affinity (KD 13 ± 3M). The similarity of the native and capped peptides may be due to the high concentration of salt in the assay buffers and was necessary for the stability of the Rev protein, but is sufficient to influence secondary structure of the peptides. Therefore, it could not be stated that the helix-inducing cap increased the affinity of the native peptide for the biologically important therapeutic target. The work conducted in this thesis firmly establishes foundations for the continued development of inhibitors against both Rev and HIV-1 PR that play key roles in the HIV-1 replication strategy. It is envisaged this work could lead to a novel synergistic therapeutic approach to treating HIV-1 infection.