Semisynthetic aminoglycoside antibiotics : toward biomimetic synthesis, evasion of bacterial resistance and reduced toxicity

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(1)Université de Montréal. Semisynthetic Aminoglycoside Antibiotics – Toward Biomimetic Synthesis, Evasion of Bacterial Resistance and Reduced Toxicity. par Juan Pablo Maianti Département de chimie Faculté des arts et des sciences. Mémoire présentée à la Faculté des Études Supérieures en vue de l'obtention du grade de Maîtrise ès Sciences (M.Sc.) en chimie. July, 2010. © Juan Pablo Maianti, 2010. i.

(2) Université de Montréal Faculté des Études Supérieures. Cette thèse intitulée:. Semisynthetic Aminoglycoside Antibiotics – Toward Biomimetic Synthesis, Evasion of Bacterial Resistance and Reduced Toxicity. présentée par Juan Pablo Maianti. est présentée à un jury composé des personnes suivantes:. Président du Jury. William Lubell. Directeur de Recherche. Membre du Jury. Stephen Hanessian. Joelle Pelletier. ii.

(3) To my family.. iii.

(4) Summary 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 aminoglycosideselective 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 -hydroxy--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. iv.

(5) 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 -substituted N1-HABA chains, modified by mono- and bis-fluorination. These chains covering a range of -N-pKa values from 10 to 7.5 were applied to advanced tetradeoxygenated neomycin antibiotics. In spite of the important reduction in -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 ,-difluoro-N1HABA 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 -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.. v.

(6) Keywords aminoglycoside, antibiotic, neomycin, sisomicin, deoxygenation, Tsuji hydrogenolysis, HABA, fluorination, biomimetic, self-assembly.. vi.

(7) Résumé 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 méthyltransférases, 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 Gramnégatives. 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--hydroxy-vii.

(8) 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 a été évalué 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 -substitutées par mono- et bis-fluoration. Des chaînes qui possèdent des -N-pKa dans l’intervale entre 10 et 7.5 ont été appliquées sur une neomycine tétra-désoxygénée pour produire des antibiotiques avancés. Malgré la réduction considérable du -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 ,-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.. viii.

(9) 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 -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.. Mots clés aminoglycoside, antibiotique, neomycine, sisomicine, désoxygénation, hydrogénolyse de Tsuji, HABA, fluorination, biomimétique, assemblage spontané.. ix.

(10) Table of Contents. Title page...................................................................................................................... i Jury Identification ........................................................................................................ ii Summary ...................................................................................................................... iv Keywords ..................................................................................................................... vi Résumé......................................................................................................................... vii Mots clés ...................................................................................................................... ix Table of Contents ......................................................................................................... x List of Sections............................................................................................................. xi List of Figures .............................................................................................................. xiv List of Schemes............................................................................................................ xvii List of Tables ............................................................................................................... xviii Abbreviations ............................................................................................................... xxi Acknowledgments........................................................................................................ xx. x.

(11) List of Sections. Chapter 1 – Introduction to Aminoglycoside Antibiotics: Structure, Nomenclature and Biosynthesis .................................................................. 1 1.1 - Discovery of Aminoglycosides .............................................................. 2 1.2 - General Aminoglycoside Nomenclature and Representation................. 3 1.3 - Aminoglycoside Classification and Structure ........................................ 4 1.4 - Aminoglycoside Biosynthesis ................................................................ 8 1.5 - Biosynthetic Deoxygenations of Ring A................................................ 9 1.6 - References .............................................................................................. 11 Chapter 2 – Mode of Action of Aminoglycoside Antibiotics: Ribosomal Target, Bactericidal Effects and Uptake.................................................... 16 2.1 - Aminoglycoside Interactions with the Ribosome................................... 17 2.2 - Aminoglycoside Effects on Translation Accuracy................................. 17 2.3 - Bactericidal Action of Antibiotics.......................................................... 23 2.4 - Aminoglycoside Uptake ......................................................................... 28 2.5 - References .............................................................................................. 26 Chapter 3 – Issues Associated with Aminoglycoside Antibiotics: Bacterial Resistance Mechanisms and Toxicity........................................................... 30 3.1 - Bacterial Resistance Overview............................................................... 31 3.2 - Aminoglycoside Modifying Enzymes.................................................... 33 3.3 - Aminoglycoside Acetyltransferases ....................................................... 36 3.4 - Aminoglycoside Phosphotransferases .................................................... 36 3.5 - Aminoglycoside Nucleotidyltransferases............................................... 38 3.6 - Efflux Pumps and Permeability.............................................................. 39 3.7 - Ribosomal Mutation and Enzymatic Modification ................................ 40 3.8 - Antimicrobial Testing – the Primary Panel ............................................ 42 3.9 - MIC Table Color-Code........................................................................... 42 3.10 - Aminoglycoside Toxicity ..................................................................... 44 3.11 - Aminoglycoside Nephrotoxicity .......................................................... 44 3.12 - Aminoglycoside Ototoxicity ................................................................ 45. xi.

(12) 3.13 - Aminoglycoside Dosing in the Clinic .................................................. 46 3.14 - References ............................................................................................ 47 Chapter 4 – Semisynthetic Hybrids of Aminoglycoside Antibiotics: Development and Application of Tsuji Palladium-Catalyzed Deoxygenation. 56. 4.1 - Design of New Aminoglycosides........................................................... 57 4.2 - Strategies for Countering Resistance – Evasion versus Inhibition ........ 58 4.3 - Deoxygenation for Evasion of Aminoglycoside Modifying Enzymes .. 59 4.4 - Synthetic Deoxygenation Methodologies .............................................. 61 4.5 - Tsuji Palladium-Catalyzed Deoxygenation............................................ 63 4.6 - Deoxygenation of Glucosamine Models of Paromomycin .................... 66 4.7 - Tsuji Palladium-Catalyzed Deoxygenation of Paromomycin................ 70 4.8 - Synthesis of Sisomicin-Hybrid Aminoglycosides ................................. 73 4.9 - Analysis of Antibacterial Activity ......................................................... 82 4.10 - Conclusions .......................................................................................... 85 4.11 - References ............................................................................................ 86 Chapter 4 – Experimental Section ............................................................................... 94 Chapter 4 – Compound Index ...................................................................................... 95 Chapter 4 – NMR spectra and HPLC purity reports.................................................... 140 Chapter 5 – Toward Neomycin Analogs with Reduced Nephrotoxic Potential: Asymmetric Synthesis of -Substituted HABA Groups.............................................. 164 5.1 - Amines and Toxicity .............................................................................. 165 5.2 - The pKa Modulation Hypothesis and Literature Precedence................. 165 5.3 - Asymmetric Synthesis of -Substituted HABA Fragments ................... 168 5.4 - Synthesis of -substituted HABA Neomycin Antibiotics ...................... 172 5.5 - Analysis of Antibacterial Activity.......................................................... 174 5.6 - Analysis of HK2 Cell Line Nephrotoxicity Models............................... 176 5.7 – Conclusions............................................................................................ 178 5.8 – References.............................................................................................. 179 Chapter 5 – Experimental Section ............................................................................... 186 Chapter 5 – Compound Index ...................................................................................... 188 Chapter 5 – NMR spectra and HPLC purity reports.................................................... 212. xii.

(13) Chapter 5 – Nonlinear fitting reports and calculations ................................................ 254 Chapter 6 – Biomimetic Synthesis, Structural Refinement and Examination of Self-Assembly of the Macrocyclic Dimer Aminoglycoside 66-40C ........................... 262 6.1 - Biosynthetic Origins of Aminoglycoside 66-40C.................................. 263 6.2 - Biomimetic Synthesis of Aminoglycoside 66-40C................................ 267 6.3 - Computer-Generated Model of Aminoglycoside 66-40C...................... 273 6.4 - Synthesis of the Sisamine Dimer Macrocyclic Core.............................. 275 6.5 - Synthesis of the 4',5'-Unsaturated Paromomycin Macrocyclic Dimer .. 276 6.6 - Synthesis of the Expanded Macrocyclic Dimer Core ............................ 277 6.7 - Examination of the Dynamics of Macrocycle Self-Assembly ............... 278 6.8 - Conclusions ............................................................................................ 281 6.9 - References .............................................................................................. 282 Chapter 6 – Experimental Section ............................................................................... 287 Chapter 6 – Compound Index ...................................................................................... 289 Chapter 6 – NMR spectra............................................................................................. 313 Annex – Crystallography Reports................................................................................ 339. xiii.

(14) List of Figures Figure 1.1 Basic numbering and representation of two classes of aminoglycoside antibiotics................................................................................................................................... 3 Figure 1.2 Structural and biosynthetic genealogy tree of the 2-deoxystreptamine derived aminoglycosides ........................................................................................................................ 5 Figure 1.3. Structures of aminoglycosides in the “kanamycin family” .................................... 5 Figure 1.4. Structures of aminoglycosides in the “neomycin family”...................................... 6 Figure 1.5. Structures of aminoglycosides in the “gentamicin family”.................................... 7 Figure 1.6. Representative aminoglycoside antibiotics not classified in the neomycin, kanamycin or gentamicin subfamilies ....................................................................................... 7 Figure 1.7. Biosynthetic pathways of glucosamine and 2-deoxystreptamine, the precursors of paromamine........................................................................................................................... 8 Figure 1.8. Representative examples of biosynthetic modifications occurring postglycosidation of the aminoglycoside frameworks ..................................................................... 9 Figure 1.9. Aminoglycosides undergoing biosynthetic deoxygenations on ring A at position 3' or both 3' and 4' ........................................................................................................ 10 Figure 1.10. A plausible 3',4'-dideoxygenation pathway accounting for 4',5'-dehydration and 3'-phosphorylation from precursor JI-20A to sisomicin by unassigned genes ................... 11 Figure 2.1. Representation of the translation cycle .................................................................. 18 Figure 2.2. Representation of the ribosome 30S subunit.......................................................... 20 Figure 2.3. Representation of the H44 A-site model showing the overlapping binding sites of neomycin, amikacin apramycin and hygromycin B.............................................................. 20 Figure 2.4. Peptide elongation cycle emphasizing reaction rates affected by aminoglycoside antibiotics ........................................................................................................ 21 Figure 2.5. Conformations of the H44 helix and G530 loop in the 30S subunit in different crystals, co-crystallized with fragment of cognate mRNA and tRNA, empty A-site and with paromomycin..................................................................................................................... 22 Figure 2.6. Contacts of neomycin co-crystallized within an oligonucleotide A-site H44 helix model ................................................................................................................................ 23 Figure 2.7. Compilation of possible mechanisms for aminoglycoside bactericidal effects ..... 24 Figure 2.8. Graphical representation of uptake for gentamicin. Representation of aminoglycoside uptake requirements ........................................................................................ 28. xiv.

(15) Figure 3.1. Resistance enzyme susceptibility of the disubstituted 2-deoxystreptamine families ...................................................................................................................................... 34 Figure 3.2. Prevalence of resistance to gentamicin .................................................................. 35 Figure 3.3. Distribution of aminoglycoside resistance mechanisms in isolates from the CID and SENTRY program collections from three time periods ............................................. 35 Figure 3.4. Active site pockets of three available aminoglycoside modifying enzymes, co-crystallized with both nucleotide cofactors and aminoglycoside ......................................... 37 Figure 3.5. Reconstruction model of a RND class efflux system, the AcrA–AcrB–TolC drug efflux pump of E. coli ....................................................................................................... 39 Figure 3.6. Representation of the H44 A-site model showing the overlapping binding sites of neomycin, amikacin apramycin............................................................................................. 41 Figure 4.1. Examples of deoxygenated aminoglycosides from natural, semisynthetic and mutasynthetic sources................................................................................................................ 60 Figure 4.2. Examples of aminoglycoside 3’,4’-dideoxygenation with variations of the Tipson-Cohen reaction .............................................................................................................. 62 Figure 4.4. Comparison of biosynthesis-inspired routes for 3’-deoxygenation ....................... 64 Figure 4.5. Mechanistic details of variants of Tsuji palladium-catalysis ................................. 65 Figure 4.6. Examples of reactions involving (-allyl)palladium complexes on unsaturated carbohydrates and glycals or enol ethers ................................................................................... 66 Figure 4.7. ORTEP drawing of 4.9 with thermal ellipsoids drawn at 30% probability level... 68 Figure 4.8. Mechanistic considerations of Tsuji deoxygenation for the generation of product 4.10 ............................................................................................................................... 70 Figure 4.9. Structures of the novel sisomicin hybrid analogs presented in this chapter, compared with natural and semisynthetic control antibiotics ................................................... 81 Figure 4.10. Comparative assessment of enzymatic resistance mechanisms targeting the gentamicin complex, amikacin and ‘Frankenmycin’ 4.34 ........................................................ 84 Figure 5.1. Examples of aminoglycoside modifications which modulate the pKa of neighboring amines.................................................................................................................... 166 Figure 5.2. Structure of Inosamycin A and proposal of -amine pKa modulation of the N1-HABA groups of next-generation 3',4'-deoxygenated neomycins ...................................... 167 Figure 5.3. Previous synthetic efforts to modulate the pKa of N1 side chains of differing structures.................................................................................................................................... 169. xv.

(16) Figure 5.4. Issues associated with fluorine substitution of -leaving groups in unsuitably protected L-HABA fragments, compared to the proposed divergent route from Sharpless desymmetrization of divinylcarbinol......................................................................................... 170 Figure 5.5. ORTEP drawing of 5.4 with thermal ellipsoids drawn at 30% probability level... 171 Figure 5.6. Luminescence caspase-3/7 assay at 31 h incubation of natural and unnatural -substituted HABA series, N1-HABA analog 5.10, gentamicin and amikacin as controls .... 176 Figure 5.7. Area under the curve analysis of Figure 5.7, for natural and unnatural -substituted HABA series, N1-HABA analog 5.10, gentamicin and amikacin as controls .... 177 Figure 5.8. Luminescence viability assay at 31 h incubation with natural and unnatural substituted HABA series, N1-HABA analog 5.10, gentamicin and amikacin as controls........ 177 Figure 6.1. Aminoglycoside 66-40C (6.1), sisomicin and their hypothetical biosynthetic relationship through a 6'-aldehyde monomer precursor (6.2). .................................................. 263 Figure 6.2. Examples of biomimetic self-assembly of dimeric or pseudodimeric natural products ..................................................................................................................................... 265 Figure 6.3. Stable N-substituted imines, hydrazides and oximino ethers................................. 266 Figure 6.4. 1H-NMR spectra of the development of a D2O solution of dimethylacetal 6.7, acidified to 0.2 N H2SO4 until aldehyde 6.2 prevailed, then neutralized with sat. Ba(OH)2 ..... 269 Figure 6.5. 1H-NMR spectra of an evolving D2O solution of aminoglycoside 66-40C 6.1 acidified with 0.2 N H2SO4 ........................................................................................................ 272 Figure 6.6. Computer model of aminoglycoside 66-40C generated on Hyperchem 8 suite ..... 273 Figure 6.7. Model of aminoglycoside 66-40C, overlaid on the skeleton of paromomycin from the A-site oligonucleotide co-crystal structure. Comparison of the exo- and endoanomeric effects in the frameworks of paromomycin and the aminoglycoside 66-40C macrocycle................................................................................................................................. 274 Figure 6.8. Sequential LCMS injections of aqueous solutions of dimer mixtures ................... 280. xvi.

(17) List of Schemes Scheme 4.1. Synthesis of 3-O-methylcarbonate substituted glucosamine models for deoxygenation............................................................................................................................ 67 Scheme 4.2. Tsuji palladium-catalyzed deoxygenation of aldehyde 4.4 .................................. 69 Scheme 4.3. Semisynthetic manipulations of paromomycin toward ,-unsaturated aldehyde intermediate 4.14 for Tsuji palladium-catalyzed 3’-deoxygenation .......................... 71 Scheme 4.4. Synthesis of sisomicin-paromomycin (4.18) and sisomicin-neomycin hybrids (4.19).......................................................................................................................................... 74 Scheme 4.5. Synthesis of sisomicin-neomycin hybrids by reductive amination ...................... 75 Scheme 4.6. Oxidative diol cleavage toward sisomicin-ribostamycin hybrids 4.25 and 4.27 ............................................................................................................................................ 76 Scheme 4.7. Selective N1-deprotection and appendage of L-HABA to sisomicin-butirosin hybrid 4.30................................................................................................................................. 78 Scheme 4.8. Selective N1-deprotection via bis-cyclic carbamate intermediates for appendage of L-HABA to sisomicin-butirosin-neomycin hybrid 4.34 ..................................... 79 Scheme 5.1. Asymmetric synthesis of -substituted HABA side chains of the natural enantiomeric series .................................................................................................................... 171 Scheme 5.2. Synthesis of tetradeoxy neomycins with N1 chains of natural and unnatural series .......................................................................................................................................... 173 Scheme 6.1 Synthesis and biomimetic self-assembly of aminoglycoside 66-40C (6.1), the refined structure, key nOe and HMBC correlations are shown................................................. 269 Scheme 6.2. Degradation of tetra-azido sisomicin ring C and synthesis of the nominal macrocyclic dimer motif (6.12). ................................................................................................ 275 Scheme 6.3. Synthesis of the 4',5'-unsaturated paromomycin macrocyclic dimer 6.14 from intermediate 4.15 presented in Chapter 4 .................................................................................. 276 Scheme 6.4. Synthesis of the 20-membered macrocycle (6.18) by two-carbon homologation of the sisamine 6'-aldehyde monomer ................................................................ 277 Scheme 6.5. Dynamic equilibrium challenge of aminoglycoside 66-40C (6.1) and dimer 6.13 ............................................................................................................................................ 278. xvii.

(18) List of Tables Table 1.1. Prominent examples of natural aminoglycosides from soil organisms ................... 2 Table 1.2. Clinically-relevant semisynthetic aminoglycosides................................................. 3 Table 3.1. Distribution of aminoglycoside resistance enzymes in “ESKAPE bacteria” ........... 34 Table 3.2. Minimum inhibitory concentration antibacterial testing against a range of susceptible and resistant isolates for antibiotics amikacin, gentamicin, neomycin B, paromomycin, N1-HABA paromomycin, ribostamycin and butirosin ..................................... 43 Table 4.1. Representative results of Tsuji deoxygenation of glucosamine models .................. 68 Table 4.2. Optimization of Tsuji palladium-catalyzed 3’-deoxygenation of paromomycin..... 72 Table 4.3. Comparison of 13C-NMR shifts of sisomicin-ribostamycin hybrid 4.27................. 77 Table 4.4. Antibacterial assessment of sisomicin hybrids and controls. Minimum inhibitory concentrations ........................................................................................................... 80 Table 4.5. Distribution of aminoglycoside resistance enzymes in “ESKAPE bacteria” ........... 84 Table 5.1. Antibacterial assessment of N1--substituted tetradeoxy neomycins and controls. Minimum inhibitory concentrations ........................................................................... 175 Table 5.2. EC50 values from nonlinear fit analysis of Figure 5.7 for assays of natural and unnatural -substituted HABA series and the respective controls ............................................ 177 Table 6.1. 1H-NMR shift comparison of natural and synthetic aminoglycoside 66-40C (6.1)............................................................................................................................................ 271 Table 6.2. 13C-NMR shift comparison of natural and synthetic aminoglycoside 66-40C (6.1) 271 Table 6.3. Comparison of 13C-NMR shifts of naturally-derived and synthetic 6.2 in acid ...... 272. xviii.

(19) Abbreviations. AAC. aminoglycoside Nacetyltransferase Ac-CoA acetyl coenzyme A ADPNP adenylyl--imidodiphosphate Amk amikacin ANT aminoglycoside Onucleotidyltransferase APH aminoglycoside Ophosphotransferase armA ribosome methyltransferase 30S N1-G1405, gene armA ATCC American Type Culture Collection ATP adenosine triphosphate AUC area under the curve Btr butirosin CAM ceric ammonium molybdate Cbe carboxyethyl Cbz carboxybenzyl CHARMM-GUI Chemistry at HARvard Macromolecular Mechanics Graphical User Interface CID Center for Infectious Diseases CLND Chemiluminescent Nitrogen Detection COSY correlation spectroscopy Cpx Cpx A/R two-component signal transduction pathway of E. coli which regulates gene transcription DAST diethylaminosulfur trifluoride dba dibenzylideneacetone DBU 1,8-Diazabicyclo[5.4.0]undec-7ene DCL dynamic combinatorial library DCM dichloromethane D-HABA D-(R)-4-amino-2-hydroxybutanoyl (or acid) DHP 3,4-dihydro-[2H]-pyran DIBAL diisobutylaluminium hydride DIPEA N,N-diisopropylethylamine (or Hünig's base) DIPHOS 1,2-bis(diphenylphosphino) ethane DIPT diisopropyl tartrate DMAP N,N-dimethyl-4-aminopyridine DMF dimethylformamide DMSO dimethyl sulfoxide DNA deoxyribonucleic acid DPPA diphenylphosphoryl azide EC50 effective concentration for halfmaximum activity ECDC European Centre for Disease Prevention and Control. EDC EDP-I and II EF-Tu EIP EMEA ESI ESKAPE. FDA FRET FT-IR FtsH GDP Gent GTP HABA HAPA HFBA HMBC HMQC H-Par HPLC HRMS HSQC IDSA iOWH KHMDS L LCMS LC-MSD LG L-HABA LRMS M-H MIC. xix. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide energy dependent phase I and II elongation factor thermo unstable energy independent phase European Medicines Agency electrospray ionization mnemonic for bacterial pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacteriaceae Food and Drug Administration Förster resonance energy transfer Fourier transform infra-red membrane-bound ATP-dependent zinc metalloprotease guanosine diphosphate gentamicin complex guanosine triphosphate (S)-4-amino-2-hydroxybutanoyl (or acid) (S)-3-amino-2-hydroxypropanoyl (or acid) heptafluorobutyric acid heteronuclear multiple bond coherence heteronuclear multiple quantum coherence N1-HABA paromomycin high pressure liquid chromatography high resolution mass spectrometry heteronuclear single quantum coherence Infectious Diseases Society of America Institute of One World Health potassium bis(trimethylsilyl)amide ligand liquid chromatography mass spectrometry liquid chromatography - mass spectrometry detection leaving group L-(S)-4-amino-2-hydroxybutanoyl (or acid) low resolution mass spectrometry metal hydride minimum inhibitory concentration.

(20) MIC-90 mit-rRNA MOM mRNA MRSA Ms NADH NeoB NHS NIAID nmpA nOe Nu ORTEP Par PCR PDB PDF py Ribo RMT rmtA-D RNA RND RRF rRNA RT SAR Sec. SENTRY TBAF TBAI TBS TEMPO TES Tf TFA THF TLC TOF tRNA UV WHO. minimum inhibitory concentration for 90% of strains mitochondrial ribosomal RNA methoxymethyl mitochondrial ribonucleic acid Methicillin-resistant Staphylococcus aureus methanesulfonyl nicotinamide adenine dinucleotide neomycin B N-hydroxysuccinimide National Institute of Allergy and Infectious Diseases (US) ribosome methyltransferase 30S N7-A1408, gene nmpA nuclear Overhauser effect nucleophile Oak Ridge thermal Ellipsoid Program paromomycin polymerase chain reaction Protein Data Bank Portable Document Format pyridine ribostamycin ribosome methyltransferase ribosome methyltransferase 30S N1-G1405, genes rmtA-D ribonucleic acid resistance-nodulation-division gene family ribosome release factor ribosomal ribonucleic acid room temperature structure-activity relationship Sec complexes, a major route of protein translocation across cell membranes Antimicrobial Surveillance Program - JMI Laboratories tetra-n-butylammonium fluoride tetrabutylammonium iodide tert-butyldimethylsilyl 2,2,6,6-tetramethylpiperidine-1oxy radical triethylsilyl trifluoromethanesulfyl trifluoroacetic acid tetrahydrofuran thin layer chromatography time of flight transfer ribonucleic acid ultra-violet World Health Organization. xx.

(21) Acknowledgements. I acknowledge Achaogen Inc. for financial support and Fonds de la Recherche en Santé du Québec (FRSQ) for a two-year fellowship.. This dissertation has taken a number of particularly interdisciplinary directions and it is certain that the bulk of this work would not have been achieved without the support of numerous talented colleagues.. Nuclear Magnetic Resonance support and 700 MHz acquisitions were performed by Dr. Phan viet Minh Tan, Dr. Sylvie Bilodeau and Dr. Cédric Malveau. I acknowledge the excellent heteronuclear correlation experiments and outstanding facilities.. Mass Spectrometry support and High-Resolution acquisitions were performed by Dr. Alexandra Furtos, Marie-Christine Tang and Karine Venne. I thank them for the outstanding level of services in the MS laboratory and Alexandra for performing the challenging LCMS analysis in Chapter 6.. Crystallographic X-ray acquisition and analysis were performed by the talented Ph.D. student Benoît Deschênes-Simard.. I acknowledge the helpful organization of the project and intellectual exchange with Achaogen scientists Adam A. Goldblum, Dr. Paola Dozzo, Dr. James Aggen, Dr. Martin Linsell and chemistry vice president Dr. Heinz Moser. I thank all the members of the Aminoglycoside Project of Achaogen for the enriching internship in San Francisco and a warm welcome to the lab by Timothy Kane, Darin Hildebrandt and Micah Gliedt.. Antibacterial testing, tissue culture and caspase assays were performed in Achaogen Inc. I acknowledge the high standard of science in their respective departments to Dr. Heinz Moser, Dr. George Miller and Dr. Eliana Armstrong.. xxi.

(22) I acknowledge the unconditional personal support throughout this work and also the careful review this dissertation to my loving wife Nicole Darricarrère.. I am very thankful to the contemporary colleagues of the Aminoglycoside Team for training and enriching exchange of ideas and protocols: Alexandre Guiguère, Dr. Justyna Grzyb and Dr. Janek Szychowski. Janek was an outstanding mentor during my early training with aminoglycosides.. Many thanks to Professor Jeffrey W. Keillor for useful discussions and encouragement.. I deeply thank all the contemporary Hanessian group members for their friendship and intellectual challenges throughout these years: Alexandre Giguère, Dr. Amy Xueling Mi, Dr. Andreas Larsson, Annmaria Baraldi, Dr. Benjamin Schroeder, Benoît DeschênesSimard, Dr. Bradley Merner, Carol Major, Elaine Fournelle, Dr. Elias J. Stoffman, Étienne Chenard, Dr. Janek Szychowski, Jeremie Tessier, John Zhang Jianbin, Dr. Justyna Grzyb, Leo Bin Chen, Dr. Luciana Auzzas, Ludivine Riber, Nancy Xiao Tian Wang, Dr. Nicolas Boyer, Michele Ammouche, Dr. Ramkrishna Reddy Vakiti, Riccardo Matera, Rupal Oza, Dr. Sébastien Guesne, Stefano Rizzo, Stéphane Dorich, Dr. Thilo Focken, Dr. Thomas Jennequin, Dr. Uday Kumar Soma, Dr. Vincent Babonneau and Vu Linh Ly. I underline the particularly helpful contributions of Dr. Janek Szychowski, Dr. Nicolas Boyer, Benoît Deschênes-Simard, and Dr. Bradley Merner for frequent and useful discussions.. Finally and inclusively, I thank Professor Stephen Hanessian for his faith, outstanding mentorship and guidance.. Thank you all most sincerely.. xxii.

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