Reversibly Photo-Crosslinkable Polycarbonate-Based Polymersomes for Drug Encapsulation and Delivery

This thesis describes the preparation of polymersomes from poly(ethylene glycol)-block-polycarbonate (PEG-PC) copolymers functionalized with pendant coumarin groups. Coumarin groups undergo photo-reversible dimerization when irradiated with specific ultraviolet wavelengths, so they can be used to prepare polymers with photo-responsive properties. In this case, the pendant coumarin groups enable stabilization of the polymersome membrane through photo-crosslinking of the hydrophobic block. Initially, several novel cinnamoyl and coumarin functionalized cyclic carbonate monomers were synthesized using ester, ether, or amide linkages. While the homopolymerization of these functionalized monomers proved challenging due to their high melting points, both cinnamoyl and coumarin functionalized monomers were successfully copolymerized with trimethylene carbonate (TMC) at 100 ℃ using a catalyst-free melt polymerization process where the TMC doubled as a solvent for the higher melting point monomer. Using this system, polycarbonate copolymers with up to 33% incorporation of the functionalized monomers were prepared. In addition, an investigation of some anomalous polymerization results identified previously unreported triethylamine-based catalysts for the melt polymerization of carbonate monomers. These studies also demonstrated that the catalyst-free polymerization of TMC occurs faster and at lower temperatures than previously reported. Subsequently, the photo-crosslinking of cinnamoyl and coumarin functionalized polycarbonates was compared and coumarin was identified as the more effective crosslinking agent when using 300-400 nm UV. An investigation of the photo-reversibility of the coumarin dimerization revealed no discernible change in the properties of crosslinked networks, but rapid photo-reversion in dilute solutions. The photo-crosslinking and photo-reversion kinetics of the coumarin functionalized polycarbonates were determined to be second-order in both cases. Finally, the self-assembly of PEG-PC diblock copolymers functionalized with coumarin was examined and both reverse solvent evaporation and solvent displacement were found to induce self-assembly, with hydrophilic mass fractions (f-factors) of 12-28% resulting in the formation of solid microparticles and nanoparticles and f-factors of 33-43% resulting in the formation of polymersomes. The stabilization of these polymersome membranes through photo-initiator-free photo-crosslinking was demonstrated with the crosslinking allowing polymersomes to withstand centrifugation at 12,000 x g. In addition, the encapsulation of calcein, as a model small molecule drug, in the stabilized polymersomes was successfully demonstrated using confocal microscopy.

Direct catalytic macrolactonization and application of the phase separation strategy in complex molecule synthesis

Les macrolactones sont des squelettes structuraux importants dans de nombreuses sphères de l’industrie chimique, en particulier dans les marchés pharmaceutiques et cosmétiques. Toutefois, la stratégie traditionnelle pour la préparation de macrolactones demeure incommode en requérant notamment l’ajout (super)stœchiométrique d’agents activateurs. Conséquemment, des quantités stœchiométriques de sous-produits sont générées; ils sont souvent toxiques, dommageables pour l’environnement et nécessitent des méthodes de purification fastidieuses afin de les éliminer. La présente thèse décrit le développement d’une macrolactonisation efficace catalysée au hafnium directement à partir de précurseurs portant un acide carboxylique et un alcool primaire, ne générant que de l’eau comme sous-produit et ne nécessitant pas de techniques d’addition lente et/ou azéotropique. Le protocole a également été adapté à la synthèse directe de macrodiolides à partir de mélanges équimolaires de diols et de diacides carboxyliques et à la synthèse de dimères tête-à-queue de seco acides. Des muscs macrocycliques ainsi que des macrolactones pertinentes à la chimie médicinale ont pu être synthétisés avec l’approche développée. Un protocole pour l’estérification directe catalysée au hafnium entre des acides carboxyliques et des alcools primaires a aussi été développé. Différentes méthodes pour la macrolactonisation catalytique directe entre des alcools secondaires et des acides carboxyliques ont été étudiées. En outre, la stratégie de séparation de phase en macrocyclisation en débit continu a été appliquée lors de la synthèse totale formelle de la macrolactone ivorenolide A. Les étapes-clés de la synthèse incluent une macrocyclisation par le couplage d’alcynes de Glaser-Hay et une réaction de métathèse d’alcènes Z-sélective.

The evolutionary history and activity of a gain-of-function polymorphism in a human antiviral gene

Thesis (Ph.D.)--University of Washington, 2016-07

FUNCTIONAL CHARACTERIZATION OF THE LINK BETWEEN CARBOHYDRATE METABOLISM AND THE PATHOGENESIS OF THE INVASIVE M1T1 GROUP A STREPTOCOCCUS

The Group A Streptococcus (GAS), or Streptococcus pyogenes, is a strict human pathogen that colonizes a variety of sites within the host. Infections can vary from minor and easily treatable, to life-threatening, invasive forms of disease. In order to adapt to niches, GAS utilizes environmental cues, such as carbohydrates, to coordinate the expression of virulence factors. Research efforts to date have focused on identifying how either components of the phosphoenolpyruvate-phosphotransferase system (PTS) or global transcriptional networks affect the regulation of virulence factors, but not the synergistic relationship between the two. The present study investigates the role of a putative PTS-fructose operon encoded by fruRBA and its role in virulence in the M1T1 strain 5448. Growth in fructose resulted in induction of fruRBA. RT-PCR showed that fruRBA formed an operon, which was repressed by FruR in the absence of fructose. Growth and carbon utilization profiles revealed that although the entire fruRBA operon was required for growth in fructose, FruA was the main fructose transporter. The ability of both ΔfruR and ΔfruB mutants to survive in whole human blood or neutrophils was impaired. However, the phenotypes were not reproduced in murine whole blood or in a mouse intraperitoneal infection, indicating a human-specific mechanism. While it is known that the PTS can affect activity of the Mga virulence regulator, further characterization of the mechanism by which sugars and its protein domains affect activity have not been studied. Transcriptional studies revealed that the core Mga regulon is activated more in a glucose-rich than a glucose-poor environment. This activation correlates with the differential phosphorylation of Mga at its PTS regulatory domains (PRDs). Using a 5448 mga mutant, transcriptome studies in THY or C media established that the Mga regulon reflects the media used. Interestingly, Mga regulates phage-encoded DNases in a low glucose environment. We also show that Mga activity is dependent on C-terminal amino acid interactions that aid in the formation of homodimers. Overall, the studies presented sought to define how external environmental cues, specifically carbohydrates, control complex regulatory networks used by GAS, contribute to pathogenesis, and aid in adaptation to various nutrient conditions encountered.

Síntese e caracterização de ligantes nitrogenados derivados de isatina, 4-benziltiossemicarbazida e seus complexos com metais d¹º

Neste trabalho são apresentadas e discutidas as estruturas cristalinas e moleculares do ligante (1), isatina-3-(toluilsulfono-hidrazona), dos complexos [bis(2-acetilpiridina-N4 - benziltiossemicarbazona-N,N,S)Cd(II)], (1), [bis (isatina-3-N4 -benziltiossemicarbazonaN,S)Hg(II)].Etanol, (2) e [bis (isatina-3-N4 -benziltiossemicarbazona-N,S,O)Zn(II)].DMF, (3). Cristais amarelos vítreos do ligante (1) foram obtidos a partir da evaporação lenta de etanol do ensaio de cristalização. Seus dados cristalográficos indicam que duas moléculas interagem através de ligações de hidrogênio do tipo N1-H···O1, formando unidades dímeras. A reação entre 2-acetilpiridina-N4 -benziltiossemicarbazona e Cd(CH3COO)2.2H2O, em presença de etanol, KOH, e após evaporação lenta da mistura de acetona e DMF(2:1), resultou em cristais amarelos do complexo (1). As interações do tipo C(10)-H(10)···S(1)···H(1)-N(4), e N(8)- H(29)⋅⋅⋅S(2) permitem a dimerização do complexo, e a formação de uma cadeia unidimensional. Os cristais laranja do complexo (2) foram obtidos da reação entre o ligante isatina-3-N4 -benziltiossemicarbazona e Hg(NO3)2.H2O, na presença de metanol, KOH, e após evaporação lenta de uma mistura de tolueno e acetona (2:1). As moléculas do complexo (2) estão associadas por ligações de hidrogênio do tipo N(63)-H(4)···O(21), essas interações centrossimétricas conduzem a formação de dímeros. A reação entre o ligante isatina-3-N4 - benziltiossemicarbazona e Zn(CH3COO)2.2H2O, em presença de etanol e KOH resultou em cristais de coloração laranja do complexo (3). A estrutura do complexo apresenta múltiplas ligações de hidrogênio, com formação de dímeros através das interações N1-H1···O1 e C3- H3···N(7). Os dímeros associam-se por interações N4-H4···O2 numa cadeia unidimensional ao longo da direção cristalográfica [100]. A polimerização bidimensional é observada 7 considerando-se as interações do tipo C20-H20···S1, N8-H8···S2 ao longo da direção cristalográfica [010], bem como das interações, N5-H5···O3DMF e C31-H31B···Car, que ocorrem através da molécula de solvente DMF.

Direct catalytic macrolactonization and application of the phase separation strategy in complex molecule synthesis

Les macrolactones sont des squelettes structuraux importants dans de nombreuses sphères de l’industrie chimique, en particulier dans les marchés pharmaceutiques et cosmétiques. Toutefois, la stratégie traditionnelle pour la préparation de macrolactones demeure incommode en requérant notamment l’ajout (super)stœchiométrique d’agents activateurs. Conséquemment, des quantités stœchiométriques de sous-produits sont générées; ils sont souvent toxiques, dommageables pour l’environnement et nécessitent des méthodes de purification fastidieuses afin de les éliminer. La présente thèse décrit le développement d’une macrolactonisation efficace catalysée au hafnium directement à partir de précurseurs portant un acide carboxylique et un alcool primaire, ne générant que de l’eau comme sous-produit et ne nécessitant pas de techniques d’addition lente et/ou azéotropique. Le protocole a également été adapté à la synthèse directe de macrodiolides à partir de mélanges équimolaires de diols et de diacides carboxyliques et à la synthèse de dimères tête-à-queue de seco acides. Des muscs macrocycliques ainsi que des macrolactones pertinentes à la chimie médicinale ont pu être synthétisés avec l’approche développée. Un protocole pour l’estérification directe catalysée au hafnium entre des acides carboxyliques et des alcools primaires a aussi été développé. Différentes méthodes pour la macrolactonisation catalytique directe entre des alcools secondaires et des acides carboxyliques ont été étudiées. En outre, la stratégie de séparation de phase en macrocyclisation en débit continu a été appliquée lors de la synthèse totale formelle de la macrolactone ivorenolide A. Les étapes-clés de la synthèse incluent une macrocyclisation par le couplage d’alcynes de Glaser-Hay et une réaction de métathèse d’alcènes Z-sélective.

Design and validation of a novel generic platform for the production of tetravalent IgG1-like bispecific antibodies

International audience

Structure of the N-terminal oligomerization domain of DnaD reveals a unique tetramerization motif and provides insights into scaffold formation

DnaD is a primosomal protein that remodels supercoiled plasmids. It binds to supercoiled forms and converts them to open forms without nicking. During this remodeling process, all the writhe is converted to twist and the plasmids are held around the periphery of large scaffolds made up of DnaD molecules. This DNA-remodeling function is the sum of a scaffold-forming activity on the N-terminal domain and a DNA-dependent oligomerization activity on the C-terminal domain. We have determined the crystal structure of the scaffold-forming N-terminal domain, which reveals a winged-helix architecture, with additional structural elements extending from both N- and C-termini. Four monomers form dimers that join into a tetramer. The N-terminal extension mediates dimerization and tetramerization, with extensive interactions and distinct interfaces. The wings and helices of the winged-helix domains remain exposed on the surface of the tetramer. Structure-guided mutagenesis and atomic force microscopy imaging indicate that these elements, together with the C-terminal extension, are involved in scaffold formation. Based upon our data, we propose a model for the DnaD-mediated scaffold formation.

Harpalycin 2 inhibits the enzymatic and platelet aggregation activities of PrTX-III, a D49 phospholipase A(2) from Bothrops pirajai venom

Background: Harpalycin 2 (HP-2) is an isoflavone isolated from the leaves of Harpalyce brasiliana Benth., a snakeroot found in northeast region of Brazil and used in folk medicine to treat snakebite. Its leaves are said to be anti-inflammatory. Secretory phospholipases A(2) are important toxins found in snake venom and are structurally related to those found in inflammatory conditions in mammals, as in arthritis and atherosclerosis, and for this reason can be valuable tools for searching new anti-phospholipase A(2) drugs.Methods: HP-2 and piratoxin-III (PrTX-III) were purified through chromatographic techniques. The effect of HP-2 in the enzymatic activity of PrTX-III was carried out using 4-nitro-3-octanoyloxy-benzoic acid as the substrate. PrTX-III induced platelet aggregation was inhibited by HP-2 when compared to aristolochic acid and p-bromophenacyl bromide (p-BPB). In an attempt to elucidate how HP-2 interacts with PrTX-III, mass spectrometry, circular dichroism and intrinsic fluorescence analysis were performed. Docking scores of the ligands (HP-2, aristolochic acid and p-BPB) using PrTX-III as target were also calculated.Results: HP-2 inhibited the enzymatic activity of PrTX-III (IC50 11.34 +/- 0.28 mu g/mL) although it did not form a stable chemical complex in the active site, since mass spectrometry measurements showed no difference between native (13,837.34 Da) and HP-2 treated PrTX-III (13,856.12 Da). A structural analysis of PrTX-III after treatment with HP-2 showed a decrease in dimerization and a slight protein unfolding. In the platelet aggregation assay, HP-2 previously incubated with PrTX-III inhibited the aggregation when compared with untreated protein. PrTX-III chemical treated with aristolochic acid and p-BPB, two standard PLA(2) inhibitors, showed low inhibitory effects when compared with the HP-2 treatment. Docking scores corroborated these results, showing higher affinity of HP-2 for the PrTX-III target (PDB code: 1GMZ) than aristolochic acid and p-BPB. HP-2 previous incubated with the platelets inhibits the aggregation induced by untreated PrTX-III as well as arachidonic acid.Conclusion: HP-2 changes the structure of PrTX-III, inhibiting the enzymatic activity of this enzyme. In addition, PrTX-III platelet aggregant activity was inhibited by treatment with HP-2, p-BPB and aristolochic acid, and these results were corroborated by docking scores.

Surface structured platinum electrodes for the electrochemical reduction of carbon dioxide in imidazolium based ionic liquids

The direct CO2 electrochemical reduction on model platinum single crystal electrodes Pt(hkl) is studied in [C2mim+][NTf2−], a suitable room temperature ionic liquid (RTIL) medium due to its moderate viscosity, high CO2 solubility and conductivity. Single crystal electrodes represent the most convenient type of surface structured electrodes for studying the impact of RTIL ion adsorption on relevant electrocatalytic reactions, such as surface sensitive electrochemical CO2 reduction. We propose here based on cyclic voltammetry and in situ electrolysis measurements, for the first time, the formation of a stable adduct [C2mimH–CO2−] by a radical–radical coupling after the simultaneous reduction of CO2 and [C2mim+]. It means between the CO2 radical anion and the radical formed from the reduction of the cation [C2mim+] before forming the corresponding electrogenerated carbene. This is confirmed by the voltammetric study of a model imidazolium-2-carboxylate compound formed following the carbene pathway. The formation of that stable adduct [C2mimH–CO2−] blocks CO2 reduction after a single electron transfer and inhibits CO2 and imidazolium dimerization reactions. However, the electrochemical reduction of CO2 under those conditions provokes the electrochemical cathodic degradation of the imidazolium based RTIL. This important limitation in CO2 recycling by direct electrochemical reduction is overcome by adding a strong acid, [H+][NTf2−], into solution. Then, protons become preferentially adsorbed on the electrode surface by displacing the imidazolium cations and inhibiting their electrochemical reduction. This fact allows the surface sensitive electro-synthesis of HCOOH from CO2 reduction in [C2mim+][NTf2−], with Pt(110) being the most active electrode studied.