Novel surface-tethered estrogen polymeric platforms in cardiovascular regenerative medicine

L’estradiol (E2) est une hormone femelle qui joue un rôle essentiel, à la fois dans la régulation et dans la détermination de certaines conditions physiologiques in vivo, telle que la différenciation et la prolifération cellulaire. Lorsque l’E2 est donné en supplément, par exemple dans le cas de thérapie hormonale, deux effets sont observés, un effet génomique et un effet non-génomique, de par son interaction avec les récepteurs à œstrogène du noyau ou de la membrane cellulaire, respectivement. L’effet non-génomique est plus difficile à étudier biologiquement parce que l’effet se produit sur une échelle de temps extrêmement courte et à cause de la nature hydrophobe de l’E2 qui réduit sa biodisponibilité et donc son accessibilité aux cellules cibles. C’est pourquoi il est nécessaire de développer des systèmes d’administration de l’E2 qui permettent de n’étudier que l’effet non-génomique de l’œstrogène. Une des stratégies employée consiste à greffer l’E2 à des macromolécules hydrophiles, comme de l’albumine de sérum bovin (BSA) ou des dendrimères de type poly(amido)amine, permettant de maintenir l’interaction de l’E2 avec les récepteurs d’œstrogène de la membrane cellulaire et d’éviter la pénétration de l’E2 dans le noyau des cellules. Toutefois, ces systèmes macromolécules-E2 sont critiquables car ils sont peu stables et l’E2 peut se retrouver sous forme libre, ce qui affecte sa localisation cellulaire. L’objectif de cette thèse est donc de développer de nouvelles plateformes fonctionnalisées avec de l’E2 en utilisant les approches de synthèses ascendantes et descendantes. Le but de ces plateformes est de permettre d’étudier le mécanisme de l’effet non-génomique de l’E2, ainsi que d’explorer des applications potentielles dans le domaine biomédical. L’approche ascendante est basée sur un ligand d’E2 activé, l’acide 17,α-éthinylestradiol-benzoïque, attaché de façon covalente à un polymère de chitosan avec des substitutions de phosphorylcholine (CH-PC-E2). L’estradiol est sous forme de pro-drogue attachée au polymère qui s’auto-assembler pour former un film. L’effet biologique de la composition chimique du film de chitosan-phosphorylcholine a été étudié sur des cellules endothéliales. Les films de compositions chimiques différentes ont préalablement été caractérisés de façon physicochimique. La topographie de la surface, la charge de surface, ainsi que la rhéologie des différents films contenant 15, 25, ou 40% molaires de phosphorylcholine, ont été étudiés par microscopie à force atomique (AFM), potentiel zêta, résonance plasmonique de surface et par microbalance à cristal de quartz avec dissipation (QCM-D). Les résultats de QCM-D ont montré que plus la part molaire en phosphorylcholine est grande moins il y a de fibrinogène qui s’adsorbe sur le film de CH-PC. Des cellules humaines de veine ombilicale (HUVECs) cultivées sur des films de CH-PC25 et de CH-PC40 forment des amas cellulaire appelés sphéroïdes au bout de 4 jours, alors que ce n’est pas le cas lorsque ces cellules sont cultivées sur des films de CH-PC15. L’attachement de l’estradiol au polymère a été caractérisé par plusieurs techniques, telles que la résonance magnétique nucléaire de proton (1H NMR), la spectroscopie infrarouge avec transformée de Fourier à réfraction totale atténuée (FTIR-ATR) et la spectroscopie UV-visible. La nature hydrogel des films (sa capacité à retenir l’eau) ainsi que l’interaction des films avec des récepteurs à E2, ont été étudiés par la QCM-D. Des études d’imagerie cellulaires utilisant du diacétate de diaminofluoresceine-FM ont révélé que les films hydrogels de CH-PC-E2 stimulent la production d’oxyde nitrique par les cellules endothéliales, qui joue un rôle protecteur pour le système cardiovasculaire. L’ensemble de ces études met en valeur les rôles différents et les applications potentielles qu’ont les films de type CH-PC-E2 et CH-PC dans le cadre de la médecine cardiovasculaire régénérative. L’approche descendante est basée sur l’attachement de façon covalente d’E2 sur des ilots d’or de 2 μm disposés en rangées et espacés par 12 μm sur un substrat en verre. Les ilots ont été préparés par photolithographie. La surface du verre a quant à elle été modifiée à l’aide d’un tripeptide cyclique, le cRGD, favorisant l’adhésion cellulaire. L’attachement d’E2 sur les surfaces d’or a été suivi et confirmé par les techniques de SPR et de QCM-D. Des études d’ELISA ont montré une augmentation significative du niveau de phosphorylation de la kinase ERK (marqueur important de l’effet non-génomique) après 1 heure d’exposition des cellules endothéliales aux motifs alternant l’E2 et le cRGD. Par contre lorsque des cellules cancéreuses sont déposées sur les surfaces présentant des motifs d’E2, ces cellules ne croissent pas, ce qui suggère que l’E2 n’exerce pas d’effet génomique. Les résultats de l’approche descendante montrent le potentiel des surfaces présentant des motifs d’E2 pour l’étude des effets non-génomiques de l’E2 dans un modèle in vitro.

Impurity profiling and synthesis of precursors to the hallucinogenic amphetamine DOB

DOB (4‐bromo‐2,5‐dimethoxyamphetamine) is a newly emerging hallucinogenic amphetamine that sparked serious health warnings in Ireland, following its first seizure back in 2003. Known more commonly as “snowball”, this drug is highly potent and may be used as a substitute to ecstasy (MDMA) and lysergic acid diethylamide (LSD). To date, the work carried out on the impurity profiling of DOB is limited in comparison to amphetamine, methamphetamine and MDMA. In this work, the impurity profile of 4‐bromo‐2,5‐dimethoxyphenyl‐2‐propanone (4‐Br‐2,5‐P2P) is explored. This ketone is a direct precursor to DOB. Its more versatile non‐bromo analogue, 2,5‐ dimethoxyphenyl‐2‐propanone (2,5‐P2P) is also examined, as in addition to DOB, it may be used in the synthesis of a range of several other hallucinogenic amphetamines. A number of different routes to both 2,5‐P2P and 4‐Br‐2,5‐P2P were investigated. For each of these routes, the impurities produced were carefully isolated. Following isolation, the impurities were fully characterised (by 1H‐NMR/13C‐NMR spectroscopy, IR, MS), in order to aid structure elucidation. Compounds not easily resolved by flash column chromatography were analysed by LC‐MS and/or independently synthesised for the purpose of attaining reference standards. Adaptation of the well‐known ‘phenylacetic acid route’ to synthesis of both 2,5‐P2P and 4‐Br‐2,5‐P2P, was found to provide low yields of the expected ketone products. Four impurities were isolated during the preparation of both ketones. The yield of one of these impurities (possessing a dibenzylketone core), was greatly influenced by the amount of acetic anhydride reagent used during the reaction. Having carried out the reaction with several different equivalents of acetic anhydride, it was found that formation of the ‘dibenzylketone’ could not be eliminated. This may increase its likelihood of being detected in the final drug product. The ‘Darzens route’, having very recently emerged as a synthetic route to amphetamine and MDMA precursors, was discovered to be a viable route for manufacture of 2,5‐P2P and 4‐Br‐2,5‐P2P. Despite execution of the reaction being more tedious, the route provides superior yields (≈50–60%) to those achieved using the ‘phenylacetic acid route’ (≈35–38%). Incorporation of a bromine atom (at the aromatic 4‐position) is required at some stage during synthesis of DOB. The bromination of many intermediates/starting materials was therefore also examined in detail. Bromination of the acid starting material 2,5‐dimethoxyphenylacetic acid (2,5‐PAA) was found to be clean and high yielding. This was in stark contrast to the bromination of the benzaldehyde starting material, the ketone precursor 2,5‐P2P and the dibenzylketone‐based impurity. Numerous brominated products were isolated from each of these reactions, many of which were novel compounds, and previously unreported as impurities in the literature. The unpredictable/nondescript nature of these brominations is likely to have a significant impact on the impurity profile of illicitly produced DOB.

Enzymatic Polymerization of High Molecular Weight DNA

The use of DNA as a polymeric building material transcends its function in biology and is exciting in bionanotechnology for applications ranging from biosensing, to diagnostics, and to targeted drug delivery. These applications are enabled by DNA’s unique structural and chemical properties, embodied as a directional polyanion that exhibits molecular recognition capabilities. Hence, the efficient and precise synthesis of high molecular weight DNA materials has become key to advance DNA bionanotechnology. Current synthesis methods largely rely on either solid phase chemical synthesis or template-dependent polymerase amplification. The inherent step-by-step fashion of solid phase synthesis limits the length of the resulting DNA to typically less than 150 nucleotides. In contrast, polymerase based enzymatic synthesis methods (e.g., polymerase chain reaction) are not limited by product length, but require a DNA template to guide the synthesis. Furthermore, advanced DNA bionanotechnology requires tailorable structural and self-assembly properties. Current synthesis methods, however, often involve multiple conjugating reactions and extensive purification steps.

The research described in this dissertation aims to develop a facile method to synthesize high molecular weight, single stranded DNA (or polynucleotide) with versatile functionalities. We exploit the ability of a template-independent DNA polymerase−terminal deoxynucleotidyl transferase (TdT) to catalyze the polymerization of 2’-deoxyribonucleoside 5’-triphosphates (dNTP, monomer) from the 3’-hydroxyl group of an oligodeoxyribonucleotide (initiator). We termed this enzymatic synthesis method: TdT catalyzed enzymatic polymerization, or TcEP.

Specifically, this dissertation is structured to address three specific research aims. With the objective to generate high molecular weight polynucleotides, Specific Aim 1 studies the reaction kinetics of TcEP by investigating the polymerization of 2’-deoxythymidine 5’-triphosphates (monomer) from the 3’-hydroxyl group of oligodeoxyribothymidine (initiator) using in situ 1H NMR and fluorescent gel electrophoresis. We found that TcEP kinetics follows the “living” chain-growth polycondensation mechanism, and like in “living” polymerizations, the molecular weight of the final product is determined by the starting molar ratio of monomer to initiator. The distribution of the molecular weight is crucially influenced by the molar ratio of initiator to TdT. We developed a reaction kinetics model that allows us to quantitatively describe the reaction and predict the molecular weight of the reaction products.

Specific Aim 2 further explores TcEP’s ability to transcend homo-polynucleotide synthesis by varying the choices of initiators and monomers. We investigated the effects of initiator length and sequence on TcEP, and found that the minimum length of an effective initiator should be 10 nucleotides and that the formation of secondary structures close to the 3’-hydroxyl group can impede the polymerization reaction. We also demonstrated TcEP’s capacity to incorporate a wide range of unnatural dNTPs into the growing chain, such as, hydrophobic fluorescent dNTP and fluoro modified dNTP. By harnessing the encoded nucleotide sequence of an initiator and the chemical diversity of monomers, TcEP enables us to introduce molecular recognition capabilities and chemical functionalities on the 5’-terminus and 3’-terminus, respectively.

Building on TcEP’s synthesis capacities, in Specific Aim 3 we invented a two-step strategy to synthesize diblock amphiphilic polynucleotides, in which the first, hydrophilic block serves as a macro-initiator for the growth of the second block, comprised of natural and/or unnatural nucleotides. By tuning the hydrophilic length, we synthesized the amphiphilic diblock polynucleotides that can self-assemble into micellar structures ranging from star-like to crew-cut morphologies. The observed self-assembly behaviors agree with predictions from dissipative particle dynamics simulations as well as scaling law for polyelectrolyte block copolymers.

In summary, we developed an enzymatic synthesis method (i.e., TcEP) that enables the facile synthesis of high molecular weight polynucleotides with low polydispersity. Although we can control the nucleotide sequence only to a limited extent, TcEP offers a method to integrate an oligodeoxyribonucleotide with specific sequence at the 5’-terminus and to incorporate functional groups along the growing chains simultaneously. Additionally, we used TcEP to synthesize amphiphilic polynucleotides that display self-assemble ability. We anticipate that our facile synthesis method will not only advance molecular biology, but also invigorate materials science and bionanotechnology.

Novel surface-tethered estrogen polymeric platforms in cardiovascular regenerative medicine

L’estradiol (E2) est une hormone femelle qui joue un rôle essentiel, à la fois dans la régulation et dans la détermination de certaines conditions physiologiques in vivo, telle que la différenciation et la prolifération cellulaire. Lorsque l’E2 est donné en supplément, par exemple dans le cas de thérapie hormonale, deux effets sont observés, un effet génomique et un effet non-génomique, de par son interaction avec les récepteurs à œstrogène du noyau ou de la membrane cellulaire, respectivement. L’effet non-génomique est plus difficile à étudier biologiquement parce que l’effet se produit sur une échelle de temps extrêmement courte et à cause de la nature hydrophobe de l’E2 qui réduit sa biodisponibilité et donc son accessibilité aux cellules cibles. C’est pourquoi il est nécessaire de développer des systèmes d’administration de l’E2 qui permettent de n’étudier que l’effet non-génomique de l’œstrogène. Une des stratégies employée consiste à greffer l’E2 à des macromolécules hydrophiles, comme de l’albumine de sérum bovin (BSA) ou des dendrimères de type poly(amido)amine, permettant de maintenir l’interaction de l’E2 avec les récepteurs d’œstrogène de la membrane cellulaire et d’éviter la pénétration de l’E2 dans le noyau des cellules. Toutefois, ces systèmes macromolécules-E2 sont critiquables car ils sont peu stables et l’E2 peut se retrouver sous forme libre, ce qui affecte sa localisation cellulaire. L’objectif de cette thèse est donc de développer de nouvelles plateformes fonctionnalisées avec de l’E2 en utilisant les approches de synthèses ascendantes et descendantes. Le but de ces plateformes est de permettre d’étudier le mécanisme de l’effet non-génomique de l’E2, ainsi que d’explorer des applications potentielles dans le domaine biomédical. L’approche ascendante est basée sur un ligand d’E2 activé, l’acide 17,α-éthinylestradiol-benzoïque, attaché de façon covalente à un polymère de chitosan avec des substitutions de phosphorylcholine (CH-PC-E2). L’estradiol est sous forme de pro-drogue attachée au polymère qui s’auto-assembler pour former un film. L’effet biologique de la composition chimique du film de chitosan-phosphorylcholine a été étudié sur des cellules endothéliales. Les films de compositions chimiques différentes ont préalablement été caractérisés de façon physicochimique. La topographie de la surface, la charge de surface, ainsi que la rhéologie des différents films contenant 15, 25, ou 40% molaires de phosphorylcholine, ont été étudiés par microscopie à force atomique (AFM), potentiel zêta, résonance plasmonique de surface et par microbalance à cristal de quartz avec dissipation (QCM-D). Les résultats de QCM-D ont montré que plus la part molaire en phosphorylcholine est grande moins il y a de fibrinogène qui s’adsorbe sur le film de CH-PC. Des cellules humaines de veine ombilicale (HUVECs) cultivées sur des films de CH-PC25 et de CH-PC40 forment des amas cellulaire appelés sphéroïdes au bout de 4 jours, alors que ce n’est pas le cas lorsque ces cellules sont cultivées sur des films de CH-PC15. L’attachement de l’estradiol au polymère a été caractérisé par plusieurs techniques, telles que la résonance magnétique nucléaire de proton (1H NMR), la spectroscopie infrarouge avec transformée de Fourier à réfraction totale atténuée (FTIR-ATR) et la spectroscopie UV-visible. La nature hydrogel des films (sa capacité à retenir l’eau) ainsi que l’interaction des films avec des récepteurs à E2, ont été étudiés par la QCM-D. Des études d’imagerie cellulaires utilisant du diacétate de diaminofluoresceine-FM ont révélé que les films hydrogels de CH-PC-E2 stimulent la production d’oxyde nitrique par les cellules endothéliales, qui joue un rôle protecteur pour le système cardiovasculaire. L’ensemble de ces études met en valeur les rôles différents et les applications potentielles qu’ont les films de type CH-PC-E2 et CH-PC dans le cadre de la médecine cardiovasculaire régénérative. L’approche descendante est basée sur l’attachement de façon covalente d’E2 sur des ilots d’or de 2 μm disposés en rangées et espacés par 12 μm sur un substrat en verre. Les ilots ont été préparés par photolithographie. La surface du verre a quant à elle été modifiée à l’aide d’un tripeptide cyclique, le cRGD, favorisant l’adhésion cellulaire. L’attachement d’E2 sur les surfaces d’or a été suivi et confirmé par les techniques de SPR et de QCM-D. Des études d’ELISA ont montré une augmentation significative du niveau de phosphorylation de la kinase ERK (marqueur important de l’effet non-génomique) après 1 heure d’exposition des cellules endothéliales aux motifs alternant l’E2 et le cRGD. Par contre lorsque des cellules cancéreuses sont déposées sur les surfaces présentant des motifs d’E2, ces cellules ne croissent pas, ce qui suggère que l’E2 n’exerce pas d’effet génomique. Les résultats de l’approche descendante montrent le potentiel des surfaces présentant des motifs d’E2 pour l’étude des effets non-génomiques de l’E2 dans un modèle in vitro.

Preparation of Polymer Nanoparticles by Ring Opening Metathesis Polymerization in Aqueous Dispersed Systems with Water-Soluble Ruthenium-based Catalyst

Ring opening metathesis polymerization (ROMP) is a variant of olefin metathesis used to polymerize strained cyclic olefins. Ruthenium-based Grubbs’ catalysts are widely used in ROMP to produce industrially important products. While highly efficient in organic solvents such as dichloromethane and toluene, these hydrophobic catalysts are not typically applied in aqueous systems. With the advancements in emulsion and miniemulsion polymerization, it is promising to conduct ROMP in an aqueous dispersed phase to generate well-defined latex nanoparticles while improving heat transfer and reducing the use of volatile organic solvents (VOCs). Herein I report the efforts made using a PEGylated ruthenium alkylidene as the catalyst to initiate ROMP in an oil-in-water miniemulsion. 1H NMR revealed that the synthesized PEGylated catalyst was stable and reactive in water. Using 1,5-cyclooctadiene (COD) as monomer, we showed the highly efficient catalyst yielded colloidally stable polymer latexes with ~ 100% conversion at room temperature. Kinetic studies demonstrated first-order kinetics with good livingness as confirmed by the shift of gel permeation chromatography (GPC) traces. Depending on the surfactants used, the particle sizes ranged from 100 to 300 nm with monomodal distributions. The more strained cyclic olefin norbornene (NB) could also be efficiently polymerized with a PEGylated ruthenium alkylidene in miniemulsion to full conversion and with minimal coagulum formation.

Sintesi di monomeri derivati da acidi carbossilici omega insaturi e successiva sintesi di poliesteri

Il presente lavoro di tesi si inserisce in un progetto basato sulla ricerca di monomeri e polimeri derivati da fonte bio, in un’ottica di transizione verso una chimica green legata alla realizzazione di prodotti ottenuti da risorse rinnovabili e con processi a basso impatto ambientale. I polimeri bio, in particolare, sono prodotti parzialmente o interamente ottenibili da risorse rinnovabili, quali oli vegetali, zuccheri, grassi, resine, proteine, amminoacidi, frazioni lignocellulosiche, etc, e non da petrolio. Tra questi, gli oli vegetali sono interessanti come reagenti di partenza perché permettono di minimizzare l’uso di derivati petrolchimici e sono disponibili in quantità maggiore rispetto al petrolio. Una possibilità di sintesi di polimeri da oli consiste nel trasformare gli acidi grassi, ottenendo composti bifunzionali che possono agire da precursori nella polimerizzazione: in questo modo si possono ottenere molti polimeri lineari, tra cui poliuretani, polieteri, poliesteri, poliammidi, etc. Questo è stato l’approccio seguito per la sintesi dei poliesteri alla base del progetto di tesi. In particolare, il lavoro si è articolato nella sintesi di monomeri e polimeri utilizzando derivati di acidi carbossilici omega insaturi. Inizialmente, trattandosi di prove esplorative sulla fattibilità delle reazioni, è stato utilizzato un precursore commerciale: visti i buoni risultati ottenuti, lo studio è poi proseguito con la sintesi di poliesteri a partire da prodotti di origine naturale. Tutte le polimerizzazioni sono state effettuate in massa. Per ogni reagente, monomero e polimero sono state effettuate analisi NMR (1H-NMR, 13C-NMR, HSQC, HMBC); su alcuni reagenti e monomeri è stata invece effettuata analisi GC-MS. Infine, su tutti i polimeri ottenuti sono state effettuate analisi ATR, GPC, DSC e TGA.

The effect of poly(lactide)-poly(carbonate) based block copolymers on the morphology and crystallization of double crystalline poly(lactide)/poly(ε-caprolactone) blends

Block copolymers of poly(lactide) and poly(carbonate) were synthetized in three different compositions and characterized by 1H-NMR and ATR analyses. The compatibilization effect of this copolymers on 80/20 (w/w%) PLA/PCL blend was evaluated. SEM micrographs show that all the blends exhibit the typical sea-island morphology characteristic of immiscible blends with PCL finely dispersed in droplets on a PLA matrix. Upon the addiction of the copolymers a reduction on PCL droplets size is observable. At the same time, a Tg depression of the PLA phase is detected when the copolymers are added in the blend. These results indicate that these copolymers are effective as compatibilizers. The copolymer that acts as the best compatibilizer is the one characterized by the same amount of PLA and PC as repeating units. As result, in the blend containing this copolymer PLA phase exhibits the highest spherulitic growth rate. An analyses on PLA phase crystallization behaviour from the glassy state within the blends was evaluated by DSC experiments. Isothermal cold crystallization of the PLA phase is enhanced up an order of magnitude upon the blending with PCL. Annealing experiments demonstrated that the crystallization of the PCL phase induces the formation of active nuclei in PLA when cooled above cooled below Tg. When the crystallization rate of PCL is retarded, a reduction on PLA nucleation is observed.

Synthesis and evaluation of temperature- and glucose-sensitive nanoparticles based on phenylboronic acid and N-vinylcaprolactam for insulin delivery

Poly N-vinylcaprolactam-co-acrylamidophenylboronic acid p(NVCL-co-AAPBA) was prepared from N-vinylcaprolactam (NVCL) and 3-acrylamidophenylboronic acid (AAPBA), using 2,2-azobisisobutyronitrile (AIBN) as initiator. The synthesis and structure of the polymer were examined by Fourier Transform infrared spectroscopy (FT-IR) and 1H-NMR. Dynamic light scattering (DLS), lower critical solution temperature (LCST) and transmission electron microscopy (TEM) were utilized to characterize the nanoparticles, CD spectroscopy was used to determine if there were any changes to the conformation of the insulin, and cell and animal toxicity were also investigated. The prepared nanoparticles were found to be monodisperse submicron particles and were glucose- and temperature-sensitive. In addition, the nanoparticles have good insulin-loading characteristics, do not affect the conformation of the insulin and show low-toxicity to cells and animals. These p(NVCL-co-AAPBA) nanoparticles may have some value for insulin or other hypoglycemic protein delivery.

Synthesis and characterization of electron deficient heterocycles as building blocks for electronic materials

This thesis describes the synthesis of a new electropolymerizable viologen derivative. A reasonably high-yielding route is reported, and a preliminary investigation of its polymerisation is described. The viologen and its precursors were examined by 1H NMR, MS, IR and elemental analysis. The energies of the band gap for the materials have been calculated using UV-vis spectroscopy, and cyclic voltammetry was also used to estimate the oxidation and the reduction potentials and to calculate the HOMO and LUMO energies. Theoretical calculations were performed using DFT. The attempted synthesis of a new flavin-functionalised phenanthroline derivative is described. Unfortunately, the protocol used failed to provide the desired compounds.

Synthesis, characterization and chemical vapor deposition of transition metal di(tert-butyl)amido compounds

Although the transition metal chemistry of many dialkylamido ligands has been well studied, the chemistry of the bulky di(tert-butyl)amido ligand has been largely overlooked. The di(tert-butyl)amido ligand is well suited for synthesizing transition metal compounds with low coordination numbers; such compounds may exhibit interesting structural, physical, and chemical properties. Di(tert-butyl)amido complexes of transition metals are expected to exhibit high volatilities and low decomposition temperatures, thus making them well suited for the chemical vapor deposition of metals and metal nitrides. Treatment of MnBr₂(THF)₂, FeI₂, CoBr₂(DME), or NiBr₂(DME) with two equivalents of LiN(t-Bu)2 in benzene affords the two-coordinate complex M[N(t-Bu)₂]₂, where M is Mn, Fe, Co, or Ni. Crystallographic studies show that the M-N distances decrease across the series: 1.9365 (Mn), 1.8790 (Fe), 1.845 (Co), 1.798 Å (Ni). The N-M- N angles are very close to linear for Mn and Fe (179.30 and 179.45°, respectively), but bent for Co and Ni (159.2 and 160.90°, respectively). As expected, the d⁵ Mn complex has a magnetic moment of 5.53 μΒ that is very close to the spin only value. The EPR spectrum is nearly axial with a low E/D ratio of 0.014. The d⁶ Fe compound has a room temperature magnetic moment of 5.55 μΒ indicative of a large orbital angular momentum contribution. It does not exhibit a Jahn-Teller distortion despite the expected doubly degenerate ground state. Applied field Mössbauer spectroscopy shows that the effective internal hyperfine field is unusually large, Hint = 105 T. The magnetic moments of Co[N(t-Bu)₂]₂ and Ni[N(t-Bu)₂]₂ are 5.24 and 3.02 μΒ respectively. Both are EPR silent at 4.2 K. Treatment of TiCl₄ with three equivalents of LiN(t-Bu)2 in pentane affords the briding imido compound Ti₂[μ-N(t-Bu)]₂Cl₂[N(t-Bu)₂]₂ via a dealkylation reaction. Rotation around the bis(tert-butyl)amido groups is hindered, with activation parameters of ΔH‡ = 12.8 ± 0.6 kcal mol-1 and ΔS‡ = -8 ± 2 cal K-1 ·mol-1, as evidenced by variable temperature 1H NMR spectroscopy. Treatment of TiCl₄ with two equivalents of HN(t-Bu)₂ affords Ti₂Cl₆[N(t-Bu)₂]₂. This complex shows a close-contact of 2.634(3) Å between Ti and the carbon atom of one of the CH₃ substituents on the tert-butyl groups. Theoretical considerations and detailed structural comparisons suggest this interaction is not agostic in nature, but rather is a consequence of interligand repulsions. Treatment of NiI₂(PPh3)₂ and PdCl₂(PPh₃)₂ with LiN(t-Bu)₂in benzene affords Ni[N(t-Bu)₂](PPh₃)I and Pd₃(μ₂-NBut₂)2(μ₂-PPh₂)Ph(PPh₃) respectively. The compound Ni[N(t-Bu)₂](PPh₃)I has distorted T-shape in geometry, whereas Pd₃(μ₂-NBut₂)₂(μ₂-PPh₂)Ph(PPh₃) contains a triangular palladium core. Manganese nitride films were grown from Mn[N(t-Bu)₂]₂ in the presence of anhydrous ammonia. The growth rate was several nanometers per minute even at the remarkably low temperature of 80⁰C. As grown, the films are carbon- and oxygen-free, and have a columnar morphology. The spacings between the columns become smaller and the films become smoother as the growth temperature is increased. The composition of the films is consistent with a stoichiometry of Mn₅N₂.

Desenvolvimento e validação de metodologias para quantificação de 3,4 metilenodioximetanfetamina (MDMA) em comprimidos de ecstasy por cromatografia gasosa e ressonância magnética nuclear

Dissertação (mestrado)—Universidade de Brasília, Instituto de Química, 2016.

Synthesis and antibacterial study of some s-substituted aliphatic analogues of 2-mercapto-5-(1-(4-toluenesulfonyl) piperidin-4-yl)-1,3,4-oxadiazole

Purpose: To synthesize a series of analogues of 1,3,4-oxadiazole and to evaluate their antibacterial activity. Methods: Ethyl piperidin-4-carboxylate (1) was mixed with 4-toluenesulfonyl chloride (2) in benignant conditions to yield ethyl 1-(4-toluenesulfonyl)piperidin-4-carboxylate (3) and then 1-(4- toluenesulfonyl)piperidin-4-carbohydrazide (4). Intermolecular cyclization of 4 into 2-mercapto-5-(1-(4- toluenesulfonyl) piperidin-4-yl)-1,3,4-oxadiazole (5) was obtained on reflux with CS2 in the presence of KOH. Molecule 5 was stirred with alkyl halides, 6a-i, in DMF in the presence of LiH to synthesize the final compounds, 7a-i. The structures of these molecules were elucidated by Fourier transform infra-red (FTIR) spectroscopy, proton nuclear magnetic resonance (1H-NMR) and electron impact mass spectrometry (EI-MS). Antibacterial activity was evaluated against five bacterial strains, namely, Salmonella typhi, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis, with ciprofloxacin used as standard antibacterial agent. Results: Out of nine synthesized derivatives, compound 7a was the most active against three bacterial strains, S. typhi, E. coli and P. aeruginosa, with minimum inhibitory concentration (MIC) of 9.11 ± 0.40, 9.89 ± 0.45 and 9.14 ± 0.72 μM, respectively, compared with 7.45 ± 0.58, 7.16 ± 0.58 and 7.14 ± 0.18 μM, respectively, for the reference standard (ciprofloxacin). Similarly, compounds 7a - 7c showed relatively good antibacterial activity against B. subtilis strain while compound 7e - 7g revealed good results against S. typhi bacterial strain. Conclusion: The results indicate that S-substituted derivatives of the parent compound are more effective antibacterial agents than the parent compound, even with minor differences in substituents

Preliminary structure-activity relationship studies on some novel s-substituted aliphatic analogues of 5-{1-[(4- chlorophenyl) sulfonyl]-3-piperidinyl}-1, 3, 4-oxadiazol-2-yl sulfide

Purpose: To study the structure-activity relationships of synthetic multifunctional sulfides through evaluation of lipoxygenase and anti-bacterial activities. Methods: S-substituted derivatives of the parent compound 5-(1-(4-chlorophenylsulfonyl) piperidin-3- yl)-1, 3, 4-oxadiazole-2-thiol were synthesized through reaction with different saturated and unsaturated alkyl halides in DMF medium, with NaH catalyst. Spectral characterization of each derivative was carried out with respect to IR, 1H - NMR, 13C - NMR and EI - MS. The lipoxygenase inhibitory and antibacterial activities of the derivatives were determined using standard procedures. Results: Compound 5e exhibited higher lipoxygenase inhibitory potential than the standard (Baicalein®), with % inhibition of 94.71 ± 0.45 and IC50 of 20.72 ± 0.34 μmoles/L. Compound 5b showed significant antibacterial potential against all the bacterial strains with % inhibition ranging from 62.04 ± 2.78, 69.49 ± 0.41, 63.38 ± 1.97 and 59.70 ± 3.70 to 78.32 ± 0.41, while MIC ranged from 8.18 ± 2.00, 10.60 ± 1.83, 10.84 ± 3.00, 9.81 ± 1.86 and 11.73 ± 5.00 μmoles/L for S. typhi, E. coli, P. aeruginosa, B. subtilis and S. aureus, respectively. Compounds 5d, 5e and 5g showed good antibacterial activity against S. typhi and B. subtilis bacterial strains. Conclusion: The results suggest that compound 5e bearing n-pentyl group is a potent lipoxygenase inhibitor, while compound 5b with n-propyl substitution is a strong antibacterial agent. In addition, compounds 5d, 5e and 5g bearing n-butyl, n-pentyl and n-octyl groups, respectively, are good antibacterial agents against S. typhi and B. subtilis.

Synthesis and antimicrobial activity of some 2- Piperidinomethylamino-4-(7-H/substituted coumarin-3-yl)- 6-chlorosubstitutedphenyl pyrimidines

Purpose: To prepare and evaluate some 2-piperidinomethylamino-4-(7-H/substitutedcoumarin-3-yl)-6- chlorosubstitutedphenyl pyrimidines as antimicrobial agents. Methods: Some 2-piperidinomethylamino-4-(7-H/substitutedcoumarin-3-yl)-6-chlorosubstitutedphenyl pyrimidines were prepared by reacting 2-amino-4-(7-H/substitutedcoumarin-3-yl)-6- (chlorosubstitutedphenyl) pyrimidines with piperidine and formaldehyde. The chemical structures of the synthesized compounds were elucidated by Fourier transform infrared (FTIR), 1H-nuclear magnetic resonance (1H-NMR), mass spectrometry and elemental analysis. These compounds were investigated for their antimicrobial activity against ten bacteria and five fungi by serial plate dilution method using standard drugs, namely, ofloxacin and ketoconazole, respectively, and their minimum inhibitory concentrations (MICs) were also determined. Results: A total of eighteen new compounds (1a-18a) were synthesized. Compound 6a (MIC = 50 μg/mL; p < 0.05 or less) displayed the highest activity against S. aureus , E. faecalis , Staphylococcus epidermidis , B. subtilis , and B. cereus . Compound 6a further showed good activity (MIC = 25 μg/mL; p < 0.05 or less) against E. coli ; P. aeruginosa K. pneumonia , B. bronchiseptica , and P. vulgaris . Compounds 6a (MIC = 25 μg/mL; p < 0.0001) and 17a (MIC = 25 μg/mL; p < 0.0001) displayed very good activity against C. albicans , A. niger , A. flavus , M. purpureous , and P. citrinum , respectively. Analysis of structure-activity relationship revealed that the presence of bromo group at 7-postion of the coumarin moiety along with the 4-chlorophenyl group at position-6 of the pyrimidine ring is critical for antimicrobial activity against Gram-positive bacteria, Gram negative bacteria and fungi. Conclusion: The synthesized 2-piperidino derivatives are better antifungal and antibacterial agents than the earlier reported 2-morpholino derivatives, but require further investigations against other microbial strains to ascertain their broad spectrum antimicrobial activity.

Physicochemical complexity in complex chemical systems

Self-replication and compartmentalization are two central properties thought to be essential for minimal life, and understanding how such processes interact in the emergence of complex reaction networks is crucial to exploring the development of complexity in chemistry and biology. Autocatalysis can emerge from multiple different mechanisms such as formation of an initiator, template self-replication and physical autocatalysis (where micelles formed from the reaction product solubilize the reactants, leading to higher local concentrations and therefore higher rates). Amphiphiles are also used in artificial life studies to create protocell models such as micelles, vesicles and oil-in-water droplets, and can increase reaction rates by encapsulation of reactants. So far, no template self-replicator exists which is capable of compartmentalization, or transferring this molecular scale phenomenon to micro or macro-scale assemblies. Here a system is demonstrated where an amphiphilic imine catalyses its own formation by joining a non-polar alkyl tail group with a polar carboxylic acid head group to form a template, which was shown to form reverse micelles by Dynamic Light Scattering (DLS). The kinetics of this system were investigated by 1H NMR spectroscopy, showing clearly that a template self-replication mechanism operates, though there was no evidence that the reverse micelles participated in physical autocatalysis. Active oil droplets, composed from a mixture of insoluble organic compounds in an aqueous sub-phase, can undergo processes such as division, self-propulsion and chemotaxis, and are studied as models for minimal cells, or protocells. Although in most cases the Marangoni effect is responsible for the forces on the droplet, the behaviour of the droplet depends heavily on the exact composition. Though theoretical models are able to calculate the forces on a droplet, to model a mixture of oils on an aqueous surface where compounds from the oil phase are dissolving and diffusing through the aqueous phase is beyond current computational capability. The behaviour of a droplet in an aqueous phase can only be discovered through experiment, though it is determined by the droplet's composition. By using an evolutionary algorithm and a liquid handling robot to conduct droplet experiments and decide which compositions to test next, entirely autonomously, the composition of the droplet becomes a chemical genome capable of evolution. The selection is carried out according to a fitness function, which ranks the formulation based on how well it conforms to the chosen fitness criteria (e.g. movement or division). Over successive generations, significant increases in fitness are achieved, and this increase is higher with more components (i.e. greater complexity). Other chemical processes such as chemiluminescence and gelation were investigated in active oil droplets, demonstrating the possibility of controlling chemical reactions by selective droplet fusion. Potential future applications for this might include combinatorial chemistry, or additional fitness goals for the genetic algorithm. Combining the self-replication and the droplet protocells research, it was demonstrated that the presence of the amphiphilic replicator lowers the interfacial tension between droplets of a reaction mixture in organic solution and the alkaline aqueous phase, causing them to divide. Periodic sampling by a liquid handling robot revealed that the extent of droplet fission increased as the reaction progressed, producing more individual protocells with increased self-replication. This demonstrates coupling of the molecular scale phenomenon of template self-replication to a macroscale physicochemical effect.