Reactivity at the membrane interface

Modulation of internal environment and maintenance of cellular structure and stability are basic requirements to ensure cell survival. These cellular functions are provided by the cell outer membrane, a phospholipid bilayer characterised by the fluid mosaic model. Chemical reactivity at the membrane interface has previously been identified between phospholipids and membrane binding species. Observed reactivity, termed intrinsic lipidation, involves non-enzymatic acyl transfer from phospholipids to a nucleophilic membrane bound molecule. Reactivity has been characterised for membrane active peptides and proteins, and been found influential to the structure and function of both the newly modified species, and the bulk membrane. Research presented within this thesis probes fundamental features of observed intrinsic lipidation reactivity at the membrane interface. This work has expanded upon previous intrinsic lipidation research, facilitated by the development of informative and robust analytical techniques for the study of reactivity. Optimised TLC has allowed improved routine high throughput reactivity screening, compared to alternative fluorescence and solution state NMR techniques. Informative analysis and mechanistic understanding of intrinsic lipidation has been achieved through LCMS and solid state NMR analysis. Synthetic protocols for preparation of isotopically labelled 15N small molecules, and 13C phospholipids, has facilitated solid state NMR in particular. Biological relevance of peptide intrinsic lipidation has also been probed to determine the role of reactivity in natural function, and disease induction. Biophysical techniques such as CD and tryptophan fluorescence revealed that solution phase intrinsically lipidated melittin adopts an α-helical structure with central proline kink, in contrast to the random coil of unmodified melittin. Furthermore, at μM concentrations, palmitoylated species were shown to undergo spontaneous micelle formation. Disease related behaviour linked to peptide intrinsic lipidation includes moderate antimicrobial activity, and possible induction of amyloid nucleation. Additionally, this study has identified novel intrinsic lipidation of small molecules in vitro utilising chromatographic and ionisation conditions optimised with synthetically prepared standards. Observed for multiple cationic amphiphilic small molecules, intrinsic lipidation was promoted by primary amines in a hydrophilic environment, due to increased proximity between reactive moieties. Small molecule intrinsic lipidation products were shown to exhibit biological relevance, including spontaneous micelle formation, membrane disruption, and phospholipidosis induction. Pharmaceutical propranolol displayed notable intrinsic lipidation in vitro, and in Hep G2 cell culture. Initial transesterification from membrane phospholipids produced O-acylated propranolol, followed by secondary N-acylated propranolol formation by intramolecular O to N migration. Study of propranolol reactivity has revealed preferential eukaryotic transfer from the sn-1 phospholipid backbone position, and reaction kinetics influenced by temperature, pH, and membrane composition

De novo development of novel DM1 toxic ncRNA targeting small molecules and its biological evaluation

La distròfia miotònica de tipus 1 (DM1) és un trastorn neuromuscular incurable causat per les transcripcions tòxiques del gen DMPK. Aquests transcrits porten expansions de repeticions CUG a les regions no traduïdes 3′ (3′UTR). La complexitat intrínseca i la falta de dades cristal·logràfiques fan que les regions d'ARN no codificant siguin objectius difícils d'estudiar en el camp del desenvolupament de nous fàrmacs. En el cas de la DM1, els transcrits tòxics tendeixen a estancar-se a l'interior dels nuclis formant complexos cossos d'inclusió anomenats foci i segrestant molts factors de splicing alternatiu essencials com el Muscleblind-like 1 (MBNL1). La majoria de les característiques fenotípiques de la DM1 es deriven de la reduïda disponibilitat de MBNL1 lliure, per la qual cosa molts esforços terapèutics es centren en recuperar la seva activitat regular. Per a això, en la present tesi, decidim utilitzar com a diana terapèutica l'ARN CUG, amb la finalitat d'alliberar MBNL1. Pel que respecta al disseny de noves estructures, es descriu el cribratge in-silico mitjançant tècniques de disseny de fàrmacs basades en estructura usant dues premisses diferents d'abordar CUG. A més, es desenvolupen vies sintètiques per als candidats seleccionats basades en química clic. Finalment, per a avaluar la seva activitat biològica, es posa a punt un assaig bioquímic ja descrit, i s'utilitzen models cel·lulars i cèl·lules musculars derivades de pacients per a avaluar els candidats més prometedors. Els resultats obtinguts poden conduir a posteriors generacions de lligands, posant de manifest un nou tractament assequible contra la DM1.La distrofia miotónica de tipo 1 (DM1) es un trastorno neuromuscular incurable causado por las transcripciones tóxicas del gen DMPK. Estos transcritos llevan expansiones de repeticiones CUG en las regiones no traducidas 3′ (3′UTR). La complejidad intrínseca y la falta de datos cristalográficos hacen que las regiones de ARN no codificante sean objetivos difíciles de estudiar en el campo del desarrollo de nuevos fármacos. En la DM1, los transcritos tóxicos tienden a estancarse en el interior de los núcleos formando complejos cuerpos de inclusión llamados foci y secuestrando muchos factores de splicing alternativo esenciales como el Muscleblind-like 1 (MBNL1). La mayoría de las características fenotípicas de la DM1 se derivan de la reducida disponibilidad de MBNL1 libre, por lo que muchos esfuerzos terapéuticos se centran en recuperar su actividad regular. Para ello, en la presente tesis, decidimos utilizar como diana terapéutica el ARN CUG, con el fin de liberar MBNL1. Por lo que respecta al diseño de nuevas estructuras, se describe el cribado in-silico mediante técnicas de diseño de fármacos basadas en estructura usando dos premisas diferentes de abordar CUG. Además, se desarrollan vías sintéticas para los candidatos seleccionados basadas en química click. Por último, para evaluar su actividad biológica, se pone a punto un ensayo bioquímico ya descrito, y se utilizan modelos celulares y células musculares derivadas de pacientes para evaluar los candidatos más prometedores. Los resultados obtenidos pueden conducir a posteriores generaciones de ligandos, poniendo de manifiesto un nuevo tratamiento asequible contra la DM1.Myotonic Dystrophy type 1 (DM1) is an incurable neuromuscular disorder caused by toxic DMPK transcripts that carry CUG repeat expansions in the 3′ untranslated regions (3′UTR). The intrinsic complexity and lack of crystallographic data make noncoding RNA regions challenging targets to study in the field of drug discovery. In DM1, toxic transcripts tend to stall in the nuclei forming complex inclusion bodies called foci and sequestering many essential alternative splicing factors such as Muscleblind-like 1 (MBNL1). Most DM1 phenotypic features stem from the reduced availability of free MBNL1, and therefore many therapeutic efforts are focused on recovering its regular activity. For that purpose, in the present thesis, we decide to target CUG RNA to free MBNL1. The in-silico screening using structure-based drug design techniques of novel candidates based on two different approaches is described. Furthermore, synthetic pathways are developed for the selected candidates based on the click chemistry approach. Finally, to assess their biological activity, an already described biochemical test is tuned, and cellular models and patient-derived muscular cells are used to evaluate the most promising candidates. The obtained results may lead to subsequent generations of ligands, highlighting a new affordable treatment against DM1

New Methodologies in Transition Metal-Mediated (11C)Radiolabelling for Positron Emission Tomography

This thesis focuses on the requirement for new methodologies to incorporate 11C into biologically active compounds for Positron Emission Tomography (PET). The use of PET has expanded over recent years as a valuable imaging technique for the study of drug delivery as well as the study of neurological diseases and oncology. The short half-life of 11C (20.4 min) limits its use to approximately 1 hour therefore rapid chemistry is required to radiolabel tracer molecules before use in vivo. Chapter 1 includes an overview of the literature in the areas of PET radiochemistry, transition metal-catalysed carbonylation, N-heterocyclic carbenes, hemilabile ligands, titanium and zirconium imido complexes and nickel-/palladium-catalysed carboxylations. Chapters 2 and 3 detail the investigations of a palladium-catalysed carbonylation reaction for the formation of amides commonly found in biologically active compounds. N-benzylbenzamide was synthesised in a model reaction which tested a range of diphosphine ligands and N-heterocyclic carbenes. The best performing systems were taken forward to be tested with [11C]CO in the radiochemistry laboratories at the GSK Clinical Imaging Centre at Hammersmith Hospital with excellent radiochemical purities of [11C]N-benzylbenzamide (99 %) and good radiochemical yields (55 %). Hemilabile phosphorus-thioether (P-S) and phosphorus-ether (P-O) bidentate ligands were also investigated in the synthesis of N-benzylbenzamide and compared to diphosphine ligands in this reaction and new palladium catalysts were prepared with novel P-S ligands. [11C]CO2 was examined as a potentially useful source of [11C] for radiolabelling as research into transition metal-mediated processes to activate CO2 for environmental reasons has grown in recent years. Chapter 4 investigates some recent literature detailing nickel- and palladium-catalysed carboxylation of organozinc halides in the synthesis of carboxylic acids. Titanium and zirconium imido complexes were synthesised with the intention of forming [11C]isocyanate precursors - this work is described in Chapter 5. A range of work has been carried out and potential future studies in all these areas are specified at the end of the thesis

Development of new strategies for the design of in situanalysis devices: nano and biomaterials

La Tesis describe el concepto de análisis in situ y biomateriales que han dado lugar al desarrollo de varios dispositivos de análisis in situ. Estos dispositivos se basan principalmente en la inmovilización de reactivos en soportes sólidos. Se han empleado nano y (bio) materiales en el desarrollo de varios (bio) sensores o kits. Para el desarrollo de los dispositivos se consideraron los siguientes dos puntos críticos: i) la selección de los materiales de soporte donde tiene lugar la inmovilización de los reactivos, ii) la reacción involucrada en el procedimiento. Es importante estudiar la reacción entre el material y los reactivos para comprender la liberación del reactivo a la disolución o la entrada de los analitos al soporte sólido. El material de soporte no debe reaccionar con el reactivo ni interferir con la medida. Algunos de los materiales propuestos en esta Tesis como soportes sólidos para el diseño de sensores in situ han sido zeína, nailon, PDMS y nanocelulosa. Asimismo, se ha realizado la caracterización de los dispositivos desarrollados. Se han propuesto nuevas metodologías de análisis in situ basadas en el empleo de (bio) sensores para la determinación de compuestos relevantes como H2O2, fosfato o fármacos en matrices reales complejas como muestras de suero, orina o agua ambiental. Estos análisis han empleado diferentes respuestas analíticas en función de la sensibilidad requerida.There is great need, in general, to develop sustainable analytical methodologies based on the miniaturization, simplification and analytical process automation, with the aim of reducing the environmental impact without compromising the selectivity and sensitivity. Specifically, biosensors, are emerging as a fast and simple method for the in situ detection of compounds in several fields such as healthcare and food and drink industry including environmental and security monitoring among others. The advantages of in situ analysis are (1) in most cases the determination is carried out without isolation of the analyte from its environment, so the sample is not altered from its original conditions and (2) the analytical process including sampling is physically carried out in space and in time reducing the time of the analysis. Also, in situ analyses generally do not require sample treatment which, according to Green Analytical Chemistry principles, minimizes waste generation. On the other hand, Green Chemistry includes the use of safe and clean material and methods to decrease the adverse effects of pollution on the environment. The use of polymer-based biomaterials such as polysaccharides and proteins appears as a responsible option since it allows microorganisms to degrade these materials and directly reduce waste generation. Moreover, biomaterials have generated great interest due to their biocompatibility in food and medical area. The Thesis describes the concept of in situ analysis and biomaterials which have been resulting in the development of several situ devices. These devices are mainly based on the reagent immobilization in solid supports. Nano and (bio)materials have been employed in the development of several (bio)sensors or kits. In order to develop the devices, the two critical points were considered: i) the selection of the support materials where the immobilization of the reagents takes place, ii) the reaction involved in the procedure. It is important to study the reaction between the material and the reagents in order to understand the reagent release to dissolution or the entry of analytes to the solid support. The support material must not react with the reagent or interfere with the measurement. Some of the materials proposed in this Thesis as solid supports for the design of in-situ sensors have been zein, nylon, PDMS and nanocellulose. The characterization of the devices has been performed. New methodologies for analysis in situ based on the employment of (bio)sensors have been proposed for the determination of relevant compounds such as H2O2, phosphate or drugs in complex real matrices like serum, urine or environmental water samples. These analyses have employed different analytical responses depending on the sensibility required. These approaches have been validated and its analytical properties have been compared with other already existing

Study of peptide-mineral interactions

The studies of peptide-mineral interactions presented within this thesis aimed to identify and understand the effect(s) induced by peptides/selected motifs on amorphous silica and crystalline zinc oxide (ZnO) formation. The effect of imidazole functionality on silica formation was studied using polyvinylimidazole (PVI) and polyhistidine (P-His). The effect of zinc oxide-binding peptides (ZnO-BPs) on the morphology and formation of ZnO were studied using G-12 (GLHVMHKVAPPR) and EM-12 (EAHVMHKVAPRP), and their derivatives, GT-16 (GLHVMHKVAPPRGGGC) and EC-12 (EAHVCHKVAPRP) respectively. The influence of these additives on reaction kinetics, their effect on the precipitates, and their level of incorporation into the precipitates were investigated. This series of studies revealed three common characteristics of peptide-mineral (ZnO-BPs-ZnO and imidazole-silica) interactions. Firstly, a specific functionality of the biomolecule was responsible for the effect induced while a supporting functionality enhanced the effect. The imidazole group of PVI and P-His catalysed the condensation of monosilicic acid but the peptide backbone and more flexible imidazole enhanced the catalytic capability of P-His with respect to PVI having a similar concentration of imidazole groups. The presence of G-12 and GT-16 reduced the aspect ratio of ZnO crystals formed via an adsorption-growth inhibition mechanism. However the addition of a GGGC-tag on GT-16 weakens the adsorption of GT-16 on the (10-10) face of ZnO crystals. This gave rise to selective adsorption of GT-16 on the (0002) face, with a greater reduction of the crystal aspect ratio. For the EM/EC-12 peptides, metal ion complexation that leads to a delay/suppression of ZnO formation was higher for EC-12 compared to EM-12 and was caused by the more efficient complexation of Zn2+ with the peptide containing cysteine. Secondly, additives can interact with different species in the reaction. Imidazole interacts with neutral monosilicic acid via hydrogen bonds but protonated species of imidazole interact with anionic polysilicic acid via electrostatic interactions. Although EM-12 only interacts with Zn2+ in solution, EC-12 was able to interact reversibly with the solid phases formed in the course of reaction. Thirdly, the type of interaction and interacting species determine the level of additive incorporation and its effect on the concentration of reactants remaining in solution when equilibrium is reached. Peptide-surface interactions generally result in incorporation of the peptide into the solid phase (G-12, GT-16, and EC-12) as opposed to the situation where complexation of reactant species in solution shown by EM-12 did not result in peptide incorporation. The use of peptides/motifs (hiomolecules) isolated from combinatorial libraries for silica and ZnO synthesis has been shown to he a promising approach for morphology and reaction control. It is particularly exciting when their use can he extended to the generation of ZnO which has non-biological origin. By understanding the phenomenal complexity of the behaviour of biomolecules in (hio)mineralising systems, the ground rules in their interactions (with species in (hio)mineralising systems) can be generated and exploited for the synthesis of novel nanomaterials with properties tailored for specific applications

Exploitation of Biomass for Applications in Sustainable Materials Science

Biorefinery may be defined as the process of accessing chemical commodities from living systems; consequently, biomass becomes the antecedent for renewable resources through biorefinery. Advantages to this process over petroleum refinery include: (1) increased potential for sustainable products, (2) increased diversity in chemical structure including heterocycles, and (3) potential for regional resource independence. Despite these clear advantages, adoption of biorefined commodities can be limited by the risk associated with small initial application portfolios and concomitant uncertainties. The strategies adopted by our dynamic and collaborative research team entail continuous engagement of those issues by: (1) preparing renewable polymers, (2) chemical diversification of biomass-derived platform chemicals, (3) direct modification of biopolymers, and (4) development of petroleum replacements. Battling the inveterate proclivity towards portents of gloom need not solely justify investigations into biorenewable feedstock chemicals; the ramifications of bioinspired molecular inquiry create opportunities to go beyond mere sustainability through innovation. This dissertation includes specific examples which illustrate utilization of three types of biomass: (1) oil seeds, (2) lignin, and (3) carbohydrates. Each class of biomass-derived materials offered unique advantages as well as challenges associated with their varied structures. The presentation has been divided into five sections: (1) biomass, sustainable chemistry and design thinking; (2) styrene replacements and their application in renewable vinyl ester thermosets; (3) catalyst-free lignin valorization by acetoacetylation; (4) chemical diversification of 5-(hydroxymethyl)furfural; (5) valorization of cellulose-derivable platform chemicals by cycloaddition with a potentially bioderivable reactive intermediate: benzyne.National Science Foundation (NSF) (Grants IIA-1330840 and IIA-1355466)ND-EPSCoR (Doctoral Dissertation Award

Expression of multidisciplinary flavour science : proceedings of the 12th Weurman Symposium

The 12th Weurman Flavour Research Symposium contributed 177 lectures and posters to the wealth of flavor knowledge; these were presented in eight sessions: biology, retention and release, psychophysics, quality, thermal generation, bioflavors, impact molecules, and analytics. Emerging topics were discussed in three workshops dealing with flavor and health, in vivo flavor research, and flavor metabolomics. It has been an excellent forum for passionate exchange of recent results obtained in traditional and emerging fields of flavor research. The symposium allowed coverage of the broad diversity of flavor-related topics: comprising odor and taste; applying targeted and holistic approaches; using sensorial, chemical, biological, physical, and chemometric techniques; as well as considering nutrition and health aspects

Expression of multidisciplinary flavour science : proceedings of the 12th Weurman Symposium

The 12th Weurman Flavour Research Symposium contributed 177 lectures and posters to the wealth of flavor knowledge; these were presented in eight sessions: biology, retention and release, psychophysics, quality, thermal generation, bioflavors, impact molecules, and analytics. Emerging topics were discussed in three workshops dealing with flavor and health, in vivo flavor research, and flavor metabolomics. It has been an excellent forum for passionate exchange of recent results obtained in traditional and emerging fields of flavor research. The symposium allowed coverage of the broad diversity of flavor-related topics: comprising odor and taste; applying targeted and holistic approaches; using sensorial, chemical, biological, physical, and chemometric techniques; as well as considering nutrition and health aspects