Nuevos ligandos poliamínicos de tipo escorpiando para aplicaciones biomédicas

La presente Tesis Doctoral nace en el marco del proyecto Consolider titulado “Aplicación de la Química Supramolecular al Diseño, Síntesis y Estudio de Compuestos Bioactivos de Acción Antiinflamatoria, Antitumoral o Antiparasitaria” (CSD2010-00065, BOE del 27 de febrero de 2010). El principal objetivo de este proyecto era la formación en España de un grupo de investigación multidisciplinar dedicado a la Química Médica Supramolecular (SUPRAMED, www.supramedic.com) que centraría sus esfuerzos, como su nombre indica, en el desarrollo de nuevos agentes terapéuticos con actividad antiparasitaria, antiinflamatoria o antitumoral. Como miembro investigador en este proyecto (dentro del grupo de Química Supramolecular de la Universidad de Valencia), el autor de la presente tesis ha orientado su trabajo a desarrollar y cumplir con las metas del mismo. Los sistemas estudiados, concretamente, corresponden a una familia de compuestos denominados poliaminas de tipo escorpiando. Estos compuestos son capaces de generar movimientos moleculares debido a cambios conformacionales inducidos por estímulos externos, tales como el pH o la presencia de cationes metálicos. Estos movimientos se asemejan a los de algunas proteínas, que modifican su actividad por un mecanismo conocido como regulación alostérica. En la presente tesis se sintetizaron once nuevos ligandos poliamínicos de tipo escorpiando. Este proceso requirió, en algunos casos, el desarrollo y puesta a punto de nuevas rutas sintéticas. Dado que las poliaminas poseen, por definición, varios grupos susceptibles a la protonación y que las características químicas y su actividad biológica pueden depender de la especie concreta que hay en disolución a un determinado valor de pH, se determinaron, para todos los ligandos sintetizados, las constantes de protonación y las constantes de estabilidad con diferentes metales de transición (cobre, zinc, manganeso y hierro) mediante técnicas potenciométricas y espectroscópicas. Para determinar su potencial como agentes antitumorales, se realizaron estudios de interacción con ácidos nucleicos, utilizando diversas secuencias de ADN y de ARN, tanto monhebra como de doble hebra. Así mismo se realizaron estudios in vitro con diferentes líneas celulares y estudios preliminares con ratones inmunodeprimidos. También se evaluó la capacidad de los complejos de manganeso (II) para mimetizar el comportamiento de la superóxido dismutasa. Para ello se determinaron los potenciales redox de los complejos de manganeso mediante técnicas electroquímicas y se realizaron estudios cinéticos indirectos para evaluar la actividad de los complejos para dismutar eficientemente el radical superóxido en disolución acuosa. Para complementar estos estudios se realizaron ensayos in vitro con cultivos de bacterias deficientes de la enzima SOD para evaluar el posible efecto protector de los complejos metálicos más prometedores. Aprovechando la red multidisciplinar de grupos de investigación organizada dentro del proyecto SUPRAMED se establecieron colaboraciones para determinar la citotoxicidad de los compuestos estudiados, así como la posibilidad de modular su actividad biológica mediante estímulos externos, como la presencia de cationes metálicos. Finalmente, en colaboración con el Grupo de Parasitología Molecular, del Departamento de Parasitología de la Universidad de Granada, se determinó la eficiencia de los compuestos sintetizados para actuar como agentes tripanocidas frente a la enfermedad de Chagas y la leishmaniasis, obteniéndose resultados prometedores. En el transcurso de esta tesis se utilizaron una gran variedad de técnicas experimentales: potenciometría, resonancia magnética nuclear, espectroscopia UV-Vis, emisión de fluorescencia, dicroísmo circular, viscosimetría, voltamperometría cíclica, difracción de rayos-X.This PhD thesis collects part of the work done under the Consolider project entitled “Supramolecular chemistry applied to the design, synthesis and evaluation of bioactive compounds of antiinflammatory, antitumour and antiparasitic activity” (CSD2010-00065, BOE from February 27, 2010). The main objective of this project was to develop a research group in Spain dedicated to the Supramolecular Medicinal Chemistry (SUPRAMED, www.supramedic.com) which would focus its efforts, as its name point out, to the development of new therapeutic agents with antiparasitic, antiinflammatory and antitumoral activity. As a research member of this project (on the Supramolecular Chemistry Group of the University of Valencia), the author of this thesis has oriented his work to achieving these objectives. The systems studied were, specifically, from a family of compounds known as scorpiand-type polyamines. These compounds are capable of generating molecular movements due to conformational changes induced by external stimuli, such as pH changes and the presence of metal cations. These movements are comparable to those of certain proteins, which modify their activity through a mechanism known as allosteric regulation. In this thesis, eleven new polyamine ligands were synthesized. This process required, in some cases, the development and optimization of new synthetic routes. Since polyamines have, by definition, several groups susceptible to be protonated and since the chemical characteristics and biological activity of these compounds might depend on the specific species present in aqueous solution at a determined pH value, the protonation constants and the stability constant with several transition metals (copper, zinc, and iron) were determined for all of them by means of potentiometric and spectroscopic titrations. The interaction with nucleic acids was studied in order to evaluate the potential of the compounds as antitumor agents, using different sequences of DNA and RNA, both single and double stranded. This studied were complemented with in vitro viability assays, using different cancer cell lines, and with preliminary studies on immunosuppressed mice. The ability of the Mn(II) complexes of this ligands to mimic the behavior of superoxide dismutase was also evaluated. With this objective, the redox potentials of the manganese complexes was determined through electrochemical measurements. The ability of this complexes to efficiently dismutate the superoxide radical in aqueous solution was determined by indirect kinetic assays. To complete this studies, in vitro assays were performed using SOD deficient bacteria to evaluate the protective effect of the most promising compounds. Taking advantage of the multidisciplinary nature of the SUPRAMED project, collaborations with different groups were stablished in order to determine the cytotoxicity of the studied compounds, as well as to investigate the possibility of controlling their biological activity through external stimuli, like the presence of metal cations. Finally, in collaboration with the Parasitology Group of the University of Granada, the potential of the synthesized compounds to act as trypanocidic agents, against Chagas disease and leishmaniasis, was determined, obtaining promising results. During the research years conducting to the realization of this thesis different experimental techniques were used: potentiometry, nuclear magnetic resonance, UV-Vis spectroscopy, steady-state fluorescence emission, circular dichroism, viscometry, cyclic voltammetry, X-ray diffraction

Molecular recognition of N-acetyltryptophan enantiomers by β-cyclodextrin

The enantioselectivity of β-cyclodextrin (β-CD) towards L- and D-N-acetyltryptophan (NAcTrp) has been studied in aqueous solution and the crystalline state. NMR studies in solution show that β-CD forms complexes of very similar but not identical geometry with both L- and D-NAcTrp and exhibits stronger binding with L-NAcTrp. In the crystalline state, only β-CD-L-NAcTrp crystallizes readily from aqueous solutions as a dimeric complex (two hosts enclosing two guest molecules). In contrast, crystals of the complex β-CD-D-NAcTrp were never obtained, although numerous conditions were tried. In aqueous solution, the orientation of the guest in both complexes is different than in the β-CD-L-NAcTrp complex in the crystal. Overall, the study shows that subtle differences observed between the β-CD-L,D-NAcTrp complexes in aqueous solution are magnified at the onset of crystallization, as a consequence of accumulation of many soft host-guest interactions and of the imposed crystallographic order, thus resulting in very dissimilar propensity of each enantiomer to produce crystals with β-CD

Ditopic Aza-Scorpiand Ligands Interact Selectively with ds-RNA and Modulate the Interaction upon Formation of Zn2+ Complexes

Nucleic acids are essential biomolecules in living systems and represent one of the main targets of chemists, biophysics, biologists, and nanotechnologists. New small molecules are continuously developed to target the duplex (ds) structure of DNA and, most recently, RNA to be used as therapeutics and/or biological tools. Stimuli-triggered systems can promote and hamper the interaction to biomolecules through external stimuli such as light and metal coordination. In this work, we report on the interaction with ds-DNA and ds-RNA of two aza-macrocycles able to coordinate Zn2+ metal ions and form binuclear complexes. The interaction of the aza-macrocycles and the Zn2+ metal complexes with duplex DNA and RNA was studied using UV thermal and fluorescence indicator displacement assays in combination with theoretical studies. Both ligands show a high affinity for ds-DNA/RNA and selectivity for ds-RNA. The ability to interact with these duplexes is blocked upon Zn2+ coordination, which was confirmed by the low variation in the melting temperature and poor displacement of the fluorescent dye from the ds-DNA/RNA. Cell viability assays show a decrease in the cytotoxicity of the metal complexes in comparison with the free ligands, which can be associated with the observed binding to the nucleic acids