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Studies of transmembrane transport by synthetic carriers for phosphate, fluoride and chloride

par Cataldo, Alessio
Président du jury Bruylants, Gilles
Promoteur Valkenier, Hennie
Publication Non publié, 2023-12-08

Thèse de doctorat

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Résumé :

In recent decades, the study of anion transport across model lipid bilayer membranes has evolved rapidly. Initially focused on chloride transport using simple synthetic transporters, the field has expanded to encompass the investigation of different anions, the development of new monitoring methods for the understanding of the process, and the creation of new transporters. This thesis touches on all these aspects and can be divided in three parts, each dealing with the transport of a different anion.After a general introduction of the concepts of anion transmembrane transport and the use of synthetic transporters, the second chapter of the thesis addresses the transport of phosphate through the lipid membranes of liposomes, a process never reported previously. The work focuses primarily on the development of two methods for monitoring the transport process: the [Eu.pBOH2]+ assay and the 31P NMR assay. The former relies on emission spectroscopy, while the latter utilizes NMR spectroscopy.Each assay came with its own distinct set of challenges. These include the research of a suitable phosphate-sensitive probe and the acquisition of consistent and clear results, in an emission-based assay. Meanwhile, for the NMR-based assay, the primary aim was to enhance sensitivity and optimize the signal-to-noise ratio. We discuss these challenges and the successful implementation of both methods, along with their application in studying phosphate transport mediated by synthetic carriers. Our results conclusively demonstrate the feasibility of phosphate transport and highlight the influence of pH on the process, given by the prevalence of different forms of the anion, namely H2PO4– (the transported form) and HPO42–. Additionally, our exploration of various efficient chloride transporters, found to be poor phosphate transporters, underscores the difficulty of transporting phosphate.Successively, in the third chapter, we focus on fluoride transport, with a primary emphasis on mechanistic studies to gain a deeper understanding of the process. The mechanisms involved in the transport of fluoride are governed by its ability to easily diffuse across membranes in the form of HF. This can lead to misleading conclusions. To address this challenge, we have adapted an existing method, the EuL1 assay, to directly monitor fluoride transport using emission spectroscopy. This assay offers advantages like sensitivity, versatility, and the ability to obtain detailed mechanistic insights. Additionally, we introduce the innovative use of time-resolved emission spectroscopy for studying anion transport, enabling the utilization of fluorescent transporters. Throughout our work, we employ three existing anion transporters, each exhibiting different behaviours, allowing us to explore diverse scenarios.The fourth chapter focuses on the study of chloride transport mediated by stimuli-responsive compounds. We initially discuss the synthesis of a photo-switchable bisazobenzene-based transporter, designed for tuneable anion transport activity under specific wavelengths of light and address the challenges encountered during its synthesis and purification (resulting in an impure product). Our results reveal that while the compound exhibits notable photoisomerization properties, it showed poor chloride binding and transport activities regardless of its isomerization state. Finally, we report for the first time the use of croconamide-based compounds as chloride transporters. Three compounds were tested, a simple croconamide, found to be inactive, a malonate-functionalised derivate active as a moderate non pH-dependent transporter and a malonitril derivate showing remarkable pH-switchable transport activity. Also, real-time in-situ pH switchable chloride transport is monitored via emission spectroscopy and the relationship between the compound structures and their transport activity is discussed.In conclusion, this thesis explores the evolving field of anion transport across lipid bilayer membranes addressing some of its main aspects: the need of studying the transport of new species, the understanding of complex transport processes, the development of new transporters and the relationship between molecular structures and transport activity.