Periodic Reporting for period 2 - ORGANITRA (Transport of phosphorylated compounds across lipid bilayers by supramolecular receptors) Reporting period: 2020-07-01 to 2021-12-31 Summary of the context and overall objectives of the project The ORGANITRA project deals with the transport of ions through biological membranes, which we find around cells and organelles. These membranes are composed of two layers of lipid molecules and ions cannot just pass through these membranes. For that reason, cell membranes contain proteins that take care of the transport of ions. Certain molecules can also transport ions across membranes and, in contrast to most proteins, they are small and can move from one side of the membrane to the other, taking a cargo along. We are developing such transport molecules, which could be used in the treatment of diseases by taking over the role of a poorly functioning transport protein or by bringing other medicines into cells. They can also be used to bring anions into or out of cells for biochemical research and they could do the same in artificial cells.The main aim of this project is to develop transporters for phosphates and biologically relevant organic phosphorylated compounds, such as nucleotides. For this, we first have to synthesize receptors, which are organic molecules that can bind these phosphates, and then study them as transporters.One of the strategies we use to synthesize receptors is dynamic combinatorial chemistry. Building blocks containing anion binding groups, such as ureas or thioureas, are connected to each other by hydrazones and other dynamic covalent groups. The dynamic character of these bonds is used to identify efficient receptors from libraries of compounds, using different phosphates as templates. With this approach, selective receptors for different nucleotides and related compounds can be obtained.The transport performance of the receptors is evaluated with newly developed assays. We use liposomes as model systems and monitor transport by fluorescence spectroscopy, using changes in the emission of an encapsulated phosphate sensitive probe, or by 1H and 31P NMR spectroscopy. Transmembrane carriers for phosphorylated compounds will make it possible to selectively introduce nucleotides into liposomes and cells, opening the way to for instance fuel enzymes with adenosine triphosphate (ATP) in liposomes as biotechnological nanoreactors or to study nucleotide-dependent biochemical processes in cells. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We have synthesized various compounds that are able to bind phosphate and other anions. However, several of these receptors turned out to be poor transporters. We have learnt that additional functional groups that are present in the structure of the receptor, and especially H-bond donors such as acylhydrazones, can lead to strong interactions with lipid head groups and thus inhibit the transport process. We have also developed methodologies to monitor the transport of anions in a direct way. Our study on bicarbonate transport, for which we have encapsulated in liposomes a europium complex of which the emission intensity increases upon binding bicarbonate, is now published. By monitoring the increase of the bicarbonate concentration inside liposomes, we were able to study the mechanisms of the transport process and found that a combination of diffusion of neutral species and pH dissipation by anion transporters could lead to apparent transport of bicarbonate, without the anion crossing the membrane. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) We will continue the development of receptors and transporters for phosphate and phosphorylated compounds and show that these substrates can not only be transported by proteins, but also by synthetic compounds.