Periodic Reporting for period 4 - ORGANITRA (Transport of phosphorylated compounds across lipid bilayers by supramolecular receptors)
Reporting period: 2023-07-01 to 2024-12-31
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 was to develop transporters for phosphates and biologically relevant organic phosphorylated compounds, such as nucleotides. This required the design and synthesis of molecules that could strongly interact with anionic phosphate groups, to be able to extract the phosphates from a solution in water into the more fat-like membrane. And then we had to test these molecules as transporters.
We have explored two ways to make such molecules. The first consisted of using ‘dynamic covalent chemistry’, which is a way to link parts of molecules together but still allowing them to exchange. Secondly, we have used 3D molecular models to design molecules that make a nice fit for phosphate using a computer and then have prepared the most promising molecules by organic synthesis. This resulted in molecules that can strongly bind inorganic phosphate, pyrophosphate, and other anions.
We have then developed methods to study these molecules as ion transporters. For this, we have used liposomes as model systems. Inside these liposomes, we encapsulated probes that become more or less fluorescent when interacting with anions. We could thus monitor the transport of phosphate by our synthetic compounds as an increase in the light released by these probes. We have used NMR spectroscopy to confirm these findings.
Overall, we have found that is it possible to transport phosphates across membrane, but that it is much more difficult compared to other anions and requires a transporter that is very well adapted for this process. Further transport studies on phosphate esters and carboxylates have provided more insight into the role of the organic moiety on the transport of anions, which is crucial for applications in drug delivery. Furthermore, we have found that dynamic covalent chemistry can be used to control the rates of anion transport across a lipid membrane.
The main aim of this project was to develop transporters for phosphates and biologically relevant organic phosphorylated compounds, such as nucleotides. This required the design and synthesis of molecules that could strongly interact with anionic phosphate groups, to be able to extract the phosphates from a solution in water into the more fat-like membrane. And then we had to test these molecules as transporters.
We have explored two ways to make such molecules. The first consisted of using ‘dynamic covalent chemistry’, which is a way to link parts of molecules together but still allowing them to exchange. Secondly, we have used 3D molecular models to design molecules that make a nice fit for phosphate using a computer and then have prepared the most promising molecules by organic synthesis. This resulted in molecules that can strongly bind inorganic phosphate, pyrophosphate, and other anions.
We have then developed methods to study these molecules as ion transporters. For this, we have used liposomes as model systems. Inside these liposomes, we encapsulated probes that become more or less fluorescent when interacting with anions. We could thus monitor the transport of phosphate by our synthetic compounds as an increase in the light released by these probes. We have used NMR spectroscopy to confirm these findings.
Overall, we have found that is it possible to transport phosphates across membrane, but that it is much more difficult compared to other anions and requires a transporter that is very well adapted for this process. Further transport studies on phosphate esters and carboxylates have provided more insight into the role of the organic moiety on the transport of anions, which is crucial for applications in drug delivery. Furthermore, we have found that dynamic covalent chemistry can be used to control the rates of anion transport across a lipid membrane.
In the first part of this project, we have synthesized many organic compounds that are able to bind phosphate and other anions. The best results were obtained with compounds with well pre-organised hydrogen bond donors connected to a scaffold which keeps them in the correct position for binding the phosphate. On the other hand, some flexibility in the compounds was required for the most effective binding.
In the second part of the project, we have tested if these compounds could transport phosphate and other anions. Unfortunately, many were not good in transporting anions, and we found out that this was because they were binding strongly to the phosphate groups in the lipids. Some other compounds were very good transporters for chloride anions, but not for phosphate. Only the compounds that could most efficiently encapsulate phosphate anions were successful in transporting phosphate. To find this out, we had to develop the required methods, as no reports on the transport of phosphate into liposomes were existing. We have managed to do this successfully using emissive probes prepared at Loughborough university , which could selectively sense different anions, including nucleotides and phosphate. Encapsulating these probes in liposomes resulted in the required methodology, which can also be used to study bicarbonate and fluoride transport.
Thirdly, we have explored applications for our transporters in (synthetic) cells.
The results of this project were shared in 19 scientific publications (and several more are being prepared) and in at least 56 contributions to international and national conferences, such as the International Symposium on Macrocyclic and Supramolecular Chemistry (ISMSC), the International Symposium on Transmembrane Transporters, the international Calixarene conference, the European Symposium on Organic Chemistry (ESOC), the EuChemS Chemistry Congress, the RSC Macrocyclic and Supramolecular Chemistry Meeting MASC), the Merck Organic Chemistry Symposium, and many more. Short research updates were also shared via social media.
In the second part of the project, we have tested if these compounds could transport phosphate and other anions. Unfortunately, many were not good in transporting anions, and we found out that this was because they were binding strongly to the phosphate groups in the lipids. Some other compounds were very good transporters for chloride anions, but not for phosphate. Only the compounds that could most efficiently encapsulate phosphate anions were successful in transporting phosphate. To find this out, we had to develop the required methods, as no reports on the transport of phosphate into liposomes were existing. We have managed to do this successfully using emissive probes prepared at Loughborough university , which could selectively sense different anions, including nucleotides and phosphate. Encapsulating these probes in liposomes resulted in the required methodology, which can also be used to study bicarbonate and fluoride transport.
Thirdly, we have explored applications for our transporters in (synthetic) cells.
The results of this project were shared in 19 scientific publications (and several more are being prepared) and in at least 56 contributions to international and national conferences, such as the International Symposium on Macrocyclic and Supramolecular Chemistry (ISMSC), the International Symposium on Transmembrane Transporters, the international Calixarene conference, the European Symposium on Organic Chemistry (ESOC), the EuChemS Chemistry Congress, the RSC Macrocyclic and Supramolecular Chemistry Meeting MASC), the Merck Organic Chemistry Symposium, and many more. Short research updates were also shared via social media.
All results described above are a significant progress beyond the state of the art prior to this project and we have made very significant contributions to the development of synthetic transporters for oxoanions. In particular, we have reported the first example of a synthetic transporter for phosphate, as well as the methodology required to demonstrate this. Furthermore, we have demonstrated that dynamic covalent chemistry can be used to control the rates of anion transport across a lipid membrane.