Final Report Summary - PHOSPHORECEPTORS (Synthesis of novel metallo-receptors for phosphorylated species via dynamic combinatorial chemistry)
This project was funded for the development of selective receptors for the recognition of phosphorylated species of biological relevance. Phosphorylation (i. e. the attachment of a phosphate group) of proteins and metabolites is an essential mechanism for a wide range of biological processes and of utmost importance for cellular function.
Therefore, it is highly desirable to develop tools for the detection, sensing and separation of this type of species. Although in recent years there have been important developments in this area, the current methods to recognise phosphorylated bio-molecules are far from idea. They often lack sensitivity, selectivity and in some instances (e. g. biosensors) the shelf life of the systems are not ideal. Therefore, we set up to investigate novel approaches based on small molecule with desirable properties such as ease of synthesis, robustness, long shelf life, fast binding kinetics, fast clearance from blood circulation, and ease of formulation for widespread use. The specific aim of the project was to develop novel chemical receptors for the efficient recognition of phosphorylated molecules of biological interest. This in turn will help us to gain a better understanding of how living organisms function at a molecular level and develop novel therapeutic agents to treat human disease, since phosphorylated proteins are important drug targets. This will also help to develop novel imaging agents to aid in diagnosis of disease and monitor of treatment. In order to achieve this task, a new approach called chemical evolution using dynamic combinatorial libraries has been investigated. The following are some of the key findings of the project throughout the past 2 years:
(a) Novel chemical receptors for phosphorylated molecules based on metallo-receptors which combine strong binding through metal-phosphate interactions with high selectivity, were synthesized. The binding properties of one the receptors based in an europium (III) complex with cell membrane models was first studied. The receptor proved to be an excellent probe for the detection of phosphatidylserine (PS, which is a universal indicator of the early/intermediate stages of cell apoptosis) on the outer envelope of this artificial membrane. This receptor with Gd (III) was then successfully used for in cellulo Magnetic Resonaance Imaging (MRI) of apoptotic cells (in collaboration with the MRC Clinical Sciences Centre, ICL). The results of this investigation have been published (Chem.commun. 2011, 47, 10245)
(b) Novel metallo-receptors for the recognition of phosphorylation sites in peptides and proteins molecules have been synthesized and showed that the complex binds strongly to the phosphopeptide under study and provides a proof-of-principle for the development of a fluorescent kinase activity probe. In this context, receptors based on terbium (III) complexes have been synthesized and characterised by NMR spectroscopy, mass spectrometry and elemental analysis. The binding ability of this metallo-receptor towards phosphorylated species was studied using phosphorylated/non phosphorylated proteins at biological pH conditions. The interaction of the receptor and phospho-peptides was monitored by the emission of the terbium complex (sensitised by tryptophan residues). These preliminary studies showed that the complex binds strongly to the phosphopeptide under study and provides a proof-of-principle for the development of a fluorescent kinase activity probe (currently under development).
(c) Metal complexes tagged with a fluorophore (fluorescein isothiocyanate) were prepared via a multi-step synthetic protocol. The novel receptor-fluorophore conjugate was characterised by NMR spectroscopy; mass spectrometry and elemental analysis. This complex was used as a probe to develop a viable method for measuring relative protein levels in single cells. This work was done in collaboration with Prof. Klug's group in Imperial College.
(d) The templating role of anions in supramolecular and coordination chemistry (including the formation of coordination networks) is an area of considerable ongoing interest. In this context, we explored the important role played by the ligands'substituents and the counteranions present in transition metal based pseudo peptide ligands. We had shown that the specific structural features on the ligand (isopropyl vs. phenyl) and the nature of the counterions (sulphate vs. bromide) have on the final structure This work was in collaboration with Prof. Santiago V. Luis from University Jaume, Spain. The results are published in CrystEngComm, 2011, 13 (23), 6997 and and the work was highlighted as a "Hot paper".
(e) As mentioned above, a new approach called chemical evolution using dynamic combinatorial library approach was adopted for the synthesis of receptors for specific biomolecules. Metallo-receptors based on transition metals and different aldehydes have been selected as building blocks for the development of the dynamic library (via imine formation between the free amine and the di-aldehydes). After the success of this dynamic library based on transition metal receptors we have extended our approach to other metal complexes (lanthanides) capable of binding phosphate anions. To overcome some experimental problems, we developed a new hydrazone based (cf. imine-based) dynamic combinatorial library. Interestingly, with this library we have very recently been able to identify a selective receptors for ATP from a pool of various potential receptor molecules (i. e. from a dynamic virtual library of receptors). We are currently finalising this work and expect to publish it soon in addition, the successful receptors will be studied in a biological context to establish their ability to interact with the target molecules under cellular environment.
In summary, we have developed novel chemical receptors for the efficient recognition of phosphorylated molecules of biological interest. We were successful in developing a new approach, namely chemical evolution using dynamic combinatorial libraries, for identifying specific receptor molecules for target biomolecules.
Therefore, it is highly desirable to develop tools for the detection, sensing and separation of this type of species. Although in recent years there have been important developments in this area, the current methods to recognise phosphorylated bio-molecules are far from idea. They often lack sensitivity, selectivity and in some instances (e. g. biosensors) the shelf life of the systems are not ideal. Therefore, we set up to investigate novel approaches based on small molecule with desirable properties such as ease of synthesis, robustness, long shelf life, fast binding kinetics, fast clearance from blood circulation, and ease of formulation for widespread use. The specific aim of the project was to develop novel chemical receptors for the efficient recognition of phosphorylated molecules of biological interest. This in turn will help us to gain a better understanding of how living organisms function at a molecular level and develop novel therapeutic agents to treat human disease, since phosphorylated proteins are important drug targets. This will also help to develop novel imaging agents to aid in diagnosis of disease and monitor of treatment. In order to achieve this task, a new approach called chemical evolution using dynamic combinatorial libraries has been investigated. The following are some of the key findings of the project throughout the past 2 years:
(a) Novel chemical receptors for phosphorylated molecules based on metallo-receptors which combine strong binding through metal-phosphate interactions with high selectivity, were synthesized. The binding properties of one the receptors based in an europium (III) complex with cell membrane models was first studied. The receptor proved to be an excellent probe for the detection of phosphatidylserine (PS, which is a universal indicator of the early/intermediate stages of cell apoptosis) on the outer envelope of this artificial membrane. This receptor with Gd (III) was then successfully used for in cellulo Magnetic Resonaance Imaging (MRI) of apoptotic cells (in collaboration with the MRC Clinical Sciences Centre, ICL). The results of this investigation have been published (Chem.commun. 2011, 47, 10245)
(b) Novel metallo-receptors for the recognition of phosphorylation sites in peptides and proteins molecules have been synthesized and showed that the complex binds strongly to the phosphopeptide under study and provides a proof-of-principle for the development of a fluorescent kinase activity probe. In this context, receptors based on terbium (III) complexes have been synthesized and characterised by NMR spectroscopy, mass spectrometry and elemental analysis. The binding ability of this metallo-receptor towards phosphorylated species was studied using phosphorylated/non phosphorylated proteins at biological pH conditions. The interaction of the receptor and phospho-peptides was monitored by the emission of the terbium complex (sensitised by tryptophan residues). These preliminary studies showed that the complex binds strongly to the phosphopeptide under study and provides a proof-of-principle for the development of a fluorescent kinase activity probe (currently under development).
(c) Metal complexes tagged with a fluorophore (fluorescein isothiocyanate) were prepared via a multi-step synthetic protocol. The novel receptor-fluorophore conjugate was characterised by NMR spectroscopy; mass spectrometry and elemental analysis. This complex was used as a probe to develop a viable method for measuring relative protein levels in single cells. This work was done in collaboration with Prof. Klug's group in Imperial College.
(d) The templating role of anions in supramolecular and coordination chemistry (including the formation of coordination networks) is an area of considerable ongoing interest. In this context, we explored the important role played by the ligands'substituents and the counteranions present in transition metal based pseudo peptide ligands. We had shown that the specific structural features on the ligand (isopropyl vs. phenyl) and the nature of the counterions (sulphate vs. bromide) have on the final structure This work was in collaboration with Prof. Santiago V. Luis from University Jaume, Spain. The results are published in CrystEngComm, 2011, 13 (23), 6997 and and the work was highlighted as a "Hot paper".
(e) As mentioned above, a new approach called chemical evolution using dynamic combinatorial library approach was adopted for the synthesis of receptors for specific biomolecules. Metallo-receptors based on transition metals and different aldehydes have been selected as building blocks for the development of the dynamic library (via imine formation between the free amine and the di-aldehydes). After the success of this dynamic library based on transition metal receptors we have extended our approach to other metal complexes (lanthanides) capable of binding phosphate anions. To overcome some experimental problems, we developed a new hydrazone based (cf. imine-based) dynamic combinatorial library. Interestingly, with this library we have very recently been able to identify a selective receptors for ATP from a pool of various potential receptor molecules (i. e. from a dynamic virtual library of receptors). We are currently finalising this work and expect to publish it soon in addition, the successful receptors will be studied in a biological context to establish their ability to interact with the target molecules under cellular environment.
In summary, we have developed novel chemical receptors for the efficient recognition of phosphorylated molecules of biological interest. We were successful in developing a new approach, namely chemical evolution using dynamic combinatorial libraries, for identifying specific receptor molecules for target biomolecules.