Design and Synthesis of Metal-Organic Cage Complexes for use as Imaging Labels in Electron Microscopy

点击翻译按钮获取中文摘要

Over the last decade electron microscopy (EM) has emerged as a promising tool for structural determination of biological molecules with the Nobel prize awarded for advancements in cryo-EM in 2017(Nature, 2017, 550, 167). Significant limitations associated with image collection and processing do however persist, and development of novel imaging labels(probes) that are able to positively identify specific structural features remains a clear goal (J. Biol. Chem., 2019, 294, 5181). We propose the design of highly symmetric metal-organic cages that undergo self-assembly incorporating multiple electron dense metals at the vertices and are capable of selectively targeting exposed residues and/ or structural features on proteinaceous biomacromolecules. We hypothesise that the spatial precision of the metal ions within supramolecular metallo-cages combined with the high overall symmetry of the complexes presents advantages over currently available commercial metal-based probes when processing the microscopy data. Moreover, the modular nature of cages will enable the binding pocket to be optimised to target specific structural features overcoming current problems with lack of probe specificity. Key objectives for this project are: development of a set of rules directing formation of metal-organic cages with multiple metal ions at their vertices; and publication of proof-of-concept studies demonstrating the utility of cages for enhancement of EM imaging. The results will be of broad interest and will be published in high impact journals.The project will be broken into two work packages (WP): WP1: Development of Novel Metal-organic Cages with Vertices Incorporating Multiple Metal IonsIn this WP ligands known to give rise to water-soluble cages will be tailored to enable increased electron density at the metal vertices. Here we will use published ligands as well as unpublished ligands developed in our lab. In parallel we will explore synthetic approaches to increase the solubility of cages which have high electron density at their vertices, looking to modify recent Zr(IV), Cu(I) and Ag(I) cages. Small molecule characterisation techniques (NMR, Mass spectrometry and single crystal X-ray diffraction) will be employed to verify the identity of the complex and assess its purity after assembly. WP2: Biomolecule Binding and Imaging StudiesCages will be evaluated for peptide binding in the first instance before looking at protein binding. Binding of short representative peptide sequences will be evaluated using typical supramolecular spectroscopic strategies (including NMR, UV-vis, fluorescence and CD measurements). Subsequent binding to protein structures may be supported through fluorescence, Raman, ICP-MS and EDX analyses before EM imaging with and without negative staining. WPs 1 and 2 detail the chemical synthesis, characterisation and host-guest potential of metallo-cages that incorporate multiple metal ions per vertex. The research outline in this proposal is thus predominantly supramolecular chemistry and falls within the EPSRC remit aligning well with the EPSRC 'directed assembly of extended structures with targeted properties', 'nanoscale design of functional materials' and 'physics of life' grand challenges.