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

在过去的十年中，电子显微镜（EM）已成为一种有前途的生物分子结构测定工具，2017年因冷冻电镜技术的进步而获得诺贝尔奖（Nature, 2017, 550, 167）。然而，与图像收集和处理相关的重大限制仍然存在，能够积极识别特定结构特征的新型成像标签（探针）的开发仍然是一个明确的目标(J. Biol。化学。浙江农业学报，2019,294,5181)。我们建议设计高度对称的金属有机笼，该笼经过自组装，在顶点包含多个电子密度金属，并且能够选择性地靶向蛋白质生物大分子上暴露的残留物和/或结构特征。我们假设，在处理显微镜数据时，超分子金属笼内金属离子的空间精度与配合物的高整体对称性相比，比目前可用的商用金属基探针具有优势。此外，笼的模块化特性将使结合袋能够针对特定的结构特征进行优化，克服目前缺乏探针特异性的问题。该项目的主要目标是：开发一套指导金属有机笼形成的规则，这些笼的顶点上有多个金属离子；并发表概念验证研究，证明笼在增强EM成像方面的效用。研究结果将引起广泛的兴趣，并将发表在高影响力的期刊上。该项目将分为两个工作包（WP）： WP1：开发具有多个金属离子顶点的新型金属有机笼。这种已知可产生水溶性笼的WP配体将被定制，以提高金属顶点的电子密度。在这里，我们将使用已发表的配体以及我们实验室开发的未发表的配体。与此同时，我们将探索提高具有高电子密度的笼的溶解度的合成方法，寻求修饰最近的Zr(IV), Cu(I)和Ag(I)笼。小分子表征技术（核磁共振、质谱和单晶x射线衍射）将用于验证复合物的身份，并在组装后评估其纯度。WP2：生物分子结合和成像研究在研究蛋白质结合之前，将首先评估cage的肽结合能力。将使用典型的超分子光谱策略（包括NMR， UV-vis，荧光和CD测量）评估短代表性肽序列的结合。随后与蛋白质结构的结合可以通过荧光、拉曼、ICP-MS和EDX分析来支持，然后进行有或没有阴性染色的EM成像。WPs 1和WPs 2详细介绍了每个顶点包含多个金属离子的金属笼的化学合成、表征和主客电位。因此，本提案中的研究大纲主要是超分子化学，属于EPSRC的职权范围，与EPSRC的“具有目标特性的扩展结构的定向组装”，“功能材料的纳米级设计”和“生命物理学”的重大挑战保持一致。

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.