Synthesis and Cellular Delivery of myo-Inositol Pyrophosphate Prometabolites; the InsP8 Challenge

自然界使用源自肌醇支架的高度磷酸化化合物作为细胞内信使分子。焦磷酸肌醇是二磷酸肌醇聚磷酸的昵称，是肌醇聚磷酸家族的特殊成员，含有比碳更多的磷，其中InsP8（一种含有两个二磷酸亚基和四个磷酸单酯的肌醇聚磷酸）是迄今为止已知的高度磷酸化的代表。它们参与多种信号传递过程，但它们的许多功能仅被描述得很差，它们如何发出信号的机制正处于激烈的争论中。这种不确定性在很大程度上是由于缺乏用于体外研究的纯材料，以及缺乏在活细胞中研究其生物学的化学生物学探针。此外，它们的不稳定性和高电荷密度，加上没有发色团，使得这类化合物在合成和分析上都特别具有挑战性。尽管对InsP7（仅含有7种磷酸盐）的研究已经克服了这些挑战，但对InsP8的研究却明显落后。尽管如此，越来越多的证据表明，InsP8可能是以前与InsP7相关的过程的真正调节器。在这个应用中，我建议立体选择性地合成InsP8的细胞渗透类似物及其光笼化变体。一旦进入细胞，就会触发裂解机制，释放生物活性或笼中的生物活性分子，激光照射后的笼去除将提供对InsP8功能的时间和空间分辨率的了解。这些特性将用于研究InsP8对信号节点激酶Akt从膜到细胞质转运的影响。此外，将评估InsP8释放后干扰素调节因子（IRF3）从细胞质到细胞核的二聚化和易位。本项目旨在开发新的立体选择性合成方法来合成InsP8及其类似物，将其有效地转移到完整细胞中，并在生物读出后进行控制释放。总的来说，这个项目可以确定InsP8是以前分配给其他信使分子的功能的真正调节器。

Nature uses highly phosphorylated compounds derived from the myo-inositol scaffold as intracellular messenger molecules. The inositol pyrophosphates, a nickname for diphosphoinositol polyphosphates, are a special member of the inositol polyphosphate family and contain more phosphorous than carbon, with InsP8 (an inositol polyphosphate containing two diphosphate subunits and four phosphate monoesters) as the most highly phosphorylated representative known to date. They are involved in diverse signaling processes, yet many of their functions are only poorly characterized and the mechanisms how they signal are under intense debate. The uncertainties are in large part due to a lack of pure material for in vitro studies, as well as a lack of chemical biology probes to study their biology in living cells. Moreover, their lability and high charge density in combination with the absence of a chromophore renders this class of compounds particularly challenging, both synthetically as well as analytically. Whereas some of these challenges have been mastered for InsP7 (containing only seven phosphates), research into InsP8 has significantly lagged behind. Notwithstanding, there is increasing evidence that InsP8 might be the true regulator of processes that have been previously associated with InsP7.In this application, I propose to stereoselectively synthesize cell-permeable analogs of InsP8 as well as photocaged variants thereof. Once inside cells, a cleavage mechanism will be triggered to release the bioactive or caged bioactive molecule and cage removal upon laser irradiation will provide insight into InsP8 function with temporal and spatial resolution. These properties will be used to study the effect of InsP8 on the signaling-node kinase Akt with regards to its translocation from the membrane to the cytoplasm. Moreover, the dimerization and translocation of interferon regulating factor (IRF3) from the cytoplasm to the nucleus upon InsP8 release will be evaluated. This project aims at the development of novel stereoselective synthetic approaches to InsP8 and its analogs, its efficient transfer into intact cells and its controlled release followed by biological readout. In its entirety, this project could establish InsP8 as the true regulator of functions that have been previously assigned to other messenger molecules.