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

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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.