Synthesis and Applications of Oligophosphate Constructs - Phosphorylated biomolecules play essential roles in human physiology, health, and medicine. Biological tar- gets for phosphorylation include nucleosides, lipids, amino acids, peptides, and proteins. A recent discovery is that protein oligo- or polyphosphorylation is an important post-translational modification, spurring researchers to synthesize chemical probes containing oligophosphate chains of specific lengths as tools to enable exploration of the human polyP-ome. This development exposes the need for well-defined chemical reagents to enable phosphate chains of a desired length to be conjugated to an organic molecule of interest. Previously we developed reagents for diphosphorylation, new methods for triphosphorylation and tetraphosphorylation, and now we will develop new reagent and methods for pentaphosphorylation and beyond. A related important innovation is the development of methods for covalently linking two (different) organic molecules by an oligophosphate chain. We will also de- velop improved strategies for synthesis of oligophosphate-organic molecule constructs having non-hydrolyzable P-C bonds. Our innovations will emphasize P(V)-based methods as these have the potential to be more efficient than existing P(III)-based ones. We will continue our collaboration with the Raines lab in which we are studying the potency of nucleoside oligophosphate constructs as inhibitors of RNase A as a model enzyme system; we plan to study the effect of increasing the oligophosphate chain length to include hexaphosphate, heptaphosphate, and even longer chain lengths. Small molecule ribonuclease inhibitors are valuable biochemical tools for studies of RNA for which success often relies on shutting down all ribonucleolytic activity. We are also targeting new con- structs bearing an electrophilic warhead linked to a nucleoside by an oligophosphate chain for covalent attachment of ligands to proteins. We are developing methods for attaching clickable moities to oligophosphate chain ends, to enable oligophosphate-organic molecule conjugates to be further attached to peptides or proteins. This is the basis for an exciting new collaborative project with the Raines lab on the decoration of proteins with oligophos- phate groups to render their surface polynegative for packaging inside of lipid nanoparticles for delivery into cells. The enzyme inhibition studies are complemented by state-of-the-art quantum chemical studies of protein-ligand interactions, studies carried out with theoretical and computational chemist Giovanni Bistoni (U. Perugia). To improve our protein crystallography capabilities we have started a new collaboration with the Drennan lab. We onboarded an exciting collaboration with the Fielder lab (FMP Berlin) to investigate the conjugation of oligophos- phate chains to polypeptides/proteins using our reagents amd methods, with the objective of elucidating the impact of these modifications on their structure and function, as well as the roles that endogenous oligophosphorylation may play in the natural regulation of enzymes. This has led us to develop new imidazolide oligophosphorylation reagents that are tolerant of water and highly selective for oligophosphate chain elongation of phosphoproteins.
