Stereochemically defined inositol pyrophosphate isomers (PP-InsP₄) were synthesized and assigned by NMR spectroscopy using a chiral solvating agent. In the form of isotopically labeled standards, they were applied in CE-MS-based profiling of plant extracts and kinase assays, enabling structural reassignment and functional insight into enzymatic inositol phosphate turnover.

Abstract

Inositol pyrophosphates (PP-InsPs) are highly phosphorylated signaling molecules that regulate diverse cellular processes, including phosphate homeostasis and energy metabolism across species. Despite extensive research on well-characterized exhaustively phosphorylated PP-InsPs, such as 5-PP-InsP₅ (5-InsP₇) and 1,5-(PP)₂-InsP₄ (1,5-InsP₈), the functional relevance of less abundant not fully phosphorylated isomers, remains largely unknown. In this study, we synthesized all unsymmetric 5-PP-InsP₄ isomers in enantiopure form and assigned their structures using ³¹P-NMR analysis in combination with a chiral solvating agent. Additionally, we developed ¹⁸O-labeled PP-InsP₄ standards for mass spectrometry in combination with capillary electrophoresis (CE-MS), enabling the assignment of PP-InsP₄ in Arabidopsis thaliana under phosphate starvation. Our findings show that the previously detected, phosphate starvation-induced root-specific PP-InsP₄ isomer does not match any 5-PP-InsP₄ isomer, contrary to previous suggestions, thus indicating an alternative phosphorylation pattern. Enzyme assays further demonstrate that Arabidopsis ITPK1 selectively phosphorylates [6-OH]-InsP₅ and [3-OH]-InsP₅ at the 5-position, while other InsP₅ isomers remain unchanged. This suggests that an unidentified enzymatic activity is involved in the formation of the elusive root PP-InsP₄ species. Our study provides a comprehensive framework for the synthesis, analysis, and functional investigation of PP-InsP₄, providing an entry point for future studies on their biochemical activity and their physiological roles.