Expanding the PP-INSP₄ Toolbox: Synthesis, Stereochemical Assignment, and Application in Plant Phosphate Signaling

28 March 2025, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

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 quantitative mass spectrometry in combination with capillary electrophoresis (CE-MS), enabling the study 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.

Keywords

inositol phosphates
inositol pyrophosphates
phosphorylation
phosphate homeostasis
ITPK1

Supplementary materials

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Supporting Information
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Procedures and Spectra
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