Expanding the Inositol Pyrophosphate Toolbox: Stereoselective Synthesis and Application of PP‐InsP ₄ Isomers in Plant Signaling

Abstract

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‐InsP 5 (5‐InsP 7 ) and 1,5‐(PP) 2 ‐InsP 4 (1,5‐InsP 8 ), the functional relevance of less abundant not fully phosphorylated isomers, remains largely unknown. In this study, we synthesized all unsymmetric 5‐PP‐InsP 4 isomers in enantiopure form and assigned their structures using 31 P‐NMR analysis in combination with a chiral solvating agent. Additionally, we developed 18 O‐labeled PP‐InsP 4 standards for mass spectrometry in combination with capillary electrophoresis (CE‐MS), enabling the assignment of PP‐InsP 4 in Arabidopsis thaliana under phosphate starvation. Our findings show that the previously detected, phosphate starvation‐induced root‐specific PP‐InsP 4 isomer does not match any 5‐PP‐InsP 4 isomer, contrary to previous suggestions, thus indicating an alternative phosphorylation pattern. Enzyme assays further demonstrate that Arabidopsis ITPK1 selectively phosphorylates [6‐OH]‐InsP 5 and [3‐OH]‐InsP 5 at the 5‐position, while other InsP 5 isomers remain unchanged. This suggests that an unidentified enzymatic activity is involved in the formation of the elusive root PP‐InsP 4 species. Our study provides a comprehensive framework for the synthesis, analysis, and functional investigation of PP‐InsP 4 , providing an entry point for future studies on their biochemical activity and their physiological roles.