Semi-enzymatic synthesis and application of C-isotopically labelled inositol-(1,4,5)-trisphosphate.

Inositol-(1,4,5)-trisphosphate (Ins(1,4,5)P) is a crucial secondary messenger that controls calcium (Ca) levels inside cells, yet many questions regarding Ins(1,4,5)P metabolism are challenging to address with current methods. Here, a semi-enzymatic milligram scale synthesis of isotopically labeled [C]Ins(1,4,5)P is reported which then served as a substrate to monitor the activity of mammalian type II inositol 1,4,5-trisphosphate 5-phosphatase INPP5B, using NMR spectroscopy in real time. In addition, the phosphorylation sequence catalyzed by inositol polyphosphate multikinase IPMK was confirmed using [C]Ins(1,4,5)P and 2D NMR spectroscopy. The method was subsequently applied to characterize the phosphorylation/dephosphorylation reactions of a putative inositol phosphate kinase from the alga (IPK2). IPK2 displayed 6-kinase activity towards [C]Ins(1,4,5)P, and dual 4/6- and 5-phosphatase activity towards [C]Ins(1,3,4,5,6)P. Finally, [C]Ins(1,4,5)P was utilized as an internal standard in hydrophilic liquid interaction chromatography mass spectrometry (HILIC-MS) experiments, to quantify dephosphorylation of Ins(1,4,5)P by INPP5B. [C]Ins(1,4,5)P therefore constitutes a broadly applicable analytical tool that should facilitate the characterization of Ins(1,4,5)P metabolism in the future.