Semi-enzymatic synthesis and application of 13C-isotopically labelled inositol-(1,4,5)-trisphosphate

Inositol-(1,4,5)-trisphosphate (Ins(1,4,5)P3) is a crucial secondary messenger that controls calcium (Ca2+) levels inside cells, yet many questions regarding Ins(1,4,5)P3 metabolism are challenging to address with current methods. Here, a semi-enzymatic milligram scale synthesis of isotopically labeled [13C6]Ins(1,4,5)P3 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 [13C6]Ins(1,4,5)P3 and 2D NMR spectroscopy. The method was subsequently applied to characterize the phosphorylation/dephosphorylation reactions of a putative inositol phosphate kinase from the alga Klebsormidium nitens (KnIPK2). KnIPK2 displayed 6-kinase activity towards [13C6]Ins(1,4,5)P3, and dual 4/6- and 5-phosphatase activity towards [13C6]Ins(1,3,4,5,6)P5. Finally, [13C6]Ins(1,4,5)P3 was utilized as an internal standard in hydrophilic liquid interaction chromatography mass spectrometry (HILIC-MS) experiments, to quantify dephosphorylation of Ins(1,4,5)P3 by INPP5B. [13C6]Ins(1,4,5)P3 therefore constitutes a broadly applicable analytical tool that should facilitate the characterization of Ins(1,4,5)P3 metabolism in the future.