Stable Isotope Phosphate Labelling of Diverse Metabolites is Enabled by a Family of ¹⁸O-Phosphoramidites

Stable isotope labelling is state-of-the-art in quantitative mass spectrometry, yet often accessing the required standards is cumbersome and very expensive. Here, a unifying synthetic concept for ¹⁸O-labelled phosphates is presented, based on a family of modified ¹⁸O₂-phosphoramidite reagents. This toolbox offers access to major classes of biologically highly relevant phosphorylated metabolites as their isotopologues including nucleotides, inositol phosphates, -pyrophosphates, and inorganic polyphosphates. ¹⁸O-enrichment ratios >95 % and good yields are obtained consistently in gram-scale reactions, while enabling late-stage labelling. We demonstrate the utility of the ¹⁸O-labelled inositol phosphates and pyrophosphates by assignment of these metabolites from different biological matrices. We demonstrate that phosphate neutral loss is negligible in an analytical setup employing capillary electrophoresis electrospray ionisation triple quadrupole mass spectrometry.