The P(III)‐Amidite Based Synthesis of Stable Isotope Labeled mRNA‐Cap‐Structures Enables their Sensitive Quantitation from Brain Tissue

The 5’ cap structure is crucial to mRNA function, with its diverse methylation patterns depending on the cellular state. Sensitive analytical methods are sought after to quantify this cap variety also referred to as cap epitranscriptome. To address a bottleneck for accurate and precise quantitation, we report a facile and fast access to high‐quality synthetic standards via a new route, involving P(III)‐amidite chemistry. A range of cap nucleotides and their stable heavy isotopic labeled analogues were derived from nucleoside diphosphates, which themselves were directly prepared in a one‐step reaction sequence starting from unprotected nucleosides using a triphosphorylating reagent in combination with ethylenediamine. Considering a wider scope, the route also enables direct access to magic spot nucleotides and diphosphates of isoprenyl‐alcohols. Stable‐isotope labeled cap nucleotides derived from this route paved the way for the development of a highly sensitive LC–MS/MS method, applied to the characterization of mouse brain cap epitranscriptomes, which turned out to be very different from those of cultured cell lines of widespread use in the life sciences. This work reports an approach to synthesize high quality cap standards using P(III)‐amidite chemistry. It enables ready access to stable isotope labeled internal standards in the milligram range. These references are the applied in the development of a highly sensitive LC–MS/MS method to study the cap epitranscriptome. The application to biological samples allowed the combined quantitation of cap structures as well as modified nucleosides in a single run.