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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. [ABSTRACT FROM AUTHOR]

Studies into the biology of condensed phosphates almost exclusively cover linear polyphosphates. However, there is evidence for the presence of cyclic polyphosphates (metaphosphates) in organisms and for enzymatic digestion of branched phosphates (ultraphosphates) with alkaline phosphatase. Further research of non‐linear condensed phosphates in biology would profit from interactome data of such molecules, however, their stability in biological media is limited. Here we present syntheses of modified, non‐hydrolysable analogues of cyclic and branched condensed phosphates, called meta‐ and ultraphosphonates, and their application in a chemical proteomics approach using yeast cell extracts. We identify putative interactors with overlapping hits for structurally related capture compounds underlining the quality of our results. The datasets serve as starting point to study the biological relevance and functions of meta‐ and ultraphosphates. In addition, we examine the reactivity of meta‐ and ultraphosphonates with implications for their "hydrolysable" analogues: Efforts to increase the ring‐sizes of meta‐ or cyclic ultraphosphonates revealed a strong preference to form trimetaphosphate‐analogue structures by cyclization and/or ring‐contraction. Using carbodiimides for condensation, the so far inaccessible dianhydro product of ultraphosphonate, corresponding to P4O112−, was selectively obtained and then ring‐opened by different nucleophiles yielding modified cyclic ultraphosphonates. [ABSTRACT FROM AUTHOR]