Non-Hydrolysable Analogues of Cyclic and Branched Condensed Phosphates: Chemistry and Chemical Proteomics.

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 P4 O11 2- , was selectively obtained and then ring-opened by different nucleophiles yielding modified cyclic ultraphosphonates.

Lost in Condensation: Poly-, Cyclo-, and Ultraphosphates.

Much like linear, branched, and cyclic alkanes, condensed phosphates exist as linear, branched, and cyclic structures. Inasmuch as alkanes are the cornerstone of organic chemistry, generating an inexplorably large chemical space, a comparable richness in structures can be expected for condensed phosphates, as also for them the concepts of isomerism apply. Little of their chemical space has been charted, and only a few different synthesis methods are available to construct isomers of condensed phosphates. Here, we will discuss the application of phosphoramidites with one, two, or three P-N bonds that can be substituted selectively to access different condensed phosphates in a highly controllable manner. Work directed toward the further exploration of this chemical space will contribute to our understanding of the fundamental chemistry of phosphates.In biology, condensed phosphates play important roles in the form of inorganic representatives, such as pyrophosphate, polyphosphate, and cyclophosphate, and also in conjugation with organic molecules, such as esters and amidates. Phosphorus is one of the six biogenic elements; the omnipresence of phosphates in biology points toward their critical involvement in prebiotic chemistry and the emergence of life itself. Indeed, it is hard to imagine any life without phosphate. It is therefore desirable to achieve through synthesis a better understanding of the chemistry of the condensed phosphates to further explore their biology.There is a rich but underexplored chemistry of the family of condensed phosphates per se, which is further diversified by their conjugation to important biomolecules and metabolites. For example, proteins may be polyphosphorylated on lysins, a very recent addition to posttranslational modifications. Adenosine triphosphate, as a representative of the small molecules, on the other hand, is well known as the universal cellular energy currency. In this Account, we will describe our motivations and our approaches to construct, modify, and synthetically apply different representatives of the condensed phosphates. We also describe the generation of hybrids composed of cyclic and linear structures of different oxidation states and develop them into reagents of great utility. A pertinent example is provided in the step-economic synthesis of the magic spot nucleotides (p)ppGpp. Finally, we provide an overview of 31P NMR data collected over the years in our laboratories, helping as a waymarker for not getting lost in condensation.

The Aryne Phosphate Reaction.

Condensed phosphates are a critically important class of molecules in biochemistry. Non-natural analogues are important for various applications, such as single-molecule real-time DNA sequencing. Often, such analogues contain more than three phosphate units in their oligophosphate chain. Consequently, investigations into phosphate reactivity enabling new ways of phosphate functionalization and oligophosphorylation are essential. Here, we scrutinize the potential of phosphates to act as arynophiles, paving the way for follow-up oligophosphorylation reactions. The aryne phosphate reaction is a powerful tool to-depending on the perspective-(oligo)phosphorylate arenes or arylate (oligo-cyclo)phosphates. Based on Kobayashi-type o-silylaryltriflates, the aryne phosphate reaction enables rapid entry into a broad spectrum of arylated products, like monophosphates, diphosphates, phosphodiesters and polyphosphates. The synthetic potential of these new transformations is demonstrated by efficient syntheses of nucleotide analogues and an unprecedented one-flask octaphosphorylation.

Stable Isotope Phosphate Labelling of Diverse Metabolites is Enabled by a Family of 18 O-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 18 O-labelled phosphates is presented, based on a family of modified 18 O2 -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. 18 O-enrichment ratios >95 % and good yields are obtained consistently in gram-scale reactions, while enabling late-stage labelling. We demonstrate the utility of the 18 O-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.

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.

The chemistry of branched condensed phosphates.

Condensed phosphates may exist as linear, cyclic or branched structures. Due to their important role in nature, linear polyphosphates have been well studied. In contrast, branched phosphates (ultraphosphates) remain largely uncharacterised, because they were already described in 1950 as exceedingly unstable in the presence of water, epitomized in the antibranching-rule. This rule lacks experimental backup, since, to the best of our knowledge, no rational synthesis of defined ultraphosphates is known. Consequently, detailed studies of their chemical properties, reactivity and potential biological relevance remain elusive. Here, we introduce a general synthesis of monodisperse ultraphosphates. Hydrolysis half-lives up to days call the antibranching-rule into question. We provide evidence for the interaction of an enzyme with ultraphosphates and discover a rearrangement linearizing the branched structure. Moreover, ultraphosphate can phosphorylate nucleophiles such as amino acids and nucleosides with implications for prebiotic chemistry. Our results provide an entry point into the uncharted territory of branched condensed phosphates.