absolute-quantitation-inositol-pyrophosphates-capillary

Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules ...

University of Freiburg

Phosphoramidite analogues of modified cyclotriphosphates provide a general and step-economical synthesis of nucleoside triphosphates and analogues on scale without the need for protecting groups. These reagents enable rapid access to pure nucleoside oligophosphates and a range of other analogues that were previously difficult to obtain (e.g., NH, CH, CCl, and CF replacements for O, phosphono- and phosphoimidazolides, -morpholidates, -azidates, and -fluoridates). DFT calculations demonstrate that the selectivity of the cyclotriphosphate opening reactions proceeds via an in-line substitution mechanism that displaces the least charged leaving group.

Synthesis of Modified Nucleoside Oligophosphates Simplified: Fast, Pure, and Protecting Group Free.

The complex phosphorylation pattern of natural and modified pentaphosphorylated magic spot nucleotides is generated in a highly efficient way. A cyclic pyrophosphoryl phosphoramidite (cPyPA) reagent is used to introduce four phosphates on nucleosides regioselectively in a one-flask key transformation. The obtained magic spot nucleotides are used to develop a capillary electrophoresis UV detection method, enabling nucleotide assignment in complex bacterial extracts.

Four Phosphates at One Blow: Access to Pentaphosphorylated Magic Spot Nucleotides and Their Analysis by Capillary Electrophoresis.

Photocages have been successfully applied in cellular signaling studies for the controlled release of metabolites with high spatio-temporal resolution. Commonly, coumarin photocages are activated by UV light and the quantum yields of uncaging are relatively low, which can limit their applications in vivo. Here, syntheses, the determination of the photophysical properties, and quantum chemical calculations of 7-diethylamino-4-hydroxymethyl-thiocoumarin (thio-DEACM) and caged adenine nucleotides are reported and compared to the widely used 7-diethylamino-4-hydroxymethyl-coumarin (DEACM) caging group. In this comparison, thio-DEACM stands out as a phosphate cage with improved photophysical properties, such as red-shifted absorption and significantly faster photolysis kinetics.

Thiocoumarin Caged Nucleotides: Synthetic Access and Their Photophysical Properties.

The analysis of myo-inositol phosphates (InsPs) and myo-inositol pyrophosphates (PP-InsPs) is a daunting challenge due to the large number of possible isomers, the absence of a chromophore, the high charge density, the low abundance, and the instability of the esters and anhydrides. Given their importance in biology, an analytical approach to follow and understand this complex signaling hub is desirable. Here, capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESI-MS) is implemented to analyze complex mixtures of InsPs and PP-InsPs with high sensitivity. Stable isotope labeled (SIL) internal standards allow for matrix-independent quantitative assignment. The method is validated in wild-type and knockout mammalian cell lines and in model organisms. SIL-CE-ESI-MS enables the accurate monitoring of InsPs and PP-InsPs arising from compartmentalized cellular synthesis pathways, by feeding cells with either [C]-myo-inositol or [C]-D-glucose. In doing so, we provide evidence for the existence of unknown inositol synthesis pathways in mammals, highlighting the potential of this method to dissect inositol phosphate metabolism and signalling.

Article	Year
Synthesis of Modified Nucleoside Oligophosphates Simplified: Fast, Pure, and Protecting Group Free. Journal of the American Chemical Society\| PubMed ID: 31512870	2019
Four Phosphates at One Blow: Access to Pentaphosphorylated Magic Spot Nucleotides and Their Analysis by Capillary Electrophoresis. The Journal of organic chemistry\| PubMed ID: 32502348	2020
Thiocoumarin Caged Nucleotides: Synthetic Access and Their Photophysical Properties. Molecules (Basel, Switzerland)\| PubMed ID: 33203096	2020
Analysis of inositol phosphate metabolism by capillary electrophoresis electrospray ionization mass spectrometry. Nature communications\| PubMed ID: 33247133	2020

Danye Qiu

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