Studies on the permethylation/dephosphorylation of inositol polyphosphates: An approach to a more sensitive assay

Abstract

Methods for the permethylation of inositol phosphates (i.e. the formation of the completely substituted C-O-methyl/P-O-methyl derivatives) have been studied as a precursor to preparing C-O-methyl inositols where the remaining inositol hydroxyl groups are at the positions originally occupied by the phosphomonoesters. Classical sodium-driven methylations, diazomethane methylations and methylation with methyl trifluoromethanesulfonate were studied and only the latter was found to produce completely alkylated inositol phosphates. Treatment of the permethylated substrates with methanolic HCl removed the dimethylphosphate groups to produce C-O-methyl inositols which are candidates for negative ion chemical ionization gas chromatographic/mass spectrometric analysis as heptafluorobutyryl C-O-methyl inositols. As an example, gas chromatographic/mass spectrometric analysis of myo-inositol 1,2,6-trisphosphate was carried out by methylation, dephosphorylation and conversion to the tris(heptafluorobutyryl) derivative. Detection at the low-femtomole level was achieved by this means. A limitation of the method may be that the methylation procedure appears to produce a variable degree of phosphate positional isomerization, with resulting loss of specificity. If stable isotope internal standards were available for the inositol polyphosphates of interest, this limitation could be compensated for.

The Chemical Biology of Phosphoinositide 3‐Kinases

“…Mass spectrometric methods for polyphosphorylated phosphoinositides up to PtdIns(4,5)P 2 were already described 20 years ago, [80] but sensitive detection of PtdIns(3,4,5)P 3 was not Figure 2. Metabolic labeling of phosphoinositides can be achieved with radioactive inorganic phosphate ( 32 P i ), which is converted to ATP in glycolysis.…”