Improved NMR Detection of Phospho-Metabolites in a Complex Mixture

Phosphorylated
metabolites are omnipresent in cells, but their
analytical characterization faces several technical hurdles. Here,
we detail an improved NMR workflow aimed at assigning the high-resolution
subspectrum of the phospho-metabolites in a complex mixture. Combining
a pure absorption J-resolved spectrum (Pell, A. J.; J. Magn. Reson. 2007, 189 (2), 293−299) with alternate on- and off-switching of the 31P coupling
interaction during the t1 evolution with
a pure in-phase (PIP) HSQMBC experiment (Castañar, L.; Angew. Chem., Int. Ed. 2014, 53 (32), 8379−8382) without or with total correlation spectroscopy (TOCSY)
transfer during the insensitive nuclei enhancement by polarization
transfer (INEPT) gives access to selective identification of the individual
subspectra of the phosphorylated metabolites. Returning to the initial J-res spectra, we can extract with optimal resolution the
full trace for the individual phospho-metabolites, which can then
be transposed on the high-resolution quantitative one dimensional
spectrum.