Analysis of Phosphorylated Metabolites in Crayfish Extracts by Two-Dimensional1H-31P NMR Heteronuclear Total Correlation Spectroscopy (heteroTOCSY)☆☆☆

In vivoand extract analyses by one-dimensional31P NMR have been a key tool in investigating energy-related metabolism. Although many phosphorylated metabolites have been observed, many of them have yet to be identified. This reflects the difficulty in identifying them using31P NMR alone. Two-dimensional1H–31P correlation experiments have been shown to be useful for assigning phosphorylated metabolites. To obtain better sensitivity and structure information,1H-detected31P–1H heteronuclear total correlation spectroscopy (heteroTOCSY) was implemented and a complete chemical shift assignment for a number of phosphorylated standards was made. The time courses of 1D heteroTOCSY signal intensity versus spin-locking time were established for these standards to aid the optimization of the 2D experiment. This method was applied to crayfish extracts for the assignment of glucose 6-phosphate, α-glycerophosphate, ribose 5-phosphate, fructose 1,6-bisphosphate, phosphocholine, phosphoethanolamine, glucose 1-phosphate, glycerophosphoethanolamine, glycerophosphocholine, ATP, ADP, and AMP. An alkyl phosphate, a hexose 1-phosphate, and a UDP-hexose were also observed. These assignments allowed the identification of many changes in the31P NMR spectra of crayfish extracts elicited by treatment with the organophosphate pesticide chlorpyrifos. The assignment of anin vivo31P spectrum of a live crayfish was also made based on the extract assignment. This approach should be a powerful tool for examining stress-associated changes in the metabolism of phosphorylated compounds.