Observation of glucose-6-phosphate anomeric exchange in real-time using dDNP hyperpolarised NMR

Abstract

A hyperpolarised-NMR acquisition approach that is sensitive to the process of glucose-6-phosphate anomerization is presented. Using selective depolarisation of one of the anomer's signals, it is possible to observe the replenishing of this signal due to the fast anomeric exchange of this compound. The forward to reverse reaction rate constants ratio was ca. 1.6.

Fig. 1. Hyperpolarised [¹³C₆,D₇]Glc in a reaction mixture with hexokinase (5.43 mg). (A) Lower panel, selective excitation and depolarisation of the C₁β position for 10 s with a TR of 1 s. Upper panel, subsequent non-selective (hard pulse) excitations (from the 13^th second) with the same TR. (B) A time course showing the signal decay for the chemical shifts of C₆-[¹³C₆,D₇]Glc and C₆-[¹³C₆,D₇]G6P determined using the non-selective excitations (upper panel in A). The dotted arrow indicates the time at which the phosphorylation reaction ended, as determined by the disappearance of the C₆-[¹³C₆,D₇]Glc signal from the spectra. (C) A time course showing the signal decay for the chemical shifts of C₁α and C₁β positions determined using the non-selective excitations (upper panel in A). The dotted arrow indicates the time at which the phosphorylation reaction ended (according to B). From this time onwards, the C₁α and C₁β signals can be considered those of [¹³C₆,D₇]G6P and were used in the analysis of [¹³C₆,D₇]G6P anomeric exchange.