Anomerisation of Fluorinated Sugars by Mutarotase Studied Using 19F NMR Two-Dimensional Exchange Spectroscopy

Abstract

Five F-19-substituted glucose analogues were used to probe the activity and mechanism of the enzyme mutarotase by using magnetisation-exchange NMR spectroscopy. The sugars (2-fluoro-2-deoxy-D-glucose, FDG2; 3-fluoro-3-deoxyD-glucose, FDG3; 4-fluoro-4-deoxy-D-glucose, FDG4; 2,3-difluoro-2,3-dideoxy-D-glucose, FDG23; and 2,2,3,3-tetrafluoro-2,3-dideoxy-D-glucose (2,3-dideoxy-2,2,3,3-tetrafluoro-D-erythro-hexopyranose), FDG2233) showed separate F-19 NMR spectroscopic resonances from their respective alpha- and beta-anomers, thus allowing two-dimensional exchange spectroscopy measurements of the anomeric interconversion at equilibrium, on the time scale of a few seconds. Mutarotase catalysed the rapid exchange between the anomers of FDG4, but not the other four sugars. This finding, combined with previous work identifying the mechanism of the anomerisation by mutarotase, suggests that the rotation around the C1-C2 bond of the pyranose ring is the rate-limiting reaction step. In addition to D-glucose itself, it was shown that all other fluorinated sugars inhibited the FDG4 anomerisation, with the tetrafluorinated FDG2233 being the most potent inhibitor. Inhibition of mutarotase by F-sugars paves the way for the development of novel fluorinated compounds that are able to affect the activity of this enzyme in vitro and in vivo.