Synthesis of a Glycosidase Inhibitor and a Potential Phosphoglucomutase Inhibitor, Expression of the Corresponding Proteins, and their Evaluation Using Kinetics or 19F-NMR Spectroscopy

Abstract

The inhibition and reactivation of several naturally prevalent enzymes has many pharmaceutical applications. A glycosidase inhibitor, 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-D-glucopyranose (6), was synthesized and its kinetics evaluated with the Family-3 retaining glycosidases, DesR, EryBI, and Hsero1941. 6 displayed quantitative inactivation of DesR and EryBI after two hours and 54% inactivation of Hsero1941 after 16 hours. Various acceptors were screened in attempts to observe transglycosylation activity from the enzymes. β-phosphoglucomutase (βPGM) is an essential enzyme for energy metabolism in cells. A W216F mutant βPGM was produced and its transition state analogue (TSA) complexation with metal fluorides and phospho- or phosphono-glucosides was monitored using 19F NMR spectroscopy. Molar ratios of metal fluoride and complexed 5FW24 were observed spectroscopically, indicating that metal fluorides are representative of the transition states. A novel potential inhibitor of βPGM, diammonium 3-deoxy-3-fluoro-β-D-glucopyranosylmethylphosphonate (21), was synthesized which is expected to lead to improved ratios of complexed: free βPGM during TSA complexation.