Synthesis of 5-fluoro-beta-D-glucopyranosyluronic acid fluoride and its evaluation as a mechanistic probe of Escherichia coli beta-glucuronidase

Synthesis of the potential mechanism-based inactivator of beta -D-glucuroni dases (5-fluoro-beta -D-glucopyranosyluronic acid fluoride) was accomplishe d via a six-step process from D-glucuronic acid that involved radical bromi nation at C-5 and displacement of the bromide by fluoride. A key step in th is process was the masking of the carboxylic acid as a phenacyl ester. This group is uniquely stable to conditions of photobromination and fluoride di splacement, yet removable under very mild conditions. Incubation of the Esc herichia coli beta -glucuronidase with 5-fluoro-beta -D-glucopyranosyluroni c acid fluoride resulted in time-dependent inactivation of the enzyme throu gh the accumulation of a covalent 5-fluoro-alpha -D-glucopyranosyluronic ac id-enzyme. Peptic digestion of the 5-fluoro-alpha -D-glucopyranosyluronic a cid-enzyme intermediate and subsequent analysis by liquid chromatography co upled to an electrospray ionization triple quadrupole mass spectrometer ind icated the presence of a 5-fluoro-alpha -D-glucopyranosyluronic acid-modifi ed peptide. This peptide was partially purified by HPLC and its sequence de termined by tandem mass spectrometry in the daughter ion scan mode, permitt ing the identification of Glu504 as the catalytic nucleophile within the se quence ITEYGVD. This new reagent is therefore useful for the specific, mech anism-based inactivation of glycuronidases and has good potential in other studies of enzymes of this general class.