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

TitleSynthesis of 5-fluoro-beta-D-glucopyranosyluronic acid fluoride and its evaluation as a mechanistic probe of Escherichia coli beta-glucuronidase
Publication TypeJournal Article
Year of Publication2001
AuthorsWong, AW, He, SM, Withers, SG
JournalCANADIAN JOURNAL OF CHEMISTRY-REVUE CANADIENNE DE CHIMIE
Volume79
Pagination510-518
Date PublishedMAY
ISSN0008-4042
Abstract

Synthesis of the potential mechanism-based inactivator of beta -D-glucuronidases (5-fluoro-beta -D-glucopyranosyluronic acid fluoride) was accomplished via a six-step process from D-glucuronic acid that involved radical bromination at C-5 and displacement of the bromide by fluoride. A key step in this process was the masking of the carboxylic acid as a phenacyl ester. This group is uniquely stable to conditions of photobromination and fluoride displacement, yet removable under very mild conditions. Incubation of the Escherichia coli beta -glucuronidase with 5-fluoro-beta -D-glucopyranosyluronic acid fluoride resulted in time-dependent inactivation of the enzyme through the accumulation of a covalent 5-fluoro-alpha -D-glucopyranosyluronic acid-enzyme. Peptic digestion of the 5-fluoro-alpha -D-glucopyranosyluronic acid-enzyme intermediate and subsequent analysis by liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer indicated the presence of a 5-fluoro-alpha -D-glucopyranosyluronic acid-modified peptide. This peptide was partially purified by HPLC and its sequence determined by tandem mass spectrometry in the daughter ion scan mode, permitting the identification of Glu504 as the catalytic nucleophile within the sequence ITEYGVD. This new reagent is therefore useful for the specific, mechanism-based inactivation of glycuronidases and has good potential in other studies of enzymes of this general class.

DOI10.1139/cjc-79-5-6-510