Synthetic β-d-Glucuronides: Substrates for Exploring Glucuronide Degradation by Human Gut Bacteria

The human gut microbiota (HGM) is a complex ecosystem subtly dependent on the interplay between hundreds of bacterial species and numerous metabolites. Dietary phenols, whether ingested (e.g., plant-derived guaiacol, mequinol, or resveratrol) or products of bacterial fermentation (e.g., p-cresol), have been attributed with influencing bacterial growth and host health. They are cleared by phase II metabolism, one form utilizing β-d-glucuronidation, but encounter bacterially derived glucuronidases capable of hydrolyzing them to release their phenolic and glucuronic acid moieties with potential effects on host cells or the surrounding bacterial population. Tools to enable the detailed study of their activity are currently lacking. Syntheses of β-d-glucuronides from methyl 1,2,3,4 tetra-acetyl β-d-glucopyranosyluronate by direct glycosylation with 2-, 3-, or 4-methoxy- and 4-fluorophenol acceptors employing trimethylsilyl triflate catalysis are reported. Yields (methoxy series) were modest. An improved route from methyl 1,2,3,4-tetra-acetyl β-d-glucopyranosyluronate via selective anomeric deprotection (N-methyl piperazine) and conversion to an α-trichloroacetimidate glycosyl donor was employed. Coupling with 2- and 3-methoxyphenol acceptors and deprotection provided 2- and 3-methoxyphenyl β-d-glucuronides in 2-fold improved overall yield. These naturally occurring methoxyphenyl glucuronides augment available model substrates of dietary glucuronides, which include 3- and 4'-linked resveratrol. The use of model glucuronides as substrates was illustrated in studies of β-d-glucuronidase activity employing cell lysates of 9 species of HGM (Bacteroidetes), revealing distinct outcomes. Contrasting effects on bacterial growth were also observed between the free phenolic components, their respective glucuronides, and glucuronic acid. The glucuronide of 4-fluorophenol provided sensitive and background-free detection of β-glucuronidase activity using ¹⁹F NMR.