Selective β-Mono-Glycosylation of a C15-Hydroxylated Metabolite of the Agricultural Herbicide Cinmethylin Using Leloir Glycosyltransferases

Cinmethylin is a well-known benzyl-ether derivative of the natural terpene 1,4-cineole that is used industrially as a pre-emergence herbicide in grass weed control for crop protection. Cinmethylin detoxification in plants has not been reported, but in animals, it prominently involves hydroxylation at the benzylic C15 methyl group. Here, we show enzymatic β-glycosylation of synthetic 15-hydroxy-cinmethylin to prepare a putative phase II detoxification metabolite of the cinmethylin in plants. We examined eight Leloir glycosyltransferases for reactivity with 15-hydroxy cinmethylin and revealed the selective formation of 15-hydroxy cinmethylin β-d-glucoside from uridine 5′-diphosphate (UDP)-glucose by the UGT71E5 from safflower (Carthamus tinctorius). The UGT71E5 showed a specific activity of 431 mU/mg, about 300-fold higher than that of apple (Malus domestica) UGT71A15 that also performed the desired 15-hydroxy cinmethylin mono-glycosylation. Bacterial glycosyltransferases (OleD from Streptomyces antibioticus, 2.9 mU/mg; GT1 from Bacillus cereus, 60 mU/mg) produced mixtures of 15-hydroxy cinmethylin mono- and disaccharide glycosides. Using UDP-glucose recycling with sucrose synthase, 15-hydroxy cinmethylin conversion with UGT71E5 efficiently provided the β-mono-glucoside (≥95% yield; ∼9 mM) suitable for biological studies.