Dual regioselective glycosylation of naringenin by Deinococcus deserti amylosucrase: regioisomers identification via methylation-LC-MS/MS.

Enzymatic α-glycosylation of flavonoids improves physicochemical and functional properties compared to β-glycosides. Amylosucrase efficiently synthesizes flavonoid α-glycosylation through transglycosylation using sucrose as a donor and flavonoids as acceptor. In this study, naringenin glycosylation by amylosucrase from Deinococcus deserti (DdAS) was investigated, and a methylation derivatization-LC-MS/MS approach was applied to characterize a trace-level regioisomer. DdAS demonstrated 2.4-fold higher transglycosylation efficiency (21.3%) toward naringenin than amylosucrase from D. geothermalis and produced two distinct monoglucoside peaks on HPLC analysis, indicating dual regioselectivity. Methylation derivatization-LC-MS/MS successfully identified both the predominant 4'-O-glucoside and the trace-level 7-O-glucoside, enabling direct structural characterization from the crude reaction mixture without requiring isolation or purification. Molecular dynamics simulations revealed that enhanced loop 3 flexibility in DdAS over DgAS facilitated dual regioselectivity through alternative substrate binding orientations. The synthesized naringenin α-glycosides exhibited 3750-fold enhanced water solubility and 4.5-fold improved antioxidant activity compared to naringenin, highlighting their potential as bioavailable functional ingredients for food and nutraceutical applications. In addition, these findings demonstrate that methylation derivatization-LC-MS/MS is an effective strategy for identifying trace-level flavonoid glycoside regioisomers.