Regioselectivity of human UDP-glucuronosyl-transferase 1A1 in the synthesis of flavonoid glucuronides determined by metal complexation and tandem mass spectrometry.
A three-part tandem mass spectrometric strategy that entails MSn analysis and a post-column LC-MS cobalt complexation method is developed to identify flavonoid monoglucuronide metabolites synthesized using the 1A1 isozyme of human UDP-glucuronosyltransferase (UGT). Ten flavonoid aglycons were used as substrates, spanning the subclasses of flavones, flavonols, and flavanones. The products were characterized by LC-MS and LC-MSn, with post-column cobalt complexation employed to pinpoint the specific sites of conjugation. The dissociation of complexes of the form [Co(II) (flavonoid glucuronide - H) (4,7-diphenyl-1,10-phenanthroline)(2)]+ allowed identification of the products and differentiation of isomers. The correlation between glycosylation site and elution order is used to provide additional structural confirmation. Flavonoids lacking a 3' hydroxyl group were glucuronidated only at position 7, while those containing this functionality also formed 3'-O-glucuronides and sometimes 4'-O-glucuronides, thus supporting the conclusion that the presence or absence of the 3'-OH group is the major determinant of the regioselectivity of glucuronidation. Moreover, the specific distribution of multiple glucuronide products (7-O, 3'-O, 4'-O) is governed by the subclass of flavonoid.
Davis
Barry D
BD
Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas 78712, USA.
Brodbelt
Jennifer S
JS
D002851:Chromatography, High Pressure Liquid; D003035:Cobalt / Q000737:chemistry; D005419:Flavonoids / Q000032:analysis* / Q000096:biosynthesis* / Q000737:chemistry; D020719:Glucuronides / Q000032:analysis* / Q000096:biosynthesis* / Q000737:chemistry; D014453:Glucuronosyltransferase / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007536:Isomerism; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D000099148:UGT1A1 Enzyme
D003035:Cobalt; D005419:Flavonoids; D020719:Glucuronides; D014453:Glucuronosyltransferase; D000099148:UGT1A1 Enzyme