Absolute Stereochemistry of a 4a-Hydroxyriboflavin Analogue of the Key Intermediate of the FAD-Monooxygenase Cycle

The biological action of flavoenzymes, such as flavin adenine dinucleotide (FAD)-containing monooxygenase, involves the formation of oxygenated flavin derivatives, such as 4a-hydroperoxyflavin and 4a-hydroxyflavin, in which a new center of chirality is created at the 4a position during the enzymatic reactions. So far, the absolute configuration of this center of chirality in natural 4a-oxygenated flavins has remained unknown in spite of its key importance for the diverse functions of flavoenzymes. Herein, we report the 4a-hydroxy adduct 3 of 3-benzyl-5-ethyl-10-(tetraacetyl-D-ribityl)flavinium (1), one of the key intermediates involved in the enantioselective organocatalytic oxidation of sulfides to sulfoxides. The 4a-hydroxyflavin diastereomers (+)-3 and (-)-3, separated by HPLC, were characterized by electronic circular dichroism (CD) spectroscopy. Their absolute configurations at the 4a position were, for the first time, determined by comparing experimental CD spectra with those calculated by means of time-dependent density functional theory (TDDFT) on DFT-optimized structures obtained after an extensive conformation analysis.