The roles of flavin-containing monooxygenases (FMOs) in the oxidation of seleno-L-methionine (SeMet) to L-methionine selenoxide (MetSeO) were investigated using cDNA-expressed human FMOs, purified rat liver FMOs, and rat liver microsomes. MetSeO and the N-2,4-dinitrophenylderivatives of SeMet and MetSeO were synthesized and characterized by 1 H-NMR and ESI/MS. These reference compounds were then used to develop a sensitive HPLC assay to monitor SeMet oxidation to MetSeO. Formation of MetSeO in rat liver microsomes was time-, protein concentration-, SeMet concentration-, and NADPH-dependent. The microsomal activity exhibited a SeMet K m value (mean ±S.D.; n=4) of 0.91 ± 0.29 mM and a V max value of 44 ± 8.0 nmol MetSeO/ mg protein/min. Inclusion of 1-benzylimidazole, superoxide dismutase or deferoxamine caused no inhibition of the rat liver microsomal activity. Because these results suggested the involvement of FMOs in the oxidation of SeMet in rat liver microsomes, formation of MetSeO was also examined using cDNA-expressed human and purified rat FMOs. The results showed that both rat and human FMO1 and FMO3 but not FMO5 can catalyze the reaction. The SeMet kinetic constants were obtained with purified rat liver FMO3 (K m = 0.11 mM, V max = 280 nmol/mg protein/min) and rat liver FMO1 (K m = 7.8 mM, V max = 1200 nmol/mg protein/min). Because SeMet has anti-cancer, chemopreventive, and toxic properties, the kinetic results suggest FMO3 is likely to play a role in the biological activities of SeMet at low exposure conditions.
The direct selective upper rim 1,3-formylation of conformationally rigid calix [4]arenes is achieved for the first time; the diametrical bis(formyl)calix[4]arene 4 is used as a key intermediate for the synthesis of new cavitands.
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