The chemical synthesis of riboflavin 5’-phosphorothioate (5‘-FMNS) is described. 5’-FMNS is obtained from the alkaline hydrolysis of riboflavin 4/,5’-cyclic phosphorothioate, which is produced upon reaction of riboflavin (RB) with thiophosphoryl chloride in trimethyl phosphate. 5’-FMNS has been tested for enzymatic reconstitution of NADPH-cytochrome P-450 reductase (EC 220.127.116.11) depleted of its FMN prosthetic group, but containing its full complement (1 equiv) of FAD. The synthesis, purification, and characterization of 5’-FMNS are reported, and documentation of its efficacy in reconstituting the reductase by fluorometric and absorbance spectrophotometric measurements, as well as enzymatic activity, is presented. Data indicate that 5’-FMNS is totally competent in reconstituting NADPH-cytochrome c reductase activity, which requires the presence of both FAD and a flavin mononucleotide, and its fluorescence is completely quenched upon addition to FMN-free NADPH-cytochrome P-450 reductase.
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