Design, synthesis, and biochemical evaluation of 1,5,6,7-tetrahydro-6,7-dioxo-9-D-ribitylaminolumazines bearing alkyl phosphate substituents as inhibitors of lumazine synthase and riboflavin synthase

The last two steps in the biosynthesis of riboflavin, an essential metabolite that is involved in electron transport, are catalyzed by lumazine synthase and riboflavin synthase. To obtain structural probes and inhibitors of these two enzymes, two ribityllumazinediones bearing alkyl phosphate substituents were synthesized. The synthesis involved the generation of the ribityl side chain, the phosphate side chain, and the lumazine system in protected form, followed by the simultaneous removal of three different types of protecting groups. The products were designed as intermediate analogue inhibitors of lumazine synthase that would bind to its phosphate-binding site as well as its lumazine binding site. Both compounds were found to be effective inhibitors of Bacillus subtilis lumazine synthase as well as Escherichia coli riboflavin synthase. Molecular modeling of the binding of one of the two compounds provided a structural explanation for how these compounds are able to effectively inhibit both enzymes. In phosphate-free buffer, the phosphate moieties of the inhibitors were found to contribute positively to their binding to Mycobacterium tuberculosis lumazine synthase, resulting in very potent inhibitors with K-i values in the low nanomolar range. The additional carbonyl in the dioxolumazine system versus the purinetrione system was found to make a positive contribution to its binding to E. coli riboflavin synthase.