Design, Synthesis, and Evaluation of Acyclic <i>C-</i>Nucleoside and <i>N</i>-Methylated Derivatives of the Ribitylaminopyrimidine Substrate of Lumazine Synthase as Potential Enzyme Inhibitors and Mechanistic Probes

Lumazine synthase and riboflavin synthase catalyze the last two steps in the biosynthesis of riboflavin, a vitamin that is involved in many critical biochemical reactions that are essential for the maintenance of life. To obtain inhibitors and structural probes that could be useful in studying the structures of bound reaction intermediates, the ribitylamino N−H moiety of the lumazine synthase substrate was replaced by CH<sub>2</sub> and N−CH<sub>3</sub> groups. The CH<sub>2</sub> replacement unexpectedly and completely abolished the affinity for lumazine synthase, thus revealing a critical, yet unexplained, role of the ribitylamino N−H moiety in conferring affinity for the enzyme. In contrast, the N−CH<sub>3</sub> replacement resulted in an inhibitor of both lumazine synthase and riboflavin synthase. Replacement of the ribitylamino N−H moiety with epimeric C−F moieties led to inhibition of lumazine synthase and riboflavin synthase when combined with the replacement of the 5-amino group with a nitro substituent.