F-19 NMR-STUDIES OF THE MECHANISM OF RIBOFLAVIN SYNTHASE - SYNTHESIS OF 6-(TRIFLUOROMETHYL)-8-(D-RIBITYL)LUMAZINE AND DERIVATIVES

6-(Trifluoromethyl)-8-(D-ribityl)lumazine (17) was synthesized in order to study its reactivity at C-7 and its binding to riboflavin synthase of Bacillus subtilis. Compound 17 was prepared by reaction of 5-amino-4-[(D-ribityl)amino]-2,4-(1H,3H)-pyrimidinedione hydrochloride (3.HCl) with trifluoropyruvaldehyde hydrate (18). NMR studies revealed that under basic conditions, 17 forms only one major anionic species in which the oxygen of the 3'-hydroxyl group on the ribityl side chain binds covalently to C-7 of the lumazine, resulting in the formation of a pyran ring. As a model for possible addition of nucleophilic groups on the enzyme to C-7 of 17, the reactions of 17 with a variety of sulfur nucleophiles were studied. Fluorolumazine 17 was found to form covalent adducts 27-31 with sulfite, sulfide, mercaptoethanol, D,L-1,4-dithiothreitol, and L-cysteine. Three molecules of 17 were found to bind per enzyme molecule (alpha subunit trimer). Equilibrium dialysis experiments and F-19 NMR spectroscopy provided dissociation constants K(D) of 38 and 100 muM, respectively. The inhibition constant K(I) was 58 muM. There was no evidence obtained for the formation of a covalent adduct between the fluorolumazine 17 and the enzyme, suggesting that the nucleophile adding to C-7 during the enzyme-catalyzed reaction is derived from water. The covalent adducts obtained from 17 were found to bind to the enzyme significantly more tightly than 17 itself. The covalent adducts 27-31 as well as 17 could be displaced from the enzyme by both riboflavin (2) and 5-nitroso-6-[(D-ribityl)-amino]-2,4(1H,3H)-pyrimidinedione (32).