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 orde r to study its reactivity at C-7 and its binding to riboflavin synthas e of Bacillus subtilis. Compound 17 was prepared by reaction of o-4-[( D-ribityl)amino]-2,4-(1H,3H)-pyrimidinedione hydrochloride (3.HCl) wit h trifluoropyruvaldehyde hydrate (18). NMR studies revealed that under basic conditions, 17 forms only one major anionic species in which th e oxygen of the 3'-hydroxyl group on the ribityl side chain binds cova lently to C-7 of the lumazine, resulting in the formation of a pyran r ing. As a model for possible addition of nucleophilic groups on the en zyme to C-7 of 17, the reactions of 17 with a variety of sulfur nucleo philes were studied. Fluorolumazine 17 was found to form covalent addu cts 27-31 with sulfite, sulfide, mercaptoethanol, D,L-1,4-dithiothreit ol, and L-cysteine. Three molecules of 17 were found to bind per enzym e molecule (alpha subunit trimer). Equilibrium dialysis experiments an d 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 betwe en the fluorolumazine 17 and the enzyme, suggesting that the nucleophi le 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 adduct s 27-31 as well as 17 could be displaced from the enzyme by both ribof lavin (2) and o-6-[(D-ribityl)-amino]-2,4(1H,3H)-pyrimidinedione (32).