SYNTHESIS OF EPIMERIC 6,7-BIS(TRIFLUOROMETHYL)-8-RIBITYLLUMAZINE HYDRATES - STEREOSELECTIVE INTERACTION WITH THE LIGHT RIBOFLAVIN SYNTHASE OF BACILLUS-SUBTILIS

Reaction of perfluorobutane-2,3-dione with 5-amino-6-(D-ribitylamino)pyrimidine-2,4(1H,3H)-dione hydrochloride (13) gave a mixture of the epimeric covalently hydrated fluorolumazines 14a and 14b, which were separated by preparative HPLC. The epimer that eluted first using a methanol-ammonium formate buffer mobile phase (epimer A) interacts stereospecifically with light riboflavin synthase of Bacillus subtilis as shown by F-19 NMR spectroscopy, which provided evidence for the formation of three enzyme-bound species that exchange with the free ligand on the NMR time scale. Dissociation constants of 13.0 and 16.2-mu-M were obtained from NMR and equilibrium dialysis experiments, respectively. Both methods showed the binding of one ligand molecule per protein subunit. Epimer A is a competitive inhibitor of the enzyme (K(I) = 38-mu-M at pH 6.8). Epimer B did not bind to the enzyme.