Synthesis and Biochemical Evaluation of Bis(6,7-dimethyl-8-D-ribityllumazines) as Potential Bisubstrate Analogue Inhibitors of Riboflavin Synthase Mark Cushman,* ,† Farahnaz Mavandadi, † Donglai Yang, † Karl Kugelbrey, ‡ Klaus Kis, ‡ and Adelbert Bacher ‡ Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy, Purdue University, West Lafayette, Indiana 47907, and Lehrstuhl fu ¨ r Organische Chemie und Biochemie, Technische Universita ¨ t Mu ¨ nchen, D-85747 Garching, Germany Received October 28, 1998 (Revised Manuscript Received March 31, 1999) The reaction catalyzed by riboflavin synthase utilizes two identical 6,7-dimethyl-8-D-ribityllumazine substrate molecules. Three bis(6,7-dimethyl-8-D-ribityllumazines) were, therefore, synthesized in which the two lumazine moieties were connected through their N-3 nitrogen atoms by polymethylene linker chains containing three, four, and five carbon atoms. The compounds with three and five carbon linkers were found to be very weak inhibitors of riboflavin synthase, having inhibition constants of 320 and >1000 µM, respectively. In contrast, the bis(lumazine) with a four-carbon linker was much more potent, with an inhibition constant of 37 µM. These results have potential implications for understanding the distance between the donor and acceptor sites of riboflavin synthase and the orientations of the two 6,7-dimethyl-8-D-ribityllumazine substrate molecules which occupy these two sites. Introduction Riboflavin synthase (E.C. 2.5.1.9) catalyzes an unusual dismutation reaction, involving the transfer of a four- carbon unit from one molecule of 6,7-dimethyl-8-(D- ribityl)lumazine (1) bound at the donor site of the enzyme to a second molecule of 1 bound at the acceptor site of the enzyme to form one molecule of riboflavin (2) and one molecule of the pyrimidinedione 3. 1-3 A working hypoth- esis concerning the reaction mechanism has been ad- vanced as shown in Scheme 1. 4-6 Addition of an uniden- tified nucleophile to 1 yields 4 at the donor site, which then undergoes nucleophilic attack by an anion 5, formed by deprotonation of the 7-methyl group of lumazine 1 bound at the acceptor site. A 1,2-elimination in 6 followed by a 1,6-elimination in 7 yields a conjugated triene system in 8, which undergoes a 3,3-sigmatropic rear- rangement to afford intermediate 9. Intermediate 9 then aromatizes by 1,2-elimination to yield the final products, riboflavin (2) and the pyrimidinedione 3. Although the overall transformation is mechanistically complex, Wood and co-workers were able to demonstrate that riboflavin (2) forms in the absence of enzyme when the lumazine 1 was boiled in phosphate buffer at neutral pH. 7,8 Beach and Plaut showed that the nonenzymatic reaction also occurs under acidic conditions. 9 Both the catalyzed and uncatalyzed reaction are characterized by strict re- giospecificity. The participation of two identical lumazine molecules 1 as enzyme substrates for riboflavin synthase suggests the possibility of bisubstrate inhibitors in which two lumazine moieties are connected by a linker chain. Two critical variables in this strategy for inhibitor design are the points of attachment of the linker chain to the two lumazine moieties and the length of the linker chain. In order for the mechanism proposed in Scheme 1 to operate, the anion 5 generated by deprotonation of the methyl group at C-7 on one lumazine molecule at the acceptor site must attack the C-6 carbon atom of another lumazine † Purdue University. ‡ Technische Universita ¨t Mu ¨ nchen. (1) Plaut, G. W. E.; Smith, C. M.; Alworth, W. L. Annu. Rev. Biochem. 1974, 43, 899-922. (2) Plaut, G. W. E. In Comprehensive Biochemistry; Florkin, M., Stotz, E. H., Eds.; Elsevier: Amsterdam, 1971; Vol. 21, pp 11-45. (3) Bacher, A.; Ladenstein, R. In Chemistry and Biochemistry of Flavoenzymes; Mu ¨ ller, F., Ed.; Chemical Rubber Co.: Boca Raton, FL, 1991; Vol. II, pp 293-316. (4) Beach, R. L.; Plaut, G. W. E. J. Am. Chem. Soc. 1970, 92, 2913- 2916. (5) Paterson, T.; Wood, H. C. S. J. Chem. Soc., Chem. Commun. 1969, 290-291. (6) Paterson, T.; Wood, H. S. C. J. Chem. Soc., Perkin Trans. 1 1972, 1051-1056. (7) Rowan, T.; Wood, H. S. C. Proc. Chem. Soc. 1963, 21-22. (8) Rowan, T.; Wood, H. S. C. J. Chem. Soc. 1968, 452-458. (9) Beach, R.; Plaut, G. W. E. Tetrahedron Lett. 1969, 3489-3492. 4635 J. Org. Chem. 1999, 64, 4635-4642 10.1021/jo9821731 CCC: $18.00 © 1999 American Chemical Society Published on Web 06/06/1999