Synthesis, Separation, Identification and Interconversion of Riboflavin Phosphates and Their Acetyl Derivatives: A Reinvestigation

Abstract

A reinvestigation of flavin phosphate synthesis, separation, identification, and interconversion was made in view of contradictory results in the literature. It has been confirmed that monochlorophosphoric acid is the best agent for selective 5′-monophosphorylation of riboflavin and derivatives. This reaction yields, however, invariably up to 20% of an isomer, which has been separated by preparative thick-layer chromatography and shown to be the 4′-monophosphate. All the earlier authors failed to detect this isomer which does not bind to flavodoxin. It equilibrates in dilute mineral acid to yield an 8:2 mixture of 5′-phosphate to 4′-phosphate by phosphate migration. The formation of 2′,3′,4′-triacetyl-flavin mononucleotide, according to Christie, S. M. H., Kenner, G. W. & Todd, A. R. (1954) J. Chem. Soc., 46–52, upon acid-catalysed acetylation of pure FMN, was confirmed. The same reaction under base catalysis, however, does not yield 2′,3′,4′-triacetyl-flavin mononucleotide as claimed by Khomutova, E. D., Shapiro, T. A., Mezentseva, M. V. & Berezovskii, V. M. (1965) Otd. Ohshch. i Tekhn. Khim., 241–244, Chem. Absir. 65, 5516a, but in fact up to 80% 2′,3′-diacetyl-flavin 4′: 5′-cyclophosphate as the main product, which is stable under neutral and weak acidic conditions and does not hydrolyse to 2′,3′-diacetyl-flavin 5′-monophosphate as claimed by McCormick, D. B. (1974) J. Heterocycl. Chem. 11, 969–974. The various flavin phosphates and their acetyl derivatives have been identified by pH-titration, electrophoresis, and proton magnetic resonance spectrometry, which permit direct analyses of crude reaction products as well as a rapid purity check of commercial FMN.