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.

Separation and structure of the prosthetic group of the blue fluorescence protein from the bioluminescent bacterium Photobacterium phosphoreum

“…The aromatic methine protons at positions 6 and 9 of the benzenoid ring of flavins exhibit singlets in the region 7.5-8.5 ppm (18,19); these resonances were not observed with the isolated compound. Though several flavins exhibit absorption spectra with maxima in the region around 400 nm (20)(21)(22)(23)(24), their absorption spectra are otherwise quite different from those of the lumazines (11).…”

Sarcosine Oxidase Contains a Novel Covalently Bound FMN

“…Peak 3 coelutes with the major contaminant in commericial FMN and appears to be a phosphorylated riboflavin derivative since it is converted to riboflavin upon treatment with alkaline phosphatase (data not shown). The major contaminant in commercial FMN has previously been identified as riboflavin 4′-phosphate (Scola-Nagelschneider & Hemmerich, 1976). A 1:1 mixture of 4′-and 5′-phosphate esters of riboflavin is formed upon acid hydrolysis of the 4′,5′-cyclic phosphate of FMN (Scola-Nagelschneider & Hemmerich, 1976), suggesting that the latter may be an intermediate in the acid hydrolysis of FAD.…”

Biosynthesis of Riboflavin Preparation of Phosphorylated Pyrimidine Intermediates

“…Since the first synthesis re ported by Kuhn and coworkers [20] in 1936, num er ous publications and patents have appeared. How ever, as reviewed in detail by Scola-Nagelschneider and Hemm erich [19], all known procedures lead to a m ixture of products containing at best about 75% of the target compound. Pure 5 '-FMN is only available by affinity chrom atography [18] or by HPLC separa tion [16].…”

“…The m ajor peak was unequivocally identified as the 5'-phosphate by binding to apoflavodoxin from M egasphaera elsdenii [16] using the method of Wassink and Mayhew [17]. It has been shown earlier that this protein shows very high selectivity for 5'-phos phoric acid esters [16,18,19].…”