Abstract
When a mixture of riboflavin (RF) and p-aminosalicylate (PAS) in a very small volume of pyridine was heated and diluted, red color was obtained. By gel-filtration with Sephadex G-15 followed by paper chromatography with butanol : acetic acid : water (4: 1: 5), formation of red substance was confirmed. In addition, some other substances were also produced, which were detected as yellow spots on paper chromatogram under UV light. In the absorption spectrum of the red product, 3 peaks were observed at 266 mμ, 525 mμ and 565 mμ. It remained at the original point by paper chromatography and was found to be non-dialysable and to be isolated from the reaction mixture through sufficient dialysis. Its melting point was shown to be 324°-326°C (dec.). Yellow solution was formed by dissolving it in conc. HCl or N, N-dimethylformamide (DMF), but turned to red by dilution. Incidentally, the spectrum having 2 peaks at 490 mμ and 264 mμ or 283 mμ changed to the original one with 3 peaks.
In the formation of the red substance, only PAS and RF are effective, but other phenolic derivatives and FMN, FAD or lumiflavin have no or less ability. Much quantities of the red substance were formed for longer reaction time and at higher temperature. Furthermore, the optimum ratio in amount of RF and PAS was observed to obtain the maximum yield. Volume and concentration of pyridine must be also restricted for the maximal production. Glacial acetic acid and DMF could be also employed as solvent. However, pinkish red substance which has Rf value of about 0.17 was formed with acetic acid differing from pyridine and DMF. On the contrary, formation of the red substance was not confirmed with butyl alcohol nor octyl alcohol.
