evaporation and sublimation (130’ at 0.05 mm.), white crystals (0.3 mg.) of m.p. 162-163’ were obtained. The melting point remained unchanged upon admixture of authentic l-carbomethoxy-8-carboline. Rt values and ultraviolet and mass spectra were identical with those of 1-carbomethoxy-&carboline. Stability Test of Tuboflavine. The alkaloid was dissolved in a mixture of chloroform and methanol; aqueous ammonium hydroxide solution, silicic acid, and alumina (Woelm, neutral) were added. This
mixture was allowed to stand at room temperature for 4 weeks with occasional shaking. After this time on separation and esterification, no l-carbomethoxy-8carboline could be found. Acknowledgment. We are indebted to Dr. Frank A! Hochstein, Chas. Pfizer and Co., for the alkaloid extracts. This work was supported by a grant from the National Institutes of Health, Public Health Service (GM 054721, and from the National Science Foundation (G-21037).
Photochemical Degradation of Flavins. 11. The Mechanism of Alkaline Hydrolysis of 6,7-Dimethyl-9-formylmethylisoalloxazine’~2 Pill-Soon Song, Eddie C. Smith, and David E. Metzler
Contributionf r o m the Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa. ReceivedMay 17,1965 The alkaline degradation of 6,7-dimethyl-9-formylmethyl- Experimental Section isoalloxazine (FMF), an intermediate in the photolysis of Materials. FMF (6,7-dimethyl-9-formylmethylisoriboflavin, was studied kinetically. The reaction folalloxazine) was made in this laboratory.’ All the inlowed pseudo-first-order kinetics to give lumipavin with a organic compounds used were analytical reagent grade less signiBcant competing reaction yielding lumichrome. obtained from Mallinckrodt Chemical Co. Silica Gel A mechanism of the carbon-carbon bond cleavage in the G for thin-layer chromatography was obtained from alkaline cleavage of FMF has been proposed on the E. Merck, A.G., and thin-layer plates were prepared as basis of the kinetics and product identiBcation. The 1 previously reported. SigniJicance of the reaction is pointed out in connection Kinetic Measurements. The rate of disappearance of with the photolysis of riboflavin. FMF in borate buffer and unbuffered solutions in the dark was followed by measuring the decrease in abIntroduction sorbance at 445 mp under various conditions of pH and ionic strength ( p ) using a Cary Model 15 recording Anaerobic photolysis of a solution of riboflavin with spectrophotometer. It must be noted that, since the visible light leads to reduction of the isoalloxazine spectrum of LF shows a marked dependence on temring with the production of a hypothetical “leucoperature, probably due to a complexation equilibrium de~teroflavin.’’**~ Subsequent aeration causes a return of LF (unpublished observation), it is important to of the yellow color of oxidized flavins and the presumed maintain the same temperature for making spectral formation of “deuteroflavin.” Recently, Smith and measurements of reaction mixture and LF solution. Metzler have isolated from a photobleached and reAll measurements were made at 25 i 1”. The pH of oxidized riboflavin solution the compound 6,7-dimethreaction mixtures was measured by the Beckman pH yl-9-formylmethylisoalloxazine‘ (9-formylmethylflavin, meter and the desired pH was obtained with 0.1 or 0.2 FMF) which possesses all of the chemical characteristics N NaOH solution. The amount of NaOH solution to of the postulated “deuteroflavin,” including a faster bring about the desired pH was predetermined. The rate of photobleaching and a rapid conversion to kinetic measurements were then followed immediately lumiflavin in basic solutions.5 The present communicaafter adjusting the pH of the solutinos. The pH of tion is concerned with the mechanism of the alkaline the reaction mixture during the reaction was nearly unhydrolysis of FMF, an important reaction in the changed both in buffered and unbuffered solutions. photochemical degradation of riboflavin. The pH of the reaction mixture in unbuffered solution (1) Part I: E. C. Smith and D. E. Metzler, J. Am. Chem. Soc., 85, at the end of the reaction was slightly lower. 3285 (1963). (2) Supported by Grant No. G-12339 from the National Science Identification of Products. In addition to thin-layer Foundation. Abbreviations used throughout this paper: RF, ribochromatography which was employed as previously flavin; FMF, 9-formylmethylflavin; LF, lumiflavin; LC, lumichrome. (3) R. Kuhn, H. Rudy, and T. Wagner-Jauregg, Ber., 66, 1150 described, 1 1. of alkali-treated reaction mixture (about (1933). 1 X molell. of FMF) was extracted three-five (4) G. Oster, J. S. Bellin, and B. Holmstrom, Experientia, 18, 249 times with about 200 ml. of chloroform, and the chloro(1962). ( 5 ) The term “deuteroflavin” is no longer adequate because the 2‘form extracts were evaporated. The residue was then keto derivative of riboflavin which also fits the description of “deuterodissolved in about 200 ml. of water and extracted with flavin” has been isolated by Terao [M. Terao, Tohoku Igaku Zassi, 59, an equal volume of chloroform. The chloroform 441 (1959)l. Song, Smith, Metzler
Hydrolysis of 6,7-Dirnethyl-9-forrnylmethylisoal[oxazine
4181
Y
SOLVENT FRONT
‘I 0
I mln
10.5mln
1
o Q 0
D C’.,
i... :
RT
,-.
...J LF
..../.
:.p
