Phenoxazinones, synthesis

The data obtained in a number of manometric experiments agree with the above formulation (Table 11). When either 3-HAA or 4-MHAA was used as substrate, three atoms of oxygen were utihzed in the formation of one equivalent of the corresponding phenoxazinone. Phenoxazinone synthesis was not observed when the reaction was carried out under anaerobic conditions. The overall reaction consists of several oxidations (removal of 6 H). It is unlikely that the enzyme is directly involved in the transfer of all of these hydrogens. One possibility is that the enzyme catalyzes only the initial activation of the substrate perhaps to a quinonimine. The activated substrate could then oxidize non-enz anatically to form the phenoxazinone. Although the data obtained are consistent with the... 

Table ll. Oxygen uptake during phenoxazinone synthesis... 

Figure 6 Assembly of 4-MHA and the five constituent amino acids in the synthesis of 4-MH.A pentapeptide lactone D. The conesponding actinomycin D arises by oxidative condensation of two of these 4-MHA pentapeptide lactone moieties, catalyzed by phenoxazinone synthase. Sar = sarcosine (N-methylglycine) MeVal M-methyl-L-valine.

The enzymatic synthesis of the actinomycin chromophore also has been described (Weissbach and Katz, 1961 Katz and Weissbach 1962 Salzman, Weissbach and Katz, unpublished results). An enzyme, designated phenoxa-zinone synthetase, was obtained from sonic extracts of S. antihioticus and was shown to catalyze the oxidative condensation of o-aminophenols to the corresponding phenoxazinone. When 3-hydroxy-4-methylanthranilic acid (4MHAA) was used as substrate, actinocin was synthesized. The enzyme has been purified approximately 25- to 30-fold from sonic or French pressure cell extracts of the organism by means of standard protein fractionation procedures (ammonium... 

The effect of chloramphenicol on synthesis of phenoxazinone sjmthetase by S. antibioticus also was examined. If chloramphenicol is added to a freshly inoculated culture or to a 12-hour old culture, no further synthesis of cell material occurs nor is there any production of actinomycin subsequently. When the inhibitor is supplied to the culture during sjmthesis of actinomycin, antibiotic formation is not affected by the inhibitor for several hours. Chloramphenicol inhibits protein synthesis including syi thesis of the enz5nne, phenoxazinone... 

Table 13. Inhibition of phenoxazinone synthetase synthesis by chloramphenicol...

Weissbach, H., and E. Katz Studies on the biosynthesis of actinomycin Enzymic synthesis of the phenoxazinone chromophore. J. Biol. Chem. 236, PC 17 (1961). 

The synthesis of 2-hydroxy-3H-phenoxazinones from o-aminophenols and quinones has been successfully adapted to the preparation of the phenothiazine analogues, e.g. (138), by condensation of 2,5-dimethoxy-l,4-benzoquinones with SH-protected o-aminobenzenethiols (137), followed by... 

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