Azidoalanine mutagenicity in Salmonella: effect of homologation and alpha-methyl substitution

Azide mutagenicity in susceptible non-mammalian systems involves the requisite formation of L-azidoalanine, a novel mutagenic amino acid. The biochemical mechanism(s) of azidoalanine-induced mutagenesis, however, is not known. Previous studies of the structural requirements for azidoalanine mutagenicity suggested the importance of free L-amino acid character, and that bioactivation of azidoalanine to the ultimate mutagenic species is required. To gain more insight into possible enzymatic processing, the alpha-methyl analogue, alpha-methyl-azidoalanine, and the homologue, 2-amino-4-azidobutanoic acid, were synthesized and tested for mutagenic potency in Salmonella typhimurium strain TA1530. In addition, azidoacetic acid, a possible azidoalanine metabolite, was prepared and tested. The results show that alpha-methyl substitution effectively blocks the mutagenic effects of azidoalanine with alpha-methyl-azidoalanine being nearly devoid of mutagenic activity. In contrast, homologation of azidoalanine to yield 2-amino-4-azidobutanoic acid produces a marked increase in molar mutagenic potency. As with azidoalanine, the mutagenic activity of this homologue is associated with the L-isomer. Azidoacetic acid, however, was only very weakly mutagenic when tested as either the free acid or ethyl ester. This low mutagenic potency may indicate that bioactivation does not involve the entry of azide-containing azidoalanine catabolite into the Kreb's cycle. The high potency of 2-amino-4-azidobutanoic acid may be indicative of more efficient bioactivation and/or greater intrinsic activity. Importantly, the latter finding clearly shows that potent azido-amino acid mutagenicity is not limited to azidoalanine alone.