N-hydroxy-2-fluorenylacetamide. Reaction of the carcinogen with guanosine, ribonucleic acid, deoxyribonucleic acid, and protein following enzymatic deacetylation or esterification.

Previous studies showed that the carcinogen N-hydroxy-2fluorenylacetamide could react with RNA following enzymatic activation of the hydroxamic acid by deacetylation or esterification. Present studies indicate that 2’-(3’)-GMP, but not 2’-(3’)-AMP, -CMP, or -UMP, competes effectively with RNA in these reactions. &(N-Z-Fluorenylamino)guanosine was isolated and identified after incubation of guanosine, N-hydroxy-2-fluorenylacetamide, and soluble rat liver enzymes in a system favoring deacetylation. This adduct is also formed from reaction of N-Z-fluorenylhydroxylamine and guanosine at pH 7.4. 2-Nitrosofluorene, an oxidation product of the hydroxylamine, does not participate in formation of the adduct. 8-(N-2-Fluorenylacetamido)guanosine was recovered following incubation of guanosine, N-hydroxy-Z-fluorenylacetamide, rat liver-soluble enzymes, and J’-phosphoadenosine 5’-phosphosulfate or ATP and MgCL. The 5’-mononucleotide analogues of the guanosine adducts were identified after nucleic acid modified by reaction with enzymatically activated derivatives of N-hydroxy-Z-fluorenylacetamide was treated with phosphodiesterase. Digestion of the nucleotide adducts with alkaline phosphatase yielded the guanosine adducts. These data, considered with results of the competition studies, implicate carbon 8 of nucleic acid guanine as the predominant site of adduct formation. Generation of the enzymatically activated derivatives of N-hydroxy-2-fluorenylacetamide in the presence of DNA or protein results in the incorporation of the carcinogen into these macromolecules. The isolation of 8-(N-2-fluorenylamino)guanosine and &(N-2-fluorenylacetamido)guanosine from aqueous solutions by adsorption onto Sephadex G-10 and elution with ethanol is detailed. A nondestructive method for the determination of the specific activity of labeled nucleic acids as their cetyltrimethylammonium salts is described.