Synthesis, characterization, and applications of oligonucleotides containing the major adducts derived from the carcinogenic nitropyrenes

Date of Completion

January 2001


Chemistry, Biochemistry




Nitropolycylic aromatic hydrocarbons are environmental pollutants that have been detected in samples such as diesel fuel exhaust, coal fly ash and cigarette smoke. Many of the compounds within this group are mutagenic and carcinogenic. 1-Nitropyrene is the most abundant nitroaromatic in many environmental samples and 1,6- and 1,8-dinitropyrene are among the most mutagenic nitroaromatics ever tested in the Ames assay. For these reasons, the nitropyrenes have been extensively studied as representative compounds from the nitroaromatic carcinogens. The major objective of this research was to synthesize oligonucleotides site-specifically modified with N-(deoxyguanosin-8-yl)-1-amino-6- (or 8-) nitropyrene, the major DNA adducts derived from the dinitropyrene isomers. In vivo, the dinitropyrenes undergo selective enzymatic reduction of one nitro group and intermediates generated along this pathway react with DNA to form adducts. In order to synthesize oligonucleotides containing these DNA adducts it was necessary to first synthesize the metabolic intermediates responsible for adduct formation. This was accomplished by a novel synthesis of aminonitropyrene isomers employing palladium-catalyzed amination. Subsequent oxidation to nitrosonitro derivatives and in situ generation of the N-hydroxyl amine derivatives in the presence of duplex oligonucleotide generated the adducted DNA fragment. The isolation of modified product was accomplished by reverse phase HPLC. ^ In order to fully characterize the modified oligonucleotides, several techniques were employed. Piperidine treatment of the modified oligonucleotides indicated that the site of adduct formation was the central G within the sequence. Enzymatic digestion of the modified oligonucleotides revealed the presence of an adducted deoxyguanosine that coelutes with a standard containing the aminonitropyrene adduct bound at the C-8 position of guanine. Additional evidence was obtained by mass spectrometry of the modified oligonucleotides. These results confirmed the synthesis of oligonucleotides containing the major adducts derived from 1,6- and 1,8-dinitropyrene. ^ Finally, translesion synthesis of the 1-nitropyrene adduct was examined using several DNA polymerases. A 50mer template containing the major adduct derived from 1-nitropyrene was constructed. The ability of several polymerases to replicate past this DNA lesion was examined. The studies indicated that the 1-nitropyrene adduct is a replication blocking lesion and polymerase bypass occurred to a limited extent. ^