Site-Specific Synthesis of Aflatoxin B₁ Adducts within an Oligodeoxyribonucleotide Containing the Human p53 Codon 249 Sequence

Abstract

This work describes the preparation of the cationic trans-8, 9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B(1) ((AFB)G) adducts at the positions corresponding to G(746) or G(747), within the oligodeoxyribonucleotide d(GGAGGCCT) containing the codon 249 sequence (underlined) of the p53 gene, using DNA triplexes to target adduction at the desired site. This approach enabled the successful preparation and purification of sufficient quantities of d(GGAG(AFB)GCCT) for NMR structural studies, using only standard phosphoramidites. The presence of multiple guanines in this oligodeoxynucleotide precluded the direct reaction of d(GGAGGCCT). d(AGGCCTCC) with aflatoxin epoxide as a method for producing large quantities of site-specific adducts for physical studies. Of the multiple potential alkylation sites at guanine N7 in d(GGAGGCCT). d(AGGCCTCC), it was found that sites G(2) and G(5) exhibited approximately equal reactivity with aflatoxin B(1)-exo-8,9-epoxide; the reactivity at site G(4) was reduced by approximately a factor of 2 as compared to that at G(2) or G(5). To successfully prepare the site-specific adducts, the p53 oligodeoxyribonucleotide was annealed with either the blocking strand d(CTCCATTTTCCT) or d(CCTCCATTTTCCTC) to form the corresponding partial triplexes which targeted AFB(1) adduction either to G(4) or to G(5). Piperidine cleavage, followed by heating, confirmed that in each instance, the product corresponded to the lone guanine not protected from adduction by the partial DNA triplex. The adducted oligodeoxyribonucleotides were examined with regard to purity by capillary electrophoresis. The primary advantage of this modified triple helix methodology is that it requires only standard phosphoramidites; thus, it is applicable to large-scale preparations that are necessary for NMR structural studies or other physical measurements.

Site-Specific Synthesis and Characterization of Oligonucleotides Containing an N6-(2-Deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine Lesion, the Ring-Opened Product from N7-Methylation of Deoxyguanosine

“…A shortcoming of the method is that methylation also occurs on O 6 of guanine, N 3 of adenine, and other sites. Direct alkylation has been used successfully for preparation of oligonucleotides containing the AFB-Fapy-dGuo adduct , . The regiospecificity of reactions of aflatoxin epoxide is much higher than that of dimethyl sulfate.…”

In Vitro Recapitulating of TP53 Mutagenesis in Hepatocellular Carcinoma Associated With Dietary Aflatoxin B1 Exposure

“…, AFB 1 - exo -8,9-epoxide 15–18. The exo -epoxide interacts with DNA predominantly through binding to guanine residues at N7 position forming trans -8,9-dihydro-8-(N7-guanyl)-9-hydoxyaflatoxin B 1 (AFB 1 -N7-Gua) adducts, although small quantities of the corresponding adenine adducts can also be formed 15, 16, 19–21. Alternatively, the highly reactive AFB 1 - exo -8,9-epoxide can be detoxified through conjugation with glutathione catalyzed by glutathione S -transferase, or conversion to AFB 1 -dihydrodiol catalyzed by microsomal epoxide hydrolase ( reviewed in 8, 9, 18).…”

Photophysics and photochemistry of aflatoxins B1 and B2

“…The principal site of covalent interaction of the epoxide with DNA is the N7 atom of guanine, leading to a positively charged adduct formation, Aflatoxin-DNA, which upon hydrolysis forms 8,9-dihydro-2-(guan-7-yl)-3hydroxy-aflatoxin. [18][19][20][21][22][23] Unlike aflatoxins B 1 and G 1 , aflatoxins B 2 Scheme 1 and G 2 show much lower carcinogenicity, which has been mainly associated to the absence of the furan ring unsaturation. 24 Recently, theoretical studies clearly demonstrated that aflatoxins, specially the B 1 form, can be responsible for phototoxic reactions involving the formation of reactive oxygen species.…”