Synthesis and Characterization of 15N5-Labeled Aflatoxin B1–Formamidopyrimidines and Aflatoxin B1–N7-Guanine from a Partial Double-Stranded Oligodeoxynucleotide as Internal Standards for Mass Spectrometric Measurements

Aflatoxin B1 (AFB1) exposure through contaminated food is a primary contributor to hepatocellular carcinogenesis worldwide. Hepatitis B viral infections in livers dramatically increase the carcinogenic potency of AFB1 exposures. Liver cytochrome P450 oxidizes AFB1 to the epoxide, which in turn reacts with N7-guanine in DNA, producing the cationic trans-8,9-dihydro-8-(N7-guanyl)–9-hydroxyaflatoxin B1 adduct (AFB1–N7-Gua). The opening of the imidazole ring of AFB1–N7-Gua under physiological conditions causes the formation of the cis- and trans-diastereomers of 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)–9-hydroxyaflatoxin B1 (AFB1–FapyGua). These adducts primarily lead to G → T mutations, with AFB1–FapyGua being significantly more mutagenic than AFB1–N7-Gua. The unequivocal identification and accurate quantification of these AFB1–Gua adducts as biomarkers are essential for a fundamental understanding and prevention of AFB1-induced hepatocellular carcinogenesis. Among a variety of analytical techniques used for this purpose, liquid chromatography–tandem mass spectrometry, with the use of the stable isotope-labeled analogues of AFB1–FapyGua and AFB1–N7-Gua as internal standards, provides the greatest accuracy and sensitivity. cis-AFB1–FapyGua-15N5, trans-AFB1–FapyGua-15N5, and AFB1–N7-Gua-15N5 have been synthesized and used successfully as internal standards. However, the availability of these standards from either academic institutions or commercial sources ceased to exist. Thus, quantitative genomic data regarding AFB1-induced DNA damage in animal models and humans remain challenging to obtain. Previously, AFB1–N7-Gua-15N5 was prepared by reacting AFB1-exo-8,9-epoxide with the uniformly 15N5-labeled DNA isolated from algae grown in a pure 15N-environment, followed by alkali treatment, resulting in the conversion of AFB1–N7-Gua-15N5 to AFB1–FapyGua-15N5. In the present work, we used a different and simpler approach to synthesize cis-AFB1–FapyGua-15N5, trans-AFB1–FapyGua-15N5, and AFB1–N7-Gua-15N5 from a partial double-stranded 11-mer Gua-15N5-labeled oligodeoxynucleotide, followed by isolation and purification. We also show the validation of these 15N5-labeled standards for the measurement of cis-AFB1–FapyGua, trans-AFB1–FapyGua, and AFB1–N7-Gua in DNA of livers of AFB1-treated mice.