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.