Structure and Oxidation of Pyrrole Adducts Formed between Aflatoxin B_2a and Biological Amines

Aflatoxin B_2a has been shown to bind to proteins through a dialdehyde intermediate under physiological conditions. The proposed structure of this adduct has been published showing a Schiff base interaction, but adequate verification using structural elucidation instrumental techniques has not been performed. In this work, we synthesized the aflatoxin B_2a amino acid adduct under alkaline conditions, and the formation of a new product was determined using high performance liquid chromatography-time-of-flight mass spectrometry. The resulting accurate mass was used to generate a novel proposed chemical structure of the adduct in which the dialdehyde forms a pyrrole ring with primary amines rather than the previously proposed Schiff base interaction. The pyrrole structure was confirmed using ¹H, ¹³C, correlation spectroscopy, heteronuclear single quantum correlation, and heteronuclear multiple bond correlation NMR and tandem mass spectrometry. Reaction kinetics show that the reaction is overall second order and that the rate increases as pH increases. Additionally, this study shows for the first time that aflatoxin B_2a dialdehyde forms adducts with phosphatidylethanolamines and does so through pyrrole ring formation, which makes it the first aflatoxin-lipid adduct to be structurally identified. Furthermore, oxidation of the pyrrole adduct produced a product that was 16 m/z heavier. When the aflatoxin B_2a-lysine (ε) adduct was oxidized, it gave a product with an accurate mass, mass fragmentation pattern, and ¹H NMR spectrum that match aflatoxin B₁-lysine, which suggest the transformation of the pyrrole ring to a pyrrolin-2-one ring. These data give new insight into the fate and chemical properties of biological adducts formed from aflatoxin B_2a as well as possible interferences with known aflatoxin B₁ exposure biomarkers.