Configurational and Conformational Equilibria of N6-(2-Deoxy-d-erythro-pentofuranosyl)-26-diamino-34-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG) Lesion in DNA

摘要

The most common lesion in DNA occurring due to clinical treatment with Temozolomide or cellular exposures to other methylating agents is 7-methylguanine (N7-Me-dG). It can undergo a secondary reaction to form N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG). MeFapy-dG undergoes epimerization in DNA to produce either α or β deoxyribose anomers. Additionally, conformational rotation around the formyl bond, C5–N⁵ bond, and glycosidic bond may occur. To characterize and quantitate the mixture of these isomers in DNA, a ¹³C-MeFapy-dG lesion, in which the CH3 group of the MeFapy-dG was isotopically labeled, was incorporated into the trimer 5′-TXT-3′ and the dodecamer 5′-CATXATGACGCT-3′ (X = ¹³C-MeFapy-dG). NMR spectroscopy of both the trimer and dodecamer revealed that the MeFapy-dG lesion exists in single strand DNA as ten configurationally and conformationally discrete species, eight of which may be unequivocally assigned. In the duplex dodecamer, the MeFapy-dG lesion exists as six configurationally and conformationally discrete species. Analyses of NMR data in the single strand trimer confirm that for each deoxyribose anomer, atropisomerism occurs around the C5–N⁵ bond to produce Ra and Sa atropisomers. Each atropisomer exhibits geometrical isomerism about the formyl bond yielding E and Z conformations. ¹H NMR experiments allow the relative abundances of the species to be determined. For the single strand trimer, the α and β anomers exist in a 3:7 ratio, favoring the β anomer. For the β anomer, with respect to the C5–N⁵ bond, the Ra and Sa atropisomers are equally populated. However, the Z geometrical isomer of the formyl moiety is preferred. For the α anomer, the E–Sa isomer is present at 12%, whereas all other isomers are present at 5–7%. DNA processing enzymes may differentially recognize different isomers of the MeFapy-dG lesion. Moreover, DNA sequence-specific differences in the populations of configurational and conformational species may modulate biological responses to the MeFapy-dG lesion.