Studies on N4-(2-Deoxy-D-pentofuranosyl)-4,6-diamino-5-formamidopyrimidine (Fapy•dA) and N6-(2-Deoxy-D-pentofuranosyl)- 6-diamino-5-formamido-4-hydroxypyrimidine (Fapy•dG) † Marc M. Greenberg,* Zsolt Hantosi, Carissa J. Wiederholt, and Christopher D. Rithner Department of Chemistry, Colorado State UniVersity, Fort Collins, Colorado 80523 ReceiVed July 17, 2001 ABSTRACT: Exposure of DNA to oxidative stress produces a variety of DNA lesions including the formamidopyrimidines, which are derived from the purines. These lesions may play important roles in carcinogenesis. We achieved the first chemical syntheses of a monomeric form of Fapy•dA (1) and oligonucleotides containing this lesion or Fapy•dG at a defined site. Monomeric Fapy•dA readily epimerized at 25 °C in phosphate buffer (pH 7.5). The -anomer was favored by a ratio of 1.33:1.0, and equilibration was achieved in less than 7 h. Deglycosylation of Fapy•dA in the monomer follows first-order kinetics from 37 to 90 °C. The rate constants for deglycosylation of Fapy•dA in the monomeric and oligonucleotide substrates were measured at a common temperature (55 °C) and found to be the same within experimental error (t 1/2 ) 20.5 h). Implementation of the activation parameters measured for the deglycosylation of 1 indicates that the half-life for deglycosylation of Fapy•dA at 37 °C is approximately 103 h. Analysis of the rate constant for deglycosylation of Fapy•dG in an oligonucleotide, revealed that this lesion is ∼25 times more resistant to hydrolysis than Fapy•dA at 55 °C. These results indicate that Fapy•dA and Fapy•dG will be sufficiently long-lived in DNA so as to warrant investigation of their genotoxicity, and both anomers will be present during this time. DNA damage is important in a variety of diseases, such as cancer and in aging. The formamidopyrimidine lesions N4-(2-deoxy-D-erythropentofuranosyl)-4,6-diamino-5-form- amidopyrimidine (Fapy•dA) and N6-(2-deoxy-D-erythropen- tofuranosyl)-6-amino-5-formamido-4-hydroxypyrimidine (Fapy•dG) are produced in DNA as a result of oxidative stress (1-4). Although the mechanism(s) for formation of these imidazole ring opened purine lesions is uncertain, they result from the formal addition of hydroxyl radical (OH•) to the C8-position of deoxyadenosine and deoxyguanosine (Scheme 1). The respective C8-hydroxyl radical adducts are also believed to be precursors of 8-oxodeoxyguanosine (OG) and 8-oxodeoxyadenosine (OA). The former is often de- scribed as the most mutagenic DNA lesion and gives rise to G f T transversions (5, 6). The structural basis of the effects of OG on polymerase enzyme activity are explained by rotation about the glycosidic bond, which enables the molecule to hydrogen bond to adenine (7). Cleavage of the purines’ imidazole rings in the formamidopyrimidine lesions provides these molecules with a greater number of degrees of freedom and increased number of possible hydrogen bonding patterns. The mutagenic potential of Fapy•dA is uncertain, but studies of damaged DNA in Escherichia coli suggest that it results in A f G transitions (8). Conclusions regarding the mutagenicity of Fapy•dG could not be drawn from these experiments. Oligonucleotides containing N- methyl Fapy•dG have been prepared. This lesion blocks polymerase activity and increases G f T and G f C transversions (9-12). However, the additional methyl sub- stituent could significantly alter the conformation about the formamido group, and it is uncertain whether one can extrapolate observations made using N-methyl Fapy•dG to the parent lesion. The impact of a modified nucleotide on polymerase enzyme activity is affected by the lifetime of the lesion in DNA. Fapy•dA and Fapy•dG are excised by the base † Supported by the NIH (CA-74954). NMR facilities were supported by NIH RR11981, NSF CHE-9610206, and the Colorado State University Research Foundation. * To whom correspondence should be addressed. E-mail: mgreenbe@ lamar.colostate.edu. Scheme 1: Formation of Formamidopyrimidine and 8-Oxopurine Lesions 15856 Biochemistry 2001, 40, 15856-15861 10.1021/bi011490q CCC: $20.00 © 2001 American Chemical Society Published on Web 11/29/2001