Synthesis of C8-arylamine-modified 2'-deoxyadenosine phosphoramidites and their site-specific incorporation into oligonucleotides.

Adducts of C8-(N-acetyl)-arylamines and 2'-deoxyadenosine were synthesised by palladium-catalysed C--N cross-coupling chemistry. These 2'-dA adducts were converted into the corresponding 3'-phosphoramidites and site-specifically incorporated into DNA oligonucleotides, which were characterised by mass spectrometry, UV thermal-stability assays and circular dichroism. These modified oligonucleotides were also used in EcoRI restriction assays and in primer-extension studies with three different DNA polymerases. The incorporation of the 2'-dA lesion close to the EcoRI restriction site dramatically reduced the susceptibility of the DNA strand to cleavage; this indicates a significant local distortion of the DNA double helix. The incorporation of the acetylated C8-2'-dA-phosphoramidites into 20-mer oligonucleotides failed, however, because the N-acetyl group was lost during the deprotection process. Instead the corresponding C8-NH-2'-dA-modified oligonucleotides were obtained. The effect of the C8-NH-arylamine-dA lesion on the replication by DNA polymerases was clearly dependent both on the polymerase used and on the arylamine-dA damage.

Synthesis of DNA strands site-specifically damaged by c8-arylamine purine adducts and effects on various DNA polymerases.

C8-Arylamine-dG and C8-arylamine-dA adducts have been prepared using palladium cross-coupling chemistry. These adducts were subsequently converted into the corresponding 5'-O-DMTr-C8-arylamine-3'-O-phosphoramidites and then used for the automated synthesis of different site-specifically modified oligonucleotides. These "damaged" oligonucleotides have been characterized by ESI-MS, UV thermal stability assays, and circular dichroism, and they have been used in EcoRI assays as well as in primer extension studies using various DNA polymerases.