2'-Deoxynucleoside 5'-triphosphates modified at α-, β- and γ-phosphates as substrates for DNA polymerases

Abstract

Replacement of α-, β- and γ-phosphate groups in 2'-deoxynucleoside 5'-triphosphates (dNTP) with phosphonate groups yields a new set of dNTP mimics with potential biological and therapeutic applications. Here, we describe the synthesis of 15 new dNTPs modified at α-, β- and γ-phosphates containing, in the case of dUTP, reporter and ligand groups at the C5 position of uracil. It was shown that γ-substituted dNTPs were substrates for AMV reverse transcriptase despite of the large size of substituent at the γ-phosphonate. On the other hand, these compounds were poorly utilized by DNA polymerase α. For dUTP analogues substituted at both γ-phosphonate and C5 of uracil, the substrate affinity was 1-2 orders of magnitude lower than for their counterparts containing substituents either at γ-phosphonate or C5 position. Meanwhile, C5-substituted β,γ-dibromomethylenediphosphonates demonstrated poor activity or were not active at all as substrates for AMV reverse transcriptase. Finally, 2'-deoxythymidine 5'-[β,γ-(methylphosphinyl)methylphosphonyl]-α-phosphate and its 3'-azido-3'-deoxy analog were substrates for AMV reverse transcriptase, but the substrate activity of these analogues was 50-100 times lower as compared with dTTP. HIV reverse transcriptase utilized these compounds 1 order of magnitude less efficiently than AMV reverse transcriptase; terminal deoxynucleotidyl transferase did not recognize them at all.