GAMMA-PHOSPHATE-SUBSTITUTED 2'-DEOXYNUCLEOSIDE 5'-TRIPHOSPHATES AS SUBSTRATES FOR DNA-POLYMERASES

Several 2'-deoxythymidine 5'-triphosphate and 3'-azido-2',3'-dideoxyth ymidine 5'-triphosphate analogs containing a hydrophobic phosphonate g roup instead of the gamma-phosphate were synthesized and evaluated as substrates for human immunodeficiency virus (HIV) and avian myeloblast osis virus reverse transcriptases, human placental DNA polymerases alp ha and beta, and calf thymus terminal deoxynucleotidyl transferase. Th ey were efficiently incorporated into the DNA chain by the retroviral enzymes but were not utilized by the mammalian ones. Also, some gamma- ester and gamma-amide derivatives of dTTP and 3'-azido-2',3'-dideoxyth ymidine 5'-triphosphate (AZTTP) were synthesized and studied. They pro ved to be substrates for both the retroviral and mammalian enzymes und er study. The K-m values for incorporation of the dTTP derivatives int o the DNA chain were close to those for dTTP and AZTTP. The K-m for th e AZTTP derivatives were one order of magnitude greater than those for dTTP and AZTTP. The results obtained indicate that HIV and avian myel oblastosis virus reverse transcriptases have no sterical obstacles for binding the triphosphate fragment bearing a bulky substituent at the gamma-position. Modification of the gamma-phosphate in AZTTP increased the selectivity of HIV reverse transcriptase inhibition versus DNA po lymerase alpha. gamma-Methylphosphonate and gamma-phenylphosphonate we re dephosphorylated in human serum much less rapidly than AZTTP. Besid es, they were shown to be markedly more hydrophobic than AZTTP. Thus, replacement of the gamma-phosphate in AZTTP with gamma-phosphonate mar kedly alters its substrate properties toward some cellular DNA polymer ases and blood dephosphorylating enzymes but does not change its subst rate activity with respect to HIV reverse transcriptase.