Dmitry G. Semizarov

Gamma-phosphate-substituted 2'-deoxynucleoside 5'-triphosphates as substrates for DNA polymerases.

Several 2′-deoxythymidine 5′-triphosphate and 3′-azido-2′,3′-dideoxythymidine 5′-triphosphate analogs containing a hydrophobic phosphonate group instead of the γ-phosphate were synthesized and evaluated as substrates for human immunodeficiency virus (HIV) and avian myeloblastosis virus reverse transcriptases, human placental DNA polymerases α and β, and calf thymus terminal deoxynucleotidyl transferase. They were efficiently incorporated into the DNA chain by the retroviral enzymes but were not utilized by the mammalian ones. Also, some γ-ester and γ-amide derivatives of dTTP and 3′-azido-2′,3′-dideoxythymidine 5′-triphosphate (AZTTP) were synthesized and studied. They proved to be substrates for both the retroviral and mammalian enzymes under study. The Km values for incorporation of the dTTP derivatives into the DNA chain were close to those for dTTP and AZTTP. The Km for the AZTTP derivatives were one order of magnitude greater than those for dTTP and AZTTP. The results obtained indicate that HIV and avian myeloblastosis virus reverse transcriptases have no sterical obstacles for binding the triphosphate fragment bearing a bulky substituent at the γ-position. Modification of the γ-phosphate in AZTTP increased the selectivity of HIV reverse transcriptase inhibition versus DNA polymerase α. γ-Methylphosphonate and γ-phenylphosphonate were dephosphorylated in human serum much less rapidly than AZTTP. Besides, they were shown to be markedly more hydrophobic than AZTTP. Thus, replacement of the γ-phosphate in AZTTP with γ-phosphonate markedly alters its substrate properties toward some cellular DNA polymerases and blood dephosphorylating enzymes but does not change its substrate activity with respect to HIV reverse transcriptase.

Selectivity of DNA polymerases toward α and β nucleotide substrates of d and l series

The substrate properties of four carbocyclic d and l nucleoside 5′‐triphosphate analogs toward HIV and AMV reverse transcriptases and terminal deoxynucleotidyl transferase were evaluated. The compounds of the d‐β and l‐β series were found to be terminating substrates for these enzymes, while the derivatives of the d‐α and l‐α series were recognized only by terminal deoxynucleotidyl transferase, suggesting that for the template‐independent enzyme the mutual orientation of the two fragments is of no significance. A hypothesis for binding of nucleotides to the DNA polymerase active center was proposed.

Effect of P-chirality of oligo(deoxyribonucleoside phosphorothioate)s on the activity of terminal deoxyribonucleotidyl transferase

Phosphorothioate analogues of oligonucleotides (PS‐oligos) of predetermined chirality at the phosphorus atom at each internucleotide linkage have been used as primers for terminal deoxyribonucleotidyl transferase (TdT, EC 2.7.7.31). The enzyme catalyzes efficient elongation of PS primers in which all phosphorothioate internucleotide linkages are uniformly of the [RP] configuration, while the presence of the linkage(s) of the [SP] configuration significantly decreases or completely inhibits the primer extension. Our results indicate that for the elongation of phosphorothioate oligomers the most important is the internucleotide bond located between the second and the third nucleoside from the 3′‐end. The presence of [SP] linkage at this position strongly reduces the enzyme activity while the [RP] bond allows for effective elongation of the primer. The activity of the enzyme is also influenced by base composition and sequence of phosphorothioate primer as well as the dNTP used for elongation process.

Synthesis, Conformation of 3′-(Tetrazole-2″-yl)-3′-deoxythymidine and its 5″-Derivatives. Substrate Properties of 3′-(Tetrazole-2″-yl)-3′-deoxythymidine 5′-Triphosphate

Abstract 5′-O-Benzoyl-3′-(tetrazole-2”-yl)-3′-deoxythymidine and its 5”-substituted derivatives were obtained by the reaction of 5′-O-benzoyl-2,3′-anhydrothymidine with triethylammonium salts of either tetrazole or 5-substituted tetrazoles. Debenzoylation of these compounds yielded 3′-(tetrazole-2”-yl)-3′-deoxythymidine and its 5”-derivatives. Structures of two of them were confirmed by X-ray analysis. Both 3′-(tetrazole-2”-yl)-3′-deoxythymidine and 3′-(5”-methyltetrazole-2”-yl)-3′-deoxythymidine have anti-conformation with respect to the glycosidic bond, and 2′-endo-3′-exo-conformation of the sugar residue with gauche + orientation relative to the C4′-C5′ bond. 3′-(Tetrazole-2”-yl)-3′-deoxythymidine 5′-triphosphate exhibited poor termination substrate properties towards avian myeloblastosis virus reverse transcriptase and did not serve as a substrate for other employed DNA polymerases.

Human DNA polymerases and retroviral reverse transcriptases: selectivity in respect to dNTPs modified at triphosphate residues.

Abstract A series of dTTP and ddTTP(3′N3) γ-phosphonates and β,γ-diphosphonates are studied as substrates or terminating substrates towards different human DNA polymerases and retroviral reverse transcriptases.

New modified substrates for discriminating between human DNA polymerases α and ϵ

Two 2′‐deoxynucleoside 5′‐α‐methylenephosphonyl‐β,γ‐diphosphates were synthesized. They were incorporated into the DNA chain by DNA polymerase α from human placenta. Meanwhile, they were not recognized by DNA polymerases ϵ and β of the same origin as well as by reverse transcriptases from human immunodeficiency virus and avian myeloblastosis virus.

Novel Open-Chain Nucleotides Imitating 2′,3′-Dideoxy-2′,3′-Didehydronucleotides: Synthesis and Substrate Properties Toward DNA Polymerases

Abstract A new series of acyclic nucleotide diphosphates was synthesized and evaluated as potential inhibitors of HIV reverse transcriptases.