Natalia B. Dyatkina

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.

Properties of 2′,3′-dideoxy-2′,3′-dehydrothymidine 5′-triphosphate in terminating DNA synthesis catalyzed by several different DNA polymerases

2′‐3′‐Dideoxy‐2′,3′‐dehydrothymidine 5′‐triphosphate (dddTTP) shows termination substrate properties in the DNA synthesis catalyzed by E. coli DNA polymerase I KF, rat liver DNA polymerase β, reverse transcriptases of avian myeloblastosis virus and Raus sarcoma virus and calf thymus terminal deoxynucleotidyl transferase. This implies that the mononucleotide residue of dddTTP incorporates into 3′‐termini of newly synthesized DNA chains. However, dddTTP has no influence on the DNA synthesis catalyzed by calf thymus DNA polymerase α. In the case of some DNA polymerases dddTTP was one order of magnitude more effective in comparison with the other known termination substrates.

STEREOCONTROLLED SYNTHESIS OF THE FOUR STEREOISOMERIC DIPHOSPHORYLPHOSPHONATES OF CARBOCYCLIC 2',3'-DIDEOXY-2',3'-DIDEHYDRO-5'-NORADENOSINE

Abstract A flexible synthesis of the four stereoisomeric enantiomerically pure 5′-nor carbocyclic adenosine analogues 4b , ent - 4b , 5b and ent - 5b starting from the common enantio-merically pure allylic monoacetate 3 has been developed. Each of the nucleoside analogues was transformed into the corresponding 4′-phosphonates and subsequently into the diphosphoryl-phosphonates 4e , ent - 4e, 5e , and ent - 5e , respectively.

Synthesis and antiviral activity of some fluorinated nucleotide derivatives

Abstract A number of 3′-fluoro-3′-deoxythymidine 5′-phosphonates and nucleoside 5′-phosphorofluoridates were prepared to study their ability to inhibit replication of HIV-1. Compounds, the 5′-phosphorofluoridates of 3′-azido-3′-deoxythymidine (VIIIc), 3′-fluoro-3′-deoxythymidine (VIIId) and 3′-deoxy-2′,3′-didehydrothymidine (VIIIe), exhibit potent anti-HIV-1 activities.

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.

Biochemical Evaluation of the Inhibition Properties of Favipiravir and 2'-C-Methyl-Cytidine Triphosphates against Human and Mouse Norovirus RNA Polymerases.

ABSTRACT Norovirus (NoV) is a positive-sense single-stranded RNA virus that causes acute gastroenteritis and is responsible for 200,000 deaths per year worldwide. No effective vaccine or treatment is available. Recent studies have shown that the nucleoside analogs favipiravir (T-705) and 2′-C-methyl-cytidine (2CM-C) inhibit NoV replication in vitro and in animal models, but their precise mechanism of action is unknown. We evaluated the molecular interactions between nucleoside triphosphates and NoV RNA-dependent RNA polymerase (NoVpol), the enzyme responsible for replication and transcription of NoV genomic RNA. We found that T-705 ribonucleoside triphosphate (RTP) and 2CM-C triphosphate (2CM-CTP) equally inhibited human and mouse NoVpol activities at concentrations resulting in 50% of maximum inhibition (IC50s) in the low micromolar range. 2CM-CTP inhibited the viral polymerases by competing directly with natural CTP during primer elongation, whereas T-705 RTP competed mostly with ATP and GTP at the initiation and elongation steps. Incorporation of 2CM-CTP into viral RNA blocked subsequent RNA synthesis, whereas T-705 RTP did not cause immediate chain termination of NoVpol. 2CM-CTP and T-705 RTP displayed low levels of enzyme selectivity, as they were both recognized as substrates by human mitochondrial RNA polymerase. The level of discrimination by the human enzyme was increased with a novel analog of T-705 RTP containing a 2′-C-methyl substitution. Collectively, our data suggest that 2CM-C inhibits replication of NoV by acting as a classic chain terminator, while T-705 may inhibit the virus by multiple mechanisms of action. Understanding the precise mechanism of action of anti-NoV compounds could provide a rational basis for optimizing their inhibition potencies and selectivities.

Synthesis and Anti-Influenza Activity of Pyridine, Pyridazine, and Pyrimidine C-Nucleosides as Favipiravir (T-705) Analogues

Influenza viruses are responsible for seasonal epidemics and occasional pandemics which cause significant morbidity and mortality. Despite available vaccines, only partial protection is achieved. Currently, there are two classes of widely approved anti-influenza drugs: M2 ion channel blockers and neuraminidase inhibitors. However, the worldwide spread of drug-resistant influenza strains poses an urgent need for novel antiviral drugs, particularly with a different mechanism of action. Favipiravir (T-705), a broad-spectrum antiviral agent, has shown potent anti-influenza activity in cell-based assays, and its riboside (2) triphosphate inhibited influenza polymerase. In one of our approaches to treat influenza infection, we designed, prepared, and tested a series of C-nucleoside analogues, which have an analogy to 2 and were expected to act by a similar antiviral mechanism as favipiravir. Compound 3c of this report exhibited potent inhibition of influenza virus replication in MDCK cells, and its triphosphate was a substrate of and demonstrated inhibitory activity against influenza A polymerase. Metabolites of 3c are also presented.

An Alternative Route for Preparation of α-Methylphosphonyl-β, γ-Diphosphates of Thymidine Derivatives

Abstract An alternative “one pot” synthesis of α-methylphosphonyl-β, γ-diphosphates of thymidine and 3′-azidothymidine is proposed. p-Toluene-sulphonic acid was used as desililating agent for triphosphate analogues.

Formation and reactivity of 2,4-ditriazolyl pyrimidine C-nucleoside derived from pseudouridine.

Abstract Thymine and 2′,3′,5′-tri-O-acetyl-Ψ-uridine (1) was converted into the corresponding 2,4-ditriazolyl derivatives 5 and 2, respectively. Of these two substituents, the C4-triazolyl group was found to be quite susceptible to nucleophilic substitution while the other triazolyl is resistant.

Synthesis of Novel Pyrene-Bearing C-Nucleoside and Its Incorporation into Oligonucleotides

Abstract Phosphoramidite of (1,2,4-triazol-1-yl)-4-[(4-pyren-1-ylbutyl)amino]-5-(2-O-methyl-5-dimethoxytrityl-β-D-ribofuranosyl)pyrimidine has been synthesized, incorporated into polypyrimidine oligoaucleotides, and studied for thier triplex forming capacity.