Synthesis, anti-HIV activity, and stability studies of 5'-phosphorofluoridate derivatives of AZT.

The synthesis, in vitro anti-HIV activity and stability studies of the 5'-fluorophosphate derivative of 3'-azido-3'-deoxythymidine (AZT) are reported. The results support the hypothesis that this phosphorylated entity exerts its biological effect via the delivery of the corresponding 5'-mononucleotide through an enzymatic process. However, the antiviral evaluation in thymidine kinase-deficient CEM cells as well as the stability studies in culture medium and cell extract showed that this bioconversion is not specific to the intracellular medium. Attempts to improve the biological activity of mononucleoside 5'-fluorophosphates by the use of the S-pivaloyl-2-thioethyl (tBuSATE) group as biolabile phosphate protection are reported.

Reasons and limits of substrate activity of modified L-dNTP in DNA biosynthesis.

Theoretical and experimental analysis of interaction of modified D- and L- dNTP as substrates for template-dependent and template-independent DNA polymerases was performed. It is shown that if the modified nucleoside 5'-triphosphates do not contain a substituent in position 3' DNA chains can be extended by both strereoisomeric series with the same kinetic parameters. But the presence of even a 3'-hydroxy group in L-dNTP prevents their incorporation into the DNA chain.

Synthesis, anti-HIV activity and stability studies of 3'-azido-2',3'-dideoxythymidine 5'-fluorophosphate.

The synthesis, in vitro anti-HIV activity, and stability studies of AZT 5'-fluorophosphate (F-AZTMP) are reported. The present results demonstrate that such compound is a bioprecursor of its parent 5'-mononucleotide (AZTMP) but its biotransformation does not allow its selective intracellular delivery. Moreover, several attempts were carried out in order to improve the biological activity of this compound by the use of a SATE prodrug strategy.

Stereoisomers of deoxynucleoside 5'-triphosphates as substrates for template-dependent and -independent DNA polymerases.

All four possible stereoisomers of dNTP with regard to deoxyribofuranose C-1' and C-4' carbon atoms were studied as substrates for several template-dependent DNA polymerases and template-independent terminal deoxynucleotidyl transferase. It was shown that DNA polymerases alpha, beta, and epsilon from human placenta and reverse transcriptases of human immunodeficiency virus and avian myeloblastosis virus incorporate into the DNA chain only natural beta-D-dNTPs, whereas calf thymus terminal deoxynucleotidyl transferase incorporates two nucleotide residues of alpha-D-dNTP and extends the resulting oligonucleotide in the presence of beta-D-dNTPs. The latter enzyme also extended alpha-anomeric D-oligodeoxynucleotide primers in the presence of beta-D-dNTPs. None of the studied enzymes utilized L-dNTPs. These data indicate that template-dependent DNA polymerases are highly stereospecific with regard to dNTPs, whereas template-independent terminal deoxynucleotidyl transferase shows less stereodifferentiation. It is likely that the active center of the latter enzyme forms no specific contacts with the nucleic bases of both nucleotide substrate and oligonucleotide primer.

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 gamma-phosphate were synthesized and evaluated as substrates for human immunodeficiency virus (HIV) and avian myeloblastosis virus reverse transcriptases, human placental DNA polymerases alpha and beta, 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 gamma-ester and gamma-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 gamma-position. Modification of the gamma-phosphate in AZTTP increased the selectivity of HIV reverse transcriptase inhibition versus DNA polymerase alpha. gamma-Methylphosphonate and gamma-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 gamma-phosphate in AZTTP with gamma-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.

Modified substrates of DNA polymerases and design of antivirals.

The results obtained in our laboratory on investigating of substrate properties of a large number of compounds towards different DNA polymerases have been summarized. On the basis of systematic analysis a directed synthesis of nucleotides with antiviral properties was performed.

Selectivity of DNA polymerases toward alpha and beta 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-beta and L-beta series were found to be terminating substrates for these enzymes, while the derivatives of the D-alpha and L-alpha 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.

Formation of phosphonester bonds catalyzed by DNA polymerase.

3'-Fluoro-2',3'-dideoxythymidine 5'-(alpha-methylphosphonyl)-beta,gamma- diphosphate and 2'-deoxythymidine-5'-(alpha-methylphosphonyl)-beta, gamma- diphosphate have been synthesized. Both compounds are incorporated into DNA chains during catalysis by reverse transcriptases of human immunodeficiency (HIV) and avian myeloblastosis (AMV) viruses, DNA polymerase beta from rat liver, terminal deoxynucleotidyl transferase from calf thymus and (at a very low rate) is by E. coli DNA polymerase I, Klenow fragment. The first compound is a termination substrate while the second is capable of multiple incorporation into the DNA chains. For instance, reverse transcriptase catalysis resulted in the appearance of 8 residues of second compound. DNA polymerases alpha and epsilon from human placenta incorporated none of the above compounds into DNA chains, although an inhibition of DNA synthesis by both compounds was observed with all enzymes mentioned. The 3'----5'-exonuclease activity of DNA polymerase I, Klenow fragment, hydrolyzed DNA fragments containing phosphonomethyl internucleoside groups, while such DNA fragments were resistant to the E. coli exonuclease III.

Nucleoside 5'-triphosphates modified at sugar residues as substrates for DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

The ability of a wide variety of nucleoside 5'-triphosphates with modified sugar moiety to serve as substrates in DNA synthesis catalyzed by DNA polymerase A from the archaebacterium Sulfolobus acidocaldarius was studied. Most of the dNTP analogs tested are shown to be specific terminating substrates for the synthesis irreversibly blocking further elongation of a nascent chain. The most powerful inhibitors were found to be 3'-amino derivatives of deoxy and arabino nucleoside triphosphates, while specific reverse transcriptase inhibitors, 3'-azido and 3'-methoxy derivatives of dNTP, were found to be inactive.

Terminating substrates of DNA polymerases: synthesis and functional study.

In order to study the mechanisms of DNA biosynthesis a number of modified nucleoside - substrates of DNA polymerases was synthesized. The absence of hydroxyl at 3'-position of ribose results in terminating properties of DNA biosynthesis of these analogues. A single step synthesis of triphosphates and alpha-thiotriphosphates of natural and 3'-modified 2'-deoxynucleosides is described.

Aminonucleosides and their derivatives. IV. Synthesis of the 3'-amino-3'-deoxynucleoside 5'-phosphates.

A new procedure has been developed for the synthesis of 3'-amino-3'-deoxyribonucleosides of adenine, cytosine and uracil by condensing the trimethylsilylated bases with peracylated 3-azido-3-deoxyribose derivative. The azido group could subsequently be reduced to amino. The 5'-phosphates of these nucleosides have been prepared and the analogues have been tested for their ability to stimulate the ribosome-catalyzed reaction of 3'(2')-O-(N-formylmethionyl) adenosine 5'-phosphate with phenylalanyl-tRNA.