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

Ludmila A. Alexandrova 0 Alexander Yu. Skoblov 0 Maxim V. Jasko 0 Lyubov S. Victorova 0 Alexander A. Krayevsky 0 0 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences , 32 Vavilov str., Moscow 117984, Russia *To whom correspondence should be addressed. Tel: +7 095 135 22 55; Fax: +7 095 135 14 05; Email: - Replacement of a -, -b and g -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 a -, -b and g -phosphates containing, in the case of dUTP, reporter and ligand groups at the C5 position of uracil. It was shown that g -substituted dNTPs were substrates for AMV reverse transcriptase despite of the large size of substituent at the g -phosphonate. On the other hand, these compounds were poorly utilized by DNA polymerase a . For dUTP analogues substituted at both g -phosphonate and C5 of uracil, the substrate affinity was 12 orders of magnitude lower than for their counterparts containing substituents either at g -phosphonate or C5 position. Meanwhile, C5-substituted b ,g -dibromomethylenediphosphonates demonstrated poor activity or were not active at all as substrates for AMV reverse transcriptase. Finally, 2-deoxythymidine 5-[,b g -(methylphosphinyl)methylphosphonyl]-a -phosphate and its 3-azido-3-deoxy analog were substrates for AMV reverse transcriptase, but the substrate activity of these analogues was 50100 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. Modified dNTPs and rNTPs are used widely in molecular biology and biochemistry as model substrates for enzymatic systems. However, these compounds cannot be used in cell biology because of their rapid dephosphorylation both, in intercellular media and during penetration through the cell membrane. Therefore, application of dNTPs and rNTPs with increased stability toward dephosphorylating enzymes would seems to have the potential to produce a high rate of success, especially for stable dNTPs and rNTPs which carry additional reporter or ligand groups. The presence of reporter (fluoresceinyl, tetramethylrhodaminyl) or ligand (biotinyl, 2,4-dinitrophenyl) groups makes it possible to monitor the diffusion of these compounds into cells and to observe their incorporation into DNA. Diffusion of modified dNTP into the cell has been poorly studied. This problem seems to be resolved by increasing the hydrophobicity or covalent attachment of ligands for transporting the modified dNTP molecule into the cell. Recently it has been shown that the replacement of the g -phosphate by methylphosphonate or phenylphosphonate in natural and glycon-modified dNTPs has little effect on the substrate activity of the compounds toward HIV and AMV Reverse Transcriptases (RTs) (1,2). In addition, dNTPs substituted at the a -phosphate (3,4), b ,g -diphosphate (5,6) and even at all three phosphates (7,8), were shown to be substrates for RTs; however, the limitations for such modifications are unknown. In this work we synthesized several novel modified dNTPs (Scheme 1, IIII) and evaluated them as substrates towards AMV RT and some other DNA polymerases. Synthesis The key nucleoside (IV) was synthesized according to Scheme 2. The principal stage was the replacement of bromine of 3,5di-O-acetyl-(5-bromomethyl)-2-deoxyuridine (Va) (9) by 1,6-hexandiol (10). To obtain 5-[(6-azidohexyl)oxymethyl]-2-deoxyuridine (IV), nucleoside Vb was methanesulfonylated and the methanesulfonyloxy group in Vc was replaced by an azide group by reaction with sodium azide in DMF. Compound Vd was deprotected with aqueous ammonia; the yield of IV was 39% starting from Va. To synthesize dNTPs modified at both, the nucleobase and the triphosphate fragment (I and II, Scheme 3), nucleoside IV was phosphorylated with POCl3 in triethylphosphate. Nucleotide VI was converted to the corresponding imidazolide, which was coupled without purification with bis-(tri-n-butylammonium) salts of phenylphosphonylphosphoric, pyrophosphoric or dibromomethylenediphosphonic acids. Triphenylphosphine, DTT and mercaptoethanol were used for reducing the azido group in Ia, Ie and IIa. The best results were obtained with DTT. The dNTP analogues containing ligand (IIc) or reporter (IId,e) groups were synthesized by coupling IIb with either N-succinimidyl N-biotinyl-6-aminohexanoate or fluorescein isothiocyanate or N-succinimidyl tetramethylrhodamine carboxylate according to (11). Compounds Ia-c were synthesized according to Scheme 4. Compound VIIb (12) was coupled either with 2,4-dinitrofluorobenzene for VIIc or with N-succinimidyl 6-N-(2,4-dinitrophenyl)aminohexanoate for VIId. Compounds VIIa,c,d were converted to VIIIa,c,d by reaction with trimethylbromosilane, then activated with CDI to yield IXa,c,d and coupled with 2-deoxythymidine 5-diphosphate to yield Ia,c,d. The yields were 21, 12 and 25%, respectively; Ia was reduced to Ib with DTT with 53% yield. 2-Deoxythymidine 5-{b ,g -[(methylphosphinyl)methylphosphonyl]-a -phosphate} (IIIa, R = OH) and its 3-azido-analog (IIIb, R = N3) were synthesized by coupling of either 2-deoxythymidine 5-phosphoimidazolide or 3-azido-2,3-dideoxythymidine 5-bis-(1,2,4-triazolyl)phosphate with (methylphosphinyl)methylphosphonate (13). The structure of all compounds was confirmed by UV, 1H- and 31P-NMR, and mass spectrometry. Several groups of modified dNTPs were studied in this work. Group A included different dTTP g -phosphonates (IaId) and dNTPs containing additional modifications at the thymine base (IeIf); compound Ig was used as a control for IeIf. Group B included compounds containing the dibromomethylenediphosphonate instead of the b ,g -diphosphate and modified at the thymine base (IIaIIe); IIf was used as a control. Compounds III contained two modifications at the g -phosphate: two of its P-O bonds were replaced by P-C bonds. It can be seen in Figure 1 that Ia (lanes 611) and Ib (lanes 1217) are substrates for AMV RT; they were incorporated into the DNA chain several times (lanes 1011 and 1617). Compound Ib was several fold less effective as a substrate than Ia (compare lanes 1213 and 67). The presence of a minor octadecanucleotide band on lanes 3, 10 and 16 is attributable to the low fidelity of RTs. It is evident from Figure 2 that DNA polymerase a incorporates Ia very weakly into the DNA chain (lanes 610), while Ib is utilized by the enzyme at 600 m M (lanes 1115). Thus, DNA polymerase a is 23 orders of magnitude more specific towards g -phosphate-substituted dNTPs than AMV RT; this observation is in agreement with our earlier data (2). Compound IIIa also elongated the primer (lanes 1618); after incorporation of one residue of IIIa the primer was efficiently extended i (...truncated)