General
For all reactions, chemicals of analytical or synthetic grade were obtained from commercial sources and were used without purification. Technical solvents were obtained from Brenntag (Deerlijk, Belgium). Analytical thin Layer Chromatography was performed on Alugram® silica gel UV254 mesh 60, 0.20 mm (Macherey-Nagel). NMR Spectra were recorded on a Brucker Avance™ II 300 MHz or 500 MHz NMR spectrometer. Chemical shifts are expressed as δ units (part per million) down field from TMS (tetramethyl silane) for 1H and 13C. 31P NMR chemical shifts are referenced to an external 85% H3PO4 standard (δ = 0.00 ppm). Exact mass spectra were obtained with a quadrupol/orthogonal-acceleration time-of-flight tandem mass spectrometer (Q-Tof 2, Micromass) equipped with a standard electrospray ionization (ESI) source. HPLC was performed on Waters 1525-2487 system using C18 column by a gradient elution of acetonitrile and 50 mM triethylammonium bicarbonate (TEAB) buffer.
Synthesis of deoxyadenosine-5'-phosphoroimidazole
General Procedure (I): Deoxyadeosine-5'-monophosphate (100 mg, 0.30 mmoles) was dissolved in dry DMSO (2 mL), pulverized triphenylphosphine (327 mg, 0.96 mmoles), 2,2-dipyridyl disulfide (211 mg, 0.96 mmoles) and imidazole (327 mg, 4.8 mmoles) were added to the solution, the resulting yellow solution was stirred at room temperature for 50 min, after completion the mixture was poured into 0.1 M sodium iodide solution in cold acetone (30 mL). The white precipitate was collected by centrifugation and washed several times with cold fresh acetone. The product was dried in desicator for 1 h.; the white solid was stored at -20°C (99 mg, yield 87%). 1H NMR (500 MHz, D2O): δ= 8.07 (s, 1H, H8), 7.92 (s, 1H, H2), 6.20 (t, 1H, J = 6.2 Hz, H1'), 4.55 (m, 1H, H3'), 4.13 (m, 3H, H4'+H5'), 2.50 (m, 1H, H2'a), 2.43 (m, 1H, H2'b); 13C NMR (125 MHz, D2O): δ = 155.1, 152.6, 148.4, 140.1, 118.4, 86.5, 84.4, 71.2, 66.0, 40.0; 31P NMR (121 MHz, D2O): δ = -11.23; HRMS: [M-H]- calculated for C20H25N10O11P2 643.1185, found: 643.1183.
Synthesis of P1, P2-bis(2'-deoxyadenosin-5'-yl) Pyrophosphate (dAppdA)
General Procedure (II): A solution of 2'-deoxyadeonosine-5'-monophosphate (100 mg, 0.30 mmoles), 2'-deoxyadeonosine-5'-phosphorimidazolide (114 mg, 0.30 mmoles), zinc chloride (35 mg, 0.30 mmoles) in 0.2 M N-ethylmorpholine buffer (3 mL, pH 7.5) was stirred at room temperature under argon for 2 days. The reaction was monitored by TLC (i-PrOH/NH3/H2O 7:1:2) and 31P NMR, quenched the reaction with 0.25 M ethylenediaminetetraacetic acid (EDTA) in order to breakdown the nucleotide-metal complex, then the mixture was lyophilized affording a white solid. The product was isolated by HPLC purification on a C18 column, running with a gradient of CH3CN in 50 mM triethylammonium (TEAB) buffer. Yield 28% (from HPLC purification profile). 1H NMR (500 MHz, D2O): δ = 8.02 (s, 2H, H8), 7.90 (s, 2H, H2), 6.16 (t, 2H, J = 5.7 Hz, H1'), 4.49 (m, 2H, H3'), 4.07 (m, 4H, H5'), 4.02 (m, 2H, H4'), 2.46 (m, 2H, H2'a), 2.40 (m, 2H, H2'b); 13C NMR (125 MHz, D2O): δ = 155.1, 152.3, 148.4, 140.1, 118.4, 86.1, 84.1, 71.2, 66.0, 40.0; 31P NMR (121 MHz, D2O): δ = -11.27; HRMS: [M-H]- calculated for C20H25N10O11P2 643.1185, found: 643.1183.
Synthesis of P1, P2-bis(2'-deoxythymidin-5'-yl) Pyrophosphate (dTppdT)
The general procedure (I) was applied using thymidine-5'-monophosphate (300 mg, 0.93 mmoles), triphenylphosphine (1.01 g, 2.98 mmoles), 2,2-dipyridyl disulfide (655 mg, 2,98 mmoles) and imidazole (1.02 g, 14.9 mmoles). After reaction the obtained product was applied to general procedure (II) using thymidine-5'-monophosphate (100 mg, 0.31 mmoles), thymidine-5'-phosphorimidazolide (115 mg, 0.31 mmoles) and zinc chloride (36 mg, 0.31 mmoles). After purification obtained white solid product (yield 35% (from HPLC purification profile)). 1H NMR (500 MHz, D2O): δ = 7.71 (s, 2H, H6), 6.29 (t, 2H, J = 7.0 Hz, H1'), 4.56-4.61 (m, 2H, H3'), 4.16-4.17 (m, 2H, H4'), 4.12-4.13 (m, 4H, H5'), 2.32-2.36 (m, 4H, H2'), 1.91 (s, 6H, CH3); 13C NMR (125 MHz, D2O): δ = 166.1, 151.3, 137.1, 111.4, 85.0, 84.9, 70.5, 65.0, 38.3, 11.4; 31P NMR (121 MHz, D2O): δ = -11.62; HRMS: [M-H]- calculated for C20H28N4O15P2 625.0953, found: 625.0962
Synthesis of P1-(2'-deoxyadenosin-5'-yl) P2-(2'-deoxythymidin-5'-yl) Pyrophosphate (dAppdT)
The general procedure (II) was applied using 2'-deoxyadeonosine- 5'-phosphorimidazolide (100 mg, 0.26 mmoles) and thymidine-5'-monophosphate (85 mg, 0.26 mmoles) and zinc chloride (30 mg, 0.26 mmoles). After purification obtained white solid product (yield 18% (from HPLC purification profile)) 1H NMR (500 MHz, D2O): δ = 8.36 (s, 1H, H8-A), 8.10 (s, 1H, H2-A), 7.31 (s, 1H, H6-T), 6.39 (t, 1H, J = 6.0 Hz, H1'-A), 6.15 (t, 1H, J = 6.0 Hz, H1'-T), 4.63-4.65 (m, 1H, H3'-A), 4.40-4.42 (m, 1H, H3'-T), 4.16-4.18 (m, 1H, H4'-A), 4.07-4.00 (m, 5H, H4'-T, H5'-A, H5'-T), 2.71-2.75 (m, 1H, H2'a-A), 2.46-2.50 (m, 1H, H2'a-T), 2.11-2.15 (m, 2H, H2'b-T), 2.04-2.08 (m, 2H, H2'b-A), 1.67 (s, 3H, T-CH3); 13C NMR (125 MHz, D2O): δ = 181.4, 172.3, 155.3, 152.4, 148.5, 139.7, 136.1, 118.4, 111.4, 85.6, 84.8, 84.6, 83.3, 71.0, 71.1, 65.4, 65.3, 38.9, 38.6, 12.3; 31P NMR (121 MHz, D2O): δ = -11.41; HRMS: [M-H]- calculated for C20H26N7O13P2 634.1069, found: 634.1063.
Synthesis of P1, P2-bis(2'-deoxyguanosin-5'-yl) Pyrophosphate (dGppdG)
The general procedure (I) was applied using using 2'-deoxyguanosine-5'-monophosphate (300 mg, 0.78 mmoles), triphenylphosphine (850 mg, 2.50 mmoles), 2,2-dipyridyl disulfide (549 mg, 2,50 mmoles) and imidazole (850 mg, 12.9 mmoles). After reaction the obtained product was applied to general procedure (II) using 2'-deoxyguanosine-5'-monophosphate (100 mg, 0.26 mmoles), 2'-deoxyguanosine-5'-phosphorimidazolide (102 mg, 0.26 mmoles) and Pd(NO3)2 (60 mg, 0.36 mmoles). After purification obtained white solid product (yield 33% (from HPLC purification profile)). 1H NMR (500 MHz, D2O): δ = 7.86 (s, 2H, H6), 6.11 (t, 2H, J = 6.5 Hz, H1'), 4.54-4.56 (m, 2H, H3'), 4.05-4.07 (m, 2H, H4'), 4.03-4.05 (m, 4H, H5'), 2.54-2.63 (m, 2H, H2'a), 2.33-2.36 (m, 2H, H2'b); 13C NMR (125 MHz, D2O): δ = 162.1, 156.3, 150.8, 136.3, 116.3, 85.1, 83.0, 70.8, 65.2, 38.4; 31P NMR (121 MHz, D2O): δ = -11.40; HRMS: [M-H]- calculated for C20H25N10O13P2 675.1083, found: 675.1092.
Synthesis of P1-(2'-deoxyguanosin-5'-yl) P2-(2'-deoxycytidin-5'-yl) Pyrophosphate (dGppdC)
The general procedure (I) was applied using using 2'-deoxyguanosine-5'-monophosphate (300 mg, 0.78 mmoles), triphenylphosphine (850 mg, 2.50 mmoles), 2,2-dipyridyl disulfide (549 mg, 2,50 mmoles) and imidazole (850 mg, 12.9 mmoles). After reaction the obtained product was applied to general procedure (II) using 2'-deoxyguanosine-5'-phosphorimidazolide (100 mg, 0.25 mmoles), 2'-deoxycytidine-5'-monophosphate (82 mg, 0.25 mmoles) and Pd(NO3)2 (58 mg, 0.25 mmoles). After purification obtained white solid product (yield 15% (from HPLC purification profile)). 1H NMR (500 MHz, D2O): δ = 8.04 (s, 1H, H8-G), 7.82 (d, 1H, J = 7.5 Hz, H6-C), 6.30 (t, 1H, J = 7.0 Hz, H1'-G), 6.25 (t, 1H, J = 6.5 Hz, H1'-C), 5.97 (d, 1H, J = 7.0 Hz, H5-C), 4.63-4.67 (m, 1H, H3'-G), 4.52-4.54 (m, 1H, H3'-C), 4.23-4.25 (m, 1H, H4'-G), 4.15-4.17 (m, 1H, H4'-C), 4.12-4.15 (m, 4H, H5'-G, H5'-C), 2.77-2.82 (m, 1H, H2'a-G), 2.48-2.50 (m, 1H, H2'a-C), 2.36-2.41 (m, 1H, H2'b-C), 2.18-2.24 (m, 1H, H2'b-G); 13C NMR (125 MHz, D2O): δ = 165.6, 161.5, 156.9, 150.9, 140.9, 136.5, 116.3, 95.9, 85.5, 85.2, 85.0, 83.0, 70.9, 70.2, 65.3, 64.8, 39.2, 38.1; 31P NMR (121 MHz, D2O): δ = -11.40; HRMS: [M-H]- calculated for C19H25N8O13P2 635.1022, found: 635.1099.
Synthesis of P1-(2'-deoxyadenosin-5'-yl) P2-(2'-deoxythymidin-5'-yl) Pyrophosphate (dAppdC)
The general procedure (II) was applied using 2'-deoxyadeonosine-5'-phosphorimidazolide (100 mg, 0.26 mmoles), 2'-deoxycytidine-5'-monophosphate (85 mg, 0.26 mmoles) and zinc chloride (30 mg, 0.26 mmoles). After purification obtained white solid product (yield 18% (from HPLC purification profile)). 1H NMR (500 MHz, D2O): δ = 8.38 (s, 1H, H8-A), 8.10 (s, 1H, H2-A), 7.53 (d, 1H, J = 6.0 Hz, H6-C), 6.40 (t, 1H, J = 5.5 Hz, H1'-A), 6.08 (t, 1H, J = 5.5 Hz, H1'-C), 5.68 (d, 1H, J = 6.5 Hz, H5-C), 4.65-4.67 (m, 1H, H3'-A), 4.37-4.39 (m, 1H, H3'-C), 4.20-4.21 (m, 1H, H4'-A), 4.09-4.05 (m, 5H, H5'-A, H5'-C, H4'-C), 2.73-2.78 (m, 1H, H2'a-A), 2.47-2.51 (m, 1H, H2'a-C), 2.22-2.26 (m, 1H, H2'b-C), 1.99-2.03 (m, 1H, H2'b-A); 13C NMR (125 MHz, D2O): δ = 165.5, 157.0, 155.3, 152.4, 148.5, 140.6, 139.6, 118.3, 95.8, 85.7, 85.6, 85.2, 83.4, 71.2, 70.4, 65.5, 65.1, 39.5, 38.8; 31P NMR (121 MHz, D2O): δ = -11.34; HRMS: [M-H]- calculated for C20H25N8O12P2 619.1073, found: 619.1089
Synthesis of P1, P2-bis(2'-deoxycytidin-5'-yl) Pyrophosphate (dCppdC)
The general procedure (I) was applied using 2'-deoxycytidine-5'-monophosphate (300 mg, 0.93 mmoles), triphenylphosphine (1.01 g, 2.98 mmoles), 2,2-dipyridyl disulfide (655 mg, 2,98 mmoles) and imidazole (1.02 g, 14.9 mmoles). After reaction the obtained product was applied to general procedure (II) using 2'-deoxycytidine-5'-monophosphate (100 mg, 0.31 mmoles), 2'-deoxycytidine-5'-phosphorimidazolide (115 mg, 0.31 mmoles) and zinc chloride (36 mg, 0.31 mmoles). After purification obtained white solid product (yield 26% (from HPLC purification profile)). 1H NMR (500 MHz, D2O): δ = 7.90 (d, J = 7.6 Hz, 2H, H6), 6.31 (t, 2H, J = 6.8 Hz, H1'), 6.05 (d, 2H, J = 7.5 Hz, H5), 4.52-4.56 (m, 2H, H3'), 4.14-4.18 (m, 6H, H4'+H5'), 2.35-2.41 (m, 2H, H2'a), 2.19-2.28 (m, 2H, H2'b); 13C NMR (125 MHz, D2O): δ = 165.7, 157.2, 141.2, 96.2, 85.5, 85.0, 70.4, 64.9, 39.2; 31P NMR (121 MHz, D2O): δ = -8.84; HRMS: [M-H]- calculated for C18H25N6O13P2 595.0960, found: 595.0954.
Oligodeoxyribonucleotides preparation
Oligodeoxyribonucleotides P1, P2, T1-T6 were purchased from Sigma Genosys. The concentrations were measured with a Varian Cary-300-Bio UV spectrophotometer. The lyophilized oligonucleotides were dissolved in diethylpyrocarbonate (DEPC)-treated water and stored at -20°C. The primer oligonucleotides were 5'-33P-labeled with 5'-[γ33P]-ATP (Perkin Elmer) using T4 polynucleotide kinase (NEB) according to standard procedures. The labelled oligonucleotide was further purified using Illustra TM Microspin TM G-25 columns (GE Healthcare).
DNA polymerase reactions
End-labelled primer was annealed to its template by combining primer and template in a molar ratio of 1:2 and heating the mixture to 70°C for 10 min followed by slow cooling to room temperature over a period of 1.5 h. For the incorporation of compound I-VII, a series of 20 μL-batch reactions were performed with the enzyme (HIV-1 RT, Taq, Vent (exo-), Therminator DNA polymerase). The final mixture contained 125 nM primer template complex, RT buffer (250 mM Tris.HCl, 250 mM KCl, 50 mM MgCl2, 2.5 mM spermidine, 50 mM dithiothreitol (DTT); pH 8.3), appropriate concentration of enzyme, and different concentrations of dinucleoside diphosphates building blocks (1 mM, 500 μM, 200 μM and 100 μM). In the control reaction a 10 μM dATP was used as reference. The mixture was incubated at 37°C or 75°C respectively, and aliquots were quenched after 10, 20, 30, 60 and 120 min. For elongation experiments, the same mixture with appropriate primer/template hybrid was incubated at 37°C or 75°C and aliquots were quenched after 15, 30, 60, 90, 120 min.
Steady-state kinetics of single nucleotide incorporation
The steady-state kinetics of single nucleotide incorporation of compound I and the natural nucleoside triphosphate (dATP) was determined by a gel-based polymerase assay. In all the experiments, the template T1 and the primer P1 were used. The primer and template in a 1:2 molar ratio were hybridised in a buffer containing 20 mM Tris.HCl, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.3 and used in an amount to provide 125 nM concentration of the primer in each 20 μL reaction. A range of building block concentrations between 10 μM and 1 mM for the phosphoramidate and between 0.1 μM and 10 μM for the natural building block was used. The final concentrations of primer-template complex and HIV-1 RT were 125 nM, and 0.025 U/μL, respectively. Reaction mixtures were incubated at 37°C and aliquots were drawn at 6 different time intervals. The reactions were quenched by addition of a buffer containing 80% formamide, 2 mM EDTA and 1X TBE buffer. The analysis of polymerase reaction was performed by polyacrylamide gel electrophoresis (see detailed protocol below). The incorporation rates (V) were calculated based on the percentage of the extended oligonucleotide in the mixture (P+1 band). The kinetic parameters (VMax and KM) were determined by plotting V (nM/min-1) versus substrate concentration (μM) and fitting the data to a non-linear Michaelis-Menten regression using GraphPad Prism Software version 5.0.