Aryl H-phosphonates. Part 13. A new, general entry to aryl nucleoside phosphate and aryl nucleoside phosphorothioate diesters

Abstract

The reaction of nucleoside H-phosphonate monoesters with phenols in the presence of a condensing agent, followed by oxidation of the in-situ-generated aryl nucleoside H-phosphonate diesters with iodine–water or with elemental sulfur, provides a new, ‘one-pot’, efficient entry to nucleoside phosphate or nucleoside phosphorothioate diesters bearing diverse aryl moieties.

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With the approval of the first oligonucleotide analogue as an antiviral drug (Vitravene of Isis Pharmaceuticals–Ciba Vision),² and with several other oligonucleotide-based therapeutics being at various stages of clinical evaluation, the development of a cost-effective method for oligonucleotide synthesis on a multi-kilogram scale becomes a matter of high importance. Although for current pharmaceutical and diagnostic uses oligonucleotides are produced on a large scale by simple scaling up the existing solid-phase protocol based on phosphoramidite chemistry,³ it seems that more suitable for this purpose would be a solution synthesis employing phosphotriester chemistry. Some recent studies in this area clearly indicate that phosphotriester methodology has still a lot to offer when it comes to a large-scale synthesis of oligonucleotides⁴ or their phosphorothioate analogues^5,6 in the anticipated industrial settings.

When the internucleotide bond is to be formed via phosphotriester methodology, the corresponding aryl nucleoside phosphodiesters are used as starting materials,⁷ since it was found that only aromatic phosphate protecting groups secure proper stability and electrophilicity of phosphorus centres.⁸ Although aryl nucleoside phosphates can be prepared using the appropriate aryl dihydrogen phosphates,^9–13 aryl chlorophosphates,^13–16 their azolides,^17,18 or hydroxybenzotriazolides,¹⁹ these syntheses always require dedicated phosphorylating reagents that in most instances have to be prepared prior to use. This might appear to be rather inconvenient particularly when a number of different aryl nucleoside phosphate diesters are required. For the preparation of aryl nucleoside phosphorothioates, the situation is even less satisfactory, since there is no method of synthetic value, comparable in terms of efficiency or availability of the phosphorylating agents, to those developed for their oxo congeners. One should note, however, that aryl phosphorothioates as nucleotidic components seemed particularly appealing, as using electron-deficient aryl derivatives in combination with highly chemoselective condensing agent^20,21 may appear to be an attractive alternative to the S-protection required in some methods for oligonucleoside phosphorothioate synthesis.^22–24

These facts, and the growing interest in recent years in the phosphotriester method as means of synthesising oligonucleotides and their analogues on a large scale, prompted us to search for a convenient method permitting preparation of aryl nucleoside phosphates and phosphorothioates bearing aryls derived from phenols of a wide range of acidity (pK_a 1.0–10.0). As part of our studies in developing new, efficient methods for the preparation of biologically important phosphate esters and their analogues using H-phosphonate methodology, we report here on a new, general entry to aryl nucleoside phosphates and their phosphorothioate analogues. As a viable approach we considered the in situ generation of bifurcated intermediates, the aryl nucleoside H-phosphonates 3, followed by their oxidative transformation to the corresponding aryl nucleoside phosphates 4 or aryl nucleoside phosphorothioates 5 (Scheme 1). The method seemed to be experimentally simple and should alleviate problems of preparing a separate phosphorylating/thiophosphorylating reagent for each aryl derivative of types 4 and 5.

Scheme 1 Abbr. DMT = 4,4′-dimethoxytrityl; DPCP = diphenyl chlorophosphate.

Results and discussion

In the transformations depicted in Scheme 1 the most critical steps were oxidation of an aryl H-phosphonate intermediate with iodine–water or elemental sulfur to produce a phosphodiester 4 or a phosphorothioate 5, respectively.

To optimise these steps, nucleoside H-phosphonate 1a was treated with the appropriate phenols 2a–j in the presence of diphenyl phosphorochloridate (DPCP), and oxidation of the product aryl nucleoside H-phosphonate 3²⁵ was followed by ³¹P NMR spectroscopy.

The generation of intermediates 3, using previously developed reaction conditions,²⁵ was uneventful and produced the desired aryl H-phosphonate 3 practically as the sole nucleotidic product. However, oxidation of compounds 3 effected by the addition of iodine (1.5 equiv.) in pyridine–water (9 : 1, v/v)²⁵ to the reaction mixtures, although rapid in all instances (<3 min), produced phosphates 4 in variable amounts, depending on the kind of the aryl moiety present in the starting H-phosphonate 3.

For aryl H-phosphonates 3aa–ac, oxidation with an aqueous solution of iodine proceeded cleanly and the corresponding aryl nucleoside phosphates 4aa–ac were obtained in >90% yield after simple work-up and purification by silica gel column chromatography (see Experimental section). The structure of products 4aa–ac was confirmed with spectral (¹H, ³¹P NMR, MS), and chromatographic (TLC) analysis.

In the instance of aryl H-phosphonates 3ad–ah bearing more electron-deficient aryl moieties, ³¹P NMR spectroscopy revealed, together with the desired phosphodiesters 4ad–ah, the presence (10–15%) also of the parent nucleoside H-phosphonate 1a [δ_P 2.89, dd (¹J_HP 628.5 Hz, ³J_HP 9.3 Hz)], from which aryl nucleoside H-phosphonates 3ad–ah had been generated. These suggested that aryl nucleoside H-phosphonates 4ad–ah, because of their enhanced reactivity as compared with 4aa–ac, apparently underwent a partial hydrolysis under the reaction conditions. To eliminate this undesired reaction path, we attempted oxidation of 3ad–ah with anhydrous iodine in pyridine, followed by the addition (after 20 s) of water. Indeed, this stepwise oxidation protocol completely eliminated hydrolysis of aryl H-phosphonate intermediates 3ad–ah during oxidation (³¹P NMR), and provided an efficient access to nucleoside phosphates 4ad–ah bearing electron-deficient aromatic rings. The two-step oxidation procedure worked well also for aryl H-phosphonates 3aa–ah, and thus all aryl nucleoside phosphodiesters 4aa–ah could be obtained in high yields (>90%, after column chromatography) using one general protocol.

With the most acidic phenols investigated (2i and 2j, pK_a < 6), we encountered some problems in preparing the corresponding aryl nucleoside phosphates 4ai and 4aj, partly due to the high susceptibility of aryl H-phosphonate intermediates 3ai and 3aj to disproportionation,^25,26 and partly due to the hydrolytic instability of products 4ai and 4aj.²⁷

For the transformation of the in-situ-produced aryl nucleoside H-phosphonates 3 to the corresponding aryl nucleoside phosphorothioates of type 5, we chose elemental sulfur as an oxidising agent. We found that synthesis of phosphorothioates 5 can be carried out as a four-components–one-pot reaction, by allowing nucleoside H-phosphonate 1a to react with phenols 2 and DPCP in the presence of elemental sulfur. This protocol simplified the experimental procedure and permitted synthesis of all phosphorothioate diesters, including those derived (5ai and 5aj) from the most acidic phenols investigated (2i and 2j) (vide infra).

By monitoring these four-components–one-pot reactions by ³¹P NMR spectroscopy we observed that in the instance of phenols 2a–h, the formation of the corresponding aryl H-phosphonate 3aa–ah (ca. 5 min) was faster than its sulfurisation, which required for completion respectively 120 min for 3aa, 30 min for 3ab, 20 min for 3ac and 10 min for 3ad, 3ae, 3af, 3ag and 3ah. For the most acidic phenols in the series (2i, 2j), ³¹P NMR spectroscopy did not reveal any transient formation of the putative intermediates 3, but instead showed direct transformation of H-phosphonate 1 into products 5. This indicated that, in these instances, the rates of formation of aryl H-phosphonates 3ai–aj were apparently lower than those for their sulfurisation, and explained why compounds 3ai and 3aj did not undergo a noticeable disproportionation^25,26 under the reaction conditions used.

In all these reactions, however, apart from the desired phosphorothioates 5, additional nucleotidic products were formed (30–40%), as was apparent from the presence in the ³¹P NMR spectra of two groups of resonances at δ_P ≈ 46 and −25 (²J_PP 18.6–22.0 Hz). Since these signals originated from a single phosphorus species (2D ³¹P NMR spectroscopy), which upon hydrolysis afforded the corresponding aryl nucleoside phosphorothioate 5 and diphenyl hydrogen phosphate, we tentatively identified these compounds as mixed pyrothiophosphates 6 (³¹P NMR data in Table 1). Their presence in the reaction mixtures were most likely due to a subsequent reaction of the produced phosphorothioates 5 with the excess of DPCP (Scheme 1) used for the formation of aryl H-phosphonates 3.²⁸ Since mixed anhydrides 6 were readily (5 min) and cleanly hydrolysed with the added water (5 molar equiv.) to produce aryl nucleoside phosphorothioates, their formation did not affect the overall yields (>80% after silica gel chromatography) of isolated aryl nucleoside phosphorothioates 5aa–aj.

Table 1 ³¹P NMR data of bifurcated intermediate aryl nucleoside H-phosphonates 3 and pyrothiophosphates of type 6

Cpd no.	δP (ppm)	^1JHP (Hz)	^3JHP (Hz)	Cpd no.	δP (ppm)	^2JPP (Hz)	^3JHP (Hz)
a Two diastereoisomers.b Multiplet with several P–F couplings.
3aa	4.44, 4.47^a	712.6 (d), 732.6 (d)	8.3 (dd)	6aa	47.43, 47.80^a	21.7 (2d)	10.2 (2d)
					−25.28	21.7 (d)
3ab	2.62, 2.70^a	738.3 (d)	9.2 (dd)	6ab	46.75, 46.81^a	20.2 (2d)	9.2 (2d)
					−25.02	20.2 (d)
3ac	4.63	737.9 (d)	8.3 (dd)	6ac	46.49, 46.68^a	21.2 (2d)	10.3 (2d)
					−25.68	21.2 (d)
3ad	3.94, 4.37^a	745.2 (d), 729.5 (d)	7.0 (dd)	6ad	46.30, 46.65^a	21.0 (2d)	10.00 (2d)
					−25.89	21.0 (d)
3ae	3.44, 3.50^a	736.3 (d), 739.0 (d)	7.3 (dd)	6ae	46.48, 46.91^a	21.0 (2d)	11.0 (2d)
					−25.96	21.0 (d)
3af	3.36, 3.42^a	740.9 (d), 738.1 (d)	7.3 (dd)	6af	46.16, 46.58^a	20.2 (2d)	10.1 (2d)
					−26.44	20.2 (d)
3ag	2.44, 2.87^a	769.3 (d), 760.6 (d)	8.3 (dd)	6ag	46.05, 46.41^a	20.4 (d)	9.3 (2d)
					−25.41	20.4 (d)
3ah	4.07	750.8 (d)	8.3 (dd)	6ah	46.18, 46.56^a	18.6 (d)	9.3 (2d)
					−26.06	18.6 (d)
3ai	1.21, 1.63^a	768.4 (d), 764.7 (d)	9.2 (dd)	6ai	46.88, 47.23^a	19.3 (2d)	11.0 (2d)
					−26.1	19.3 (d)
3aj	3.61, 3.77^a	767.4 (d), 763.8 (d)	dm	6aj	49.02, 49.17^a	20.2 (2m)	m^b
					−26.20	20.2 (d)
3bc	3.42, 3.55^a	732.6 (d), 729.9 (d)	8.7 (dd)	6bc	46.17, 47.08^a	22.0 (2d)	9.7 (2d)
					−25.90	22.0 (d)
3bh	2.90, 2.97^a	742.7 (d), 740.0 (d)	8.2 (dd)	6bh	44.78, 45.74^a	21.1 (2d)	10.1 (2d)
					−25.91	21.1 (d)
3cc	3.55, 3.61^a	730.8 (d), 733.6 (d)	7.3 (dd)	6cc	45.35, 45.77^a	20.2 (2d)	10.1 (2d)
					−25.95	20.2 (d)
3ch	2.99, 3.05^a	743.6 (d)	8.2 (dd)	6ch	45.51, 45.71^a	20.2 (2d)	10.1 (2d)
					−25.94	20.2 (d)
3dc	3.65, 3.98^a	732.6 (d), 730.8 (d)	7.3 (dd)	6dc	45.77, 46.76^a	19.2 (2d)	9.6 (2d)
					−25.94	19.2 (d)
3dh	3.50, 3.85^a	743.6 (d), 742.7 (d)	8.25 (dd)	6dh	44.44, 45.49^a	17.9 (2d)	9.6 (2d)
					−25.89	17.9 (d)

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To confirm the generality of the developed protocols, the above transformations were carried out on suitably protected H-phosphonate monoesters derived from 2′-deoxyadenosine 1b, 2′-deoxycytidine 1c, and 2′-deoxyguanosine 1d, using two arbitrarily chosen phenols, 4-chlorophenol 2c and 4-nitrophenol 2h. In all instances the reactions proceeded similarly to that described for thymidine derivative 1a, and the products, aryl nucleoside phosphate diesters 4bc, 4cc, 4dc, 4bh, 4ch, 4dh and the corresponding phosphorothioate diesters, 5bc, 5cc, 5dc, 5bh, 5ch and 5dh, were obtained in high yields (see Experimental section).

In conclusion, we have developed a new, efficient method for the synthesis of aryl nucleoside phosphate and phosphorothioate diesters, based on H-phosphonate chemistry. Starting from one type of substrates—easily accessible nucleoside H-phosphonates and phenols—and using simple ‘one-pot’ reaction, it was possible by changing the oxidation protocol to obtain, in high yields, aryl nucleoside phosphate or phosphorothioate diesters, derived from phenols of a wide range of acidity.²⁹ The method seems to be rather general and thus applicable to the preparation of other phosphorus-containing natural products and their analogues, or various alkyl aryl phosphates and phosphorothioates for synthetic and biochemical applications.

Experimental

¹H and ³¹P NMR spectra were recorded at 300 MHz and 121 MHz, respectively, on a Varian Unity BB VT spectrometer. The ³¹P NMR experiments were carried out at 25 °C in 5 mm tubes using 0.1 M concentrations of phosphorus-containing compounds in appropriate solvents (0.6 mL) and the spectra were referenced to 2% H₃PO₄ in D₂O (external standard). Mass spectra were recorded with liquid secondary-ion mass technique (LSIMS) using Cs⁺ (12 keV) for ionisation. Molecular mass of compounds containing chlorine atom(s) was calculated and measured for ³⁵Cl isotope. TLC analyses were carried out on Merck silica gel 60 F₂₅₄ precoated plates using the following solvent systems: (A) i-PrOH–NH₄OH–water 85 ∶ 10 : 5 (v/v/v), (B) CH₂Cl₂–CH₃OH–Et₃N 85 : 10 : 5 (v/v/v). TLC mobilities (R_fTPH) are reported relative to 5′-O-(dimethoxytrityl)thymidine 3′-H-phosphonate. Pyridine (LabScan Ltd.) was stored over molecular sieves 4 Å until the amount of water was below 20 ppm (Karl Fischer coulometric titration). Dichloromethane (POCH, Poland) was dried with P₂O₅, distilled, and stored over molecular sieves 4 Å until the amount of water was below 10 ppm. Phenols 2a–j, diphenyl chlorophosphate (DPCP) and sulfur were commercial grade from Aldrich. Nucleoside H-phosphonates of type 1 were obtained according to the published method.³⁰ In all experiments 0.5 mmol of substrate 1 was used and the yields of products 4 and 5 were not optimised. The assignments of signals in the ³¹P NMR spectra to particular products or intermediates were done on the basis of their chemical shifts, multiplicity of the signals in ¹H-coupled and ¹H-decoupled spectra, by spiking the reaction mixtures with appropriate species and, if possible, by isolation of the compound in question from reaction mixtures. Multiplicity of some signals in ¹H NMR spectra of products 5 and 6 is due to P-diastereomers.

General procedure for synthesis of aryl nucleoside phosphates of type 4

Nucleoside H-phosphonate 1 (0.5 mmol) and phenol 2 (1.5 molar equiv.) were made anhydrous by evaporation (twice) of added pyridine, dissolved in CH₂Cl₂ containing pyridine [9 : 1 (v/v)] (0.5 mmol of 1/5 mL of solvent), and then DPCP (1.5 molar equiv.) was added. When the formation of aryl H-phosphonate 3 was complete (15 min), iodine (1.5 molar equiv.) followed by (after 20 s) water (5 molar equiv.) was added. After 5 min the excess of iodine was neutralised with ethanethiol, the reaction mixture was diluted with CH₂Cl₂ (15 mL/0.5 mmol of nucleotide), and the organic phase was extracted with saturated aq. NaHCO₃ (one third of volume of organic layer) and dried with anhydrous Na₂SO₄. The solvent was removed by evaporation and the resulting glassy residue was dissolved in the minimum volume of CH₂Cl₂ and loaded on a silica gel column prepared in the same solvent. Products of type 4 were isolated using linear gradient of methanol (0–10%) in CH₂Cl₂ containing triethylamine (1% of volume). Fractions containing pure product were collected and, after evaporation of solvents, triethylammonium salts of aryl nucleoside phosphate diesters of type 4 were obtained as non-hygroscopic, dry foams, which were solidified by freeze-drying from benzene. Purity of the isolated compounds was >97% (¹H NMR spectroscopy).

Phenyl 5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphate triethylammonium salt 4aa. Yield 0.377 g, 94%; R_fTPH 1.26 (A), 1.74 (B); δ_H (CDCl₃) 1.28 (3H, s, 5-CH₃), 1.85 (9H, t, J = 6.9 Hz, CH₂CH₃), 2.35, 2.64 (2H, 2m, 2′-H₂), 2.82 (6H, q, J = 6.9 Hz, CH₂CH₃), 3.35 (2H, m, 5′-H₂), 3.37 (6H, s, 2 × OCH₃), 4.42 (1H, m, 4′-H), 5.11 (1H, m, 3′-H), 6.46 (1H, m, 1′-H), 6.79–7.37 (18H, m, ArH of DMT and Ph groups), 7.59 (1H, s, 6-H), 8.62 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) −6.14 (d, ³J_HP = 6.1 Hz); [M⁻] m/z 699.2106. Calc. for [C₃₇H₃₆N₂O₁₀P]⁻: 699.2108.

2-Chlorophenyl 5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphate triethylammonium salt 4ab. Yield 0.376 g, 90% {for 5′-O-[o-(dibromomethyl)benzoyl]thymidine derivative 95%¹⁷}; R_fTPH 1.30 (A), 1.71 (B); δ_H (CDCl₃) 1.27 (3H, s, 5-CH₃) 1.28 (9H, t, J = 7.2 Hz, 3 × CH₂CH₃), 2.38, 2.68 (2H, 2m, 2′-H₂), 3.02 (6H, q, J = 7.2 Hz, 3 × CH₂CH₃), 3.38 (2H, m, 5′-H₂), 3.77 (6H, s, 2 × OCH₃), 4.31 (1H, m, 4′-H), 5.18 (1H, m, 3′-H), 6.47 (1H, m, 1′-H), 6.79–7.36 (17H, m, ArH of DMT and 2-chlorophenyl groups), 7.96 (1H, s, 6-H), 8.47 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) −6.59 (d, ³J_HP = 7.3 Hz); [M⁻] (m/z) 733.1716. Calc. for [C₃₇H₃₅ClN₂O₁₀P]⁻: 733.1718.

4-Chlorophenyl 5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphate triethylammonium salt 4ac. Yield 0.385 g, 92% (95%³¹); R_fTPH 1.37 (A), 1.71 (B); δ_H (CDCl₃) 1.18 (3H, s, 5-CH₃), 1.3 (9H, t, J = 7.2 Hz, 3 × CH₂CH₃), 2.35, 2.64 (2H, 2m, 2′-H₂), 3.05 (6H, q, J = 7.2 Hz, 3 × CH₂CH₃), 3.23, 3.41 (2H, 2m, 5′-H₂), 3.79 (6H, s, 2 × OCH₃), 4.22 (1H, m, 4′-H), 5.07 (1H, m, 3′-H), 6.45 (1H, m, 1′-H), 6.79–7.37 (17H, m, ArH of DMT and 4-chlorophenyl), 7.59 (1H, s, 6-H), 8.62 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) −6.30 (d, ³J_HP = 6.1 Hz); [M⁻] m/z 733.1716. Calc. for [C₃₇H₃₅ClN₂O₁₀P]⁻: 733.1718.

2,4-Dichlorophenyl 5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphate triethylammonium salt 4ad. Yield 0.409 g, 94%; R_fTPH 1.44 (A), 1.71 (B); δ_H (CDCl₃) 1.34 (3H, s, 5-CH₃), 1.35 (9H, t, J = 7.5 Hz, 3 × CH₂CH₃) 2.40, 2.66 (2H, 2m, 2′-H₂), 3.00 (6H, q, J = 7.5 Hz, 3 × CH₂CH₃), 3.40 (2H, m, 5′-H₂), 3.78 (6H, s, 2 × OCH₃), 4.39 (1H, m, 4′-H), 5.15 (1H, m, 3′-H), 6.45 (1H, m, 1′-H), 6.79–7.60 (16H, m, ArH of DMT and 2,4-dichlorophenyl), 7.60 (1H, s, 6-H), 8.63 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) −7.14 (d, ³J_HP = 6.1 Hz); [M⁻] m/z 767.1339. Calc. for [C₃₇H₃₄Cl₂N₂O₁₀P]⁻: 767.1328.

3,4-Dichlorophenyl 5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphate triethylammonium salt 4ae. Yield 0.392 g, 90%; R_fTPH 1.48 (A), 1.71 (B); δ_H (CDCl₃) 1.18 (9H, t, J = 7.2 Hz, 3 × CH₂CH₃), 1.22 (3H, s, 5-CH₃), 2.54, 2.66 (2H, 2m, 2′-H₂), 2.90 (6H, q, J = 7.2 Hz, 3 × CH₂CH₃), 3.17, 3.25 (2H, m, 5′-H₂), 3.68 (6H, s, 2 × OCH₃), 4.15 (1H, 2m, 4′-H), 4.98 (1H, m, 3′-H), 6.35 (1H, m, 1′-H), 6.69–7.28 (16H, m, ArH of DMT and 3,4-dichlorophenyl groups), 7.49 (1H, s, 6-H), 8.60 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) −6.64 (d, ³J_HP = 6.4 Hz); [M⁻] m/z 767.1329. Calc. for [C₃₇H₃₄Cl₂N₂O₁₀P]⁻: 767.1328.

3,5-Dichlorophenyl 5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphate triethylammonium salt 4af. Yield 0.387 g, 89%; R_fTPH 1.56 (A), 1.77 (B); δ_H (CDCl₃) 1.28 (9H, t, J = 7.2 Hz, 3 × CH₂CH₃), 1.35 (3H, s, 5-CH₃), 2.09, 2.36 (2H, 2m, 2′-H₂), 3.00 (6H, q, J = 7.2 Hz, CH₂CH₃), 3.38 (2H, m, 5′-H₂), 3.76 (6H, s, 2 × OCH₃), 4.28 (1H, m, 4′-H), 5.10 (1H, m, 3′-H), 6.44 (1H, m, 1′-H), 6.78–7.36 (16H, m, ArH of DMT and 3,5-dichlorophenyl groups), 7.59 (1H, s, 6-H), 8.76 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) −7.70 (d, ³J_HP = 7.3 Hz); [M⁻] m/z 767.1315. Calc. for [C₃₇H₃₄Cl₂N₂O₁₀P]⁻: 767.1328.

2,4,6-Trichlorophenyl 5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphate triethylammonium salt 4ag. Yield 0.407 g, 90%; R_fTPH 1.44 (A), 1.81 (B); δ_H (CDCl₃) 1.26 (9H, t, J = 7.2 Hz, 3 × CH₂CH₃), 1.30 (3H, s, 5-CH₃), 2.43, 2.86 (2H, 2m, 2′-H₂), 2.99 (6H, q, J = 7.2 Hz, 3 × CH₂CH₃), 3.47 (2H, m, 5′-H₂), 3.77 (6H, s, 2 × OCH₃), 4.44 (1H, m, 4′-H), 5.30 (1H, m, 3′-H), 6.50 (1H, m, 1′-H), 6.80–7.40 (15H, m, ArH of DMT and 2,4,6-trichlorophenyl groups), 7.65 (1H, s, 6-H), 11.94 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) −7.06 (d, ³J_HP = 6.1 Hz); [M⁻] m/z 801.0935. Calc. for [C₃₇H₃₃Cl₃N₂O₁₀P]⁻: 801.0938.

4-Nitrophenyl 5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphate triethylammonium salt 4ah. Yield 0.377 g, 89%; R_fTPH 1.48 (A), 1.68 (B); δ_H (CDCl₃) 1.29 (3H, s, 5-CH₃), 1.32 (9H, t, J = 7.5 Hz, 3 × CH₂CH₃), 2.43, 2.81 (2H, 2m, 2′-H₂), 3.47 (2H, m, 5′-H₂), 3.05 (6H, q, J = 7.5 Hz, 3 × CH₂CH₃), 3.69 (6H, s, 2 × OCH₃), 4.44 (1H, m, 4′-H), 5.30 (1H, m, 3′-H), 6.50 (1H, m, 1′-H), 6.80–7.40 (17H, m, ArH of DMT and 4-nitrophenyl groups), 7.65 (1H, s, 6-H), 8.63 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) −6.78 (d, ³J_HP = 6.1 Hz); [M⁻] m/z 744.1957. Calc. for [C₃₇H₃₅N₃O₁₂P]⁻: 744.1958.

4-Chlorophenyl 5′-O-(4,4′-dimethoxytrityl)-N⁶-benzoyl-2′-deoxyadenosin-3′-yl phosphate triethylammonium salt 4bc. Yield 0.370 g, 78%; R_fTPH 1.82 (A), 1.77 (B); δ_H (CDCl₃) 1.28 (9H, t, J = 7.2 Hz, 3 × CH₂CH₃), 2.87 (2H, m, 2′-H₂), 3.05 (6H, q, J = 7.2 Hz, 3 × CH₂CH₃), 3.34 (2H, m, 5′-H₂), 3.76 (6H, s, 2 × OCH₃), 5.15 (1H, m, 4′-H), 6.53 (1H, m, 3′-H), 6.53 (1H, m, 1′-H), 6.74–7.63 (22H, m, ArH of DMT, N⁶-benzoyl and 4-chlorophenyl groups), 8.02 (1H, s, 2-H), 8.05 (1H, s, 8-H), 9.14 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) −7.31 (d, ³J_HP = 6.4 Hz); [M⁻] m/z 846.2096. Calc. for [C₄₄H₃₈ClN₅O₉P]⁻: 846.2096.

4-Nitrophenyl 5′-O-(4,4′-dimethoxytrityl)-N⁶-benzoyl-2′-deoxyadenosin-3′-yl phosphate triethylammonium salt 4bh. Yield 0.374 g, 78%; R_fTPH 1.74 (A), 1.77 (B); δ_H (CDCl₃) 1.32 (9H, t, J = 7.4 Hz, 3 × CH₂CH₃), 2.90 (2H, m, 2′-H₂), 3.06 (6H, q, J = 7.4 Hz, 3 × CH₂CH₃), 3.40 (2H, m, 5′-H₂), 3.76 (6H, s, 2 × OCH₃), 4.40 (1H, m, 4′-H), 5.16 (1H, m, 3′-H), 6.52 (1H, m, 1′-H), 6.76–8.20 (22H, m, ArH of DMT, N⁶-benzoyl and 4-nitrophenyl groups), 8.14 (1H, s, 2-H), 8.71 (1H, s, 8-H), 9.07 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) −7.31 (d, ³J_HP = 6.4 Hz); [M⁻] m/z 857.2352. Calc. for [C₄₄H₃₈N₆O₁₁P]⁻: 857.2336.

4-Chlorophenyl 5′-O-(4,4′-dimethoxytrityl)-N⁴-benzoyl-2′-deoxycytidin-3′-yl phosphate triethylammonium salt 4cc. Yield 0.384 g, 83%; R_fTPH 1.78 (A), 1.81 (B); δ_H (CDCl₃) 1.35 (9H, t, J = 7.5 Hz, 3 × CH₂CH₃), 2.30, 2.95 (2H, 2m, 2′-H₂), 3.07 (6H, q, J = 7.5 Hz, 3 × CH₂CH₃), 3.39 (2H, m, 5′-H₂), 3.78 (6H, s, 2 × OCH₃), 4.35 (1H, m, 4′-H), 5.04 (1H, m, 3′-H), 6.28 (1H, m, 1′-H), 6.80–7.91 (22H, m, ArH of DMT, Bz and 4-chlorophenyl groups), 8.15 (1H, s, 6-H), 8.84 (1H, br s, NH, exch. D₂O). As judged from a COSY experiment, signals of H-5 overlapped with aromatic proton resonances; δ_P (CH₂Cl₂) −6.84 (d, ³J_HP = 6.4 Hz); [M⁻] m/z 822.1983. Calc. for [C₄₃H₃₈ClN₃O₁₀P]⁻: 823.1984.

4-Nitrophenyl 5′-O-(4,4′-dimethoxytrityl)-N⁴-benzoyl-2′-deoxycytidin-3′-yl phosphate triethylammonium salt 4ch. Yield 0.380 g, 81%; R_fTPH 1.70 (A), 1.77 (B); δ_H (CDCl₃) 1.35 (9H, t, J = 7.4 Hz, 3 × CH₂CH₃), 2.46, 2.99 (2H, 2m, 2′-H₂), 3.10 (6H, q, J = 7.4 Hz, 3 × CH₂CH₃), 3.46 (2H, m, 5′-H₂), 3.78 (6H, s, 2 × OCH₃), 4.44 (1H, m, 4′-H), 5.33 (1H, m, 3′-H), 6.32 (1H, t, J = 6.3 Hz, 1′-H), 6.80–8.15 (22H, m, ArH of DMT, Bz and 4-nitrophenyl groups), 8.19 (1H, d, J = 7.5 Hz, 6-H), 8.57 (1H, br s, NH, exch. D₂O). As judged from a COSY experiment, signals of H-5 overlapped with aromatic proton resonances; δ_P (CH₂Cl₂) −7.26 (d, ³J_HP = 6.4 Hz); [M⁻] m/z 833.2221. Calc. for [C₄₃H₃₈N₄O₁₂P]⁻: 833.2224.

4-Chlorophenyl 5′-O-(4,4′-dimethoxytrityl)-N²-isobutyryl-2′-deoxyguanosin-3′-yl phosphate triethylammonium salt 4dc. Yield 0.382 g, 82%; R_fTPH 1.59 (A), 1.68 (B); δ_H (CDCl₃) 0.83, 1.08 [6H, 2d, J = 6.9 Hz, CH(CH₃)₂], 1.21 (9H, t, J = 7.2 Hz, 3 × CH₂CH₃) 2.24, 2.62 (2H, 2m, 2′-H₂), 2.96 (6H, q, J = 7.2 Hz, 3 × CH₂CH₃), 3.05 [1H, m, J = 6.9 Hz, CH(CH₃)₂], 3.19, 3.36 (2H, 2m, 5′-H₂), 3.75 (6H, s, 2 × OCH₃), 4.24 (1H, m, 4′-H), 5.46 (1H, m, 3′-H), 6.13 (1H, t, J = 6.6 Hz, 1′-H), 6.70–7.36 (17H, m, ArH of DMT and 4-chlorophenyl groups), 7.72 (1H, s, 8-H), 9.52 (1H, br s, NH, exch. D₂O), 12.03 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) −6.88 (d, ³J_HP = 7.3 Hz); [M⁻] m/z 828.2204. Calc. for [C₄₁H₄₀ClN₅O₁₀P]⁻: 828.2201.

4-Nitrophenyl 5′-O-(4,4′-dimethoxytrityl)-N²-isobutyryl-2′-deoxyguanosin-3′-yl phosphate triethylammonium salt 4dh. Yield 0.348 g, 74%; R_fTPH 1.52 (A), 1.65 (B); δ_H (CDCl₃) 1.03 [6H, d, J = 6.9 Hz, CH(CH₃)₂], 1.27 (9H, t, J = 7.5 Hz, 3 × CH₂CH₃), 2.24, 2.62 (2H, 2m, 2′-H₂), 3.01 (6H, q, J = 7.5 Hz, 3 × CH₂CH₃), 3.07 [1H, m, J = 6.9 Hz, CH(CH₃)₂], 3.26 (2H, m, 5′-H₂), 3.76 (6H, s, 2 × OCH₃), 4.24 (1H, m, 4′-H), 5.42 (1H, m, 3′-H), 6.14 (1H, t, J = 6.3 Hz, 1′-H), 6.71–8.10 (17H, m, ArH of DMT and 4-nitrophenyl groups), 7.72 (1H, s, 8-H), 9.40 (1H, br s, NH, exch. D₂O), 12.03 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) −7.69 (d, ³J_HP = 7.3 Hz); [M⁻] m/z 839.2442. Calc. for [C₄₁H₄₀N₆O₁₂P]⁻: 839.2442.

General procedure for synthesis of aryl nucleoside phosphorothioates of type 5

Nucleoside H-phosphonate 1 (0.5 mmol), the appropriate phenol 2 (1.5 molar equiv.) and sulfur (3 molar equiv.) were made anhydrous by evaporation (twice) of added excess of pyridine, dissolved in CH₂Cl₂ containing pyridine [9 : 1 (v/v)] (0.5 mmol of 1/5 mL of solvent) and then, to the stirred mixture, was added DPCP (1.5 molar equiv.). After sulfurisation of the produced aryl H-phosphonate 3 was complete (ca. 120 min for 3aa, 30 min for 3ab, 3ac, 3bc, 3cc, 3dc, 20 min for 3ad–af, and 10 min for 3ag–3aj, 3bh, 3ch, 3dh), water and Et₃N (5 molar equiv. each) were added to hydrolyse the pyrothiophosphate of type 6. After 5 min the reaction mixture was diluted with CH₂Cl₂ (15 mL/0.5 mmol of nucleotide), and the organic phase was extracted with saturated aq. NaHCO₃ (one-third of volume of organic layer). Further work-up and isolation of pure triethylammonium salt of aryl nucleoside phosphorothioates of type 5 was as described above for aryl nucleoside phosphates of type 4. Compounds 5 were obtained as white solids (purity >97%, ¹H NMR spectroscopy).

Phenyl 5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphorothioate triethylammonium salt 5aa. Yield 0.372 g, 91%; R_fTPH 1.44 (A), 1.94 (B); δ_H (CDCl₃) 1.21 (9H, t, J = 6.9 Hz, 3 × CH₂CH₃), 1.33, 1.35 (3H, 2s, 5-CH₃), 2.34, 2.57 (2H, 2m, 2′-H₂), 2.89 (6H, q, J = 6.9 Hz, 3 × CH₂CH₃), 3.36, 3.56 (2H, 2m, 5′-H₂), 3.77 (6H, s, 2 × OCH₃), 4.29, 4.37 (1H, 2m, 4′-H), 5.46 (1H, m, 3′-H), 6.17 (1H, m, 1′-H), 6.48–7.42 (18H, m, ArH of DMT and phenyl groups), 7.61 (1H, s, 6-H), 8.63 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) 53.61, 53.77 (d, ³J_HP = 9.4 Hz); [M⁻] m/z 715.1877. Calc. for [C₃₇H₃₆N₂O₉PS]⁻: 715.1879.

2-Chlorophenyl 5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphorothioate triethylammonium salt 5ab. Yield 0.392 g, 92%; R_fTPH 1.52 (A), 1.87 (B); δ_H (CDCl₃) 1.30 (3H, s, 5-CH₃), 1.31 (9H, t, J = 7.2 Hz, 3 × CH₂CH₃), 2.38, 2.59 (2H, 2m, 2′-H₂), 3.08 (6H, q, J = 7.2 Hz, 3 × CH₂CH₃), 3.80 (6H, s, 2 × OCH₃), 3.96 (2H, m, 5′-H₂), 4.17 (1H, m, 4′-H), 5.27 (1H, m, 3′-H), 6.21 (1H, m, 1′-H), 6.41–7.36 (17H, m, ArH of DMT and 2-chlorophenyl groups), 7.60 (1H, m, 6-H), 8.12 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) 53.67, 53.74 (2d, ³J_HP = 9.2 Hz); [M⁻] m/z 749.1490. Calc. for [C₃₇H₃₅ClN₂O₉PS]⁻: 749.1489.

4-Chlorophenyl 5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphorothioate triethylammonium salt 5ac. Yield 0.324 g, 76%; R_fTPH 1.56 (A), 1.87 (B); δ_H (CDCl₃): 1.29 (9H, t, J = 7.5 Hz, 3 × CH₂CH₃), 1.34, 1.38 (3H, s, 5-CH₃), 2.37, 2.65 (2H, 2m, 2′-H₂), 3.04 (6H, q, J = 7.5 Hz, 3 × CH₂CH₃), 3.37, 3.51 (2H, 2m, 5′-H₂), 3.77 (6H, s, 2 × OCH₃), 4.28, 4.40 (1H, 2m, 4′-H), 5.41 (1H, m, 3′-H), 6.47 (1H, m, 1′-H), 6.81–7.41 (17H, m, ArH of DMT and 4-chlorophenyl groups), 7.60 (m, 1H, 6-H), 8.32 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) 54.82, 54.90 (2d, ³J_HP = 11.0 Hz); [M⁻] m/z 749.1490. Calc. for [C₃₇H₃₅ClN₂O₉PS]⁻: 749.1489.

2,4-Dichlorophenyl 5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphorothioate triethylammonium salt 5ad. Yield 0.412 g, 93%; R_fTPH 1.59 (A), 1.87 (B); δ_H (CDCl₃) 1.23 (9H, t, J = 7.5 Hz, 3 × CH₂CH₃), 1.36 (3H, s, 5-CH₃), 2.45, 2.85 (2H, 2m, 2′-H₂), 3.08 (6H, q, J = 7.5 Hz, 3 × CH₂CH₃), 3.40, 3.59 (2H, 2m, 5′-H₂), 3.76 (6H, s, 2 × OCH₃), 4.44, 4.57 (1H, 2m, 4′-H), 5.62, 5.76 (1H, m, 3′-H), 6.47 (1H, m, 1′-H), 6.52–7.44 (16H, m, ArH of DMT and 2,4-dichlorophenyl groups), 7.67 (1H, m, 6-H), 8.63 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) 55.02, 55.73 (2d, ³J_HP = 9.7 Hz); [M⁻] m/z 783.1120. Calc. for [C₃₇H₃₄Cl₂N₂O₉PS]⁻: 783.1100.

3,4-Dichlorophenyl 5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphorothioate triethylammonium salt 5ae. Yield 0.395 g, 89%; R_fTPH 1.67 (A), 1.90 (B); δ_H (CDCl₃) 1.26 (9H, t, J = 7.5 Hz, 3 × CH₂CH₃), 1.34, 1.38 (3H, 2s, 5-CH₃), 2.39, 2.66 (2H, 2m, 2′-H₂), 3.00 (6H, q, J = 7.5 Hz, 3 × CH₂CH₃), 3.38, 3.52 (2H, 2m, 5′-H₂), 3.76 (6H, s, 2 × OCH₃), 4.30, 4.39 (1H, 2m, 4′-H), 5.41 (1H, m, 3′-H), 6.47 (1H, m, 1′-H), 6.79–7.45 (16H, m, ArH of DMT and 3,4-dichlorophenyl groups), 7.59, 7.61 (1H, 2br s, 6-H), 9.13 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) 53.28, 53.33 (d, ³J_HP = 10.1 Hz); [M⁻] m/z 783.1110. Calc. for [C₃₇H₃₄Cl₂N₂O₉PS]⁻: 783.1100.

3,5-Dichlorophenyl 5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphorothioate triethylammonium salt 5af. Yield 0.404 g, 91%; R_fTPH 1.67 (A), 1.94 (B); δ_H (CDCl₃) 1.30 (9H, t, J = 7.2 Hz, 3 × CH₂CH₃), 1.35, 1.39 (3H, 2s, 5-CH₃), 2.42, 2.68 (2H, 2m, 2′-H₂), 3.05 (6H, q, J = 7.2 Hz, 3 × CH₂CH₃), 3.39, 3.56 (2H, 2m, 5′-H₂), 3.77, 3.78 (6H, 2s, 2 × OCH₃), 4.39, 4.41 (1H, 2m, 4′-H), 5.44 (1H, m, 3′-H), 6.48 (1H, m, 1′-H), 6.81–7.31 (16H, m, ArH of DMT and 3,5-dichlorophenyl groups), 7.62, 7.63 (1H, 2s, 6-H), 8.26 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) 53.25 (d, ³J_HP = 10.1 Hz); [M⁻] m/z 783.1087. Calc. for [C₃₇H₃₄Cl₂N₂O₉PS]⁻: 783.1100.

2,4,6-Trichlorophenyl 5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphorothioate triethylammonium salt 5ag. Yield 0.438 g, 95%; R_fTPH 1.67 (A), 1.87 (B); δ_H (CDCl₃) 1.30 (9H, t, J = 7.2 Hz, 3 × CH₂CH₃), 1.35, 1.36 (3H, 2s, 5-CH₃), 2.43, 2.81 (2H, 2m, 2′-H₂), 3.07 (6H, q, J = 7.2 Hz, 3 × CH₂CH₃), 3.46 (2H, m, 5′-H₂), 3.76 (6H, s, 2 × OCH₃), 4.45 (1H, m, 4′-H), 5.62 (1H, m, 3′-H), 6.46 (1H, m, 1′-H), 6.48–7.41 (15H, m, ArH of DMT and 2,4,6-trichlorophenyl groups), 7.70 (1H, br s, 6-H), 11.58 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) 53.58, 53.74 (2d, ³J_HP = 9.7 Hz); [M⁻] m/z 817.0706. Calc. for [C₃₇H₃₃Cl₃N₂O₉PS]⁻: 817.0710.

4-Nitrophenyl 5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphorothioate triethylammonium salt 5ah. Yield 0.410 g, 95%; R_fTPH 1.63 (A), 1.87 (B); δ_H (CDCl₃) 1.27 (9H, t, J = 7.2 Hz, 3 × CH₂CH₃), 1.35, 1.40 (3H, s, 5-CH₃), 2.39, 2.66 (2H, 2m, 2′-H₂), 2.99 (6H, q, J = 7.2 Hz, 3 × CH₂CH₃), 3.41 (2H, m, 5′-H₂), 3.77 (6H, s, 2 × OCH₃), 4.29, 4.38 (1H, m, 4′-H), 5.41 (1H, m, 3′-H), 6.47 (1H, m, 1′-H), 6.81–7.41 (17H, m, ArH of DMT and 4-nitrophenyl groups), 7.62 (1H, m, 6-H), 8.62 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) 55.00, 55.66 (2d, ³J_HP = 9.7 Hz); [M⁻] m/z 760.1730. Calc. for [C₃₇H₃₅N₃O₁₁PS]⁻: 760.1730.

Pentachlorophenyl 5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphorothioate triethylammonium salt 5ai. Yield 0.420 g, 85%; R_fTPH 1.70 (A), 1.94 (B); δ_H (CDCl₃) 1.12 (9H, t, J = 7.2 Hz, 3 × CH₂CH₃), 1.34, 1.38 (3H, 2br s, 5-CH₃), 2.46, 2.84 (2H, 2m, 2′-H₂), 2.69 (6H, q, J = 7.2 Hz, 3 × CH₂CH₃), 3.52 (2H, m, 5′-H₂), 3.76, 3.78 (6H, 2s, 2 × OCH₃), 4.44, 4.53 (1H, 2m, 4′-H), 5.56, 5.66 (1H, 2m, 3′-H), 6.50 (1H, m, 1′-H), 6.80–7.42 (13H, m, ArH of DMT), 7.66, 7.68 (1H, 2br s, 6-H), 8.68 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) 52.61, 53.67 (2d, ³J_HP = 9.2 and 11.0 Hz); [M⁻] m/z 884.9914. Calc. for [C₃₇H₃₁Cl₅N₂O₉PS]⁻: 884.9931.

Pentafluorophenyl 5′-O-(4,4′-dimethoxytrityl)thymidin-3′-yl phosphorothioate triethylammonium salt 5aj. Yield 0.409 g, 90%; R_fTPH 1.63 (A), 1.87 (B); δ_H (CDCl₃) 1.24 (9H, t, J = 7.5 Hz, 3 × CH₂CH₃), 1.35, 1.37 (3H, 2br s, 5-CH₃), 2.42, 2.76 (2H, 2m, 2′-H₂), 2.91 (6H, q, J = 7.5 Hz, 3 × CH₂CH₃), 3.47 (2H, m, 5′-H₂), 3.78 (6H, s, 2 × OCH₃), 4.43 (1H, m, 4′-H), 5.40, 5.41 (1H, 2m, 3′-H), 6.51 (1H, m, 1′-H), 6.81–7.44 (13H, m, ArH of DMT), 7.63, 7.68 (1H, 2br s, 6-H), 8.64 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) 54.89, 55.85 (2m); [M⁻] m/z 805.1413. Calc. for [C₃₇H₃₁F₅N₂O₉PS]⁻: 805.1408.

4-Chlorophenyl 5′-O-(4,4′-dimethoxytrityl)-N⁶-benzoyl-2′-deoxyadenosin-3′-yl phosphorothioate triethylammonium salt 5bc. Yield 0.396 g, 82%; R_fTPH 2.00 (A), 1.94 (B); δ_H (CDCl₃) 1.28 (9H, t, J = 7.5 Hz, 3 × CH₂CH₃), 2.87, 2.94 (2H, 2m, 2′-H₂), 3.04 (6H, q, J = 7.5 Hz, 3 × CH₂CH₃), 3.37, 3.44 (2H, m, 5′-H₂), 3.76 (6H, s, 2 × OCH₃), 4.47, 4.56 (1H, 2m, 4′-H), 5.44 (1H, m, 3′-H), 6.57 (1H, m, 1′-H), 6.75–7.61 (22H, m, ArH of DMT, Bz and 4-chlorophenyl groups), 8.02, 8.04 (1H, 2s, 2-H), 8.16, 8.19 (1H, 2s, 8-H), 9.07 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) 53.10, 53.13 (2d, ³J_HP = 9.2 Hz); [M⁻] m/z 862.1854. Calc. for [C₄₄H₃₈ClN₅O₈PS]⁻: 862.1867.

4-Nitrophenyl 5′-O-(4,4′-dimethoxytrityl)-N⁶-benzoyl-2′-deoxyadenosin-3′-yl phosphorothioate triethylammonium salt 5bh. Yield 0.405 g, 83%; R_fTPH 1.93 (A), 1.97 (B); δ_H (CDCl₃) 1.32 (9H, t, J = 7.5 Hz, 3 × CH₂CH₃), 2.94 (2H, m, 2′-H₂), 3.10 (6H, q, J = 7.5 Hz, 3 × CH₂CH₃), 3.39, 3.44 (2H, m, 5′-H₂), 3.76 (6H, s, 2 × OCH₃), 4.50 (1H, m, 4′-H), 5.44, 5.48 (1H, 2m, 3′-H), 6.57 (1H, m, 1′-H), 6.76–8.19 (22H, m, ArH of DMT, Bz and 4-nitrophenyl groups), 8.17, 8.20 (1H, 2s, 2-H), 8.72, 8.73 (1H, 2s, 8-H), 9.09 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) 52.34 (d, ³J_HP = 9.2 Hz); [M⁻] m/z 873.2099. Calc. for [C₄₄H₃₈N₆O₁₀PS]⁻: 873.2108.

4-Chlorophenyl 5′-O-(4,4′-dimethoxytrityl)-N⁴-benzoyl-2′-deoxycytidin-3′-yl phosphorothioate triethylammonium salt 5cc. Yield 0.410 g, 87%; R_fTPH 1.89 (A), 1.97 (B); δ_H (CDCl₃) 1.30 (9H, t, J = 7.5 Hz, 3 × CH₂CH₃), 2.30, 2.98 (2H, 2m, 2′-H₂), 3.06 (6H, q, J = 7.5 Hz, 3 × CH₂CH₃), 3.45 (2H, m, 5′-H₂), 3.78 (6H, s, 2 × OCH₃), 4.48 (1H, m, 4′-H), 5.34 (1H, m, 3′-H), 6.33 (1H, t, J = 6.2 Hz, 1′-H), 6.82–7.91 (22H, m, ArH of DMT, Bz and 4-chlorophenyl groups), 8.19 (1H, d, J = 7.2 Hz, 6-H), 8.70 (1H, br s, NH, exch. D₂O). As judged from a COSY experiment, the signal of H-5 overlaps with aromatic proton resonances; δ_P (CH₂Cl₂) 55.00, 55.66 (2d, ³J_HP = 9.7 Hz); δ_P (CH₂Cl₂) 53.06, 53.12 (2d, ³J_HP = 11.0 Hz); [M⁻] m/z 838.1760. Calc. for [C₄₃H₃₈ClN₃O₉PS]⁻: 838.1755.

4-Nitrophenyl 5′-O-(4,4′-dimethoxytrityl)-N⁴-benzoyl-2′-deoxycytidin-3′-yl phosphorothioate triethylammonium salt 5ch. Yield 0.419 g, 88%; R_fTPH 1.78 (A), 1.97 (B); δ_H (CDCl₃) 1.36 (9H, t, J = 7.4 Hz, 3 × CH₂CH₃), 2.46, 2.99 (2H, 2m, 2′-H₂), 3.10 (6H, q, J = 7.4 Hz, 3 × CH₂CH₃), 3.46 (2H, m, 5′-H₂), 3.78 (6H, s, 2 × OCH₃), 4.48 (1H, m, 4′-H), 5.33 (1H, m, 3′-H), 6.32 (1H, t, J = 6.3 Hz, 1′-H), 6.81–7.94 (22H, m, ArH of DMT, Bz and 4-nitrophenyl groups), 8.16 (1H, m, 6-H), 8.60 (1H, br s, NH, exch. D₂O). As judged from a COSY experiment, the signal of H-5 overlaps with aromatic proton resonances; δ_P (CH₂Cl₂) 52.20, 52.32 (2d, ³J_HP = 9.1 Hz); [M⁻] m/z 849.1978. Calc. for [C₄₃H₃₈N₄O₁₁PS]⁻: 849.1955.

4-Chlorophenyl 5′-O-(4,4′-dimethoxytrityl)-N²-isobutyryl-2′-deoxyguanosin-3′-yl phosphorothioate triethylammonium salt 5dc. Yield 0.398 g, 84%; R_fTPH 1.70 (A), 1.81 (B); δ_H (CDCl₃) 0.99 [m, 6H, CH(CH₃)₂], 1.26 (9H, t, J = 7.5 Hz, 3 × CH₂CH₃), 2.05, 2.65 (2H, 2m, 2′-H₂), 3.03 (6H, q, J = 7.5 Hz, 3 × CH₂CH₃), 3.16 [1H, m, CH(CH₃)₂], 3.26, 3.41 (2H, 2m, 5′-H₂), 3.74, 3.75 (6H, 2s, 2 × OCH₃), 4.29 (1H, m, 4′-H), 5.71, 5.87 (1H, 2m, 3′-H), 6.15 (1H, m, 1′-H), 6.69–7.40 (17H, m, ArH of DMT and 4-chlorophenyl groups), 7.74, 7.76 (1H, 2s, 8-H), 8.48 (1H, br s, NH, exch. D₂O), 11.89 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) 53.09, 53.80 (2d, ³J_HP = 11.0 Hz); [M⁻] m/z 844.1969. Calc. for [C₄₁H₄₀ClN₅O₉PS]⁻: 844.1973.

4-Nitrophenyl 5′-O-(4,4′-dimethoxytrityl)-N²-isobutyryl-2′-deoxyguanosin-3′-yl phosphorothioate triethylammonium salt 5dh. Yield 0.378 g, 79%; R_fTPH 1.67 (A), 1.81 (B); δ_H (CDCl₃) 0.93, 1.06 [6H, 2d, J = 6.9 Hz, CH(CH₃)₂], 1.32 (9H, t, J = 7.5 Hz, 3 × CH₂CH₃), 2.57, 2.68 (2H, 2m, 2′-H₂), 2.91, 3.20 [1H, 2m, J = 6.9 Hz, CH(CH₃)₂], 3.11 (6H, q, J = 7.5 Hz, 3 × CH₂CH₃), 3.75 (2H, m, 5′-H₂), 3.80 (6H, s, 2 × OCH₃), 4.32 (1H, m, 4′-H), 5.53, 5.68 (1H, 2m, 3′-H), 6.15 (1H, m, 1′-H), 6.71–7.43 (17H, m, ArH of DMT and 4-nitrophenyl groups), 8.17, 8.20 (1H, 2s, 8-H), 11.23 (1H, br s, NH, exch. D₂O); δ_P (CH₂Cl₂) 52.19, 52.74 (2d, ³J_HP = 11.0 and 10.1 Hz); [M⁻] m/z 855.2221. Calc. for [C₄₁H₄₀N₆O₁₁PS]⁻: 855.2213.

Acknowledgements

Financial support from the State Committee for Scientific Research, Republic of Poland and the Swedish Natural Science Research Council is gratefully acknowledged.

References

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