Syntheses of Two 5-Hydroxymethyl-2′-DeoxyCytidine Phosphoramidites with TBDMS as the 5-hydroxyl protecting group and Their Incorporation into DNA

Abstract

5-Hydroxymethylcytosine (5-hmC) is a newly discovered DNA base modification in mammalian genomic DNA that is proposed to be a major epigenetic mark. We report here the syntheses of two new versions of phosphoramidites III and IV from dU in 18 and 32% overall yields, respectively, with TBDMS as the 5-hydroxyl protecting group. Phosphoramidites III and IV allow efficient incorporation of 5-hmC into DNA and a “one-step” deprotection procedure to cleanly remove all the protecting groups. A “two-step” deprotection strategy is compatible with ultra-mild DNA synthesis, which enables the synthesis of 5hmC-containing DNA with additional modifications.

5-Methylcytosine (5-mC) is an important DNA modification found in eukaryotes and is referred to as the fifth base besides dA, dC, dG, and dT. It constitutes ∼2-8% of the total cytosine in human genomic DNA, and impacts a broad range of biological functions including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, cancer, and so on.¹ In 2009, 5-hydroxymethylcytosine (5-hmC), an oxidized form of 5-mC, was discovered in substantial amounts in mammalian genome of certain cell types as the “sixth” base.² Recent studies have shown that 5-hmC is a widespread DNA modification in the brain tissues and stem cells, and its abundance is dependent on tissue type.³ A group of Tet dioxygenases have been shown to utilize dioxygen to oxidize 5-mC to 5-hmC in the mammalian genome, and display important functions in the maintenance and normal myelopoiesis of embryonic stem cells (ES cells).^2b,4 We have recently developed an efficient method to specifically label and pull down 5-hmC-containing DNA fragments for subsequent deep sequencing to reveal the genome-wide distribution of 5-hmC, which in mouse cerebellum appears to be enriched in a gene expression level-dependent manner.⁵ All results so far suggested that 5-hmC is an important epigenetic modification.⁶ In order to facilitate the biological study of 5-hmC in DNA, an efficient synthesis of 5-hmC-containing DNA as model substrates is a prerequisite. In addition, DNA with other labeling such as fluorophores or biotin is usually needed for biochemical characterizations, whose incorporation into DNA often requires or prefers the use of ultra-mild DNA synthesis. Therefore, it is also highly desirable to develop 5-hmC phosphoramidite building blocks which is compatible with ultra-mild DNA synthesis.

So far, two phosphoramidite building blocks (I⁷ and II⁸, Fig. 1) have been developed for the incorporation of 5-hmC into DNA. These methods are currently used in the synthesis of 5-hmC-containing DNAs for biological studies. However, both methods pose limitations in the post-synthetic removal of the protecting groups.

Fig. 1. Two reported phosphoramidite building blocks of 5-hydroxymethyl-2′-deoxycytidine.

Phosphoramidite I uses Ac as the protecting group for the 5-hydroxymethyl (5-CH₂OH) group. After its incorporation into DNA, the post-synthetic treatment with NH₄OH generates an amide byproduct, which is produced by the S_N2 reaction of ammonia attacking the pseudobenzylic carbon with the OAc group as the leaving group to give an amine intermediate followed by migration of the benzoyl group.⁷ To avoid the formation of this byproduct, strong base treatment (0.1 M NaOH in dioxane/H₂O) followed by ammonolysis is required. Phosphoramidite II with a cyanoethyl as the protecting group of 5-CH₂OH is more widely used since it is commercially available. However, the post-synthetic removal of the cyanoethyl group turned out to be troublesome.⁸ Treatment of synthetic DNA with NH₄OH at 65 °C for 60 h could not completely remove the protecting group. An additional treatment with stronger base (NaOMe in MeOH) is necessary. Obviously, neither of the phosphoramidites reported is suitable for synthesizing 5-hmC-containing DNA with additional base-labile modifications. Therefore, it is highly desirable to develop new 5-hmC phosphoramidites using protecting groups that can be cleanly removed under mild conditions. Herein we report the efficient syntheses of two 5-hmC phosphoramidite building blocks (III and IV), and demonstrated advantages in the removal of the protecting groups following oligo synthesis.

We first chose to synthesize phosphoramidite III with TBDMS and Bz as 5-CH₂OH and exocyclic amino protecting groups, respectively (Scheme 1). Unlike OAc at the pseudobenzylic position, O-TBDMS is not a good leaving group and thus will not result in the formation of the S_N2 byproduct during NH₄OH treatment. TBDMS can be readily removed using by fluoride treatment such as TBAF.

Scheme 1. Synthesis of intermediate 5.

5-Hydroxymethyl-2′-deoxyuridine (5-hmdU, 2) is commercially available, but relatively expensive;⁹ therefore, we chose to start from 2′-deoxyuridine (dU, 1) and converted it to 2 in 60% yield (Scheme 1).¹⁰ Both the 5- and 5′-hydroxyl of 2 are primary alcohols; however, the 5-hydroxyl is more reactive than 5′-hydroxyl since it is in the pseudobenzylic position.¹¹ Conte et al. reported that 5-CH₂OH of 2 could be selectively protected with TBDMS. When 2 was treated with AgNO₃ and TBDMS-Cl in the presence of pyridine and THF, a mixture of the desired 3 and its regio-isomer 4 was obtained in a 4:1 ratio in 82% combined yield.¹² Unfortunately, these two isomers could not be resolved by silica gel chromatography using various eluent systems.¹³ Instead we successfully resolved the two isomers by treating the mixture of 3 and 4 with di-tert-butylsilyl bistriflate in DMF. A cyclic six member ring was formed for 3 to generate 5; in contrast, a cyclic silyl ring between 3′ and 5 hydroxyls of 4 could not form. Thus, compound 5 was obtained in pure form in 40% yield from 2 after silica gel chromatography (overall yield from 1 was 24%).

Although we were able to prepare the key intermediate 5 using the above method, several drawbacks such as the difficulty to purify 2 due to its high polarity, the formation of the undesired regio-isomer 4, and the unsatisfactory overall yield prompted us to explore more efficient synthesis of 5. Crouch et al. reported that 5-iodo-3′,5′-di-O-TBDMS-2′-deoxyuridine could be converted to the corresponding 5-formyl-dU analogue in 82% yield using standard Stille coupling conditions.¹⁴ We reasoned that the reduction of the aldehyde analogue should generate the corresponding alcohol. Our new synthesis started from a commercial reagent, 5-iodo-2′-deoxyuridine (6, Scheme 2). We chose to protect the 3′ and 5′-hydroxyls with di-t-butylsilyl group so that it can be selectively removed after 5-CH₂OH is protected by TBDMS. Thus, 6 was converted to 7 in 92% yield. Stille reaction generated the corresponding 5-formyl-dU analogue 8 in 85% yield. Reduction of 8 with NaBH₄ in the presence of CeCl₃ gave the corresponding 5-hmdC analogue 9 in 78% yield. Protection of the hydroxyl group with TBDMS provided 5 in 95% yield. The product for each step could be easily purified using low polarity eluents. The overall yield of 5 was improved to 58%.

Scheme 2. Synthesis of phosphoramidite III from 6.

To prepare 5-hmC phosphoramidite III, we next converted 5 into the 5-hmdC analogue by first treating 5 with POCl₃ and 1,2,4-triazole to generate a 4-triazolyl intermediate, followed by reacting with NH₄OH in dioxane to generate the amine 10 in 65% yield in two steps. Protection of the exocyclic amine provided 11 in 80% yield. Selective removal of the 3′,5′-silyl protecting group of 11 with HF in pyridine generated 12 in 84% yield. Selective protection of the 5′-OH of 12 with DMTr (13, 82%) and subsequent phosphitylation of the 3′-OH by standard procedure gave phosphoramidite III (87%). The synthesis entails 9 steps in 18% overall yield from 6.

So far, all the syntheses of phosphoramidite building blocks I, II, and III required a multiple-step transformation of converting 5-hmU derivatives to the corresponding 5-hmC analogues in two steps and an additional step to protect the resulting exocyclic amine with Bz. To simplify the synthesis, we chose to synthesize another version of phosphoramidite IV that bears 4-triazoylide instead of 4-BzNH. Since 4-triazolide can be converted simultaneously into 4-NH₂ during post-synthetic NH₄OH treatment, two steps of reaction could therefore be spared. The same strategy has been successfully used for synthesizing nucleotides containing 4-NH₂, 4-MeO, 4-EtO, 4-Me₂N-NH and 4-SH analogues.¹⁵ Besides allowing for the incorporation of 5-hmdC into DNA, phosphoramidite IV may also find applications in synthesizing DNA containing 4-substituted 5-hydroxymethylpyrimidine derivatives.

The synthesis of phosphoramidite IV started from intermediate 5 (Scheme 3). Selective removal of the 3′,5′-silyl protecting group of 5 with HF in pyridine generated 14 in 88% yield. Selective protection of the 5′-OH of 14 with DMTr (15, 85%) and subsequent phosphitylation of the 3′-OH gave phosphoramidite 16 in 92% yield. Phosphoramidite 16 was quantitatively converted to phosphoramidite IV although it has a silylated hydroxymethyl group in the adjacent 5-position.⁸ Phosphoramidite IV was also stable during silica gel chromatography and it was isolated in 81% yield. The synthesis entails 8 steps and the overall yield from 1 reached 32%.

Scheme 3. Synthesis of phosphoramidite IV.

Phosphoramidites III and IV were then incorporated into a short model sequence 5′-TCXGA (X = 5-hmC) using ultra mild reagents with the modified phosphoramidites using double coupling to give oligodeoxynucleotides ODN1 and ODN2, respectively. As comparion, phosphoramidite II from Glen Research was incorporated into the same sequence under the same conditions to give ODN3. After the post-synthetic treatment of ODN2 with NH₄OH at room temperature overnight, reverse phase HPLC analysis (Fig. 2A) showed that, besides DNA with a TBDMS protecting group (peak c, t_R = 34 min), over 20% of DNA without TBDMS group was also produced (peak b, t_R = 22 min). It is interesting to note that the 5-CH₂O-TBDMS group is much more labile to the NH₄OH treatment than the regular 2′-O-TBDMS protecting group in RNA synthesis, which can survive the overnight treatment at 55 °C. This observation is consistent with the enhanced reactivity of the 5-CH₂OH in 2 as indicated by selective protection of 5-CH₂OH by TBDMS or Ac against the 5′-hydroxyl.^11,12 The liability of the 5-CH₂O-TBDMS ether to the NH₄OH treatment suggested that it is possible to fully remove it from the oligo at elevated temperature so that the extra fluoride treatment might be spared. Indeed, when ODN 2 was treated with NH₄OH at 65 °C for 16 h, HPLC analysis indicated that TBDMS was close to fully removed (Fig. 2, B). The same treatment of ODN 1 also gave the same major desired product. In contrast, ODN-3 only gave about 56% of the desired product, while another 44% with the 5-cyanoethyl protecting group still on (Fig. 2, C), suggesting that the TBDMS is a superior protecting group to the cyanotheyl group.

Fig. 2.

(A) ODN-2 was deprotected by treatment of NH₄OH overnight at rt. Peak a is a failed sequence dimer, 5′-CT (Maldi ms: [MH]⁺= 531.1); peak b is the fully deprotected 5mer 5′-GAXCT (Maldi ms: [MH]⁺=1493); peak c is the 5mer with TBDMS protecting group on 5hmC (Maldi ms: [MH]⁺=1607). (B) ODN-2 was deprotected by treatment of NH₄OH overnight at 65 °C. (C) ODN-3 was deprotected by treatment of NH₄OH overnight at 65 °C; peak d is the 5mer with cyanoethyl protecting group on 5hmC (Maldi ms: [MH]⁺=1546). (D) ODN-2 was deprotected by treatment of 0.5 M NH₄F in MeOH overnight followed by NH₄OH treatment at rt for 2 h.

The above one-step deprotection procedure is very convenient for the synthesis of DNA containing 5-hmC. For DNA containing additional labeling modifications that requires milder deprotection, a prior treatment with fluoride to remove TBDMS group may allow the use of ultra mild deprotection of the synthetic DNA. Thus, after treating the solid resin bearing newly synthesized ODN-2 with NH₄F (0.5 M in MeOH) overnight at room temperature to remove the TBDMS group and rinsing with MeOH to get rid of excess NH₄F, the ultra mild deprotection condition was applied (NH₄OH treatment at rt for 2 h). HPLC analysis showed that all the protecting groups were cleanly removed and the desired product (peak b) was produced as the major product (Fig. 2, D). The same treatment of ODN-1 also gave fully deprotected oligo. This is surprising because 4-benzoyl protecting group of cytidine is usually not compatible with ultra mild deprotection conditions; probably the 5-CH₂OH facilitate the removal of benzoyl group in the presence of NH₄OH.

In summary, we have achieved the syntheses of two novel 5-hmC phosphoramidites III and IV with TBDMS as the 5-hydroxyl protecting group from 2′-dU in good overall yields (18% for III and 32% for IV). Complete removal of the TBDMS protection group was accomplished conveniently by a “one-step” procedure (overnight treatment with NH₄OH at 65 °C) or a “two-step” procedure (NH₄F treatment followed by the ultra mild NH₄OH treatment). These two deprotection procedures are complementary. While the “one-step” procedure can be used conveniently for the synthesis of DNA with the 5-hmC modification, the “two-step” procedure is more suitable for ultra mild synthesis of DNA containing 5-hmC and additional base labile modifications. The application of the new phosphoramidite building blocks will greatly facilitate the biological study of 5-hmC in DNA. While the current work was going on, another 5-hmC phosphoramidite using a carbamate to protect simultaneously the amino and hydroxyl groups was reported.¹⁶ The synthesis derives from 5-iodo-deoxycytidine and entails seven steps of reaction and gave the phosphoramidite in 7.5% overall yield. While being a significant advance compared to the commercial available phosphoramidite, the synthesis is still not compatible with ultra mild deprotection conditions.

Experimental Section

3′,5′-O-Di-t-butylsilyl-5-iodo-2′-deoxyuridine (7)

To a cloudy solution of 6 (2.05 g, 5.79 mmol) in DMF (15 mL) at 0 °C were added di-tert-butylsilyl-bis(trifluoromethanesulfonate) (2.39 mL, 1.1 eq.). After stirring at rt for 10 min, imidazole (0.98 g, 2.5 eq.) was added and the mixture was stirred at rt for 0.5 h. After removal of DMF under high vacuum, the residue was dissolved in ethyl acetate and washed with water, 5% NaHCO₃ solution and brine and dried over Na₂SO₄. After evaporation of the solvent under reduced pressure, the residue was purified by silica gel chromatography, eluting with 1-3% MeOH in CH₂Cl₂, to give 7 (2.63g, 92%) as white foam. ¹H NMR (500 MHz) (CDCl₃) δ: 9.86 (br., 1H), 7.66 (s, 1H), 6.13 (m, 1H), 4.46 (m, 1H), 4.20 (m, 1H), 4.01 (m, 1H), 3.71 (m, 1H), 2.41 (m, 2H), 1.08 (s, 9H), 0.99 (s, 9H). ¹³C NMR (125.8 MHz) (CDCl₃) δ: 160.2, 150.0, 144.2, 84.8, 78.3, 74.6, 69.0, 67.3, 39.0, 27.5, 27.2, 22.8, 20.2. HRMS calculated for C₁₇H₂₈IN₂O₅Si, [MH⁺] 495.0812 (calcd.), 495.0807 (found).

3′,5′-O-Di-t-butylsilyl-5-formyl-2′-deoxyuridine (8)

To a solution of 7 (2.01 g, 4.06 mmol) in anhydrous THF (50 mL) in a flask with a self-contained glass coupling apparatus equipped with a pressure equalizing addition funnel were added triphenylphosphine (0.64 g, 0.6 eq.) and Pd₂(dba)₃*CHCl₃ (0.42 g, 0.40 mmol, 0.10 eq.). The apparatus was charged with 50 psi of CO and heated to 70 °C and Bu₃SnH (1.19 mL, 1.05 eq.) was added slowly with a syringe within 1 h. After that, the mixture was kept on stirring for 2 h at 70 °C. After cooled to rt, the small amount of solid was removed by filtration. The solvent of filtrate was removed under reduced pressure and the residue was purified by silica gel chromatography, eluting with 2-3% MeOH in CH₂Cl₂, to give 8 (1.37g, 85%) as pale foam. ¹H NMR (500 MHz) (CDCl₃) δ: 10.03 (s, 1H), 8.60 (br., 1H), 8.21 (s, 1H), 6.13 (m, 1H), 4.49 (m, 1H), 4.17 (m, 1H), 4.08 (m, 1H), 3.77 (m, 1H), 2.45 (m, 2H), 1.07 (s, 9H), 1.04 (s, 9H). ¹³C NMR (125.8 MHz) (CDCl₃) δ: 186.7, 162.1, 149.7, 145.3, 112.1, 86.5, 79.6, 75.1, 68.1, 40.1, 28.4, 28.1, 23.6, 21.1. HRMS calculated for C₁₈H₂₉N₂O₆Si, [MH⁺] 397.1795, (calcd.), 397.1789 (found).

3′,5′-O-Di-t-butylsilyl-5-hydroxymethyl-2′-deoxyuridine (9)

To a solution of 8 (1.50 g, 3.79 mmol) in MeOH (40 mL) were added CeCl₃.7H₂O (4.24 g, 3.0 eq.) under argon and the mixture was cooled to 0 °C with an ice-bath. NaBH₄ (144 mg, 1.0 eq.) was added slowly within 15 min. After that, ice-bath was removed and the mixture was stirred at rt for 0.5 h and TLC show that 8 was completely consumed. Silica gel (5.0 g) was added and MeOH was removed under reduced pressure. The residue was purified by silica gel chromatography, eluting with 8-12% MeOH in CH₂Cl₂, to give 9 as pale foam (1.18 g, 78%). ¹H NMR (500 MHz) (CDCl₃) δ: 10.10 (br., 1H), 7.34 (s, 1H), 6.20 (t, J=5.0 Hz, 1H), 4.34-4.45 (m, 3H), 4.21 (m, 1H), 4.01 (m, 1H), 3.68 (m, 1H), 2.36 (m, 2H), 1.06 (s, 9H), 1.00 (s, 9H). ¹³C NMR (125.8 MHz) (CDCl₃) δ: 164.7, 151.1, 138.1, 115.3, 85.0, 79.0, 75.7, 68.2, 59.2, 39.6, 28.4, 28.1, 23.6, 21.1. HRMS calculated for C₁₈H₃₁N₂O₆Si, [MH⁺] 399.1951 (calcd.), 399.1946 (found).

3′,5′-O-Di-t-butylsilyl-5-t-butyldimethylsiloxymethyl-2′-deoxyuridine (5)

To a solution of 9 (1.00 g, 2.51 mmol) in DMF (20 mL) were added TBDMS-Cl (454 mg, 1.2 eq.) and imidazole (427 mg, 2.5 eq.). The mixture was heated to 60 °C for 2 h under argon. DMF was then removed under high vacuum and the residue was added ethyl acetate (100 mL). The solution was washed with water, 5% NaHCO₃ and brine, dried over Na₂SO₄. After filtration, the solvent of the filtrate was removed under reduced pressure and the residue was purified by silica gel chromatography, eluting with 1-2% MeOH in CH₂Cl₂, to give 5 (1.22 g, 95%) as white foam. ¹H NMR (500 MHz) (CDCl₃) δ: 9.69 (br., 1H), 7.33 (s, 1H), 6.30 (m, 1H), 4.39-4.43 (m, 3H), 4.20 (m, 1H), 3.99 (m, 1H), 3.68 (m, 1H), 2.36 (m, 2H), 1.05 (s, 9H), 1.01 (s, 9H), 0.93 (s, 9H), 0.08 (s, 6H). ¹³C NMR (125.8 MHz) (CDCl₃) δ: 163.3, 151.2, 135.6, 116.1, 84.6, 78.9, 75.9, 68.2, 59.1, 39.6, 28.4, 28.1, 26.9, 23.6, 21.1, 19.2, −4.48, −4.49. HRMS calculated for C₂₄H₄₅N₂O₆Si, [MH⁺] 485.3047 (calcd.), 485.3041 (found).

3′,5′-O-Di-t-butylsilyl-5-t-butyldimethylsiloxymethyl-2′-deoxycytidine (10)

To a solution of 5 (1.00 g, 2.51 mmol) in CH₃CN (20 mL) were added Et₃N (8.56 mL, 55.3 mmol, 22 eq.), 1,2,4-triazole (3.47 g, 50.5 mmol, 20.0 eq.) and the mixture was cooled to 0 °C. POCl₃ (0.60 mL, 6.27 mmol, 2.5 eq.) was added and the mixture was stirred at 0 °C for 0.5 h and at rt for 1 h. CH₂Cl₂ (100 mL) was added and the mixture was washed with water, 5% NaHCO₃ and brine, dried over Na₂SO₄, concentrated to dryness. The residue was dissolved in 1,4-dioxane (10 mL), cooled to 0 °C and NH₄OH (1 mL) was then added. The mixture was stirred at 0 °C for 1 h and then concentrated to dryness. The residue was purified by silica gel chromatography, eluting with 2-4% MeOH in CH₂Cl₂, to give 10 (835 mg, 65%) as white foam. ¹H NMR (500 MHz) (CDCl₃) δ: 7.28 (s, 1H), 6.17 (m, 1H), 4.36-4.46 (m, 3H), 4.06 (m, 1H), 3.97 (m, 1H), 3.71 (m, 1H), 2.36 (m, 2H), 1.02 (s, 9H), 0.98 (s, 9H), 0.86 (s, 9H), 0.06 (s, 6H). ¹³C NMR (125.8 MHz) (CDCl₃) δ: 165.9, 156.3, 138.0, 106.5, 85.9, 79.0, 75.5, 68.5, 61.4, 40.3, 28.4, 28.1, 26.8, 23.6, 21.0, 19.2, −4.3. HMRS calculated for C₂₄H₄₆N₃O₅Si₂, [MH⁺] 512.2976 (calcd.), 512.2971 (found).

3′,5′-O-Di-t-butylsilyl-5-t-butyldimethylsiloxymethyl-N⁴-benzoyl-2′-deoxycytidine (11)

To a solution of 10 (800 mg, 1.56 mmol) in pyridine (12 mL) were added benzoyl chloride (0.22 mL, 1.87 mmol, 1.2 eq.) under argon. The mixture was stirred overnight at rt. MeOH (1 mL) was added to quench the reaction. After removal of the solvents under the reduced pressure, the residue was dissolved in CH₂Cl₂ (100 mL) and washed with water, 5% NaHCO₃ and brine. The organic phase was dried over Na₂SO₄ and concentrated to dryness. The residue was purified by silica gel chromatography, eluting with 1-2% MeOH in CH₂Cl₂, to give 11 (769 mg, 80%) as white foam. ¹H NMR (500 MHz) (CDCl₃) δ: 8.24 (m, 2H), 7.58 (s, 1H), 7.54 (m, 1H), 7.45 (m, 2H), 6.29 (m, 1H), 4.70 (m, 2H), 4.48 (m, 1H), 4.22 (m, 1H), 4.04 (m, 1H), 3.85 (m, 1H), 2.41 (m, 2H), 1.08 (s, 9H), 1.06 (s, 9H), 0.96 (s, 9H), 0.18 (s, 6H). ¹³C NMR (125.8 MHz) (CD₃OD) δ: 158.4, 148.7, 137.9, 136.5, 133.6, 131.6, 130.8, 129.9, 129.1, 116.3, 85.3, 79.2, 75.7, 68.2, 59.4, 39.9, 28.4, 28.1, 27.0, 23.6, 21.1, 19.3, −4.36, −4.38. HRMS calculated for C₃₁H₅₀N₃O₆Si₂, [MH⁺] 616.3238 (calcd.), 616.3233 (found).

5-t-Butyldimethylsiloxymethyl-N⁴-benzoyl-2′-deoxycytidine (12)

To a solution of 11 (720 mg, 1.17 mmol) in THF (20 mL) were added HF in pyridine (1.0 eq.) under argon and the mixture was stirred at rt for 1 h. Silica gel (4.0 g) was added and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography, eluting with 3-7% MeOH in CH₂Cl₂, to give 12 (476 mg, 84%) as white foam. ¹H NMR (500 MHz) (Aceton-d₆) δ: 8.26 (m, 2H), 8.15 (s, 1H), 7.56 (m, 1H), 7.47 (m, 2H), 6.34 (m, 1H), 4.73 (s, 2H), 4.52 (m, 1H), 4.03 (m, 1H), 3.80 (m, 1H), 2.39 (m, 1H), 2.30 (m, 1H), 0.97 (s, 9H), 0.18 (s, 6H). ¹³C NMR (125.8 MHz) (Aceton-d₆) δ: 159.7, 148.6, 139.6, 138.2, 133.3, 130.6, 129.0, 114.9, 89.1, 87.0, 72.3, 63.0, 59.4, 41.47, 26.5, 19.1, −5.0. HRMS calculated for C₂₃H₃₄N₃O₆Si, [MH⁺] 476.2217 (calcd.), 476.2211 (found).

5′-O-(4,4′-Dimethoxytrityl)-5-t-butyldimethylsiloxymethyl-N⁴-benzoyl-2′-deoxycytidine (13)

To a solution of 12 (412 mg, 0.87 mmol) in pyridine (12 mL) was added DMTr-Cl (352 mg, 1.04 mmol, 1.2 eq.) under argon. The mixture was stirred overnight at rt. MeOH (1 mL) was added to quench the reaction. After concentrated to dryness, the residue was dissolved in CH₂Cl₂ (100 mL) and the mixture was washed with water, 5% NaHCO₃ and brine, dried over Na₂SO₄ and concentrated to dryness. The residue was purified by silica gel chromatography, eluting with 1-3% MeOH in CH₂Cl₂, to give 13 (553 mg, 82%) as white foam. ¹H NMR (500 MHz) (CD₃CN) δ: 8.24 (d, J=8.0 Hz, 2H), 7.71 (s, 1H), 7.24-7.58 (m, 12H), 6.87 (m, 4H), 6.22 (m, 1H), 4.57 (m, 1H), 4.40 (m, 1H), 4.28 (m, 1H), 3.98 (m, 1H), 3.76 (s, 6H), 3.34 (m, 1H), 3.29 (m, 1H), 3.22 (m, 1H), 2.39 (m, 1H), 2.18 (m, 1H), 0.83 (s, 9H), 0.05 (s, 3H), 0.04 (s, 3H). ¹³C NMR (125.8 MHz) (CD₃CN) δ: 159.7, 146.0, 139.3, 137.8, 136.9, 136.8, 133.5, 131.0, 130.9, 130.4, 129.2, 129.0, 128.9, 127.9, 114.1, 87.3, 86.8, 86.6, 71.9, 64.8, 59.5, 55.9, 40.8, 26.3, 18.9, −5.1. HRMS calculated for C₄₄H₅₂N₃O₈Si, [MH⁺] 778.3524 (calcd.), 778.3525 (found).

5′-O-(4,4′-Dimethoxytrityl)-5-t-butyldimethylsiloxymethyl-N⁴-benzoyl-2′-deoxycytidine 3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (III)

To a stirring solution of 13 (120 mg, 0.15 mmol) in CH₂Cl₂ (8 mL) were added 1-methylimidazole (3.40 mg, 41.5 μmol), N,N-diisopropylethylamine (0.2 mL) under argon followed by 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (71.7 mg, 0.30 mmol). After the reaction mixture was stirred at room temperature for 0.5 h, CH₂Cl₂ (80 mL) was added. The mixture was washed with 5% aqueous NaHCO₃ and brine, dried over Na₂SO₄ and concentrated to dryness. The residue was purified by silica gel chromatography, eluting with 6-9% acetone in CH₂Cl₂ containing 0.2% Et₃N, to give III (131 mg, 87%) as white foam. ¹H NMR (500 MHz) (CD₃CN) δ: 8.30 (m, 2H), 7.31-7.72 (m, 13H), 6.87 (m, 4H), 6.18-6.28 (m, 1H), 4.62 (m, 1H), 4.40-4.50 (m, 2H), 4.15 (m, 1H), 3.76 (m, 6H), 3.65 (m, 4H), 3.32 (m, 2H), 2.65 (m, 2H), 2.54 (m, 2H), 2.28 (m, 1H), 2.16 (m, 1H), 1.17 (m, 9H), 1.05 (m, 3H), 0.84 (s, 9H), 0.04 (s, 6H). ³¹P NMR (202.5 MHz) (CD₃CN) 148.44 ppm. HRMS calcd. for C₅₃H₆₉N₅O₉PSi, [MH]⁺ 978.4602 (calcd.), 978.4611 (found).

5-t-Butyldimethylsiloxymethyl-2′-deoxyuridine (14)

To a solution of 5 (440 mg g, 0.86 mmol) in THF (20 mL) were added HF in pyridine (1.0 eq.) under argon and the mixture was stirred at rt for 1 h. Silica gel (4.0 g) was added and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography, eluting with 3-7% MeOH in CH₂Cl₂, to give 14 (281 mg, 88%) as pale foam. ¹H NMR (500 MHz) (Aceton-d₆) δ: 10.07 (br., 1H), 7.83 (s, 1H), 6.34 (m, 1H), 4.50 (s, 1H), 4.42 (s, 3H), 4.12 (m, 1H), 3.95 (m, 1H), 3.75 (m, 2H), 2.26 (m, 2H), 0.92 (s, 9H), 0.12 (s, 6H). ¹³C NMR (125.8 MHz) (Aceton-d₆) δ: 163.0, 151.3, 137.7, 114.7, 88.7, 85.9, 72.5, 63.2, 59.2, 41.0, 26.4, 19.0, −5.1. HRMS calculated for C₁₆H₂₉N₂O₆Si, [MH⁺] 373.1795 (calcd.), 373.1789 (found).

5′-O-(4,4′-Dimethoxytrityl)-5-t-butyldimethylsiloxymethyl-2′-deoxyuridine (15)

To a solution of 14 (260 mg, 0.70 mmol) in pyridine (8 mL) was added DMTr-Cl (284 mg, 0.84 mmol, 1.2 eq.) under argon. The mixture was stirred overnight at rt. MeOH (1 mL) was added to quench the reaction. The solvents were removed under reduced pressure and the residue was dissolved with CH₂Cl₂ (100 mL). The mixture was washed with water, 5% NaHCO₃ and brine, dried over Na₂SO₄ and concentrated to dryness. The residue was purified by silica gel chromatography, eluting with 1-3% MeOH in CH₂Cl₂, to give 15 (400 mg, 85%) as pale foam. ¹H NMR (500 MHz) (CD₃CN) δ: 9.34 (br., 1H), 7.46 (m, 3H), 7.24-7.35 (m, 7H), 6.87 (m, 4H), 6.23 (m, 1H), 4.29 (m, 2H), 4.12 (m, 1H), 3.95 (m, 1H), 3.77 (s, 6H), 3.29 (m, 1H), 3.20 (m, 1H), 2.30 (m, 1H), 2.19 (m, 1H), 0.84 (s, 9H), 0.02 (s, 3H), 0.01 (s, 3H). ¹³C NMR (125.8 MHz) (CD₃CN) δ: 163.3, 159.7, 151.3, 146.0, 137.6, 136.8, 131.0, 128.98, 128.90, 127.89, 118.3, 115.0, 114.1, 87.3, 86.0, 85.4, 72.1, 65.0, 59.0, 55.9, 40.5, 26.3, 18.9, 7.5. HRMS calculated for C₃₇H₄₇N₂O₈Si, [MH⁺] 675.3102 (calcd.), 675.3096 (found).

5′-O-(4,4′-Dimethoxytrityl)-5-t-butyldimethylsiloxymethyl-2′-deoxyuridine 3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (16)

To a stirring solution of 15 (120 mg, 0.18 mmol) in CH₂Cl₂ (8 mL) were added 1-methylimidazole (3.40 mg, 41.5 μmol), N,N-diisopropylethylamine (0.2 mL) under argon followed by 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (60 μL, 0.27 mmol, 1.5 eq.). After the reaction mixture was stirred at room temperature for 0.5 h, CH₂Cl₂ (80 mL) was added. The mixture was washed with 5% aqueous sodium carbonate and brine, dried over sodium sulfate and concentrated to dryness. The residue was purified by silica gel chromatography, eluting with 6-9% acetone in CH₂Cl₂ containing 0.2% Et₃N, to give 16 (143 mg, 92%) as white foam. ¹H NMR (500 MHz) (CD₃CN) δ: 9.04 (br., 1H), 7.03-7.46 (m, 10H), 6.89 (m, 4H), 6.23 (m, 1H), 4.50 (m, 1H), 4.28 (m, 1H), 4.08 (m, 2H), 3.78 (s, 3H), 3.77 (s, 3H), 3.62 (m, 4H), 3.28 (m, 2H), 2.63 (m, 1H), 2.53 (m, 1H), 2.44 (m, 1H), 2.26 (m, 1H), 1.17 (m, 9H), 1.05 (d, 3H), 0.82 (s, 9H), 0.02 (s, 3H), 0.00 (s, 3H). ³¹P NMR (202.5 MHz) (CD₃CN) 148.34, 148.27 ppm. HRMS calculated for C₄₆H₆₆N₄O₉PSi, [MH⁺] 877.4337 (calcd.), 877.4331 (found).

5′-O-(4,4′-Dimethoxytrityl)-5-t-butyldimethylsiloxymethyl-4-triazolothymidine 3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (IV)

To a solution of 16 (110 mg, 0.13 mmol) in CH₃CN (5 mL) were added Et₃N (0.43 mL), 1,2,4-triazole (174 mg) and the mixture was cooled to 0 °C. POCl₃ (30 μL) was added and the mixture was stirred at 0 °C for 0.5 h and at rt for 1 h. Added another portion of POCl₃ (16 μL) and the mixture was stirred at rt for 5 h. CH₂Cl₂ (100 mL) was added and the mixture was washed with water, 5% NaHCO₃ and brine, dried over Na₂SO₄, concentrated to dryness. The residue was purified by silica gel chromatography, eluting with 6-10% acetone in CH₂Cl₂ containing 0.2% Et₃N, to give IV (94.5 mg, 81%) as white foam. ¹H NMR (500 MHz) (CD₃CN) δ: 9.18 (s, 1H), 8.50 (s, 0.5H), 8.38 (s, 0.5H), 8.15 (s, 1H), 7.24-7.42 (m, 9H), 6.87 (m, 4H), 6.18 (m, 1H), 4.92 (m, 1H), 4.55 (m, 2H), 4.22 (m, 1H), 3.75 (s, 6H), 3.73 (m, 4H), 3.40 (m, 2H), 2.66 (m, 1H), 2.56 (m, 1H), 2.25 (m, 2H), 1.17 (m, 9H), 1.07 (d, 3H), 0.79 (s, 9H), −0.04, −0.03 (s, 3H), 0.00 (s, 3H). ¹³C NMR (125.8 MHz) (CD₃CN) δ: 159.7, 154.5, 154.5, 147.1, 147.0, 145.6, 136.6, 131.0, 131.0, 129.0, 128.9, 127.98, 127.95, 114.2, 89.9, 88.8, 89.0, 88.1, 88.0, 74.6, 74.4, 64.2, 60.9, 60.9, 59.5, 59.3, 55.9, 55.9, 44.1, 44.02, 44.00, 40.1, 40.1, 26.3, 26.2, , 25.0, 24.9, 24.8, 21.0, 18.6, −5.15, −5.16. ³¹P NMR (202.5 MHz) (CD₃CN) 148.31, 148.24 ppm. HMRS calculated for C₄₈H₆₅N₇O₈PSi, [MH⁺] 926.4402 (calcd.), 926.4396 (found).