SYNTHESIS OF 2′-O-PHOTOCAGED RIBONUCLEOSIDE PHOSPHORAMIDITES

Abstract

The chemical synthesis and incorporation of the phosphoramidite derivatives of 2′-O-photocaged ribonucleosides (A, C, G and U) with o-nitrobenzyl, α-methyl-o-nitrobenzyl or 4,5-dimethoxy-2-nitrobenzyl group into oligoribonucleotides are described. The efficiency of UV irradiated uncaging of these 2′-O-photocaged oligoribonucleotides was found in the order of α-methyl-o-nitrobenzyl > 4,5-dimethoxy-2-nitrobenzyl > 2′-O-o-nitrobenzyl.

INTRODUCTION

Photolabile protecting groups have been extensively investigated over the years to protect hydroxyl or amino groups in organic synthesis.^[1,2] Photocaging provides a well-established strategy to cage functions of biologically important molecules until released by UV irradiation.^[3–5] 2′-O-Photocaged oligonucleotides including the 3′-S-oligonucleotide and 5′-S-oligonucleotide phosphorothiolates as useful RNA analogues have been successfully utilized to investigate the mechanism of RNA catalyzed reactions^[6–10] and pre-mRNA splicing.^[11,12] Although a number of photolabile groups have been introduced into photocaged molecules for biological studies, to the best of our knowledge, only o-nitrobenzyl group has been introduced to 2′-O-caged RNAs.^[6,10,13,14] For example, the 2′-O-o-nitrobenzyl derivatives of adenosine,^[15] cytidine,^[15] guanosine,^[16] and uridine^[17] were synthesized by Ohtsuka et al. and applied to the solution synthesis of 2′-O-caged dinucleotides and trinucleotides.^[15,17,18] The first solid-phase synthesis of 2′-O-photocaged oligoribonucleotides was achieved by using 2′-O-o-nitrobenzyl-3′-H-phosphonate chemistry.^[13] Later, the phosphoramidite derivatives of 2′-O-o-nitrobenzyl-N⁶-benzoyladenosine^[6] and 2′-O-o-nitrobenzyl-N⁴-benzoylcytidine^[10] were prepared and applied to synthesize the 2′-O-caged RNAs via phosphoramidite chemistry. These 2′-O-o-nitrobenzyl RNAs have proven useful as tools to probe RNA reactivity and dynamics via a photochemically-controlled manner.^[6–12] The 2′-O-photolabile group and ultramild N-phenoxyacetyl group protected phosphoramidites are useful for the synthesis of oligoribonucleotides containing a 5′-S-phosphorothiolate linkage since these modified oligoribonucleotides are more susceptible to alkaline cleavage compared to the wild type RNAs.^[7,19] Only three 2′-O-photolabile phosphoramidite derivatives (with 2′-O-o-nitrobenzyl and N-benzoyl or N-isobutyryl protection) have been synthesized and reported in the literature.^[10,14,20] 2′-O-o-Nitrobenzyl caged RNAs synthesized by either solid phase synthesis or a two-step ligation method have been successfully applied to investigate the mechanisms of ribozyme catalyzed reactions.^[7–9,19] However, the relatively long UV irradiation time (4 to 6 minutes)^[9,19] for the full removal of o-nitrobenzyl group from 2′-O-photocaged RNAs may limit its applications in the case of unstable RNAs (or ribozymes) with UV exposure for a long time. To overcome this limitation, here we report the general synthetic methods to obtain 2′-O-photocaged phosphoramidites containing a number of more efficient 2′-O-photolabile and N-phenoxyacetyl protected ribonucleoside phosphoramidites.

Results and Discussion

The 2′-O-photolabile 5′-O-DMTr-N²-butyrylguanosine derivatives (2a and 2b) were prepared in 21% and 18% yields, respectively, according to our previously improved procedures by the reactions of N²-butyrylguanosine (1a) with o-nitrobenzyl bromide or α-methyl-o-nitrobenzyl bromide, followed by the 5′-O-DMTr protection.^[16,19] The corresponding 2′-O-photolabile 5′-O-DMTr-N²-phenoxyacetylguanosine derivatives (2c and 2d) were prepared from N²-phenoxyacetylguanosine (1b) in 20% and 5% yields, respectively (Scheme 1). It is worthy to note the free guanosine could not be selectively 2′-O-benzylated by the reaction with o-nitrobenzyl bromide due to the side reaction at N1 of the guanine ring.

Scheme 1.

The 2′-O-photolabile 5′-O-DMTr-N-phenoxyacetyl adenosine and cytidine derivatives (6a-6d) could be prepared from their respective free ribonucleoside precursors (i.e. adenosine and cytidine) in three steps (Scheme 2). Without any protection both adenosine and cytidine could be selectively 2′-O-benzylated with 4,5-dimethoxy-2-nitrobenzyl bromide or α-methyl-o-nitrobenzyl bromide to give 4a-4d. The amino group of nucleobases of 4a-4d was then selectively protected by a phenoxyacetyl group to give compounds 5a-5d. 5′-O-DMTr protection of 5a-5d gave the corresponding phophoramidite precursors 6a-6d in three steps from free nucleosides 3a and 3b with 11–44% overall yields.

Scheme 2.

Ohtsuka and Ikehara reported the synthesis of 2′-O-o-nitrobenzyluridine (5e) in 24% yield by the reaction of 2′,3′-O-(dibutylstannyl)uridine^[21] with o-nitrobenzyl bromide in DMF at 110 ºC for 4 hours (Scheme 3).^[17] DMTr protection of 5e then yielded the corresponding 2′-O-photocaged uridine phosphoramidite precursor 6e in 75% yield. Unfortunately, we found this method cannot be extended for the preparation of 2′-O-α-methyl-o-nitrobenzyluridine by reacting of 2′,3′-O-(dibutylstannyl)uridine with α-methyl-o-nitrobenzyl bromide. No desired product was formed after this reaction, even at 110 ºC for 24 hours. According to the procedure for the synthesis of 2a, direct deprotonation of uridine with NaH in DMF, followed by reaction with o-nitrobenzyl bromide generated only O⁴-o-nitrobenzyluridine.

Scheme 3.

Phosphitylation of 2a-3d and 6a-6e with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite yielded the corresponding 2′-O-photocaged nucleoside phosphoramidites 7a-7i including the 2′-O-photolabile N-phenoxyacetylribonucleoside phosphoramidites 7c-7h in good yields (Scheme 4 & Table 1).

Scheme 4.

Table 1.

Phosphoramidites 7a-7j prepared by the phosphitylation of 2a-2d and 6a-6f.

Product	Precursor	X	Y	Z	B	Yield (%)
7a	2a	H	H	H	Gi-Bu	95
7b	2b	Me	H	H	Gi-Bu	86
7c	2c	H	H	H	GPhOAc	77
7d	2d	Me	H	H	GPhOAc	63
7e	6a	H	OMe	OMe	APhOAc	88
7f	6b	Me	H	H	APhOAc	92
7g	6c	H	H	H	CPhOAc	92
7h	6d	Me	H	H	CPhOAc	91
7i	6e	H	H	H	U	86
7j	6f	H	H	H	ABz	82

To compare the efficiency of the UV irradiated decaging of RNAs containing various photolabile groups, the known phosphoramidite derivative of 2′-O-o-nitrobenzyl-N⁶-benzoyladenosine 7j was also prepared (Scheme 4).^[6,14] The adenosine phosphoramidites 7e (with 3,4-dimethoxy-2-nitrobenzyl group), 7f (with α-methyl-o-nitrobenzyl group), and 7j (with o-nitrobenzyl group) were then incorporated into a 30-mer RNA sequence (an oligomer fragment for the construction of glms ribozyme):^[22] 5′-GGU AAA UUA UAG AXG CGC CAG AAC UAC ACC-3′, where X represented the coupling of 2′-O-photocaged phosphoramidites (7e, 7f or 7h) at the ribozyme cleavage site. The solid-phase synthesis was carried out on Expedite 8900 Nucleic Acid Synthesizer with a modified RNA 1 μmole protocol. The standard RNA 1 μmole protocol for X was modified to double coupling before capping and oxidation. The trityl yields for the coupling of 7e, 7f or 7h were comparable to the commercially available phosphoramidites. After deprotection with ammonium hydroxide, and desilylation with 1-methyl-2-pyrrolidinone (NMP)/Et₃N/Et₃N-3HF,^[23] these RNAs were 5′-radiolabeled and purified by dPAGE gel as usual.^[19] The progress of UV irradiated decaging of these RNAs was analyzed after alkaline hydrolysis and quantified by phosphorimager according to our reported method.^[9,19] As expected, no alkaline hydrolysis was observed at the caged nucleotide site until UV irradiation (Figure 1). These data were then fitted into a first-order kinetics equation (y = y₀ – A*e^-kt); and the resulting kinetics rates are shown in Table 2. From Table 2, we conclude that 2′-O-caged RNA containing an α-methyl-o-nitrobenzyl group has the most prominent photolabile properties. An RNA oligomer with α-methyl-o-nitrobenzyl group is uncaged by UV at 365 nm about 10 times faster compared to an RNA oligomer with o-nitrobenzyl group, whereas an RNA strand with 3,4-dimethoxy-2-nitrobenzyl group is uncaged by UV about 5 times faster than an RNA oligomer with o-nitrobenzyl group at 365 nm. The electronic donating groups (α-methyl and 3,4-dimethoxy) may have stabilized the resonance isomer (intermediate) formation thus facilitating the UV irradiated deprotection.^[24]

Figure 1. UV initiated deprotection and alkaline hydrolysis of 2′-O-caged RNAs containing 2′-O-photolabile groups.

A: Gel image corresponds to the RNA containing α-methyl-o-nitrobenzyl protecting group. B: Gel image corresponds to the RNA containing o-nitrobenzyl protecting group. Method: A 5′-radiolabeled 30 mer RNA fragment in water (20 μL) was irradiated at 365 nm and 0.5-μL aliquots were taken and diluted in water (3 μL). Alkaline hydrolysis ladders were performed by adding sodium bicarbonate, pH 9, and incubating at 90 °C for 15 min. The resulting ladder of RNA fragments was separated by denaturing PAGE, and deprotection was quantitated by measuring the intensity of the band corresponding to cleavage by the deprotected nucleophile. The first lane in the depicted gel corresponds to deprotected substrate prior to alkaline hydrolysis.

Table 2.

Efficiency of the UV (365 nm) irradiated uncaging of 2′-O-caged RNAs containing various 2′-O-photolabile groups.

2′-O-Caged RNAs	k (min−1)a	t1/2 (min)b	Time (min)c
2′-O-α-methyl-o-nitrobenzyl-RNA	3.0	0.23	0.83
2′-O-2-nitro-3,4-(MeO)2C6H3CH2-RNA	1.6	0.43	1.5–3
2′-o-nitrobenzyl-RNA	0.27	2.6	4–6

^ak is obtained from alkaline hydrolysis data of UV initiated uncaging of RNAs by fitting to the equation: y = y₀ – A*e^−kt.

^bt_1/2 is the time when 50% RNAs are uncaged.

^cTime estimated for full uncaging.

In summary, we have described the synthesis of the phosphoramidite derivatives of adenosine, cytidine and guanosine with more 2′-O-photolabile and N-phenoxyacetyl protections. We also prepared the phosphoramidite derivative of 2′-O-o-nitrobenzyluridine. Solid phase synthesis using these phosphoramidites together with commercially available 2′-O-TBS-N-phenoxyacetylribonucleoside phosphoramidites would provide a general method to synthesize 2′-O-caged RNA oligonucleotides featured with ultramild deprotection conditions (NH₄OH/rt, 2 hours). Application of this new class of ribonucleoside phosphoramidites in the synthesis of RNA oligomers containing a 2′-O-photocaged-5′-S-phosphorothiolate linkage are currently being further evaluated and the syntheses are expected to be reported elsewhere later. Overall, 2′-O-photocaged RNAs containing an α-methyl-o-nitrobenzyl group seems to be the most photolabile amongst the three photocaged groups studied.

Experimental Section

5′-O-(Dimethoxytrityl)-N²-isobutyryl-2′-O-(o-nitrobenzyl)guanosine (2a).^[19]

Compound 2a was prepared from N²-isobutyrylguanosine (1a) in two steps (21% yield). Under argon N²-Isobutyrylguanosine (1a) (1.717 g, 4.86 mmol) was treated with NaH (292 mg, 12.2 mmol) in dry DMF (40 mL) at 0 ºC. After hydrogen gas generation ceased (about 40 min), o-nitrobenzyl bromide (1.575 g, 7.29 mmol) was added and the mixture was stirred for 5 h at 0 ºC. The reaction was quenched with EtOH and neutralized with 1 N HCl. The mixture was evaporated and the crude product was purified by silica gel chromatography, eluting with 4–6% methanol in dichloromethane, to give N²-isobutyryl-2′-O-(o-nitrobenzyl)guanosine^[16,19] as a yellow foam: 0.76 g (32% yield). To a solution of N²-isobutyryl-2′-O-(o-nitrobenzyl)guanosine (192 mg, 0.393 mmol) in dry pyridine (4.0 mL) under dry argon, DMTrCl (400 mg, 1.18 mmol) was added. After the mixture was stirred at rt overnight, methanol (2.0 mL) was added to quench the reaction. The solvent was removed by rotary evaporation, and the crude product was purified by silica gel chromatography, eluting with 2% MeOH in CH₂Cl₂ containing 0.5% Et₃N to give compound 2a^[19] as a yellow foam (206 mg, 66% yield). ¹H NMR (CDCl₃) δ 12.02 (br s, 1H), 8.61 (br s, 1H), 8.01 (d, J = 8 Hz, 1H), 7.87 (s, 1H), 7.67–7.18 (m, 12H), 6.8 (m, 4H), 6.01 (d, J = 3.5 Hz, 1H), 5.37 (d, J = 15 Hz, 1H), 5.22 (d, J = 15 Hz, 1H), 4.62 (t, J = 4.5 Hz, 1H), 4.55 (m, 1H), 4.27 (m, 1H), 3.77 (s, 3H), 3.767 (s, 3H), 3.55 (d, J = 10.5 Hz, 3H), 3.35 (m, 1H), 3.21 (br s, 1H), 2.29 (m, 1H), 1.12 (d, J = 7 Hz, 1H), 1.01 (d, J = 7 Hz, 1H); ¹³C NMR (DMSO-d₆) δ 231.0, 178.8, 158.6, 155.5, 147.5, 147.4, 146.9, 144.6, 137.5, 135.8, 135.6, 134.1, 133.8, 130.0, 129.9, 128.6, 128.5, 128.0, 127.9, 127.0, 124.7, 121.9, 113.2, 113.1, 87.0, 86.4, 83.6, 82.3, 69.4, 69.3, 63.1, 55.2, 36.2, 18.7, 18.6; HRMS calcd for C₄₂H₄₃N₆O₁₀ [MH⁺] 791.3035, found 791.3041.

5′-O-(Dimethoxytrityl)-N²-isobutyryl-2′-O-(α-methyl-o-nitrobenzyl)guanosine (2b).^[19]

According to the procedure described for the synthesis of 2a, N²-isobutyryl-2′-O-(α-methyl-o-nitrobenzyl)guanosine^[19] (636 mg, 26% yield) as a yellow foam was prepared from 1a (1.717 g, 4.86 mmol), NaH (292 mg, 12.2 mmol) and α-methyl-o-nitrobenzyl bromide (1.677 g, 7.29 mmol). Compound 2b was then prepared by the reaction of N²-isobutyryl-2′-O-(α-methyl-o-nitrobenzyl)guanosine (307 mg, 0.61 mmol) with DMTrCl (621 mg, 1.83 mmol) in dry pyridine at rt overnight. Silica gel chromatography (1–2% methanol in dichloromethane containing 0.5% triethylamine) of the crude product yielded 2b^[19] (345 mg, 70% yield) as a yellow foam. ¹H NMR (CDCl₃) δ 12.04 (s, 1H), 8.57 (s, 1H), 7.92–7.70 (m, 3H), 7.47–7.19 (m, 10H), 6.82–6.79 (m, 4H), 6.01 (d, J = 1.2 Hz, 1H), 5.77 (m, 1H), 4.33–4.26 (m, 2H), 3.91 (m, 1H), 3.770 (s, 3H), 3.767 (s, 3H), 3.57–3.45 (m, 2H), 2.72 (d, J = 8.3 Hz, 1H), 2.60 (m, 1H), 1.66 (d, J = 6.3 Hz, 3H), 1.27 (d, J = 6.9 Hz, 3H), 1.21 (d, J = 6.8 Hz, 3H); ¹³C NMR (CDCl₃) δ 178.8, 158.5, 155.3, 147.8, 147.6, 147.0, 144.4, 139.0, 136.6, 135.6, 135.5, 134.7, 130.0, 128.8, 128.0, 127.9, 127.0, 124.6, 121.9, 113.2, 87.6, 86.6, 82.7, 80.4, 72.4, 69.1, 62.5, 55.2, 36.5, 24.1, 19,2, 18.4; HRMS calcd for C₄₃H₄₅N₆O₁₀ [MH⁺] 805.3192, found 805.3209.

5′-O-(Dimethoxytrityl)-2′-O-(o-nitrobenzyl)-N²-phenoxyacetylguanosine (2c).

According to the procedure described for the synthesis of 2a, 2′-O-(o-nitrobenzyl)-N²-phenoxyacetylguanosine (0.81 g, 30% yield) as a yellow foam was prepared from N²-phenoxyacetylguanosine (1b)^[25] (2.027 g, 4.86 mmol), NaH (292 mg, 12.2 mmol) and o-nitrobenzyl bromide (1.575 g, 7.29 mmol). Compound 2c was then prepared by the reaction of 2′-O-(o-nitrobenzyl)-N²-phenoxyacetylguanosine (308 mg, 0.56 mmol) with DMTrCl (565 mg, 1.68 mmol) in dry pyridine at rt overnight. Silica gel chromatography (1–2% methanol in dichloromethane containing 0.5% triethylamine) of the crude product yielded 2c (317 mg, 67% yield) as a yellow foam. ¹H NMR (DMSO-d₆) δ 8.11 (s, 1H), 8.01 (d, J = 8.1 Hz, 1H), 7.73–7.69 (m, 2H), 7.55 (t, J = 6.6 Hz, 1H), 7.35–7.20 (m, 11H), 7.00–6.96 (m, 3H), 6.85–6.81 (m, 4H), 6.06 (d, J = 5.2 Hz, 1H), 5.47 (d, J = 2.6 Hz, 1H, disappeared with D₂O), 5.06 (d, J = 14.7 Hz, 1H), 4.97 (d, J = 14.6 Hz, 1H), 4.85 (d, J = 2.5 Hz, 2H), 4.59 (t, J = 5.1 Hz, 1H), 4.42 (m, 1H), 4.12 (m, 1H), 3.72 (s, 6H), 3.28–3.18 (m, 2H); ¹³C NMR (CDCl₃) δ 169.7, 158.6, 156.6, 147.7, 147.4, 144.3, 136.8, 135.5, 135.4, 133.8, 133.1, 130.0, 129.8, 128.9, 128.7, 128.1, 127.9, 127.0, 124.7, 122.6, 122.0, 114.8, 113.2, 86.7, 86.3, 83.6, 82.7, 69.8, 69.3, 67.0, 63.0, 55.2; HRMS calcd for C₄₆H₄₃N₆O₁₁ [MH⁺] 855.2984, found 855.2996.

5′-O-(Dimethoxytrityl)-2′-O-(α-methyl-o-nitrobenzyl)-N²-phenoxyacetylguanosine (2d).

According to the procedure described for the synthesis of 2a, a crude 2′-O-(α-methyl-o-nitrobenzyl)-N²-phenoxyacetylguanosine (530 mg, 19% yield) with some impurity as a yellow foam was prepared from 1b (2.09 g, 5.01 mmol), NaH (301 mg, 12.54 mmol) and α-methyl-o-nitrobenzyl bromide (1.729 g, 7.52 mmol). Compound 2d was then prepared by the reaction of 2′-O-(α-methyl-o-nitrobenzyl)-N²-phenoxyacetylguanosine (107 mg, 0.19 mmol) with DMTrCl (192 mg, 0.57 mmol) in dry pyridine at rt overnight. Silica gel chromatography (1% methanol in dichloromethane containing 0.5% triethylamine) of the crude product yielded 2d (47 mg, 28% yield) as a yellow foam. ¹H NMR (DMSO-d₆) δ 7.76 (d, J = 7.8 Hz, 1H), 7.70 (s, 1H), 7.45–7.17 (m, 15H), 7.07–6.74 (m, 7H), 5.80 (d, J = 4.7 Hz, 1H), 5.43 (m, 1H), 4.63 (s, 2H), 4.53 (t, J = 4.8 Hz, 1H), 4.40 (t, J = 4.8 Hz, 1H), 4.21 (m, 1H), 3.75 (s, 6H), 3.42 (dd, J = 2.4, 10.7 Hz, 1H), 3.35 (dd, J = 2.4, 10.7 Hz, 1H), 1.59 (d, J = 6.3 Hz, 3H); ¹³C NMR (CDCl₃) δ 169.8, 158.5, 156.7, 147.4, 144.3, 138.7, 137.4, 135.5, 133.8, 129.9, 129.9, 129.7, 128.6, 128.0, 127.8, 127.4, 126.9, 123.8, 122.5, 114.7, 113.1, 86.5, 85.9, 84.1, 81.1, 73.9, 69.4, 67.0, 63.3, 55.1, 23.7; HRMS calcd for C₄₇H₄₅N₆O₁₁ [MH⁺] 869.3141, found 869.3158.

2′-O-(4,5-Dimethoxy-2-nitrobenzyl)adenosine (4a).

According to the procedure described for the synthesis of 2a, compound 4a was prepared from adenosine (1.51 g, 5.43 mmol), NaH (196 mg, 8.17 mmol) and 4,5-dimethoxy-2-nitrobenzyl bromide (2.248 g, 8.14 mmol). Silica gel chromatography (0–8% methanol in dichloromethane) of the crude product yielded 4a (1.41 g, 54% yield) as a yellow foam. ¹H NMR (DMSO-d₆) δ 8.35 (s, 1H), 8.06 (s, 1H), 7.56 (s, 1H), 7.35 (brs, 2H), 7.20 (s, 1H), 6.13 (d, J = 5.0 Hz, 1H), 5.50 (d, J = 5.0 Hz, 1H), 5.42 (m, 1H), 5.07 (d, J = 15.0 Hz, 1H), 4.87 (d, J = 15.0 Hz, 1H), 4.56 (t, J = 5.0 Hz, 1H), 4.40 (m, 1H), 4.05 (m, 1H), 3.82 (s, 3H), 3.71 (s, 3H), 3.70 (m, 1H), 3.59 (m, 1H); ¹³C NMR (DMSO-d₆) δ 156.1, 153.2, 152.4, 148.9, 147.3, 139.5, 139.0, 129.4, 119.3, 110.1, 107.8, 86.3, 86.1, 81.3, 68.9, 67.9, 61.3, 56.0, 55.8; HRMS calcd for C₁₉H₂₃N₆O₈ [MH⁺] 463.1572, found 463.1560.

2′-O-(4,5-Dimethoxy-2-nitrobenzyl)-N⁶-phenoxyacetyladenosine (5a).

Compound 4a (0.68 g, 1.47 mmol) was dried by co-evaporation with dry pyridine (3 × 50 mL) under vacuum, and resuspended in dry pyridine (30 mL) under Ar. Chlorotrimethylsilane (1.5 mL) was added to the suspension at 0 ºC. After the reaction mixture was stirred at rt for 45 min, a solution of phenoxyacetyl chloride (310 μL, 2.21 mmol) and 1,2,4-triazole (155 mg, 2.26 mmol) in dry pyridine/acetonitrile (20 mL, 1:1) was slowly added. After the mixture was stirred at 55 ºC overnight, it was cooled to rt, and the reaction was quenched by addition of H₂O (1.5 mL). After stirring for 5 min, concentrated aqueous NH₄OH (1 mL) was added at 0 ºC, and the mixture was stirred for 30 min. The solvent was removed, the crude product was purified by silica gel chromatography, eluting with 3–5 % MeOH in CH₂Cl₂ to give 5a (797 mg, 91% yield) as a yellow foam. ¹H NMR (CDCl₃) δ 9.67 (br s, 1H), 8.71 (s, 1H), 8.17 (s, 1H), 7.44 (s, 1H), 7.37–7.33 (m, 2H), 7.07–7.04 (m, 3H), 6.76 (s, 1H), 6.07 (d, J = 7.3 Hz, 1H), 5.03 (d, J = 12.8 Hz, 1H), 4.96–4.93 (m, 3H), 4.71–4.65 (m, 2H), 4.40 (s, 1H), 3.99 (dd, J = 1.5, 13.0 Hz, 1H), 3.87 (s, 3H), 3.82 (s, 3H), 3.81 (dd, J = 1.5, 13.0 Hz, 1H); ¹³C NMR (CDCl₃) δ 167.3, 157.1, 153.1, 152.0, 150.4, 149.1, 148.5, 143.7, 140.2, 129.9, 129.7, 126.7, 123.8, 115.0, 114.6, 111.4, 108.1, 89.1, 88.1, 81.2, 70.7, 70.1, 68.3, 65.1, 63.1, 56.5, 56.4; HRMS calcd for C₂₇H₂₉N₆O₁₀ [MH⁺] 597.1940, found 597.1937.

2′-O-(o-Nitrobenzyl)-N⁴-phenoxyacetylcytidine (5c).^[19]

2′-O-(o-Nitrobenzyl)cytidine (4c)^[15] (3.58 g, 9.46 mmol) was dried by co-evaporation with dry pyridine (3 × 100 mL) under vacuum and resuspended in dry pyridine (180 mL) under argon. Chlorotrimethylsilane (9.0 mL) was added to the suspension at 0 ºC. After the mixture was stirred at rt for 45 min, a solution of phenoxyacetyl chloride (2.00 mL, 14.4 mmol) and 1,2,4-triazole (0.980 g, 14.3 mmol) in pyridine-acetonitrile (120 mL, 1:1) was slowly added. After the mixture was stirred at 55 ºC overnight, it was cooled to rt, and the reaction was quenched by addition of H₂O (10 mL). After stirring for 5 min, concentrated aqueous NH₄OH (6.5 mL) was added at 0 ºC, and the mixture was stirred for 30 min. The solvent was removed, and the crude product was purified by silica gel chromatography, eluting with 3–5 % MeOH in CH₂Cl₂ to give 5c^[19] (3.44 g, 71% yield) as a yellow foam. ¹H NMR (DMSO-d₆) δ 11.04 (s, 1H), 8.51 (d, 1H, J = 7.5 Hz), 8.08 (dd, 1H, J = 1.3, 8.2 Hz), 7.94 (d, 1H, J = 7.7 Hz), 7.77 (m, 1H), 7.57 (m, 1H), 7.35–7.25 (m, 2H), 7.11 (d, 1H, J = 7.5 Hz), 6.96 (m, 1H), 6.92 (m, 2H), 5.94 (d, 1H, J = 2.4 Hz), 5.34 (d, 1H, J = 6.7 Hz), 5.25 (t, 1H, J = 5.0 Hz), 5.15 (s, 2H), 4.83 (s, 2H), 4.12 (m, 1H), 4.15 (s, 1H), 4.03–3.95 (m, 3H), 3.79 (m, 1H), 3.64 (m, 1H); HRMS calcd for C₂₄H₂₅N₄O₉ [MH⁺] 513.1622, found 513.1621.

5′-O-(Dimethoxytrityl)-2′-O-(4,5-dimethoxy-2-nitrobenzyl)-N⁶-phenoxyacetyladenosine (6a).

According to the procedure described for the synthesis of 2a, compound 6a was prepared by the reaction of 5a (797 mg, 1.34 mmol) with DMTrCl (1.25 g, 4.02 mmol) in dry pyridine at rt overnight. Silica gel chromatography (1% methanol in dichloromethane containing 0.5% triethylamine) of the crude product yielded 6a (864 mg, 72% yield) as a yellow foam. ¹H NMR (CDCl₃) δ 9.47 (brs, 1H), 8.66 (s, 1H), 8.26 (s, 1H), 7.59 (s, 1H), 7.44–6.81 (m, 19H), 6.30 (d, J = 4.1 Hz, 1H), 5.24 (d, J = 14.0 Hz, 1H), 5.08 (d, J = 14.0 Hz, 1H), 4.86 (s, 2H), 4.74 (t, J = 4.6 Hz, 1H), 4.57 (t, J = 4.9 Hz, 1H), 4.32 (m, 1H), 3.96 (s, 3H), 3.91 (s, 3H), 3.78 (s, 6H), 3.57 (dd, J = 3.2, 10.8 Hz, 1H), 3.46 (dd, J = 4.1, 10.7 Hz, 1H), 2.92 (b, 1H); ¹³C NMR (CDCl₃) δ 158.7, 157.1, 153.6, 152.6, 151.4, 148.4, 148.3, 144.5, 141.9, 140.1, 135.62, 135.60, 130.2, 130.0, 128.2, 128.0, 127.8, 127.1, 123.2, 122.5, 115.0, 113.3, 110.6, 108.2, 87.1, 86.9, 84.3, 81.9, 70.1, 69.8, 68.2, 62.9, 56.5, 56.4, 55.3; HRMS calcd for C₄₈H₄₇N₆O₁₂ [MH⁺] 899.3246, found 899.3244.

5′-O-(Dimethoxytrityl)-2′-O-(α-methyl-o-nitrobenzyl)-N⁶-phenoxyacetyladenosine (6b).

According to the procedure described for the synthesis of 2a, a crude 2′-O-(α-methyl-o-nitrobenzyl)adenosine (4b) (1.42 g) as a yellow foam was prepared from adenosine (2.00 g, 7.48 mmol), NaH (269 mg, 11.2 mmol) and α-methyl-o-nitrobenzyl bromide (2.51 g, 10.9 mmol). Compound 5b was then prepared from the crude 4b (1.42 g, 3.41 mmol), TMSCl (3.5 mL), and phenoxyacetyl chloride (0.71 mL, 5.12 mmol) as described for the synthesis of 5a. Silica gel chromatography (5% methanol in dichloromethane) of the crude product yielded 5b (1.20 g, 65% yield, two steps from adenosine) as a yellow foam. ¹H NMR (CDCl₃) δ 9.55 (s, 1H), 8.71 (s, 1H), 8.13 (s, 1H), 7.72–7.06 (m, 9H), 6.01 (d, J = 7.0 Hz, 1H), 5.42 (m, 1H), 5.11 (m, 1H), 4.93 (s, 2H), 4.85 (m, 1H), 4.40 (d, J = 4.6 Hz, 1H), 4.32 (d, J = 1.3 Hz, 1H), 3.90 (m, 1H), 3.76 (m, 1H), 2.69 (s, 1H), 1.33 (d, J = 6.4 Hz, 3H). Compound 6b was then prepared by the reaction of 5b (295 mg, 0.538 mmol) with DMTrCl (546 mg, 1.61 mmol) according to the procedure described for synthesis of 2a. Silica gel chromatography (1% methanol in dichloromethane containing 0.5% triethylamine) of the crude product yielded 6b (305 mg, 67% yield, or 44% from 3a) as a yellow foam. ¹H NMR (CDCl₃) δ 9.36 (brs, 1H), 8.68 (s, 1H), 8.63–8.61 (m, 3H), 8.25 (s, 1H), 7.94–6.81 (m, 18H), 6.25 (d, J = 2.1 Hz, 1H), 5.68 (m, 1H), 4.86 (s, 2H), 4.34 (m, 1H), 4.29–4.25 (m, 2H), 3.79 (s, 6H), 3.55 (dd, J = 2.7, 10.8 Hz, 1H), 3.44 (dd, J = 4.0, 10.8 Hz, 1H), 2.56 (d, J = 8.0 Hz, 1H), 1.65 (d, J = 6.3 Hz, 3H); HRMS calcd for: C₄₇H₄₅N₆O₁₀, [MH⁺]: 853.3197, found 853.3200.

5′-O-(Dimethoxytrityl)-2′-O-(o-nitrobenzyl)-N-phenoxyacetylcytidine (6c).^[19]

According to the procedure described for the synthesis of 2a, compound 6c was prepared by the reaction of 5c (1.24 g, 2.42 mmol) with DMTrCl (2.46 g, 7.26 mmol) in dry pyridine at rt overnight. Silica gel chromatography (1% methanol in dichloromethane containing 0.5% triethylamine) of the crude product yielded 6c^[19] (1.67 g, 86% yield) as a yellow foam. ¹H NMR (DMSO-d₆) δ 11.03 (s, 1H), 8.34 (d, J = 7.5 Hz, 1H), 8.08 (d, J = 8.2 Hz, 1H), 7.98 (d, J = 7.7 Hz, 1H), 7.76 (m, 1H), 7.57 (m, 1H), 7.40–6.85 (m, 19H), 5.94 (s, 1H), 5.4 (d, J = 7.3 Hz, 1H), 5.21 (dd, J = 15.2, 25.4 Hz, 2H), 4.82 (s, 2H), 4.35 (m, 1H), 4.15 (s, 1H), 4.01 (d, J = 4.8 Hz, 1H), 3.70 (s, 3H), 3.69 (s, 3H), 3.42–3.36 (m, 2H); HRMS calcd for C₄₅H₄₃N₄O₁₁ [MH⁺] 815.2928, found 815.2930.

5′-O-(Dimethoxytrityl)-2′-O-(α-methyl-o-nitrobenzyl)-N-phenoxyacetylcytidine (6d).

According to the procedure described for the synthesis of 2a, a crude 2′-O-(α-methyl-o-nitrobenzyl)cytidine (4d) (~1.42 g) containing impurities as a yellow foam was prepared from cytidine (0.97 g, 4.0 mmol), NaH (192 mg, 8.00 mmol) and α-methyl-o-nitrobenzyl bromide (1.43 g, 6.22 mmol). The molecular weight of 4d was confirmed by MS (ES-API), calcd for C₁₇H₂₁N₄O₇ [MH⁺] 393.1, found 393.2. According to the procedure described for the synthesis of 5c and 6c, intermediate 4d (~1.42 g, 3.60 mmol) was first protected with phenoxyacetyl group to give a crude 2′-O-(α-methyl-o-nitrobenzyl)-N⁴-phenoxyacetylcytidine (5d) (1.35 g). The molecular weight of 5d was also confirmed by MS (ES-API), calcd for C₂₅H₂₇N₄O₉ [MH⁺] 527.2, found 527.2. Compound 6d was then prepared by the reaction of crude 5d (1.35 g) with DMTrCl (2.57 g, 7.58 mmol) in dry pyridine at rt overnight. Silica gel chromatography (1% methanol in dichloromethane containing 0.5% triethylamine) of the crude product yielded 6d (459 mg, three steps 14% yield from cytidine) as a yellow foam. ¹H NMR (CDCl₃) δ 9.78 (brs, 1H), 8.36 (d, J = 7.5 Hz, 1H), 7.86 (d, J = 8.5 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.59 (m, 1H), 7.40–7.20 (m, 14H), 7.09 (d, J = 7.5 Hz, 1H), 7.01 (t, J = 7.5 Hz, 1H), 6.86 (d, J = 8.5 Hz, 4H), 5.61 (q, J = 6.5 Hz, 1H), 5.56 (s, 1H), 4.65 (s, 2H), 4.35 (m, 1H), 4.11 (m, 1H), 3.92 (m, 1H), 3.81 (s, 6H), 3.62–3.52 (m, 2H), 1.64 (d, J = 6.5 Hz, 3H); ¹³C NMR (CDCl₃) δ 168.5, 161.8, 158.8, 156.9, 154.8, 148.0, 145.0, 144.2, 139.4, 135.6, 135.3, 133.3, 130.18, 130.15, 129.8, 128.3, 128.1, 127.2, 124.4, 122.4, 114.6, 113.4, 96.4, 88.5, 87.2, 83.4, 82.2, 75.0, 68.2, 67.6, 61.2, 55.3, 23.4; HRMS calcd for C₄₆H₄₄N₄O₁₁Na [MNa⁺] 851.2904, found 851.2874.

5′-O-(Dimethoxytrityl)-2′-O-(o-nitrobenzyl)uridine (6e).

According to the procedure described for the synthesis of 2a, compound 6e was prepared by the reaction of 2′-O-(o-nitrobenzyl)uridine (5e)^[17] (540 mg, 1.42 mmol) with DMTrCl (1.083 g, 3.20 mmol) in dry pyridine at rt overnight. Silica gel chromatography (1.5–2% methanol in dichloromethane) of the crude product yielded 6e (727 mg, 75% yield) as a yellow foam. ¹H NMR (CDCl₃) δ 10.05 (brs, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 8.5 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.56 (m, 1H), 7.44–7.35 (m, 3H), 7.33–7.25 (m, 7H), 6.83 (d, 4H, J = 8.5 Hz), 6.05 (d, J = 1.8 Hz, 1H), 5.31 (d, J = 8.5 Hz, 1H), 5.28 (d, J = 13.5 Hz, 1H), 5.15 (d, J = 13.5 Hz, 1H), 4.53 (dd, J = 7.5, 5.5 Hz, 1H, 1H), 4.13–4.08 (m, 2H), 3.77 (s, 6H), 3.58 (m, 2H); ¹³C NMR (CDCl₃) δ 163.8, 158.7, 150.5, 147.9, 144.4, 139.9, 135.3, 135.1, 133.6, 133.0, 130.2, 130.1, 129.8, 128.8, 128.2, 128.0, 127.2, 124.8, 113.3, 102.2, 87.4, 87.1, 83.2, 82.6, 69.4, 68.7, 61.3, 55.3; HRMS calcd for C₃₇H₃₅N₃O₁₀Na [MNa⁺] 704.2220, found 704.2200.

5′-O-(Dimethoxytrityl)-N²-isobutyryl-2′-O-(o-nitrobenzyl)guanosine 3′-N,N-Diisopropyl(cyanoethyl)phosphoramidite (7a).^[20]

To a solution of compound 2a^[19] (47 mg, 0.059 mmol) in dry CH₂Cl₂ (5.0 mL) under Ar, N,N-diisopropylethylamine (52 μL, 0.30 mmol), 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (42 mg, 0.18 mmol) and 1-methylimidazole (5.0 μL, 0.059 mmol) were added. The mixture was stirred at rt until all starting material was consumed (1 h). The reaction was quenched with MeOH (1 mL) and stirred for 5 min. After the solvent was removed, the crude product was purified by silica gel chromatography, eluting with 1 % MeOH in CH₂Cl₂ containing 0.5% Et₃N to give phosphoramidite 7a^[20] (56 mg, 95% yield) as a yellow foam. ³¹P NMR (CD₃CN) δ 152.8, 152.6; HRMS calcd for C₅₁H₆₀N₈O₁₁P [MH⁺]: 991.4119, found: 991.4110.

5′-O-(Dimethoxytrityl)-N²-isobutyryl-2′-O-(α-methyl-o-nitrobenzyl)guanosine 3′-N,N-Diisopropyl(cyanoethyl)phosphoramidite (7b).

Phosphoramidite 7b was prepared from 5′-O-(dimethoxytrityl)-N²-isobutyryl-2′-O-(α-methyl-o-nitrobenzyl)guanosine (2b)^[19] (255 mg, 0.317 mmol), N,N-diisopropylethylamine (277 μL, 1.59 mmol), 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (224 mg, 0.95 mmol), and 1-methylimidazole (27 μL, 0.32 mmol) as described for 7a. Silica gel chromatography (1% methanol in dichloromethane containing 0.5% triethylamine) of the crude product yielded 7b (275 mg, 86% yield) as a yellow foam. ³¹P NMR (CD₃CN) δ 152.7, 152.4; HRMS calcd for C₅₂H₆₂N₈O₁₁P [MH⁺]: 1005.4276, found: 1005.4276.

5′-O-(Dimethoxytrityl)-2′-O-(o-nitrobenzyl)-N²-phenoxyacetylguanosine 3′-N,N-Diisopropyl(cyanoethyl)phosphoramidite (7c).

hosphoramidite 7c was prepared from 2c (284 mg, 0.332 mmol), N,N-diisopropylethylamine (290 μL, 1.66 mmol), 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (234 mg, 0.996 mmol), and 1-methylimidazole (28 μL, 0.33 mmol) as described for 7a. Silica gel chromatography (1% methanol in dichloromethane containing 0.5% triethylamine) of the crude product yielded 7c (270 mg, 77% yield) as a yellow foam. ³¹P NMR (CD₃CN) δ 153.0, 152.7; HRMS calcd for C₅₅H₆₀N₈O₁₂P [MH⁺]: 1055.4069, found: 1055.4075.

5′-O-(Dimethoxytrityl)-2′-O-(α-methyl-o-nitrobenzyl)-N²-phenoxyacetylguanosine 3′-N,N-Diisopropyl(cyanoethyl)phosphoramidite (7d).

Phosphoramidite 7d was prepared from 2d (156 mg, 0.18 mmol), N,N-diisopropylethylamine (157 μL, 0.90 mmol), 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (127 mg, 0.54 mmol), and 1-methylimidazole (15 μL, 0.18 mmol) as described for 7a. Silica gel chromatography (1% methanol in dichloromethane containing 0.5% triethylamine) of the crude product yielded 7d (121 mg, 63% yield) as a yellow foam. ³¹P NMR (CD₃CN) δ 152.6, 152.5; HRMS calcd for C₅₆H₆₂N₈O₁₂P [MH⁺]: 1069.4225, found: 1069.4235.

5′-O-(Dimethoxytrityl)-2′-O-(4,5-dimethoxy-2-nitrobenzyl)-N⁶-phenoxyacetyladenosine 3′-N,N-diisopropyl (cyanoethyl)phosphoramidite (7e).

Phosphoramidite 7e was prepared from 6a (210 mg, 0.234 mmol), N,N-diisopropylethylamine (194 μL, 1.11 mmol), 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (156 mg, 0.66 mmol), and 1-methylimidazole (18 μL, 0.21 mmol) as described for 7a. Silica gel chromatography (1% methanol in dichloromethane containing 0.5% triethylamine) of the crude product yielded 7e (225 mg, 88% yield) as a yellow foam. ³¹P NMR (CD₃CN) δ 152.8, 152.7; HRMS calcd for C₅₇H₆₄N₈O₁₃P [MH⁺] 1099.4325, found 1099.4309.

5′-O-(Dimethoxytrityl)-2′-O-(α-methyl-o-nitrobenzyl)-N⁶-phenoxyacetyladenosine 3′-N,N-Diisopropyl(cyanoethyl)phosphoramidite (7f).

Phosphoramidite 7f was prepared from 6b (284 mg, 0.33 mmol), N,N-diisopropylethylamine (276 μL, 1.58 mmol), 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (221 mg, 0.94 mmol), and 1-methylimidazole (26 μL, 0.30 mmol) as described for 7a. Silica gel chromatography (1% methanol in dichloromethane containing 0.5% triethylamine) of the crude product yielded 7f (322 mg, 92% yield) as a yellow foam. ³¹P NMR (CD₃CN) δ 152.6, 152.0; HRMS calcd for C₅₆H₆₂N₈O₁₁P [MH⁺]: 1053.4276, found: 1053.4277.

5′-O-(Dimethoxytrityl)-2′-O-(o-nitrobenzyl)-N-phenoxyacetylcytidine 3′-N,N-Diisopropyl-(cyanoethyl)phosphoramidite (7g).

Phosphoramidite 7g was prepared from 5′-O-(dimethoxytrityl)-2′-O-(o-nitrobenzyl)-N-phenoxyacetylcytidine (6c)^[19] (185 mg, 0.23 mmol), N,N-diisopropylethylamine (192 μL, 1.10 mmol), 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (154 mg, 0.66 mmol), and 1-methylimidazole (18 μL, 0.21 mmol) as described for 7a. Silica gel chromatography (0–4% acetone in dichloromethane containing 0.5% triethylamine) of the crude product yielded 7g (215 mg, 92% yield) as a yellow foam. ³¹P NMR (CD₃CN) δ 152.4, 151.6; HRMS calcd for C₅₄H₆₀N₆O₁₂P, [MH⁺]: 1015.4007, found: 1015.4011.

5′-O-(Dimethoxytrityl)-2′-O-(α-methyl-o-nitrobenzyl)-N-phenoxyacetylcytidine 3′-N,N-Diisopropyl(cyanoethyl)phosphoramidite (7h).

Phosphoramidite 7h was prepared from 6d (150 mg, 0.18 mmol), N,N-diisopropylethylamine (158 μL, 0.91 mmol), 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (129 mg, 0.54 mmol), and 1-methylimidazole (15 μL, 0.18 mmol) as described for 7a. Silica gel chromatography (0–1% acetone in dichloromethane containing 0.5% triethylamine) of the crude product yielded 7h (169 mg, 91% yield) as a white foam. ³¹P NMR (CD₃CN) δ 151.2, 149.4; HRMS calcd for C₅₅H₆₂N₆O₁₂P, [MH⁺]: 1029.4163, found: 1029.4143.

5′-O-(Dimethoxytrityl)-2′-O-(o-nitrobenzyl)uridine 3′-N,N-Diisopropyl(cyanoethyl)-phosphoramidite (7i).

Phosphoramidite 7i was prepared from 6e (121 mg, 0.18 mmol), N,N-diisopropylethylamine (158 μL, 0.91 mmol), 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (129 mg, 0.54 mmol), and 1-methylimidazole (15 μL, 0.18 mmol) as described for 7a. Silica gel chromatography (0–2% acetone in dichloromethane containing 0.5% triethylamine) of the crude product yielded 7i (135 mg, 86% yield) as a white foam. ³¹P NMR (CD₃CN) δ 150.9, 150.1; HRMS calcd for C₄₆H₅₃N₅O₁₁P, [MH⁺]: 882.3479, found: 882.3469.

5′-O-(Dimethoxytrityl)-2′-O-(o-nitrobenzyl)-N⁶-benzoyladenosine 3′-N,N-Diisopropyl-(cyanoethyl)phosphoramidite (7j).^[6,14]

Phosphoramidite 7j was prepared from 5′-O-(dimethoxytrityl)-2′-O-(o-nitrobenzyl)-N⁶-benzoyladenosine (6f)^[6,14,19] (99 mg, 0.12 mmol), N,N-diisopropylethylamine (106 μL, 0.61 mmol), 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (221 mg, 0.36 mmol), and 1-methylimidazole (10 μL, 0.12 mmol) as described for 7a. Silica gel chromatography (1% methanol in dichloromethane containing 0.5% triethylamine) of the crude product yielded 7j^[6,14] (101 mg, 82% yield) as a yellow foam. ³¹P NMR (CD₃CN) δ 152.9, 152.7.