Synthesis and Biophysical Characterization of RNAs Containing (R

Article Cite This: J. Org. Chem. XXXX, XXX, XXX−XXX

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Synthesis and Biophysical Characterization of RNAs Containing (R)and (S)‑5′‑C‑Aminopropyl-2′‑O‑methyluridines Ryohei Kajino,† Yusuke Maeda,‡ Hisae Yoshida,∥ Kenji Yamagishi,∥ and Yoshihito Ueno*,†,‡,§ †

Department of Life Science and Chemistry, the Graduate School of Natural Science and Technology, ‡Course of Applied Life Science, Faculty of Applied Biological Sciences, and §Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University, 1-1 Yanagido, Gifu, Gifu 501-1193, Japan ∥ Department of Chemical Biology and Applied Chemistry, College of Engineering, Nihon University, 1 Azanakagawara, Tokusada, Tamuramachi, Koriyama, Fukushima 963-8642, Japan

J. Org. Chem. Downloaded from pubs.acs.org by WEBSTER UNIV on 03/04/19. For personal use only.

S Supporting Information *

ABSTRACT: We designed and synthesized (R)-5′-C-aminopropyl-2′-O-methyluridine and (S)-5′-C-aminopropyl-2′-Omethyluridine, which are applicable to small interfering RNAs (siRNAs). We have evaluated the properties of siRNAs containing (R)-5′-C-aminopropyl-2′-O-methyl and (S)-5′-Caminopropyl-2′-O-methyl modifications and have compared them to that of the 4′-C-aminopropyl-2′-O-methyl modification. Although these modifications decreased the thermal stability of double-stranded RNAs (dsRNAs) and siRNAs, the dsRNA containing the (S)-5′-C-aminopropyl-2′-O-methyl modification showed the highest melting temperature (Tm) among them. Silencing activity of the modified siRNAs was assessed by a dual luciferase reporter assay using HeLa cells. Incorporation of the (R)-5′-C-aminopropyl-2′-O-methyl and (S)-5′-C-aminopropyl-2′-O-methyl modifications on a passenger and guide strand was found to be tolerated for the silencing activity of siRNAs except for in the seed region on the guide strand. Furthermore, these modifications significantly increased the stability of single-stranded RNAs (ssRNAs) and siRNAs in a buffer containing bovine serum.

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INTRODUCTION RNA interference (RNAi) is an endogenous biological mechanism, triggered by small RNAs such as small interfering RNA (siRNA) and microRNA (miRNA). siRNA is a doublestranded RNA (dsRNA) composed of 19−21 base pairs and two 3′ nucleotide overhangs.1−3 One strand of siRNA, which has the same sequence as the target mRNA (mRNA), is called the passenger strand, and the strand complementary to the target mRNA sequence is called the guide strand. The siRNA interacts with Argonaute 2 protein (Ago2) to form an RNA-induced silencing complex (RISC) in which the passenger strand is cleaved and removed from the RISC. The remaining guide strand then forms a duplex with the target mRNA. Finally, the target mRNA is cleaved by the endonuclease activity of Ago2 in the RISC.4−7 Since siRNAs can suppress the expression of target mRNAs in a sequence-specific manner, they have the potential to be used as new therapeutic agents for unmet medical needs. However, there are several challenges in the therapeutic application of siRNAs. It is known that siRNAs are highly vulnerable to nucleases present inside and outside of the cells, stimulate immune responses, and have low cell membrane permeability.8,9 Thus, chemical modification of siRNAs is needed to overcome these challenges.10−13 So far, it has been reported that 2′-O-methyl and 2′-fluoro modifications increase © XXXX American Chemical Society

the stability of siRNAs in serum, reduce immune response by the toll-like receptor (TLR), and are well-tolerated for RISC formation in the RNAi pathway.14−18 Furthermore, it was reported that aminoalkyl modifications enhanced nuclease resistance and cell membrane permeability of siRNAs.19−22 Recently, we reported the synthesis of siRNAs containing 4′-Caminoalkyl-2′-O-methyluridines where we observed that the introduction of these nucleoside analogs into siRNAs, especially 4′-C-aminopropyl-2′-O-methyluridine (1), dramatically enhanced the stability of siRNAs in serum.23 Furthermore, incorporation of these nucleoside analogs into the passenger strand was found to be tolerable in RNAi activity. However, 4′C-aminoalkyl modifications decreased the thermal stability of double-stranded RNAs (dsRNAs) and siRNAs. The thermal destabilization of dsRNAs and siRNAs could be attributed to a conformational change in the sugar component of the nucleoside analogs due to steric hindrance between the 4′-Caminoalkyl and 2′-O-methyl functional groups. On the other hand, Manoharan and co-workers have reported that 5′-Cmethyl modifications enhanced the nuclease resistance of RNAs toward snake venom phosphodiesterases (SVPD) without Received: December 28, 2018 Published: February 26, 2019 A

DOI: 10.1021/acs.joc.8b03277 J. Org. Chem. XXXX, XXX, XXX−XXX

Article

The Journal of Organic Chemistry

Figure 1. Structure of 4′-C-aminopropyl-2′-O-methyl uridine (1), (R)-5′-C-aminopropyl-2′-O-methyluridine (2), and (S)-5′-C-aminopropyl-2′-Omethyluridine (3).

changing the sugar conformation of the nucleoside analogs.24 On the basis of this information, we designed the (R)-5′-Caminopropyl-2′-O-methyluridine (2) and (S)-5′-C-aminopropyl-2′-O-methyluridine (3) nucleoside analogs (Figure 1). We hypothesized that the aminoalkyl modifications at the 5′position of a nucleoside would greatly enhance nuclease resistance and stability of siRNAs in serum without decreasing thermal stability. In this study, we report the synthesis of (R)-5′-C-aminopropyl-2′-O-methyluridine (2) and (S)-5′-C-aminopropyl-2′-Omethyluridine (3), along with the biophysical and biological properties of the siRNAs containing these nucleoside analogs.

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uridine derivative 25 was prepared according to a reported method.27 Oxidation of the primary hydroxyl function of 25, followed by an allylation reaction using allyltrimethylsilane and BF3·OEt2, gave 5′-C-allyluridine derivatives 27 and 28 in 43% and 2%, respectively. Configurations of the 5′-carbons in 27 and 28 were determined by nuclear Overhauser effect spectroscopy (NOESY) measurement. To fix the conformations of the sugar moieties of 27 and 28, the 3′- and 5′-hydroxy functions of 27 and 28 were protected by cyclic silyl groups. After removing the 3′O-silyl groups of 27 and 28, the resulting 3′,5′-dihydroxy compounds were treated with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPDSCl2) in pyridine to give 3′,5′-O-TIPDS derivatives 36 and 37 in 56% and 54% yields, respectively (Scheme 3), followed by NOESY measurement (Supporting Information). As a result, the NOE was observed between H-3′ and H-5′ of 37 but not between H-3′ and H-5′ of 36. Thus, we concluded that the nucleoside derivative 27 is an (S)-isomer while the nucleoside derivative 28 is an (R)-isomer. This result is consistent with that for similar nucleoside analogs previously reported by Nielsen and co-workers.28 The 5′-hydroxyl function of 27 was then protected with the DMTr group to give 29 in 83% yield. Hydroboration and oxidation of the allyl moiety of 29 produced a 5′-Chydroxypropyluridine derivative 30 in 49% yield. Tosylation and azidation of the hydroxy function of 30 afforded an azidopropyl derivative 32 in 50% yield from 30. After converting the azide function of 32 to an amino function by treatment with PPh3, the resulting amino function was protected with the TFA group to give 33 in 94% yield. Deprotection of the 3′-O-silyl group of 33 by treating with 1 M TBAF in THF gave a 3′hydroxy derivative 34 in 84% yield. Finally, 34 was phosphorylated by a standard procedure to give the corresponding phosphoramidite 35 in 89% yield. Furthermore, to incorporate the analogs 2 and 3 into the 3′end of RNAs, 23 and 34 were modified to produce the corresponding 3′-succinates 38 and 40, which were then reacted with controlled pore glass (CPG) to produce the solid supports 39 and 41 containing 38 (25 μmol/g) and 40 (45 μmol/g), respectively (Scheme 4). Oligonucleotide Synthesis. The nucleoside analogs 2 and 3 were incorporated into RNA oligomers using the phosphoramidites 24 and 35 by a DNA/RNA synthesizer. After the synthesis, to prevent the additional reaction of acrylonitrile with the 5′-C-aminopropyl functional groups, the CPG beads were first treated with 10% diethylamine in CH3CN at room temperature for 5 min and then with concentrated NH3 solution/40% methylamine (1:1, v/v) solution at 65 °C for 10 min. 2′-O-TBDMS groups were removed by treatment with Et3N·3HF in DMSO at 65 °C for 1.5 h. RNAs were purified using 20% denaturing polyacrylamide gel electrophoresis

RESULTS AND DISCUSSION

Synthesis of Nucleoside Analogs. The synthetic route of a phosphoramidite of (R)-5′-C-aminopropyl-2′-O-methyluridine (2) is shown in Scheme 1. A (R)-5-C-hydroxymethyl ribofuranose derivative 4, which was prepared according to a reported method,25 was treated with tert-butyldiphenylsilyl chloride (TBDPSCl) in pyridine to give a 6-O-TBDPS derivative 5 in 92% yield. 5-O-Benzylation and desilylation of 5 resulted in a 6-hydroxyl derivative 7 in 89% yield. Oxidation of the 6-hydroxyl function followed by the Horner−Wadsworth− Emmons olefination reaction produced 9 in 80% yield. Olefinselective hydrogenation of 9 using Pd/C and ammonium formate, followed by reduction of an ethyl ester moiety using LiAlH4, gave an alcohol 11 in 99% yield. Tosylation and azidation of 11 afforded an azide derivative 13 in 78% yield from 11. Acetolysis of the 1,2-acetonide 13, followed by the Vorbrüggen reaction26 with a silylated uracil derivative, gave an 5′-C-azidopropyluridine derivative 15 in 91% yield. Treatment of 15 with K2CO3 in CH3OH produced a 2′-hydroxyl derivative 16 in 95% yield. Methylation of the hydroxyl group of 16 by treatment with CH3I, followed by deprotection of the pmethoxybenzyl (PMB) group using 2,3-dichloro-5,6-dicyano-pbenzoquinone (DDQ), gave a 3′-hydroxyl derivative 18 in 79% yield. Protection of the 3′-hydroxyl function by the TBDPS group gave 19 in 95% yield. Subsequent deprotection of the 5′O-benzyl group gave a 5′-hydroxyl derivative 20 in 98% yield. The 5′-hydroxyl function of 20 was protected by a 4, 4′dimethoxytrityl (DMTr) group to give 21 in 75% yield. After converting the azide function of 21 to an amino function by treatment with PPh3, the resulting amino function was protected by a trifluoroacetyl (TFA) group to produce 22 in 87% yield from 21. Deprotection of the 3′-O-TBDPS group of 22 by treatment with 1 M n-tetrabutylammonium fluoride (TBAF) in THF gave a 3′-hydroxy derivative 23 in 85% yield. Finally, 23 was phosphorylated by a standard procedure to give the corresponding phosphoramidite 24 in 73% yield. The synthetic route of a phosphoramidite of (S)-5′-Caminopropyl-2′-O-methyluridine (3) is shown in Scheme 2. A B


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The Journal of Organic Chemistry Scheme 1. Synthesis Scheme of (R)-5′-C-Aminopropyl-2′-O-methyluridine Phosphoramidite 24a

a Reagents and conditions: (a) TBDPSCl, pyridine, r.t., 17 h, 92%; (b) BnBr, NaH, NaI, DMF, 90 °C, 5 h, 66%; (c) TBAF/THF, THF, r.t., 19 h, 89%; (d) TEMPO, KBr, NaHCO3, NaClO aq., CH2Cl2, 0 °C, 40 min; (e) (EtO)2P(O)CH2CO2Et, NaH, THF, r.t., 30 min, 80% (2 steps); (f) 5% Pd/C, ammonium formate, EtOAc, r.t., 5.5 h, 90%; (g) LiAlH4, THF, 0 °C, 30 min, 99%; (h) p-TsCl, pyridine, CH2Cl2, r.t., 3 h, 96%; (i) NaN3, DMF, 60 °C, 4 h, 81%; (j) (i) 50% AcOH aq., 70 °C, 24 h, (ii) Ac2O, pyridine, r.t., 6 h, 50%; (k) uracil, N,O-bis(trimethylsilyl)acetamide, TMSOTf, CH3CN, 55 °C, 2 h, 91%; (l) K2CO3, CH3OH, r.t., 1 h, 95%; (m) CH3I, NaH, THF, 0 °C, 4.5 h, 90%; (n) DDQ, CH2Cl2/H2O (19:1 v/ v), 0 °C to r.t., 4 h, 79%; (o) TBDPSCl, imidazole, DMF, r.t., 18 h, 95%; (p) BCl3/CH2Cl2, CH2Cl2, −78 to −30 °C, 3 h, 98%; (q) DMTrCl, 2,6lutidine, pyridine, 40 °C, 48 h, 75%; (r) (i) Ph3P, H2O, THF, 40 °C, 3 h, (ii) CF3CO2Et, Et3N, CH2Cl2, r.t., 25 h, 87%; (s) TBAF/THF, THF, r.t., 6 h, 85%; (t) 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphoroamidite, 1H-tetrazole, 1-methylimidazole, DMF, r.t., 2.5 h, 73%.

(PAGE). The RNA sequences synthesized in this study are shown in Tables 1−4 and S1−S5. Thermal Stability of Duplexes. First, we evaluated the effect of the (R)-5′-C-aminopropyl and (S)-5′-C-aminopropyl modifications on thermal stability of double-stranded RNAs (dsRNA1−4). Temperature-induced melting was measured by

ultraviolet (UV) spectroscopy in 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl. The melting temperature (Tm) values are shown in Table 1. The Tm value of the unmodified dsRNA 1 was 45.6 °C, while that of dsRNAs 2, 3, and 4 containing the nucleoside analogs 1, 2, and 3 were 39.1, 37.4, and 42.7 °C, respectively. Incorporation of the 4′-CC


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The Journal of Organic Chemistry Scheme 2. Synthesis Scheme of (S)-5′-C-Aminopropyl-2′-O-methyluridine Phosphoramidite 35a

Reagents and conditions: (a) EDC·HCl, CHCl2CO2H, DMSO/CH2Cl2 (1:1 v/v), −5 °C, 1.5 h; (b) allyltrimethylsilane, BF3·OEt2, CH2Cl2, 0 °C, 1 h, 27: 43% (2 steps); 28: 2% (2 steps); (c) DMTrCl, 2,6-lutidine, pyridine, 40 °C, 67 h, 83%; (d) 9-BBN, THF then 30% H2O2 aq., 3 N NaOH aq., 30 °C, 15 min, 49%; (e) p-TsCl, pyridine, CH2Cl2, r.t., 7 h, 56%; (f) NaN3, DMF, 60 °C, 11 h, 90%; (g) (i) Ph3P, H2O, THF, 40 °C, 16 h, (ii) CF3CO2Et, Et3N, CH2Cl2, r.t., 5 h, 94%; (h) TBAF/THF, THF, r.t., 14 h, 84%; (i) 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphoroamidite, 1Htetrazole, 1-methylimidazole, DMF, r.t., 1 h, 89%. a

67.0, and 71.6 °C, respectively. The ΔTm values of siRNAs 2, 3, and 4 were −1.2, −1.3, and −0.8 °C/modification. Thus, it was found that the (S)-5′-C-aminopropyl modification improved the thermal stability of siRNAs compared to the 4′-C-aminopropyl modification. We calculated thermodynamic parameters of RNA duplex formation to evaluate the effects of the 4′-C-aminopropyl, (R)5′-C-aminopropyl, and (S)-5′-C-aminopropyl modifications on the stability of dsRNA in detail. We determined the thermodynamic parameters from calculations based on the slope of the plot of 1/Tm versus (CT/4), with CT being the total concentration of single-stranded RNA (ssRNA) (Table 3). The ΔG°37 °C value of the unmodified dsRNA 1 was −11.1 kcal/mol, whereas those of dsRNAs 2−4 containing the 4′-C-aminopropyl, (R)-5′-C-aminopropyl, and (S)-5′-C-aminopropyl modifications were −9.4, −8.9, and −10.4 kcal/mol, respectively. The ΔH° values of dsRNAs 1−4 were −93.3, −97.0, −90.0, and −88.0 kcal/mol, while the ΔS° values of dsRNAs 1−4 were −265.4, −282.4, −261.6 and −250.3 cal/mol·K, respectively. These results indicate that the thermodynamic destabilization of dsRNA by incorporating the 4′-C-aminopropyl modification was caused by unfavorable entropy (ΔS°), whereas the thermodynamic destabilization of dsRNAs by the introduction

aminopropyl analog 1 decreased the Tm value of dsRNA by 2.2 °C/modification,20,23 whereas the introduction of (R)-5′-Caminopropyl and (S)-5′-C-aminopropyl analogs 2 and 3 reduced the Tm values of dsRNAs by 2.7 and 1.0 °C/ modification, respectively. The effects of the 5′-C-aminopropyl modifications on thermal stability of dsRNAs were found to be dependent on stereochemistry at the C5′-positions of the nucleoside analogs. Although both analogs reduced thermal stability of the dsRNAs, the (R)-isomer 2 thermally destabilized dsRNA more than the (S)-isomer 3. It was reported that a (R)5′-C-methyl modification on a nucleoside interferes with hydration around the phosphate backbone of dsRNA since the (R)-5′-C-methyl group is located closer to the phosphate linkage than a (S)-5′-C-methyl group.24 Therefore, it was considered that the (R)-5′-C-aminopropyl modification also interferes with hydration around the phosphate backbone, resulting in destabilization of the dsRNA. It was revealed that the dsRNA containing the (S)-5′-C-aminopropyl analog 3 was thermally more stable than that containing the 4′-C-aminopropyl analog 1. Next, we measured the thermal stability of siRNAs. Incorporation of the nucleoside analogs 1, 2, and 3 to siRNA (siRNA 2−4) decreased the thermal stability of siRNAs (Figure S2 and Table 2). The Tm value of the unmodified siRNA (siRNA 1) was 77.7 °C, while those of siRNAs 2, 3, and 4 were 68.4, D


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Table 1. Sequences of ssRNAs, dsRNAs, and Tm Values of dsRNAs

Scheme 3. Synthesis Scheme of the 3′,5′-O-TIPDS Derivatives 36 and 37a

F, 1 (green), 2 (blue), and 3 (red) denote fluorescein, 4′-Caminoalkyl-2′-O-methyl uridine, (R)-5′-C-aminopropyl-2′-O-methyluridine, and (S)-5′-C-aminopropyl-2′-O-methyluridine, respectively. b The Tm’s were measured in a buffer containing 10 mM sodium phosphate (pH 7.0) and 100 mM NaCl. The concentrations of the duplexes were 3 μM. All experiments were carried out three times, and data are represented as mean ± SD. cΔTm represents [Tm (dsRNAmod) − Tm (dsRNAunmod)]. a

Molecular Modeling Study. It is known that sugar puckering in nucleosides is one of the determinants of the global conformation of a double-stranded nucleic acid. We have previously reported that the 4′-C-aminoethyl-2′-O-methyl and 4′-C-aminopropyl-2′-O-methyl modifications drove the sugar conformations of nucleosides to adopt the south-type C2′-endo pucker. Thus, to analyze the effects of the 5′-C-aminoalkyl-2′-Omethyl modifications on sugar puckering, we calculated the percentage of (R)-5′-C-aminoalkyl-2′-O-methyluridine (2) and (S)-5′-C-aminoalkyl-2′-O-methyluridine (3) in the C3′-endo sugar conformation on the basis of the 500 ns MD trajectories and compared them to that of 4′-C-aminopropyl-2′-Omethyluridine (1). As shown in Figures S4 and S5 and Table S1, it was found that the percentages of the analogs 2 and 3 harboring the 5′-C-aminoalkyl modifications in the C3′-endo sugar conformation were higher than that of the analog 1 with the 4′-C-aminopropyl modification. This suggests that, as the population of the C3′-endo sugar puckering in the 5′-Caminoalkyl analogs increased in comparison to the 4′-Caminoalkyl analog, the duplex formation of the RNAs containing

a

Reagents and conditions: (a) (i) TBAF/THF, THF, r.t., 2 h, (ii) TIPDSCl2, pyridine, r.t., 10 h, 56%; (b) (i) TBAF/THF, THF, r.t., 6 h, (ii) TIPDSCl2, pyridine, r.t., 40 h, 54%.

of the (R)-5′-C-aminopropyl and (S)-5′-C-aminopropyl modifications was due to unfavorable enthalpy (ΔH°). Next, we examined the base-discriminating ability of the nucleoside analogs 2 and 3 in dsRNAs (Table 4). The ΔTm (Tm of a dsRNA containing mismatched base pair − Tm of a fully matched dsRNA) values for the unmodified dsRNA (dsRNA 5− 8) ranged from −4.6 to −12.1 °C. The ΔTm values for the modified dsRNA (dsRNA 9−12, dsRNA 13−16, dsRNA 17− 20) also ranged from −5.6 to −11.9 °C. Thus, it was revealed that the nucleoside analogs 2 and 3 had base-discriminating ability in dsRNAs.

Scheme 4. Synthesis Scheme of the Controlled-Pore Glasses (CPGs) 39 and 41 Carrying 23 and 34a

a Reagents and condition: (a) succinic anhydride, DMAP, pyridine, r.t., 20 h; (b) CPG 500 Å, EDC·HCl, DMF, r.t., 5 days, loading: 25 μmol/g; (c) succinic anhydride, DMAP, pyridine, r.t., 15 h; (d) CPG 500 Å, EDC·HCl, DMF, r.t., 5 days, loading: 45 μmol/g.

E


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The Journal of Organic Chemistry Table 2. Sequences of ssRNAs, siRNAs, and Tm Values of siRNAs

F, 1 (green), 2 (blue), and 3 (red) denote fluorescein, 4′-C-aminoalkyl-2′-O-methyl uridine, (R)-5′-C-aminopropyl-2′-O-methyluridine, and (S)5′-C-aminopropyl-2′-O-methyluridine, respectively. bThe Tm’s were measured in a buffer containing 10 mM sodium phosphate (pH 7.0) and 100 mM NaCl. The concentrations of the duplexes were 3 μM. All experiments were carried out three times, and data are represented as mean ± SD. c ΔTm represents [Tm (dsRNAmod) − Tm (dsRNAunmod)]. a

Table 3. Thermodynamic Parameters of dsRNAsa

Thermodynamics parameters were determined from the plot of 1/Tm versus (CT/4). bF, 1 (green), 2 (blue), and 3 (red) denote fluorescein, 4′-Caminoalkyl-2′-O-methyl uridine, (R)-5′-C-aminopropyl-2′-O-methyluridine, and (S)-5′-C-aminopropyl-2′-O-methyluridine, respectively.

a

(Figure 3). The results exhibited that the incorporation of one analog at the passenger strand was well tolerated for forming the RISC and eliciting RNAi activity. Then, we assessed the silencing activity of the siRNAs (siRNA 14−19) containing four analogs at the passenger strand. As shown in Figure 4, siRNA 14−19 also showed RNAi activity that was equal to or slightly lower than that of the unmodified siRNA 1. The results indicated that the incorporation of four analogs at the passenger strand was also tolerable. Next, we measured the RNAi activity of siRNAs that were modified at the guide strands (siRNAs 20−28). As shown in Figure 5, it was found that incorporation of the (R)-5′-Caminopropyl-2′-O-methyl modification at position 8 to form the 5′-end of the guide strand reduced the RNAi activity of siRNA (siRNA 21). The region of the nucleotide from positions 2−8 from the 5′-end of the guide strand is known as the seed region.29 It was reported that the seed region is important for forming the duplex with the target mRNA in the RISC and is specifically recognized by Ago2.23,30 The silencing activity of the siRNAs containing the analogs at position 8 were in the following order: the siRNA 20 containing the 4′-C-aminoalkyl2′-O-methyl modification, siRNA 22 containing the (S)-5′-Caminopropyl-2′-O-methyl modification, and siRNA 21 containing the (R)-5′-C-aminopropyl-2′-O-methyl modification. These results indicated that the position and configuration of the modifications at the sugar moieties are important for the silencing activities of siRNAs. Since the 5′-position in the sugar moiety is closer to the 5′-end of the strand than the 4′-position, the former is located further within the seed region compared to the latter, and therefore, the 5′-C-aminopropyl modifications

the 5′-C-aminoalkyl modifications became entropically more favored than the RNA containing the 4′-C-aminoalkyl modification. Circular Dichroism (CD) Spectra. Next, we measured the CD spectra of siRNAs 1−4 to compare the global structures of the siRNAs containing three 4′-C-aminopropyl-2′-O-methyl, three (R)-5′-C-aminopropyl-2′-O-methyl, or three (S)-5′-Caminopropyl-2′-O-methyl modifications. As shown in Figure 2, all siRNAs exhibited positive CD bands at around 260 nm and negative CD bands at around 210 nm which could be attributed to A-type duplexes. However, the positive CD band of siRNA 3 containing the (R)-5′-C-aminopropyl modifications was smaller and had a shoulder at around 285 nm as compared to the others. These results suggested that, although all modified siRNAs could form A-type duplexes, the (R)-5′-C-aminopropyl modification changed the global structure of the siRNA duplex slightly. RNAi Activity. We evaluated the RNAi activity of the modified and unmodified siRNAs by a dual luciferase reporter assay using HeLa cells, in which the target luciferase genes were constitutively expressed. All siRNAs targeted the Renilla luciferase genes, while expression of firefly luciferase genes were used as controls. The HeLa cells were transfected with the siRNAs using RNAiMAX, and the expression of both luciferase genes were analyzed after 24 h of incubation. The relative percentages of Renilla and firefly luciferase activities compared to the controls containing no siRNAs are shown Figures 3−5. The siRNAs (siRNA 5−13), which contained one analog at the passenger strand efficiently, suppressed the expression of the Renilla luciferase gene similar to the unmodified siRNA 1 F


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The Journal of Organic Chemistry Table 4. Base-Discriminating Ability of the Nucleoside Analogs 2 and 3 in dsRNAs

Underlined letters denote mismatched base pairs. bF, 1 (green), 2 (blue), and 3 (red) represent fluorescein, 4′-C-aminoalkyl-2′-O-methyl uridine, (R)-5′-C-aminopropyl-2′-O-methyluridine, and (S)-5′-C-aminopropyl-2′-O-methyluridine, respectively. The Tm’s were measured in a buffer containing 10 mM sodium phosphate (pH 7.0) and 100 mM NaCl. The concentrations of the duplexes were 3 μM. cΔTm represents [Tm (dsRNAmod) − Tm (dsRNAunmod)]. a

might have a greater effect on the RNAi activities of siRNAs. Furthermore, since the (R)-5′-C-aminopropyl modification influenced the global conformation of siRNA more than the (S)-5′-C-aminopropyl modification, the (R)-5′-C-aminopropyl modification might decrease the RNAi activity of siRNA to a greater extent. However, RNAi activity of siRNAs 24, 25, 27, and 28 containing the (S)-5′-C-aminopropyl or the (R)-5′-Caminopropyl modification at other positions was equal to that of the unmodified siRNA 1. The results suggested that the 5′-Caminopropyl modifications at the guide strand were tolerated in eliciting the RNAi activity except for in the seed region. Serum Stability. Since unmodified RNAs are highly unstable in vivo due to their vulnerability to nucleases, chemical modifications are required to enhance the biological stability of siRNAs and prolong RNAi activity.31,32 Thus, we next evaluated the effect of the 4′-C-aminopropyl, the (R)-5′-C-aminopropyl, and the (S)-5′-C-aminopropyl modifications on the stability of ssRNA and siRNA in serum. First, fluorescein-labeled ssRNAs were incubated in a buffer containing 3% bovine serum, and subsequently, the reaction mixtures at various incubation times (0, 0.5, 1, 5, and 30 min and 1, 3, and 6 h) were analyzed by

Figure 2. CD spectra of the modified and unmodified siRNAs in a buffer containing 10 mM sodium phosphate (pH 7.0) and 100 mM NaCl at 15 °C. The concentrations of duplexes were 4 μM.

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Figure 3. RNAi activity of siRNA modified by one analog at the passenger strand. siRNAs were transfected into HeLa cells at concentrations of 1 and 10 nM. After a 24 h incubation, the activities of Renilla and firefly luciferases in the cells were determined with a Dual-Luciferase Reporter Assay System. The results were confirmed by at least three independent transfection experiments with two cultures each and are expressed as the average from four experiments as mean ± SD.

Figure 4. RNAi activity of siRNA modified by four analogs at the passenger strand. siRNAs were transfected into HeLa cells at concentrations of 1 and 10 nM. After a 24 h incubation, the activities of Renilla and firefly luciferases in the cells were determined with a Dual-Luciferase Reporter Assay System. The results were confirmed by at least three independent transfection experiments with two cultures each and are expressed as the average from four experiments as mean ± SD.

Figure 5. RNAi activity of siRNA modified by one analog at the guide strand. siRNAs were transfected into HeLa cells at concentrations of 1 and 10 nM. After a 24 h incubation, the activities of Renilla and firefly luciferases in the cells were determined with a Dual-Luciferase Reporter Assay System. The results were confirmed by at least three independent transfection experiments with two cultures each and are expressed as the average from four experiments as mean ± SD.

denaturing PAGE. As shown in Figure 6, the 4′-C-aminopropyl, (R)-5′-C-aminopropyl, and (S)-5′-C-aminopropyl-modified ssRNAs remained intact after 6 h of incubation, whereas the unmodified ssRNA was completely degraded by nucleases in serum within 1 h of incubation. Thus, it was found that incorporation of the (R)-5′-C-aminopropyl and (S)-5′-Caminopropyl analogs 2 and 3 highly enhanced the stability of ssRNA in a buffer containing bovine serum. The half-lives of RNA 1, RNA 2, RNA 3, and RNA 4 were 0.09, 2.9, 1.6, and 3.9 h, respectively. Next, we assessed the nuclease resistance of the

modified siRNAs (Table S5) in a buffer containing 10% bovine serum. As shown in Figure 7, the unmodified siRNA was completely degraded within 1 h of incubation, while the fulllength siRNAs remained unaffected in the 4′-C-aminoalkyl, (R)5′-C-aminoalkyl, and (S)-5′-C-aminoalkyl-modified siRNAs even after 6 h of incubation.

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CONCLUSIONS We successfully synthesized the (R)-5′-C-aminopropyl-2′-Omethyl- and (S)-5′-C-aminopropyl-2′-O-methyl-modified urH


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Figure 6. PAGE analysis of ssRNA (8 μM) treated in a buffer containing 3% bovine serum. Fluorescein-labeled RNAs 1−4 (300 pmol) were incubated in a buffer containing 3% bovine serum, and subsequently, the reaction mixtures at various incubation times (0, 0.5, 1, 5, and 30 min and 1, 3, and 6 h) were analyzed by denaturing PAGE.

Figure 7. PAGE analysis donated stability of siRNA (6 μM) in 10% bovine serum. Fluorescein-labeled siRNAs 29−32 (600 pmol) were incubated in a buffer containing 10% bovine serum, and subsequently, the reaction mixtures at various incubation times (0, 0.5, 1, 5, and 30 min and 1, 3, and 6 h) were analyzed by denaturing PAGE. Chemical shifts (δ) are given in parts per million (ppm) from CDCl3 (7.26 ppm) for 1H NMR spectra and from CDCl3 (77.2 ppm) for 13C NMR spectra. The abbreviations s, d, t, q, and m signify singlet, doublet, triplet, quadruplet, and multiplet, respectively. High resolution mass spectra (HRMS) were obtained in positive ion electrospray ionization (ESI-TOF) mode. 6-O-[(1,1-Dimethylethyl)diphenylsilyl]-1,2-O-isopropylidene-3-O-(4-methoxybenzyl)-α-D-allofuranose (5). TBDPSCl (6.09 mL, 23.4 mmol) was added to a solution of compound 4 (7.25 g, 21.3 mmol) in pyridine (72 mL) under argon. The reaction mixture was stirred for 17 h at room temperature; the mixture was extracted with ethyl acetate and saturated sodium bicarbonate, and the organic layer was washed water and brine and dried by Na2SO4. After concentration, the resulting residue was purified by column chromatography (25% ethyl acetate in hexane) to afford desired product 5 as a colorless oil (11.34 g, 19.6 mmol, 92%). 1H NMR (600 MHz, CDCl3) δ 7.69−7.65 (m, 4 H), 7.44−7.35 (m, 6 H), 7.19−7.17 (m, 2 H), 6.81−6.80 (m, 2 H), 5.71 (d, J = 4.2 Hz, 1 H), 4.58 (d, J = 11.0 Hz, 1 H), 4.48 (t, J = 4.1 Hz, 1 H), 4.43 (d, J = 11.7 Hz, 1 H), 4.06 (dd, J = 8.6, 3.4 Hz, 1 H), 4.02−4.04 (m, 1 H), 3.89 (dd, J = 8.6, 4.8 Hz, 1 H), 3.79 (s, 3 H), 3.75− 3.73 (m, 2H), 2.55 (d, J = 3.5 Hz, 1 H), 1.55 (s, 3 H), 1.34 (s, 3 H), 1.06 (s, 9 H); 13C {1H} NMR (151 MHz, CDCl3) δ 159.5, 135.8, 135.7, 129.9, 129.9, 129.8, 127.9, 113.9, 113.0, 104.2, 78.0, 77.9, 77.3, 72.1, 71.9, 64.7, 55.4, 27.0, 27.0, 26.7, 19.4; HRMS (ESI-TOF) m/z Calcd for C33H42NaO7Si (M + Na)+, 601.2598; found, 601.2581. 5-O-Benzyl-6-O-[(1,1-dimethylethyl)diphenylsilyl]-1,2-Oisopropylidene-3-O-(4-methoxybenzyl)-α-D-allofuranose (6). NaH (1.57 g, 39.2 mmol) was added to a solution of compound 5 (11.34 g, 19.6 mmol) in DMF (113 mL) under argon. After the mixture was stirred for 30 min at room temperature, BnBr (4.66 mL, 39.2 mmol) and NaI (0.59 g, 3.92 mmol) were added to the reaction mixture in an ice bath. The reaction mixture was stirred for 5 h at 90 °C; methanol (20 mL) was added to the reaction mixture in an ice bath. The mixture was extracted with ethyl acetate and saturated sodium bicarbonate; the organic layer was washed with brine and dried by Na2SO4. After concentration, the resulting residue was purified by

idines and evaluated the properties of siRNAs containing these nucleoside analogs and compared them to that of the 4′-Caminopropyl-2′-O-methyl-modified uridine. It was found that the (S)-5′-C-aminopropyl-2′-O-methyl modification improved the thermal stability of dsRNA and siRNA compared to the 4′-Caminopropyl-2′-O-methyl and (R)-5′-C-aminopropyl-2′-Omethyl modifications. Incorporation of the (R)-5′-C-aminopropyl-2′-O-methyl- and (S)-5′-C-aminopropyl-2′-O-methyl modifications at the passenger strand was well tolerated for forming the RISC and eliciting RNAi activity. The 5′-Caminopropyl-2′-O-methyl modifications at the guide strand were also tolerable for eliciting RNAi activity except for in the seed region. It was revealed that the 4′-C-aminopropyl-2′-Omethyl and 5′-C-aminopropyl-2′-O-methyl modifications significantly increased the stability of ssRNAs and siRNAs in a buffer containing bovine serum. Among all the modifications, (S)-5′-C-aminopropyl-2′-O-methyl modification enhanced the stability of ssRNAs in serum to the highest degree. Hence, the (S)-5′-C-aminopropyl modification might be a potential candidate for the application of siRNAs as therapeutics.

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EXPERIMENTAL SECTION

General Remark. All chemicals and dry solvents (THF, DMF, DMSO, CH2Cl2, CH3CN, and pyridine) were obtained from commercial sources and used without any further purification. Thin layer chromatography (TLC) was performed on silica gel plates precoated with fluorescent indicator with visualization by UV light or by dipping into a solution of 5% (v/v) concentrated H2SO4 in mixture of p-anisaldehyde and methanol and then heating. Silica gel (63-210 mesh) was used for column chromatography. 1H NMR (400, 500, or 600 MHz), 13C {1H} NMR (101, 126, or 151 MHz), 31P NMR (162 MHz) were recorded on 400, 500, or 600 MHz NMR equipment. CDCl3 or DMSO-d6 was used as a solvent for obtaining NMR spectra. I


Article


7.34−7.27 (m, 5H), 7.23−7.22 (m, 2H), 6.85−6.83 (m, 2H), 5.65 (d, J = 3.4 Hz, 1H), 4.67 (d, J = 11.7 Hz, 1H), 4.65 (d, J = 11.7 Hz, 1H), 4.52 (d, J = 11.7 Hz, 1H), 4.50 (d, J = 11.0 Hz, 1H), 4.48 (t, J = 4.1 Hz, 1H), 4.17 (dd, J = 8.9, 2.1 Hz, 1H), 4.08−4.04 (m, 2H), 3.94 (dd, J = 8.6, 4.1 Hz, 1H), 3.78 (s, 3H), 3.73−3.70 (m, 1H), 2.42−2.37 (m, 1H), 2.33− 2.28 (m, 1H), 1.96−1.90 (m, 1H), 1.80−1.74 (m, 1H), 1.57 (s, 3H), 1.33 (s, 3H), 1.20 (t, J = 6.9 Hz, 3H); 13C NMR {1H} (151 MHz, CDCl3) δ 173.5, 159.5, 138.9, 129.9, 129.8, 128.4, 127.8, 127.5, 113.9, 113.0, 104.0, 81.1, 77.9, 77.3, 77.1, 73.5, 71.9, 60.5, 55.4, 31.0, 27.1, 26.8, 26.2, 14.3; HRMS (ESI-TOF) m/z Calcd for C28H36KO8 (M + K)+, 539.2047; found, 539.2026. (R)-5-O-Benzyl-5-C-hydroxypropyl-1,2-O-isopropylidene-3O-(4-methoxybenzyl)-α-D-ribose (11). Under argon atmosphere, LiAlH4 (0.55 g, 14.4 mmol) was added to THF (35 mL); the mixture was stirred in ice bath. A solution of compound 10 (3.61 g, 7.20 mmol) in THF (10 mL) was added slowly to the stirring solution. The reaction mixture was stirred for 30 min in an ice bath. Saturated potassium sodium tartrate aqueous solution was added to the stirring solution; the mixture was stirred for 30 min in an ice bath. The mixture was extracted with ethyl acetate and water; the organic layer was washed with brine. The organic layer was dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (50% ethyl acetate in hexane) to afford desired product 11 as a colorless oil (3.28 g, 7.14 mmol, 99%). 1H NMR (400 MHz, CDCl3) δ 7.35−7.27 (m, 7H), 6.87−6.85 (m, 2H), 5.67 (d, J = 3.7 Hz, 1H), 4.71 (d, J = 11.9 Hz, 1H), 4.68 (d, J = 12.8 Hz, 1H), 4.56 (d, J = 11.5 Hz, 1H), 4.51 (t, J = 4.1 Hz, 1H), 4.49 (d, J = 11.0 Hz, 1H), 4.20 (dd, J = 8.7, 1.8 Hz, 1H), 3.96 (dd, J = 8.7, 4.6 Hz, 1H), 3.80 (s, 3H), 3.72−3.69 (m, 1H), 3.57−3.53 (m, 2H), 1.70−1.63 (m, 2H), 1.59 (s, 3H), 1.56−1.52 (m, 1H), 1.45−1.42 (m, 1H), 1.36 (s, 3H); 13C {1H} NMR (101 MHz, CDCl3) δ 159.5, 138.9, 129.9, 129.8, 128.4, 127.9, 127.6, 113.9, 113.0, 104.0, 81.2, 78.2, 77.9, 77.0, 73.5, 71.8, 62.8, 55.4, 29.6, 27.4, 27.1, 26.8; HRMS (ESITOF) m/z Calcd for C26H34NaO7 (M + Na)+, 481.2202; found, 481.2208. (R)-5-O-Benzyl-1,2-O-isopropylidene-3-O-(4-methoxybenzyl)-5-C-p-toluenesulfonyloxypropyl-α-D-ribose (12). Under argon atmosphere, p-TsCl (0.15 g, 0.79 mmol) and pyridine (0.13 mL, 1.58 mmol) were added to a solution of compound 11 (0.10 g, 0.23 mmol) in CH2Cl2 (1.0 mL) at 0 °C. The mixture was stirred for 3 h at room temperature. The mixture was extracted with CHCl3 and saturated NaHCO3 aqueous solution; organic layer was washed with brine. The organic layer was dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (25% ethyl acetate in hexane) to afford desired product 12 as a colorless oil (0.13 g, 0.22 mmol, 96%). 1H NMR (600 MHz, CDCl3) δ 7.74 (d, J = 8.3 Hz, 2H), 7.32−7.27 (m, 5H), 7.25−7.21 (m, 4H), 6.88−6.85 (m, 2H), 5.65 (d, J = 4.1 Hz, 1H), 4.64 (d, J = 11.7 Hz, 2H), 4.50 (t, J = 4.1 Hz, 1H), 4.46 (d, J = 11.6 Hz, 1H), 4.46 (d, J = 12.4 Hz, 1H), 4.10 (dd, J = 6.5 Hz, 2.1 Hz, 1H), 3.94−3.87 (m, 3H), 3.81 (s, 3H), 3.59−3.58 (m, 1H), 2.42 (s, 3H), 1.82−1.74 (m, 1H), 1.64−1.59 (m, 2H), 1.56 (s, 3H), 1.47−1.42 (m, 1H), 1.35 (s, 3H); 13C {1H} NMR (101 MHz, CDCl3) δ 159.6, 144.8, 138.8, 133.2, 129.9, 129.9, 129.7, 128.4, 128.0, 127.8, 127.6, 113.9, 113.0, 104.0, 81.1, 77.8, 77.4, 77.0, 73.4, 71.8, 70.6, 55.4, 27.1, 26.9, 26.8, 25.7, 21.8; HRMS (ESITOF) m/z Calcd for C33H40NaO9S (M + Na)+, 635.2291; found, 635.2282. (R)-5-C-Azidopropyl-5-O-benzyl-1,2-O-isopropylidene-3-O(4-methoxybenzyl)-α-D-ribose (13). Under argon atmosphere, NaN3 (3.65 g, 56.2 mmol) was added to a solution of compound 12 (4.10 g, 6.70 mmol) in DMF (40 mL). The mixture was stirred for 4 h at 60 °C. The mixture was extracted with ethyl acetate and brine. The organic layer was dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (15% ethyl acetate in hexane) to afford desired product 13 as a colorless oil (2.63 g, 5.43 mmol, 81%). 1H NMR (600 MHz, CDCl3) δ 7.34−7.27 (m, 7H), 6.88−6.85 (m, 2H), 5.68 (d, J = 3.4 Hz, 1H), 4.70 (d, J = 11.6 Hz, 1H), 4.67 (d, J = 11.7 Hz, 1H), 4.54 (d, J = 11.7 Hz, 1H), 4.52 (t, J = 4.1 Hz, 1H), 4.49 (d, J = 11.6, 1H), 4.17 (dd, J = 8.6, 1.4 Hz, 1H), 3.96 (dd, J = 8.6, 4.8 Hz, 1H), 3.80 (s, 3H), 3.70−3.68 (m, 1H), 3.17 (t, J = 6.8 Hz, 2H), 1.75−1.64 (m, 2H), 1.59 (s, 3H), 1.53−1.46 (m, 2H), 1.36 (s,

column chromatography (10−15% ethyl acetate in hexane) to afford desired product 6 as a yellow oil (8.69 g, 13.0 mmol, 66%). 1H NMR (600 MHz, CDCl3) δ 7.68−7.64 (m, 4 H), 7.44−7.39 (m, 2H), 7.35− 7.31 (m, 5H), 7.30−7.26 (m, 4H), 7.12−7.10 (m, 2H), 6.76−6.74 (m, 2 H), 5.67 (d, J = 4.1 Hz, 1 H), 4.74 (d, J = 11.6 Hz, 1 H), 4.68 (d, J = 11.6 Hz, 1 H), 4.54 (d, J = 11.6 Hz, 1 H), 4.47 (t, J = 4.1 Hz, 1 H), 4.38 (d, J = 11.0 Hz, 1 H), 4.26 (dd, J = 8.2, 2.1 Hz, 1 H), 4.00−3.96 (m, 2H), 3.80−3.79 (m, 2H), 3.78 (s, 3 H), 1.57 (s, 3 H), 1.34 (s, 3 H), 1.04 (s, 9 H); 13C {1H} NMR (151 MHz, CDCl3) δ 159.4, 139.3, 135.8, 135.8, 133.7, 133.6, 129.9, 129.7, 128.3, 127.8, 127.8, 127.6, 127.4, 113.8, 113.0, 104.2, 79.6, 79.3, 78.0, 74.1, 71.8, 64.1, 55.4, 27.1, 27.0, 26.8, 19.3; HRMS (ESI-TOF) m/z Calcd for C40H48NaO7Si (M + Na)+, 691.3067; found, 691.3074. 5-O-Benzyl-1,2-O-isopropylidene-3-O-(4-methoxybenzyl)α-D-allofuranose (7). n-Tetrabuthylammonium fluoride (8.24 mL of a 1 M solution in THF) was added to a solution of compound 6 (3.67 g, 5.49 mmol) in THF (37 mL). The reaction mixture was stirred for 19 h at room temperature; the mixture was extracted with ethyl acetate and water, and the organic layer was washed with brine and dried by Na2SO4. After concentrating, the resulting residue was purified by column chromatography (25% ethyl acetate in hexane) to afford desired product 7 as a colorless oil (2.11 g, 4.91 mmol, 89%). 1H NMR (600 MHz, CDCl3) δ 7.34−7.32 (m, 2H), 7.30−7.28 (m, 5H), 6.88− 6.86 (m, 2H), 5.72 (d, J = 4.1 Hz, 1H), 4.72 (d, J = 12.4 Hz, 1H), 4.70 (d, J = 11.0 Hz, 1H), 4.64 (d, J = 11.7 Hz, 1H), 4.56 (t, J = 3.4 Hz, 1H), 4.50 (d, J = 11.7 Hz, 1H), 4.21 (dd, J = 8.9, 2.0 Hz, 1H), 4.03 (dd, J = 8.9, 4.1 Hz, 1H), 3.89−3.87 (m, 1H), 3.80 (s, 3H), 3.67−3.65 (m, 2H), 2.42 (t, J = 5.5 Hz, 1H), 1.59 (s, 1H), 1.36 (s, 1H); 13C {1H} NMR (151 MHz, CDCl3) δ 159.6, 138.5, 129.9, 128.9, 128.3, 127.6, 127.5, 113.8, 112.9, 104.0, 80.0, 77.9, 77.2, 76.3, 73.3, 71.7, 61.9, 55.2, 26.8, 26.5; HRMS (ESI-TOF) m/z Calcd for C24H30NaO7 (M + Na)+, 453.1889; found, 453.1864. (R)-5-O-Benzyl-5-C-[2-ethoxycarbonyl-(E)-vinyl]-1,2-O-isopropylidene-3-O-(4-methoxybenzyl)-α-D-ribose (9). TEMPO (16 mg, 0.10 mmol) and KBr (0.5 mL of a 2 M solution in water) were added to a solution of compound 7 (4.47 g, 10.4 mmol) in CH2Cl2 (17 mL); the mixture was stirred in an ice bath. After NaHCO3 (0.15 g, 0.018 mmol) in 1.5 M NaClO aq. (8.5 mL) was added to the mixture, the reaction mixture was stirred for 40 min in an ice bath. The mixture was extracted with CHCl3 and water; the organic layer was washed with 10% aq. HCl, 10% aq. Na2S2O3, water, and brine. The organic layer was dried by Na2SO4, filtered, and concentrated. Under argon, NaH (0.49 g, 12.2 mmol) was added to a solution of (EtO)2P(O)CH2COOEt (2.43 mL, 12.2 mmol) in THF (25 mL), and the mixture was stirred for 10 min in an ice bath. The residue in THF (25 mL) was added slowly to the stirring solution. The reaction mixture was stirred for 30 min at room temperature. The mixture was extracted with ethyl acetate and water; the organic layer was washed with brine. The organic layer was dried by Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (25% ethyl acetate in hexane) to afford desired product 9 as a yellow oil (4.15 g, 8.33 mmol, 80%). 1H NMR (600 MHz, CDCl3) δ 7.34−7.32 (m, 2H), 7.29−7.27 (m, 3H), 7.23− 7.22 (m, 2H), 6.86−6.83 (m, 3H), 5.88 (dd, J = 15.6, 1.2 Hz, 1H), 5.71 (d, J = 4.1 Hz, 1H), 4.63 (d, J = 11.7 Hz, 1H), 4.62 (d, J = 11.6 Hz, 1H), 4.51−4.48 (m, 2H), 4.26−4.22 (m, 1H), 4.23−4.21 (m, 1H), 4.19 (q, J = 6.9 Hz, 2H), 3.82−3.80 (m, 1H), 3.80 (s, 3H), 1.58 (s, 3H), 1.35 (s, 3H), 1.29 (t, J = 7.6 Hz, 3H); 13C {1H} NMR (151 MHz, CDCl3) δ 165.9, 159.6, 143.6, 138.0, 130.0, 129.6, 128.5, 127.8, 127.8, 123.8, 114.0, 113.3, 104.4, 80.8, 77.6, 77.2, 76.6, 72.2, 71.8, 60.6, 55.4, 27.1, 26.8, 14.4; HRMS (ESI-TOF) m/z Calcd for C28H34NaO8 (M + Na)+, 521.2151; found, 521.2158. (R)-5-O-Benzyl-5-C-(2-ethoxycarbonylethyl)-1,2-O-isopropylidene-3-O-(4-methoxybenzyl)-α-D-ribose (10). 5% Pd/C (1.45 g, 13.6 mmol) and ammonium formate (3.18 g, 50.5 mmol) were added to a solution of compound 9 (5.04 g, 10.1 mmol) in ethyl acetate (25 mL); the mixture was stirred for 5.5 h at room temperature. The mixture was filtered through Celite, and the filtrate was concentrated. The crude material was purified by column chromatography (25% ethyl acetate in hexane) to afford desired product 10 as a colorless oil (4.57 g, 9.13 mmol, 90%). 1H NMR (600 MHz, CDCl3) δ J


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The Journal of Organic Chemistry 3H); 13C NMR {1H} (151 MHz, CDCl3) δ 159.6, 138.9, 129.9, 129.8, 128.4, 127.9, 127.6, 113.9, 113.0, 104.0, 81.2, 77.9, 77.5, 77.1, 73.5, 71.8, 55.4, 51.5, 28.2, 27.1, 26.8, 25.7; HRMS (ESI-TOF) m/z Calcd for C26H33N3NaO6 (M + Na)+, 506.2267; found, 506.2294. (R)-5-C-Azidopropyl-5-O-benzyl-1,2-O-diacetyl-3-O-(4-methoxybenzyl)-α-D-ribose (14). Compound 13 (3.79 g, 7.84 mmol) was dissolved in 50% AcOH aqueous solution (68 mL); the mixture was stirred for 24 h at 70 °C. EtOH was added to the mixture and concentrated. The crude material was purified by column chromatography (50% ethyl acetate in hexane) to afford a colorless oil compound. This compound was dissolved in pyridine (10 mL), and Ac2O (6.7 mL, 72.4 mmol) was added. The mixture was stirred for 6 h at room temperature. The mixture was poured into water in an ice bath and extracted with ethyl acetate and saturated NaHCO3 aqueous solution; organic layer was washed with brine. The organic layer was dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (25% ethyl acetate in hexane) to afford desired product 14 as a colorless oil (2.05 g, 3.89 mmol, 50%). 1H NMR (600 MHz, CDCl3) δ 7.36−7.27 (m, 5H), 7.20 (d, J = 8.3 Hz, 2H), 6.86−6.85 (m, 2H), 6.11 (s, 1H), 5.33 (d, J = 4.8 Hz, 1H), 4.71 (d, J = 11.7 Hz, 1H), 4.55 (d, J = 11.7 Hz, 1H), 4.53 (d, J = 10.3 Hz, 1H), 4.41 (d, J = 10.3 Hz, 1H), 4.40 (dd, J = 8.3, 4.8 Hz, 1H), 4.15 (dd, J = 7.6, 2.8 Hz, 1H), 3.80 (s, 3H), 3.67−3.65 (m, 1H), 3.18−3.15 (m, 2H), 2.13(s, 3H), 1.85 (s, 3H), 1.70−1.50 (m, 4H); 13C {1H} NMR (151 MHz, CDCl3) δ 170.0, 169.5, 159.7, 138.6, 130.1, 129.4, 128.5, 127.7, 127.6, 114.0, 98.6, 83.8, 77.5, 75.9, 74.0, 73.1, 73.0, 55.4, 51.5, 28.0, 25.3, 21.0, 20.9; HRMS (ESI-TOF) m/z Calcd for C27H33N3NaO8 (M + Na)+, 550.2165; found, 550.2175. 2′-O-Acetyl-(R)-5′-C-azidopropyl-5′-O-benzyl-3′-O-(4methoxybenzyl)uridine (15). N,O-Bis(trimethylsilyl)acetamide (3.6 mL, 13.9 mmol) was added to a solution of compound 14 (2.04 g, 3.87 mmol) and uracil (0.74 g, 6.60 mmol) in CH3CN (20 mL). The mixture was stirred for 1 h at 55 °C. This solution was cooled to 0 °C, and TMSOTf (1.6 mL, 8.75 mmol) was added in dropwise; the mixture was warmed to 55 °C and stirred for 2 h. The reaction was quenched with saturated NaHCO3 aqueous solution and extracted with ethyl acetate and brine. The organic layer was dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (50% ethyl acetate in hexane) to afford desired product 15 as a colorless oil (2.04 g, 3.51 mmol, 91%). 1H NMR (600 MHz, CDCl3) δ 8.82 (s, 1H), 7.39−7.29 (m, 6H), 7.22 (d, J = 8.3 Hz, 2H), 6.88 (d, J = 8.2 Hz, 2H), 6.04 (d, J = 4.1 Hz, 1H), 5.23 (t, J = 5.5 Hz, 1H), 5.18 (dd, J = 7.9, 2.1 Hz, 1H), 4.73 (d, J = 11.6 Hz, 1H), 4.53 (d, J = 11.0 Hz, 1H), 4.45 (d, J = 11.0 Hz, 1H), 4.40 (d, J = 11.0 Hz, 1H), 4.30 (t, J = 6.2 Hz, 1H), 4.10 (dd, J = 5.5, 2.8 Hz, 1H), 3.81 (s, 3H), 3.75−3.74 (m, 1H), 3.24 (t, J = 6.8 Hz, 2H), 2.12 (s, 3H), 1.77− 1.72 (m, 1H), 1.66−1.52 (m, 3H); 13C {1H} NMR (151 MHz, CDCl3) δ 170.1, 162.9, 159.7, 150.2, 140.1, 137.7, 130.0, 129.2, 128.8, 128.3, 127.7, 114.0, 102.7, 87.4, 83.8, 78.4, 74.5, 74.2, 73.0, 72.9, 55.4, 51.4, 27.4, 25.4, 20.9; HRMS (ESI-TOF) m/z Calcd for C29H33KN5O8 (M + Na)+, 618.1966; found, 618.1988. (R)-5′-C-Azidopropyl-5′-O-benzyl-3′-O-(4-methoxybenzyl)uridine (16). K2CO3 (0.38 g, 2.75 mmol) was added to a solution of compound 15 (0.53 g, 0.91 mmol) in methanol (5.2 mL). The mixture was stirred for 1 h at room temperature. The mixture was extracted with ethyl acetate and water; the organic layer was washed with brine. The organic layer was dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (70% ethyl acetate in hexane) to afford desired product 16 as a white solid (0.46 g, 0.86 mmol, 95%). 1H NMR (400 MHz, CDCl3) δ 8.04 (s, 1H), 7.40− 7.34 (m, 3H), 7.29−7.27 (m, 3H), 7.25−7.21 (m, 2H), 6.91−6.89 (m, 2H), 5.85 (d, J = 5.5 Hz, 1H), 5.26 (dd, J = 8.3, 2.3 Hz, 1H), 4.74 (d, J = 11.0 Hz, 1H), 4.60 (d, J = 11.4 Hz, 1H), 4.53 (d, J = 11.0 Hz, 1H), 4.41 (d, J = 11.0 Hz, 1H), 4.17−4.07 (m, 3H), 3.82 (s, 3H), 3.71−3.69 (m, 1H), 3.29−3.27 (m, 2H), 2.87 (d, J = 7.3 Hz, 1H), 1.78−1.59 (m, 4H); 13 C {1H} NMR (151 MHz, CDCl3) δ 163.0, 159.9, 150.6, 140.3, 137.7, 130.0, 128.8, 128.7, 128.3, 127.7, 114.2, 102.7, 89.4, 83.7, 78.8, 75.9, 73.5, 72.8, 72.6, 55.4, 51.5, 27.5, 25.2; HRMS (ESI-TOF) m/z Calcd for C28H33N5NaO7 (M + Na)+, 560.2121; found, 560.2129.

(R)-5′-C-Azidopropyl-5′-O-benzyl-3′-O-(4-methoxybenzyl)2′-O-methyluridine (17). NaH (0.10 g, 2.55 mmol) was added to a solution of compound 16 (0.46 g, 0.86 mmol) in THF (4.6 mL) at 0 °C. Then, CH3I (0.27 mL, 4.31 mmol) was added in dropwise and stirred at 0 °C. Saturated NaHCO3 aqueous solution was added. The mixture was extracted with ethyl acetate and saturated NaHCO3 aqueous solution. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (60% ethyl acetate in hexane) to afford desired product 17 as a white solid (0.43 g, 0.78 mmol, 90%). 1H NMR (600 MHz, CDCl3) δ 8.31 (s, 1H), 7.55 (d, J = 7.5 Hz, 1H), 7.39−7.33 (m, 4H), 7.28−7.26 (m, 3H), 6.90−6.88 (m, 2H), 5.94 (d, J = 2.1 Hz, 1H), 4.96 (dd, J = 8.3, 2.1 Hz, 1H), 4.77 (d, J = 11.0 Hz, 1H), 4.56 (d, J = 11.6 Hz, 1H), 4.49 (d, J = 11.0 Hz, 1H), 4.41 (d, J = 11.0 Hz, 1H), 4.21 (dd, J = 7.2, 2.8 Hz, 1H), 4.11 (dd, J = 7.2, 4.8 Hz, 1H), 3.83−3.82 (m, 1H), 3.82 (s, 3H), 3.64 (dd, J = 4.8, 2.8 Hz, 1H), 3.49 (s, 3H), 3.31−3.24 (m, 2H), 1.88−1.83 (m, 1H), 1.72−1.65 (m, 3H); 13C {1H} NMR (151 MHz, CDCl3) δ 163.4, 159.7, 150.1, 140.3, 137.7, 129.9, 129.2, 128.8, 128.3, 127.7, 114.0, 102.0, 87.9, 82.5, 82.5, 78.2, 73.9, 72.6, 72.1, 58.6, 55.4, 51.5, 27.1, 25.3; HRMS (ESI-TOF) m/z Calcd for C28H33N5NaO7 (M + Na)+, 574.2278; found, 574.2253. (R)-5′-C-Azidopropyl-5′-O-benzyl-2′-O-methyluridine (18). H2O (96 μL) was added to a solution of compound 17 (0.43 g, 0.78 mmol) in CH2Cl2 (1.85 mL), and was cooled in ice bath. DDQ (0.21 g, 0.94 mmol) was added to the mixture, the mixture was stirred for 2 h in ice bath. After 2 h, the reaction mixture was warmed from 0 °C to room temperature and stirred for 2 h. Saturated NaHCO3 aqueous solution (5 mL) was added to the stirring solution, and the solution was filtered through a Celite pad. The filtrate was extracted with CHCl3 and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The crude material was purified by column chromatography (60−70% ethyl acetate in hexane) to afford desired product 18 as a white solid (0.27 g, 0.62 mmol, 79%). 1H NMR (600 MHz, CDCl3) δ 8.33 (s, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.41−7.32 (m, 5H), 5.96 (d, J = 2.3 Hz, 1H), 4.98 (dd, J = 8.2, 2.3 Hz, 1H), 4.81 (d, J = 11.0 Hz, 1H), 4.45 (d, J = 11.0 Hz, 1H), 4.41−4.36 (m, 1H), 3.99 (dd, J = 6.9, 2.3 Hz, 1H), 3.91−3.87 (m, 1H), 3.63 (dd, J = 5.5, 2.3 Hz, 1H), 3.58 (s, 3H), 3.34 (t, J = 6.9 Hz, 2H), 2.75 (d, J = 8.2 Hz, 1H), 1.96−1.68 (m, 4H); 13C {1H} NMR(151 MHz, CDCl3) δ 163.3, 150.2, 140.0, 137.6, 128.8, 128.3, 127.8, 102.2, 86.7, 84.7, 84.2, 78.3, 72.9, 67.8, 58.8, 51.5, 27.2, 25.4; HRMS (ESITOF) m/z Calcd for C20H25N5NaO6 (M + Na)+, 454.1703; found, 454.1676. (R)-5′-C-Azidopropyl-5′-O-benzyl-3′-O-[(1,1-dimethylethyl)diphenylsilyl]-2′-O-methyluridine (19). Under argon atmosphere, imidazole (0.42 g, 6.17 mmol), TBDPSCl (0.80 mL, 3.08 mmol) was added to a solution of compound 18 (0.27 g, 0.62 mmol) in DMF (2.7 mL); the mixture was stirred for 18 h at room temperature. The mixture was extracted with ethyl acetate and water. The organic layer was washed with saturated NaHCO3 aqueous solution and brine, dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (40% ethyl acetate in hexane) to afford desired product 19 as a white solid (0.39 g, 0.59 mmol, 95%). 1H NMR (600 MHz, CDCl3) δ 7.91 (s, 1H), 7.73−7.71 (m, 2H), 7.67−7.66 (m, 2H), 7.44 (t, J = 7.6 Hz, 2H), 7.39−7.33 (m, 8H), 7.23−7.22 (m, 2H), 5.88 (d, J = 3.5 Hz, 1H), 4.94 (dd, J = 8.2, 2.8 Hz, 1H), 4.74 (d, J = 11.9 Hz, 1H), 4.41 (dd, J = 6.2, 4.8 Hz, 1H), 4.38 (d, J = 11.0 Hz, 1H), 4.22 (dd, J = 6.2, 2.1 Hz, 1H), 3.74−3.72 (m, 1H), 3.24−3.15 (m, 2H), 3.10 (dd, J = 4.8, 3.4 Hz, 1H), 3.07 (s, 3H), 1.80−1.75 (m, 1H), 1.64−1.56 (m, 3H), 1.09 (s, 9H); 13C {1H} NMR (151 MHz, CDCl3) δ 163.1, 149.9, 140.1, 137.7, 136.2, 136.0, 133.0, 130.2, 130.2, 128.8, 128.2, 127.9, 127.8, 127.5, 101.9, 86.5, 84.1, 83.3, 78.7, 72.7, 69.9, 57.6, 51.3, 27.1, 27.1, 25.8, 19.5; HRMS (ESI-TOF) m/z Calcd for C36H43KN5O6Si (M + K)+, 708.2620; found, 708.2625. (R)-5′-C-Azidopropyl-3′-O-[(1,1-dimethylethyl)diphenylsilyl]-2′-O-methyluridine (20). Under argon atmosphere, BCl3 (3.50 mL of a 1 M solution in CH2Cl2) was added to a solution of compound 19 (0.39 g, 0.59 mmol) in CH2Cl2 (6.0 mL) at −78 °C. The mixture was stirred at −78 °C. After 3 h, the reaction mixture was warmed to −30 °C and 50% methanol in CH2Cl2 (10 mL) was added. The mixture was extracted with CHCl3 and saturated NaHCO3 K


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product 23 as a white solid (0.55 g, 0.77 mmol, 85%). 1H NMR (600 MHz, DMSO-d6) δ 11.39 (s, 1H), 9.26 (t, J = 5.4 Hz, 1H), 7.43 (d, J = 7.8 Hz, 2H), 7.31−7.28 (m, 7H), 7.23−7.21 (m, 1H), 6.89 (dd, J = 9.0, 2.4 Hz, 4H), 5.69 (d, J = 6.0 Hz, 1H), 5.30−5.29 (m, 1H), 5.15 (d, J = 6.6 Hz, 1H), 4.12 (dd, J = 10.8, 6.0 Hz,1H), 3.76−3.75 (m, 1H), 3.74 (s, 6H), 3.59 (t, J = 5.4 Hz, 1H), 3.30−3.29 (m, 1H), 3.27 (s, 3H), 2.92− 2.86 (m, 2H), 1.35−1.23 (m, 4H); 13C {1H} NMR (151 MHz, DMSOd6) δ 162.8, 158.2, 150.4, 146.3, 140.6, 136.2, 136.1, 130.3, 130.2, 127.9, 127.7, 126.7, 113.0, 102.0, 86.1, 85.7, 84.4, 81.2, 72.7, 67.7, 57.6, 55.0, 55.0, 27.1, 24.2; HRMS (ESI-TOF) m/z Calcd for C36H38F3N3NaO9 (M + Na)+, 736.2458; found, 736.2473. 3′-O-[2-Cyanoethoxy(diisopropylamino)phosphino]-(R)-5′O-(4,4′-dimethoxytrityl)-2′-O-methyl-5′-C-trifluoroacetylaminopropyluridine (24). Under argon atmosphere, 1H-tetrazole (17 mg, 0.25 mmol) in DMF (0.5 mL), 1-methylimidazole (7.8 μL, 0.098 mmol), and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphoroamidite (0.13 mL, 0.40 mmol) were added to a solution of compound 23 (0.19 g, 0.27 mmol) in DMF (1.5 mL). After being stirred at room temperature for 2.5 h, the mixture was extracted with ethyl acetate and saturated NaHCO3 aqueous solution. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (50% ethyl acetate in hexane) to afford desired product 24 as a white solid (0.18 g, 0.20 mmol, 73%). 1H NMR (600 MHz, CDCl3) δ 8.51 (s, 1H), 7.46−7.44 (m, 2H), 7.36−7.32 (m, 4H), 7.28−7.24 (m, 2H), 7.21−7.18 (m, 1.5H), 7.10 (d, J = 8.2 Hz, 0.5H), 6.84−6.80 (m, 4H), 6.65 (t, J = 5.4 Hz, 0.5H), 6.47 (t, J = 5.4 Hz, 0.5H), 5.85 (d, J = 5.5 Hz, 0.5H), 5.79 (d, J = 4.1 Hz, 0.5H), 5.07 (dd, J = 8.3, 2.1 Hz, 1H), 4.81−4.78 (m, 0.5H), 4.60−4.58 (m, 0.5H), 4.08−4.05 (m, 1H), 3.96−3.89 (m, 1H), 3.83− 3.81 (m, 1H), 3.78 (d, J = 2.1 Hz, 6H), 3.76−3.71 (m, 0.5H), 3.69− 3.63 (m, 1.5H), 3.46 (s, 1.5H), 3.46−3.44 (m, 0.5H), 3.43 (s, 1.5H), 3.32−3.30 (m, 0.5H), 3.11−2.95 (m, 2H), 2.70−2.58 (m, 2H), 1.56− 1.33 (m, 4H), 1.22−1.20 (m, 10H), 1.15−1.11 (m, 1H); 13C {1H} NMR (151 MHz, CDCl3) δ 162.9, 159.0, 158.8, 157.0, 150.2, 146.3, 146.2, 140.6, 140.4, 136.0, 135.9, 135.8, 130.8, 130.5, 130.5, 128.2, 128.0, 127.2, 118.2, 118.1, 113.4, 113.4, 113.3, 113.3, 102.5, 87.9, 87.6, 87.4, 86.7, 84.5, 83.8, 82.6, 82.1, 73.8, 73.1, 70.8, 70.7, 69.9, 69.8, 58.5, 57.7, 57.6, 55.4, 43.6, 43.5, 43.4, 43.3, 28.2, 25.3, 25.1, 25.0, 24.7, 20.7, 20.6; 31P NMR (162 MHz, CDCl3) δ 150.7, 148.9; HRMS (ESI-TOF) m/z Calcd for C45H55F3KN5O10P (M + K)+, 952.3276; found, 952.3250. (S)-5′-C-Allyl-3′-O-[(1,1-dimethylethyl) Dimethylsilyl]-2′-Omethyluridine (27) and (R)-5′-C-Allyl-3′-O-[(1,1-dimethylethyl) Dimethylsilyl]-2′-O-methyluridine (28). 50% DMSO in CH2Cl2 (26 mL) was added to the mixture of compound 25 (1.92 g, 5.15 mmol) and 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC, 3.95 g, 20.6 mmol) under argon atmosphere, and the mixture was stirred at −5 °C. Then, CHCl2CO2H (0.25 mL, 1.94 mmol) was added dropwise to the mixture and stirred for 1.5 h at −5 °C. The mixture was extracted with cold ethyl acetate and 5% sodium bicarbonate; the organic layer was washed with brine, dried by Na2SO4, filtered, and concentrated. The residue was dissolved in CH2Cl2 (38 mL) under argon atmosphere, and the mixture was stirred at 0 °C. Then, allyltrimethylsilane (4.1 mL, 25.8 mmol) and the boron trifluoride−ethyl ether complex (3.3 mL, 25.8 mmol) were added to the mixture, and it was stirred for 1 h at 0 °C. The mixture was extracted with CHCl3 and saturated sodium bicarbonate; the organic layer was washed with brine, dried by Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (50% ethyl acetate in hexane) to afford desired product 27 as a white solid (0.92 g, 2.22 mmol, 43%). 1H NMR (600 MHz, CDCl3) δ 9.31 (s, 1H), 7.85 (d, J = 8.4 Hz, 1H), 5.84−5.79 (m, 1H), 5.75 (d, J = 3.4 Hz, 1H), 5.73 (dd, J = 7.6, 1.4 Hz, 1H), 5.20−5.17 (m, 2H), 4.32 (t, J = 5.5 Hz, 1H), 3.96− 3.95 (m, 1H), 3.88 (t, J = 4.1 Hz, 1H), 3.77−3.74 (m, 1H), 3.49 (s, 3H), 2.63 (d, J = 5.5 Hz, 1H), 2.44−2.36 (m, 2H), 0.90 (s, 9H), 0.10 (s, 3H), 0.09 (s, 3H); 13C {1H} NMR (151 MHz, CDCl3) δ 163.6, 150.4, 141.9, 134.2, 119.1, 102.3, 90.2, 86.1, 82.9, 70.6, 69.1, 58.5, 39.1, 25.8, 18.3, −4.6, −4.7; HRMS (ESI-TOF) m/z Calcd for C19H32N2NaO6Si (M + Na)+, 435.1927; found, 435.1957.

aqueous solution. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (60−70% ethyl acetate in hexane) to afford desired product 20 as a white solid (0.34 g, 0.58 mmol, 98%). 1H NMR (600 MHz, CDCl3) δ 8.09 (s, 1H), 7.73−7.72 (m, 2H), 7.68−7.66 (m, 2H), 7.48−7.45 (m, 2H), 7.42−7.38 (m, 5H), 5.73 (dd, J = 8.3, 2.1 Hz, 1H), 5.55 (d, J = 6.9 Hz, 1H), 4.35 (dd, J = 4.8, 2.0 Hz, 1H), 4.09 (dd, J = 6.9, 4.8 Hz, 1H), 4.00 (t, J = 2.1 Hz, 1H), 3.66−3.62 (m, 1H), 3.54 (d, J = 2.1 Hz, 1H), 3.49 (d, J = 4.8 Hz, 1H), 3.19 (s, 3H), 3.17−3.11 (m, 2H), 1.64−1.60 (m, 1H), 1.46−1.41 (m, 1H), 1.09 (s, 9H), 1.02−0.98 (m, 1H), 0.92−0.88 (m, 1H); 13C {1H} NMR (151 MHz, CDCl3) δ 163.1, 150.4, 143.8, 136.2, 136.1, 133.2, 133.0, 130.3, 130.2, 128.0, 127.8, 102.7, 92.7, 89.9, 81.0, 71.3, 70.2, 58.5, 51.2, 29.4, 27.0, 25.7, 19.5; HRMS (ESI-TOF) m/z Calcd for C29H37N5NaO6Si (M + Na)+, 602.2411; found, 602.2389. (R)-5′-C-Azidopropyl-5′-O-(4,4′-dimethoxytrityl)-3′-O-[(1,1dimethylethyl)diphenylsilyl]-2′-O-methyluridine (21). Under argon atmosphere, DMTrCl (2.07 g, 6.11 mmol) and 2,6-lutidine (0.85 mL, 7.34 mmol) were added to a solution of compound 20 (0.71 g, 1.22 mmol) in pyridine (4.1 mL). After being stirred at 40 °C for 48 h, the mixture was extracted with ethyl acetate and water. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (40% ethyl acetate in hexane) to afford desired product 21 as a yellow solid (0.81 g, 0.92 mmol, 75%). 1H NMR (600 MHz, CDCl3) δ 8.52 (s, 1H), 7.76−7.75 (m, 2H), 7.72−7.71 (m, 2H), 7.51−7.47 (m, 2H), 7.43−7.40 (m, 4H), 7.30−7.28 (m, 2H), 7.21−7.18 (m, 7H), 6.97 (d, J = 8.4 Hz, 1H), 6.75 (d, J = 7.2 Hz, 4H), 5.93 (d, J = 4.8 Hz, 1H), 5.06 (dd, J = 7.8, 1.8 Hz, 1H), 4.50−4.49 (m, 1H), 4.12−4.11 (m, 1H), 3.77 (s, 3H), 3.77 (s, 3H), 3.29−3.27 (m, 2H), 3.04 (s, 3H), 2.85−2.81 (m, 1H), 2.75−2.71 (m, 1H), 1.38−1.34 (m, 1H), 1.20−1.13 (m, 1H), 1.10 (s, 9H), 1.05−0.99 (m, 1H); 13C {1H} NMR (151 MHz, CDCl3) δ 163.3, 158.8, 158.7, 150.2, 146.2, 140.2, 136.4, 136.0, 135.9, 135.8, 133.5, 133.0, 130.7, 130.4, 130.2, 130.1, 128.1, 127.9, 127.7, 127.0, 113.3, 113.2, 113.1, 102.4, 87.4, 86.4, 84.9, 82.6, 73.7, 70.7, 57.9, 55.3, 55.3, 51.1, 27.8, 27.0, 25.3, 19.5; HRMS (ESI-TOF) m/z Calcd for C50H55KN5O8Si (M + K)+, 920.3457; found, 920.3458. (R)-5′-O-(4,4′-Dimethoxytrityl)-3′-O-[(1,1-dimethylethyl)diphenylsilyl]-2′-O-methyl-5′-C-trifluoroacetylaminopropyluridine (22). Under argon atmosphere, Ph3P (0.13 g, 0.50 mmol) and water (0.15 mL, 8.32 mmol) were added to a solution of compound 21 (0.17 g, 0.19 mmol) in THF (1.7 mL). After being stirred at 40 °C for 3 h, the mixture was concentrated. The residue was dissolved in CH2Cl2 (1.7 mL). Et3N (40 μL, 0.29 mmol) and CF3CO2Et (70 μL, 0.59 mmol) were added to the mixture. After being stirred at room temperature for 25 h, the mixture was extracted with ethyl acetate and water. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (40% ethyl acetate in hexane) to afford desired product 22 as a yellow solid (0.16 g, 0.17 mmol, 87%). 1H NMR (600 MHz, CDCl3) δ 8.43 (d, J = 1.8 Hz, 1H), 7.75−7.74 (m, 2H), 7.70− 7.69 (m, 2H), 7.51−7.47 (m, 2H), 7.41 (t, J = 7.6 Hz, 4H), 7.29−7.27 (m, 2H), 7.21−7.18 (m, 7H), 6.95 (d, J = 7.8 Hz, 1H), 6.74 (d, J = 8.4 Hz, 4H), 5.94 (s, 1H), 5.89 (d, J = 5.4 Hz, 1H), 5.14 (dd, J = 7.8, 1.8 Hz, 1H), 4.45 (t, J = 3.6 Hz, 1H), 4.11 (t, J = 3.0 Hz, 1H), 3.77 (s, 3H), 3.76 (s, 3H), 3.31−3.26 (m, 2H), 3.05 (s, 3H), 2.88−2.84 (m, 2H), 1.28− 1.24 (m, 1H), 1.13−1.11 (m, 1H), 1.09 (s, 9H), 1.04−0.99 (m, 2H); 13 C {1H} NMR (151 MHz, CDCl3) δ 62.8, 158.9, 158.8, 150.1, 146.1, 140.6, 136.4, 136.1, 135.8, 133.8, 132.8, 130.6, 130.4, 130.3, 130.1, 128.1, 128.0, 127.9, 127.9, 127.1, 113.3, 113.2, 102.5, 87.4, 87.1, 85.2, 82.3, 73.5, 70.7, 58.0, 55.4, 55.4, 39.6, 27.7, 27.0, 25.1, 19.6; HRMS (ESI-TOF) m/z Calcd for C52H56F3KN3O9Si (M + K)+, 990.3375; found, 990.3391. (R)-5′-O-(4,4′-Dimethoxytrityl)-2′-O-methyl-5′-C-trifluoroacetylaminopropyluridine (23). Under argon atmosphere, ntetrabuthylammonium fluoride (1.40 mL of a 1 M solution in THF) was added to a solution of compound 22 (0.86 g, 0.91 mmol) in THF (8.6 mL). After being stirred at room temperature for 6 h, the mixture was concentrated. The resulting residue was purified by column chromatography (60−70% ethyl acetate in hexane) to afford desired L


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The Journal of Organic Chemistry Another product 28 as a white solid (0.049 g, 0.12 mmol, 2%). 1H NMR (600 MHz, CDCl3) δ 9.72 (s, 1H), 7.21 (d, J = 8.4 Hz, 1H), 5.88−5.84 (m, 1H), 5.76 (dd, J = 7.2, 1.2 Hz, 1H), 5.69 (d, J = 4.8 Hz, 1H), 5.16−5.13 (m, 2H), 4.49 (t, J = 4.2 Hz, 1H), 4.09−4.07 (m, 2H), 4.00−3.96 (m, 1H), 3.42 (s, 3H), 2.52−2.48 (m, 1H), 2.45 (d, J = 3.6 Hz, 1H), 2.21−2.16 (m, 1H), 0.92 (s, 9H), 0,13 (s, 3H), 0.12 (s, 3H); 13 C {1H} NMR (151 MHz, CDCl3) δ 163.8, 150.3, 141.4, 134.3, 118.5, 102.7, 91.4, 83.9, 83.4, 71.9, 68.5, 58.9, 38.1, 25.9, 25.8, 18.5, −4.4, −4.9; HRMS (ESI-TOF) m/z Calcd for C19H32N2NaO6Si (M + Na)+, 435.1927; found, 435.1954. (S)-5′-C-Allyl-5′-O-(4,4′-dimethoxytrityl)-3′-O-[(1,1dimethylethyl)dimethylsilyl]-2′-O-methyluridine (29). 4,4′-Dimethoxytrityl chloride (DMTrCl, 0.90 g, 2.65 mmol) and 2,6-lutidine (0.37 mL, 3.18 mmol) were added to a solution of compound 28 (0.22 g, 0.53 mmol) in pyridine (1.3 mL). After being stirred at 40 °C for 67 h, the mixture was extracted with ethyl acetate and water. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (70% ethyl acetate in hexane) to afford desired product 29 as a yellow solid (0.32 g, 0.44 mmol, 83%). 1H NMR (600 MHz, CDCl3) δ 8.92 (s, 1H), 8.39 (d, J = 8.3 Hz, 1H), 7.36 (d, J = 7.6 Hz, 2H), 7.29− 7.24 (m, 6H), 6.83 (dd, J = 8.9, 6.2 Hz, 4H), 6.05 (d, J = 3.4 Hz, 1H), 5.57 (dd, J = 8.3, 2.0 Hz, 1H), 5.27−5.21 (m, 1H), 4.88 (d, J = 10.3 Hz, 1H), 4.79 (d, J = 17.2 Hz, 1H), 4.27 (t, J = 4.8 Hz, 1H), 4.01 (d, J = 5.5 Hz, 1H), 3.82−3.80 (m, 7H), 3.46 (s, 3H), 3.45−3.44 (m, 1H), 2.42− 2.37 (m, 1H), 2.10−2.07 (m, 1H), 0.77 (s, 9H), −0.03 (s, 3H), −0.20 (s, 3H); 13C {1H} NMR (151 MHz, CDCl3) δ 163.4, 158.9, 150.4, 145.8, 140.4, 136.3, 136.2, 133.5, 130.7, 130.7, 128.6, 127.9, 127.4, 118.2, 113.2, 102.4, 87.6, 86.7, 85.2, 84.2, 73.6, 70.0, 58.1, 55.4, 36.1, 25.8, 25.7, 18.1, −4.3; HRMS (ESI-TOF) m/z Calcd for C40H50KN2O8Si (M + K)+, 753.2974; found, 753.2979. (S)-5′-O-(4,4′-Dimethoxytrityl)-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-5′-C-hydroxypropyl-2′-O-methyluridine (30). 9Borabicyclo [3.3.1] nonane (9-BBN, 0.5 M in THF, 7.0 mL) was added dropwise to a solution of compound 29 (0.88 g, 1.22 mmol) in THF (18 mL) and stirred for 17 h at room temperature. Water was added to the reaction mixture until evolution of gas ceased. 3 N NaOH solution (2.6 mL) was added, and then, slowly 30% aqueous hydrogen peroxide solution (1.1 mL) was added while keeping the temperature between 30 and 50 °C. The mixture was stirred and extracted with water and ethyl acetate. The organic layer was washed with neutral phosphate buffer solution and brine, dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (50% ethyl acetate in hexane) to afford desired product 30 as a white solid (0.43 g, 0.59 mmol, 49%). 1H NMR (600 MHz, DMSO-d6) δ 11.46 (s, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.37 (d, J = 7.6 Hz, 2H), 7.32−7.22 (m, 7H), 6.90 (dd, J = 8.9, 6.2 Hz, 4H), 5.84 (d, J = 4.8 Hz, 1H), 5.58 (d, J = 8.3 Hz, 1H), 4.23 (dd, J = 8.2, 4.8 Hz, 2H), 3.99 (t, J = 4.8 Hz, 1H), 3.86 (dd, J = 4.9, 3.0 Hz, 1H), 3.74 (s, 3H), 3.74 (s, 3H), 3.32 (s, 3H), 3.29− 3.25 (m, 1H), 3.01 (dd, J = 11.6, 6.8 Hz, 2H), 1.50−1.44 (m, 1H), 1.25−1.21 (m, 1), 1.16−1.10 (m, 1H), 0.83−0.80 (m, 1H), 0.77 (s, 9H), −0.02 (s, 3H), −0.16 (s, 3H); 13C {1H} NMR (151 MHz, DMSOd6) δ 163.0, 158.2, 150.4, 146.1, 139.7, 136.2, 136.0, 130.2, 130.1, 128.0, 127.7, 126.8, 113.1, 102.0, 86.5, 85.7, 84.9, 82.2, 73.5, 69.8, 60.3, 57.3, 55.1, 28.6, 27.2, 25.6, 25.5, 17.6, −5.0, −5.2; HRMS (ESI-TOF) m/z Calcd for C40H52N2NaO9Si (M + Na)+, 755.3340; found, 755.3369. (S)-5′-O-(4,4′-Dimethoxytrityl)-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-2′-O-methyl-5′-C-p-toluenesulfonyloxypropyluridine (31). Pyridine (0.35 mL, 4.14 mmol) and p-toluenesulfonyl chloride (p-TsCl, 0.40 g, 2.07 mmol) were added to a solution of 30 (0.43 g, 0.59 mmol) in CH2Cl2 (4.5 mL) under argon atmosphere at 0 °C, and the mixture was stirred at room temperature. The mixture was extracted with CHCl3 and saturated NaHCO3 aqueous solution; the organic layer was washed with brine. The organic layer was dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (30−40% ethyl acetate in hexane) to afford desired product 31 as a white solid (0.29 g, 0.33 mmol, 56%). 1H NMR (600 MHz, CDCl3) δ 8.27 (d, J = 7.6 Hz, 1H), 8.12 (d, J = 2.0 Hz, 1H), 7.65 (d, J = 8.3 Hz, 2H), 7.32−7.21 (m, 11H), 6.84−6.80 (m, 4H), 5.97 (d, J = 3.4 Hz, 1H), 5.56 (dd, J = 8.2, 2.8 Hz, 1H), 4.24 (t, J = 5.5 Hz,

1H), 3.91 (dd, J = 6.2, 2.0 Hz, 1H), 3.82 (s, 3H), 3.81 (s, 3H), 3.77 (dd, J = 5.5, 3.4 Hz, 1H), 3.71−3.68 (m, 1H), 3.62−3.58 (m, 1H), 3.46 (s, 3H), 3.41 (dt, J = 10.3, 2.8 Hz, 1H), 2.44 (s, 3H), 1.65−1.58 (m, 1H), 1.44−1.36 (m, 1H), 1.35−1.29 (m, 1H), 1.04−0.96 (m, 1H), 0.78 (s, 9H), −0.03 (s, 3H), −0.21 (s, 3H); 13C {1H} NMR (151 MHz, CDCl3) δ 163.1, 159.0, 150.2, 146.0, 144.8, 140.3, 136.2, 136.0, 133.1, 130.7, 129.9, 128.5, 127.9, 127.4, 113.3, 102.4, 87.6, 86.9, 85.1, 84.1, 73.3, 69.9, 69.9, 58.1, 55.4, 27.4, 25.8, 25.7, 25.2, 21.8, 18.1, −4.3; HRMS (ESI-TOF) m/z Calcd for C47H58N2NaO11SSi (M + Na)+, 909.3428; found, 909.3427. (S)-5′-C-Azidopropyl-5′-O-(4,4′-dimethoxytrityl)-3′-O-[(1,1dimethylethyl)dimethylsilyl]-2′-O-methyluridine (32). NaN3 (0.18 g, 2.76 mmol) was added to a solution of 31 (0.29 g, 0.33 mmol) in DMF (3.0 mL) under argon atmosphere, and the mixture was stirred for 11 h at 60 °C. The mixture was extracted with ethyl acetate and brine. The organic layer was dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (30−40% ethyl acetate in hexane) to afford desired product 32 as a white solid (0.22 g, 0.29 mmol, 90%). 1H NMR (400 MHz, CDCl3) δ 8.34 (d, J = 8.2 Hz, 1H), 8.17 (s, 1H), 7.35−7.33 (m, 2H), 7.30−7.28 (m, 4H), 7.25−7.23 (m, 2H), 6.84−6.81 (m, 4H), 6.01 (d, J = 3.2 Hz, 1H), 5.56 (dd, J = 8.2, 2.3 Hz, 1H), 4.28 (t, J = 5.5 Hz, 1H), 4.00 (dd, J = 6.0, 1.8 Hz, 1H), 3.80 (s, 3H), 3.80 (s, 3H), 3.48 (s, 3H), 3.00−2.96 (m, 1H), 2.91−2.86 (m, 1H), 1.73−1.68 (m, 1H), 1.36−1.34 (m, 2H), 0.95−0.91 (m, 1H), 0.79 (s, 9H), −0.02 (s, 3H), −0.20 (s, 3H); 13C {1H} NMR (151 MHz, DMSO-d6) δ 163.0, 158.3, 150.4, 146.0, 139.9, 136.1, 135.9, 130.2, 130.1, 127.9, 127.7, 126.8, 113.1, 102.0, 99.5, 86.5, 86.0, 84.7, 82.1, 73.0, 69.8, 57.3, 55.1, 50.4, 27.9, 25.6, 25.5, 24.4, 17.6, −4.8; HRMS (ESI-TOF) m/z Calcd for C40H51N5NaO8Si (M + Na)+, 780.3405; found, 780.3388. (S)-5′-O-(4,4′-Dimethoxytrityl)-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-2′-O-methyl-5′-C-trifluoroacetylaminopropyluridine (33). Ph3P (0.19 g, 0.74 mmol) and H2O (0.22 mL, 11.8 mmol) were added to a solution of compound 32 (0.22 g, 0.29 mmol) in THF (4.5 mL). After being stirred at 40 °C for 16 h, the mixture was concentrated in vacuo. The residue was dissolved in CH2Cl2 (2.1 mL). Et3N (61 μL, 0.44 mmol) and CF3CO2Et (100 μL, 0.88 mmol) were added to the mixture. After being stirred at room temperature for 5 h, the mixture was extracted with ethyl acetate and water. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (40% ethyl acetate in hexane) to afford desired product 33 as a white solid (0.23 g, 0.28 mmol, 94%). 1H NMR (600 MHz, CDCl3) δ 8.76 (d, J = 1.4 Hz, 1H), 8.32 (d, J = 8.3 Hz, 1H), 7.34−7.21 (m, 9H), 6.82 (dd, J = 8.9, 6.2 Hz, 4H), 6.11 (s, 1H), 5.98 (d, J = 3.5 Hz, 1H), 5.55 (dd, J = 8.2, 2.0 Hz, 1H), 4.29 (t, J = 5.5 Hz, 1H), 3.99 (dd, J = 6.2, 2.1 Hz, 1H), 3.80 (s, 3H), 3.80 (s, 3H), 3.49 (s, 3H), 3.47 (s, 1H), 3.06−2.99 (m, 2H), 1.70−1.65 (m, 1H), 1.35−1.26 (m, 2H), 0.99− 0.94 (m, 1H), 0.79 (s, 9H), −0.02 (s, 3H), −0.20 (s, 3H); 13C {1H} NMR (151 MHz, CDCl3) δ 163.2, 159.0, 150.3, 145.8, 140.3, 136.3, 136.0, 130.7, 130.6, 128.6, 128.0, 127.5, 113.3, 102.4, 87.6, 87.0, 85.1, 84.1, 73.1, 69.9, 58.1, 55.4, 39.7, 28.7, 25.8, 25.7, 25.0, 18.1, −4.8; HRMS (ESI-TOF) m/z Calcd for C42H52F3N3NaO9Si (M + Na)+, 850.3323; found, 850.3299. (S)-5′-O-(4,4′-Dimethoxytrityl)-2′-O-methyl-5′-C-trifluoroacetylaminopropyluridine (34). n-Tetrabuthylammonium fluoride (TBAF, 0.40 mL of a 1 M solution in THF) was added to a solution of compound 33 (0.21 g, 0.26 mmol) in THF (2.0 mL) at room temperature under argon atmosphere. After being stirred at room temperature for 14 h, the mixture was concentrated. The residue was purified by column chromatography (70−80% ethyl acetate in hexane) to afford desired product 34 as a white solid (0.16 g, 0.23 mmol, 84%). 1 H NMR (600 MHz, DMSO-d6) δ 11.44 (d, J = 2.1 Hz, 1H), 9.21 (t, J = 5.5 Hz, 1H), 7.76 (d, J = 7.6 Hz, 1H), 7.40 (d, J = 7.6 Hz, 2H), 7.30− 7.27 (m, 6H), 7.21 (t, J = 6.9 Hz, 1H), 6.87 (t, J = 8.9 Hz, 4H), 5.79 (d, J = 5.5 Hz, 1H), 5.60 (dd, J = 8.3, 2.0 Hz, 1H), 5.05 (d, J = 6.8 Hz, 1H), 4.11 (dd, J = 12.4, 5.5 Hz, 1H), 3.86−3.84 (m, 2H), 3.74 (s, 3H), 3.73 (s, 3H), 3.36 (s, 3H), 3.35−3.34 (m, 1H), 2.82−2.79 (m, 2H), 1.28− 1.20 (m, 3H), 1.07−1.01 (m, 1H); 13C {1H} NMR (151 MHz, DMSOd6) δ 163.0, 158.2, 158.1, 150.4, 146.2, 140.1, 136.3, 136.2, 130.2, 127.9, M


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organic layer was washed with brine. The organic layer was dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (100% CHCl3) to afford desired product 37 as a white solid (0.10 g, 0.19 mmol, 54%). 1H NMR (400 MHz, CDCl3) δ 8.02 (s, 1H), 7.17 (d, J = 7.6 Hz, 1H), 5.94−5.85 (m, 1H), 5.80 (d, J = 7.2 Hz, 1H), 5.74 (dd, J = 7.6, 2.4 Hz, 1H), 5.12−5.08 (m, 2H), 4.52 (dd, J = 3.6, 1.6 Hz,), 4.17 (dd, J = 8.0, 3.2 Hz, 1H), 4.10 (dd, J = 9.6, 2.0 Hz, 1H), 4.06−4.02 (m, 1H), 3.38 (s, 3H), 2.49−2.43 (m, 1H), 2.34− 2.26 (m, 1H), 1.13−1.01 (m, 24H); 13C {1H} NMR (151 MHz, CDCl3) δ 163.0, 150.0, 141.8, 133.9, 118.0, 102.9, 91.0, 84.2, 83.7, 70.0, 67.7, 58.9, 37.9, 17.6, 17.5, 17.5, 17.4, 17.3,17.3, 17.1, 13.6, 13.4, 12.7, 12.6; HRMS (ESI-TOF) m/z Calcd for C25H44NaN2O7Si2 (M + Na)+, 563.2574; found, 563.2585. Synthesis of the Controlled Pore Glass Support 39 or 41. N,N-Dimethyl-4-aminopyridine (DMAP, 34 mg, 0.28 mmol) and succinic anhydride (56 mg, 0.56 mmol) were added to a solution of 23 (100 mg, 0.14 mmol) in pyridine (1.0 mL) under argon atmosphere, and the reaction mixture was stirred for 20 h at room temperature. The mixture was extracted with ethyl acetate and water. The organic layer was washed with saturated NaHCO3 aqueous solution and brine, dried over Na2SO4, filtered, and concentrated. The residue was dissolved in DMF (1.4 mL) under argon atmosphere. Aminopropyl controlled pore glass (210 mg, CPG) and 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 27 mg, 0.14 mmol) were added to a solution of 38, and the mixture was kept at room temperature for 5 days. After the resin was washed with pyridine, 15 mL of capping solution (0.1 M DMAP in pyridine/Ac2O 9:1) was added to the resin and the mixture was kept at room temperature for 1 day. The resin was washed with pyridine, EtOH, and CH3CN and dried under vacuum to give solid support 39. The amount of loaded nucleoside 38 to the solid support is 25 μmol/g from the calculation of released 4,4′demethoxytrityl cation by a solution of 70% HClO4/EtOH (3:2, v/ v). In a similar manner, the solid support 41 was obtained in 45 μmol/g of loading amount. Solid-Phase Oligonucleotide Synthesis. The synthesis was carried out with a DNA/RNA synthesizer by the phosphoramidite method. After the synthesis, the CPG beads were treated with 10% dimethylamine in CH3CN for 5 min followed by a rinse with CH3CN to selectively remove cyanoethyl groups. Then, the oligomers were cleaved from CPG beads and deprotected by treatment with concentrated NH3 solution/40% methylamine (1:1, v/v) for 10 min at 65 °C. 2′-O-TBDMS groups were removed by Et3N·3HF (125 μL) in DMSO (100 μL) at 65 °C for 1.5 h. The reaction was quenched with 0.1 M TEAA buffer (pH 7.0), and the mixture was desalted using a Sep-Pak C18 cartridge. The oligonucleotides were purified by 20% PAGE containing 7 M urea to give highly purified oligonucleotides. MALDI-TOF/MS Analysis of ONs. The spectra were obtained with a time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). A solution of 3-hydroxypicolinic acid (3-HPA) and diammonium hydrogen citrate in H2O was used as the matrix. Data of synthetic ONs: RNA 1 m/z = 3839.92 (Calcd for C126H149N26O92P11 [M − H] − , 3840.68); RNA 3 m/z = 4053.58 (Calcd for C138H177N29O92P11 [M − H]−, 4054.10); RNA 4 m/z = 4055.41 (Calcd for C138H177N29O92P11 [M − H]−, 4054.10); RNA 5 m/z = 3606.31 (Calcd for C110H133N55O67P10 [M − H]−, 3607.68); RNA 6 m/z = 6505.97 (Calcd for C194H245N65O150P20 [M − H]−, 6506.43); RNA 8 m/z = 7075.93 (Calcd for C226H317N73O150P20 [M − H]−, 7075.55); RNA 9 m/z = 7075.02 (Calcd for C226H317N73O150P20 [M − H]−, 7075.55); RNA 10 m/z = 6817.01 (Calcd for C203H247N86O144P20 [M − H]−, 6815.76); RNA 19 m/z = 6578.68 (Calcd for C198H254N66O150P20 [M − H]−, 6577.57); RNA 20 m/z = 6577.53 (Calcd for C198H254N66O150P20 [M − H]−, 6577.57); RNA 22 m/z = 6577.91 (Calcd for C198H254N66O150P20 [M − H]−, 6577.57); RNA 23 m/z = 6577.40 (Calcd for C198H254N66O150P20 [M − H]−, 6577.57); RNA 25 m/z = 6577.78 (Calcd for C198H254N66O150P20 [M − H]−, 6577.57); RNA 26 m/z = 6576.53 (Calcd for C198H254N66O150P20 [M − H] − , 6577.57); RNA 27 m/z = 6792.03 (Calcd for C210H281N69O150P20 [M − H]−, 6790.99); RNA 28 m/z = 6790.44 (Calcd for C210H281N69O150P20 [M − H]−, 6790.99); RNA 29 m/z = 6791.55 (Calcd for C210H281N69O150P20 [M − H]−, 6790.99); RNA 30

127.6, 126.7, 113.0, 102.0, 86.3, 85.9, 84.4, 82.0, 73.0, 68.2, 59.8, 55.0, 27.7, 24.1, 20.8; HRMS (ESI-TOF) m/z Calcd for C36H38F3N3NaO9 (M + Na)+, 736.2458; found, 736.2443. 3′-O-[2-Cyanoethoxy(diisopropylamino)phosphino]-(S)-5′O-(4,4′-dimethoxytrityl)-2′-O-methyl-5′-C-trifluoroacetylaminopropyluridine (35). 1H-Tetrazole (71 mg, 1.02 mmol) in DMF (1.0 mL), N-methylimidazole (30 μL, 0.40 mmol), and 2-cyanoethylN,N,N′,N′-tetraisopropylphosphoroamidite (0.52 mL, 1.65 mmol) were added to a solution of 34 in DMF (3.0 mL) under argon atmosphere. After being stirred at room temperature for 1 h, the mixture was extracted with ethyl acetate and saturated NaHCO3 aqueous solution. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (60−70% ethyl acetate in hexane) to afford desired 35 as a white solid (0.45 g, 0.49 mmol, 89%). 1 H NMR (600 MHz, CDCl3) δ 8.65 (s, 1H), 8.16 (d, J = 8.3 Hz, 0.5H), 8.11 (d, J = 8.2 Hz, 0.5H), 7.39−7.22 (m, 9H), 6.85−6.81 (m, 4H), 6.43−6.42 (m, 0.5H), 6.24 (s, 0.5H), 6.02 (d, J = 4.8 Hz, 0.5H), 5.99 (d, J = 4.1 Hz, 0.5H), 5.66 (d, J = 8.2 Hz, 0.5H), 5.60 (d, J = 8.2 Hz, 0.5H), 4.34−4.29 (m, 1H), 4.16 (dd, J = 4.8, 2.0 Hz, 0.5H), 4.11 (dd, J = 5.5, 2.0 Hz, 0.5H), 4.06−4.03 (m, 1H), 3.81−3.67 (m, 7H), 3.59− 3.43 (m, 6.5H), 3.40−3.38 (m, 0.5H), 3.03−2.95 (m, 2H), 2.58 (dt, J = 6.2, 1.4 Hz, 1H), 2.35−2.23 (m, 1H), 1.73−1.63 (m, 1.5H), 1.38−1.22 (m, 4.5H), 1.17−1.13 (m, 9H), 1.01 (d, J = 6.8 Hz, 2H); 13C {1H} NMR (151 MHz, CDCl3) δ 163.1, 163.0, 159.0, 150.4, 150.3, 146.0, 145.8, 140.1, 136.2, 136.1, 136.0, 130.7, 130.6, 128.6, 128.5, 127.9, 127.4, 127.3, 117.8, 113.2, 102.6, 102.5, 87.8, 87.7, 87.3, 86.8, 84.5, 84.1, 83.8, 82.7, 73.6, 73.4, 70.5, 70.4, 58.8. 58.5, 58.4, 58.1, 55.4, 43.4, 43.4, 43.3, 39.7, 39.7, 28.5, 28.3, 24.8, 24.7, 20.5, 20.3; 31P NMR (162 MHz, CDCl3) δ 151.4, 150.6. HRMS (ESI-TOF) m/z Calcd for C45H55F3KN5O10P (M + K)+, 952.3276; found, 952.3273. (S)-5′-C-Allyl-2′-O-methyl-3′,5′-O-(1,1,3,3-tetraisopropyl1,3-disiloxandiyl)uridine (36). TBAF (0.50 mL of a 1 M solution in THF) was added to a solution of 27 in THF (5.0 mL) at room temperature under argon atmosphere. After being stirred at room temperature for 2 h, the mixture was concentrated. The residue was extracted with ethyl acetate and saturated NaHCO3 aqueous solution. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. After the crude material was coevaporated with pyridine for three times, the residue was dissolved in pyridine (1.5 mL). 1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPDSCl2, 0.16 mL, 0.50 mmol) was added to the reaction mixture in an ice bath under argon atmosphere. After being stirred at room temperature for 10 h, the mixture was concentrated. The residue was extracted with CHCl3 and saturated NaHCO3 aqueous solution; the organic layer was washed with brine. The organic layer was dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (20−30% ethyl acetate in hexane) to afford desired product 36 as a white solid (76 mg, 0.14 mmol, 56%). 1H NMR (400 MHz, DMSOd6) δ 11.39 (s, 1H), 7.63 (d, J = 7.8 Hz, 1H), 5.88−5.81 (m, 1H), 5.60 (s, 1H), 5.53 (dd, J = 7.8, 1.8 Hz, 1H), 5.17−5.08 (m, 2H), 4.14 (dd, J = 9.2, 4.1 Hz, 1H), 3.98−3.94 (m, 1H), 3.92−3.88 (m, 2H), 3.51 (s, 3H), 2.54−2.51 (m, 1H), 2.44−2.39 (m, 1H), 1.08−0.97 (m, 24H); 13C {1H} NMR (151 MHz, CDCl3) δ 164.1, 150.2, 139.7, 133.6, 118.5, 101.7, 88.3, 84.2, 81.7, 69.5, 69.0, 59.3, 38.3, 17.7, 17.7, 17.6, 17.3, 17.2, 17.1, 16.9, 13.6, 13.2, 12.5; HRMS (ESI-TOF) m/z Calcd for C25H44KN2O7Si2 (M + K)+, 579.2324; found, 579.2349. (R)-5′-C-Allyl-2′-O-methyl-3′,5′-O-(1,1,3,3-tetraisopropyl1,3-disiloxandiyl)uridine (37). TBAF (0.84 mL of a 1 M solution in THF) was added to a solution of 28 in THF (7.8 mL) at room temperature under argon atmosphere. After being stirred at room temperature for 6 h, the mixture was concentrated. The residue was extracted with ethyl acetate and saturated NaHCO3 aqueous solution. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. After the crude material was coevaporated with pyridine for three times, the residue was dissolved in pyridine (7.0 mL). TIPDSCl2 (0.16 mL, 0.50 mmol) was added to the reaction mixture in an ice bath under argon atmosphere. After being stirred at room temperature for 40 h, the mixture was concentrated. The residue was extracted with CHCl3 and saturated NaHCO3 aqueous solution; the N


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The Journal of Organic Chemistry m/z = 6791.47 (Calcd for C210H281N69O150P20 [M − H]−, 6790.99); RNA 31 m/z = 6791.81 (Calcd for C210H281N69O150P20 [M − H]−, 6790.99); RNA 32 m/z = 6791.69 (Calcd for C210H281N69O150P20 [M − H] − , 6790.99); RNA 34 m/z = 6887.36 (Calcd for C207H256N87O144P20 [M − H]−, 6886.90); RNA 35 m/z = 6886.27 (Calcd for C207H256N87O144P20 [M − H]−, 6886.90); RNA 37 m/z = 6887.45 (Calcd for C207H256N87O144P20 [M − H]−, 6886.90); RNA 38 m/z = 6888.03 (Calcd for C207H256N87O144P20 [M − H]−, 6886.90); RNA 39 m/z = 6887.79 (Calcd for C207H256N87O144P20 [M − H]−, 6886.90); RNA 40 m/z = 6888.96 (Calcd for C207H256N87O144P20 [M − H] − , 6886.90); RNA 41 m/z = 6888.03 (Calcd for C207H256N87O144P20 [M − H]−, 6886.90); RNA 44 m/z = 7709.07 (Calcd for C250H317N92O153P21 [M − H]−, 7710.19); RNA 45 m/z = 7709.37 (Calcd for C250H317N92O153P21 [M − H]−, 7710.19). Thermal Denaturation Study. The solution containing 3.0 μM duplex in a buffer of 10 mM sodium phosphate (pH 7.0) containing 100 mM NaCl was heated at 100 °C and then cooled gradually to room temperature and used for the thermal denaturation study. Thermally induced transitions were monitored at 260 nm with a UV/vis spectrometer fitted with temperature controller in quartz cuvettes with a path length of 1.0 cm. The sample temperature was increased by 0.5 °C/min. The thermodynamic parameters of the duplexes on duplex formation were determined by calculations based on the slope of a 1/Tm vs ln(CT/4) plot, where CT (1, 3, 6, 12, 15, 21, 16, 30, and 60 μM) is the total concentration of single strands. Molecular Modeling Study. The starting structures of 4′-Caminopropyl-2′-O-methyluridine, (R)-5′-C-aminopropyl-2′-O-methyluridine, and (S)-5′-C-aminopropyl-2′-O-methyluridine were modeled using the BIOPOYMER module implemented in SYBYL package. The geometry of each nucleoside was optimized using Gaussian0933 at the HF/6-31G* level of theory, and RESP charges34 were derived using the Antechamber RESP fitting procedure. The force field parameters of the modified nucleosides were obtained using the general AMBER force field (GAFF).35 Each nucleoside was solvated in TIP3P water model36 using the LEaP module of AMBER 1637 and placed with periodic boundary conditions in a cubic box with side length of 42.0 Å. Counterion (Cl−) was added into the solution to neutralize the charge of the systems. The solvent was energy minimized by 2500 steps of the steepest descent method and then followed by 2500 steps of the conjugate gradient method, applying a restraint force constant of 2 kcal/ (mol·Å2) to the nucleoside. After that, the minimization was carried for the entire system by 5000 steps of the conjugate gradient method without any restraints. Initially, the temperature of the system was heated gradually from 0 to 300 K over a period of 100 ps of NVT dynamics, with 2 kcal/ (mol·Å2) constraints for the nucleoside. This was followed by 50 ps of NPT equilibration at 300 K and 1 bar pressure with 2 kcal/ (mol·Å2) constraints for the nucleoside. After the equilibration phase, 500 ns productive MD simulations were performed in an NPT ensemble at 300 K and 1 bar pressure without any restraints. During all MD simulations, a time step of 2 fs was used. The cutoff of the van der Waals interaction was set to be 10 Å; the SHAKE algorithm38 was used to constrain all bonds involving hydrogen atoms, and particle-mesh Ewald (PME)39 was used for long-range electrostatic interactions. The temperature is regulated by the Langevin dynamics40 with a collision frequency of 2.0 ps−1. All the MD simulations were performed using the PMEMD module of AMBER 16 package on NVIDIA GPU cluster. Trajectory analysis was done using the cpptraj module in AMBER 16 and examined visually using VMD 1.9.2. CD Spectroscopy. All CD spectra were recorded at 25 °C. The following instrument settings were used: resolution, 0.1 nm; response, 1.0 s; speed, 50 nm/min; accumulation, 10. Dual-Luciferase Assay. HeLa cells were transfected with the psiCHECK-2 (Promega) reporter and the pcDNA3.1 containing a hygromycin resistance gene (Thermo Fisher Scientific). Cells were cultured in the presence of 0.5 mg/mL hygromycin for 1 week. Stable HeLa-psiCHECK-2 cells expressing both Renilla and firefly luciferases were grown in Dulbecco’s Modified Eagle Medium (d-MEM) supplemented with 10% bovine serum (BS) and 0.25 mg/mL hygromycin at 37 °C. HeLa-psiCHECK-2 cells (8.0 × 104/mL) were cultured on a 96-well plate (100 μL/well) for 24 h and transfected with

siRNA targeting the Renilla luciferase gene using lipofectamine RNAiMax in Opti-MEM reduced serum medium. Transfection without siRNA was used as a control. After 1 h, D-MEM (50 μL) containing 10% BS was added to each well and cells were further incubated for another 24 h. The activities of Renilla and firefly luciferases in the cells were determined with the Dual-Luciferase Reporter Assay System (Promega) according to a manufacture’s protocol. The activity of Renilla luciferase was normalized by the firefly luciferase activity. The results were confirmed by at least three independent transfection experiments with two cultures each and are expressed as the average from four experiments as mean ± SD. Nuclease Resistance of Single-Stranded RNA. Fluorescein labeled RNAs (300 pmol) were dissolved in Opti-MEM (37.5 μL) and used for the serum stability test. 1.2 μL of bovine serum was added, and the mixture was incubated at 37 °C for the required time. Aliquots of 2.4 μL were diluted with a stop solution (10 mM EDTA in formamide, 10 μL). Samples were subjected to electrophoresis in 20% PAGE containing 7 M urea and quantified by a Luminescent Image analyzer LAS-4000 (Fujifilm). Nuclease Resistance of siRNA. Fluorescein labeled siRNAs (600 pmol) were dissolved in 20 μL of buffer of 10 mM sodium phosphate (pH 7.0) containing 100 mM NaCl, and the solutions were heated at 100 °C and then cooled gradually to room temperature and used for the serum stability test. 100 μL of Opti-MEM and 12 μL of bovine serum were added, and the mixture was incubated at 37 °C for the required time. Aliquots of 6.7 μL were diluted with a stop solution (65 mM EDTA, 12% glycerol, 8.0 μL). Samples were subjected to electrophoresis in 15% polyacrylamide-TBE under nondenaturing conditions and quantified by a Luminescent Image analyzer LAS-4000 (Fujifilm).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.8b03277. UV melting profiles of duplexes and graphical data of 1/ Tm versus ln(CT/4) plots; time-dependent rmsd (Å) of the analogs 1, 2, and 3 heavy atoms over 500 ns of MD simulation; sequences of siRNAs and their abilities to suppress gene expression; sequences of siRNAs used in the serum stability test; 1H NMR spectra of compounds 5−24 and 27−37; 13C NMR spectra of compounds 5−24 and 27−37; 31P NMR spectra of compounds 24 and 35 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-58-293-2919. Fax: +81-58-293-2919. E-mail: [email protected]. ORCID

Yoshihito Ueno: 0000-0003-3492-4677 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Japan Agency for Medical Research and Development (AMED) through its Funding Program for Basic Science and Platform Technology Program for Innovative Biological Medicine, development of siRNA conjugates with tissue-specific delivery functions (18am0301022h0004). The authors thank Professor Yoko Hirata (Gifu University) for supplying cells and giving technical advice. O


Article


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Synthesis and Biophysical Characterization of RNAs Containing (R

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