Practical and Reliable Synthesis of

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS

, VOL. , NO. , –

http://dx.doi.org/./..

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS 65

Figure .Approaches for the synthesis of dR^H.

and furanose erythro-glycals (furanoid glycals) (d).^[25,26] However, most of the existing protocols are not practical. Chenaultet al.synthesized dR^Hfrom 2-deoxy-d-ribose (a) in high yield, although an excess of ion exchange resin is required to obtain dR^H.^[23]Beigelman and co-workers reported a simple synthetic method for dR^H precursors via a furanoid glycal, but preparation of the furanoid glycal was challenging and derivation into dR^Hwas not reported.^[27] Ferreroet al.described a biocatalytic procedure for preparing dR^Hprecursors from readily available mate-rials that is useful in ON synthesis, but the protocol requires multiple steps.^[28] To study several ONs containing dR^Hfor ON-based therapeutics, a practical and reli-able synthesis of dR^His needed.

Here we describe a practical and reliable procedure for synthesizing dR^H (1) from 5-O-silylated thymidine via the corresponding furanoid glycal by elimina-tion of nucleobase (Scheme 1). We also studied the biological properties of small interfering RNAs (siRNAs) bearing dR^Hdimer instead of the natural nucleotides at the 3-overhang region of siRNAs.

Results and discussion

Synthesis of 1,2-dideoxy-D-ribofuranose and its derivatives

First, 5-O-(tert-butyldiphenylsilyl)thymidine (2),^[25] which can be easily prepared from thymidine, was reacted with ammonium sulfate ((NH₄)₂SO₄) in 1,1,1,3,3,3 -hexamethyldisilazane (HMDS) under reflux to give the corresponding 3,5-O-disilylglycal 3. Although Pedersen’s protocol^[25] gave the corresponding furan derivative3^[29] as a major product rather than the desired glycal3, optimization of concentration, amount of (NH₄)₂SO₄, and reaction time provided the desired glycal3almost quantitatively. Subsequently, the trimethylsilyl (TMS) group at the 3-position was removed with potassium carbonate to produce 5-mono-silylated gly-cal4. Han and Lowary reported obtaining5in 87% yield from4with Pd/C-catalyzed hydrogenation in MeOH;^[30] in contrast, we found that hydrogenation of olefin in 4 under the above conditions gave the desired 5-O-TBDPS-dR^H 5 in 36% yield, along with a tetrahydrofuran derivative 5^[29] (31%) produced from the Ferrier rearrangement^[31] followed by hydrogenation of resulting olefin. We could

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66 Y. NAGAYA ET AL.

completely suppress the side reaction by using i-PrOH, a bulkier alcohol as sol-vent, and could isolate 5 (95% yield from 2) as a sole product. Finally, desi-lylation by treatment with tetra-n-butylammonium fluoride (TBAF) provided 1 quantitatively.

For incorporation of 1 into ONs by the standard phosphoramidite solid-phase method, 1 was converted to the corresponding phosphoramidite deriva-tive 7 and the solid support 8 on controlled pore glass (CPG). Treatment of 1 with 4,4-dimethoxytrityl (DMTr) chloride in DMF/pyridine gave the corre-sponding 5-DMTr derivative6, which was phosphorylated with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite to produce7 quantitatively. Compound6was succinated, and the resulting succinate was linked with CPG to generate solid sup-port8(54.5μmol/g).

RNA interfering (RNAi) study

Small interfering RNAs inhibit gene suppression by RNAi and thus hold great promise as nucleic acid drugs. We have reported the synthesis and biological prop-erties of siRNAs chemically modified at their 3-overhang regions.^[32–37]In an RNAi study, an overhang of two thymidine residues (dTdT) at the 3-end of siRNA is gener-ally used for nuclease protection. Here we synthesized siRNA incorporating an aba-sic deoxyribonucleoside (dR^H) dimer at this position to investigate the effects of lack of nucleobase moiety at the two nucleotides of the 3-overhang region (Figure 2).

Scheme .Reagents and conditions: (a) (NH_)_SO_, HMDS, reflux; (b) K_CO_, MeOH, rt; (c) H_, Pd/C, i-PrOH, rt, %; (d) TBAF, THF, rt, %; (e) DMTrCl, pyridine, rt, quant; (f ) (i-Pr_N)P(Cl)O(CH_)_CN,i-Pr_NEt, THF, rt, quant; (g) (i) succinic anhydride, DMAP, pyridine, rt; (ii) CPG, EDC·HCl, DMF, rt, .μmol/g.

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NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS 67

Figure .Structures of modified siRNAs.

Oligonucleotide synthesis

Using 7 and 8, ONs containing dR^H were synthesized using a DNA/RNA synthesizer (Table 1). The fully protected ONs linked to a solid support were treated with concentrated NH₄OH/EtOH (3:1, v/v) at room temperature for 12 h and with TBAF (1.0 M solution in THF) at room temperature for 12 h. The ONs were puri-fied by denaturing 20% polyacrylamide gel electrophoresis (PAGE) to isolate the deprotected ONs bearing abasic deoxyribonucleoside dR^H. These ONs were ana-lyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrom-etry (MALDI-TOF/MS), and the observed molecular weights were in good agree-ment with their structures.^[38]

Table .Sequences of ONs and siRNAs used in this study.

No. of siRNA No. of ON Sequence^a,b

siRNA9 ON13 -GGCCUUUCACUACUCCUACdTdT-

ON14 -dTdTCCGGAAAGUGAUGAGGAUG-

siRNA10 ON15 -GGCCUUUCACUACUCCUACdR^HdR^H-

ON16 -dR^HdR^HCCGGAAAGUGAUGAGGAUG-

siRNA11 ON13 -GGCCUUUCACUACUCCUACdTdT-

ON16 -dR^HdR^HCCGGAAAGUGAUGAGGAUG-

siRNA12 ON15 -GGCCUUUCACUACUCCUACdR^HdR^H-

ON14 -dTdTCCGGAAAGUGAUGAGGAUG-

ON17 F--GUAGGAGUAGUGAAAGGCCdTdT-

ON18 F--GUAGGAGUAGUGAAAGGCCdR^HdR^H-

aCapital letters indicate ribonucleosides.

bF denotes fluorescein.

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68 Y. NAGAYA ET AL.

Figure .Dual-luciferase assay.

Dual-luciferase assay

The silencing activity of siRNAs 9–12 was examined by a dual-reporter assay using a psiCHECK-2 vector in HeLa cells. This vector contains the Renilla and firefly luciferase genes, and the siRNA sequences were designed to target theRenilla luciferase gene. HeLa cells were co-transfected with the vector and the indicated amount of siRNAs. The signal levels ofRenilla luciferase were normalized by the signal levels of firefly luciferase. All siRNAs effectively reduced luciferase activity in a dose-dependent manner (Figure 3). The silencing activity of siRNA10with dR^H dimers was slightly less than that of siRNA9, which possesses natural thymidines, at 0.1 and 10 nM. Of siRNAs9–12, the lowest knockdown activity was exhibited by scrambled siRNA11, which possesses dR^Hat the 3-end of the antisense strand, and the highest was exhibited by scrambled siRNA12, which bears dR^Hat the 3-end of the sense strand.

Nuclease resistance

We next investigated the enzymatic stability of ONs against 3-exonucleases, which are dominant in human serum. The susceptibility of ONs to snake venom phospho-diesterase (SVPD), which degrades ONs from their 3-end, was examined. Unmod-ified ON17 and modified ON18, labeled with fluorescein at their 5-ends, were incubated with SVPD, and the reactions were analyzed by PAGE under denatur-ing conditions. The half-life (t_1/2) of unmodified ON17was <5 min, and that of modified ON 18 was 15 min, indicating that ON 18 carrying an dR^H dimer at its 3-end was significantly more resistant to SVPD than was unmodified ON17 (Figure 4).

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NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS 69

Figure .Nuclease resistance of ON17and ON18against SVPD.

Experimental

General methods

All reactions were carried out under an argon atmosphere, unless otherwise noted.

All reagents and solvents were purchased from commercial vendors and used without further purification unless indicated otherwise. Pyridine was distilled over CaH₂ and stored over activated molecular sieves 4 ˚A. ¹H and¹³C NMR spectra were recorded on a JEOL JNM AL-400 spectrometer or JNM ECS-400 spectrom-eter (400 MHz for¹H NMR, 100 MHz for¹³C NMR, and 162 MHz for³¹P NMR).

Chemical shifts (δ) were expressed in parts per million (ppm) and internally refer-enced (7.26 ppm for CDCl₃or 3.31 for CD₃OD for¹H NMR, 77.0 ppm for CDCl₃or 49.0 ppm for CD₃OD for¹³C NMR and 0.00 ppm for H₃PO₄/CDCl₃for³¹P NMR).

Direct analysis in real time (DART) coupled with time-of-flight (TOF) mass spectra were taken on a JMS T100TD instrument. Electron impact (EI) mass spectra were taken on a Shimadzu GCMS-QP2010A instrument. Matrix-assisted laser desorp-tion/ionization (MALDI) coupled with TOF mass spectra were taken on a Shimadzu AXIMA-CFR plus instrument. Flash column chromatography was performed using silica gel 60 N (spherical neutral [63–210µm]) from Kanto Chemical Co. Inc.

5-O-(tert-Butyldiphenylsilyl)thymidine (2)^[25]

TBDPSCl (600 μL, 2.34 mmol) was added to a solution of thymidine (486 mg, 2.01 mmol) and DMAP (49.2 mg, 403μmol) in pyridine (10 mL), and the mixture was stirred at room temperature for 15 h. The mixture was partitioned as EtOAc and H₂O. The organic layer was washed with saturated aqueous NH₄Cl solution and brine, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (n-hexane/EtOAc, 3:1˜0:1) to give2as a white solid (882 mg, 91%);¹H NMR (CDCl₃)δ9.25 (s, 1H, NH), 7.67–

7.64 (m, 4H, H-Ph), 7.50 (d,J=0.9 Hz, 1H, H-6), 7.47–7.37 (m, 6H, H-Ph), 6.43 (dd, J=8.4 Hz, 5.8 Hz, 1H, H-1), 4.58–4.56 (m, 1H, H-3), 4.05–4.03 (m, 1H, H-4), 3.98 (dd,J=11.8 Hz, 2.7 Hz, 1H, H-5_α), 3.85 (dd,J=11.8 Hz, 2.7 Hz, 1H, H-5_β), 2.43

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70 Y. NAGAYA ET AL.

(ddd,J=13.8 Hz, 5.8 Hz, 2.3 Hz, 1H, H-2_α), 2.20 (ddd,J=13.8 Hz, 8.4 Hz, 5.9 Hz, 1H, H-2_β), 1.62 (d,J=0.9 Hz, 3H, CH₃of thymine), 1.08 (s, 9H, C(CH₃)₃);¹³C NMR (CDCl₃)δ163.8, 150.5, 135.5, 135.3, 132.8, 132.3, 130.1, 130.0, 128.0, 127.9, 111.2, 87.1, 84.7, 72.3, 64.1, 41.0, 27.0, 19.3, 12.1; MS (DART)m/z504 [M+Na]⁺, HRMS (DART) Calcd. for C₂₆H₃₂N₂NaO₅Si [M+Na]⁺: 503.6180. Found: 503.6163.

1,4-Anhydro-5-O-(tert-butyldiphenyl)-2-deoxy-D-erythro-pent-1-enitol (5)^[30]

A mixture of2(482 mg, 1.00 mmol) and (NH₄)₂SO₄(265 mg, 2.01 mmol) in HMDS (8 mL) was refluxed for 1.5 h. The solvent was removed under reduced pressure, and the residue was diluted with EtOAc and H₂O. The organic layer was washed with brine and dried over Na₂SO₄, and concentrated under reduced pressure. A suspen-sion of K₂CO₃(28.5 mg, 206μmol) in MeOH (15 mL) was added to the residue containing 3 and stirred at room temperature for 2 h. The reaction mixture was partitioned as EtOAc and H₂O. The organic layer was washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was filtered through column chromatography on silica gel (n-hexane/EtOAc, 3:1˜2:1) to give4as a yel-low oil. A mixture of4and 10% Pd/C (15 wt%) ini-PrOH (10 mL) was vigorously stirred at room temperature under ambient pressure of hydrogen for 24 h. The reac-tion mixture was filtered through a celite pad, and the filtrate was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel (n-hexane/EtOAc, 7:1˜2:1) to give5as a yellow oil (338 mg, 94%);¹H NMR (CDCl₃) δ7.70–7.66 (m, 4H, H-Ph), 7.46–7.37 (m, 6H, H-Ph), 4.43–4.40 (m, 1H, H-3), 3.96 (dd,J=8.4 Hz, 5.7 Hz, 2H, H-5), 3.85–3.82 (m, 1H, H-4), 3.78 (dd,J=10.7 Hz, 4.3 Hz, 1H, H-1_α), 3.60 (dd, J= 10.7 Hz, 6.4 Hz, 1H, H-1_β), 2.20–2.10 (m, 1H, H-2_α), 1.93–1.86 (m, 1H, H-2_β), 1.07 (s, 9H, C(CH₃)₃);¹³C NMR (CDCl₃)δ135.6, 135.5, 133.2, 133.1, 129.8, 129.8, 127.7, 86.1, 77.2, 74.3, 67.1, 64.8, 34.8, 26.8, 19.2; MS (DART)m/z379 [M+Na]⁺, HRMS (DART) Calcd. for C₂₁H₂₈NaO₃Si [M+Na]⁺: 379.1705. Found: 379.1750.

1,2-Dideoxy-D-ribofuranose (1)^[28]

Tetra-n-butylammonium fluoride (1.0 M in THF, 660μL) was added to a solution of 5(215 mg, 603μmol) in THF (2.7 mL), and the mixture was stirred at room temper-ature for 12 h. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (CH₂Cl₂/acetone, 5:1˜2:1) to give1as a yellow oil (68.8 mg, 97%);¹H NMR (CD₃OD)δ4.21–4.18 (m, 1H, H-3), 3.97–3.87 (m, 2H, H-1), 3.75–3.72 (m, 1H, H-4), 3.57–3.49 (m, 2H, H-5), 2.18–2.03 (m, 1H, H-2_α), 1.88–1.81 (m, 1H, H-2_β); ¹³C NMR (CD₃OD)δ 88.1, 73.8, 68.0, 63.6, 35.9; MS (DART)m/z141 [M+Na]⁺, HRMS (DART) Calcd. for C₅H₁₀NaO₃ [M+Na]⁺: 141.1209. Found: 141.1250.

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NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS 71

1,2-Dideoxy-5-O-(4,4-dimethoxytrityl)-D-ribofuranose (6)^[24]

A mixture of1(101 mg, 853μmol) and DMTrCl (376 mg, 1.11 mmol) in pyridine (3.3 mL) was stirred at room temperature for 12 h. The reaction mixture was par-titioned as CH₂Cl₂and H₂O. The organic layer was washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by col-umn chromatography on silica gel (CH₂Cl₂/acetone, 20:1˜10:1) to give7as a yellow oil (265 mg, quant);¹H NMR (400 MHz, CDCl₃)δ7.43 (d,J=7.8 Hz, 2H, H-Ph), 7.36–7.16 (m, 7H, H-Ph), 6.82 (d,J=9.2 Hz, 4H, H-Ph), 4.30–4.27 (m, 1H, H-3), 3.99–3.96 (m, 2H, H-1), 3.90–3.86 (m, 1H, H-4), 3.79 (s, 6H, OCH₃), 3.23 (dd,J= 9.8 Hz, 4.8 Hz, 1H, H-5_α), 3.07 (dd,J=9.8 Hz, 6.2 Hz, 1H, H-5_β), 2.19–2.10 (m, 1H, H-2_α), 1.92–1.84 (m, 1H, H-2_β);¹³C NMR (CDCl₃)δ158.4, 144.8, 136.0, 136.0, 130.0, 128.1, 127.8, 126.8, 113.1, 86.2, 85.0, 74.6, 67.0, 64.5, 55.2, 34.8; MS (EI)m/z 420 (M⁺)

3-O-[(2-Cyanoethoxy)(N,N-diisopropylamino)phosphanyl]-1,2-dideoxy-5-O-(4,4-dimethoxytrityl)-D-ribofuranose (7)

i-Pr₂NEt (730 μL, 4.18 mmol) and 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (250 μL, 1.12 mmol) were added to a solution of 6 (292 mg, 694 mmol) in THF, and stirred at room temperature for 2 h. The reaction mixture was partitioned as CHCl₃and saturated NaHCO₃aqueous solution. The organic layer was washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (n-hexane/EtOAc, 4:1) to give7as a yellow oil;³¹P NMR (162 MHz, CDCl₃)δ 148.3, 148.6

Solid support synthesis

A mixture of7(102 mg, 243μmol), succinic anhydride (112 mg, 1.12 mmol), and DMAP (30.4 mg, 249μmol) in pyridine (2.4 mL) was stirred for 23 h at room tem-perature. The reaction mixture was partitioned as EtOAc and saturated NaHCO₃ aqueous solution. The organic layer was washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure to give the corresponding succinate. Amino-propyl controlled pore glass (546 mg, 53.5μmol) was added to a solution of suc-cinate and EDC·HCl (41.6 mg, 217μmol) in DMF (5.3 mL), and the mixture was kept for 72 h at room temperature. After the resin was washed with pyridine, a cap-ping solution (30 mL, 0.1 M DMAP in pyridine/Ac₂O, 9:1, v/v) was added and the whole mixture was kept for 72 h at room temperature. The resin was washed with pyridine, EtOH, MeCN, and driedin vacuo. The amount of loaded6to the solid support was 54.5μmol/g from calculation of released dimethoxytrityl cation by a solution of 70% HClO₄/EtOH, 3:2 (v/v).

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72 Y. NAGAYA ET AL.

Oligonucleotide synthesis

Synthesis was carried out with a DNA/RNA synthesizer (ABI Expedite 3400) by the phosphoramidite method. Deprotection of bases and phosphates was performed in concentrated NH₄OH/EtOH, 3:1 (v/v) at room temperature for 12 h. 2-TBDMS groups were removed by TBAF (1.0 M in THF) at room temperature for 12 h. The reaction was quenched with 0.1 M triethylammonium acetate buffer (pH 7.0) and desalted on a Sep-Pak C18 cartridge. Deprotected ONs were purified by 20% PAGE containing 7.0 M urea to give a highly purified ON15(8), ON16(7), and ON18 (12). The yields are indicated in parentheses as OD units at 260 nm starting from 1.0-μmol scale. The extinction coefficients of ONs were calculated from those of the mononucleotides and dinucleotides according to the nearest neighbor approxima-tion method.^[38]

Mass spectrometric analyses of ONs

Spectra were obtained by MALDI-TOF/MS (negative mode). ON 15: calculated mass, 6250.8; observed mass, 6251.0. ON 16: calculated mass, 6559.9; observed mass, 6559.8. ON18: calculated mass, 7098.0; observed mass, 7099.4.

Dual-luciferase assay

HeLa cells were grown at 37°C in a humidified atmosphere of 5% CO₂ in air in D-MEM (Wako) supplemented with 10% bovine serum (Sigma). HeLa cells (4× 10⁴/mL) were transferred to 96-well plates (100μL per well) 24 h before transfec-tion. They were transfected using TransFast (Promega) according to instructions for transfection of adherent cell lines. Cells in each well were transfected with a solu-tion (35μL) of 20 ng of psi-CHECK-2 vector (Promega), the indicated amounts of siRNAs, and 0.3μg of TransFast in Opti-MEM I reduced-serum medium (Invit-rogen), and incubated at 37°C. Transfection without siRNA was used as a control.

After 1 h, D-MEM (100μL) containing 10% bovine serum was added to each well, and the whole was further incubated at 37°C. After 24 h, cell extracts were frozen at−80°C. Activities of firefly andRenillaluciferases in cell lysates were determined with a dual-luciferase assay system (Promega). The results were confirmed by at least three independent transfection experiments with two cultures each and expressed as an average of mean±SD from three experiments.

Nuclease resistance

Each ON (300 pmol) labeled with fluorescein at the 5-end was incubated with SVPD (1 unit/mL) in PBS(–) (100μL) at 37°C. At appropriate periods, aliquots (5μL) of the reaction mixture were separated and added to a solution of 7.0 M urea (15μL).

The resulting mixtures were heated at 90°C for 10 min and analyzed by electrophore-sis on 20% PAGE containing 7.0 M urea. The labeled ONs in the gel were visualized with a lumino image analyzer.

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NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS 73

Conclusions

We demonstrated a practical and reliable method for preparing dR^H. To synthe-size ONs bearing dR^Hby the standard phosphoramidite solid phase method, the corresponding phosphoramidite derivative and a solid support were prepared. The silencing activity of siRNAs containing dR^Hin their 3-overhang region was exam-ined using a dual-luciferase assay. Introducing dR^Hto the 3-end of the guide strand of siRNA was found to attenuate its gene silencing effect in an in vitro dual-luciferase experiment. It was also found that incorporating dR^H into 3-end enhances the nuclease resistance of ONs. Further study of dR^Hwill contribute to the development of ON-based therapeutics.

Acknowledgment

We acknowledge the Division of Instrumental Analysis, Life Science Research Center, Gifu Uni-versity, for the maintenance of instruments.

Funding

This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS).

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Introduction of 2-O-benzyl abasic nucleosides to the 3

⁰

-overhang regions of siRNAs greatly improves nuclease resistance

Yuki Nagaya^a, Yoshiaki Kitamura^b, Aya Shibata^b, Masato Ikeda^a,b,c, Yukihiro Akao^a, Yukio Kitade^b,d,⇑

aUnited Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan

bDepartment of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan

cGifu Center for Highly Advanced Integration of Nanosciences and Life Sciences (G-CHAIN), Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan

dDepartment of Applied Chemistry, Faculty of Engineering, Aichi Institute of Technology, 1247 Yachigusa, Yakusa-cho, Toyota, Aichi 470-0392, Japan

a r t i c l e i n f o

Article history:

Received 6 October 2017 Revised 26 October 2017 Accepted 27 October 2017 Available online 28 October 2017

Keywords:

RNA siRNA RNAi

Abasic nucleoside analogues Nuclease resistance

a b s t r a c t

Chemically modified siRNAs containing 2-O-benzyl-1-deoxy-D-ribofuranose (R^HOBn) in their 3⁰-overhang region were significantly more resistant towards serum nucleases than siRNAs possessing the natural nucle-oside in this region. The knockdown efficacies and binding affinities of these modified siRNAs to the recom-binant human Argonaute protein 2 (hAgo2) PAZ domain were comparable with that of siRNA with a thymidine dimer at the 3⁰-end.

Ó2017 Elsevier Ltd. All rights reserved.

Small interfering RNAs (siRNAs) inhibit gene expression by RNA interference (RNAi) and thus have great potential as nucleic acid drugs.¹RNAi technology is a useful strategy in the fight against cancers,² viral infections,³and other diseases.⁴ However, natural RNA strands have many problems that complicate their therapeu-tic application, such as rapid degradation in biological media, non-specific gene silencing (off-target effects), and poor administration using existing drug delivery systems. To overcome these problems, artificially modified siRNAs have been developed extensively. An siRNA is a short (18–26 nucleotides) double-stranded RNA (dsRNA) containing a 2-nucleotide overhang at the 3⁰-end of each strand.⁵ Once siRNAs are introduced into a cell by transfection, they are incorporated into a RNA-induced silencing complex (RISC). Each RISC contains a helicase that unwinds the siRNA helix. Upon unwinding, one of the strands, known as an antisense strand (guide strand), is retained in the RISC. This antisense RNA-RISC, called mature RISC, binds to and degrades the complementary mRNA tar-get through base-pairing interactions. Eukaryotic translation initi-ation factor 2C2 (EIF2C2, Argonaute protein 2, Ago2), the core component of RISC, is considered to be the major player in RNAi.

Ago2 has a conserved structure and includes PAZ, MID, and PIWI domains. The PAZ domain specifically recognizes the antisense

strand of dsRNA through binding to the 3⁰-overhang region.^6,7 The binding site is a hydrophobic pocket composed of aromatic amino acids.^7–10

On the basis of these findings, it has been suggested that chem-ical modification of the 3⁰-overhang region is an effective tech-nique for improving the functionality of siRNA for RNAi-based therapy.^11–25We previously reported the design and synthesis of various chemically modified functional RNAs bearing nucleic acid mimics at their 3⁰-end.11–15,17,23 As part of our ongoing studies, we found that siRNAs containing 2-O-benzyl-1-deoxy-D -ribofura-nose (R^H_OBn;1) at the 3⁰-ends showed high nuclease resistance and a desirable knockdown effect (Fig. 1).

Synthesis:To synthesize the desired RNAs via the conventional phosphoramidite method using a DNA/RNA synthesizer, we pre-pared the phosphoramidite derivative of R^HOBn(2) (Scheme 1). First, 1-deoxy-D-ribofuranose (R^H,3), which can be prepared from com-mercially available 1-O-acetyl-2,3,5-tri-O-benzoyl-b-D-ribofuranose via reductive cleavage of the anomeric position,¹⁵ was converted to TIPDS-R^H4using a disiloxane protection strategy. Subsequently, benzylation with benzaldehyde, Et3SiH, and FeCl3in CH3NO2,²⁶ fol-lowed by desilylation by treatment with Et3N3HF, gave1in moder-ate yield. Treatment of1with 4,4⁰-dimethoxytrityl (DMTr) chloride in pyridine gave the corresponding 5-DMTr derivative 5, which was phosphorylated with 2-cyanoethyl N,N-diisopropylchlorophos-phoramidite to produce2in 97% yield. Oligonucleotides (ONs) con-taining R^HOBn were synthesized using an automated nucleic acid

https://doi.org/10.1016/j.bmcl.2017.10.070 0960-894X/Ó2017 Elsevier Ltd. All rights reserved.

⇑Corresponding author at: Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.

E-mail address:[email protected](Y. Kitade).

Bioorganic & Medicinal Chemistry Letters 27 (2017) 5454–5456

Contents lists available atScienceDirect

Bioorganic & Medicinal Chemistry Letters

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b m c l

synthesizer with phosphoramidite derivative2(Table 1). The fully protected ONs linked to a solid support were treated with NH4OH/

MeNH2(40% in H2O), 1:1 (v/v) at 65°C for 30 min and with Et3N3HF in DMSO at 65°C for 2.5 h. The crude product can be precipitated by adding 3 M NaOAc, followed byn-BuOH. The mixture was cooled at 80°C for 12 h and centrifuged at 4°C, 12,500 rpm, for 30 min. After removal of the supernatant, 70% EtOH was added to the pellet. The resulting mixture was centrifuged at 4°C, 12,500 rpm, for 15 min.

The supernatant was removed, and the washing step was repeated.

Deprotected ONs were purified by denaturing 20% polyacrylamide gel electrophoresis (PAGE) to isolate the desired ONs bearing the R^H

-OBnmodification. These ONs were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS), and the observed molecular weights were in good agree-ment with their expected structures.²⁷

Gene silencing of Renilla luciferase:The silencing activities of the siRNAs were examined by a dual-reporter assay using the psi-CHECK-2 vector in HeLa cells. This vector contains theRenillaand firefly luciferase genes, and the siRNA sequences were designed

to target theRenillaluciferase gene. HeLa cells were co-transfected with the vector and the indicated amount of each siRNA. The signal levels ofRenillaluciferase were normalized to those of firefly luci-ferase.Fig. 2shows the silencing activities of the siRNAs.Renilla luciferase was suppressed by each siRNA in a dose-dependent manner. At 1.0 nM and 10 nM, the silencing activity of siRNA 7 with R^H_OBndimers was almost equal to that of siRNA6with natural thymidines.

Nuclease resistance: The susceptibilities of the ONs to snake venom phosphodiesterase (SVPD), a highly active 3⁰-exonuclease, were examined. Thymidine-modified ON 14 and R^HOBn-modified ON15, each labeled with fluorescein at their 5⁰-end, were incubated with SVPD and the reactions were analyzed by PAGE under denatur-ing conditions. The half-life (t1/2) of ON14was <4 min, and that of ON15carrying a R^HOBndimer was 19 min. ON15was at least 5 times more resistant to the enzyme than thymidine-modified ON 14 (Fig. 3). Furthermore, the R^H_OBn-modified ON15was more resistant to nucleolytic hydrolysis by SVPD than the corresponding modified Fig. 1.Conceptual diagram of this study.

Scheme 1.Reagents and conditions: (a) TIPDSCl2, imidazole, DMF, rt, 82%; (b) i) PhCHO, Et3SiH, FeCl3, MeNO2, rt; ii) Et3N3HF, THF, rt, 47%; (c) DMTrCl, pyridine, rt, 67%; (d) (i-Pr2N)P(Cl)O(CH2)2CN,i-Pr2NEt, THF, rt, 97%.

Table 1

Sequences of ONs and siRNAs used in this study.

No. of siRNA No. of ON Sequence

siRNA6 ON10 5⁰-GGCCUUUCACUACUCCUACtt-3⁰ ON11 3⁰-ttCCGGAAAGUGAUGAGGAUG-5⁰ siRNA7 ON12 5⁰-GGCCUUUCACUACUCCUACR^HOBnR^HOBn-3⁰

ON13 3⁰-R^HOBnR^HOBnCCGGAAAGUGAUGAGGAUG-5⁰

ON14 F-5⁰-GUAGGAGUAGUGAAAGGCCtt-3⁰

ON15 F-5⁰-GUAGGAGUAGUGAAAGGCCdR^HdR^H-3⁰ asiRNA8 ON16 5⁰-GGCCUUUCACUACUCCUAC-3⁰

ON11 3⁰-ttCCGGAAAGUGAUGAGGAUG-5⁰ asiRNA9 ON16 5⁰-GGCCUUUCACUACUCCUAC-3⁰

ON13 3⁰-R^HOBnR^HOBnCCGGAAAGUGAUGAGGAUG-5⁰

aCapital letters indicate ribonucleosides and small letters show 2⁰ -deoxyribonucleosides.

bF denotes fluorescein.

Fig. 2.Dual-luciferase assay.

Fig. 3.Nuclease resistance of ONs against SVPD.

Y. Nagaya et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 5454–5456 5455

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