Liquid-Phase Synthesis of N-Functionalized Azanucleoside-Incorporated Oligonucleotides and Development of Anodic C(sp3)–H Acetoxylation Reaction for Direct Preparation of Azaribose

Kazuhiro Okamoto; Naoki Shida; Mizuki Tsutsui; Kazuhiro Chiba

doi:10.1055/s-0037-1611550

Synlett, Table of Contents

Synlett 2019; 30(11): 1303-1307
DOI: 10.1055/s-0037-1611550

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Liquid-Phase Synthesis of N-Functionalized Azanucleoside-Incorporated Oligonucleotides and Development of Anodic C(sp³)–H Acetoxylation Reaction for Direct Preparation of Azaribose

Authors

Kazuhiro Okamoto
Naoki Shida
Mizuki Tsutsui
Kazuhiro Chiba*

Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509, Japan Email: chiba@cc.tuat.ac.jp

Abstract

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Abstract

We report the synthesis of pyrene-conjugated azanucleoside-incorporated oligodeoxynucleotides (aza-ODNs). Combination of liquid-phase synthesis by alkyl-chain-soluble-support (ACSS) and electrochemical C–H activation realized efficient access to aza-ODNs without requiring an excess amount of reagent or solvent. The fluorescent properties of pyrene-conjugated aza-ODNs were also investigated. The resulting fluorescence spectrum indicated that the modification of the position of the nitrogen atom was suitable for the preparation of artificial functionalized oligonucleotides. A synthetic route to azaribose, as a precursor of aza-ODNs, was also reinvestigated to realize more efficient production. Electrochemical N-α-acetoxylation in 0.1 M lithium perchlorate/nitromethane/50 mM AcOH medium was found to be a suitable medium for this route. These results represent a new efficient synthetic route to aza-ODNs.

Key words

electrochemistry - C–H activation - nucleoside - nucleoside analogue - nucleic acid - acetoxylation Lewis acid

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References

References and Notes
1 New address: Naoki Shida, School of Materials and Chemical Technology, Tokyo Institute of Technology, 226-8502, Nagatsuta-cho, Yokohama, Kanagawa, Japan.

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11 Spectral data for compound 6 and 7 were reported in Ref. 7a and 7c.
12 Elongation of oligonucleotides by ACSS; General Procedure 5′-DMTr-protected oligonucleotides were treated with 3% dichloroacetic acid in CH₂Cl₂. After 3 min, the reaction mixture was neutralized with triethylamine and quenched with MeOH. The resulting precipitate was collected by filtration. Condensation with phosphoramidite (2 equiv) was conducted in 10:1 (v/v) CH₂Cl₂/MeCN while stirring for 10 min under Ar atmosphere. A capping solution [1:1:1 (v/v/v) Ac₂O/pyridine/N-methylimidazole in CH₂Cl₂] was added to the resulting mixture and stirring was continued for 10 min. The oxidizing solution (final 0.67% butanoneperoxide/dimethylphthalate solution in CH₂Cl₂) was added and the solution was stirred for 30 min. Methanol was added and the precipitate was collected and dried in vacuo.
13 ((2R,3S,5S)-3-Acetoxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-1-(3-((pyren-1-ylmethyl)thio)propanoyl)pyrrolidin-2-yl)methyl Acetate (2) Compound 1 (0.27 mmol, 102 mg) was dissolved in CH₂Cl₂/MeOH (v/v = 1:1, 10 mL). Compound 11 (0.8 mmol, 199 mg) and DIPEA (1.2 mmol, 209 μL) were added and the resulting solution and stirred for 12 h under Ar atmosphere in the dark. The resulting mixture was concentrated in vacuo and the crude product was purified by column chromatography on silica gel (Hex/EtOAc, 1:5) to give 2 (0.25 mmol, 93% yield) as a yellow solid. ¹H NMR (600 MHz, CDCl₃): δ = 9.43 (s, 1 H), 8.25–8.35 (m, 1 H), 7.86–8.22 (m, 8 H), 7.65–7.68 (m, 1 H), 7.50 (m, 1 H), 7.08–7.11 (m, 1 H), 6.31 (dd, J = 13.4, 7.9 Hz, 1 H), 5.06–5.23 (m, 1 H), 4.28–4.60 (m, 3 H), 4.16–4.23 (m, 1 H), 2.48–2.90 (m, 4 H), 1.76–2.11 (m, 10 H). ¹³C NMR (151 MHz, CDCl₃): δ = 171.33, 170.17, 170.08, 169.20, 163.45, 150.43, 135.20, 134.64, 132.05, 131.99, 131.91, 131.26, 131.15, 130.76, 130.68, 128.76, 128.72, 128.49, 128.42, 127.77, 127.67, 127.62, 127.44, 127.33, 127.29, 127.24, 126.17, 125.99, 125.42, 125.19, 125.11, 125.05, 124.61, 124.55, 124.50, 123.18, 123.09, 123.00, 110.95, 74.78, 74.17, 73.77, 73.63, 68.77, 68.58, 64.83, 63.09, 62.84, 62.42, 60.97, 38.87, 36.13, 35.21, 34.87, 34.78, 34.44, 32.88, 28.40, 27.19, 26.61, 20.88, 20.82, 20.75, 20.65, 20.58, 12.57, 12.50, 12.43. HRMS (ESI⁺): m/z calcd for C₃₄H₃₃N₃NaO₇S⁺: 650.1973; found: 650.1930.
14 1-((2S,4S,5R)-5-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-1-(3-((pyren-1-ylmethyl)thio)propanoyl)pyrrolidin-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (3) Compound 2 (156 mg, 0.25 mmol) was dissolved in MeOH/H₂O (10:1 v/v, 5 mL). K₂CO₃ (69.1 mg, 0.5 mmol) was added to the solution and stirred at room temperature for 1 h in the dark. The reaction mixture was neutralized with Amberlite and filtrated. The filtrate was concentrated in vacuo and dissolved in CH₂Cl₂. A catalytic amount of DMAP, NEt₃ (116 μL, 0.83 mmol), and DMTr-Cl (281.2 mg, 0.83 mmol) were added to the solution and stirred at 45 °C for 12 h under Ar atmosphere in the dark. The reaction was quenched with MeOH (15 mL) and stirred for 15 min. After concentrated in vacuo, the crude product was purified by column chromatography on the silica gel (Hex/EtOAc, 1:5) to give compound 3 (0.21 mmol, 84% yield) as a yellow solid. ¹H NMR (600 MHz, CDCl₃): δ = 10.00–10.49 (m, 1 H), 7.80–8.30 (m, 10 H), 7.34–7.41 (m, 1 H), 7.25 (m, 3 H), 7.07–7.22 (m, 4 H), 6.68–6.82 (m, 5 H), 6.02–6.31 (m, 1 H), 4.30–4.48 (m, 4 H), 3.59–3.78 (m, 8 H), 3.29–3.41 (m, 1 H), 2.45–2.94 (m, 5 H), 1.87–2.38 (m, 4 H). ¹³C NMR (151 MHz, CDCl₃): δ = 171.13, 170.99, 164.45, 164.15, 158.54, 158.40, 151.11, 151.02, 144.72, 144.10, 136.90, 135.61, 135.50, 135.17, 134.96, 131.59, 131.15, 130.76, 130.70, 130.67, 129.88, 129.85, 129.79, 128.72, 128.67, 127.94, 127.85, 127.77, 127.70, 127.64, 127.32, 127.13, 126.98, 126.81, 126.10, 125.89, 125.30, 125.26, 125.13, 125.07, 124.64, 124.61, 124.55, 124.51, 123.15, 113.21, 113.08, 109.71, 86.93, 86.42, 73.17, 71.92, 70.11, 68.26, 67.56, 63.06, 60.66, 55.12, 55.07, 55.05, 40.56, 38.24, 35.17, 34.77, 34.69, 27.89, 26.38, 12.41, 12.30. HRMS (ESI⁺): m/z calcd for C₅₁H₄₇N₃NaO₇S⁺: 868.3032; found: 868.3058.
15 5′-DMTr-dT^(Aza)-3′-ACSS (4) Compound 3 (62 mg, 0.073 mmol) was dissolved in CH₂Cl₂ (5 mL). NEt₃ (71.2 μL, 0.51 mmol), a catalytic amount of DMAP, and succinic anhydride (33 mg, 0.33 mmol) were added to the solution and stirred at room temperature for 12 h under Ar atmosphere in the dark. To the resulting mixture was added saturated NH₄Cl aq. and extracted with CH₂Cl₂ (3 × 5 mL). The organic layer was dried over anhydrous Na₂SO₄ and concentrated in vacuo. The resulting crude material was dissolved in CH₂Cl₂ (5 mL) and DIPEA (38.3 μL, 0.22 mmol), ACSS·HCl (74.3 mg, 0.073 mmol), DIPCI (20.1 μL, 0.13 mmol), HOBt (12.2 mg, 0.09 mmol), and a catalytic amount of DMAP were added and stirred at room temperature for 14 h. After the reaction, solvent was removed in vacuo to half volume and MeOH was added. After this cycle was repeated two times, the resulting precipitate was filtrated and washed with MeOH. The precipitate was collected and dried in vacuo. Compound 4 (0.06 mmol, 82% yield) was obtained as a white solid. ¹H NMR (600 MHz, CDCl₃): δ = 7.84–8.30 (m, 8 H), 7.30 (m, 2 H), 7.11–7.24 (m, 5 H), 6.78 (dd, J = 8.6, 6.5 Hz, 3 H), 6.58 (t, J = 6.9 Hz, 2 H), 5.20 (m, 1 H), 4.36–4.54 (m, 2 H), 3.93–3.97 (m, 7 H), 3.63–3.77 (m, 6 H), 3.06–3.17 (m, 1 H), 2.58–2.85 (m, 3 H), 2.28–2.47 (m, 2 H), 2.04–2.22 (m, 1 H), 1.72–1.85 (m, 10 H), 1.26–1.30 (m, 109 H), 0.88 (dd, J = 7.6, 6.2 Hz, 11 H). ¹³C NMR (151 MHz, CDCl₃): δ = 172.53, 172.13, 170.68, 170.53, 163.95, 158.58, 158.44, 153.18, 153.05, 150.49, 144.35, 143.82, 139.64, 135.47, 135.42, 135.34, 134.89, 134.83, 131.11, 130.74, 130.70, 129.86, 129.81, 129.78, 128.73, 128.61, 127.99, 127.84, 127.78, 127.73, 127.61, 127.31, 127.25, 127.18, 127.02, 126.87, 126.03, 125.92, 125.22, 125.15, 125.06, 124.65, 124.56, 124.49, 123.10, 113.27, 113.13, 108.74, 105.57, 105.49, 87.18, 86.63, 76.43, 74.98, 73.45, 69.46, 69.21, 65.97, 64.32, 55.11, 55.05, 35.01, 34.62, 31.86, 30.25, 29.66, 29.60, 29.55, 29.36, 29.32, 29.27, 27.58, 26.28, 26.04, 22.63, 14.07, 12.48, 12.34. HRMS (ESI⁺): m/z calcd for C₁₂₀H₁₇₁N₅NaO₁₃S⁺: 1945.2492; found: 1945.2460.
16 5′-OH-dT^(Aza)-3′-ACSS (8) The deprotection process of the DMTr group was carried out by using the general procedure. Compound 4 (84.3 mg, 0.052 mmol) was dissolved in CH₂Cl₂ (10 mL) and 3% DCA solution in CH₂Cl₂ (4.1 mL) was added. After stirring at room temperature for 3 min, MeOH was added to the resulting mixture and neutralized with NEt₃ (1.49 mmol, 209 μL). After concentration in vacuo, the resulting precipitate was filtrated and washed with MeOH. Compound 8 (0.049 mmol, 94% yield) was obtained as a pink solid. ¹H NMR (600 MHz, CDCl₃): δ = 8.69 (s, 1 H), 8.30 (d, J = 9.6 Hz, 1 H), 7.98–8.19 (m, 7 H), 7.86–7.90 (m, 1 H), 7.40 (m, 1 H), 6.58 (m, 3 H), 6.34 (m, 1 H), 5.08–5.30 (m, 3 H), 4.41–4.49 (m, 3 H), 3.95 (q, J = 6.4 Hz, 8 H), 3.70 (d, J = 9.6 Hz, 1 H), 3.46 (s, 1 H), 3.10–3.16 (m, 1 H), 2.73–2.86 (m, 2 H), 2.57 (m, 1 H), 2.44 (m, 1 H), 2.28–2.37 (m, 2 H), 1.84 (s, 2 H), 1.71–1.81 (m, 9 H), 1.44 (q, J = 7.3 Hz, 7 H), 1.27 (s, 93 H), 0.88 (t, J = 6.9 Hz, 6 H). ¹³C NMR (151 MHz, CDCl₃): δ = 172.33, 171.76, 170.70, 170.14, 169.87, 168.61, 163.80, 153.23, 139.59, 135.56, 131.24, 131.02, 130.74, 128.81, 127.77, 127.36, 126.08, 125.32, 124.59, 123.07, 110.64, 105.64, 75.46, 73.53, 69.29, 67.62, 60.56, 53.41, 38.65, 34.78, 34.30, 31.91, 30.29, 29.71, 29.64, 29.41, 29.35, 28.32, 27.27, 26.08, 22.67, 14.10, 12.48. HRMS (ESI⁺): m/z calcd. for C₉₉H₁₅₃N₅NaO₁₁S⁺: 1643.1185; found: 1643.1197.
17 5′-DMTr-d[A^(Bz)T^(Aza)]-3′-ACSS (9) The condensation process with phosphoramidite was carried out by using the general procedure. Compound 8 (0.049 mmol, 79.4 mg) was dissolved in CH₂Cl₂ (4.5 mL). 0.25 M BMT solution in MeCN (500 μL), and dA^(Bz) phosphoramidite (0.1 mmol, 85.8 mg) were added to the resulting solution and stirred for 10 min under Ar. Capping solution (2.4 mL, premix of 25 μL pyridine, Ac₂O, NMI in CH₂Cl₂) was added and stirred for 10 min. Oxidation premix (2.7 mL; 23 μL of 55% BPO sol. in CH₂Cl₂) was added and stirred for 30 min. After these reactions, to the reaction mixture was added MeOH. After concentration in vacuo, the precipitate was filtrated and washed with MeOH. Compound 9 (0.047 mmol, 96% yield) was obtained as a pink solid. ¹H NMR (600 MHz, CDCl₃): δ = 8.63–8.65 (m, 1 H), 7.87–8.28 (m, 9 H), 7.32–7.61 (m, 4 H), 7.18–7.25 (m, 2 H), 6.77 (m, 3 H), 6.56–6.59 (m, 2 H), 5.26 (m, 1 H), 4.65 (s, 1 H), 4.34–4.42 (m, 1 H), 3.93–3.96 (m, 6 H), 3.72–3.77 (m, 4 H), 3.37–3.63 (m, 5 H), 2.50–2.81 (m, 4 H), 1.99–2.33 (m, 2 H), 1.72–1.83 (m, 7 H), 1.60 (s, 34 H), 1.45 (s, 6 H), 1.25–1.29 (m, 90 H), 0.87 (t, J = 6.9 Hz, 10 H). ¹³C NMR (101 MHz, CDCl₃): δ = 169.23, 158.64, 153.26, 145.78, 143.88, 139.72, 137.99, 136.93, 132.50, 130.03, 128.84, 128.11, 127.92, 127.35, 125.27, 124.60, 113.20, 92.35, 86.47, 77.61, 73.53, 69.32, 55.24, 34.94, 31.92, 29.72, 29.66, 29.36, 26.10, 22.68, 14.11. HRMS (ESI⁺): m/z calcd. for C₁₄₀H₁₉₀N₁₁NaO₁₉PS⁺: 2415.3595; found: 2415.3577.
18 5′-OH-d[C^(Bz)A^(Bz)T^(Aza)]-3′-ACSS (5) The same elongation cycle was carried out for compound 9 with dC^(Bz) phosphoramidite to give 5 (94% yield) as a white solid. ¹H NMR (600 MHz, CDCl₃): δ = 8.74 (m, 1 H), 7.86–8.27 (m, 9 H), 7.36–7.60 (m, 4 H), 6.57–6.59 (m, 2 H), 5.29 (m, 1 H), 4.16–4.44 (m, 3 H), 3.91–3.96 (m, 7 H), 3.53–3.83 (m, 5 H), 3.13 (q, J = 7.3 Hz, 1 H), 2.65–2.82 (m, 5 H), 2.17 (s, 1 H), 1.72–1.83 (m, 23 H), 1.45 (s, 6 H), 1.25–1.33 (m, 105 H), 0.88 (t, J = 6.9 Hz, 10 H). ¹³C NMR (151 MHz, CDCl₃): δ = 170.56, 157.93, 153.27, 139.76, 135.25, 132.93, 128.79, 128.01, 127.77, 127.33, 126.13, 125.37, 124.62, 123.14, 105.71, 73.56, 69.34, 45.43, 31.93, 30.33, 29.73, 29.67, 29.44, 29.38, 26.12, 22.70, 14.13, 8.51. HRMS (ESI⁺): m/z calcd for C₁₃₈H₁₉₁N₁₅NaO₂₄P₂S⁺: 2559.3280; found: 2559.3212.

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