Synlett 2019; 30(11): 1303-1307
DOI: 10.1055/s-0037-1611550
letter
© Georg Thieme Verlag Stuttgart · New York

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*
Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509, Japan   Email: [email protected]
› Author Affiliations
This work was partially supported by JSPS KAKENHI Grant Numbers 15H04494 (to K.C.), 16J07350 (to N.S.).
Further Information

Publication History

Received: 03 April 2019

Accepted after revision: 29 April 2019

Publication Date:
15 May 2019 (online)


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.

Supporting Information

 
  • 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.