A Facile Synthesis of 3′-Fluoro Hexitol Adenosine and Guanosine Phosphoramidites

Dedicated to Prof. Har Gobind Khorana

Abstract

We report a convenient and scalable synthetic approach for the synthesis of 3′-fluoro hexitol adenosine and guanosine nucleoside analogues and corresponding phosphoramidites in good yield. 1,5-Anhydro-4,6-O-benzylidene-d-glucitol was converted into 1,5-anhydro-4,6-O-benzylidene-3-deoxy-3-fluoro-2-O-trifluoromethanesulfonyl-d-altritol in a three-step process. Glycosylation using adenosine or 2-amino-6-iodopurine yielded the corresponding nucleoside analogues in excellent yield. Based on this strategy, a highly concise and scalable synthesis of 3′-fluoro hexitol purine nucleosides (1–2 g, 18–21% overall yield) was accomplished, which will enable the use of 3′-fluoro hexitol nucleic acids for genetic medicine development and diagnostic applications.

Key words

antisense oligonucleotides - FHNA adenosine nucleoside - FHNA guanosine nucleoside - hexitol nucleic acids - chemically modified siRNAs

Publication History

Received: 23 July 2023

Accepted after revision: 19 September 2023

Article published online:
19 October 2023

• References and Notes

• 1 Crooke ST, Baker BF, Crooke RM, Liang X.-h. Nat. Rev. Drug Discovery 2021; 20: 427 DOI: 10.1038/s41573-021-00162-z.
• 2 Egli M, Manoharan M. Nucleic Acids Res. 2023; 51: 2529
• 3 Wan WB, Seth PP. J. Med. Chem. 2016; 59: 9645
• 4 Egli M, Manoharan M. Acc. Chem. Res. 2019; 52: 1036
• 5 Prakash TP. In Chemistry and Biology of Artificial Nucleic Acids . Egli M, Herdewijn P. Wiely-VCH; Weinheim: 2012: 233
• 6 Egli M, Pallan PS, Allerson CR, Prakash TP, Berdeja A, Yu J.-H, Lee S, Watt A, Gaus H, Bhat B, Swayze EE, Seth PP. J. Am. Chem. Soc. 2011; 133: 16642
• 7 Purser S, Moore PR, Swallow S, Gouverneur V. Chem. Soc. Rev. 2008; 37: 320
• 8 Hagmann WK. J. Med. Chem. 2008; 51: 4359
• 9 Kirk KL. J. Fluorine Chem. 2006; 127: 1013
• 10 Prakash TP, Lima WF, Murray HM, Li W, Kinberger GA, Chappell AE, Gaus H, Seth PP, Bhat B, Crooke ST, Swayze EE. Nucleic Acids Res. 2017; 45: 6994
• 11 Parmar RG, Brown CR, Matsuda S, Willoughby JL. S, Theile CS, Charisse K, Foster DJ, Zlatev I, Jadhav V, Maier MA, Egli M, Manoharan M, Rajeev KG. J. Med. Chem. 2011; 61: 734
• 12 Guzaev AP, Manoharan M. J. Am. Chem. Soc. 2003; 125: 2380
• 13 1,5-Anhydro-4,6-O-benzylidene-3-deoxy-3-fluoro-2-O-pivaloyl-d-altritol (6). A solution of trifluoromethanesulfonic anhydride (6.0 mL, 35.6 mmol) in dichloromethane (6 mL) was added to a cold (0 °C) solution of 4 (10.0 g, 29.7 mmol) in dichloromethane (45 mL) and pyridine (4.7 mL, 58.5 mmol). After stirring for 1 h the reaction was quenched by adding water and the organic layer was washed with water (100 mL) and brine (100 mL), then dried (Na₂SO₄) and concentrated to provide the 3-O-trifluoromethyl derivative, which was used without further purification. ¹H NMR (300 MHz, CDCl₃): δ = 7.53–7.42 (m, 2 H), 7.42–7.32 (m, 3 H), 5.59 (s, 1 H), 5.10 (s, 2 H), 4.48–4.33 (m, 1 H), 4.32–4.15 (m, 1 H), 3.90–3.69 (m, 2 H), 3.57–3.42 (m, 1 H), 3.40–3.22 (m, 1 H), 1.24 (s, 9 H). A solution of 3-O-trifluoromethanesulfonyl derivative from above and cesium fluoride (16.7 g, 109.9 mmol) in tert-butanol (110 mL) was heated at 70 ° for 2 h. The reaction mixture was then cooled to room temperature, diluted with ethyl acetate (200 mL) and the organic layer was washed with water (200 mL) and brine (220 mL), then dried (Na₂SO₄) and concentrated. Purification by column chromatography (silica gel, eluting with 10 to 20% ethyl acetate in hexanes) provided compound 6 (9.4 g, 94%). ¹H NMR (300 MHz, CDCl₃): δ = 7.49 (m, 2 H), 7.37 (m, 3 H), 5.56 (s, 1 H), 5.29–5.02 (m, 1 H), 5.02–4.81 (m, 1 H), 4.49–4.32 (m, 1 H), 4.22–4.04 (m, 1 H), 3.99–3.54 (m, 7 H), 1.23 (s, 9 H). ¹⁹F NMR (282 MHz, CDCl₃): δ = –217. 53 (m, H-F coupled). HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₂₄FO₅: 339.1608; found: 339.1609.
• 14 1,5-Anhydro-4,6-O-benzylidene-3-deoxy-3-fluoro-d-altritol (7). Potassium carbonate (9.9 g, 71.6 mmol) was added to a solution of compound 6 (9.0 g, 26.6 mmol) in methanol (100 mL). After stirring at room temperature for 3 h, the solvent was evaporated under reduced pressure and the residue was partitioned between ethyl acetate (100 mL) and water (100 mL). The organic layer was dried (Na₂SO₄) and concentrated to yield 7 (6.5 g, 96%), which was used without further purification. ¹H NMR (300 MHz, CDCl₃): δ = 7.58–7.30 (m, 5 H), 5.54 (s, 1 H), 5.23–4.94 (m, 1 H), 4.39 (dd, J = 4.7, 10.0 Hz, 1 H), 4.02–3.43 (m, 6 H), 2.25–2.08 (m, 1 H). ¹³C NMR (75.47 MHz, CDCl₃): δ = 136.8, 129.3, 128.4, 126.3, 102.2, 90.5, 88.1, 77.7, 69.2, 66.9, 65.8. ¹⁹F NMR (282 MHz, DMSO-d₆): δ = –217. 53 (m, H-F coupled). MS (ESI): m/z [M + H]⁺ calcd for C₁₃H₁₆FO₄: 254.26; found: 254.16.
• 15 1,5-Anhydro-4,6-O-benzylidene-3-deoxy-3-fluoro-2-O-trifluoromethanesulfonyl-d-altritol (8). Trifluoromethanesulfonic anhydride (5.2 mL, 30.8 mmol) was added to a cold (0 °C) solution of compound 7 (6.5 g, 25.7 mmol) in dichloromethane (39 mL) and pyridine (6.1 mL, 75.9 mmol). After stirring for 1 h, the reaction was quenched by adding water (10 mL) and the organic layer was washed with water (50 mL) and brine (50 mL), then dried (Na₂SO₄) and concentrated to provide crude 8 (9.4 g, 95%), which was used without further purification. ¹H NMR (300 MHz, CDCl₃): δ = 7.58–7.32 (m, 5 H), 5.55 (s, 1 H), 5.28 (1 H, d, J = 55 Hz), 5.02–4.85 (m, 1 H), 4.42 (dd, J = 4.9, 10.4 Hz, 1 H), 4.09 (dd, J = 5.7, 10.8 Hz, 1 H), 4.01–3.80 (m, 2 H), 3.78–3.50 (m, 2 H). ¹³C NMR (75 MHz, CDCl₃): δ = 136.4, 129.5, 128.4, 126.2, 102.3, 87.5, 85.0, 78.4, 78.1, 77.1, 76.9, 68.8, 66.1, 66.0, 63.5. ¹⁹F NMR (282 MHz, CDCl₃): δ = –74.77 (s, 3 F), –215. 96 (m, 1 F, H-F coupled). MS (ESI): m/z [M + H]⁺ calcd for C₁₄H₁₅F₄O₆S: 387.04; found: 387.01
• 16 1,5-Anhydro-4,6-O-benzylidene-2,3-dideoxy-3-fluoro-2-(N-6-benzoyl-adenin-9-yl)-d-altritol (13). Compound 12 (3.9 g 10.5 mmol) was dissolved in anhydrous pyridine (50 mL), then cooled to 0 °C and the solution was treated with benzoyl chloride (1.97 mL, 16.9 mmol). Stirring was continued at 0 °C for 15 min at which time the mixture was warmed to room temperature over 2.5 h. The mixture was cooled to 0 °C, quenched with H₂O (20 mL) and stirred for 15 min. Concentrated aqueous NH₄OH (20 mL) was added to the mixture with stirring for 30 min. The mixture was concentrated under reduced pressure to approximately 40 mL and poured into ethyl acetate (200 mL). The mixture was washed with saturated aqueous NaCl (3 × 500 mL), dried (Na₂SO₄), filtered, and evaporated. The residue was purified by silica gel chromatography (methanol/dichloromethane, 1–5%) to yield 13 (3.6 g, 72%). ¹H NMR (300 MHz, CDCl₃): δ = 9.23 (s, 1 H), 8.77 (s, 1 H), 8.55 (s, 1 H), 8.03 (d, J = 9.0 Hz, 2 H), 7.68–7.28 (m, 8 H), 5.51 (s, 1 H), 5.31 (d, J = 48.0 Hz, 1 H), 5.04 (br. s, 1 H), 4.59–4.35 (m, 3 H), 4.21–4.07 (m, 1 H), 3.86–3.59 (m, 2 H). ¹³C NMR (75 MHz, CDCl₃): δ = 164.8, 152.9, 152.3, 149.8, 141.4, 136.5, 133.3, 132.9, 129.3, 128.8, 128.3, 128.0, 126.1, 122.7, 102.6, 85.8, 83.4, 75.2, 75.9, 68.8, 67.6, 65.0, 53.7, 53.3. ¹⁹F NMR (282 MHz, CDCl₃): δ = –200.8 (m, H-F coupled). MS (ESI): m/z [M + H]⁺ calcd for C₂₅H₂₃FN₅O₄: 476.17; found: 476.02.
• 17a 1,5-Anhydro-(2-amino-6-iodopurin-9-yl)-4,6-O-benzylidene-2,3-dideoxy-3-fluoro-2-d-altritol (17). Compound 8 (2.9 g, 7.51 mmol) and 6-iodo-2-aminopurine tetrabutylammonium salt 16 (8.5 g, 17.6 mmol, prepared as described in ref. 17b), were dissolved in anhydrous hexamethylphosphoramide (HMPA, 26 mL). After stirring at room temperature for 18 h, the reaction mixture was poured into ethyl acetate (100 mL) and washed with water (100 mL) and brine (100 mL). The organic phase was dried over anhydrous Na₂SO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/hexanes, 50%) to yield 17 (2.8, 75%). ¹H NMR (300 MHz, DMSO-d ₆): δ = 8.17 (s, 1 H), 7.36 (s, 5 H), 7.00 (s, 2 H), 5.68 (s, 1 H), 4.99–5.31 (d, J = 48 Hz, 1 H), 4.70–4.84 (m, 1 H), 4.35–4.53 (m, 1 H), 4.14–4.35 (m, 2 H), 3.56–4.10 (m, 3 H). ¹³C NMR (75.47 MHz, CDCl₃): δ = 159.0, 149.8, 139.7, 13654, 131.7, 129.3, 128.3, 126.1, 123.0, 102.6, 85.6, 83.1, 75.2, 74.9, 68.8, 67.5, 64.8, 53.5, 53.1. ¹⁹F NMR (282 MHz, DMSO-d ₆): δ = –201.8 (m, H-F coupled). MS (ESI): m/z [M + H]⁺ calcd for C₁₈H₁₈FIN₅O₃: 498.27; found: 498.20.
• 17b Bisacchi GS, Singh J, Godfrey JD. Jr, Kissick TP, Mitt T, Malley MF, Di Marco JD, Gougoutas JZ, Mueller RH, Zahler R. J. Org. Chem. 1995; 60: 2902
• 18 1,5-Anhydro-4,6-O-benzylidene-2,3-dideoxy-3-fluoro-2-(guanin-9-yl)-d-altritol (18). Compound 17 (2.7 g, 5.43 mmol) was dissolved in 1,4-dioxane (44 mL) and 1 M aqueous NaOH (30 mL) was added, and reaction mixture was heated at 55 °C for 18 h. The reaction mixture was cooled then neutralized with 1N HCl. The reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (methanol/dichloromethane, 5%) to yield 18 (1.9 g, 90%). ¹H NMR (300 MHz, DMSO-d ₆): δ = 10.65 (s, 1 H), 7.83 (s, 1 H), 7.37 (d, J = 1.32 Hz, 5 H), 6.61 (br s, 2 H), 5.72 (s, 1 H), 5.00–5.33 (d, J = 45 Hz, 1 H), 4.56–4.76 (m, 1 H), 4.05–4.46 (m, 3 H), 3.75–4.03 (m, 3 H). ¹³C NMR (75.47 MHz, DMSO-d ₆): δ = 156.8, 153.9, 151.3, 137.3, 135.2, 128.9, 128.0, 126.1, 116.1, 101.1, 86.4, 84.4, 74.0, 67.8, 67.0, 64.7, 52.6, 52.2. ¹⁹F NMR (282 MHz, DMSO-d ₆): δ = –200.39 (m, H-F coupled). HRMS (ESI): m/z [M + H]⁺ calcd for C₁₈H₁₉FN₅O₄: 388.1421; found: 388.1429.
• 19 1,5-Anhydro-2,3-dideoxy-6-O-(4,4′-dimethoxytrityl)-3-fluoro-2-(N-2-isobutyrylguanin-9-yl)-d-altritol (21). Compound 20 (1.4 g 3.79 mmol) was dissolved in anhydrous pyridine (32 mL), 4,4′-dimethoxytrityl chloride (1.7 g, 5.02 mmol) was added and the mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated in vacuo and the residue was redissolved in ethyl acetate (50 mL). The organic solution was washed with saturated aqueous NaHCO₃ (50 mL) and brine (50 mL), dried over anhydrous Na₂SO₄, filtered, and evaporated under reduced pressure. Purification by silica gel column chromatography (methanol/dichloromethane, 0–5%) yielded 21 (1.6 g, 88%). ¹H NMR (300 MHz, DMSO-d ₆): δ = 12.12 (s, 1 H), 11.74 (s, 1 H), 8.04–8.34 (m, 1 H), 7.36–7.50 (m, 2 H), 7.16–7.35 (m, 7 H), 6.89 (dd, J = 2.74, 8.93 Hz, 4 H), 5.75 (s, 1 H), 5.18–5.32 (m, 1 H), 4.98–5.18 (m, 1 H), 4.96 (br d, J = 3.59 Hz, 1 H), 4.77 (br d, J = 1.97 Hz, 1 H), 4.32 (br d, J = 12.93 Hz, 1 H), 4.02–4.23 (m, 1 H), 3.65–3.88 (m, 8 H), 3.47–3.65 (m, 1 H), 2.64–2.90 (m, 1 H), 1.13 (d, J = 6.73 Hz, 6 H). ¹³C NMR (75 MHz, CDCl₃): δ = 180.4, 158.5, 158.5, 158.2, 155.8, 149.6, 148.8, 144.7, 136.2, 130.1, 128.3, 128.1, 127.8, 127.7, 127.5, 126.5, 119.5, 118.1, 113.1, 112.8, 85.9, 62.9, 62.8, 57.7, 55.2, 54.9, 36.1, 21.3, 19.1, 18.9, 18.7. ¹⁹F NMR (282 MHz, CDCl₃): δ = –203.96. HRMS (ESI): m/z [M + H]⁺ calcd for C₃₆H₃₉FN₅O₇: 672.2834; found: 672.2836.