Evaluation of Transiently O-6-Protected Guanosine Morpholino Nucleosides in Phosphorodiamidate Morpholino Oligonucleotide Synthesis

Abstract

Figure 1 (A) O-6-Protected DNA and RNA monomers. (B) O-6-Protected morpholino monomers.

A novel strategy is presented for the synthesis of morpholino guanosine monomers protected at O-6 with 1-(4-azidophenyl)ethan-1-ol, p-methoxybenzyl alcohol and trimethylsilylethyl groups. The introduction of these protecting groups increases the solubility of the morpholino nucleosides which is crucial during the synthesis of phosphorodiamidate morpholino oligonucleotides (PMOs). HPLC analysis shows that the trimethylsilylethyl-protected monomer gives better coupling efficiency in PMO synthesis compared to the regular monomer. Moreover the nonpolar nature of the O-6-protected monomer facilitates the preparation of guanosine-rich oligomer in solution.

Key words

antisense agents - bioorganic chemistry - oligonucleotides - protecting groups - solid-phase synthesis

Publication History

Received: 24 July 2023

Accepted: 08 May 2024

Accepted Manuscript online:
08 May 2024

Article published online:
27 May 2024

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• 21 To a stirred solution of 4a (202 mg, 0.3 mmol, 1 equiv.) in dry (1:1) CH₃CN: DCM at 0 °C were added LiBr (1.12 mmol, 4 equiv.), tetramethylguanidine (1.12 mmol, 4 equiv.) and POCl₂NMe ₂ (0.84 mmol, 3 equiv.). Then the reaction mixture was left for 15 min at 0° C. TLC showed complete consumption of the starting material. After completion of the reaction (TLC analysis), the solvent was evaporated in vacuo and re-dissolved in EtOAc. The organic layer was washed with water and saturated aq. NH₄Cl. The organic layer was then dried on anhydrous Na₂SO₄ and solvent was evaporated in vacuo. The crude mixture was purified by silica gel (60-120 mesh) column chromatography eluting with Acetone-DCM to obtain the compound 5a in 60% yield (143 mg) (Rf = 0.6 in 3.5% MeOH-DCM).1H NMR (300 MHz, CDCl₃): δ 7.84 – 7.72 (m, 2H), 7.59 – 7.34 (m, 10H), 7.34 – 7.27 (m, 6H), 7.19 (q, J = 5.8 Hz, 3H), 7.01 – 6.89 (m, 2H), 6.42 (q, J = 6.6 Hz, 1H), 6.21 (dt, J = 9.7, 2.3 Hz, 1H), 4.54 – 4.41 (m, 1H), 4.11 (ddd, J = 8.9, 6.0, 3.4 Hz, 1H), 3.44 (d, J = 11.6 Hz, 1H), 3.26 – 3.08 (m, 2H), 2.62 (dd, J = 13.9, 4.9 Hz, 6H), 1.74 – 1.69 (m, 3H), 1.55 (dd, J = 10.7, 3.2 Hz, 1H), 1.35 (d, J = 6.9 Hz, 3H), 1.31 – 1.24 (m, 7H) ppm. 13C NMR (75 MHz, CDCl₃): δ 178.0, 176.1, 160.3, 152.3, 152.0, 147.4, 139.5, 139.0, 138.9, 138.7, 136.5, 135.6, 135.6, 129.9, 129.2, 128.1, 128.0, 127.9, 127.8, 127.0, 126.8, 126.6, 119.1, 119.1, 118.0, 80.6, 80.5, 77.6, 77.4, 77.2, 77.0, 76.7, 74.7, 74.6, 69.6, 67.2, 64.6, 53.9, 53.1, 49.1, 49.0, 36.7, 35.5, 31.8, 29.8, 29.4, 26.8, 22.8, 19.6, 19.5, 19.4, 19.3, 19.1, 19.0, 14.2 ppm. 31P NMR (121 MHz, CDCl₃): δ 18.5, 18.2 ppm. IR (CHCl₃, cm-1): 2980, 2929, 2102, 1716, 1601, 1508, 1249, 1050, 712.HRMS(ESI)[M+H]+: Mass calculated for C₄₃H₄₇ClN10O5P was 849.3152, found 849.3159.Synthesis of compound 5b: Compound 5b was synthesized from 4b (265 mg, 0.38 mmol, 1 equiv.) following the procedure of 5a in 60% yield (188mg). Rf: 0.5 (3.5% MeOH-DCM).1H NMR (300 MHz, CDCl₃): δ 7.80 (s, 1H), 7.75 (d, J = 3.0 Hz, 1H), 7.53 – 7.38 (m, 7H), 7.36 – 7.26 (m, 7H), 7.19 (t, J = 7.3 Hz, 3H), 6.94 – 6.81 (m, 3H), 6.39 – 6.06 (m, 1H), 5.61 – 5.47 (m, 2H), 4.54 – 4.41 (m, 1H), 4.10 (tt, J = 7.9, 3.8 Hz, 2H), 3.79 (s, 3H), 3.46 (d, J = 11.4 Hz, 1H), 3.22 (d, J = 11.9 Hz, 2H), 2.61 (dd, J = 13.9, 5.8 Hz, 6H), 1.35 (t, J = 6.5 Hz, 6H) ppm. 13C NMR (75 MHz, CDCl₃): δ 160.5, 159.3, 151.9, 151.7, 138.6, 138.5, 132.8, 129.9, 128.8, 128.3, 127.7, 127.6, 126.4, 117.5, 113.7, 113.5, 80.0, 77.1, 76.9, 76.7, 76.3, 74.3, 74.2, 68.3, 66.8, 64.8, 55.0, 52.6, 48.6, 36.3, 36.3, 35.2, 29.4, 19.1, -0.32 ppm. 31P NMR (121 MHz, CDCl3): δ 18.5, 18.2 ppm. IR (CHCl₃, cm-1): 2931, 1709, 1607, 1513, 1242, 1027, 747. HRMS. (ESI) [M + Na]+: Mass calculated for C₄₃H₄₇ClN₇NaO₆P was 846.2906, found 846.2910.
• 22 Synthesis of compound 11: Compound 11 was synthesized from compound 10 (484 mg, 0.7 mmol, 1 equiv.) following the procedure of 5a in 66% yield (369mg). Rf: 0.6 (3.5% MeOH-DCM). 1H NMR (300 MHz, CDCl₃): δ 7.87 (s, 1H), 7.75 (d, J = 3.1 Hz, 1H), 7.47 (d, J = 7.6 Hz, 6H), 7.29 (t, J = 7.6 Hz, 6H), 7.18 (t, J = 7.3 Hz, 3H), 6.25 (dt, J = 9.7, 2.2 Hz, 1H), 4.69 – 4.53 (m, 2H), 4.53 – 4.42 (m, 1H), 4.18 – 4.03 (m, 2H), 3.52 – 3.41 (m, 1H), 3.37 (s, 1H), 3.23 (dd, J = 11.6, 2.4 Hz, 1H), 2.60 (dd, J = 13.9, 5.7 Hz, 6H), 1.77 (ddd, J = 11.3, 9.9, 3.0 Hz, 1H), 1.66 – 1.51 (m, 1H), 1.34 (t, J = 6.8 Hz, 6H), 1.26 – 1.16 (m, 3H), 0.08 (s, 9H) ppm. 13C NMR (75 MHz, CDCl₃): δ 176.8, 161.1, 152.2, 152.1, 138.8, 138.8, 129.2, 128.0, 126.7, 117.8, 80.3, 77.6, 77.4, 77.2, 77.0, 76.7, 74.7, 74.6, 67.2, 67.2, 65.9, 53.0, 49.0, 36.7, 36.6, 35.1, 19.5, 19.4, 17.5, -1.3, -1.7 ppm. 31P NMR (121MHz, CDCl₃): δ 18.5, 18.1 ppm. IR (CHCl₃, cm-1): 2953, 1688, 1603, 1250, 1030, 712. HRMS (ESI) [M + H]+: Mass calculated for C₄₀H₅₂N₇O₅SiP was 804.3220 , found 804.3224.