Modified GSMP Synthesis Greatly Improves the Disulfide Crosslink of T7 Run-Off siRNAs with Cell Penetrating Peptides

Abstract

Preventing intramolecular cyclization greatly improves 5′-deoxy-5′-thioguanosine monophosphorothioate (GSMP) synthesis and its application as a potent initiator nucleotide for T7 run-off transcription of noncoding RNAs. GSMP was efficiently incorporated into the 5′-end of siRNA sense strands and the resulting thiol-modified siRNA was crosslinked with a free cysteine of cell penetrating peptide Penetratin as monitored by mass spectrometry. Cellular uptake and the knockdown potential of the peptide-coupled siRNA (pepsiRNA) were evaluated in primary cells.

guanosine - T7 RNA - siRNA - Penetratin - disulfide crosslink - knockdown

References and Notes

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N , N -Dimethylaminomethylene-2′,3′- O , O -isopro-pylideneguanosine (8): 2′,3′-O,O-Isopropylideneguanosine (2; 5.75 g, 17.8 mmol) was resuspended in DMF (60 mL) and N,N-dimethylformamide dimethyl acetal (8.91 mL) was added under argon to yield an orange-brown solution. The reaction mixture was stirred at 50 ˚C for 4 h. The solvent was removed under reduced pressure and at elevated temperatures (ca. 55 ˚C), the white residue was then removed by filtration. The filtrate was dried under reduced pressure, redissolved in MeOH (25 mL) yielding a fluorescent green solution and precipitated with 50 mL of EtOAc. After storage overnight at 4 ˚C, the residue was removed by filtration and the combined filtrates were thoroughly washed with EtOAc and dried under reduced pressure. The product was obtained as a white powder in 80% yield (5.38 g, 14.2 mmol). All steps were carried out under protection from light; mp >200 ˚C; R _f 0.47 (CHCl₃-MeOH, 8:1), 0.08 (CHCl₃-MeOH, 19:1). ^¹H NMR (400 MHz, DMSO-d ₆): δ = 11.32 (br s, 1 H, NH), 8.56 (s, 1 H, H-imine), 8.00 (s, 1 H, H-8), 6.03 (d, J = 3.0 Hz, 1 H, H-1′), 5.26 (dd, J = 3.0, 6.3 Hz, 1 H, H-2′), 5.03 (dd, J = 5.4, 5.4 Hz, 1 H, 5′-OH), 4.95 (dd, J = 2.9, 6.3 Hz, 1 H, H-3′), 4.13 (ddd, J = 2.9, 4.9, 4.9 Hz, 1 H, H-4′), 3.59-3.47 (m, 2 H, H-5′), 3.15 (s, 3 H, NMe), 3.03 (s, 3 H, NMe), 1.53 (s, 3 H, Me), 1.33 (s, 3 H, Me). ^¹³C NMR (101 MHz, DMSO-d ₆): δ = 158.3 (C-imine), 157.6, 157.5 (C-4, C-6), 149.6 (C2), 136.0 (C-8), 119.9 (C-5), 113.2 (C-quat), 88.6, 86.4, 83.6, 81.2 (C-1′ to C-4′), 61.5 (C-5′), 40.8 (NMe), 34.8 (NMe), 27.2 (Me), 25.3 (Me). MS (EI; 70 eV): m/z (%) = 378 (89%) [M]⁺, 363 (6%) [M - CH]⁺, 348 (6%) [M - 2 Me]⁺, 333 (2%) [M - NH(Me)₂]⁺, 206 (100%) [M - DAMG]⁺, 191 (26%) [DAMG - Me]⁺, 176 (3%) [DAMG - 2 Me]⁺, 150 (6%) [guanine]⁺. HRMS: m/z calcd for C₁₆H₂₂N₆O₅: 378.1652; found: 378.1654 (±0.0095); (DAMG = 2-N′,N-dimethylaminomethylene guanine).
N , N -Dimethylaminomethylene-2′,3′- O , O -isopro-pylidene-5′-deoxy-5′-iodoguanosine (9): Ground 8 (2.20 g, 5.81 mmol) was resuspended in anhyd THF (110 mL) under argon and cooled to -70 ˚C by a mixture of acetone and dry ice. Methyltriphenoxyphosphonium iodide (3.94 g, 8.71 mmol; 1.5 equiv) was added. Due to the light sensitivity of the reactant and the product all subsequent steps were carried out under exclusion of light. After 30 min of stirring the reaction mixture was allowed to warm to r.t. and stirred for another 4 h. The reaction was stopped by the addition of MeOH (5 mL) and the solvent was removed under reduced pressure. The dark red residue was dissolved in MeOH-CHCl₃ (1:4; 2.5 mL) and CHCl₃ (7 mL) was added. The solution of the crude product was subjected to chromatography on silica gel (Merck 0.40-0.65 µm; eluent: CHCl₃-MeOH, 9:1). After removal of the solvent, 9 was obtained as an orange solid in 99% yield (2.81 g, 5.75 mmol); mp 95-97 ˚C; R _f 0.74 (CHCl₃-MeOH, 5:1), 0.22 (CHCl₃-MeOH, 19:1). ^¹H NMR (400 MHz, DMSO-d ₆): δ = 11.39 (br s, 1 H, NH), 8.58 (s, 1 H, H-imine), 8.01 (s, 1 H, H-8), 6.15 (d, J = 2.1 Hz, 1 H, H-1′), 5.42 (dd, J = 2.1, 6.3 Hz, 1 H, H-2′), 5.02 (dd, J = 2.8, 6.3 Hz, 1 H, H-3′), 4.28 (ddd, J = 2.8, 5.7, 9.0 Hz, 1 H, H-4′), 3.49 (dd, J = 9.0, 9.9 Hz, 1 H, H-5′), 3.30 (dd, J = 5.7, 9.9 Hz, 1 H, H-5′), 3.19 (s, 3 H, NMe), 3.04 (s, 3 H, NMe), 1.53 (s, 3 H, Me), 1.35 (s, 3 H, Me). ^¹³C NMR (101 MHz, DMSO-d ₆): δ = 158.2 (C-imine), 157.7, 157.5 (C-4, C-6), 149.2 (C2), 137.9 (C-8), 120.2 (C-5), 113.5 (C-quart), 89.4, 86.4, 84.0, 83.9 (C-1′ to C-4′), 41.1 (NMe), 34.9 (NMe), 27.2 (Me), 25.3 (Me), 6.7 (C-5′). MS (EI; 70 eV): m/z = 488 (13%) [M]⁺, 361 (7%)
[M - I]⁺, 360 (35%) [M - HI]⁺, 345 (1%) [M - HI, Me]⁺, 206 (3%) [DAMG]⁺, 188 (2%) [DAMG - H₂O]⁺, 176 (26%) [DAMG - 2 Me]⁺, 149 (12%) [guanine - H]⁺, 128 (100%) [HI]. HRMS: m/z calcd for C₁₆H₂₁N₆O₄I: 488.0669; found: 488.06631; (DAMG = 2-N′,N-dimethylaminomethylene guanine).

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