Synthesis and Evaluation of 3′-Oleyl–Oligonucleotide Conjugates as Potential Cellular Uptake Enhancers

Abstract

The field of therapeutic oligonucleotides has experienced significant growth in recent years, both in terms of approved drugs and those undergoing clinical trials. This expansion has transformed it into a rapidly evolving area of research. However, their cellular internalization remains a major limitation for the clinical application of oligonucleotides. To address this limitation, we report different strategies for the synthesis of specialized solid supports for the direct synthesis of 3′-oleyl-oligonucleotides by means of an l-threoninol derivative. A series of in vitro cell experiments were conducted to evaluate the potential of this strategy for enhanced cellular uptake. The results suggest that lipid conjugation enhances cellular uptake and facilitates oligonucleotide intracellular trafficking. Given these findings, the modification of therapeutic oligonucleotides through the attachment of lipidic moieties using a threoninol linker emerges as a valuable strategy to enhance their cellular internalization.

Key words

oligonucleotides - lipid conjugates - oleic acid - solid-phase synthesis - cellular uptake - therapeutics

Publikationsverlauf

Eingereicht: 20. Juli 2023

Angenommen nach Revision: 30. Oktober 2023

Artikel online veröffentlicht:
01. Dezember 2023

• References and Notes

• 1 Present address: Department of Organic Chemistry Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS), Santiago de Compostela University CIQUS, Spain.
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• 26 l-Threoninol (5 mmol) was dissolved in DMF (5 mL), treated with HMDS (10 mmol) and then stirred at rt for 30 min. The solution was evaporated to dryness. The crude residue was dissolved in pyridine (15 mL), oleic chloride (2 mL) was added dropwise and the mixture was stirred at rt for 1 h. The pyridine was evaporated and the crude residue was dissolved in CH₂Cl₂ and washed with brine. After concentration of the organic layer, the residue was dissolved in dioxane/MeOH (1:1 + NH₃ 25%) and evaporated. Purification by flash chromatography (CH₂Cl₂/MeOH 1% to 4%) yielded N-threoninol-oleamide 2. ¹H NMR (400 MHz, CDCl₃): δ = 6.34 (d, J = 8.5 Hz, 1 H, CH-OH), 5.39–5.25 (m, 2 H, CH=CH), 4.20 (s, 1 H, CH₃-CH-OH), 3.81 (s, 4 H, CH₂-NH, CH₂-OH), 3.04 (s, 1 H, NH), 2.27 (t, 4 H, CH₂CH=CH), 2.02 (m, 2 H, CH₂CO), 1.64 (m, 2 H, CH₂CH₂CO), 1.37–1.29 (m, 20 H, CH₂-oleic), 1.21 (d, J = 6.4 Hz, 3 H, CH₃-threoninol), 0.91(t, J = 13.9 Hz, 3 H, CH₃-oleyl). ¹³C NMR (125 MHz, CDCl₃): δ = 174.42 (CO), 130.0–129.72 (CH=CH), 68.53 (CH-NH), 64.85 (CH₃CH-OH), 54.63 (CH₂-O), 36.9 (CH₂-CO), 31.91 (CH₂-CH=CH), 29.78, 29.73, 29.34, 29.32, 29.29, 29.18, 27.24, 27.19, 25.88 (CH₂, alkyl chain), 22.69 (CH₂-CH₃), 20.50 (CH₃-threoninol), 14.13 (CH₃-oleyl).
• 27 Oleic acid (2.19 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (3.29 mmol), 1-hydroxybenzotriazole (HOBt) (3.29 mmol), and diisopropylethylamine (DIPEA) (3.29 mmol) in anhydrous DMF (20 mL) were mixed together under argon. After 5 minutes of stirring, l-threoninol (1.35 mmol) was added and the mixture was stirred overnight at rt. The solvent was evaporated under reduced pressure. The crude was dissolved in CH₂Cl₂ and washed twice with 10% aqueous NaHCO₃ and brine, dried and evaporated. The crude product was purified using flash chromatography on silica gel with a methanol gradient (1% to 4% in CH₂Cl₂) to yield N-threoninol-oleamide 2 as a yellowish oil.
• 28 4-Nitrophenol (10 mmol) was dissolved in CH₂Cl₂ (10 mL) with Et₃N (40 mmol) and oleyl chloride (10 mmol) added under ice-bath conditions. The resulting mixture was stirred at rt for 1 h after removal of the ice bath and then washed sequentially with water, NaHCO₃, and water again. The organic layer was separated, dried and concentrated to give the desired product in 85% yield.¹H NMR (400 MHz, CDCl₃): δ = 8 2 (d, 2 H, HAr), 7.2 (d, 2 H, HAr), 5.2 (m, 2 H, CH=CH), 2.5 (t, 2 H, CH₂-CO), 2 (m, 4 H, CH₂-alkene), 1.7 (t, 2 H, CH₂-CH₂-CO), 1.3-1.2 (n, dd, m, 20H, alkyl chain), 0.8 (t, 3 H, CH₃). 0,82-0,81 (m, 3 H, CH3), C NMR (101 MHz, CDCl₃): δ = 171.29 (CO), 155.54 (Ar-O), 145.26 (Ar-NO₂), 130.11–129.66 (CH=CH), 125.19 (Ar), 122.43 (Ar), 34.34 (CH₂-CO), 31.92 (CH₂-alkene), 29.77, 29.67, 29.54, 29.34, 29.13, 29.07, 29.03, 27.24, 27.23, 27.15, 24.74, 22.69 (CH₂-CH₃, alkyl chain), 14.12 (CH₃). MS (MALDI-TOF): m/z = 503.5 (M + Et₃N).l-threoninol (1 equiv) was dissolved in dioxane (5 mL) under an Ar atmosphere, and Et₃N (4 equiv) and the above-prepared 4-nitrophenyl oleate (2 equiv) were added. The reaction was left to stir overnight at 40 °C. After work-up, the crude product was purified via silica gel column chromatography (gradient 0–3% MeOH/CH₂Cl₂) to afford product 2 as a yellowish oil [R_f = 0 15 (MeOH/CH₂Cl₂, 5:95)].
• 29a Product 2 (1 equiv) and DMAP (0.5 equiv) were dissolved in pyridine (5 mL). DIPEA (2 equiv) was added dropwise and the resulting mixture was stirred for 5 min at rt. Trityl chloride (TrCl) (1.5 equiv) was added and the mixture was heated to 45 °C and stirred overnight in the dark. Further TrCl (0.5 equiv) was added to ensure a complete reaction. Following this, MeOH (1 mL) was added and the mixture was stirred for 5 min. The pyridine was evaporated and work-up was performed. The solution medium was neutralized using saturated NaHCO₃ (30 mL), and subsequently washed with DCM (3 x 30 mL). The organic layer was separated, dried out with MgSO₄, filtered, and concentrated to dryness. The resulting crude residue was purified by flash column chromatography (CH₂Cl₂ to CH₂Cl₂/MeOH 4%), resulting in the expected trityl derivative 3 as a white solid in a yield of 78%.¹H NMR (400 MHz, CDCl₃): δ = 8.54 (s, 1 H, NH), 7.35–7.29 (m, 6 H), 7.27–7.13 (m, 9 H), 6.01 (d, J = 8.7 Hz, 1 H, CH₃CH-OH), 5.31–5.24 (m, 2 H, CH=CH), 4.03–3.98 (m, 1 H, CH₃CH-OH), 3.88–3.83 (m, 1 H, CH-NH), 3.71–3.64 (m, 2 H, CH₂OH), 3.36 (dd, J = 9.6, 4.4 Hz, 1 H), 3.20 (dd, J = 9.6, 3.5 Hz, 1 H), 2.15 (dd, J = 8.5, 6.8 Hz, 4 H, CH₂CH=CH), 1.94–1.93 (m, 2 H, CO-CH₂-CH₂), 1.83–1.75 (m, 2 H, CO-CH₂-CH₂), 1.27–1.20 (m, 20 H, alkyl chain), 1.05 (d, J = 6.3 Hz, 3 H, CH₃-threoninol), 0.86–0.72 (m, 3 H, CH₃-oleyl). ¹³C NMR (101 MHz, CDCl₃): δ = 172.53 (CO), 148.76 , 142.28 (Carom), 134.98 (Carom), 128.98–128.72 (CH=CH), 127.43 (Carom), 127.04 (Carom), 126.88 (Carom), 126.33 (Carom), 122.74 (Carom), 86.28 (Cq), 76.21, 67.67 (CH-NH), 66.95 (CH₃CH-OH), 64.47 (CH₂-O), 52.16 (CH₂-CO), 35.90 (CH₂-CH=CH), 30.88, 28.75, 28.71, 28.68, 28.50, 28.33, 28.30, 28.13, 26.20, 26.17, 24.86, 24.58 (CH₂, alkyl chain), 21.67 (CH₂-CH₃), 18.86 (CH₃-threoninol), 13.11 (CH₃-oleyl).
• 30 Compound 3 (1 equiv) was dried (2 × toluene, 2 × MeCN) and dissolved in anhydrous CH₂Cl₂. Succinic anhydride (1.3 equiv) and DMAP (1.3 equiv) were added and the reaction mixture was stirred overnight at rt. The organic layer was washed with NaH₂PO₃ (0.1 M) and dried. The resulting crude residue was used in the next step without further purification.
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