Synthesis of 2-Aminopyridine-Modified Peptide Nucleic Acids

Abstract

Triplex-forming peptide nucleic acids (PNAs) require chemical modifications for efficient sequence-specific recognition of DNA and RNA at physiological pH. Our research groups have developed 2-aminopyridine (M) as an effective mimic of protonated cytosine in C+•G-C triplets. M-modified PNAs have a high binding affinity and sequence specificity as well as promising biological properties for improving PNA applications. This communication reports the optimization of synthetic procedures that give PNA M monomer in seven steps, with minimal need for column chromatography and in good yields and high purity. The optimized route uses inexpensive reagents and easily performed reactions, which will be useful for the broad community of nucleic acid chemists. Thought has also been given to the potential for future development of industrial syntheses of M monomers.

Key words

peptide nucleic acids - aminopyridine - RNA recognition - triple helix - solid-phase synthesis

• References and Notes

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• 18 Ethyl 2-(6-Aminopyridin-3-yl)acetate TFA (47.0 mL, 610 mmol, 10 equiv) was added to a solution of diester 7 (19.0 g, 61.1 mmol) in CH₂Cl₂ (200 mL) at 0 °C (ice bath). After 23 h at RT, the solvent was evaporated. The residue was co-evaporated with toluene (2 × 50 mL) under reduced pressure to afford acid 8 (15.1 g), which was dissolved in EtOH (160 mL). N₂ gas was bubbled through this solution for 10 min, and then 10% w/w Pd/C (1.60 g) was added and the mixture was bubbled with H₂ gas. After 30 h, N₂ gas was bubbled through this solution for 10 min. The mixture was then filtered through a pad of Celite and washed with EtOH (3 × 15 mL). The combined filtrates were evaporated under reduced pressure to give a dark-yellow oil [yield: 10.3 g (estimated 93% over 2 steps)], which was used in the next step without purification.
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• 20 {6-[(tert-Butoxycarbonyl)amino]pyridin-3-yl}acetic Acid (5) 1 M aq LiOH (49 mL, 48.7 mmol, 2 equiv) was added to a solution of ester 4 (6.83 g, 24.4 mmol) in EtOH (60 mL). After 6 h at RT, the solvent was partly evaporated and the residue was acidified with 1 M aq HCl to pH ~5. The resulting precipitate was collected by filtration and washed with cold H₂O (3 × 10 mL) to give a white solid; yield: 4.89 g (80%). The characterization data matched those previously reported.¹³
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• 22 11b 2-{6-[(tert-Butoxycarbonyl)amino]pyridin-3-yl}acetic acid (5) (3.26 g, 12.9 mmol, 1 equiv), O-benzyl-protected PNA backbone 10b (5.56 g, 12.9 mmol), and HBTU (4.90 g, 12.9 mmol) were dissolved in anhyd DMF (50 mL). The solution was cooled on ice, and DIPEA (5.6 mL, 32.3 mmol, 2.5 equiv) was added. The ice bath was removed, and the solution was stirred overnight at RT. The mixture was then concentrated under reduced pressure, and the residue was dissolved in EtOAc (70 mL) and extracted with sat. aq NaHCO₃ (2 × 20 mL). The organic layer was dried (Na₂SO₄) and concentrated under reduced pressure. The product was purified by column chromatography [silica gel, EtOAc–hexanes (linear gradient 50–100%)] to give a slightly yellow oil; yield: 6.14 g (71% yield); R_f = 0.44 (hexane–EtOAc, 3:7). ¹H NMR (400 MHz, DMSO-d ₆) (mixture of rotamers): δ = 9.74 (s, 1 H), 8.10 (d, J = 2.0 Hz, 0.6 H), 8.03 (d, J = 2.0 Hz, 0.4 H), 7.91–7.85 (m, 2 H), 7.77–7.61 (m, 3 H), 7.55–7.28 (m, 11 H), 5.16 (s, 0.6 H), 5.12 (s, 1.4 H), 4.45–4.27 (m, 2.6 H), 4.22 (t, J = 5.9 Hz, 1 H), 4.11 (s, 1.4 H), 3.70 (s, 1.4 H), 3.56 (s, 0.6 H), 3.48 (t, J = 6.5 Hz, 1.4 H), 3.39 (t, J = 6.5 Hz, 1 H, 0.6 H), 3.23 (q, J = 6.4 Hz, 1.4 H), 3.15 (q, J = 6.4 Hz, 0.6 H), 1.47 (s, 9 H). ¹³C{¹H} NMR (101 MHz, DMSO-d ₆): δ = 171.0, 170.7, 169.7, 169.2, 156.4, 156.1, 152.7, 151.0, 151.0, 150.9, 148.1, 148.0, 143.9, 143.8, 140.8, 140.7, 138.9, 138.7, 135.8, 135.6, 128.5, 128.4, 128.2, 128.1, 128.0, 127.8, 127.6, 127.0, 125.5, 125.5, 125.1, 125.0, 120.1, 120.1, 111.8, 111.8, 82.5, 79.5, 66.5, 65.8, 65.4, 65.4, 50.1, 48.0, 47.8, 46.7, 35.6, 35.1, 28.0, 27.4. HRMS (ESI/Q-TOF): m/z [M + H]⁺ calcd for C₃₈H₄₁N₄O₇: 665.2970; found: 665.2988. M Monomer 12 A solution of intermediate 11b (6.14 g, 9.2 mmol) in MeOH (70 mL) was bubbled with N₂ gas for 10 min, and then Pd/C (600 mg) was added and H₂ gas (1 atm) was bubbled through the reaction mixture. After 1 h, N₂ gas was bubbled through this solution for 10 min. The mixture was filtered through a pad of Celite, and the pad was washed with MeOH (4 × 15 mL). The combined filtrates were evaporated under reduced pressure to give a white foam; yield: 5.0 g (95%). ¹H NMR (400 MHz, DMSO-d ₆) (mixture of rotamers): δ = 12.7 (br s, 1 H), 9.78 (s, 1 H), 8.1 (d, J = 2.4 Hz, 0.7 H), 8.06 (d, J = 2.4 Hz, 0.3 H), 7.88 (d, J = 7.5 Hz, 2 H), 7.74 (d, J = 8.6 Hz, 1 H), 7.68 (d, J = 7.5 Hz, 2 H), 7.59–7.52 (m, 1 H), 7.50–7.21 (m, 5 H), 4.48–4.15 (m, 3.7 H), 3.98 (s, 1.3 H), 3.70 (s, 1.3 H), 3.56 (s, 0.7 H), 3.45 (t, J = 6.6 Hz, 1.3 H), 3.37 (t, J = 6.6 Hz, 0.7 H), 3.23 (q, J = 6.3 Hz, 1.3 H), 3.15 (q, J = 6.3 Hz, 0.7 H), 1.46 (s, 9 H). ¹³C{¹H} NMR (101 MHz, DMSO-d ₆): δ = 206.5, 171.3, 171.0, 170.8, 170.5, 156.4, 156.2, 152.8, 151.0, 150.9, 148.2, 148.0, 143.9, 143.9, 140.8, 140.8, 139.1, 138.8, 127.6, 127.1, 125.7, 125.2, 125.1, 124.8, 120.2, 111.8, 111.8, 79.5, 65.5, 65.4, 50.1, 47.9, 47.6, 46.8, 46.7, 35.5, 35.2, 30.7, 28.0. HRMS (ESI/Q-TOF): m/z [M + H]⁺ calcd for C₃₁H₃₅N₄O₇: 575.2500; found: 575.2524.
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