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

Publication History

Received: 26 August 2023

Accepted after revision: 13 October 2023

Accepted Manuscript online:
13 October 2023

Article published online:
17 November 2023

• References and Notes

• 1 Nielsen PE, Egholm M, Berg RH, Buchardt O. Science 1991; 254: 1497
• 2a Brodyagin N, Katkevics M, Kotikam V, Ryan CA, Rozners E. Beilstein J. Org. Chem. 2021; 17: 1641
• 2b Saarbach J, Sabale PM, Winssinger N. Curr. Opin. Chem. Biol. 2019; 52: 112
• 3 Egholm M, Christensen L, Dueholm KL, Buchardt O, Coull J, Nielsen PE. Nucleic Acids Res. 1995; 23: 217
• 4a Devi G, Yuan Z, Lu Y, Zhao Y, Chen G. Nucleic Acids Res. 2014; 42: 4008
• 4b Hildbrand S, Blaser A, Parel SP, Leumann CJ. J. Am. Chem. Soc. 1997; 119: 5499
• 4c Bates PJ, Laughton CA, Jenkins TC, Capaldi DC, Roselt PD, Reese CB, Neidle S. Nucleic Acids Res. 1996; 24: 4176
• 4d Cassidy SA, Slickers P, Trent JO, Capaldi DC, Roselt PD, Reese CB, Neidle S, Fox KR. Nucleic Acids Res. 1997; 25: 4891
• 5 Devi G, Yuan Z, Lu Y, Zhao Y, Chen G. Nucleic Acids Res. 2014; 42: 4008
• 6a Zengeya T, Gupta P, Rozners E. Angew. Chem. Int. Ed. 2012; 51: 12593
• 6b Hnedzko D, McGee DW, Karamitas YA, Rozners E. RNA 2017; 23: 58
• 7 Ryan CA, Brodyagin N, Lok J, Rozners E. Biochemistry 2021; 60: 1919
• 8 Kim KT, Chang D, Winssinger N. Helv. Chim. Acta 2018; 101: e1700295
• 9 Endoh T, Hnedzko D, Rozners E, Sugimoto N. Angew. Chem. Int. Ed. 2016; 55: 899
• 10 Endoh T, Brodyagin N, Hnedzko D, Sugimoto N, Rozners E. ACS Chem. Biol. 2021; 16: 1147
• 11 Annoni C, Endoh T, Hnedzko D, Rozners E, Sugimoto N. Chem. Commun. 2016; 52: 7935
• 12a Kumpina I, Baskevics V, Nguyen KD, Katkevics M, Rozners E. ChemBioChem 2023; 24: e202300291
• 12b Brodyagin N, Maryniak AL, Kumpina I, Talbott JM, Katkevics M, Rozners E, MacKay JA. Chem. Eur. J. 2021; 27: 4332
• 12c Talbott JM, Tessier BR, Harding EE, Walby GD, Hess KJ, Baskevics V, Katkevics M, Rozners E, MacKay JA. Chem. Eur. J. 2023; e202302390
• 13 Hnedzko D, Cheruiyot SK, Rozners E. Curr. Protoc. Nucleic Acid Chem. 2014; 58: 4.60.1
• 14 Burns CJ, Goswami R, Jackson RW, Lessen T, Li W.-P, Pevear D, Tirunahari PK, Xu H.-Y. WO 2010130708, 2010
• 15a Qin L, Cui H, Zou D, Li J, Wu Y, Zhu Z, Wu Y. Tetrahedron Lett. 2010; 51: 4445
• 15b Surry DS, Buchwald SL. Chem. Sci. 2011; 2: 27
• 15c Bhagwanth S, Waterson AG, Adjabeng GM, Hornberger KR. J. Org. Chem. 2009; 74: 4634
• 16 Kumpina I, Brodyagin N, MacKay JA, Kennedy SD, Katkevics M, Rozners E. J. Org. Chem. 2019; 84: 13276
• 17 (tert-Butyl) Ethyl 2-(6-Nitropyridin-3-yl)malonate (7) tert-Butyl ethyl malonate (31.7 mL, 167 mmol, 2 equiv) was added dropwise to a suspension of NaH (60% dispersion in mineral oil; 6.70 g, 167 mmol, 2 equiv) in dry DMF (100 mL) at 0 °C (ice bath) under an argon atmosphere. The suspension was stirred at RT until the mixture formed a clear solution and then 5-bromo-2-nitropyridine (6) (17.0 g, 83.7 mmol) was added. The mixture was stirred at 80 °C (oil bath) for 20 h, cooled to RT, and sat. aq NH₄Cl (100 mL) was added. The pH was adjusted to 4 with 1 N aq HCl, and the aqueous phase was extracted with EtOAc (4 × 70 mL). The combined organic layers were washed with sat. brine (70 mL), dried (Na₂SO₄), and concentrated under reduced pressure. The crude product was purified by column chromatography [silica gel, EtOAc–hexanes (linear gradient; 10–25%)] to give a light-yellow oil; yield: 19.0 g (73%). The characterization data matched those previously reported.¹⁶
• 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.
• 19 Ethyl {6-[(tert-Butoxycarbonyl)amino]pyridin-3-yl}acetate (4) Boc₂O (6.28 g, 28.8 mmol, 1.5 equiv) and DMAP (0.24 g, 1.92 mmol, 0.1 equiv) were added to a solution of ethyl 2-(6-aminopyridin-3-yl)acetate (3.46 g, 19.2 mmol) in dry THF (50 mL). After 15 h at RT, the solvent was partly evaporated and the oily residue was purified by column chromatography [silica gel, EtOAc–hexanes (linear gradient, 0–30%)] to give a white solid; yield: 4.07 g (68%). The characterization data matched those previously reported.¹³
• 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
• 21 Tähtinen V, Granqvist L, Murtola M, Strömberg R, Virta P. Chem. Eur. J. 2017; 23: 7113
• 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.
• 23 Brodyagin N, Hnedzko D, MacKay JA, Rozners E. In Peptide Nucleic Acids: Methods and Protocols . Nielsen PE. Springer; New York: 2020: 157
• 24 Hansen AM, Bonke G, Hogendorf WF. J, Björkling F, Nielsen J, Kongstad KT, Zabicka D, Tomczak M, Urbas M, Nielsen PE, Franzyk H. Eur. J. Med. Chem. 2019; 168: 134
• 25a Hansen AM, Shaikh AY, Franzyk H. In Peptide Nucleic Acids: Methods and Protocols . Nielsen PE. Springer; New York: 2020: 97
• 25b Shaikh AY, Hansen AM, Franzyk H. In Peptide Nucleic Acids: Methods and Protocols . Nielsen PE. Springer; New York: 2020: 1
• 26 Ryan CA, Rahman MM, Kumar V, Rozners E. J. Am. Chem. Soc. 2023; 145: 10497