Two-Step Synthesis of a 5′-Azidothymidine Building Block for the Assembly of Oligonucleotides for Triazole-Forming Ligations

Abstract

A two-step synthesis converting thymidine into a phosphotriester building block of 5′-azido-5′-deoxythymidine in 60% overall yield is presented. The building block was used to assemble an oligonucleotide with an azido group at its 5′-terminus, which underwent ligation–cycloaddition, producing a strand with PCR-compatible linkage in high yield.

Key words

azides - phosphotriester chemistry - click reaction - oligonucleotide - ligation

• References

• 1 Reese C. Org. Biomol. Chem. 2005; 3: 3851
• 2 Dillo PJ, Rosen CA. BioTechniques 1990; 9: 298
• 3 Stemmer WP. C, Crameri A, Ha D, Brennan TM, Heyneker L. Gene 1995; 164: 49
• 4 Engels JW. Angew. Chem. Int. Ed. 2005; 44: 7166
• 5 Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO. Nat. Methods 2009; 6: 343
• 6 El-Sagheer AH, Sanzone AP, Gao R, Tavassoli A, Brown T. Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 11343
• 7 El-Sagheer AH, Brown T. Chem. Commun. 2011; 47: 12057
• 8 Kumar R, El-Sagheer A, Tumpane J, Lincoln P, Wilhelmsson LM, Brown T. J. Am. Chem. Soc. 2007; 129: 6859
• 9 El-Sagheer AH, Brown T. J. Am. Chem. Soc. 2009; 131: 3958
• 10a Miller GP, Kool ET. Org. Lett. 2002; 4: 3599
• 10b Miller GP, Kool ET. J. Org. Chem. 2004; 69: 2404
• 11 Bayer C, Wagenknecht HA. Chem. Commun. 2010; 46: 2230
• 12 Letsinger RL, Lunsford WB. J. Am. Chem. Soc. 1976; 98: 767
• 13 Sproat BS, Gait M. J. Am. Chem. Soc. 1976; 98: 3655
• 14a Aigner M, Hartl M, Fauster K, Steger J, Bister K, Micura R. ChemBioChem 2011; 12: 47
• 14b Fauster K, Hartl M, Santner T, Aigner M, Kreutz C, Bister K, Ennifar E, Micura R. ACS Chem. Biol. 2012; 7: 581
• 15a Bannwarth W. Helv. Chim. Acta 1988; 71: 1517
• 15b Mag M, Engels JW. Nucleic Acids Res. 1989; 17: 5973
• 16 5′-Azido-5′-deoxythymidine (1): The preparation of compound 1 used a slight modification of the protocol described in reference 15, using NaN₃ instead of LiN₃. Briefly, triphenylphosphine (3.21 g, 12.24 mmol, 1.2 equiv), NaN₃ (1.95 g, 30.04 mmol, 3 equiv) and CBr₄ (4.01 g, 12.09 mmol, 1.2 equiv) were added to a solution of thymidine (2.48 g, 10.23 mmol, 1 equiv) in anhyd DMF (40 mL). The reaction mixture was stirred at r.t. for 24 h under argon. When TLC (CH₂Cl₂–MeOH, 9:1) showed complete conversion, the reaction mixture was treated with sat. NaHCO₃ solution (50 mL). After extracting with CHCl₃ (3 × 50 mL), the organic solution was washed once with H₂O and twice with brine (2 × 100 mL), followed by drying over Na₂SO₄. The reaction mixture was filtered and the solvents were evaporated. The crude product was purified by column chromatography on silica, eluting with a step gradient of MeOH in CH₂Cl₂ (0–6%) to give compound 1 (2.14 g, 78%). The spectroscopic data were in agreement with the literature.¹⁵ 5′-Azidothymidine-3′-O-(2-chlorophenyl)monophosphate (2): 1,2,4-Triazole (0.757 g, 10.3 mmol, 5.5 equiv) was dissolved in anhyd THF (23 mL) and treated with Et₃N (1.3 mL, 9.3 mmol, 5 equiv). To this, 2-chlorophenyl phosphorodichloridate (800 μL, 4.62 mmol, 2.5 equiv) was added, immediately leading to a white precipitate. The suspension was stirred at 20 °C for 30 min. Then, a solution of 5′-azido-5′-deoxythymidine (0.5 g, 1.87 mmol, 1 equiv) and 1-methylimidazole (600 μL, 7.42 mmol, 4 equiv) in anhyd THF (8 mL) was added, and the reaction mixture was stirred at r.t. for 45 min under Ar. When TLC (CH₂Cl₂–MeOH, 9:1) showed complete conversion, the reaction was quenched with H₂O (400 μL) and Et₃N (1.5 mL). The solvents were evaporated, and the residue was dissolved in CH₂Cl₂ (50 mL) and sat. NaHCO₃ (50 mL). The product was extracted in CH₂Cl₂ (3 × 100 mL). The combined organic layers were washed with brine (3 × 100 mL) and dried over Na₂SO₄. After evaporation of the solvents, the crude product was purified by column chromatography on silica (CH₂Cl₂–MeOH–Et₃N, 98:1:1 for packing the column), eluting with a step gradient of 0% to 9% MeOH–0.5% Et₃N to give 2 (0.803 g, 77%) as an orange-colored foam. ¹H NMR (300 MHz, DMSO-d ₆): δ = 1.15 (t, ³ J = 7.3 Hz, 9 H, CH₃CH₂NH), 1.79 (s, 3 H, thymidine-Me), 2.23 (m, 2 H, 2′-H, 2′′-H), 3.03 (q, ³ J = 7.3 Hz, 6 H, CH₃CH₂NH), 3.54 (m, 2 H, 5′-H, 5′′-H), 4.04 (m, 1 H, 4′-H), 4.62 (m, 1 H, 3′-H), 6.14 (t, ³ J = 6.6 Hz, 1 H, 1′-H), 6.93 (t, ³ J = 7.8 Hz, 1 H, ArH), 7.18 (t, ³ J = 7.2 Hz, 1 H, ArH), 7.35 (d, ³ J = 7.9 Hz, 1 H, ArH), 7.51 (s, 1 H, 6-H), 7.58 (d, ³ J = 7.8 Hz, 1 H, ArH), 9.5 (br s, 1 H, CH₃CH₂NH), 11.35 (s, 1 H, NH). ³¹P NMR (121.5 MHz, DMSO-d ₆): δ = –6.87. MS (ESI^–; MeOH–H₂O, 1:1): m/z calcd for C₁₆H₁₆ClN₅O₇P [M–H]^–: 456.05, found: 456.02.
• 17 Synthesis of 5′-Azide Oligonucleotide (4): Azido building block 2 was coupled to the 5′-terminus of the fully protected oligonucleotide attached on controlled pore glass. The immobilized octamer oligodeoxynucleotide on cpg (3, 10 mg, approx. 0.2 μmol loading, DMT-off state), had been purchased from Biomers Inc. (Ulm, Germany), where it had been assembled via conventional automated DNA synthesis. The support was dried at 0.1 mbar for 1 h. A solution of 5′-azido nucleotide 2 (8.5 mg, 15 μmol), previously co-evaporated twice from anhydrous pyridine and dried at 0.1 mbar, was treated with 1-(2-mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT; 22.2 mg, 75 μmol) in anhydrous pyridine (100 μL) at r.t. for 15 min. Then, 1-methyl-imidazole (10 μL, 126 μmol) was added and the mixture was transferred to a polypropylene cup containing the DNA-bearing cpg. After 50 min, the supernatant was carefully aspirated, and the cpg was washed with anhyd pyridine (3 × 200 μL) and MeCN (5 × 200 μL). The support was treated with a solution of syn-2-nitrobenzaldoxime (18 mg, 10 mmol) and 1,1,3,3-tetramethylguanidine (20 μL) in dioxane–H₂O (250 μL, 1:1). The mixture was left at r.t. for 16 h and the support was then washed with dioxane–H₂O (1:1; 5 × 200 μL). After drying at 0.1 mbar, the cpg was treated with NH₄OH (25% aq NH₃, 500 μL) for 5 h at 55 °C. The mixture was cooled to r.t. and excess NH₃ was removed by gently blowing a stream of nitrogen onto the surface until the solution was odorless. The solution was lyophilized and the oligonucleotide was purified by reversed-phase HPLC with a step gradient of MeCN (0% for 5 min to 25% in 35 min, t _R = 24 min) in 0.1 M triethylammonium acetate buffer (pH 7.0). Elution was monitored by UV-absorption at λ = 260 nm.
• 18 Synthesis of 3′-Alkyne Oligonucleotide 6: The oligonucleotide was synthesized via standard phosphoramidite synthesis. The first nucleoside 5′-O-(4,4′-dimethoxytrityl)-3′-O-propargyl-5-methyldeoxycytidine was attached to the solid support (33 μmol/g loading, AM polystyrene, Applied Biosystems) according to the method reported in reference 20. The support was transferred into a DNA synthesizer column and the assembly of the sequence was performed in the 3′- to 5′-direction, followed by cleavage, deprotection, and HPLC purification.
• 19 Template-Directed Ligation of Oligonucleotides: Oligonucleotides 4, 6 and template strand 7 (0.5 nmol each) were mixed, lyophilized and dissolved in 0.2 M NaCl (125 μL). For annealing, the solution was heated to 85 °C for 5 min and cooled to 4 °C in the course of 2 h. A solution of tris-hydroxypropyltriazole²¹ (2.8 μmol in 0.2 M NaCl, 38 μL), sodium ascorbate (4 μmol in 0.2 M NaCl, 8 μL) and CuSO₄·5H₂O (0.04 μmol in 0.2 M NaCl, 4 μL) was added to the oligonucleotides, and the reaction mixture was kept at 0 °C for 1 h and at r.t. for 1 h. Then, the sample was desalted by gel filtration (Sephadex G-25 column) and analyzed by MALDI–TOF–MS and 20% denaturing polyacrylamide gel electrophoresis.
• 20 El-Sagheer AH, Brown T. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 15329
• 21 Chan TR, Hilgraf R, Sharpless KB, Fokin VV. Org. Lett. 2004; 6: 2853