Synlett 2012; 23(20): 2923-2926
DOI: 10.1055/s-0032-1317605
letter
© Georg Thieme Verlag Stuttgart · New York

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

Hassan Said
a   Institut für Organische Chemie, Universität Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany   Fax: +49(0)71168564321   Email: lehrstuhl-2@oc.uni-stuttgart.de
,
Claudia Guttroff
a   Institut für Organische Chemie, Universität Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany   Fax: +49(0)71168564321   Email: lehrstuhl-2@oc.uni-stuttgart.de
,
Afaf H. El-Sagheer
b   School of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, UK
c   Chemistry Branch, Department of Science and Mathematics, Faculty of Petroleum and Mining Engineering, Suez Canal University, Suez 43721, Egypt
,
Clemens Richert*
a   Institut für Organische Chemie, Universität Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany   Fax: +49(0)71168564321   Email: lehrstuhl-2@oc.uni-stuttgart.de
› Author Affiliations
Further Information

Publication History

Received: 18 September 2012

Accepted after revision: 19 October 2012

Publication Date:
16 November 2012 (online)


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.

Supporting Information

 
  • References

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  • 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