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J. Milecki et al. / Bioorg. Med. Chem. 19 (2011) 6098–6106
5.3. Oligonucleotide synthesis
of Bioorganic Chemistry, Polish Academy of Sciences, for his help
with MALDI spectra. This work was supported by the funds from
the Ministry of Science and Higher Education, Republic of Poland.
Syntheses were performed on ABI 391PCR Mate instrument
applying built-in 1 lmol protocol and commercial CPG supports.
Iodine solution of 0.05 M concentration was used in the oxidation
References and notes
step.
1. (a) Favre, A. In Bioorganic Photochemistry; Morrison, H., Ed.; Wiley: New York,
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A. L.; Taunton, J. Curr. Protocols Chem. Biol. 2009, 55; online version
5.4. Deprotection and purification of oligonucleotides
Column containing solid support with the synthesized oligonu-
cleotide was attached to a syringe containing 2 mL of 0.4 M solu-
tion of DBU in anhydr CH3CN. Approx. 0.7 mL of the solution was
pushed into the column, replaced by a new portion after 5 min
and another one after next 5 min. After total time of 15 min the
support was washed with dry CH3CN (10 mL) and dried in vacuo.
The dried support was placed into a vial, 1 mL of approx. 22%
methanolic ammonia was added and the vial closed tightly. After
3 h at rt the solid was filtered off, washed with methanol and water
and the filtrates were evaporated. The residue was dissolved in
0.1 m ammonium acetate (1 mL) and passed through a NAP 25 col-
umn. The column was washed with the same solution and 6 frac-
tions of 1.4 mL were collected and checked by UV. The oligomer
was present mainly in the fractions 3 and 4, with some residue
in 5th fraction. Combined fractions were evaporated, dissolved in
H2O/acetonitrile, 95/5, v/v (1 mL) and purified by HPLC (two
2. (a) McGregor, A.; Rao, M. V.; Duckworth, G.; Stockley, P. G.; Connolly, B. A.
Nucleic Acids Res. 1996, 24, 3173; (b) Favre, A.; Saintome, C.; Fourrey, J.-L.;
Clivio, P.; Laguaa, P. J. Photochem. Photobiol. 1998, 42, 109; (c) Wenska, G.;
´
´
Taras-Goslinska, K.; Lamparska-Kupsik, K.; Skalski, B.; Gdaniec, M.; Gdaniec, Z.
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500 ll injections, Waters XBridge OST C18 Column, 2.5 lm,
10 ꢂ 50 mm, phase A = 0.1 M TEAA, B = 0.1 M TEAA/Acetonitrile,
50/50, v/v, flow rate 1 ml/min, T = 40 °C, gradient 17–20%B in
10 min, then to 50%B in 5 min. Combined fractions were concen-
trated to approx. 0.5 mL and desalted by passage through HPLC col-
umn. Yields of the oligomers BS3–BS6 see Table 1. The oligomers
were verified by enzymatic digestion, results see Table 3. Enzy-
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_
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matic digestion conditions: 0.2 OD of oligonucleotide in 150 lL
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manteus venom (0.0055 units, Sigma–Aldrich, Purified) for 18 h
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Acknowledgments
The excellent technical help of Ms. Krystyna Sternal is greatly
appreciated. The authors thank Dr. Łukasz Marczak of the Institute