The 4‐Methylthio‐1‐Butyl Group for Phosphate/Thiophosphate Protection in Oligodeoxyribonucleotide Synthesis

Jacek Cieślak1, Andrzej Grajkowski1, Victor Livengood2, Serge L. Beaucage1

1 Food and Drug Administration, Bethesda, Maryland, 2 National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 3.11
DOI:  10.1002/0471142700.nc0311s19
Online Posting Date:  December, 2004
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Abstract

The detailed preparation of deoxyribonucleoside phosphoramidites functionalized with a 4‐methylthio‐1‐butyl group for P(III) protection is described, along with the incorporation of these phosphoramidites into DNA oligonucleotides via solid‐phase techniques. The versatility of the thermolabile 4‐methylthio‐1‐butyl phosphate/thiophosphate‐protecting group is exemplified through its facile removal from oligonucleotides under neutral conditions or under standard basic conditions. The sulfonium salt that is produced during the thermolytic deprotection of oligonucleotides did not alter DNA nucleobases or desulfurize phosphorothioate diesters to a significant extent.

Keywords: Deoxyribonucleoside phosphoramidites; 4‐methylthio‐1‐butanol; phosphorus trichloride; bis(N,N‐diisopropylamino)chlorophosphine; O‐(4‐methylthio‐1‐butyl)‐N,N,N′,N′‐tetraisopropylphosphorodiamidite; intramolecular cyclodeesterification; thermolytic deprotection; S‐methyltetrahydrothiophenium chloride

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of 4‐Methylthio‐1‐Butyl Deoxyribonucleoside Phosphoramidites and their use in Solid‐Phase DNA Synthesis
  • Support Protocol 1: Preparation of O‐(4‐Methylthio‐1‐Butyl)‐N,N,N′,N′‐Tetraisopropylphosphorodiamidite
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of 4‐Methylthio‐1‐Butyl Deoxyribonucleoside Phosphoramidites and their use in Solid‐Phase DNA Synthesis

  Materials
  • Protected deoxyribonucleosides ( S.1a‐d; Chem‐Impex International):
    • 5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxythymidine
    • N4‐benzoyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxycytidine
    • N6‐benzoyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxyadenosine
    • N2‐isobutyryl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxyguanosine
  • Argon source
  • Anhydrous dichloromethane (CH 2Cl 2; Aldrich)
  • O‐(4‐Methylthio‐1‐butyl)‐N,N,N′,N′‐tetraisopropylphosphorodiamidite ( S.2; see protocol 2)
  • 1H‐Tetrazole, sublimed
  • Triethylamine (TEA; Aldrich)
  • Benzene (Aldrich)
  • Silica gel (60 Å, 230 to 400 mesh; Merck)
  • Hexane (−20°C)
  • Dry ice/acetone bath
  • Reagents recommended for automated solid‐phase oligonucleotide synthesis (Applied Biosystems and/or Glen Research):
    • Standard 2‐cyanoethyl deoxyribonucleoside phosphoramidites (T, CBz, ABz and Gi‐Bu)
    • Activator solution: 1H‐tetrazole in acetonitrile
    • Oxidation solution: 0.02 M iodine in THF/pyridine/water
    • Cap A solution: acetic anhydride in THF/pyridine
    • Cap B solution: 1‐methylimidazole in THF
    • Deblocking solution: trichloroacetic acid in dichloromethane
  • Acetonitrile
  • 3H‐1,2‐Benzodithiol‐3‐one‐1,1‐dioxide (Glen Research)
  • Concentrated ammonium hydroxide (NH 4OH; Fisher Scientific)
  • 2 M triethylammonium acetate (TEAA) buffer, pH 7.0 (Applied Biosystems)
  • 80% acetic acid (AcOH)
  • Loading buffer: 1:4 (v/v) 10× TBE electrophoresis buffer ( appendix 2A) in formamide containing 2 mg/mL bromphenol blue
  • 20 × 40–cm, 7 M urea/20% polyacrylamide gel (unit 10.4 and appendix 3B)
  • Staining buffer: 1:5:20:0.1 (v/v/v/v) formamide/isopropyl alcohol/ddH 2O/3.0 M Tris⋅Cl, pH 8.8
  • 1 mg/mL Stains‐All (Aldrich) in formamide
  • 1.0 M Tris⋅Cl, pH 9.0 ( appendix 2A)
  • 1.0 M MgCl 2 (Sigma)
  • Snake venom phosphodiesterase (SVPD, Crotalus adamanteus; Sigma)
  • Bacterial alkaline phosphatase (BAP, E. coli; Sigma)
  • 50‐, 100‐, and 250‐mL round‐bottom flasks
  • Rubber septa for 14/20‐ and 24/40‐glass joints
  • Vacuum desiccator
  • High‐vacuum oil pump
  • 1‐ and 10‐mL glass syringes
  • 5‐mm NMR tube
  • Rotary evaporator connected to a vacuum pump
  • 2.5 × 20–cm disposable Flex chromatography columns (Kontes)
  • 2.5 × 7.5–cm EMD TLC plates precoated with a 250‐µm layer of silica gel 60 F 254
  • Lyophilizer
  • 392 DNA/RNA synthesizer (Applied Biosystems)
  • 1‐mL Luer‐tipped syringes
  • 4‐mL screw‐cap glass vials
  • Heating block (VWR)
  • 25 cm × 4.6–mm Supelcosil LC‐18S HPLC column (5‐µm; Supelco)
  • 1.5‐mL microcentrifuge tubes
  • 37°C water bath
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D), column chromatography ( appendix 3E), automated DNA synthesis ( appendix 3C), DNA quantification by UV spectrophotometry (unit 10.3), reversed‐phase HPLC (unit 10.5), and polyacrylamide gel electrophoresis (PAGE; unit 10.4& appendix 3B)

Support Protocol 1: Preparation of O‐(4‐Methylthio‐1‐Butyl)‐N,N,N′,N′‐Tetraisopropylphosphorodiamidite

  • Phosphorus trichloride (Aldrich)
  • N,N‐Diisopropylamine (Aldrich)
  • 4‐Methylthio‐1‐butanol (Aldrich)
  • Drierite, 8 mesh (Aldrich)
  • 10‐µm stainless‐steel frit (20 × 10–mm o.d.; Kontes)
  • 3‐mm‐i.d. Teflon canula
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Figures

Videos

Literature Cited

Literature Cited
   Barone, A.D., Tang, J.‐T., and Caruthers, M.H. 1984. In situ activation of bis‐dialkylaminophosphines—A new method for synthesizing deoxyoligonucleotides on polymer supports. Nucl. Acids Res. 12:4051‐4061.
   Boal, J.H., Wilk, A., Harindranath, N., Max, E.E., Kempe, T., and Beaucage, S.L. 1996. Cleavage of oligodeoxyribonucleotides from controlled‐pore glass supports and their rapid deprotection by gaseous amines. Nucl. Acids Res. 24:3115‐3117.
   Byrne, B. and Lafleur Lawter, L.M. 1986. The preparation of trimethylsulfonium chloride from methyl chloroformate and dimethyl sulfide. Tetrahedron Lett. 27:1233‐1236.
   Chmielewski, M.K., Marchán, V., Cieślak, J., Grajkowski, A., Livengood, V., Münch, U., Wilk, A., and Beaucage, S.L. 2003. Thermolytic carbonates for potential 5′‐hydroxyl protection of deoxyribonucleosides. J. Org. Chem. 68:10003‐10012.
   Cieślak, J. and Beaucage, S.L. 2003. Thermolytic properties of 3‐(2‐pyridyl)‐1‐propyl and 2‐[N‐methyl‐N‐(2‐pyridyl)]aminoethyl phosphate/thiophosphate protecting groups in solid‐phase synthesis of oligodeoxyribonucleotides. J. Org. Chem. 68:10123‐10129.
   Cieślak, J., Grajkowski, A., Livengood, V., and Beaucage, S.L. 2004. Thermolytic 4‐methylthio‐1‐butyl group for phosphate/thiophosphate protection in solid‐phase synthesis of DNA oligonucleotides. J. Org. Chem. 69:2509‐2515.
   Grajkowski, A., Wilk, A., Chmielewski, M.K., Phillips, L.R., and Beaucage, S.L. 2001. The 2‐(N‐formyl,N‐methyl)aminoethyl group as a potential phosphate/thiophosphate protecting group in solid‐phase oligodeoxyribonucleotide synthesis. Org. Lett. 3:1287‐1290.
   Grajkowski, A., Cieślak, J., Chmielewski, M.K., Marchán, V., Phillips, L.R., Wilk, A., and Beaucage, S.L. 2003. Conceptual “heat‐driven” approach to the synthesis of DNA oligonucleotides on microarrays. Ann. N.Y. Acad. Sci. 1002:1‐11.
   Guo, Q.M. 2003. DNA microarray and cancer. Curr. Opin. Oncol. 15:36‐43.
   Iyer, R.P., Phillips, L.R., Egan, W., Regan, J.B., and Beaucage, S.L. 1990. The automated synthesis of sulfur‐containing oligodeoxyribonucleotides using 3H‐1,2‐benzodithiol‐3‐one‐1,1‐dioxide as a sulfur‐transfer reagent. J. Org. Chem. 55:4693‐4699.
   Rhodes, D.R. and Chinnaiyan, A.M. 2002. DNA microarrays: Implications for clinical medicine. J. Invest. Surg. 15:275‐279.
   Sinha, N.D., Biernat, J., McManus, J., and Köster, H. 1984. Polymer support oligonucleotide synthesis. 18. Use of β‐cyanoethyl‐N,N,‐dialkylamino/‐N‐morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product. Nucl. Acids Res. 12:4539‐4557.
   Wilk, A., Grajkowski, A., Phillips, L.R., and Beaucage, S.L. 1999a. The 4‐[N‐methyl‐N‐(2,2,2‐trifluoroacetyl)amino]butyl group as an alternative to the 2‐cyanoethyl group for phosphate protection in the synthesis of oligodeoxyribonucleotides. J. Org. Chem. 64:7515‐7522.
   Wilk, A., Grajkowski, A., Srinivasachar, K., and Beaucage, S.L. 1999b. Improved chemistry for the production of synthetic oligodeoxyribonucleotides. Antisense Nucleic Acid Drug Dev. 9:361‐366.
   Wilk, A., Chmielewski, M.K., Grajkowski, A., Phillips, L.R., and Beaucage, S.L. 2001. The 4‐oxopentyl group as a labile phosphate/thiophosphate protecting group for synthetic oligodeoxyribonucleotides. Tetrahedron Lett. 42:5635‐5639.
   Wilk, A., Chmielewski, M.K., Grajkowski, A., Phillips, L.R., and Beaucage, S.L. 2002. The 3‐[(N‐tert‐butyl)carboxamido]‐1‐propyl group as an attractive phosphate/thiophosphate protecting group for solid‐phase oligodeoxyribonucleotide synthesis. J. Org. Chem. 67:6430‐6438.
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