Deoxyribonucleoside Phosphoramidites

Andrzej Wilk1, Andrzej Grajkowski1, Marcin K. Chmielewski1, Serge L. Beaucage1, Lawrence R. Phillips2

1 Food and Drug Administration, Bethesda, Maryland, 2 National Cancer Institute, Frederick, Maryland
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 2.7
DOI:  10.1002/0471142700.nc0207s04
Online Posting Date:  May, 2001
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The detailed preparation of deoxyribonucleoside phosphoramidites bearing a 4‐[N‐methyl‐N‐(2,2,2‐trifluoroacetyl)amino]butyl group for P(III) protection is presented. The use of this group circumvents nucleobase alkylation during oligonucleotide deprotection. Two syntheses of phosphoramidites starting from either a phosphordichloridite precursor or a bis‐(N,N‐diisopropylamino)chlorophosphine intermediate are described for the phosphinylation of suitably protected deoxyribonucleosides.

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Table of Contents

  • Basic Protocol 1: Preparation of 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐ (N,N‐Diisopropylamino)‐{4‐[N‐Methyl‐N‐(2,2,2‐Trifluoroacetyl)Amino]Butoxy}Phosphinyl‐2′‐Deoxyribonucleosides
  • Support Protocol 1: Preparation of N,N,N′,N′‐Tetraisopropyl‐O‐{4‐[N‐Methyl‐N‐ (2,2,2‐Trifluoroacetyl)Amino]Butyl}Phosphordiamidite
  • Support Protocol 2: Preparation of 4‐[N‐Methyl‐N‐(2,2,2‐Trifluoroacetyl)Amino]Butan‐1‐ol
  • Alternate Protocol 1: Preparation of N,N,N′,N′‐Tetraisopropyl‐O‐ {2‐[(N‐Formyl‐N‐Methyl)Amino]Ethyl}Phosphordiamidite
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation of 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐ (N,N‐Diisopropylamino)‐{4‐[N‐Methyl‐N‐(2,2,2‐Trifluoroacetyl)Amino]Butoxy}Phosphinyl‐2′‐Deoxyribonucleosides

  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐deoxythymidine (S.1a; Chem‐Impex International)
  • N4‐Benzoyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxycytidine (S.1b; Chem‐Impex International)
  • N6‐Benzoyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxyadenosine (S.1c; Chem‐Impex International)
  • N2‐Isobutyryl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxyguanosine (S.1d; Chem‐Impex International)
  • Anhydrous methylene chloride (Aldrich)
  • N,N,N′,N′‐Tetraisopropyl‐O‐{4‐[N‐methyl‐N‐(2,2,2‐trifluoroacetyl)‐ amino]butyl}phosphordiamidite (S.2; see protocol 2)
  • Sublimed 1H‐tetrazole (Aldrich)
  • 9:1 (v/v) benzene/triethylamine (both available from Aldrich)
  • 230‐ to 400‐mesh silica gel 60Å (Merck)
  • 50‐mL three‐necked round‐bottom flask and rubber septa
  • 15‐mL powder addition funnel (Labglass)
  • Vacuum desiccator
  • Rotary evaporator
  • High vacuum pump
  • Dry argon gas cylinder
  • 10‐mL syringe
  • 2.5 × 20–cm disposable Flex chromatography columns (Kontes)
  • Fraction collector
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D) and column chromatography ( appendix 3E)

Support Protocol 1: Preparation of N,N,N′,N′‐Tetraisopropyl‐O‐{4‐[N‐Methyl‐N‐ (2,2,2‐Trifluoroacetyl)Amino]Butyl}Phosphordiamidite

  • Phosphorus trichloride (Aldrich), freshly distilled
  • Anhydrous acetonitrile (Aldrich)
  • 4‐[N‐Methyl‐N‐(2,2,2‐trifluoroacetyl)amino]butan‐1‐ol (S.4; see protocol 3)
  • Anhydrous petroleum ether (Aldrich), freshly distilled from phosphorus pentoxide
  • Drierite, 8 mesh (Aldrich)
  • Anhydrous N,N‐diisopropylamine (Aldrich)
  • 250‐ and 1000‐mL round‐bottom flasks
  • 25‐mL pressure‐equalizing dropping funnel
  • Vacuum distillation head 24/40 and appropriate thermometer
  • Three‐way stopcock
  • Reflux condenser
  • Drying tube
  • Rubber septum
  • 250‐mL sintered glass funnel (coarse porosity)
  • Rotary evaporator/vacuum pump system
  • NOTE: Phosphordichloridites are very sensitive to moisture. Reaction yields depend on the dryness of the reaction conditions. It is recommended that all glassware be oven‐dried overnight at 120°C. The dried glassware should then be cooled to ambient temperature under an inert gas atmosphere in a desiccator. Acetonitrile is refluxed over calcium hydride for ≥2 hr prior to distillation.

Support Protocol 2: Preparation of 4‐[N‐Methyl‐N‐(2,2,2‐Trifluoroacetyl)Amino]Butan‐1‐ol

  • γ‐Butyrolactone (S.6; Aldrich)
  • Anhydrous gaseous methylamine (Aldrich)
  • Diethyl ether (Malinckrodt), freshly distilled from sodium
  • Drierite (Aldrich)
  • Lithium aluminum hydride powder (LAH, Aldrich)
  • Triethanolamine (Aldrich)
  • Methyl trifluoroacetate (Aldrich)
  • 250‐mL pressure vessel (Barrskogen)
  • 2‐L three‐necked round‐bottom flask
  • Mechanical stirrer (Arrow)
  • Pressure‐equalizing dropping funnel
  • Reflux condenser connected to a drying tube
  • Dry argon gas cylinder
  • 250‐mL sintered glass funnel (coarse porosity)
  • Rotary evaporator/water aspirator system
  • 50‐mL round‐bottom flask

Alternate Protocol 1: Preparation of N,N,N′,N′‐Tetraisopropyl‐O‐ {2‐[(N‐Formyl‐N‐Methyl)Amino]Ethyl}Phosphordiamidite

  • 2‐(Methylamino)ethanol (Aldrich)
  • Ethyl formate (Aldrich)
  • Anhydrous benzene
  • Freshly distilled phosphorous trichloride
  • Anhydrous N,N‐diisopropylamine
  • 95:5 (v/v) anhydrous benzene/triethylamine
  • Toluene
  • 100‐ and 250‐mL round‐bottom flasks equipped with a reflux condenser
  • Heating mantle
  • 60‐mL sintered glass funnel (coarse porosity)
  • 3 × 20–cm chromatography column ( appendix 3E)
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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.
   Beaucage, S.L. 1993. Oligodeoxyribonucleotides synthesis—Phosphoramidite approach. In Methods in Molecular Biology, Vol. 20: Protocols for Oligonucleotides and Analogs (S. Agrawal, ed.) pp. 33‐61. Humana Press, Totowa, N.J.
   Beaucage, S.L. and Caruthers, M.H. 1981. Deoxynucleoside phosphoramidite—A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22:1859‐1862.
   Beaucage, S.L. and Iyer, R.P. 1992. Advances in the synthesis of oligonucleotides by the phosphoramidite approach. Tetrahedron. 48:2223‐2311.
   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.
   Chambers, R.W. 1965. The chemistry of pseudouridine. IV. Cyanoethylation. Biochemistry. 4:219‐226.
   Crippa, S., Di Gennaro, P., Lucini, R., Orlandi, M., and Rindone, B. 1993. Characterization of adducts of nucleic bases and acrylic‐monomers. Gazz. Chim. Ital. 123:197‐203.
   Gardrat, C., Latxague, L., and Picard, J.P. 1990. A new synthesis of dl‐5‐vinyloxazolidine‐2‐thione, a natural antithyroid factor. J. Het. Chem. 27:811‐812.
   Grajkowski, A., Wilk, A., Chmielewski, M.K., and Beaucage, S.L. 2000. unpublished results.
   Pon, R.T., Buck, G.A., Niece, R.L., Robertson, M., Smith, A.J., and Spicer, E. 1994. A survey of nucleic‐acid services in core laboratories. BioTechniques. 17:526‐534.
   Powell, J., James, N., and Smith, S.J. 1986. Lithium aluminum hydride reductions: A new hydrolysis method for intractable products. Synthesis. 338‐340.
   Prokopczyk, B., Bertinato, P., and Hoffman, D. 1988. Synthesis and kinetics of decomposition of 7‐(2‐cyanoehtyl)guanine and O‐6‐(2‐cyanoethyl)guanine, markers for reaction of acrylonitrile and 3‐(methylnitrosamino)propionitrile with DNA. Carcinogenesis. 9:2125‐2128.
   Sinha, N.D., Biernat, J., McManus, J., and Köster, H. 1984. Polymer support oligonucleotide synthesis. 18. Use of β‐cyanoethyl‐N,N.,‐dialkyl‐amino/‐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.
   Solomon, J.J., Cote, I.L., Wortman, M., Decker, K., and Segal, A. 1984. In vitro alkylation of calf thymus DNA by acrylonitrile—Isolation of cyanoethyl adducts of guanine and thymine and carboxyethyl adducts of adenine and cytosine. Chem.‐Biol. Interactions. 51:167‐190.
   Tener, G.M. 1961. 2‐Cyanoethyl phosphate and its use in the synthesis of phosphate esters. J. Am. Chem. Soc. 83:159‐168.
   Wilk, A., Grajkowski, A., Phillips, L.R., and Beaucage, S.L. 1999. 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.
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