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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Abstract

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

  • Unit Introduction
  • Basic Protocol: 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: Preparation of N,N,N¢,N¢-Tetraisopropyl-O- {2-[(N-Formyl-N-Methyl)Amino]Ethyl}Phosphordiamidite
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol: 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

 Materials
  • 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 Support Protocol 1)
  • 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

 Materials
  • Phosphorus trichloride (Aldrich), freshly distilled
  • Anhydrous acetonitrile (Aldrich)
  • 4-[N-Methyl-N-(2,2,2-trifluoroacetyl)amino]butan-1-ol (S.4; see Support Protocol 2)
  • 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 ³2 hr prior to distillation.

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

 Materials
  • -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: Preparation of N,N,N¢,N¢-Tetraisopropyl-O- {2-[(N-Formyl-N-Methyl)Amino]Ethyl}Phosphordiamidite

 Additional Materials (also see Basic Protocol)
  • 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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Figures

  •  FigureFigure 2.7.1 Preparation of deoxyribonucleoside phosphoramidites from N- and 5¢-O-protected deoxyribonucleosides.
    View Image
  •  FigureFigure 2.7.2 Preparation of N,N,N¢,N¢-tetraisopropyl-O-{4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl}phosphordiamidite.
    View Image
  •  FigureFigure 2.7.3 Preparation of 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butan-1-ol.
    View Image
  •  FigureFigure 2.7.4 Preparation of N,N,N¢,N¢-tetraisopropyl-O-{2-[(N-methyl-N-formyl)amino]ethyl}-phosphordiamidite.
    View Image
  •  FigureFigure 2.7.5 Deprotection of oligodeoxyribonucleotides carrying the 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl phosphate protecting group under standard basic conditions.
    View Image

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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 -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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