3‐(N‐tert‐Butylcarboxamido)‐1‐propyl and 4‐Oxopentyl Groups for Phosphate/Thiophosphate Protection in Oligodeoxyribonucleotide Synthesis

Andrzej Wilk1, Marcin K. Chmielewski1, Andrzej Grajkowski1, 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 3.9
DOI:  10.1002/0471142700.nc0309s11
Online Posting Date:  February, 2003
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Abstract

This unit provides procedures for the preparation of deoxyribonucleoside phosphoramidites and appropriate phosphordiamidite precursors with P(III) protecting groups different than the standard 2‐cyanoethyl group. Specifically, these phosphoramidites are functionalized with the 3‐(N‐tert‐butylcarboxamido)‐1‐propyl or 4‐oxopentyl groups. The usefulness of these novel deoxyribonucleoside phosphoramidites in the solid‐phase synthesis of a 20‐mer DNA oligonucleotide and its phosphorothioated analog is demonstrated. It is also shown that removal of the 3‐(N‐tert‐butylcarboxamido)‐1‐propyl phosphate/thiophosphate‐protecting group from these oligonucleotides is rapidly effected under thermolytic conditions at neutral pH, whereas the 4‐oxopentyl group is preferably removed by treatment with pressurized ammonia gas or concentrated ammonium hydroxide at ambient temperature. These detailed methods constitute an economical and alkylation‐free approach to large‐scale preparations of therapeutic oligonucleotides.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Oligodeoxyribonucleotides Using 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐(N,N‐Diisopropylamino)‐[3‐(N‐tert‐Butylcarboxamido)‐1‐Propyloxy]Phosphinyl‐2′‐Deoxyribonucleosides
  • Support Protocol 1: Preparation of N,N,N′,N′‐Tetraisopropyl‐O‐[3‐(N‐tert‐Butyl‐Carboxamido)‐1‐Propyl]Phosphordiamidite
  • Alternate Protocol 1: Preparation of Oligodeoxyribonucleotides Using 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐(N,N‐Diisopropylamino)‐(4‐OXO‐1‐Pentyloxy)Phosphinyl‐2′‐Deoxyribonucleosides
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Oligodeoxyribonucleotides Using 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐(N,N‐Diisopropylamino)‐[3‐(N‐tert‐Butylcarboxamido)‐1‐Propyloxy]Phosphinyl‐2′‐Deoxyribonucleosides

  Materials
  • Protected deoxyribonucleoside (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
  • Sublimed 1H‐tetrazole (Aldrich)
  • Argon gas
  • Anhydrous methylene chloride (Aldrich)
  • N,N,N′,N′‐Tetraisopropyl‐O‐[3‐(Ntert‐butylcarboxamido)‐1‐propyl]phosphor‐ diamidite (S.2; see protocol 2)
  • Benzene (Aldrich; optional)
  • Triethylamine (Aldrich)
  • Methylene chloride
  • Hexane
  • D 2O (Aldrich)
  • Silica gel (60 Å, 230 to 400 mesh; Merck)
  • Anhydrous acetonitrile
  • Reagents recommended for automated solid‐phase oligonucleotide synthesis (PE Biosystems):
  •  Standard 2‐cyanoethyl deoxyribonucleoside phosphoramidites (T, CBz, ABz, Gi‐Bu)
  •  Activator solution (1H‐tetrazole in acetonitrile)
  •  Oxidation solution (0.02 M iodine in tetrahydrofuran/pyridine/water)
  •  Cap A solution (acetic anhydride in tetrahydrofuran/pyridine)
  •  Cap B solution (1‐methylimidazole in tetrahydrofuran)
  •  Deblocking solution (trichloroacetic acid in dichloromethane)
  • 3H‐1,2‐Benzodithiol‐3‐one‐1,1‐dioxide (Glen Research)
  • Ammonia gas (LB, Aldrich)
  • 1× PBS, pH 7.2 ( appendix 2A)
  • Loading buffer: 1:4 (v/v) 10 × TBE electrophoresis buffer ( appendix 2A) in formamide containing 2 mg/mL bromphenol blue
  • 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 in formamide (both from Aldrich)
  • 1.0 M Tris⋅Cl buffer, pH 9.0 ( appendix 2A)
  • 1.0 M MgCl 2 (Sigma)
  • Snake venom phosphodiesterase (SVP, Crotallus durissus, Boehringer)
  • Bacterial alkaline phosphatase (BAP, E. coli, Sigma)
  • 0.1 M triethylammonium acetate buffer, pH 7.0 (2 M stock from Applied Biosystems)
  • 50‐mL three‐necked round‐bottom flask with rubber septa
  • High vacuum pump
  • 15‐mL powder‐addition funnel
  • 10‐mL syringe
  • 2.5 × 7.5–cm Whatman TLC plates precoated with a 250‐µm layer of Diamond MK6F silica gel (60 Å)
  • 2.5 × 20–cm disposable Flex chromatography columns (Kontes)
  • DNA synthesizer (e.g., Applied Biosystems 380B)
  • 200‐mL pressure vessel container (Barrskogen)
  • 1‐mL syringes with Luer tip
  • 4‐mL screw‐capped glass vials
  • Heating block (VWR), 90° ± 2°C
  • UV spectrophotometer
  • 1.5‐mL microcentrifuge tubes
  • 37°C water bath
  • 5‐µm Supelcosil LC‐18S HPLC column (25 cm × 4.6 mm, Supelco)
  • Additional reagents and equipment for polyacrylamide gel electrophoresis (PAGE; unit 10.4 and appendix 3B), thin‐layer chromatography (TLC; appendix 3D), column chromatography ( appendix 3E), solid‐phase oligonucleotide synthesis ( appendix 3C), SVP and BAP digestion (unit 10.6), and reversed‐phase HPLC (RP‐HPLC; unit 10.5)
NOTE: The composition of DNA synthesis reagents (i.e., activator, oxidation, capping, and deblocking solutions) varies by manufacturer and by instrument. It is important to use the DNA synthesis reagents recommended for the specific synthesizer used, according to manufacturer's instructions.

Support Protocol 1: Preparation of N,N,N′,N′‐Tetraisopropyl‐O‐[3‐(N‐tert‐Butyl‐Carboxamido)‐1‐Propyl]Phosphordiamidite

  • γ‐Butyrolactone (S.6; Aldrich)
  • tert‐Butylamine (Aldrich)
  • Benzene
  • Phosphorus trichloride (Aldrich), freshly distilled
  • N,N‐Diisopropylamine (Aldrich), anhydrous
  • 90°C water bath
  • 100‐mL round‐bottom flask, oven dried
  • 60‐mL sintered glass funnel (coarse porosity)

Alternate Protocol 1: Preparation of Oligodeoxyribonucleotides Using 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐(N,N‐Diisopropylamino)‐(4‐OXO‐1‐Pentyloxy)Phosphinyl‐2′‐Deoxyribonucleosides

  • 3‐Acetyl‐1‐propanol (Aldrich)
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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.
   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 Organic Lett. 3:1287‐1290.
   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.
   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.
   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. 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., Grajkowski, A., Phillips, L.R., and Beaucage, S.L. 2000. Deoxyribonucleoside cyclic N‐acylphosphoramidites as a new class of monomers for the stereocontrolled synthesis of oligothymidylyl‐ and oligocytidylyl‐phosphorothioates. J. Am. Chem. Soc. 122:2149‐2156.
   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‐butylcarboxamido)‐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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