DNA Synthesis Without Base Protection Using the Phosphoramidite Approach

Akihiro Ohkubo1, Kohji Seio1, Mitsuo Sekine1

1 Tokyo Institute of Technology, Tokyo, Japan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 3.15
DOI:  10.1002/0471142700.nc0315s26
Online Posting Date:  October, 2006
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Abstract

In the strategy of DNA synthesis without base protection, a recent O‐selective phosphorylation procedure known as the “activated phosphite method” is used to generate reactive phosphite intermediates from N‐unprotected phosphoramidite building blocks. This new strategy proved to be advantageous over previous strategies because the cleavage of the N‐P(III) bond, which can be formed by the significant phosphitylation of the nucleobase amino groups, can be omitted. The activated phosphite method provides an attractive route to high‐throughput synthesis of DNA and the synthesis of alkali‐labile modified DNAs such as oligonucleotides containing DNA lesions. This unit presents two convenient methods for the synthesis of N‐unprotected deoxynucleoside 3′‐phosphoramidite units and detailed procedures for automated synthesis of oligodeoxyribonucleotides from these phosphoramidite units.

Keywords: N-unprotected phosphoramidite; base protection; activated phosphite method; DNA; O-selective phosphorylation; alkali-labile modified DNA

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐Deoxycytidine 3′‐(2‐Cyanoethyl‐N,N‐Diisopropylphosphoramidite) from 2′‐Deoxycytidine
  • Alternate Protocol 1: Synthesis of the Deoxycytidine 3′‐Phosphoramidite by Removal of Benzoyl Protection
  • Alternate Protocol 2: Synthesis of the Deoxycytidine 3′‐Phosphoramidite by Removal of Acetyl Protection
  • Basic Protocol 2: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl‐2′‐Deoxyadenosine 3′‐(2‐Cyanoethyl‐N,N‐Diisopropylphosphoramidite) from 2′‐Deoxyadenosine
  • Alternate Protocol 3: Synthesis of the Deoxyadenosine 3′‐Phosphoramidite by Removal of Benzoyl Protection
  • Alternate Protocol 4: Synthesis of the Deoxyadenosine 3′‐Phosphoramidite by Removal of Phenoxyacetyl Protection
  • Basic Protocol 3: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐Deoxyguanosine 3′‐(2‐Cyanoethyl‐N,N‐Diisopropylphosphoramidite) from 2′‐Deoxyguanosine
  • Alternate Protocol 5: Synthesis of the Deoxyguanosine 3′‐Phosphoramidite by Removal of Isobutyryl Protection
  • Alternate Protocol 6: Synthesis of the Deoxyguanosine 3′‐Phosphoramidite by Removal of Isopropylphenoxyacetyl Protection
  • Basic Protocol 4: Typical Procedure for Solid‐Phase Synthesis of Oligodeoxyribonucleotides
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐Deoxycytidine 3′‐(2‐Cyanoethyl‐N,N‐Diisopropylphosphoramidite) from 2′‐Deoxycytidine

  Materials
  • 2′‐Deoxycytidine hydrochloride ( S.1a; Yamasa Shoyu)
  • Pyridine (anhydrous, dried over 4‐Å molecular sieves, distilled from CaH 2)
  • Argon source
  • Triethylamine (Wako, distilled from CaH 2)
  • Dichloroacetic acid (Wako)
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl)
  • Chloroform (CHCl 3)
  • Methanol (MeOH; Wako, anhydrous, dried over 4‐Å molecular sieves)
  • Brine (saturated aqueous NaCl)
  • Sodium sulfate (Na 2SO 4, anhydrous)
  • Hexane
  • Silica gel (Wakogel C‐200, 75 to 150 µm)
  • Toluene (anhydrous, dried over 4‐Å molecular sieves)
  • Dichloromethane (CH 2Cl 2, anhydrous, dried over 4‐Å molecular sieves)
  • Tetrahydrofuran (THF, anhydrous, dried over 4‐Å molecular sieves, no stabilizer)
  • Diisopropylethylamine (DIPEA, anhydrous, dried over CaH 2, distilled from CaH 2)
  • 2‐Cyanoethoxy‐(N,N‐diisopropylamino)chlorophosphine (synthesized as described in Nagai et al., ; Tanimura et al., )
  • 200‐mL round‐bottom flasks with argon‐filled balloon
  • Rotary evaporator and vacuum pump
  • Thin‐layer chromatography (TLC) silica plates (Kieselgel 60 F 254; 0.2 mm thick; Merck)
  • 500‐mL separatory funnel
  • No. 2 filter paper and glass funnel
  • 5 × 6– and 5 × 7.5–cm chromatography columns
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D) and column chromatography ( appendix 3E)

Alternate Protocol 1: Synthesis of the Deoxycytidine 3′‐Phosphoramidite by Removal of Benzoyl Protection

  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐4‐N‐benzoyl‐2′‐deoxycytidine 3′‐(2‐cyanoethyl‐N,N‐diisopropylphosphoramidite) ( S.6a; Glen Research)
  • 2 M methylamine (MeNH 2; Aldrich) in THF
  • 5 × 8–cm chromatography column

Alternate Protocol 2: Synthesis of the Deoxycytidine 3′‐Phosphoramidite by Removal of Acetyl Protection

  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐4‐N‐acetyl‐2′‐deoxycytidine 3′‐(2‐cyanoethyl‐N,N‐diisopropylphosphoramidite) ( S.7a; Glen Research)
  • 2 M NH 3 in MeOH (Aldrich)
  • 5 × 8–cm chromatography column

Basic Protocol 2: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl‐2′‐Deoxyadenosine 3′‐(2‐Cyanoethyl‐N,N‐Diisopropylphosphoramidite) from 2′‐Deoxyadenosine

  Materials
  • 2′‐Deoxyadenosine ( S.1b; Yamasa Shoyu)
  • Pyridine (anhydrous, dried over 4‐Å molecular sieves, distilled from CaH 2)
  • Argon source
  • Triethylamine (Wako, distilled from CaH 2)
  • Dichloroacetic acid (Wako)
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl)
  • Chloroform (CHCl 3)
  • Methanol (MeOH; Wako, anhydrous, dried over 4‐Å molecular sieves)
  • Brine (saturated aqueous NaCl)
  • Sodium sulfate (Na 2SO 4, anhydrous)
  • Hexane
  • Silica gel (Wakogel C‐200, 75 to 150 µm)
  • Toluene (anhydrous, dried over 4‐Å molecular sieves)
  • Dichloromethane (CH 2Cl 2, anhydrous, dried over 4‐Å molecular sieves)
  • Tetrahydrofuran (THF, anhydrous, dried over 4‐Å molecular sieves, no stabilizer)
  • Diisopropylethylamine (DIPEA, anhydrous, dried over CaH 2, distilled from CaH 2)
  • 2‐Cyanoethoxy‐(N,N‐diisopropylamino)chlorophosphine (synthesized as described in Nagai et al., ; Tanimura et al., )
  • 200‐mL round‐bottom flasks with argon‐filled balloon
  • Rotary evaporator and vacuum pump
  • Thin‐layer chromatography (TLC) silica plates (Kieselgel 60 F 254; 0.2 mm thick; Merck)
  • 500‐mL separatory funnel
  • No. 2 filter paper and glass funnel
  • 5 × 6– and 5 × 7.5–cm chromatography columns
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D) and column chromatography ( appendix 3E)

Alternate Protocol 3: Synthesis of the Deoxyadenosine 3′‐Phosphoramidite by Removal of Benzoyl Protection

  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐6‐N‐benzoyl‐2′‐deoxyadenosine 3′‐(2‐cyanoethyl‐N,N‐diisopropylphosphoramidite) ( S.6b; Glen Research)
  • 2 M methylamine (MeNH 2; Aldrich) in THF
  • 5 × 8–cm chromatography column

Alternate Protocol 4: Synthesis of the Deoxyadenosine 3′‐Phosphoramidite by Removal of Phenoxyacetyl Protection

  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐6‐N‐phenoxyacetyl‐2′‐deoxyadenosine 3′‐(2‐cyanoethyl‐N,N‐diisopropylphosphoramidite) ( S.8b; Glen Research)
  • 2 M NH 3 in MeOH (Aldrich)

Basic Protocol 3: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐Deoxyguanosine 3′‐(2‐Cyanoethyl‐N,N‐Diisopropylphosphoramidite) from 2′‐Deoxyguanosine

  Materials
  • 2′‐Deoxyguanosine ( S.1c; Yamasa Shoyu)
  • Pyridine (anhydrous, dried over 4‐Å molecular sieves, distilled from CaH 2)
  • Methanol (MeOH; Wako, anhydrous, dried over 4‐Å molecular sieves)
  • Argon source
  • N,N‐Dimethylformamide dimethyl acetal (Tokyo Kasei Co. Ltd.; http://www.tokyokasei.co.jp/)
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl)
  • Chloroform (CHCl 3)
  • Brine (saturated aqueous NaCl)
  • Sodium sulfate (Na 2SO 4, anhydrous)
  • Diisopropyl ether
  • Ammonium hydroxide
  • Silica gel (Wakogel C‐200, 75 to 150 µm)
  • Hexane
  • Toluene (anhydrous, dried over 4‐Å molecular sieves)
  • Dichloromethane (CH 2Cl 2, anhydrous, dried over 4‐Å molecular sieves)
  • Diisopropylethylamine (DIPEA, anhydrous, dried over CaH 2, distilled from CaH 2)
  • 2‐Cyanoethoxy‐(N,N‐diisopropylamino)chlorophosphine (synthesized as described in Nagai et al., ; Tanimura et al., )
  • Triethylamine (Wako, distilled from CaH 2)
  • 100‐ and 200‐mL round‐bottom flasks with argon‐filled balloon
  • Rotary evaporator and vacuum pump
  • Thin‐layer chromatography (TLC) silica plates (Kieselgel 60 F 254; 0.2 mm thick; Merck)
  • 60‐mm Buchner funnel with filter paper
  • 500‐ml separatory funnel
  • No. 2 filter paper and glass funnel
  • 5 × 6– and 5 × 7.5–cm chromatography columns
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D) and column chromatography ( appendix 3E)

Alternate Protocol 5: Synthesis of the Deoxyguanosine 3′‐Phosphoramidite by Removal of Isobutyryl Protection

  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2‐N‐isobutyryl‐2′‐deoxyguanosine 3′‐(2‐cyanoethyl‐N,N‐diisopropylphosphoramidite) ( S.6c; Glen Research)
  • 2 M methylamine (MeNH 2; Aldrich) in THF
  • 5 × 8–cm chromatography column

Alternate Protocol 6: Synthesis of the Deoxyguanosine 3′‐Phosphoramidite by Removal of Isopropylphenoxyacetyl Protection

  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2‐N‐(4‐isopropylphenoxyacetyl)‐2′‐deoxyguanosine 3′‐(2‐cyanoethyl‐N,N‐diisopropylphosphoramidite) ( S.9c; Glen Research)
  • 2 M NH 3 in MeOH (Aldrich)
  • 5 × 8–cm chromatography column

Basic Protocol 4: Typical Procedure for Solid‐Phase Synthesis of Oligodeoxyribonucleotides

  Materials
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐deoxynucleoside 3′‐(2‐cyanoethyl‐N,N‐diisopropylphosphoramidite)s: C ( S.5a), A ( S.5b), G ( S.5c), and T
  • Thymidine‐loaded HCP (28 µmol/g, succinate linker)
  • 3% trichloroacetic acid in CH 2Cl 2 (Glen Research)
  • Dichloromethane (CH 2Cl 2, anhydrous, dried over 4‐Å molecular sieves)
  • Acetonitrile (CH 3CN, anhydrous, dried over 3‐Å molecular sieves, distilled from CaH 2)
  • N‐Methyl‐2‐pyrrolidone (NMP, anhydrous, Wako, dried over 4‐Å molecular sieves)
  • 1‐Hydroxybenzotriazole (HOBt, Dojindo Molecular Technologies)
  • Oxidizing solution: 0.1 M I 2 in 9:1 (v/v) pyridine/H 2O (for manual operation) or 0.1 M I 2 in 2:1:7 (v/v/v) pyridine/H 2O/THF (Glen Research; for automated operation)
  • Pyridine (anhydrous, dried over 4‐Å molecular sieves, distilled from CaH 2)
  • Concentrated NH 3, aqueous
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU, anhydrous, dried over CaH 2, distilled from CaH 2)
  • Tetrabutylammonium fluoride (TBAF)
  • Acetic acid (AcOH; Wako)
  • Tetrahydrofuran (THF, anhydrous, dried over 4‐Å molecular sieves, no stabilizer)
  • 1‐Hydroxy‐6‐(trifluoromethyl)benzotriazole (HOtfBt, Aldrich)
  • Benzimidazolium triflate (BIT; Hayakawa et al., )
  • ABI 392 DNA synthesizer
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Figures

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Literature Cited

Literature Cited
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   Gryaznov, S.M. and Letsinger, R.L. 1992. Selective O‐phosphitylation with nucleoside phosphoramidite reagents. Nucl. Acids Res. 20:1879‐1882.
   Hayakawa, Y., and Kataoka, M. 1998. Facile synthesis of oligodeoxyribonucleotides via the phosphoramidite method without nucleoside base protection. J. Am. Chem. Soc. 120:12395‐12401.
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   Ohkubo, A., Aoki, K., Ezawa, Y., Sato, Y., Taguchi, H., Seio, K., and Sekine, M. 2005a. Synthesis of oligodeoxyribonucleotides containing hydroxymethylphosphonate bonds in the phosphoramidite method and their hybridization properties. Tetrahedron Lett. 46:8953‐8957.
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