A Universal and Recyclable Solid Support for Oligonucleotide Synthesis

François Morvan1, Albert Meyer1, Jean‐Jacques Vasseur1

1 Université Montpellier, Montpellier, France
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 3.16
DOI:  10.1002/0471142700.nc0316s30
Online Posting Date:  September, 2007
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This unit provides a modified phosphoramidite method to synthesize oligodeoxyribonucleotides onto a universal and reusable hydroxyl solid support thanks to the use of deoxyribonucleoside tert‐butyl and cyanoethyl phosphoramidites. The nucleoside tert‐butyl phosphoramidite allows the introduction of an H‐phosphonate diester linkage using the phosphoramidite method. After elongation, the H‐phosphonate diester linker is cleaved by transesterification under mild basic conditions to yield an oligonucleotide with free 3′‐ and 5′‐hydroxyls and the starting solid support. Thus, the solid support is easily recycled and used for a subsequent synthesis. In addition, a nucleoside tert‐butyl phosphoramidite could be introduced inside the oligonucleotide chain during the elongation to yield a second H‐phosphonate diester linkage. After elongation, the two H‐phosphonate diester linkages are cleaved, producing two oligonucleotides with free 3′‐ and 5′‐hydroxyls. Curr. Protoc. Nucleic Acid Chem. 30:3.16.1‐3.16.19. © 2007 by John Wiley & Sons, Inc.

Keywords: phosphoramidite; H‐phosphonate; automatized synthesis; tandem synthesis

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Deoxyribonucleoside tert‐Butyl Phosphoramidites
  • Basic Protocol 2: Preparation of the Hexanol‐LCAA‐CPG Solid Support
  • Basic Protocol 3: Oligonucleotide Synthesis
  • Alternate Protocol 1: Tandem Oligonucleotide Synthesis
  • Alternate Protocol 2: Tandem Oligonucleotide Synthesis with Selective Release of Each Oligonucleotide
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Preparation of Deoxyribonucleoside tert‐Butyl Phosphoramidites

  • 5′‐O‐DMTr‐N‐protected nucleoside (Samchully Pharmaceuticals):
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐N6‐benzoyl‐2′‐deoxyadenosine (S.1a)
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐N4‐benzoyl‐2′‐deoxycytidine (S.1b)
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐N2‐isobutyryl‐2′‐deoxyguanosine (S.1c)
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐deoxythymine (S.1d)
  • Diisopropylammonium tetrazolide
  • Acetonitrile (dried over CaH 2 and distilled)
  • Anhydrous methylene chloride (CH 2Cl 2; freshly distilled over P 2O 5)
  • tert‐Butyl tetraisopropylphosphorodiamidite (Aldrich)
  • Argon
  • Molybde stain (see recipe), optional
  • Ethyl acetate
  • Saturated aqueous sodium chloride solution (brine)
  • Anhydrous sodium sulfate
  • Cyclohexane
  • Triethylamine (Et 3N)
  • Silica gel (0.04 to 0.06 mm)
  • Dioxane
  • 50‐mL round‐bottom flask equipped with stir bar and CaCl 2 guard
  • TLC silica plates (Kieselgel 60 F‐254; Merck)
  • 254‐nm UV lamp
  • 250‐mL separatory funnel
  • 5‐cm‐diameter chromatography column
  • Vacuum evaporator
  • Lyophilizer
  • Additional reagents and equipment thin‐layer chromatography (TLC; appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 2: Preparation of the Hexanol‐LCAA‐CPG Solid Support

  • Long‐chain alkylamine controlled‐pore glass (LCAA‐CPG, 500 Å, 80 to 120 mesh, amino group, 80 to 90 µmol/g)
  • 3% trichloroacetic acid (TCA) in methylene chloride (commercial deblocking solution)
  • Triethylamine (Et 3N)
  • Diisopropylethylamine (DIPEA)
  • Anhydrous methylene chloride (CH 2Cl 2)
  • Diethyl ether
  • Phosphorous pentoxide (P 2O 5)
  • Anhydrous pyridine
  • Succinic anhydride
  • 4‐Dimethylaminopyridine (DMAP)
  • N‐(3‐Dimethylaminopropyl)‐N′‐ethylcarbodiimide (DEC)
  • N‐Hydroxysuccinimide
  • 6‐Aminohexan‐1‐ol
  • Anhydrous piperidine
  • Methanol
  • Deoxyribonucleoside phosphoramidite
  • 0.1 M p‐toluenesulfonic acid solution in acetonitrile
  • 25‐mL round‐bottom flasks
  • Frits (porosity 3)
  • Vacuum desiccator
  • DNA synthesis column
  • 10‐mL volumetric flasks
  • Spectrophotometer
  • Additional reagents and equipment for automated oligonucleotide synthesis and the trityl assay ( appendix 3C)

Basic Protocol 3: Oligonucleotide Synthesis

  • 5‐Benzylthio‐1H‐tetrazole (BMT)
  • Anhydrous acetonitrile (<10 ppm H 2O)
  • Anhydrous tert‐butyl hydroperoxide (tBuOOH; 5.5 M in decane; Fluka)
  • Anhydrous methylene chloride (CH 2Cl 2; freshly distilled over P 2O 5)
  • 3H‐1,2‐Benzodithiole‐3‐one 1,1‐dioxide
  • Bis(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐N‐protected‐2′‐deoxynucleoside 3′‐O‐(tert‐butyl)‐N,N‐diisopropylphosphoramidite (S.2a‐d; see protocol 1)
  • Standard 3′‐O‐(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidites of 5′‐O‐(4,4′‐dimethoxytrityl)‐N‐protected‐2′‐deoxyribonucleosides:
    • 5′‐O‐DMTr‐N6‐benzoyl‐2′‐deoxyadenosine (Pierce)
    • 5′‐O‐DMTr‐N4‐benzoyl‐2′‐deoxycytidine (Pierce)
    • 5′‐O‐DMTr‐N2‐isobutyryl‐2′‐deoxyguanosine (Pierce)
    • 5′‐O‐DMTr‐2′‐deoxythymine (Pierce)
    • 5′‐O‐DMTr‐N6‐phenoxyacetyl‐2′‐deoxyadenosine (Glen Research)
    • 5′‐O‐DMTr‐N4‐acetyl‐2′‐deoxycytidine (Glen Research)
    • 5′‐O‐DMTr‐N2‐(isopropylphenoxyacetyl)‐2′‐deoxyguanosine (Glen Research)
  • Hexanol‐LCAA‐CPG solid support (S.5; see protocol 2)
  • 3% Dichloracetic acid (DCA) in CH 2Cl 2 (Biosolve)
  • 10 mM potassium carbonate (K 2CO 3) in methanol (70 mg in 50 mL)
  • Concentrated aqueous ammonia
  • Methanol
  • Phosphorous pentoxide (P 2O 5)
  • ABI 381A DNA synthesizer
  • 1‐mL syringes
  • 2‐mL microcentrifuge tubes
  • Speed vacuum
  • Sealed vial (vial with Teflon septum and screw top for HPLC sample preparations)
  • 55°C dry bath
  • Vacuum desiccator
  • Additional reagents and equipment for automated oligonucleotide synthesis and the trityl assay ( appendix 3C)

Alternate Protocol 1: Tandem Oligonucleotide Synthesis

  • Commercial 3′‐O‐succinylated solid support
  • Concentrated ammonium hydroxide
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Literature Cited

Literature Cited
   Alvarez, K., Vasseur, J.J., Beltran, T., and Imbach, J.L. 1999. Photocleavable protecting groups as nucleobase protections allowed the solid‐phase synthesis of base‐sensitive SATE‐prooligonucleotides. J. Org. Chem. 64:6319‐6328.
   Beaucage, S.L. and Caruthers, M.H. 1981. Deoxynucleoside phosphoramidites—A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22:1859‐1862.
   Bologna, J.C., Morvan, F., and Imbach, J.L. 1999. The prooligonucleotide approach: Synthesis of mixed phosphodiester and SATE phosphotriester prooligonucleotides using H‐phosphonate and phosphoramidite chemistries. Eur. J. Org. Chem. 2353‐2358.
   Dittmer, J.C. and Lester, R.L. 1964. Simple specific spray for detection of phospholipids on thin‐layer chromatograms. J. Lipid Res. 5:126‐127.
   Ferreira, F., Meyer, A., Vasseur, J.J., and Morvan, F. 2005. Universal solid supports for the synthesis of oligonucleotides via a transesterification of H‐phosphonate diester linkage. J. Org. Chem. 70:9198‐9206.
   Hardy, P.M., Holland, D., Scott, S., Garman, A.J., Newton, C.R., and Mclean, M.J. 1994. Reagents for the preparation of two oligonucleotides per synthesis (TOPS). Nucl. Acids Res. 22:2998‐3004.
   Hayakawa, Y., Uchiyama, M., and Noyori, R. 1986. Nonaqueous oxidation of nucleoside phosphite to the phosphates. Tetrahedron Lett. 27:4191‐4194.
   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.
   Jung, P.M., Histand, G., and Letsinger, R.L. 1994. Hybridization of alternating cationic/anionic oligonucleotides to RNA segments. Nucleosides Nucleotides 13:1597‐1605.
   Meyer, A., Morvan, F., and Vasseur, J.‐J. 2004. H‐Phosphonate oligonucleotides from phosphoramidite chemistry. Tetrahedron Lett. 45:3745‐3748.
   Natt, F. and Haner, R. 1997. Lipocap—A lipophilic phosphoramidite‐based capping reagent. Tetrahedron 53:9629‐9636.
   Pon, R.T., Yu, S., and Sanghvi, Y.S. 2002. Tandem oligonucleotide synthesis on solid‐phase supports for the production of multiple oligonucleotides. J. Org. Chem. 67:856‐864.
   Sobkowski, M., Stawinski, J., Sobkowska, A., and Kraszewski, A. 1994. Studies on reactions of nucleoside H‐phosphonates with bifunctional reagents. 2. Stability of nucleoside H‐phosphonate diesters in the presence of amino alcohols. J. Chem. Soc., Perkin Trans. 11803‐1808.
   Wu, X.L. and Pitsch, S. 1998. Synthesis and pairing properties of oligoribonucleotide analogues containing a metal‐binding site attached to beta‐D‐allofuranosyl cytosine. Nucl. Acids Res. 26:4315‐4323.
   Yu, D., Tang, J.Y., Iyer, R.P., and Agrawal, S. 1994. Diethoxy N,N‐diisopropyl phosphoramidite as an improved capping reagent in the synthesis of oligonucleotides using phosphoramidite chemistry. Tetrahedron Lett. 35:8565‐8568.
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