Methoxyoxalamido Chemistry in the Synthesis of Tethered Phosphoramidites and Functionalized Oligonucleotides

Alan M. Morocho1, Nikolai N. Polushin1

1 Fidelity Systems, Inc., Gaithersburg, Maryland
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.29
DOI:  10.1002/0471142700.nc0429s25
Online Posting Date:  July, 2006
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Abstract

A general approach to phosphoramidites tethered with single and multiple linkers through the use of methoxyoxalamido (MOX) chemistry is described. The approach utilizes readily available and inexpensive primary aliphatic amino alcohols and diamines to produce a rich and diverse variety of tethered phosphoramidites. Furthermore, the use of MOX chemistry in a modular fashion enables fairly rapid assembly of compound tethers. All novel phosphoramidites described have been successfully used in automated syntheses of 5′‐modified oligonucleotides.

Keywords: methoxyoxalamido (MOX) chemistry; tethered phosphoramidites; 5′‐modified oligonucleotides

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Phosphoramidites Tethered with Single Linkers
  • Alternate Protocol 1: Preparation of Phosphoramidites Tethered with Multiple Linkers
  • Basic Protocol 2: Synthesis, Deprotection, and Purification of Oligonucleotides Derivatized with Tethered Phosphoramidites
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Phosphoramidites Tethered with Single Linkers

  Materials
  • trans‐4‐Amino‐1‐cyclohexanol hydrochloride (97%; Aldrich)
  • Triethylamine (Et 3N), ≥99%
  • Methanol (MeOH), HPLC grade
  • Dimethyl oxalate, 99% (Aldrich)
  • Diethyl ether, anhydrous
  • Chloroform (CHCl 3), HPLC grade
  • 60 Å silica gel, 200 to 400 mesh (EM Science)
  • 5′‐Amino‐5′‐deoxythymidine (Berry & Associates, http://www.berryassoc.com or Fidelity Systems, http://www.fidelitysystems.com)
  • Aliphatic primary diamines
    • Ethylenediamine (EDA), ≥99.5% (Aldrich) for S.3a
    • 2,4,8,10‐Tetraoxaspiro[5.5]undecane‐3,9‐dipropanamine (TUDA), 97% (Aldrich) for S.4a
    • 4,7,10‐Trioxa‐1,13‐tridecanediamine (TTDD), ≥98% (Aldrich) for S.5a and S.6a
  • Aliphatic primary amino alcohols
    • 6‐Amino‐1‐hexanol (AH), 97% (Aldrich) for S.11a
    • 2‐(2‐Aminoethoxy)ethanol (AEE), 98% (Aldrich) for S.10a
  • Dichloromethane (CH 2Cl 2), HPLC grade
  • N,N‐Dimethylformamide (DMF), anhydrous
  • Pyridine, anhydrous, 99.8% (Aldrich)
  • 4‐Monomethoxytrityl chloride (MMTr‐Cl; ChemGenes)
  • Saline solution: 25% to 30% (w/v) aqueous NaCl
  • Na 2SO 4, reagent grade, anhydrous
  • Pentane, HPLC grade
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl; ChemGenes)
  • Tetrazole, dried
  • Argon gas
  • 2‐Cyanoethyl‐N,N,N′,N′‐tetraisopropylphosphorodiamidite (ChemGenes)
  • 10% (w/v) aqueous sodium hydrogen carbonate (NaHCO 3)
  • Dichloromethane (CH 2Cl 2), anhydrous
  • Ethyl acetate (EtOAc), HPLC grade
  • Toluene, anhydrous
  • Phosphorus pentoxide (P 2O 5)
  • Rotary evaporator equipped with a vacuum pump or water aspirator (5 to 10 Torr)
  • Buchner funnel, 140‐mL capacity with glass frit (porosity 4 to 8 µm)
  • Paper filters (coarse porosity)
  • 4 × 40–cm sintered glass columns
  • Vacuum oil pump (0.05 to 0.5 Torr)
  • Separatory funnel
  • Thin‐layer chromatography (TLC) Kieselgel 60 F 254 plates (EM Science)
  • I 2‐silica chamber
  • 254‐nm UV lamp
  • Heat gun
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Alternate Protocol 1: Preparation of Phosphoramidites Tethered with Multiple Linkers

  Materials
  • Tethered phosphoramidites ( S.3d‐S.14d; see protocol 1 and protocol 2)
  • Acetonitrile (CH 3CN), anhydrous, DNA synthesis grade
  • Standard 2′‐deoxyribonucleoside phosphoramidites (Transgenomic)
  • 0.5 M tetrazole in CH 3CN (Glen Research) or 0.25 M 5‐ethylthio‐1H‐tetrazole (ETT, Glen Research)
  • Ethanolamine (EA), ≥99% (Aldrich)
  • 10% (w/v) LiClO 4 in ethanol (EtOH)
  • Ethanol (EtOH), 200 proof
  • 7 M urea
  • 15% (w/v) polyacrylamide gel ( appendix 3B) containing 7 M urea in 0.5× TBE electrophoresis buffer ( appendix 2A)
  • 0.25 M triethyl ammonium bicarbonate (TEAB), aqueous solution
  • 1.7‐mL microcentrifuge tubes
  • 70°C incubator or water bath
  • Microcentrifuge
  • Speedvac evaporator (Savant)
  • Sephadex G‐25 NAP‐10 columns (Pharmacia)
  • Lyophilizer
  • Water aspirator or vacuum pump (5 to 10 Torr)
  • Additional reagents and equipment for automated solid‐phase oligonucleotide synthesis ( appendix 3C), purification of oligonucleotides (units 10.1, 10.4, 10.5, 10.7& APPENDIX 3.NaN), and determination of molecular mass (unit 10.1)
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Figures

Videos

Literature Cited

Literature Cited
   Bannwarth, W. 1988. Solid phase synthesis of oligonucleotides containing phosphoramidate internucleotide linkages and their specific chemical cleavage. Helv. Chim. Acta 71:1517‐1527.
   Jaschke, A., Furste, J.P., Nordhoff, E., Hillenkamp, F., Cech, D., and Erdmann, V.A. 1994. Synthesis and properties of oligodeoxyribonucleotide‐polyethylene glycol conjugates. Nucl. Acids Res. 22:4810‐4817.
   Polushin, N.N. 2000. The precursor strategy: Terminus methoxyoxalamido modifiers for single and multiple functionalization of oligodeoxyribonucleotides. Nucl. Acids Res. 28:3125‐3133.
   Polushin, N.N., Morocho, A.M., Chen, B.C., and Cohen, J.S. 1994. On the rapid deprotection of synthetic oligonucleotides and analogs. Nucl. Acids Res. 22:639‐645.
   Reddy, M.P., Hanna, N.B., and Farooqui, F. 1994. Fast cleavage and deprotection of oligonucleotides. Tetrahedron Lett. 35:4311‐4314.
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