Synthesis of Endcap Dimethoxytrityl Phosphoramidites for Endcapped Oligonucleotides

Maneesh R. Pingle1, Pei‐Sze Ng1, Xiaolin Xu1, Donald E. Bergstrom1

1 Purdue University, West Lafayette, Indiana
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 5.6
DOI:  10.1002/0471142700.nc0506s12
Online Posting Date:  May, 2003
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Abstract

Endcaps may be either aromatic or aliphatic molecules that specifically cross‐link the 5′ end of one strand with the 3′ end of the complementary strand in a DNA duplex. Endcaps may be viewed as a replacement of the loop region nucleotides of a DNA hairpin, with the added advantage of increased thermal stability. An endcap is incorporated into the sequence during oligonucleotide synthesis. Three endcaps are described in this unit. The naphthalene diimide endcap prefers to base stack with GC base pairs. The terthiophene endcap has higher lipophilicity than the naphthalene diimide endcap and provides higher stability when stacked over an AT base pair. The 2,2′‐oxydiacetamide endcap provides lower enhancement in stability but a more rigid and well‐defined structure than the oligo(ethylene glycol) endcaps. Synthesis of endcapped oligonucleotides can be carried out using standard automated synthesis protocols with only minor modifications.

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis of N‐[3‐O‐(2‐Cyanoethyl‐N,N‐Diisopropylphoshoramidite)Propyl]‐N′‐[3‐(4,4′‐Dimethoxytrityloxy)Propyl]‐Naphthalene‐1,4,5,8‐Tetracarboxylic Diimide
  • Basic Protocol 2: Synthesis of 5‐[3‐O‐(2‐Cyanoethyl‐N,N‐Diisopropylphosphoramidite)Propyl]‐5″‐[3‐(4,4′‐Dimethoxytrityloxy)Propyl]‐2,2′:5′,2″‐Terthiophene
  • Basic Protocol 3: Synthesis of N‐[3‐O‐(2‐Cyanoethyl‐N,N‐Diisopropylphosphoramidite)Propyl]‐N′‐[3‐(4,4′‐Dimethoxytrityloxy)Propyl]‐2,2′‐Oxydiacetamide
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of N‐[3‐O‐(2‐Cyanoethyl‐N,N‐Diisopropylphoshoramidite)Propyl]‐N′‐[3‐(4,4′‐Dimethoxytrityloxy)Propyl]‐Naphthalene‐1,4,5,8‐Tetracarboxylic Diimide

  Materials
  • Napthalene‐1,4,5,8‐tetracarboxylic dianhydride (S.1; Figure )
  • 3‐Aminopropanol
  • 2 M sodium carbonate
  • Chloroform, reagent grade
  • Activated charcoal
  • Methanol, reagent grade
  • Dichloromethane, reagent grade
  • Pyridine, anhydrous
  • 4,4′‐Dimethoxytrityl chloride (DMTr⋅Cl)
  • 5% (w/v) sodium bicarbonate
  • Sodium sulfate, anhydrous
  • Silica gel (230 to 400 mesh, 60 Å, E Merck)
  • Triethylamine, reagent grade
  • Hexanes, reagent grade
  • Ethyl acetate, reagent grade
  • Dichloromethane, anhydrous
  • Diisopropylethylamine
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite
  • Ethyl acetate, prewashed with 5% (w/v) sodium bicarbonate
  • 25‐, 50‐, 100‐, and 250‐mL round‐bottom flasks
  • Buchner funnel
  • Filtration flask
  • Whatman no. 1 filter paper
  • Vacuum oven
  • Water‐cooled reflux condenser
  • Oil bath
  • Filter funnel, prewarmed (∼50° to 60°C)
  • Rotary evaporator equipped with a water aspirator and vacuum pump
  • Nitrogen atmosphere (see inert atmosphere/vacuum manifold in UNIT , Fig. )
  • 125‐mL Erlenmeyer flasks
  • 125‐mL separatory funnels
  • Glass funnel
  • Glass wool
  • 2 × 20–cm and 3 × 18–cm glass chromatography columns
  • 25‐mL pear‐shaped flask
  • 1‐mL syringe and stainless steel needle
  • Additional reagents and solutions for thin‐layer chromatography (TLC; appendix 3D) and flash chromatography ( appendix 3E)
CAUTION: Exposure to pyridine and its vapors should be minimized. All reactions should be performed in a fume hood. The reactions for dimethoxytrityl protection and the phosphitylation of the dimethoxytrityl‐protected endcap are sensitive to moisture and the glassware used for the reactions must be scrupulously dry.NOTE: All listed reagents are available from Sigma‐Aldrich.

Basic Protocol 2: Synthesis of 5‐[3‐O‐(2‐Cyanoethyl‐N,N‐Diisopropylphosphoramidite)Propyl]‐5″‐[3‐(4,4′‐Dimethoxytrityloxy)Propyl]‐2,2′:5′,2″‐Terthiophene

  Materials
  • 2,2′:5′,2′‐Terthiophene (S.5; Fig. )
  • Tetrahydrofuran (THF), anhydrous
  • Dry ice/acetone freezing bath
  • n‐Butyllithium
  • Boron trifluoride diethyl etherate
  • Trimethylene oxide
  • Saturated sodium bicarbonate
  • Diethyl ether, reagent grade
  • Brine solution (saturated aqueous NaCl)
  • Sodium sulfate, anhydrous
  • Silica gel (230 to 400 mesh, 60 Å, E Merck)
  • Hexanes, reagent grade
  • Ethyl acetate, reagent grade
  • Pyridine, anhydrous
  • 4,4′‐Dimethoxytrityl chloride (DMTr⋅Cl)
  • Dichloromethane, anhydrous
  • 5% (w/v) sodium bicarbonate
  • Triethylamine
  • Diisopropylethylamine
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite
  • 25‐mL round‐bottom flasks
  • Nitrogen atmosphere (see inert atmosphere/vacuum manifold in unit 1.1, Fig )
  • 1‐mL syringe and stainless steel needle
  • 125‐mL separatory funnels
  • 125‐mL Erlenmeyer flasks
  • Glass funnel
  • Glass wool
  • Rotary evaporator with water aspirator and vacuum pump
  • 2 × 20–cm and 4 × 20–cm glass chromatography columns
  • Additional reagents and equipment for column chromatography ( appendix 3E) and thin‐layer chromatography (TLC; appendix 3D)
NOTE: All listed reagents are available from Sigma‐Aldrich.

Basic Protocol 3: Synthesis of N‐[3‐O‐(2‐Cyanoethyl‐N,N‐Diisopropylphosphoramidite)Propyl]‐N′‐[3‐(4,4′‐Dimethoxytrityloxy)Propyl]‐2,2′‐Oxydiacetamide

  Materials
  • 3‐Aminopropanol
  • Dichloromethane, anhydrous
  • Triethylamine (TEA), anhydrous (preferably freshly distilled)
  • tert‐Butyldimethylsilyl chloride (TBDMS⋅Cl)
  • 4,4‐Dimethylaminopyridine
  • Brine solution (saturated NaCl)
  • Sodium sulfate, anhydrous
  • Diglycolyl chloride
  • 5% (v/v) acetic acid
  • 5% (w/v) sodium bicarbonate
  • Concentrated HCl
  • 95% (v/v) ethanol
  • Methanol, reagent grade
  • Hexanes, reagent grade
  • Pyridine, anhydrous (preferably freshly distilled)
  • 4,4′‐Dimethoxytrityl chloride (DMTr⋅Cl)
  • Silica gel (230 to 400 mesh, 60 Å, E Merck)
  • Dichloromethane, reagent grade
  • 1% (w/v) sodium hydroxide (optional)
  • Acetonitrile, anhydrous
  • 2‐Cyanoethyl‐N,N,N′,N′‐tetraisopropylphosphorodiamidite
  • 1H‐Tetrazole
  • 25‐, 50‐, 100‐ and 250‐mL round‐bottom flasks
  • Nitrogen atmosphere (see inert atmosphere/vacuum manifold in unit 1.1, Fig. )
  • 1‐mL syringe and stainless steel needles
  • 125‐ and 250‐mL separatory funnels
  • 125‐ and 250‐mL Erlenmeyer flasks
  • Glass funnels
  • Glass wool
  • Rotary evaporator with a water aspirator and vacuum pump
  • 2 × 20–cm and 4 × 25–cm glass chromatography columns
  • Additional reagents and equipment for column chromatography ( appendix 3E) and thin‐layer chromatography (TLC; appendix 3E)
NOTE: All listed reagents are available from Sigma‐Aldrich.
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Figures

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Literature Cited

Literature Cited
   Altmann, S. , Labhardt, A.M. , Bur, D. , Lehmann, C. , Bannworth, W. , Billeter, M. , Wuthrich, K. , and Leupin, W. 1995. NMR studies of DNA duplexes singly cross‐linked by different synthetic linkers Nucl. Acids Res. 23:4827‐4835.
   Bevers, S. , O'Dea, T.P. , and McLaughlin, L.W. 1998. Perylene‐ and naphthalene‐based linkers for duplex and triplex stabilization. J. Am. Chem. Soc. 120:11004‐11005.
   Bevers, S. , Schutte, S. , and McLaughlin, L.W. 2000. Naphthalene‐ and perylene‐based linkers for the stabilization of hairpin triplexes. J. Am. Chem. Soc. 122:5905‐5915.
   Letsinger, R.L. , and Wu, T.F. 1995. Use of a stilbenedicarboxamide bridge in stabilizing, monitoring, and photochemically altering folded conformations of oligonucleotides. J. Am. Chem. Soc. 117:7323‐7328.
   Lewis, F.D. , Wu, T.F. , Burch, E.L. , Bassani, D.M. , Yang, J.S. , Schneider, S. , Jager, W. , and Letsinger, R.L. 1995. Hybrid oligonucleotides containing stilbene units—Excimer fluorescence and photodimerization. J. Am. Chem. Soc. 117:8785‐8792.
   Lewis, F.D. , Wu, T.F. , Zhang, Y.F. , Letsinger, R.L. , Greenfield, S.R. , and Wasielewski, M.R. 1997. Distance‐dependent electron transfer in DNA hairpins. Science. 277:673‐676.
   Lewis, F.D. , Liu, X.Y. , Wu, Y. , Miller, S.E. , Wasielewski, M.R. , Letsinger, R.L. , Sanishvili, R. , Joachimiak, A. , Tereshko, V. , and Egli, M. 1999. Structure and photoinduced electron transfer in exceptionally stable synthetic DNA hairpins with stilbenediether linkers. J. Am. Chem. Soc. 121:9905‐9906.
   Lewis, F.D. , Letsinger, R.L. , Wasielewski, M.R. , and Egli, M. 2000a. Structure and electron transfer in synthetic DNA hairpins. Biophys. J. 78:817Symp.
   Lewis, F.D. , Wu, T.F. , Liu, X.Y. , Letsinger, R.L. , Greenfield, S.R. , Miller, S.E. , and Wasielewski, M.R. 2000b. Dynamics of photoinduced charge separation and charge recombination in synthetic DNA hairpins with stilbenedicarboxamide linkers. J. Am. Chem. Soc. 122:2889‐2902.
   Lewis, F.D. , Letsinger, R.L. , and Wasielewski, M.R. 2001. Dynamics of photoinduced charge transfer and hole transport in synthetic DNA hairpins. Accounts Chem. Res. 34:159‐170.
   Nelson, J.S. , Giver, L. , Ellington, A.D. , and Letsinger, R.L. 1996. Incorporation of a non‐nucleotide bridge into hairpin oligonucleotides capable of high‐affinity binding to the Rev protein of HIV‐1. Biochemistry. 35:5339‐5344.
   Yamana, K. , Yoshikawa, A. , and Nakano, H. 1996. Synthesis of a new photoisomerizable linker for connecting two oligonucleotide segments. Tetrahedron Lett. 37:637‐640.
   Yamana, K. , Noda, R. , and Nakano, H. 1998. Synthesis and binding properties of oligonucleotides containing an azobenzene linker. Nucleosides Nucleotides. 17:233‐242.
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