Synthesis of Oligoribonucleotides Using the 2‐Nitrobenzyloxymethyl Group for 2′‐Hydroxyl Protection

Tod J. Miller1, Miriam E. Schwartz1, Geoffrey R. Gough1

1 Purdue University, West Lafayette, Indiana
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 3.7
DOI:  10.1002/0471142700.nc0307s03
Online Posting Date:  May, 2001
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Abstract

RNA can be rapidly synthesized using protected ribonucleoside phosphoramidite monomers that carry 2‐nitrobenzyloxymethyl groups on their 2‐hydroxyls. These protecting groups are removed from the oligoribonucleotide products by exposure to long‐wave UV light.

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis and Deprotection of N‐Protected 2′‐O‐(2‐Nitrobenzyloxymethyl) Oligoribonucleotides
  • Support Protocol 1: Synthesis and Purification of 3′‐O‐(2‐Cyanoethyl‐N,N‐Diisopropyl) Phosphoramidites of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(2‐Nitrobenzyloxymethyl) Ribonucleosides
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis and Deprotection of N‐Protected 2′‐O‐(2‐Nitrobenzyloxymethyl) Oligoribonucleotides

  Materials
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(2‐nitrobenzyloxymethyl)‐3′‐O‐(2‐cyanoethyl‐N,N‐diisopropyl) phosphoramidites (U, C, A, and G; see protocol 2)
  • Phosphorus pentoxide
  • Anhydrous acetonitrile (Aldrich)
  • 0.45 M tetrazole (Amersham Pharmacia Biotech) in anhyrdous acetonitrile
  • Pyridine, HPLC grade (Aldrich)
  • Concentrated ammonium hydroxide, 4°C
  • 50% (v/v) aqueous pyridine
  • 50% (v/v) aqueous 2‐methyl‐2‐propanol, HPLC grade
  • 0.2 M formic acid
  • 0.2 M ammonium hydroxide
  • DNA synthesizer with ancillary reagents
  • Amidite vials fitted with septa
  • 5‐mL syringes and 21‐G needles
  • Synthesis column containing support derivatized with O‐acyl ribonucleosides (Glen Research; see Critical Parameters)
  • 10.2‐cm pressure tube (e.g., Ace Glass) fitted with Teflon screw‐in cap and Teflon‐encapsulated O ring
  • Yellow lights
  • Covered water bath, 55°C
  • 30‐mL Buchner funnel (medium porosity)
  • 500‐mL filter flask
  • Vacuum aspirator
  • 500‐mL round‐bottom flask
  • Rotary evaporator connected to water aspirator, with bath temperature set <40°C
  • 250‐mL Pyrex Erlenmeyer flasks
  • Long‐wave (365‐nm) UV lamp, containing two 15‐W black‐light tubes behind a UV‐transmitting glass filter measuring ∼10 × 30 cm
NOTE: The amidites are sensitive to UV light (see Critical Parameters).

Support Protocol 1: Synthesis and Purification of 3′‐O‐(2‐Cyanoethyl‐N,N‐Diisopropyl) Phosphoramidites of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(2‐Nitrobenzyloxymethyl) Ribonucleosides

  Materials
  • N‐Protected 5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐O‐(2‐nitrobenzyloxymethyl) ribonucleosides (unit 2.5)
  • Pyridine, HPLC grade (Aldrich)
  • Anhydrous toluene (Aldrich)
  • Anhydrous tetrahydrofuran (Aldrich)
  • N,N‐Diisopropylethylamine (Aldrich)
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite (Aldrich)
  • 99:1 (v/v) chloroform/methanol containing 3% (v/v) triethylamine
  • Ethyl acetate
  • 1 M sodium carbonate (for U, A, G) or 1 M sodium bicarbonate (for C), ice cold
  • Merck 60 silica gel (230 to 400 mesh ASTM)
  • 4:1 (v/v) ethyl acetate/hexane containing 0.1% (v/v) triethylamine (for A, C, and U)
  • 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, and 3.0% (v/v) methanol in dichloromethane containing 5% (v/v) triethylamine (for G)
  • Anhydrous diethyl ether
  • Dry argon
  • 100‐mL flask fitted with a septum
  • Rotary evaporator, with its air inlet connected to a Drierite gas‐drying unit, connected interchangeably to water aspirator and vacuum pump
  • 1‐mL syringe and 21‐C needle
  • 3 × 60–cm flash chromatography column, with reservoir and flow controller
  • 100‐mL round‐bottom flask
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D)
NOTE: Triethylamine, N,N‐diisopropylethylamine, and pyridine should be kept dry over calcium hydride granules.
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Figures

Videos

Literature Cited

Literature Cited
   Schwartz, M.E., Breaker, R.R., Asteriadis, G.T., deBear, J.S., and Gough, G.R. 1992. Rapid synthesis of oligoribonucleotides using 2′‐O‐(2‐nitrobenzyloxymethyl)‐protected monomers. BioMed. Chem. Lett. 2:1019‐1024.
   Schwartz, M.E., Breaker, R.R., Asteriadis, G.T., and Gough, G.R. 1995. A universal adapter for chemical synthesis of DNA or RNA on any single type of solid support. Tetrahedron Lett. 36:27‐30.
   Sinha, N.D., Biernat, J., McManus, J., and Köster, H. 1984. Polymer support oligonucleotide synthesis XVIII: 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.
   Vinayak, R., Colonna, F., Tsou, D., Mullah, B., Andrus, A., and Sproat, B. 1994. arge scale chemical synthesis and purification of RNA. Nucl. Acids Symp. Ser. 31:165–166
Key References
   Schwartz, M.E., Breaker, R.R., Asteriadis, G.T., deBear, J.S., and Gough, G.R. 1992. Rapid synthesis of oligoribonucleotides using 2′‐O‐(2‐ nitrobenzyloxymethyl)‐protected monomers. Bio. Med. Chem. Lett. 2:1019‐1024.
  Provides an overview of the use of 2‐nitrobenzyl‐oxymethyl as a 2′‐hydroxyl‐protecting group in oligoribonucleotide synthesis.
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