Preparation of 5′‐Silyl‐2′‐Orthoester Ribonucleosides for Use in Oligoribonucleotide Synthesis

Stephen A. Scaringe1, David Kitchen1, Robert J. Kaiser1, William S. Marshall1

1 Dharmacon Inc., Lafayette, Colorado
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 2.10
DOI:  10.1002/0471142700.nc0210s16
Online Posting Date:  May, 2004
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Abstract

The recent discovery that small interfering RNAs (siRNAs) induce gene suppression in mammalian cells has sparked tremendous interest in using siRNA‐based assays and high‐throughput screens to study gene function. As a result, research programs at leading academic and commercial institutions have become a substantial and rapidly growing market for synthetic RNA. Important considerations in synthesizing RNA for biological gene function studies are sequence integrity, purity, scalability, and resistance to nucleases; ease of chemical modification, deprotection, and handling; and cost. Of the well‐established RNA synthesis methods, 2′‐ACE chemistry is the only one that meets all of these criteria. 2′‐ACE technology employs a unique class of silyl ethers to protect the 5′‐hydroxyl, in combination with an acid‐labile orthoester protecting group on the 2′‐hydroxyl (2′‐ACE). 2′‐ACE‐protected phosphoramidite monomers are joined using standard solid‐phase technology to achieve RNA synthesis at efficiencies rivaling those for DNA. This unit describes the synthesis of standard 5′‐silyl‐2′‐ACE‐protected phosphoramidites.

Keywords: RNA synthesis; silyl protection; orthoester; ribonucleoside; phosphoramidite

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis of 2′‐ACE‐Protected Uridine Ribonucleoside Phosphoramidite
  • Alternate Protocol 1: Synthesis of 2′‐ACE‐Protected Adenosine Ribonucleoside Phosphoramidite
  • Alternate Protocol 2: Synthesis of 2′‐ACE‐Protected Guanosine Ribonucleoside Phosphoramidite
  • Alternate Protocol 3: Synthesis of 2′‐ACE‐Protected Cytidine Ribonucleoside Phosphoramidite
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of 2′‐ACE‐Protected Uridine Ribonucleoside Phosphoramidite

  Materials
  • Uridine (S.1; 244 g/mol; Monomer Sciences)
  • Pyridine
  • 1,3‐Dichloro‐1,1,3,3‐tetraisopropyldisiloxane (TIPS‐Cl 2; 315 g/mol; d = 0.986 g/mL; Monomer Sciences)
  • Toluene
  • Dichloromethane
  • 5% and 8% (w/v) aqueous sodium bicarbonate (NaHCO 3)
  • Saturated aqueous sodium chloride (NaCl)
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Silica gel 60 (for column chromatography)
  • Hexane
  • Ethyl acetate (EtOAc)
  • Silica gel thin‐layer chromatography (TLC) plates
  • Methanol (MeOH)
  • Acetonitrile (MeCN)
  • Tris(2‐acetoxyethyl) orthoformate (322 g/mol; Dharmacon Inc.)
  • Pyridinium p‐toluenesulfonate (251 g/mol)
  • Triethylamine (TEA)
  • 4‐tert‐Butyldimethylsiloxy‐3‐penten‐2‐one (238 mL/mol; Silar)
  • N,N,N′,N′‐Tetramethylethylene diamine (TEMED; 150 mL/mol)
  • 48% (w/v) aqueous hydrofluoric acid (HF; 32 mL/mol)
  • Diisopropylamine (140 mL/mol)
  • Benzhydroxy‐bis(trimethylsilyloxy)chlorosilane (BzH‐Cl; 425 g/mol; Dharmacon Inc.)
  • Acetone
  • Bis(N,N‐diisopropylamino)methoxyphosphine (262 g/mol; Monomer Sciences)
  • 0.45 M 1H‐tetrazole in MeCN (AIC)
  • Ethanol (EtOH)
  • Rotary evaporator
  • 1‐L separatory funnel
  • Glass‐fritted Buchner funnel (coarse porosity) and side‐arm Erlenmeyer flask
  • High‐vacuum pump
  • Water aspirator
  • 50 × 600–mm chromatography column
  • Additional reagents and equipment for flash chromatography ( appendix 3E) and TLC ( appendix 3D)
NOTE: For solvent evaporation on a rotary evaporator, the required vacuum source will depend on the boiling point of the solvent involved. For dichloromethane and hexane, a water aspirator or diaphragm pump will suffice. For all other solvents, a high‐vacuum oil pump is needed.NOTE: The efficiency of reactions as well as the results of column chromatography at each step are assessed by TLC on silica gel plates. It is suggested that the TLC plates be spotted in three horizontal locations at the origin of the plate as follows: (1) starting material alone, (2) co‐spot of starting material plus crude reaction mixture or product fraction, and (3) crude reaction mixture or product fraction alone. In this way, the conversion of starting material to product, or the purity of the product fraction, can be conveniently monitored.

Alternate Protocol 1: Synthesis of 2′‐ACE‐Protected Adenosine Ribonucleoside Phosphoramidite

  • N6‐Benzoyladenosine (337 g/mol; Monomer Sciences)

Alternate Protocol 2: Synthesis of 2′‐ACE‐Protected Guanosine Ribonucleoside Phosphoramidite

  • N2‐Isobutyrylguanosine (353 g/mol; Monomer Sciences)

Alternate Protocol 3: Synthesis of 2′‐ACE‐Protected Cytidine Ribonucleoside Phosphoramidite

  • N4‐Acetylcytidine (285 g/mol; Monomer Sciences)
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Figures

Videos

Literature Cited

Literature Cited
   Caruthers, M.H. 1985. Gene synthesis machines: DNA chemistry and its uses. Science 230:281‐285.
   Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. 2001. Duplexes of 21‐nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494‐498.
   Matteucci, M.D. and Caruthers, M.H. 1981. Synthesis of deoxyoligonucleotides on a polymer support. J. Am. Chem. Soc. 103:3185‐3191.
   Scaringe, S.A., Wincott, F.E., and Caruthers, M.H. 1998. Novel RNA synthesis method using 5′‐silyl‐2′‐orthoester protecting groups. J. Am. Chem. Soc. 120:11820‐11821.
   Usman, N.O., Ogilvie, K.K., Jiang, M.Y., and Cedergren, R.J. 1987. The automated chemical synthesis of long oligoribonucleotides using 2′‐O‐silylated ribonucleoside 3′‐O‐phosphoramidites on a controlled‐pore glass support: Synthesis of a 43‐nucleotide sequence similar to the 3′‐half molecule of an Escherichia coli formylmethionine tRNA. J. Am. Chem. Soc. 109:7845‐7854.
   Wu, X. and Pitsch, S. 1998. Synthesis and pairing properties of oligoribonucleotide analogues containing a metal‐binding site attached to β‐D‐allofuranosyl cytosine. Nucl. Acids Res. 26:4315‐4323.
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