Chemical Synthesis of Oligoribonucleotides with 2′‐O‐(2‐Cyanoethoxymethyl)‐Protected Phosphoramidites

Tadaaki Ohgi1, Hidetoshi Kitagawa1, Junichi Yano1

1 Discovery Research Laboratories, Nippon Shinyaku Co., Ltd., Ibaraki, Japan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 2.15
DOI:  10.1002/0471142700.nc0215s34
Online Posting Date:  September, 2008
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Abstract

An RNA synthetic method with 2‐cyanoethoxymethyl (CEM) as the 2′‐hydroxyl protecting group allows the synthesis of long oligoribonucleotides from CEM‐amidites with an efficiency and final purity comparable to that obtained in DNA synthesis. The CEM‐amidites give a high coupling efficiency, because the CEM group minimizes steric hindrance in the coupling reaction. The CEM group shows satisfactory stability under solid‐phase synthetic conditions, avoids the generation of asymmetric centers, and is easily cleaved to give the final product. This unit describes the synthesis of the four CEM‐amidites, the preparation of reagents, the solid‐phase synthesis of oligoribonucleotides on an automated DNA synthesizer, and their deprotection. Curr. Protoc. Nucleic Acid Chem. 34:2.15.1‐2.15.19. © 2008 by John Wiley & Sons, Inc.

Keywords: 2‐cyanoethoxymethyl; CEM; RNA; amidite; TBAF

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of the 2′‐O‐CEM‐Uridine Phosphoramidite
  • Alternate Protocol 1: Synthesis of the 2′‐O‐CEM Cytidine Phosphoramidite
  • Alternate Protocol 2: Synthesis of the 2′‐O‐CEM‐Guanosine Phosphoramidite
  • Alternate Protocol 3: Synthesis of the 2′‐O‐CEM‐Adenosine Phosphoramidite
  • Basic Protocol 2: Automated Solid‐Phase Synthesis of CEM‐Protected Oligoribonucleotides
  • Support Protocol 1: Preparation of N‐Protected 3′,5′‐O‐Tips Ribonucleosides
  • Support Protocol 2: Preparation of 2‐Cyanoethyl Methylthiomethyl Ether
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of the 2′‐O‐CEM‐Uridine Phosphoramidite

  Materials
  • Argon gas
  • 3′,5′‐O‐(1,1,3,3‐Tetraisopropyldisiloxane‐1,3‐diyl)uridine (3′,5′‐O‐TIPS‐uridine, S.2a; see protocol 6)
  • Tetrahydrofuran, anhydrous (THF; H 2O <50 ppm)
  • 2‐Cyanoethyl methylthiomethyl ether (S.1; see protocol 7)
  • Trifluoromethanesulfonic acid (TfOH; e.g., Wako Pure Chemical Industries)
  • N‐Iodosuccinimide (NIS; e.g., Aldrich)
  • Triethylamine (TEA)
  • Ethyl acetate (EtOAc)
  • Saturated aqueous sodium thiosulfate (Na 2S 2O 3)
  • Saturated aqueous sodium bicarbonate (NaHCO 3)
  • Saturated aqueous sodium chloride (NaCl)
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Methanol, anhydrous (MeOH; H 2O <50 ppm)
  • Ammonium fluoride (NH 4F)
  • Acetonitrile, anhydrous (CH 3CN; H 2O <50 ppm)
  • n‐Hexane
  • Pyridine, anhydrous (H 2O <50 ppm)
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl; e.g., Junsei Chemical, http://www.junsei.co.jp)
  • Dichloromethane (CH 2Cl 2)
  • Silica gel (35 to 100 mesh; e.g., Wako Pure Chemical Industries)
  • Acetone
  • Diisopropylammonium tetrazolide (see recipe)
  • 2‐Cyanoethyl N,N,N′,N′‐tetraisopropylphosphordiamidite (e.g., Aldrich)
  • 50‐mL pressure‐equalizing dropping funnels
  • 1‐L round‐bottom flasks
  • Cold bath (dry ice in acetonitrile)
  • 1‐L and 2‐L separatory funnels
  • Rotary evaporator
  • TLC sheets (Merck, silica gel 60 F254 aluminum sheets)
  • 6‐cm‐diameter Buchner funnel fitted with filter paper
  • Vacuum pump
  • 11.5 × 30–cm chromatography column
  • 9.5 × 30–cm chromatography column
  • Additional reagents and equipment for thin‐layer chromatography ( appendix 3D)

Alternate Protocol 1: Synthesis of the 2′‐O‐CEM Cytidine Phosphoramidite

  • N4‐Ac‐3′,5′‐O‐TIPS‐cytidine (S.2b; see protocol 6)
  • Triethylamine trihydrofluoride (TEA·3HF; e.g., Daikin Chemicals; http://www.daikin.cc)
  • Isopropyl acetate (AcOiPr; e.g., Nacalai Tesque, http://www.nacalai.co.jp)
  • 500‐mL round‐bottom flask

Alternate Protocol 2: Synthesis of the 2′‐O‐CEM‐Guanosine Phosphoramidite

  • N2‐Pac‐3′,5′‐O‐TIPS‐guanosine (S.2c; see protocol 6)
  • Triethylamine trihydrofluoride (TEA·3HF; e.g., Daikin Chemical, http://www.daikin.cc)
  • Isopropyl acetate (AcOiPr; e.g., Nacalai Tesque, http://www.nacalai.co.jp)
  • Diethyl ether (Et 2O)

Alternate Protocol 3: Synthesis of the 2′‐O‐CEM‐Adenosine Phosphoramidite

  • N6‐Ac‐3′,5′‐O‐TIPS‐adenosine (S.2d; see protocol 6)
  • Dimethyl sulfoxide (DMSO)
  • Acetic anhydride (Ac 2O)
  • Acetic acid (AcOH)
  • Magnesium sulfate (MgSO 4), anhydrous
  • 3‐Hydroxypropionitrile (e.g., Tokyo Chemical Industry, http://www.tciamerica.com)
  • 4A molecular sieves (e.g., Wako Pure Chemical Industries)
  • 5‐L conical flask
  • 3‐L separatory funnel
  • 200‐ and 500‐mL round‐bottom flasks
  • 9.5 × 35–cm chromatography column
  • 30‐mL pressure‐equalizing dropping funnel

Basic Protocol 2: Automated Solid‐Phase Synthesis of CEM‐Protected Oligoribonucleotides

  Materials
  • Controlled‐pore glass (CPG) with attached nucleoside (30 to 50 µmol/g; Glen Research)
  • 0.05 M CEM‐amidite solution (see recipe)
  • 0.25 M 5‐benzylthio‐1H‐tetrazole (5‐benzylmercaptotetrazole or BMT) in CH 3CN (e.g., Wako Pure Chemical Industries)
  • Capping solutions A and B (see recipes)
  • Oxidizing solution: 0.1 M I 2 in 7:1:2 (v/v/v) THF/pyridine/H 2O (Wako Pure Chemical Industries)
  • Deblocking solution: 3% TCA in CH 2Cl 2 (Wako Pure Chemical Industries)
  • Helium gas (argon or nitrogen can also be used)
  • Ethanol (EtOH)
  • 28% aqueous ammonia
  • Methanol (MeOH)
  • Nitromethane (CH 3NO 2; e.g., Nacalai Tesque, http://www.nacalai.co.jp)
  • 1 M tetrabutylammonium fluoride (TBAF) solution (see recipe)
  • 1 M Tris·Cl, pH 7.5 ( appendix 2A)
  • Empty 1‐µmol synthesis column fitted with a filter (Glen Research)
  • Automated DNA synthesizer (e.g., Expedite 8909, Applied Biosystems)
  • Vacuum pump
  • 5‐mL screw‐cap vials
  • 30‐mL round‐bottom flasks
  • 2.1‐cm‐diameter Buchner funnel fitted with filter paper
  • Rotary evaporator
  • 15‐mL centrifuge tubes
  • Additional reagents and equipment for HPLC (unit 10.5)

Support Protocol 1: Preparation of N‐Protected 3′,5′‐O‐Tips Ribonucleosides

  Materials
  • 3‐Hydroxypropionitrile (e.g., Tokyo Chemical Industry, http://www.tciamerica.com)
  • Dimethyl sulfoxide (DMSO)
  • Acetic anhydride (Ac 2O)
  • Acetic acid (AcOH)
  • n‐Hexane
  • Ethyl acetate (EtOAc)
  • Anisaldehyde reagent (unit 3.8)
  • Sodium bicarbonate (NaHCO 3)
  • Magnesium sulfate (MgSO 4)
  • Silica gel (100 µm; e.g., Fuji Silysia Chemical, http://fuji‐silysia.co.jp)
  • 2‐L round‐bottom flask with stir bar
  • TLC sheets (Merck, silica gel 60 F254 aluminum sheets)
  • 10‐L conical flask with stir bar
  • 3‐L separatory funnel
  • Rotary evaporator
  • 5.0 × 30–cm chromatography column
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D)
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Figures

Videos

Literature Cited

Literature Cited
   Gough, G.R., Miller, T.J., and Mantick, N.A. 1996. p‐Nitrobenzyloxymethyl: A new fluoride‐removable protecting group for ribonucleoside 2′‐hydroxyls. Tetrahedron Lett. 37:981‐982.
   Kurz, M., Göbel, K., Hartel, K., and Göbel, M.W. 1998. Acridine‐labeled primers as tools for the study of nonenzymatic RNA oligomerization. Helv. Chim. Acta 81:1156‐1180.
   Matysiak, S., Fitznar, H.‐P., Schnell, R., and Pfleiderer, W. 1998. Acetals as new 2′‐O‐protecting functions for the synthesis of oligoribonucleotides: Synthesis of uridine building blocks and evaluation of their relative acid stability. Helv. Chim. Acta 81:1545‐1566.
   Micura, R. 2002. Small interfering RNAs and their chemical synthesis. Angew. Chem. Int. Ed. 41:2265‐2269.
   Ohgi, T., Masutomi, Y., Ishiyama, K., Kitagawa, H., Shiba, Y., and Yano, J. 2005. A new RNA synthetic method with a 2′‐O‐(2‐Cyanoethoxymethyl) Protecting Group (CEM). Org. Lett. 7:3477‐3480.
   Pitsch, S., Weiss, P.A., Jenny, L., Stutz, A., and Wu, X. 2001. Reliable chemical synthesis of oligoribonucleotides (RNA) with 2′‐O‐[(triisopropylsilyl)oxy]methyl (2′‐O‐tom)‐protected phosphoramidites. Helv. Chim. Acta 84:3773‐3795.
   Reese, C.B. 2002. The chemical synthesis of oligo‐ and poly‐nucleotides: A personal commentary. Tetrahedron 58:8893‐8920.
   Scaringe, S.A., Wincott, F.E., and Caruthers, M.H. 1998. Novel RNA synthesis method using 5′‐O‐silyl‐2′‐O‐orthoester protecting groups. J. Am. Chem. Soc. 120:11820‐11821.
   Semenyuk, A., Földesi, A., Johansson, T., Estmer‐Nilsson, C., Blomgren, P., Brännvall, M., Kirsebom, L.A., and Kwiatkowski, M. 2006. Synthesis of RNA using 2′‐O‐DTM protection. J. Am. Chem. Soc. 128:12356‐12357.
   Shiba, Y., Masuda, H., Watanabe, N., Ego, T., Takagaki, K., Ishiyama, K., Ohgi, T., and Yano, J. 2007. Chemical synthesis of a very long oligoribonucleotide with 2‐cyanoethoxymethyl (CEM) as the 2′‐O‐protecting group: Structural identification and biological activity of a synthetic 110mer precursor‐microRNA candidate. Nucleic Acids Res. 35:3287‐3296.
   Umemoto, T. and Wada, T. 2004. Oligoribonucleotide synthesis by the use of 1‐(2‐cyanoethoxy)ethyl (CEE) group as a 2′‐hydroxy protecting group. Tetrahedron Lett. 45:9529‐9531.
   Usman, N., Ogilvie, K.K., Jiang, M.‐Y., and Cedergren, R.J. 1987. The automated chemical synthesis of long oligoribonucleotides using 2′‐O‐silylated ribonucleotides 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.
   Welz, R. and Müller, S. 2002. 5‐(Benzylmercapto)‐1H‐tetrazole as activator for 2′‐O‐TBDMS phosphoramidite building blocks in RNA synthesis. Tetrahedron Lett. 43:795‐797.
   Zhou, C., Honcharenko, D., and Chattopadhyaya, J. 2007. 2‐(4‐Tolylsulfonyl)ethoxymethyl (TEM)‐a new 2′‐OH protecting group for solid‐supported RNA synthesis. Org. Biomol. Chem. 5:333‐343.
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