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Chromophoric 5′‐O‐Silyl Protection of N‐Protected 2′‐ACE Ribonucleosides for Solid‐Phase RNA Synthesis

Michael O. Delaney1,  Angela Thomas1,  Chris Ricketts1,  David E. Kitchen1,  Robert J. Kaiser1

1ThermoFisher Scientific, Dharmacon Products, Lafayette, Colorado

Publication Name: 
Current Protocols in Nucleic Acid Chemistry
Unit Number: 
UNIT 2.14
DOI: 
10.1002/0471142700.nc0214s32
Online Posting Date: 
March, 2008
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Abstract

Recent advances in the understanding of the pivotal roles played by endogenous small RNAs in gene regulation have resulted in a substantial and rapidly growing market for synthetic RNA. 5¢-Silyl-2¢-ACE chemistry has proven to be a robust and reliable technology for the synthesis of oligoribonucleotides. This unit describes an important improvement to this chemistry, by adding a cycle-to-cycle traceability analogous to that inherent in 5¢-dimethoxytrityl–based approaches. This is achieved by first regioselectively introducing a 5¢-alkynylsilyl protecting group onto the 2¢-ACE-protected nucleosides. The 5¢-alkynylsilyl group is then reacted with an azide derivative of the chromophore Disperse Red 1, which enables spectrophotometric interrogation of each coupling step following 5¢-deprotection. Finally, the protected nucleosides are elaborated into their 3¢-phosphoramidite derivatives for use in solid-phase RNA synthesis. Curr. Protoc. Nucleic Acid Chem. 32:2.14.1-2.14.26. © 2008 by John Wiley & Sons, Inc.

Keywords: RNA synthesis; 5¢-silyl-2¢-ACE chemistry; phosphoramidite; “click” reaction; alkyne; azide

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: 5¢-O-Silylation of N-Protected 2¢-ACE-Adenosine Ribonucleoside with a Chloroalkynylsilane
  • Alternate Protocol 1: 5¢-O-Silylation of 2¢-ACE-Uridine Ribonucleoside with a Chloroalkynylsilane
  • Alternate Protocol 2: 5¢-O-Silylation of N-Protected 2¢-ACE-Guanosine Ribonucleoside with a Chloroalkynysilane
  • Alternate Protocol 3: 5¢-O-Silylation of N-Protected 2¢-ACE-Cytidine Ribonucleoside with a Chloroalkynylsilane
  • Basic Protocol 2: Coupling of a Chromophoric Azide to 5¢-O-Alkynylsilyl-N-Protected 2¢-ACE-Adenosine Ribonucleoside
  • Alternate Protocol 4: Coupling of a Chromophoric Azide to 5¢-O-Alkynylsilyl 2¢-ACE-Uridine Ribonucleoside
  • Alternate Protocol 5: Coupling of a Chromophoric Azide to 5¢-O-Alkynylsilyl-N-Protected 2¢-ACE-Guanosine Ribonucleoside
  • Alternate Protocol 6: Coupling of a Chromophoric Azide to 5¢-O-Alkynylsilyl-N-Protected 2¢-ACE-Cytidine Ribonucleoside
  • Basic Protocol 3: Preparation of Chromophoric 5¢-O-Silyl-N-Protected 2¢-ACE-Adenosine Ribonucleoside Phosphoramidite
  • Alternate Protocol 7: Preparation of Chromophoric 5¢-O-Silyl 2¢-ACE-Uridine Ribonucleoside Phosphoramidite
  • Alternate Protocol 8: Preparation of Chromophoric 5¢-O-SILYL-N-Protected 2¢-ACE-Guanosine Ribonucleoside Phosphoramidite
  • Alternate Protocol 9: Preparation of Chromophoric 5¢-O-Silyl-N-Protected 2¢-ACE-Cytidine Ribonucleoside Phosphoramidite
  • Support Protocol 1: Synthesis of the Chloroalkynylsilane
  • Support Protocol 2: Synthesis of the Chromophoric Azide
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: 5¢-O-Silylation of N-Protected 2¢-ACE-Adenosine Ribonucleoside with a Chloroalkynylsilane

 Materials
  • 2¢-ACE-N6-isobutyryladenosine (S.1a; unit 2.10)
  • Methylene chloride (CH2Cl2)
  • Diisopropylamine (³99.5%; Sigma-Aldrich)
  • Chlorodiisopropoxy(2-methylbut-3-yn-2-yloxy)silane (see Support Protocol 1)
  • Methanol (MeOH)
  • Silica gel 60 adsorbent, 230- to 400-mesh, 40- to 63-µm (EMD Chemicals)
  • Ethyl acetate (EtOAc)
  • Acetone
  • Hexanes
  • Triethylamine (TEA; Acros Organics), 99.7% extra-pure
  • 500-ml and 2-L round-bottom, single-neck reaction flasks
  • Teflon-coated magnetic stir bar and stir plate
  • 500-mL pressure-equalizing addition funnels
  • Rubber septa, Suba-Seal, sizes to fit glassware joints (Aldrich)
  • Rotary evaporator
  • Diaphragm vacuum pump
  • High vacuum oil pump
  • Vacuum gauge and regulator
  • Additional reagents and equipment for flash column chromatography (appendix 3E) and thin-layer chromatography (appendix 3D)

Basic Protocol 2: Coupling of a Chromophoric Azide to 5¢-O-Alkynylsilyl-N-Protected 2¢-ACE-Adenosine Ribonucleoside

 Materials
  • 5¢-Sil-2¢-ACE-N6-isobutyryladenosine (S.2a; see Basic Protocol 1)
  • Toluene
  • N-Ethyl-N-(2-azidoethyl)-4-(4-nitrophenyldiazenyl)aniline (see Support Protocol 2)
  • N,N-Diisopropylethylamine (DIPEA; Acros Organics), 99.5+%
  • Copper(I) iodide (Acros Organics), 99.995%
  • Ethyl acetate (EtOAc)
  • Saturated aqueous sodium chloride (NaCl)
  • Silica gel 60 adsorbent, 230- to 400-mesh, 40- to 63-µm (EMD Chemicals)
  • Acetone
  • Hexanes
  • Triethylamine (TEA; Acros Organics), 99.7% extra-pure
  • Methylene chloride (CH2Cl2)
  • 3- and 5-L round-bottom, single-neck reaction flasks
  • Sonicating bath
  • Teflon-coated magnetic stir bar and stir plate
  • 4-L separatory funnel
  • Rotary evaporator
  • High vacuum oil pump
  • Vacuum gauge and regulator
  • Additional reagents and equipment for flash column chromatography (appendix 3E) and thin-layer chromatography (appendix 3D)

Basic Protocol 3: Preparation of Chromophoric 5¢-O-Silyl-N-Protected 2¢-ACE-Adenosine Ribonucleoside Phosphoramidite

 Materials
  • 5¢-DRSil-2¢-ACE-N6-isobutyryladenosine (S.3a; see Basic Protocol 2)
  • Acetonitrile, anhydrous for DNA synthesis (ThermoFisher Scientific)
  • Methylene chloride (CH2Cl2)
  • Diisopropylamine, ³99.5% (Aldrich)
  • Methyl N,N,N¢,N¢-tetraisopropylphosphorodiamidite (Digital Specialty Chemicals)
  • 0.5 M 5-ethylthio-1H-tetrazole (see recipe)
  • Absolute ethanol (200 proof)
  • Silica gel 60 adsorbent, 230- to 400-mesh, 40- to 63-µm (EMD Chemicals)
  • Hexanes
  • Acetone
  • Triethylamine (TEA; Acros Organics), 99.7% extra-pure
  • Toluene
  • Inert gas (argon or nitrogen), dry
  • 1-L round-bottom, single-neck reaction flasks
  • Rotary evaporator
  • High-vacuum oil pump
  • Teflon-coated magnetic stir bar and magnetic stir plate
  • Rubber septa, Suba-Seal, sizes to fit glassware joints (Aldrich)
  • Diaphragm vacuum pump
  • Vacuum gauge and regulator
  • Additional reagents and equipment for flash column chromatography (appendix 3E) and thin-layer chromatography (appendix 3D)

Support Protocol 1: Synthesis of the Chloroalkynylsilane

 Materials
  • Inert gas (argon or nitrogen), dry
  • Silicon(IV) chloride (Acros Organics), 99.8+%
  • Drierite
  • 25% (w/v) aqueous potassium hydroxide
  • 2-Propanol (Aldrich), 99.5%, anhydrous
  • Methylene chloride (CH2Cl2)
  • Triethylamine (TEA; Acros Organics), 99.7% extra-pure
  • 2-Methyl-3-butyn-2-ol (Acros Organics), 98%
  • Molecular sieves, 4-Å, 8- to 12-mesh beads (Acros Organics)
  • 2- and 3-L round-bottom single- and three-neck reaction flasks
  • Teflon-coated magnetic stir bar and magnetic stir plate
  • 1-L pressure-equalizing addition funnels
  • Hose inlet adapters
  • Tygon tubing (diameter to fit barb on hose inlet adapters and drying tubes)
  • Polyethylene drying tubes (ThermoFisher Scientific)
  • 1-L Erlenmeyer flask
  • Rubber septa, Suba-Seal, sizes to fit glassware joints (Sigma-Aldrich)
  • Pasteur pipets or other disposable glass tubes
  • Silicone oil bath with thermometer
  • Glassware for vacuum distillation, including:
    • Distilling flask
    • Short-path distilling head with condenser
    • Four-position rotating receiver with flasks
    • Thermometer
    • 30-cm distilling column packed with 3-mm glass beads
  • Aluminum foil
  • Overhead mechanical stirrer with stainless steel paddle
  • Filtration adapter to fit 24/40 joint
  • 150- and 600-mL sintered glass filter funnels, coarse frit
  • Large conical glass funnel
  • Diaphragm vacuum pump
  • Rotary evaporator
  • High-vacuum oil pump
  • Vacuum gauge and regulator

Support Protocol 2: Synthesis of the Chromophoric Azide

 Materials
  • N-Ethyl-N-hydroxyethylaniline (Acros Organics), 96%
  • Methylene chloride (CH2Cl2)
  • Triethylamine (TEA; Acros Organics), 99.7% extra-pure
  • Methanesulfonyl chloride (Acros Organics), 99.50%
  • 0.5 M hydrochloric acid
  • Saturated aqueous sodium chloride (NaCl)
  • Sodium sulfate, anhydrous
  • Methyl sulfoxide
  • Sodium azide (Acros Organics), 99% extra-pure
  • Diethyl ether
  • Sulfuric acid, concentrated
  • Sodium nitrite (Acros Organics), 97+%
  • Glacial acetic acid
  • Propionic acid (Acros Organics), 99%
  • 4-Nitroaniline (Acros Organics), 99+%
  • Sodium acetate, anhydrous
  • 20% (w/v) aqueous sodium acetate, 4°C
  • 95% ethanol
  • 1-, 2-, 3-, and 5-L round-bottom, single- and three-neck reaction flasks
  • Teflon-coated magnetic stir bars and magnetic stirrer
  • 100-, 500-, and 1000-mL pressure-equalizing addition funnels
  • Glass stoppers
  • 3-L separatory funnels
  • Filtration adapter to fit 24/40 joint
  • 300-, 600-, and 2000-mL sintered glass filter funnels, coarse frit
  • Diaphragm vacuum pump
  • Rotary evaporator
  • 70°C oil bath
  • Hose inlet adapters
  • Rubber septa, Suba-Seal, sizes to fit glassware joints (Sigma-Aldrich)
  • Overhead mechanical stirrer with stainless steel paddle
  • Vacuum desiccator and high-vacuum oil pump
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 2.14.1
    Synthetic scheme for preparation of N-protected 5¢-DRSil-2¢-ACE ribonucleoside 3¢-phosphoramidites S.4a-d from N-protected 2¢-ACE ribonucleosides S.1a-d. (a) Chlorodiisopropoxy(2-methylbut-3-yn-2-yloxy)silane, diisopropylamine, CH2Cl2; (b) N-ethyl-N-(2-azidoethyl)-4-[(nitrophenyl)diazenyl)aniline, copper(I) iodide, N,N-diisopropylethylamine, toluene; (c) methyl N,N,N¢,N¢-tetraisopropylphosphorodiamidite, 5-ethylthio-1H-tetrazole, diisopropylamine, CH3CN, CH2Cl2.

    View Image
  • Figure 2.14.2
    Synthetic scheme for preparation of chlorodiisopropoxy(2-methylbut-3-yn-2-yloxy)silane. (a) 2-propanol; (b) 2-methyl-3-butyn-2-ol, triethylamine, CH2Cl2.

    View Image
  • Figure 2.14.3
    Synthetic scheme for preparation of N-ethyl-N-(2-azidoethyl)-4-[(nitrophenyl)diazenyl]aniline. (a) Methanesulfonyl chloride, triethylamine, CH2Cl2; (b) sodium azide, methyl sulfoxide, 70°C; (c) sodium nitrite, sulfuric acid, acetic acid, proprionic acid, 4-nitroaniline, 0° to 2°C.

    View Image

Literature Cited

Literature Cited
    Bock, V.D., Hiemstra, H., and van Maarseveen, J.H. 2006. CuI catalyzed alkyne-azide “Click” cycloadditions from a mechanistic and synthetic perspective. Eur. J. Org. Chem. 1: 51-68.
    Chappelow, C.C., Elliott, R.L., and Goodwin, J.T. 1960. The phenylation and methylation of alkoxychlorosilanes. J. Org. Chem. 25: 435-459.
    Hartsel, S.A., Kitchen, D.E., Scaringe, S.A., and Marshall, W.S. 2004. "RNA oligonucleotide synthesis via 5¢-silyl-2¢-orthoester chemistry". In Methods in Molecular Biology, Vol. 288: Oligonucleotide Synthesis: Methods and Applications (P. Herdewijn, ed.) pp. 33-49. Humana Press, Totowa, New Jersey.
    Kolb, H.C. and Sharpless, K.B. 2003. The growing impact of click chemistry on drug discovery. Drug Discov. Today 8: 1128-1137.
    Moses, J.E. and Moorhouse, A.D. 2007. The growing applications of click chemistry. Chem. Soc. Rev. 36: 1249-1262.
    Vogel, A.I., Tatchell, A.R., Furmis, B.S., Hanna-ford, A.J., and Smith, P.W.G. 1996. Vogel's Textbook of Practical Organic Chemistry, 5th edition. p. 1552. Prentice Hall, Upper Saddle River, New Jersey.
     
 
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