Synthesis of Oligoribonucleotides Containing N6‐Alkyladenosine and 2‐Methylthio‐N6‐Alkyladenosine

Elzbieta Kierzek1, Ryszard Kierzek1

1 Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.23
DOI:  10.1002/0471142700.nc0423s17
Online Posting Date:  September, 2004
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Abstract

The N6‐alkyladenosines and 2‐methylthio‐N6‐alkyladenosines are the most common modified adenosine nucleosides, and transfer ribonucleic acids (tRNA) are particularly rich in these modified nucleosides. They are present at position 37 of the anticodon arm, and the contributions of these hypermodified nucleosides to codon‐anticodon interactions as well as to translation are significant, although they are not fully understood. This unit describes a new chemical synthesis method for oligoribonucleotides containing N6‐alkyladenosines and 2‐methylthio‐N6‐alkyladenosines via postsynthetic modifications of precursor oligoribonucleotides. To obtain oligoribonucleotides containing N6‐alkyladenosines, a precursor oligoribonucleotide carrying 6‐methylthiopurine riboside residues was used, whereas for the synthesis of oligoribonucleotides containing 2‐methylthio‐N6‐alkyladenosines, a precursor oligoribonucleotide carrying the 2‐methylthio‐6‐chloropurine riboside was applied. This allowed synthesis of modified oligoribonucleotides containing naturally occurring modified nucleosides such as N6‐isopentenyladenosine (i6A), N6‐methyladenosine (m6A), 2‐methylthio‐N6‐isopentenyladenosine (ms2i6A), and 2‐methylthio‐N6‐methyladenosine (ms2m6A), as well as several unnaturally modified adenosine derivatives.

Keywords: N6‐alkyladenosine; 2‐methylthio‐N6‐alkyladenosine; phosphoramidites of modified nucleosides; postsynthetic modification of oligoribonucleotides

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis of the 6‐Methylthiopurine Riboside Phosphoramidite
  • Alternate Protocol 1: Synthesis of the 2‐Methylthio‐6‐Chloropurine Riboside Phosphoramidite
  • Basic Protocol 2: Synthesis of Oligoribonucleotides Containing N6‐Alkyladenosine or 2‐Methylthio‐N6‐Alkyladenosine
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of the 6‐Methylthiopurine Riboside Phosphoramidite

  Materials
  • Inosine ( S.1)
  • Pyridine (reagent grade or better)
  • Acetic anhydride
  • Dichloromethane (anhydrous)
  • Methanol (anhydrous)
  • Saturated aqueous sodium bicarbonate solution
  • Sodium sulfate (anhydrous)
  • Toluene
  • 1,4‐Dioxane (anhydrous)
  • Lawesson's reagent (Aldrich)
  • N,N‐Dimethylformamide (DMF; anhydrous)
  • Potassium carbonate (anhydrous), ground
  • Methyl iodide
  • Celite 545
  • Silica gel 60H (Merck)
  • Concentrated aqueous ammonium hydroxide (NH 4OH; 28% to 30%)
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl)
  • Imidazole
  • tert‐Butyldimethylsilyl chloride (TBDMS‐Cl)
  • Ethyl acetate
  • Hexanes (anhydrous)
  • 10% (w/v) aqueous sodium phosphate, monobasic (NaH 2PO 4)
  • Benzene (anhydrous)
  • Dry ice/ethanol bath
  • Acetonitrile (commercial with <20 ppm water or dried over 3Å  molecular sieves)
  • Diisopropylethylamine (DIPEA; anhydrous)
  • 2‐Cyanoethyl diisopropylchlorophosphoramidite
  • Acetone
  • Triethylamine (TEA; anhydrous)
  • 250‐ and 25‐mL round‐bottom flasks
  • Silica gel 60F thin‐layer chromatography (TLC) plates (Merck)
  • 250‐mL and 1‐L separatory funnels
  • Rotary evaporator
  • Vacuum pump and water aspirator
  • Reflux condenser
  • 100° and 50°C oil baths (silicone oil)
  • 60‐ and 150‐mL sintered glass funnels
  • Rubber septa
  • Vacuum adaptor for 250‐mL round‐bottom flask and appropriate traps
  • Vacuum desiccator
  • 1‐mL and 10‐mL glass syringes with needles
  • Silanized silica gel 60F TLC plates (Merck), optional
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography (unit 2.4& APPENDIX )

Alternate Protocol 1: Synthesis of the 2‐Methylthio‐6‐Chloropurine Riboside Phosphoramidite

  • Guanosine ( S.10; anhydrous)
  • 4‐Dimethylaminopyridine (DMAP)
  • Phosphorus oxychloride (POCl 3), freshly distilled
  • N,N‐Dimethylaniline
  • Dimethyl disulfide
  • Isoamyl nitrite
  • 500‐mL and 1‐L round‐bottom flasks
  • 140° and 60°C oil baths (silicone oil)
  • 250‐µL and 5‐mL glass syringes with needles

Basic Protocol 2: Synthesis of Oligoribonucleotides Containing N6‐Alkyladenosine or 2‐Methylthio‐N6‐Alkyladenosine

  Materials
  • Commercial phosphoramidites: 5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐Otert‐butyldimethylsilyl‐3′‐O‐[(2‐cyanoethoxy)‐(N,N‐diisopropylamino)]phosphinyl uridine and N‐protected cytidine, adenosine, and guanosine
  • Modified phosphoramidite: 5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐O‐tert‐butyldimethylsilyl‐3′‐O‐[(2‐cyanoethoxy)‐(N,N‐diisopropyl)]phosphinyl‐6‐methylthiopurine riboside ( S.9; see protocol 1) or ‐2‐thiomethyl‐6‐chloropurine riboside ( S.18; see protocol 2)
  • Acetonitrile, anhydrous
  • Magnesium monoperoxyphthalate hexahydrate (for oligomers containing S.9)
  • Dioxane (for oligomers containing S.9)
  • Primary amine: e.g., isopentenylamine hydrochloride and triethylamine (TEA; for i6A or ms2i6A) or methylamine (for m6A or ms2m6A)
  • Pyridine (reagent grade or better)
  • 2 M methylamine in tetrahydrofuran (for m6A or ms2m6A)
  • Concentrated aqueous ammonia (28% to 32%)
  • Ethanol
  • 1 M triethylammonium fluoride in pyridine (or other desilylating agent)
  • 10 mM ammonium acetate
  • Automated oligonucleotide synthesizer (Applied Biosystems 392)
  • 1.5‐ and 5‐mL screw‐top tubes
  • 15‐mL sintered glass funnels
  • Water aspirator
  • 55°C water bath
  • Speedvac evaporator (Savant)
  • Spin filters
  • Sep‐Pak cartridges (Waters)
  • 10‐mL disposable syringes
  • Additional reagents and equipment for automated oligoribonucleotide synthesis ( appendix 3C)
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Figures

Videos

Literature Cited

Literature Cited
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   Beaucage, S.L. and Caruthers, M.H. 1981. Deoxynucleoside phosphoramidites—a new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22:1859‐1862.
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   Grosjean, H., Houssier, C., Romby, P., and Marquet, R. 1998. Modulatory role of modified nucleotides in RNA loop‐loop interaction. In Modification and Editing of RNA (H. Grosjean and  R. Benne, eds.) pp. 113‐133. American Society of Microbiology Press, Washington, D.C.
   Kierzek, E. and Kierzek, R. 2003a. The synthesis of oligoribonucleotides containing N6‐alkyladenosines and 2‐methylthio‐N6‐alkyladenosines via post‐synthetic modifications of precursor oligomers. Nucl. Acids Res. 31:4461‐4471.
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   Sochacka, E. 1998. The chemical synthesis of E.coli tRNALys anticodon loop fragment and its analogues. Nucleosides Nucleotides 17:327‐338.
   Stuart, J.W., Gdaniec, Z., Guenther, R., Marszalek, M., Sochacka, E., Malkiewicz, A., and Agris, P.F. 2000. Functional anticodon architecture of human tRNALys3 includes disruption of intraloop hydrogen bonding by the naturally occurring amino acid modification, t6A. Biochemistry 39:13396‐13404.
   Sundaram, M., Crain, P.F., and Davis, D.R. 2000. Synthesis and characterization of the native anticodon domain of E. coli. Simultaneous incorporation of modified nucleosides mnm5s2U, t6A, and pseudouridine using phosphoramidite chemistry. J. Org. Chem. 65:5609‐5614.
   Wetzel, R. and Eckstein, F. 1975. Synthesis and reactions of 6‐methylsulfonyl‐9‐β‐D‐ribofuranosylpurine. J. Org. Chem. 40:658‐660.
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