A Base‐Labile Protecting Group (Fluorenylmethoxycarbonyl) for the 5′‐Hydroxy Function of Nucleosides

Michael J. Gait1, Christian Lehmann2

1 MRC Laboratory of Molecular Biology, Cambridge, United Kingdom, 2 Institute of Organic Chemistry, University of Lausanne, Lausanne, Switzerland
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 2.4
DOI:  10.1002/0471142700.nc0204s00
Online Posting Date:  May, 2001
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Abstract

Many popular synthesis strategies look for appropriate 2′‐O‐protection methods to use in conjunction with 5′‐O‐trityl chemistry. In contrast, this unit describes the use of FMOC as a 5′‐protecting group in conjunction with a ketal‐type 2′‐O‐protecting group, 4‐methoxytetrahydropyran‐4‐yl (MTHP). The synthesis of all four 2′‐O‐MTHP‐5′‐O‐FMOC‐protected ribonucleosides and 5′‐O‐FMOC‐2′‐deoxythymidine is described, as is the preparation of the N‐protected, 2′‐O‐MTHP‐protected starting nucleosides.

     
 
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Table of Contents

  • Basic Protocol 1: Acylation of the 5′‐Hydroxy Group of 2′‐O‐(4‐Methoxytetrahydropyran‐4‐yl)‐Ribonucleosides with 9‐Fluorenylmethoxycarbonyl Chloride
  • Support Protocol 1: Preparation of 2′‐O‐(4‐Methoxytetrahydropyran‐4‐yl)‐ Uridine (S.1a) from Uridine
  • Support Protocol 2: Preparation of 2′‐O‐(4‐Methoxytetrahrdropyran‐4‐yl)‐4‐N‐Benzoylcytidine (S.1b) from Cytidine
  • Support Protocol 3: Preparation of 2′‐O‐(4‐Methoxytetrahydropyran‐4‐yl)‐6‐N‐Benzoyladenosine (S.1c) from Adenosine
  • Support Protocol 4: Preparation of 2′‐O‐(4‐Methoxytetrahydropyran‐4‐yl)‐2‐N‐ Isobutyrylguanosine (S.1d) from Guanosine
  • Support Protocol 5: Preparation of 1,3‐Dichloro‐1,1,3,3‐ Tetraisopropyldisiloxane
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Acylation of the 5′‐Hydroxy Group of 2′‐O‐(4‐Methoxytetrahydropyran‐4‐yl)‐Ribonucleosides with 9‐Fluorenylmethoxycarbonyl Chloride

  Materials
  • 2′‐Deoxythymidine or 2′‐O‐MTHP‐ribonucleoside: 2′‐O‐(4‐methoxytetrahydropyran‐4‐yl)‐rU, ‐rCBz, ‐rABz, or ‐rGi‐Bu (see protocol 2 to protocol 5)
  • Dry pyridine, freshly distilled from calcium hydride (5 g/L) after refluxing for 2 hr under an inert atmosphere
  • Nitrogen gas
  • 9‐Fluorenylmethoxycarbonyl (FMOC) chloride (Cambridge Research Biochemicals), recrystallized from ether/pentane in a large desiccator (vapor diffusion method)
  • Diethyl ether (analytical grade)
  • Chloroform stabilized with 1% ethanol (commercially available analytical grade)
  • Methanol (analytical grade)
  • Anisaldehyde reagent (see recipe)
  • Ethane‐1,2‐diol (analytical grade)
  • Saturated aqueous sodium bicarbonate solution
  • Sodium sulfate (anhydrous)
  • Toluene (analytical grade)
  • Ethanol (analytical grade)
  • Pentane (analytical grade)
  • Silica‐coated thin‐layer chromatography (TLC) plate with fluorescent indicator Kieselgel 60F 254 (Merck 5554 glass plates or 5744 aluminium foils)
  • UV light source
  • D4 glass‐filter crucible
  • Short column (diameter 5 cm; length ∼10 cm) containing 50 g Kieselgel 60H without calcium sulfate (Merck 7736 or Fluka 60770, particle size 5‐40 µm; or Merck 11677, particle size 15 µm), preconditioned with chloroform
  • Glass‐fiber tissue
CAUTION: Wear gloves and perform all operations involving TLC solvents and reagents in a well‐ventilated fume hood.

Support Protocol 1: Preparation of 2′‐O‐(4‐Methoxytetrahydropyran‐4‐yl)‐ Uridine (S.1a) from Uridine

  • Uridine (Sigma or Fluka), dried before use for 2 hr at 50°C over phosphorus pentoxide in vacuo
  • Toluene‐p‐sulfonic acid monohydrate (analytical grade)
  • Dioxane (analytical grade), dried by keeping over activated basic aluminum oxide, then distilled before use from sodium/benzophenone after refluxing until the blue color indicates complete dryness
  • Trimethyl orthoacetate (purum, 97%), dried by refluxing over calcium hydride (5 g/L) and distilled under inert atmosphere
  • Pyridine (analytical grade), dried by refluxing over calcium hydride (5 g/L) and then distilled under inert atmosphere
  • Acetic anhydride (analytical grade)
  • Methylene chloride (analytical grade)
  • Saturated (∼1 M) aqueous sodium hydrogen carbonate
  • Magnesium sulfate
  • Formic acid (analytical grade)
  • 5,6‐Dihydro‐4‐methoxy‐2H‐pyran (analytical grade; Sigma or Fluka pract., ∼90%; b.p. 20 59° to 61°C)
  • Half‐saturated methanolic ammonia solution (see recipe)
  • 500‐mL separatory funnel

Support Protocol 2: Preparation of 2′‐O‐(4‐Methoxytetrahrdropyran‐4‐yl)‐4‐N‐Benzoylcytidine (S.1b) from Cytidine

  • Cytidine (S.7; Sigma or Fluka), dried before use for 2 hr at 50°C over phosphorus pentoxide in vacuo
  • N,N‐Dimethylformamide, dried by stirring overnight at room temperature with calcium hydride (5 g/L) and subsequent distillation under reduced pressure (b.p. 70° to 80°C, 20 to 30 mmHg)
  • Dry pyridine (see protocol 2 for drying procedure)
  • 1,3‐Dichloro‐1,1,3,3‐tetraisopropyldisiloxane (see protocol 6)
  • 2 M triethylammonium bicarbonate buffer (see recipe)
  • Acetone (analytical grade)
  • 1‐Hydroxybenzotriazole (Fluka), dried before use for 72 hr at 50°C over phosphorus pentoxide in vacuo
  • Triethylamine, dried by refluxing 2 hr over calcium hydride (5 g/L) followed by distillation
  • Benzoyl chloride (Fluka, puriss.)
  • Acetonitrile (analytical grade)
  • 1 M n‐tetrabutylammonium fluoride (Aldrich/Fluka) in acetonitrile
CAUTION: 1‐Hydroxybenzotriazole may explode at higher temperatures.

Support Protocol 3: Preparation of 2′‐O‐(4‐Methoxytetrahydropyran‐4‐yl)‐6‐N‐Benzoyladenosine (S.1c) from Adenosine

  • Adenosine (S.10; Sigma or Fluka), dried before use for 2 hr at 50°C over phosphorus pentoxide in vacuo
  • Dry pyridine (see protocol 2 for drying procedure)
  • Acetic anhydride, fractionally distilled with 10% toluene to remove acetic acid
  • 25% (w/v) sodium methoxide in methanol (pract., Aldrich, Fluka)
  • Acetic acid (analytical grade)
  • Diethyl ether (analytical grade)
  • Imidazole (analytical grade)
  • 0.1 M hydrochloric acid

Support Protocol 4: Preparation of 2′‐O‐(4‐Methoxytetrahydropyran‐4‐yl)‐2‐N‐ Isobutyrylguanosine (S.1d) from Guanosine

  • Guanosine (S.14; Sigma or Fluka), dried before use for 2 hr at 50°C over phosphorus pentoxide in vacuo
  • Dry pyridine (see protocol 2 for drying procedure)
  • Trimethylsilyl chloride (analytical grade)
  • Isobutyryl chloride (analytical grade)
  • Concentrated aqueous ammonia (∼25%)
  • Methylene chloride (analytical grade)
  • Phosphorus pentoxide

Support Protocol 5: Preparation of 1,3‐Dichloro‐1,1,3,3‐ Tetraisopropyldisiloxane

  Materials
  • Magnesium curls
  • Dry diethyl ether, distilled from phosphorus pentoxide (30 g/L)
  • Isopropyl bromide, distilled from calcium hydride (5 g/L)
  • Trichlorosilane, freshly distilled
  • 0.1 N hydrochloric acid
  • Magnesium sulfate
  • Methylene chloride (analytical grade), dried by passage through activated basic alumina
  • Chlorine gas, dried over concentrated sulfuric acid
  • NaCl plates for infrared (IR) spectroscopy
  • Additional reagents and equipment for IR spectroscopy
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Figures

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Literature Cited

Literature Cited
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   Lehmann, C., Xu, Y.‐Z., Christodoulou, C., Tan, Z.‐K., and Gait, M.J. 1989. Solid‐phase synthesis of oligoribonucleotides using 9‐fluorenylmethoxy‐carbonyl (Fmoc) for 5′‐hydroxyl protection. Nucl. Acids Res. 17:2379‐2390.
   Lehmann, C., Xu, Y.‐Z., Christodoulou, C., Gait, M.J., Van Meervelt, L., Moore, M., and Kennard, O. 1991. 3′/5′‐Regioselectivity of introduction of the 9‐fluorenylmethoxy‐carbonyl group to 2′‐O‐tetrahydropyran‐2‐yl and 2′‐O‐(4‐methoxytetrahydropyran‐4‐yl)‐nucleosides: Useful intermediates for solid‐phase RNA synthesis. Nucleosides Nucleotides 10:1599‐1614.
   Ma, Y. and Sonveaux, E. 1987. The 9‐fluorenylmethyloxycarbonyl (Fmoc) group as a 5′‐O base labile protecting group in solid supported oligonucleotide synthesis. Nucleosides Nucleotides 6:491‐493.
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   Reese, C.B. and Skone, P.A. 1985. Action of acid on oligoribonucleotide phosphotriester intermediates. Effect of released vicinal hydroxy functions. Nucl. Acids Res. 13:5215‐5231.
   Reese, C.B., Saffhill, R., and Sulston, J. 1970. 4‐Methoxytetrahydropyran‐4‐yl: A symmetrical alternative to the tetrahydropyranyl group. Tetrahedron 26:1023‐1030.
   Schaller, H., Weiman, G., Lerch, B., and Khorana, H.G. 1963. Protected derivatives of deoxyribonucleotides and new syntheses of deoxyribonucleotide‐3′ phosphates. J. Am. Chem. Soc. 85:3821‐3827.
   Schwartz, M.E., Breaker, R.R., Asteriadis, G.T., deBear, J.S., and Gough, G.R. 1992. Rapid synthesis of oligoribonucleotides using 2′‐O‐(o‐nitrobenzyloxymethyl)‐protected monomers. Bioorg. Med. Chem. Lett. 2:1019‐1024.
   Still, W.C., Kahn, M., and Mitra, A. 1978. Rapid chromatographic technique for preparative separations with moderate resolution. J. Org. Chem. 43:2923‐2925.
   Ti, G.S., Gaffney, B.L., and Jones, R.A. 1982. Transient protection: Efficient one‐flask synthesis of protected deoxynucleosides. J. Am. Chem. Soc. 104:1316‐1319.
   van Boom, J.H. and Wreesmann, C.T.J. 1984. Chemical synthesis of small oligoribonucleotides in solution. In Oligonucleotide Synthesis: A Practical Approach (M.J. Gait, ed.) pp. 153‐183. IRL Press, Oxford.
Key References
   Blackburn, M. and Gait, M.J. (eds.) 1996. Nucleic Acids in Chemistry and Biology. Oxford University Press, New York.
  In particular,chapter 3 on chemical synthesis is very recommendable as an illustrative overview to the present topics.
  Gait, M.J. (ed.) 1984. Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford.
  Basic principles of oligonucleotide synthesis are illustrated by practical advice through further step‐by‐step protocols; some of the chapters may be regarded as primers for units in the present volume.
   Lehmann et al., 1991. See above.
  The procedure in the is first described for the four 2′‐O‐MTHP‐protected ribonucleosides. For more background information on particular complications arising when the chiral 2′‐O‐tetrahydropyranyl (THP) group is used, see Lehmann et al., .
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