2′‐Hydroxyl‐Protecting Groups that are Either Photochemically Labile or Sensitive to Fluoride Ions

Tod J. Miller1, Miriam E. Schwartz1, Geoffrey R. Gough1

1 Purdue University, West Lafayette, Indiana
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 2.5
DOI:  10.1002/0471142700.nc0205s03
Online Posting Date:  May, 2001
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Abstract

Protected ribonucleotide monomers are more difficult to obtain than their 2′‐deoxy counterparts because of the need to protect the 2′‐hydroxy function. This unit describes the stepwise preparation of suitably 2′‐protected ribonucleosides using two protecting groups: 2‐nitrobenzyloxymethyl (NBOM) and tert‐butyldimethylsilyl (TBDMS). In addition, details are given for protecting the 5′‐hydroxyl and the nucleobase, yielding nucleosides that are easily converted to phosphoramidite or H‐phosphonate derivatives for automated oligoribonucleotide synthesis.

     
 
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Table of Contents

  • Preparation of N‐Protected 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(2‐Nitrobenzyloxymethyl) Nucleosides
  • Basic Protocol 1: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(2‐nitrobenzyloxymethyl)uridine
  • Basic Protocol 2: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(2‐nitrobenzyloxymethyl)‐N4‐benzoylcytidine
  • Basic Protocol 3: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(2‐nitrobenzyloxymethyl)‐N6‐benzoyl‐adenosine
  • Basic Protocol 4: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(2‐nitrobenzyloxymethyl)‐N2‐isobutyrylguanosine
  • Preparation of N‐Protected 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl) Nucleosides
  • Alternate Protocol 1: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)uridine
  • Alternate Protocol 2: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)‐N4‐benzoyl‐cytidine
  • Alternate Protocol 3: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)‐N6‐phenoxy‐acetyladenosine
  • Alternate Protocol 4: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)‐N2‐phenoxy‐acetylguanosine
  • Support Protocol 1: Preparation of 2‐Nitrobenzyl Chloromethyl Ether
  • Support Protocol 2: Preparation of Pentafluorophenyl Benzoate
  • Support Protocol 3: Chromatographic Techniques
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(2‐nitrobenzyloxymethyl)uridine

  Materials
  • Methanol
  • Uridine
  • Dibutyltin oxide
  • Phosphorus pentoxide
  • Anhydrous tetra‐n‐butylammonium bromide
  • Anhydrous dimethylformamide, store over 4A molecular sieves
  • 2‐Nitrobenzyl chloromethyl ether, make fresh (see protocol 9)
  • Anhydrous pyridine, store over coarse granules of calcium hydride
  • 9:1 and 95:5 (v/v) chloroform/methanol
  • 66% (v/v) aqueous pyridine
  • Silica gel 60, 70 to 230 mesh ASTM (e.g., EM Science)
  • 2.5 × 30–cm and 3 × 60–cm glass chromatography columns packed with silica gel 60, 70 to 230 mesh ASTM, in chloroform to a bed height of 25 and 50 cm, respectively (see protocol 11)
  • Chloroform
  • Anhydrous triethylamine, store over coarse granules of calcium hydride
  • 4,4′‐Dimethoxytrityl chloride
  • Ethyl acetate
  • 1 M NaHCO 3
  • 2 M NaCl
  • Anhydrous sodium sulfate
  • 2:1 (v/v) ethyl acetate/hexane
  • 0% to 1% (v/v) methanol in chloroform, containing 0.25% (v/v) pyridine
  • 250‐mL and 2‐L round‐bottom flasks
  • Boiling chips
  • Water‐cooled reflux condenser
  • Heating mantle
  • Rotary evaporator connected interchangeably to water aspirator and vacuum pump
  • 50‐mL pressure‐equalizing dropping funnel
  • Large petri plate
  • Filter paper, coarse porosity and fast flow rate (e.g., Quantitative Q8, Fisher)
  • Additional reagents and equipment for TLC and column chromatography (see protocol 11)

Basic Protocol 2: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(2‐nitrobenzyloxymethyl)‐N4‐benzoylcytidine

  Materials
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(2‐nitrobenzyloxymethyl)uridine (see protocol 1)
  • Anhydrous pyridine, stored over coarse granules of calcium hydride
  • Acetic anhydride
  • Ethyl acetate
  • 1 M NaHCO 3
  • 2 M NaCl
  • Anhydrous sodium sulfate
  • 1,2,4‐Triazole
  • 4‐Chlorophenyl phosphorodichloridate
  • 3:1 (v/v) pyridine/concentrated ammonium hydroxide
  • 2.5 × 30–cm glass chromatography column packed with silica gel 60, 70 to 230 mesh ASTM, in chloroform to a bed height of 25 cm (see protocol 11)
  • 0% to 1% and 0% to 4% (v/v) methanol in chloroform, containing 0.25% (v/v) pyridine
  • 2:1 (v/v) ethyl acetate/hexane
  • Pentafluorophenyl benzoate (see protocol 10)
  • 250‐ and 500‐mL separatory funnels
  • Filter paper, coarse porosity and fast flow rate (e.g., Quantitative Q8, Fisher)
  • Rotary evaporator connected interchangeably to water aspirator and vacuum pump
  • 10‐ and 25‐mL round‐bottom flasks
  • Additional reagents and equipment for TLC and column chromatography (see protocol 11)

Basic Protocol 3: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(2‐nitrobenzyloxymethyl)‐N6‐benzoyl‐adenosine

  Materials
  • Methanol
  • Adenosine
  • Dibutyltin oxide
  • Phosphorous pentoxide
  • Anhydrous tetra‐n‐butylammonium bromide
  • Anhydrous dimethylformamide, stored over 4A molecular sieves
  • 2‐Nitrobenzyl chloromethyl ether, make fresh (see protocol 9)
  • Anhydrous pyridine, store over coarse granules of calcium hydride
  • 9:1 and 95:5 (v/v) chloroform/methanol
  • 66% (v/v) aqueous pyridine
  • Silica gel 60, 70 to 230 mesh ASTM (e.g., EM Science)
  • 2.5 × 25–cm, 2.5 × 30–cm, and 3 × 60–cm glass chromatography columns packed with silica gel 60, 70 to 230 mesh ASTM, in chloroform (see protocol 11)
  • 1% to 5% (v/v) methanol in chloroform
  • 80% (v/v) aqueous acetonitrile
  • Acetonitrile
  • Trimethylchlorosilane
  • Benzoyl chloride
  • Concentrated ammonium hydroxide
  • Ethyl acetate
  • 1 M NaHCO 3
  • 2 M NaCl
  • Anhydrous sodium sulfate
  • Chloroform
  • Triethylamine, stored over coarse granules of calcium hydride
  • 4,4′‐Dimethoxytrityl chloride
  • 0% to 1% (v/v) methanol in chloroform containing 0.25% (v/v) pyridine
  • 2:1 (v/v) ethyl acetate/hexane
  • 100‐mL, 250‐mL, and 2‐L round‐bottom flasks
  • Boiling chips
  • Water‐cooled reflux condenser
  • Heating mantle
  • Rotary evaporator connected interchangeably to water aspirator and vacuum pump
  • Oil bath, 60°C
  • 50‐mL pressure‐equalizing dropping funnel
  • 500‐mL separatory funnel
  • Filter paper, coarse porosity and fast flow rate (e.g., Quantitative Q8, Fisher)
  • Additional reagents and equipment for TLC and column chromatography (see protocol 11)

Basic Protocol 4: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(2‐nitrobenzyloxymethyl)‐N2‐isobutyrylguanosine

  Materials
  • Guanosine hydrate
  • Anhydrous pyridine, stored over coarse granules of calcium hydride
  • Isobutyryl chloride
  • Ethyl acetate
  • 1 M NaHCO 3
  • 2 M NaCl
  • Anhydrous sodium sulfate
  • Ethanol
  • 2 N NaOH, ice cold
  • Dowex AG50W‐X8 ion exchange resin (pyridinium form)
  • 5 × 60–cm glass chromatography column filled with 50 mL Dowex AG50W‐X8 ion‐exchange resin (pyridinium form) in water
  • 4:1 (v/v) chloroform/methanol
  • Phosphorus pentoxide
  • Dibutyltin oxide
  • Methanol
  • Anhydrous dimethylformamide, stored over 4A molecular sieves
  • 2‐Nitrobenzyl chloromethyl ether, make fresh (see protocol 9)
  • Silica gel 60, 70 to 230 mesh ASTM (e.g., EM Science)
  • 2.5 × 25–cm and 2.5 × 30–cm glass chromatography columns packed with silica gel 60, 70 to 230 mesh ASTM, in chloroform to a bed height of 20 and 25 cm, respectively (see protocol 11)
  • Chloroform
  • 2%, 4%, 6%, and 8% (v/v) methanol in chloroform
  • 9:1 (v/v) chloroform/methanol
  • 80% (v/v) aqueous acetonitrile
  • Acetonitrile
  • Triethylamine, stored over coarse granules of calcium hydride
  • 4,4′‐Dimethoxytrityl chloride
  • 0% to 1% (v/v) methanol in chloroform, containing 0.25% (v/v) pyridine
  • 2:1 (v/v) ethyl acetate/hexane
  • Oven, 130°C
  • 500‐mL and 2‐L round‐bottom flasks
  • 100‐mL pressure‐equalizing dropping funnel
  • Rotary evaporator connected interchangeably to water aspirator and vacuum pump
  • 1‐L separatory funnel
  • Filter paper, coarse porosity and fast flow rate (e.g., Quantitative QB, Fisher)
  • Water‐cooled reflux condenser
  • Heating mantle
  • Additional reagents and equipment for TLC and column chromatography (see protocol 11)
NOTE: The pyridinium form of Dowex AG50W‐X8 ion‐exchange resin is made by washing the hydrogen form with several changes of 20% aqueous pyridine.

Alternate Protocol 1: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)uridine

  Materials
  • Uridine
  • Anhydrous pyridine, stored over coarse granules of calcium hydride
  • 4,4′‐Dimethoxytrityl chloride
  • Methanol
  • Methylene chloride
  • 1 M NaHCO 3
  • Anhydrous sodium sulfate
  • 1:20 (v/v) chloroform/hexane
  • 3 × 30–cm and 3 × 60–cm flash chromatography columns (with reservoirs and flow controller), packed with silica gel 60, 230 to 400 Mesh ASTM (see protocol 11)
  • Diethyl ether containing 0.25% (v/v) pyridine
  • Ethyl acetate containing 0.25% (v/v) pyridine
  • 9:1 (v/v) chloroform/methanol
  • Anhydrous tetrahydrofuran
  • Silver nitrate
  • tert‐Butyldimethylsilyl chloride
  • 3:1 (v/v) diethyl ether/hexane
  • 2 M NaCl
  • Diethyl ether
  • 30% (v/v) ethyl acetate in hexane
  • 100‐ and 250‐mL round‐bottom flasks
  • Rotary evaporator connected interchangeably to water aspirator and vacuum pump
  • 250‐mL separatory funnels
  • Filter paper, coarse porosity and fast flow rate (e.g., Quantitative Q8, Fisher)
  • Additional reagents and equipment for TLC and column chromatography (see protocol 11)

Alternate Protocol 2: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)‐N4‐benzoyl‐cytidine

  • Cytidine
  • Benzoic anhydride
  • Chloroform
  • 19:1 (v/v) chloroform/methanol
  • 1‐L round‐bottom flasks
  • Water‐cooled reflux condenser
  • Heating mantle
  • Coarse sintered funnel

Alternate Protocol 3: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)‐N6‐phenoxy‐acetyladenosine

  Materials
  • Adenosine
  • Anhydrous pyridine, stored over coarse granules of calcium hydride
  • Trimethylchlorosilane
  • 1‐Hydroxybenzotriazole
  • Anhydrous acetonitrile
  • Phenoxyacetyl chloride
  • Concentrated ammonium hydroxide
  • Chloroform
  • 90% (v/v) ethanol
  • 4,4′‐Dimethoxytrityl chloride
  • 9:1 (v/v) methylene chloride/methanol
  • Methanol
  • Ethyl acetate
  • 1 M NaHCO 3, 5°C
  • 2 M NaCl
  • Anhydrous sodium sulfate
  • 3 × 30–cm and 3 × 60–cm flash chromatography columns (with reservoirs and flow controller), packed with silica gel 60, 230 to 400 Mesh ASTM (see protocol 11)
  • 2% to 10% (v/v) ethanol in ethyl acetate
  • Imidazole
  • tert‐Butyldimethylsilyl chloride
  • 1:1 (v/v) methylene chloride/ethyl acetate
  • 1% and 35% (v/v) ethyl acetate in hexane
  • Rotary evaporator connected interchangeably to water aspirator and vacuum pump
  • 250‐mL round‐bottom flasks with rubber septa
  • 5‐ and 50‐mL syringes and vent needles
  • 100‐mL pressure‐equalizing dropping funnel
  • 500‐mL separatory funnel
  • Filter paper, coarse porosity and fast flow rate (e.g., Quantitative Q8, Fisher)
  • Additional reagents and materials for TLC and column chromatography (see protocol 11)

Alternate Protocol 4: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)‐N2‐phenoxy‐acetylguanosine

  • Guanosine hydrate
  • 50% (v/v) ethyl acetate in hexane
  • Oven, 130°C

Support Protocol 1: Preparation of 2‐Nitrobenzyl Chloromethyl Ether

  Materials
  • 2‐Nitrobenzyl alcohol
  • Dimethyl sulfoxide, dried over 4A molecular sieves
  • Acetic anhydride
  • Acetic acid
  • 1:1 (v/v) diethyl ether/hexane
  • Sodium bicarbonate
  • 1:1 (v/v) ethyl acetate/hexane
  • Saturated sodium bicarbonate
  • 2 M NaCl
  • Anhydrous sodium sulfate
  • 5 × 60–cm chromatography column packed with silica gel 60, 70 to 230 mesh ASTM, in hexane to a bed height of 56 cm (see protocol 11)
  • 1%, 5%, 10%, and 15% (v/v) diethyl ether in hexane
  • Anhydrous methylene chloride
  • Ethanol (for dry ice/ethanol bath)
  • 1 M SO 2Cl 2 in methylene chloride
  • Anhydrous dimethylformamide, store over 4A molecular sieves
  • 500‐mL separatory funnel
  • Filter paper, coarse porosity and fast flow rate (e.g., Quantitative Q8, Fisher)
  • Rotary evaporator connected to water aspirator
  • 250‐mL round‐bottom flask
  • 50‐mL pressure‐equalizing dropping funnel
  • Additional reagents and equipment for TLC and column chromatography (see protocol 11)
CAUTION: The sulfuryl chloride (SO 2Cl 2) solution is corrosive and extremely toxic.

Support Protocol 2: Preparation of Pentafluorophenyl Benzoate

  Materials
  • 2,3,4,5,6‐Pentafluorophenol
  • Anhydrous dimethylformamide, store over 4A molecular sieves
  • Benzoic acid
  • 1,3‐Dicyclohexylcarbodiimide
  • Ethyl acetate
  • Ethanol
  • Phosphorus pentoxide
  • Filter paper, coarse porosity and fast flow rate (e.g., Quantitative Q8, Fisher)
  • Rotary evaporator connected interchangeably to water aspirator and vacuum pump
  • Coarse sintered funnel
CAUTION: Pentafluorophenol, pentafluorophenyl benzoate, and 1,3‐dicyclohexylcarbodiimide are toxic irritants. Avoid skin contact.
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Literature Cited

Literature Cited
   Benneche, T., Strande, P., and Undheim, K. 1983. A new synthesis of chloromethyl benzyl ethers. Synthesis. 1983:762‐763.
   Chaix, C., Duplaa, A.M., Molko, D., and Teoule, R. 1989. Solid phase synthesis of the 5′‐half of the initiator t‐RNA from B. subtilis. Nucl. Acids Res. 17:7381‐7393.
   Fromageot, H.P.M., Griffin, B.E., Reese, C.B., Sulston, J.E., and Trentham, D.R. 1966. Orientation of ribonucleoside derivatives by proton magnetic resonance spectroscopy. Tetrahedron 22:705‐710.
   Hakimelahi, G.H., Proba, Z.A., and Ogilvie, K.K. 1982. New catalysts and procedures for the dimethoxytritylation and selective silylation of ribonucleosides. Can. J. Chem. 60:1106‐1113.
   Igolen, J. and Morin, C. 1980. Rapid synthesis of protected 2′‐deoxycytidine derivatives. J. Org. Chem. 45:4802‐4804.
   Ohtsuka, E., Nakagawa, E., Tanaka, T., Markham, A.F., and Ikehara, M. 1978. Studies on transfer ribonucleic acids and related compounds. XXI. Synthesis and properties of guanine rich fragments from E. coli tRNAfMet 5′ end. Chem. Pharm. Bull. 26:2998‐3006.
   Schwartz, M.E., Breaker, R.R., Asteriadis, G.T., deBear, J.S., and Gough, G.R. 1992. Rapid synthesis of oligoribonucleotides using 2′‐O‐(2‐nitrobenzyloxymethyl)‐protected monomers. BioMed. Chem. Lett. 2:1019‐1024.
   Sinha, N.D., Biernat, J., McManus, J., and Köster, H. 1984. Polymer support oligonucleotide synthesis XVIII: Use of β‐cyanoethyl‐N,N‐dialkylamino‐/N‐morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product. Nucl. Acids Res. 12:4539‐4557.
   Sinha, N.D., Davis, P., Usman, N., Perez, J., Hodge, R., Kremsky, J., and Casale, R. 1993. Labile exocyclic amine protection of nucleosides in DNA, RNA and oligonucleotide analog synthesis facilitating N‐deacylation, minimizing depurination and chain degradation. Biochimie 75:13‐23.
   Sung, W.L. 1982. Synthesis of 4‐(1,2,4‐triazol‐1‐yl)pyrimidin‐2(1H)‐one ribonucleotide and its application in synthesis of oligoribonucleotides. J. Org. Chem. 47:3623‐3628.
   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.
   Usman, N., Ogilvie, K.K., Jiang, M.‐Y., and Cedergren, R.J. 1987. 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.
   Wagner, D., Verheyden, J.P.H., and Moffatt, J.G. 1974. Preparation and synthetic utility of some organotin derivatives of nucleosides. J. Org. Chem. 39:24‐30.
   Watanabe, K.A. and Fox, J.J. 1966. A simple method for selective acylation of cytidine on the 4‐amino group. Angew. Chem. Int. Ed. Engl. 5:579‐580.
   Wincott, F., DiRenzo, A., Shaffer, C., Grimm, S., Tracz, D., Workman, C., Sweedler, D., Gonzalez, C., Scaringe, S., and Usman, N. 1995. Synthesis, deprotection, analysis and purification of RNA and ribozymes. Nucl. Acids Res. 23:2677‐2684.
Key References
   Schwartz et al., 1992. See above.
  The general strategy involved in using the 2′‐O‐(2‐nitrobenzyloxymethyl) protecting group is presented by this method's developers.
   Usman et al., 1987. See above.
  The general strategy involved in using the 2′‐O‐(tert‐butyldimethylsilyl) protecting group is presented by this method's developers.
   Wincott et al., 1995. See above.
  This publication describes some recent advances in RNA synthesis using the tert‐butyldimethylsilyl group.
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