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

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

1Purdue 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

  • Unit Introduction
  • Preparation of N-Protected 5¢-O-(4,4¢-Dimethoxytrityl)-2¢-O-(2-Nitrobenzyloxymethyl) Nucleosides
  • Preparation of N-Protected 5¢-O-(4,4¢-Dimethoxytrityl)-2¢-O-(tert-butyldimethylsilyl) Nucleosides
  • Support Protocol 1: Preparation of 2-Nitrobenzyl Chloromethyl Ether
  • Support Protocol 2: Preparation of Pentafluorophenyl Benzoate
  • Support Protocol 3: Chromatographic Techniques
  • Commentary
  • Bibliography
  • 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 Support Protocol 1)
  • 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 Support Protocol 3)
  • Chloroform
  • Anhydrous triethylamine, store over coarse granules of calcium hydride
  • 4,4¢-Dimethoxytrityl chloride
  • Ethyl acetate
  • 1 M NaHCO3
  • 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 Support Protocol 3)
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 Basic Protocol 1)
  • Anhydrous pyridine, stored over coarse granules of calcium hydride
  • Acetic anhydride
  • Ethyl acetate
  • 1 M NaHCO3
  • 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 Support Protocol 3)
  • 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 Support Protocol 2)
  • 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 Support Protocol 3)
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 Support Protocol 1)
  • 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 Support Protocol 3)
  • 1% to 5% (v/v) methanol in chloroform
  • 80% (v/v) aqueous acetonitrile
  • Acetonitrile
  • Trimethylchlorosilane
  • Benzoyl chloride
  • Concentrated ammonium hydroxide
  • Ethyl acetate
  • 1 M NaHCO3
  • 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 Support Protocol 3)
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 NaHCO3
  • 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 Support Protocol 1)
  • 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 Support Protocol 3)
  • 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 Support Protocol 3)

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 NaHCO3
  • 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 Support Protocol 3)
  • 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 Support Protocol 3)
Alternate Protocol 2:  Synthesis of 5¢-O-(4,4¢-Dimethoxytrityl)-2¢-O-(tert-butyldimethylsilyl)-N4-benzoyl-cytidine
 Additional Materials (also see Alternate Protocol 1)
  • 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 NaHCO3, 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 Support Protocol 3)
  • 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 Support Protocol 3)
Alternate Protocol 4:  Synthesis of 5¢-O-(4,4¢-Dimethoxytrityl)-2¢-O-(tert-butyldimethylsilyl)-N2-phenoxy-acetylguanosine
 Additional Materials (also see Alternate Protocol 3)
  • 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 Support Protocol 3)
  • 1%, 5%, 10%, and 15% (v/v) diethyl ether in hexane
  • Anhydrous methylene chloride
  • Ethanol (for dry ice/ethanol bath)
  • 1 M SO2Cl2 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 Support Protocol 3)

CAUTION: The sulfuryl chloride (SO2Cl2) 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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Figures

  • Figure 2.5.1
    The four 5¢-O-(4,4¢-dimethoxytrityl)-2¢-O-(2-nitrobenzyloxymethyl) ribonucleosides. The general structure of these ribonucleosides is in the upper-left corner and the four bases (B) are shown below. DMTr is the 4,4¢-dimethoxytrityl group.

    View Image
  • Figure 2.5.2
    Preparation of 5¢-O-(4,4¢-dimethoxytrityl)-2¢-O-(2-nitrobenzyloxymethyl)uridine from uridine. Bu is n-butyl.

    View Image
  • Figure 2.5.3
    Preparation of 5¢-O-(4,4¢-dimethoxytrityl)-2¢-O-(2-nitrobenzyloxymethyl)-N4-benzoyl-cytidine from 5¢-O-(4,4¢-dimethoxytrityl)-2¢-O-(2-nitrobenzyloxymethyl)uridine.

    View Image
  • Figure 2.5.4
    Preparation of 5¢-O-(4,4¢-dimethoxytrityl)-2¢-O-(2-nitrobenzyloxymethyl)-N6-benzoyl-adenosine from adenosine. Bu is n-butyl.

    View Image
  • Figure 2.5.5
    Preparation of 5¢-O-(4,4¢-dimethoxytrityl)-2¢-O-(2-nitrobenzyloxymethyl)-N2-isobutyrylguanosine from guanosine. Bu is n-butyl.

    View Image
  • Figure 2.5.6
    The four 5¢-O-(4,4¢-dimethoxytrityl)-2¢-O-(tert-butyldimethylsilyl) ribonucleosides. The general structure of these ribonucleosides is in the upper-left corner and the four bases (B) are shown below. DMTr is the 4,4¢-dimethoxytrityl group (Fig. 2.5.1), and TBDMS is the tert-butyldimethylsilyl group.

    View Image
  • Figure 2.5.7
    Preparation of 5¢-O-(4,4¢-dimethoxytrityl)-2¢-O-(tert-butyldimethylsilyl)uridine from uridine.

    View Image
  • Figure 2.5.8
    Preparation of 5¢-O-(4,4¢-dimethoxytrityl)-2¢-O-(tert-butyldimethylsilyl)-N4-benzoylcytidine from cytidine.

    View Image
  • Figure 2.5.9
    Preparation of 5¢-O-(4,4¢-dimethoxytrityl)-2¢-O-(tert-butyldimethylsilyl)-N6-phenoxyacetyladenosine from adenosine.

    View Image
  • Figure 2.5.10
    Preparation of 5¢-O-(4,4¢-dimethoxytrityl)-2¢-O-(tert-butyldimethylsilyl)-N2-phenoxyacetylguanosine from guanosine.

    View Image
  • Figure 2.5.11
    Preparation of 2-nitrobenzyl chloromethyl ether from 2-nitrobenzyl alcohol.

    View Image

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 -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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