Deoxyribo‐ and Ribonucleoside H‐Phosphonates

Jacek Stawinski1, Roger Strömberg2

1 Stockholm University, Stockholm, Sweden, 2 Karolinska Institutet, Stockholm, Sweden
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 2.6
DOI:  10.1002/0471142700.nc0206s04
Online Posting Date:  May, 2001
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Abstract

Most methods for preparing H‐phosphonate monoesters suffer from variable yields and are often incompatible with common protecting groups used in oligonucleotide synthesis. This unit describes four procedures that consistently give high yields of the desired products. Taken together, they provide an arsenal of phosphonylation prodecures that it compatible with most common protecting groups.

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis of Protected Deoxyribonucleoside or Ribonucleoside 3′‐H‐Phosphonates Using the Phosphorus Trichloride/Imidazole/Triethylamine Reagent
  • Alternate Protocol 1: Synthesis of Ribonucleoside 3′‐H‐Phosphonates Via in Situ 2′‐O‐2‐Chlorobenzoylation Followed by Phosphonylation
  • Basic Protocol 2: Synthesis of Protected Deoxyribonucleoside 3′‐H‐Phosphonates Using H‐Pyrophosphonate
  • Basic Protocol 3: Synthesis of Protected Nucleoside 3′‐H‐Phosphonates Using Diphenyl H‐Phosphonate
  • Basic Protocol 4: Synthesis of Protected Nucleoside 3′‐H‐Phosphonates Using 2‐Chloro‐4H‐1,3,2‐Benzo‐Dioxaphosphinan‐4‐One
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of Protected Deoxyribonucleoside or Ribonucleoside 3′‐H‐Phosphonates Using the Phosphorus Trichloride/Imidazole/Triethylamine Reagent

  Materials
  • Imidazole, made anhydrous by repeated evaporation of added dry acetonitrile
  • Dichloromethane (99.9+%; HPLC grade), stored over 3A molecular sieves
  • Acetone (for dry ice/acetone bath)
  • Phosphorus trichloride, freshly distilled
  • Triethylamine, distilled, stored over calcium hydride
  • Protected deoxyribo‐ or ribonucleoside (see Chapter 2), dried by repeated evaporation of added pyridine
  • 9:1 (v/v) chloroform/methanol
  • 2 M TEAB, pH 7.5 (see recipe)
  • Sodium sulfate
  • 5 × 25–cm glass chromatography column, packed with Merck silica gel 60, 230 to 400 mesh ASTM (e.g., EM Science) or matrix silica, 35 to 70 µm (Millipore) in chloroform (bed height, 15 to 20 cm)
  • Methanol
  • Chloroform
  • 0.5:1:8.5 or 1:2:7 (v/v/v) concentrated ammonia/water/isopropanol
  • 1:1 (v/v) hexane/diethyl ether
  • TLC silica‐gel plates (e.g., Merck silica gel 60 F254, EM Science)
  • Rotary evaporator connected to water aspirator
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D) and column chromatography ( appendix 3E)

Alternate Protocol 1: Synthesis of Ribonucleoside 3′‐H‐Phosphonates Via in Situ 2′‐O‐2‐Chlorobenzoylation Followed by Phosphonylation

  • Protected ribonucleoside with free 2′‐ and 3′‐hydroxyls (see Chapter 2), dried by repeated evaporation of added pyridine
  • Pyridine (HPLC grade; e.g., LabScan), dried by and stored over 4A molecular sieves
  • 2‐Chlorobenzoyl chloride

Basic Protocol 2: Synthesis of Protected Deoxyribonucleoside 3′‐H‐Phosphonates Using H‐Pyrophosphonate

  Materials
  • Protected deoxyribonucleoside (see Chapter 2), dried by repeated evaporation of added pyridine
  • 2 M phosphorous acid (H 3PO 3) solution (see recipe)
  • Condensing agent (select one):
  •  5,5‐Dimethyl‐2‐oxo‐2‐chloro‐1,3,2‐dioxaphosphinane (NEPCl; see recipe)
  •  Pivaloyl chloride (PV⋅Cl), commercial grade (e.g., Aldrich), distilled before use (store <2 months at −20°C)
  • 9:1 (v/v) chloroform/methanol
  • 1 M and 2 M TEAB, pH 7.5 (see recipe)
  • Dichloromethane (HPLC grade), stored over 3A molecular sieves
  • Sodium sulfate
  • 4× 25–cm glass chromatography column, packed with Merck silica gel 60, 230 to 400 mesh ASTM (e.g., EM Science) or Matrix silica, 35 to 70 µm (Millipore) in chloroform (bed height, 10 to 15 cm)
  • Methanol
  • Chloroform
  • 0.5:1:8.5 or 1:2:7 (v/v/v) concentrated ammonia/water/isopropanol
  • 1:1 (v/v) hexane/diethyl ether
  • TLC silica‐gel plates (e.g., Merck silica gel 60 F254, EM Science)
  • Rotary evaporator connected to water aspirator
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 3: Synthesis of Protected Nucleoside 3′‐H‐Phosphonates Using Diphenyl H‐Phosphonate

  Materials
  • Protected deoxyribo‐ or ribonucleoside (see Chapter 2), dried by repeated evaporation of added pyridine
  • Pyridine (HPLC grade; e.g., Labscan), dried by and stored over 4A molecular sieves
  • Diphenyl H‐phosphonate, commercial grade (e.g., Aldrich)
  • 9:1 (v/v) chloroform/methanol
  • Triethylamine, distilled, stored over calcium hydride
  • 5% (w/v) sodium bicarbonate
  • Dichloromethane, distilled
  • Sodium sulfate
  • 4 ×25–cm glass chromatography column, packed with Merck silica gel 60, 230 to 400 mesh ASTM (e.g., EM Science) or Matrix chloroform (bed height, 10 to 15 cm)
  • Methanol
  • Chloroform
  • 0.5:1:8.5 or 1:2:7 (v/v/v) concentrated ammonia/water/isopropanol
  • 1:1 (v/v) hexane/diethyl ether
  • TLC silica‐gel plates (e.g., Merck silica gel 60 F254, EM Science)
  • Rotary evaporator connected to water aspirator
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 4: Synthesis of Protected Nucleoside 3′‐H‐Phosphonates Using 2‐Chloro‐4H‐1,3,2‐Benzo‐Dioxaphosphinan‐4‐One

  Materials
  • Protected deoxyribo‐ or ribonucleoside (see Chapter 2), dried by repeated evaporation of added pyridine
  • 2:1 (v/v) dichloromethane/pyridine
  • 1.25 M 2‐chloro‐4H‐1,3,2‐benzo‐dioxaphosphinan‐4‐one solution (see recipe)
  • 9:1 (v/v) chloroform/methanol
  • 1 M TEAB, pH 7.5 (see recipe)
  • Dichloromethane (99%; HPLC grade), stored over 3A molecular sieves
  • Sodium sulfate
  • 5 × 25–cm glass chromatography column, packed with Merck silica gel 60, 230 to 400 mesh ASTM (e.g., EM Science) or Matrix silica, 35 to 70 µm (Millipore) in chloroform (bed height, 10 to 15 cm)
  • Methanol
  • Chloroform
  • 0.5:1:8.5 or 1:2:7 (v/v/v) concentrated ammonia/water/isopropanol
  • 1:1 (v/v) hexane/diethyl ether
  • TLC silica‐gel plates (e.g., Merck silica gel 60 F254, EM Science)
  • Rotary evaporator connected to water aspirator
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)
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Figures

Videos

Literature Cited

Literature Cited
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   Gaffney, B.L. and Jones, R.A. 1988. Large‐scale oligonucleotide synthesis by the H‐phosphonate method. Tetrahedron Lett. 29:2619‐2622.
   Garegg, P.J., Regberg, T., Stawinski, J., and Strömberg, R. 1985. Formation of internucleotidic bond via phosphonate intermediates. Chem. Scr. 25:280‐282.
   Garegg, P.J., Lindh, I., Regberg, T., Stawinski, J., Strömberg, R., and Henrichson, C. 1986a. Nucleoside H‐Phosphonates. III. Chemical synthesis of oligodeoxyribonucleotides by the hydrogenphosphonate approach. Tetrahedron Lett. 27:4051‐4054.
   Garegg, P.J., Lindh, I., Regberg, T., Stawinski, J., Strömberg, R., and Henrichson, C. 1986b. Nucleoside H‐phosphonates. IV. Automated solid Phase synthesis of oligoribonucleotides by the hydrogenphosphonate approach. Tetrahedron Lett. 27:4055‐4058.
   Garegg, P.J., Regberg, T., Stawinski, J., and Strömberg, R. 1986c. Nucleoside hydrogenphosphonates in oligonucleotide synthesis. Chem. Scr. 26:59‐62.
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   Jankowska, J., Sobkowski, M., Stawinski, J., and Kraszewski, A. 1994. Studies on aryl H‐phosphonates. I. Efficient method for the preparation of deoxyribo‐ and ribonucleoside 3′‐H‐phosphonate monoesters by transesterification of diphenyl H‐phosphonate. Tetrahedron Lett. 35:3355‐3358.
   Kers, A., Kers, I., Stawinski, J., Sobkowski, M., and Kraszewski, A. 1996. Studies on aryl H‐phosphonates. 3. Mechanistic investigations related to the disproportionation of diphenyl H‐phosphonate under anhydrous basic conditions. Tetrahedron 52:9931‐9944.
   Marugg, J.E., Tromp, M., Kuyl‐ Yeheskiely, E., van der Marel, G.A., and van Boom, J.H. 1986. A convenient and general approach to the synthesis of properly protected d‐nucleoside‐3′ hydrogenphosphonates via phosphite intermediates. Tetrahedron Lett. 27:2661‐2664.
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   Rozners, E., Strömberg, R., and Bizdena, E. 1995a. Synthesis of oligoarabinonucleotides using H‐phosphonates. Nucleosides Nucleotides 14:851‐853.
   Rozners, E., Strömberg, R., and Bizdena, E. 1995b. Synthesis of RNA fragments using the H‐phosphonate method and 2′‐(2‐chlorobenzoyl) protection. Nucleosides Nucleotides 14:855‐857.
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   Sekine, M., Narui, S., and Hata, T. 1988. A convenient method for the synthesis of deoxyribonucleoside 3′‐hydrogenphosphonates. Tetrahedron Lett. 29:1037‐1040.
   Stawinski, J. and Thelin, M. 1990a. Nucleoside H‐Phosphonates. XI. A convenient method for the preparation of nucleoside H‐phosphonates. Nucleosides Nucleotides 9:129‐135.
   Stawinski, J. and Thelin, M. 1990b. Studies on the activation pathway of phosphonic acid using acyl chlorides as activators. J. Chem. Soc. Perkin Trans. 2. (1990):849‐853.
   Stawinski, J., Strömberg, R., Thelin, M., and Westman, E. 1988. Studies on the t‐butyldimethylsilyl group as 2′‐O‐protection in oligoribonucleotide synthesis via the H‐phosphonate approach. Nucl. Acids Res. 16:9285‐9298.
   Szabò, T., Almer, H., Strömberg, R., and Stawinski, J. 1995. 2‐Cyanoethyl H‐phosphonate. A reagent for the mild preparation of nucleoside H‐phosphonate monoesters. Nuceosides Nucleotides 4:715‐716.
   Takaku, H., Yamakage, S., Sakatsume, O., and Ohtsuki, M. 1988. A convenient approach to the synthesis of deoxyribonucleoside 3′‐hydrogenphosphonates via bis(1,1,1,3,3,3‐hexafluoro‐2‐propyl) phosphonate intermediate. Chem. Lett. (1988):1675‐1678.
   Wada, T., Sato, Y., Honda, F., Kawahara, S., and Sekine, M. 1997. Chemical synthesis of oligodeoxyribonucleotides using N‐unprotected H‐phosphonate monomers and carbonium and phosphonium condensing reagents: O‐Selective phosphonylation and condensation. J. Am. Chem. Soc. 119:12710‐12721.
   Young, R.W. 1952. A re‐examination of the reaction between phosphorus trichloride and salicylic acid. J. Am. Chem. Soc. 74:1672‐1673.
   Zhang, X., Abad, J.‐L., Huang, Q., Zeng, F., Gaffney, B., and Jones, R. 1997. High yield protection of purine ribonucleosides for H‐phosphonate RNA synthesis. Tetrahedron. Lett. 38:7135‐7138.
Key References
   Froehler et al., 1986. See above.
  Provide primary sources with complete experimental details for .
   Garegg et al., 1986c. See above.
  Provides primary sources with complete experimental details for .
   Rozners et al., 1995b. See above.
  Provides primary sources with complete experimental details for .
   Stawinski et al., 1988. See above.
  Provides primary sources with complete experimental details for .
   Stawinski and Thelin, 1990a. See above.
  Provide general aspects of the underlying chemistry in the context of nucleotide and oligonucleotide synthesis.
   Jankowska et al., 1994. See above.
   Marugg et al., 1986. See above.
   Stawinski, J. 1992. Some Aspects of H‐Phosphonate Chemistry. In Handbook of Organophosphorus Chemistry (R. Engel ed.) pp.377‐434. Marcel Dekker, New York.
   Stawinski, J. and Strömberg, R. 1993. H‐Phosphonates in Oligonucleotide Synthesis. Trends Org. Chem. 4:31‐67.
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