Synthesis of Altritol Nucleoside Phosphoramidites for Oligonucleotide Synthesis

Mikhail Abramov1, Piet Herdewijn1

1 Rega Institute for Medical Research, Leuven, Belgium
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.18
DOI:  10.1002/0471142700.nc0118s30
Online Posting Date:  September, 2007
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Abstract

This unit describes in detail the optimized preparations of altritol nucleoside phosphoramidite building blocks for oligonucleotide synthesis (aA, aG, aC, aU). D‐Altritol nucleosides with adenine and uracil bases are obtained by nucleophilic opening of the epoxide ring of 1,5:2,3‐dianhydro‐4,6‐O‐benzylidene‐D‐allitol using the 1,8‐diazabicyclo[5.4.0]undec‐7‐ene salts of the above‐mentioned salts, while phase transfer catalysis (18‐crown‐6, K2CO3) is optimal for alkylation of 2‐amino‐6‐chloropurine. The cytosine nucleoside is synthesized starting from the uracil congener. The 3′‐hydroxyl function of hexitol sugar is protected with the benzoyl group. Curr. Protoc. Nucleic Acid Chem. 30:1.18.1‐1.18.21. © 2007 by John Wiley & Sons, Inc.

Keywords: altritol nucleic acid (ANA); altritol nucleoside phosphoramidites; aA; aG; aC; aU

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of 1′,5′‐Anhydro‐3′‐O‐benzoyl‐6′‐O‐monomethoxytrityl‐2′‐deoxy‐2′‐(uracil‐1‐yl)‐D‐altro‐hexitol
  • Basic Protocol 2: Synthesis of 1′,5′‐Anhydro‐3′‐O‐benzoyl‐6′‐O‐monomethoxytrityl‐2′‐deoxy‐2′‐(N4‐benzoylcytosin‐1‐yl)‐d‐altro‐hexitol
  • Basic Protocol 3: Synthesis of 1′,5′‐Anhydro‐3′‐O‐benzoyl‐6′‐O‐monomethoxytrityl‐2′‐deoxy‐2′‐(N6‐benzoyladenin‐9‐yl)‐D‐altro‐hexitol
  • Basic Protocol 4: Synthesis of 1′,5′‐Anhydro‐3′‐O‐benzoyl‐6′‐O‐monomethoxytrityl‐2′‐deoxy‐2′‐(N2‐(dimethylamino)methylene‐guanin‐9‐yl)‐D‐altro‐hexitol
  • Basic Protocol 5: General Procedure for Phosphoramidite Synthesis
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of 1′,5′‐Anhydro‐3′‐O‐benzoyl‐6′‐O‐monomethoxytrityl‐2′‐deoxy‐2′‐(uracil‐1‐yl)‐D‐altro‐hexitol

  Materials
  • Uracil, 99+%
  • 1,5:2,3‐Dianhydro‐4,6‐O‐benzylidene‐D‐allitol (CMS Chemical, http://www.cms‐chemicals.com, or Brockway et al., )
  • N,N‐Dimethylformamide (DMF), anhydrous
  • Nitrogen gas
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU)
  • Dry ice (solid CO 2 for cooling bath)
  • Acetic acid (AcOH)
  • Brine (saturated aqueous sodium chloride solution)
  • Ethyl acetate (EtOAc)
  • Sodium sulfate (Na 2SO 4)Silica gel (0.060‐ to 0.200‐nm)
  • Dichloromethane PA (DCM)
  • Methanol (MeOH)
  • Anhydrous pyridine
  • Benzoyl chloride (BzCl)
  • Toluene
  • Saturated aqueous sodium bicarbonate solution (sat. aq. NaHCO 3)
  • Chloroform
  • Hexane PA
  • 1,2‐Dichloroethane, 99.8+% (DCE)
  • Trifluoroacetic acid (TFA)
  • Monomethoxytrityl chloride (MMTr‐Cl)
  • 100‐, 250‐, and 500‐mL round‐bottom flasks
  • Gas balloon
  • Dropping funnel
  • Rotary evaporator equipped with a vacuum pump
  • 5 × 35–cm and 5 × 70–cm chromatography columns
  • 500‐ and 1000‐mL separatory funnels
  • Glass filters (porosity 3)
  • Vacuum desiccator
  • Additional reagents and equipment for column chromatography ( appendix 3E)
NOTE: All reactions are carried out with anhydrous solvents. All starting materials are commercially available. All evaporations are accomplished in vacuo using a rotary evaporator at 40°C. All filtrations are performed using a glass filter under vacuum.

Basic Protocol 2: Synthesis of 1′,5′‐Anhydro‐3′‐O‐benzoyl‐6′‐O‐monomethoxytrityl‐2′‐deoxy‐2′‐(N4‐benzoylcytosin‐1‐yl)‐d‐altro‐hexitol

  Materials
  • 1,2,4‐1H‐Triazole, 99.5%
  • Phosphorus oxychloride (POCl 3)
  • Anhydrous pyridine
  • Dry ice (solid CO 2 for cooling bath)
  • Triethylamine
  • 1′,5′‐Anhydro‐3′‐O‐benzoyl‐4′,6′‐O‐benzylidene‐2′‐deoxy‐2′‐(uracil‐1‐yl)‐D‐altro‐hexitol (S.2; see protocol 1)
  • Toluene PA
  • Dichloromethane PA (DCM)
  • Brine (saturated aqueous sodium chloride solution)
  • Sodium sulfate (Na 2SO 4)Dioxane
  • Concentrated aqueous ammonia (25% NH 3)
  • Methanol (MeOH)
  • Celite
  • Silica gel (0.060‐ to 0.200‐nm)
  • Benzoyl chloride (BzCl)1,2‐Dichloroethane, 99.8+% (DCE)
  • Trifluoroacetic acid (TFA)
  • Monomethoxytrityl Chloride (MMTr‐Cl)
  • Hexane PA
  • 100‐, 250‐, and 500‐mL round‐bottom flasks
  • Dropping funnel
  • 5 × 35–cm and 5 × 70–cm chromatography columns
  • Gas balloon
  • Rotary evaporator equipped with a vacuum pump
  • Glass filters (porosity 3)
  • Vacuum desiccator
  • Additional reagents and equipment for column chromatography ( appendix 3E)
NOTE: All reactions are carried out with anhydrous solvents. All starting reagents are commercially available. All evaporations are accomplished in vacuo using a rotary evaporator at 40°C. All filtrations are performed using a glass filter under vacuum.

Basic Protocol 3: Synthesis of 1′,5′‐Anhydro‐3′‐O‐benzoyl‐6′‐O‐monomethoxytrityl‐2′‐deoxy‐2′‐(N6‐benzoyladenin‐9‐yl)‐D‐altro‐hexitol

  Materials
  • Adenine, 99+%
  • 1,5:2,3‐Dianhydro‐4,6‐O‐benzylidene‐D‐allitol (CMS Chemical, http://www.cms‐chemicals.com, or Brockway et al., )
  • N,N‐Dimethylformamide (DMF), dry
  • Nitrogen gas
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU)Dry ice (solid CO 2 for cooling bath)
  • Acetic acid (AcOH)
  • Anhydrous pyridine
  • Benzoyl chloride (BzCl)
  • Concentrated aqueous ammonia (25% NH 3)
  • Toluene PA
  • Dichloromethane PA (DCM)
  • Silica gel (0.060‐ to 0.200‐nm)
  • Hexane PA
  • Ethyl acetate (EtOAc)
  • 1,2‐Dichloroethane 99.8+% (DCE), dry
  • Trifluoroacetic acid (TFA)
  • Monomethoxytrityl chloride (MMTr‐Cl)
  • Methanol (MeOH)
  • Sodium sulfate (Na 2SO 4)
  • 100‐, 250‐, and 500‐mL round‐bottom flasks
  • Gas balloon
  • Dropping funnel
  • Ice bath
  • 500‐mL and 1‐L separatory funnels
  • 5 × 35–cm and 5 × 70–cm chromatography columns
  • Glass filters (porosity 3)
  • Oven at 70° to 80°C
  • Rotary evaporator equipped with a vacuum pump
  • Vacuum desiccator
  • Additional reagents and equipment for column chromatography ( appendix 3E)
NOTE: All reactions are carried out with anhydrous solvents. All starting reagents are commercially available. All evaporations are accomplished in vacuo using a rotary evaporator at 40°C. All filtrations are performed using a glass filter under vacuum.

Basic Protocol 4: Synthesis of 1′,5′‐Anhydro‐3′‐O‐benzoyl‐6′‐O‐monomethoxytrityl‐2′‐deoxy‐2′‐(N2‐(dimethylamino)methylene‐guanin‐9‐yl)‐D‐altro‐hexitol

  Materials
  • 2‐Amino‐6‐chloropurine, 99+%
  • 1,5:2,3‐Dianhydro‐4,6‐O‐benzylidene‐D‐allitol (CMS Chemical, http://www.cms‐chemicals.com, or Brockway et al., )
  • Hexamethylenephosphorotriamide (HMPA), anhydrous
  • Nitrogen gas
  • Aqueous potassium carbonate (K 2CO 3), anhydrous
  • 18‐Crown‐6
  • Dichloromethane PA (DCM)
  • Sodium sulfate (Na 2SO 4)
  • Silica gel (0.060‐ to 0.200‐nm)
  • Methanol (MeOH)
  • 1,8‐Diazabicyclo[2.2.2]octane (DABCO)
  • 1 N NaOH
  • Dry ice (solid CO 2 for cooling bath)
  • 1 N HCl
  • N,N‐Dimethylformamide diethyl acetal
  • N,N‐Dimethylformamide (DMF), anhydrous
  • p‐Xylene
  • Silica gel (0.060 to 0.200 nm)
  • Chloroform (CHCl 3)
  • Tri‐n‐butylamine
  • Benzoyl cyanide
  • 1,2‐Dichloroethane, 99.8+% (DCE)
  • Trifluoroacetic acid (TFA)
  • Pyridine (Py), anhydrous
  • Monomethoxytrityl chloride (MMTr‐Cl)
  • Hexane PA
  • 100‐, 250‐, and 500‐mL round‐bottom flasks
  • Gas balloon
  • 5 × 35–cm and 5 × 70–cm chromatography columns
  • Glass filters (porosity 3)
  • Vacuum desiccator
  • Rotary evaporator equipped with a vacuum pump
  • 500‐mL and 1‐L separatory funnels
  • Additional reagents and equipment for column chromatography ( appendix 3E)
NOTE: All reactions are carried out with anhydrous solvents. All starting reagents are commercially available. All evaporations are accomplished in vacuo using a rotary evaporator at 40°C. All filtrations are performed using a glass filter under vacuum.

Basic Protocol 5: General Procedure for Phosphoramidite Synthesis

  Materials
  • Altritol nucleoside S.4 ( protocol 1), S.8 ( protocol 2), S.12 ( protocol 3), or S.18 ( protocol 3)
  • Dioxane, dry
  • 2,4,6‐Collidine
  • N‐Methylimidazole
  • N,N‐Diisopropyl(cyanoethyl)phosphonamidic chloride (CEPA)
  • Dichloromethane PA (DCM)
  • Acetone PA
  • Triethylamine PA (TEA)
  • 5% Aqueous sodium bicarbonate solution (NaHCO 3)
  • Sodium sulfate (Na 2SO 4)
  • Silica gel (0.035‐ to 0.060‐mm)
  • Hexane PA
  • Glass syringe
  • Gas balloon
  • Glass filter (porosity 3)
  • Rotary evaporator equipped with a vacuum pump
  • Vacuum desiccator
  • 2 × 35–cm chromatography column
  • 100‐mL round bottom flasks
  • 250‐mL separatory funnel
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)
NOTE: The reactions are carried out with dry dioxane under nitrogen in 1 to 2 mmol scale. All starting reagents are commercially available. All evaporations are accomplished in vacuo using a rotary evaporator at 25°C. All filtrations are performed using a glass filter under vacuum.
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Figures

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Literature Cited

Literature Cited
   Abramov, M., Marchand, A., and Herdewijn, P. 2004. Synthesis of D‐altritol nucleosides with a 3′‐O‐tert‐butyldimethylsilyl protecting group. Nucleosides, Nucleotides and Nucleic Acids 23:439‐455.
   Abramov, M., Schepers, G., Van Aerschot, A., and Herdewijn, P. 2007a. Fmoc‐protected altritol phosphoramidite building blocks and their application in the synthesis of altritol nucleic acids (ANAs). Eur. J. Org. Chem. 1446‐1456.
   Abramov, M., Schepers, G., Van Aerschot, A., and Herdewijn, P. 2007b. HNA and ANA high‐affinity arrays for detections of DNA and RNA single‐base mismatches. Biosensors and Bioelectronics, in press.
   Allart, B., Busson, R., Rozenski, J., Van Aerschot, A., and Herdewijn, P. 1999a. Synthesis of protected D‐altritol nucleosides as building blocks for oligonucleotide synthesis. Tetrahedron 55:6527‐6546.
   Allart, B., Khan, K., Rosemeyer, H., Schepers, G., Hendrix, C., Rothenbacher, K., Seela, F., Van Aerschot, A., and Herdewijn, P. 1999b. D‐Altritol nucleic acids (ANA): Hybridization properties, stability, and initial structural analysis. Chem. Eur. J. 5:2424‐2431.
   Brockway, C., Kocienski, P., and Pant, C. 1984. Unusual stereochemistry in the copper‐catalysed ring opening of a carbohydrate oxirane with vinylmagnesium bromide. J. Chem. Soc. Perkin Trans. I 875‐878.
   De Bouvere, B., Kerremans, L., Rozenski, J., Janssen, G., Van Aerschot, A., Claes, P., Busson, R., and Herdewijn, P. 1997. Improved synthesis of anhydrohexitol building blocks for oligonucleotide synthesis. Liebigs Ann. 1453‐1461.
   Fisher, M., Abramov, M., Van Aerschot, A., Xu, D., Juliano, R.L., and Herdewijn, P. 2007. Inhibition of MDR1 expression with altritol‐modified siRNAs. Nucl. Acids Res. 35:1064‐1074.
   Kozlov, I.A., Zielinski, M., Allart, B., Kerremans, L., Van Aerschot, A., Busson, R., Herdewijn, P., and Orgel, L.E. 2000. Nonenzymatic template‐directed reactions on altritol oligomers, preorganized analogues of oligonucleotides. Chem. Eur. J. 6:151‐155.
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