Synthesis of Threose Nucleic Acid (TNA) Phosphoramidite Monomers and Oligonucleotide Polymers

Su Zhang1, John C. Chaput1

1 Center for Evolutionary Medicine and Informatics, The Biodesign Institute and Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.51
DOI:  10.1002/0471142700.nc0451s50
Online Posting Date:  September, 2012
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Abstract

This unit describes the preparation of dimethoxytrityl (DMTr)−protected α‐L‐threofuranosyl nucleic acid (TNA) phosphoramidite monomers for A, C, G, T, and diaminopurine, as well as their incorporation into TNA oligonucleotides by solid‐phase synthesis. Starting from commercially available L‐ascorbic acid, the protected threofuranosyl sugar is obtained in four steps. Vorbrüggen‐Hilbert‐Johnson glycosylation affords the desired threofuranosyl nucleosides, which are converted to their corresponding DMTr‐protected phosphoramidite nucleosides in four additional steps. Phosphoramidite monomers are then used to construct TNA oligonucleotides by solid‐phase synthesis using a standard DNA synthesizer. Curr. Protoc. Nucleic Acid Chem. 50:4.51.1‐4.51.26. © 2012 by John Wiley & Sons, Inc.

Keywords: alternative nucleic acids (ANA); threose nucleic acid (TNA); phosphoramidite; oligonucleotide; chemical synthesis; solid‐phase synthesis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Protected L‐Threofuranose, 1‐O‐Acetyl‐2,3‐Di‐O‐Benzoyl‐L‐Threofuranose, and 1,2,3‐Tri‐O‐Benzoyl‐L‐Threofuranose
  • Basic Protocol 2: Synthesis of the Protected TNA Cytidine Nucleoside Phosphoramidite
  • Basic Protocol 3: Synthesis of the Protected TNA Diaminopurine Nucleoside Phosphoramidite
  • Basic Protocol 4: Synthesis of the Protected TNA Guanosine Nucleoside Phosphoramidite
  • Basic Protocol 5: Synthesis of the TNA Thymidine Nucleoside Phosphoramidite
  • Basic Protocol 6: Synthesis of the Protected TNA Adenosine Nucleoside Phosphoramidite
  • Basic Protocol 7: Synthesis, Isolation, and Characterization of Threose Nucleic Acid (TNA) Oligonucleotides
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of Protected L‐Threofuranose, 1‐O‐Acetyl‐2,3‐Di‐O‐Benzoyl‐L‐Threofuranose, and 1,2,3‐Tri‐O‐Benzoyl‐L‐Threofuranose

  Materials
  • L‐Ascorbic acid ( S.1)
  • Ultra pure water (H 2O)
  • Calcium carbonate (CaCO 3)
  • Hydrogen peroxide (H 2O 2)
  • Activated charcoal
  • Methanol (MeOH)
  • Dowex 50WX4‐50 resin, acidic form
  • Acetonitrile (MeCN), anhydrous
  • p‐Toluenesulfonic acid (TsOH)
  • Argon gas
  • Pyridine, anhydrous
  • Benzoyl chloride (BzCl)
  • Dichloromethane (DCM)
  • 1 M aq. HCl
  • Saturated aqueous sodium bicarbonate solution (sat. aq. NaHCO 3)
  • Brine (sat. aq. NaCl)
  • Magnesium sulfate (MgSO 4)
  • Hexanes
  • Ethyl acetate (EtOAc)
  • Tetrahydrofuran (THF), anhydrous
  • 1.5 M diisobutylaluminium hydride (DIBAL‐H) in toluene
  • Saturated aqueous sodium potassium tartrate
  • Acetic anhydride (Ac 2O)
  • 4‐Dimethylaminopyridine (DMAP)
  • Silica gel (60 Å, 230‐400 mesh, Whatman)
  • Ethanol (EtOH)
  • 250‐, 500‐, 1000‐, and 2000‐mL round‐bottom flasks
  • Dropping funnels
  • Quantofix peroxide test sticks (Sigma‐Aldrich)
  • Filter paper
  • Büchner funnel
  • 250‐, 500‐, 1000‐, and 2000‐mL separatory funnels
  • Rotary evaporator equipped with a vacuum pump
  • Gas balloon
  • Allihn condenser (reflux condenser)
  • Thin‐layer chromatography (TLC) plate, EMD silica gel 60 F 254
  • UV lamp, 254 nm
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 2: Synthesis of the Protected TNA Cytidine Nucleoside Phosphoramidite

  Materials
  • 1‐O‐Acetyl‐2,3‐di‐O‐benzoyl‐L‐threofuranose ( S.5; see protocol 1)
  • Acetonitrile (MeCN), anhydrous
  • N,O‐Bis(trimethylsilyl)acetamide (BSA)
  • N4‐Benzoylcytosine (CBz)
  • Trimethylsilyl trifluoromethanesulfonate (TMSOTf)
  • Ethyl acetate (EtOAc)
  • Saturated aqueous sodium bicarbonate solution (sat. aq. NaHCO 3)
  • Brine (sat. aq. NaCl)
  • Magnesium sulfate (MgSO 4)
  • Silica gel (60 Å, 230‐400 mesh, Whatman)
  • Hexanes
  • Dichloromethane (DCM), anhydrous
  • Tetrahydrofuran (THF)
  • Methanol (MeOH)
  • Sodium hydroxide (NaOH)
  • 0.1 M aq. HCl
  • Toluene
  • N,N‐Dimethylformamide (DMF), anhydrous
  • 4,4′‐Dimethoxytriphenylmethyl chloride (DMTr‐Cl)
  • 2,6‐Lutidine
  • Silver trifluoromethanesulfonate (AgOTf)
  • Ultra pure water (H 2O)
  • Sodium sulfate (Na 2SO 4)
  • Triethylamine (Et 3N)
  • N,N‐Diisopropylethylamine (DIPEA)
  • Chloro(2‐cyanoethoxy)(diisopropylamino)phosphine
  • Argon gas
  • Benzene
  • 50‐, 100‐, and 250‐mL round‐bottom flasks
  • Allihn condenser (reflux condenser)
  • Rotary evaporator equipped with a vacuum pump
  • Filter paper
  • Büchner funnel
  • 250‐mL separatory funnel
  • Gas balloon
  • Thin‐layer chromatography (TLC) plate, EMD silica gel 60 F 254
  • UV lamp, 254 nm
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 3: Synthesis of the Protected TNA Diaminopurine Nucleoside Phosphoramidite

  Materials
  • N2,N6‐Dibenzoyl‐2,6‐diaminopurine (D(Bz)2)
  • Acetonitrile (MeCN), anhydrous
  • Argon gas
  • N,O‐Bis(trimethylsilyl)acetamide (BSA)
  • 1‐O‐Acetyl‐2,3‐di‐O‐benzoyl‐L‐threofuranose ( S.5; see protocol 1)
  • Trimethylsilyl trifluoromethanesulfonate (TMSOTf)
  • Ethyl acetate (EtOAc)
  • Saturated aqueous sodium bicarbonate solution (sat. aq. NaHCO 3)
  • Brine (sat. aq. NaCl)
  • Magnesium sulfate (MgSO 4)
  • Silica gel (60 Å, 230‐400 mesh, Whatman)
  • Hexanes
  • Tetrahydrofuran (THF)
  • Methanol (MeOH)
  • Ultra pure water (H 2O)
  • Sodium hydroxide (NaOH)
  • Ammonium chloride (NH 4Cl)
  • Dichloromethane (DCM)
  • N,N‐Dimethylformamide (DMF)
  • 4,4′‐Dimethoxytriphenylmethyl chloride (DMTr‐Cl)
  • 2,6‐Lutidine
  • 4Å molecular sieves (freshly activated by heating at 300°C for at least 3 hr)
  • Silver trifluoromethanesulfonate (AgOTf)
  • 0.1 M aq. HCl
  • Sodium sulfate (Na 2SO 4)
  • Triethylamine (Et 3N)
  • Benzene
  • N,N‐Diisopropylethylamine (DIPEA)
  • Chloro(2‐cyanoethoxy)(diisopropylamino)phosphine
  • 50‐, 100‐, and 500‐mL round‐bottom flasks
  • Gas balloon
  • Rotary evaporator equipped with a vacuum pump
  • Filter paper
  • Büchner funnel
  • 250‐mL separatory funnel
  • Allihn condenser (reflux condenser)
  • Thin layer chromatography (TLC) plate, EMD silica gel 60 F 254
  • UV lamp, 254 nm
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 4: Synthesis of the Protected TNA Guanosine Nucleoside Phosphoramidite

  Materials
  • N2‐Acetyl‐O6‐diphenylcarbamoylguanine (GPAC)
  • 1,2‐Dichloroethane (DCE), anhydrous
  • N,O‐Bis(trimethylsilyl)acetamide (BSA)
  • Argon gas
  • Toluene, anhydrous
  • Trimethylsilyl trifluoromethanesulfonate (TMSOTf)
  • 1‐O‐Acetyl‐2,3‐di‐O‐benzoyl‐L‐threofuranose ( S.5; see protocol 1)
  • Ethyl acetate (EtOAc)
  • Saturated aqueous sodium bicarbonate solution (sat. aq. NaHCO 3)
  • Brine (sat. aq. NaCl)
  • Magnesium sulfate (MgSO 4)
  • Silica gel (60 Å, 230‐400 mesh, Whatman)
  • Dichloromethane (DCM), anhydrous
  • Triethylamine (Et 3N)
  • Methanol (MeOH)
  • Tetrahydrofuran (THF)
  • Ultra pure water (H 2O)
  • Sodium hydroxide (NaOH)
  • Ammonium chloride (NH 4Cl)
  • N,N‐Dimethylformamide (DMF)
  • 2,6‐Lutidine
  • 4,4′‐Dimethoxytriphenylmethyl chloride (DMTr‐Cl)
  • N,N‐Diisopropylethylamine (DIPEA)
  • Chloro(2‐cyanoethoxy)(diisopropylamino)phosphine
  • Pentane
  • 25‐, 100‐, 250‐, and 500‐mL round‐bottom flasks
  • Allihn condenser
  • Gas balloon
  • Rotary evaporator equipped with a vacuum pump
  • Filter paper
  • Büchner funnel
  • Thin layer chromatography (TLC) plate, EMD silica gel 60 F 254
  • UV lamp, 254 nm
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 5: Synthesis of the TNA Thymidine Nucleoside Phosphoramidite

  Materials
  • 1‐O‐Acetyl‐2,3‐di‐O‐benzoyl‐L‐threofuranose ( S.5; see protocol 1)
  • Thymine
  • Acetonitrile, anhydrous (MeCN)
  • N,O‐Bis(trimethylsilyl)acetamide (BSA)
  • Argon gas
  • Trimethylsilyl trifluoromethanesulfonate (TMSOTf)
  • Saturated aqueous sodium bicarbonate solution (sat. aq. NaHCO 3)
  • Ethyl acetate (EtOAc)
  • Brine (sat. aq. NaCl)
  • Magnesium sulfate (MgSO 4)
  • Silica gel (60 Å, 230‐400 mesh, Whatman)
  • Dichloromethane (DCM), anhydrous
  • 2 M NH 3 in MeOH
  • Ultra pure water (H 2O)
  • N,N‐Dimethylformamide (DMF), anhydrous
  • 4,4′‐Dimethoxytriphenylmethyl chloride (DMTr‐Cl)
  • 2,6‐Lutidine
  • Silver trifluoromethanesulfonate (AgOTf)
  • Methanol (MeOH)
  • 0.1 M aq. HCl
  • Sodium sulfate (Na 2SO 4)
  • Triethylamine (Et 3N)
  • N,N‐Diisopropylethylamine (DIPEA)
  • Chloro(2‐cyanoethoxy)(diisopropylamino)phosphine
  • Hexanes
  • 50‐, 100‐, and 500‐mL round‐bottom flasks
  • Allihn condenser (reflux condenser)
  • Gas balloon
  • 250‐mL separatory funnel
  • Rotary evaporator equipped with a vacuum pump
  • Filter paper
  • Büchner funnel
  • Thin layer chromatography (TLC) plate, EMD silica gel 60 F 254
  • UV lamp, 254 nm
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 6: Synthesis of the Protected TNA Adenosine Nucleoside Phosphoramidite

  Materials
  • 1,2,3‐Tri‐O‐benzoyl‐L‐threofuranose ( S.6; see protocol 1)
  • Acetonitrile (MeCN), anhydrous
  • N6‐Benzoyladenine (ABz)
  • N,O‐Bis(trimethylsilyl)acetamide (BSA)
  • Tin (IV) chloride (SnCl 4)
  • Argon gas
  • Ethyl acetate (EtOAc)
  • Saturated aqueous sodium bicarbonate solution (sat. aq. NaHCO 3)
  • Magnesium sulfate (MgSO 4)
  • Silica gel (60 Å, 230‐400 mesh, Whatman)
  • Dichloromethane (DCM), anhydrous
  • Tetrahydrofuran (THF)
  • Methanol (MeOH)
  • Ultra pure water (H 2O)
  • Sodium hydroxide (NaOH)
  • 2 N and 0.1 M aq. HCl
  • Toluene
  • Ethanol (EtOH)
  • N,N‐Dimethylformamide (DMF), anhydrous
  • 4,4′‐Dimethoxytriphenylmethyl chloride (DMTr‐Cl)
  • 2,6‐Lutidine
  • Silver trifluoromethanesulfonate (AgOTf)
  • Brine (sat. aq. NaCl)
  • Sodium sulfate (Na 2SO 4)
  • Triethylamine (Et 3N)
  • N,N‐Diisopropylethylamine (DIPEA)
  • Chloro(2‐cyanoethoxy)(diisopropylamino)phosphine
  • Hexanes
  • 10‐, 100‐, and 250‐mL round‐bottom flasks
  • Allihn condenser
  • Gas balloon
  • 250‐mL separatory funnel
  • Rotary evaporator equipped with a vacuum pump
  • Filter paper
  • Büchner funnel
  • Thin layer chromatography (TLC) plate, EMD silica gel 60 F 254
  • UV lamp, 254 nm
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 7: Synthesis, Isolation, and Characterization of Threose Nucleic Acid (TNA) Oligonucleotides

  Materials
  • TNA phosphoramidite monomers (see Basic Protocols protocol 22 to protocol 66), pre‐dried under high vacuum
  • Acetonitrile (MeCN), anhydrous (Applied Biosystems)
  • Argon gas (ultra high purity)
  • 4Å molecular sieves (freshly activated by heating at 300°C for at least 3 hr)
  • Detritylation solution: 6% dichloroacetic acid in 1,2‐dichloroethane (DCE)
  • Coupling solution: 0.35 M (ethylthio)‐1H‐tetrazole
  • Concentrated ammonium hydroxide (conc. NH 4OH)
  • n‐Butanol
  • Nanopure water
  • 25‐mL round‐bottom flasks
  • 0.45‐µm disposable syringe filters
  • CPG‐column precharged with Universal Support II (Glen Research)
  • 2‐mL screw‐cap microcentrifuge tube
  • 15‐mL screw‐cap conical centrifuge tube (Falcon)
  • Additional reagents and equipments for automated solid‐phase oligonucleotide synthesis ( appendix 3C) and isolation and characterization of synthetic nucleic acids (units 10.1& 10.4)
NOTE: All glassware, syringes, and needles should be pre‐dried in a drying oven and cooled to room temperature in a desiccator prior to use. All TNA phosphoramidites should be dried under high vacuum for 48 hr prior to use.
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Literature Cited

Literature Cited
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   Chaput, J.C., Ichida, J.K., and Szostak, J.W. 2003. DNA polymerase‐mediated DNA synthesis on a TNA template. J. Am. Chem. Soc. 125:856‐857.
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