Synthesis of Glycerol Nucleic Acid (GNA) Phosphoramidite Monomers and Oligonucleotide Polymers

Su Zhang1, John C. Chaput1

1 The Biodesign Institute at Arizona State University, Tempe, Arizona
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.40
DOI:  10.1002/0471142700.nc0440s42
Online Posting Date:  September, 2010
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Abstract

This unit describes a straightforward method for preparing glycerol nucleic acid (GNA) phosphoramidite monomers and oligonucleotide polymers using standard cyanoethyl phosphoramidite chemistry. GNA is an unnatural nucleic acid analog composed of an acyclic three‐carbon sugar‐phosphate backbone that contains one stereogenic center per repeating unit. GNA has attracted significant attention as a nucleic acid derivative due to its unique ability to form stable Watson‐Crick anti‐parallel duplex structures with thermal and thermodynamic stabilities rivaling those of natural DNA and RNA. The chemical simplicity of this nucleic acid structure provides access to enantiomerically pure forms of right‐ and left‐handed helical structures that can be used as unnatural building blocks in DNA nanotechnology. Curr. Protoc. Nucleic Acid Chem. 42:4.40.1‐4.40.18. © 2010 by John Wiley & Sons, Inc.

Keywords: glycerol nucleic acid (GNA); phosphoramidite; oligonucleotide; chemical synthesis; solid‐phase synthesis; thermal stability; nanotechnology

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Enantiomerically Pure Dimethoxytrityl‐O‐(S)‐Glycidol
  • Basic Protocol 2: Synthesis of 2′‐O‐(2‐Cyanoethoxy)(Diisopropylamino)Phosphino‐3′‐O‐(4,4′‐Dimethoxytriphenyl)Methyl‐N6‐Benzoyl‐(S)‐9‐(2,3‐Dihydroxypropyl)Adenine
  • Basic Protocol 3: Synthesis of 2′‐O‐(2‐Cyanoethoxy)(Diisopropylamino)Phosphino‐3′‐O‐(4,4′‐Dimethoxytriphenyl)Methyl‐N4‐Benzoyl‐(S)‐1‐(2,3‐Dihydroxypropyl)Cytosine
  • Basic Protocol 4: Synthesis of 2′‐O‐(2‐Cyanoethoxy)(Diisopropylamino)Phosphino‐3′‐O‐(4,4′‐Dimethoxytriphenyl)Methyl‐N2‐Isobutyryl‐(S)‐9‐(2,3‐Dihydroxypropyl)Guanine
  • Basic Protocol 5: Synthesis of 2′‐O‐(2‐Cyanoethoxy)(Diisopropylamino)Phosphino‐3′‐O‐(4,4′‐Dimethoxytriphenyl)Methyl‐(S)‐1‐(2,3‐Dihydroxypropyl)Thymine
  • Basic Protocol 6: Synthesis, Isolation, and Characterization of Glycerol Nucleic Acid (GNA) Oligonucleotides
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of Enantiomerically Pure Dimethoxytrityl‐O‐(S)‐Glycidol

  Materials
  • (R)‐(+)‐Glycidol
  • Dichloromethane (DCM)
  • Triethylamine (Et 3N), 99.5%
  • 4,4′‐Dimethoxytritylchloride (DMT‐Cl), 95%
  • Argon source
  • Saturated aqueous sodium bicarbonate solution (sat. aq. NaHCO 3)
  • Brine (sat. aq. NaCl)
  • Sodium sulfate (Na 2SO 4)
  • Silica gel (60 Å, 230 to 400 mesh; Whatman)
  • Hexanes
  • Ethyl acetate (AcOEt)
  • 100‐mL round‐bottomed flasks
  • Magnetic stir plate and stir bar
  • Büchner funnels
  • 200‐mL separatory funnels
  • Filter paper
  • Gas balloon
  • Rotary evaporator equipped with a vacuum pump
  • 2.5 × 25–cm chromatography column
  • Thin layer chromatography (TLC) plate, EMD silica gel 60 F 254
  • 254‐nm UV lamp
  • Additional reagents and equipment for thin layer chromatography (TLC; appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 2: Synthesis of 2′‐O‐(2‐Cyanoethoxy)(Diisopropylamino)Phosphino‐3′‐O‐(4,4′‐Dimethoxytriphenyl)Methyl‐N6‐Benzoyl‐(S)‐9‐(2,3‐Dihydroxypropyl)Adenine

  Materials
  • Adenine
  • 60% sodium hydride in mineral oil (NaH)
  • Dimethylformamide (DMF), anhydrous
  • Argon source
  • Benzoyl chloride (BzCl)
  • DMT‐O‐(S)‐glycidol ( S.2; protocol 1)
  • Ethyl acetate (AcOEt)
  • Silica gel (60 Å, 230 to 400 mesh)
  • Dichloromethane (DCM)
  • Triethylamine (Et 3N)
  • Methanol (MeOH)
  • Pyridine, anhydrous
  • Trimethylsilyl chloride (TMS‐Cl)
  • Ammonium hydroxide (concentrated NH 4OH)
  • Hexanes
  • Diisopropylethylamine, redistilled (DIPEA)
  • Chloro(2‐cyanoethoxy)‐(diisopropylamino)phosphine
  • 50‐ and 100‐mL round‐bottomed flasks
  • Magnetic stir plate and stir bar
  • Graham condenser
  • Büchner funnel
  • Filter paper
  • Rotary evaporator equipped with a vacuum pump
  • 6.4 × 45–cm and 1.3 × 30–cm chromatography columns
  • TLC plate, EMD silica gel 60 F 254
  • 254‐nm UV lamp
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 3: Synthesis of 2′‐O‐(2‐Cyanoethoxy)(Diisopropylamino)Phosphino‐3′‐O‐(4,4′‐Dimethoxytriphenyl)Methyl‐N4‐Benzoyl‐(S)‐1‐(2,3‐Dihydroxypropyl)Cytosine

  Materials
  • N4‐benzoyl‐protected cytosine
  • 60% sodium hydride in mineral oil (NaH)
  • Dimethylformamide (DMF), anhydrous
  • Argon source
  • DMT‐O‐(S)‐glycidol ( S.2; protocol 2)
  • Ethyl acetate (AcOEt)
  • Silica gel (60 Å, 230 to 400 mesh)
  • Hexanes
  • Triethylamine (Et 3N)
  • Acetone
  • Diisopropylethylamine (DIPEA)
  • Dichloromethane (DCM)
  • Chloro(2‐cyanoethoxy)‐(diisopropylamino)phosphine
  • 50‐ and 100‐mL round‐bottomed flasks
  • Magnetic stir plate and stir bar
  • Gas balloon
  • Graham condenser
  • Rotary evaporator equipped with a vacuum pump
  • Büchner funnel
  • 200‐mL separatory funnel
  • Filter paper
  • 1.3 × 30–cm and 6.4 × 45–cm chromatography columns
  • 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 2′‐O‐(2‐Cyanoethoxy)(Diisopropylamino)Phosphino‐3′‐O‐(4,4′‐Dimethoxytriphenyl)Methyl‐N2‐Isobutyryl‐(S)‐9‐(2,3‐Dihydroxypropyl)Guanine

  Materials
  • Dimethylformamide (DMF), anhydrous
  • R‐(+)‐glycidol ( S.1)
  • 2‐Amino‐6‐chloropurine
  • Potassium carbonate (K 2CO 3)
  • Methanol (MeOH)
  • Silica gel (60 Å, 230 to 400 mesh)
  • Ethyl acetate (AcOEt)
  • 1 N hydrochloric acid solution (HCl)
  • Ammonium hydroxide (concentrated NH 4OH)
  • Pyridine, anhydrous
  • Argon source
  • Trimethylsilyl chloride (TMS‐Cl)
  • Isobutyryl chloride (i‐PrOCl)
  • DMT‐Cl
  • Hexanes
  • Triethylamine (Et 3N)
  • Diisopropylethylamine (DIPEA)
  • Tetrahydrofuran (THF)
  • Chloro(2‐cyanoethoxy)‐(diisopropylamino)phosphine
  • Pentane
  • 25‐, 50‐, 100‐, and 250‐mL round‐bottomed flasks
  • Graham condenser
  • Magnetic stir plate and stir bar
  • Büchner funnel
  • Filter paper
  • Rotary evaporator equipped with a vacuum pump
  • 1.3 × 30–cm and 6.4 × 45–cm chromatography columns
  • TLC plate, EMD silica gel 60 F 254
  • UV lamp, 254 nm
  • Balloon
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 5: Synthesis of 2′‐O‐(2‐Cyanoethoxy)(Diisopropylamino)Phosphino‐3′‐O‐(4,4′‐Dimethoxytriphenyl)Methyl‐(S)‐1‐(2,3‐Dihydroxypropyl)Thymine

  Materials
  • Thymine
  • 60% sodium hydride in mineral oil (NaH)
  • Dimethylformamide (DMF), anhydrous
  • Argon source
  • DMT‐O‐(S)‐glycidol ( S.2; protocol 1)
  • Ethyl acetate (AcOEt)
  • Silica gel (60 Å, 230‐400 mesh)
  • Diisopropylethylamine (DIPEA)
  • Dichloromethane (DCM)
  • Chloro(2‐cyanoethoxy)‐(diisopropylamino)phosphine
  • Hexanes
  • Triethylamine (Et 3N)
  • Methanol (MeOH)
  • 25‐ and 50‐mL round‐bottomed flasks
  • Magnetic stir plate and stir bar
  • Gas balloon
  • Graham condenser
  • Rotary evaporator equipped with a vacuum pump
  • 1.3 × 30–cm and 6.4 × 45–cm chromatography columns
  • TLC plate, EMD silica gel 60 F 254
  • 200‐mL separatory funnel
  • UV lamp, 254 nm
  • Büchner funnel
  • Filter paper
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 6: Synthesis, Isolation, and Characterization of Glycerol Nucleic Acid (GNA) Oligonucleotides

  Materials
  • GNA phosphoramidite monomers bearing nucleobases A, C, G, and T ( S.5, S.7, S.12, and S.14, respectively; Basic Protocols protocol 22, protocol 33, protocol 44, and protocol 55, respectively)
  • Anhydrous acetonitrile (MeCN; Applied Biosystems)
  • Standard 5′‐O‐(4,4′‐dimethoxytrityl)phosphoramidites (Glen Research)
  • Ammonium hydroxide (concentrated NH 4OH)
  • n‐Butanol
  • Nanopure water
  • Ethanol
  • 3‐Hydroxypicolinc acid solution
  • 25‐mL round‐bottomed flasks
  • 4‐Å molecular sieves (freshly activated by heating 3 hr at 300°C)
  • 0.45‐µm disposable syringe filter
  • Applied Biosystems 3400 DNA synthesizer
  • 2‐mL screw‐capped microcentrifuge tubes (Eppendorf)
  • 15‐mL screw‐capped centrifuge tubes (Falcon)
  • Spectrophotometer
  • MALDI‐TOF mass spectrometer
  • Additional reagents and equipments for automated solid‐phase oligodeoxyribonucleotide synthesis ( appendix 3C), isolation and characterization of synthetic nucleic acids (units 10.1& 10.4)
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Figures

Videos

Literature Cited

Literature Cited
   Acevedo, O.L. and Andrews, R.S. 1996. Synthesis of propane‐2,3‐diol combinatorial monomers. Tetrahedron Lett. 37:3913‐3934.
   Holý, A. 1975. Aliphatic analogues of nucleosides, nucleotides, and oligonucleotides. Collect. Czech. Chem. Commun. 40:187‐214.
   Kallenbach, N.R., Ma, R.‐I., and Seeman, N.C. 1983. An immobile nucleic acid junction constructed from oligonucleotides. Nature 305:829‐831.
   Schlegel, M.K. and Meggers, E. 2009. Improved phosphoramidite building blocks for the synthesis of the simplified nucleic acid GNA. J. Org. Chem. 74:4615‐4618.
   Schlegel, M.K., Peritz, A.E., Kittigowittana, K., Zhang, L., and Meggers, E. 2007. Duplex formation of the simplified nucleic acid GNA. Chem. Bio. Chem. 8:927‐932.
   Schlegel, M.K., Essen, L.‐O., and Meggers, E. 2008. Duplex formation of a minimal nucleic acid. J. Am. Chem. Soc. 130:8158‐8159.
   Schlegel, M.K., Xie, X., Zhang, L., and Meggers, E. 2009. Insight into the high duplex stability of the simplified nucleic acid GNA. Angew. Chem. Int. Ed. 48:960‐963.
   Schlegel, M.K., Essen, L.‐O., and Meggers, E. 2010. Atomic resolution duplex structure of the simplified nucleic acid GNA. Chem. Commun. 46:1094‐1096.
   Tsai, C.‐H., Chen, J., and Szostak, J.W. 2007. Enzymatic synthesis of DNA on glycerol nucleic acid templates without stable duplex formation between product and template. Proc. Natl. Acad. Sci. U.S.A. 104:14598‐14603.
   Yang, Y.‐W., Zhang, S., McCullum, E.O., and Chaput, J.C. 2007. Experimental evidence that GNA and TNA were not sequential polymers in the prebiotic evolution of RNA. J. Mol. Evol. 65:289‐295.
   Zhang, L., Peritz, A., and Meggers, E. 2005. A simple glycol nucleic acid. J. Am. Chem. Soc. 127:4174‐4175.
   Zhang, L., Peritz, A.E., Carroll, P.J., and Meggers, E. 2006. Synthesis of glycol nucleic acids. Synthesis 4:645‐653.
   Zhang, R.S., McCullum, E.O., and Chaput, J.C. 2008. Synthesis of two mirror image 4‐helix junctions derived from glycerol nucleic acid. J. Am. Chem. Soc. 130:5846‐5647.
   Zhang, S., Switzer, C., and Chaput, J.C. 2010. The resurgence of acyclic nucleic acids. Chem. Biodivers. 7:245‐258.
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