Solid‐Phase Synthesis of 2′‐Deoxy‐2′‐fluoro‐ β‐D‐Oligoarabinonucleotides (2′F‐ANA) and Their Phosphorothioate Derivatives

Ekaterina Viazovkina1, Maria M. Mangos1, Mohamed I. Elzagheid1, Masad J. Damha1

1 McGill University, Montreal
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.15
DOI:  10.1002/0471142700.nc0415s10
Online Posting Date:  November, 2002
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Abstract

This unit describes the chemical synthesis of 2'‐deoxy‐2'‐fluoro‐b‐D‐oligoarabinonucleotides (2'F‐ANA), both with phosphodiester and phosphorothioate linkages. The protocols described herein include araF phosphoramidite preparation, assembly on DNA synthesizers, and final deprotection and purification of oligonucleotides.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of araF Phosphoramidites
  • Basic Protocol 2: Solid‐Phase Assembly of Protected araF Phosphoramidites
  • Basic Protocol 3: Deprotection and Purification of araF Oligonucleotides
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of araF Phosphoramidites

  Materials
  • Protected araF nucleosides (unit 1.7):
    • N6‐Benzoyl‐9‐[2‐deoxy‐2‐fluoro‐5‐O‐(4‐methoxytrityl)‐β‐D‐arabinofuranosyl] adenine (S.1a)
    • N2‐Isobutyryl‐9‐[2‐deoxy‐2‐fluoro‐5‐O‐(4‐methoxytrityl)‐β‐D‐arabinofuranosyl]guanine (S.1b)
    • N4‐Benzoyl‐1‐[2‐deoxy‐2‐fluoro‐5‐O‐(4‐methoxytrityl)‐β‐D‐arabinofuranosyl] cytosine (S.1c)
    • 1‐[2‐Deoxy‐2‐fluoro‐5‐O‐ (4‐methoxytrityl)‐β‐D‐arabinofuranosyl]thymine (S.1d)
  • THF, anhydrous (see recipe)
  • N‐Ethyl‐N,N‐diisopropylamine (DIPEA; Aldrich), double distilled
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite (Chem Genes)
  • Dichloromethane
  • 1:9 and 1:19 (v/v) methanol/dichloromethane
  • Saturated sodium bicarbonate solution
  • Magnesium sulfate, anhydrous
  • Solvent system:
  •  1:99 (v/v) triethylamine/chloroform (for araF‐T)
  •  1:9:10 (v/v) triethylamine/dichloromethane/hexanes (for araF‐A and araF‐C)
  •  1:99 (v/v) triethylamine/dichloromethane (for araF‐G)
  • Silica gel (230 to 400 mesh)
  • Diethyl ether
  • 50‐mL round‐bottom flask equipped with stir bar and rubber septum
  • Syringe
  • TLC Merck silica plates (Kieselgel 60 F‐254)
  • 254‐nm UV lamp
  • 500‐mL separatory funnel
  • Vacuum evaporator (e.g., Savant Speedvac)
  • 3‐cm‐diameter chromatography column
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 2: Solid‐Phase Assembly of Protected araF Phosphoramidites

  Materials
  • AraF phosphoramidites (see protocol 1)
  • Argon gas
  • Acetonitrile, anhydrous (see recipe)
  • Liquid Reagent kit for the Expedite 8909 instrument (PerSeptive Biosystems):
  •  Acetonitrile wash and amidite diluent: anhydrous acetonitrile
  •  Activator solution: dissolve 1.8 g sublimed tetrazole (0.5 M) in 50 mL
  •  acetonitrile; store up to 2 wks at room temperature.
  •  Cap A (see recipe)
  •  Cap B (see recipe)
  •  Oxidizer solution (see recipe)
  •  Deblock solution: 15 g trichloroacetic acid in 500 mL dichloromethane; store up to 1 yr at room temperature
  • Sulfurization reagent (see recipe)
  • Amidite column (see recipe)
  • Synthesizer vials with caps
  • Vacuum desiccator containing phosphorus pentoxide
  • Syringe
  • Automated DNA synthesizer (e.g., Expedite 8909, Perseptive Biosystems) with trityl monitor
  • Additional reagents and equipment for oligonucleotide synthesis ( appendix 3C)

Basic Protocol 3: Deprotection and Purification of araF Oligonucleotides

  Materials
  • Fully protected oligonucleotides, attached to the solid support of a synthesis column (see protocol 2)
  • Ethanol, anhydrous
  • 29% ammonium hydroxide
  • Denaturing acrylamide gel stock solution (see recipe)
  • Loading buffer (e.g., formamide/dye mix, unit 10.4)
  • Sephadex G‐25 column (Amersham Pharmacia Biotech)
  • 1 M NaClO 4
  • Anhydrous and 25% or 50% (v/v) acetonitrile
  • 1.5 mL microcentrifuge tubes or screw‐cap microcentrifuge tubes with O‐ring seal
  • Platform shaker
  • 55°C heating block or water bath (optional)
  • Vacuum evaporator (e.g., Savant Speedvac) with low and high vacuum sources
  • UV spectrophotometer and cuvette
  • 0.75‐ and 1.5‐mm‐thick gel plates
  • TLC Merck silica plate (Kieselgel 60 F‐254)
  • Hand‐held 254‐nm UV lamp
  • Camera with UV filter (optional)
  • 0.45 µm hydrophilic fluid filter (Creative Medical)
  • 0.22 µm membrane filter (Millipore; optional)
  • Anion‐exchange high‐performance liquid chromatograph (HPLC) with:
  •  Gradient maker
  •  0.5 mm × 7.5 cm Protein Pak DEAE 5PW column (Waters)
  •  Column heater
  • Sep‐Pak C18 cartridges (Waters Chromatography)
  • 10 mL syringe
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis ( appendix 3B and unit 10.4)
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Figures

Videos

Literature Cited

Literature Cited
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   Iyer, R.P., Phillips, L.R., Egan, W., Regan, J.B., and Beaucage, S.L. 1990. The automated synthesis of sulfur‐containing oligodeoxyribonucleotides using 3H‐1,2‐benzodithiol‐3‐one 1,1‐dioxide as a sulfur‐transfer reagent. J. Org. Chem. 55:4693‐4699.
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   Mangos, M.M. and Damha, M.J. 2002. Flexible and frozen sugar‐modified nucleic acids: modulation of biological activity through furanose ring dynamics in the antisense strands. Curr. Topics Med. Chem. 2:1145‐1169.
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   Noronha, A.M., Wilds, C.J., Lok, C.‐N., Viazovkina, K., Arion, D., Parniak, M.A., and Damha, M.J. 2000. Synthesis and biophysical properties of arabinonucleic acids (ANA): Circular dichroic spectra, melting temperatures and ribonuclease H susceptibility of ANA:RNA hybrid duplexes. Biochemistry 39:7050‐7062.
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   Trempe, J.F., Wilds, C.J., Denisov, A.Y., Pon, R.T., Damha, M.J., and Gehring, K. 2001. NMR solution structure of an oligonucleotide hairpin with a 2′F‐ANA/RNA stem: Implications for RNase H specificity toward DNA/RNA hybrid duplexes. J. Am. Chem. Soc. 123:4896‐4903.
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   Venkateswarlu, D. and Ferguson, D.M. 1999. Effects of C2′‐substitution on arabinonucleic acid structure and conformation. J. Am. Chem. Soc. 121:5609‐5610.
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Key References
   Crouch and Toulmé, 1998. See above
  A comprehensive book on ribonucleases H, their sources, properties, biological utility, and antisense applications.
   Damha et al., 2001. See above.
  Highlights the role of the 2′‐sugar position in ANA and 2′F‐ANA conformations, and the origins of RNase H activity.
   Freier, S.M. and Altmann, K.‐H. 1997. The ups and downs of nucleic acid duplex stability: Structure‐stability studies on chemically‐modified DNA:RNA duplexes. Nucl. Acids Res. 25:4429‐4443.
  An extensive resource that relates duplex stabilities to nucleotide structure with 197 examples of oligonucleotide modifications.
   Kvaernø, L. and Wengel, J. 2001. Antisense molecules and furanose conformations—is it really that simple? Chem. Commun. 1419‐1424.
  A concise review briefly comparing RNA binding versus cleavage with promising antisense candidates.
   Lebedeva and Stein, 2001. See above.
  Insightful discussion of antisense design that focuses on in vivo toxicity from nonspecific interactions and potential irrelevant cleavage of nontargeted RNA.
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