Synthesis of Protected 2′‐Deoxy‐2′‐fluoro‐β‐D‐arabinonucleosides

Mohamed I. Elzagheid1, Ekaterina Viazovkina1, Masad J. Damha1

1 McGill University, Montreal, Canada
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.7
DOI:  10.1002/0471142700.nc0107s10
Online Posting Date:  November, 2002
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Abstract

This unit describes in detail the preparation of protected 2'‐deoxy‐2'‐fluoroarabinonucleosides. These building blocks are required for the synthesis of 2'‐deoxy‐2'‐fluoroarabinonucleic acid (2'F‐ANA), an oligonucleotide analog exhibiting very promising antisense properties. The preparation of phosphoramidites from these building blocks and the synthesis of 2'F‐ANA are described elsewhere in the manual.

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis and Characterization of N2‐Isobutyryl‐9‐[2‐Deoxy‐2‐Fluoro‐5‐O‐(4‐Methoxytrityl)‐β‐D‐Arabinofuranosyl]Guanine
  • Basic Protocol 2: Synthesis and Characterization of N4‐Benzoyl‐1‐[2‐Deoxy‐2‐Fluoro‐5‐O‐(4‐Methoxytrityl)‐β‐D‐Arabinofuranosyl]Cytosine
  • Basic Protocol 3: Synthesis and Characterization of N6‐Benzoyl‐9‐[2‐Deoxy‐2‐ Fluoro‐5‐O‐(4‐Methoxytrityl)‐β‐D‐Arabinofuranosyl]Adenine
  • Basic Protocol 4: Synthesis and Characterization of 1‐[2‐Deoxy‐2‐Fluoro‐5‐O‐(4‐Methoxytrityl)‐β‐D‐Arabinofuranosyl]Thymine
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis and Characterization of N2‐Isobutyryl‐9‐[2‐Deoxy‐2‐Fluoro‐5‐O‐(4‐Methoxytrityl)‐β‐D‐Arabinofuranosyl]Guanine

  Materials
  • Nitrogen gas source
  • 1,3,5‐Tri‐O‐benzoyl‐α‐D‐ribofuranose (S.1; Pfanstiehl)
  • Dichloromethane, dry (see recipe)
  • [Bis(2‐methoxyethyl)amino]sulfur trifluoride (MAST; Aldrich) or (diethylamino)sulfur trifluoride (DAST; Aldrich)
  • Saturated aqueous sodium bicarbonate
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Silica gel (230 to 400 mesh)
  • Chloroform
  • Sand
  • Merck thin‐layer chromatography (TLC) silica plates (Kieselgel 60 F‐254; 0.2 mm thick)
  • 30% (w/v) HBr in acetic acid
  • Chlorotrimethylsilane (TMSCl)
  • 2,6‐Dichloropurine
  • Hexamethyldisilazane (HMDS)
  • Acetonitrile, dry (see recipe)
  • Trimethylsilyl trifluoromethanesulfonate (TMS‐Tfl)
  • 5:1 (v/v) toluene/ethyl acetate
  • Sodium hydride, as a 60% (w/v) solution in mineral oil (Aldrich)
  • Sodium azide (NaN 3)
  • 95% (v/v) ethanol
  • Tin dichloride (SnCl 2)
  • Methanol
  • 29.7% (w/w) aqueous ammonia (Fisher)
  • 5% and 10% (v/v) methanol in dichloromethane
  • Adenosine deaminase solution in 50% glycerol (from calf intestine mucosa, specific activity 160 to 200 U/mg protein; Sigma)
  • Phosphorus pentoxide (P 2O 5)
  • Pyridine, dry (see recipe)
  • Isobutyryl chloride
  • 2:1:1 (v/v/v) water/ethyl acetate/ether
  • 4‐Dimethylaminopyridine (4‐DMAP)
  • p‐Anisylchlorodiphenylmethane (monomethoxytrityl chloride or MMTr⋅Cl)
  • 0% to 5% (v/v) gradient of methanol in dichloromethane
  • Oven‐dried glassware, including:
  •  25‐, 100‐, and 250‐mL round‐bottom flasks
  •  1‐L Erlenmeyer flasks
  • Reflux condenser
  • Oil bath, 40° to 50°C
  • 1‐L separatory funnels
  • Rotary evaporator equipped with a vacuum pump or water aspirator
  • Chromatography columns: 5 × 50 cm, 3 × 15 cm, and 3 × 20 cm
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 2: Synthesis and Characterization of N4‐Benzoyl‐1‐[2‐Deoxy‐2‐Fluoro‐5‐O‐(4‐Methoxytrityl)‐β‐D‐Arabinofuranosyl]Cytosine

  Materials
  • Nitrogen gas source
  • Chlorotrimethylsilane (TMSCl)
  • N4‐Acetylcytosine
  • Hexamethyldisilazane (HMDS)
  • 2‐Deoxy‐2‐fluoro‐3,5‐di‐O‐benzoyl‐α‐D‐arabinofuranosyl bromide (S.3; see protocol 1)
  • Carbon tetrachloride (CCl 4), anhydrous (Aldrich)
  • Merck thin‐layer chromatography (TLC) silica plates (Kieselgel 60 F‐254; 0.2 mm thick)
  • Dichloromethane, dry (see recipe)
  • Saturated aqueous sodium bicarbonate
  • Sodium sulfate (Na 2SO 4), anhydrous
  • 29.7% (w/w) aqueous ammonia (Fisher)
  • Methanol
  • 5% (v/v) ethanol in chloroform
  • Silica gel (230 to 400 mesh)
  • Chloroform
  • 3:1 (v/v) chloroform/ethanol
  • Benzoic anhydride
  • N,N‐Dimethylformamide (DMF), anhydrous (Aldrich)
  • Pyridine, dry (see recipe)
  • p‐Anisylchlorodiphenylmethane (monomethoxytrityl chloride or MMTr⋅Cl)
  • 4‐Dimethylaminopyridine (4‐DMAP)
  • 0% to 1% (v/v) gradient of methanol in chloroform
  • 250‐mL round‐bottom flasks, oven dried
  • Reflux condenser
  • Oil bath, 120°C and 77°C
  • Rotary evaporator equipped with a vacuum pump or water aspirator
  • 3 × 20–cm chromatography column
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 3: Synthesis and Characterization of N6‐Benzoyl‐9‐[2‐Deoxy‐2‐ Fluoro‐5‐O‐(4‐Methoxytrityl)‐β‐D‐Arabinofuranosyl]Adenine

  Materials
  • 2‐Deoxy‐2‐fluoro‐3,5‐di‐O‐benzoyl‐α‐D‐arabinofuranosyl bromide (S.3; see protocol 1)
  • Dichloromethane, dry (see recipe)
  • N6‐Benzoyladenine, anhydrous
  • Activated molecular sieves (type 4A)
  • Pyridine, dry (see recipe)
  • Chloroform
  • Silica gel (230 to 400 mesh)
  • 7:3 (v/v) chloroform/dichloromethane
  • Merck thin‐layer chromatography (TLC) silica plates (Kieselgel 60 F‐254; 0.2 mm thick)
  • Ethanol
  • 29.7% (w/w) aqueous ammonia (Fisher)
  • p‐Anisylchlorodiphenylmethane (monomethoxytrityl chloride or MMTr⋅Cl)
  • Trimethylsilyl chloride (TMSCl)
  • Benzoyl chloride
  • Brine (saturated aqueous NaCl)
  • Magnesium sulfate, anhydrous
  • 33:1 (v/v) dichloromethane/methanol
  • 250‐ and 500‐mL round‐bottom flasks, oven dried
  • Reflux condenser
  • Oil bath, 40° to 50°C
  • Rotary evaporator equipped with vacuum pump
  • 7 × 15–cm chromatography column
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 4: Synthesis and Characterization of 1‐[2‐Deoxy‐2‐Fluoro‐5‐O‐(4‐Methoxytrityl)‐β‐D‐Arabinofuranosyl]Thymine

  Materials
  • Thymine
  • Ammonium sulfate
  • Phosphorous pentoxide (P 2O 5)
  • Acetonitrile, dry (see recipe)
  • Hexamethyldisilazane (HMDS)
  • 2‐Deoxy‐2‐fluoro‐3,5‐di‐O‐benzoyl‐α‐D‐arabinofuranosyl bromide (S.3; see protocol 1)
  • Magnesium sulfate, anhydrous
  • Carbon tetrachloride (CCl 4), anhydrous (Aldrich)
  • Dichloromethane, dry (see recipe)
  • Ethanol
  • Concentrated aqueous ammonia
  • Merck thin‐layer chromatography (TLC) silica plates (Kieselgel 60 F‐254; 0.2 mm thick)
  • Pyridine, dry (see recipe)
  • p‐Anisylchlorodiphenylmethane (monomethoxytrityl chloride or MMTr⋅Cl)
  • Chloroform
  • Brine (aqueous saturated NaCl)
  • Silica gel (230 to 400 mesh)
  • 19:1 (v/v) dichloromethane/methanol
  • 250‐ and 500‐mL round‐bottom flasks, oven dried
  • Oil bath, 100°
  • Rotary evaporator attached to a vacuum pump
  • 7 × 15–cm chromatography column chromatography ( appendix 3E)
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)
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Figures

Videos

Literature Cited

Literature Cited
   Burchenal, J.H., Leyland‐Jones, B., Watanabe, B., Klein, R., Lopez, C., and Fox, J.J. 1983. Experimental and clinical studies on 2′‐fluoroarabinosyl pyrimidines and purine‐like C‐nucleosides. In Nucleosides, Nucleotides, and Their Biological Applications, Proceedings 5th International Round Table, pp. 47‐65. International Society for Nucleosides, Nucleotides, and Nucleic Acids, Montpellier, France.
   Chu, C.K., Matulic‐Adamic, J., Huang, J.‐T., Chou, T.‐C., Burchenal, J.H., Fox, J.J., and Watanabe, K.A. 1989. Synthesis of some 9‐(2‐deoxy‐2‐fluoro‐β‐D‐arabinofuranosyl)‐9H‐purines and their biological activities. Chem. Pharm. Bull. 37:336‐339.
   Damha, M.J., Wilds, C.J., Novonha, A., Brunker, I., Borkow, G., Arion, D., and Parniak, M.A. 1998. Hybrids of RNA and arabinonucleic acids (ANA and 2′F‐ANA) are substrates of ribonuclease H. J. Am. Chem. Soc. 120:12976‐12977.
   Eberhardt, E.S., Panasik, N. Jr., and Raines, R.T. 1996. Inductive effects on the energetics of prolyl peptide bond isomerization: Implications for collagen folding and stability. J. Am. Chem. Soc. 118:12261‐12266.
   Herdewijn, P., VanAerschot, A., and Kerremans, L. 1989. Synthesis of nucleosides fluorinated in the sugar moiety. The application of diethylaminosulfur trifluoride to the synthesis of fluorinated nucleosides. Nucleosides Nucleotides 8:65‐96.
   Holmgren, S.K., Bretscher, L.E., Taylor, K.M., and Raines, R.T. 1999. A hyperstable collagen mimic. Chem. Biol. 6:63‐70.
   Howell, H.G., Brodfuehrer, P.R., Brundidge, S.P., Benigni, D.A., and Sapino, C. Jr. 1988. Antiviral nucleosides. A stereospecific, total synthesis of 2′‐fluoro‐2′‐deoxy‐β‐D‐arabinofuranosyl nucleosides. J. Org. Chem. 53:85‐88.
   Kierzek, R. 1985. The synthesis of 5′‐O‐dimethoxytrityl‐N‐acetyl‐2′‐deoxynucleosides. Improved “transient protection” approach. Nucleosides Nucleotides 4:641‐649.
   Ma, T., Lin, J.‐S., Newton, M.G., Cheng, Y.‐C., and Chu, C.K. 1997. Synthesis and anti hepatitis B virus activity of 9‐(2‐deoxy‐2‐fluoro‐β‐L‐arabinofuranosyl)purine nucleosides. J. Med. Chem. 40:2750‐2754.
   Maruyama, T., Takamatsu, S., Kozai, S., Satoh, Y., and Izawa, K. 1999. Synthesis of 9‐(2‐deoxy‐2‐fluoro‐β‐D‐arabinofuranosyl)adenine bearing a selectively removable protecting group. Chem. Pharm. Bull. 47:966‐970.
   Middleton, W.J. 1975. New fluorinating reagents. Dialkylaminosulfur fluorides. J. Org. Chem. 40:574‐578.
   Lok, C.‐N., Viazovkina, E., Min, K.‐L., Nagy, E., Wilds, C.J., Damha, M.J., and Parniak, M.A. 2002. Potent gene‐specific inhibitory properties of mixed‐backbone antisense oligonucleotides comprised of 2′‐deoxy‐2′‐fluoro‐D‐arabinose and 2′‐deoxyribose nucleotides. Biochemistry. 41:3457‐3467.
   Pankiewicz, K.W. 2000. Fluorinated nucleosides. Carbohydr. Res. 327:87‐105.
   Pankiewicz, K.W., Krzeminski, J., Cizewski, L.A., Ren, W.‐Y., and Watanabe, K.A. 1992. Synthesis of 9‐(2‐deoxy‐2‐fluoro‐β‐D‐arabinofuranosyl)adenine and hypoxanthine. An effect of C3′‐endo to C2′‐endo conformational shift on the reaction course of 2′‐hydroxyl group with DAST. J. Org. Chem. 57:553‐559.
   Scharer, O.D. and Verdine, G.L. 1995. A designed inhibitor of base‐excision DNA repair. J. Am. Chem. Soc. 117:10781‐10782.
   Still, W.C., Kahn, M., and Mitra, A. 1978. Rapid chromatographic technique for preparative separation with moderate resolution. J. Org. Chem. 43:2923‐2925.
   Tann, C.H., Brodfuehrer, P.R., Brundidge, S.P., Sapino, C. Jr., and Howell, H.G. 1985. Fluorocarbohydrate in synthesis. An efficient synthesis of 1‐(2‐deoxy‐2‐fluoro‐β‐D‐arabinofuranosyl)‐5‐iodouracil (β‐FIAU) and 1‐(2‐deoxy‐2‐fluoro‐β‐D‐arabinofuranosyl)thymine (β‐FMAU). J. Org. Chem. 50:3644‐3647.
   Tennila, T., Azhayev, E., Vepsalainen, J., Laatikainen, R., Azhayev, A., and Mikhailopulo, I.A. 2000. Oligonucleotides containing 9‐(2‐deoxy‐2‐fluoro‐β‐D‐arabinofuranosyl)‐adenine and guanine: Synthesis, hybridization and antisense properties. Nucleosides Nucleotides Nucleic Acids. 19:1861‐1884.
   Wilds, C.J. and Damha, M.J. 2000. 2′‐Deoxy‐2′‐fluoro‐β‐D‐arabinonucleosides and oligonucleotides (2′F‐ANA): Synthesis and physicochemical studies. Nucl. Acids Res. 28:3625‐3635.
   Watanabe, K.A., Chu, C.K., and Fox, J.J. June, 1988. 2‐Fluoro‐arabinofuranosyl purine nucleosides. U.S. patent 47,551,221.
   Wower, J., Hixson, S.S., Sylvers, L.A., Xing, Y., and Zimmermann, R.A. 1994. Synthesis of 2,6‐diazido‐9‐(β‐D‐ribofuranosyl)purine 3′,5′‐bisphosphate: Incorporation into transfer RNA and photochemical labelling of Escherichia coli ribosomes. Bioconjug. Chem. 5:158‐161.
Key References
   Herdewijn et al., 1989. See above.
  This article reviews the application of DAST to the synthesis of fluorinated nucleosides.
   Howell et al., 1988. See above.
  This article describes key connections and synthetic strategies of araF‐nucleosides that form the basis of the procedures outlined in these protocols.
   Pankiewicz, 2000. See above.
  This review article focuses on the synthesis of sugar fluorinated nucleosides.
   Tann et al., 1985. See above.
  This article also describes synthetic strategies of araF‐nucleosides.
   Wilds and Damha, 2000. See above.
  This article provides procedures for the synthesis of araF‐nucleosides and 2′F‐ANA.
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