Synthesis of a North‐Methanocarba‐Thymidine (N‐MCT) Analog

Andrew Thompson1, Victor E. Marquez2

1 J‐Star Research, Inc., South Plainfield, New Jersey, 2 Chemical Biology Laboratory, Frederick National Laboratory for Cancer Research, NIH, Frederick, Maryland
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.29
DOI:  10.1002/0471142700.nc0129s51
Online Posting Date:  December, 2012
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A detailed protocol for the synthesis of North‐methanocarba‐thymidine (N‐MCT), a potent antiviral nucleoside with a restricted bicyclo[3.1.0]hexane pseudosugar conformation, is presented. The process is described in two parts. The first basic protocol deals with the synthesis of the carbobicyclic pseudosugar precursor that can be utilized in the syntheses of other bicyclo[3.1.0]hexane nucleosides with natural and non‐natural nucleobases. The second basic protocol describes the specific construction of the thymine base in a linear fashion from the carbobicyclic intermediate. Curr. Protoc. Nucleic Acid Chem. 51:1.29.1‐1.29.14. © 2012 by John Wiley & Sons, Inc.

Keywords: carbocyclic nucleosides; bicyclo[3.1.0]hexane nucleosides; conformationally locked; antiviral

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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Carbobicyclic Pseudosugar Precursor
  • Basic Protocol 2: Linear Construction of the Thymine Base
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Synthesis of Carbobicyclic Pseudosugar Precursor

  • 60% sodium hydride (mol. wt. 24.00; Aldrich)
  • N 2 source
  • Tetrahydrofuran (THF, anhydrous, sieves; Pharmco‐AAPER)
  • Compound S.1 (mol. wt. 204.26; Ash Stevens)
  • Tetra‐n‐butylammonium iodide (mol. wt. 369.37; Aldrich)
  • Benzyl bromide (mol. wt. 171.03; Aldrich)
  • Hexanes (Pharmco‐AAPER)
  • Silica gel (70 to 230 mesh; Silicycle)
  • 10% ethyl acetate (EtOAc; Pharmco‐AAPER)
  • Dimethyl sulfoxide (DMSO, anhydrous, sieves; Acros)
  • Phenylselenyl chloride (mol. wt. 191.52; Aldrich)
  • Silver trifluoroacetate (mol. wt. 220.9; Aldrich)
  • 95% ethanol/5% KOH solution
  • Ammonium chloride (half‐saturated NH 4Cl)
  • Brine
  • MgSO 4
  • Methanol (MeOH; Pharmco‐AAPER)
  • Sodium periodate (NaIO 4, mol. wt. 213.89; Aldrich)
  • Dichloromethane (CH 2Cl 2, Pharmco‐AAPER)
  • Triphenylphosphine (mol. wt. 262.29; Aldrich)
  • Benzoic acid (mol. wt. 122.12; Aldrich)
  • Benzene (anhydrous; Aldrich)
  • Diisopropyl azodicarboxylate (DIAD, mol. wt. 202.21; Aldrich)
  • Potassium carbonate (powdered, mol. wt. 138.21; Aldrich)
  • 1 M diethylzinc ((Et) 2Zn, heptanes; Aldrich)
  • Diiodomethane (mol. wt. 267.84; Aldrich)
  • 3‐L, 5‐L, and 12‐L, 4‐neck flasks with overhead stirrer, N 2 inlet, type‐J Teflon‐covered thermocouple, and addition funnel
  • 18°, 20°, and 25°C water baths
  • Sintered glass Büchner funnels (medium porosity)
  • Vacuum
  • Silica and thin layer chromatography equipment
  • Rotary evaporator
  • Celite pads

Basic Protocol 2: Linear Construction of the Thymine Base

  • N 2 source
  • S.7 (see protocol 1)
  • Tetrahydrofuran (THF; Pharmco‐AAPER)
  • Diphenyl phosphoryl azide (Sigma‐Aldrich)
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU; Sigma‐Aldrich)
  • Methyl tert‐butyl ether (MTBE; Pharmco‐AAPER)
  • Hydrochloric acid (37% aqueous HCl solution; Pharmco‐AAPER)
  • Sodium sulfate (Na 2SO 4; Dawn Scientific Inc.)
  • Biotage SNAP 340 g column (Biotage)
  • Ethyl acetate (Pharmco‐AAPER)
  • Hexanes (Fisher)
  • Sodium hydroxide (NaOH; Acros)
  • Triphenylphosphine (Acros)
  • Diethyl ether (Et 2O)
  • Dichloromethane (CH 2Cl 2; Fisher)
  • Benzene (anhydrous; Aldrich)
  • Silver cyanate (Aldrich)
  • 3‐Methoxy‐2‐methylacryloyl chloride (Aldrich)
  • DMF (anhydrous; Aldrich)
  • Silica gel (70 to 230 mesh; Sylicycle)
  • Ethyl acetate (EtOAc; Pharmco‐AAPER)
  • Hexanes (Pharmco‐AAPER)
  • Ethanol
  • NaHCO 3 (saturated; Pharmco‐AAPER)
  • MgSO 4
  • Brine
  • Methanol (MeOH; Pharmco‐AAPER)
  • HPLC‐grade water (Pharmco‐AAPER)
  • Acetone (Pharmco‐AAPER)
  • 1‐L, 4‐neck flasks with an overhead stirrer, an N 2 inlet, a type‐J‐Teflon covered thermocouple, and an addition funnel
  • Stir plate
  • Sintered glass funnels (medium porosity)
  • Rotary evaporator
  • 3‐L, 3‐neck flasks with an overhead stirrer, a type‐J Teflon‐covered thermocouple, and an addition funnel
  • 3‐L, 4‐neck flasks with an overhead stirrer, an N 2 inlet, a type‐J Teflon‐covered thermocouple, a reflux condenser, and an addition funnel
  • Ceramic Büchner (Whatman no. 1)
  • Krapcho funnel
  • 5‐L, 4‐neck flasks with an overhead stirrer, an N 2 inlet, a type‐J Teflon‐covered thermocouple, and an addition funnel
  • Vacuum
  • 2‐L hydrogenation bottle
  • Shaker hydrogenation apparatus (Parr)
  • Celite pad (1‐cm; Aldrich)
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Literature Cited

Literature Cited
   Biggadike, K., Borthwick, A.D., Exall, A.M., Kirk, B.E., Roberts, S.M., Youds, P., Slawin, A.M.Z., and Williams, D.J. 1987. Synthesis of fluorinated carbocyclic nucleosides: Preparation of carbocyclic 1‐(2′‐deoxy‐6′‐fluororibofuranosyl)‐5‐iodouracils. J. Chem. Soc. Chem. Commun. 255‐256.
   Biggadike, K., Borthwick, A.D., Evans, D., Exall, A.M., Kirk, B.E., Roberts, S.M., Stephenson, L., and Youds, P. 1988. Use of diethylaminosulphur trifluoride (DAST) in the preparation of carobycyclic nucleosides. J. Chem. Soc. Perkin Trans. 1:549‐554.
   Ezzitouni, A., Russ, P., and Marquez, V.E. 1997. (1S,2R)‐[(Benzyloxy)methyl]cyclopent‐3‐enol. A versatile synthon for the preparation of 4′,1′a‐methano‐ and 1′,1′a‐methanocarbocyclic nucleosides. J. Org. Chem. 62:4870‐4873.
   Ludek, O.R. and Marquez, V.E. 2007. Convergent or linear? A challenging question in carbocyclic nucleoside chemistry. Synthesis 3451‐3460.
   Madeira, M., Shenoy, S., Van, Q.N., Marquez, V.E., and Barchi, J.J. Jr. 2007. Biophysical studies of DNA modified with conformationally constrained nucleotides: Comparison of 2′‐exo (north) and 3′‐exo (south) ‘locked’templates. Nucleic Acids Res. 35:1978‐1991.
   Marquez, V.E., Siddiqui, M.A., Ezzitouni, A., Russ, P., Wang, J., Wagner, R.W., and Matteucci, M.D. 1996. Nucleosides with a twist. Can fixed forms of sugar ring pucker influence biological activity in nucleosides and oligonucleotides? J. Med. Chem. 39:3739‐3747.
   Marquez, V.E., Russ, P., Alonso, R., Siddiqui, M.A., Hernandez, S., George, C., Nicklaus, M.C., Dai, F., and Ford, H. Jr. 1999. Synthesis of conformationally restricted carbocyclic nucleosides: The role of the O(4′)‐atom in the key hydration step of adenosine deaminase. Helv. Chim. Acta 82:2119‐2129.
   Marquez, V.E., Hughes, S.H., Sei, S., and Agbaria, R. 2006. The history of N‐methanocarbathymidine: The investigation of a conformational concept leads to the discovery of a potent and selective nucleoside antiviral agent. Antiviral Res. 71:268–275.
   Pallan, P.S., Marquez, V.E., and Egli, M. 2012. The conformationally constrained N‐methanocarba‐dT analogue adopts an unexpected C4′‐exo sugar pucker in the structure of a DNA hairpin. Biochemistry 51:2639–2641.
   Terrazas, M., Ocampo, S.M., Perales, J.C., Marquez, V.E., and Eritja, R. 2011. Effect of North bicyclo[3.1.0]hexane 2′‐deoxysugars on RNA interference: A novel class of siRNA modification. ChemBioChem 12:1056‐1065.
   Wu, Z., Madeira, M., Barchi, J.J. Jr., Marquez, V.E., and Bax, A. 2005. Changes in DNA bending induced by restricting nucleotide ring pucker studied by weak alignment NMR spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 102:24‐28.
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