Gram‐Scale Chemical Synthesis of 2′‐Deoxynucleoside‐5′‐O‐Triphosphates

Anilkumar R. Kore1, Muthian Shanmugasundaram1, Annamalai Senthilvelan1, Balasubramanian Srinivasan1

1 Bioorganic Chemistry Division, Life Technologies Corporation, Austin, Texas
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 13.10
DOI:  10.1002/0471142700.nc1310s49
Online Posting Date:  June, 2012
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Abstract

A simple, straightforward, reliable, and efficient method for the chemical synthesis of sodium salt of 2′‐deoxynucleoside‐5′‐O‐triphosphates (dNTPs), starting from the corresponding nucleoside, is described. This improved “one‐pot, three‐step” synthetic strategy involves the monophosphorylation of nucleoside, followed by reaction with tributylammonium pyrophosphate and hydrolysis of the resulting cyclic intermediate to provide the corresponding dNTP in good yields (65% to 70%). It is noteworthy that the protocol holds good for both the purine deoxynucleotides, such as 2′‐deoxyguanosine‐5′‐O‐triphosphate (dGTP) and 2′‐deoxyadenosine‐5′‐O‐triphosphate (dATP), and pyrimidine deoxynucleotides, such as 2′‐deoxycytidine‐5′‐O‐triphosphate (dCTP), thymidine‐5′‐O‐triphosphate (TTP), and 2′‐deoxyuridine‐5′‐O‐triphosphate (dUTP). Curr. Protoc. Nucleic Acid Chem. 49:13.10.1‐13.10.12. © 2012 by John Wiley & Sons, Inc.

Keywords: 2′‐deoxyribonucleotides; chemical synthesis; one‐pot synthesis; phosphorylation; proton sponge; triphosphate; dNTP

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of 2′‐Deoxynucleoside‐5′‐O‐Triphosphates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of 2′‐Deoxynucleoside‐5′‐O‐Triphosphates

  Materials
  • Tributylammonium pyrophosphate (prepared by the procedure in Ludwig and Eckstein, )
  • Acetonitrile, anhydrous (Aldrich)
  • Tributylamine (Aldrich)
  • 2′‐Deoxyguanosine >98% ( S.1; Rasayan)
  • Trimethyl phosphate, >97% pure (Aldrich)
  • Argon gas cylinder
  • Ice/NaCl bath (−5° to −10°C)
  • Phosphorous oxychloride (Acros)
  • Dichloromethane, technical grade (DCM; Aldrich)
  • Ammonium hydroxide (28% NH 3 in water; Aldrich)
  • DEAE Sepharose (GE Healthcare)
  • 2 M NaCl (see recipe)
  • Isopropanol
  • TEAB buffer (see recipe)
  • HPLC mobile phase A: 5 mM ammonium phosphate monobasic, pH 2.8 (see recipe)
  • HPLC mobile phase B: 750 mM ammonium phosphate monobasic, pH 3.7 (see recipe)
  • Sodium perchlorate (Aldrich)
  • Acetone
  • 2′‐Deoxyadenosine >98% ( S.3; Rasayan)
  • 2′‐Deoxycytidine >98% ( S.5; Rasayan)
  • 2′‐Deoxythymidine >98% ( S.7; Rasayan)
  • Proton sponge [1,8‐bis(dimethylamino)naphthalene] (Aldrich)
  • Nuclease‐free water
  • 2′‐Deoxyuridine >98% ( S.9; Rasayan)
  • 500‐mL and 1.0‐L Centrifuge bottles
  • 1.0‐L round‐bottom flasks, oven dried (Chemglass)
  • Teflon‐coated magnetic stirring bar (oval shaped, Aldrich)
  • Rubber septa for 24/40‐glass joints (Chemglass)
  • Magnetic stir plate (VWR)
  • Vacuum/nitrogen (or argon) gas manifold
  • 1‐mL, 2‐mL and 5‐mL sealed glass syringes (Aldrich)
  • 2‐L separatory funnel
  • 4.0‐L Conical flask
  • pH meter (VWR)
  • Chromatography column: (10‐cm × 78.5‐cm)
  • FPLC ÄKTA purifier (GE Healthcare)
  • UV detector ranging from 254‐ to 272‐nm
  • HPLC system (Waters) including:
    • Detector module
    • Hypersil SAX column (4.6‐mm × 25‐cm)
  • Rotary evaporator
  • Sorvall RC‐3B centrifuge
  • Additional reagents and equipment for column chromatography ( appendix 3E)
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Figures

Videos

Literature Cited

Literature Cited
   Baddiley, J., Michelson, A.M., and Todd, A.R. 1949. Nucleotides. Part II. A synthesis of adenosine triphosphate. J. Chem. Soc. 582‐586.
   Burgess, K. and Cook, D. 2000. Syntheses of nucleoside triphosphates. Chem. Rev. 100:2047‐2059.
   Catson‐Wiliams, J., Smith, M., Carrasco, N., and Huang, Z. 2011. Protection‐free one‐pot synthesis of 2′‐deoxynucleoside 5′‐triphosphates and DNA polymerization. Org. Lett. 13:4156‐4159.
   Davisson, V.J., Davis, D.R., Dixit, V.M., and Poulter, C.D. 1987. Synthesis of nucleotide‐5′‐diphosphates from 5′‐O‐tosyl nucleosides. J. Org. Chem. 52:1794‐1801.
   Franca, L.T.C., Carrilho, E., and Kist, T.B.L. 2002. A review of DNA sequencing techniques. Q. Rev. Biophy. 35:169‐200.
   Gandhi, V.V. and Samuels, D.C. 2011. A review comparing deoxyribonucleoside triphosphate (dNTP) concentrations in the mitochondrial and cytoplasmic compartments of normal and transformed cells. Nucleosides Nucleotides Nucleic Acids 30:317‐339.
   Gillerman, I. and Fischer, B. 2010. An improved one‐pot synthesis of nucleoside 5′‐triphosphate analogues. Nucleosides Nucleotides Nucleic Acids 29:245‐256.
   Guo, J., Yu, L., Turro, N.J., and Ju, J. 2010. An integrated system for DNA sequencing by synthesis using novel nucleotide analogues. Acc. Chem. Res. 43:551‐563.
   Hoard, D.E. and Ott, D.G. 1965. Conversion of mono‐ and oligodeoxyribonucleotides to 5′‐triphosphates. J. Am. Chem. Soc. 87:1785‐1788.
   Ludwig, J. 1981. A new route to nucleoside 5′‐triphosphates. Acta Biochim. Biophys. Acad. Hung. 16:131‐133.
   Ludwig, J. and Eckstein, F. 1989. Rapid and efficient synthesis of nucleoside 5′‐O‐(1‐thiotriphosphates),5′‐triphosphates and 2′,3′‐cyclophosphorothiolates using 2‐chloro‐4H‐1,3,2‐benzodioxaphosphorin‐4‐one. J. Org. Chem. 54:631‐635.
   Moffatt, J.G. 1964. General synthesis of nucleoside 5′‐triphosphate. Can. J. Chem. 42:599‐604.
   Niida, H., Shimada, M., Murakami, H., and Nakanishi, M. 2010. Mechanisms of dNTP supply that play an essential role in maintaining genome integrity in eukaryotic cells. Cancer Sci. 101:2505‐2509.
   Simoncsits, A. and Tomasz, J. 1975. Nucleoside 5′‐phosphordiamidates, synthesis and some properties. Nucleic Acids Res. 2:1223‐1233.
   Yoshikawa, M., Kato, T., and Takenishi, T. 1967. A novel method for phosphorylation of nucleosides to 5′‐nucleotides. Tetrahedron Lett. 50:5065‐5068.
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