Use of a Novel 5′‐Regioselective Phosphitylating Reagent for One‐Pot Synthesis of Nucleoside 5′‐Triphosphates from Unprotected Nucleosides

Julianne Caton‐Williams1, Rudiona Hoxhaj1, Bilal Fiaz1, Zhen Huang1

1 Department of Chemistry and Department of Biology, Georgia State University, Atlanta, Georgia
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.30
DOI:  10.1002/0471142700.nc0130s52
Online Posting Date:  March, 2013
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

5′‐Triphosphates are building blocks for enzymatic synthesis of DNA and RNA. This unit presents a protocol for convenient synthesis of 2′‐deoxyribo‐ and ribonucleoside 5′‐triphosphates (dNTPs and NTPs) from any natural or modified base. This one‐pot synthesis can also be employed to prepare triphosphate analogs with a sulfur or selenium atom in place of a non‐bridging oxygen atom of the α‐phosphate. These S‐ or Se‐modified dNTPs and NTPs can be used to prepare diastereomerically pure phosphorothioate or phosphoroselenoate nucleic acids. Even without extensive purification, the dNTPs or NTPs synthesized by this method are of high quality and can be used directly in DNA polymerization or RNA transcription. Synthesis and purification of the 5′‐triphosphates, as well as analysis and confirmation of natural and sulfur‐ or selenium‐modified nucleic acids, are described in this protocol unit. Curr. Protoc. Nucleic Acid Chem. 52:1.30.1‐1.30.21. © 2013 by John Wiley & Sons, Inc.

Keywords: nucleoside 5′‐triphosphate; sulfur modification; selenium modification; phosphorothioate; phosphoroselenoate; diastereomer

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: One‐Pot Synthesis of Native Nucleoside 5′‐Triphosphates
  • Basic Protocol 2: One‐Pot Synthesis of Nucleoside 5′‐(α‐P‐Thio)Triphosphates
  • Basic Protocol 3: One‐Pot Synthesis of Nucleoside 5′‐(α‐P‐Seleno)Triphosphates
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: One‐Pot Synthesis of Native Nucleoside 5′‐Triphosphates

  Materials
  • Starting nucleosides:
    • Adenosine (5a) or 2′‐deoxyadenosine monohydrate (4a) (Sigma‐Aldrich)
    • Cytidine (5c), 2′‐deoxycytidine monohydrochloride (4c), guanosine (5g), 2′‐deoxyguanosine monohydrate (4g), or uridine (5u) (ChemGenes)
    • 2′‐Thymidine (4t), 99% (Alfa Aesar)
  • Tributylammonium pyrophosphate ( 2) (Sigma‐Aldrich)
  • 2‐Chloro‐1,3,2‐benzodioxaphosphorin‐4‐one (salicyl phosphorochloridite, 1, Sigma‐Aldrich)
  • Argon gas (dried)
  • Anhydrous N,N‐dimethylformamide (DMF, Sigma‐Aldrich)
  • Tributylamine (TBA, Sigma‐Aldrich)
  • Anhydrous dimethyl sulfoxide (DMSO, Sigma‐Aldrich)
  • Methanol (MeOH)
  • Dichloromethane (methylene chloride, CH 2Cl 2)
  • Iodine solution (Glen Research)
  • Deionized water
  • Isopropanol
  • Ammonium hydroxide (NH 4OH)
  • 3 M sodium chloride (NaCl)
  • Ethanol (200 proof, KOPTEC)
  • 20 mM triethylammonium acetate (TEAA) buffer, pH 7.1
  • Anhydrous acetonitrile, 99.8% (CH 3CN, Sigma‐Aldrich)
  • Ribonucleoside or 2′‐deoxyribonucleoside 5′‐triphosphate standard (Epicentre)
  • 5‐, 10‐, and 15‐mL oven‐dried, round‐bottom flasks
  • 8 × 1.5−mm magnetic stir bars
  • Rubber septa
  • Parafilm
  • High‐vacuum pump
  • Argon‐filled balloons: connect a deflated balloon to the top end of a 1‐mL syringe (Norm Ject) and seal the connection with Parafilm
  • 1‐ and 3‐mL syringes
  • 23‐G, 1.5‐in. (∼38‐mm) IM needles (Becton Dickinson)
  • Silica‐coated thin‐layer chromatography (TLC) plates with fluorescent indicator Kieselgel 60F 254 (Dynamic Adsorbents and Sorbent Technologies)
  • UV lamp
  • 9‐in. disposable glass pipets (Pasteur pipets)
  • 15‐ or 50‐mL Falcon tubes
  • UV‐vis spectrophotometer
  • Reversed‐phase HPLC system with 21.2 × 250−mm Welchrom (or Ultisil) C18 column
  • Lyophilizer
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D)

Basic Protocol 2: One‐Pot Synthesis of Nucleoside 5′‐(α‐P‐Thio)Triphosphates

  Materials
  • 3‐[(Dimethylaminomethylidene)amino]‐3H‐1,2,4,dithiazole‐3‐thione (sulfurizing reagent II, Glen Research)
  • Pyridine (Sigma‐Aldrich)
  • 20 mM triethylammonium acetate (TEAA) buffer, pH 6.5
  • Guanosine and 2′‐deoxyguanosine 5′‐(α‐P‐thio)triphosphate standards (GTPαS and dGTPαS, TriLinks)
  • 4.6 × 250−mm Welchrom (or Ultisil) C18 RP‐HPLC column
  • Additional reagents and equipment for one‐pot synthesis of native 5′‐triphosphates (see protocol 1)

Basic Protocol 3: One‐Pot Synthesis of Nucleoside 5′‐(α‐P‐Seleno)Triphosphates

  Materials
  • 3H‐1,2‐Benzothiaselenol‐3‐one (BTSe, SeNA Research)
  • Dioxane (Sigma‐Aldrich)
  • Triethylamine (TEA, Sigma‐Aldrich)
  • 20 mM triethylammonium acetate (TEAA) buffer, pH 6.5
  • 4.6 × 250−mm Welchrom (or Ultisil) C18 RP‐HPLC column
  • Additional reagents and equipment for one‐pot synthesis of native 5′‐triphosphates (see protocol 1)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Baddiley, J., Michelson, A.M., and Todd, A.R.J. 1949. Nucleosides Part II. A synthesis of adenosine triphosphate. Chem. Soc. 1949:582‐586.
   Bogdanov, A.A., Sumbatyan, N.V., Shishkina, A.V., Karpenko, V.V., and Korshunova, G.A. 2010. Ribosomal tunnel and translation regulation. Biochemistry 75:1501‐1516.
   Brandt, G., Carrasco, N., and Huang, Z. 2006. Efficient substrate cleavage catalyzed by hammerhead ribozymes derivatized with selenium for X‐ray crystallography. Biochemistry 45:8972‐8977.
   Brody, R.S., Adler, S., Modrich, P., Stec, W.J., Leznikowski, Z.J., and Frey, P.A. 1982. Stereochemical course of nucleotidyl transfer catalyzed by bacteriophage T7 induced DNA polymerase. Biochemistry 21:2570‐2572.
   Burgers, P.M. and Eckstein, F. 1978. Absolute configuration of the diastereomers of adenosine 5′‐O‐(1‐thio‐triphosphate): Conse‐ quences for the stereochemistry of polymerization by DNA‐dependent RNA polymerase from Escherichia coli. Proc Natl. Acad. Sci. U.S.A. 75:4798‐4800.
   Burgers, P.M. and Eckstein, F. 1979. A study of the mechanism of DNA polymerase I from Escherichia coli with diastereomeric phosphorothioate analogs of deoxyadenosine triphosphate. J. Biol. Chem. 254:6889‐6893.
   Burgers, P.M., Eckstein, F., and Hunneman, D.H. 1979. Stereochemistry of hydrolysis by snake venom phosphodiesterase. J. Biol. Chem. 254:7476‐7478.
   Burgess, K. and Cook, D. 2000. Syntheses of nucleoside triphosphates. Chem. Rev. 100:2047‐2060.
   Buzin, Y., Carrasco, N., and Huang, Z. 2004. Synthesis of selenium‐derivatized cytidine and oligonucleotides for X‐ray crystallography using MAD. Org. Lett. 6:1099‐1102.
   Carrasco, N. and Huang, Z. 2004. Enzymatic synthesis of phosphoroselenoate DNA using thymidine 5′‐(α‐P‐seleno)triphosphate and DNA polymerase for X‐ray crystallography via MAD. J. Am. Chem. Soc. 126:448‐449.
   Carrasco, N., Ginsburg, D., Du, Q., and Huang, Z. 2001. Synthesis of selenium‐derivatized nucleosides and oligonucleotides for X‐ray crystallography. Nucleosides Nucleotides Nucleic Acids 20:1723‐1734.
   Carrasco, B., Buzin, B., Tyson, E., Halpert, E., and Huang, Z. 2004. Selenium derivatization and crystallization of DNA and RNA oligonucleotides for X‐ray crystallography using multiple anomalous dispersion. Nucleic Acids Res. 32:1638‐1646.
   Carrasco, N., Caton‐Williams, J., Brandt, G., Wang, S., and Huang, Z. 2005. Efficient enzymatic synthesis of phosphoroselenoate RNA by using adenosine 5′‐(α‐P‐sleno)triphosphosphate. Angew. Chem. Int. Ed. Engl. 45:94‐97.
   Caton‐Williams, J. and Huang, H. 2008a. Biochemistry of naturally‐occurring and unatural selenium‐derivatized nucleic acids. Chem. Biodivers. 5:396‐407.
   Caton‐Williams, J. and Huang, H. 2008b. Synthesis and DNA polymerase incorporation of colored 4 seleno‐thymidine triphosphate with a single atom substitution. Angew. Chem. Int. Ed. Engl. 47:1723‐1725.
   Caton‐Williams, J., Lin, L., Smith, M., and Haung, Z. 2011a. Convenient synthesis of nucleoside 5′‐triphosphates for RNA transcription. Chem. Commun. 47:8142‐8144.
   Caton‐Williams, J., Smith, M., Carrasco, N., and Huang, Z. 2011b. Protection‐free one‐pot synthesis of 2′‐deoxynucleoside 5′‐triphosphates and DNA polymerization. Org. Lett. 13:4156‐4159.
   Caton‐Williams, J., Fiaz, B., Hoxhaj, R., Smith, M., and Huang, Z. 2012. Convenient synthesis of nucleoside 5′‐(α‐P‐thio)triphosphates and phosphorothioate nucleic acids (DNA and RNA). Sci. China Chem. 55:80‐89.
   Cheek, M.A., Dobrikov, M.I., Wennefors, C.K., Xu, Z., Hashmi, S.N., Shen, X., and Shaw, B.R. 2008. Synthesis and properties of (alpha‐P‐borano)nucleoside 5′‐triphosphate analogues as potential antiviral agents. Nucleic Acids Symp. Ser. 52:81‐82.
   Eckstein, F. 1979. Phosphorothioate analogues of nucleotides. Acc. Chem. Res. 12:204‐210.
   Eckstein, F. 1985. Nucleoside phosphorothioates. Annu. Rev. Biochem. 54:367‐402.
   Eckstein, F. and Gindl, H. 1983. Assignment of resonances in the phosophorus‐31 nuclear magnetic resonance spectrum of poly[d(A‐T)] from phosphorothioate substitution. Biochemistry 22:4546‐4550.
   Ferre‐D'Amare, A.R. 2010. Use of the spliceosomal protein U1A to facilitate crystallization and structure determination of complex RNAs. Methods 52:159‐167.
   Ferre‐D'Amare, A.R., Zhou, K., and Doudna, J.A. 1998. Crystal structure of a hepatitis delta virus ribozyme. Nature 395:567‐574.
   Frey, P.A. 1982. Practical enzymatic synthesis of adenosine 5′‐O‐(3‐thiotriphosphate) (ATP‐γ‐S). Tetrahedron 38:1547‐1567.
   Gan, J., Sheng, J., and Huang, Z. 2011. Chemical and structural biology of nucleic acids and their protein complexes for novel drug discovery. Sci. China Chem. 54:3‐23.
   Gillerman, I. and Fischer, B. 2010. An improved one‐pot synthesis of nucleoside 5′‐triphosphate analogues. Nucleosides Nucleotides Nucleic Acids 29:245‐256.
   Golubeva, N.A., Ivanov, A.V., Vanov, M.A., Batyunina, O.A., Shipitsyn, A.V., Tuniskaya, V.L., and Alexandrova, L.A. 2008. New nucleoside analogues and their 5′‐triphosphates: Synthesis and biological properties. Moscow Univ. Chem. Bull. 63:89‐93.
   Hassan, A.E.A., Sheng, J., Jiang, J., Zhang, W., and Huang, Z. 2009. Synthesis and crystallographic analysis of 5‐Se‐thymidine DNAs. Org. Lett. 11:2503‐2506.
   He, K., Hasan, A., Krzyzanowska, B., and Shaw, B.R. 1998. Synthesis and separation of diastereomers of ribonucleoside 5′‐(alpha‐P‐borano)triphosphates. J. Org. Chem. 63:5769‐5773.
   Horhota, A.T., Szostak, J.W., and McLaughlin, L.W. 2006. Glycerol nucleoside triphosphates: Synthesis and polymerase substrate activities. Org. Lett. 8:5345‐5347.
   Jansen, R.S., Rosing, H., Schellens, J.H., and Beijnen, J.H. 2010. Facile small scale synthesis of nucleoside 5′‐phosphate mixtures. Nucleosides Nucleotides Nucleic Acids 29:14‐26.
   Juliano, R., Alam, M.R., Dixit, V., and Kang, H. 2008. Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides. Nucleic Acids Res. 36:4158‐4171.
   Ke, A. and Doudna, J.A. 2004. Crystallization of RNA and RNA‐protein complexes. Methods 34:408‐414.
   Keel, A.Y., Rambo, R.P., Batey, R.T., and Kieft, J.S. 2007. A general strategy to solve the phase problem in RNA crystallography. Structure 15:761‐772.
   Koldobskaya, Y., Duguid, E.M., Shechner, D.M., Suslov, N.B., Ye, J., Sidhu, S.S., Bartel, D.P., Koide, S., Kossiakoff, A.A., and Piccirili, J.A. 2011. A portable RNA sequence whose recognition by a synthetic antibody facilitates structural determination. Nat. Struct. Mol. Biol. 18:100‐106.
   Krishna, H. and Caruthers, M.H. 2011. Solid‐phase synthesis, thermal denaturation studies, nuclease resistance, and cellular uptake of (oligodeoxyribonucleoside) methylborane phosphine‐DNA chimeras. J. Am. Chem. Soc. 133:9844‐9854.
   Kunkel, T.A., Eckstein, F., Mildvan, A.S., Koplitz, R.M., and Loeb, L.A. 1981. Deoxynucleoside (1‐thio)triphosphates prevent proof reading during in vitro DNA synthesis. Proc. Natl. Acad. Sci. U.S.A. 78:6734‐6738.
   Levin, A.A. 1999. Antisense drug technology: Principles, strategies, and applications. Biochim. Biophys. Acta 1489:69‐84.
   Lin, J. and Shaw, B.R. 2000. Synthesis of a novel triphosphate analogue: Nucleoside alpha‐P‐borano, alpha‐P‐thiotriphosphate. Chem. Commun. 2000:2115‐2116.
   Lin, L., Sheng, J., Momin, R.K., Du, Q., and Huang, Z. 2009. Facile synthesis and anti‐tumor cell activity of Se‐containing nucleosides. Nucleosides, Nucleotides, Nucleic Acids 28:56‐66.
   Lin, L., Caton‐Williams, J., Kaur, M., Patino, A.M., Sheng, J., Punetha, J. and Huang, Z. 2011a. Facile synthesis of nucleoside 5′‐(alpha‐P‐seleno)triphosphates and phoshoroselenoate RNA transcription. RNA 17:1932‐1938.
   Lin, L., Sheng, J., and Huang, Z. 2011b. Nucleic acid X‐ray crystallography via direct selenium derivatization. Chem. Soc. Rev. 40:4591‐4602.
   Lorenz, P., Baker, B.F, Bennett, C.F., and Spector, D.L. 1998. Phosphorothioate antisense oligonucleotides induce the formation of nuclear bodies. Mol. Biol. Cell 9:1007‐1023.
   Ludwig, J. and Eckstein, F. 1989. Rapid and efficient synthesis of nucleoside 5′‐O‐(1‐thiotriphosphates), 5′‐triphosphates and 2′,3′‐cyclophosphorothioates using 2‐chloro‐4H‐1,3,2‐benzo‐dioxaphosphorin‐4‐one. J. Org. Chem. 54:631‐635.
   Ludwig, J. and Eckstein, F. 1991. Synthesis of nucleoside 5′‐O‐(1,3‐dithiotriphosphates) and 5′‐O‐(1,1‐dithiotriphosphates). J. Org. Chem. 56:1777‐1783.
   Misiura, K., Szymanowicz, D., and Stec, W.J. 2005. Synthesis of nucleoside alpha‐thiotriphosphates via an oxathiaphospholane approach. Org Lett. 7:2217‐2220.
   Mori, K.B., Cazenave, C., Matsukura, M., Subasinghe, C., Cohen, J.S., Broder, S., Toulmé, J.J., and Stein, C.A. 1989. Phosphoroselenoate oligodeoxynucleotides: Synthesis, physico‐chemical characterization, antisense inhibitory properties and anti‐HIV activity. Nucleic Acids Res. 17:8207‐8219.
   Romaniuk, P.J. and Eckstein, F. 1982. A study of the mechanism of T4 DNA polymerase with diastereomeric phosphorothioate analogues of deoxyadenosine triphosphate. J. Biol. Chem. 257:7684‐7688.
   Salon, J., Sheng, J., Jiang, J., Chen, G., Caton‐Williams, J., and Huang, Z. 2007. Oxygen replacement with selenium at the thymidine 4‐position for the Se‐base‐paring and crystal structure studies. J. Am. Chem. Soc. 129:4862‐4863.
   Salon, J., Jiang, J., Sheng, J., Gerlits, O.O., and Huang, Z. 2008. Derivatization of DNAs with selenium at 6‐position of guanine for function and crystal structure studies. Nucleic Acids Res. 36:7009‐7018.
   Schultheisz, H.L., Szymczyna, B.R., Scott, L.G., and Williamson, J.R.J. 2010. Enzymatic de novo pyrimidine nucleotide synthesis. J. Am. Chem. Soc. 133:297‐304.
   Sheng, J. and Huang, Z. 2008. Selenium derivatization of nucleic acids for phase determination in nucleic acid X‐ray crystallography. Int. J. Mol. Sci. 9:258‐271.
   Soutourina, J., Wydau, S., Ambroise, Y., Boschiero, C., and Werner, M. 2011. Direct interaction of RNA polymerase II and mediator required for transcription in vivo. Science 331:1451‐1454.
   Storz, G. 2002. An expanding universe of noncoding RNAs. Science 296:1260‐1263.
   Summerton, J., Stein, D., Huang, S.B., Matthews, P., Weller, D., and Partridge, M. 1997. Morpholino and phosphorothioate antisense oligomers compared in cell‐free and in‐cell systems. Antisense Nucleic Acid Drug Dev. 7:63‐70.
   Sun, Q., Edathil, J.P., Wu, R., Smidansky, E.D., Cameron, C.E., and Peterson, B.R. 2008. One‐pot synthesis of nucleoside 5′‐triphosphates from nucleoside 5′‐H‐phosphonates. Org. Lett. 10:1703‐1706.
   Ueda, T., Tohda, H., Chikazumi, N., Eckstein, F., and Watanabe, K. 1991. Phosphorothioate‐containing RNAs show mRNA activity in the prokaryotic translation systems in vitro. Nucleic Acids Res. 19:547‐552.
   Warnecke, S. and Meier, C.J. 2009. Synthesis of nucleoside di‐ and triphosphates and dinucleoside polyphosphates with cycloSal‐ nucleotides. J. Org. Chem. 74:3024‐3030.
   Wu, W., Freel Meyers, C.L. and Borch, R.F. 2004. A novel method for the preparation of nucleoside triphosphates from activated nucleoside phosphoramidates. Org. Lett. 6:2257‐2260.
   Yoshikawa, M., Kato, T., and Takenishi, T. 1967. A novel method for phosphorylation of nucleosides to 5′‐nucleotides. Tetrahedron Lett. 50:5065‐5068.
   Zou, K., Horhota, A., Yu, B., Szostak, J.W., and McLaughlin, L.W. 2005. Synthesis of alpha‐l‐threofuranosyl nucleoside triphosphates (tNTPs). Org. Lett. 7:1485‐1487.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library