Chemoenzymatic Preparation of Nucleoside Triphosphates

Weidong Wu1, Donald E. Bergstrom1, V. Jo Davisson1

1 Purdue University, West Lafayette, Indiana
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 13.2
DOI:  10.1002/0471142700.nc1302s16
Online Posting Date:  May, 2004
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Abstract

The design and synthesis of alternative nucleoside triphosphate substrates for DNA and RNA polymerases holds continued promise to create biochemical probes and precursors for synthesis of nucleic acid mimics. The azole carboxamide nucleotides are of particular interest, as they display multiple conformations in the context of DNA replication. An efficient chemoenzymatic preparation of azole carboxamide deoxyribo‐ and ribonucleoside triphosphates is presented. Nucleoside diphosphate is prepared from nucleoside 5′‐O‐tosylate by displacement with tris(tetra‐n‐butylammonium) pyrophosphate. Enzymatic phosphorylation of the azole carboxamide deoxyribonucleoside diphosphate to its triphosphate is based on ATP as the phosphate donor and nucleoside diphosphate kinase as the catalyst, coupled with phospho(enol)pyruvate (PEP) and pyruvate kinase as an ATP regeneration system. Enzymatic phosphorylation of the azole carboxamide ribonucleoside diphosphate requires PEP as the phosphate donor and pyruvate kinase as the catalyst. The optimized purification uses boronate affinity gel to yield highly purified nucleoside triphosphate.

Keywords: nucleoside diphosphate; nucleoside triphosphate; synthesis, kinase

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis of Azole Carboxamide Deoxyribonucleoside Triphosphates
  • Basic Protocol 2: Synthesis of Azole Carboxamide Ribonucleoside Triphosphates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of Azole Carboxamide Deoxyribonucleoside Triphosphates

  Materials
  • Azole carboxamide deoxyribonucleoside (Fig. ): S.1 (N = TzA 4; Makabe et al., ), S.2 (N = TzA 3; Witkowski et al., ), or S.3 (N = Tz 2A 4; Makabe et al., )
  • Pyridine, anhydrous
  • Argon
  • p‐Toluenesulfonyl chloride (TsCl)
  • Ethyl acetate, ACS reagent grade
  • 5% (w/v) phosphomolybdic acid solution (see recipe)
  • Methanol, ACS reagent grade
  • 200‐ to 400‐mesh silica gel 60
  • Hexanes, ACS reagent grade
  • Phosphorus pentoxide (P 2O 5)
  • Dowex AG 50W‐X8 cation‐exchange resin (100 to 200 mesh, Bio‐Rad)
  • 1 M HCl
  • Disodium dihydrogen pyrophosphate
  • 1 M and concentrated (28%) ammonium hydroxide (NH 4OH)
  • 40% (w/v) tetra‐n‐butylammonium hydroxide solution (Aldrich)
  • Acetonitrile, anhydrous
  • Deuterated acetonitrile (Aldrich)
  • CF11 fibrous cellulose powder (Whatman)
  • 50 mM, 100 mM, and 500 mM ammonium bicarbonate (NH 4HCO 3) solutions
  • (no pH adjustment)
  • 1% (w/v) sulfosalicylic solution (see recipe)
  • 0.2% (w/v) ferric chloride solution (see recipe)
  • Triethanolamine hydrochloride (Sigma)
  • Magnesium chloride hexahydrate (MgCl·6H 2O)
  • Potassium chloride (KCl)
  • Adenosine triphosphate (ATP, disodium salt, Sigma)
  • Sodium phosphoenolpyruvate monohydrate (PEP; Sigma)
  • 1 M sodium hydroxide (NaOH)
  • Nucleoside diphosphate kinase (see recipe)
  • Pyruvate kinase (see recipe)
  • Mobile phase A: 0.1 M potassium phosphate buffer (pH 6.0; appendix 2A) containing 4 mM tetrabutylammonium dihydrogenphosphate (TBAP, Aldrich; added from a 1.0 M stock)
  • Mobile phase B: 70:30 (v/v) mobile phase A/methanol, pH 7.2
  • Adenosine diphosphate (ADP, sodium salt, Sigma)
  • Affi‐Gel 601 boronate affinity gel (Bio‐Rad)
  • 1 M ammonium bicarbonate (NH 4HCO 3), pH 8.5 (see recipe)
  • Carbon dioxide source (e.g., dry ice)
  • Q Sepharose FF anion‐exchange resin (Bio‐Rad)
  • 1 M potassium chloride (KCl)
  • 10‐ and 20‐mL oven‐dried one‐neck round‐bottom flasks with rubber septa
  • Rotary evaporator equipped with a water aspirator and a temperature‐controlled water bath (45°C)
  • Capillary tubes
  • TLC plates:
    • Silica gel 60 F 254 polyester‐backed TLC plates (Aldrich)
    • Cellulose TLC plates (EM Science)
  • Heat gun
  • 254‐nm UV lamp
  • 2.0 × 25–cm, 2.5 × 5–cm, 2.5 × 10–cm, 2.5 × 20–cm, and 2.5 × 25–cm chromatography columns
  • Vacuum desiccator
  • 250‐mL and 1‐L beakers
  • 250‐mL round‐bottom flasks
  • Lyophilizer
  • TLC sprayer (Analtech)
  • 15‐mL polystyrene round‐bottom tube
  • HPLC system (e.g., Beckman System Gold) including:
    • 128 solvent module
    • 166 detector set at 230 nm
    • 25‐cm × 4.6‐mm × 5‐µm‐i.d. Supelcosil LC‐18‐T column
  • 25‐mm‐diameter, 0.2‐µm nylon syringe filter (Fisher)
  • Peristaltic pump
  • Medium‐pressure liquid chromatography (MPLC) system (e.g., ISCO LC system) with following equipment:
    • Model 2360 gradient programmer
    • Model 2350 HPLC pump
    • V4 variable wavelength absorbance detector
  • Additional reagents and equipment for thin‐layer chromatography (TLC, appendix 3D), column chromatography ( appendix 3E), and 1H NMR, 13C NMR, 31P NMR, and ESI‐MS
NOTE: All the nucleosides used in the reaction are dried under reduced pressure (0.1 Torr) in the presence of phosphorus pentoxide at 50°C in an Abderhalden drying apparatus (Ace Glass) overnight prior to reaction.

Basic Protocol 2: Synthesis of Azole Carboxamide Ribonucleoside Triphosphates

  Materials
  • Azole carboxamide ribonucleoside (Fig. ): S.13 (N = rTz 2A 4; Lehmkuhl et al., ) or S.14 (N = rTzA 4; Lehmkuhl et al., )
  • p‐Toluenesulfonic acid monohydrate (TsOH; Aldrich)
  • Tetrahydrofuran (THF), anhydrous
  • Trimethyl orthoformate [(CH 3O) 3CH; Aldrich], anhydrous
  • Methylene chloride, anhydrous (distill from phosphorus pentoxide and store over 4‐Å molecular sieves)
  • Methanol, ACS reagent grade
  • 5% phosphomolybdic acid solution (see recipe)
  • Pyridine, anhydrous
  • p‐Toluenesulfonyl chloride (TsCl)
  • 4‐N,N‐Dimethylaminopyridine (DMAP)
  • Ethyl acetate, ACS reagent grade
  • 200‐ to 400‐mesh silica gel 60
  • Hexane, ACS reagent grade
  • Phosphorus pentoxide (P 2O 5)
  • Acetonitrile, anhydrous
  • Argon
  • Tris(tetra‐n‐butylammonium) hydrogen pyrophosphate (see protocol 1)
  • Deuterated acetonitrile (Aldrich)
  • Dowex AG 50W‐X8 cation‐exchange resin (100 to 200 mesh, Bio‐Rad)
  • 1 M HCl
  • 1 M and concentrated (28%) ammonium hydroxide (NH 4OH)
  • Trifluoroacetic acid (TFA)
  • Mobile phase A: 0.1 M potassium phosphate buffer, pH 6.0 ( appendix 2A) containing 4 mM tetrabutylammonium dihydrogenphosphate (TBAP, Aldrich; added from a 1.0 M stock)
  • Mobile phase B: 70:30 (v/v) mobile phase A/methanol, pH 7.2
  • CF11 fibrous cellulose (Whatman)
  • 50, 100, and 500 mM ammonium bicarbonate (NH 4HCO 3) solutions
  • (no pH adjustment)
  • 1% (w/v) sulfosalicylic solution (see recipe)
  • 0.2% (w/v) ferric chloride solution (see recipe)
  • Triethanolamine
  • Magnesium chloride hexahydrate (MgCl·6H 2O)
  • Potassium chloride (KCl)
  • Sodium phosphoenolpyruvate monohydrate (PEP; Sigma)
  • 1 M sodium hydroxide (NaOH)
  • Pyruvate kinase (see recipe)
  • Ammonium bicarbonate
  • Ammonium hydroxide, concentrated
  • Affi‐Gel 601 boronate affinity gel (Bio‐Rad)
  • 1 M ammonium bicarbonate (NH 4HCO 3), pH 8.5 (see recipe)
  • Carbon dioxide source (e.g., dry ice)
  • Q Sepharose FF anion‐exchange resin (Bio‐Rad)
  • 1 M potassium chloride (KCl)
  • 20‐mL one‐neck round‐bottom flasks, oven dried
  • TLC plates:
    • Silica gel 60 F 254 polyester‐backed TLC plates (Aldrich)
    • Cellulose TLC plates (EM Science)
  • Heat gun
  • 254‐nm UV light lamp
  • Rotary evaporator with adjustable temperature and water aspirator
  • 2 × 25–cm, 2.5 × 5–cm, 2.5 × 10–cm, 2.5 × 20‐cm, and 2.5 × 25–cm chromatography columns
  • Vacuum desiccator
  • 250‐mL round‐bottom flask
  • Lyophilizer
  • HPLC system (e.g., Beckman System Gold) including:
    • 128 Solvent Module
    • 166 Detector set at 230 nm
    • 25‐cm × 4.6‐mm × 5‐µm‐i.d. Supelcosil LC‐18‐T column
  • 1‐L beaker
  • TLC sprayer (Analtech)
  • 25‐mm‐diameter, 0.2‐µm nylon syringe filter (Fisher)
  • Peristaltic pump
  • Medium‐pressure liquid chromatography (MPLC) system (e.g., ISCO LC system) with following equipment:
    • Model 2360 gradient programmer
    • Model 2350 HPLC pump
    • V4 variable wavelength absorbance detector
  • Additional reagents and equipment for thin‐layer chromatography (TLC, appendix 3D), column chromatography ( appendix 3E), and 1H NMR, 13C NMR, 31P NMR, and ESI‐MS
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Figures

Videos

Literature Cited

   Burgess, K. and Cook, D. 2000. Syntheses of nucleoside triphosphates. Chem. Rev. 100:2047‐2060.
   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.
   Hirschbein, B.L., Mazenod, F.P., and Whitesides, G.M. 1982. Synthesis of phosphoenolypyruvate and its use in ATP cofactor regeneration. J. Org. Chem. 47:3765‐3766.
   Hoard, D.E. and Ott, D.G. 1965. Conversion of mono‐ and oligodeoxyribonucleotides to 5′‐triphosphates. J. Am. Chem. Soc. 87:1785‐1788.
   Hoops, G.C., Zhang, P., Johnson, W.T., Paul, N., Bergstrom, D.E., and Davisson, V.J. 1997. Template directed incorporation of nucleotide mixtures using azole‐nucleobase analogs. Nucl. Acids Res. 25:4866‐4871.
   Kamiya, H. and Kasai, H. 1999. Preparation of 8‐hydroxy‐dGTP and 2‐hydroxy‐dATP by a phosphate transfer reaction by nucleoside‐diphosphate kinase. Nucleos. Nucleot. Nucl. Acids 18:307‐310.
   Lehmkuhl, F.A., Witkowski, J.T., and Robins, R.K. 1972. Synthesis of 1,2,3‐triazole nucleosides via the acid‐catalyzed fusion procedure. J. Heterocyclic Chem. 9:1195‐1201.
   Ludwig, J. 1981. A new route to nucleoside 5′‐triphosphates. Acta Biochim. Biophys. Acad. Sci. 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′‐cyclophosphorothioates using 2‐chloro‐4H‐1,3,2‐benzodioxaphosphorin‐4‐one. J. Org. Chem. 54:631‐635.
   Makabe, O., Suzuki, H., and Umezawa, S. 1977. Syntheisi of D‐arabinofuranosyl and 2′‐deoxy‐D‐ribofuranosyl 1,2,3‐triazolecarboxamides. Bull. Chem. Soc. Jpn. 50:2689‐2693.
   Miller, W.H., Daluge, S.M., Garvey, E.P., Hopkins, S., Reardon, J.E., Boyd, F.L., and Miller, R.L. 1992. Phosphorylation of carbovir enantiomers by cellular enzymes determines the stereoselectivity of antiviral activity. J. Biol. Chem. 267:21220‐21224.
   Mishra, N.C. and Broom, A.D. 1991. A novel synthesis of nucleoside triphosphates. J. Chem. Soc. Chem. Comm. 1276‐1277.
   Moffatt, J.G. and Khorama, H.G. 1961. Nucleoside polyphosphates. X1. The synthesis and some reactions of nucleoside‐5′ phosphoromorpholidates and related compounds. Improved methods for the preparation of nucleoside‐5′ polyphosphates. J. Am. Chem. Soc. 83:649‐658.
   Mourad, N. and Parks, R.E. Jr. 1966. Erythrocytic nucleoside diphosphokinase. II. Isolation and kinetics. J. Biol. Chem. 241: 271‐278.
   Ratliff, R.C., Weaver, R.H., Lardy, H.A., and Kuby, S.A. 1964. Nucleoside triphosphate‐nucleoside diphosphate transphosphorylase (nucleoside diphosphokinase). I. Isolation of the crystalline enzyme from brewer's yeast. J. Biol. Chem. 239:301‐309.
   Schott, H. 1972. New dihydroxyboryl‐substituted polymers for column‐chromatographic separation of ribonucleoside‐deoxyribonucleoside mixtures. Angew. Chem. Int. Ed. Engl. 11:824‐825.
   Schott, H., Rudloff, E., Schmidt, P., Roychoudhury, R., and Kossel, H. 1973. A dihydroxyboryl‐substituted methacrylic polymer for the column chromatographic separation of mononucleotides, oligonucleotides, and transfer ribonucleic acid. Biochemistry 12:932‐938.
   Simon, E.S., Bednarski, M.D., and Whitesides, G.M. 1988. Generation of cytidine 5′‐triphosphate using adenylate kinase. Tetrahedron Lett. 29:1123‐1126.
   Simoncsits, A. and Tomasz, J. 1975. Nucleoside 5′‐phosphordiamidates, synthesis and some properties. Nucl. Acids Res. 2:1223‐1233.
   Tomasz, J., Simoncsits, A., Kajtar, M., Krug, R.M., and Shatkin, A.J. 1978. Chemical synthesis of 5′‐pyrophosphate and triphosphate derivatives of 3′‐5′ ApA, ApG, GpA and GpG. CD study of the effect of 5′‐phosphate groups on the conformation of 3′‐5′ GpG. Nucl. Acids Res. 5:2945‐2957.
   van Boom, J.H., Crea, R., Luyten, W.C., and Vink, A.B. 1975. 2,2,2‐Tribromoethyl phosphoromorpholinochloridate: A convenient reagent for the synthesis of ribonucleoside mono‐, di‐ and tri‐phosphates. Tetrahedron Lett. 16:2779‐2782.
   Witkowski, J.T., Fuertes, M., Cook, P.D., and Robins, R.K. 1975. Nucleosides of 1,2,4‐triazole‐3‐carboxamide. Synthesis of certain pentofuranosyl, deoxypentofuranosyl, and pentopyranosyl 1,2,4‐triazoles. J. Carbohydr. Nucleos. Nucleot. 2:1‐36.
   Wong, C.H., Haynie, S.L., and Whitesides, G.M. 1983. Preparation of a mixture of nucleoside triphosphates from yeast RNA: Use in enzymic synthesis requiring nucleoside triphosphate regeneration and conversion to nucleoside diphosphate sugars. J. Am. Chem. Soc. 105:115‐117.
   Wu, W., Bergstrom, D.E., and Davisson, V.J. 2003. A combination chemical and enzymatic approach for the preparation of azole carboxamide nucleoside triphosphate. J. Org. Chem. 68:3860‐3865.
   Yoshikawa, M., Kato, T., and Takenishi, T. 1967. A novel method for phosphorylation of nucleosides to 5′‐nucleotides. Tetrahedron Lett. 50:5065‐5068.
   Zhang, P., Johnson, W.T., Klewer, D., Paul, N., Hoops, G., Davisson, V.J., and Bergstrom, D.E. 1998. Exploratory studies on azole carboxamides as nucleobase analogs: Thermal denaturation studies on oligodeoxyribonucleotide duplexes containing pyrrole‐3‐carboxamide. Nucl. Acids Res. 26:2208‐2215.
Key References
  Burgess and Cook, 2000. See above.
  This paper gives a review on syntheses of nucleoside triphosphates.
  Davisson et al., 1987. See above.
  This paper describes synthesis of nucleoside diphosphates from 5′‐O‐tosyl nucleosides.
  Wu et al., 2003. See above.
  This paper describes synthesis of the azole nucleoside triphosphate and compound characterization.
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