Synthesis and Polymerase Incorporation of 5′‐Amino‐2′,5′‐Dideoxy‐5′‐N‐Triphosphate Nucleotides

Jia Liu Wolfe1, Tomohiko Kawate1

1 Massachusetts General Hospital, Cambridge, Massachusetts
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 13.3
DOI:  10.1002/0471142700.nc1303s18
Online Posting Date:  October, 2004
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit presents synthetic procedures for the preparation of 5′‐amino‐2′,5′‐dideoxy analogs of adenosine, cytidine, guanosine, and thymidine, as well as corresponding 5′‐N‐triphosphate nucleotides, using commercially available reagents. The modified nucleosides are prepared in high yields from naturally occurring 2′‐deoxynucleosides using robust chemical reactions including tosylation, azide exchange, and the Staudinger reaction. Efficient conversion of these 5′‐amino nucleosides to corresponding 5′‐N‐triphosphate nucleotides is achieved through a one‐step reaction with trimetaphosphate in Tris‐buffered aqueous solution. The 5′‐amino modification renders these nucleoside and nucleotide analogs markedly increased reactivity, which is useful for a variety of biochemical, pharmaceutical, and genomic applications. Also included in this unit are protocols for polymerase incorporation of the 5′‐amino nucleotides, either partially or completely replacing their naturally occurring counterparts, and subsequent sequence‐specific cleavage at the modified nucleotides under mildly acidic conditions.

Keywords: amine; azide; modified nucleosides; modified nucleotide triphosphates; chemical synthesis; polymerase incorporation; sequence specific DNA cleavage

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

Table of Contents

  • Basic Protocol 1: Preparation of 5′‐Azido‐2′,5′‐Dideoxypurines
  • Basic Protocol 2: Preparation of 5′‐Azido‐2′,5′‐Dideoxypyrimidines
  • Basic Protocol 3: Preparation of 5′‐Amino‐2′,5′‐Dideoxynucleosides
  • Basic Protocol 4: Preparation and HPLC Analysis of 5′‐Amino‐2′,5′‐Dideoxy‐5′‐N‐Triphosphate Nucleotides
  • Basic Protocol 5: Polymerase Incorporation of NH2‐dNTPS
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of 5′‐Azido‐2′,5′‐Dideoxypurines

  Materials
  • Dry nitrogen (N 2) or argon (Ar)
  • N6‐Benzoyl‐2′‐deoxyadenosine (N6‐Bz‐dA; S.1)
  • Pyridine, anhydrous
  • p‐Toluenesulfonyl chloride (TsCl)
  • N2‐Isobutyryl‐2′‐deoxyguanosine (N2i‐Bu‐dG; S.5)
  • Ethyl acetate (EtOAc)
  • Saturated aqueous sodium bicarbonate (NaHCO 3)
  • Saturated aqueous sodium chloride (brine)
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Dichloromethane (CH 2Cl 2)
  • Silica gel 60, 230 to 400 mesh
  • Methanol (MeOH)
  • Lithium azide (LiN 3), concentrated to dryness from a 20% solution in dH 2O
  • Dimethyl sulfoxide (DMSO), anhydrous
  • Ammonium hydroxide (NH 4OH), concentrated aqueous solution
  • Chloroform (CHCl 3)
  • Dry ice
  • 250‐, 100‐, 50‐, and 25‐mL round‐bottom flasks, oven dried
  • Inert atmosphere/vacuum manifold with dry ice/2‐propanol trap
  • Balloons
  • Tubing adaptor
  • 10‐mL syringes
  • 18‐G needles
  • Rotary evaporator with a built‐in dry ice/2‐propanol trap, attached to a vacuum pump
  • 250‐mL separatory funnels
  • 250‐mL Erlenmeyer flasks
  • Fritted funnels
  • 250‐mL glass chromatography columns
  • Test tubes
  • Thin‐layer chromatography (TLC) plates, 0.25‐mm silica gel 60F‐254 on glass plates
  • UV light source
  • 60° and 50°C oil baths
  • Vacuum pump
  • 10‐mL screw‐capped micro‐vials (e.g., Accuform vials, Kimble)
  • Lyophilizer
  • Additional reagents and equipment for column chromatography ( appendix 3E) and TLC ( appendix 3D)

Basic Protocol 2: Preparation of 5′‐Azido‐2′,5′‐Dideoxypyrimidines

  Materials
  • N4‐Benzoyl‐2′‐deoxycytidine (N4‐Bz‐dC; S.9)
  • Dimethylformamide (DMF), anhydrous
  • Lithium azide (LiN 3), concentrated to dryness from a 20% solution in dH 2O
  • Triphenylphosphine (Ph 3P)
  • Dry nitrogen (N 2) or argon (Ar)
  • Carbon tetrabromide (CBr 4)
  • Silica gel 60, 230 to 400 mesh
  • Chloroform (CHCl 3)
  • Methanol (MeOH)
  • Dichloromethane (CH 2Cl 2)
  • Pyridine, anhydrous
  • Ammonium hydroxide (NH 4OH), concentrated aqueous solution
  • Ethyl acetate (EtOAc)
  • Diethyl ether (Et 2O)
  • Dry ice
  • 25‐, 100‐, and 250‐mL round‐bottom flasks, oven dried
  • Rotary evaporator with dry ice/2‐propanol trap, attached to a vacuum pump
  • Vacuum pump
  • Inert atmosphere/vacuum manifold with dry ice/2‐propanol trap
  • Tubing adaptor
  • Balloons
  • 250‐mL glass chromatography column
  • Thin‐layer chromatography (TLC) plates, 0.25‐mm silica gel 60F‐254 on glass
  • UV light source
  • 10‐mL screw‐capped micro‐vials (e.g., Accuform vials, Kimble)
  • 60°C oil bath
  • 100‐mL separatory funnel
  • Lyophilizer
  • Additional reagents and equipment for column chromatography ( appendix 3E) and TLC ( appendix 3D)

Basic Protocol 3: Preparation of 5′‐Amino‐2′,5′‐Dideoxynucleosides

  Materials
  • N 3‐dA, N 3‐dG, and N 3‐dC (see Basic Protocols protocol 11 and protocol 22)
  • Pyridine, anhydrous
  • Triphenylphosphine (Ph 3P)
  • Dry nitrogen (N 2) or argon (Ar)
  • Ammonium hydroxide (NH 4OH), concentrated aqueous solution
  • Ethyl acetate (EtOAc)
  • 25‐mL round‐bottom flask
  • Rotary evaporator with dry ice/2‐propanol trap, attached to a vacuum pump
  • Inert atmosphere/vacuum manifold with dry ice/2‐propanol trap
  • Tubing adaptor
  • Balloon
  • 55°C oil bath
  • Fritted funnel
  • Lyophilizer

Basic Protocol 4: Preparation and HPLC Analysis of 5′‐Amino‐2′,5′‐Dideoxy‐5′‐N‐Triphosphate Nucleotides

  Materials
  • NH 2‐dA, NH 2‐dC, NH 2‐dG (see protocol 3), and NH 2‐dT (Sigma)
  • Trisodium trimetaphosphate (TMP)
  • 0.5 M aqueous Tris base
  • HPLC buffers A and B (see reciperecipes)
  • 1.5‐mL microcentrifuge tubes
  • Vortex mixer
  • C18 column (Waters Nova‐pak; 3.9 × 150 mm, 4 µm)
  • Additional reagents and equipment for HPLC (unit 10.5)

Basic Protocol 5: Polymerase Incorporation of NH2‐dNTPS

  Materials
  • 20 µM oligonucleotide primer
  • 1.67 µM [γ‐32P]ATP (6000 Ci/mmol; Perkin‐Elmer)
  • 10 U/µL T4 polynucleotide kinase and 10× buffer (New England Biolabs)
  • TE buffer, pH 8.0 ( appendix 2A)
  • Single‐stranded DNA templates: ∼100 or 500 nucleotides in length (for complete or partial NH 2‐dNTP substitution, respectively)
  • 200 mM MgCl 2
  • 500 mM sodium acetate (NaOAc)
  • 10× polymerase extension buffers A and B (see reciperecipes)
  • 100 mM NH 2‐dNTP solution(s) in 0.5 M Tris (see protocol 4)
  • 2′‐Deoxynucleoside triphosphates (dATP, dCTP, dGTP, dTTP)
  • 5 U/µL Klenow fragment of DNA polymerase (exo; New England Biolabs)
  • 1% and 10% (v/v) acetic acid
  • Formamide loading buffer (Life Technologies)
  • Denaturing polyacrylamide gel mix and buffer solutions (National Diagnostics)
  • 1× TBE buffer ( appendix 2A)
  • 10 mM EDTA, pH 8 ( appendix 2A)
  • 0.5‐mL microcentrifuge tubes
  • Heat block at 37° and 90°C
  • Sephadex G‐50 columns, preswollen in dH 2O
  • Speedvac evaporator
  • Additional reagents and equipment for polyacrylamide gel electrophoresis (PAGE; unit 10.4)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Bergmann, F. and Bannwarth, W. 1995. Solid‐phase synthesis of directly linked peptide‐oligodeoxynucleotide hybrids using standard synthesis protocols. Tetrahedron Lett. 36:1839‐1842.
   Koppitz, M., Nielsen, P.E., and Orgel, L.E. 1998. Formation of oligonucleotide‐PNA‐chimeras by template‐directed ligation. J. Am. Chem. Soc. 120:4563‐4569.
   Letsinger, R.L., Hapke, B., Petersen, G.R., and Dumas, L.B. 1976a. Enzymatic synthesis of duplex circular phiX174 DNA containing phosphoramidate bonds in the (−) strand. Nucl. Acids Res. 3:1053‐1063.
   Letsinger, R.L., Wilkes, J.S., and Dumas, L.B. 1976b. Incorporation of 5′‐amino‐5′‐deoxythymidine‐5′‐phosphate in polynucleotides by use of DNA polymerase I and a phiX174 DNA template. Biochemistry 15:2810‐2816.
   Luo, P.Z., Leitzel, J.C., Zhan, Z.Y.J., and Lynn, D.G. 1998. Analysis of the structure and stability of a backbone‐modified oligonucleotide: Implications for avoiding product inhibition in catalytic template‐directed synthesis. J. Am. Chem. Soc. 120:3019‐3031.
   Lutz, M.J., Benner, S.A., Hein, S., Breipohl, G., and Uhlmann, E. 1997. Recognition of uncharged polyamide‐linked nucleic acid analogs by DNA polymerases and reverse transcriptases. J. Am. Chem. Soc. 119:3177‐3178.
   Mag, M. and Engels, J.W. 1989. Synthesis and selective cleavage of oligodeoxyribonucleotides containing non‐chiral internucleotide phosphoramidate linkages. Nucl. Acids Res. 17:5973‐5988.
   Mungal, W.S., Greene, G.L., Heavner, G.A., and Letsinger, R.L. 1975. Use of the azido group in the synthesis of 5′‐terminal aminodeoxythymidine oligonucleotides. J. Org. Chem. 40:1659‐1662.
   Shchepinov, M.S., Denissenko, M.F., Smylie, K.J., Worl, R.J., Leppin, A.L., Cantor, C.R., and Rodi, C.P. 2001. Matrix‐induced fragmentation of P3′‐N5′ phosphoramidate‐containing DNA: High‐throughput MALDI‐TOF analysis of genomic sequence polymorphisms. Nucl. Acids Res. 29:3864‐3872.
   Tetzlaff, C.N., Schwope, I., Bleczinski, C.F., Steinberg, J.A., and Richert, C. 1998. A convenient synthesis of 5′‐amino‐5′‐deoxythymidine and preparation of peptide‐DNA hybrids. Tetrahedron Lett. 39:4215‐4218.
   Wolfe, J.L., Kawate, T., Belenky, A., and Stanton, V. Jr. 2002. Synthesis and polymerase incorporation of 5′‐amino‐2′,5′‐dideoxy‐5′‐N‐triphosphate nucleotides. Nucl. Acids Res. 30:3739‐3747.
   Wolfe, J.L., Wang, B., Kawate, T., and Stanton, V.P. Jr. 2003. Sequence‐specific dinucleotide cleavage promoted by synergistic interactions between neighboring modified nucleotides in DNA. J. Am. Chem. Soc. 125:10500‐10501.
   Yamamoto, I., Sekine, M., and Hata, T. 1980. One‐step synthesis of 5′‐azido‐nucleosides. J. Chem. Soc. Perkin I 306‐310.
   Zhan, Z.Y.J. and Lynn, D.G. 1997. Chemical amplification through template‐directed synthesis. J. Am. Chem. Soc. 119:12420‐12421.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library