Palladium‐Mediated C5 Substitution of Pyrimidine Nucleosides

Mohammad Ahmadian1, Douglas A. Klewer2, Donald E. Bergstrom3

1 Cerus Corp., Concord, California, 2 Texas A&M University, College Station, Texas, 3 Purdue University, West Lafayette, Indiana
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.1
DOI:  10.1002/0471142700.nc0101s00
Online Posting Date:  May, 2001
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Abstract

One of the most efficient ways to link a reporter group to oligonucleotides is through the incorporation of a modified nucleoside during automated oligonucleotide synthesis. To be useful, it is important that the reporter group not interfere in hybridization reactions. This unit describes two linkers that can be used for the incorporation of a reporter group at the C5 position of deoxyuridine: a flexible aminoethylthioether linker, and a rigid amidopropynyl linker. The latter is suffciently long and positioned so that the reporter group lies outside the major groove of the DNA duplex.

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis of 5‐(3‐Nicotinamidopropyn‐1‐yl)‐5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐Deoxyuridine
  • Basic Protocol 2: Synthesis of 5‐(3‐Acetamido‐1‐Thiapropyl)‐2′‐Deoxyuridine
  • Support Protocol 1: Synthesis of N,N′‐Bis (Trifluoroacetyl)Cystamine
  • Support Protocol 2: Synthesis of N‐Acetoxysuccinimide
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of 5‐(3‐Nicotinamidopropyn‐1‐yl)‐5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐Deoxyuridine

  Materials
  • Nicotinoyl chloride hydrochloride
  • Pyridine, anhydrous
  • Nitrogen (N 2) stream
  • Triethylamine, freshly distilled (dried and purified by distillation at atmospheric pressure over calcium hydride; boiling point = 89° to 90°C)
  • Propargylamine, reagent grade (typically 99% pure)
  • Dichloromethane, reagent grade
  • 10% (w/v) hydrochloric acid in water
  • Sodium sulfate, anhydrous
  • Silica gel (230 to 400 mesh)
  • Methanol, reagent grade
  • 5‐Iodo‐2′‐deoxyuridine
  • 4,4′‐Dimethoxytrityl chloride
  • Diethyl ether, anhydrous
  • N,N‐Dimethylformamide, anhydrous
  • Argon gas (optional)
  • Tetrakis(triphenylphosphine)palladium, [(C 6H 5) 3P] 4Pd
  • Copper(I) iodide
  • 5% (w/v) Na 2EDTA in water
  • Ethyl acetate, reagent grade
  • 25‐ and 50‐mL round‐bottom flasks
  • Inert atmosphere/vacuum manifold (see Fig. )
  • 500‐µL and 1‐mL syringes with stainless steel needles
  • 125‐ and 250‐mL Ehrlenmeyer flask
  • 100‐mL separatory funnel
  • Filter funnel and Whatman no. 1 filter paper
  • Chromatotron and radial chromatography plate coated with silica gel (2‐mm thickness; Harrison Research)
  • Rotary evaporator with vacuum pump and water aspirator
  • Glass column (2‐cm i.d. × ≥20‐cm length) with stopcock
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 4D)

Basic Protocol 2: Synthesis of 5‐(3‐Acetamido‐1‐Thiapropyl)‐2′‐Deoxyuridine

  Materials
  • 2′‐Deoxyuridine
  • Mercury(II) acetate
  • 30% (w/v) and 0.1 M sodium chloride (reagent grade) in water
  • Ethanol, anhydrous
  • Diethyl ether, anhydrous
  • N,N′‐Bis(trifluoroacetyl)cystamine (see protocol 3)
  • 0.1 M Li 2PdCl 4 solution (see recipe)
  • Hydrogen sulfide (H 2S)
  • Methanol, reagent grade
  • Chloroform, reagent grade
  • Silica gel (230 to 400 mesh)
  • Concentrated ammonium hydroxide
  • Dry ice/acetone (for freezing)
  • N‐Acetoxysuccinimide (see protocol 4)
  • Triethylamine, freshly distilled (dried and purified by distillation at atmospheric pressure over calcium hydride; boiling point = 89° to 90°C)
  • Tetrahydrofuran, anhydrous
  • Ethyl acetate, reagent grade
  • 100‐ and 200‐mL round‐bottom flasks
  • Temperature‐controlled magnetic stirrer
  • Mortar and pestle
  • Whatman no. 1 filter paper
  • 100‐mm‐diameter porcelain Buchner funnel
  • Rotary evaporator with water aspirator
  • Glass chromatography column (2‐cm i.d. × ≥20‐cm length) with stopcock
  • Lyophilizer

Support Protocol 1: Synthesis of N,N′‐Bis (Trifluoroacetyl)Cystamine

  Materials
  • Chloroform, reagent grade
  • Cystamine dihydrochloride
  • Triethylamine, freshly distilled (dried and purified by distillation at atmospheric pressure over calcium hydride; boiling point = 89° to 90°C)
  • Trifluoroacetic anhydride
  • 10% (w/v) NaHCO 3
  • 2 N HCl
  • Sodium sulfate, anhydrous
  • Methanol, reagent grade
  • Ethyl acetate, reagent grade
  • Hexane, reagent grade
  • 1‐liter round‐bottom flask
  • Drying tube containing Drierite
  • 5‐mL syringe
  • 1‐liter separatory funnel
  • Rotary evaporator with water aspirator
  • Vacuum oven at 35°C
  • Buchner funnel and Whatman no. 1 filter paper

Support Protocol 2: Synthesis of N‐Acetoxysuccinimide

  Materials
  • N‐Hydroxysuccinamide
  • Tetrahydrofuran, anhydrous
  • Nitrogen (N 2) gas
  • Glacial acetic acid
  • Dicyclohexylcarbodiimide (DCC)
  • Silica gel (optional; 230 to 400 mesh)
  • Ethyl acetate, reagent grade
  • Methanol, reagent grade
  • Vacuum manifold apparatus (Fig. ) modified with a 10‐mL conical flask and a 500‐µL syringe
  • Filter funnel and Whatman no. 1 filter paper
  • Glass chromatography column (optional; 2‐cm i.d. × 10‐cm length) with stopcock
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Figures

Videos

Literature Cited

Literature Cited
   Ahmadian, M., Zhang, P., and Bergstrom, D.E. 1998. A comparative study of the thermal stability of oligodeoxyribonucleotides containing 5‐substituted‐2′‐deoxyuridines. Nucl. Acids Res. 26:3127‐3135.
   Bashkin, J.K., Sondhi, S.M., Sampath, U., d'Avignon, D.A., and Modak, A.S. 1994. Synthesis and connectivity assignment (by 2D‐NMR) of a nucleoside‐dipeptide: 5‐[3‐[[2‐[[2‐[[[2‐Amino]‐1‐oxo‐3‐[1H‐imidazol‐4‐yl]propyl]amino]‐1‐ oxo‐3‐[1H‐imidazol‐4‐yl]propyl]amino]ethyl] amino]‐3‐oxopropyl]‐2′‐deoxyuridine. New J. Chem. 18:305‐318.
   Bergstrom, D.E. and Chen, J. 1996. Sequence‐specific oligodeoxyribonucletide cleavage by a major‐groove‐positioned metal‐binding ligand tethered to C‐5 of deoxyuridine. Bioorg. Med. Chem. Lett. 6:2211‐2214.
   Bergstrom, D.E. and Ogawa, M.K. 1978. C‐5 substituted pyrimidine nucleosides. 2. Synthesis via olefin coupling to organopalladium intermediates derived from uridine and 2′‐deoxyuridine. J. Am. Chem. Soc. 100:8106‐8112.
   Bergstrom, D.E. and Ruth, J.L. 1977. Preparation of C‐5 mercurated pyrimidine nucleosides. J. Carbohydrates Nucleotides Nucleosides 42:257‐269.
   Bergstrom, D.E., Beal, P., Jenson, J., and Lin, X. 1991. Palladium‐mediated synthesis of C‐5 pyrimidine nucleoside thioethers from disulfides and mercurinucleosides. J. Org. Chem. 56:5598‐5602.
   Chaudhuri, N.C. and Kool, E.T. 1995. Very high affinity DNA recognition by bicyclic and cross‐linked oligonucleotides. J. Am. Chem. Soc. 117:10434‐10442.
   Cook, A.F., Vuocolo, E., and Brakel, C.L. 1988. Synthesis and hybridization of a series of bio‐tinylated oligonucleotides. Nucl. Acids. Res. 16:4077‐4095.
   Dreyer, G.B. and Dervan, P.B. 1985. Sequence‐specific cleavage of single‐stranded DNA: Oligodeoxynucleotide‐EDTA.Fe(II). Proc. Natl. Acad. Sci. U.S.A. 82:968‐972.
   Gibson, K.J. and Benkovic, S.J. 1987. Synthesis and application of derivatizable oligonucleotides. Nucl. Acids Res. 15:6455‐6467.
   Goodchild, J. 1990. Conjugates of oligonucleotides and modified oligonucleotides: A review of their synthesis and properties. Bioconjugate Chem. 1:165‐187.
   Hagmar, P., Bailey, M., Tong, G., Haralambidis, J., Sawyer, W.H., and Davidson, B.E. 1995. Synthesis and characterization of fluorescent oligonucleotides. Effect of internal labelling on protein recognition. Biochim. Biophys. Acta 1244:259‐268.
   Hobbs, F.W. Jr. 1989. Palladium‐catalyzed synthesis of alkynylamino nucleosides. A universal linker for nucleic acids. J. Org. Chem. 54:3420‐3422.
   Jones, R.A. 1984. Preparation of protected deoxyribonucleosides. In Oligonucleotide Synthesis: A Practical Approach (M.J. Gait, ed.) pp. 27‐28. IRL Press, Washington,D.C.
   Kirchner, J.J., Hustedt, E.J., Robinson, B.H., and Hopkins, P.B. 1990. DNA dynamics from a spin probe:Dependence of probe motion on tether length. Tetrahedron Lett. 31:593‐596.
   Kwiatkowski, M., Samiotaki, M., Lamminmaki, U., Mukkala, V.‐M., and Landegren, U. 1994. Solid‐phase synthesis of chelate‐labelled oligonucleoties:Application in triple‐color ligase‐mediated gene analysis. Nucl. Acids Res. 22:2604‐2611.
   Langer, P.R., Waldrop, A.A., and Ward, D.C. 1981. Enzymatic synthesis of biotin‐labeled polynucleotides: Novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. U.S.A. 78:6633‐6637.
   Meyer, K.L. and Hanna, M.M. 1996. Synthesis and characterization of a new 5‐thiol‐protected deoxyuridine for site‐specific modification of DNA. Bioconjugate Chem. 7:401‐412.
   Prober, J.M., Trainor, G.L., Dam, R.J., Hobbs, F.W., Robertson, C.W., Zagursky, R.J., Cocuzza, A.J., Jensen, M.A., and Baumeister, K. 1987. A system for rapid DNA sequencing with fluorescent chain‐terminating dideoxynucleotides. Science 238:336‐341.
   Robins, M.J. and Barr, P.J. 1981. Nucleic acid related compounds. 31. Smooth and efficient palladium‐copper catalyzed coupling of terminal alkynes with 5‐iodouracil nucleosides. Tetrahedron Lett. 22:421‐424.
   Robins, M.J. and Barr, P.J. 1983. Nucleic acid related compounds. 39. Efficient conversation of 5‐iodo to 5‐alkynyl and derived 5‐substituted uracil bases and nucleosides. J. Org. Chem. 48:1854‐1862.
   Sagi, J., Szemzo, A., Ebinger, K., Szabolcs, A., Sagi, G., Ruff, E., and Otvos, L. 1993. Base‐modified oligodeoxynucleotides. I. Effect of 5‐alkyl, 5‐(1‐alkenyl) and 5‐(1‐alkynyl) substitution of the pyrimidines on duplex stability and hydrophobicity. Tetrahedron Lett. 34:2191‐2194.
   Shah, K., Neenhold, H., Wang, Z., and Rana, T.M. 1996. Incorporation of an artificial protease and nuclease at the HIV‐1 Tat binding site of trans‐activation responsive RNA. Bioconjugate Chem. 7:283‐289.
   Shimkus, M., Levy, J., and Herman, T. 1985. A chemically cleavable biotinylated nucleotide: Usefulness in the recovery of protein‐DNA complexes from avidin affinity columns. Proc. Natl. Acad. Sci. U.S.A. 82:2593‐2597.
   Spaltenstein, A., Robinson, B.H., and Hopkins, P.B. 1988. A rigid and nonperturbing probe for duplex DNA motion. J. Am. Chem. Soc. 110:1299‐1301.
   Spaltenstein, A., Robinson, B.H., and Hopkins, P.B. 1989. Sequence‐and structure‐dependent DNA base dynamics: Synthesis,structure, and dynamics of site and sequence specifically spin‐labeled DNA. Biochemistry 28:9484‐9495.
   Tabone, J.C., Stamm, M.R., Gamper, H.B., and Meyer, R.B. Jr. 1994. Factors influencing the extent and regiospecificity of cross‐link formation between single‐stranded DNA and reactive complementary oligodeoxynucleotides. Biochemistry 33:375‐383.
   Tesler, J., Cruickshank, K.A., Morrison, L.E., and Netzel, T. 1989. Synthesis and characterization of DNA oligomers and duplex containing covalently attached molecular labels: Comparison of biotin,fluorescin, and pyrene labels by thermodynamic and optical spectroscopic measurements. J. Am. Chem. Soc. 111:6966‐6976.
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