Synthesis, Characterization, and Application of Substituted Pyrazolopyrimidine Nucleosides

Robert O. Dempcy1, Mikhail A. Podyminogin1, Michael W. Reed1

1 Epoch Biosciences, Bothell, Washington
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.8
DOI:  10.1002/0471142700.nc0108s11
Online Posting Date:  February, 2003
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Abstract

This unit describes, in detail, the preparation of 3‐aminopropyl‐substituted pyrazolo[3,4‐d]pyrimidine analogs of the purines deoxyadenosine (dA) and deoxyguanosine (dG). Phosphoramidite reagents of these so‐called aminopropyl‐PPA and ‐PPG nucleosides (AP‐PPA and AP‐PPG, respectively) allow introduction of amino linkers into internal positions of synthetic DNA strands. Synthesis of suitably protected AP‐PPA and AP‐PPG phosphoramidites are described. The stepwise alkynylation, hydrogenation, selective protection, and phosphoramidite synthesis is similar for both the PPA and PPG analogs. To demonstrate the application of these reagents, a protocol is given in which a simple DNA strand is synthesized and conjugated to a lipophilic activated ester (dabcyl‐SE) to form a stable amide linkage. Utility of this chemistry for preparing internally modified DNA conjugates is discussed.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Protected Aminopropyl‐PPG Phosphoramidite
  • Basic Protocol 2: Preparation of Protected Aminopropyl‐PPA Phosphoramidite
  • Basic Protocol 3: Synthesis of DNA Conjugates Using AP‐PPA and AP‐PPG Phosphoramidites
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Protected Aminopropyl‐PPG Phosphoramidite

  Materials
  • 6‐Amino‐1‐(2‐deoxy‐β‐D‐erythro‐pentofuranosyl)‐3‐iodopyrazolo[3,4‐d]pyrimidin‐4(5H)‐one (Seela and Zulauf, )
  • CuI
  • Tetrakis[triphenylphosphine]palladium[0] (Aldrich)
  • Argon
  • Anhydrous dimethylformamide (DMF), deoxygenated (i.e., argon purged)
  • Anhydrous triethylamine
  • N‐Trifluoroacetylpropargylamine (Cruickshank and Stockwell, )
  • Chloroform
  • 230‐ to 400‐mesh (40‐ to 63‐µm) silica gel (EM Sciences)
  • Methanol
  • Ethyl acetate
  • Diethyl ether
  • recipe20% (w/v) palladium hydroxide on carbon, preactivated (see reciperecipe)
  • 4 M triethylammonium formate buffer, pH 6.5 (see recipe)
  • Hydrogen gas
  • Celite (1.5‐cm pad)
  • N,N‐Dimethylformamide dimethylacetal (Aldrich)
  • Xylenes
  • Anhydrous pyridine
  • 4,4′‐Dimethoxytrityl chloride
  • 5% (w/v) sodium bicarbonate
  • Sodium sulfate
  • Anhydrous methylene chloride
  • N,N‐Diisopropylethylamine
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite
  • Hexane
  • 100‐mL round‐bottom flask
  • Rotary evaporator equipped with a vacuum pump
  • 5 × 35–cm chromatography columns
  • Silica gel 60 F254 aluminum‐backed TLC plates (EM Science)
  • 254‐nm UV lamp
  • Oil pump (>1 mmHg)
  • 500‐mL Parr hydrogenation bottle
  • 250‐mL separatory funnel
  • Additional reagents and equipment for column chromatography ( appendix 3E) and thin‐layer chromatography (TLC; appendix 3D)
NOTE: 1H NMR spectra were obtained at 300 MHz on a Varian VXR‐300 spectrometer. Two‐dimensional COSY and NOE experiments assisted in the assignment of proton resonances. Elemental analyses were performed by Quantitative Technologies.NOTE: Like most phosphoramidite reagents, AP‐PPA and ‐PPG are sensitive to hydrolysis and oxidation and must be handled with care after isolation. Desiccated storage at −20°C is recommended. Performance in DNA synthesis depends on good quality reagents; only reagent‐grade chemicals should be used to synthesize these phosphoramidites. Characterization of the compounds as described here helps ensure purity.

Basic Protocol 2: Preparation of Protected Aminopropyl‐PPA Phosphoramidite

  Materials
  • 4‐Amino‐1‐(2‐deoxy‐β‐D‐erythro‐pentofuranosyl)‐3‐iodo‐1H‐pyrazolo[3,4‐d]pyrimidine (Seela and Zulauf, )
  • Celite (1.5–cm pad)
  • N,N‐Dimethylacetamide dimethylacetal (TCI America)
  • Anhydrous triethylamine
  • Anhydrous dimethylformamide (DMF)
  • Xylenes
  • 4,4′‐Dimethoxytrityl chloride
  • Anhydrous pyridine
  • 230‐ to 400‐mesh (40‐ to 63‐µm) silica gel (EM Sciences)
  • Ethyl acetate
  • Methylene chloride
  • Methanol
  • 5 × 35–cm chromatography column
  • Additional reagents and equipment for synthesizing AP‐PPG (see protocol 1)

Basic Protocol 3: Synthesis of DNA Conjugates Using AP‐PPA and AP‐PPG Phosphoramidites

  Materials
  • AP‐PPA‐modified 9‐mer oligonucleotide from a 1‐µmol‐scale synthesis, trityl‐on (e.g., Epoch Biosciences)
  • 3 M sodium acetate
  • Butanol
  • Ethanol
  • Solvent A: 0.1 M triethylammonium acetate, pH 7.5
  • Solvent B: acetonitrile
  • Anhydrous dimethylsulfoxide (DMSO; e.g., Aldrich Sure‐seal; store over calcium hydride)
  • Anhydrous triethylamine (Fluka; store over calcium hydride)
  • N‐Hydroxysuccinimidyl ester of dabcyl (dabcyl‐SE; Molecular Probes)
  • 2% (w/v) sodium perchlorate in acetone
  • Acetone
  • Centrifugal evaporator (i.e., Savant Speedvac) with high vacuum source (<1 mmHg)
  • High‐performance liquid chromatograph (HPLC) with:
  •  4.6 × 250–mm C18 analytical column and appropriate guard column (Varian Dynamax)
  •  UV‐Vis or photodiode array detector
  • 14‐mL polypropylene tubes
  • UV‐Vis spectrophotometer
  • Additional reagents and equipment for RP‐HPLC and detritylation (unit 10.5)
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Figures

Videos

Literature Cited

Literature Cited
   Beaucage, S.L. and Iyer, R.P. 1993. The synthesis of modified oligonucleotides by the phosphoramidite approach and their applications. Tetrahedron 49:6123‐6194.
   Cruickshank, K.A. and Stockwell, D.L. 1988. Oligonucleotide labeling: A concise synthesis of a modified thymidine phosphoramidite. Tetrahedron Lett. 29:5221‐5224.
   Goodchild, J. 1990. Conjugates of oligonucleotides and modified oligonucleotides: A review of their synthesis and properties. Bioconjugate Chem. 1:165‐187.
   Karamychev, V.N., Panyutin, I.G., Reed, M.W., and Neumann, R.D. 1997. Effect of radionuclide linker structure on DNA cleavage by 125I‐labeled oligonucleotides. Antisense Nucl. Acid Drug Dev. 7:549‐557.
   Kutyavin, I.V., Afonina, I.A., Mills, A., Gorn, V.V., Lukhtanov, E.A., Belousov, E.S., Singer, M.J., Walburger, D.K., Lokhov, S.G., Gall, A.A., Dempcy, R., Reed, M.W., Meyer, R.B.J., and Hedgpeth, J. 2000. 3′‐Minor groove binder‐DNA probes increase sequence specificity at PCR extension temperatures. Nucl. Acids Res. 28:655‐661.
   Milesi, D., Kutyavin, I.V., Lukhtanov, E.A., Gorn, V.V., and Reed, M.W. 1999. Synthesis of oligonucleotides in anhydrous dimethyl sulfoxide. Methods Enzymol. 313:164‐173.
   Petrie, C.R., Adams, A.D., Stamm, M., Van Ness, J., Watanabe, S.M., and Meyer, R.B.J. 1991. A novel biotinylated adenylate analogue derived from pyrazolo[3,4‐d]pyrimidine for labeling DNA probes. Bioconjugate Chem. 2:441‐446.
   Podyminogin, M.A., Meyer, R.B.J., and Gamper, H.B. 1996. RecA‐catalyzed, sequence‐specific alkylation of DNA by cross‐linking oligonucleotides. Effects of length and nonhomologous base substitutions. Biochemistry 35:7267‐7274.
   Seela, F. and Becher, G. 1998. Synthesis of 7‐halogenated 8‐aza‐7‐deaza‐2′‐deoxyguanosines and related pyrazolo[3,4‐d]pyrimidine 2′‐deoxyribonucleotides. Synthesis (9):207‐214.
   Seela, F. and Becher, G. 1999. Oligonucleotides containing pyrazolo[3,4‐d]pyrimidines: The influence of 7‐substituted 8‐aza‐7‐deaza‐2′‐deoxyguanosines on the duplex structure and stability. Helv. Chim. Acta 82:1640‐1655.
   Seela, F. and Zulauf, M. 1998. Synthesis of 7‐alkynylated 8‐aza‐7‐deaza‐2′‐deoxyadenosines via the Pd‐catalysed cross‐coupling reaction. J. Chem. Soc. Perkin Trans. 1(19):3233‐3240.
   Seela, F. and Zulauf, M. 1999. Synthesis of oligonucleotides containing pyrazolo[3,4‐d]pyrimidines: The influence of 7‐substituted 8‐aza‐7‐deazaadenines on the duplex structure and stability. J. Chem. Soc. Perkin Trans. 1(4):479‐488.
   Seela, F., Ramzaeva, N., and Becher, G. 1996. 7‐Deazapurine DNA: Oligonucleotides containing 7‐substituted 7‐deaza‐2′‐deoxyguanosine and 8‐aza‐7‐deaza‐2′‐deoxyguanosine. Collect. Czech. Chem. Commun. 61:258‐261.
   Tyagi, S., Bratu, D.P., and Kramer, F.R. 1998. Multicolor molecular beacons for allele discrimination. Nature Biotech. 16:49‐53.
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