Synthesis of Building Blocks and Oligonucleotides with {G}O6‐Alkyl‐O6{G} Cross‐Links

Christopher J. Wilds1, Jason D. M. Booth1, Anne M. Noronha1

1 Concordia University, Montreal, Quebec, Canada
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 5.9
DOI:  10.1002/0471142700.nc0509s44
Online Posting Date:  March, 2011
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Abstract

This unit describes two methods for preparing oligonucleotides containing an O6‐2′‐deoxyguanosine‐alkyl‐O6‐2′‐deoxyguanosine interstrand cross‐link by a solid‐phase synthesis approach. Depending on the desired orientation of the cross‐link in the DNA duplex, either a bis‐ or a mono‐phosphoramidite synthesis strategy can be employed. Both procedures require the preparation of a protected 2′‐deoxyguanosine‐containing dimer where the two nucleosides are attached at the O6‐atoms by an alkyl linker. This linker is introduced as a protected diol using two successive Mitsunobu reactions to produce a cross‐linked amidite that is incorporated into an oligonucleotide via solid‐phase synthesis. The use of a protected diol lends versatility to this method, as cross‐links of variable chain length may be prepared. The bis‐phosphoramidite approach is a direct method to preparing the cross‐linked duplex, whereas the mono‐phosphoramidite strategy requires additional manipulation of the solid support to prepare cross‐linked oligonucleotides. Once all synthetic steps are completed, these oligonucleotides can then be removed from the solid support and deprotected, and then purified via ion‐exchange HPLC to produce sufficient quantities of substrates that can be used in DNA repair studies. Curr. Protoc. Nucleic Acid Chem. 44:5.9.1‐5.9.19. © 2011 by John Wiley & Sons, Inc.

Keywords: interstrand cross‐link; phosphoramidite; oligonucleotide synthesis; chemical synthesis; solid‐phase synthesis; DNA repair

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Protection of the Diol with a tert‐Butyldiphenyl Silyl Group
  • Basic Protocol 2: Synthesis of O6‐2′‐Deoxyguanosine‐Alkyl‐O6‐2′‐Deoxyguanosine‐3′‐O‐bis‐Phosphoramidites
  • Basic Protocol 3: Synthesis of 1‐{O6‐[3′‐O‐tert‐Butyldimethysilyl‐5′‐O‐Dimethoxytrityl‐N2‐Phenoxyacetyl‐2′‐Deoxyguanidyl]}‐7‐{O6‐[5′‐O‐Dimethoxytrityl‐N2‐Phenoxyacetyl‐2′‐Deoxyguanidyl‐3′‐O‐(β‐2‐Cyanoethyl‐N,N′‐Diisopropyl)Phosphoramidite]}‐Heptane
  • Basic Protocol 4: Solid‐Phase Synthesis and Deprotection of O6‐2′‐Deoxyguanosine‐Alkyl‐O6‐2′‐Deoxyguanosine Oligonucleotides
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Protection of the Diol with a tert‐Butyldiphenyl Silyl Group

  Materials
  • Argon gas
  • 1,4‐butanediol or 1,7‐heptanediol (Aldrich Chemical)
  • Imidazole (Aldrich Chemical)
  • Anhydrous tetrahydrofuran (THF; Aldrich Chemical)
  • Tert‐butyldiphenylchlorosilane (TBDPS‐Cl; Aldrich Chemical)
  • Dichloromethane (DCM, reagent grade; Caledon Laboratory Chemicals)
  • 5% (w/v) Aqueous sodium bicarbonate (NaHCO 3)
  • Magnesium sulfate (MgSO 4)
  • Hexanes (reagent grade; Caledon Laboratory Chemicals)
  • Ethyl acetate (EtOAc, reagent grade)
  • 50‐mL round‐bottom flask with a rubber septum
  • Magnetic stir plate and stir bar
  • 25‐mL glass syringe
  • TLC plate, EMD silica gel 60 F 254
  • Rotary evaporator and chemically resistant dry vacuum pump
  • 500‐mL separatory funnel
  • Glass funnel and filter paper
  • Silica gel (60 Å, 230 to 400 mesh)
  • 3 × 50–cm and 5 × 50–cm chromatography column
  • Vacuum manifold
  • Vacuum pump
  • Additional reagents and equipment for thin‐layer chromatography ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 2: Synthesis of O6‐2′‐Deoxyguanosine‐Alkyl‐O6‐2′‐Deoxyguanosine‐3′‐O‐bis‐Phosphoramidites

  Materials
  • 3′‐O‐alloxycarbonyl‐5′‐O‐dimethoxytrityl‐N2‐phenoxyacetyl‐2′‐deoxyguanosine (see McManus et al., )
  • Anhydrous 1,4‐dioxane
  • 1‐Otert‐Butyldiphenylsilylbutanol ( S.1; protocol 1)
  • Triphenylphosphine (Aldrich Chemical)
  • Argon gas
  • Diisopropylazodicarboxylate (Aldrich Chemical)
  • Dichloromethane (DCM; reagent grade)
  • 5% (w/v) Aqueous sodium bicarbonate (NaHCO 3)
  • Magnesium sulfate (MgSO 4)
  • Silica gel (60 Å, 230 to 400 mesh)
  • Hexanes (reagent grade)
  • Ethyl acetate (EtOAc; reagent grade)
  • Anhydrous tetrahydrofuran (THF)
  • Tetrabutylammonium fluoride (1 M in THF)
  • Sodium sulfate (Na 2SO 4)
  • Palladium (0) tetrakistriphenylphosphine (Aldrich Chemical)
  • Butylamine (Aldrich Chemical)
  • Formic acid (Aldrich Chemical)
  • Diisopropylethylamine (Aldrich Chemical)
  • N,N‐diisopropylamino cyanoethyl phosphonamidic chloride (ChemGenes)
  • 10‐, 50, 100, and 250‐mL round‐bottom flasks with a rubber septum
  • Magnetic stir plate and stir bar
  • 1‐, 2‐, 10‐, and 25‐mL glass syringes
  • Rotary evaporator and chemically resistant dry vacuum pump
  • 100‐ and 250‐mL Separatory funnel
  • Glass funnel and filter paper
  • 2 × 50–cm and 10 × 70–cm flash chromatography columns
  • Vacuum manifold
  • Vacuum pump
  • Additional reagents and equipment for column chromatography ( appendix 3E)

Basic Protocol 3: Synthesis of 1‐{O6‐[3′‐O‐tert‐Butyldimethysilyl‐5′‐O‐Dimethoxytrityl‐N2‐Phenoxyacetyl‐2′‐Deoxyguanidyl]}‐7‐{O6‐[5′‐O‐Dimethoxytrityl‐N2‐Phenoxyacetyl‐2′‐Deoxyguanidyl‐3′‐O‐(β‐2‐Cyanoethyl‐N,N′‐Diisopropyl)Phosphoramidite]}‐Heptane

  Materials
  • 3′‐O‐alloxycarbonyl‐5′‐O‐dimethoxytrityl‐N2‐phenoxyacetyl‐2′‐deoxyguanosine (see McManus et al., )
  • Anhydrous 1,4‐dioxane (Aldrich Chemical)
  • 1‐O‐tert‐Butyldiphenylsilylheptanol ( S.2; protocol 1)
  • Triphenylphosphine (Aldrich Chemical)
  • Argon gas
  • Dichloromethane (DCM; reagent grade)
  • 5% (w/v) Aqueous sodium bicarbonate (NaHCO 3)
  • Magnesium sulfate (MgSO 4)
  • Silica gel (60 Å, 230 to 400 mesh)
  • Anhydrous tetrahydrofuran (THF)
  • Tetrabutylammonium fluoride (1 M in THF)
  • Sodium sulfate (Na 2SO 4)
  • Hexanes (reagent grade)
  • Ethyl acetate (EtOAc; reagent grade)
  • Diisopropylazodicarboxylate (Aldrich Chemical)
  • Palladium (0) tetrakistriphenylphosphine (Aldrich Chemical)
  • Butylamine (Aldrich Chemical)
  • Formic acid (Aldrich Chemical)
  • Diisopropylethylamine (Aldrich Chemical)
  • N,N‐diisopropylamino cyanoethyl phosphonamidic chloride (ChemGenes)
  • 10‐, 25‐, 50‐ and 100‐mL round‐bottom flasks with a rubber septum
  • Magnetic stir plate and stir bar
  • 1‐, 2‐, and 10‐mL glass syringes
  • Rotary evaporator and chemically resistant dry vacuum pump
  • 100‐ and 250‐mL Separatory funnels
  • Glass funnel and filter paper
  • Vacuum manifold
  • Vacuum pump
  • 3 × 70–cm and 4 × 70cm Flash chromatography columns
  • Additional reagents and equipment for column chromatography ( appendix 3E)

Basic Protocol 4: Solid‐Phase Synthesis and Deprotection of O6‐2′‐Deoxyguanosine‐Alkyl‐O6‐2′‐Deoxyguanosine Oligonucleotides

  Materials
  • 5′‐O‐DMT‐2′‐deoxynucleoside‐3′‐O‐succinate long‐chain alkylamine controlled‐pore glass (LCAA‐CPG, 500 Å; Glen Research)
  • Protected 2′‐deoxyribonucleoside 3′‐phosphoramidites (Glen Research)
  • Anhydrous acetonitrile (MeCN; Applied Biosystems)
  • O6‐2′‐deoxyguanosine‐alkyl‐O6‐2′‐deoxyguanosine bis‐ or mono‐phosphoramidite ( S.7 or S.12; Basic Protocols protocol 22 and protocol 33, respectively)
  • 4Å activated molecular sieves
  • Argon
  • Ammonium hydroxide, concentrated (conc. NH 4OH)
  • Anhydrous ethanol (EtOH)
  • Triethylamine (TEA)
  • Triethylamine trihydrofluoride (TEA⋅3HF; Aldrich Chemical)
  • Protected 2′‐deoxyribonucleoside 5′‐phosphoramidites (ChemGenes)
  • Screw‐capped columns
  • Brown bottles
  • DNA synthesizer (Applied Biosystems)
  • 2‐mL screw‐capped microcentrifuge tubes
  • 55°C sand bath
  • DNA concentrator
  • 5′‐O‐DMT‐2′‐deoxynucleoside polystyrene columns (Glen Research)
  • 3‐mL disposable syringes
  • Vacuum pump
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Figures

Videos

Literature Cited

Literature Cited
   Borowy‐Borowski, H. and Chambers, R.W. 1987. A study of side reactions occurring during synthesis of oligodeoxynucleotides containing O6‐alkyldeoxyguanosine residues at preselected sites Biochemistry 26:2465‐2471.
   Braich, R.S. and Damha, M.J. 1997. Regiospecific solid‐phase synthesis of branched oligonucleotides. Effect of vicinal 2′,5′‐ (or 2′,3′‐) and 3′,5′‐phosphodiester linkages on the formation of hairpin DNA. Bioconjug. Chem. 8:370‐377.
   Damha, M.J. and Ogilvie, K.K. 1993. Oligoribonucleotide synthesis. In Protocols for Oligonucleotides and Analogues: Synthesis and Properties, Methods in Molecular Biology (S. Agrawal, ed.) pp. 84‐114. Humana Press, Totowa, N.J.
   Fang, Q., Noronha, A.M., Murphy, S.P., Wilds, C.J., Tubbs, J.L., Tainer, J.A., Chowdhury, G., Guengerich, F.P. and Pegg, A.E. 2008. Repair of O6‐G‐alkyl‐O6‐G interstrand cross‐links by human O6‐alkylguanine‐DNA alkyltransferase. Biochemistry 47:10892‐10903.
   McManus, F.P., Fang, Q., Booth, J.D.M., Noronha, A.M., Pegg, A.E. and Wilds, C.J. 2010. Synthesis and characterization of oligonucleotides containing an O6‐2′‐deoxyguanosine‐alkyl‐O6‐2′‐deoxyguanosine interstrand cross‐link in a 5′‐GNC motif and repair by human O6‐alkylguanine‐DNA alkyltransferase. Org. Biomol. Chem. 8:4414‐4426.
   Mitsunobu, O. 1981. The use of diethyl azodicarboxylate and triphenylphosphine in synthesis and transformation of natural products. Synthesis 1:1‐28.
   Noll, D.M., Mason, T.M., and Miller, P.S. 2006. Formation and repair of interstrand cross‐links in DNA. Chem. Rev. 106:277‐301.
   Spratt, T.E. and Campbell, C.R. 1994. Synthesis of oligodeoxyribonucleotides containing analogs of O6‐methylguanine and reaction with O6‐alkylguanine‐DNA alkyltransferase. Biochemistry 33:11364‐11371.
   Wilds, C.J., Noronha, A.M., Robidoux, S., and Miller, P.S. 2004. Mispair‐aligned N3T‐alkyl‐N3T interstrand cross‐linked DNA: Synthesis and characterization of duplexes with interstrand cross‐links of variable lengths. J. Am. Chem. Soc. 126:9257‐9265.
   Wilds, C.J., Booth, J.D., and Noronha, A.M. 2006. Synthesis of oligonucleotides containing an O6‐G‐alkyl‐O6‐G interstrand cross‐link. Tetrahedron Lett. 47:9125‐9128.
   Zhu, Q., Delaney, M.O., and Greenberg, M.M. 2001. Observation and elimination of N‐acetylation of oligonucleotides prepared using fast‐deprotecting phosphoramidites and ultra‐mild deprotection. Bioorg. Med. Chem. Lett. 11:1105‐1107.
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