Synthesis of Building Blocks and Oligonucleotides with {T}N3‐Alkylene‐N3{T} Cross‐Links

Gang Sun1, Anne M. Noronha1, Paul S. Miller2, Christopher J. Wilds1

1 Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada, 2 Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 5.11
DOI:  10.1002/0471142700.nc0511s51
Online Posting Date:  December, 2012
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Abstract

This unit describes two methods to directly prepare oligonucleotide duplexes containing an N3thymidine‐alkylene‐N3thymidine inter‐strand cross‐link. The inter‐strand cross‐link can be engineered into the duplex with a number of possible orientations. Both methods require the preparation of a protected thymidine dimer where the N3 atoms of the two nucleosides are covalently attached by an alkyl linker. This linker is prepared starting from a protected diol using two successive alkylation reactions under basic conditions to accomplish the alkylation selectively at the N3 atom of the nucleoside. The chain length of the cross‐link can be varied based on the selection of the diol used in the dimer synthesis. The solid‐phase mono‐phosphoramidite approach involves oligonucleotide synthesis with 3′‐O‐phosphoramidites, on‐column removal of a 3′‐Otert‐butyldimethylsilyl protecting group, and continued oligonucleotide synthesis with 5′‐O‐phosphoramidites. The bis‐phosphoramidite approach does not require synthesis with 5′‐O‐phosphoramidites. At the end of synthesis using either method, the N3thymidine‐alkylene‐N3thymidine inter‐strand cross‐linked oligonucleotides can be removed from the solid‐support and purified using standard techniques (ion‐exchange HPLC) in yields sufficient for various structural studies and repair assays. Curr. Protoc. Nucleic Acid Chem. 51:5.11.1‐5.11.17. © 2012 by John Wiley & Sons, Inc.

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

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of the 4‐Iodo‐Butyl Phenoxyacetate Linker
  • Basic Protocol 2: Synthesis of 1‐{N3‐[3′‐O‐Tert‐Butyldimethylsilyl‐5′‐O‐(4,4′‐Dimethoxytrityl)‐Thymidinyl]}‐4‐{N3‐[3′‐O‐(Diisopropylamino)‐(2‐Cyanoethoxy)Phosphino‐ 5′‐O‐(4,4′‐Dimethoxytrityl)‐Thymidinyl]}‐Butane
  • Basic Protocol 3: Synthesis of N3‐Thymidine‐Alkylene‐N3‐Thymidine 3′‐O‐Bis‐Phosphoramidite
  • Basic Protocol 4: Synthesis and Deprotection of N3‐Thymidine‐Alkylene‐N3‐Thymidine Oligonucleotides via the Mono‐ or Bis‐Phosphoramidite Strategy
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of the 4‐Iodo‐Butyl Phenoxyacetate Linker

  Materials
  • Argon gas
  • Triethylamine (TEA)
  • 1,4‐butanediol
  • Phenoxyacetyl chloride (Pac‐Cl)
  • 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)
  • Triphenylphosphine
  • Imidazole
  • Anhydrous tetrahydrofuran (THF)
  • Iodine (I 2)
  • 10% (w/v) aqueous sodium thiosulfate
  • 50‐ and 100‐mL round‐bottom flasks with rubber septa
  • Magnetic stir plate and stir bar
  • 3‐mL glass syringe
  • TLC plate, silica gel 60 F 254 (EMD Millipore)
  • UV lamp, 254 nm
  • 250‐mL separatory funnel
  • Glass funnel and filter paper
  • Rotary evaporator and chemically resistant dry vacuum pump
  • 3 × 50–cm and 5×50–cm chromatography columns
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D)

Basic Protocol 2: Synthesis of 1‐{N3‐[3′‐O‐Tert‐Butyldimethylsilyl‐5′‐O‐(4,4′‐Dimethoxytrityl)‐Thymidinyl]}‐4‐{N3‐[3′‐O‐(Diisopropylamino)‐(2‐Cyanoethoxy)Phosphino‐ 5′‐O‐(4,4′‐Dimethoxytrityl)‐Thymidinyl]}‐Butane

  Materials
  • Argon gas
  • 5′‐O‐dimethoxytrityl‐thymidine (ChemGenes, Inc.)
  • Anhydrous tetrahydrofuran (THF)
  • Triethylamine (TEA)
  • Phenoxyacetyl chloride (Pac‐Cl)
  • 5% (w/v) aqueous sodium bicarbonate (NaHCO 3)
  • Magnesium sulfate (MgSO 4)
  • Silica gel (60 Å, 230‐400 mesh)
  • Anhydrous dichloromethane (DCM, Reagent Grade)
  • Methanol (MeOH, Reagent Grade)
  • 3′‐O‐(tert‐butyldimethylsilyl)‐5′‐O‐(4,4′‐dimethoxytrityl)‐thymidine (Wilds et al., )
  • S.2 ( protocol 1)
  • Anhydrous acetonitrile (ACN)
  • Hexanes (Reagent Grade)
  • Ethyl acetate (EtOAc, Reagent Grade)
  • Propylamine
  • Imidazole
  • Triphenylphosphine
  • Iodine (I 2)
  • 10% (w/v) sodium thiosulfate
  • 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU)
  • Diisopropylamine tetrazolide (ChemGenes Inc.)
  • Bis(diisopropylamino)(2‐cyanoethoxy)phosphine (ChemGenes Inc.)
  • 50‐, 100‐, and 250‐mL round‐bottom flasks with rubber septa
  • Magnetic stir plate and stir bar
  • 3‐mL glass syringe
  • TLC plate, silica gel 60 F 254 (EMD Millipore)
  • UV lamp, 254 nm
  • Rotary evaporator and chemically resistant dry vacuum pump
  • 150‐ and 250‐mL Separatory funnel
  • Filter paper
  • Glass funnel
  • 5×50–cm Flash chromatography columns
  • 1‐, 2‐ and 25‐mL glass syringes
  • Vacuum pump

Basic Protocol 3: Synthesis of N3‐Thymidine‐Alkylene‐N3‐Thymidine 3′‐O‐Bis‐Phosphoramidite

  Materials
  • S.7 ( protocol 2)
  • Argon gas
  • Anhydrous tetrahydrofuran (THF)
  • Tetrabutylammonium fluoride (1 M in THF)
  • Dichloromethane (DCM, Reagent Grade)
  • 5% (w/v) aqueous sodium bicarbonate (NaHCO 3)
  • Silica gel (60 Å, 230‐400 mesh)
  • Diisopropylethylamine
  • N,N‐diisopropylamino cyanoethyl phosphonamidic chloride (ChemGenes Inc.)
  • Ethyl acetate (EtOAc, Reagent Grade)
  • Hexanes (Reagent Grade)
  • 250‐, 100‐, and 10‐mL round‐bottom flasks with rubber septa
  • Magnetic stir plate and stir bar
  • 1‐mL glass syringe
  • TLC plate, silica gel 60 F 254 (EMD Millipore)
  • UV lamp, 254 nm
  • Rotary evaporator and chemically resistant dry vacuum pump
  • 100‐mL separatory funnels
  • Glass funnel and filter paper
  • 3 × 50 cm chromatography column
  • Vacuum pump

Basic Protocol 4: Synthesis and Deprotection of N3‐Thymidine‐Alkylene‐N3‐Thymidine Oligonucleotides via the Mono‐ or Bis‐Phosphoramidite Strategy

  Materials
  • 5′‐O‐DMT‐2′‐deoxynucleoside‐3′‐O‐succinate long chain alkylamine controlled‐pore glass (500 Å; Glen Research)
  • Protected 2′‐deoxyribonucleoside 3′‐phosphoramidites (Glen Research)
  • S.8 ( protocol 2)
  • Low‐water acetonitrile (ACN, EMD Millipore)
  • 4‐Å activated molecular sieves
  • Argon
  • Anhydrous triethylamine (TEA)
  • Anhydrous tetrahydrofuran (THF)
  • Tetrabutylammonium fluoride (1 M in THF, dried over molecular sieves)
  • Protected 2′‐deoxyribonucleoside 5′‐phosphoramidites (ChemGenes Inc.)
  • Ammonium hydroxide (NH 4OH), concentrated
  • Anhydrous ethanol (EtOH)
  • N3‐thymidine‐alkylene‐ N3‐thymidine mono‐ or bis‐phosphoramidite ( S.8, protocol 2or S.10, protocol 3)
  • Synthesis columns (Glen Research, cat. no. 20‐0021‐01)
  • Syringes and needles
  • Vacuum pump
  • Sand bath
  • Heat gun
  • Screw‐capped microcentrifuge tubes (2 mL)
  • Additional reagents and equipment for automated solid‐phase oligodeoxyribonucleotide synthesis, and isolation, purification, and characterization of synthetic nucleic acids (see appendix 3C)
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Figures

Videos

Literature Cited

Literature Cited
   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, New Jersey.
   Deans, A.J. and West, S.C. 2011. DNA interstrand crosslink repair and cancer. Nat. Rev. Cancer. 11:467‐480.
   Dronkert, M.L.G. and Kanaar, R. 2001. Repair of DNA interstrand cross‐links. Mutat. Res. 486:217‐247.
   Helleday, T., Petermann, E., Lundin, C., Hodgson, B., and Sharma, R.A. 2008. DNA repair pathways as targets for cancer therapy. Nat. Rev. Cancer 8:193‐204.
   McHugh, P.J., Spanswick, V.J., and Hartley, J.A. 2001. Repair of DNA interstrand crosslinks: Molecular mechanisms and clinical relevance. Lancet Oncol. 2:483‐490.
   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.
   Noll, D.M., Noronha, A.M., Wilds, C.J., and Miller, P.S. 2004. Preparation of interstrand cross‐linked DNA oligonucleotide duplexes. Front. Biosci. 9:421‐437.
   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.
   Räschle, M., Knipscheer, P., Enoiu, M., Angelov, T., Sun, J., Griffith, J.D., Ellenberger, T.E., Schärer, O.D., and Walte, J.C. 2008. Mechanism of replication‐coupled DNA interstrand crosslink repair. Cell 134:969‐980.
   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., Palus, E., and Noronha, A.M. 2006. An approach for the synthesis of duplexes containing N3T‐Alkyl‐N3T interstrand cross‐links via a bisphosphoramidite strategy. Can. J. Chem. 85:249‐256.
   Wilds, C.J., Xu, F., and Noronha, A.M. 2008. Synthesis and characterization of DNA containing an N1‐2′‐deoxyinosine‐ethyl‐N3‐thymidine interstrand cross‐link: A structural mimic of the cross‐link formed by 1,3‐bis‐(2‐chloroethyl)‐1‐nitrosourea. Chem. Res. Toxicol. 21:686‐695.
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