DNA Interstrand Cross‐Link Formation Using Furan as a Masked Reactive Aldehyde

Lieselot L.G. Carrette1, Ellen Gyssels1, Annemieke Madder1

1 Ghent University, Krijgslaan, Ghent
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 5.12
DOI:  10.1002/0471142700.nc0512s54
Online Posting Date:  October, 2013
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Abstract

This unit describes a method for interstrand cross‐linking between a furan‐modified oligonucleotide and its unmodified complement. The synthesis of two furan‐modified phosphoramidites, selected based on high cross‐linking yield versus improved cross‐linking selectivity, is described. The methods allow gram‐scale synthesis starting from stable and readily available furan derivatives. Cross‐linking requires selective oxidation of the furan moiety to an aldehyde. The masked nature of the latter avoids undesired and off‐target reactions, resulting in clean and high‐yield cross‐link formation. Curr. Protoc. Nucleic Acid Chem. 54:5.12.1‐5.12.16. © 2013 by John Wiley & Sons, Inc.

Keywords: cross‐linking; oligonucleotide; nucleoside; furan

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Selective 2′‐Furan‐Modified Uridine Nucleoside Phosphoramidite
  • Alternate Protocol 1: Synthesis of 2′‐Furan‐Modified Acyclic Nucleoside Phosphoramidite
  • Basic Protocol 2: Synthesis of Furan‐Containing Oligodeoxynucleotides
  • Basic Protocol 3: Selective Formation of DNA Interstrand Cross‐Links
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of Selective 2′‐Furan‐Modified Uridine Nucleoside Phosphoramidite

  Materials
  • Uridine
  • Diphenylcarbonate ((PhO) 2CO)
  • N,N‐Dimethylformamide (DMF), extra dry, on molecular sieves
  • Sodium bicarbonate (NaHCO 3)
  • Dichloromethane (CH 2Cl 2)
  • Methanol (MeOH)
  • Lithium fluoride (LiF)
  • Azidotrimethylsilane (Me 3SiN 3)
  • Tetramethylethylenediamine (TMEDA)
  • Ethyl acetate (EtOAc)
  • Triphenylphosphine (PPh 3)
  • Toluene
  • Furfurylisocyanate (Sigma‐Aldrich)
  • Pyridine, extra dry, on molecular sieves
  • 4,4′‐Dimethoxytritylchloride (DMTrCl)
  • Saturated aqueous sodium bicarbonate (NaHCO 3)
  • Brine (saturated aqueous NaCl)
  • Sodium sulfate (Na 2SO 4)
  • Isooctane
  • Triethylamine (TEA)
  • Diisopropylethylamine (DIPEA)
  • 2‐Cyanoethyl N,N‐diisopropylchlorophosphoramidite
  • 25‐, 50‐, and 100‐mL round‐bottom flasks
  • 10‐ and 25‐mL oven‐dried round‐bottom flask
  • Liebig condensers
  • No. 4 sintered glass filter
  • Rotary evaporator
  • Vacuum pump (dry ice + isopropanol)
  • Distillation apparatus for CH 2Cl 2
  • Additional reagents and equipment for TLC ( appendix 3D) and flash chromatography ( appendix 3E)

Alternate Protocol 1: Synthesis of 2′‐Furan‐Modified Acyclic Nucleoside Phosphoramidite

  Materials
  • (4S)‐(+)‐4‐(2‐Hydroxyethyl)‐2,2‐dimethyl‐1,3‐dioxolane (8; Sigma‐Aldrich)
  • Pyridine, extra dry, on molecular sieves
  • p‐Toluenesulfonyl chloride (pTsCl)
  • Argon gas
  • Ethyl acetate (EtOAc)
  • Pentane
  • 1 M and 4 M aqueous HCl
  • Diethyl ether (Et 2O)
  • Saturated aqueous sodium bicarbonate (NaHCO 3)
  • Anhydrous sodium sulfate (Na 2SO 4)
  • N,N‐Dimethylformamide (DMF), extra dry, on molecular sieves
  • Lithium bromide (LiBr), made anhydrous by heating under vacuum for 1 min
  • Isooctane
  • Tetrahydrofuran (THF), extra dry, on molecular sieves
  • Isopropanol
  • Dry ice
  • n‐Butyllithium (n‐BuLi)
  • Hexane
  • Furan
  • 4,4′‐Dimethoxytritylchloride (DMTrCl)
  • Cyclohexane
  • Methanol (MeOH)
  • Triethylamine (TEA)
  • Toluene
  • Dichloromethane (CH 2Cl 2)
  • Diisopropylethylamine (DIPEA)
  • 2‐Cyanoethyl N,N‐diisopropylchlorophosphoramidite
  • 25‐ and 50‐mL two‐neck flasks
  • 25‐ and 50‐mL oven‐dried round‐bottom flasks
  • Rotary evaporator
  • Vacuum pump (dry ice + isopropanol)
  • Distillation apparatus for CH 2Cl 2
  • Additional reagents and equipment for TLC ( appendix 3D) and flash chromatography ( appendix 3E)

Basic Protocol 2: Synthesis of Furan‐Containing Oligodeoxynucleotides

  Materials
  • Furan‐modified phosphoramidite 7 (see protocol 1) or 14 (see protocol 2Alternate Protocol)
  • 4,5‐Dicyanoimidazole (DCI)
  • Acetonitrile (MeCN), extra dry, on molecular sieves
  • Nitrogen and argon gas
  • Controlled‐pore glass (CPG) derivatized with desired 3′ nucleoside (Proligo)
  • Unmodified phosphoramidites, configured for DNA synthesizer (Proligo): DMTr‐dABz, DMTr‐dCBz, DMTr‐dGiBu, and DMTr‐T
  • Activated molecular sieves
  • Reagents for automated ODN synthesis, configured for DNA synthesizer (Proligo): activator, oxidizer, cap A and B solutions, TCA deblock
  • 50 mM aqueous ammonia (NH 4OH)
  • DMTr‐selective cartridge (Sep‐pak from Waters)
  • Rotary evaporator
  • Vacuum pump (dry ice + isopropanol)
  • Microcentrifuge tube
  • Additional reagents and equipment for automated DNA synthesis ( appendix 3C) and ODN purification using a DMTr‐selective cartridge (unit 10.7)
NOTE: ODN synthesis should be performed under anhydrous conditions using oven‐dried glassware, needles, and syringes, or new plastic syringes and needles that have been dried in a desiccator for 2 days.

Basic Protocol 3: Selective Formation of DNA Interstrand Cross‐Links

  Materials
  • Furan‐modified ODN (see protocol 3)
  • Complementary ODN
  • 100 mM (10×) phosphate buffer, pH 7
  • 1 M (10×) NaCl
  • Milli‐Q‐purified water
  • N‐Bromosuccinimide (NBS)
  • Acetonitrile (MeCN), HPLC grade, >99.9
  • 0.1 M triethylammonium acetate (TEAA) buffer, pH 7
  • Formamide
  • GelRed stock solution (Biotium): 10,000× in dimethyl sulfoxide (DMSO)
  • Microcentrifuge tube
  • Eppendorf thermomixer
  • Additional reagents and equipment for RP‐HPLC (unit 10.5) and PAGE (unit 10.4 & appendix 3B)
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Figures

Videos

Literature Cited

Literature Cited
  Bakhiya, N. and Appel, K.E. 2010. Toxicity and carcinogenity of furan in human diet. Arch. Toxicol. 84:563‐578.
  Grillari, J., Katinger H., and Voglauer, R. 2007. Contributions of DNA interstrand cross‐links to aging of cells and organisms. Nucleic Acids Res. 35:7566‐7576.
  Guainazzi, A. and Scharer, O.D. 2010. Using synthetic DNA interstrand crosslinks to elucidate repair pathways and identify new therapeutic targets for cancer chemotherapy. Cell. Mol. Life Sci. 67:3683‐3697.
  Halila, S., Velasco, T., De Clercq, P., and Madder, A. 2005. Fine‐tuning furan toxicity: Fast and quantitative DNA interchain crosslink formation upon selective oxidation of a furan containing oligonucleotide. Chem. Commun. 7:936‐938.
  Jawalekar, A.M., Op de Beeck, M., van Delft, F.L., and Madder, A. 2011. Synthesis and incorporation of a furan‐modified adenosine building block for DNA interstrand crosslinking. Chem. Commun. 47:2796‐2798.
  Kirschenheuter, G.P., Zhai, Y.S., and Pieken, W.A. 1994. An improved synthesis of 2′‐azido‐2′‐deoxyuridine. Tetrahedron Lett. 35:8517‐8520.
  Lin, F.L., Hoyt, H.M., van Halbeek, H., Bergman, R.G., and Bertozzi, C.R. 2005. Mechanistic investigation of the Staudinger ligation. J. Am. Chem. Soc. 127:2686‐2695.
  McGee, D.P.C., VaughnSettle, A., Vargeese, C., and Zhai, Y.S. 1996. 2′‐Amino‐2′‐deoxyuridine via an intramolecular cyclization of a trichloroacetimidate. J. Org. Chem. 61:781‐785.
  Noll, D.M., McGregor Mason, T., and Miller, P.S. 2006 Formation and repair of interstrand cross‐links in DNA. Chem. Rev. 106:277‐301.
  Op de Beeck, M. and Madder, A. 2011. Unprecedented C‐selective interstrand cross‐linking through in situ oxidation of furan‐modified oligodeoxynucleotides. J. Am. Chem. Soc. 133:796‐807.
  Op de Beeck, M. and Madder, A. 2012. Sequence specific DNA cross‐linking triggered by visible light. J. Am. Chem. Soc. 134:10737‐10740.
  Peterson, L.A. 2006. Electrophilic intermediates produced by bioactivation of furan. Drug Metab. Rev. 38:615‐626.
  Singh, I., Vyle, J.S., and Heaney, F. 2009 Fast, copper‐free click chemistry: A convenient solid‐phase approach to oligonucleotide conjugation. Chem. Comm. 22:3276‐3278.
  Stevens, K. and Madder, A. 2009. Furan‐modified oligonucleotides for fast, high‐yielding and site‐selective DNA inter‐strand cross‐linking with non‐modified complements. Nucleic Acid. Res. 37:1555‐1565.
  Stevens, K., Claeys, D.D., Catak, S., Figaroli, S., Hocek, M., Tromp, J.M., Schurch, S., Van Speybroeck, V., and Madder, A. 2011. Furan‐oxidation‐triggered inducible DNA cross‐linking: Acyclic versus cyclic furan‐containing building blocks‐on the benefit of restoring the cyclic sugar backbone. Chem. Eur. J. 17:6940‐6953.
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