Synthesis of 5′‐Aldehyde Oligonucleotide

Rémy Lartia1

1 Département de Chimie Moléculaire, Université Grenoble Alpes, Grenoble
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.70
DOI:  10.1002/0471142700.nc0470s64
Online Posting Date:  March, 2016
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Abstract

Synthesis of oligonucleotide ending with an aldehyde functional group at their 5′‐end (5′‐AON) is possible for both DNA (5′‐AODN) and RNA (5′‐AORN) series irrespectively of the nature of the last nucleobase. The 5′‐alcohol of on‐support ODN is mildly oxidized under Moffat conditions. Transient protection of the resulting aldehyde by N,N′‐diphenylethylenediamine derivatives allows cleavage, deprotection, and RP‐HPLC purification of the protected 5′‐AON. Finally, 5′‐AON is deprotected by usual acetic acid treatment. In the aggregates, 5′‐AON can be now synthesized and purified as routinely as non‐modified ODNs, following procedures similar to the well‐known “DMT‐On” strategy. © 2016 by John Wiley & Sons, Inc.

Keywords: oligonucleotide; aldehyde; protecting group

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of 5′‐AODN
  • Alternate Protocol 1: DDQ‐Assisted Protection of CPG‐Linked 5′‐AODN
  • Alternate Protocol 2: Synthesis of 5′‐AORN Using N,N′‐bis‐(4‐Bromophenyl)‐Ethylenediamine
  • Alternate Protocol 3: Synthesis of 5′‐AORN Using N,N′‐bis‐(2,5‐Dichlorophenyl)‐Ethylenediamine
  • Alternate Protocol 4: Cleavage and Deprotection of 5′‐AORN
  • Support Protocol 1: Synthesis of N,N′‐bis‐(4‐Bromophenyl)‐Ethylenediamine
  • Support Protocol 2: Synthesis of N,N′‐bis‐(2,5‐Dichlorophenyl)‐Ethylenediamine
  • Support Protocol 3: Derivatization of 5′‐AON Prior to Mass Spectroscopy Analysis
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of 5′‐AODN

  Materials
  • Fully protected ODN anchored to CPG beads synthesized using DMT‐off mode (final DMT group had to be removed at the end of ODN elongation).AODN 5a are synthesized using a succinyl linker and BzdA, T, dmfdG, and AcdC phosphoramidites. For AORN (5b) synthesis, 2′‐hydroxyl groups are protected as tBDMS derivatives, and BzA, U, dmfG, and AcC phosphoramidites are used.
  • 19% (v/v) diisopropylcarbodiimide in DMSO
  • 1.7% (v/v) dichloroacetic acid in DMSO
  • Acetonitrile (MeCN), synthesis grade
  • Diethylether, synthesis grade
  • 0.2% (w/v) p‐toluenesulphonic acid (PTSA) in DMF
  • N,N′‐diphenylethylenediamine
  • 28% to 30% aqueous ammonia solution, or 40% aqueous methylamine solution
  • Diisopropylethylamine
  • 80% (v/v) aqueous acetic acid solution
  • Centrifuge
  • 1.5‐mL plastic transparent vial
  • 20‐ to 200‐μL pipettor
  • Vortex agitator
  • Centrifuge evaporator
  • Freeze dryer
  • Additional reagents and equipment for column chromatography ( appendix 3E)

Alternate Protocol 1: DDQ‐Assisted Protection of CPG‐Linked 5′‐AODN

  Additional Materials (see also Basic Protocol)
  • N,N′‐diphenylethylenediamine solution (23 mg in 1 mL 96% ethanol)
  • dichlorodicyanoquinone (DDQ) solution (26 mg in 1 mL 96% ethanol)

Alternate Protocol 2: Synthesis of 5′‐AORN Using N,N′‐bis‐(4‐Bromophenyl)‐Ethylenediamine

  Additional Materials (see also Basic Protocol)
  • N,N′bis‐(4‐bromophenyl)‐ethylenediamine 9 (Fig.  ; see protocol 6)

Alternate Protocol 3: Synthesis of 5′‐AORN Using N,N′‐bis‐(2,5‐Dichlorophenyl)‐Ethylenediamine

  Additional Materials (see also Basic Protocol)
  • N,N′bis‐(2,5‐dichlorophenyl)‐ethylenediamine 10 (Fig.  ; see protocol 7)

Alternate Protocol 4: Cleavage and Deprotection of 5′‐AORN

  Additional Materials (see also Basic Protocol)
  • 40% (v/v) aqueous methylamine solution
  • DMSO
  • 70% (w/v) aqueous glycolic acid solution
  • 40% (w/v) aqueous ammonium fluoride solution
    The ammonium fluoride solution must be prepared in a plastic vial.

Support Protocol 1: Synthesis of N,N′‐bis‐(4‐Bromophenyl)‐Ethylenediamine

  Materials
  • N,N′‐diphenylethylenediamine
  • Chloroform (synthesis grade)
  • Tetraethylammonium tribromide
  • Dichloromethane
  • Pentane
  • 100‐mL round bottom flask
  • Magnetic stirrer
  • Silica gel
  • Additional reagents and equipment for column chromatography ( appendix 3E)

Support Protocol 2: Synthesis of N,N′‐bis‐(2,5‐Dichlorophenyl)‐Ethylenediamine

  Materials
  • 3,5‐dichloroaniline
  • 1,2‐dibromoethane
  • Dimethylacetamide (synthesis grade)
  • Ethyl acetate
  • 5% (w/v) NaHCO 3 solution
  • Brine (NaCl, saturated solution)
  • Sodium sulfate
  • 9:1 (v/v) cyclohexane/ethyl acetate
  • Two‐necked, 50‐mL round‐bottom flask
  • Additional reagents and equipment for column chromatography ( appendix 3E)

Support Protocol 3: Derivatization of 5′‐AON Prior to Mass Spectroscopy Analysis

  Materials
  • 50 mM phosphate buffered saline (PBS), pH 4.3
  • n‐decyloxiamine (easily synthesized according to Ewan et al., )
  • Dioxanne
  • Acetonitrile
  • Additional reagents and equipment for column chromatography ( appendix 3E)
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Figures

Videos

Literature Cited

Literature Cited
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  Angeloff, A., Dubey, I., Pratviel, G., Bernadou, J., and Meunier, B. 2001. Characterization of a 5′‐aldehyde terminus resulting from the oxidative attack at C5′ of a 2‐deoxyribose on DNA. Chem. Res. Toxicol. 14:1413‐1420. doi: 10.1021/tx0100800.
  Beaucage, S.L. and Caruthers, M.H. 2001. Synthetic Strategies and parameters involved in the synthesis of oligodeoxyribonucleotides according to the phosphoramidite method. Curr. Protoc. Nucleic Acid Chem. 00:3.3.1‐3.3.20. doi: 10.1002/0471142700.nc0303s00.
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  Lartia, R. and Constant J. 2015. Synthetic access to the chemical diversity of DNA and RNA 5′‐aldehyde lesions. J. Org. Chem. 80:705‐710. doi: 10.1021/jo502170e.
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  Pitié, M., Burrows, C.J., and Meunier, B. 2000. Mechanisms of DNA cleavage by copper complexes of 3‐Clip‐Phen and of its conjugate with a distamycin analogue. Nucleic Acids Res. 28:4856‐4864. doi: 10.1093/nar/28.24.4856.
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