Modification of the 5′ Terminus of Oligodeoxyribonucleotides for Conjugation with Ligands

Ulysse Asseline1, Nguyen T. Thuong1

1 Centre de Biophysique Moléculaire, CNRS, Orléans, France
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.9
DOI:  10.1002/0471142700.nc0409s05
Online Posting Date:  August, 2001
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Abstract

Ligands can be introduced at the 5′ terminus of an oligonucleotide by adding a linker to the ligand and modifying the 5′ terminus of the oligonucleotide. These are then reacted to give the ligand‐oligonucleotide conjugate. This unit describes the addition of carboxylated and aminoalkylated linkers, and phosphorothioate, phosphate, and masked thiol groups to the 5′ terminus of an oligonucleotide. The addition of linkers to ligands and the final reaction that produces the ligand‐conjugated oligonucleotide are described elsewhere in the series. This approach is particularly useful when there is a limited amount of ligand available, when the ligand is sensitive to chemical conditions required for oligonucleotide deprotection, or when the ligand is weakly soluble in solvents required for phosphoramidite‐ or H‐phosphonate‐mediated oligonucleotide synthesis.

     
 
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Table of Contents

  • Basic Protocol 1: Addition of a Carboxylated or Aminoalkylated Linker to the 5′ End of Oligodeoxyribonucleotides
  • Basic Protocol 2: Addition of a Phosphorothioate or Phosphate Group to the 5′ Terminus of Oligodeoxyribonucleotides
  • Basic Protocol 3: Addition of a Masked Thiol Group to the 5′ Terminus of Oligodeoxyribonucleotides Using an S‐Diphenylphosphinate Phosphoramidite
  • Alternate Protocol 1: Addition of a Masked Thiol Group to the 5′ Terminus of Oligodeoxyribonucleotides Using an S‐Acetyl Phosphoramidite
  • Alternate Protocol 2: Addition of a Masked Thiol Group to the 5′ Terminus of Oligodeoxyribonucleotides Using a Disulfide Phosphoramidite Derivative
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Addition of a Carboxylated or Aminoalkylated Linker to the 5′ End of Oligodeoxyribonucleotides

  Materials
  • 5′‐Detritylated oligodeoxyribonucleotide bound to a controlled‐pore glass (CPG) support
  • Nitrogen gas
  • 40 mg/mL anhydrous 1,1′‐carbonyldiimidazole in anhydrous dioxane
  • Anhydrous dioxane
  • Amino acid or bis amine (select one):
  •  10 mg/mL 5‐aminovaleric acid or 6‐aminocaproic acid,
  •  1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU) salt (Aldrich), in pyridine
  •  12 mg/mL 1,5‐diaminopentane or 1,6‐diaminopentane in pyridine
  • Pyridine, redistilled from p‐toluenesulfonyl chloride, stored over 3A molecular sieves
  • Acetonitrile (CH 3CN), DNA synthesis grade, stored over 3A molecular sieves, and HPLC grade
  • Concentrated ammonium hydroxide (25%)
  • Ethyl acetate, distilled
  • 1.5 M NaCl
  • 25 mM Tris⋅Cl, pH 7 ( appendix 2A), containing 10% (v/v) distilled HPLC‐grade methanol
  • 1 M triethylammonium acetate (TEAA) buffer, pH 7 (stock solution)
  • 8‐mL vial with septum and screw cap
  • 22‐G hypodermic needle
  • Desiccator containing P 2O 5 and KOH
  • 50° and 55°C ovens or water baths
  • 0.45‐µm filter attached to a disposable syringe
  • UV spectrophotometer
  • Rotary evaporator with water bath and a water aspirator
  • Ion‐exchange chromatography system and column (select one):
  •  Mono Q HR 5/5 or HR 10/10 column (Amersham Pharmacia Biotech)
  •  DEAE column (8 µm, 100 × 10 mm; Waters)
  • High‐performance liquid chromatograph (HPLC) equipped with multiwavelength detector and reversed‐phase column (select one):
  •  Lichrospher 100 RP 18 column (5 µm; 125 mm × 4 mm; Merck)
  •  Lichrospher 100 RP 18 column (10 µm; 250 mm × 10 mm; Merck)
  •  Delta‐Pak C 4 column (5 µm, 100 Å; 150 × 3.9 mm; Waters)
  • Lyophilizer
  • Additional reagents and equipment for analytical and preparative ion‐exchange chromatography and reversed‐phase HPLC (RP‐HPLC), and for sample purification (unit 4.3)

Basic Protocol 2: Addition of a Phosphorothioate or Phosphate Group to the 5′ Terminus of Oligodeoxyribonucleotides

  Materials
  • Diisopropylethylamine, distilled from KOH
  • 2‐Cyanoethanol
  • Diethyl ether dried over sodium wires
  • Nitrogen source
  • N,N′‐Diisopropylphosphoramidous dichloride (Aldrich)
  • Argon atmosphere
  • 5′‐Detritylated oligodeoxyribonucleotide bound to a controlled‐pore glass (CPG) support
  • 0.5 M tetrazole in anhydrous CH 3CN
  • Anhydrous CH 3CN
  • 10 mg/mL Beaucage reagent in anhydrous CH 3CN or 100 mg/mL tetraethylthiuram disulfide in anhydrous CH 3CN (optional; for sulfurization)
  • Iodine solution (same composition as for DNA synthesis; optional; for oxidation)
  • Concentrated ammonium hydroxide (NH 4OH; 25%)
  • Dithiothreitol (DTT; optional)
  • Ethyl acetate, distilled
  • Isopropanol
  • 2.5 mg/mL 2,6‐dibromo‐4‐benzoquinone‐N‐chloroimine (DBPNC; Prolabo) in ethanol
  • 1‐liter three‐neck round‐bottom flask
  • Dropping funnel
  • Reflux condenser with a calcium chloride drying tube
  • Gas inlet adapter
  • Glass filter (porosity 4)
  • Rotary evaporator with a water aspirator and a water bath
  • Falling‐film distillation head (Aldrich) or other vacuum distillation apparatus
  • 50° and 55°C ovens or water baths
  • Kieselgel 60F plates for analytical TLC (Merck)
  • Additional reagents and equipment for automated oligonucleotide synthesis ( appendix 3C), thin‐layer chromatography (TLC; appendix 3D), and purification and characterization of the product (see protocol 1)

Basic Protocol 3: Addition of a Masked Thiol Group to the 5′ Terminus of Oligodeoxyribonucleotides Using an S‐Diphenylphosphinate Phosphoramidite

  Materials
  • Methyldiphenylphosphinite (S.11; Aldrich)
  • Toluene
  • Elemental sulfur (S 8)
  • Hexane, distilled
  • Ethyl acetate, distilled
  • Triethylamine, distilled from KOH
  • 2.5 mg/mL 2,6‐dibromo‐4‐benzoquinone‐N‐chloroimine (DBPNC; Prolabo) in ethanol.
  • Celite 521 (Aldrich)
  • 20% (w/v) trimethylamine/acetonitrile
  • Diethyl ether, dried over sodium wires, ice cold and room temperature
  • Dichloromethane (CH 2Cl 2) distilled over P 2O 5 and passed through a column of basic alumina
  • Methanol, distilled
  • 2‐[2‐(2‐Chloroethoxy)ethoxy]ethanol (S.15; Fig. ; Aldrich)
  • NaI
  • Sodium bicarbonate (NaHCO 3)
  • Acetone, anhydrous
  • Anhydrous acetonitrile (CH 3CN)
  • Diisopropylethylamine, distilled from KOH
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite (Aldrich)
  • Nitrogen gas
  • 10% (w/v) aqueous sodium carbonate (Na 2CO 3)
  • Saturated aqueous NaCl, ice cold
  • Sodium sulfate, anhydrous
  • 5′‐Detritylated oligodeoxyribonucleotide bound to a controlled‐pore glass (CPG) support
  • 2,2′‐Dithiodipyridine
  • Phenol
  • Concentrated ammonium hydroxide
  • 1 M triethylammonium acetate (TEAA) buffer, pH 7 (stock solution)
  • Tris‐(2‐carboxyethyl)phosphine (TCEP), hydrochloride
  • 250‐mL three‐necked round‐bottom flask
  • Reflux condenser
  • Magnetic stirrer with heating element
  • Kieselgel 60F plates for analytical TLC (Merck)
  • Rotary evaporator with a water aspirator
  • Calcium chloride drying tube
  • 3 × 50–cm chromatography column containing 40 g silica gel (e.g., Kieselgel 60; Merck) and 1.6 × 45–cm column containing 25 g silica gel
  • 10‐mL vial and stoppers
  • Separatory funnel
  • Spectrophotometer
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D); column chromatography ( appendix 3E); direct addition of an acridinyl phosphoramidite (unit 4.3); and analysis, purification, and characterization of product (see protocol 1)

Alternate Protocol 1: Addition of a Masked Thiol Group to the 5′ Terminus of Oligodeoxyribonucleotides Using an S‐Acetyl Phosphoramidite

  • Potassium thioacetate
  • 1.6 × 45–cm column containing 20 g silica gel (e.g., Kieselgel 60; Merck)

Alternate Protocol 2: Addition of a Masked Thiol Group to the 5′ Terminus of Oligodeoxyribonucleotides Using a Disulfide Phosphoramidite Derivative

  • 2‐[2‐(2‐Acetylthioethoxy)ethoxy]ethanol S.16 (see protocol 4)
  • 5% ammonium hydroxide
  • Anhydrous pyridine
  • Dimethoxytrityl chloride (DMTr⋅Cl)
  • 10% perchloric acid
  • MgSO 4
  • 80% (v/v) acetic acid
  • Ethanol
  • 1 M triethylammonium acetate (TEAA) buffer, pH 6 (stock solution)
  • Dithiothreitol (DTT)
  • Methanol, HPLC grade
  • 1.6 × 45–cm chromatography column containing 20 g silica gel (e.g., Kieselgel 60; Merck)
  • 10‐mL gel‐filtration column
  • Lyophilizer
  • Additional reagents and equipment for gel‐filtration chromatography
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Figures

Videos

Literature Cited

Literature Cited
   Aubert, Y., Bourgerie, S., Meunier, L., Mayer, R., Roche, A.‐C., Monsigny, M., Thuong, N.T., and Asseline, U. 2000. Optimized synthesis of phosphorothioate oligodeoxyribonucleotides substituted with a 5′‐protected thiol function and a 3′‐amino group. Nucleic Acids Res. 28:818‐825.
  We would like to express our appreciation to our past and present collaborators for their contribution to the development of varied oligonucleotide families over the past years. The work was supported by Rhône‐Poulenc, Agence Nationale de Recherche contre le SIDA and Bio‐Mérieux.
   Bonfils, E. and Thuong, N.T. 1991. Solid‐phase synthesis of 5′‐3′‐bifunctional oligodeoxyribonucleotides bearing a masked thiol group at their 3′‐end. Tetrahedron Lett. 35:3053‐3056.
   Burns, J.A., Butler, J.C., Moran, J., and Whitesides, G.M. 1991. Selective reduction of disulfides by tris(2‐carboxyethyl)phosphine. J. Org. Chem. 56:2648‐2650.
   Carlsson, J., Drevin, H., and Axen, R. 1978. Protein‐thiolation and reversible protein‐protein conjugation. Biochem. J. 173:723‐737.
   Eckstein, F. 1983. Phosphorothioate analogues of nucleotides. Tools for investigation of biochemical processes. Angew. Chem. Int. Ed. Engl. 22:423‐506.
   Gottikh, M., Asseline, U., and Thuong, N.T. 1990. Synthesis of oligonucleotides containing a carboxyl group at either their 5′‐end or their 3′‐end and their subsequent derivatization by an intercalating agent. Tetrahedron Lett. 31:6657‐6660.
   Grimm, G.N., Boutorine, A.S., and Hélène, C. 2000. Rapid routes of synthesis of oligonucleotide conjugates from non‐protected oligonucleotides and ligands possessing different nucleophilic or electrophilic functional groups. Nucleosides Nucleotides and Nucleic Acids 19:1943‐1965.
   Kuijpers, W.H. and Van Boeckel, C.A. 1993. A new strategy for the solid‐phase synthesis of 5′‐thiolated oligodeoxyribonucleotides. Tetrahedron 49:10944‐10944.
   Kurfürst, R., Roig, V., Chassignol, M., Asseline, U., and Thuong, N.T. 1993. Oligo‐α‐deoxyribonucleotides with a modified nucleic acid base and covalently linked to reactive agent. Tetrahedron 32:6975‐6990.
   Raynaud, F., Asseline, U., Roig, V., and Thuong, N.T. 1996. Synthesis and characterization of O6 ‐modified deoxyguanosine‐containing oligodeoxyribonucleotides for triple‐helix formation. Tetrahedron 52:2047‐2064.
   Thuong, N.T. and Asseline, U. 1991. Oligodeoxyribonucleotides attached to intercalators, photoreactive and cleavage agents. In Oligodeoxyribonucleotides and Analogues: A Practical Approach (F. Eckstein, ed.) pp. 283‐308. IRL Press, Oxford.
   Wachter, L., Jablonski, J.A., and Ramachandran, K.L. 1986. A simple and efficient procedure for the synthesis of 5′‐aminoalkyl oligonucleotides. Nucleic Acids Res. 14:7985‐7994.
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