Incorporation of Halogenoalkyl, 2‐Pyridyldithioalkyl, or Isothiocyanate Linkers into Ligands

Ulysee Asseline1, Nguyen T. Thuong1

1 Centre de Biophysique Moléculaire, CNRS, Orléans, France
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.8
DOI:  10.1002/0471142700.nc0408s05
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. The addition of appropriate linkers to ligands is described in this unit. 5'Modification of the oligonucleotide 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: Functionalization of an Acridine Derivative with a Bromoalkyl Linker
  • Basic Protocol 2: Functionalization of a Psoralen Derivative with an Iodoalkyl or 2‐Pyridyldithioalkyl Linker
  • Basic Protocol 3: Functionalization of an Orthophenanthroline Derivative with a Bromoalkyl Linker or an Isothiocyanate Group
  • Basic Protocol 4: Functionalization of a Thiazole Orange Derivative with an Iodoalkyl Linker
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Functionalization of an Acridine Derivative with a Bromoalkyl Linker

  Materials
  • 2‐Methoxy‐6‐chloro‐9‐(ω‐hydroxyhexylamino)acridine S.1b (unit 4.3)
  • Acetonitrile distilled from P 2O 5, stored over 3A molecular sieves
  • N,N‐Dimethylformamide (DMF) redistilled in vacuo over ninhydrin, stored over 4A molecular sieves
  • Triphenylphosphine (Aldrich)
  • CBr 4 (Aldrich)
  • Dichloromethane (CH 2Cl 2) distilled from P 2O 5 and passed over basic aluminum oxide
  • Methanol, distilled or synthesis grade
  • Nitrogen source
  • 10‐mL round‐bottom flask
  • Magnetic stirrer with Teflon stir bars
  • Vacuum pump (oil pump) capable of creating <0.1 mmHg pressure, with manifold and cold trap
  • Preparative 20 × 20–cm glass‐backed silica TLC plates (2‐mm thickness; Merck)
  • Short‐wave UV light box
  • Mortar and pestle
  • 1.5 × 15–cm chromatography column (empty)
  • 2.5 × 7–cm Kieselgel 60F analytical TLC plates (Merck)
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D)

Basic Protocol 2: Functionalization of a Psoralen Derivative with an Iodoalkyl or 2‐Pyridyldithioalkyl Linker

  Materials
  • 5‐Hydroxypsoralen S.2b (unit 4.3)
  • N,N‐Dimethylformamide redistilled in vacuo over ninhydrin, stored over 4A molecular sieves
  • 1,6‐Diiodohexane (Aldrich)
  • Anhydrous potassium carbonate
  • Argon source
  • CH 2Cl 2
  • Distilled pentane (CE Instruments)
  • Distilled methanol
  • Nitrogen source
  • Potassium thioacetate (Aldrich)
  • Distilled acetone
  • Distilled ethyl acetate
  • Distilled hexane (CE Instruments)
  • Sodium sulfate (Aldrich)
  • 2,2′‐Dipyridyl disulfide (Aldrich)
  • Acetonitrile distilled from P 2O 5, stored over 3A molecular sieves
  • 29% (v/v) aqueous ammonium hydroxide
  • 2.5 mg/mL 2,6‐dibromo‐4‐benzoquinone‐N‐chloroimine (DBPNC; Merck) in ethanol
  • 100‐mL and 25‐mL round‐bottom flasks and stoppers
  • Reflux condenser
  • CaCl 2 drying tube
  • Magnetic stirrer with temperature‐controlled oil bath and Teflon stir bars
  • 5‐cm glass filter funnel (porosity 4)
  • Vacuum pump (oil pump) capable of creating <0.1 mmHg pressure, with manifold and cold trap
  • Kieselgel 60F chromatography column (e.g., 3‐cm diameter, 50‐cm height, 50 g silica gel; Merck)
  • 2.5 × 7–cm analytical TLC plates (e.g., Kieselgel 60F plates, Merck)
  • Short‐wave UV light box
  • Rotary evaporator with a water aspirator
  • Desiccator containing P 2O 5
  • 7‐cm filter funnel with filter paper
  • 25‐mL flask with stopper
  • Preparative 20 × 20–cm glass‐backed silica gel TLC plates (2‐mm thickness; e.g. Merck)
  • Mortar and pestle
  • 1.5 × 15–cm chromatography column (empty)
  • Additional reagents and equipment for thin‐layer chromatography (TLC, appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 3: Functionalization of an Orthophenanthroline Derivative with a Bromoalkyl Linker or an Isothiocyanate Group

  Materials
  • 20% (w/v) aqueous ammonium sulfide
  • Nitrogen source
  • 5‐Nitro‐1,10‐phenanthroline (S.3a; Aldrich)
  • Absolute ethanol, stored over 4A molecular sieves
  • Chloroform
  • Sodium sulfate, anhydrous (Aldrich)
  • Acetonitrile distilled from P 2O 5, stored over 3A molecular sieves (for S.3c only)
  • N,N‐Diisopropylethylamine (Aldrich; for S.3c only)
  • 6‐Bromohexanoyl chloride (Aldrich; for S.3c only)
  • 5% (v/v) aqueous NaHCO 3 (for S.3c only)
  • Dichloromethane (CH 2Cl 2) distilled from P 2O 5 and passed over basic aluminum oxide
  • Methanol
  • Pyridine redistilled from p‐toluenesulfonylchloride, stored over 3A molecular sieves (for S.3d only)
  • 1,3‐Dicyclohexylcarbodiimide (Aldrich; DCC; for S.3d only)
  • CS 2 (Merck; for S.3d only)
  • Dioxane, freshly distilled (Aldrich; for S.3d only)
  • 500‐mL three‐necked flask
  • Reflux condenser
  • 100‐mL dropping funnel
  • 0.5‐cm nitrogen inlet tube
  • Magnetic stirrer with temperature‐controlled oil bath and Teflon stir bars
  • 500‐mL separatory funnel
  • 7‐cm filter funnel and filter paper
  • Rotary evaporator with water aspirator
  • 5‐cm glass filter funnel (porosity 4)
  • Desiccator containing P 2O 5
  • 25‐mL flask with rubber stopper
  • Neutral‐activated chromatography column, 1.6‐cm diameter, 50‐cm height (e.g., 40 g of Kieselgel 60 or Aluminumoxid 90; Merck; for S.3c only)
  • 2.5 × 7–cm analytical TLC plates (e.g., Kieselgel 60F or Aluminumoxid 60F 254 neutral, Type E; Merck)
  • Short‐wave UV light box
  • 10‐mL round‐bottom flask with rubber septa and glass stoppers (for S.3d only)
  • Silica gel column, 1.5‐cm diameter, 45‐cm height (17 g Kieselgel 60; Merck; for S.3d only)
  • Additional reagents and equipment for thin‐layer chromatography (TLC, appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 4: Functionalization of a Thiazole Orange Derivative with an Iodoalkyl Linker

  Materials
  • 3‐Methyl benzothiazole‐2‐thione S.4a
  • Methyl iodide (Fluka)
  • Distilled methanol
  • Diethyl ether, anhydrous
  • 1,8‐Diiodooctane (Aldrich)
  • Dioxane, freshly distilled (Aldrich)
  • Lepidine (Aldrich)
  • CH 2Cl 2
  • Nitrogen source
  • Ethanol, prewarmed (55°C)
  • Triethylamine (Merck)
  • 100‐mL and 10‐mL round‐bottom flask and rubber septa/glass stoppers
  • Magnetic stirrer with temperature‐controlled oil bath and Teflon stir bars
  • Reflux condenser
  • 5‐cm glass filter funnel (porosity 4)
  • Desiccator containing P 2O 5
  • Two‐necked round‐bottom flask
  • 10‐mL dropping funnel
  • Silica gel chromatography columns: 35 g, 2.5‐cm diameter, 45‐cm height; and 25 g, 1.2‐cm diameter, 40‐cm height (e.g., Kieselgel 60, Merck)
  • 2.5 × 7–cm analytical TLC plates (e.g., Kieselgel 60F, Merck)
  • Short‐wave UV light box
  • Additional reagents and equipment for thin‐layer chromatography (TLC, appendix 3D) and column chromatography ( appendix 3E)
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Figures

Videos

Literature Cited

Literature Cited
   Asseline, U., Bonfils, E., Kurfürst, R., Chassignol, M., Roig, V., and Thuong, N.T. 1992. Solid‐phase preparation of 5′,3′‐heterobifunctional oligodeoxyribonucleotides using modified solid supports. Tetrahedron 48:1233‐1254
  The authors would like to express their appreciation to their past and present collaborators for their contribution to the development of various families of oligonucleotide‐ligand conjugates. This work was supported by Rhne‐Poulenc, the Agence Nationale de Recherches sur le SIDA and bio‐Mrieux.
   Asseline, U., Bonfils, E., Dupret, D., and Thuong, N.T. 1996. Synthesis and binding properties of oligonucleotides covalently linked to an acridine derivative. A new study of the influence of the dye attachment site. Bioconjugate Chem. 7:369‐379
   Benson, S.C., Singh, P., and Glazer, A.N. 1993 Heterodimeric DNA‐binding dyes designed for energy transfer: Synthesis and spectroscopic properties. Nucl. Acids Res. 21:5727‐5735
   Brooker, L.G., Keyer, G.H., and Williams, W.W. 1942 The absorption of unsymmetrical cyanines. Resonance as a basis for classification of dyes. J. Am. Chem. Soc. 64:199‐210
   Chassignol, M. and Thuong, N.T. 1998. Phosphodisulfide bond: A new linker for the oligonucleotide conjugation. Tetrahedron Lett. 39:8271‐8274
   Costes, B., Girodon, E., Ghanem, N., Chassignol, M., Thuong, N.T., Dupret, D., and Goossens, M. 1993. Psoralen‐modified oligonucleotide primers improve detection of mutations by denaturing gradient gel electrophoresis and provide an alternative to GC‐clamping. Hum. Mol. Genet. 2:393‐397
   François, J.‐C., Saison‐Behmoaras, T., Barbier, C., Chassignol, M., Thuong, N.T., and Hélène, C. 1989a. Sequence‐specific recognition and cleavage of duplex DNA via triple‐helix formation by oligonucleotides covalently linked to a phenanthroline‐copper chelate. Proc. Natl. Acad. Sci.U.S.A. 86:9702‐9706
   François, J.‐C., Saison‐Behmoaras, T., Chassignol, M., Thuong, N.T., and Hélène, C. 1989b. Sequence‐targeted cleavage of single‐ and double‐stranded DNA by oligothymidylates covalently linked to 1,10‐phenanthroline. J. Biol. Chem. 264:5891‐5898
   Giovannangeli, C., Thuong, N.T., and Hélène, C. 1992. Oligodeoxynucleotide‐directed photo‐induced cross‐linking of HIV proviral DNA via triple‐helix formation. Nucl. Acids Res. 20:4275‐4281
   Giovannangeli, C., Perrouault, L., Escudé, C., Thuong, N.T., and Hélène, C. 1996. Specific inhibition of in vitro transcription elongation by triplex‐forming oligonucleotide‐intercalator conjugates targeted to HIV proviral DNA. Biochemistry 35:10539‐10548
   Grigoriev, M., Praseuth, D., Guieysse, A.L., Robin, P., Thuong, N.T., Hélène, C., and Harel‐Bellan, A. 1993. Inhibition of gene expression by triple helix‐directed DNA cross‐linking at specific sites. Proc. Natl. Acad. Sci. U.S.A. 90:3501‐3505
   Sun, J.S., François, J.C., Montenay‐Garestier, T., Saison‐Behmoaras, T., Roig, V., Thuong, N.T., and Hélène, C. 1989. Sequence‐specific intercalating agents. Intercalation at specific sequences on duplex DNA via major groove recognition by oligonucleotide‐intercalator conjugates. Proc. Natl. Acad. Sci. U.S.A. 86:9198‐9202
   Takasugi, M., Guendouz, A., Chassignol, M., Decout, J.L., Lhomme, J., Thuong, N.T., and Hélène, C. 1991. Sequence‐specific photo‐induced cross‐linking of the two strands of double‐helical DNA by a psoralen covalently linked to a triple helix forming oligonucleotide. Proc. Natl. Acad. Sci. U.S.A. 88:5602‐5606
   Thuong, N.T. and Asseline, U. 1991. Oligonucleotides attached to intercalators, photoreactive and cleavage agents. Oligonucleotides and Analogues: A Practical Approach (F. Eckstein, ed.) pp.283‐308 IRL Press, Oxford.
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