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

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 P₂ O₅ , stored over 3A molecular sieves
N ,N ‐Dimethylformamide (DMF) redistilled in vacuo over ninhydrin, stored over 4A molecular sieves
Triphenylphosphine (Aldrich)
CBr₄ (Aldrich)
Dichloromethane (CH₂ Cl₂ ) distilled from P₂ O₅ 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
CH₂ Cl₂
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 P₂ O₅ , 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
CaCl₂ 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 P₂ O₅
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 P₂ O₅ , 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 NaHCO₃ (for S.3c only)
Dichloromethane (CH₂ Cl₂ ) distilled from P₂ O₅ 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)
CS₂ (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 P₂ O₅
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 60F₂₅₄ 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)
CH₂ Cl₂
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 P₂ O₅
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

Figure 4.8.1 Bromination of an acridine derivative. DMF, N , N ‐dimethylformamide.

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Figure 4.8.2 Functionalization of 5‐hydroxypsoralen (S.2b ) with an iodoalkyl linker (S.2f ) or a 2‐pyridyldithioalkyl linker (S.2h ). DMF, N , N ‐dimethylformamide.

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Figure 4.8.3 Derivatization of orthophenanthroline (S.3a ) with a bromoalkyl linker (S.3c ) or an isothiocyanate group (S.3d ). DCC, 1,3‐dicyclohexylcarbodiimide; DIPEA, N , N ‐diisopropylethyl‐amine.

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Figure 4.8.4 Thiazole orange derivative functionalized with an iodoalkyl linker (S.4e ). TEA, triethylamine.

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

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited