Synthesis of Oligonucleotide Conjugates via Aqueous Diels‐Alder Cycloaddition

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

The conjugation of maleimide reporter groups to 5??diene?modified oligonucleotides via aqueous Diels?Alder cycloaddition is described. Detailed basic protocols are provided for the preparation of a diene?amidite (5??diene modifier), for the attachment of a diene group to the 5??terminus of oligonucleotides, and for the conjugation of maleimide reporter groups to diene?modified oligonucleotides.

Keywords: diene?amidite; 5??diene modifier; Diels?Alder conjugation; 5??labeling of oligonucleotides

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Basic Protocol 1: Synthesis of the Diene‐Amidite
Basic Protocol 2: Synthesis of 5′‐Diene‐Modified Oligonucleotides
Basic Protocol 3: Labeling of A 5′‐Diene‐Modified Oligonucleotide with Tamra‐5‐Maleimide
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Synthesis of the Diene‐Amidite

Materials

3‐Cyclohexene‐1‐methanol (S.1 ; Aldrich)
Imidazole
Argon source
N ,N ‐Dimethylformamide (DMF)
tert ‐Butyldimethylsilyl chloride (TBDMS·Cl; Aldrich)
Hexanes
Ethyl acetate (EtOAc)
TLC stain (see recipe )
Brine (saturated aqueous NaCl)
Magnesium sulfate (MgSO₄ )
Bromine
Dichloromethane (CH₂ Cl₂ )
10% (w/v) sodium thiosulfate (Na₂ S₂ O₃ )
Aliquat 336 (tricaprylylmethylammonium chloride; Aldrich)
Tetrahydrofuran (THF)
Potassium tert ‐butoxide (Aldrich)
Saturated ammonium chloride (NH₄ Cl)
Dowex 50WX4‐50 strongly acidic ion‐exchange resin (Aldrich)

Methanol
1,1′‐Carbonyldiimidazole (CDI; Aldrich)
6‐Amino‐1‐hexanol (Aldrich)
N ,N ‐Diisopropylethylamine (DIPEA; Aldrich)
2‐Cyanoethyl diisopropylchlorophosphoramidite (Aldrich)
Saturated sodium bicarbonate (NaHCO₃ )

100‐ and 250‐mL round‐bottom flasks with outlet stopcock adapters for gas line
TLC plates: 7.5 × 5–mm silica gel 60 F₂₅₄ precoated aluminum‐backed TLC sheets (EM Science)
Heat gun
1‐L and 500‐mL separatory funnels
Rotary evaporator equipped with a vacuum pump and vacuum controller
Rubber septa

10‐ and 50‐mL vials
3‐ and 5‐mL syringes with 2‐in., 20‐G stainless steel needles
Shaker
Short‐path distillation apparatus (Aldrich)
Biotage Flash 40 chromatography system with Flash 40M silica cartridge

Additional reagents and equipment for TLC ( appendix 3D ) and flash chromatography ( appendix 3E )

Basic Protocol 2: Synthesis of 5′‐Diene‐Modified Oligonucleotides

Materials

Dry acetonitrile (DNA synthesis grade; H₂ O content <30 µg/mL)
Deblock solution: trichloroacetic acid (TCA) in dichloromethane (Proligo)
Cap A solution: acetic anhydride in tetrahydrofuran (Proligo)
Cap B solution: 1‐methylimidazole in tetrahydrofuran/pyridine (Proligo)
Oxidizer solution: iodine in tetrahydrofuran/water/pyridine (Proligo)
Activator: 0.25 M 4,5‐dicyanoimidazole (DCI) in acetonitrile (Proligo)
50 mg/mL standard phosphoramidites [dT, dC(bz), dA(bz), and dG(ib); Proligo] in dry acetonitrile
50 mg/mL diene‐amidite (S.7 ; see protocol 1 ) in dry acetonitrile
Helium source
Concentrated ammonium hydroxide (NH₄ OH)
3 M sodium acetate ( appendix 2A )
Isopropanol
70% (v/v) ethanol

Automated DNA synthesizer (e.g., Expedite 8909, PerSeptive Biosystems)
Synthesis column for 1‐µmol scale (Proligo): controlled‐pore glass (CPG; 500 Å for <50‐mers, 1000 Å for ≥50‐mers)
Heating block
Speedvac evaporator

Additional reagents and equipment for quantitating oligonucleotides by OD₂₆₀ measurement (unit 10.3 )

Basic Protocol 3: Labeling of A 5′‐Diene‐Modified Oligonucleotide with Tamra‐5‐Maleimide

Materials

Diene‐modified oligonucleotide (see protocol 2 )
100 mM sodium acetate buffer, pH 4.5 ( appendix 2A )
40 mM TAMRA‐5‐maleimide solution: 5.0 mg TAMRA‐5‐maleimide (Molecular Probes) in 268 µL dimethylformamide (DMF)

Heating block
NAP‐5 columns (Amersham Pharmacia Biotech)
Speedvac evaporator

Additional reagents and equipment for quantitating oligonucleotides by OD₂₆₀ measurement (unit 10.3 )

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Figures

Figure 4.18.1 Preparation of diene‐amidite (S.7 ). Abbreviations: Aliquat 336, tricaprylylmethylammonium chloride; CDI, 1,1′‐carbonyldiimidazole; DIPEA, N , N ‐diisopropylethylamine; TBDMS·Cl, tert ‐butyldimethylsilyl chloride.

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Figure 4.18.2 Synthesis of diene‐modified oligonucleotides. Standard synthesis cycle: deblocking (deblock solution); coupling (N‐amidite/DCI); capping (Cap A and B solutions); oxidation (oxidizer solution). N‐amidite: standard dT, dC(bz), dA(bz), and dG(ib) phosphoramidites.

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Figure 4.18.3 Labeling of diene‐modified oligonucleotide with TAMRA‐5‐maleimide via aqueous Diels‐Alder cycloaddition.

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Figure 4.18.4 Common oligonucleotide conjugation methods. Abbreviations: FG, functional group; MMTr, 4‐monomethoxytrityl; TFA, 2,2,2‐trifluoroacetyl.

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Videos

Literature Cited

Literature Cited
	Bowtell, D. and Sambrook, J. (eds.) 2002. DNA Microarrays: A Molecular Cloning Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
	Goodchild, J. 1990. Conjugates of oligonucleotides and modified oligonucleotides: A review of their synthesis and properties. Bioconjugate Chem. 1: 165‐187.
	Hill, K.W., Taunton‐Rigby, J., Carter, J.D., Kropp, E., Vagle, K., Pieken, W., McGee, D.P.C., Husar, G., Leuck, M., Anziano, D., and Sebesta, D.P. 2001. Diels‐Alder bioconjugation of diene‐modified oligonucleotides. J. Org. Chem. 66: 5352‐5358.
	Letsinger, R.L., Elghanian, R., Biswanadham, G., and Mirkin, C. 2000. Use of a steroid cyclic disulfide anchor in constructing gold nanoparticle‐oligonucleotide conjugates. Bioconjugate Chem. 11: 289‐291.
	Smith, L.M., Fung, S., Hunkapiller, M.W., Hunkapiller, T.J., and Hoo, L.E. 1985. The synthesis of oligonucleotides containing an aliphatic amino group at the 5′ terminus: Synthesis of fluorescent DNA primers for use in DNA sequence analysis. Nucl. Acids Res. 13: 2399‐2412.
	Telser, J., Cruickshank, K.A., Morrison, L.E., and Netzel, T.L. 1989. Synthesis and characterization of DNA oligomers and duplexes containing covalently attached molecular labels: Comparison of biotin, fluorescein, and pyrene labels by thermodynamic and optical spectroscopic measurements. J. Am. Chem. Soc. 111: 6966‐6976.