Methoxyoxalamido Chemistry in the Synthesis of Tethered Phosphoramidites and Functionalized Oligonucleotides

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

A general approach to phosphoramidites tethered with single and multiple linkers through the use of methoxyoxalamido (MOX) chemistry is described. The approach utilizes readily available and inexpensive primary aliphatic amino alcohols and diamines to produce a rich and diverse variety of tethered phosphoramidites. Furthermore, the use of MOX chemistry in a modular fashion enables fairly rapid assembly of compound tethers. All novel phosphoramidites described have been successfully used in automated syntheses of 5??modified oligonucleotides.

Keywords: methoxyoxalamido (MOX) chemistry; tethered phosphoramidites; 5??modified oligonucleotides

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Basic Protocol 1: Preparation of Phosphoramidites Tethered with Single Linkers
Alternate Protocol 1: Preparation of Phosphoramidites Tethered with Multiple Linkers
Basic Protocol 2: Synthesis, Deprotection, and Purification of Oligonucleotides Derivatized with Tethered Phosphoramidites
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Preparation of Phosphoramidites Tethered with Single Linkers

Materials

trans ‐4‐Amino‐1‐cyclohexanol hydrochloride (97%; Aldrich)
Triethylamine (Et₃ N), ≥99%
Methanol (MeOH), HPLC grade
Dimethyl oxalate, 99% (Aldrich)
Diethyl ether, anhydrous
Chloroform (CHCl₃ ), HPLC grade
60 Å silica gel, 200 to 400 mesh (EM Science)
5′‐Amino‐5′‐deoxythymidine (Berry & Associates, http://www.berryassoc.com or Fidelity Systems, http://www.fidelitysystems.com)
Aliphatic primary diamines
- Ethylenediamine (EDA), ≥99.5% (Aldrich) for S.3a
- 2,4,8,10‐Tetraoxaspiro[5.5]undecane‐3,9‐dipropanamine (TUDA), 97% (Aldrich) for S.4a
- 4,7,10‐Trioxa‐1,13‐tridecanediamine (TTDD), ≥98% (Aldrich) for S.5a and S.6a
Aliphatic primary amino alcohols
- 6‐Amino‐1‐hexanol (AH), 97% (Aldrich) for S.11a
- 2‐(2‐Aminoethoxy)ethanol (AEE), 98% (Aldrich) for S.10a
Dichloromethane (CH₂ Cl₂ ), HPLC grade
N ,N ‐Dimethylformamide (DMF), anhydrous
Pyridine, anhydrous, 99.8% (Aldrich)
4‐Monomethoxytrityl chloride (MMTr‐Cl; ChemGenes)
Saline solution: 25% to 30% (w/v) aqueous NaCl
Na₂ SO₄ , reagent grade, anhydrous
Pentane, HPLC grade
4,4′‐Dimethoxytrityl chloride (DMTr‐Cl; ChemGenes)
Tetrazole, dried
Argon gas
2‐Cyanoethyl‐N ,N ,N ′,N ′‐tetraisopropylphosphorodiamidite (ChemGenes)
10% (w/v) aqueous sodium hydrogen carbonate (NaHCO₃ )
Dichloromethane (CH₂ Cl₂ ), anhydrous
Ethyl acetate (EtOAc), HPLC grade
Toluene, anhydrous
Phosphorus pentoxide (P₂ O₅ )

Rotary evaporator equipped with a vacuum pump or water aspirator (5 to 10 Torr)
Buchner funnel, 140‐mL capacity with glass frit (porosity 4 to 8 µm)
Paper filters (coarse porosity)
4 × 40–cm sintered glass columns
Vacuum oil pump (0.05 to 0.5 Torr)
Separatory funnel
Thin‐layer chromatography (TLC) Kieselgel 60 F₂₅₄ plates (EM Science)
I₂ ‐silica chamber
254‐nm UV lamp
Heat gun

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

Alternate Protocol 1: Preparation of Phosphoramidites Tethered with Multiple Linkers

Materials

Tethered phosphoramidites ( S.3d‐S.14d ; see protocol 1 and protocol 2 )
Acetonitrile (CH₃ CN), anhydrous, DNA synthesis grade
Standard 2′‐deoxyribonucleoside phosphoramidites (Transgenomic)
0.5 M tetrazole in CH₃ CN (Glen Research) or 0.25 M 5‐ethylthio‐1H ‐tetrazole (ETT, Glen Research)
Ethanolamine (EA), ≥99% (Aldrich)
10% (w/v) LiClO₄ in ethanol (EtOH)
Ethanol (EtOH), 200 proof
7 M urea
15% (w/v) polyacrylamide gel ( appendix 3B ) containing 7 M urea in 0.5× TBE electrophoresis buffer ( appendix 2A )
0.25 M triethyl ammonium bicarbonate (TEAB), aqueous solution

1.7‐mL microcentrifuge tubes
70°C incubator or water bath
Microcentrifuge
Speedvac evaporator (Savant)
Sephadex G‐25 NAP‐10 columns (Pharmacia)
Lyophilizer
Water aspirator or vacuum pump (5 to 10 Torr)

Additional reagents and equipment for automated solid‐phase oligonucleotide synthesis ( appendix 3C ), purification of oligonucleotides (units 10.1 , 10.4 , 10.5 , 10.7 & APPENDIX 3.NaN ), and determination of molecular mass (unit 10.1 )

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Figures

Figure 4.29.1 Synthetic pathway for phosphoramidites tethered with a single linker. MOX, methoxyoxalamido; DMTr, dimethoxytrityl; MMTr, monomethoxytrityl.

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Figure 4.29.2 Synthetic pathway to phosphoramidites tethered with multiple linkers. MOX, methoxyoxalamido; DMTr, dimethoxytrityl; MMTr, monomethoxytrityl; n = number of cycles.

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Figure 4.29.3 Initial MOX precursors.

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Figure 4.29.4 Phosphoramidites and precursors with simple or multiple aminated linkers. MMTr, monomethoxytrityl.

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Figure 4.29.5 Phosphoramidites and precursors with simple or multiple hydroxylated linkers. DMTr, dimethoxytrityl.

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Figure 4.29.6 PAGE analysis of crude 5′‐modified T₁₀ oligonucleotides. Lane 1 of each gel corresponds to the unmodified T₁₀ oligonucleotide. The other lanes correspond to oligonucleotides modified at the 5′ terminus with the following phosphoramidites: S.3d (A2), S.4d (A3), S.5d (A4), S.6d (A5), S.8d (A6), S.7d (A7), S.9d (A8), S.10d (B2), S.11d (B3), S.13d (B4), S.12d (B5), S.14d (B6).

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Literature Cited

Literature Cited
	Bannwarth, W. 1988. Solid phase synthesis of oligonucleotides containing phosphoramidate internucleotide linkages and their specific chemical cleavage. Helv. Chim. Acta 71:1517‐1527.
	Jaschke, A., Furste, J.P., Nordhoff, E., Hillenkamp, F., Cech, D., and Erdmann, V.A. 1994. Synthesis and properties of oligodeoxyribonucleotide‐polyethylene glycol conjugates. Nucl. Acids Res. 22:4810‐4817.
	Polushin, N.N. 2000. The precursor strategy: Terminus methoxyoxalamido modifiers for single and multiple functionalization of oligodeoxyribonucleotides. Nucl. Acids Res. 28:3125‐3133.
	Polushin, N.N., Morocho, A.M., Chen, B.C., and Cohen, J.S. 1994. On the rapid deprotection of synthetic oligonucleotides and analogs. Nucl. Acids Res. 22:639‐645.
	Reddy, M.P., Hanna, N.B., and Farooqui, F. 1994. Fast cleavage and deprotection of oligonucleotides. Tetrahedron Lett. 35:4311‐4314.