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An Aminooxy‐Functionalized Non‐Nucleosidic Phosphoramidite for the Construction of Multiantennary Oligonucleotide Glycoconjugates on a Solid Support

Johanna Katajisto¹, Pasi Virta¹, Harri Lönnberg¹

¹University of Turku, Turku, Finland

Publication Name:

Current Protocols in Nucleic Acid Chemistry

Unit Number:

Unit 4.26

DOI:

10.1002/0471142700.nc0426s21

Online Posting Date:

July, 2005

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Abstract

In this unit, a method is described that allows construction of multiantennary oligonucleotide glycoconjugates on a solid support. A bis(hydroxymethyl)malondiamide-based phosphoramidite that contains two phthaloyl-protected aminooxy groups compatible with normal chain assembly is prepared. The aminooxy functions can be deblocked with a hydrazinium acetate treatment and subsequently oximated on-support with fully acetylated 4-oxobutyl -d-mannopyranoside. The resulting reagent is then used to prepare a conjugate containing two non-nucleosidic building blocks (i.e., four -d-mannopyranosyl units) close to the 5¢ terminus of the oligonucleotide.

Keywords: Solid support; oligonucleotides; glycoconjugates; oximation; phosphoramidites

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Unit Introduction
Basic Protocol 1: Preparation of the Aminooxy-Functionalized Phosphoramidite from Diethyl 2,2-Bis(Hydroxymethyl)Malonate
Basic Protocol 2: Preparation of the Mannosyl Aldehyde Ligand
Basic Protocol 3: Synthesis of Oligonucleotide Glycoconjugates by On-Support Oximation
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Preparation of the Aminooxy-Functionalized Phosphoramidite from Diethyl 2,2-Bis(Hydroxymethyl)Malonate

Materials

Diethyl 2,2-bis(hydroxymethyl)malonate (S.1.), 95% pure (Acros)
Dry tetrahydrofuran (THF), freshly distilled from sodium (store over 4-Å molecular sieves)
Trimethyl orthoformate, 98% pure (Aldrich)
p-Toluenesulfonic acid monohydrate, ³98.5% pure (Aldrich)
5% (w/v) aqueous sodium hydrogen carbonate (NaHCO₃)
Diethyl ether
Saturated aqueous sodium chloride (NaCl)
Sodium sulfate (Na₂SO₄), anhydrous
Silica gel: 0.040- to 0.063-mm Fluka Kieselgel 60 (dry overnight in 150°C oven)
Dichloromethane (CH₂Cl₂), ³99% pure
Bromocresol green indicator: dissolve 0.04 g bromocresol green (Merck) in 100 mL ethanol and add 0.1 M aqueous NaOH until the blue color appears (store up to 1 month at room temperature)
3-Aminopropanol, 99% pure (Aldrich)
Methanol (MeOH), ³99.8% pure
Dry benzene, distilled from powdered CaH₂ (store over 4-Å molecular sieves)
Triphenylphosphine, 99% pure (Aldrich)
N-Hydroxyphthalimide, 97% pure (Aldrich)
Diethyl azodicarboxylate (DEAD), ³97% (Fluka)
2-Propanol (i-PrOH), Baker analyzed
80% (v/v) aqueous acetic acid (AcOH)
Dry pyridine, distilled from powdered CaH₂ (store over 4-Å molecular sieves)
4,4¢-Dimethoxytrityl chloride (DMTr-Cl), 97% (Aldrich)
Dry toluene, distilled from powdered CaH₂ (store over 4-Å molecular sieves)
Dry acetonitrile, HPLC grade (store over 4-Å molecular sieves)
Phosphorus pentoxide (P₂O₅), 97% pure (Aldrich)
Dry nitrogen (or argon)
Anhydrous triethylamine, distilled from powdered CaH₂ (store over 4-Å molecular sieves)
2-Cyanoethyl N,N-diisopropylphosphonamidic chloride (TRC)
Ethyl acetate (EtOAc), analytical grade
Hexane, HPLC grade

Separatory funnels
Rotary evaporator equipped with a water aspirator
5 × 35–cm sintered glass chromatography column, porosity 2
TLC plate: silica-coated aluminium plate with fluorescent indicator (Merck silica gel 60 F₂₅₄)
3 × 20–cm sintered glass chromatography column, porosity 3
254-nm UV lamp
Vacuum desiccator

Additional reagents and equipment for column chromatography (appendix 3E) and thin-layer chromatography (TLC, appendix 3D)

Basic Protocol 2: Preparation of the Mannosyl Aldehyde Ligand

Materials

-d-Mannose pentaacetate (S.7; Sigma)
Dry benzene, distilled from powdered CaH₂ (store over 4-Å molecular sieves)
Dry nitrogen (or argon)
Dry acetonitrile (MeCN), HPLC grade (store over 4-Å molecular sieves)
1,4-Butanediol, 99% pure (Aldrich)
Boron trifluoride etherate (Merck)
Dichloromethane (CH₂Cl₂), ³ 99% pure
Sodium sulfate (Na₂SO₄), anhydrous
Silica gel: 0.040- to 0.063-mm Fluka Kieselgel 60 (dry overnight in oven at 150°C)
Ethyl acetate (EtOAc), analytical reagent
Hexane, HPLC grade
10% (v/v) H₂SO₄
Oxalyl chloride, 99% pure (Aldrich)
Dry argon
Dry ice/isopropanol bath
Dry dimethyl sulfoxide (DMSO; store over 4-Å molecular sieves)
Triethylamine (TEA), freshly distilled from powdered CaH₂
Diethyl ether
1 M aqueous HCl, ice cold
Saturated aqueous sodium hydrogen carbonate (NaHCO₃)
Saturated aqueous sodium chloride (NaCl)
Methanol (MeOH), ³99.8% pure

Separatory funnels
Rotary evaporator equipped with a water aspirator
3 × 20–cm sintered glass chromatography column, porosity 3
TLC plate: silica-coated aluminium plate with fluorescent indicator (Merck silica gel 60 F₂₅₄)
254-nm UV lamp

Additional reagents and equipment for column chromatography (appendix 3E) and thin-layer chromatography (TLC, appendix 3D)

Basic Protocol 3: Synthesis of Oligonucleotide Glycoconjugates by On-Support Oximation

Materials

Phosphoramidite S.6 (see Basic Protocol 1)
Dry acetonitrile (MeCN), HPLC grade (store over 4-Å molecular sieves)
2¢-Deoxyribonucleoside phosphoramidites (Glen Research)
0.5 M hydrazinium acetate solution: 0.124:4:1 (v/v/v) mixture of hydrazine monohydrate (>98% pure, Aldrich), analytical-grade pyridine, and glacial acetic acid
Pyridine, analytical grade
Acetonitrile (MeCN), analytical grade
Mannosyl aldehyde ligand S.9 (see Basic Protocol 2)
33% aqueous ammonia
Mobile phase A: 0.05 M ammonium acetate, pH 7.0, in H₂O
Mobile phase B: 0.05 M ammonium acetate in 65% (v/v) aqueous MeCN

55°C water bath
Speedvac evaporator
HPLC system with 4.6 × 150–mm analytical ThermoHypersil C18 column and 260-nm detector
Desalting column: 7.5 mm × 30 cm TSKgel G 2000 size-exclusion chromatography column, particle size 10 µm (Toso-Haas)
UV spectrophotometer

Additional reagents and equipment for automated solid-phase oligonucleotide synthesis (appendix 3C) and for purification (unit 10.5) and characterization (unit 10.2) of oligonucleotides

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Figures

Figure 4.26.1

Synthesis of the non-nucleosidic phosphoramidite S.6 (see Basic Protocol 1). DMTr, 4,4¢-dimethoxytrityl. Reprinted from Katajisto et al. (2004) with permission from the American Chemical Society.

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Figure 4.26.2

Synthesis of the sugar ligand S.9 (see Basic Protocol 2). Ac, acetyl. Reprinted from Katajisto et al. (2004) with permission from the American Chemical Society.

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Figure 4.26.3

Synthesis of the oligonucleotide glycoconjugate S.12 utilizing on-support oximation. Ac, acetyl; Pht, phthaloyl.

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Figure 4.26.4

RP-HPLC profile of crude S.12 at 260 nm. Reprinted from Katajisto et al. (2004) with permission from the American Chemical Society.

View Image

Literature Cited

Literature Cited
	Adinolfi, M., Barone, G., De Napoli, L., Guariniello, L., Iadonisi, A., and Piccialli, G. 1999. Solid-phase glycosidation of oligonucleotides. Tetrahedron Lett. 40:2607-2610.
	Akhtar, S., Routledge, A., and Patel, R. 1995. Synthesis of mono- and dimannoside phosphoramidate derivatives for the solid-phase conjugation to oligonucleotides. Tetrahedron Lett. 36:7333-7336.
	De Kort, M., Ebrahimi, E., Wijsman, E.R., Van der Marel, G.A., and Van Boom, J.H. 1999. Synthesis of oligodeoxynucleotides containing 5-(-d-glycopyranosyloxymethyl)-2¢-deoxyuridine, a modified nucleoside in the DNA of Trypanosoma Brucei. Eur. J. Chem. 9:2337-2344.
	Defrancq, E. and Lhomme, J. 2001. Use of an aminooxy linker for the functionalization of oligodeoxyribonucleotides. Bioorg. Med. Chem. Lett. 11:931-933.
	Dubber, M. and Fréchet, J.M.J. 2003. Solid-phase synthesis of multivalent glycoconjugates on a DNA synthesizer. Bioconjugate Chem. 14:239-246.
	Guzaev, A., Salo, H., Azhayev, A., and Lönnberg, H. 1996. Novel nonnucleosidic building blocks for the preparation of multilabeled oligonucleotides. Bioconjugate Chem. 7:240-248.
	Hamma, T. and Miller, P.S. 2003. 4-(2-Aminoethoxy)-2-ethylureido)quinoline-oligonucleotide conjugates: Synthesis, binding interactions, and derivatization with peptides. Bioconjugate Chem. 14:320-330.
	Hunziker, J. 1999. Synthesis of 5-(2-amino-2-deoxy-d-glucopyranosyloxymethyl)-2¢-deoxyuridine and its incorporation into oligothymidylates. Bioorg. Med. Chem. Lett. 9:201-204.
	Katajisto, J., Virta, P., and Lönnberg, H. 2004. Solid-phase synthesis of multiantennary oligonucleotide glycoconjugates utilizing on-support oximation. Bioconjugate Chem. 15:890-896.
	Mancuso, A.J., Huang, S., and Swern, D. 1978. Oxidation of long-chain and related alcohols to carbonyls by dimethyl sulfoxide “activated” by oxalyl chloride. J. Org. Chem. 43:2480-2482.
	Matsuura, K., Hibino, M., Yamada, Y., and Kobayashi, K. 2001. Construction of glycoclusters by self-organization of site-specifically glycosylated oligonucleotides and their cooperative amplification of lectin-recognition. J. Am. Chem. Soc. 123:357-358.
	Mitsunobu, O. 1981. The use of diethyl azodicarboxylate and triphenylphosphine in synthesis and transformation of natural products. Synthesis 1-28.
	Salo, H., Virta P., Hakala, H., Prakash, T.P., Kawasaki, A.M., Manoharan, M., and Lönnberg, H. 1999. Aminoxy functionalized oligonucleotides: Preparation, on-support derivatization, and post-synthetic attachment to polymer support. Bioconjugate Chem. 10:815-823.
	Salvador, L.A., Elofsson, M., and Kihlberg, J. 1995. Preparation of building blocks for glycopeptide synthesis by glycosylation of Fmoc amino acids having unprotected carboxyl groups. Tetrahedron 51:5643-5656.
	Sheppard, T.L., Wong, C., and Joyce, G.F. 2000. Neoglycoconjugates: Design and synthesis of a new class of DNA-carbohydrate conjugates. Angew. Chem. Int. Ed. Engl. 39:3660-3663.
	Wang, L., Prakash, R.K., Stein, C.A., Koehn, R.K., and Ruffner, D.E. 2003. Progress in the delivery of therapeutic oligonucleotides: Organ/cellular distribution and targeted delivery of oligonucleotides in vivo. Antisense Nucleic Acid Drug Dev. 13:169-189.