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

Johanna Katajisto1, Pasi Virta1, Harri Lönnberg1

1 University of Turku, Turku
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 α‐D‐mannopyranoside. The resulting reagent is then used to prepare a conjugate containing two non‐nucleosidic building blocks (i.e., four α‐D‐mannopyranosyl units) close to the 5′ terminus of the oligonucleotide.

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

     
 
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Table of Contents

  • 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, ≥98.5% pure (Aldrich)
  • 5% (w/v) aqueous sodium hydrogen carbonate (NaHCO 3)
  • Diethyl ether
  • Saturated aqueous sodium chloride (NaCl)
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Silica gel: 0.040‐ to 0.063‐mm Fluka Kieselgel 60 (dry overnight in 150°C oven)
  • Dichloromethane (CH 2Cl 2), ≥99% 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), ≥99.8% pure
  • Dry benzene, distilled from powdered CaH 2 (store over 4‐Å molecular sieves)
  • Triphenylphosphine, 99% pure (Aldrich)
  • N‐Hydroxyphthalimide, 97% pure (Aldrich)
  • Diethyl azodicarboxylate (DEAD), ≥97% (Fluka)
  • 2‐Propanol (i‐PrOH), Baker analyzed
  • 80% (v/v) aqueous acetic acid (AcOH)
  • Dry pyridine, distilled from powdered CaH 2 (store over 4‐Å molecular sieves)
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl), 97% (Aldrich)
  • Dry toluene, distilled from powdered CaH 2 (store over 4‐Å molecular sieves)
  • Dry acetonitrile, HPLC grade (store over 4‐Å molecular sieves)
  • Phosphorus pentoxide (P 2O 5), 97% pure (Aldrich)
  • Dry nitrogen (or argon)
  • Anhydrous triethylamine, distilled from powdered CaH 2 (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 F 254)
  • 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
  • α‐D‐Mannose pentaacetate ( S.7; Sigma)
  • Dry benzene, distilled from powdered CaH 2 (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 (CH 2Cl 2), ≥ 99% pure
  • Sodium sulfate (Na 2SO 4), 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) H 2SO 4
  • 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 CaH 2
  • Diethyl ether
  • 1 M aqueous HCl, ice cold
  • Saturated aqueous sodium hydrogen carbonate (NaHCO 3)
  • Saturated aqueous sodium chloride (NaCl)
  • Methanol (MeOH), ≥99.8% 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 F 254)
  • 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 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 protocol 2)
  • 33% aqueous ammonia
  • Mobile phase A: 0.05 M ammonium acetate, pH 7.0, in H 2O
  • 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

Videos

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‐(β‐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.
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