Synthesis of 5′‐O‐Phosphoramidites with a Photolabile 3′‐O‐Protecting Group

Markus Beier1, Jörg D. Hoheisel1

1 Deutsches Krebsforschungszentrum, Heidelberg
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 12.3
DOI:  10.1002/0471142700.nc1203s17
Online Posting Date:  September, 2004
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Abstract

This unit describes the chemical synthesis of phosphoramidite building blocks that carry a protecting group at the 3′ position. These inversely oriented synthons expose a 2‐(2‐nitrophenyl)propoxycarbonyl (NPPOC) group as the photolabile protecting group of choice. Among other applications, the building blocks can be employed for light‐controlled in situ synthesis of DNA microarrays, producing arrayed oligonucleotides that are attached to the support via their 5′ ends, leaving their 3′ termini available to act as substrates for polymerases.

Keywords: photolabile protecting group; phosphoramidite; DNA microarray

     
 
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Table of Contents

  • Basic Protocol 1: Introduction of the Photolabile 2‐(2‐Nitrophenyl)Propoxycarbonyl Group Into 5′‐O‐Dimethoxytritylated Nucleosides
  • Basic Protocol 2: Preparation of 3′‐O‐[2‐(2‐Nitrophenyl)Propoxycarbonyl]‐ Protected 5′‐O‐Phosphoramidites
  • Support Protocol 1: Preparation of 2‐(2‐Nitrophenyl)Propoxycarbonyl‐N‐Methylimidazolium Chloride
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Introduction of the Photolabile 2‐(2‐Nitrophenyl)Propoxycarbonyl Group Into 5′‐O‐Dimethoxytritylated Nucleosides

  Materials
  • Nitrogen source
  • 2‐(2‐Nitrophenyl)propoxycarbonyl‐N‐methylimidazolium chloride solution ( S.6; see protocol 3), prepare fresh
  • Dichloromethane, anhydrous
  • N‐[(4‐tert‐Butylphenoxy)acetyl]‐5′‐O‐(4,4′‐dimethoxytrityl) deoxyribonucleosides:
    • N6‐tac‐5′‐O‐DMTr‐2′‐deoxyadenosine ( S.1a; e.g., Proligo, ChemGenes; see )
    • N4‐tac‐5′‐O‐DMTr‐2′‐deoxycytidine ( S.1b; e.g., Proligo, ChemGenes; see )
    • N2‐tac‐5′‐O‐DMTr‐2′‐deoxyguanosine ( S.1c; e.g., Proligo, ChemGenes; see )
    • 5′‐O‐DMTr‐thymidine ( S.1d; e.g., Proligo)
  • Toluene
  • Ethyl acetate
  • 5% (v/v) aqueous HCl
  • Sodium sulfate (Na 2SO 4)
  • 10% (v/v) trichloroacetic acid in dichloromethane
  • Methanol
  • Saturated aqueous sodium bicarbonate (NaHCO 3) solution
  • Silica gel (30 to 60 µm; e.g., Baker) for flash chromatography
  • 100‐mL three‐neck round‐bottom flask with drying tube
  • Dropping funnel
  • Balloons
  • 100‐mL two‐neck flask
  • Thin‐layer chromatography (TLC) silica gel 60 plates (Merck)
  • 254‐nm UV lamp
  • 500‐mL separatory funnels
  • Rotary evaporator equipped with a water aspirator
  • Additional reagents and equipment for TLC ( appendix 3D), flash chromatography ( appendix 3E), nuclear magnetic resonance (NMR), and mass spectrometry (MS)

Basic Protocol 2: Preparation of 3′‐O‐[2‐(2‐Nitrophenyl)Propoxycarbonyl]‐ Protected 5′‐O‐Phosphoramidites

  Materials
  • Nitrogen source
  • 3′‐O‐NPPOC‐protected nucleosides ( S.2a S.2d; see protocol 1)
  • Acetonitrile, anhydrous
  • 2‐Cyanoethyl‐N,N,N,N‐tetraisopropylphosphorodiamidite
  • 0.5 M pyridine hydrochloride in anhydrous acetonitrile, dried over 4 Å  molecular sieves
  • Toluene
  • Ethyl acetate
  • Dichloromethane, anhydrous
  • Saturated aqueous sodium bicarbonate (NaHCO 3) solution
  • Sodium sulfate (Na 2SO 4)
  • Silica gel for flash chromatography (30 to 60 µm; Baker)
  • 100‐mL three‐neck round‐bottom flask with drying tube
  • Dropping funnel
  • Balloons
  • 100‐mL two‐neck flask
  • Thin‐layer chromatography (TLC) silica gel 60 plates (Merck)
  • 254‐nm UV lamp
  • 500‐mL separatory funnel
  • Rotary evaporator equipped with a water aspirator
  • Additional reagents and equipment for TLC ( appendix 3D), flash chromatography ( appendix 3E), nuclear magnetic resonance (NMR), and mass spectrometry (MS)

Support Protocol 1: Preparation of 2‐(2‐Nitrophenyl)Propoxycarbonyl‐N‐Methylimidazolium Chloride

  Materials
  • Nitrogen source
  • Tetrahydrofuran (THF), dry
  • Trichloromethyl chloroformate (diphosgene) (e.g., Fluka, Sigma)
  • 2‐(2‐Nitrophenyl)propanol ( S.4; see Uhlmann and Pfleiderer, )
  • N‐Methylmorpholine
  • Toluene
  • Ethyl acetate
  • Liquid nitrogen
  • Methanolic KOH solution: 5% (w/v) KOH in MeOH, prepare fresh
  • N‐Methylimidazole
  • Dichloromethane, dry
  • Molecular sieves, 4 Å
  • 100‐ and 250‐mL three‐neck round‐bottom flasks
  • Drying tubes and septa for 100‐ and 250‐mL flasks
  • Dropping funnel for 250‐mL flasks
  • Balloons
  • 20‐mL syringes and 18‐ to 22‐G needles
  • Thin‐layer chromatography (TLC) silica gel 60 plates (Merck)
  • Glass frit
  • 250‐ and 500‐mL two‐neck Schlenk‐type flasks with glass stoppers (neoLab)
  • Vacuum tubing
  • Gas inlets
  • Vacuum pump with appropriate tubing
  • 2‐mL gas‐tight syringe with disposable needles
  • Additional reagents and equipment for TLC ( appendix 3D)
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Figures

Videos

Literature Cited

Literature Cited
   Baum, M., Bielau, S., Rittner, N., Schmid, K., Eggelbusch, K., Dahms, M., Schlauersbach, A., Tahedl, H., Beier, M., Güimil, R., Scheffler, M., Hermann, C., Funk, J.M., Wixmerten, A., Rebscher, H., Hönig, M., Andreae, C., Büchner, D., Moschel, E., Glathe, A., Jäger, E., Thom, M., Greil, A., Bestvater, F., Obermeier, F., Burgmaier, J., Thome, K., Weichert, S., Hein, S., Binnewies, T., Foitzik, V., Müller, M., Stähler, C.F., and Stähler, P.F. 2003. Validation of a novel, fully integrated and flexible microarray benchtop facility for gene expression profiling. Nucl. Acids Res. 31:e151.
   Beier, M. and Hoheisel, J.D. 1999. Versatile derivatisation of solid support media for covalent bonding on DNA‐microchips. Nucl. Acids Res. 27:1970‐1977.
   Beier, M. and Hoheisel, J.D. 2000. Production by quantitative photolithographic synthesis of individually quality checked DNA microarrays. Nucl. Acids Res. 28:e11.
   Beier, M. and Pfleiderer, W. 1999. Pyridinium salts—an effective class of catalysts for oligonucleotide synthesis. Helv. Chim. Acta 82:879‐887.
   Beier, M., Stephan, A., and Hoheisel, J.D. 2001. Synthesis of photolabile 5′‐O‐phosphoramidites for the production of microarrays of inversely oriented oligonucleotides. Helv. Chim. Acta 84:2089‐2095.
   Giegrich, H., Eisele‐Bühler, S., Hermann, C., Kvasyuk, E., Charubala, R., and Pfleiderer, W. 1998. New photolabile protecting groups in nucleoside and nucleotide chemistry—synthesis, cleavage mechanisms and applications. Nucleosides Nucleotides 17:1987‐1996.
   Hasan, A., Stengele, K.P., Giegrich, H., Cornwell, P., Isham, K.I., Sachleben, R., Pfleiderer, W., and Foote, R.S. 1997. Photolabile protecting groups for nucleosides: Synthesis and photodeprotection rates. Tetrahedron 53:4247‐4264.
   Pease, A.C., Solas, D., Sullivan, E.J., Cronin, M.T., Holmes, C.P., and Fodor, S.P.A. 1994. Light‐generated oligonucleotide arrays for rapid DNA sequence analysis. Proc. Natl. Acad. Sci. U.S.A. 91:5022‐5026.
   Singh‐Gasson, S., Green, R.D., Yue, Y.J., Nelson, C., Blattner, F., Sussman, M.R., and Cerrina, F. 1999. Maskless fabrication of light‐directed oligonucleotide microarrays using a digital micromirror array. Nat. Biotechnol. 17:974‐978.
   Sinha, N.D., Davis, P., Usman, N., Perez, J., Hodge, R., Kremsky, J., and Casale, R. 1993. Labile exocyclic amine protection of nucleosides in DNA, RNA, and oligonucleotide analog synthesis facilitating N‐deacylation, minimizing depurination and chain degradation. Biochimie 75:13‐23.
   Uhlmann, E., and Pfleiderer, W. 1981. Helv. Chim. Acta 64:1688‐1704.
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