Release of DNA Oligonucleotides and Their Conjugates from Controlled‐Pore Glass Under Thermolytic Conditions

Andrzej Grajkowski1, Jacek Cieślak1, Scott Norris2, Darón I. Freedberg2, Jon S. Kauffman3, Robert J. Duff3, Serge L. Beaucage1

1 Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, 2 Division of Bacterial, Parasitic & Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, 3 Lancaster Laboratories, Lancaster, Pennsylvania
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 3.17
DOI:  10.1002/0471142700.nc0317s35
Online Posting Date:  December, 2008
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


The sequential functionalization of long‐chain alkylamine controlled‐pore glass (CPG) with a 3‐hydroxypropyl‐(2‐cyanoethyl)thiophosphoryl linker and a dinucleoside phosphorotetrazolide leads to a uniquely engineered support for solid‐phase synthesis. Unlike conventional succinylated‐CPG supports, this support is designed to allow oligonucleotide deprotection and elimination of deprotection side‐products to proceed without release of the oligonucleotide. When needed, the DNA oligonucleotide can be thermolytically released in 2 hr under essentially neutral conditions. The modified CPG support has been successfully employed in the synthesis of both native and fully phosphorothioated DNA 20‐mers. On the basis of reversed‐phase HPLC and electrophoretic analyses, the purity of the released oligonucleotides is comparable to that of identical oligonucleotides synthesized from succinylated‐CPG supports, in terms of both shorter‐than‐full‐length oligonucleotide contaminants and overall yields. The detailed preparation of DNA oligonucleotides conjugated with exemplary reporter or functional groups, either at the 3′‐terminus or at both 3′‐ and 5′‐termini, is also described. Curr. Protoc. Nucleic Acid Chem. 35:3.17.1‐3.17.21. © 2008 by John Wiley & Sons, Inc.

Keywords: deoxyribonucleoside phosphorodiamidites; solid‐phase synthesis; modified CPG support; thermolytic conditions; DNA oligonucleotides conjugates

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Functionalization of CPG with Covalently Linked Dinucleotides and Its Application to Solid‐Phase Synthesis of Native and Modified DNA Oligonucleotides
  • Support Protocol 1: Preparation of Deoxyribonucleoside Phosphorodiamidites
  • Basic Protocol 2: Application of the Dinucleotide‐Bound Support for Synthesis of a DNA Oligonucleotide with 5′‐ and 3′‐Functional Groups
  • Alternate Protocol 1: Application of the Hydroxylated CPG Support for Synthesis of a DNA Oligonucleotide with a 3′‐Reporter Group
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: Functionalization of CPG with Covalently Linked Dinucleotides and Its Application to Solid‐Phase Synthesis of Native and Modified DNA Oligonucleotides

  • Long‐chain alkylamine controlled‐pore glass, 500 Å (CPG)
  • Triethylamine (TEA)
  • Anhydrous acetonitrile (MeCN; Glen Research)
  • Dry argon gas cylinder
  • O‐(2‐Cyanoethyl)‐O‐[3‐(4,4′‐dimethoxytrityl)oxy‐1‐propyl]‐N,N‐diisopropylphosphoramidite (S.1; Glen Research)
  • 0.25 M 5‐ethylthio‐1H‐tetrazole in dry acetonitrile (ETT/MeCN; Glen Research)
  • 3H‐1,2‐Benzodithiol‐3‐one 1,1‐dioxide (Glen Research)
  • Reagents recommended for automated solid‐phase oligonucleotide synthesis (Glen Research):
    • Standard 2‐cyanoethyl deoxyribonucleoside phosphoramidites (T, CBz, ABz, and GiBu)
    • Activator solution: 1H‐tetrazole in acetonitrile
    • Oxidation solution: 0.02 M iodine in THF/pyridine/water
    • 3H‐1,2‐Benzodithiol‐3‐one 1,1‐dioxide
    • Cap A solution: acetic anhydride in THF/pyridine
    • Cap B solution: 1‐methylimidazole in THF
    • Deblocking solution: trichloroacetic acid (TCA) in dichloromethane
  • Deoxyribonucleoside phosphorodiamidites (S.4; see protocol 2):
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐bis(N,N‐diisopropylamino)phosphinyl‐2′‐deoxythymidine
    • N4‐Benzoyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐3′‐O‐bis(N,N‐diisopropylamino)phosphinyl‐2′‐deoxycytidine
    • N6‐Benzoyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐3′‐O‐bis(N,N‐diisopropylamino)phosphinyl‐2′‐deoxyadenosine
    • N2‐Isobutyryl‐5′‐O‐(4,4′‐dimethoxytrityl)‐3′‐O‐bis(N,N‐diisopropylamino)phosphinyl‐2′‐deoxyguanosine
  • 3′‐O‐Levulinyl‐2′‐deoxyribonucleosides (S.5; Rasayan):
    • 3′‐O‐Levulinyl‐2′‐deoxythymidine
    • N4‐Benzoyl‐3′‐O‐levulinyl‐2′‐deoxycytidine
    • N6‐Benzoyl‐3′‐O‐levulinyl‐2′‐deoxyadenosine
    • N2‐Isobutyryl‐3′‐O‐levulinyl‐2′‐deoxyguanosine
  • Anhydrous ammonia cylinder (Aldrich)
  • Phosphate‐buffered saline
  • 2 M triethylammonium acetate (TEAA) buffer (Applied Biosystems)
  • Loading buffer: 1:4 (v/v) 10× TBE, pH 8.3 ( appendix 2A) in formamide, containing 2 mg/mL bromphenol blue
  • 20 × 40–cm 7 M urea/20% polyacrylamide gel (unit 10.4 and appendix 3B)
  • Stains‐all (Aldrich)
  • 4‐ and 8‐mL screw‐thread glass vials (Wheaton)
  • 15‐mL glass sintered funnel (coarse and medium porosities)
  • Rubber septa of assorted sizes
  • 16‐ and 21‐G hypodermic needles
  • 0.5‐, 1‐, 5‐, and 10‐mL glass syringes
  • Spectrophotometer
  • 1‐mL Luer‐tipped syringe
  • Empty synthesis columns (Glen Research)
  • DNA/RNA synthesizer
  • 250‐mL stainless steel pressure vessel (Parr Instrument)
  • 90°C heating block (VWR)
  • 0.45‐µm syringe filters
  • 5‐µm Supelcosil LC‐18S HPLC column (25 cm × 4.6 mm, Supelco)
  • 1.5‐mL microcentrifuge tubes
  • Platform agitator
  • Additional reagents and equipment for quantitative trityl assay (unit 3.2), automated oligonucleotide synthesis ( appendix 3C), and polyacrylamide gel electrophoresis (unit 10.4 and appendix 3B)
NOTE: All filtrations, unless otherwise indicated, are performed using a water aspirator as a vacuum source.

Support Protocol 1: Preparation of Deoxyribonucleoside Phosphorodiamidites

  • 5′‐O‐Protected deoxyribonucleosides (Chem‐Impex International):
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐deoxythymidine
    • N4‐Benzoyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxycytidine
    • N6‐Benzoyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxyadenosine
    • N2‐Isobutyryl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxyguanosine
  • Anhydrous pyridine
  • Anhydrous methylene chloride (CH 2Cl 2; Aldrich)
  • N,N‐Diisopropylethylamine (DIPEA; Aldrich)
  • Argon source
  • Bis(N,N‐diisopropylamino)chlorophosphine (Aldrich)
  • Benzene (Aldrich)
  • Triethylamine (TEA; Aldrich)
  • Silica gel (60Å, 230 to 400 mesh; Merck)
  • 2.5 × 7.5–cm EMD TLC plates precoated with a 250‐µm layer of Silica Gel 60 F 254
  • 100‐mL round‐bottom flasks
  • High vacuum oil pump
  • Stir bars
  • Rubber septa
  • 5‐ and 10‐mL glass syringes
  • Separatory funnels
  • Rotary evaporator connected to a vacuum pump
  • 2.5 × 20–cm disposable Flex chromatography columns (Kontes)
  • Fraction collector
  • Chromaflex TLC developing jars (Kontes)

Basic Protocol 2: Application of the Dinucleotide‐Bound Support for Synthesis of a DNA Oligonucleotide with 5′‐ and 3′‐Functional Groups

  • CPG‐linked dinucleotide S.7 (see protocol 1)
  • 0.5 M hydrazine monohydrate (Aldrich)
  • Pyridine
  • Acetic acid
  • Dry MeCN
  • Argon
  • O‐(2‐Cyanoethyl)‐O‐(5‐hexyn‐1‐yl)‐N,N‐diisopropylphosphoramidite (Glen Research)
  • 0.45 M 1H‐tetrazole in dry MeCN
  • Cap A solution (see protocol 1)
  • Cap B solution (see protocol 1)
  • 0.02 M iodine in THF/pyridine/H 2O
  • O‐(2‐Cyanoethyl)‐O‐[6‐(4‐methoxytrityl)‐amino‐1‐hexyl]‐N,N‐diisopropylphosphoramidite (Glen Research)
  • 3% TCA in CH 2Cl 2
  • 1:1 (v/v) TEA/MeCN
  • 1:1:8 (v/v/v) azidoacetic anhydride/pyridine/THF
  • Synthesis columns
  • 1‐, 3‐, and 10‐mL syringes
  • Rubber septa
  • 4‐mL glass vials
  • Luer‐tipped syringes
  • 16‐G hypodermic needles
  • DNA/RNA synthesizer

Alternate Protocol 1: Application of the Hydroxylated CPG Support for Synthesis of a DNA Oligonucleotide with a 3′‐Reporter Group

  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐bis(N,N‐diisopropylamino)phosphinyl‐2′‐deoxythymidine (S.4, see protocol 2)
  • 1‐Pyrenebutanol (Aldrich)
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Beaucage, S.L. 1999. Attachment of reporter and conjugate groups to DNA. In Comprehensive Natural Products Chemistry, Volume 7: DNA and Aspects of Molecular Biology (D. Barton, K. Nakanishi, O. Meth‐Cohn, and E.T. Kool, eds.) pp. 153‐249. Elsevier Science Publishing, New York.
   Beaucage, S.L. and Iyer, R.P. 1992. Advances in the synthesis of oligonucleotides by the phosphoramidite approach. Tetrahedron 48:2223‐2311.
   Berner, S., Mühlegger, K., and Seliger, H. 1989. Studies on the role of tetrazole in the activation of phosphoramidites. Nucleic Acids Res. 17:853‐864.
   Boal, J.H., Wilk, A., Harindranath, N., Max, E.E., Kempe, T., and Beaucage, S.L. 1996. Cleavage of oligodeoxyribonucleotides from controlled‐pore glass supports and their rapid deprotection by gaseous amines. Nucleic Acids Res. 24:3115‐3117.
   Cieślak, J. and Beaucage, S.L. 2003. Thermolytic properties of 3‐(2‐pyridyl)‐1‐propyl and 2‐[N‐methyl‐N‐(2‐pyridyl)]aminoethyl phosphate/thiophosphate protecting groups in solid‐phase synthesis of oligodeoxyribonucleotides. J. Org. Chem. 68:10123‐10129.
   Cieślak, J., Grajkowski, A., Livengood, V., and Beaucage, S.L. 2004. The thermolytic 4‐methylthio‐1‐butyl group for phosphate/thiophosphate protection in solid‐phase synthesis of DNA oligonucleotides. J. Org. Chem. 69:2509‐2515.
   Dyke, J.M., Groves, A.P., Morris, A., Ogden, J.S., Dias, A.A., Oliveira, A.M.S., Costa, M.L., Barros, M.T., Cabral, M.H., and Moutinho, A.M.C. 1997. Study of the thermal decomposition of 2‐azidoacetic acid by photoelectron and matrix isolation infrared spectroscopy. J. Am. Chem. Soc. 119:6883‐6887.
   Grajkowski, A., Wilk, A., Chmielewski, M.K., Phillips, L.R., and Beaucage, S.L. 2001. The 2‐(N‐formyl‐N‐methyl)aminoethyl group as a potential phosphate/thiophosphate protecting group in solid‐phase oligodeoxyribonucleotide synthesis. Org. Lett. 3:1287‐1290.
   Grajkowski, A., Ausín, C., Kauffman, J.S., Snyder, J., Hess, S., Lloyd, J.R., and Beaucage, S.L. 2007. Solid‐phase synthesis of thermolytic DNA oligonucleotides functionalized with a single 4‐hydroxy‐1‐butyl or 4‐phosphato/‐thiophosphato‐1‐butyl thiophosphate protecting group. J. Org. Chem. 72:805‐815.
   Grajkowski, A., Cieślak, J., Kauffman, J.S., Duff, R.J., Norris, S., Freedberg, D., and Beaucage, S.L. 2008. Thermolytic release of covalently linked DNA oligonucleotides and their conjugates from controlled‐pore glass at near neutral pH. Bioconjugate Chem. 19:1696‐1706.
   Hassner, A., Strand, G., Rubinstein, M., and Patchornik, A. 1975. Levulinic esters. Alcohol protecting group applicable to some nucleosides. J. Am. Chem. Soc. 97:1614‐1615.
   Iyer, R.P., Phillips, L.R., Egan, W., Regan, J.B., and Beaucage, S.L. 1990. The automated synthesis of sulfur‐containing oligodeoxyribonucleotides using 3H‐1,2‐benzodithiol‐3‐one 1,1‐dioxide as a sulfur‐transfer reagent. J. Org. Chem. 55:4693‐4699.
   Kumar, R., El‐Sagheer, A., Tumpane, J., Lincoln, P., Wilhelmsson, L.M., and Brown, T. 2007. Template‐directed oligonucleotide strand ligation, covalent intramolecular DNA circularization and catenation using click chemistry. J. Am. Chem. Soc. 129:6859‐6864.
   Letsinger, R.L. and Mahadevan, V. 1965. Oligonucleotide synthesis on a polymer support. J. Am. Chem. Soc. 87:3526‐3527.
   Letsinger, R.L. and Mahadevan, V. 1966. Stepwise synthesis of oligodeoxyribonucleotides on an insoluble polymer support. J. Am. Chem. Soc. 88:5319‐5324.
   Marugg, J.E., Burik, A., Tromp, M., van der Marel, G.A., and van Boom, J.H. 1986. A new and versatile approach to the preparation of valuable deoxynucleoside 3′‐phosphite intermediates. Tetrahedron Lett. 27:2271‐2274.
   Merrifield, R.B. 1963. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149‐2154.
   Merrifield, R.B. 1965. Automated synthesis of peptides. Science 150:178‐185.
   Regan, J.B., Phillips, L.R., and Beaucage, S.L. 1992. Large‐scale preparation of the sulfur‐transfer reagent 3H‐1,2‐benzodithiol‐3‐one 1,1‐dioxide. Org. Prep. Proc. Int. 24:488‐492.
   Seeberger, P.H. and Haase, W.‐C. 2000. Solid‐phase oligosaccharide synthesis and combinatorial carbohydrate libraries. Chem. Rev. 100:4349‐4393.
   Seeberger, P.H. and Werz, D.B. 2005. Automated synthesis of oligosaccharides as a basis for drug discovery. Nature Rev. Drug Discov. 4:751‐763.
   Virta, P., Katajisto, J., Niittymäki, T., and Lönnberg, H. 2003. Solid‐supported synthesis of oligomeric bioconjugates. Tetrahedron 59:5137‐5174.
   Wilk, A., Grajkowski, A., Phillips, L.R., and Beaucage, S.L. 2000. Deoxyribonucleoside cyclic N‐acylphosphoramidites as a new class of monomers for the stereocontrolled synthesis of oligothymidylyl‐ and oligocytidylyl‐phosphorothioates. J. Am. Chem. Soc. 122:2149‐2156.
   Wilk, A., Chmielewski, M.K., Grajkowski, A., Phillips, L.R., and Beaucage, S.L. 2001. The 4‐oxopentyl group, as a labile phosphate/thiophosphate protecting group for synthetic oligodeoxyribonucleotides. Tetrahedron Lett. 42:5635‐5639.
   Wilk, A., Chmielewski, M.K., Grajkowski, A., Phillips, L.R., and Beaucage, S.L. 2002. The 3‐[(N‐tert‐butyl)carboxamido]‐1‐propyl group as an attractive phosphate/thiophosphate protecting group for solid‐phase oligodeoxyribonucleotide synthesis. J. Org. Chem. 67:6430‐6438.
PDF or HTML at Wiley Online Library