Oligodeoxyribonucleotide Analogs Functionalized with Phosphonoacetate and Thiophosphonoacetate Diesters

Douglas J. Dellinger1, Christina M. Yamada2, Marvin H. Caruthers2

1 Agilent Laboratories, Boulder, Colorado, 2 University of Colorado, Boulder, Colorado
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.24
DOI:  10.1002/0471142700.nc0424s18
Online Posting Date:  October, 2004
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Oligodeoxyribonucleotides with phosphonoacetate or thiophosphonoacetate internucleotide linkages can be made in high yield by solid‐phase synthesis and possess many advantages. They are highly stable to nucleases, water‐soluble, and anionic at neutral pH. They form stable duplexes with DNA and RNA, and stimulate RNase H degradation of complementary RNA. The preparation of the N,N‐(diisopropylamino)phosphinyl acetate monomers from standard protected nucleosides is described here, followed by the synthesis of phosphonoacetate and thiophosphonoate oligodeoxyribonucleotides, as well as chimeric oligomers that have these modified linkages in combination with natural or phosphorothioate linkages. Purification and characterization of these oligomers is also presented.

Keywords: phosphonoacetate DNA synthesis; thiophosphonoacetate DNA synthesis

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Table of Contents

  • Basic Protocol 1: Synthesis of Protected 2′‐Deoxynucleoside‐3′‐O‐(N,N‐Diisopropylamino)Phosphinyl Acetates
  • Basic Protocol 2: Automated Synthesis of Phosphonoacetate and Thiophosphonoacetate DNA
  • Support Protocol 1: Drying of Phosphonoacetate and Thiophosphonoacetate Monomers
  • Basic Protocol 3: Automated Synthesis of Modified DNA Chimeras
  • Basic Protocol 4: Deprotection, Purification, and Characterization of Modified DNA
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Synthesis of Protected 2′‐Deoxynucleoside‐3′‐O‐(N,N‐Diisopropylamino)Phosphinyl Acetates

  • Argon, dry
  • Toluene, anhydrous (Aldrich)
  • Bromoacetyl bromide (S.1, Fig. ; Aldrich)
  • Nitrogen, dry (optional)
  • 50‐g bottle of 3‐hydroxy‐3‐methylbutyronitrile (Fluka)
  • Bis(diisopropylamino)chlorophosphine (Digital Specialty Chemicals)
  • Tetrahydrofuran, anhydrous (THF; Aldrich)
  • Zinc metal, granular (Aldrich)
  • Acetonitrile, anhydrous
  • Phosporic acid/CD 3CN NMR standard
  • Reagent‐grade hexanes, anhydrous
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐protected 2′‐deoxynucleoside (S.4; ChemGenes):
    • 5′‐O‐DMTr‐N6‐benzoyl‐2′‐deoxyadenosine
    • 5′‐O‐DMTr‐N4‐acetyl‐2′‐deoxycytidine
    • 5′‐O‐DMTr‐N2‐isobutyryl‐2′‐deoxyguanosine
    • 5′‐O‐DMTr‐2′‐deoxythymidine
  • Dichloromethane, anhydrous (Aldrich)
  • 0.45 M tetrazole in acetonitrile (Glen Research)
  • Ethyl acetate, reagent grade
  • Diisopropylethylamine, anhydrous (DIPEA; Aldrich)
  • Silica gel 60 for medium‐pressure liquid chromatography (MPLC; 230 to 400 mesh; Aldrich)
  • 120°C oven
  • 500‐mL and 1‐liter round‐bottom flasks with 24/40 joints
  • Drying tubes containing calcium sulfate (Drierite) and equipped with a 24/40 joint
  • PTFE‐coated stir bar
  • 125‐mL, 250‐mL, and 2‐L Erlenmeyer flasks
  • Top‐loading balance
  • Addition funnels with 24/40 joints, pressure‐equalization arms (100‐ and 500‐mL capacity), and PTFE stopcocks
  • 325‐mm Friedrich's condensers with 24/40 joints (Labglass)
  • Acid vapor trap and acid‐resistant tubing
  • Heating mantle to fit a 1‐liter round‐bottom flask, with controller
  • Rotary evaporator with solvent‐resistant Teflon head pump
  • 1‐ and 2‐liter recovery flasks (Labglass)
  • Rotary evaporator trap (Labglass)
  • Short‐path distillation apparatus (Labglass)
  • Three‐flask distribution receiver
  • Vacuum pump with an inlet acid vapor trap
  • Capillary bleed tube (Kontes Glass) or needle valve attached to a Y‐connector
  • Powder funnels
  • 24/40 rubber septa
  • Hand‐held heat gun (Aldrich)
  • 500‐mL separatory funnel
  • Ground‐glass 24/40 joint
  • 50‐mL glass pipet
  • Glass‐backed silica‐gel TLC plates with fluorescent indicator (Aldrich)
  • TLC developing tank (Aldrich)
  • UV viewing cabinet (Aldrich)
  • Flash‐chromatography column (Aldrich cat) and flow controller
  • Sea sand
  • 350‐mL fritted glass Buchner funnel, medium porosity, fitted with a 24/40 vacuum adapter (Labglass)
  • Vacuum desiccator and solid PTFE vacuum pump with maximum vacuum of 1.5 torr (Aldrich)
  • Additional reagents and equipment for 31P NMR, thin‐layer chromatography (TLC; appendix 3D), column chromatography ( appendix 3E), and fast atom bombardment mass spectrometry (FAB‐MS)

Basic Protocol 2: Automated Synthesis of Phosphonoacetate and Thiophosphonoacetate DNA

  • 1,1‐Dimethyl‐2‐cyanoethyl 5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxynucleoside‐3′‐O‐(N,N‐diisopropylamino)phosphinyl acetates (S.5a‐d; see protocol 1; also available from MetaSense Technologies)
  • Argon or nitrogen, anhydrous
  • Anhydrous acetonitrile, synthesis grade (Fisher Biotech)
  • Amber serum vials with rubber septa
  • 18‐G × 1‐in. needle
  • Vacuum desiccator
  • Two‐stage vacuum pump or high vacuum line, with trap
  • Syringe, dry

Support Protocol 1: Drying of Phosphonoacetate and Thiophosphonoacetate Monomers

  • DNA synthesis column containing oligodeoxyribonucleotides (ODN) linked to controlled‐pore glass (CPG)
  • Acetonitrile, anhydrous
  • Argon or nitrogen, anhydrous
  • 1.5% (v/v) 1,8‐diazabicyclo‐[5.4.0]undec‐7‐ene (DBU; Aldrich) in anhydrous acetonitrile
  • 40% methylamine, aqueous (Aldrich)
  • RP‐HPLC mobile phases:
    • A: 100 mM triethylammonium acetate, pH 8.0
    • B: acetonitrile
  • 10 mM Tris⋅Cl, pH 8.0 ( appendix 2A)
  • 80% acetic acid
  • 50 mM triethylammonium acetate, pH 8
  • TE buffer, pH 8.0 ( appendix 2A)
  • IEX‐HPLC mobile phases:
    • A: 10 mM NaOH/80 mM NaBr
    • B: 10 mM NaOH/1.5 M NaBr
  • 2,4,6‐Trihydroxyacetophenone monohydrate (THAP) matrix
  • Ammonium citrate
  • 1:1 (v/v) acetonitrile/H 2O
  • 10‐mL Luer‐tip syringe
  • Male Luer‐to‐tubing connector (Aldrich)
  • Conical vial: 3‐mL Reacti‐Vial sealed with Teflon‐bonded silicon Tuf‐Bond discs (Pierce), preferred
  • 55°C heating block (e.g., Reacti‐Block Aluminum Heat‐Block, Pierce) or oven
  • Speedvac evaporator (Savant)
  • RP‐HPLC apparatus with 25‐cm × 9.4‐mm‐i.d. Zorbax 300SB‐C18 column (Agilent Technologies)
  • Ion‐exchange (IEX)‐HPLC apparatus with 1‐mL RESOURCE Q column (Amersham Pharmacia Biotech)
  • 100‐well plate, gold plated
  • Voyager‐DE STR Biospectrometry Workstation mass analyzer (PerSeptive Biosystems)
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Literature Cited

   Arbuzov, A.E. and Dunin, A.A. 1927. Über phosphon‐carbonsäuren. Ber. 60B:291‐295.
   Bayless, P.L. and Hauser, C.R. 1954. A Reformatskii type condensation of aroyl chlorides with ethyl 2‐bromoisobutyrate by means of zinc to form β‐keto esters. J. Am. Chem. Soc. 76:2306‐2308.
   Caruthers, M.H., Barone, A.D., Beaucage, S.L., Dodds, D.R., Fisher, E.F., McBride, L.J., Matteucci, M., Stabinsky, Z., and J.‐Y. Tang. 1987. Chemical synthesis of deoxyoligonucleotides by the phosphoramidite method. Methods Enzymol. 154:287‐313.
   Dellinger, D.J., Sheehan, D.M., Christensen, N.K., Lindberg, J.G., and Caruthers, M.H. 2003. Solid phase chemical synthesis of phosphonoacetate and thiophosphonoacetate oligodeoxyribonucleotides. J. Am. Chem. Soc. 125:940‐950.
   Glen Research. 1996. Non‐aqueous oxidation with 10‐camphorsulfonyl‐oxziridine. Glen Research Corporation Technical Report 9:8‐9.
   Greef, C.H., Seeberger, P.H., Caruthers, M.H., Beaton, G., and Bankaitis‐Davis, D. 1996. Synthesis of phosphorodithioate RNA by the H‐phosphonothioate method. Tetrahedron Lett. 37:4451‐4454.
   Griengl, H., Hayden, W., Penn, G., Declercq, E., and Rosenwirth, B. 1988. Phosphonoformate and phosphonoacetate derivatives of 5‐substituted 2′‐deoxyuridines—synthesis and antiviral activity. J. Med. Chem. 31:1831‐1839.
   Hogrefe, R.I., Reynolds, M.A., Vaghefi, M.M., Young, K.M., Riley, T.A., Klem, R.E., and Arnold, L.J. Jr. 1993. An improved method for the synthesis and deprotection of methylphosphonate oligonucleotides. In Protocols for Oligonucleotides and Analogs, Vol. 20 (S. Agrawal, ed.) pp. 143‐164. Humana Press, Totowa, New Jersey.
   Lambert, R.W., Martin, J.A., Thomas, G.J., Duncan, I.B., Hall, M.J., and Heimer, E.P. 1989. Synthesis and antiviral activity of phosphonoacetic and phosphonoformic acid‐esters of 5‐bromo‐2′‐deoxyuridine and related pyrimidine nucleosides and acyclonucleosides. J. Med. Chem. 32:367‐374.
   Matrosov, E.I., Tsvetlsov, E.N., Malevannaya, R.A., and Kabachnik, M.I. 1972. Infrared spectra and association of phosphinylacetic acid. Zh. Obshch. Khim. 42:1695‐1700.
   Overby, L.R., Duff, R.G., and Mao, J.C. 1977. Antiviral potential of phosphonoacetic acid. Ann. N.Y. Acad. Sci. 284:310‐320.
   Reddy, M.P., Hanna, N.B., and Farooqui, F. 1994. Fast cleavage and deprotection of oligonucleotides. Tetrahedron Lett. 35:4311‐4314.
   Rudolph, M.J., Reitman, M.S., MacMillan, E.W., and Cook. A.F. 1996. Phosphonoacetate derivatives of oligodeoxyribonucleotides. Nucleosides Nucleotides 15:1725‐1739.
   Sheehan, D., Lunstad, B., Yamada, C.M., Stell, B., Caruthers, M.H., and Dellinger, D.J. 2003. Biochemical properties of phosphonoacetate and thiophosphonoacetate oligodeoxyribonucleotides. Nucl. Acids Res. 31:4109‐4118.
Key References
   Dellinger et al., 2003. See above.
  This reference outlines the chemistry used to prepare phosphonoacetate and thiophosphonoacetate ODNs from protected 2′‐deoxynucleoside‐3′‐O‐(N,N‐diisopropylamino)phosphinyl acetate synthons.
   Sheehan et al., 2003. See above.
  This reference presents initial biochemical and biophysical results with phosphonoacetate and thiophosphonoacetate ODNs.
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