Synthesis and Purification of Oligonucleotide N3′→P5′ Phosphoramidates and their Phosphodiester and Phosphorothioate Chimeras

Karen L. Fearon1, Jeffrey S. Nelson1

1 Lynx Therapeutics, Hayward, California
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.7
DOI:  10.1002/0471142700.nc0407s03
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes the synthesis and purification of oligonucleotide N3′→P5′ phosphoramidates, wherein each 3′‐oxygen is replaced by a 3′‐amine in the 2′‐deoxyribose ring. The synthesis of required monomers and application of the method to preparation of phosphodiester‐ and phosphorothioate‐containing chimera of phosphoramidate is also reported.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Solid‐Phase Chain Assembly of Oligonucleotide N3′→P5′ Phosphoramidates for IEC Purification
  • Alternate Protocol 1: Solid‐Phase Chain Assembly for RP‐HPLC Purification
  • Basic Protocol 2: IEC Purification, Isolation, and Analysis
  • Alternate Protocol 2: Reversed‐Phase HPLC Purification, Isolation, and Analysis
  • Support Protocol 1: Synthesis of 3′‐Tritylamino‐2′,3′‐Deoxythymidine
  • Support Protocol 2: Synthesis of N4‐Benzoyl‐3′‐Tritylamino‐2′,3′‐Dideoxycytidine
  • Support Protocol 3: Synthesis of N2‐Isobutyryl‐O6‐(N,N‐Diphenylcarbamoyl)‐ 3′‐Tritylamino‐2′,3′‐Dideoxyguanosine
  • Support Protocol 4: Synthesis of N6‐Benzoyl‐3′‐Tritylamino‐ 2′,3′‐Dideoxyadenosine
  • Support Protocol 5: Synthesis of 3′‐O‐(4,4′‐Dimethoxytrityl)‐Protected Deoxyribonucleosides
  • Support Protocol 6: Synthesis of 3′‐Aminonucleoside‐Containing Solid Support
  • Support Protocol 7: Phosphoramidite Synthesis
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Solid‐Phase Chain Assembly of Oligonucleotide N3′→P5′ Phosphoramidates for IEC Purification

  Materials
  • 3% (v/v) dichloroacetic acid (Cl 2CHCO 2H) in dichloromethane (CH 2Cl 2; see recipe)
  • Acetonitrile (CH 3CN; ≤0.001% H 2O)
  • 0.1 M phosphoramidite monomer solutions (see recipe):
    • 0.1 M 3′‐tritylamino‐5′‐O‐[(cis‐2,6‐dimethylpiperidino)(2‐cyanoethoxy)]phosphinyl‐2′,3′‐dideoxynucleoside monomers (ABz, CBz, Gi‐Bu,DPC, T; S.1; see protocol 11)
    • 0.1 M 3′‐O‐(4,4′‐dimethoxytrityl)‐5′‐O‐[(cis‐2,6‐dimethylpiperidino)(2‐cyanoethoxy)]phosphinyl‐2′‐deoxyribonucleoside monomers (ABz, CBz, Gi‐Bu,DPC, T; S.2; see protocol 11; for phosphodiester or phosphorothioate chimera)
  • 0.167 M 1H‐tetrazole in CH 3CN (see recipe)
  • 20% (v/v) pyridine in CH 3CN (see recipe)
  • 1.5:3.5:20:75 (v/v/v/v) H 2O 2/H 2O/pyridine/THF (see recipe)
  • 0.2 M S‐Tetra (Stec et al., ) in pyridine, prepared under argon, or 3H‐1,2‐benzodithiol‐3‐one‐1,1‐dioxide in acetonitrile (Beaucage reagent; Glen Research)
  • 1:1:8 (v/v/v) isobutyric anhydride/2,6‐lutidine/THF (see recipe)
  • 16.5% (v/v) N‐methylimidazole (NMI)/THF (see recipe)
  • 3′‐Tritylamino‐2′,3′‐dideoxynucleoside‐5′‐O‐hemisuccinate conjugated to aminopropyl‐controlled‐pore glass (CPG; ABz, CBz, Gi‐Bu,DPC, or T; see protocol 10)
  • Concentrated aqueous ammonia
  • Column‐mode DNA synthesizer capable of 1‐µmol‐scale syntheses (e.g., 392 or 394, PE Biosystems) with at least four monomer positions (preferably eight for synthesis of chimeras)
  • Empty 1‐µmol synthesis columns
  • Desiccator with vacuum
  • 4‐mL glass screw‐cap vials
  • Heat block or oven set at 58°C
  • Additional reagents and equipment for automated DNA synthesis (see manufacturer's instructions and appendix 3C)
NOTE: This chemistry is extremely water sensitive. Oven‐dry all bottles and syringes used for transferring solvents and solutions. Dissolve solids using a manifold or firestone valve to maintain an argon atmosphere. Perform all transfers under argon.

Alternate Protocol 1: Solid‐Phase Chain Assembly for RP‐HPLC Purification

  • 0.1 M 5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)‐3′‐O‐ [(N,N‐diisopropylamino)(2‐cyanoethoxy)]phosphinyl uridine (see recipe)
  • 0.5 M 1H‐tetrazole in CH 3CN
  • 3:1 (v/v) concentrated aqueous ammonia/ethanol

Basic Protocol 2: IEC Purification, Isolation, and Analysis

  Materials
  • Deprotected oligonucleotide solution (see protocol 1), 4°C
  • Buffer A: 0.01 M aqueous NaOH/0.01 M NaCl, pH 12
  • Buffer B: 0.01 M aqueous NaOH/1.5 M NaCl, pH 12
  • Concentrated aqueous ammonia, 4°C
  • 0.5 M aqueous NaOH solution
  • 100% ethanol
  • UV/visible spectrometer
  • Analytical IEC column (preferably a 4 × 250–mm Dionex PA‐100 NucleoPac column)
  • HPLC or FPLC system compatible with high pH buffer systems equipped with a UV detector, data collection system, and a 1‐ or 2‐mL sample injection loop
  • 3‐mL disposable syringe with luer lock
  • 0.45‐µm filter that fits the end of a luer lock syringe
  • Speedvac evaporator (Savant)
  • Preparative IEC column (preferably a Pharmacia MonoQ 10/10 column)
  • Sample holder with 1.5‐mL centrifuge tubes or fraction collector
  • Sephadex G‐25 column (e.g., Pharmacia NAP‐10; optional)

Alternate Protocol 2: Reversed‐Phase HPLC Purification, Isolation, and Analysis

  • Deprotected oligonucleotide solution (see protocol 2), 4°C
  • Buffer C: acetonitrile
  • Buffer D (see recipe): 0.1 M TEAB/2% acetonitrile, pH 8
  • 3:1 (v/v) concentrated aqueous ammonia/ethanol
  • 1 M TEAB buffer, pH 8 (see recipe)
  • Acetonitrile
  • 1 M aqueous NaF (0.45‐µm filtered)
  • Analytical RP‐HPLC column (e.g., Polymer Laboratories 0.46 × 15–cm PLRP‐S column)
  • HPLC system compatible with reversed‐phase buffers and solvents, equipped with a UV detector, data collection system, and a 2‐mL sample injection loop
  • Semipreparative RP‐HPLC column (e.g., Polymer Labs 0.8 × 30–cm PLRP‐S column)
  • Heat block or oven set at 58°C

Support Protocol 1: Synthesis of 3′‐Tritylamino‐2′,3′‐Deoxythymidine

  Materials
  • Thymidine
  • N,N‐Dimethylformamide (DMF)
  • Triphenylphosphine
  • p‐Anisic acid
  • Diisopropylazodicarboxylate (DIAD)
  • Diethyl ether, 5°C
  • Lithium azide (LiN 3)
  • Ethyl acetate
  • Saturated aqueous NaCl
  • Sodium sulfate (Na 2SO 4, anhydrous)
  • 8:2 (v/v) ethyl acetate/hexane
  • 1:1 (v/v) ethanol/dichloromethane (CH 2Cl 2)
  • Hydrogen
  • 10% Pd/C catalyst (Aldrich)
  • Pyridine (anhydrous)
  • Triethylamine
  • Trityl chloride
  • Chromatography‐grade silica gel, 70‐230 mesh 60 Å (Aldrich)
  • 0.5% to 5% (v/v) triethylamine in 2% (v/v) methanol/CH 2Cl 2
  • 2% to 5% (v/v) methanol/CH 2Cl 2
  • 5:95, 1:9, and 2:8 (v/v) methanol/CH 2Cl 2
  • 57:43 (v/v) 1,4‐dioxane/methanol
  • 2 M aqueous NaOH ( appendix 2A)
  • Dowex 50W‐X8 cation‐exchange resin (pyridinium H+ form; see recipe)
  • Saturated aqueous NaHCO 3
  • 1:1 (v/v) ethyl ether/hexane
  • Heating mantle, variac, and temperature controller
  • Mechanical overhead stirrer (Fisher)
  • TLC plates (e.g., 0.2‐mm‐thick precoated Merck silica gel 60 F254 plates)
  • Rotary evaporator
  • Parr shaker‐type hydrogenator able to hold pressures up to 60 psi
  • Additional reagents and equipment for thin‐layer chromatography (TLC, appendix 3D) and column chromatography ( appendix 3E)

Support Protocol 2: Synthesis of N4‐Benzoyl‐3′‐Tritylamino‐2′,3′‐Dideoxycytidine

  • 2′‐Deoxyuridine
  • 4‐Dimethylaminopyridine
  • tert‐Butyldimethylsilyl (TBDMS) chloride
  • 2:1 (v/v) ethanol/CH 2Cl 2
  • 1,2,4‐Triazole
  • Phosphorus oxychloride (POCl 3)
  • 1,4‐Dioxane
  • Benzoyl chloride
  • Concentrated aqueous ammonia (28%), 4°C
  • Tetrahydrofuran (THF)
  • 1 M tetra‐n‐butylammonium fluoride (TBAF) in THF
  • 1% (v/v) triethylamine in 30% to 50% (v/v) ethyl acetate/hexane

Support Protocol 3: Synthesis of N2‐Isobutyryl‐O6‐(N,N‐Diphenylcarbamoyl)‐ 3′‐Tritylamino‐2′,3′‐Dideoxyguanosine

  • N2‐Isobutyryl‐2′‐deoxyguanosine
  • Benzoyl chloride
  • Trifluoromethanesulfonic anhydride
  • 4‐Dimethylaminopyridine
  • tert‐Butyldimethylsilyl chloride
  • 1:1 (v/v) methanol/1,4‐dioxane
  • 1 M aqueous HCl
  • Diethylazodicarboxylate
  • Argon
  • N,N‐Diisopropylethylamine
  • N,N‐Diphenylcarbamyl chloride
  • Triethylamine trihydrofluoride
  • Toluene
  • 7:3 and 6:4 (v/v) ethyl acetate/hexane
  • 2‐liter large‐mouth Erlenmeyer flask

Support Protocol 4: Synthesis of N6‐Benzoyl‐3′‐Tritylamino‐ 2′,3′‐Dideoxyadenosine

  • (−)‐Adenosine
  • Dibutyltin oxide
  • p‐Toluenesulfonyl chloride
  • tert‐Butyldimethylsilyl chloride
  • 1 M lithium triethyl borohydride in THF
  • Ammonium chloride
  • Benzoyl chloride
  • 7:10 (v/v) methanol/1,4‐dioxane
  • Pyridinium hydrochloride
  • Diethylazodicarboxylate
  • Argon
  • 4:6 (v/v) ethyl acetate/hexane
  • Tetrahydrofuran (THF)
  • 1.0 M tetrabutylammonium fluoride (TBAF) in THF

Support Protocol 5: Synthesis of 3′‐O‐(4,4′‐Dimethoxytrityl)‐Protected Deoxyribonucleosides

  • N6‐Benzoyl‐2′‐deoxyadenosine
  • N4‐Benzoyl‐2′‐deoxycytidine
  • N2‐Isobutyryl‐2′‐deoxyguanosine
  • Thymidine
  • 4‐Dimethylaminopyridine
  • tert‐Butyldimethylsilyl chloride
  • Tetrahydrofuran (THF)
  • 1 M tetrabutylammonium fluoride (TBAF) in THF
  • N,N‐Diisopropylethylamine
  • N,N‐Diphenylcarbamyl chloride
  • 9:1 (v/v) CH 2Cl 2/pyridine
  • Triethylamine trihydrofluoride
  • 50% to 70% ethyl acetate/hexane

Support Protocol 6: Synthesis of 3′‐Aminonucleoside‐Containing Solid Support

  • 3′‐Tritylamino‐2′,3′‐dideoxynucleosides (S.5, S.9, S.13, or S.18; see Support Protocols protocol 51 to protocol 84)
  • 4‐Dimethylaminopyridine
  • Succinic anhydride
  • 10% (v/v) aqueous citric acid, cold
  • 1‐Hydroxybenzotriazole
  • 1:1 (v/v) 1‐methyl‐2‐pyrrolidinone (anhydrous)/dimethyl sulfoxide (anhydrous)
  • N,N‐Diisopropylethylamine
  • 2‐(1H‐Benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate
  • Aminopropyl‐conjugated controlled‐pore glass (aminopropyl‐CPG; Sigma)
  • 1:1:8 (v/v/v) acetic anhydride/2,6‐lutidine/THF
  • 16.5% (v/v) N‐methylimidazole in THF (see recipe)
  • Mechanical shaker

Support Protocol 7: Phosphoramidite Synthesis

  • 3′‐Tritylamino‐2′,3′‐dideoxynucleosides (see Support Protocols protocol 51 to protocol 84) or 3′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxyribonucleosides (see protocol 9)
  • Phosphorus trichloride
  • 3‐Hydroxypropionitrile
  • 10% (w/v) aqueous KOH
  • 1:4 (v/v) toluene/hexane
  • cis‐2,6‐Dimethylpiperidine
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU), distilled from CaH 2 before use
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Chen, J.‐K., Schultz, R.G., Lloyd, D.H., and Gryaznov, S.M. 1995. Synthesis of oligodeoxyribonucleotide N3′→P5′ phosphoramidates. Nucl. Acids Res. 23 2661‐2668.
   Czernecki, S. and Valéry, J.M. 1991. An efficient synthesis of 3′‐azido‐3′‐deoxythymidine (AZT). Synthesis 1991:329‐240.
   DeDionisio, L. and Gryaznov, S.M. 1995. Analysis of a ribonuclease H digestion of N3′→P5′ phosphoramidate‐RNA duplexes by capillary gel electrophoresis. J. Chromatogr. B Biomed. Appl. 669:125‐131.
   Escudé, C., Giovannangeli, C., Sun, J.‐S., Lloyd, D.H., Chen, J.‐K., Gryaznov, S.M., Garestier, T., and Hélène, C. 1996. Stable triple helices formed by oligonucleotide N3′→P5′ phosphoramidates inhibit transcription elongation. Proc. Natl. Acad. Sci. U.S.A. 93:4365‐4369.
   Fearon, K.L., Nelson, J.S., Hirschbein, B.L., Foy, M.F., Nguyen, M.Q., McCurdy, S.N., Frediani, J.E., Okruszek, A., DeDionisio, L.A., Raible, A.M. ,and Boyd, V. 1998. An improved synthesis of oligonucleotide N3′→P5′ phosphoramidates and their chimera using hindered phosphoramidite monomers and a novel handle for reverse phase purification. Nucl. Acids Res. 26:3813‐3824.
   Giovannangeli, C., Diviacco, S., Labrousse, V., Gryaznov, S., Charneau, P., and Hélène, C. 1997. Accessibility of nuclear DNA to triplex‐forming oligonucleotides:The integrated HIV‐1 provirus as a target. Proc. Natl. Acad. Sci. U.S.A. 94:79‐84.
   Gryaznov, S. and Chen, J.‐K. 1994. Oligodeoxyribonucleotide N3′→P5′ phosphoramidates: Synthesis and hybridization properties. J. Am. Chem. Soc. 116:3143‐3144.
   Gryaznov, S.M., Lloyd, D.H., Chen, J.‐K., Schultz, R.G., DeDionisio, L.A., Ratmeyer, L., and Wilson, W.D. 1995. Oligonucleotide N3′→P5′ phosphoramidates. Proc. Natl. Acad. Sci. U.S.A. 92:5798‐5802.
   Gryaznov, S., Skorski, T., Cucco, C., Nieborowska‐Skorska, M., Chiu, C.Y., Lloyd, D., Chen, J.‐K., Koziolkiewicz, M., and Calabretta, B. 1996. Oligonucleotide N3′→P5′ phosphoramidates as antisense agents. Nucl. Acids Res. 24:1508‐1514.
   Hansske, F. and Robins, M.J. 1983. A deoxygenative [1,2]‐hydride shift rearrangement converting cyclic cis‐diol monotosylates to inverted secondary alcohols. J. Am. Chem. Soc 105:6736‐6737.
   Heidenreich, O., Gryaznovv, S., and Nerenberg, M. 1997. RNase H‐independent antisense activity of oligonucleotide N3′→P5′ phosphoramidates. Nucl. Acids Res. 25:776‐780.
   Herdewijn, P. and Van Aerschot, A. 1989. Synthesis of 9‐ 3‐ azido‐2,3‐dideoxy‐β‐D‐erythro‐pentofuranosyl)‐2,6‐diaminopurine (AzddDPA). Tetrahedron Lett. 30:855‐858.
   McCurdy, S.N., Nelson, J.S., Hirschbein, B.L., and Fearon, K.L. 1997. An improved method for the synthesis of N3′→P5′ phosphoramidate oligonucleotides. Tetrahedron Lett. 38:207‐210.
   Nelson, J.S., Fearon, K.L., Nguyen, M.Q., McCurdy, S.N., Frediani, J.E., Foy, M.F., and Hirschbein, B.L. 1997. N3′→P5′ Oligodeoxyribonucleotide phosphoramidates: A new method of synthesis based on a phosphoramidite amine‐exchange reaction. J. Org. Chem. 62:7278‐7287.
   Nishino, S., Yamamoto, H., Nagato, Y., and Ishido, Y. 1986. Partial protection of carbohydrate derivatives. Part 19. Highly regioselective 5′‐O‐aroylation of 2′‐deoxyribonucleosides in terms of dilution‐drop‐by‐drop‐addition procedure. Nucleosides Nucleotides 5:159‐168.
   Reese, C.B. and Skone, P.A. 1984. The protection of thymine and guanine residues in oligodeoxyribonucleotide synthesis. J. Chem. Soc. Perkin Trans. I 1263‐1271.
   Skorski, T., Perrotti, D., Nieborowska‐Skorska, M., Gryaznov, S., and Calabretta, B. 1997. Antileukemia effect of c‐.myc N3′→P5′ phosphoramidate antisense oligonucleotides in vivo . Proc. Natl. Acad. Sci. U.S.A. 94:3966‐3971.
   Stec, W.J., Uznanski, B., Wilk, A., Hirschbein, B.L., Fearon, K.L., and Bergot, B.J. 1993. Bis(O, O‐diisopropoxy phosphinothioyl) disulfide: A highly efficient sulfurizing reagent for cost‐effective synthesis of oligo (nucleoside phosphorothiaote)s. Tetrahedron Lett. 34:5317‐5320.
   Stein, C.A. and Cheng, Y.‐C. 1993. Antisense oligonucleotides as therapeutic agents: Is the bullet really magical? Science 261:1004‐1012.
   Stultz, J.T. and Marsters, J.C. 1991. Improved electrospray ionization of synthetic oligonucleotides. Rapid Commun. Mass. Spectrom. 5:359‐363.
   Thuong, N.T. and Hélène, C. 1993. Sequence‐specific recognition and modification of double‐helical DNA by oligonucleotides. Angew. Chem. Int. Ed. Engl. 32:666‐690.
   Uhlman, E. and Peyman, A. 1990. Antisense oligonucleotides: A new therapeutic principle. Chem. Rev. 90:544‐584.
   Wagner, D., Verheyden, J.P.H., and Moffatt, J.G. 1974. Preparation and synthetic utility of some organotin derivatives of nucleosides. J. Org. Chem. 39:24‐30.
Key References
   Nelson et al., 1997. See above.
  This reference details the couple‐oxidize‐couple‐oxidize amine‐exchange method for synthesis of pnODNs, as well as the synthesis of the 3′‐trityl‐amino protected nucleosides.
   Fearon et al., 1998. See above.
  This reference describes the optimized amine‐exchange method for the synthesis of pnODNs and the synthesis of the 3′‐O‐DMTr‐protected nucleosides and the cis‐2,6‐dimethylpiperidinyl phosphoramidite monomers.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library