Preparation of DNA and RNA Fragments Containing Guanine N2‐Thioalkyl Tethers

Xiaorong Hou1, Gang Wang1, Barbara L. Gaffney1, Roger A. Jones1

1 Rutgers, The State University of New Jersey, Piscataway, New Jersey
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 5.8
DOI:  10.1002/0471142700.nc0508s41
Online Posting Date:  June, 2010
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Abstract

This unit describes procedures for preparation of deoxyguanosine and guanosine derivatives in which the guanine N2 contains a thiopropyl tether, protected as a tert-butyl disulfide. After incorporation into a DNA or RNA fragment, this tether allows site-specific cross-linking to a thiol of a protein or another nucleic acid. Curr. Protoc. Nucleic Acid Chem. 41:5.8.1-5.8.23. © 2010 by John Wiley & Sons, Inc.

Keywords: oligonucleotides; disulfide cross-links

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Diisopropyl-1-(tert-Butylthio)-1,2-Hydrazinedicarboxylate (4)
  • Basic Protocol 2: Preparation of the 2¢-Deoxyguanosine N2-Propyl-tert-Butyl Disulfide Phosphoramidite Derivative (12)
  • Basic Protocol 3: Preparation of the Guanosine N2-Propyl-tert-Butyl Disulfide Phosphoramidite Derivative (20)
  • Basic Protocol 4: Preparation of DNA Fragments Containing N2-Propyl-tert-Butyl Disulfide Guanine
  • Alternate Protocol: Preparation of RNA Fragments Containing N2-Propyl-tert-Butyl Disulfide Guanine
  • Basic Protocol 5: Procedures for Converting the N2-Propyl-tert-Butyl Disulfide to the Ethylamine Disulfide, 5-Thio-2-Nitrobenzoic Acid (TNB) Disulfide, or Free Thiol
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Diisopropyl-1-(tert-Butylthio)-1,2-Hydrazinedicarboxylate (S.4)

 Materials
  • 3-Chloropropylammonium hydrochloride (S.1; Aldrich)
  • Sodium thiosulfate pentahydrate (Na2S2O3), reagent grade
  • Iodine
  • Methanol (MeOH), reagent grade
  • 6 N NaOH
  • Methylene chloride
  • Toluene
  • Dithiothreitol (DTT; Aldrich)
  • Concentrated HCl
  • 2-Methyl-2-propanethiol (tert-butyl thiol)
  • Diisopropyl azodicarboxylate (S.5; Aldrich)
  • Diethyl ether, anhydrous
  • Nitrogen and argon sources
  • 25% (w/v) sodium methoxide in methanol
  • 6 N HCl
  • Dimethylformamide (DMF), anhydrous reagent, argon degassed
  • Triethylamine, anhydrous
  • Magnetic stir bars
  • Magnetic stir plate
  • Reflux condenser
  • Addition funnel
  • Rotary evaporator with diaphragm pump or water aspirator
  • 125-mL separatory funnel
  • Falling film distillation apparatus (available from Aldrich; see Sigma-Aldrich, 2001)
  • Shaker
  • Silica gel column: 40 to 63 µm in 40 × 150–mm prepackaged cartridges (SciPro, cat. no. CO2001; http://www.scipro.com/)

Basic Protocol 2: Preparation of the 2¢-Deoxyguanosine N2-Propyl-tert-Butyl Disulfide Phosphoramidite Derivative (S.12)

 Materials
  • 2¢-Deoxyguanosine monohydrate (S.7; Aldrich)
  • 1,4-Dioxane
  • Trimethylsilylimidazole (Aldrich)
  • Trimethylsilylethanol, Aldrich
  • Diethyl azodicarboxylate (DEAD; Aldrich)
  • Concentrated aqueous ammonia
  • Methylene chloride
  • Methanol (MeOH), reagent grade
  • 0.1 M triethylammonium chloride (TEAA), pH 6.8
  • Acetonitrile (CH3CN)
  • Phosphorus pentoxide (P2O5)
  • Pyridine, anhydrous
  • Toluene, anhydrous
  • 70% HF×pyridine (Aldrich)
  • tert-Butyl nitrite (Aldrich)
  • Dry ice/acetone bath
  • K2CO3
  • Ethyl acetate
  • Triethylamine
  • 4, 4¢-Dimethoxytrityl chloride (Aldrich)
  • Nitrogen source
  • 5% (w/v) sodium bicarbonate
  • Diethyl ether
  • S.4 (Basic Protocol 1)
  • 1 M NaOH
  • Hexane
  • 2-Cyanoethyl (N,N,N¢,N¢-tetraisopropyl)phosphordiamidite (Cambridge Major, http://www.c-mlabs.com)
  • Pyridinium trifluoroacetate (Aldrich)
  • Magnetic stir bars
  • Magnetic stir plate
  • 250-mL separatory funnel
  • Rotary evaporator with diaphragm pump or water aspirator
  • Silica gel column: 40 to 63 µm in 40 × 150–mm prepackaged cartridges (SciPro, cat. no. CO2001; http://www.scipro.com/)
  • Reversed-phase HPLC system with ESI-MS
  • XTerra MS or Atlantis µC18 reversed-phase columns

Basic Protocol 3: Preparation of the Guanosine N2-Propyl-tert-Butyl Disulfide Phosphoramidite Derivative (S.20)

 Materials
  • Guanosine monohydrate (S.13; Aldrich)
  • 1,4-Dioxane
  • Trimethylsilylimidazole (Aldrich)
  • Nitrogen (or argon) source
  • Triphenylphosphine (Aldrich)
  • Trimethylsilylethanol (Aldrich)
  • Diethyl azodicarboxylate (DEAD; Aldrich)
  • Concentrated aqueous ammonia
  • Methylene chloride
  • Ethyl acetate
  • Pyridine, anhydrous
  • Toluene, anhydrous
  • 70% HF×pyridine (Aldrich)
  • Dry ice/acetone bath
  • tert-Butyl nitrite (Aldrich)
  • K2CO3
  • Methanol
  • Triethylamine
  • P2O5
  • S.4 (Basic Protocol 1)
  • 1 M NaOH
  • Dimethylformamide (DMF), anhydrous reagent, argon degassed
  • Di-tert-butylsilyl ditriflate (Aldrich)
  • Imidazole
  • tert-Butyldimethylchlorosilane (Aldrich)
  • NaHCO3
  • 4,4¢-Dimethoxytrityl chloride (Aldrich)
  • Acetone, HPLC grade
  • 2-Cyanoethyl (N,N,N¢,N¢-tetraisopropyl)phosphordiamidite (Cambridge Major, http://www.c-mlabs.com)
  • Pyridinium trifluoroacetate (Aldrich)
  • Magnetic stir bars
  • Magnetic stir plate
  • 250-mL separatory funnel
  • Rotary evaporator with diaphragm pump or water aspirator
  • Filter for vacuum filtration
  • Silica gel column: 40 to 63 µm in 40 × 150–mm prepackaged cartridges (SciPro, cat. no. CO2001; http://www.scipro.com/)
  • Vacuum desiccator

Basic Protocol 4: Preparation of DNA Fragments Containing N2-Propyl-tert-Butyl Disulfide Guanine

 Materials
  • Phosphoramidite S.12 (Basic Protocol 2)
  • Reagents specified for the synthesizer (also see appendix 3D)
  • Concentrated aqueous ammonia
  • 10% aqueous ammonia
  • Triethylamine (TEA) dried over CaH2
  • Acetonitrile
  • 0.1 M triethylammonium acetate (TEAA)
  • 0.6 M acetic acid, HPLC grade
  • NH4HCO3, solid
  • 0.1 M NH4HCO3, pH 6.8 to 7.0
  • AG 50W-X2 ion-exchange resin (BioRad)
  • DNA/RNA synthesizer (appendix 3D)
  • 50-mL and 15-mL conical centrifuge tubes
  • Sintered-glass funnel, medium porosity
  • Reversed-phase HPLC system (see unit 10.1) with ESI-MS
  • UV/Vis spectrophotometer
  • 3-mL syringes
  • Syringe filters, 4.5 mm, 0.45-µm pore size
  • Semi-preparative reversed-phase HPLC system (see unit 10.1)
  • Anion-exchange HPLC system
  • Glass column
  • Silica gel column: 40 to 63 µm in 40 × 150–mm prepackaged cartridges (SciPro, cat. no. CO2001; http://www.scipro.com/)
  • Additional reagents and equipment for oligonucleotide synthesis (appendix 3D) and HPLC purification of oligonucleotides (discussed in unit 10.1)

Alternate Protocol: Preparation of RNA Fragments Containing N2-Propyl-tert-Butyl Disulfide Guanine

 Additional Materials (also see Basic Protocol 4)
  • Phosphoramidite S.20 (Basic Protocol 3)
  • 40% (w/v) methylamine (aqueous)
  • 50% (v/v) ethanol
  • Triethylamine×HF (Aldrich)
  • Methyl-2-pyrrolidinone (NMP; Aldrich)
  • Triethylamine (TEA) dried over CaH2
  • Isopropoxytrimethylsilane (Aldrich)
  • Ethyl ether, anhydrous
  • 65°C water bath
  • Large rubber septum
  • Vent needle connected to bubbler
  • Tabletop centrifuge
  • Spatula cleaned with nitric acid and rinsed well

Basic Protocol 5: Procedures for Converting the N2-Propyl-tert-Butyl Disulfide to the Ethylamine Disulfide, 5-Thio-2-Nitrobenzoic Acid (TNB) Disulfide, or Free Thiol

 Materials
  • Dithiothreitol (DTT; Aldrich)
  • Concentrated aqueous ammonia
  • Purified S.21(Basic Protocol 4 or Alternate Protocol)
  • 5,5¢-Dithiobis(2-nitrobenzoic acid) (Aldrich)
  • 0.1 M potassium phosphate buffer, pH 8.0 (appendix 2A)
  • Cysteamine hydrochloride (2-aminoethanethiol hydrochloride; Aldrich)
  • Cystamine dihydrochloride (2,2¢-diaminodiethyl disulfide dihydrochloride; Aldrich)
  • 40°C water bath
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Figures

  •  FigureFigure 5.8.1 Preparation of S.4. 3-chloropropylammonium chloride (S.1) is reacted with sodium thiosulfate, followed by treatment with iodine, to give S.2 (Doi and Musker, 1985). The free thioalkylamine (S.3) is obtained by treating S.2 with dithiothreitol (DTT). The conversion to the tert-butyl disulfide is carried out by reacting S.3 with diisopropyl-1-(tert-butylthio)-1,2-hydrazinedicarboxylate (S.6). S.6 is prepared from diisopropylazodicarboxylate (S.5) and tert-butyl thiol (Wunsch et al., 1982; Virgilio and Ellman, 1994).
    View Image
  •  FigureFigure 5.8.2 Conversion of 2¢-deoxyguanosine (S.7) to the N2-propyl-tert-butyl disulfide phosphoramidite derivative S.12. S.7 is treated with trimethylsilyl (TMS) imidazole, followed by reaction with trimethylsilylethanol, diisopropyl azodicarboxylate (DEAD), and triphenylphosphine (PPh3; Gao et al., 1986; DeCorte et al., 1996). The trimethylsilyl (TMS) groups are cleaved with aqueous dilute NH3, and silica gel column chromatography is performed to give S.8. The next step is a nonaqueous diazotization using tert-butyl nitrite and pyridine×HF (Robins and Uznanski, 1981), to give S.9. Protection of the 5¢ hydroxyl by reaction with 4,4¢-dimethoxytrityl chloride gives S.10. Displacement of the 2-fluoro group of S.10 is carried out by reaction with 3-aminopropyl-tert-butyl disulfide (S.4) for 3 days at 60° to give S.11. Conversion to the phosphoramidite S.12 is carried out using 2-cyanoethyl (N,N,N¢,N¢-tetraisopropyl)phosphordiamidite and pyridinium trifluoroacetate (Sanghvi et al., 2000).
    View Image
  •  FigureFigure 5.8.3 Conversion of guanosine (S.13) to the N2-propyl-tert-butyl disulfide phosphoramidite derivative S.20. The initial reactions to get to the 2-fluoro-O6-trimethylsilylethyl derivative (S.15) are the same as for the conversion of 2¢-deoxyguanosine to the N2-propyl-tert-butyl disulfide phosphoramidite derivative (Fig. 5.8.2). However, displacement of the 2-fluoro group to give S.16 must be done before introduction of the 2¢-O-tert-butyldimethylsilyl (TBS) group, to avoid cleavage of the TBS by the released fluoride. Protection of the 2¢ hydroxyl is carried out after protection of the 3¢ and 5¢ hydroxyls as a di-tert-butylsilylene (Serebryany and Beigelman, 2002). Cleavage of the di-tert-butylsilylene followed by dimethoxytritylation gives S.19. Conversion to the phosphoramidite S.20 is carried out using 2-cyanoethyl (N,N,N¢,N¢-tetraisopropyl)phosphordiamidite and pyridinium trifluoroacetate (Sanghvi et al., 2000).
    View Image
  •  FigureFigure 5.8.4 Conversion of purified DNA or RNA fragments (S.21) to more reactive species. Treatment with dithiothreitol (DTT) gives the free thiol (S.22), which can be used itself for cross-linking, or it may be converted to the nitrobenzoic acid (S.23) disulfide by treatment with 5,5¢-dithiobis(2-nitrobenzoic acid). Reaction with a mixture of cysteamine hydrochloride (2-aminoethanethiol hydrochloride) and cystamine dihydrochloride (2,2¢-diaminodiethyl disulfide dihydrochloride) gives the ethylamine disulfide (S.24).
    View Image

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Literature Cited

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