2′‐Hydroxy Protection of Ribonucleosides as 2‐Cyano‐2,2‐Dimethylethanimine‐N‐Oxymethyl Ethers in Solid‐Phase Synthesis of RNA Sequences

Jacek Cieślak1, Cristina Ausín1, Andrzej Grajkowski1, Serge L. Beaucage1

1 Food and Drug Administration, Bethesda, Maryland
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 3.22
DOI:  10.1002/0471142700.nc0322s54
Online Posting Date:  October, 2013
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Abstract

The reaction of 2′‐O‐aminooxymethylribonucleosides with 2‐cyano‐2‐methyl propanal leads to the formation of stable and yet reversible 2′‐O‐(2‐cyano‐2,2‐dimethylethanimine‐N‐oxymethyl)ribonucleosides in post‐purification yields of 54% to 82%. Phenoxyacetylation of the exocyclic amino functions of these ribonucleosides proceeds in yields of 74% to 89%, and subsequent 5′‐O‐dimethoxytritylation and 3′‐O‐phosphitylation of the corresponding N‐phenoxyacetylated ribonucleosides provide the fully protected ribonucleoside phosphoramidite monomers in isolated yields of 69% to 88%. These ribonucleoside phosphoramidites are employed in solid‐phase synthesis of three chimeric RNA sequences, each differing in purine/pyrimidine content. The stepwise coupling efficiency of the ribonucleoside phosphoramidites (as 0.15 M solutions in acetonitrile) averages 99% over a coupling time of 180 s when 5‐benzylthio‐1H‐tetrazole is used as an activator. Upon completion of RNA chain assembly, removal of the nucleobase‐ and phosphate‐protecting groups and release of sequences from the solid support are carried out under standard basic conditions. Finally, the 2′‐O‐(2‐cyano‐2,2‐dimethylethanimine‐N‐oxymethyl) protective groups are cleaved from the RNA sequences by treatment with 0.5 M tetra‐n‐butylammonium fluoride in dry DMSO for 24 to 48 hr at 55°C without releasing RNA‐alkylating side‐products. Characterization of the fully deprotected RNA sequences by PAGE, enzymatic hydrolysis, and MALDI‐TOF mass spectrometry confirms the identity and high quality of these sequences. Curr. Protoc. Nucleic Acid Chem. 54:3.22.1‐3.22.28. © 2013 by John Wiley & Sons, Inc.

Keywords: 2′‐O‐aminooxymethyl ribonucleosides; 2′‐O‐(2‐cyano‐2,2‐dimethyl‐ethanimine‐N‐oxymethyl)ribonucleosides; solid‐phase RNA synthesis; fluoride‐mediated 2′‐deprotection; chimeric RNA sequences

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis, Purification, and Characterization of 2′‐O‐(2‐Cyano‐2,2‐Dimethylethanimine‐N‐Oxymethyl)Uridine
  • Alternate Protocol 1: Synthesis, Purification, and Characterization of Nucleobase‐Protected 2′‐O‐(2‐Cyano‐2,2‐Dimethylethanimine‐N‐Oxymethyl)Cytidine, Adenosine, and Guanosine
  • Support Protocol 1: Synthesis and Characterization of 2‐Cyano‐2‐Methyl Propanal
  • Basic Protocol 2: Synthesis, Purification, and Characterization of RNA Sequences
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis, Purification, and Characterization of 2′‐O‐(2‐Cyano‐2,2‐Dimethylethanimine‐N‐Oxymethyl)Uridine

  Materials
  • 3′,5′‐O‐(1,1,3,3‐Tetraisopropyldisiloxane‐1,3‐diyl)uridine (1a; Rasayan)
  • Dimethylsulfoxide (DMSO; Acros)
  • Glacial acetic acid (AcOH; Acros)
  • Acetic anhydride (Ac 2O; Acros)
  • Drierite (20‐40 mesh; Aldrich)
  • Potassium carbonate, anhydrous (K 2CO 3; Fisher)
  • Tetrahydrofuran (THF; Acros)
  • Pyridine, dry (Acros)
  • Toluene (Acros)
  • Methylene chloride, anhydrous (CH 2Cl 2; Fisher)
  • Silica gel (60 Å, 230 to 400 mesh; Merck)
  • Methanol (MeOH; Fisher)
  • Chloroform (CHCl 3; Fisher)
  • Benzene, dry (Aldrich)
  • Dry ice
  • Acetone (Fisher)
  • Sulfuryl chloride (Aldrich), freshly distilled
  • N‐Hydroxyphthalimide (Acros), vacuum‐dried
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene, dry (DBU; Acros)
  • Saturated aqueous sodium hydrogen carbonate (NaHCO 3; Fisher)
  • Sodium sulfate, anhydrous (Na 2SO 4; Fisher)
  • Ammonium fluoride (Aldrich)
  • 2‐Cyano‐2‐methyl propanal (6; see protocol 3Support Protocol)
  • Concentrated hydrochloric acid (HCl; Fisher)
  • 50‐, 100‐, and 250‐mL round‐bottom flasks (Kontes)
  • Rubber septa for 14/20 and 24/40 glass joints (Aldrich)
  • 10‐mL plastic syringes (B‐D)
  • 21‐G stainless steel syringe needles
  • Heating mantle (VWR)
  • 500‐ and 2000‐mL Erlenmeyer flasks (Kimax)
  • 250‐mL addition funnel (Kontes)
  • 25‐mL pipets
  • 10‐mL glass syringe (Hamilton)
  • Rotary evaporator (Büchi) connected to a vacuum pump (KNF)
  • 5 × 20−cm glass chromatography columns (Labglass)
  • 2.5 × 7.5‐cm TLC plates precoated with a 250‐µm layer of silica gel 60 F 254 (EMD)
  • High‐vacuum oil pump (Savant)
  • 500‐mL separatory funnel (Kontes)
  • 60‐mL sintered glass funnel (coarse porosity; Kontes)
  • Reflux condenser (Kontes)
  • 100‐mL beaker
  • Additional reagents and equipment for column chromatography ( appendix 3E) and TLC ( appendix 3D)

Alternate Protocol 1: Synthesis, Purification, and Characterization of Nucleobase‐Protected 2′‐O‐(2‐Cyano‐2,2‐Dimethylethanimine‐N‐Oxymethyl)Cytidine, Adenosine, and Guanosine

  Additional Materials (also see protocol 1)
  • Protected starting nucleosides (Rasayan):
  • N4‐Phenoxyacetyl‐3′,5′‐O‐(1,1,3,3‐tetraisopropyldisiloxane‐1,3‐diyl)cytidine (1b)
  • N6‐Phenoxyacetyl‐3′,5′‐O‐(1,1,3,3‐tetraisopropyldisiloxane‐1,3‐diyl)adenosine (1c)
  • N2‐Phenoxyacetyl‐3′,5′‐O‐(1,1,3,3‐tetraisopropyldisiloxane‐1,3‐diyl)guanosine (1d)
  • Ammonium hydroxide (conc. aqueous NH 3; Aldrich)
  • Chlorotrimethylsilane (Aldrich)
  • Phenoxyacetyl chloride (Aldrich)
  • Phenoxyacetic anhydride (TCI)
  • Triethylamine trihydrofluoride (TEA·3HF; Aldrich)
  • 250‐mL Erlenmeyer flask (Kimax)
  • 60‐mm funnel (Nalgene)
  • Filter paper, no.1 (Whatman)
  • 2.5 × 20−cm disposable Flex chromatography column (Kontes)
For Cytidine Derivative

Support Protocol 1: Synthesis and Characterization of 2‐Cyano‐2‐Methyl Propanal

  Materials
  • Isobutyraldehyde (9; Aldrich)
  • 37% aqueous formaldehyde (Aldrich)
  • Potassium carbonate (K 2CO 3; Fisher)
  • Methylene chloride (CH 2Cl 2; Fisher)
  • Brine: saturated aqueous NaCl
  • Anhydrous sodium sulfate (Na 2SO 4; Fisher)
  • Hydroxylamine hydrochloride (Fisher)
  • Ethanol (Acros)
  • Sodium chloride (NaCl, Fisher)
  • Diethyl ether (Acros)
  • Acetonitrile, anhydrous (MeCN; Acros)
  • Acetone (Fisher)
  • Trifluoroacetic anhydride (Fisher)
  • Calcium chloride, anhydrous (CaCl 2; Aldrich)
  • Methanol (Fisher)
  • Silica gel (60 Å, 230 to 400 mesh; Merck)
  • Chloroform (CHCl 3; Fisher)
  • Iodine (Aldrich)
  • Dimethyl sulfoxide, anhydrous (DMSO; Acros)
  • Oxalyl chloride (Aldrich)
  • Dry ice
  • Triethylamine (TEA; Aldrich)
  • 2 M hydrochloric acid (HCl; Fisher)
  • 50‐, 250‐, 500‐, and 1000‐mL round‐bottom flasks (Kontes)
  • 100‐ and 250‐mL addition funnels (Kimax)
  • 500‐ and 1000‐mL separatory funnels (Kontes)
  • 500‐ and 1000‐mL Erlenmeyer flasks (Kimax)
  • 60‐mm funnel (Nalgene)
  • Whatman no. 1 filter paper
  • Rotary evaporator (Büchi) connected to a vacuum pump (KNF)
  • Heating mantle (VWR)
  • 100‐mL sintered glass funnel (coarse porosity, Kontes)
  • High‐vacuum oil pump (Savant)
  • Reflux condensers (Kontes)
  • 100‐ and 500‐mL pressure‐equalizing addition funnels (Kontes)
  • Drying tubes (Kontes)
  • 10‐mL glass Hamilton syringes with 21‐G needles
  • 5 × 20–cm glass chromatography column (LabGlass)
  • 2.5 × 7.5−cm TLC plates precoated with a 250‐µm layer of silica gel 60 F 254 (EMD)
  • 500‐mL three‐necked round‐bottom flask (Kontes)
  • Rubber septum for a 24/40 glass joint (Aldrich)
  • Additional reagents and equipment for column chromatography ( appendix 3E) and TLC ( appendix 3D)

Basic Protocol 2: Synthesis, Purification, and Characterization of RNA Sequences

  Materials
  • 2′‐O‐(2‐Cyano‐2,2‐dimethylethanimine‐N‐oxymethyl) ribonucleosides (7a, see protocol 1; 8b‐d, see protocol 2Alternate Protocol)
  • Pyridine, dry (Acros)
  • 4,4′‐Dimethoxytrityl chloride (Chem‐Impex International)
  • Methylene chloride (CH 2Cl 2; Fisher)
  • Saturated aqueous sodium hydrogen carbonate (NaHCO 3; Fisher)
  • Sodium sulfate, anhydrous (Na 2SO 4; Fisher)
  • Silica gel (60 Å, 230 to 400 mesh; Merck)
  • Methanol (Fisher)
  • Triethylamine (TEA; Aldrich)
  • Chloroform (CHCl 3; Fisher)
  • Dry argon gas cylinder (Matheson)
  • 2‐Cyanoethyl N,N‐diisopropylchlorophosphoramidite (Aldrich)
  • Benzene (C 6H 6; Aldrich)
  • Hexane (Fisher)
  • Dry ice
  • Acetone (Fisher)
  • 2′‐O‐TBDMS‐protected phosphoramidites (ChemGenes, for synthesis of control sequences):
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐[(N,N‐diisopropylamino)(2‐cyanoethyloxy)] phosphinyl‐2′‐O‐(tert‐butyldimethylsilyl)uridine
    • N4‐Phenoxyacetyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐3′‐O‐[(N,N‐diisopropylamino)(2‐ cyanoethyloxy)]phosphinyl‐2′‐O‐(tert‐butyldimethylsilyl)cytidine
    • N6‐Phenoxyacetyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐3′‐O‐[(N,N‐diisopropylamino)(2‐ cyanoethyloxy)]phosphinyl‐2′‐O‐(tert‐butyldimethylsilyl)adenosine
    • N2‐Phenoxyacetyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐3′‐O‐[(N,N‐diisopropylamino)(2‐ cyanoethyloxy)]phosphinyl‐2′‐O‐(tert‐butyldimethylsilyl)guanosine
  • Acetonitrile, dry (MeCN; Acros)
  • Reagents for oligonucleotide synthesis (all available from Glen Research):
    • 0.25 M 5‐Benzylthio‐1H‐tetrazole in MeCN
    • Cap A solution: acetic anhydride in tetrahydrofuran (THF)/pyridine
    • Cap B solution: 1‐methylimidazole in THF
    • Deblocking solution: 3% trichloroacetic acid in CH 2Cl 2
    • Oxidation solution: 0.02 M iodine in THF/pyridine/water
  • Ammonium hydroxide (conc. aqueous NH 3; Aldrich)
  • Dimethyl sulfoxide (DMSO; Acros)
  • Tetra‐n‐butylammonium fluoride (TBAF; Aldrich)
  • Triethylamine trihydrofluoride (TEA·3HF; Aldrich)
  • 2 M triethylammonium acetate (TEAA) buffer, pH 7.0 (Applied Biosystems)
  • Diethylpyrocarbonate (DEPC)−treated water (Research Genetics)
  • Loading buffer: 1:4 (v/v) 10× TBE electrophoresis buffer ( appendix 2A) in formamide containing 2 mg/mL bromphenol blue
  • 20 × 40–cm, 7 M urea/20% polyacrylamide gel
  • Staining buffer: 1:5:20:0.1 (v/v/v/v) formamide/isopropyl alcohol/ddH 2O/3.0 M Tris·Cl, pH 8.8
  • 1 mg/mL Stains‐All in formamide (both from Aldrich)
  • 1 M Tris·Cl, pH 9.0 ( appendix 2A)
  • 1.0 M magnesium chloride (MgCl 2; Sigma)
  • Snake venom phosphodiesterase (Crotalus adamanteus; Sigma)
  • Bacterial alkaline phosphatase (E. coli; Sigma)
  • 100‐ and 250‐mL round‐bottom flasks (Kontes)
  • Rotary evaporator (Büchi) connected to a vacuum pump (KNF)
  • Rubber septa for 14/20 glass joints (Aldrich)
  • 250‐mL separatory funnels with stoppers (Kontes)
  • 250‐mL Erlenmeyer flasks (Kimax)
  • 60‐mm funnel (Nalgene)
  • Whatman no. 1 filter paper
  • 2.5 × 20−cm disposable Flex chromatography columns (Kontes)
  • 2.5 × 7.5−cm TLC plates precoated with a 250‐µm layer of silica gel 60 F 254 (EMD)
  • Speedvac evaporator connected to a vacuum pump
  • Automated DNA/RNA synthesizer (Applied Biosystems model 392)
  • Synthesis columns with long‐chain alkylamine controlled‐pore glass (LCAA‐CPG, 500 Å) support loaded with 5′‐O‐(4,4‐dimethoxytrityl)‐2′‐deoxythymidine covalently bound through a 3′‐O‐hemisuccinate linker (Glen Research)
  • 4‐mL screw‐cap glass vials (Fisher)
  • UV spectrophotometer
  • Heat block (VWR)
  • PD‐10 columns (Sephadex G‐25M, GE Healthcare)
  • 1.5‐mL microcentrifuge tubes
  • 50‐µL and 10‐mL glass Hamilton syringes
  • 1‐mL plastic syringe (B‐D)
  • 37°C water bath
  • 5‐µm Supelcosil LC‐18S HPLC column (25 cm × 4.6 mm, Supelco)
  • Additional reagents and equipment for column chromatography ( appendix 3E), TLC ( appendix 3D), automated oligonucleotide synthesis ( appendix 3C), SDS‐PAGE (unit 10.4 & appendix 3B), and RP‐HPLC (unit 10.5)
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Literature Cited

Literature Cited
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