Synthesis of Geranyl‐2‐Thiouridine‐Modified RNA

Rui Wang1, Phensinee Haruehanroengra1, Jia Sheng1

1 Department of Chemistry and The RNA Institute, University at Albany, State University of New York, Albany, New York
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.72
DOI:  10.1002/cpnc.22
Online Posting Date:  March, 2017
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This unit describes the chemical synthesis of the S‐geranyl‐2‐thiouridine (ges2U) phosphoramidite and its incorporation into RNA oligonucleotides through solid‐phase synthesis. Starting from the 2‐thiouracil nucleobase and the protected ribose, the 2‐thiouridine is synthesized and the geranyl functionality is introduced into the 2‐thio position by using geranyl bromide as the geranylating reagent before the conversion of this modified nucleoside into a phosphoramidite building block. The modified phosphoramidite is used to make the geranyl‐RNA oligonucleotides with a solid‐phase DNA synthesizer. These RNA strands are then purified by ion‐exchange HPLC before further structural and functional studies, such as base pairing and enzyme recognition, can be done. © 2017 by John Wiley & Sons, Inc.

Keywords: nucleic acids; natural RNA modification; tRNA; geranylation

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of S‐Geranyl‐2‐Thiouridine Phosphoramidite
  • Basic Protocol 2: Synthesis, Purification, and Characterization of Geranyl‐Modified RNA Oligonucleotides
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation of S‐Geranyl‐2‐Thiouridine Phosphoramidite

  • 2‐Thiouracil (≥99%, Sigma‐Aldrich)
  • Argon
  • Hexamethyldisilazane (ReagentPlus hexamethyldisilazane, 99.9%, Sigma‐Aldrich)
  • Trimethylsilyl chloride (purified by redistillation, ≥99%, Sigma‐Aldrich)
  • 1‐O‐Acetyl‐2,3,5‐tri‐O‐benzoyl β‐D‐ribofuranose (99.5% pure, ChemGenes)
  • 1,2‐Dichloroethane (anhydrous, 99.8%, Sigma‐Aldrich)
  • NaCl (aqueous, saturated)
  • Tin(IV) chloride (99.995% trace metals basis, Sigma‐Aldrich)
  • Saturated aqueous sodium bicarbonate (NaHCO 3)
  • Methylene chloride (dichloromethane, CH 2Cl 2; purity >99.5%, Fluka)
  • Anhydrous magnesium sulfate (MgSO 4)
  • Methanol (anhydrous MeOH, Sigma‐Aldrich)
  • Sodium methoxide (99.995% trace metals basis, Sigma‐Aldrich)
  • DOWEX 50WX8‐400 ion‐exchange resin (H+ form) (Sigma‐Aldrich)
  • Ethyl acetate (EtOAc)
  • Ethanol (absolute)
  • N,N‐Dimethylformamide (DMF; anhydrous, 99% pure, Sigma‐Aldrich)
  • Di‐tert‐butylsilyl bis(trifluoromethanesulfonate) (97%, Sigma‐Aldrich)
  • Imidazole (ACS reagent, ≥99%, Sigma‐Aldrich)
  • tert‐Butyldimethylsilyl chloride (>97%, Sigma‐Aldrich)
  • HCl (aqueous, 1.0 M)
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Silica gel (60‐Å porosity; 40‐ to 63‐μm particle size; 400 mesh)
  • HF‐pyridine (∼30% pyridine/∼70% hydrogen fluoride, Sigma‐Aldrich)
  • Pyridine (anhydrous, purity >99%, Sigma‐Aldrich)
  • 4,4′‐Dimethoxytrityl chloride (95%, Sigma‐Aldrich)
  • Triethylamine (>99%, Sigma‐Aldrich)
  • N,N‐Diisopropylethylamine (DIPEA; 99% pure, Sigma‐Aldrich)
  • Geranyl bromide (95%, Sigma‐Aldrich)
  • 2‐Cyanoethyl N,N‐diisopropylchlorophosphoramidite (ChemGenes Corporation)
  • 250‐mL three‐neck, round‐bottom flasks, oven‐dried
  • Magnetic stir bars
  • Reflux apparatus
  • 250‐mL one‐neck, round‐bottom flasks, oven‐dried
  • Rotary evaporator
  • Vacuum oil pump
  • 1‐, 5‐, and 10‐mL syringes
  • 1000‐mL beakers
  • Filter paper
  • pH paper
  • Lyophilizer
  • 10‐, 25‐, 50‐, and 100‐mL round‐bottom flasks
  • Thermometer
  • Hot plate
  • Separatory funnels
  • Rubber septum
  • 22 × 457–mm silica gel chromatography columns
  • Additional reagents and equipment for performing thin‐layer chromatography (TLC) and column chromatography

Basic Protocol 2: Synthesis, Purification, and Characterization of Geranyl‐Modified RNA Oligonucleotides

  • Geranylated 2‐thiouridine phosphoramidite (ges2U phosphoramidite; protocol 1)
  • Anhydrous dichloromethane
  • Acetonitrile (CH 3CN), anhydrous, HPLC‐grade
  • Unmodified RNA phosphoramidites: rA‐CE, rG‐CE, Ac‐rC‐CE, rU‐CE (ChemGenes)
  • Methylamine solution (40% wt. in H 2O)
  • Ammonium hydroxide solution (28.0% to 30.0% NH 3 basis)
  • Ammonium acetate (>99%, Sigma‐Aldrich)
  • 2.0 M triethylammonium acetate (TEAA) buffer, pH 7.0
  • RNA synthesizer and columns (e.g., Oligo‐800 DNA synthesizer or equivalent)
  • Vacuum pump
  • 2‐mL screw‐cap tubes or vials
  • Hotplate
  • 13‐mm syringe filter with 0.2‐μm nylon membrane (Life Sciences)
  • Ion exchange‐HPLC columns (Dionex, cat. no. PA‐100 or PA‐200)
  • HPLC system with detector at 260 nm
  • Lyophilizer
  • Sep‐Pac C18 columns (Waters)
  • Microcentrifuge tubes
  • 2‐mL autoclaved vials
  • UV spectrophotometer (Nanodrop‐1000)
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Literature Cited

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