A New Nucleic Acid Prodrug Responsive to High Thiol Concentration: Synthesis of 2′‐O‐Methyldithiomethyl‐Modified Oligonucleotides by Post‐Synthetic Modification

Yosuke Ochi1, Osamu Nakagawa2, Junsuke Hayashi1, Shun‐ichi Wada1, Hidehito Urata1

1 Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka, 2 Present address: Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.63
DOI:  10.1002/0471142700.nc0463s62
Online Posting Date:  September, 2015
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Abstract

This unit describes the synthesis of 2′‐O‐methyldithiomethyluridine‐containing oligonucleotides, which can be deprotected to yield the parental oligoribonucleotides under high concentrations of glutathione similar in cytoplasm. The 2′‐O‐methyldithiomethyl group is sensitive to reductive conditions, so that it is incompatible to 3′‐O‐phosphoramidite modification in nucleosides. Thus, a novel post‐synthetic approach to obtain 2′‐O‐methyldithiomethyluridine‐containing oligonucleotides was developed, in which 2′‐O‐(2,4,6‐trimethoxybenzylthiomethyl)uridine‐modified oligonucleotides are readily converted by treatment with dimethyl(methylthio)sulfonium tetrafluoroborate to the 2′‐O‐methyldithiomethyluridine‐modified oligonucleotides. The 2′‐O‐methyldithiomethyluridine‐modified oligonucleotides are readily and cleanly converted to the parental oligonucleotides under high thiol conditions, such as 10 mM glutathione and dithiothreitol. © 2015 by John Wiley & Sons, Inc.

Keywords: modified oligonucleotide; 2,4,6‐trimethoxybenzylthiomethyl group; methyldithiomethyl group; post‐synthetic modification; prodrug RNA; conversion in reducing environment

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of 2′‐O‐(2,4,6‐Trimethoxybenzylthiomethyl)Uridine
  • Support Protocol 1: Synthesis of 2,4,6‐Trimethoxybenzylmercaptan
  • Basic Protocol 2: Synthesis and Purification of 2′‐O‐(2,4,6‐Trimethoxybenzylthiomethyl)Uridine‐Containing Oligonucleotides
  • Support Protocol 2: Synthesis of Diisopropylammonium Tetrazolide
  • Basic Protocol 3: Synthesis of 2′‐O‐Methyldithiomethyluridine‐Containing Oligonucleotides (12‐15) and their Reductive Deprotection
  • Support Protocol 3: Nuclease Resistance of 2′‐O‐Methyldithiomethyl‐Modified Oligonucleotide
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of 2′‐O‐(2,4,6‐Trimethoxybenzylthiomethyl)Uridine

  Materials
  • Uridine
  • N,N‐Dimethylformamide (DMF)
  • Argon
  • Di(tert‐butyl)silyl ditriflate (Aldrich)
  • TLC (Silica gel 60 F 254; Merck Millipore)
  • Chloroform (CHCl 3)
  • Methanol (MeOH)
  • Triethylamine (Et 3N)
  • Sodium hydrogen carbonate (NaHCO 3)
  • Ethyl acetate (AcOEt)
  • Deionized water
  • Brine
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Dimethyl sulfoxide (DMSO)
  • Acetic acid (AcOH)
  • Acetic anhydride (Ac 2O)
  • n‐Hexane
  • Silica gel (Wakosil C‐200; particle size, 64 to 210 μm; Wako Pure Chemical Industries)
  • Dichloromethane (CH 2Cl 2), anhydrous
  • Sulfuryl chloride (SO 2Cl 2)
  • 2,4,6‐Trimethoxybenzylmercaptan (see protocol 2)
  • 60% Sodium hydride (NaH) in mineral oil
  • Potassium dihydrogen phosphate (KH 2PO 4)
  • Silica gel (Wakogel C‐400HG; particle size, 20 to 40 μm; Wako Pure Chemical Industries)
  • Tetrahydrofuran (THF)
  • Triethylamine trihydrofluoride (Et 3N·3HF; Aldrich)
  • 50‐, 100‐, and 500‐mL round‐bottom flasks
  • Teflon‐coated magnetic stirring bars
  • Rubber septum for 15/25 glass joint
  • Gas balloon
  • 10‐ and 50‐mL syringes
  • Magnetic stirrer
  • Separatory funnel
  • Rotary evaporator
  • High‐vacuum oil pump
  • 4.5 × 18‐cm silica gel (Wakosil C‐200) column
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D)

Support Protocol 1: Synthesis of 2,4,6‐Trimethoxybenzylmercaptan

  Materials
  • 2,4,6‐Trimethoxybenzaldehyde
  • Methanol (MeOH)
  • Sodium hydroxide (NaOH)
  • Sodium borohydride (NaBH 4)
  • TLC (Silica gel 60 F 254; Merck Millipore)
  • Ethyl acetate (AcOEt)
  • n‐Hexane
  • Diethyl ether (Et 2O)
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Acetone
  • Thiourea
  • Hydrochloric acid (HCl)
  • Silica gel (Wakosil C‐200; particle size, 64 to 120 μm; Wako Pure Chemical Industries)
  • 200‐, 500‐mL Round bottom flasks
  • Teflon‐coated magnetic stirring bars
  • Magnetic stirrer
  • Separatory funnel
  • Condenser
  • Rotary evaporator
  • High‐vacuum oil pump
  • Magnetic stirrer heat block (Tokyo Rikakikai)
  • pH paper
  • Additional reagents and equipment for thin‐layer chromatography ( appendix 3D)

Basic Protocol 2: Synthesis and Purification of 2′‐O‐(2,4,6‐Trimethoxybenzylthiomethyl)Uridine‐Containing Oligonucleotides

  Materials
  • 2′‐O‐(2,4,6‐Trimethoxybenzylthiomethyl)uridine (2′‐O‐TMBTM‐uridine, 5; see protocol 1)
  • Pyridine, anhydrous
  • 4,4′‐Dimethoxytrityl chloride (DMT‐Cl; Aldrich)
  • TLC (Silica gel 60 F 254; Merck Millipore)
  • Ethyl acetate (AcOEt)
  • n‐Hexane
  • Ethanol (EtOH)
  • Chloroform (CHCl 3)
  • Sodium hydrogen carbonate (NaHCO 3)
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Silica gel (Wakogel C‐400HG; particle size, 20 to 40 μm; Wako Pure Chemical Industries)
  • Acetonitrile (CH 3CN)
  • Dichloromethane (CH 2Cl 2)
  • Argon
  • 2‐Cyanoethyl N,N,N,N′‐tetraisopropylphosphordiamidite (Wako Pure Chemical Industries)
  • Diisopropylammonium tetrazolide (see protocol 4)
  • Monomers and reagents for oligonucleotide synthesis:
    • dT‐CPG‐support (Glen Research)
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐[(N,N‐diisopropylamino)(2‐cyanoethyloxy)]phosphinyl‐2′‐O‐(2,4,6‐trimethoxybenzylthiomethyl)uridine (7)
    • N4‐Acetyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐3′‐O‐[(N,N‐diisopropylamino)(2‐cyanoethyloxy)] phosphinyl‐2′‐deoxycytidine (Glen Research)
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐[(N,N‐diisopropylamino)(2‐cyanoethyloxy)]phosphinyl‐2′‐deoxythymidine (Glen Research)
    • N2‐Dimethylformamidine‐5′‐O‐(4,4′‐dimethoxytrityl)‐3′‐O‐[(N,N‐diisopropylamino)(2‐cyanoethyloxy)]phosphinyl‐2′‐deoxyguanosine (Glen Research)
    • 0.25 M 5‐Ethylthio‐1 H‐tetrazole (Glen Research)
    • Deblocking solution: 3% trichloroacetic acid in CH 2Cl 2 (Glen Research)
    • Cap A solution: tetrahydrofuran/pyridine/acetic anhydride (8/1/1) (Glen Research)
    • Cap B solution: 10% 1‐methylimidazole in tetrahydrofuran (Glen Research)
    • Oxidation solution: 0.02 M iodine in THF/pyridine/water (Glen Research)
  • 28% Ammonium hydroxide (NH 4OH)
  • Triethylammonium acetate (TEAA)
  • Deionized water
  • Trifluoroacetic acid (TFA)
  • 2′,4′,6′‐Trihydroxyacetophenone (Aldrich)
  • Diammonium hydrogen citrate (Wako Pure Chemical Industries)
  • 50‐, 100‐, and 200‐mL Round bottom flasks
  • Rubber septum for 15/25 glass joint (Aldrich)
  • Gas balloon
  • Teflon‐coated magnetic stirring bars
  • Magnetic stirrer
  • Separatory funnel
  • Rotary evaporator
  • High‐vacuum oil pump
  • DNA/RNA synthesizer (ABI‐392; Applied Biosystems)
  • 4‐mL Screw‐cap glass vial
  • Millex‐LG (0.20 μm) filter cartridge (Merck Millipore)
  • COSMOSIL 5C18‐MS‐II column (φ 10.0 × 250 mm; Nacalai tesque)
  • Reversed‐phase HPLC system with UV detector at 260 nm
  • Centrifugal evaporator connected to vacuum pump
  • UV spectrophotometer
  • Sep‐Pak Plus C18 (Waters)

Support Protocol 2: Synthesis of Diisopropylammonium Tetrazolide

  Materials
  • 1 H‐Tetrazole
  • Acetonitrile (CH 3CN)
  • Diisopropylamine
  • 500‐mL Round‐bottom flasks
  • Stir bar
  • Vacuum apparatus

Basic Protocol 3: Synthesis of 2′‐O‐Methyldithiomethyluridine‐Containing Oligonucleotides (12‐15) and their Reductive Deprotection

  Materials
  • 2′‐O‐(2,4,6‐Trimethoxybenzylthiomethyl) (TMBTM)‐modified oligonucleotides (8‐11; protocol 3)
  • Sodium acetate
  • Dimethyl(methylthio)sulfonium tetrafluoroborate (DMTSF)
  • Acetonitrile (CH 3CN)
  • Triethylammonium acetate (TEAA)
  • Deionized water
  • 2′,4′,6′‐Trihydroxyacetophenone (Aldrich)
  • Diammonium hydrogen citrate (Wako Pure Chemical Industries)
  • DL‐1,4‐Dithiothreitol (DTT)
  • Tris·Cl, pH 8.0 ( appendix 2A)
  • Glutathione (GSH), optional
  • Ethanol (EtOH)
  • 50 mM Sodium phosphate (pH 7.0)
  • 1.5‐mL Micro tubes
  • COSMOSIL 5C18‐MS‐II column (φ 4.6 × 250 mm; Nacalai tesque)
  • NAP‐25 gel filtration column (GE Healthcare)
  • COSMOSIL 5C18‐MS‐II column (φ 10.0 × 250 mm; Nacalai tesque)
  • Centrifugal evaporator connected to vacuum pump
  • Spectrophotometer

Support Protocol 3: Nuclease Resistance of 2′‐O‐Methyldithiomethyl‐Modified Oligonucleotide

  Materials
  • Magnesium chloride (MgCl 2)
  • Tris·Cl, pH 8.0 ( appendix 2A)
  • Snake venom phosphodiesterase (SVPD)
  • Fetal bovine serum (FBS)
  • Formamide
  • Acetonitrile (CH 3CN)
  • Triethylammonium acetate (TEAA)
  • Lyophilizer
  • 1.5‐mL micro tubes
  • 37°C incubator
  • Heat block
  • μBondasphere 5C18 100 Å column (φ 3.9 × 150 mm; Waters)
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Figures

Videos

Literature Cited

Literature Cited
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