Synthesis of Branched DNA Using Oxidatively Cleavable Tritylsulfenyl as a Hydroxy Protecting Group

Kohji Seio1, Takashi Kanamori2, Mitsuo Sekine1

1 Department of Life Science, Tokyo Institute of Technology, Tokyo, 2 Education Academy of Computational Life Sciences, Tokyo Institute of Technology, Tokyo
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 2.18
DOI:  10.1002/0471142700.nc0218s58
Online Posting Date:  September, 2014
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Abstract

The application of oxidatively cleavable tritylsulfenyl (TrS) group to the synthesis of branched DNA is described. The TrS protecting group can be removed by treatment with 1 M aqueous iodine, while it is stable toward an oxaziridine‐type oxidant. At the same time, the sulfur‐oxygen linkage showed sufficient stability under the acidic and basic conditions used in oligonucleotide synthesis. These properties of the TrS group enabled the synthesis of branched DNA using a branched phosphoramidite in which the two hydroxy groups are protected by a 4,4′‐dimethoxytrityl (DMTr) group or a TrS group. In this unit, we describe an example of the synthesis of a three‐way branched DNA using a branched phosphoramidite. Curr. Protoc. Nucleic Acid Chem. 58:2.18.1‐2.18.19. © 2014 by John Wiley & Sons, Inc.

Keywords: branched DNA; hydroxy protecting group; tritylsulfenyl group

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Protected Amines 6, 10, and Trimesic Acid Dimethyl Ester (13)
  • Basic Protocol 2: Synthesis of Branched Phosphoramidite 1 Protected by DMTR and TRS Groups for the Synthesis of Branched DNA 2
  • Basic Protocol 3: Synthesis of Branched DNA 2 Using Branched Phosphoramidite 1
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Protected Amines 6, 10, and Trimesic Acid Dimethyl Ester (13)

  Materials
  • Sodium hydride (NaH, 60% in oil)
  • Propane‐1,3‐diol
  • Tritylsulfenyl chloride (TrSCl)
  • Anhydrous tetrahydrofuran (THF, dried over 4 Å molecular sieves, no stabilizer)
  • Hexane
  • Ethyl acetate
  • 28% aqueous ammonia (28% NH 4OH)
  • Magnesium sulfate (MgSO 4), anhydrous
  • Silica gel (Wakogel C‐200, 75–150 µm)
  • Dichloromethane
  • Triphenylphosphine (PPh 3)
  • Phthalimide
  • Argon gas
  • Diisopropyl azodicarboxylate (DIAD, 40% in toluene)
  • Methanol
  • Chloroform
  • Hydrazine monohydrate
  • NH‐silica gel (Fuji silysia)
  • Sodium hydroxide (NaOH)
  • Triethylamine
  • 3‐Amino‐propan‐1‐ol
  • Ethyl trifluoroacetate
  • Ninhydrin reagent (0.3 g ninhydrin in 100 mL n‐butyl alcohol containing 3 mL acetic acid)
  • Anhydrous pyridine (dried over 4 Å molecular sieves)
  • 4,4′‐Dimethoxytrityl chloride (DMTrCl)
  • Saturated aqueous sodium bicarbonate (saturated aq. NaHCO 3)
  • Trimesic acid
  • 95% sulfuric acid
  • Brine
  • Hydrochloric acid (concentrated HCl)
  • Sodium sulfate (Na 2SO 4), anhydrous
  • 10‐mL, 50‐mL, 100‐mL, 500‐mL, and 1‐L round‐bottom flasks
  • 200‐mL two‐neck, round‐bottom flask
  • Merck thin‐layer chromatography (TLC) silica plates (Kieselgel 60 F‐254; 0.2 mm thick)
  • TLC plate NH‐silica (Fuji silysia)
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 2: Synthesis of Branched Phosphoramidite 1 Protected by DMTR and TRS Groups for the Synthesis of Branched DNA 2

  Materials
  • Anhydrous tetrahydrofuran (THF, dried over 4 Å molecular sieves, no stabilizer)
  • 1,1′‐Carbonyldiimidazole (CDI)
  • Hexane
  • Ethyl acetate
  • Magnesium sulfate (MgSO 4), anhydrous
  • Pyridine
  • Toluene
  • Chloroform
  • Silica gel (Wakogel C‐200, 75–150 µm)
  • Methanol
  • Triethylamine
  • Sodium hydroxide (NaOH)
  • 3‐Aminopropan‐1‐ol
  • Anhydrous pyridine (dried over 4 Å molecular sieves)
  • Anhydrous dichloromethane
  • 2‐Cyanoethyl N,N,N′,N′‐tetraisopropylphosphorodiamidite
  • N,N‐Diisopropylamine
  • 1H‐Tetrazole
  • Acetonitrile
  • Isopropyl ether
  • NH‐silica gel (Fuji silysia)
  • 30‐mL, 50‐mL, 100‐mL, and 300‐mL round‐bottom flasks
  • TLC NH‐silica plates (Fuji silysia)
  • Merck thin‐layer chromatography (TLC) silica plates (Kieselgel 60 F‐254; 0.2 mm thick)
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 3: Synthesis of Branched DNA 2 Using Branched Phosphoramidite 1

  Materials
  • C unit: 4‐N‐Acetyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxycytidine 3′‐(2‐cyanoethyl N,N‐diisopropylphosphoramidite)
  • T unit: 5′‐O‐(4,4′‐Dimethoxytrityl)thymidine 3′‐(2‐cyanoethyl N,N‐diisopropylphosphoramidite)
  • A unit: 5′‐O‐(4,4′‐Dimethoxytrityl)‐6‐N‐phenoxyacetyl‐2′‐deoxyadenosine 3′‐(2‐cyanoethyl N,N‐diisopropylphosphoramidite)
  • G unit: 5′‐O‐(4,4′‐Dimethoxytrityl)‐2‐N‐[(4‐isopropylphenoxy)acetyl]‐2′‐deoxyguanosine 3′‐(2‐cyanoethyl N,N‐diisopropylphosphoramidite)
  • Dichlorodimethylsilane
  • Dichloromethane
  • 3% Trichloroacetic acid in dichloromethane
  • Trifluoroacetic acid
  • Acetonitrile (anhydrous, dried over 4 Å molecular sieves)
  • Argon balloon
  • Activator‐42 (5‐[4,6‐bis(trifluoromethyl)phenyl]‐1H‐tetrazole, Glen Research)
  • Acetic anhydride
  • 4‐Dimethylaminopyridine (DMAP)
  • (+)‐camphorsulfonyloxaziridine (CSO)
  • Iodine (I 2)
  • 28% NH 4OH
  • MiliQ water
  • Pyridine
  • Anhydrous pyridine (dried over 4 Å molecular sieves)
  • Ammonium acetate
  • Sodium phosphate buffer
  • Acetonitrile (HPLC grade)
  • Controlled pore glass (CPG) support functionalized with 4‐N‐acetyl‐2′‐deoxycytidine
  • Reversed‐phase HPLC column (Waters Xbridge C18, 5 µm, 4.6 × 150 mm)
  • Reversed‐phase cartridge column (Waters OASIS HLB, 6 cc, 200 mg)
  • Anion‐exchange HPLC column (Waters, Gen‐pak FAXTM)
  • Additional reagents and equipment for synthesis and purification of oligonucleotides ( appendix 3E) and column chromatography ( appendix 3E)
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Figures

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Literature Cited

Literature Cited
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