Preparation of Oligodeoxyribonucleotides Containing the Pyrimidine(6–4)pyrimidone Photoproduct by Using a Dinucleotide Building Block

Shigenori Iwai1

1 Division of Chemistry, Graduate School of Engineering Science, Osaka University, Osaka, Japan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.56
DOI:  10.1002/0471142700.nc0456s53
Online Posting Date:  June, 2013
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Abstract

This unit describes procedures for the synthesis of a dinucleotide‐type building block of the pyrimidine(6–4)pyrimidone photoproduct [(6–4) photoproduct], which is one of the major DNA lesions induced by ultraviolet (UV) light, and its incorporation into oligodeoxyribonucleotides. Although this type of lesion is frequently found at thymine‐cytosine sites, the building block of the (6–4) photoproduct formed at thymine‐thymine sites can be synthesized much more easily. The problem in the oligonucleotide synthesis is that the (6–4) photoproduct is labile under alkaline conditions. Therefore, building blocks with an amino‐protecting group that can be removed by a brief treatment with ammonia water at room temperature must be used for the incorporation of the normal bases. Byproduct formation by the coupling of phosphoramidites with the N3 of the 5′ component should also be considered. This side reaction can be avoided by using benzimidazolium triflate as an activator. Curr. Protoc. Nucleic Acid Chem. 53:4.56.1‐4.56.18. © 2013 by John Wiley & Sons, Inc.

Keywords: oligonucleotides; UV damage; DNA repair; xeroderma pigmentosum

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Oligonucleotides Containing the (6–4) Photoproduct
  • Alternate Protocol 1: Synthesis of Oligonucleotides Using Tetrazole as an Activator
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Thymidine ( 1)
  • Pyridine, dehydrated
  • 4,4′‐Dimethoxytrityl chloride (DMT chloride)
  • Chloroform
  • Methanol
  • Sat. aq. NaHCO 3
  • Sat. aq. NaCl
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Toluene
  • Hexane
  • Benzene
  • 5′‐O‐(4,4′‐Dimethoxytrityl)thymidine 3′‐(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite ( 3)
  • Phosphorus pentoxide
  • 1,4‐Dioxane, dehydrated
  • 1,3‐Dicyclohexylcarbodiimide (DCC)
  • 4‐(Dimethylamino)pyridine (DMAP)
  • Levulinic acid
  • Silica gel for chromatography
  • Acetic acid
  • Acetonitrile, dehydrated
  • 1H‐Tetrazole
  • Iodine
  • Tetrahydrofuran
  • Deionized water
  • Sodium thiosulfate (Na 2S 2O 3)
  • Ethyl acetate
  • Ethanol
  • Diethyl ether
  • 500‐mL round‐bottom or pear‐shaped flasks
  • Magnetic stirrer and stirring bars
  • Silica gel TLC plates
  • Compact UV lamp, for the detection of compounds on TLC plates
  • Rotary evaporator with a diaphragm pump and a recirculating chiller, for the removal of solvents with boiling points lower than 100°C
  • Rotary evaporator with an oil‐sealed rotary vacuum pump and a recirculating chiller, for the removal of solvents with boiling points higher than 100°C
  • Vacuum desiccator

Basic Protocol 2:

  Materials
  • Partially protected thymidylyl(3′–5′)thymidine 4 ( protocol 1)
  • Distilled water or ultrapure water
  • Acetonitrile, HPLC grade
  • Nitrogen source
  • Triethylammonium acetate (TEAA, 2 M, pH 7.0)
  • Dimethyloctadecylsilyl‐bonded amorphous silica (Waters Preparative C18; 125Å 55‐105 µm)
  • Phosphorus pentoxide
  • Aluminum trays
  • Ice bath
  • UV crosslinker (Spectronics Spectrolinker XL‐1500)
  • Rotary evaporator with an oil‐sealed rotary vacuum pump and a recirculating chiller
  • Gradient‐type HPLC system, preferably equipped with a PDA detector
  • Reversed‐phase HPLC column [Waters µBondasphere C18 5 µm 300A (3.9 ×150–mm) or XBridge C18 5 µm (4.6 ×150–mm)]
  • 1.5 ×50–cm glass columns
  • Low‐pressure chromatography system (Bio‐Rad BioLogic LP System)

Basic Protocol 3:

  Materials
  • Partially protected dinucleoside monophosphate containing the (6–4) photoproduct 5 ( protocol 2)
  • Pyridine, dehydrated
  • DMT chloride
  • Silica gel TLC plates
  • Methanol
  • Chloroform
  • Toluene
  • Deionized water
  • Silica gel for chromatography
  • Phosphorus pentoxide
  • Hydrazine monohydrate
  • Acetic acid, glacial
  • Acetone
  • Sodium bicarbonate (NaHCO 3)
  • Sodium chloride
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Tetrahydrofuran, dehydrated
  • N,N‐Diisopropylethylamine
  • 2‐Cyanoethyl N,N‐diisopropylchlorophosphoramidite
  • Ethyl acetate
  • Pentane
  • Acetonitrile
  • 30‐mL round‐bottom or pear‐shaped flasks
  • Magnetic stirrer and stirring bars
  • Compact UV lamp, for the detection of compounds on TLC plates
  • Rotary evaporator with a diaphragm pump and a recirculating chiller, for the removal of solvents with boiling points lower than 100°C
  • Rotary evaporator with an oil‐sealed rotary vacuum pump and a recirculating chiller, for the removal of solvents with boiling points higher than 100°C
  • Vacuum desiccator

Basic Protocol 4: Synthesis of Oligonucleotides Containing the (6–4) Photoproduct

  Materials
  • Phosphoramidite building block of the (6–4) photoproduct 8 (see protocol 3)
  • Acetonitrile, dehydrated
  • BIT (see recipe)
  • Phenoxyacetic anhydride in tetrahydrofuran and pyridine for DNA synthesizers
  • 1‐Methylimidazole in tetrahydrofuran and pyridine for DNA synthesizers
  • Iodine in tetrahydrofuran, pyridine, and water for DNA synthesizers
  • Trichloroacetic acid in dichloromethane for DNA synthesizers
  • Dichloromethane for DNA synthesizers
  • Acetonitrile for DNA synthesizers
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐6‐N‐phenoxyacetyl‐2′‐deoxyadenosine 3′‐O‐(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite for DNA synthesizers
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐4‐N‐acetyl‐2′‐deoxycytidine 3′‐O‐(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite for DNA synthesizers
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2‐N‐(4‐isopropylphenoxy)acetyl‐2′‐deoxyguanosine 3′‐O‐(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite for DNA synthesizers
  • 5′‐O‐(4,4′‐Dimethoxytrityl)thymidine 3′‐O‐(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite for DNA synthesizers
  • Universal Support III PS (Glen Research)
  • 2.0 M Ammonia in methanol
  • 28% Ammonia water
  • Ethyl acetate
  • Distilled water or ultrapure water
  • Acetonitrile, HPLC grade
  • TEAA (2 M, pH 7.0)
  • Disposable membrane filter unit, optional
  • DNA synthesizer (e.g., Applied Biosystems Model 3400)
  • Rotary evaporator with a diaphragm pump and a recirculating chiller
  • Rotary evaporator with an oil‐sealed rotary vacuum pump and a recirculating chiller
  • Gradient‐type HPLC system, preferably equipped with a PDA detector
  • Reversed‐phase HPLC column [Waters µBondasphere C18 5 µm 300A (3.9 ×150–mm) or XBridge C18 5 µm (4.6 ×150–mm)]

Alternate Protocol 1: Synthesis of Oligonucleotides Using Tetrazole as an Activator

  Materials
  • Phosphoramidite building block of the (6–4) photoproduct 8 (see protocol 3)
  • Acetonitrile, dehydrated
  • Tetrazole in acetonitrile for DNA synthesizers
  • Phenoxyacetic anhydride in tetrahydrofuran and pyridine for DNA synthesizers
  • 1‐Methylimidazole in tetrahydrofuran and pyridine for DNA synthesizers
  • Iodine in tetrahydrofuran, pyridine, and water for DNA synthesizers
  • Trichloroacetic acid in dichloromethane for DNA synthesizers
  • Dichloromethane for DNA synthesizers
  • Acetonitrile for DNA synthesizers
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐6‐N‐phenoxyacetyl‐2′‐deoxyadenosine 3′‐O‐(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite for DNA synthesizers
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐4‐N‐acetyl‐2′‐deoxycytidine 3′‐O‐(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite for DNA synthesizers
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2‐N‐(4‐isopropylphenoxy)acetyl‐2′‐deoxyguanosine 3′‐O‐(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite for DNA synthesizers
  • 5′‐O‐(4,4′‐Dimethoxytrityl)thymidine 3′‐O‐(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite for DNA synthesizers
  • UltraMild CPG (Glen Research) for DNA synthesizers
  • 28% ammonia water
  • Ethyl acetate
  • Distilled water or ultrapure water
  • Acetonitrile, HPLC grade
  • TEAA (2 M, pH 7.0)
  • Disposable membrane filter unit, optional
  • DNA synthesizer
  • Rotary evaporator with a diaphragm pump and a recirculating chiller
  • Rotary evaporator with an oil‐sealed rotary vacuum pump and a recirculating chiller
  • Gradient‐type HPLC system, preferably equipped with a PDA detector
  • Reversed‐phase HPLC column [Waters µBondasphere C18 5 µm 300A (3.9 ×150–mm) or XBridge C18 5 µm (4.6 ×150–mm)]
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Figures

Videos

Literature Cited

Literature Cited
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