Synthesis of 5‐Formyl‐2′‐Deoxyuridine and Its Incorporation into Oligodeoxynucleotides

Kousuke Sato1, Wataru Hirose1, Akira Matsuda1

1 Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.21
DOI:  10.1002/0471142700.nc0121s35
Online Posting Date:  December, 2008
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Abstract

A straightforward, efficient method for the synthesis of 5-formyl-2¢-deoxyuridine (dfU) and solid-phase synthesis of oligodeoxynucleotides containing dfU using a phosphoramidite method are described. The synthesis of dfU is achieved by oxidation of the 5-methyl group in thymidine derivatives. However, incorporation of the dfU 3¢-O-phosphoramidite into oligodeoxynucleotides proceeds in low yield, due to instability of the 5-formyl group under conditions used for automated DNA synthesis. Therefore, oligodeoxynucleotides containing a 5-(1,2-dihydroxyethyl)uracil derivative are first prepared and finally oxidized by periodate to give the desired oligodeoxynucleotides containing 5-formyluracil. Curr. Protoc. Nucleic Acid Chem. 35:1.21.1-1.21.19. © 2008 by John Wiley & Sons, Inc.

Keywords: 5-formyl-2¢-deoxyuridine; thymidine; oligodeoxynucleotide; oxidation; oxidative damage; chemical synthesis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of 5-Formyl-2¢-Deoxyuridine from Thymidine Derivatives
  • Basic Protocol 2: Preparation of 5-(1,2-Diacetoxyethyl)-5¢-O-(4,4¢-Dimethoxytrityl)-2¢-Deoxyuridine 3¢-O-Phosphoramidite
  • Basic Protocol 3: Synthesis, Isolation, and Characterization of Oligodeoxynucleotides Containing 5-Formyl-2¢-Deoxyuridine
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of 5-Formyl-2¢-Deoxyuridine from Thymidine Derivatives

 Materials
  • Thymidine (S.1; Yamasa Co.)
  • N,N-Dimethylformamide (DMF), 99.5% (Junsei Chemical), drying with 4A molecular sieves
  • Imidazole, >99% (Kishida Chemical)
  • tert-Butyldimethylchlorosilane (TBDMS-Cl), 95% (Kishida Chemical)
  • Argon gas (>99.99% pure)
  • Methanol (MeOH), 99.6% (Junsei Chemical)
  • Ethyl acetate (EtOAc), 99% (Junsei Chemical)
  • Saturated aqueous NaHCO3
  • Saturated aqueous NaCl (brine)
  • Anhydrous Na2SO4, 99% (Junsei Chemical)
  • Silica gel 60 (0.063 to 0.2-mm, 70 to 230 mesh, Merck)
  • Hexane, 95% (Wako Pure Chemical)
  • Anhydrous pyridine, distilled from KOH and stored with 4A molecular sieves
  • Acetic anhydride (Ac2O), 99% (Junsei Chemical)
  • Chloroform (CHCl3), 98% (Junsei Chemical)
  • Acetonitrile (CH3CN), 99.5% (Junsei Chemical)
  • K2S2O8, 95% (Junsei Chemical)
  • CuSO4·5H2O, 99.5% (Wako Pure Chemical)
  • 2,6-Lutidine, 98% (Kanto Chemical)
  • EDTA, 99.5% (Junsei Chemical)
  • 0.5 N HCl
  • Silica gel 60N (spherical, neutral, 63 to 210 µm, Kanto Chemical)
  • Tetrahydrofuran (THF), 99.5% (Junsei Chemical)
  • Tetrabutylammonium fluoride (TBAF), 1 M THF solution (TCI)
  • Triethylamine (TEA), 98% (Junsei Chemical)
  • Rotary evaporator equipped with a diaphragm pump
  • 5 × 10–cm, 10 × 15–cm, 1 × 6–cm, 2 × 10–cm, and 1 × 5–cm glass columns
  • Vacuum oil pump
  • TLC plate, Merck silica gel 60 F254
  • 254-nm UV lamp (for TLC)
  • Celite pad
  • Additional reagents and equipments for TLC (appendix 3D) and column chromatography (appendix 3E)

Basic Protocol 2: Preparation of 5-(1,2-Diacetoxyethyl)-5¢-O-(4,4¢-Dimethoxytrityl)-2¢-Deoxyuridine 3¢-O-Phosphoramidite

 Materials
  • 5-Iodo-2¢-deoxyuridine (S.7, Yamasa Co. Ltd.)
  • Anhydrous pyridine, distilled from KOH and stored with 4A molecular sieves
  • 4,4¢-Dimethoxytrityl chloride (DMTr-Cl), >97% (TCI)
  • Argon gas (>99.99% pure)
  • Methanol (MeOH), 99.6% (Junsei Chemical)
  • Ethyl acetate (EtOAc), 99% (Junsei Chemical)
  • Saturated aqueous NaHCO3
  • Saturated aqueous NaCl (brine)
  • Anhydrous Na2SO4, 99% (Junsei Chemical)
  • Chloroform (CHCl3), 98% (Junsei Chemical)
  • N,N-Dimethylformamide (DMF), 99.5% (Junsei Chemical) drying with 4A molecular sieves
  • Imidazole, >99% (Kishida Chemical)
  • tert-Butyldimethylchlorosilane (TBDMS-Cl), 95% (Kishida Chemical)
  • Silica gel 60 (0.063 to 0.2-mm, 70 to 230 mesh; Merck)
  • Hexane, 95% (Wako Pure Chemical)
  • (Ph3P)2PdCl2, 98% (Aldrich)
  • Tributyl(vinyl)tin (Bu3SnCH = CH2), 97% (Wako Pure Chemical)
  • Acetone, 99% (Junsei Chemical)
  • t-Butanol (t-BuOH), 99% (Wako Pure Chemical)
  • N-Methylmorpholine N-oxide (NMO, Wako Pure Chemical)
  • Osmium tetroxide (OsO4), 99.8% (Aldrich)
  • N,N-Dimethylaminopyridine (DMAP), 99% (Aldrich)
  • Triethylamine (TEA), 98% (Junsei Chemical)
  • Acetic anhydride (Ac2O), 99% (Junsei Chemical)
  • Tetrahydrofuran (THF), 99.5% (Junsei Chemical)
  • Tetrabutylammonium fluoride (TBAF), 1 M THF solution (TCI)
  • Dichloromethane (CH2Cl2), 98% (Junsei Chemical) distilled from P2O5 and stored with 3A molecular sieves
  • N,N-Diisopropylethylamine (DIPEA), 97% (Wako Pure Chemical)
  • 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite, 97% (Wako Pure Chemical)
  • Silica gel 60N (spherical, neutral) 63- to 210-µm, (Kanto Chemical)
  • Rotary evaporator equipped with a diaphragm pump
  • 3 × 12–cm, 2 × 7–cm, 1 × 10–cm, 3 × 10–cm and 2 × 10–cm glass columns
  • Vacuum oil pump
  • TLC plate, Merck silica gel 60 F254
  • 254-nm UV lamp (for TLC)
  • Celite pad
  • Additional reagents and equipments for TLC (appendix 3D) and column chromatography (appendix 3E)

Basic Protocol 3: Synthesis, Isolation, and Characterization of Oligodeoxynucleotides Containing 5-Formyl-2¢-Deoxyuridine

 Materials
  • 5-(1,2-Diacetoxyethyl)-2¢-deoxyuridine phosphoramidite (S.14; see Basic Protocol 2)
  • Anhydrous acetonitrile (CH3CN), 99% (Dojindo)
  • Standard 5¢-O-(4,4¢-dimethoxytrityl) phosphoramidites (Glen Research):
    • Thymidine phosphoramidite
    • N-Acetyl-2¢-deoxycytidine phosphoramidite
    • N-Benzoyl-2¢-deoxyadenosine phosphoramidite
    • N-Isobutylyl-2¢-deoxyguanosine phosphoramidite
  • Argon gas (>99.99% pure)
  • 28% Aqueous ammonia (Junsei Chemical)
  • 2 M triethylammonium acetate (TEAA), pH 7.0
  • Trifluoroacetic acid (TFA), >99% (Kishida Chemical)
  • Deionized H2O
  • Buffer A: 5% CH3CN in 0.1 M TEAA (reversed-phase HPLC)
  • Buffer B: 50% CH3CN in 0.1 M TEAA (reversed-phase HPLC)
  • Buffer C: 20% CH3CN in H2O (anion-exchange HPLC)
  • Buffer D: 20% CH3CN in 2 M ammonium formate (anion-exchange HPLC)
  • NaIO4, >99.5% (Nacalai Tesque)
  • Glycerol, 99% (Wako Pure Chemical)
  • Snake venom phosphodiesterase (SVPD; Funakoshi)
  • Nuclease P1 (MP Biochemicals)
  • Alkaline phosphatase (calf intestine; Takara Bio)
  • 200 mM Tris·Cl, pH 7.7 (appendix 2A)
  • MgCl2×6H2O, 98% (Junsei Chemical)
  • Screw-capped vial
  • Tape
  • Rotary evaporator
  • 55°C incubator
  • Filtration device
  • Sep-Pak Plus C18 cartridge (Waters)
  • UV detector
  • 0.45-µm disposable syringe filter
  • 1.5-mL tube
  • HPLC columns:
    • 4.6 × 150–mm YMC J'sphere ODS-M80
    • 4.6 × 250–mm YMC J'sphere ODS-M80
    • 4.6 × 250–mm TOSOH TSK-gel DEAE-2SW
  • Centrifugal filter device (e.g., Micropure-EZ, Millipore)
  • Additional reagents and equipments for automated solid-phase oligodeoxynucleotide synthesis (appendix 3C), and purification of oligodeoxynucleotides (units 10.1, 10.4, 10.5, & 10.7)
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Figures

  •  FigureFigure 1.21.1 Scheme for synthesis of 5-formyl-2¢-deoxyuridine (see Basic Protocol 1).
    View Image
  •  FigureFigure 1.21.2 Scheme for synthesis of 5-(1,2-diacetoxyethyl)-2¢-deoxyuridine 3¢-O-phosphoramidite (see Basic Protocol 2).
    View Image
  •  FigureFigure 1.21.3 Time-course chromatograms of reversed-phase HPLC of conversion reaction from 5-(1,2-dihydroxyethyl)-2¢-deoxyuridine to 5-formyl-2¢-deoxyuridine by treatment with NaIO4 for 2 min (A) and 30 min (B).
    View Image
  •  FigureFigure 1.21.4 (A) Reversed-phase HPLC chromatograms following enzymatic digestion and (B) MALDI-TOF mass spectrum of oligodeoxynucleotide containing 5-formyl-2¢-deoxyuridine.
    View Image

Videos

Literature Cited

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