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 NaHCO 3
  • Saturated aqueous NaCl (brine)
  • Anhydrous Na 2SO 4, 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 (Ac 2O), 99% (Junsei Chemical)
  • Chloroform (CHCl 3), 98% (Junsei Chemical)
  • Acetonitrile (CH 3CN), 99.5% (Junsei Chemical)
  • K 2S 2O 8, 95% (Junsei Chemical)
  • CuSO 4·5H 2O, 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 F 254
  • 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 NaHCO 3
  • Saturated aqueous NaCl (brine)
  • Anhydrous Na 2SO 4, 99% (Junsei Chemical)
  • Chloroform (CHCl 3), 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)
  • (Ph 3P) 2PdCl 2, 98% (Aldrich)
  • Tributyl(vinyl)tin (Bu 3SnCH = CH 2), 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 (OsO 4), 99.8% (Aldrich)
  • N,N‐Dimethylaminopyridine (DMAP), 99% (Aldrich)
  • Triethylamine (TEA), 98% (Junsei Chemical)
  • Acetic anhydride (Ac 2O), 99% (Junsei Chemical)
  • Tetrahydrofuran (THF), 99.5% (Junsei Chemical)
  • Tetrabutylammonium fluoride (TBAF), 1 M THF solution (TCI)
  • Dichloromethane (CH 2Cl 2), 98% (Junsei Chemical) distilled from P 2O 5 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 F 254
  • 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 protocol 2)
  • Anhydrous acetonitrile (CH 3CN), 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 H 2O
  • Buffer A: 5% CH 3CN in 0.1 M TEAA (reversed‐phase HPLC)
  • Buffer B: 50% CH 3CN in 0.1 M TEAA (reversed‐phase HPLC)
  • Buffer C: 20% CH 3CN in H 2O (anion‐exchange HPLC)
  • Buffer D: 20% CH 3CN in 2 M ammonium formate (anion‐exchange HPLC)
  • NaIO 4, >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)
  • MgCl 2⋅6H 2O, 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 ).
  •   FigureFigure 1.21.2 Scheme for synthesis of 5‐(1,2‐diacetoxyethyl)‐2′‐deoxyuridine 3′‐ O‐phosphoramidite (see ).
  •   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).
  •   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.

Videos

Literature Cited

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