Enzymatic Synthesis of M1G‐Deoxyribose

Nathalie C. Schnetz‐Boutaud1, Marie‐Christine Chapeau1, Lawrence J. Marnett1

1 Vanderbilt University, Nashville, Tennessee
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.2
DOI:  10.1002/0471142700.nc0102s00
Online Posting Date:  May, 2001
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Abstract

Adducts formed between electrophiles and nucleic acid bases are believed to play a key role in chemically induced mutations and cancer. M1G‐dR is an endogenous exocyclic DNA adduct formed by the reaction of the dicarbonyl compound malondialdehyde with a dG residue in DNA. It is an intermediate in the synthesis of a class of modified oligodeoxyribonucleotides that are used to study the mutagenicity and repair of M1G. This unit presents methods for synthesizing M1G‐dR by enzymatic coupling.

     
 
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Table of Contents

  • Basic Protocol 1: Enzymatic Coupling Using Nucleoside 2′‐Deoxyribosyltransferase
  • Support Protocol 1: Preparation of Nucleoside 2′‐Deoxyribosyltransferase
  • Alternate Protocol 1: Enzymatic Coupling Using Purine Nucleoside Phosphorylase and Thymidine Phosphorylase
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Enzymatic Coupling Using Nucleoside 2′‐Deoxyribosyltransferase

  Materials
  • Guanine hydrochloride (Sigma)
  • 1 N HCl
  • Tetraethoxypropane (Aldrich)
  • Methanol (MeOH; Aldrich)
  • Potassium carbonate (Aldrich)
  • Nanopure water (water purified using Nanopure system from Barnstead/Thermolyne)
  • MES (2‐[N‐morpholino]ethanesulfonic acid; Sigma)
  • 2′‐Deoxycytidine (dC; Sigma)
  • 1 N NaOH
  • Nucleoside 2′‐deoxyribosyltransferase (transferase; see protocol 2)
  • Dichloromethane (CH 2Cl 2; Fisher)
  • 250‐mL round‐bottom flask
  • Oil bath, 70°C
  • Magnetic stir plate and stir bar
  • Ice bath
  • pH indicator strips
  • Büchner funnel
  • Whatman No. 1 filter paper
  • Shaking incubator, 37°C
  • Silica‐gel thin‐layer chromatography (TLC) plates
  • Lyophilizer
  • Silica gel (60 to 100 mesh; Fisher)
  • 8 × 50–cm chromatography column

Support Protocol 1: Preparation of Nucleoside 2′‐Deoxyribosyltransferase

  Materials
  • Lactobacillus broth AOAC (see recipe)
  • Lactobacillus helveticus culture
  • 0.15 M NaCl (4°C)
  • 50 mM potassium phosphate buffers, pH 6.0 and 6.9 (see appendix 2A; dilute with Nanopure water to desired molarity)
  • 50 mM potassium phosphate, pH 5.1, containing 10 g/L NaCl
  • 250‐mL Erlenmeyer flask
  • Centrifuge and rotors (e.g., Sorvall GS‐3 and SS‐34)
  • Microtip sonicator (Virsonic 100)
  • BCA Protein Assay (Pierce; optional) or equivalent

Alternate Protocol 1: Enzymatic Coupling Using Purine Nucleoside Phosphorylase and Thymidine Phosphorylase

  • Thymidine (e.g., Sigma)
  • Purine nucleoside phosphorylase (PNPase; Sigma)
  • Thymidine phosphorylase (TPase; Sigma)
  • 20 mM potassium phosphate, pH 7.3 ( appendix 2A)
  • MPLC buffer: 20% methanol in water
  • UV lamp (254 and 365 nm)
  • mPLC column (Baker C 18‐40 µm, 30 × 500 mm)
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Figures

Videos

Literature Cited

Literature Cited
   Basu, A.K. and Essigmann, J.M. 1988. Site‐specifically modified oligonucleotides as probes for the structural and biological effects of DNA‐damaging agents. Chem. Res. Toxicol. 1:1‐18.
   Beck, W.S. and Levin, M. 1963. Purification, kinetics, and repression control of bacterial trans‐N‐deoxyribosylase. J. Biol. Chem. 238:702.
   Carson, D.A. and Wasson, D.B. 1988. Synthesis of 2′,3′‐dideoxynucleosides by enzymatic trans‐glycosylation. Biochem. Biophys. Res. Comm. 155:829‐834.
   Garner, P. and Ramakanth, S. 1988. A regiocontrolled synthesis of N7‐and N9‐guanine nucleosides. J. Org. Chem. 53:1294‐1298.
   Holy, A. and Votruba, I. 1987. Facile preparation of purine and pyrimidine 2‐deoxy‐β‐D‐ribonucleosides by biotransformation on encapsulated cells. Seventh symposium on the Chemistry of Nucleic Acid Components August 30–September 5, 1987. Nucleic Acids Symp. Ser. 18:69‐72.
   Krenitsky, T.A., Kozallka, G.W., and Tuttle, J.V. 1981. Purine nucleoside synthesis, an efficient method employing nucleoside phosphorylases. Biochemistry 20:3615‐3621.
   Krenitsky, T.A., Rideout, J.L., Chao, E.Y., Koszalka, G.W., Gurney, F., Crouch, R.C., Cohn, N.K., Wolberg, G., and Vinegar, R. 1986. Imidazo[4,5‐c]pyridines (3‐deazapurines) and their nucleosides as immunosuppresive and antiinflammatory agents. J. Med. Chem. 29:138‐143.
   McNutt, W.S. 1952. The enzymically catalysed transfer of the deoxyribosyl group from one purine or pyrimidine to another. Biochem. J. 50:384.
   Muller, M., Hutchinson, L.K., and Guengerich, F.P. 1996. Addition of deoxyribose to guanine and modified DNA bases by Lactobacillus helveticus trans‐N‐deoxyribosylase. Chem. Res. Toxicol. 9:1140‐1144.
   Singer, B. and Grunnenberger, D. 1983. Molecular Biology of Mutagens and Carcinogens Plenum, New York.
   Srivasta, P.C., Robins, R.K., and Meyer, R.B. 1988. Synthesis and properties of purine nucleosides and nucleotides. In Chemistry of Nucleosides and Nucleotides (L.B. Townsend, ed.) pp. 113‐281. Plenum, New York.
   Uerkvitz, W. 1971. Purification of nucleoside 2‐deoxyribosyltransferase from Lactobacillus helveticus. Eur. J. Biochem. 23:387‐395.
Key Reference
   Uerkvitz, 1971. See above.
  Describes the purification and crystallization of the transferase.
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