Alpha,beta-methylene-2'-deoxynucleoside 5'-triphosphates as noncleavable substrates for DNA polymerases: isolation, characterization, and stability studies of novel 2'-deoxycyclonucleosides, 3,5'-cyclo-dG, and 2,5'-cyclo-dT

J Med Chem. 2008 Oct 23;51(20):6460-70. doi: 10.1021/jm800692a. Epub 2008 Sep 24.


We report synthesis and characterization of a complete set of alpha,beta-methylene-2'-dNTPs (alpha,beta-m-dNTP; N = A, C, T, G, 12-15) in which the alpha,beta-oxygen linkage of natural dNTP was replaced by a methylene group. These nucleotides were designed to be noncleavable substrates for DNA polymerases. Synthesis entails preparation of 2'-deoxynucleoside 5'-diphosphate precursors, followed by an enzymatic gamma-phosphorylation. All four synthesized alpha,beta-m-dNTPs were found to be potent inhibitors of polymerase beta, with K i values ranging 1-5 microM. During preparation of the dG and dT derivatives of alpha,beta-methylene diphosphate, we also isolated significant amounts of 3,5'-cyclo-dG (16) and 2,5'-cyclo-dT (17), respectively. These novel 2'-deoxycyclonucleosides were formed via a base-catalyzed intramolecular cyclization (N3 --> C5' and O2 --> C5', respectively). In acidic solution, both 16 and 17 underwent glycolysis, followed by complete depurination. When exposed to alkaline conditions, 16 underwent an oxidative deamination to produce 3,5'- cyclo-2'-deoxyxanthosine (19), whereas 17 was hydrolyzed exclusively to dT.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Chromatography, High Pressure Liquid
  • DNA-Directed DNA Polymerase / metabolism*
  • Deoxycytosine Nucleotides / chemistry*
  • Deoxycytosine Nucleotides / isolation & purification
  • Deoxycytosine Nucleotides / metabolism*
  • Deoxyribonucleotides / chemical synthesis*
  • Deoxyribonucleotides / chemistry
  • Deoxyribonucleotides / metabolism*
  • Hydrolysis
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Molecular Structure
  • Substrate Specificity


  • Deoxycytosine Nucleotides
  • Deoxyribonucleotides
  • DNA-Directed DNA Polymerase