Side-chain conformational restriction in template-competitive inhibitors of E. coli DNA polymerase I Klenow fragment: synthesis, structural characterization and inhibition activity

Nucleosides Nucleotides Nucleic Acids. 2004;23(11):1751-65. doi: 10.1081/NCN-200034042.


Nucleotide triphosphate alpha-(4-azidophenyl)-1,N6-etheno-dATP 3 and its monophosphate 3m were synthesized by condensation of 2-halo-2-(4-azidophenyl)acetaldehyes with dATP and dAMP, respectively. Structure analysis shows that the azidophenyl side chain is attached to the alpha-position of the etheno ring (i.e., the carbon attached to N1 of the purine), and conformation calculations show minima in the etheno-phenyl bond rotation at 50 and 130 degrees where the bulk of the phenyl ring projects out from the plane of the etheno group. Like DNA Pol inhibitor 2-(4-azidophenacyl)thio-2'-deoxyadenosine 5'-triphosphate 1, nucleotide 3 is a template-competitive DNA polymerase inhibitor (TCPI), with a competitive Ki for Pol I KF of 3.41 microM, but has only weak activity as an HIV RT inhibitor relative to the template-competitive reverse transcriptase inhibitor 2-(4-azidophenacyl)thio-1,N6-etheno-2'-deoxyadenosine 5'-triphosphate 2. Additionally, 3 photoinactivates KF in a time-dependent manner, confirming the kinetic data that 3 binds to the free form of KF. The TCPI activity of 3 provides evidence for an extended side chain conformational preference in the combined substrate polymerase inhibitors.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine / analogs & derivatives
  • Azides / chemistry
  • Azides / metabolism
  • Binding, Competitive
  • DNA Polymerase I / antagonists & inhibitors*
  • DNA Polymerase I / chemistry
  • Enzyme Inhibitors / chemical synthesis*
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology*
  • Escherichia coli / enzymology
  • HIV Reverse Transcriptase / antagonists & inhibitors
  • Humans
  • Phenol / chemistry
  • Phenol / metabolism
  • Protein Conformation
  • Reverse Transcriptase Inhibitors / chemical synthesis
  • Reverse Transcriptase Inhibitors / chemistry
  • Reverse Transcriptase Inhibitors / pharmacology


  • Azides
  • Enzyme Inhibitors
  • Reverse Transcriptase Inhibitors
  • Phenol
  • HIV Reverse Transcriptase
  • DNA Polymerase I
  • Adenosine