Deoxyadenosine-based DNA polymerase photoprobes: design, synthesis, and characterization as inhibitors of the Escherichia coli DNA polymerase I Klenow fragment.
DNA polymerase photoprobes 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate (1), 2-[(4-azidophenylsulfenyl)thio]-2'-deoxyadenosine 5'-triphosphate (2), and 2-[(4-azido-2-nitrophenyl)-thio]-2'-deoxyadenosine 5'-triphosphate (3) were designed from a thermodynamic model of DNA polymerase 1-substrate interactions such that the triphosphate would anchor the inhibitor and allow the phenyl azide to interact with the complementary template binding site. Photoprobes 1-3 were synthesized by condensation of 2-thio-2'-deoxyadenosine or its phosphate with p-azidophenacyl bromide, N-(4-azidophenylsulfenyl)phthalimide, and 4-azido-1-fluoro-2-nitrobenzene, respectively, and characterized as reversible and photoinduced irreversible inhibitors of the DNA polymerase I Klenow fragment and HIV I reverse transcriptase. The aryl azides decomposed with irradiation at 300 and 350 nm with half-lives ranging from 0.98 to 2.33 min and 2.15 to 5.38 min, respectively, with quantum efficiencies ranging from 0.29 to 0.55 and no apparent photodecomposition of the 2-thio-2'-deoxyadenosine nucleotide. Photoprobes 1-3 showed mixed noncompetitive inhibition of the Klenow fragment polymerase activity versus poly(dA).(T)10 as variable substrate with apparent competitive inhibition constants of 2.1, 36, and 29 microM, respectively, evidence suggesting that these photoprobes bind to both the free enzyme form and the enzyme-template-primer binary complex. Of the three photoprobes, only nucleotide 1 photoinactivates the Klenow fragment; in the presence of a 200-fold excess of nitrene scavenger, photoprobe 1 inactivates 92% of the Klenow fragment polymerase activity with saturation observed at 9.7 microM and an IC50 of about 2 microM. This evidence demonstrates that photoprobe 1 does bind to the Klenow fragment in the absence of template-primer and that it is an efficient photoprobe.
Moore
B M
BM
Department of Medicinal Chemistry, University of Kansas, Lawrence 66045, USA.
Li
K
K
Doughty
M B
MB
D000345:Affinity Labels / Q000138:chemical synthesis; D001386:Azides; D004256:DNA Polymerase I / Q000037:antagonists & inhibitors*; D003838:Deoxyadenine Nucleotides / Q000138:chemical synthesis* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D015335:Molecular Probes / Q000138:chemical synthesis* / Q000378:metabolism; D015394:Molecular Structure; D010777:Photochemistry; D010782:Photolysis; D013056:Spectrophotometry, Ultraviolet
D000345:Affinity Labels; D001386:Azides; D003838:Deoxyadenine Nucleotides; D004791:Enzyme Inhibitors; D015335:Molecular Probes; D004256:DNA Polymerase I