DEOXYADENOSINE-BASED DNA-POLYMERASE PHOTOPROBES - DESIGN, SYNTHESIS, AND CHARACTERIZATION AS INHIBITORS OF THE ESCHERICHIA-COLI DNA-POLYMERASE-I KLENOW FRAGMENT

DNA polymerase photsprobes 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate (1), 2-[(4-azidophenylsulfenyl)thio]-2'-deoxy 5'-trip hosphate (2), and 2-[(4-azido-2-nitrophenyl)thio]-2'-deoxyadenosine 5- triphosphate (3) were designed from a thermodynamic model of DNA polym erase I-substrate interactions such that the triphosphate would anchor the inhibitor and allow the phenyl azide to interact with the complem entary template binding site. Photoprobes 1-3 were synthesized by cond ensation of 2-thio-2'-deoxyadenosine or its phosphate with p-azidophen acyl bromide, N-(4-azidophenyfsulfenyl)phthalimide, and 3-azido-1-fluo ro-2-nitrobenzene, respectively, and characterized as reversible and p hotoinduced irreversible inhibitors of the DNA polymerase I Klenow fra gment 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 r anging from 0.29 to 0.55 and no apparent photodecomposition of the 2-t hio-2'-deoxyadenosine nucleotide. Photoprobes 1-3 showed mixed noncomp etitive inhibition of the Klenow fragment polymerase activity versus p oly(dA).(T)(10) as variable substrate with apparent competitive inhibi tion constants of 2.1, 36, and 29 mu M, respectively, evidence suggest ing that these photoprobes bind to both the free enzyme form and the e nzyme-template-primer binary complex. Of the three photoprobes, only n ucleotide 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 mu M and an IC50 of about 2 mu M. This evidence demonstrates that p hotoprobe 1 does bind to the Klenow fragment in the absence of templat e-primer and that it is an efficient photoprobe.