Deoxyadenosine-based DNA polymerase photoprobes: design, synthesis, and characterization as inhibitors of the Escherichia coli DNA polymerase I Klenow fragment.
Deoxyadenosine-based DNA polymerase photoprobes: design, synthesis, and characterization as inhibitors of the Escherichia coli DNA polymerase I Klenow fragment.
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基于脱氧腺苷的 DNA 聚合酶光探针：作为大肠杆菌 DNA 聚合酶 I Klenow 片段抑制剂的设计、合成和表征。
DOI：
10.1021/bi952514u
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Doughty,MB
中科院分区：
文献类型：
--
作者：
Moore2nd,BM;Li,K;Doughty,MB
DNA聚合酶光探针2-[（4-叠氮苯甲酰基）硫代]-2 '-脱氧腺苷5'-三磷酸（1），2-[（4-叠氮基苯硫基）硫代]-2 '-脱氧腺苷5'-三磷酸（2），2-[（4-叠氮基-2-硝基苯基）硫代]-2 '-脱氧腺苷5'-三磷酸（3）根据DNA聚合酶I-底物相互作用的热力学模型设计，使得三磷酸盐将锚抑制剂，苯基叠氮化物与互补模板结合位点相互作用。Photoprobes 1 - 3分别由2-硫代-2 '-脱氧腺苷或其磷酸盐与对叠氮苯酰溴、N-（4-叠氮苯硫基）邻苯二甲酰亚胺和4-叠氮基-1-氟-2-硝基苯缩合而成，其特征在于是DNA聚合酶I Klenow片段和HIV I逆转录酶的可逆和光诱导不可逆抑制剂。芳基叠氮化合物在300和350 nm处的辐照下分解，半衰期分别为0.98 - 2.33 min和2.15 - 5.38 min，量子效率为0.29 - 0.55，2-硫代-2 '-脱氧腺苷核苷酸没有明显的光分解。Photoprobes 1 − 3显示了Klenow片段聚合酶活性与作为可变底物的poly（dA）·（T）10的混合非竞争性抑制作用，表观竞争性抑制常数分别为2.1、36和29 μM，这表明这些光探针既与游离酶形式结合，也与酶-模板-引物二元复合物结合。在三种光探针中，只有核苷酸1使Klenow片段光失活;在存在200倍过量的氮宾清除剂的情况下，光探针1使92%的Klenow片段聚合酶活性失活，在9.7 μM下观察到饱和，IC 50约为2 μM。这一证据表明，photoprobe 1结合Klenow片段在模板引物的情况下，它是一个有效的光探针。
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 I−substrate interactions such that the triphosphate would anchor the inhibitor and allow the phenyl azide to interact with the complementary template binding site. Photoprobes1−3were synthesized by condensation of 2-thio-2‘-deoxyadenosine or its phosphate withp-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. Photoprobes1−3showed mixed noncompetitive inhibition of the Klenow fragment polymerase activityversuspoly(dA)·(T)10as variable substrate with apparent competitive inhibition constants of 2.1, 36, and 29 μM, 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 nucleotide1photoinactivates the Klenow fragment; in the presence of a 200-fold excess of nitrene scavenger, photoprobe1inactivates 92% of the Klenow fragment polymerase activity with saturation observed at 9.7 μM and an IC50of about 2 μM. This evidence demonstrates that photoprobe1does bind to the Klenow fragment in the absence of template-primer and that it is an efficient photoprobe.