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首页> 外文学位 >Deoxynucleotide analog probes and model compounds for studying DNA polymerase structure and mechanism: Synthesis and evaluation of alkyl-, azido-, and halomethylene bisphosphonate-substituted triphosphates.
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Deoxynucleotide analog probes and model compounds for studying DNA polymerase structure and mechanism: Synthesis and evaluation of alkyl-, azido-, and halomethylene bisphosphonate-substituted triphosphates.

机译：用于研究DNA聚合酶结构和机理的脱氧核苷酸类似物探针和模型化合物：烷基，叠氮基和卤代亚甲基双膦酸酯取代的三磷酸酯的合成和评估。

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摘要

A variety of triphosphate analogs that replace a natural phosphate anhydride P-O-P linkage with a phosphonate P-CXY-P moiety have been synthesized for the characterization of DNA polymerase beta (pol beta) mechanism and structure. A suite of beta,gamma-CXY-dTTP compounds (X,Y = H, F, Cl, Br) was synthesized to complement a previous study conducted with series of beta,gamma-CXY-dGTP analogs. The high purity of the beta,gamma-CXY-dTTP following dual-pass HPLC purification is demonstrated by NMR, MS, and HPLC data and illustrates the merit of the DCC-mediated morpholidate coupling approach to Pbeta -CXY-Pgamma triphosphate mimics.;Novel alpha-azido bisphosphonates [(RO)2P(O)]2CXN 3 (R = i-Pr, X = Me; R = i-Pr, X = H; R = H, X = Me; and R = H, X = H) were developed to examine the impact of the unique azido steric profile on the synthesis and bioactivity of alpha,beta-CXN3 and beta,gamma-CXN 3 dNTP analogs. For one example, alpha,beta-CMeN3-dATP, RP HPLC separated dADP precursors that were used to generate the first examples of nucleoside triphosphate analogs with isolated stereochemistry at an asymmetrically substituted bridging carbon. The acidity constants of the alpha-azido bisphosphonic acids were determined by potentiometric titration and selected reactions including reduction and UV photolysis are introduced.;The alpha,beta-C(Me)2-dATP and (R/S)-alpha,beta-CHF-dATP analogs were synthesized and, together with the alpha,beta-CXN3 dATP analogs, the binding affinities (Kd) with pol beta were determined. (R/S)-alpha,beta-CHF-dATP was crystallized with a DNA-pol beta binary complex. X-ray structure analysis revealed only the (S)-alpha,beta-CFH-dATP diastereomer was present in the enzyme active site. Analysis of this structure provides evidence for a non-covalent stabilizing interaction between an active site water bound to Asp276 and the fluorine in (S)-alpha,beta-CFH-dATP. Molecular docking studies further demonstrate the importance of this structural water by suggesting a steric clash with the methyl group of alpha,beta-C(Me) 2-dATP.;A series of novel beta,gamma-CH2, -CHF, and -CF2 bisphosphonophosphate alkyl monoesters were synthesized and used as model compounds intended to estimate the activation parameters for the non-enzymatic hydrolysis of the triphosphate Palpha-O-Pbeta moiety. 18O-labeled water experiments showed that the exclusive site of nucleophilic attack in these systems is at Pbeta. Thus, the catalytic efficiency for a small class of diphosphokinases could be approximated and the upper limit for the rate of non-enzymatic hydrolysis at Palpha established. Supplementary compounds that increase Pbeta-O bond stability by substituting Pgamma; with a phenyl ring are introduced. Preliminary hydrolysis experiments of these compounds highlight the relative stability of the Palpha-O anhydride bond in triphosphates.
机译：为了表征DNA聚合酶β（pol beta）的机理和结构，已经合成了多种用膦酸酯P-CXY-P部分代替天然磷酸酐P-O-P键的三磷酸酯类似物。合成了一组β，γ-CXY-dTTP化合物（X，Y = H，F，Cl，Br），以补充先前对一系列β，γ-CXY-dGTP类似物进行的研究。 NMR，MS和HPLC数据证实了双程HPLC纯化后的β，γ-CXY-dTTP的高纯度，并说明了DCC介导的吗啉酸酯偶联方法与Pbeta-CXY-γ三磷酸酯类似物的优点。新型α-叠氮基双膦酸酯[（RO）2P（O）] 2CXN 3（R = i-Pr，X = Me； R = i-Pr，X = H； R = H，X = Me；并且R = H， X = H）被开发来检查独特的叠氮立体位构型对α，β-CXN3和β，γ-CXN3 dNTP类似物的合成和生物活性的影响。对于一个实例，α，β-CMeN3-dATP，RP HPLC分离了dADP前体，其用于在不对称取代的桥联碳上产生具有分离的立体化学的核苷三磷酸类似物的第一实例。通过电位滴定法确定α-叠氮基双膦酸的酸度常数，并引入包括还原和UV光解在内的所选反应。;α，β-C（Me）2-dATP和（R / S）-α，β-合成CHF-dATP类似物，并与α，β-CXN3dATP类似物一起确定与polβ的结合亲和力（Kd）。 （R / S）-α，β-CHF-dATP用DNA-polβ二元复合物结晶。 X射线结构分析表明，酶活性位点中仅存在（S）-α，β-CFH-dATP非对映异构体。对这种结构的分析提供了证据，证明与Asp276结合的活性位点水与（S）-α，β-CFH-dATP中的氟之间存在非共价稳定相互作用。分子对接研究通过暗示与α，β-C（Me）2-dATP的甲基发生空间碰撞，进一步证明了这种结构水的重要性。;一系列新型的β，γ-CH2，-CHF和-CF2合成了双膦酰基磷酸烷基单酯并将其用作模型化合物，用于估计三磷酸Palpha-O-Pbeta部分的非酶水解的活化参数。 18O标记的水实验表明，这些系统中亲核攻击的唯一位置是Pbeta。因此，可以估算一小类二磷酸激酶的催化效率，并确定在Palpha处非酶水解速率的上限。通过取代Pgamma增加Pbeta-O键稳定性的补充化合物；引入具有苯环的环。这些化合物的初步水解实验突出了三磷酸酯中Palpha-O酸酐键的相对稳定性。

著录项

  • 作者

    Chamberlain, Brian Thomas.;

  • 作者单位

    University of Southern California.;

  • 授予单位 University of Southern California.;
  • 学科 Chemistry Biochemistry.;Chemistry Organic.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 253 p.
  • 总页数 253
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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