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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
by
Brian Thomas Chamberlain
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMISTRY)
August 2012
Copyright 2012 Brian Thomas Chamberlain
Object Description
| Title | Deoxynucleotide analog probes and model compounds for studying DNA polymerase structure and mechanism: synthesis and evaluation of alkyl-, azido-, and halomethylene bisphosphonate-substituted triphosphates |
| Author | Chamberlain, Brian Thomas |
| Author email | brian.t.chamberlain@gmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Chemistry |
| School | College of Letters, Arts And Sciences |
| Date defended/completed | 2012-04-09 |
| Date submitted | 2012-05-31 |
| Date approved | 2012-05-31 |
| Restricted until | 2012-05-31 |
| Date published | 2012-05-31 |
| Advisor (committee chair) | McKenna, Charles E. |
| Advisor (committee member) |
Goodman, Myron F. Haworth, Ian S. |
| Abstract | 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 β (pol β) mechanism and structure. A suite of β,γ-CXY-dTTP compounds (X,Y = H, F, Cl, Br) was synthesized to complement a previous study conducted with series of β,γ-CXY-dGTP analogs. The high purity of the β,γ-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 Pᵦ-CXY-Pᵧ triphosphate mimics. ❧ Novel α-azido bisphosphonates [(RO)₂P(O)]₂CXN₃ (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 α,β-CXN₃ and β,γ-CXN₃ dNTP analogs. For one example, α,β-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 α-azido bisphosphonic acids were determined by potentiometric titration and selected reactions including reduction and UV photolysis are introduced. ❧ The α,β-C(Me)₂-dATP and (R/S)-α,β-CHF-dATP analogs were synthesized and, together with the α,β-CXN₃ dATP analogs, the binding affinities (Kd) with pol β were determined. (R/S)-α,β-CHF-dATP was crystallized with a DNA-pol β binary complex. X-ray structure analysis revealed only the (S)-α,β-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)-α,β-CFH-dATP. Molecular docking studies further demonstrate the importance of this structural water by suggesting a steric clash with the methyl group of α,β-C(Me)2-dATP. ❧ A series of novel β,γ-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 Pα-O-Pᵦ moiety. ¹⁸O-labeled water experiments showed that the exclusive site of nucleophilic attack in these systems is at Pᵦ. 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 Pα established. Supplementary compounds that increase Pᵦ-O bond stability by substituting Pᵧ with a phenyl ring are introduced. Preliminary hydrolysis experiments of these compounds highlight the relative stability of the Pα-O anhydride bond in triphosphates. |
| Keyword | bisphosphonates;organoazides; nucleotides; polymerase |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m |
| Contributing entity | University of Southern California |
| Rights | Chamberlain, Brian Thomas |
| Access conditions | The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given. |
| Repository name | University of Southern California Digital Library |
| Repository address | USC Digital Library, University of Southern California, University Park Campus MC 7002, 106 University Village, Los Angeles, California 90089-7002, USA |
| Repository email | cisadmin@usc.edu |
| Archival file | uscthesesreloadpub_Volume4/etd-Chamberlai-871.pdf |
Description
| Title | Page 1 |
| Contributing entity | University of Southern California |
| Full text | DEOXYNUCLEOTIDE ANALOG PROBES AND MODEL COMPOUNDS FOR STUDYING DNA POLYMERASE STRUCTURE AND MECHANISM: SYNTHESIS AND EVALUATION OF ALKYL-, AZIDO-, AND HALOMETHYLENE BISPHOSPHONATE-SUBSTITUTED TRIPHOSPHATES by Brian Thomas Chamberlain A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (CHEMISTRY) August 2012 Copyright 2012 Brian Thomas Chamberlain |
