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DESIGN AND SYNTHESIS OF A SERIES OF METHYLENEBISPHOSPHONATES:
A NUCLEOTIDE ANALOGUE TOOLKIT TO PROBE NUCLEIC ACID
POLYMERASE STRUCTURE AND FUNCTION
by
Thomas George Upton
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMISTRY)
August 2008
Copyright 2008 Thomas George Upton
Object Description
| Title | Design and synthesis of a series of methylenebisphosphonates: a nucleotide analogue toolkit to probe nucleic acid polymerase structure and function |
| Author | Upton, Thomas George |
| Author email | tupton@usc.edu; tom.upton@cenomedbio.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Chemistry |
| School | College of Letters, Arts and Sciences |
| Date defended/completed | 2008-06-23 |
| Date submitted | 2008 |
| Restricted until | Restricted until 4 Feb. 2009. |
| Date published | 2009-02-04 |
| Advisor (committee chair) | McKenna, Charles E. |
| Advisor (committee member) |
Goodman, Myron F. Haworth, Ian |
| Abstract | A stereoelectronically varied series of alpha-substituted methylenebisphosphonic acids (X,Y = H, F, Cl, Br, CH3) was synthesized and used to prepare corresponding dNTP beta,y-CXY and also certain alpha,beta-analogues. Improved analytical and preparative HPLC methods are described for the NTP analogues. The pKa4 values of the bisphosphonic acids were determined under self-consistent conditions, enabling a "toolkit" of pyrophosphate analogues for kinetic and structural studies of DNA polymerase mechanisms and fidelity. The dNTP analogues are substrates for DNA pol beta and the kpol values of the beta,y-CXY analogues were compared to the pKa4 of the relevant bisphosphonate (BP) leaving group. For BPs with a pKa4 less than or equal to that of pyrophosphoric acid, log kpol values were similar or decreased moderately with increasing pKa4. For BPs with a pKa4 greater than that of pyrophosphate a significant trend of decreasing log kpol was apparent. This observation, and the absence of an analogous effect on ground state analog binding (Kd values, except for the CBr2 analogue), points to an influence of the leaving group aptitude on the energy of the transition state. Reduced catalysis rates were observed with the dihalo-substituted substrates particularly for the T-G mispair incorporation. X-ray crystallographic studies with several of Palpha-Z-Pbeta (Z = NH, CH2, CF2) and Pbeta-CXY-Py dNTP analogues in ternary complex with DNA pol beta and DNA primer-template strand have been carried out. Evidence consistent with a docking study was obtained for a C-F⋅⋅⋅H-N-Arg interaction in the beta,y-CHF-dGTP analogue -- active site complex, in which only the (R)-diastereomer of the analogue is observed. The synthesis of new bisphosphonates incorporating a CF3-group was also investigated. The dNTP analogue "toolkit" should prove useful to probe structure and function in other DNA polymerases and to refine theoretical studies of the enzymes mechanisms. |
| Keyword | DNA polymerase beta; bisphosphonates; titrations; fluorination; trifluoromethylation; halogenation; nucleotide analogues; stereoselectivity; autodock; docking; inhibitor design; HIV reverse transcriptase; polymerase kinetics |
| 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-m1540 |
| Rights | Upton, Thomas George |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | http://www.usc.edu/isd/libraries/services/ask_a_librarian/email/ |
| Filename | etd-Upton-2311 |
| Archival file | uscthesesreloadpub_Volume17/etd-Upton-2311.pdf |
Description
| Title | Page 1 |
| Full text | DESIGN AND SYNTHESIS OF A SERIES OF METHYLENEBISPHOSPHONATES: A NUCLEOTIDE ANALOGUE TOOLKIT TO PROBE NUCLEIC ACID POLYMERASE STRUCTURE AND FUNCTION by Thomas George Upton A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (CHEMISTRY) August 2008 Copyright 2008 Thomas George Upton |
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