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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

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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
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