Year of Award

2008

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Chemistry

Department or School/College

Department of Chemistry

Committee Chair

Charles M. Thompson

Commitee Members

Mark Cracolice, C. Sean Esslinger, J. B. Alexander (Sandy) Ross, Holly Thompson

Keywords

asymmetric phosphonates

Abstract

Organophosphates (OPs) react with acetylcholinesterases (AChEs) to form a covalent bond at a specific serine residue in the active gorge, thereby providing a highly precise modification of this enzyme and an opportunity to explore protein structure, function and mechanism. The goal of this study is: 1) to show how covalent modification of AChEs by chromophore-linked OPs can be used to probe the active and peripheral sites of the protein and its local environment; 2) to differentiate AChE stereoselectivity with asymmetric phosphonothiolates.

Click chemistry was used as a key transformation method to prepare an array of chromophores linked to a reactive fluorophosphonate (FP) head group. Chromophore-linked FPs varying in length and chromophore were synthesized and computer-modeled to visualize and calculate positioning of the chromophore-FP-AChE relative to the protein active gorge. The inhibition rates of chromophore-FPs against recombinant mouse AChE (rMAChE) and electric eel AChE (EEAChE) were determined via colorimetric assay and the dansyl containing FPs were demonstrated to be the most potent. In addition, the binding effects of the chromophore and FP moiety to protein were evaluated and results demonstrated that the size and structure of chromophore and the length of the ligands mutually affect the inhibition potency. Dansyl, dabsyl and pyrene were the best chromophores with least interaction with AChEs.

To examine the interactions of asymmetric analogs of the OP compounds within the steric confines of AChEs, the synthesis of phosphonothiolate enantiomers as anti-AChEs was conducted using a chiral auxiliary for separation. X-ray analysis and 31P NMR were used to show an exclusively separation of the two diastereomers. The kinetic parameters ki and KD for the inhibition of recombinant human AChE (rHuAChE) were determined. A 4-fold difference in anti-AChE potency was observed between Sp (ki = 1.7 x 103 M-1min-1) and Rp (ki = 9.0 x 103 M-1min-1). These enantiomers link to the chromophores via click reaction to form chromophore-linked asymmetric phosphonothiolates (CLAPs), which can be used to study AChE stereospecificity.

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© Copyright 2008 Lilu Guo