Document Type


Date of Degree

Fall 2013

Degree Name

PhD (Doctor of Philosophy)

Degree In


First Advisor

Daniel M. Quinn


The essential role that AChE plays in terminating the action potential triggered by ACh's pre-synaptic release is recognized by its astounding catalytic turnover rate, ca. kcat/Km = 108 M-1 s-1. AChE's adeptness at rapid hydrolysis of choline esters is largely attributed to the enzyme's two step mechanism of action initiated by acylation of the enzyme and a subsequent deacylation step. Understanding the inhibition of AChE plays an essential role in both organophosphate (OP) nerve agent poisoning as well as developing new drug treatments for neurological disorders.

Novel sarin and soman analogues were prepared that all bear a fluorescent leaving group (R = 4-methylumbelliferyl, 4-trifluoromethylumbelliferyl, or N-methyl quinolinium) to produce non-volatile inhibitors that all show irreversible inhibition for hAChE. Bimolecular inhibition rate constants (k¬i/KI = 103 to 107 M-1 min-1 range) were determined by stop-time and continuous assays, where the quarternized N-methylquinolinium sarin analogue showed the highest inhibitory potency. 4-methylumbelliferyl functionalized sarin and soman analogues were assayed for rate of aging kinetics. Tert-butoxy substituted OP analogue aged faster, agingt1/2 = 60 min at 27ºC, than its isopropyloxy and methoxy counterparts (agingt1/2 = 660 min and 9 days, respectively).

AChE exposed to an OP nerve agent undergoes rapid and irreversible inhibition in which acute exposure can be fatal. A secondary intermolecular process called aging can occur to completely deaden enzymatic activity which is unrecoverable by conventional methods. The synthesized OP analogue of sarin, (R,R' = 4-methylumbelliferyl, isopropyloxy) was utilized in the development of a bioassay procedure to evaluate the ability of novel alkylating or acylating AChE ligands to reactivate aged-AChE, termed the "resurrection" assay. Several families of compounds designed to alkylate or acylate a phosphonic oxyanion adduct of aged-AChE were screened for inhibition of apo hAChE and reactivation of aged-hAChE character. To date, no recovery of aged-AChE was observed following treatment with alkylating agents and evaluation in the "resurrection" assay.

Novel β-lactam and 6-fluoro-2-methoxy pyridinium compounds were evaluated as time dependent inhibitors of hAChE. Each inhibitor showed time-dependent inhibition of hAChE (IC50 = 24-150 µM). For the β-lactam family of inhibitors, inhibition was characterized as competitive and supported by computational docking and reaction modeling. Additional evaluation of hydrolytic stability and spontaneous reactivation of hAChE activity provided useful insight on observed biphasic inhibitory behavior for the β-lactam inhibitors. 6-fluoro-2-methoxy pyridinium showed reversible inhibition followed by a proposed nucleophilic aromatic substitution (SNAr) mechanism. Irreversible inhibition was characterized for 6-fluoro-2-methoxy pydinium via stop-time and dose response assay (ki/Ki = 6.6 x 104 M-1 min-1 and IC50 = 63 µM). Evaluation of initial rates to resolve Michaelis-Menten kinetic parameters showed non-competitive mode of inhibition, suggests slow nucleophilic attack by the active site serine supporting the SNAr mechanism.

Additionally, several [2.2] paracylcophane compounds were evaluated as potential inhibitors of AChE. Neutral and quaternized [2.2] paracylophanes (pCp) showed moderate, reversible inhibitory character (IC50 = 10-5-10-6 M range) for hAChE. Evaluation of quaternized pCp, tetrakis(4-(tetra-N-methylpyridinium)cylcobutyl) paracylcophane iodide, showed non-linear, non-competitive inhibition. Further evaluation as a peripheral active site inhibitor was confirmed with inhibition evaluation using the slow hydrolyzing substrate, p-nitrophenyl acetate.


xxii, 242 pages


Includes bibliographical references (pages 237-242).


Copyright 2013 Alexander M. Lodge

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