Date of Award

5-12-2012

Degree Type

Thesis

Degree Name

Honors Thesis

Department

Chemistry

First Advisor

Jeremy Johnson

Abstract

Mycobacterium tuberculosis is the pathogenic bacterial agent commonly responsible for tuberculosis, or TB. Although treatment exists for the active form of tuberculosis, no method has been developed for eliminating M tuberculosis in its dormant state. One hypothesized method for the elimination of dormant TB is to develop an inhibitor specific for M tuberculosis esterases and lipases, as these esterases and lipases are essential to the survival of dormant TB infection. In this research, the substrate specificity of the Rv0045c esterase from M tuberculosis was studied due to the essential role of Rv0045c in TB metabolism and its dissimilarity to other esterases. Two fluorogenic substrates, fluorescein dieethylbutyloxymethyl ether) and fluorescein di(phenylbutyloxymethyl ether), were designed to match the binding pocket structure of Rv0045c and to test the substrate specificity of the Rv0045c esterase. Both fluorogenic substrates were synthesized via a three step synthetic process with reasonable yields (1.63% and 5.6%) and purified using column chromatography. Correct purification of one final product was confirmed using liquid chromatography mass spectrometry (LCMS) and HNMR, giving the expected masses and NMR spectrum. This substrate was then tested for its stability to hydrolyze in water and the kinetics for activation by Rv0045c. Michaelis-Menten kinetic values were determined for the interaction between the substrate and both Rv0045c and pig liver esterase (PLE), a common esterase. The specificity constants for Rv0045c and PLE were 2.95 M-1 S-I and 20.99 M-1 s', respectively; these results indicate that the synthesized substrate shows no specificity to Rv0045c and that it would be inappropriate to use for the development of a prodrug against TB. 5

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