Novel enzyme substrates and cell labelling reagents for use in diagnostic microbiology

This thesis is divided into two parts; the first part describing the synthesis and evaluation of novel fluorogenic enzymatic substrates for microorganism detection, and the second part investigating the synthesis of thiophenes with extended conjugation as fluorescent labels with potential applications in antimicrobial susceptibility testing. ‘Spacer’ substrates were identified as a method of incorporating a phenolic fluorophore (ArOH) into an enzymatic substrate designed at targeting aminopeptidase activity. Substrates of the general structure ArO-spacer-AA were therefore synthesised (AA = amino acid). The action of aminopeptidases on these substrates would therefore result in fragmentation, liberating the phenolic fluorophore. The spacer group of choice was para-aminobenzyl alcohol (PABA), as it has been widely used as such for the synthesis of prodrugs. After condensation of the amine group of PABA with either Boc-L-alanine or pyroglutamic acid and subsequent chlorination of the benzylic alcohol giving the corresponding benzyl chloride, 2-(2-hydroxyphenyl)benzothiazole and 2-(2-hydroxyphenyl)benzoxazole were attached using a Williamson ether synthesis to produce the Boc-protected substrates. After removal of the Boc groups, the substrates produced strongly fluorescent colonies with Gram-negative bacteria. Also investigated were a series of fluorogenic substrates, both with and without ‘spacer’ methodology, designed for the detection of nitroreductase activity but little activity was observed with bacteria. Three BODIPY-derived substrates were also prepared and evaluated for detecting nitroreductase and esterase activity. These substrates showed good activity in agar media when the agar plates were post-treated with acid resulting in the generation of strong colour and fluorescence. The second section of this thesis focuses on antimicrobial susceptibility testing. Two types of substrates possessing highly conjugated thiophene cores were synthesised: hydrazides and D-amino acids. The hydrazides are expected to label dead cells by reacting with aldehyde groups that are produced by protein carbonylation. The D-amino acid series of compounds are expected to be incorporated into the cell walls of growing bacteria, and hence live cells can be labelled. The thiophene core structures were chosen to have excitation wavelengths of approximately 488 nm because this excitation wavelength is commonly used in flow cytometry. Results from fluorescence measurement testing indicate that both sets of cell labelling substrates synthesised have an excitation wavelength of roughly 450 – 460 nm with emission wavelengths ranging from 480 nm to as high as 520 nm.