Abstract/Summary

The increasing level of threat of antimicrobial resistance due to less effective treatment of, and prevention against, bacterial infections is of growing concern. The Review on Antimicrobial Resistance predicts an estimated death toll of 10 million people a year by 2050, which is higher than the estimated mortality rate for cancer and diabetes combined. For decades, only a few classes of antibiotics have been developed by pharmaceutical companies. The lack of new antibiotics has led to the emergence of resistance for older drugs. Inappropriate and overconsumption of antibiotics are still common in healthcare and agricultural settings. To preserve the efficacy of existing antibiotics and to circumvent the urgency for discovering new drug treatments, one of the most effective ways to manage antimicrobial resistance is through developing rapid, point-of-care diagnostic tests. One of the most common and well-established protocols to identify bacteria is the incorporation of synthetic enzyme substrates in solid culture media. Indoxyl glycosides have been widely and successfully used as chromogenic substrates for the detection and presumptive identification of bacterial pathogens. Based on the presence of glycosidase enzymes in the bacteria, the overall aim of this work was therefore to synthesise and analyse two previously synthesised fluorinated indoxyl β-galactosides, and four novel fluorinated indoxyl β-galactoside and β-glucosides to complement the existing range of commercially available substrates. Preparation of indoxyl glycosides is recognised to be challenging. Therefore, in this project, one of the main objectives was to develop an efficient synthesis of the substrates by following the method by Böttcher and co-workers. Glycosidation via phase-transfer catalysis proved to be effective and this was successfully achieved for 1,2-trans glycosides. The per-O-acetyl fluorinated indoxylic acid allyl ester β-galactosides and β-glucosides were isolated with yields of 85-95% and 78-90%, respectively. The acetylated substrates were deprotected via decarboxylation using silver-mediated reaction, followed by Zemplén de-O-acetylation to furnish the final fluorinated indoxyl β-galactosides and β-glucosides with varying yields from 19-31% and 48-90%, respectively. The overall yield for the glycosylation route for β-galactosides and β-glucosides varied from 7-21% and 21-58%, respectively. Attempts to synthesise unsubstituted indoxyl rhamnoside via phase-transfer catalysis and using trimethylsilyl trifluoromethanesulfonate as a promoter were unsuccessful. The chromogenic properties of the fluorinated indoxyl β-glycosides were evaluated against several microorganisms in Nutrient Agar and Tryptone Soya Agar. Hydrolysis of the six fluorinated derivatives normally produced dark olive green and midnight blue colours that were highly restricted within microbial colonies. The new substrates colours are different from conventional substrates. Interestingly, the β-galactosides and β-glucosides were more highly sensitive for the detection of Escherichia and Staphylococcus species compared to commercially well-known chromogenic substrates 5-bromo-4-chloro-3-indolyl β-D-glucoside and 5-bromo-4-chloro-3-indolyl β-D-galactoside in Tryptone Soya Agar. Therefore, the colours and identification profiles of the derivatives complement the existing chromogenic substrates. Furthermore, the hydrolysis of two substrates 5-bromo-4-chloro-3-indolyl β-Dglucoside and 6-trifluormethyl-3-indolyl β-D-glucoside were analysed using quantitative HPLC analysis. The study revealed that 5-bromo-4-chloro-3-indolyl β-D-glucoside (X-β-D-gluc) formed a stronger affinity to the β-glycosidase enzyme than 6-(trifluormethyl)- 3-indolyl β-D-glucoside (CF3-β-D-gluc), suggesting that X-β-D-gluc reached saturation quicker than CF3-β-D-gluc. Therefore, the research work detailed in this project provided new and further insights on the applications of chromogenic substrates in culture media.