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Bedernjak, Alexandre; Zaytsev, A.V.; Babolat, M.; Cellier, M.; James, A.L.; Orenga, S.; Perry, J.D.; Groundwater, Paul; Anderson, Rosaleen (2016)
Publisher: American Chemical Society
Languages: English
Types: Article
Subjects: top_sciences
A series of novel 8-aminophenoxazin-3-one and 7-aminophenoxazin-3-one chromogens and their corresponding β-alanine derivatives were synthesized and evaluated for their ability to detect β-alanyl aminopeptidase activity in bacteria known to hydrolyse β-alanine derivatized substrates. The results provided insight into the structural requirements for effective visualization of enzymatic activity and the mechanism of formation of phenoxazinon-3-ones. 8-Aminophenoxazin-3-one substrates 23c, 23d and 23e were prepared in good to high overall yield and were selective for β-alanyl aminopeptidase activity in bacteria, producing a lighter agar background coloration facilitating visualization of colored colonies, with variable localization to the colonies, but had lower sensitivities for the detection of Pseudomonas aeruginosa in comparison to the analogous 7-aminophenoxazin-3-one substrates. The synthetic methodology employed here allows the preparation of a range of substrates for evaluation and the establishment of structure-activity relationships. For example, the 2-pentyl substituted aminophenoxazin-3-one 22b performed with analogous sensitivity to the corresponding 1-pentyl-7-aminophenoxazin-3-one substrate 1 used commercially, highlighting that the position of the pentyl substituent can be varied while maintaining detection sensitivity.
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    • 4.2.5. Individual Bacterial Species Streaked Plates. For each strain tested, a bacterial suspension was prepared in saline 0.85% at a standard inoculum of 0.5 McFarland (1.5 × 108 colony forming units per mL), using a densitometer. Using a sterile loop, 10 μL of the bacterial suspension was inoculated onto a test plate containing a particular substrate. The plates were then incubated aerobically at 37 °C and visual inspections made at 24 h and 48 h of incubation.
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