Synthesis and evaluation of novel 7- and 8-aminophenoxazinones for the detection of β-alanine aminopeptidase activity and the rapid identification of Pseudomonas aeruginosa in clinical samples

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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)

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.
  • References (12)
    12 references, page 1 of 2

    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.

    (1) Zaytsev, A. V.; Anderson, R. J.; Bedernjak, A. L.; Groundwater, P. W.; Huang, Y.; Perry, J. D.; Orenga, S.; Roger-Dalbert, C.; James, A. L. Synthesis and testing of chromogenic phenoxazinone substrates for β- alanyl aminopeptidase. Org. Biomol. Chem. 2008, 6, 682−692.

    (2) Komeda, H.; Asano, Y. A DmpA-homologous protein from Pseudomonas sp. is a dipeptidase specific for beta-alanyl dipeptides. FEBS J. 2005, 272, 3075−3084.

    (3) James, A. L.; Perry, J. D.; Rigby, A.; Stanforth, S. P. Synthesis and evaluation of novel chromogenic aminopeptidase substrates for microorganism detection and identification. Bioorg. Med. Chem. Lett. 2007, 17, 1418−1421.

    (4) Anderson, R. J.; Groundwater, P. W.; Huang, Y.; James, A. L.; Orenga, S.; Rigby, A.; Roger-Dalbert, C.; Perry, J. D. Synthesis and evaluation of novel chromogenic peptidase substrates based on 9-(40- aminophenyl)-10-methylacridinium salts as diagnostic tools in clinical bacteriology. Bioorg. Med. Chem. Lett. 2008, 18, 832−835.

    (5) Pendleton, J. N.; Gorman, S. P.; Gilmore, B. F. Clinical relevance of the ESKAPE pathogens. Expert Rev. Anti-Infect. Ther. 2013, 11, 297−308.

    (6) Shafikhani, S. H.; Morales, C.; Engel, J. The Pseudomonas aeruginosa type III secreted toxin ExoT is necessary and sufficient to produce apoptosis in epithelial cells. Cell. Microbiol. 2008, 10, 994− 1007.

    (7) Laine, L.; Perry, J. D.; Lee, J.; Oliver, M.; James, A. L.; De LaFoata, C.; Halimi, D.; Orenga, S.; Galloway, A.; Gould, F. K. A novel chromogenic medium for isolation of Pseudomonas aeruginosa from the sputa of cystic fibrosis patients. J. Cystic Fibrosis 2009, 8, 143−149.

    (8) Reinheimer, J. D.; Douglass, J. P.; Leister, H.; Voelkel, M. B. Aromatic Nucleophilic Substitution Reaction in Qualitative Organic Chemistry: The Reaction of 2,4-Dinitrofluorobenzene with Phenols. J. Org. Chem. 1957, 22, 1743−1745.

    (9) Kubo, I.; Kim, M.; Ganjian, I.; Kamikawa, T.; Yamagiwa, Y. Isolation, structure and synthesis of maesanin, a host defense stimulant from an African medicinal plant maesalanceolata. Tetrahedron 1987, 43, 2653−2660.

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