Synthesis and Evaluation of Novel 7- and 8‑Aminophenoxazinones for the Detection of β‑Alanine Aminopeptidase Activity and the Reliable Identification of Pseudomonas aeruginosa in Clinical Samples

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 hydrolyze 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.