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

Abstract

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.

The First Synthesis of Baphicacanthin A, a Natural Phenoxazinone Alkaloid Derived from Baphicacanthus cusia

“…Finally, to provide hydroxyquinone and aniline moieties for the construction of the central 1,4‐oxazine ring of 1 , global hydrogenolysis/hydrogenation of biaryl ether 7 was performed using catalytic amount of palladium on activated carbon under hydrogen atmosphere. And we could obtain the purposed baphicacanthin A ( 1 ) after 30 h in a moderate yield (66%) without further ring‐closure reaction steps . The spectral data for the synthesized baphicacanthin A ( 1 ) were identical to the reported data.…”

“…The benzaldehyde 4 was prepared from 6 via Vilsmeier reaction in 84% yield. Baeyer–Villiger oxidation using m ‐CPBA of benzaldehyde 4 provided phenol 3 in a moderate yield (71%). Then, phenol 3 was reacted with 1‐fluoro‐2‐nitrobenzene through nucleophilic aromatic substitution to afford key intermediate biaryl ether 7 in 75% yield.…”

Pharmacological Activities of Aminophenoxazinones

“…The final study presented in this section will be the finding of new chromog nophenoxazinone derivatives with applicability for the detection of Pseudomon ginosa (a pathogenic Gram-negative bacterium) by detecting β-alanyl aminopepti tivity in clinical samples [104]. This family of compounds is characterized by the p of a β-alanyl moiety bonded to the first ring of aminophenoxazinones via an amid The agar well diffusion method was employed for the sensitivity study of 8-15.…”

Detection of β-alanyl aminopeptidase as a biomarker for Pseudomonas aeruginosa in the sputum of patients with cystic fibrosis using exogenous volatile organic compound evolution

“…28 The presence of b-alanyl aminopeptidase activity is well documented in P. aeruginosa, 29,30 and has subsequently led to studies on the development of chromogenic and uorogenic substrates targeting b-alanyl aminopeptidase activity as a method for the detection of P. aeruginosa. 31,32 There is also further evidence for b-alanyl aminopeptidase activity in Burkholderia cepacia complex and Serratia marcescens in studies using the b-alanyl based 7-N-(b-alanyl)aminophenoxazin-3-one, 31…”