The Synthesis of L-Alanyl and β-Alanyl Derivatives of 2-Aminoacridone and Their Application in the Detection of Clinically-Important Microorganisms.

Marie Cellier,Arthur L James,Stephen P Stanforth,Sylvain Orenga,John D Perry,Graeme Turnbull

doi:10.1371/journal.pone.0158378

Marie Cellier, Arthur L James + Show 4 more

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https://doi.org/10.1371/journal.pone.0158378

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Abstract

In clinical microbiology the speed with which pathogenic microorganisms may be detected has a direct impact on patient health. One important strategy used in the laboratory is the growth of cultures in the presence of an enzymatic substrate which, once transformed by the appropriate microbial enzyme, generates a detectable colour or fluorescence output. Such substrates have previously been prepared by our group and others and are available as commercial diagnostic kits, however they all suffer from some degree of diffusion when used in a solid growth medium. This diffusion complicates the detection and differentiation of species in polymicrobial cultures and so we sought to improve on our previous work. In this work we have prepared and evaluated a series of novel fluorogenic enzyme substrates based on N-substituted-2-aminoacridones. All of the prepared substrates were found to be suitable for the detection and differentiation of certain microorganisms, however those based on the 2-amino-10-benzylacridone core in particular showed no apparent diffusion when incorporated into solid growth media. On transformation these substrates generated brightly fluorescent colonies that are clearly contrasted with the background medium due to the difference in emission wavelength (λem 445–450 nm for the substrate, λem 550 nm for the product). Here we have shown that our L-alanyl aminopeptidase substrate, 2-(N-L-alanylamino)-10-benzylacridone, is particularly suited to the detection of Gram-negative bacteria, and our β-alanyl aminopeptidase substrate, 2-(N- β-alanylamino)-10-benzylacridone, to the detection of Pseudomonas aeruginosa and Serratia marcescens when grown on solid media incorporating these substrates. The resulting fluorophore shows no apparent diffusion from the colonies of interest, and the enhanced sensitivity offered by fluorescent emission may allow for the detection of these organisms as microcolonies using automated fluorescence microscopy.

Highlights

The use of enzymatic substrates as tools for the detection and identification of clinically-important microorganisms is a subject of particular interest in the health-care sector [1,2,3]
This enzyme is ubiquitous in Gram-negative microorganisms whereas it is usually absent from most Gram-positive microorganisms. β-Alanyl aminopeptidase has been detected in Pseudomonas aeruginosa, a common respiratory pathogen in cystic fibrosis patients [6]
One important strategy that has been utilised for microorganism detection and identification is to grow the microorganisms in the presence of an enzymatic substrate; microorganisms which possess the appropriate enzymes can transform the substrate into a detectable product

Summary

Introduction

The use of enzymatic substrates as tools for the detection and identification of clinically-important microorganisms is a subject of particular interest in the health-care sector [1,2,3]. Whether derived from the culture of food, clinical or environmental samples are polymicrobial in nature (i.e. they contain more than one species), and diffusion of a liberated fluorophore into the media can reduce the sensitivity of the test In this communication we report the synthesis and initial evaluation of L-alanyl and β-alanyl aminopeptidase substrates derived from N-substituted-2-aminoacridones 5a-c. These generate a highly fluorescent product which is yellow in colour (reducing the impact of cellular autofluorescence) and which does not appear to diffuse (providing high contrast to the growth medium)

Methods

Background