The synthetic route chosen for the preparation of
The synthetic route chosen for the preparation of the substrates 18 is shown in Scheme 3. Commercially available 4-aminobenzyl alcohol 13 was found to be relatively unstable to storage and hence it was prepared immediately before use by reduction of the readily available and inexpensive 4-nitrobenzyl alcohol 12. A mixed anhydride condensation of amine 13 with Boc-protected L-alanine furnished ApexPrep DNA Plasmid Miniprep Kit 14 which was reacted with methanesulfonyl chloride giving the benzylic chloride 15 directly, presumably via displacement of the mesylate group in the initially formed mesylate derivative by chloride. Compound 15 was then reacted with an appropriate heterocyclic phenol 16 under basic conditions affording the Boc-protected derivatives 17a–e. Treatment of these compounds with hydrogen chloride produced the required substrates 18a–e respectively as their hydrochloride salts.
Based upon previous studies, di-l-alanyl aminopeptidase substrates were found to be less inhibitory to Gram-positive bacteria. Thus, the substrate 22 was also prepared as part of this study in order to assess its scope of activity (Scheme 4). Removal of the Boc-group in compound 15 under acidic conditions gave compound 19 which was then subjected to a mixed anhydride coupling reaction with Boc-L-alanine affording the protected di-l-alanyl derivative 20. The reaction of compound 20 with 6-(1,3-benzothiazol-2-yl)naphthalene-2-ol 16e under basic conditions yielded compound 21 from which the required substrate 22 was obtained by treatment with hydrogen chloride (Scheme 4).
Evaluation of substrates Each substrate was evaluated in Columbia agar media (37°C in air for 18h) on a single plate against 20 clinically important microorganisms, including 10 Gram-negative bacteria, 8 Gram-positive bacteria and 2 yeasts (substrate concentration 100mgL−1). The growth of the microorganisms was compared to control plates in which no substrate was present. The Gram-negative microorganisms all grew well on the control plates whereas the Gram-positive microorganisms and the yeasts showed only moderate growth. Figure 2 depicts the arrangement of microorganisms on the agar plates and shows a representative example of an agar plate produced by incorporation of substrate 18e into the media. The coumarin substrate 18a gave intense, blue fluorescent colonies with most of the Gram-negative bacteria and moderately intense, blue fluorescent colonies with four of the Gram-positive bacteria (Streptococcus pyogenes, Listeria monocytogenes, Enterococcus faecium and Enterococcus faecalis) and also with the yeast species, Candida albicans (Table 2). There was some diffusion of the fluorescence from the colonies into the surrounding media with this substrate and this could be a potential disadvantage when investigating polymicrobial cultures obtained from pathological specimens because the diffusion of fluorescence through the agar media into surrounding colonies may not allow a clear differentiation of species that demonstrate enzyme activity. The 7-hydroxyflavone derived substrate 18b produced moderately intense, yellow fluorescent colonies with most of the Gram-negative bacteria (Table 2). Growth of the majority of the Gram-positive bacteria was inhibited by this substrate and hence no fluorescence was observed. Growth of the yeast species, Candida glabrata, was also inhibited by this substrate whereas C. albicans did show moderate growth but only produced very weak fluorescence. The substrates 18c–e all produced highly fluorescent colonies with the panel of Gram-negative microorganisms (Table 3). Thus, the benzothiazole derivative 18d gave intensely yellow fluorescent colonies with all of the Gram-negative bacteria. This substrate was inhibitory towards the Gram-positive bacteria and no growth was apparent and hence no fluorescent colonies were produced. The yeast species, C. albicans, grew moderately well and produced blue-fluorescent colonies. The substrates 18c and 18e both gave similar results to substrate 18d, except that the colonies of Gram-negative bacteria were associated with an intense green fluorescence (substrate 18c, data not shown) and a strong blue fluorescence (substrate 18e). The observed fluorescence with substrates 18c–e was restricted to the microorganism colonies and this is an advantage over our previously described substrates 10 in which noticeable diffusion of the fluorophore into the surrounding media was apparent. Additionally, some diffusion of the fluorophore is also observed with the aminocoumarin-derived substrate 8 in agar media. In accord with expectation, the di-l-alanyl substrate 22 was less inhibitory towards most of the selection of Gram-positive microorganisms compared to the mono-l-alanyl substrate 18e and consequently blue fluorescent colonies were produced with growing Gram-positive microorganisms and also with both yeasts. It is interesting to note that a chromogenic l-alanylaminopeptidase substrate based on a 9-(4-aminophenyl)-10-methylacridinium core was non-inhibitory to five of a panel of ten Gram-positive microorganisms and did not undergo hydrolysis even when microorganism growth occurred. The di-l-alanyl-analogue was even less inhibitory, allowing growth of nine of the same panel of Gram-positive microorganisms. This contrasts with our work described here and elsewhere, in which the l-alanyl fluorogenic substrates were often inhibitory to most Gram-positive microorganisms. This difference in detection profile between these two sets of fluorogenic and chromogenic substrates might be attributed to a variety of factors, including for example, the greater sensitivity of fluorogenic substrates, the higher degree of toxicity of the fluorogenic substrates to Gram-positive microorganisms, the degree of permeation of the substrates into the cell and structural differences between the substrates; the chromogenic substrates are quaternised heterocycles whereas the fluorogenic substrates are not.