The synthetic routes to the substrates based on the
The synthetic routes to the substrates based on the 2,3-dihydroxynaphthalene and 6,7-dibromo-2,3-dihydroxynaphthalene cores are depicted in Scheme 3. A Michael-type glycosylation of 2,3-dihydroxynaphthalene 16a gave the acetylated sugar 17a which was deprotected giving the required β-glucosidase substrate 18a.28, 29, 30 A photoluminescence approach for the detection of β-d-glucosidase activity (but not in microorganisms) using IWR-1-endo 18a has recently been reported. The physical and spectral data of compounds 17a and 18a were in good accord with published data. Similarly prepared were the novel dibrominated analogues 17b and 18b, both of which were associated with the large anomeric proton coupling constants and negative optical rotations confirming the presence of the β-isomers. The novel β-galactosidase substrates 20a and 20b were prepared from compounds 19a and 19b respectively by analogous procedures. The β-glucosaminidase substrate 22a was synthesised from the reaction of 2,3-dihydroxynaphthalene 16a and α-acetochloroglucosamine under basic conditions (giving the intermediate 21a) followed by deprotection. The β-glucuronidase substrate 24a was synthesised using a similar procedure to that shown in Scheme 2 for the preparation of substrate 15. The intermediate 23b was produced from the reaction of naphthalene 16b with a glucuronide trichloroacetimidate in the presence of boron trifluoride etherate. Deprotection of compound 23b, followed by treatment with cyclohexylamine then afforded the substrate 24b. The reaction of either α-d-ribofuranosyl trichloroacetimidate or 1,2,3,5-tetra-O-acetyl-β-d-ribofuranose with naphthalenes 16a and 16b in the presence of boron trifluoride etherate produced the β-anomers of the acetylated intermediates 25a and 25b. Deprotection of these two compounds afforded the required ribofuranoside substrates 26a and 26b respectively.
Evaluation of substrates In order to simplify the microbiological evaluation of the substrates, each assay comprised a representative panel of 20 clinically important microorganisms which were inoculated simultaneously onto a single Columbia agar plate. A standardised inoculum of approximately 108 colony forming units (CFU)/mL was prepared using a densitometer and 1 µL was delivered onto the agar surface using a multipoint inoculation device (final inoculum: approximately 106 CFU/spot). All assays were conducted at 37 °C in air for 18 h. For each assay, the panel of microorganisms comprised 10 Gram-negative bacteria, 8 Gram-positive bacteria and 2 yeasts. Each substrate was incorporated into the agar medium at a concentration of 300 mgL− in the presence of ammonium iron(III) citrate (500 mgL−). The substrates were added to molten agar at 50 °C, after the agar had been sterilised by autoclaving. Microorganism growth was compared to control plates in which no substrate or metal salt was present. Control plates were also prepared containing metal ions (500 mgL−) in the absence of substrates. All of the collection of microorganisms exhibited good growth on the substrate-free control plates and no growth inhibition was apparent in the presence of the metal salt. For illustrative purposes, selected substrates were subjected to additional testing in a broth-based medium. For this purpose, we selected tryptone soya broth (Oxoid) that was supplemented with ammonium iron(III) citrate (500 mgL−) before sterilization by autoclaving. The broth was then supplemented aseptically with 300 mgL− of substrate and inoculated with 106 CFU of test organism before incubation for 18 h at 37 °C. Strong growth was observed for the whole panel of microorganisms in the presence of the catechol-derived substrates 11, 13 and 15 exemplifying the non-inhibitory nature of these substrates (Table 1). Where hydrolysis of the substrates had occurred and catechol was liberated, the brown colour of the resulting iron chelate was extensively dispersed around the border of the colonies and this was attributed to diffusion of the catechol into the surrounding medium. The microbial strains showed mostly expected activity with the glucopyranoside substrate 11 with the exception of an unexpected weak positive reaction with Salmonella typhimurium, which is not known to produce β-glucosidase. The weak reaction observed with E. coli is consistent with the low level of inducible enzyme known to be produced by this species.9, 34 For substrate 13, coloration was only generated by known producers of β-galactosidase and hydrolysis of the glucuronide substrate 15 only occurred with E. coli as expected.