In this paper we report the synthesis and microbiological
In this paper, we report the synthesis and microbiological evaluation of a series of novel chromogenic sugar-based enzyme substrates based upon catechol, 2,3-dihydroxynaphthalene and 6,7-dibromo-2,3-dihydroxynaphthalene 9 cores (Fig. 1).14, 15 2,3-Dihydroxynaphthalene is inexpensive and available in large quantities (>100 g) from several commercial suppliers thus making this an ideal starting material for the synthesis of enzyme substrates. Additionally, halogen atoms can be introduced into this ring-system remote from the hydroxyl-groups, i.e. at the 6,7-positions, whereas the introduction of halogen atoms into known substrates such as compounds 6 and 7 would only be possible adjacent to the hydroxy-groups which may have a detrimental effect on glycosidase activity. We anticipated that the introduction of halogen atoms would be beneficial for reducing triphosphate of chelates in solid (agar) media. We envisaged that catechol-derived substrates would have potential applications in liquid media (where the resulting metal chelates would require appreciable aqueous solubility) and that the increased size of the naphthalene-derived substrates 9 would potentially generate a more insoluble end-point better suited for use in solid (agar) media, where diffusion of the chelate must be localised within colonies of microorganisms. The sugar components of structures 9 have been chosen to target a broad range of enzymatic activities across a range of clinically important pathogenic microorganisms. The sugar moieties together with illustrative applications in diagnostic microbiology include: (i) β-d-glucopyranosides (for the detection of enterococci and Listeria monocytogenes), (ii) β-d-galactopyranosides (for the detection of coliforms), β-d-glucuronides (for the detection of Escherichia coli), N-acetylhexosaminides (for the detection of the pathogenic yeast, Candida albicans) and β-d-ribofuranosides (for the detection of Staphylococcus aureus, including MRSA). Catechol β-d-ribofuranoside has previously shown efficacy for S. aureus detection in liquid media.
Synthesis of substrates Catechol 2,3,4,6-tetra-O-acetyl-β-d-glucopyranoside 1018, 19, 20, 21 was prepared from catechol in low yield using a Michael-type glycosidation procedure (Scheme 2). A Zemplén deprotection of compound 10 gave the required β-glucosidase substrate 11. The proton-NMR spectral data of compounds 10 and 11 were consistent with those reported in the literature with large anomeric coupling constants confirming the β-configurations at the anomeric centres.20, 22 The direct reaction of glucose and catechol has been reported to give a 95:5 ratio of α:β anomers in low (11%) overall yield. 2,3,4,6-Tetra-O-acetyl-β-d-galactopyranoside 12 was prepared using a Michael-type glycosidation reaction and after deprotection, the β-galactosidase substrate 13 was obtained. The coupling constant for the anomeric proton (7.7 Hz, d6-DMSO) and the chemical shift of C-1 (104.0 ppm, d6-DMSO) in the proton and carbon NMR spectra of compound 13 respectively, confirmed the presence of a β-glycoside. The tetraacetyl derivative 12 has been described previously in the literature and was reported as comprising a mixture of both α- and β-anomers. Somewhat surprisingly, the substrate 13 appears to be novel although the synthesis of its isomer with the α-configuration has been claimed but no NMR-spectral data was disclosed to support this assignment. Additionally, the large negative optical rotation (–33° in DMSO) reported for this proposed α-anomer structure is more aligned to the value expected from a β-d-galactopyranoside. The protected glucuronide derivative 1425, 26 was prepared from catechol following a similar procedure to that reported in the literature. Deprotection of compound 14 under basic conditions, followed by acidification using an ion-exchange resin, afforded the required β-glucuronic acid derivative which was conveniently isolated as the cyclohexylamine salt 15. The impure glucuronic acid has been proposed as a catechol metabolite produced from rabbits but this compound was not characterised directly. Methylation, per-acylation and finally hydrolysis of the metabolite gave 2-methoxyphenol (guaiacol) which suggested the rabbit metabolite was a mono-glucuronide.